Extended Abstracts forthe 46th International Symposium of
the Princess Takamatsu Cancer Research Fund

ONCO-METABOLOMICS; A NEW CLUE TO
UNDERSTAND CARCINOGENESIS,CANCER BIOLOGY AND TO DEVELOP NOVELDIAGNOSTICS AND THERAPEUTICS
November 17-19, 2015
Tokyo, Japan
Edited by

Hiroyasu Esumi
Tak W. Mak
Tomoyoshi Soga
Makoto Suematsu
Masaki Mori

Princess Takamatsu Cancer Research Fund

CopyrightⒸ2016 by the Princess Takamatsu Cancer Research Fund
No part of this Extended Abstracts may be reproduced, stored in retrieval
systems or transmitted, in any form or by any means, without permission
from the Princess Takamatsu Cancer Research Fund.

Princess Takamatsu Cancer Research Fund

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Telephone: +81-3-3441-0111
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E-mail:info@ptcrf.or.jp
Printed in Japan
ISSN 2186-8603

B

CONTENTS
KEYNOTE LECTURE

Cancer Metabolism: Back to the Future
Tak W. Mak .........................................................................................................................................1

RECASTING THE WARBURG EFFECT

The Role of Metabolism in Cancer Progression
Matthew G. Vander Heiden ..................................................................................................15
Metabolic Heterogeneity in Cancer Cells and Tumors
Ralph J. Deberardinis.................................................................................................................21
The Metabolic Regulator AMPK - Friend or Foe in Cancer
D. Grahame Hardie.....................................................................................................................26
MYC-mediated Perturbation of Metabolism and Therapy
Chi V. Dang.......................................................................................................................................33

Metabolic Regulation by RB Tumor Suppressor Gene
Chiaki Takahashi ..........................................................................................................................37iMETABOLIC REGULATION OF GENE FUNCTION

Fumarate Metabolism in Cancer and Type 2 Diabetes
Julie Adam ........................................................................................................................................40
IDH Mutations and Therapeutic Strategy in Cancer
Issay Kitabayashi ..........................................................................................................................47
Epigenetic Mechanisms in Hematologic Malignancies Linked to
Metabolic Pathways
Ari M. Melnick ...............................................................................................................................53

GASEOUS REGULATION AND METABOLISM

Carbon Monoxide-Responsive Heme Proteins Regulate Cancer
Proliferation and Chemoresistance
Makoto Suematsu ........................................................................................................................58
Heme Oxygenase-1 and Metabolic Reprogramming in Response to
Ischemia
Roland Stocker ...............................................................................................................................62
Protein-Bound Reactive Sulfur Species and Its Translational Biosynthesis:
Potential Implication for Oxidative Stress and Tumor Biology
Takaaki Akaike ...............................................................................................................................65

ii

STRESS RESPONSE, OXYSTRESS

Identification of a Metabolic Pathway for Activation of the Hypoxia Gene
Regulatory Response in Cancer
Lorenz Poellinger .........................................................................................................................69
Cytoprotection and Metabolic Reprogramming Governed by KEAP1NRF2 System
Hozumi Motohashi .....................................................................................................................71
Mitochondria Regulate Cancer
Navdeep S. Chandel...................................................................................................................76

MICROENVIRONMENT AND
METABOLIC HETEROGENEITY

mTOR Growth Signaling and Metabolism
David M. Sabatini ........................................................................................................................82
Serine Metabolism in Cancer Development
Karen H. Vousden ........................................................................................................................84
Tumor Microenvironment and Cancer Metabolism
Tsuyoshi Osawa ............................................................................................................................88iii

METABOLIC ASPECTS OF CELL-CELL INTERACTION

Dialogues between Macrophage and Tumor Cells Promote Malignancy
Jeffrey W. Pollard ..........................................................................................................................92
Cell Competition and Warburg EffectYasuyuki Fujita ..............................................................................................................................99
Innovative Medicine for Metabolic Reprograming of Intractable Cancer
Stem Cells
Hideshi Ishii ...................................................................................................................................103

NAKAHARA MEMORIAL LECTURE

PI Kinases and Cancer Metabolism
Lewis C. Cantley .........................................................................................................................109

TARGETING METABOLISM: CLINICAL AND
PRECLINICAL CHALLENGE

Targeting Cancer Microenvironment-Specific Metabolism Reorganizes
Tumor Vasculature
Hiroyasu Esumi ...........................................................................................................................113

Inhibitors of Mutated Isocitrate Dehydrogenase as a Potential New
Therapy for AML: from the Bench to the Clinic
David P. Schenkein ....................................................................................................................117iv

Large-Scale Pooled Shrna Screens and the Discovery of Novel Cancer
Targets for Drug Discovery
William R. Sellers........................................................................................................................125

CLOSE ENCOUNTERS OF METABOLISM,
THE THIRD KIND?

What Causes Altered Metabolism in Colon Cancer Cells?
Tomoyoshi Soga ..........................................................................................................................127
Nutrient Sensing and Metabolic Reprograming in the Tumor Stroma
Jorge Moscat...................................................................................................................................129
The Microbiome and Microbial-Derived Metabolites in Carcinogenesis:
The Fiber-Microbiota-Butyrate Axis in Histone Acetylation and Tumor
Suppression as a Case Study
Scott J. Bultman ...........................................................................................................................132

CONCLUDING KEYNOTE LECTURE

Beyond Glucose and Glutamine: Some Cancer Cells Utilize Extracellular
Macromolecules to Fuel Cell Growth
Craig B. Thompson ...................................................................................................................134

CONCLUDING REMARKS

Hiroyasu Esumi ...........................................................................................................................138v

List of Participants ................................................................................................................................140
List of Previous International Symposia Series .............................................................167

vi

1

CANCER METABOLISM: BACK TO THE FUTURE

Tak W. Mak

The Campbell Family Institute for Breast Cancer Research
Ontario Cancer Institute, University Health Network
610 University Avenue, 9th Floor, Room 406, Toronto, Ontario M5G 2M9, Canada(tmak@uhnres.utoronto.ca)

Why has it been so difficult to cure cancer? The answer lies in our inability to define all

1. Introduction

the elements that contribute to the transformation and survival of tumor cells, andchallenges in dissecting the body’s responses to these malignant growths. As far back as

1924, Otto Warburg proposed that “The cause of cancer is the replacement of therespiration of oxygen in normal body cells by a fermentation of sugar”, a concept now

widely known as the “Warburg effect” 1,2) . Because this change would not alter the“antigenic face” of a cancer cell, the immune system would not be able to recognize theseaberrant cells and remove them, necessitating externally imposed therapies.

Oncologistsof the day therefore focused on eliminating all fast-replicating cells, normal or cancerous,

by radiation or chemotherapy, a trend that remained firmly in place into the 1990s (Table1). In the 1970s, Warburg’s hypothesis was sidelined as scientists became convinced that
Table 1 Chemotherapeutic agents approved.
Year Approved

Agent

1942

Nitrogen mustard

1948

6-Mercaptopurine

1958

Methotrexate

1959

Cyclophosphamide

1975

5-Fluorouracil

1978

Cisplatin

1992

Pacitaxel

1996

Gemcitabine

1996

Topotecan

2004

Pemetrexed

Chemotherapeutic agents approved.

2

T. W. Mak

the underlying cause of cancer was aberrant function of either oncogenes or tumor

suppressor genes. This mindset led to the development of numerous therapeutic agentstargeting specific oncogenes or other relevant molecules or structures (Table 2). Althoughanti-oncogene agents are undeniably helpful, it has become clear that the cancer cell
genome is too varied and oncogenes too numerous for these strategies to be able toeradicate all tumors. Researchers have instead returned to Warburg’s hypothesis in theirsearch for ways to disrupt tumor cell metabolism and mitosis.
Table 2

Targeted agents approved.

Year of approval

TargetDrug name

1998

Her-2

Herceptin

2001

Bcr-ablImatinib

2003

EGFR

Gefitinib

2003

Proteasome

Bortezomib

2004

VEGF

Bevacizumab

2006

HDAC

Vorinostat

2007

mTOR

Temsirolimus

2011

CTLA-4

Ipilimumab

2011

ALK

Crizotinib

2011

B-Raf

Vemurafenib

2012

Hedgehog

Vismodegib

2013

Btk

Ibrutinib

2014

PI3K-G

Idelalisib

2014

anti-PD1

Nivolumab

2014

PARPi

Olaparib

2015

CDK4/6

Abemaciclib

T

In addition to their genetic and epigenetic alterations, cells undergoing transformation

2. Targeting Cancer Cell Metabolism

implement specific metabolic adaptations that are induced by their alteredmicroenvironment. These adaptations lead to upregulation of signaling pathways that are

not inherently tumorigenic but allow developing tumor cells to survive under conditions
that would kill normal cells3). This “metabolic addiction” of precancerous and ultimately

cancerous cells provides new opportunities for therapeutic intervention, since normal cells,

which have not had to endure the same stresses, should be unaffected by agents targetingcancer cell metabolic adaptations.

Keynote Lecture

3

CPT1C, a brain-specific metabolic enzyme, may be involved in tumor cell metabolic

2.1 Targeting Carnitine Palmitoyltransferase-1C (CPT1C)

adaptation to heightened stress4). CPT1C expression correlates inversely with mTOR

pathway activation in tumor cells4,5) and contributes to rapamycin resistance in murine

primary tumors. CPT1C is overexpressed in human non-small-cell lung carcinomas4), andincreases fatty acid oxidation, ATP production, and resistance to glucose deprivation or
hypoxia in cultured human cancer cells4). CPT1C depletion reduces tumor growth inmouse xenograft models4) and increases mouse survival in a neurofibromatosis type I

tumor model6-8). CPT1C is a p53 target gene, and CPT1C expression is induced by hypoxia

or glucose deprivation in a p53- and AMPK-dependent manner8). Thus, p53 may initially

protect cells from metabolic stress via CPT1C induction, but excessive CPT1C expression

then promotes carcinogenesis (Figure 1)8). Because CPT1C expression is normally restricted

to the brain, and most drugs cannot penetrate the blood–brain barrier, a small-molecule
CPT1C inhibitor may be ideal for treatment of hypoxic and otherwise treatment-resistantcancers5,8).

Figure 1 The role of CPT1c in tumor cell metabolic adaptation.

IDH1 and IDH2 govern the NADP/NADPH ratio in a cell’ s cytoplasm and

2.2 Targeting Mutated Isocitrate Dehydrogenases (IDH)

mitochondria, respectively. IDH1/2 normally convert isocitrate to a-ketoglutarate (aKG)

while reducing NADP to NADPH and liberating CO29). Oncogenic IDH mutations werefirst discovered during cancer genome sequencing projects10-12). IDH1 mutations occur athigh frequency in glioblastoma multiforme (GBM)11), acute myeloid leukemia (AML)10),

cholangiocarcinoma13,14), and chondrosarcoma15), and less frequently in melanoma, NSLLC,
and prostate and colon cancers16). IDH2 mutations9,17) occur in cholangiocarcinoma13,14),

myelodysplastic syndrome (MDS) and myeloproliferative disorder (MPD)18-20), AML10),

4

T. W. Mak

chondrosarcoma15), angioimmunoblastic T-cell lymphoma (AITL)21), and D-2HG aciduria22)(Figure 2). In 2009, scientists at Agios Pharmaceuticals made the breakthrough discovery
that the tumorigenic effect of IDH1/2 mutations is not due to a loss-of-function of these

proteins. Instead, the mutant IDH enzymes acquire a neomorphic activity in which thenormal product aKG is converted to 2-hydroxyglutarate (D2HG) in a reaction that

consumes, rather than produces, NADPH 9 , 2 3 ) . D2HG competitively inhibits 2-OGdependent dioxygenases (2OGD) 9) , more than 60 of which are involved in collagenbiosynthesis, fatty acid metabolism, DNA repair, RNA and chromatin modifications, andhypoxia detection24). Additional potential targets of D2HG inhibition include TET proteins

involved in DNA methylation, JumonjiC domain-containing histone demethylases, prolylhydroxylases (PHD) and lysyl hydroxylases (LHD) required for collagen folding andmaturation, and PHDs that regulate hypoxia-inducible factor (HIF) signaling9).

Our group generated conditional knock-in mouse strains allowing expression of themutant IDH1 protein from the endogenous locus25). Systemic expression of mutant IDH1

leads to D2HG production that is embryonic lethal9). When expressed solely in the myeloid
compartment, mutant IDH1 causes splenomegaly, decreased bone marrow cellularity, andextramedullary hematopoiesis 25) . Mutant hematopoietic progenitors show increased

hypermethylation of CpG sites and histones25), consistent with the DNA methylation

changes observed in human IDH1/2 gliomas26) and AML27). In early 2014, an oral inhibitor(AG-221) of mutant IDH2 underwent clinical evaluation for patients with advanced IDH2-

mutant hematologic malignancies. AG-221 treatment reduced D2HG levels anddemonstrated a dose-dependent survival benefit28). Pursuit of a similar compound tocombat IDH1-mutant cancers is ongoing.

Figure 2

Percentages of patients with the indicated disorders that have the indicated IDH1 or IDH2
mutations.

Keynote Lecture

5

An important stressor in cancer cells is the level of reactive oxygen species (ROS). ROS

3. Targeting Reactive Oxygen Species (ROS)

regulation is critical for normal cellular functions and survival, and the accelerated growthof tumor cells generates increased ROS. Cancers therefore need to adjust signaling

pathways linked to ROS regulation to cope with their enhanced ROS. Elevated ROS are
generated by hypoxia, defective metabolism, endoplasmic reticulum (ER) stress, andoncogene activity29). Conversely, ROS are eliminated routinely via NADPH, glutathioneand dietary antioxidants, and under stress conditions through the activation oftranscription factors such as NRF2 and the activity of tumor suppressors such as BRCA1,

p53, PTEN and ATM29). During transformation, cellular ROS levels progressively increase,

triggering the cell to upregulate antioxidant pathways. Targeted therapeutics that interferewith this upregulation may thus induce apoptotic death. Alternatively, agents that increase

ROS production beyond the capacity of the upregulated antioxidant mechanisms to cope

may kill the tumor cell while sparing normal cells in which these pathways are notactivated.

Our group has shown that ROS regulation may explain the tissue specificity of BRCA1-

related cancers, which occur almost exclusively in breast and ovary. BRCA1 deficiency

enhances ROS levels in breast cancer cells and impairs Nrf2-driven antioxidant pathways30).
BRCA1 directly interacts with Nrf2 in a manner that affects Keap1-mediated Nrf2

ubiquitination, stability and activation30). Estrogen treatment partially restores Nrf2 levels

and enhances tumor growth in the absence of BRCA130,31). We hypothesize that, in tissues

lacking estrogen, BRCA1 deficiency impairs Nrf2 antioxidant signaling, leading to an

accumulation of ROS in BRCA1-deficient cells that kills them. However, in breast andovary, estrogen activates Nrf2 via a mechanism that depends on PI3K–AKT and protects

BRCA1-deficient cells from ROS-induced death. If such a BRCA1-deficient cell also loses

PTEN, the PI3K–AKT pathway may reinforce estrogen-mediated Nrf2 signaling. Mitogenic

and antioxidant pathways acting downstream of AKT, coupled with the genomic

instability caused by a lack of BRCA1-mediated DNA repair, might then drive complete
transformation of BRCA1-deficient cells31) (Figure 3). Exploitation of the altered ROS

regulation in these cells may point to an effective future therapy.

Other antioxidant pathways appear to play the opposite role. We have demonstrated

that GCLM-driven synthesis of the antioxidant glutathione (GSH) is required for cancer
initiation. Genetic loss of Gclm in mice prevents precancerous cells from undergoingtransformation. Buthionine sulfoximine (BSO), an inhibitor of GSH synthesis, has the sameanticancer effect but only if delivered prior to tumor onset. This result suggests that GSH
becomes dispensable during tumor progression, perhaps due to compensation byalternative antioxidant pathways. Combined inhibition of GSH and thioredoxin (TXN)

6

T. W. Mak

antioxidant pathways leads to synergistic cancer cell death in vitro and in vivo, pinpointing

these two antioxidants as potential therapeutic targets32). In the clinic, use of BSO alone to

inhibit GSH synthesis and treat malignancies has failed. However, we have demonstrated

in mice that use of sulfasalazine (SSA) and auranofin (AUR), which are TXN inhibitors
currently used to treat inflammatory diseases, can be combined with BSO to reducetumorigenesis32).

Figure 3

Model of the roles of Nrf2, estrogen, ROS and p53 in the survival of BRCA1-deficient cells inbreast/ovary vs other tissues. Estrogen-mediated support of antioxidation allows precancerous BRCA1-deficient cells to survive high ROS levels and progress to breast or ovariancancer.

An alteration shared by many advanced cancer cells but not found in normal cells is

4. Exploiting Cancer Cell Aneuploidy

aneuploidy. By combining RNAi screening with gene expression analysis in human breast

cancers and cell lines while focusing on cancer cell aneuploidy, we identified polo-likekinase-4 (PLK4), an enzyme critical for aneuploidy maintenance, as a promisingtherapeutic target33). Our drug discovery program culminated in the isolation of CFI-

400945, a potent and selective small-molecule PLK4 inhibitor33). In vitro treatment of humancancer cells with CFI-400945 results in mitotic defects, centriole duplication and cell death33).

In in vivo mouse models based on human ovarian or breast cancer xenografts, tumor

growth is significantly inhibited by CFI-400945 in a manner influenced by the PTEN statusof the tumor33). PTEN-deficient xenografts show a greater response to CFI-400945 thanxenografts expressing wild type PTEN, making PTEN status a potential predictive

biomarker for therapy with this first-in-class agent33).

A second target emerging from our screen was TTK (MPS1), which is a conserved dual-

specificity kinase essential for spindle assembly checkpoint (SAC) maintenance during cellKeynote Lecture

7

division34). TTK is overexpressed in many solid tumors exhibiting genomic instability or

aneuploidy35-41). Elevated TTK levels correlate with aggressive subtypes and high histologicalgrade36), and are associated with adverse outcome in ER+ breast cancer42). Overexpression of

TTK may promote both tumor initiation and survival of genomically unstable and aneuploidbreast cancer cells43,44). TTK also has several non-mitotic roles, including participation in DNA

damage signaling pathways 45-48) . We have generated a series of small-molecule TTK
inhibitors 49,50) that not only have potential utility as single anticancer agents but also

demonstrate synergy with taxanes in vitro. Taxanes trigger SAC activation but this signal isoverridden by TTK inhibition, resulting in chromosome missegregation and mitotic
catastrophe. This chain of events has been independently identified using a chemicallydistant TTK inhibitor51) and is under investigation in a Phase I clinical trial52). Preclinical

toxicology studies of our TTK inhibitor CFI-402257 are now complete and FDA/Health
Canada IND/CTA submissions are in preparation. Phase I trials are slated for 2016 (Figure 4).

Figure 4

Image of a dividing cell showing where the indicated small-molecule inhibitors of mitosis
could allow exploitation of cancer cell aneuploidy as therapy.

This presentation has briefly outlined three innovative anticancer approaches under

5. Conclusion

investigation in our laboratory. By concentrating on unique aspects of tumor biology, we

strive to identify strategies and targets that are applicable to a broad range of cancers andless likely to induce damaging side-effects in normal tissues. By looking back to Warburg’s
hypothesis, we hope to come closer to a future free of cancer.

8

T. W. Mak

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23. Dang, L., D.W. White, S. Gross, B.D. Bennett, M.A. Bittinger, E.M. Driggers, V.R. Fantin,
H.G. Jang, S. Jin, M.C. Keenan, K.M. Marks, R.M. Prins, P.S. Ward, K.E. Yen, L.M. Liau,
J.D. Rabinowitz, L.C. Cantley, C.B. Thompson, M.G. Vander Heiden, and S.M. Su,
Cancer-associated IDH1 mutations produce 2-hydroxyglutarate. Nature, 2009.
462(7274): p. 739-44
24. Rose, N.R., M.A. McDonough, O.N. King, A. Kawamura, and C.J. Schofield, Inhibition of
2-oxoglutarate dependent oxygenases. Chem Soc Rev, 2011. 40(8): p. 4364-97
25. Sasaki, M., C.B. Knobbe, J.C. Munger, E.F. Lind, D. Brenner, A. Brustle, I.S. Harris, R.
Holmes, A. Wakeham, J. Haight, A. You-Ten, W.Y. Li, S. Schalm, S.M. Su, C. Virtanen, G.
Reifenberger, P.S. Ohashi, D.L. Barber, M.E. Figueroa, A. Melnick, J.C. Zuniga-Pflucker,
and T.W. Mak, IDH1(R132H) mutation increases murine haematopoietic progenitors
and alters epigenetics. Nature, 2012. 488(7413): p. 656-9
26. Noushmehr, H., D.J. Weisenberger, K. Diefes, H.S. Phillips, K. Pujara, B.P. Berman, F. Pan,
C.E. Pelloski, E.P. Sulman, K.P. Bhat, R.G. Verhaak, K.A. Hoadley, D.N. Hayes, C.M.
Perou, H.K. Schmidt, L. Ding, R.K. Wilson, D. Van Den Berg, H. Shen, H. Bengtsson, P.
Neuvial, L.M. Cope, J. Buckley, J.G. Herman, S.B. Baylin, P.W. Laird, and K. Aldape,
Identification of a CpG island methylator phenotype that defines a distinct subgroup ofglioma. Cancer Cell, 2010. 17(5): p. 510-22

Keynote Lecture

11

27. Figueroa, M.E., O. Abdel-Wahab, C. Lu, P.S. Ward, J. Patel, A. Shih, Y. Li, N. Bhagwat, A.
Vasanthakumar, H.F. Fernandez, M.S. Tallman, Z. Sun, K. Wolniak, J.K. Peeters, W.
Liu, S.E. Choe, V.R. Fantin, E. Paietta, B. Lowenberg, J.D. Licht, L.A. Godley, R. Delwel,
P.J. Valk, C.B. Thompson, R.L. Levine, and A. Melnick, Leukemic IDH1 and IDH2
mutations result in a hypermethylation phenotype, disrupt TET2 function, and impair
hematopoietic differentiation. Cancer Cell, 2010. 18(6): p. 553-67
28. Stein, E., M. Tallman, D.A. Pollyea, I.W. Flinn, A.T. Fathi, R.M. Stone, R.A. Levine, S.
Agresta, D. Schenkein, H. Yang, B. Fan, K. Yen, and S. De Botton, Clinical safety and activity in a phase I trial of AG-221, a first in class, potent inhibitor of the IDH2-mutant protein,in patients with IDH2 mutant positive advanced hematologic malignancies.
Proceedings of the 105th Annual Meeting of the American Association for Cancer
Research, 2014. Apr 5-9; : p. San Diego, CA
29. Gorrini, C., I.S. Harris, and T.W. Mak, Modulation of oxidative stress as an anticancer
strategy. Nat Rev Drug Discov, 2013. 12(12): p. 931-47
30. Gorrini, C., P.S. Baniasadi, I.S. Harris, J. Silvester, S. Inoue, B. Snow, P.A. Joshi, A.
Wakeham, S.D. Molyneux, B. Martin, P. Bouwman, D.W. Cescon, A.J. Elia, Z.
Winterton-Perks, J. Cruickshank, D. Brenner, A. Tseng, M. Musgrave, H.K. Berman, R.
Khokha, J. Jonkers, T.W. Mak, and M.L. Gauthier, BRCA1 interacts with Nrf2 to regulate
antioxidant signaling and cell survival. J Exp Med, 2013. 210(8): p. 1529-44
31. Gorrini, C., B.P. Gang, C. Bassi, A. Wakeham, S.P. Baniasadi, Z. Hao, W.Y. Li, D.W.
Cescon, Y.T. Li, S. Molyneux, N. Penrod, M. Lupien, E.E. Schmidt, V. Stambolic, M.L.
Gauthier, and T.W. Mak, Estrogen controls the survival of BRCA1-deficient cells via aPI3K-NRF2-regulated pathway. Proc Natl Acad Sci USA, 2014. 111(12): p. 4472-7
32. Harris, I.S., A.E. Treloar, S. Inoue, M. Sasaki, C. Gorrini, K.C. Lee, K.Y. Yung, D.
Brenner, C.B. Knobbe-Thomsen, M.A. Cox, A. Elia, T. Berger, D.W. Cescon, A. Adeoye, A.
Brustle, S.D. Molyneux, J.M. Mason, W.Y. Li, K. Yamamoto, A. Wakeham, H.K.
Berman, R. Khokha, S.J. Done, T.J. Kavanagh, C.W. Lam, and T.W. Mak, Glutathione andthioredoxin antioxidant pathways synergize to drive cancer initiation and progression.
Cancer Cell, 2015. 27(2): p. 211-22
33. Mason, J.M., D.C. Lin, X. Wei, Y. Che, Y. Yao, R. Kiarash, D.W. Cescon, G.C. Fletcher, D.E.
Awrey, M.R. Bray, G. Pan, and T.W. Mak, Functional Characterization of CFI-400945, a
Polo-like Kinase 4 Inhibitor, as a Potential Anticancer Agent. Cancer Cell, 2014
34. Liu, X. and M. Winey, The MPS1 family of protein kinases. Annu Rev Biochem, 2012. 81:
p. 561-85
35. Chng, W., E. Braggio, G. Mulligan, B. Bryant, E. Remstein, R. Valdez, A. Dogan, and R.
Fonseca, The centrosome index is a powerful prognostic marker in myeloma and identifies a cohort of patients that might benefit from aurora kinase inhibition. Blood, 2008.
111(3): p. 1603-9

12

T. W. Mak

36. Daniel, J., J. Coulter, J. Woo, K. Wilsbach, and E. Gabrielson, High levels of the Mps1
checkpoint protein are protective of aneuploidy in breast cancer cells. Proc Natl Acad Sci
USA, 2011. 108(13): p. 5384-9
37. Kilpinen, S., K. Ojala, and O. Kallioniemi, Analysis of kinase gene expression patterns
across 5681 human tissue samples reveals functional genomic taxonomy of the kinome.
PLoS One, 2010. 5(12): p. e15068
38. Landi, M., T. Dracheva, M. Rotunno, J. Figueroa, H. Liu, A. Dasgupta, F. Mann, J.
Fukuoka, M. Hames, A. Bergen, S. Murphy, P. Yang, A. Pesatori, D. Consonni, P.
Bertazzi, S. Wacholder, J. Shih, N. Caporaso, and J. Jen, Gene expression signature of cigarette smoking and its role in lung adenocarcinoma development and survival. PLoS
One, 2008. 3(2): p. e1651
39. Mills, G., R. Schmandt, M. McGill, A. Amendola, M. Hill, K. Jacobs, C. May, A.
Rodricks, S. Campbell, and D. Hogg, Expression of TTK, a novel human protein kinase, isassociated with cell proliferation. J Biol Chem, 1992. 267(22): p. 16000-6
40. Salvatore, G., T. Nappi, P. Salerno, Y. Jiang, C. Garbi, C. Ugolini, P. Miccoli, F. Basolo, M.
Castellone, A. Cirafici, R. Melillo, A. Fusco, M. Bittner, and M. Santoro, A cell proliferationand chromosomal instability signature in anaplastic thyroid carcinoma. Cancer Res,2007. 67(21): p. 10148-58
41. Yuan, B., Y. Xu, J. Woo, Y. Wang, Y. Bae, D. Yoon, R. Wersto, E. Tully, K. Wilsbach, and E.
Gabrielson, Increased expression of mitotic checkpoint genes in breast cancer cells withchromosomal instability. Clin Cancer Res, 2006. 12(2): p. 405-10
42. Gyorffy, B., G. Bottai, J. Lehmann-Che, G. Keri, L. Orfi, T. Iwamoto, C. Desmedt, G.
Bianchini, N.C. Turner, H. de The, F. Andre, C. Sotiriou, G.N. Hortobagyi, A. Di Leo, L.
Pusztai, and L. Santarpia, TP53 mutation-correlated genes predict the risk of tumor
relapse and identify MPS1 as a potential therapeutic kinase in TP53-mutated breast cancers. Mol Oncol, 2014. 8(3): p. 508-19
43. Carter, S., A. Eklund, I. Kohane, L. Harris, and Z. Szallasi, A signature of chromosomal
instability inferred from gene expression profiles predicts clinical outcome in multiple
human cancers. Nat Genet, 2006. 38(9): p. 1043-8
44. Fisk, H., C. Mattison, and M. Winey, Human Mps1 protein kinase is required for centrosome duplication and normal mitotic progression. Proc Natl Acad Sci USA, 2003.
100(25): p. 14875-80
45. Leng, M., D. Chan, H. Luo, C. Zhu, J. Qin, and Y. Wang, MPS1-dependent mitotic BLM
phosphorylation is important for chromosome stability. Proc Natl Acad Sci USA, 2006.
103(31): p. 11485-90
46. Nihira, K., N. Taira, Y. Miki, and K. Yoshida, TTK/Mps1 controls nuclear targeting of cAbl by 14-3-3-coupled phosphorylation in response to oxidative stress. Oncogene, 2008.
27(58): p. 7285-95

Keynote Lecture

13

47. Wei, J., Y. Chou, Y. Ou, Y. Yeh, S. Tyan, T. Sun, C. Shen, and S. Shieh, TTK/hMps1 participates in the regulation of DNA damage checkpoint response by phosphorylating
CHK2 on threonine 68. J Biol Chem, 2005. 280(9): p. 7748-57
48. Yeh, Y., Y. Huang, T. Lin, and S. Shieh, The cell cycle checkpoint kinase CHK2 mediates
DNA damage-induced stabilization of TTK/hMps1. Oncogene, 2009. 28(10): p. 1366-78
49. Liu, Y., Y. Lang, N.K. Patel, G. Ng, R. Laufer, S.W. Li, L. Edwards, B. Forrest, P.B.
Sampson, M. Feher, F. Ban, D.E. Awrey, I. Beletskaya, G. Mao, R. Hodgson, O.
Plotnikova, W. Qiu, N.Y. Chirgadze, J.M. Mason, X. Wei, D.C. Lin, Y. Che, R. Kiarash, B.
Madeira, G.C. Fletcher, T.W. Mak, M.R. Bray, and H.W. Pauls, The Discovery of Orally
Bioavailable Tyrosine Threonine Kinase (TTK) Inhibitors: 3-(4-(heterocyclyl)phenyl)1H-indazole-5-carboxamides as Anticancer Agents. J Med Chem, 2015. 58(8): p. 3366-92
50. Laufer, R., G. Ng, Y. Liu, N.K. Patel, L.G. Edwards, Y. Lang, S.W. Li, M. Feher, D.E.
Awrey, G. Leung, I. Beletskaya, O. Plotnikova, J.M. Mason, R. Hodgson, X. Wei, G.
Mao, X. Luo, P. Huang, E. Green, R. Kiarash, D.C. Lin, M. Harris-Brandts, F. Ban, V.
Nadeem, T.W. Mak, G.J. Pan, W. Qiu, N.Y. Chirgadze, and H.W. Pauls, Discovery of
inhibitors of the mitotic kinase TTK based on N-(3-(3-sulfamoylphenyl)-1H-indazol-5-yl)acetamides and carboxamides. Bioorg Med Chem, 2014. 22(17): p. 4968-97
51. Jemaa, M., L. Galluzzi, O. Kepp, L. Senovilla, M. Brands, U. Boemer, M. Koppitz, P.
Lienau, S. Prechtl, V. Schulze, G. Siemeister, A.M. Wengner, D. Mumberg, K.
Ziegelbauer, A. Abrieu, M. Castedo, I. Vitale, and G. Kroemer, Characterization ofnovel MPS1 inhibitors with preclinical anticancer activity. Cell Death Differ, 2013.
20(11): p. 1532-45 clinicaltrials.gov, NCT02138812. NCT02138812

14

T. W. Mak

Tak W. Mak, OC, PhD, DSc (Hons), MD, FRSC, FRS

1972-1973
1974-present
1984-present
1991-1993

1993-2002
1995-2001

2002-present

2004-present

2004-present
2008-present

Postdoctoral Fellow, The Ontario Cancer InstituteSenior Scientific Staff, Ontario Cancer Institute
Professor, Departments of Medical Biophysics &Immunology, University of Toronto
Head, Division of Cellular & Molecular Biology,
Ontario Cancer InstituteFounding Director, Amgen Institute
Graduate Secretary, Department of Medical Biophysics,
University of Toronto
Director, Advanced Medical Discovery Institute,
University Health Network
Honorary Professor, Department of Pathology,
University of Hong Kong
Director, The Campbell Family for Breast Cancer
Research Institute, Princess Margaret Cancer Centre,
University Health Network
Professor, Department of Medicine, University ofTorontoSpecialty and Present Interest:
Cancer, Metabolism, Immunology, Transgenic Mouse Models, Genetics, Immunotherapy,
Drug Discovery, Cell Signaling Pathways

15

THE ROLE OF METABOLISM IN CANCER PROGRESSION

Matthew G. Vander Heiden

Koch Institute for Integrative Cancer Research
Massachusetts Institute of Technology
77 Massachusetts Avenue, 76-561, Cambridge, MA 02139, USA(mvh@mit.edu)

Complex regulatory mechanisms enable cell metabolism to match physiological state1).

Proliferating cells have metabolic requirements that differ from non-proliferating cells toallow production of biomass 2,3) , and tissue specialization imposes further metabolic

requirements. For cancer cells to proliferate and survive in a pathological tissue context

they must adapt metabolism to meet the distinct requirements for cancer initiation andprogression. Therefore, cancer cells likely have altered metabolism relative to most normal

tissues in order to support the accumulation of biomass and to allow adaptation to cellstresses associated with the tumor environment2,4-6). Understanding how the nutrient

requirements of cancer cells differ from that of normal tissues can define new cancer targets

and suggest novel combinations of new and existing therapies7-9).

Glucose metabolism is increased in tumors2,4). Most glucose consumed by cancer cells in

culture is converted to lactate, while glutamine is the major source of tricarboxylic acid

(TCA) cycle carbon6,10) (Figure 1). However, nutrients levels and oxygen in cell culture

differ from those found in tumors6), and emerging data suggests that tissue of origin andenvironment can also have an impact on metabolic phenotypes11-14). To determine whether
tumor tissue exhibits the same metabolic phenotype as cells in culture, and how the

metabolism of tumors relates to that of normal tissues, we traced glucose and glutamine
fate in tumor and normal tissue in mice25). Using several mouse cancer models, including

lung and pancreatic cancer models driven by oncogenic Kras and loss of Tp5315-17), we foundthat tumor metabolism in vivo differed from that observed in cell culture. Tumors exhibit

increased lactate production from glucose, but the cancers analyzed unexpectedly used
minimal glutamine. Instead glucose was a major source of TCA cycle carbon (Figure 2).

16

M. G. V. Heiden

Figure 1

Glucose and glutamine in cancer cells.
A schematic showing the major fates of glucose and glutamine in central carbon metabolism,and how they can be used to generate lactate and aspartate. Most mammalian cells must synthesize aspartate, and this amino acid is required for de novo nucleotide and protein synthesis. Select enzymes considered in this study are included. (GLS, glutaminase; PC, pyruvatecarboxylate; PDH, pyruvate dehydrogenase; OAA, oxaloacetate; aKG, a-ketoglutarate)

Figure 2

Nutrient use in tumors can differ from cancer cells in culture.
A. 13C-glucose was infused into mice with KrasG12D Tp53-/- lung tumors, and the fate of glucose
determined in the tumor and normal lung by mass spectrometry. Two carbon citrate andaspartate labeling (M2, M4) is indicative of glucose entry in to the TCA cycle via pyruvate
dehydrogenase (PDH), while three carbon labeling of these metabolites (M3) suggests
pyruvate carboxylase (PC) activity. Minimal labeling of TCA cycle intermediates fromglucose is observed in lung cancer cells in culture (not shown) .
B. 13C-glutamine use by lung tumors and normal lung tissue was also assessed. Unlike lungcancer cells in culture, minimal glutamate (glu) labeling (M5) is observed despite deliveryof labeled glutamine (gln) . *p<0.05. Date adapted from Davidson et al., submitted.

Recasting the Warburg Effect17

Because the cancer cell lines generated from the autochthonous arising tumors in themouse models exhibit a similar metabolic phenotype to other cultured cells, we questioned

whether genetic adaptation to cell culture or tissue environment was responsible for thedifferent metabolic phenotypes and re-implanted cancer cells to form tumors in syngeneic

mice. The resulting tumors exhibited a similar metabolic phenotype to the autochthonoustumors, suggesting that tissue environment is an important determinant of how nutrientsare used. This finding was confirmed by comparing the metabolism of human cancer cells

lines and xenograft tumors derived from these cancer cells. The xenograft tumors alsorelied on glucose to support TCA cycle metaobolism, while the cells used to generate thetumors used glutamine in culture. This finding was evident in xenograft tumors derived

from both Kras-mutant and EGFR-mutant cancers, arguing that the tissue environment is

an important determinant of tumor metabolism.

Comparison of metabolism in mouse lung and pancreatic tumors that both arose as aresult of KrasG12D expression and loss of Tp53 show that these tumors share some common
features, but also differ in the metabolism of amino acids. Pancreatic tumors rely heavily

on catabolism of extracellular protein18,19), while lung tumors appear to directly utilize somefree amino acids. These findings suggest that tissue of origin can also contribute to themetabolic phenotype of cancer cells, and is consistent with previous analyses of metabolic

gene expression demonstrating tumor metabolic networks are more similar to the tissue oforigin than they are to tumors arising in a different organ20).

To determine whether the differential use of glucose and glutamine in tumor and in cell

culture represented context-specific metabolic dependencies, we utilized CRISPR/Cas9 to

disrupt enzymes required for glutamine or glucose entry into the TCA cycle25). Deletion of
pyruvate carboxylate (PC) had no effect on proliferation in culture, but completely blockedlung tumor formation in mice (Figure 3), findings consistent with other studies21). A similar
dependency on pyruvate dehydrogenase (PDH) was observed in vivo, while loss of

Figure 3

Understanding nutrient use in tumors can be predictive of metabolic dependencies.
Pyruvate carboxlase (PC) was delered in cancer cells derived from KrasG12D Tp53-/- lungtumors (sgPC). Proliferation of sgPC cells versus control cells was compared in culture(left) and after implantation to from allograft tumors (right).
Date adapted from Davidson et al., submitted.

18

M. G. V. Heiden

glutaminase (GLS, required for glutamine metabolism) had no effect on tumor growth

despite being required for cell proliferation in culture. These findings argue that

understanding nutrient utilization can predict metabolic dependencies.

One major difference between tissues and cell culture is oxygen availability, as oxygen

tensions are much lower in tissues, even when well perfused6, 22). A major role of respirationin cell proliferation is use of oxygen as an electron acceptor to generate aspartate23,24).

Aspartate is more oxidized than the nutrients cells consume, so electron acceptors areneeded to regenerate oxidized cofactors used for aspartate synthesis. Because aspartate isrequired for protein, purine and pyrimidine synthesis3), and most mammalian cells lack theability to acquire exogenous aspartate 23,24) , the metabolism of tumors in vivo may be
explained in part by differences in how tumors acquire aspartate in different environmental

contexts.

The success of chemotherapies that target nucleotide metabolism demonstrates thataltered cancer metabolism can be exploited to have an impact on patient outcomes7). In

some cases drugs targeting metabolic pathways can contribute to patient cures, but because

these drugs were developed and deployed based entirely on empiric evidence, how to

identify new metabolic targets and rationally select patients is not known. The phenotypes

we observe tracing metabolism in mouse tumor models align well with nutrient tracing
studies in human tumors14,21). This may begin to explain why chemotherapies targeting

metabolism have been successful despite selecting patients based on the cancer tissue oforigin. Better defining the metabolic dependencies of tumors will uncover new metabolic

enzyme targets, and could suggest how to combine new and existing drugs to improvecancer therapy.

References
1. Metallo CM, Vander Heiden MG. Understanding metabolic regulation and its influenceon cell physiology. Molecular cell. 2013;49(3):388-398.
2. Vander Heiden MG, Cantley LC, Thompson CB. Understanding the Warburg effect: themetabolic requirements of cell proliferation. Science. 2009;324(5930):1029-1033.
3. Lunt SY, Vander Heiden MG. Aerobic glycolysis: meeting the metabolic requirements ofcell proliferation. Annu Rev Cell Dev Biol. 2011;27:441-464.
4. Cairns RA, Harris IS, Mak TW. Regulation of cancer cell metabolism. Nat Rev Cancer.
2011;11(2):85-95.
5. White E. Exploiting the bad eating habits of Ras-driven cancers. Genes Dev.
2013;27(19):2065-2071.
6. Mayers JR, Vander Heiden MG. Famine versus feast: understanding the metabolism oftumors in vivo. Trends in Biochemical Sciences. 2015;40(3):130-140.

Recasting the Warburg Effect19

7. Vander Heiden MG. Targeting cancer metabolism: a therapeutic window opens. Nature
reviews Drug discovery. 2011;10(9):671-684.
8. Galluzzi L, Kepp O, Vander Heiden MG, Kroemer G. Metabolic targets for cancer therapy. Nature reviews Drug discovery. 2013;12(11):829-846.
9. Vander Heiden MG. Exploiting tumor metabolism: challenges for clinical translation. J
Clin Invest. 2013;123(9):3648-3651.
10. Hensley 2013 Glutamine and cancer: cell biology, physiology, and clinical opportunities. In J Clin Invest, pp. 3678-3684.
11. Yuneva MO, Fan TW, Allen TD, Higashi RM, Ferraris DV, Tsukamoto T, Mates JM,
Alonso FJ, Wang C, Seo Y, Chen X, Bishop JM. The metabolic profile of tumors depends
on both the responsible genetic lesion and tissue type. Cell Metab. 2012;15(2):157-170.
12. Davidson SM, Vander Heiden MG. METabolic adaptations in the tumor
MYCroenvironment. Cell Metab. 2012;15(2):131-133.
13. Marin-Valencia I, Yang C, Mashimo T, Cho S, Baek H, Yang X-L, Rajagopalan KN,
Maddie M, Vemireddy V, Zhao Z, Cai L, Good L, Tu BP, Hatanpaa KJ, Mickey BE,
Matés JM, Pascual JM, Maher EA, Malloy CR, Deberardinis RJ, Bachoo RM. Analysis oftumor metabolism reveals mitochondrial glucose oxidation in genetically diverse
human glioblastomas in the mouse brain in vivo. Cell Metab. 2012;15(6):827-837.
14. Maher EA, Marin-Valencia I, Bachoo RM, Mashimo T, Raisanen J, Hatanpaa KJ, Jindal A,
Jeffrey FM, Choi C, Madden C, Mathews D, Pascual JM, Mickey BE, Malloy CR,
DeBerardinis RJ. Metabolism of [U-13 C]glucose in human brain tumors in vivo. NMR inbiomedicine. 2012;25(11):1234-1244.
15. DuPage M, Dooley AL, Jacks T. Conditional mouse lung cancer models using adenoviralor lentiviral delivery of Cre recombinase. Nature protocols. 2009;4(7):1064-1072.
16. Bardeesy N, Aguirre AJ, Chu GC, Cheng KH, Lopez LV, Hezel AF, Feng B, Brennan C,
Weissleder R, Mahmood U, Hanahan D, Redston MS, Chin L, Depinho RA. Bothp16(Ink4a) and the p19(Arf)-p53 pathway constrain progression of pancreatic adenocarcinoma in the mouse. Proc Natl Acad Sci USA. 2006;103(15):5947-5952.
17. Mayers JR, Wu C, Clish CB, Kraft P, Torrence ME, Fiske BP, Yuan C, Bao Y, Townsend
MK, Tworoger SS, Davidson SM, Papagiannakopoulos T, Yang A, Dayton TL, Ogino S,
Stampfer MJ, Giovannucci EL, Qian ZR, Rubinson DA, Ma J, Sesso HD, Gaziano JM,
Cochrane BB, Liu S, Wactawski-Wende J, Manson JE, Pollak MN, Kimmelman AC,
Souza A, Pierce K, Wang TJ, Gerszten RE, Fuchs CS, Vander Heiden MG, Wolpin BM.
Elevation of circulating branched-chain amino acids is an early event in human pancreatic
adenocarcinoma development. Nat Med. 2014;20(10):1193-1198.
18. Commisso C, Davidson SM, Soydaner-Azeloglu RG, Parker SJ, Kamphorst JJ, Hackett S,
Grabocka E, Nofal M, Drebin JA, Thompson CB, Rabinowitz JD, Metallo CM, Vander
Heiden MG, Bar-Sagi D. Macropinocytosis of protein is an amino acid supply route in
Ras-transformed cells. Nature. 2013;497(7451):633-637.

20

M. G. V. Heiden

19. Kamphorst JJ, Nofal M, Commisso C, Hackett SR, Lu W, Grabocka E, Vander Heiden MG,
Miller G, Drebin JA, Bar-Sagi D, Thompson CB, Rabinowitz JD. Human pancreatic cancer
tumors are nutrient poor and tumor cells actively scavenge extracellular protein.
Cancer Res. 2015;75(3):544-553.
20. Hu J, Locasale JW, Bielas JH, O'Sullivan J, Sheahan K, Cantley LC, Heiden MG, Vitkup D.
Heterogeneity of tumor-induced gene expression changes in the human metabolic network. Nat Biotechnol. 2013;31(6):522-529.
21. Sellers K, Fox MP, Bousamra M, 2nd, Slone SP, Higashi RM, Miller DM, Wang Y, Yan J,
Yuneva MO, Deshpande R, Lane AN, Fan TW. Pyruvate carboxylase is critical for nonsmall-cell lung cancer proliferation. J Clin Invest. 2015;125(2):687-698.
22. Bertout JA, Patel SA, Simon MC. The impact of O2 availability on human cancer. Nat Rev
Cancer. 2008;8(12):967-975.
23. Sullivan LB, Gui DY, Hosios AM, Bush LN, Freinkman E, Vander heiden MG.
Supporting aspartate biosynthesis is an essential function of respiration in proliferating
cells. . Cell. 2015;162(3):552-563.
24. Birsoy K, Wang T, Freinkman E, Remaileh MA, Chen W, Sabatini DM. An Essential
Role of the Mitochondrial Electron Transport Chain in Cell Proliferation Is to Enable
Aspartate Synthesis. Cell. 2015;25. Davidson SM, Papagiannakopoulos T, Olenchock BA, Heyman JE, Keibler MA, Luengo
A, Bauer MR, Jha AK, O'Brien JP, Pierce KA, Gui DY, Sullivan LB, Wasylenko TM,
Subbaraj L, Chin CR, Stephanopolous G, Mott BT, Jacks T, Clish CB, Vander Heiden
MG. Environment impacts the metabolic dependencies of ras-driven non-small celllung cancer. Cell Metab. 2016 doi: 10.1016/j.cmet.2016.01.007.

Matthew G. Vander Heiden, MD, PhD

2007-present

2009- present
2009-20142014- present

2014- present

Instructor in Medicine, Dana-Farber Cancer Institute /Harvard Medical School
Associate Member, Broad Institute of Harvard andMassachusetts Institute of Technology (MIT)Assistant Professor, Department of Biology , Koch
Institute for Integrative Cancer Research, MIT
Associate Professor of Biology, Koch Institute ofIntegrative Cancer Research, MIT
Eisen and Chang Career Development Professor, Koch
Institute of Integrative Cancer Research, MIT

Specialty and Present Interest:
Cell Metabolism, Cancer Metabolism, Cancer Biology

21

METABOLIC HETEROGENEITY
IN CANCER CELLS AND TUMORS

Ralph J. DeBerardinisChildren's Medica1 Center Research lnstitute (CRI)UT Southwestem Medica1 Center
5323 Harry Hines Boulevard, Dallas,Texas,75390-8502, USA(ralph.deberardinis@utsouthwestern.edu)

In cancer, metabolic reprogramming – the regulated alteration of metabolism in

response to tumorigenic mutations and other factors – is viewed as an essential componentof malignancy. Research in cancer metabolism is motivated by the hope that

understanding the basis of metabolic reprogramming will stimulate the development of
new approaches in cancer imaging and therapy, both of which have historically capitalizedon altered metabolic states in tumors. Cancer cells use abundant nutrients like glucose andglutamine to feed metabolic pathways that promote energetics, biosynthesis, and redox

homeostasis. There is now substantial information about how these pathways are

regulated in conventional culture, but we know comparatively little about how metabolic

phenotypes are established under more complex and stressful conditions, including intact

human tumors in vivo. Closing this gap in knowledge should help us understand therelative roles of cell-intrinsic and -extrinsic influences on tumor cell metabolism.

I will discuss progress in understanding how metabolic phenotypes are specified incancer cells. First, I will briefly discuss a large phenotyping effort to assess the breadth ofcell-intrinsic metabolic diversity in non-small cell lung cancer (NSCLC) cell lines. By

culturing over 80 cell lines under identical conditions and quantifying ~100 metabolic
features in each, we generated a large, mineable database which can be used to assess

metabolic diversity across the lines and identify novel correlations between metabolic andmolecular features. We find that the metabolic effects of specific mutations/combinationsof mutations can be observed when the data are viewed in aggregate. Examples involving
carbon utilization in the TCA cycle will be discussed.

22

R. J. DeBerardinis

We are also making progress in analyzing patterns of nutrient utilization in living

human tumors, where both cell-intrinsic and cell-extrinsic processes influence themetabolic phenotype. In humans, NSCLC is highly heterogeneous both in the genetic andenvironmental parameters that influence cell metabolism in culture. However, the impactof these factors on human NSCLC metabolism in vivo is unknown. We are conducting
intra-operative infusions with 13C-glucose in NSCLC patients to compare metabolism

between tumors and benign lung. In addition, we perform extensive multi-parametric pre-surgical imaging to non-invasively assess areas of biological heterogeneity expected toinfluence the metabolic phenotype. Careful review of the imaging data prior to surgery

then allows us to identify areas of heterogeneity to be sampled during the tumor resection.

A fragment-by-fragment analysis of histopathology, gene expression and metabolism isthen performed. We find that tumors of diverse grade, stage, histology and oncogenotype

displayed enhanced glycolysis and glucose oxidation relative to the surrounding lung
(Figure 1A, B). Importantly, although glycolysis appeared to be activated in the tumors,

Figure 1

Recasting the Warburg Effect23

there was no evidence for systematic suppression of glucose oxidation, indicating that both

aerobic and anaerobic glucose metabolism occur concomitantly in the same tissue in vivo.

Metabolic flux analysis revealed that pyruvate dehydrogenase (PDH) was significantly

more active in the tumors than surrounding lung (Figure 1C), and activity of this enzyme

was positively correlated with the Ki67 fraction observed on quantitative histopathology(Figure 1D).

Interestingly, all tumors had evidence for oxidation of alternative nutrients in addition

to glucose, as indicated by the relatively low 13C enrichment in TCA cycle metabolites

compared to glucose. We find evidence that both primary human NSCLC and xenografts

derived from NSCLC cell lines take up lactate from the circulation and use it as a fuel for

Figure 2

24

R. J. DeBerardinis

the tricarboxylic acid cycle. Additionally, metabolically heterogeneous regions wereidentified within and between tumors using dynamic gadolinium enhancement (DCE) as asurrogate for perfusion (Figure 2A, B). Regions of higher contrast enhancement (i.e. higher

perfusion) demonstrated lower 13C enrichment, likely reflecting contributions of unlabeled

nutrients in well-perfused areas (Figure 2C). Consistent with this idea, RNA sequencing

data from the same tumor fragments used for analysis of 13 C enrichment revealed

alternative patterns of metabolic gene expression. Gene sets involving glycolysis, oxidative

phosphorylation and pyruvate oxidation were enriched in tumor fragments with low DCE

Figure 3

Recasting the Warburg Effect25

phenotypes (Figure 3, large arrowheads), consistent with the enhanced 13C enrichment inmetabolites from these tumors. In contrast, fragments with high DCE phenotypes wereenriched for gene sets involving the metabolism of other nutrients, particularly nitrogenous

metabolites including amino acids (Figure 3, small arrowheads). Altogether, the datasuggest that heterogeneous metabolism of these NSCLC tumors is highly and predictably

influenced by cell-extrinsic effects, including perfusion. They also emphasize the potential

for existing and new imaging approaches to report alterations of metabolic flux in vivo.

Ralph J. DeBerardinis, MD, PhD

2008-20112011-20132013-present
2013-present

2013-present

Assistant Professor, Department of Pediatrics, UTSouthwestern
Assistant Professor, CRI, UT Southwestern
Associate Professor, CRI, UT Southwestern
Director, Genetic and Metabolic Disease Program, CRI,UT Southwestern
Joel B. Steinberg, MD Professor in Pediatrics, UTSouthwesternSpecialty and Present Interest:
Cancer Metabolism, Metabolic Imaging, Inborn Errors of Metabolism

26

THE METABOLIC REGULATOR AMPK –FRIEND OR FOE IN CANCER

D. Grahame Hardie, Diana Vara Ciruelos and Fiona M. Russell
School of Life Sciences, University of Dundee
Dow Street, Dundee, DD1 5EH, Scotland, UK(d.g.hardie@dundee.ac.uk)

The AMP-activated protein kinase (AMPK) is a sensor of cellular energy status

expressed ubiquitously in eukaryotic cells1-3). AMPK exists as heterotrimeric complexes

comprising catalytic a subunits and regulatory b and g subunits. In mammals, there are
multiple genes encoding isoforms of each subunit (PRKAA1/2, encoding a1/a2,

PRKAB1/2, encoding b1/b2, and PRKAG1/2/3, encoding g1/g2/g3) - these combine to form

up to twelve heterotrimeric combinations.

In the yeast Saccharomyces cerevisiae, the AMPK ortholog is required for the switch fromrapid glycolysis (fermentation) and rapid growth that occurs when the glucose

concentration in the medium is high, to the oxidative metabolism and slower growth that

occurs when glucose runs low4). Fermentation is related to the Warburg effect that occurs
in most rapidly proliferating tumor cells, with the exception that glycolysis in yeast results

in production of ethanol rather than lactate.

AMPK heterotrimers can be activated >100-fold by phosphorylation at a conserved

threonine residue (Thr172) within the a subunit kinase domain. The major upstreamkinase phosphorylating Thr172 is a complex containing the protein kinase LKB1, although
it can also be phosphorylated by the calmodulin-dependent kinase, CaMKKb, in response

to a rise in intracellular Ca2,5). The findings that LKB1 was the principal upstream kinaseprovided the first links between AMPK and cancer6,7), because STK11 (encoding LKB1) had

previously been shown to be the gene in which mutations cause an inherited susceptibility

to cancer termed Peutz-Jeghers syndrome. Subjects with this syndrome carry monoallelicloss-of-function mutations in STK11, and develop numerous benign intestinal polyps, butalso have an increased risk of developing malignant tumors at other sites8). Mutations inRecasting the Warburg Effect27

the STK11 gene are also quite frequent in spontaneous, non-inherited cancers, especially in

non-small cell lung cancer where the frequency may be as high as 20-30%9,10).

Unlike CaMKKb which is Ca 2 + -dependent, the LKB1 complex appears to beconstitutively active, and modulation of the phosphorylation of Thr172 (and hence theactivity) of AMPK is due instead to the fact that binding of AMP or ADP to the g subunit of

AMPK causes conformational changes that promote Thr172 phosphorylation by LKB111, 12),

as well as inhibiting Thr172 dephosphorylation13,14). AMPK complexes containing g1 or g2that are phosphorylated on Thr172 are also allosterically activated up to 10-fold by AMP,

although not ADP12). All three effects of AMP binding (of which two are mimicked by

ADP) are antagonized by ATP, making the AMPK system an exquisitely sensitive sensor ofcellular energy status.

In 2001 it was reported that the biguanide drug metformin, now the front line agent fortreatment of Type 2 diabetes, activated AMPK15). Metformin activates AMPK indirectly byinhibiting complex I of the respiratory chain, thus increasing cellular AMP:ATP and
ADP:ATP ratios, a mechanism shared by other AMPK activators derived from traditional

herbal medicines, such as berberine16) or arctigenin17). Although some of the acute effects of

metformin on hepatic glucose production are AMPK-independent 18) , its longer-term
insulin-sensitizing effects appear to be mediated by actions of AMPK on lipid

metabolism19). Intriguingly, use of metformin to treat diabetes is associated with a reduced

risk of cancer in humans20).

Another AMPK-activating drug of interest with respect to cancer is the antifolate

pemetrexed21, 22), licensed for use in non-small cell lung cancer where AMPK may often be
down-regulated due to loss of LKB1. Pemetrexed is unique among antifolates in inhibiting

AICAR transformylase, the enzyme catalysing the first step in the conversion of theintermediate ZMP to purine nucleotides. It therefore activates AMPK by causing

accumulation of ZMP, an AMP analog that mimics all three effects of AMP on AMPK. The

primary target of pemetrexed is in fact thought to be thymidylate synthase, required for

DNA synthesis. Interestingly, however, if thymidylate is provided in the medium of cells
to circumvent inhibition of thymidylate synthesis, pemetrexed remains cytostatic due to
AMPK activation, although it is no longer cytotoxic22).

Once activated by energy stress in mammalian cells, AMPK acts to restore energybalance by switching on catabolic pathways that produce ATP, while switching off anabolic
pathways that consume ATP23). Catabolic pathways switched on acutely include glucose

uptake and glycolysis; while this would promote rapid glycolysis, i.e. the Warburg effect,in the longer term AMPK activation tends to promote instead the more energy-efficient andglucose-sparing oxidative metabolism, by increasing fatty acid oxidation, expression ofoxidative enzymes, and mitochondrial biogenesis. Another critical catabolic pathway

28

D. G. Hardie et al.

switched on by AMPK is autophagy24) which, like its upstream activator AMPK, can haveboth positive and negative effects in cancer25). AMPK also switches off most anabolic

pathways including the synthesis of fatty acids, sterols, triglycerides, phospholipids,
glycogen and rRNA23), and inhibits protein synthesis by inactivating mTOR complex-1(mTORC1)26,27). Finally, it causes cell cycle arrest in G1 phase28,29).

AMPK would therefore be expected to limit cell proliferation, suggesting that it would

suppress tumor growth. Is there any other evidence that AMPK mediates the tumor

suppressive effects of LKB1? LKB1 also phosphorylates and activates 12 AMPK-related
kinases (ARKs) in addition to AMPK30), and it is possible that some of its tumor suppressor

effects might be mediated by one or more of these (e.g.31)). However, unlike AMPK, none of
the ARKs are known to oppose the metabolic changes observed in tumor cells or cause cell

cycle arrest, and none are activated by metformin. Finally, whole body knockout of AMPK-

a1 (the sole a subunit isoform expressed in lymphocytes) accelerated the formation of B

cell lymphomas caused by B cell-specific over-expression of Myc32), while T cell-specificknockout of AMPK-a1 accelerated the development of T cell lymphomas triggered byknockout of the tumor suppressor PTEN in T cells (DGH/DVC, unpublished). In theformer case, loss of AMPK was associated with a metabolic shift towards aerobic glycolysis(i.e. the Warburg effect), while in the latter case it was associated with up-regulation of themTORC1 pathway and increased expression of HIF-1a, which would also be expected topromote the Warburg effect.

These findings support the idea that AMPK-a1 is indeed

acting as a tumor suppressor in these mouse models of lymphoma.

If AMPK does suppress tumor growth like its upstream kinase LKB1, you would expect

there to be selection pressure for the pathway to be down-regulated in human cancers,which would reduce its restraining influence on tumor growth and proliferation. Clearly,

this occurs in the 20-30% of non-small cell lung cancers that have lost expression of

LKB19,10). In addition, histochemical evidence suggests that the phosphorylated form of
AMPK is reduced in tumor cells, compared with adjacent regions of normal epithelium, in
up to 90% of biopsies taken for diagnosis of breast cancer33). Moreover, the a2 isoform of

AMPK is down-regulated in many cases of hepatocellular carcinoma, and this is associatedwith poor prognosis34). Although the mechanisms underlying AMPK down-regulation inthese cases are not known, one intriguing mechanism for down-regulation of AMPK-a1 hasrecently been reported35). MAGE-A3 and -A6 are closely related proteins encoded byneighbouring genes on the X chromosome. In common with most other MAGE proteins,they are normally only expressed in the testis, but become aberrantly re-expressed in many

tumours (up to 25% of breast invasive carcinomas, 50% of colon adenocarcinomas and 75%
of lung squamous cell carcinomas). MAGE-A3/-A6 bind the E3 ubiquitin ligase TRIM28,

and this complex has recently been shown to target AMPK-a1 for proteasomal degradation

Recasting the Warburg Effect29

by promoting its poly-ubiquitylation35). A second mechanism for down-regulation of

AMPK-a1 has recently been described by our laboratory, in tumor cells in which the Aktpathway is hyperactivated due to loss of the tumor suppressor PTEN 3 6 ) . Akt

phosphorylates a conserved serine residue (Ser487) within a serine/threonine-rich loop of

the C-terminal domain of AMPK (although not the equivalent residue of AMPK-a2,

Ser491). Phosphorylation of Ser487 blocks the phosphorylation of Thr172 (and consequent

activation) by the upstream kinases LKB1 or CaMKKb, most likely due to a simple physicalexclusion mechanism36).

The evidence discussed above suggests that AMPK is a tumor suppressor that is often

down-regulated in cancers. Paradoxically, however, a low level of AMPK may bebeneficial to tumor cells to help them to survive the lack of oxygen and nutrients that often

occurs while they are establishing a blood supply, or to withstand the stresses caused bycytotoxic anti-cancer treatments. AMPK may therefore be a “friend” or a “foe” in cancer,

depending on the context. Consistent with the idea that AMPK may sometimes be a “foe”,

H-Ras mutant transformed mouse embryo fibroblasts (MEFs) that were deficient in bothisoforms of AMPK (a1 and a2) grew normally in culture in vitro but very poorly as

xenografts in immunodeficient mice in vivo, where they might be subjected to hypoxic

and/or nutritional stresses37). By contrast, MEFs with knockout of a2 alone displayed
increased susceptibility to H-Ras mutant transformation in vitro and tumorigenesis invivo38). It has also been pointed out, following analysis of cancer genome databases, thatthe genes encoding the a1 and b2 isoforms of AMPK subunit are quite frequently amplified,rather than mutated or deleted, in human cancers39) - this would be expected if they were

oncogenes, rather than tumor suppressors. Since this behaviour was not observed with thegenes encoding the other five subunits of AMPK, it suggests that the protective effects of

AMPK in tumor cells might be confined to specific isoforms. A final example of AMPK

acting as a “foe” in cancer comes from some of our own recent unpublished work, where

we have shown that the anti-cancer drug etoposide activates AMPK-a1 in the cell nucleus
by a mechanism involving phosphorylation of Thr172 by CaMKKb. Etoposide causes

double strand breaks during replication of DNA in S phase, and this can lead to apoptosisof rapidly proliferating cells while quiescent cells are less sensitive. We have shown thatprior activation of AMPK by the Ca2+ ionophore A23187 can protect cells against thecytotoxic effects of etoposide. The likely mechanism is that AMPK causes a G1 cell cycle

arrest and thus prevents the cells from entering S phase, where they are more vulnerable toetoposide. In this context, AMPK is clearly protecting the cells against the cytotoxic stresses
caused by etoposide, and it is therefore possible that an AMPK inhibitor might be useful forcancer treatment, perhaps as an adjunct to etoposide.

30

D. G. Hardie et al.

References
1. Hardie, D. G. Molecular Pathways: Is AMPK a friend or a foe in cancer? Clin. Cancer Res.
2015;21: 3836-3840.
2. Hardie, D. G. AMPK--sensing energy while talking to other signaling pathways. Cell
Metab. 2014;20: 939-952.
3. Hardie, D. G., Schaffer, B. & Brunet, A. AMP-activated protein kinase: an energy-sensing
pathway with multiple inputs and outputs. Trends Cell Biol. 2015: in press.
4. Haurie, V., Boucherie, H. & Sagliocco, F. The Snf1 protein kinase controls the induction ofgenes of the iron uptake pathway at the diauxic shift in Saccharomyces cerevisiae. J.
Biol. Chem. 2003;278: 45391-45396.
5. Hawley, S. A. et al. Calmodulin-dependent protein kinase kinase-beta is an alternative
upstream kinase for AMP-activated protein kinase. Cell Metab. 2005;2: 9-19.
6. Hawley, S. A. et al. Complexes between the LKB1 tumor suppressor, STRADa/b andMO25a/b are upstream kinases in the AMP-activated protein kinase cascade. J. Biol.
2003;2: 28.
7. Woods, A. et al. LKB1 is the upstream kinase in the AMP-activated protein kinase cascade. Curr. Biol. 2003;13: 2004-2008.
8. Alessi, D. R., Sakamoto, K. & Bayascas, J. R. Lkb1-dependent signaling pathways.
Annu. Rev. Biochem. 2006;75: 137-163.
9. Sanchez-Cespedes, M. et al. Inactivation of LKB1/STK11 is a common event in adenocarcinomas of the lung. Cancer Res. 2002;62: 3659-3662.
10. Ji, H. et al. LKB1 modulates lung cancer differentiation and metastasis. Nature 2007;448:
807-810.
11. Oakhill, J. S. et al. AMPK is a direct adenylate charge-regulated protein kinase. Science
2011;332: 1433-1435.
12. Gowans, G. J., Hawley, S. A., Ross, F. A. & Hardie, D. G. AMP is a true physiological regulator of AMP-activated protein kinase by both allosteric activation and enhancing net
phosphorylation. Cell Metab. 2013;18: 556-566.
13. Davies, S. P., Helps, N. R., Cohen, P. T. W. & Hardie, D. G. 5'-AMP inhibits dephosphorylation, as well as promoting phosphorylation, of the AMP-activated protein kinase.
Studies using bacterially expressed human protein phosphatase-2Ca and native bovine
protein phosphatase-2AC. FEBS Lett. 1995;377: 421-425.
14. Xiao, B. et al. Structure of mammalian AMPK and its regulation by ADP. Nature
2011;472: 230-233.
15. Zhou, G. et al. Role of AMP-activated protein kinase in mechanism of metformin
action. J. Clin. Invest. 2001;108: 1167-1174.

Recasting the Warburg Effect31

16. Hawley, S. A. et al. Use of cells expressing gamma subunit variants to identify diverse
mechanisms of AMPK activation. Cell Metab. 2010;11: 554-565.
17. Huang, S. L. et al. Arctigenin, a natural compound, activates AMP-activated protein
kinase via inhibition of mitochondria complex I and ameliorates metabolic disorders in
ob/ob mice. Diabetologia 2012;55: 1469-1481.
18. Foretz, M. et al. Metformin inhibits hepatic gluconeogenesis in mice independently of the
LKB1/AMPK pathway via a decrease in hepatic energy state. J. Clin. Invest. 2010;120:2355-2369.
19. Fullerton, M. D. et al. Single phosphorylation sites in ACC1 and ACC2 regulate lipid
homeostasis and the insulin-sensitizing effects of metformin. Nat. Med. 2013;19: 16491654.
20. Evans, J. M., Donnelly, L. A., Emslie-Smith, A. M., Alessi, D. R. & Morris, A. D.
Metformin and reduced risk of cancer in diabetic patients. BMJ 2005;330: 1304-1305.
21. Agarwal, S., Bell, C. M., Rothbart, S. B. & Moran, R. G. AMPK control of mTORC1 is p53and TSC2-independent in pemetrexed-treated carcinoma cells. J. Biol. Chem. 2015.
22. Racanelli, A. C., Rothbart, S. B., Heyer, C. L. & Moran, R. G. Therapeutics by cytotoxic
metabolite accumulation: pemetrexed causes ZMP accumulation, AMPK activation,and mammalian target of rapamycin inhibition. Cancer Res. 2009;69: 5467-5474.
23. Hardie, D. G., Ross, F. A. & Hawley, S. A. AMPK: a nutrient and energy sensor thatmaintains energy homeostasis. Nature Rev. Mol. Cell Biol. 2012;13: 251-262.
24. Egan, D. F. et al. Phosphorylation of ULK1 (hATG1) by AMP-activated protein kinase
connects energy sensing to mitophagy. Science 2011;331: 456-461.
25. Mathew, R. & White, E. Autophagy in tumorigenesis and energy metabolism: friend byday, foe by night. Curr. Opin. Genet. Dev. 2011;21: 113-119.
26. Inoki, K., Zhu, T. & Guan, K. L. TSC2 mediates cellular energy response to control cell
growth and survival. Cell 2003;115: 577-590.
27. Gwinn, D. M. et al. AMPK phosphorylation of raptor mediates a metabolic checkpoint.
Mol. Cell 2008;30: 214-226.
28. Imamura, K., Ogura, T., Kishimoto, A., Kaminishi, M. & Esumi, H. Cell cycle regulation
via p53 phosphorylation by a 5'-AMP activated protein kinase activator, 5-aminoimidazole- 4-carboxamide-1-beta-d- ribofuranoside, in a human hepatocellular carcinoma cellline. Biochem. Biophys. Res. Commun. 2001;287: 562-567.
29. Jones, R. G. et al. AMP-activated protein kinase induces a p53-dependent metabolic
checkpoint. Mol. Cell 2005;18: 283-293.
30. Lizcano, J. M. et al. LKB1 is a master kinase that activates 13 protein kinases of the
AMPK subfamily, including the MARK/PAR-1 kinases. EMBO J. 2004;23: 833-843.

32

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31. Goodwin, J. M. et al. An AMPK-independent signaling pathway downstream of the
LKB1 tumor suppressor controls Snail1 and metastatic potential. Mol. Cell 2014;55: 436450.
32. Faubert, B. et al. AMPK Is a negative regulator of the Warburg effect and suppresses
tumor growth In vivo. Cell Metab. 2012;17: 113-124.
33. Hadad, S. M. et al. Histological evaluation of AMPK signalling in primary breast cancer.
BMC Cancer 2009;9: 307.
34. Lee, C. W. et al. AMPK promotes p53 acetylation via phosphorylation and inactivation of
SIRT1 in liver cancer cells. Cancer Res. 2012;72: 4394-4404.
35. Pineda, C. T. et al. Degradation of AMPK by a cancer-specific ubiquitin ligase. Cell
2015;160: 715-728.
36. Hawley, S. A. et al. Phosphorylation by Akt within the ST loop of AMPK-a1 down-regulates its activation in tumour cells. Biochem. J. 2014;459: 275-287.
37. Laderoute, K. R. et al. 5'-AMP-activated protein kinase (AMPK) is induced by low-oxygenand glucose deprivation conditions found in solid-tumor microenvironments. Mol.
Cell. Biol. 2006;26: 5336-5347.
38. Phoenix, K. N., Devarakonda, C. V., Fox, M. M., Stevens, L. E. & Claffey, K. P.
AMPKalpha2 suppresses murine embryonic fibroblast transformation and tumorigenesis.
Genes Cancer 2012;3: 51-62.
39. Monteverde, T., Muthalagu, N., Port, J. & Murphy, D. J. Evidence of cancer promoting
roles for AMPK and related kinases. FEBS J. 2015.

D. Grahame Hardie, PhD, FRS, FRSE, FMedSci

1977-1987
1987-1990
1990-1994
1994-present

Lecturer, University of Dundee
Senior Lecturer, University of Dundee
Reader, University of Dundee
Professor, University of Dundee

Speciality and Present Interest:
Cell Signalling Regulation, Physiological and Pathological Roles of AMP-activatedProtein Kinase

33

MYC-MEDIATED PERTURBATION OF
METABOLISM AND THERAPY

Chi V. Dang

Abramson Cancer Center, University of Pennsylvania
Philadelphia, PA 19104, USA(dangvchi@exchange.upenn.edu)

The MYC oncogene belongs to the family of MYC genes, including MYCN (N-MYC)

and MYCL (L-MYC), which are linked to human cancers such as Burkitt’s lymphoma,
neuroblastoma and lung cancer, respectively. MYC proteins belong to the MAX-MLX

network of heterodimeric transcription factors that bind ‘E-boxes’ (5’-CACGTG-3’) toregulate genes involved in cell proliferation, differentiation and metabolism. MYC is

downstream of many signal transduction pathways in normal cells; when stimulated

through pathways such as tyrosine receptor kinases, MYC is induced to produce the MYC

protein that dimerizes with MAX to bind DNA and regulate a transcriptional program of

metabolism, cell growth and proliferation1). MYC is under the control of growth factor
signaling as well as nutrient availability, such that nutrient deprivation could result in

diminished normal MYC expression and cell growth arrest. By contrast, deregulation of

MYC in normal cells result in MYC binding not only to high affinity physiological bindingsites but also invading into low-affinity binding sites and enhancers, resulting in an
imbalance amplification of gene expression that triggers stress and cell death throughactivation of checkpoints such as p53. Elimination of p53, however, in tumorigenesis canunleash MYC’s transcriptional power to drive deregulated cell growth that renders cells
addicted to nutrients, such that deprivation of glucose or glutamine result in cell death.

The conceptual framework has been exploited to target metabolism for therapy against

MYC-driven cancers, particularly aiming at glycolysis and glutaminolysis – processes thatare increased by MYC. While LDHA inhibition could curb MYC-mediated tumorigenesis2),

glutaminolysis remains an alternative survival pathway that could be targeted throughinhibition of glutaminase3-9). Glutaminase is normally expressed from two different genes,

34

C. V. Dang

GLS that is normally expressed in kidney and brain, and GLS2 that is normally expressedin liver. Indeed, we have shown that knockdown of glutaminase (GLS) or inhibition with asmall molecule (BPTES) diminishes the progression of a MYC-inducible human lymphoma

xenograft model3-7). We further documented that glutaminase (Gls) is induced by MYC andis required for early tumor development in a MYC-inducible model of mouse liver cancer8),

which demonstrates a decrease in the expression of normal liver Gls2. In this regard, theisoform switch from Gls2 to Gls1 in mouse (and also human) liver cancer, renders tumors

vulnerable to loss of one copy of Gls, which delayed tumorigenesis. We further showedthat treatment with BPTES as a single agent was sufficient to prolong survival of micebearing these MYC-induced liver cancers, providing proof-of-concept that targeting asingle enzyme, in this case GLS, could change the course of the disease8).

Targeting GLS, however, has limitations, since the oncogenotypes of cancers result indifferent re-wiring of metabolism that is best illustrated by metabolomics studies todifferent mouse models of liver cancer10). The MET oncogene-driven liver cancer model

largely relies on glucose, whereas MYC oncogene-driven liver or lung cancers rely on bothglutamine and glucose. As such, glutaminase inhibition should be considered in thecontext of the oncogenotype and metabolic profile of specific cancer types. Further,targeting metabolism could be constrained by the circadian regulation of cellular

metabolism, rendering proliferating normal cells more sensitive to interference of

metabolism at specific times of the day. In this regard, we tested the hypothesis that highoncogenic MYC would ectopically invade the circadian Clock regulated genes that are also

driven by E-boxes11). The cell intrinsic clock machinery comprises of the central ClockBmal1 transcription factor, which induces Rev-erb’s, Cry’s, and Per’s that in turn negatively

regulated Bmal1 expression or Clock-Bmal1 levels, resulting in a circadian oscillation of

Clock-Bmal1 function. Oscillation of the central clock transcription, which drive metabolic

genes with E-boxes, results in oscillatory metabolism. Indeed, using an inducible MYC-ER
system, we demonstrate that MYC could activate the negative clock regulators PER, CRY,

and REV-ERBS and documented that the suppression of Bmal1 expression by MYC is

mediated transcriptionally through REV-ERBs. Metabolic profiling in time-series

experiments reveals oscillation of glucose and glutamine metabolic in the MYC-OFF state.

In the MYC-ON state, intracellular glucose ceases to oscillate and when barely detectableby NMR and additional evidence suggest that it is converted toward lactate and biomass

for cell growth. MYC, hence, can invade and disrupt the molecular circadian clock as wellas metabolism, in favor of a growth metabolic program.

These observations have key implications for targeting metabolic enzymes that havebeen documented as being induced by Clock-Bmal1 and MYC. For example, ODC and

NAMPT are both common targets for these transcription factors. As such, we hypothesize

Recasting the Warburg Effect35

that the dose-limiting toxicity of NAMPT inhibition, being thrombocytopenia, could be

curbed in a lymphoma xenograft model by applying chronotherapy. In this regard, ourpreliminary studies illustrate that while NAMPT inhibition at two different times of the

day resulted in similar efficacy in reducing lymphoma xenograft growth, one ofadministration time resulted in thrombocytopenia while the other time of drug
administration was indistinguishable from the control treated animals (unpublished).

These observations indicate that the combination of identifying metabolic vulnerabilities of

MYC-driven cancer along with understanding circadian could strategically guide the use of

metabolism-targeted drugs in the clinic through oncogenotyping and potentiallychronotherapy.

References
1. Stine ZE, Walton ZE, Altman BJ, Hsieh AL, Dang CV. MYC, Metabolism, and Cancer.
Cancer Discov. 2015 Oct;5(10):1024-39.
2. Le A, Cooper CR, Gouw AM, Dinavahi R, Maitra A, Deck LM, Royer RE, Vander Jagt DL,
Semenza GL, Dang CV. Inhibition of lactate dehydrogenase A induces oxidative stressand inhibits tumor progression. Proc Natl Acad Sci USA. 2010 Feb 2;107(5):2037-42.
3. Gao P, Tchernyshyov I, Chang TC, Lee YS, Kita K, Ochi T, Zeller KI, De Marzo AM, Van
Eyk JE, Mendell JT, Dang CV. c-Myc suppression of miR-23a/b enhances mitochondrial
glutaminase expression and glutamine metabolism. Nature. 2009 Apr 9;458(7239):762-5.
4. Wang JB, Erickson JW, Fuji R, Ramachandran S, Gao P, Dinavahi R, Wilson KF,
Ambrosio AL, Dias SM, Dang CV, Cerione RA. Targeting mitochondrial glutaminase
activity inhibits oncogenic transformation. Cancer Cell. 2010 Sep 14;18(3):207-19.
5. Le A, Lane AN, Hamaker M, Bose S, Gouw A, Barbi J, Tsukamoto T, Rojas CJ, Slusher BS,
Zhang H, Zimmerman LJ, Liebler DC, Slebos RJ, Lorkiewicz PK, Higashi RM, Fan TW,
Dang CV. Glucose-independent glutamine metabolism via TCA cycling for proliferation and survival in B cells. Cell Metab. 2012 Jan 4;15(1):110-21.
6. Shukla K, Ferraris DV, Thomas AG, Stathis M, Duvall B, Delahanty G, Alt J, Rais R,
Rojas C, Gao P, Xiang Y, Dang CV, Slusher BS, Tsukamoto T. Design, synthesis, andpharmacological evaluation of bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide
3 (BPTES) analogs as glutaminase inhibitors. J Med Chem. 2012 Dec 13;55(23):10551-63.
7. Dutta P, Le A, Vander Jagt DL, Tsukamoto T, Martinez GV, Dang CV, Gillies RJ.
Evaluation of LDH-A and glutaminase inhibition in vivo by hyperpolarized 13C-pyruvatemagnetic resonance spectroscopy of tumors. Cancer Res. 2013 Jul 15;73(14):4190-5.

36

C. V. Dang.

8. Xiang Y, Stine ZE, Xia J, Lu Y, O'Connor RS, Altman BJ, Hsieh AL, Gouw AM, Thomas
AG, Gao P, Sun L, Song L, Yan B, Slusher BS, Zhuo J, Ooi LL, Lee CG, Mancuso A,
McCallion AS, Le A, Milone MC, Rayport S, Felsher DW, Dang CV. Targeted inhibition oftumor-specific glutaminase diminishes cell-autonomous tumorigenesis. J Clin Invest.
2015 Jun;125(6):2293-306.
9. Shroff EH, Eberlin LS, Dang VM, Gouw AM, Gabay M, Adam SJ, Bellovin DI, Tran PT,
Philbrick WM, Garcia-Ocana A, Casey SC, Li Y, Dang CV, Zare RN, Felsher DW. MYC
oncogene overexpression drives renal cell carcinoma in a mouse model through glutamine metabolism. Proc Natl Acad Sci USA. 2015 May 26;112(21):6539-44.
10. Yuneva MO, Fan TW, Allen TD, Higashi RM, Ferraris DV, Tsukamoto T, Matés JM,
Alonso FJ, Wang C, Seo Y, Chen X, Bishop JM. The metabolic profile of tumors depends
on both the responsible genetic lesion and tissue type. Cell Metab. 2012 Feb 8;15(2):157-70.
11. Altman BJ, Hsieh AL, Sengupta A, Krishnanaiah SY, Stine ZE, Walton ZE, Gouw AM,
Venkataraman A, Li B, Goraksha-Hicks P, Diskin SJ, Bellovin DI, Simon MC, Rathmell JC,
Lazar MA, Maris JM, Felsher DW, Hogenesch JB, Weljie AM, Dang CV. MYC Disrupts the
Circadian Clock and Metabolism in Cancer Cells. Cell Metab. 2015 Sep 16. pii: S15504131(15)00460-X. doi:10.1016/j.cmet. 2015.09.003.

Chi V. Dang, MD, PhD

1982-1985

1985-1987

1987-1991

1993-2003
1997-2011

2001-20112005-20112011-present

Intern & Resident, Internal Medicine, Johns Hopkins
Hospital
Hematology-Oncology Fellow, University of California,
San Francisco
Assistant Professor of Medicine, Division of
Hematology, Johns Hopkins
Chief, Division of Hematology, Johns Hopkins
Professor of Medicine, Cell Biology, Oncology andPathology, Johns Hopkins
Vice Dean for Research, Johns Hopkins University
School of Medicine
Johns Hopkins Family Endowed Chair Professor
Director, Abramson Cancer Center, John GlickEndowed Director’s Chair Professor, University ofPennsylvania

Specialty and Present Interest:
Hematological Malignancies, MYC Oncogene, Transcriptional Regulation of Cancer
Metabolism

37

METABOLIC REGULATION BY
RB TUMOR SUPPRESSOR GENE

Chiaki Takahashi

Division of Oncology and Molecular Biology
Cancer Research Institute, Kanazawa University
Kakuma-machi, Kanazawa, Ishikawa 920-1192, Japan(chtakaha@staff.kanazawa-u.ac.jp)

Control of biomass volume tightly synchronizes with cell cycle progression. This

represents one of the mechanisms whereby cells maintain their size after rounds of cell

division. Then, what is the mechanism by which biomass synthesis is coupled to cell cycle?

Many of mitogenic signals mediated by Ras, AKT, Myc, etc., have been implicated in theregulation of metabolic pathways. In addition, tumor suppressing machineries mediatedby p53 or PTEN appeared to direct rewiring in cellular metabolism. Then, what about pRB,

which is the central molecule in the cell cycle control? pRB has been recently implicated in

the control of glutaminolysis and OXPHOS. We, by using cell systems derived fromvarious genetically engineered mice, discovered that pRB controls several other metabolic

pathways. Determination of pRB targets in various genetic backgrounds significantly

contributed to the following discoveries. pRB inactivation in thyroid C cells and MEFs

induced N-Ras-dependent cellular senescence. This explained why C cell tumor developedin Rb-heterozygous mice stays in adenoma or low grade medullary carcinoma. The

mechanism appeared to involve most of isoprenyltransferases, those are essential for the

first step of Ras maturation. We further demonstrated that E2Fs, SREBPs and AKT wereinvolved in this pathway (Ref #3, 5, Muranaka et al., in preparation, and Sasaki et al., in
preparation) (Figure 1). Particularly in a p53-deficient background, RB status criticallydetermines self-renewal activity without impact on cell proliferation (Ref#2). In ARFdeficient breast cancers, RB inactivation increased fatty acid oxidation (FAO). This via

increase in mitochondrial superoxide production and Jun kinase (JNK) activity, triggers afeed forward loop between IL-6 and STAT3 leading to increase in self-renewal and drug

resistance (Kitajima et al., submitted). The analysis of a srcoma system revealed that pRB

38

C. Takahashi

positively regulates transcription of phosphglycerate mutase (PGAM) 1 and 2, thereby
directs rewiring in glycolysis and TCA activity (Ref#1, and Kohno et al., in preparation).

This transcriptional regulation appeared to involve C/EBPb and MEF2C,D. Thorough thisfunctional interaction with PGAMs, pRB appeared to control the undifferentiated status ofcancer stem-like cells. We will further demonstrate the evidence indicating that the RB-

dependent myogenic differentiation in C2C12 cells depend on PGAM2. These findings

indicated a case that pRB controls differentiation primarily by regulating cellular

metabolism. I am going to introduce you to previously unexpected functions of pRB incellular metabolism (Ref#4) unveiled by our unique experimental systems (Figure 2).

Figure 1

The mechanism whereby pRB regulates mevalonate (MVA) pathway

Figure 2

Possible roles of pRB in controlling central carbon metabolism and the relationship with othercancer-related genes.

Recasting the Warburg Effect

39

References
1. Okahashi N, Kohno S, Kitajima S, Matsuda F, Takahashi C and H.Shimizu. Metabolic
characterization of cultured mammalian cells by mass balance analysis, tracer labelingexperiments and computer-aided simulations. J Biosci Bioeng. 2015 Apr 27.
2. Kitajima S, Kohno S, Kondoh A, Sasaki N. Nishimoto Y, Li F, Mohammed SA,
Muranaka H, Nagatani N, Suzuki M, Kido Y and Takahashi C. Undifferentiated state
induced by Rb-p53 double inactivation in mouse thyroid neuroendocrine cells andembryonic fibroblasts. Stem Cells. 2015;33: 1657-1669.
3. Shamma A, Takegami Y, Miki T, Kitajima S, Noda M, Obara T, Okamoto T and
Takahashi C. Rb Regulates DNA damage response and cellular senescence throughE2F-dependent suppression of N-ras isoprenylation. Cancer Cell. 2009;15: 255-269.
4. Takahashi C, Sasaki N and Kitajima S. Twists in views on RB functions in cellular signaling, metabolism and stem cells. Cancer Sci. 2012;103: 1182-1188.
5. Takahashi C, Contreras B, Iwanaga T, Takegami Y, Bakker A, Bronson R T, Noda M, Loda
M, Hunt J L and Ewen M E. Nras loss induces metastatic conversion of Rb1-deficient neuroendocrine thyroid tumor. Nat Genet. 2006;38: 118-123.

Chiaki Takahashi, MD, PhD

1996-2003

1999-2002

2003-20082009-present

Assistant Professor, Kyoto University School of
Medicine
Research Fellow, Dana-Farber Cancer Institute, Harvard
University School of Medicine
Associate Professor, Kyoto University School of
Medicine
Professor, Kanazawa University, Cancer ResearchInstitute

Specialty and Present Interest:
Cancer Cell Biology, Molecular Biology, Tumor Suppressors, Metabolism, Stem Cells

40

FUMARATE METABOLISM IN CANCER AND
TYPE 2 DIABETES

Julie Adam1, Tomoyoshi Soga2, Norma Frizzell3, Andrew Silver4,
Patrik Rorsman1and Patrick J. Pollard5*

Oxford Centre for Diabetes, Endocrinology & Metabolism (OCDEM)
Churchill Hospital, University of Oxford, Oxford, OX3 7LJ, UK(julie.adam@ocdem.ox.ac.uk)
2Institute for Advanced Biosciences, Keio University
246-2 Mizukami, Tsuruoka, Yamagata 997-0052, Japan3
Department of Pharmacology, Physiology & Neuroscience, School of Medicine
University of South Carolina, Columbia, SC 29208, USA
4
Blizard Institute, Barts and The London School of Medicine and Dentistry
Queen Mary University of London, London E1 2AT, UK
5
Department of Physiology, Institute of Neuroscience and Physiology
Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden
1* This abstract is dedicated to Dr. Patrick John Pollard who died unexpectedly on 10th June 2015.
Patrick (Paddy) was an enthusiastic and exceptionally gifted scientist. We will remember him
fondly as a friend and colleague (Figure 1).

Dr. PatrickJohnPollard
2nd January1974– 10th June2015
Figure 1

Metabolic Regulation of Gene Function

41

Altered intermediary metabolism is a hallmark of cancer and the abnormal

accumulation of certain metabolites has linked dysregulated metabolism and cellularfunctions to tumorigenesis. Recently the abnormal accumulation of certain metabolites has

been linked to dysregulated metabolism and cellular functions leading to tumorigenesis1-3).

Such ‘oncometabolites’ are exemplified by fumarate, which accumulates as a consequenceof loss of activity of the Krebs cycle enzyme Fumarate hydratase (FH). FH catalyzes thehydration of fumarate to malate and has been identified as a tumor suppressor; it ismutated in hereditary leiomyomatosis and renal cell cancer (HLRCC)4). Elevated fumarate
has multiple cellular consequences including the stabilization of hypoxia-inducible factor(HIF1a) and activation of HIF dependent pathways such as angiogenesis, increased

glycolysis and glucose metabolism5,6). In addition, metabolic pathways are dysregulated, themitochondria of FH-deficient cells are dysfunctional, there is evidence of epigenetic

deregulation, blocks in cell differentiation and the posttranslational modification ofcysteine residues in proteins to form (S)-2-succinocysteine (2SC); an irreversible chemical

process termed ‘succination’ 7 ) (Figure 2). Originally identified in adipocytes andgastrocnemius muscle in some rodent diabetic models, succination in these cells might becaused by glucotoxicity, but has functional consequences, including inactivation of theglycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase. Succination in FHdeficient cells and tissues is ubiquitous and occurs in multiple proteins involved in avariety of cellular processes, such as redox homeostasis, iron-sulphur cluster assembly8),

cell metabolism and the ubiquitin conjugation pathway. Previously we have shown that

2SC is a sensitive biomarker for HLRCC9). In FH-deficiency succination of the Kelch-like

ECH-associated protein 1 (KEAP1), a key component of the ubiquitin-ligase complex
responsible for degradation of transcriptional factor Nuclear factor (erythroid-derived 2)-

like 2 (NRF2), abrogates its interaction with NRF2. This results in the constitutive

M



 

 





HIF
stabilisation

Succination

LossofFH
activity

Elevated
fumarate

Alteredmetabolism
Eg Purine&Urea/arginine
Mitochondrial
Dysfunction
Epigenetic
regulation

Figure 2

Multiple consequences of loss of Fumarate Hydratase activity

42

J. Adam et al.

activation of the potentially oncogenic NRF2-mediated antioxidant defence pathway10).

Also, mitochondrial aconitase is succinated on three critical cysteine residues in FHdeficient mouse embryonic fibroblasts (MEFs) and consequently displays impaired

enzymatic activity8) and succination of glutathione leads to an elevation of reactive oxygen

species (ROS) signalling.

Previously we demonstrated that fumarate accumulation causes dysregulation of theurea cycle and some dependence on arginine, potentially highlighting an ‘Achilles heel’ forthese tumours. Normalization of fumarate levels in the cytosol by re-expression ofcytoplasmic-specific FH ameliorates the defects associated with renal specific FH deletionin mice11) (Figure 3).

Building on our studies into the loss of FH (Fh1 in mice) associated with renal cancer,

we have generated a novel murine diabetic model in which Fh1 is deleted in pancreatic

beta cells (Fh1bKO) and in which glucose intolerance is related to dysregulated
metabolism. This is particularly interesting given the additional links to HIF1a, glucose-

induced insulin secretion (GIIS) and succination. These animals develop progressive








 



diabetes and impaired GIIS. Analyses have shown that insulin secretion is reduced,







Control
Fh1ɴKO

Pyruvate PDH

Glycolysis

Aspartate

AcetylCoA

AcetylCoA

CitrateCitrateOxaloacetate
CS

MDH
Argininosuccinate

Citrulline

Malate

ACL

ASS
FH
OCT
Ornithine

Urea
cycle

ASL

ACO2

Arginine

SDH
ARG

Krebs
Cycle

Succinate

Urea

cis Aconitate

ACO2

ACO1

Isocitrate

Isocitrate

AMP
ADSL

AICAR

IDH2,3SUC
SuccͲCoA

0.03
2.5
2.0
1.5
1.0
0.5
0.0

0.02
0.01
0.00

Purine synthesis

CO2

CO2

2OG

IDH1
2OG

N.D.

Adenylosuccinate

Figure 3

ACO1

cis Aconitate

Fumarate

SAICAR

OGDH
CO2

GLDH
Gln

GLS

Fumarate accumulation alters metabolism in both the mitochondria and the cytosol

GluMetabolic Regulation of Gene Function

43

without significant differences in the KATP channel, calcium influx and exocytosis responses

compared to control islets. Also, the pancreatic a-cells show significantly reduced
glucagon secretion and increased glucagon content. These Fh1bKO islet cells exhibit

dysregulated metabolism as determined by metabolite analysis, measurement of glucose

oxidation and utilisation (measured by production of 14CO2 and 3H20 respectively) andaccumulation of fumarate, particularly in high glucose concentrations (20mM). A screen of

Fh1bKO islets revealed succination in a number of proteins, including Glyceraldehyde 3-

phosphate dehydrogenase and other interesting targets, notably Park7 (also known as DJ1),

which acts as a sensor for oxidative stress, a recognised driver of tumorigenesis and link todiabetes and to Parkinson’s disease. Surprisingly glucose stimulated ATP: ADP ratio in

Fh1bKO islets is normal in the early stages of disease (9-12 weeks); but drops significantly

4-6weeks later in very diabetic animals. This is mirrored in altered electrophysiologicalresponses. Fh1bKO cells also show mitochondrial dysfunction exhibited by altered size anddistribution.

Very significantly, analysis of human pancreatic islets has revealed evidence forsuccination of proteins in glucagon-secreting a-cells in Type 2 diabetes, indicating elevationof fumarate resulting from dysregulated cell metabolism. DJ1 was also identified as asuccination target for human islets from both normal and type-2-diabetic donors.

Fumarate is elevated in both mouse and human islets exposed to hyperglycemic

conditions for as little as 1 hour. Protein succination is increased in diabetic murine andhuman pancreatic islets and systemically; it is observed in the renal tubular cells andadipocytes of Fh1bKO mice. Interestingly there is evidence from matched, paired samples

of increased succination in adenomas and colorectal cancers compared to normal mucosa of

non-diabetic and diabetic individuals, none of whom have FH mutations. The mostsignificant increased levels of succinaton are seen in the colorectal cancer samples of type-2

diabetic patients.

Globally we face the challenge of tackling diabetes, obesity and their consequences12),

such as predisposition to some cancers, there are compelling reasons to investigate links

between these diseases. Currently, the connections between dysregulated metabolism andcancer are attracting significant interest fuelled in part by exploitation of mass spectrometryadvances to facilitate high-resolution metabolic profiling of cells and tumours13-15).

Our studies indicate that diabetes via hyperglycemia and elevated fumarate impairs a-cell function, and raise the exciting possibility that hyperglycemia-induced accumulation of

fumarate links diabetes and co-morbidities including colorectal cancer and in the kidney.

Here we propose that the insights into the myriad consequences of elevated fumarate

conferred by our Fh1KO mouse models, linked to our studies on renal and colorectal cancer

and diabetes will allow us to elucidate potentially common mechanisms evoked in cells as

44

J. Adam et al.

a stress response (Figure 4). We hope that this work will not only provide novel insights

into a serious global disease, diabetes, and a rare but aggressive disease, HLRCC; but also

will increase our understanding of cellular metabolism and inform on potential targets forintervention.

I



 









fumarate

Figure 4

Co-morbidities?

Is elevated fumarate a molecular link between hyperglycaemia, diabetes and co-morbidities?

References
1. Yang, M., Soga, T., and Pollard, P.J. 2013. Oncometabolites: linking altered metabolism
with cancer. J Clin Invest 123:3652-3658.
2. Adam, J., Yang, M., Soga, T., and Pollard, P.J. 2013. Rare insights into cancer biology.
Oncogene doi: 10.1038/onc.2013.222.
3. Yang, M., Soga, T., Pollard, P.J., and Adam, J. 2012. The emerging role of fumarate as an
oncometabolite. Front Oncol 2:85.
4. Tomlinson, I.P., Alam, N.A., Rowan, A.J., Barclay, E., Jaeger, E.E., Kelsell, D., Leigh, I.,
Gorman, P., Lamlum, H., Rahman, S., Roylance, R.R., Olpin, S., Bevan, S., Barker, K.,
Hearle, N., Houlston, R.S., Kiuru, M., Lehtonen, R., Karhu, A., Vilkki, S., Laiho, P.,
Eklund, C., Vierimaa, O., Aittomaki, K., Hietala, M., Sistonen, P., Paetau, A., Salovaara, R.,
Herva, R., Launonen, V., and Aaltonen, L.A. 2002. Germline mutations in FH predispose
to dominantly inherited uterine fibroids, skin leiomyomata and papillary renal cell cancer.
Nat Genet 30:406-410.

Metabolic Regulation of Gene Function

45

5. Pollard, P.J., Spencer-Dene, B., Shukla, D., Howarth, K., Nye, E., El-Bahrawy, M.,
Deheragoda, M., Joannou, M., McDonald, S., Martin, A., Igarashi, P., Varsani-Brown, S.,
Rosewell, I., Poulsom, R., Maxwell, P., Stamp, G.W., and Tomlinson, I.P. 2007. Targeted
inactivation of fh1 causes proliferative renal cyst development and activation of thehypoxia pathway. Cancer Cell 11:311-319.
6. Pollard, P.J., Briere, J.J., Alam, N.A., Barwell, J., Barclay, E., Wortham, N.C., Hunt, T.,
Mitchell, M., Olpin, S., Moat, S.J., Hargreaves, I.P., Heales, S.J., Chung, Y.L., Griffiths, J.R.,
Dalgleish, A., McGrath, J.A., Gleeson, M.J., Hodgson, S.V., Poulsom, R., Rustin, P., andTomlinson, I.P. 2005. Accumulation of Krebs cycle intermediates and over-expression of
HIF1alpha in tumours which result from germline FH and SDH mutations. Hum Mol
Genet 14:2231-2239.
7. Nagai, R., Brock, J.W., Blatnik, M., Baatz, J.E., Bethard, J., Walla, M.D., Thorpe, S.R.,
Baynes, J.W., and Frizzell, N. 2007. Succination of protein thiols during adipocyte maturation: a biomarker of mitochondrial stress. J Biol Chem 282:34219-34228.
8. Ternette, N., Yang, M., Laroyia, M., Kitagawa, M., O'Flaherty, L., Wolhulter, K.,
Igarashi, K., Saito, K., Kato, K., Fischer, R., Berquand, A., Kessler, B.M., Lappin, T.,
Frizzell, N., Soga, T., Adam, J., and Pollard, P.J. 2013. Inhibition of mitochondrial aconitaseby succination in fumarate hydratase deficiency. Cell Rep 3:689-700.
9. Bardella, C., El-Bahrawy, M., Frizzell, N., Adam, J., Ternette, N., Hatipoglu, E.,
Howarth, K., O'Flaherty, L., Roberts, I., Turner, G., Taylor, J., Giaslakiotis, K., Macaulay,
V.M., Harris, A.L., Chandra, A., Lehtonen, H.J., Launonen, V., Aaltonen, L.A., Pugh,
C.W., Mihai, R., Trudgian, D., Kessler, B., Baynes, J.W., Ratcliffe, P.J., Tomlinson, I.P., andPollard, P.J. 2011. Aberrant succination of proteins in fumarate hydratase-deficient miceand HLRCC patients is a robust biomarker of mutation status. J Pathol 225:4-11.
10. Adam, J., Hatipoglu, E., O'Flaherty, L., Ternette, N., Sahgal, N., Lockstone, H., Baban, D.,
Nye, E., Stamp, G.W., Wolhuter, K., Stevens, M., Fischer, R., Carmeliet, P., Maxwell,
P.H., Pugh, C.W., Frizzell, N., Soga, T., Kessler, B.M., El-Bahrawy, M., Ratcliffe, P.J., andPollard, P.J. 2011. Renal cyst formation in Fh1-deficient mice is independent of the
Hif/Phd pathway: roles for fumarate in KEAP1 succination and Nrf2 signaling. Cancer
Cell 20:524-537.
11. Adam, J., Yang, M., Bauerschmidt, C., Kitagawa, M., O'Flaherty, L., Maheswaran, P.,
Ozkan, G., Sahgal, N., Baban, D., Kato, K., Saito, K., Iino, K., Igarashi, K., Stratford, M.,
Pugh, C., Tennant, D.A., Ludwig, C., Davies, B., Ratcliffe, P.J., El-Bahrawy, M.,
Ashrafian, H., Soga, T., and Pollard, P.J. 2013. A role for cytosolic fumarate hydratase inurea cycle metabolism and renal neoplasia. Cell Rep 3:1440-1448.
12. Ashcroft, F.M., and Rorsman, P. 2012. Diabetes mellitus and the beta cell: the last ten
years. Cell 148:1160-1171.

46

J. Adam et al.

13. Soga, T., and Imaizumi, M. 2001. Capillary electrophoresis method for the analysis of inorganic anions, organic acids, amino acids, nucleotides, carbohydrates and other anioniccompounds. Electrophoresis 22:3418-3425.
14. Soga, T. 2007. Capillary electrophoresis-mass spectrometry for metabolomics. Methods
Mol Biol 358:129-137.
15. Soga, T., Igarashi, K., Ito, C., Mizobuchi, K., Zimmermann, H.P., and Tomita, M. 2009.
Metabolomic profiling of anionic metabolites by capillary electrophoresis mass spectrometry. Anal Chem 81:6165-6174.

Julie Adam, PhD

1982-1985

1986-1989

1989-2001

2001-2008

2008-20132013-present

Research Assistant in Experimental Haematology,
University College London
Post-doctoral Fellowship in Experimental Haematology,
Radiobiology Unit, Harwell
Post-doctoral Research Fellow in Developmental
Biology, ICRF, Oxford University and LIF
Post-doctoral Fellow & Investigator Scientist, MRC,RAGSU
Post-doctoral Research Scientist, NDM, Henry
Wellcome Building for Molecular Physiology,
University of Oxford
Post-doctoral Research Scientist, RDM, OCDEM,
University of Oxford

Specialty and Present Interest:
Role of Fumarate Hydratase, Fumarate, Succination, Dysregulated Metabolism, Cancer,
Oncometabolites, Diabetes and Diabetic Co-morbidities, Clinical Biomarkers, HLRCC

47

IDH MUTATIONS AND
THERAPEUTIC STRATEGY IN CANCER

Issay Kitabayashi

Division of Hematological Malignancy
National Cancer Center Research Institute5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan(ikitabay@ncc.go.jp)

Mutations in isocitrate dehydrogenase (IDH) 1 and 2 are frequently observed in acute

myeloid leukemia (AML), glioma, and many other cancers. While wild-type IDHs convert

isocitrate to a-ketoglutarate (a-KG), mutant IDHs convert a-KG to oncometabolite 2-hydroxyglutarate (2-HG), which dysregulates a set of a-KG-dependent dioxygenases, suchas TETs, histone demethylases, EGLNs, and other enzymes. Because the role of mutant IDH

is not necessary for normal cells, inhibitors directed against mutant IDH are expected tohave minimum side effects as those of anti-cancer agents.

To determine whether mutant IDH enzymes are valid targets for cancer therapy, we

created a mouse model of mutant IDH-dependent AML. Previously, the IDH mutation

alone was shown to be insufficient for the induction of AML, and IDH mutations occur
simultaneously with mutations in other genes such as NPM, DNMT3A, and FLT3. In

accordance with these observations, we found that NPM+/- hematopoietic progenitor cells

transduced with IDH2/R140Q, NPMc, DNMT3A/R882H, and FLT3/ITD cooperatively
induced AML in a mouse model (Figure 1). However, when only three of these mutant

genes were transduced, myeloproliferative neoplasms (MPNs) rather than AML was more

frequently induced and their onset was delayed in any combinations of the mutant genes.

These results clearly indicate that all four mutations are necessary for the efficientinduction of AML. By using a combination of AML model mice with cre-loxp, weconditionally deleted IDH2/R140Q from AML mice, which blocked 2-HG production andresulted in the loss of leukemia stem cells. Accordingly, the progression of AML wassignificantly delayed (Figure 2). These results indicate that the function of IDH2 mutationis critical for the development and maintenance of AML stem cells, and that mutant IDHs
are promising targets for anticancer therapy.

48

I. Kitabayashi

A

day1
NPM+/- c-Kit+ BM cells

day2
NPMcires-EGFP

IDH2/R140Qires-NGFR

day3
BMT

DNMT3A/R882H

FLT3-ITD

serialinfection

B
100

80
OverallSurvival(%)

genecombination
60

NPMc

IDH2/
R140Q

DNMT3A/
R882H

FLT3Ͳ
ITD

IDF

Ͳ

+

+

+

40

NDF

+

Ͳ

+

+

INF

+

+

Ͳ

+

20

IND

+

+

+

Ͳ

INDF

+

+

+

+

0

Figure 1

0

50

100
150
200
Daysaftertransplantation

250

300

Establishment of an AML model harboring IDH2/R140Q.
A) Scheme of the experiments. c-Kit+ hematopoietic progenitor cells were isolated from
Npm1+/- mouse bone marrow (BM) and serially infected with three or four of the following
constructs:
pMy-NPMc-ires-EGFP,pGCDN-IDH2/R140Q-ires-NGFR,pMSCVDNMT3A/R882H-puro, and pMSCV-FLT3/ITD-neo. When cells were infected withthree mutant genes, the infection of corresponding empty vector for the absent mutant
gene was performed. The infectants were transplanted into irradiated mice.
B) NPMc, IDH2/R140Q, DNMT3A/R882H, and FLT3/ITD (NIDF) cooperatively induce
AML in mice. The abbreviations for the gene combinations (IDF, NDF, NIF, NID, and
NIDF) are indicated. IDF vs. NIDF, P < 0.0001; NDF vs. NIDF, P < 0.0001; NIF vs.
NIDF, P = 0.0009; NID vs. NIDF, P = 0.0006; log-rank test.

Metabolic Regulation of Gene Function

A

B
㼼TAM

4weeksof+TAMtreatment

IDH2/R140Q(iresͲNGFR)

80

60

40

+TAM
PB

+TAM

NPMc (iresͲEGFP)

NPMc (iresEGFP)+TAM BM

IDH2/R140Q(iresͲNGFR)

BM

100

OverallSurvival(%)

49c

NPMc (iresͲEGFP)

20

ͲTAM

LͲGMP

0
0

20

40

60

80

100

120

3

Daysaftertransplantation%ofAMLcells

2.5
2
1.5
1
0.5
0

Figure 2

IDH2/R140Q is necessary for the maintenance of AML.
A) c-Kit+ hematopoietic progenitor cells were isolated from ERT2-Cre+ Npm1+/- mice, serially
infected with pMy-NPMc-ires-EGFP, pGCDN-flox-IDH2/R140Q-ires-NGFR, pMSCVDNMT3A/R882H-puro, and pMSCV-FLT3/ITD-neo [Nf(I)DF], and then transplanted
into irradiated mice. The Nf(I)DF-induced AML cells were transplanted into secondary
recipient mice. After 2 weeks, the secondary recipient mice were injected with corn oil ortamoxifen (TAM). The inset panels show the percentage of EGFP+ leukemic cells in the PB
at the start of tamoxifen treatment.
B) Deletion of IDH2/R140Q decreased the fraction of cells expressing LSC markers andexhausted EGFP+ cells in BM. TAM treatment began 7 weeks after transplantation. BM
cells were isolated from the mice and analyzed for NPMc, EGFP, and NGFR by flow
cytometry. The mean percentages of EGFP+ cells in the BM of three mice are shown. Bargraphs show the populations of L-GMP (Lin-, Sca1-, cKit+, CD16/32+, CD34+) among
EGFP+ cells after TAM treatment. Genotyping of IDH2/R140Q was performed with BM
cells at 2 weeks after TAM treatment.
+Oil

+TAM

I. Kitabayashi

Figure 3BM4weeksafter3rd BMT

3rd BMTNPMc (iresͲEGFP)

PB

IDH2/R140Q(iresͲNGFR) IDH2/R140Q(iresͲNGFR)+TAM

IDH2/R140Q(iresͲNGFR)

ͲTAM

BM

IDH2/R140Q(iresͲNGFR)

2weeksof㼼TAMtreatment

Overall Survival (%)

50

PB

+TAM

100

50

0

0
50
100
days after 3rd transplantation

NPMc (iresͲEGFP)

Deletion of IDH2/R140Q decreased the LSC population. BM cells treated or untreated with
TAM for 2 weeks were isolated from the mice shown in Figure 3I. The third BMT was performed using these BM cells. The mice were not treated with tamoxifen after the third
BMT. PB cells were isolated from mice 4 weeks after the third BMT and were analyzed for
NPMc (EGFP) and IDH2/R140Q (NGFR) expression by flow cytometry. The number represents the mean percentage of EGFP+ cells in the PB of 5 mice.

A

Spib

Ebf1
1.2

RelativeExpression

RelativeExpression

1.2
1
0.8
0.6
0.4
0.2

1
0.8
0.6
0.4
0.2
0

0

controlOilcontrol

TAM

Oil

B

Spiblocus
0.3

0.25

0.25

%ofinput%ofinput

Ebf1locus
0.3
0.2
0.15
0.1
0.05

TAM
Nf(I)DF

Nf(I)DF

0.2
0.15
0.1
0.05
0

0
controFigure 4

ͲTAM

oil

TAM
Nf(I)DF

controoil

TAM
Nf(I)DF

IDH2/R140Q negatively regulates the 5hmC modification and expression of differentiation-inducing factors.
A) Expression of Spib and Ebf1 in NBM cells, untreated Nf(I)DF-AML cells, and TAMtreated Nf(I)DF-AML cells was analyzed by real-time PCR.
B) 5hmC modification of Ebf1 and Spib loci in NBM cells, untreated Nf(I)DF-AML cells,
and TAM-treated Nf(I)DF-AML cells was analyzed by real-time PCR. (n=3 per group)

Metabolic Regulation of Gene Function

51

To confirm the decrease in the number of LSCs, we performed a third transplantation

using BM cells isolated from mice treated with or without tamoxifen for 2 weeks. When PB

cells were analyzed 4 weeks later, the number of AML cells was increased in micetransplanted with untreated BM cells (Figure 3). Conversely, AML cells were almost

undetectable in mice transplanted with tamoxifen-treated BM cells (Figure 3). All micetransplanted with tamoxifen-untreated BM cells died of AML by 127 days after the third

BMT, whereas none of the mice transplanted with tamoxifen-treated BM cells died by 140

days (Figure 3. These results strongly indicate that IDH2/R140Q is necessary for the

maintenance of the LSC population.

Based on these findings, we developed potent and specific inhibitors of mutant IDH1

and tested their effects on the mutant IDH1-dependent AML mouse model, created byintroducing four mutant genes including mutant IDH1. The 2HG level was promptly anddramatically decreased in AML cells soon after treatment with the mutant IDH1 inhibitors,

and the number of leukemia cells was reduced after a 4-week treatment. We also tested theeffects of the inhibitors on patient-derived xenograft (PDX) models and cell lines ofglioblastoma and other cancers with IDH1 mutations. The inhibitors decreased the 2HGlevels in the tumors and inhibited the growth of the tumors. These results indicate that

IDH1 mutant inhibitors are effective for the treatment for AML as well as glioblastoma
with IDH1 mutations.

NPMc and IDH2/R140Q cooperatively activated the Hoxa9/Meis1 pathway, andIDH2/R140Q activated the hypoxia pathway. These two pathways are likely to beimportant for IDH2/R140Q-mediated engraftment/survival of NPMc+ cells in mice. In
addition to IDH2/R140Q and NPMc, expression of DNMT3A/R882H and FLT3/ITD isalso required for efficient induction of AML. DNMT3A/R882H further upregulated theexpression levels of Meis1. Furthermore, DNMT3A/R882H promoted the maintenance ofcells in an undifferentiated state. Taken together, our results suggest that the activation ofmultiple signaling pathways is required for NIDF cells to induce AML.

Because IDH mutations and TET2 mutations are mutually exclusive in AML, theinhibition of TET-mediated conversion of 5mC to 5hmC is considered one of the main rolesof mutant IDH. We found that levels of 5hmC on differentiation-inducing genes, such asEbf1, Spib and Pax5 were decreased in AML cells with IDH2/R140Q and recovered byconditional deletion of IDH2/R140Q. In consistent with levels of 5hmC, expressions ofthese genes are upregulated in the AML cells and revreased by deletion of IDH2/R140Q.

Gene expression analysis revealed that IDH2/R140Q up-regulates a set of genes that is

activated in response to hypoxia as well as Meis1. As 2HG inhibits EGLN thathydroxylates and marks HIF1a for ubiquitin-proteasomal degradation, it is probable thatmutant IDH2-produced 2HG stabilizes HIF1a through inhibition of EGLN. Furthermore, it52

I. Kitabayashi

was reported that Meis1 activates the transcription of HIF1a. In consistent with theseinformations, we showed that IDH2/R140Q increased the protein levels of HIF1a in
cultured cells.

Issay Kitabayashi, PhD

1992-1995
1995-1998

1998-2002

2002-present
2011-20132007-present

Special Researcher for Basic Science, RIKEN
Staff Scientist, National Cancer Center ResearchInstitute
Section Head, National Cancer Center ResearchInstitute
Chief, National Cancer Center Research Institute
Deputy Director, National Cancer Center ResearchInstitute
Guest Professor, Tokyo University of Science

Specialty and Present Interest:
Cancer Stem Cells, Hematological Malignancy, Cancer Epigenetics

53

EPIGENETIC MECHANISMS IN HEMATOLOGICMALIGNANCIES LINKED TO METABOLIC PATHWAYS

Ari M. Melnick1, Ross L. Levine2 and Maria E. Figueroa3
Sackler Center for Biomedical and Physical Sciences
Weill Cornell Medical College
413 East 69th Street, New York, NY 10021, USA(Amm2014@med.cornell.edu)
2
Memorial Sloan Kettering, 3University of Michigan

1

The instructions that endow cells with the capacity to manifest unique phenotypes

reside in the chemical modifications, proteins, and RNA molecules that constitute theepigenome. It is the particular distribution of epigenetic marks that enables cells sharing

the same genomic DNA sequence to differentiate into distinct cell lineages with specificfunctions. Just like in normal cells the epigenome also encodes the phenotype of tumor

cells. Indeed, aberrant epigenetic patterning of the genome is emerging as a hallmark ofacute myeloid leukemia, a malignancy characterized by a relative paucity of genetic

lesions. AML can be classified into disease subtypes based on epigenetic signatures1).

These signatures were established by performing integrative epigenomic profiling ofalmost 800 patients enrolled in clinical trials in Europe and the US1,2). More recently thenature of these signatures was further explored using next generation sequencing of themethylomes of these patients (3 and unpublished data) .

Careful examination of thesemethylomes revealed that the most informative methylcytosine residues driving epigenetic
clustering of AML patients are located in enhancer elements, positioned outside of CpG

islands. Hence perturbation of enhancer function through aberrant cytosine methylation

may play a key role in encoding the unique phenotypes and clinical outcomes of subsets of
AML patients.

Importantly, some of these signatures cannot be attributed to any

particular somatic mutation suggesting that at least some epigenetic signatures arise due toother influences.

Based on these considerations we predicted that epigenetic signatures could be used as

molecular blueprints that could point towards causative mechanisms that drive leukemia
phenotypes. Along these lines we found that a subset of AMLs that share a particular

54

A. M. Melnick et al.

DNA hypermethylation signature exhibit somatic mutations of the IDH1 and IDH2 genes2).

These enzymes normally catalyze the conversion of isocitrate to a-ketoglutarate (aKG).

Various lines of investigation revealed that mutant IDH enzymes generate theoncometabolite 2-hydroxyglutarate, which can inhibit aKG dependent dioxygenases

including the TET family of enzymes that convert 5-methylcytosine (5mC) into 5hydroxymethlcytosine (5hmC)4). Indeed patients with TET2 mutations share the aberrant

DNA methylation profile of IDH mutant AML patients2). TET2 is not a DNA binding

proteins and must be recruited to DNA by other proteins to carry out its actions. We foundthat WT1, a transcriptional regulator often mutated in AML interacts with TET25). Notably,

somatic mutations in IDH1, IDH2, TET2 and WT1 are almost universally mutually

exclusive of each other in AML. Patients with all four of these mutations exhibit reduced

global levels of 5hmC and in genome wide profiling studies display overlapping 5hmC

distribution profiles5). Whereas 5mC distribution shows weak inverse correlation with gene

expression, 5hmC displays very strong positive correlation5). Collectively these data define
a functional axis whereby WT1 mutation defines a subset of the effects emanating from

TET2 loss of function, which in turn mediates a subset of the actions of 2HG generated bymutant IDH1 and IDH25,6). The link between gene expression and 5hmC points towards
perturbation of this mark as perhaps representing the dominant effect of this functional

axis. Given that IDH mutants potentially affect many epigenetic enzymes including all the

TET family proteins and jumanji histone demethylases4), it would be informative to dissect

out the contributions of TET2 to further distinguish its biological and epigenetic

contribution to the IDH phenotype.

Further characterization of the epigenetic actions of TET2 was pursued by crossing

TET2 deficient mice with FLT3ITD transgenic animals. The two mutations often occur

together in AML patients and are associated with an unfavorable clinical outcome. Whileneither mutation alone causes leukemia, the combination of both together yielded a lethal

phenotype7). Analysis of the methylomes of single vs. double mutant mice revealed that

TET2 alone resulted in a small hypermethylated signature, and FLT3 in a small

hypomethylated signature. However the combination of both together resulted in massive
epigenetic dysregulation of genes distinct from those affected by either mutation alone7).

Most of this perturbation consisted of DNA hypermethylation. Among the top methylated
genes was GATA2, which was silenced in the double but not single mutant animals.
Similar effects were observed in human AML patients with TET2 + FLT3ITD mutation7).

Restoration of GATA2 rescued the TET2-FLT3ITD leukemia phenotype, demonstrating thatloss of GATA2 plays a critical role in the biological effect of the combined mutations. To
determine if epigenetic synergy is reversible we treated TET2-FLT3ITD leukemic mice with a

specific FLT3 inhibitor (unpublished). This treatment only modestly affected the leukemia

Metabolic Regulation of Gene Function

55

phenotype and had little effect on epigenetic signatures. In contrast treatment of mice with

DNA methyltransferase inhibitor drugs led to complete resolution of the leukemic

phenotype. Leukemia blasts disappeared from blood and the mutant hematopoietic stem

cells recovered the ability to differentiate normally and contributed to normal

hematopoiesis.

GATA2 methylation and gene expression normalized. However the

mutant leukemia stem cells could not be eradicated. Hence purely reversing the DNA

methylation effects reprogrammed leukemia stem cells to resume the hematopoietic

program but could not kill them. Perhaps combination therapies would achieve thisendpoint.

Finally, we wondered whether in addition to altering epigenetic programming, somatic

mutations of IDH would affect AML epigenetic heterogeneity. We considered that
epigenetic heterogeneity among populations of leukemia cells would be potentially resultin biological actions such as improved population fitness and hence greater tendency torelapse. We developed an algorithm called MethClone that enables epigenetic allelicdiversity to be measured8). We found that epigenetic allele burden is linked to unfavorable

clinical outcome in AML, independent of somatic mutations or other biomarkers(unpublished). Genetic and epigenetic allelic diversity did not track together and appeared

to be distinct biological phenomenon. AMLs could be classified into those with highepiallele burden with low somatic mutations, and a second group with low epiallele
burden and higher somatic mutation burden. Surprisingly, IDH mutations were linked to

the former, suggesting that IDH epigenetic effects are quite specific and direct, and do notdisplay much random dispersion. The presence of epialleles was accompanied bytranscriptional deregulation, as evidence from RNA-seq and single cell RNA-seq(unpublished). Perhaps shifting of epialleles thus enables AMLs to sample different

transcriptional states. In summary, exploring AML epigenomes has provided theopportunity to discover novel disease mechanisms and develop new therapies, including

the discovery of the IDH-TET2-WT1 axis and their downstream effects. Ongoing

examination of the layers of the AML epigenome should continue to provide valuable
insights into this mostly fatal disease.

References
1. Figueroa, M.E., S. Lugthart, Y. Li, C. Erpelinck-Verschueren, X. Deng, P.J. Christos, E.
Schifano, J. Booth, W. van Putten, L. Skrabanek, F. Campagne, M. Mazumdar, J.M.
Greally, P.J. Valk, B. Lowenberg, R. Delwel, and A. Melnick, DNA methylation signatures
identify biologically distinct subtypes in acute myeloid leukemia. Cancer Cell, 2010.
17(1): p. 13-27.

56

A. M. Melnick et al.

2. Figueroa, M.E., O. Abdel-Wahab, C. Lu, P.S. Ward, J. Patel, A. Shih, Y. Li, N. Bhagwat, A.
Vasanthakumar, H.F. Fernandez, M.S. Tallman, Z. Sun, K. Wolniak, J.K. Peeters, W.
Liu, S.E. Choe, V.R. Fantin, E. Paietta, B. Lowenberg, J.D. Licht, L.A. Godley, R. Delwel,
P.J. Valk, C.B. Thompson, R.L. Levine, and A. Melnick, Leukemic IDH1 and IDH2
mutations result in a hypermethylation phenotype, disrupt TET2 function, and impair
hematopoietic differentiation. Cancer Cell, 2010. 18(6): p. 553-67.
3. Akalin, A., F.E. Garrett-Bakelman, M. Kormaksson, J. Busuttil, L. Zhang, I.
Khrebtukova, T.A. Milne, Y. Huang, D. Biswas, J.L. Hess, C.D. Allis, R.G. Roeder, P.J.
Valk, B. Lowenberg, R. Delwel, H.F. Fernandez, E. Paietta, M.S. Tallman, G.P. Schroth,
C.E. Mason, A. Melnick, and M.E. Figueroa, Base-pair resolution DNA methylation
sequencing reveals profoundly divergent epigenetic landscapes in acute myeloid
leukemia. PLoS Genet, 2012. 8(6): p. e1002781.
4. Garrett-Bakelman, F.E. and A.M. Melnick, Differentiation therapy for IDH1/2 mutant
malignancies. Cell Res, 2013. 23(8): p. 975-7.
5. Rampal, R., A. Alkalin, J. Madzo, A. Vasanthakumar, E. Pronier, J. Patel, Y. Li, J. Ahn, O.
Abdel-Wahab, A. Shih, C. Lu, P.S. Ward, J.J. Tsai, T. Hricik, V. Tosello, J.E. Tallman, X.
Zhao, D. Daniels, Q. Dai, L. Ciminio, I. Aifantis, C. He, F. Fuks, M.S. Tallman, A.
Ferrando, S. Nimer, E. Paietta, C.B. Thompson, J.D. Licht, C.E. Mason, L.A. Godley, A.
Melnick, M.E. Figueroa, and R.L. Levine, DNA hydroxymethylation profiling reveals
that WT1 mutations result in loss of TET2 function in acute myeloid leukemia. Cell Rep,
2014. 9(5): p. 1841-55.
6. Wang, Y., M. Xiao, X. Chen, L. Chen, Y. Xu, L. Lv, P. Wang, H. Yang, S. Ma, H. Lin, B.
Jiao, R. Ren, D. Ye, K.L. Guan, and Y. Xiong, WT1 recruits TET2 to regulate its target gene
expression and suppress leukemia cell proliferation. Mol Cell, 2015. 57(4): p. 662-73.
7. Shih, A.H., Y. Jiang, C. Meydan, K. Shank, S. Pandey, L. Barreyro, I. Antony-Debre, A.
Viale, N. Socci, Y. Sun, A. Robertson, M. Cavatore, E. de Stanchina, T. Hricik, F.
Rapaport, B. Woods, C. Wei, M. Hatlen, M. Baljevic, S.D. Nimer, M. Tallman, E. Paietta, L.
Cimmino, I. Aifantis, U. Steidl, C. Mason, A. Melnick, and R.L. Levine, Mutational
cooperativity linked to combinatorial epigenetic gain of function in acute myeloid
leukemia. Cancer Cell, 2015. 27(4): p. 502-15.
8. Li, S., F. Garrett-Bakelman, A.E. Perl, S.M. Luger, C. Zhang, B.L. To, I.D. Lewis, A.L.
Brown, R.J. D'Andrea, M.E. Ross, R. Levine, M. Carroll, A. Melnick, and C.E. Mason,
Dynamic evolution of clonal epialleles revealed by methclone. Genome Biol, 2014. 15(9): p.
472.

Metabolic Regulation of Gene Function

Ari M. Melnick, MD

2002-2008

2004-2008
2008-2012
2006-present
2008-present

2009-present

2011-present
2013-present

2013-present

2014-present

Assistant Professor, Albert Einstein College ofMedicine (AECOM)
Co-leader, Immuno-Oncology Program, AECOM
Associate Professor, Weill Cornell Medical College
Chair, ECOG Leukemia Laboratory Committee
Faculty Director, Epigenomics Core Facility, Weill
Cornell Medical College (WCMC)
Chair, Sackler Center for Biomedical and PhysicalSciences, WCMC
Standing member, NIH CAMP study section
Gebroe Family Professor, Weill Cornell Medical
College
Chair, Hematologic Malignancies Program, Weill
Cornell Cancer Center
NCI steering committee on lymphomasSpeciality and Present Interests:
Cancer Epigenetics, Hematologic Malignancies, Tumorigenesis, Treatment

57

58

CARBON MONOXIDE-RESPONSIVE HEME PROTEINSREGULATE CANCER PROLIFERATION AND
CHEMORESISTANCE

Makoto Suematsu

Department of Biochemistry
Keio University School of Medicine
35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582(gasbiology@keio.jp)

Gases constitute a group of smallest metabolites in biological systems. Among gases,

Introduction

carbon monoxide (CO) is generated by heme oxygenase that is inducible in cancer cells in

response to exposure to stimuli such as radiation, hypoxia and anti-cancer reagents. Sincegases are too small to handle for mining the specific receptors in biological systems, only

little information has been available for CO-specific receptors. Previously, metabolome
analyses allowed us to find lists of metabolites that are up- or down-regulated in response

to CO-inducing conditions1), since CO has the ability to bind to proteins with metalcentered prosthetic groups, that is, enzymes. Of interest is observation that exogenous CO

application to cultured cell lines acutely causes modest elevation of remethylation
metabolites such as methionine and S-adenosylhomocysteine (SAH), a potent inhibitor of

methyltransferation, and decreases in cystathionine and total amounts of glutathione incells. These lines of observations led us to hypothesize that cystathionine b-synthase (CBS)serves as a CO-responsive target. Interestingly, this hypothesis was actually proven byobservation that recombinant human CBS is inhibited by CO but not by NO1), suggestingthat CBS accounts for a CO receptor.

Such an inhibitory action of CO on CBS causes alterations in methylation/

CO regulates carbohydrate metabolism through CBS inhibition

demethylation of many proteins in cells. The surveillance of molecular targets affected by

CO led us to determine that a glycolytic enzyme PFKFB3 that produces fructose 2,6-

bisphosphate serves as a target molecule regulated be CO-sensitive methylation. In humanGaseous Regulation and Metabolism

59

colon cancer HCT116 cells, CO induction or CBS knockdown turned out to down-regulate
methylation of PFKFB3.

This event might result from a metabolic response of SAH, aninhibitor of methyltransferase, by either CO-mediated CBS inhibition or CBS knockdown.

Interestingly, PFKFB3 expressed in cancer cells is mostly arginine-methylated2), and stressresponses of CO causes demethylation of the enzyme. De-methylation of PFKFB3 reduces

fructose 2,6-bisphosphate, a potent activator of PFK1, the rate-limiting enzyme forglycolysis, and resultantly up-regulates glucose bio-transformation towards pentose

phosphate pathway (PPP) rather than glycolysis. Such responses to CO-mediatedinhibition of CBS benefit increases in NADPH/GSH system to enhance cancer survival

against anti-cancer reagents2). Simultaneously, the response of this kind might benefit
synthesis of biomass such as nucletic acids through the PPP activation.

In a xenograft transplantation model using super-immunodeficient mice, cancer cells

inplanted in spleen are metastasized in the liver. Under these circumstances, GSH wasremarkably elevated in tumors to increase anti-oxidative properties, as judged by

microscopic imaging microscopy2,3). To note is that CBS knockdown in the cancer cells

accelerates cancer growth with increased GSH. Considering the inhibitory action of CO on

CBS, GSH elevation in CBS-knockdown cancer cells appears to be inconsistent. However,this event might result from a back-up mechanism for supplying cystine/cysteine as a

substrate of glutathione through CD44/xCT system that helps uptake of extracellularcystine in HCT116 cancer cells3,4). Although the relationship between increased amounts of
GSH and cancer survival remains unknown, it is not unreasonable to speculate that

increased GSH might benefit escaping from oxidative stress during cell division, so far as

judged by increases in O2 consumption and reactive oxygen species during G2/M phase in
human-derived cancer cell lines5). The CO/CBS-dependent modulatory action on PFKFB3

methylation in cancer cells appears to determine directional glucose utilization tostrengthen their anti-oxidative capacity for survival.

Use of metabolome analyses for mining gas-responsive receptors allowed us to explore

CO-responsive receptors besides CBS ?

CBS as a CO-responsive switch in metabolic systems. However, technical limitation is thefact that putative CO-responsive macromolecules mined by this method might be enzymes.
We are challenging to explore other candidates of macromolecules that directly capturegases like CO to regulate function of the target molecules using an approach of chemical

biology. Among products of oncogenes with haem-binding motif, progesterone receptor
membrane component 1 (PGRMC1/Sigma-2 receptor) has attracted great interests incancer biology6-8). Suppression of the gene inhibits cancer growth, although the function of

PGRMC1 has not fully been elucidated. Since CO has the ability to bind to the prosthetic

60

M. Suematsuhaem of PGRMC1, it is not unreasonable to speculate that this protein serves as a COresponsive target that may explains anti-cancer effects of CO9-11). Further investigation is
now underway in our laboratory.

The author thanks Takehiro Yamamoto, PhD and Naoharu Takano, PhD for collecting

Acknowledgments

data on CO-CBS axis, and Yasuaki Kabe, PhD, who is an expert of chemical biology tomining CO-responsive haem proteins and their function in biological systems.
References

1. Shintani T, Iwabuchi T, Soga T, Kato Y, Yamamoto T, Takano N, Hishiki T, Ueno Y,
Ikeda S, Sakuragawa T, Ishikawa K, Goda N, Kitagawa Y, Kajimura M, Matsumoto K, andSuematsu M: Cystathionine b-synthase as a carbon monoxide-sensitive regulator of bile
excretion. Hepatology 2009; 49: 141-150.
2. Yamamoto T, Takano N, Ishiwata K, Ohmura M, Nagahata Y, Matsuura T, Kamata A,
Sakamoto K, Nakanishi T, Kubo A, Hishiki T, Suematsu M. Reduced methylation of
PFKFB3 in cancer cells shunts glucose towards the pentose phosphate pathway. Nat
Commun. 2014; 5, 3480.
3. Ohmura M, Hishiki T, Yamamoto T, Nakanishi T, Kubo A, Tsuchihashi K, Tamada M,
Toue S, Kabe Y, Saya H, Suematsu M. Impacts of CD44 knockdown in cancer cells on
tumor and host metabolic systems revealed by quantitative imaging mass spectrometry.
Nitric Oxide. 2015, 46, 102-113.
4. Ishimoto T, Nagano O, Yae T, Tamada M, Oshima H, Oshima M, Ikeda T, Asaba R,
Matsuko T, Shimizu T, Ishikawa T, Kai K, Takahashi E, Imamura Y, Baba Y, Ohmura M,
Suematsu M, Baba H, Saya H. CD44 variant regulates redox status in cancer cells bystabilizing the xCT subunit of system xc - and thereby promotes tumor growth. Cancer
Cell 2011, 19(3), 387-400.
5. Bao Y, Mukai K, Hishiki T, Kubo A, Ohmura M, Sugiura Y, Matsuura T, Nagahata Y,
Hayakawa N, Yamamoto T, Fukuda R, Saya H, Suematsu M, Minamishima YA. Energy
management by enhanced glycolysis in G1-phase in human colon cancer cells in vitro andin vivo. Mol Cancer Res. 2013, 11(9):973-985.
6. Craven, R.J. PGRMC1: a new biomarker for the estrogen receptor in breast cancer.
Breast Cancer Res 2008, 10, 113.
7. Peluso, J.J., Liu, X., Saunders, M.M., Claffey, K.P. & Phoenix, K. Regulation of ovarian cancer cell viability and sensitivity to cisplatin by progesterone receptor membrane component-1. J Clin Endocrinol Metab 2008, 93, 1592-1599.

Gaseous Regulation and Metabolism

61

8. Friel, A.M. et al. Progesterone receptor membrane component 1 deficiency attenuates
growth while promoting chemosensitivity of human endometrial xenograft tumors.
Cancer Lett 2015, 356, 434-442.
9. Kaluka, D., Batabyal, D., Chiang, B.Y., Poulos, T.L. & Yeh, S.R. Spectroscopic and mutagenesis studies of human PGRMC1. Biochemistry 2015, 54, 1638-1647.
10. Wegiel, B. et al. Carbon monoxide expedites metabolic exhaustion to inhibit tumor
growth. Cancer Res. 2013, 73, 7009-7021.
11. Suematsu M, Nakamura T, Tokumoto Y, Yamamoto T, Kajimura M, Kabe Y. CO-CBS-H2S
axis: From vascular mediator to cancer regulator. Microcirculation. 2015 doi: 10.1111/
micc.12253

Makoto Suematsu, MD, PhD

1983
1988
1991

2001

2007-2015
2015-present

Graduated from Keio University School of Medicine
Instructor, Department of Medicine (Gastroenterology)
Research Bioengineer, Institute for Biomedical
Engineering, University of California, San Diego
Professor, Department of Biochemistry, Keio University
School of Medicine
Dean, Keio University School of Medicine
President, Japan Agency for Medical Research andDevelopment (AMED), Visiting Professor, Keio
University School of Medicine

Speciality and Present Interests:
Biochemistry, Gastroenterology, Gas Biology, Systems Biology

62

HEME OXYGENASE-1 AND METABOLICREPROGRAMMING IN RESPONSE TO ISCHEMIALouise Dunn1, Kim Hoe Chan2, Stephanie Kong1, Emma Collinson3,James Cantley4, Ghassan Maghzal1, Martin Ng2 and Roland Stocker1
Vascular Biology Division, The Victor Chang Cardiac Research Institute
Lowy Packer Building, 405 Liverpool St, Darlinghurst NSW 2010, Australia
2
Royal Prince Alfred Hospital and 3University of Sydney
Camperdown, NSW 2050, Australia
4
Department of Physiology and Anatomy, Oxford University
Oxford OX1 3PT, UK(r.stocker@victorchang.edu.au)
1

Heme oxygenase-1 (Hmox1) is an inducible stress protein that is increasingly

recognized as an important mediator of cellular homeostasis. Hmox1 is a known down-

stream target of hypoxia-inducible factor 1 (HIF1), and it is induced by both hypoxia andglucose deprivation1,2). Here, we investigated a potential role of Hmox1 in metabolic

reprogramming in ischemia after vascular occlusion, a key problem associated withcardiovascular diseases.

Initial studies using yeast as a model system revealed that the absence of the Hmox1

ortholog, Hmx1, results in altered gene expression, including those involved in cellular

redox control (e.g., antioxidant enzymes), glucose transport (HKT2) and mitochondrial
respiration (COQ7) 3 ) . Phenotypically, Hmx1-deficient yeast cells had decreased

mitochondrial membrane potential and respiration that was rescued by overexpression of

HMX1, Hmox1, and COQ7. Hmx1-deficiency also decreased mitochondrial coenzyme Q
content that was similarly rescued by over-expression of HMX1, Hmox1, and COQ7. Thesestudies suggested that Hmox1-deficiency affects cellular energy metabolism.

We next confirmed the putative role of Hmox1 in energy metabolism using skinfibroblasts obtained from littermate Hmox1 –/– , Hmox1 +/– and Hmox1 +/+ mice (Balb/c

background). Moreover, compared with wild type cells, Hmox1-deficient fibroblasts
showed abnormal metabolic adaptation in response to hypoxia (1% O 2 ), including

attenuated HIF1a stabilization. Hypoxia resulted in rapid induction of Hmox1 protein inwild type cells that preceded stabilization of HIF1a, suggesting that Hmox1 may be
upstream of HIF1.

Gaseous Regulation and Metabolism

63

A unilateral hind limb arterial resection model was used to examine the role of Hmox1

in ischemia-mediated energy adaptation in vivo. Compared with littermate wild typecontrol animals, Hmox1–/– mice were observed to have impaired blood flow recovery(Figure 1) and neovascularization, and to suffer from auto-amputation in response toischemia (Figure 2). Auto-amputation preceded blood flow recovery and was not rescuedby bone marrow transfer experiments. We therefore explored a role for Hmox1 in theskeletal muscle response to ischemia. Compared with Hmox1+/+ mice, skeletal muscle fibresof Hmox1–/– mice had decreased oxidative phosphorylation capacity and elevated steadystate glucose uptake. Ischemia was associated with induction of Hmox1 transcript thatpreceded that of known HIF1 target genes, such as glucose transporter-1 and vascular

(

Pre

D21

D0

Hmox1+/+

Hmox1–/–
S

I

S

I

S

I

Figure 1

Laser Doppler images of sham (S) and ischemic (I) limbs of Hmox1+/+ and Hmox1-/- mice
before (Pre), immediately (D0) and 21 days (D21) after hind limb ischemia.

Figure 2

Images of sham and ischemic limbs of Hmox1+/+, Hmox1+/- and Hmox1-/- mice 21 days after
hind limb ischemia, illustrating tissue injury and auto-amputation in mice deficient in
Hmox1.

64

R. Stocker et al.

endothelial growth factor. Reminiscent of the situation in fibroblasts, ischemia induced theexpression of Hmox1 protein in skeletal muscle before HIF1a. Together, these data suggestthat Hmox1 is upstream of HIF1, in addition to being a known down-stream target of HIF1.

This scenario is analogous to Hmox1 being up- and down-stream of the transcription factor

YY14).

We propose a novel role for Hmox1 in cellular energy reprogramming in response tohypoxia, and a new paradigm for the interplay between Hmox1 and HIF1a in which

Hmox1 is both downstream and upstream of HIF1. Insights obtained from our ischemia

model may have important implications not only for cardiovascular diseases, but also otherpathologies in which oxygen supply and energy metabolism are perturbed, such as cancer.
References

1. Ryter SW, Alam J, Choi AM. Heme oxygenase-1/carbon monoxide: from basic science totherapeutic applications. Physiol Rev 2006;86:583-650.
2. Dunn LL, Midwinter RG, Ni J, Hamid HA, Parish CR, Stocker R. New insights into
intracellular locations and functions of heme oxygenase-1. Antioxid Redox Signal
2014;20:1723-1742.
3. Collinson EJ, Wimmer-Kleikamp S, Gerega SK, Yang YH, Parish CR, Dawes IW, Stocker
R. The yeast homolog of heme oxygenase-1 affords cellular antioxidant protection via thetranscriptional regulation of known antioxidant genes. J Biol Chem 2011;286:2205-2214.
4. Beck K, Wu BJ, Ni J, Santiago FS, Malabanan KP, Li C, Wang Y, Khachigian LM, Stocker
R. Interplay between heme oxygenase-1 and the multifunctional transcription factor yinyang 1 in the inhibition of intimal hyperplasia. Circ Res 2010;107:1490-1497.

Roland Stocker, PhD

1988-2002
2002-2006
2007-20122012-presentGroup Leader, Heart Research Institute
Professor, University of New South Wales
Professor, University of Sydney
Head, Vascular Biology Division
Victor Chang Cardiac Research InstituteSpeciality and Present Interests:
Vascular Biology, Redox Processes in Health and Disease

65

PROTEIN-BOUND REACTIVE SULFUR SPECIES ANDITS TRANSLATIONAL BIOSYNTHESIS:POTENTIAL IMPLICATION FOR OXIDATIVE STRESS AND
TUMOR BIOLOGY
Takaaki Akaike

Department of Environmental Health Sciences and Molecular Toxicology
Tohoku University Graduate School of Medicine
2-1 Seiryo-machi, Aobaku, Sendai, Miyagi 980-8575, Japan(takaike@med.tohoku.ac.jp)

Many organisms utilize high-level energy metabolism that involves the chemical

reactivity of molecular oxygen, i.e., redox activity. The oxygen-dependent redox reaction,

mostly mediated by reactive oxygen species, then modulates and optimizes biological

functions of various protein effector molecules. The energy metabolism of aerobic
organisms makes use of the redox activity of molecular oxygen. Reactive oxygen species(e.g., superoxide anion [O 2 - ] and hydrogen peroxide [H 2 O 2 ]) are reduced metabolites
derived from molecular oxygen that are produced during mitochondrial respiration or

defense responses during infection of cells and tissues. Some reactive oxidants were
thought to be harmful agents that mediate oxygen toxicity (oxidant toxicity theory).

Indeed, these oxidant species may be involved in the pathogenesis of different diseases
associated with oxidative stress1-5). These disorders include infections; inflammations;cancer; lifestyle-related diseases and metabolic diseases such as arteriosclerosis anddiabetes mellitus; and neurological disorders such as Alzheimer’s disease. The clinicalapplication of antioxidant agents to prevent and treat these diseases has not yet achieved
the hoped-for results, however.

Reactive sulfur species (RSS) like cysteine hydropersulfide (CysSSH) has been long time

known to be physiologically formed in various organisms including prokaryotes andeukaryotes including mammals6). Abundant endogenous formation of CysSSH recently

reported indicates pivotal physiological roles of these RSS derivatives6,7). In fact, ourcurrent study identified RSS as a major component of low-molecular-weight polysulfur

compounds endogenously generated in cells. Its biosynthesis is effectively catalyzed
mainly by two enzymes such as cystathionine g-lyase (CSE) and cystathionine b-synthase

66

T. Akaike(CBS) by use of cystine (CysSSCys) as a substrate (Figure 1). We have unequivocallyverified generation of appreciable amount of CysSSH, and its derivative glutathionehydropersulfide (GSSH) in cultured cells and tissues in vivo7). Cysteine persulfide thus

formed is highly nucleophilic and a stronger antioxidant than is cysteine. This uniquefeature of reactive cysteine persulfide relies on adjacent electron pairs in what is called thea-effect (Figure 1)6,8). In fact, hydropersulfides, i.e., reduced forms of these RSS were potent

scavengers for reactive oxygen species, especially for hydrogen peroxide. RSS also showeda strong redox signaling regulatory function via electrophile thiolation. A prime exampleof this is interaction of RSS with an endogenously generated electrophile 8-nitroguanosine

3’,5’-cyclic monophosphate (8-nitro-cGMP)7, 9-11). Here, we clarified that extremely highlevels of protein-bound cysteinyl persulfide or RSS formation in most Cys-containing
proteins expressed in cells and tissues, of which extension reaching almost 100% at

particular Cys residues of many proteins. This fact suggested that CysSSH might beincorporated into nascent polypeptides and/or synthesized at a translational level and thus

initially expressed to function as a natural amino acid residue in proteins. Surprisingly, aclear translation-coupled Cys polysulfuration (TCysPS) was revealed herein, directly

involved in a remarkable translational CysSSH biosynthesis and its incorporation into

Figure 1

Cysteine persulfide formation mediated by CBS and CSE.
C-S bond cleavage in cystine by CBS and CSE results in formation of cysteine persulfide.
Antioxidant and nucleophilic activities of terminal thiol residues are enhanced by an effectbrought about by lone-pair electrons of adjacent sulfur atoms.

Gaseous Regulation and Metabolism

67

proteins ubiquitously occurring among different organisms. Exploring such a unique

TCysPS pathway may advance a new paradigm of molecular biology and especiallycellular translational mechanism, evolving an innovative era of the modern redox biology.

Also, several recent reports suggest a potential involvement of CBS and CSE and theirproduct hydrogen sulfide in tumor promotion and progression, and even in drug
resistance12-14). These tumor-promoting effects of CSE/CBS may be caused via strong

antioxidant activity of RSS. Therefore, TCysPS, if occurring extensively in situ in tumor

tissues, may contribute to the tumor progression. This discovery of versatile physiological

functions of the protein-bound RSS produced via TCysPS may warrant its potential
medicinal and pharmaceutical application for many diseases including cancer15).
References
1. Beckman JS, Beckman TW, Chen J, Marshall PA, Freeman BA. Apparent hydroxyl radical
production by peroxynitrite: implications for endothelial injury from nitric oxide andsuperoxide. Proc Natl Acad Sci USA. 1990; 87:1620-1624.
2. van der Vliet A, Cross CE. Oxidants, nitrosants, and the lung. Am J Med. 2000;109:398421.
3. Forman HJ, Fukuto JM, Torres M. Redox signaling: thiol chemistry defines which reactive
oxygen and nitrogen species can act as second messengers. Am J Physiol Cell Physiol.
2004;287:C246-256.
4. Halliwell B. Biochemistry of oxidative stress. Biochem Soc Trans. 2007;35:1147-1150.
5. Trachootham D, Alexandre J, Huang P. Targeting cancer cells by ROS-mediated mechanisms: a radical therapeutic approach? Nat Rev Drug Discov. 2009;8:579-591.
6. Ono K, Akaike T, Sawa T, Kumagai Y, Wink DA, Tantillo DJ, Hobbs AJ, Nagy P, Xian M,
Lin J, Fukuto JM. Redox chemistry and chemical biology of H2S, hydropersulfides, andderived species: implications of their possible biological activity and utility. Free Radic
Biol Med. 2014;77:82-94.
7. Ida T, Sawa T, Ihara H, Tsuchiya Y, Watanabe Y, Kumagai Y, Suematsu M, Motohashi H,
Fujii S, Matsunaga T, Yamamoto M, Ono K, Devarie-Baez NO, Xian M, Fukuto JM,
Akaike T. Reactive cysteine persulfides and S-polythiolation regulate oxidative stressand redox signaling. Proc Natl Acad Sci USA. 2014;111:7606-7611.
8. Edwards JO, Pearson RG. The factors determining nucleophilic reactivities. J Am Chem
Soc. 1962;84:16-24.
9. Ito C, Saito Y, Nozawa T, Fujii S, Sawa T, Inoue H, Matsunaga T, Khan S, Akashi S,
Hashimoto R, Aikawa C, Takahashi E, Sagara H, Komatsu M, Tanaka K, Akaike T,
Nakagawa I, Arimoto H. Endogenous nitrated nucleotide is a key mediator of
autophagy and innate defense against bacteria. Mol Cell. 2013;52:794-804.

68

T. Akaike

10. Nishida M, Sawa T, Kitajima N, Ono K, Inoue H, Ihara H, Motohashi H, Yamamoto M,
Suematsu M, Kurose H, van der Vliet A, Freeman BA, Shibata T, Uchida K, Kumagai Y,
Akaike T. Hydrogen sulfide anion regulates redox signaling via electrophile sulfhydration. Nat Chem Biol. 2012;8:714-724.
11. Sawa T, Zaki MH, Okamoto T, Akuta T, Tokutomi Y, Kim-Mitsuyama S, Ihara H,
Kobayashi A, Yamamoto M, Fujii S, Arimoto H, Akaike T. Protein S-guanylation by the
biological signal 8-nitroguanosine 3',5'-cyclic monophosphate. Nat Chem Biol. 2007;3:
727-735.
12. Szabo C, Coletta C, Chao C, Módis K, Szczesny B, Papapetropoulos A, Hellmich MR.
Tumor-derived hydrogen sulfide, produced by cystathionine-b-synthase, stimulates
bioenergetics, cell proliferation, and angiogenesis in colon cancer. Proc Natl Acad Sci
USA. 2013;110:12474-12479.
13. Bhattacharyya S, Saha S, Giri K, Lanza IR, Nair KS, Jennings NB, Rodriguez-Aguayo C,
Lopez-Berestein G, Basal E, Weaver AL, Visscher DW, Cliby W, Sood AK, Bhattacharya R,
Mukherjee P. Cystathionine beta-synthase (CBS) contributes to advanced ovarian cancer
progression and drug resistance. PLoS One. 2013;8:e79167.
14. Panza E, De Cicco P, Armogida C, Scognamiglio G, Gigantino V, Botti G, Germano D,
Napolitano M, Papapetropoulos A, Bucci M, Cirino G, Ianaro A. Role of the cystathionine
g lyase/hydrogen sulfide pathway in human melanoma progression. Pigment Cell
Melanoma Res. 2015;28:61-72.
15. Fujii S, Sawa T, Nishida M, Ihara H, Ida T, Motohashi H, Akaike T. Redox signaling
regulated by an electrophilic cyclic nucleotide and reactive cysteine persulfides. Arch
Biochem Biophys. 2015; in press.

Takaaki Akaike, MD, PhD

1991
1993

1994
2001
2001
2005
2011

2013-present

Assistant Professor at Kumamoto Univ. Sch. Med.
Visiting Professor at Center for Neurovirology, Thomas
Jefferson Univ.
Associate Professor at Kumamoto Univ. Sch. Med.
Visiting Professor at Center Free Radic. Res., UAB
Program Officer at the MEXT of Japan
Professor, Grad. Sch. Med. Sci., Kumamoto Univ.
Vice Dean & Director of Center Med. Educ. Res.,
Kumamoto Univ. Sch. Med.
Professor, Tohoku Univ. Grad. Sch. Med.

Specialty and Present Interest:
Chemical Biology, Oxidative Stress, Microbial Pathogenesis, NO & Redox Biology,
Redox Signal Transduction, Polysulfur Chemistry & Biology

69

IDENTIFICATION OF A METABOLIC PATHWAY FOR
ACTIVATION OF THE HYPOXIA GENE REGULATORYRESPONSE IN CANCER

1

Lorenz Poellinger1,2 and Hiroyuki Kato2

Dept. of Cell and Molecular Biology, Karolinska Institutet, 17177 Stockholm, Sweden
Cancer Science Institute of Singapore, National University of Singapore, Singapore(lorenz.poellinger@ki.se)

2

Cancer research has traditionally focused on the biology of transformed cells and thegenetic changes that drive tumor malignancy. However, it has become increasingly clearthat tumours develop within - and actively shape – specialized microenvironments that are

typically characterized by low oxygen tension (hypoxia). The hypoxic microenvironment

drives transcriptional responses mediated by hypoxia inducible transcription factors (HIFs)

that enable oncogenic adaptation. Our laboratory is interested in elucidating the mode ofaction of HIFs, epigenetic mechanisms of gene regulation in hypoxia and how the
integrated function of these mechanisms determines the cellular response to hypoxia in
physiology and disease, notably cancer. The von Hippel-Lindau tumor suppressor protein(VHL) plays a critical role in regulating the cellular hypoxic response by functioning as an

E3 ubiquitin ligase determining the rapid degradation of HIFs in normoxia. Thus, inhypoxia, the cellular hypoxic response is initiated by release of VHL and stabilization of the

HIFs. Unexpectedly, we have discovered a new mechanism for activation and stabilizationof HIFs that is VHL-dependent but independent of oxygen-sensing. This mechanism

involves metabolically active enzyme complexes that are up-regulated in many cancers andthat we have identified as hitherto unknown VHL-interacting proteins. Thus, thesefindings link the tumor metabolic phenotype to regulation of the activity and function of
hypoxia-inducible transcription factors.

70

L. Poellinger et al.

Lorenz Poellinger, MD, PhD

1987-1989
1989-1996

1996-

2008-2012
2012-

Postdoctoral fellow, Prof. Robert G. Roeder,
Rockefeller Univ.
Associate Professor, Dept. of Medical Nutrition,
Karolinska Institute
Professor, Dept. of Cell and Molecular Biology,
Karolinska Institute
Distinguished Visiting Professor, Cancer Science
Institute, National University of Singapore
Professor, Cancer Science Institute, National Universityof Singapore

Speciality and Present Interests:
Hypoxia Signaling, Cancer Epigenetics, Transcriptional Regulation, Cancer Genomics

71

CYTOPROTECTION AND METABOLIC REPROGRAMMING
GOVERNED BY KEAP1-NRF2 SYSTEM

Hozumi Motohashi

Department of Gene Expression Regulation
Institute of Development, Aging and Cancer (IDAC), Tohoku University
4-1 Seiryo-cho, Aoba-ku, Sendai, Miyagi 980-8575, Japan(hozumi.motohashi.a7@tohoku.ac.jp)

NRF2 is a master transcriptional activator playing a critical role in the defense

mechanism against oxidative insults 1 - 3 ) (Figure 1). NRF2 activates genes encoding

cytoprotective enzymes and antioxidant proteins in response to electrophiles and reactive

oxygen species (ROS) of exogenous and endogenous origins. Under unstressed conditions,

NRF2 is constantly ubiquitinated by KEAP1 and degraded in the proteasome. Duringexposure to electrophiles or ROS, KEAP1 is inactivated, and NRF2 is stabilized.

Consequently, NRF2 activates transcription, conferring resistance against xenobiotic andoxidative stress. NRF2 inducers have been found especially effective for improvement of㻌㻌 㻌 ischemia
㻌diabetic nephropathy, multiple㻌 sclerosis,
andreperfusion injury.
㻌

㻌

㻌 㻌

KEAP1

KEAP1

Ub

㻱㼘㼑㼏㼠㼞㼛㼜㼔㼕㼘㼑㼟㻾㼑㼍㼏㼠㼕㼢㼑㻌㻻㼤㼥㼓㼑㼚㻌㻿㼜㼑㼏㼕㼑㼟㻿㼠㼍㼎㼕㼘㼕㼦㼍㼠㼕㼛㼚㻰㼑㼓㼞㼍㼐㼍㼠㼕㼛㼚㼠㼔㼞㼛㼡㼓㼔㻌㻼㼞㼛㼠㼑㼍㼟㼛㼙㼑
Itoh et al., BBRC 1997
Itoh et al. Genes & Dev 1999
Wakabayashi et al. Nat Genet 2003
Motohashi et al. PNAS 2004
Kobayashi et al. MCB 2004 & 2006
Tong et al. MCB 2006 & 2007
Suzuki et al, JBC 2008
Ogura et al, PNAS 2010㻯㼥㼠㼛㼜㼞㼛㼠㼑㼏㼠㼕㼢㼑㻌㻳㼑㼚㼑㼟
Small
MAF

NRF2
Pol II㻝㻚㻌㻳㼘㼡㼠㼍㼠㼔㼕㼛㼚㼑㻌㻿㼥㼚㼠㼔㼑㼟㼕㼟㻌㻔㻳㼏㼘㼏㻘㻌㻳㼏㼘㼙㻕㻞㻚㻌㻾㻻㻿㻌㻱㼘㼕㼙㼕㼚㼍㼠㼕㼛㼚㻌㻔㼀㼤㼚㼞㼐㻝㻘㻌㻼㼞㼐㼤㻝㻕㻟㻚㻌㻰㼑㼠㼛㼤㼕㼒㼕㼏㼍㼠㼕㼛㼚㻌㻔㻳㼟㼠㻘㻌㻺㼝㼛㻝㻕㻠㻚㻌㻰㼞㼡㼓㻌㻱㼤㼏㼞㼑㼠㼕㼛㼚㻌㻔㻹㼞㼜㻕㻌㻭㼚㼠㼕㼛㼤㼕㼐㼍㼚㼠㻌㻾㼑㼟㼜㼛㼚㼟㼑㻌㻱㼘㼑㼙㼑㼚㼠㻌㻔㻭㻾㻱㻕㻭㼏㼝㼡㼕㼞㼑㻌㼞㼑㼟㼕㼟㼠㼍㼚㼏㼑㻌㼍㼓㼍㼕㼚㼟㼠㻌㼔㼍㼞㼙㼒㼡㼘㻌㼕㼚㼟㼡㼘㼠㼟㻌

Figure 1㻹㼍㼕㼚㼠㼍㼕㼚㻌㼠㼔㼑㻌㼞㼑㼐㼛㼤 㼔㼛㼙㼑㼛㼟㼠㼍㼟㼕㼟

KEAP1-NRF2 System Plays a Central Role in the Maintenance of Redox Homeostasis

72

H. Motohashi

While NRF2 activation is beneficial to our health, NRF2 is responsible for the malignant

progression of various human cancers. Recent cancer genome projects revealed that

KEAP1 gene and NRF2 gene are often mutated in human cancers4,5).

According to the

COSMIC database, non-synonymous mutations of KEAP1 gene are distributed widely inthe coding region, while those of NRF2 gene are clustered in the two motifs in the Nterminal region, which are necessary for the interaction between KEAP1 and NRF2. In

either case, NRF2 cannot be ubiquitinated and stabilized, resulting in the constitutive
activation of its target genes. Because NRF2 activates many cytoprotective genes,

constitutive stabilization of NRF2 confers chemo- and radio-resistance and leads to poor
prognosis6).

We found that NRF2 not only enhances survival of cancers by activating cytoprotective

genes but also redirects glucose and glutamine into anabolic pathways by activating
metabolic genes, which are advantageous for cancer proliferation7) (Figure 2). Enhanced

activity of PI3K-AKT signaling enables NRF2 to induce the metabolic genes and modulate

metabolism. We observed that NRF2 stabilization under the sustained activation of PI3K-

AKT pathway in hepatocytes results in the liver enlargement 8) . Intriguingly, it was
accompanied by the marked increase of bile duct-like structures, suggesting that the robust

activation of NRF2 affects the cell type specification during the liver development inaddition to cell proliferation. We also found that NRF2 activation promotes hepatocyte

proliferation during compensatory liver hypertrophy after partial portal vein ligation,

implying that NRF2 contributes to proliferation under the transient and physiologicalenhancement of proliferative signals9).

㻌 㻌

㻌 㻌

㻌

㻽㼡㼕㼟㼏㼑㼚㼠 㻯㼑㼘㼘㼟㻼㼞㼛㼘㼕㼒㼑㼞㼍㼠㼕㼚㼓㻌㻯㼑㼘㼘㼟

Glucose
G
l
y
c
o
l
y
s
i
s

Detoxification
ROS
Elimination
NRF2

GlucoseDeNicola et al.
Nat Genet 2015

G
p53 l
NRF2
HIF1 ycmTOR olSerineATF4ysi
Lactic Acids

Synthesis of
nucleotides, aminoacids, and lipids

Pentose Phosphate
Pathway (PPP)
NRF2

Nucleotide

Mitsuishi et al.
Cancer Cell 2012

NRF2

Lipid
Amino Acid㼀㻯㻭㻌㼏㼏㼥㼏㼘㼑㼀㻯㻭㻌㼏㼏㼥㼏㼘㼑
ATP
NADH

Figure 2c-Myc
Glutamine

Glutathione
NRF2

Contribution of Nrf2 in Metabolic Reprogramming

Stress Response, Oxystress73

To understand the molecular basis of the potent transcriptional activation mediated by

NRF2 under the sustained activation for PI3K-AKT pathway, we purified endogenous

NRF2 complex from Keap1: Pten double deficient liver. NRF2 and its heterodimericpartner molecule, small MAF, were obtained. CBP and p300, which are already known

coactivators of NRF2, were also obtained. In addition to these factors, many subunits ofMediator complex and SWI/SNF chromatin remodeling complex were obtained.

Intriguingly, among the subunits of the Mediator complex, MED16 turned out to directlyinteract with NRF2 through its transactivation domains, Neh4 and Neh510). Approximately

three fourths of the NRF2 target genes exhibited blunted induction in response toelectrophilic stimuli. Moreover, MED16 disruption significantly sensitized cells tooxidative stress. These results indicate that KEAP1-NRF2-MED16 axis is essential for the

NRF2-mediated cell protection (Figure 3). Our on-going analysis of the endogenous NRF2

complex purified from Keap1 single deficient liver will give more clear answer to themechanism how the sustained activation of PI3K-AKT signaling enhances the NRF2
activity.

From these results, we propose that NRF2 is a facultative accelerator of cellproliferation, activating cytoprotective genes for the maintenance of redox homeostasis andmetabolic genes for the contribution to the metabolic reprogramming in cooperation withother oncogenic signals (Figure 4).

㻌

㻌

㻌

㻌

㻌
㻻㼤㼕㼐㼍㼠㼕㼢㼑㻌㻿㼠㼞㼑㼟㼟㻱㼘㼑㼏㼞㼛㼜㼔㼕㼘㼑㼟

㻌

㻌

㻌

㻼㻵㻟㻷㻙㻭㼗㼠㻌㻿㼕㼓㼚㼍㼘㼕㼚㼓㻼㼞㼛㼘㼕㼒㼑㼞㼍㼠㼕㼚㼓㻌㻯㼑㼘㼘㼟㻫㻭㼏㼠㼕㼢㼍㼠㼑

㻴㻭㼀

㻺㻾㻱
㻞
㻹㻱㻰㻝
㻢

㻿㼃㻵㻛㻿㻺㻲㻌
㻯㼛㼙㼜㼘㼑㼤

㻹㼑㼐㼕㼍㼠㼛㼞㻌㻯㼛㼙㼜㼘㼑㼤㻺㻾㻱 㻹㻭㻲㻞 㻭㻾㻱㻾㻺㻭㻼㻌㻵㻵
㼔㼛㼘㼛㼏㼛㼙㼜㼘㼑㼤㻾㼑㼐㼛㼤 㻴㼛㼙㼑㼛㼟㼠㼍㼟㼕㼟

Figure 3㻹㼑㼠㼍㼎㼛㼘㼕㼏㻌㻳㼑㼚㼑㼟㻯㼥㼠㼛㼜㼞㼛㼠㼑㼏㼠㼕㼢㼑㻌㻳㼑㼚㼑㼟㼀㼍㼞㼓㼑㼠㻌㻳㼑㼚㼑㼟㻭㼚㼍㼎㼛㼘㼕㼏㻌㻼㼍㼠㼔㼣㼍㼥㻹㼑㼠㼍㼎㼛㼘㼕㼏㻌㻾㼑㼜㼞㼛㼓㼞㼍㼙㼙㼕㼚㼓㻌

KEAP1-NRF2-MED16 Axis Transduces Oxidative Stress into Cytoprotective/Metabolic
Gene Expression

74

H. Motohashi

㻌 㻌 㻌

㻌

㻌 㻌

㻌
㻌

㻌

㻌 㻌

㻌
㻌
㻼㼞㼛㼘㼕㼒㼑㼞㼍㼠㼕㼚㼓㻌㻯㼑㼘㼘㼟㻽㼡㼕㼟㼏㼑㼚㼠 㻯㼑㼘㼘㼟㻻㼤㼕㼐㼍㼠㼕㼢㼑㻌㻿㼠㼞㼑㼟㼟㻱㼘㼑㼏㼞㼛㼜㼔㼕㼘㼑㼟㻻㼤㼕㼐㼍㼠㼕㼢㼑㻌㻿㼠㼞㼑㼟㼟㻱㼘㼑㼏㼞㼛㼜㼔㼕㼘㼑㼟

Activate

Activate
NRF2 MAF㻭㻾㻱㻼㻵㻟㻷㻙㻭㼗㼠㻌㻿㼕㼓㼚㼍㼘㼕㼚㼓㻹㼑㼠㼍㼎㼛㼘㼕㼏㻌㻳㼑㼚㼑㼟㻯㼥㼠㼛㼜㼞㼛㼠㼑㼏㼠㼕㼢㼑㻌㻳㼑㼚㼑㼟㼀㼍㼞㼓㼑㼠㻌㻳㼑㼚㼑㼟NRF2MAF㻭㻾㻱㻾㼑㼐㼛㼤 㻴㼛㼙㼑㼛㼟㼠㼍㼟㼕㼟

Figure 4㻯㼥㼠㼛㼜㼞㼛㼠㼑㼏㼠㼕㼢㼑㻌㻳㼑㼚㼑㼟㼀㼍㼞㼓㼑㼠㻌㻳㼑㼚㼑㼟㻾㼑㼐㼛㼤 㻴㼛㼙㼑㼛㼟㼠㼍㼟㼕㼟㻭㼚㼍㼎㼛㼘㼕㼏㻌㻼㼍㼠㼔㼣㼍㼥㻹㼑㼠㼍㼎㼛㼘㼕㼏㻌㻾㼑㼜㼞㼛㼓㼞㼍㼙㼙㼕㼚㼓㻌NRF2 Is a “Facultative Accelerator of Cell Proliferation ” Maintaining Redox Homeostasis
and Reprogramming Metabolism

References
1. Motohashi H, Yamamoto M. 2004. Nrf2-Keap1 defines a physiologically importantstress response mechanism. Trends Mol Med. 2004;10:549-557.
2. Uruno A, Motohashi H. 2011. The Keap1-Nrf2 system as an in vivo sensor for electrophiles. Nitric Oxide. 2011;25:153-160.
3. Taguchi K, Motohashi H, Yamamoto M. Molecular mechanisms of the Keap1–Nrf2
pathway in stress response and cancer evolution. Genes Cells. 2011;16:123-140.
4. Cancer Genome Atlas Research Network. Comprehensive genomic characterization ofsquamous cell lung cancers. Nature. 2012;489:519-525.
5. Kandoth C, McLellan MD, Vandin F, Ye K, Niu B, Lu C, Xie M, Zhang Q, McMichael JF,
Wyczalkowski MA, Leiserson MD, Miller CA, Welch JS, Walter MJ, Wendl MC, Ley TJ,
Wilson RK, Raphael BJ, Ding L. Mutational landscape and significance across 12 major
cancer types. Nature. 2013;502:333-339.
6. Inoue D, Suzuki T, Mitsuishi Y, Miki Y, Suzuki S, Sugawara S, Watanabe M, Sakudara A,
Endo C, Uruno A, Sasano H, Nakagawa T, Satoh K, Tanaka N, Kubo H, Motohashi H,
Yamamoto M. Accumulation of p62/SQSTM1 is associated with poor prognosis inpatients with lung adenocarcinoma. Cancer Sci. 2012;103:760-766.
7. Mitsuishi Y, Taguchi K, Kawatani Y, Shibata T, Nukiwa T, Aburatani H, Yamamoto M,
Motohashi H. Nrf2 redirects glucose and glutamine into anabolic pathways in metabolic
reprogramming. Cancer Cell. 2012;22:66-79.
8. Taguchi K, Hirano I, Itoh T, Tanaka M, Miyajima A, Suzuki A, Motohashi H,
Yamamoto M. Nrf2 enhances cholangiocyte expansion in Pten-deficient livers. Mol Cell
Biol. 2014;34:900-913.

Stress Response, Oxystress75

9. Shirasaki K, Taguchi K, Unno M, Motohashi H, Yamamoto M. Nrf2 promotes compensatory liver hypertrophy after portal vein branch ligation in mice. Hepatology.
2014;59:2371-2382.
10. Sekine H, Okazaki K, Ota N, Shima H, Katoh Y, Suzuki N, Igarashi K, Ito M, Motohashi
H, Yamamoto M. The Mediator subunit MED16 transduces NRF2-activating signals
into antioxidant gene expression. Mol Cell Biol. in press.

Hozumi Motohashi, MD, PhD

1996-2000

2000
2001-2006

2006-20132013-present

Assistant Professor, Center for Tsukuba Advanced
Research Alliance, University of Tsukuba
Visiting Scholar, Northwestern University
Lecturer and Associate Professor, Comprehensive
Human Sciences, University of Tsukuba
Associate Professor, Graduate School of Medicine,
Tohoku University
Professor, IDAC, Tohoku University

Specialty and Present Interest:
Transcriptional Regulation Mechanism for Antioxidant Response, Development of
Anticancer Therapy for NRF2-addicted Tumors

76

MITOCHONDRIA REGULATE CANCER

Navdeep S. Chandel

Department of Medicine, Northwestern University
240 East Huron Avenue, McGaw M-334, Chicago, IL 60611-2909, USA(nav@northwestern.edu)

Historically, mitochondrial metabolism has been erroneously viewed as inconsequential

for the metabolic demands of rapidly proliferating cells1,2). Furthermore, the mitochondria

have been ascribed to produce copious amount of reactive oxygen species (ROS) that

promote DNA damage and genetic instability3). This view mainly stems from two long-standing observations. The first observation, made in the 1920s, states that cancer cells takeup glucose and produce large quantities of lactate in the presence of ample oxygen4). Thisis referred to as aerobic glycolysis or the Warburg effect. This observation led to the

hypothesis that mitochondria are damaged in tumors; thus, cancer cells primarily utilize
glycolysis as the major metabolic pathway for proliferation2). A second observation is thattumors frequently produce high levels of mitochondrial ROS in order to invoke genetic

instability and ultimately tumorigenesis5). Consequently, mitochondrial dysfunction was
designated as a metabolic hallmark of cancer cells.

The notion that mitochondria in tumor cells are dysfunctional was challenged in the

1950s 6) by the demonstration that like normal tissues such as the liver, tumor cells

effectively oxidized the fatty acid palmitate7). Moreover, enzymes of the tricarboxylic acid

(TCA) cycle had similar activities in the mitochondria of tumors as the mitochondria of
normal tissues8) and that the high rate of glycolysis is a hallmark of both normal andneoplastic cells9). Importantly, these observations and models have largely been validated
over the past 50 years. Recent studies provided genetic evidence that mitochondrial

metabolism is essential for tumorigenesis10-12).

It is not surprising that mitochondrial metabolism would be necessary fortumorigenesis. Mitochondria are well appreciated as biosynthetic and bioenergetic

Stress Response, Oxystress77

organelles for their role in producing metabolites and ATP, which are byproducts of thetricarboxylic acid (TCA) cycle and the mitochondrial membrane potential, respectively. The

TCA cycle metabolites such as oxaloacetate and citrate generate cytosolic aspartate andacetyl-CoA that are required for pyrimidine and fatty acid synthesis, respectively. The
TCA cycle produces reducing equivalents NADH and FADH2, which deliver theirelectrons to the electron transport chain (ETC) that ultimately utilizes oxygen as the final

acceptor (respiration). An emerging idea is that mitochondria also function as signalingorganelles. Two notable mitochondrial-dependent signaling mechanisms involve therelease of reactive oxygen species (ROS) for protein thiol oxidation and the release of

citrate, which generates acetyl-CoA used for histone acetylation, an important mechanism

to regulate epigenetics in cancer cells. Mitochondrial ROS-dependent signaling controls

numerous biological responses including proliferation, differentiation, and adaptation tostress as well as physiological and pathological outcomes such as immunity, cancer, andaging. For example, mitochondrial-derived ROS have been implicated in hypoxic signaltransduction and activation of hypoxia-inducible transcription factor 1 (HIF-1), which isnecessary for tumor cells to metabolically adapt to hypoxia. Thus, mitochondria provide

ATP, building blocks for macromolecular synthesis and important signaling molecules fortumor cell proliferation, survival and metabolic adaptation13).

The recognition that mitochondrial metabolism is essential for tumorigenesis has led toidea of targeting mitochondria for cancer therapy. Intriguingly, the anti-diabetic drug

metformin has emerged as an anti-cancer agent. In diabetic patients, the therapeutic effectof metformin is a result of significantly decreasing hepatic gluconeogenesis to diminish

circulating insulin levels14). Epidemiological studies have suggested that patients on
metformin, in order to control their blood glucose levels, are less likely to develop cancer15).

In addition, metformin increases the survival rate of patients that have already developed

cancer16). Furthermore, multiple laboratory-based studies have also provided evidence that

metformin may serve as an anti-cancer agent17-19). Due to the strong, retrospective clinicalevidence and laboratory-based experiments there are more than 100 ongoing clinical trials

assessing metformin’s anti-cancer effects in combination with current standard
treatments20).

There are two not necessarily mutually exclusive mechanisms to explain the anti-tumor

effects of metformin 21) . First, metformin decreases blood glucose and consequently

circulating insulin levels. Insulin is a known mitogen for many cancer cells. Insulin andinsulin growth factors (IGFs) stimulate the pro-tumorigenic PIK3 signaling pathway intumor cells that are insulin and/or IGF receptor positive22). It is important to note that not

all cancers are insulin sensitive therefore reduction of insulin levels would be irrelevant. A

second possible mechanism by which metformin acts as an anti-cancer agent is through78

N. S. Chandel

inhibition of mitochondrial ETC complex I to diminish tumor growth. In 2000, two studies

demonstrated that metformin in vitro inhibits mitochondrial complex I23,24). Recent work has
provided mechanistic understanding of how metformin and related biguanides inhibit

complex I25). Yet, it was only recently shown in experimental models of cancer that the invivo anti-tumorigenic effects of metformin are also dependent on inhibiting mitochondrialcomplex I26). Specifically, metformin inhibits mitochondrial ATP production and inducescell death when glycolytic ATP diminishes due to limited glucose availability. In thepresence of ample glucose, metformin decreases proliferation through mechanisms that are

not fully understood. Metformin similar to other complex I inhibitors in prostate cancer
cells does not limit the ability to generate TCA cycle intermediates due to induction of
glutamine-dependent reductive carboxylation27). However, in breast cancer cells metformin

through complex I inhibition diminishes TCA cycle intermediate production28). A leadingmechanism to explain metformin’s anti-proliferative effect is that metformin decreases
mitochondrial ATP production, resulting in AMPK activation and diminished mTOR
activity, a necessity for the anabolism of proliferating cells 29) . It is likely that thismechanism cannot solely explain the anti-proliferative effect of metformin since many

cancers that are unable to activate AMPK are still susceptible to the drug30).

An important concern is whether metformin, as an anti-cancer agent, has a favorable

therapeutic index. Metformin requires organic cation transporters (OCTs) that are present

in a few tissues such as liver and kidney31). Thus, the current dosing of metformin fordiabetes has a remarkable safety profile. Tumor cells can also express OCTs to allow theuptake of metformin. However, not all tumor cells express OCTs; thus, metformin would

not accumulate in these tumors and inhibit mitochondrial complex I. The uptake of thepositively charged metformin at normal pH into the mitochondrial matrix to inhibit

complex I requires a robust mitochondrial membrane potential26). In going forward, clinical
trials using metformin as an anti-cancer agent should assess the expression levels of OCTs

and mitochondrial genes in the tumors in order to identify those likely to be susceptible tometformin, those with highest expression. The combination therapy of metformin with

PI3K inhibitors that reduce glucose uptake and glycolysis will also be more efficacious than

metformin alone. Lastly, it is not clear whether the anti-diabetic dosing of metformin usedin current clinical trials will allow for accumulation to levels necessary to inhibit

mitochondrial complex I in tumors. It might be possible to escalate the dosing of

metformin to higher levels than currently used for diabetes without causing toxicity.

Stress Response, Oxystress

79

References
1. Bonnet, S. et al. A mitochondria-K+ channel axis is suppressed in cancer and its normalization promotes apoptosis and inhibits cancer growth. Cancer cell 11, 37-51 (2007).
2. Warburg, O. On the origin of cancer cells. Science 123, 309-314 (1956).
3. Ames, B.N., Shigenaga, M.K. & Hagen, T.M. Oxidants, antioxidants, and the degenerative
diseases of aging. Proceedings of the National Academy of Sciences of the United States ofAmerica 90, 7915-7922 (1993).
4. Koppenol, W.H., Bounds, P.L. & Dang, C.V. Otto Warburg's contributions to current
concepts of cancer metabolism. Nature reviews. Cancer 11, 325-337 (2011).
5. Cross, C.E. et al. Oxygen radicals and human disease. Annals of internal medicine 107,526-545 (1987).
6. Weinhouse, S. On respiratory impairment in cancer cells. Science 124, 267-269 (1956).
7. Weinhouse, S., Millington, R.H. & Wenner, C.E. Metabolism of neoplastic tissue. I. The
oxidation of carbohydrate and fatty acids in transplanted tumors. Cancer research 11, 845850 (1951).
8. Wenner, C.E., Spirtes, M.A. & Weinhouse, S. Metabolism of neoplastic tissue. II. A surveyof enzymes of the citric acid cycle in transplanted tumors. Cancer research 12, 44-49(1952).
9. Weinhouse, S. Studies on the fate of isotopically labeled metabolites in the oxidative
metabolism of tumors. Cancer research 11, 585-591 (1951).
10. Weinberg, F. et al. Mitochondrial metabolism and ROS generation are essential for
Kras-mediated tumorigenicity. Proceedings of the National Academy of Sciences of theUnited States of America 107, 8788-8793 (2010).
11. Fogal, V. et al. Mitochondrial p32 Protein Is a Critical Regulator of Tumor Metabolism via
Maintenance of Oxidative Phosphorylation. Molecular and cellular biology 30, 13031318 (2010).
12. Guo, J.Y. et al. Activated Ras requires autophagy to maintain oxidative metabolism andtumorigenesis. Genes & development 25, 460-470 (2011).
13. Martinez-Reyes, I. et al. TCA Cycle and Mitochondrial Membrane Potential Are
Necessary for Diverse Biological Functions. Molecular cell (2015).
14. Bailey, C.J. & Turner, R.C. Metformin. The New England journal of medicine 334, 574-579(1996).
15. Evans, J.M., Donnelly, L.A., Emslie-Smith, A.M., Alessi, D.R. & Morris, A.D. Metformin
and reduced risk of cancer in diabetic patients. Bmj 330, 1304-1305 (2005).
16. Dowling, R.J., Niraula, S., Stambolic, V. & Goodwin, P.J. Metformin in cancer: translational challenges. J Mol Endocrinol 48, R31-43 (2012).

80

N. S. Chandel

17. Buzzai, M. et al. Systemic treatment with the antidiabetic drug metformin selectively
impairs p53-deficient tumor cell growth. Cancer research 67, 6745-6752 (2007).
18. Memmott, R.M. et al. Metformin prevents tobacco carcinogen--induced lung tumorigenesis. Cancer prevention research 3, 1066-1076 (2010).
19. Tomimoto, A. et al. Metformin suppresses intestinal polyp growth in ApcMin/+ mice.
Cancer Sci 99, 2136-2141 (2008).
20. Pollak, M. Overcoming Drug Development Bottlenecks With Repurposing:
Repurposing biguanides to target energy metabolism for cancer treatment. Nature
medicine 20, 591-593 (2014).
21. Birsoy, K., Sabatini, D.M. & Possemato, R. Untuning the tumor metabolic machine:
Targeting cancer metabolism: a bedside lesson. Nature medicine 18, 1022-1023 (2012).
22. Pollak, M. The insulin and insulin-like growth factor receptor family in neoplasia: an
update. Nature reviews. Cancer 12, 159-169 (2012).
23. El-Mir, M.Y. et al. Dimethylbiguanide inhibits cell respiration via an indirect effect targeted on the respiratory chain complex I. The Journal of biological chemistry 275, 223-228(2000).
24. Owen, M.R., Doran, E. & Halestrap, A.P. Evidence that metformin exerts its anti-diabetic
effects through inhibition of complex 1 of the mitochondrial respiratory chain. The
Biochemical journal 348 Pt 3, 607-614 (2000).
25. Bridges, H.R., Jones, A.J., Pollak, M.N. & Hirst, J. Effects of metformin and otherbiguanides on oxidative phosphorylation in mitochondria. The Biochemical journal(2014).
26. Wheaton, W.W. et al. Metformin inhibits mitochondrial complex I of cancer cells to
reduce tumorigenesis. eLife 3, e02242 (2014).
27. Fendt, S.M. et al. Metformin Decreases Glucose Oxidation and Increases the
Dependency of Prostate Cancer Cells on Reductive Glutamine Metabolism. Cancer
research (2013).
28. Janzer, A. et al. Metformin and phenformin deplete tricarboxylic acid cycle and glycolytic intermediates during cell transformation and NTPs in cancer stem cells.
Proceedings of the National Academy of Sciences of the United States of America 111,
10574-10579 (2014).
29. Hardie, D.G. AMPK: a target for drugs and natural products with effects on both diabetes
and cancer. Diabetes 62, 2164-2172 (2013).
30. Storozhuk, Y. et al. Metformin inhibits growth and enhances radiation response of nonsmall cell lung cancer (NSCLC) through ATM and AMPK. British journal of cancer 108,2021-2032 (2013).

Stress Response, Oxystress81

31. Emami Riedmaier, A., Fisel, P., Nies, A.T., Schaeffeler, E. & Schwab, M. Metformin andcancer: from the old medicine cabinet to pharmacological pitfalls and prospects. Trends
Pharmacol Sci 34, 126-135 (2013).

Navdeep S. Chandel, PhD

2000-2006

2006-20102010-20142014-present

Assistant Professor, Department of Medicine,Northwestern University
Associate Professor, Department of Medicine,Northwestern University
Professor, Department of Medicine, Northwestern
University
David Cugell Distinguished Professor, Department ofMedicine, Northwestern University

Specitaly and Present Interest:
Mitochondria as Signaling Organelles

82

mTOR GROWTH SIGNALING AND METABOLISMDavid M. Sabatini

Whitehead InstituteMIT
Howard Hughes Medical InstituteBroad Institute
Koch Institute for Integrative Cancer Research at MIT
Sabatini Laboratory, 9 Cambridge Center, Cambridge, MA 02142-1479, USA(sabatini@wi.mit.edu)

mTOR is the target of the immunosuppressive drug rapamycin and the centralcomponent of a nutrient- and hormone-sensitive signaling pathway that regulates cell
growth and proliferation. We now appreciate that this pathway becomes deregulated inmany human cancers and has an important role in the control of metabolism and aging.

We have identified two distinct mTOR-containing proteins complexes, one of which

regulates growth through S6K and another that regulates cell survival through Akt. These

complexes, mTORC1 and mTORC2, define both rapamycin-sensitive and insensitive

branches of the mTOR pathway. I will discuss new results from our lab on the regulationand functions of the mTORC1 and mTORC2 pathways.

Microenvironment and Metabolic Heterogeneity

David M. Sabatini, MD, PhD

1997-2002

2002-present

2008-present
2012-present

83Whitehead Fellow, Whitehead Institute for Biomedical
Research
Member, Whitehead Institute for Biomedical Research
Senior Associate Member, Broad InstituteMember, Koch Institute for Integrative Cancer Researchat MIT
Associate Professor, Dept. of Biology, Massachusetts
Institute of Technology
Investigator of the Howard Hughes Medical Institute
Professor, Dept. of Biology, Massachusetts Institute ofTechnology

Specialty and Present Interest:
Physiological and Pathological mTOR Pathway Activation in Cellular and WholeOrganism Context

84

SERINE METABOLISM IN CANCER DEVELOPMENT

Karen H. Vousden

CRUK Beatson Institute
Switchback Road, Glasgow G61 1BD, UK(k.vousden@beatson.gla.ac.uk)

Cancer cells show changes in metabolism that help to support enhanced proliferationand tumour growth in abnormal environments and under conditions of fluctuating

nutrient availability. However, these metabolic alterations can also impose vulnerabilities

that may be exploited for therapy. Recent studies have shown that many cancer cells havea high dependency on serine, which can be synthesized de novo or taken up from themedium. The serine synthesis pathway (SSP) enzymes are overexpressed in several

tumour types1,2) but those cancer cells that do not show up-regulation of this pathway aremuch more dependent on exogenous serine3). Following serine starvation, intracellularserine levels drop dramatically in these cells and a period of adaptation is required to allow

the up-regulation of the de novo SSP. Intracellular serine is an allosteric activator of
pyruvate kinase M2 (PKM2), the isoform of this glycolytic enzyme that is expressed incancers and other proliferating cells. Following starvation, the reduction in serine levels

results in lower PKM2 activity, a decreased flux through this step of glycolysis and thediversion of glucose derived carbons into the SSP4). This response is accompanied by atranscriptional activation of the key SSP enzymes, allowing the cell to switch fromexogenous to de novo synthesized serine supply. However, the decrease in glycolytic ratethat is a result of the reduced PKM2 activity results in less ATP production, and tocompensate cells channel more pyruvate into mitochondria, allowing an increase OXPHOS

for more efficient energy production. This increased mitochondrial respiration isaccompanying increase in ROS that must be limited to prevent oxidative damage and cell

death. Interestingly, we found that the tumour suppressor p53 can help to support theadaptation of cancer cells to serine starvation3). The activation of p53 in response to serine

Microenvironment and Metabolic Heterogeneity85

depletion leads to a transient cell cycle arrest, allowing the cells to channel metabolites into

glutathione synthesis to provide increased antioxidant defense. Cells lacking p53 are lessable to survive the switch to de novo serine synthesis because they are unable to adequately

limit ROS.

An appreciation of the importance of serine in cancer cell growth has prompted us tolook more closely at the fate of serine in the cell. Several biosynthetic pathways utilise
serine, including protein synthesis, the one carbon cycle, and the synthesis of other aminoacids, sphingosine and phospholipids. Serine can be converted into glycine, which is also

required for nucleotide and glutathione synthesis. Although serine and glycine can bereadily interconverted so that either might be used for nucleotide synthesis and one-carbon
metabolism, we found that exogenous glycine cannot replace serine to support tumour cell

growth in culture, with higher concentrations of glycine inhibiting proliferation5). Further

analysis revealed than in the absence of serine, glycine was converted to serine, a reactionthat results in the depletion the one-carbon pool. Accordingly, glycine-only fed cells were

rescued by addition of folate to replenish the one carbon pool.

Our observations that p53 can help limit redox stress and so support cell survival underserine starvation mirrors a general observation that excessive ROS can be detrimental tocell growth. The antioxidant activity of p53 is mediated through the transcriptional
activation of a number of genes encoding proteins with ROS limiting activities. In humancells, one of these is TIGAR, an enzyme that regulates the level of fructose-2, 6-bisphosphate (F-2,6-BP) and helps to support antioxidant defense by increasing NADPH
and GSH levels6). Although the ability of p53 to drive the expression of TIGAR suggests arole for TIGAR in limiting tumour development – possibly by preventing the accumulationof oxidative damage – studies have shown that oncogenic transformation promotes

increases in oxidative stress that can be damaging to the cell, and that the ability to limit

ROS can also be important for cancer cell survival7). Interestingly, although many tumours

lack p53 activity, we detected high levels of TIGAR in many human cancers and cancer celllines that lack wild-type p53, suggesting that TIGAR expression can be uncoupled from p53
and provide antioxidant functions that help to promote tumour development. p53-

independent expression of TIGAR is also seen in In mouse models, where TIGAR

expression is only weakly responsive to p53, but is induced in response to oncogenic

signals such as loss of APC, Myc activation and increased ROS itself8). In vivo, loss of
TIGAR leads to increased ROS levels that impede survival in regenerating epithelial tissue

and inhibit tumour development in several cancer models9).

Although ROS can be detrimental to cell growth, ROS are also required for mitogenic

signaling and cell proliferation. This dual activity in both promoting proliferation and cell
death makes prediction of the outcome of ROS limiting treatments in cells or in vivo

86

K. H. Vousdendifficult10). Our results support a role for TIGAR in limiting damaging ROS in intestinal

regeneration and proliferation induced by APC loss. However, previous studies haveshown that loss of APC also leads to the increased expression of Rac1, which promotes
proliferative ROS through NOX11). In these studies, loss of Rac1 led to decreased ROS andinhibited proliferation. Rac1 and TIGAR therefore have opposing effects on ROS, althoughboth are important to support hyperproliferation resulting from APC deletion in intestinal

crypts. To test whether TIGAR simply functions to limit ROS induced by Rac1, or whether

TIGAR limits a pool of damaging ROS that is functionally distinct from the proliferative
ROS induced by Rac1, we simultaneous deleted both Rac1 and TIGAR in APC null crypts.

This resulted in a more profound inhibition of crypt proliferation than seen following lossof either Rac1 or TIGAR alone, suggesting that the pro- and anti-proliferative effects of ROS

can be independently modulated in the same cell. Our results show that two key targets

induced by the Wnt pathway function to integrate the different ROS signals for optimal cellproliferation.

As many cancer cells were found to be sensitive to serine depletion, we sought todevelop a mechanism to deplete circulating serine levels, to assess whether this might havea selectively detrimental effect on tumour development in vivo. Dietary depletion of serine

does not significantly impact the general health of mice, although a clear reduction inserum serine and glycine levels can be detected in these animals3). In xenograft models,tumours grew more slowly in animals maintained on a serine/glycine free diet, with anaccompanying increase in survival. More recently we have been testing the effect of thisdiet on cancer progression in genetically engineered mouse models of cancer, including an

EuMyc lymphoma model, an intestinal adenoma models based on APC deletion and apancreatic cancer model driven by mutations in KRas and p53. To test the hypothesis that

serine starvation will further enhance the vulnerability of cancer cells to increases inoxidative stress or depleted antioxidant functions, we are examining the effect of

combining dietary serine/glycine starvation with TIGAR depletion on cancer development.
References

1. Locasale, J.W., et al., Phosphoglycerate dehydrogenase diverts glycolytic flux and contributes to oncogenesis. Nat Genet, 2011. 43(9): p. 869-74.
2. Possemato, R., et al., Functional genomics reveal that the serine synthesis pathway isessential in breast cancer. Nature, 2011. 476(7360): p. 346-50.
3. Maddocks, O.D., et al., Serine starvation induces stress and p53-dependent metabolic
remodelling in cancer cells. Nature, 2013. 493(7433): p. 542-6.
4. Chaneton, B., et al., Serine is a natural ligand and allosteric activator of pyruvate kinase
M2. Nature, 2012. 491(7424): p. 458-62.

Microenvironment and Metabolic Heterogeneity87

5. Labuschagne, C.F., et al., Serine, but not glycine, supports one-carbon metabolism andproliferation of cancer cells. Cell Rep, 2014. 7(4): p. 1248-58.
6. Bensaad, K., et al., TIGAR, a p53-inducible regulator of glycolysis and apoptosis. Cell,
2006. 126: p. 107-120.
7. Gorrini, C., I.S. Harris, and T.W. Mak, Modulation of oxidative stress as an anticancer
strategy. Nat Rev Drug Discov, 2013. 12(12): p. 931-47.
8. Lee, P., et al., p53- and p73-independent activation of TIGAR expression in vivo. Cell
Death Dis, 2015. 6: p. e1842.
9. Cheung, E.C., et al., TIGAR is required for efficient intestinal regeneration and tumorigenesis. Dev Cell, 2013. 25(5): p. 463-77.
10. Weinberg, F. and N.S. Chandel, Reactive oxygen species-dependent signaling regulates
cancer. Cell Mol Life Sci, 2009. 66(23): p. 3663-73.
11. Myant, K.B., et al., ROS production and NF-kappaB activation triggered by RAC1 facilitate WNT-driven intestinal stem cell proliferation and colorectal cancer initiation. Cell
Stem Cell, 2013. 12(6): p. 761-73.

Karen H. Vousden, PhD, CBE, FRS

1987-1995
1995-2002
2002-presentGroup Leader, LICR at St Mary’s Hospital
Laboratory Chief, NCI
Director, CRUK Beatson InstituteSpecialty and Present Interest:
Mechanisms of Oncogenesis, Regulation and Function of the p53 Tumour Suppressor,
Cancer Cell Metabolism

88

TUMOR MICROENVIRONMENT AND
CANCER METABOLISM

Tsuyoshi Osawa

Systems Biology and Medicine, RCAST, The University of Tokyo
4-6-1 Komaba, Meguro, Tokyo 153-8904, Japan(osawa@lsbm.org)

Angiogenesis, formation of new blood vessels, is essential for tumor progression,

invasion, and metastasis. Vascular endothelial growth factors (VEGFs) and their receptors(VEGFRs) are primary regulators of angiogenesis. Anti-VEGF antibodies (e.g.,

bevacizumab) and small molecular inhibitors of VEGFRs (e.g., sunitinib and sorafenib)

were validated as the first cancer therapeutic agents targeting the tumor
microenvironment. However, the effectiveness of anti-angiogenic treatments is limited tocertain types of cancer. In addition, they may not completely eradicate tumor growth andmay elicit malignant progression by, for example, inducing resistance to chemotherapy.

Thus, the molecular mechanism underlying the elimination of such resistant and refractory

cancer cells needs to be elucidated. The adaptation of cancer cells to tumor
microenvironments such as in response to hypoxia, nutrient deficiency, acidosis, andreactive oxygen species can be achieved via alteration of metabolic states to glycolysis,

glutaminolysis, and other metabolic pathways, but metabolic pathways utilized underhypoxia and nutrient starvation is unknown.

We have previously reported that cancer cells resistant to hypoxia and nutrient

starvation stimulated AKT phosphorylation, anchorage independent growth, cell migrationand invasion in culture and increased angiogenesis and infiltration of macrophages into
tumor tissues in vivo, partly under epigenetic regulations 1-5) . To elucidate metabolic

pathways utilized in cancer cells under hypoxia and nutrient starvation, we conducted

integrated analysis of epigenome, transcriptome and metabolome under control, hypoxia,

nutrient starvation, and hypoxia and nutrient starvation in vitro. Glycolytic metabolites
were accumulated under hypoxia, in contrast, accumulation of fatty acids and late

Microenvironment and Metabolic Heterogeneity

89

glycolytic metabolites were accumulated under nutrient starvation and hypoxia andnutrient starvation double stress (Figure 1). Interestingly, phosphoethanolamine was mostaccumulated in cancer cells under nutrient starvation and hypoxia and nutrient starvation

by up-regulation of ethanolamine kinases (ETNK1/CHKA) and down-regulation of thefollowing rate-limiting enzyme PCYT2 in the Kennedy pathway (Figure 2). Moreover,Growth medium in pO2 21%/1%

Starvation medium in pO2 21%/1%

3
2
G6P
F6P
F16P
G3H
13P
DG2
3PG
2PG
PEP
Pyruvate
Lactate
GGL
ARG
SER
ASP
TYR
MET
ALA
GLY
GL
Y
LEU
ACO (C2)BUA (C4)
6NA (C6)OCA (C8)
DKA (C10)DAO (C12)
3butyratecarnitine
PEtn

1
0
í
í
í
í
í
í

HeLa
HepG2
T98G
A431
HeLa
HepG2
T98G
A431

HepG2
T98G
A431

HeLa

Fatty acids

0.1% FBS
1/100 Glucose
1/100 Amino acids

Ethanolamine

CHKA

ETNK1
CHKA

***
***

Phosphoetanolamine
h t
l

****

PCYT2

60
50
40
30
20
10
0

Cont Starv. Cont Starv.

HeLa

T98G

Cont Starv. Cont Starv.

HeLa

PE

PSS2
PSD

PS

PCYT2

PC

PEMT

6
5
4
3
2
1
0

T98G

5

EPT

mRNA expression

Stravation

Hypoxia &Stravation

Hypoxia

CDP-Etn
Control

fmol/Cell

Phosphoethanolamine
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0

ETNK

C

mRNA expression

B

mRNA expression

Accumulation of Fatty acids were observed under nutrient starvation, hypoxia and nutrient
starvation in cancer cells in vitro. (A) Ingredients of growth rich- and nutrient starvedmedium. (B) Metabolome analysis of cancer cells under hypxia, starvation, hypoxia andstarvation.

A

Figure 2

Hypoxia &Starvation

Amino
acids

10% FBS
High-Glucose
Amino acids

Figure 1

Starvation

Hypoxia

B
Glycolysis

A

4
3
2
1
0

Phosphoethanolamine was accumulated in cancer cells under starvation.
(A) Accumulation of phosphoethanolamine (fmol/cell) in cancer cells (n=3).
(B) Biosynthesis of phosphoethanolamine. (C) Expression of ethanolamine kinases ETNK,
CHKA, and rate limiting PCYT2 by real time PCR.
Cont Starv. Cont Starv.

HeLa

T98G

90

T. Osawa

phosphoethanolamine (EAP) was accumulated in tumor tissue and stimulated tumor
growth both in vivo (Figure 3), suggesting that phosphoethanolamine can play animportant role in cancer cell survival under nutrient starvation.

Our results form the foundation of a strategy to attack hypoxia- and nutrient starvation-

resistant cancer cells as an approach to leverage conventional chemotherapy and limit

resistance to them.

B

Phosphoethanolamine
Phosphoethanolamine

nmol/g of tumor

2500

**
**

2000
1500
1000

**
**

500

**
**

0

3000

B16 Placebo
B16 EA 1000mg/kg
1000mg/
B16 EAP 10mg/kg
B16 EAP 100mg/kg
B16 EAP 1000mg/kg

2500
2000

1500 Placebo EAP1000
1000

**

**
**

*

500

Day6

Day4

Day2

0

Day0

Figure 3C3

in vivo Tu
Tumor
mor Samples

T
Tumor
volume
l
( m)
(mm

A

0

5

10

15

20

Days after injection (days)

Phosphoetanolamine was accumulated and stimulated tumor growth in vivo.
(A) Preparation of in vivo tumor samples (n=4). (B) Phosphoethanolamine was accumulated
in vivo tumor tissue. (C) Phosphoethanolamine stimulated in vivo tumor growth (n=4, B6
mice i.p. per every 2 days)

References
1. Osawa T, Muramatsu M, Watanabe M and Shibuya M: Hypoxia and low nutrition double
stress induces aggressiveness in a murine model of melanoma, Cancer Science, 2009,
May 9;100:844-51.
2. Osawa T, Tsuchida R, Muramatsu M, Yuasa Y, and Shibuya M: Human glioblastoma cells
exposed to long-term hypoxia and nutrient starvation stimulated induction of secondary
T cell leukemia in mice, Blood Cancer Journal, 2011, Feb 25; 1, e6.
3. Osawa T, Muramatsu M, Tsuchida R, Wang F, Kodama T, Minami T and Shibuya M*:
Increased expression of histone demethylase JHDM1D under nutrient starvation suppresses tumor growth via downregulating angiogenesis, Proc Nattl Acad Sci USA,2011, Dec 20;108:20725-9.
4. Osawa T, Tsuchida R, Muramatsu M, Shimamura T, Wang F, Suehiro J, Kanki Y, Wada Y,
Yuasa Y, Aburatani H, Miyano S, Minami T, Kodama T, and Shibuya M: Inhibition of histone demethylase JMJD1A improves anti-angiogenic therapy and reduces tumor associated macrophages. Cancer Research, 2013, May 15;73:3019-3028.

Microenvironment and Metabolic Heterogeneity91

5. Osawa T* and Shibuya M*: Targeting cancer cells resistant to hypoxia and nutrient starvation to improve anti-angiogeneic therapy. Cell Cycle 2013, Aug 15;12:2519-20.
6. Tsuchida R*, Osawa T*, Wang F, Nishii R, Tsuchida S, Muramatsu M, Das B, Takahashi T,
Yuasa Y, Inoue T, Wada Y, Minami T, and Shibuya M: BMP4/Thrombospondin-1 loop
paracrinically inhibits tumor angiogenesis and suppresses the growth of solid tumors.
Oncogene 2014 Jul 17;33:3803-11.

Tsuyoshi Osawa, PhD

2006-2007

2007-20112011-present

Research fellow, Division of Genetics, IMS, The
University of Tokyo
Assistant Professor, Department of Oncology, Tokyo
Medical and Dental University
Assistant Professor, Systems Biology andMedicine, RCAST, The University of Tokyo

Specialty and Present Interest:
Cancer Epigenetics, Angiogenesis, Hypoxia, Nutrient Starvation

92

DIALOGUES BETWEEN MACROPHAGE AND
TUMOR CELLS PROMOTE MALIGNANCY

Jeffrey W. Pollard

MRC Centre for Reproductive Health
The Queen’s Medical Research Institute, The University of Edinburgh
47 Little France Crescent, Edinburgh, EH16 4TJ, UK and
Department of Developmental and Molecular Biology
Albert Einstein College of Medicine
1300 Morris Park Avenue, Bronx, NY 10461, USA(jeff.pollard@ed.ac.uk)

The progression of tumors to malignancy is associated with a dynamically changing

microenvironment that supports tumor growth and survival1). This microenvironment ispopulated with a wide variety of immune cells from both the innate and acquired system2).

Indeed as soon as tumors emerge they engage the immune system suggesting that thetissue damage and possibly presentation of neo- or re-expressed foetal antigens results inimmune cell activation3). However, in most cases this immune response does not result in

the destruction of the tumor. Instead the data supports the contention that these immune

cells are polarized to provide support to the tumor and thus perversely enhance its growthand progression3,4). Indeed a hallmark of cancer appears not only to be immune evasion but
also an immune response that enhances malignancy.

Macrophages are one of the major immune cell types that are found in tumors often

achieving surprising concentrations1). Early studies showed that their accumulation wasusually associated with markers of poor prognosis 1) . Similarly the local tumor andcirculating concentrations of macrophage growth factors such as colony stimulating factor1 (CSF1) were also found to be associated with poor prognosis5). A good example of thiswas our perspective study in endometrial cancer which indicated that CSF1 was over-expressed in cancer and positively correlated with grade and stage and in a follow up
study that tumor cell produced CSF1 was an independent predictor of survival 6,7) .

Subsequently genetic removal of CSF1 and thereby tumor associated macrophages (TAM)

in a mouse model of breast cancer caused by the mammary epithelial restricted expressionof the Polyoma Middle T oncoprotein (PyMT) caused a reduction in tumor progression tomalignancy and a dramatic decrease in metastasis8). Similar studies have been performed inMetabolic Aspects of Cell-Cell Interaction

93

several models of cancer with comparable results9,10). These studies have given impetus totranslation from mouse models into the human with the results that some reagents usuallytargeted to the CSF1 receptor (neutralizing antibodies and small molecule inhibitors) havebeen introduced into clinical trials with early efficacy reported11).

Most studies of the functions of TAMs have been focused upon the primary tumor. In

this context they have been shown to stimulate stimulating angiogenesis, induce tumor cell

migration, invasion and intravasation and by suppress anti-tumor immunity12). In eachcase they are involved in reciprocal dialogues with the tumor cells adding support to the

hypothesis that tumor cells develop mutations that result in enhancement of theirmalignancy through engagement of the immune system within the tumor

microenvironment. A good example of this is in the TAM promotion of tumor cell invasion

and intravasation. In several models from the PyMT one to patient derived xenografts(PDX) this activity involves a paracrine interaction between tumor cell synthesized CSF1

that induces macrophage expression of EGF that drives tumor cell migration, invasion andintravasation13). Blockage individually of either growth factors or their receptor signaling

inhibits migration of both cell types14). Furthermore the macrophages act to open up vessels

through the expression of VEGFa at a perivascular site15). This unique site of tumor cell

escape into the circulation is characterized by a trio of cells, endothelial cells, macrophageand tumor cells expressing an isoform of Mena that can be identified histologically and thathas been termed the “Tumor Microenvironment of Metastasis (TMEM)”16,17). This structure
is predictive of the metastatic capacity of human breast cancers18). Interestingly and anotherexample of the complex dynamics found in tumors, is that the ability of the TAMs toengage in this migration-promoting activity is dependent on IL4 that in the PyMT model isproduced by CD4 positive T cells19). Thus a dynamic of cancer progression is immune cells

engagement results in a TH2 skewed cytokine environment that promotes metastatic

capacity2).

Another prominent activity of TAMs is to induce angiogenesis20) and in part the effectsof their ablation causing a slower transition to malignancy in several mouse models ofcancer is because they are involved in the angiogenic switch that is considered to be

essential for the malignant state21). These TAMs in mouse models have been identified as

being TIE2 Hi, a ligand that allows close apposition to vessels22). Thus ablation of Tie2 in the

myeloid lineage results in the loss of this close association and a reduction inangiogenesis23). This angiogenic activity is in part mediate by VEGFa, a molecule highly

expressed by macrophages. Indeed in human breast cancer macrophage VEGF expression
is associated with markers of poor prognosis 24) . However, surprisingly myeloid cell

ablation of VEGFa in the PyMT model although blocking the angiogenic switch, resulted intumors progressing faster to more malignant state defined by histology 25) . This was

94

J. W. Pollard

attributed to external selection pressure that selected for tumor cells less dependent on thetri-carboxylic acid cycle and more on glycolysis, that is a hallmark of advanced cancers.

Recent studies in the PyMT model however have defined Wnt7b as an upstream
macrophage regulator of VEGFa expression either in endothelial cells or TAMs 2 6 ) .
Consequently, unlike the result obtained following genetic ablation of VEGF that

accelerated tumour progression, in this case Wnt7b ablation inhibited the angiogenic
switch but also slowed the progression to malignancy and inhibited metastasis26). These

data suggest that Wnt7b is upstream of VEGFa but has more functions than simply

stimulating the macrophage stimulation of angiogenesis. Wnt7b is highly unregulated inseveral human cancer types and is also expressed in macrophages at least in human breast

cancer26). Thus these data also suggest given the relative failure of therapies based upon
inhibition of VEGFa that the inhibition of WNT7b might be better strategy for control ofprimary tumors.

Many workers have contributed to understanding the activities of TAMs in tumors, notonly through the biology described above but also through their production of a multitudeof molecules such as proteases, chemokines and growth factors and immunosuppressive

molecules4,10,27,28). The general consensus for these studies is that tumors recruit macrophageand polarize them to a state that reflects those seen during tissue repair or during

development29). These two states may not be that different since macrophage play roles indevelopment pertaining to branching complexity and the maintenance and function ofstem cells and these roles may well be important to repair tissues to the normal state

following injury29). However, to view macrophages as just two basic sub-types is a mistakethat belies the complexity of these cells both in their origin and function. In fact most of thefunctions described above are performed by different sub-populations of macrophages that

express canonical macrophage markers but display distinct expression of surface markers

and unique transcriptomes 12) . Thus macrophages involved in promoting tumor cell
invasion are different from those stimulating angiogenesis despite being within the same

tumor. Nevertheless some rules do apply at least in mice that have led macrophages to be
described as two polar opposites called M1, for those involved in pathogen and tumor
rejection and M2, for those involved in tumor promotion. These states were originallydefined by their arginine metabolism profile with the M1-type using arginine to generate

the cytotoxic molecule inducible nitric oxide (iNOS) while the M2 macrophages metabolize

arginine through the induction of arginase that has the consequence of suppressing
cytotoxic T cell activity30,31).

Insight into these differences in macrophage behavior and function have been obtained

through viewing the tumor microenvironment using multiphoton microscopy32). This has

led to observations of macrophage-tumor cell streaming to blood vessels, to theMetabolic Aspects of Cell-Cell Interaction

95

visualization of tumor cell extravasation at the TMEN sites where macrophages cause

pulsatile permeability events in blood vessels15,33). An additional characteristic of tumors as

they grow is that they have significant areas of necrosis and hypoxia. These necrotic areas

are highly populated by very motile macrophages that are engulfing dead cells but also

probably attempting to vascularize these areas. While these macrophages have not been
well characterized these observations mean that macrophage must function in areas thatare highly hypoxic and suggest that they can readily switch to a glycolytic metabolism.

Indeed these macrophages have high expression of HIF1a and HIF2. A characteristic ofanaerobic metabolism is the generation of lactate. Tumor cell derived lactate drives

macrophage polarization to a tumor trophic state through up-regulating the transcription

factor HIF1a34). These HIF transcription factor in many contexts, controls a transcriptional
programme that promotes angiogenesis and thus may play a role in the re-vascularizationof necrotic area as tumors grow.

Many of these studies have been performed in mice and clinical translation has

suggested similarities to human TAMs. However, there is a paucity of information onhuman macrophages from tumors. Indeed the canonical rules derived from the mousestudies immediately break down in humans as human macrophages do not express

arginase and highly restricted mouse macrophage surface markers such as F4/80 are notfound on human macrophages29). To address this issue we have profiled human breast
cancer TAMs compared to normal resident macrophages and have defined newtranscriptional signatures. Striking in these results is a significant shift in metabolic profiles

particularly in lipolysis suggesting unique adaptation to the tumor microenvironment in

humans. Such studies should enhance the ability to manipulate macrophages in humans

from pro-tumoral to anti-tumoral functions as a potential therapeutic approach to enhancechemo-, radio- or immuno-therapy.

References
1. Pollard JW. (2004). Tumor educated macrophages promote tumor progression andmetastasis. Nature Reviews Cancer. 4:71-8.
2. Ruffell B, DeNardo DG, Affara NI, Coussens LM. (2010). Lymphocytes in cancer development: polarization towards pro-tumor immunity. Cytokine Growth Factor Rev.
21(1):3-10. PMID: 20005150/PMCID: PMC2834837.
3. Noy R, Pollard JW. (2014). Tumor-associated macrophages: from mechanisms to therapy.
Immunity. 41(1):49-61. PMID: 25035953/PMCID: PMC4137410.
4. Ruffell B, Affara NI, Coussens LM. (2012). Differential macrophage programming in thetumor microenvironment. Trends in immunology. 33(3):119-26. PMID: 22277903/
PMCID: PMC3294003.

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J. W. Pollard

5. Lin EY, Gouon-Evans V, Nguyen AV, Pollard JW. (2002). The macrophage growth factor,
CSF-1, in mammary gland development and tumor progression. J Mammary Gland
Biol Neoplasia. 7(2):147-62.
6. Smith HO, Anderson PS, Kuo DY, Goldberg GL, DeVictoria CL, Boocock CA, Jones JG,
Runowicz CD, Stanley ER, Pollard JW. (1995). The role of colony-stimulating factor 1 andits receptor in the etiopathogenesis of endometrial adenocarcinoma. Clin Cancer Res.
1(3):313-25. PMID: 9815987.
7. Smith HO, Stephens ND, Qualls CR, Fligelman T, Wang T, Lin CY, Burton E, Griffith JK,
Pollard JW. (2013). The clinical significance of inflammatory cytokines in primary cell culture in endometrial carcinoma. Mol Oncol. 7(1):41-54. PMID: 22944067.
8. Lin EY, Nguyen AV, Russell RG, Pollard JW. (2001). Colony-stimulating factor 1 promotes
progression of mammary tumors to malignancy. J Exp Med. 193(6):727-40. PMID:
11257139.
9. Joyce JA, Pollard JW. (2009). Microenvironmental regulation of metastasis. Nature
reviews Cancer. 9(4):239-52. PMID: 19279573.
10. Quail DF, Joyce JA. (2013). Microenvironmental regulation of tumor progression andmetastasis. Nat Med. 19(11):1423-37. PMID: 24202395/PMCID: PMC3954707.
11. Ries CH, Cannarile MA, Hoves S, Benz J, Wartha K, Runza V, Rey-Giraud F, Pradel LP,
Feuerhake F, Klaman I, Jones T, Jucknischke U, Scheiblich S, Kaluza K, Gorr IH, Walz A,
Abiraj K, Cassier PA, Sica A, Gomez-Roca C, de Visser KE, Italiano A, Le Tourneau C,
Delord JP, Levitsky H, Blay JY, Ruttinger D. (2014). Targeting Tumor-AssociatedMacrophages with Anti-CSF-1R Antibody Reveals a Strategy for Cancer Therapy.
Cancer Cell. 25, 1-14. PMID: 24898549.
12. Qian BZ, Pollard JW. (2010). Macrophage diversity enhances tumor progression andmetastasis. Cell. 141(1):39-51. PMID: 20371344.
13. Kitamura T, Qian BZ, Pollard JW. (2015). Immune cell promotion of metastasis. Nature
reviews Immunology. 15(2):73-86. PMID: 25614318/PMCID: PMCPMC4470277.
14. Wyckoff J, Wang W, Lin EY, Wang Y, Pixley F, Stanley ER, Graf T, Pollard JW, Segall J,
Condeelis J. (2004). A paracrine loop between tumor cells and macrophages is required fortumor cell migration in mammary tumors. Cancer Res. 64(19):7022-9. PMID: 15466195.
15. Harney AS, Arwert EN, Entenberg D, Wang Y, Guo P, Qian BZ, Oktay MH, Pollard JW,
Jones JG, Condeelis JS. (2015). Real-Time Imaging Reveals Local, Transient Vascular
Permeability, and Tumor Cell Intravasation Stimulated by TIE2hi Macrophage-Derived
VEGFA. Cancer Discovery. 5 (9): 932-943. PMID: 26269515.
16. Gertler F, Condeelis J. (2011). Metastasis: tumor cells becoming MENAcing. Trends Cell
Biol. 21(2):81-90. PMID: 21071226.

Metabolic Aspects of Cell-Cell Interaction

97

17. Oktay MH, Gertler FB, Liu YF, Rohan TE, Condeelis JS, Jones JG. (2012). Correlated
immunohistochemical and cytological assays for the prediction of hematogenous dissemination of breast cancer. J Histochem Cytochem. 60(3):168-73. PMID: 22215635/
PMCID: PMC3351127.
18. Rohan TE, Xue X, Lin HM, D'Alfonso TM, Ginter PS, Oktay MH, Robinson BD,
Ginsberg M, Gertler FB, Glass AG, Sparano JA, Condeelis JS, Jones JG. (2014). Tumor
microenvironment of metastasis and risk of distant metastasis of breast cancer. J NatlCancer Inst. 106(8). PMID: 24895374.
19. DeNardo DG, Barreto JB, Andreu P, Vasquez L, Tawfik D, Kolhatkar N, Coussens LM.
(2009). CD4(+) T cells regulate pulmonary metastasis of mammary carcinomas byenhancing protumor properties of macrophages. Cancer Cell. 16(2):91-102. PMID:
19647220.
20. Murdoch C, Muthana M, Coffelt SB, Lewis CE. (2008). The role of myeloid cells in the promotion of tumour angiogenesis. Nature reviews Cancer. 8(8):618-31. PMID: 18633355.
21. Lin EY, Pollard JW. (2007). Tumor-associated macrophages press the angiogenic switch inbreast cancer. Cancer Res. 67(11):5064-6. PMID: 17545580.
22. De Palma M, Murdoch C, Venneri MA, Naldini L, Lewis CE. (2007). Tie2-expressing
monocytes: regulation of tumor angiogenesis and therapeutic implications. Trends inimmunology. 28(12):519-24. PMID: 17981504.
23. Mazzieri R, Pucci F, Moi D, Zonari E, Ranghetti A, Berti A, Politi LS, Gentner B, Brown JL,
Naldini L, De Palma M. (2011). Targeting the ANG2/TIE2 axis inhibits tumor growth andmetastasis by impairing angiogenesis and disabling rebounds of proangiogenic
myeloid cells. Cancer Cell. 19(4):512-26. PMID: 21481792.
24. Leek RD, Lewis CE, Whitehouse R, Greenall M, Clarke J, Harris AL. (1996). Association ofmacrophage infiltration with angiogenesis and prognosis in invasive breast carcinoma.
Cancer Res. 56(20):4625-9. PMID: 8840975.
25. Stockmann C, Doedens A, Weidemann A, Zhang N, Takeda N, Greenberg JI, Cheresh
DA, Johnson RS. (2008). Deletion of vascular endothelial growth factor in myeloid cells
accelerates tumorigenesis. Nature. 456:814-8. PMID: 18997773.
26. Yeo EJ, Cassetta L, Qian BZ, Lewkowich I, Li JF, Stefater JA, 3rd, Smith AN,
Wiechmann LS, Wang Y, Pollard JW, Lang RA. (2014). Myeloid WNT7b mediates theangiogenic switch and metastasis in breast cancer. Cancer Res. 74(11):2962-73. PMID:
24638982/PMCID: PMC4137408.
27. Balkwill FR, Mantovani A. (2012). Cancer-related inflammation: common themes andtherapeutic opportunities. Semin Cancer Biol. 22(1):33-40. PMID: 22210179.
28. Biswas SK, Allavena P, Mantovani A. (2013). Tumor-associated macrophages: functional
diversity, clinical significance, and open questions. Seminars in immunopathology.
35(5):585-600. PMID: 23657835.

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29. Wynn TA, Chawla A, Pollard JW. (2013). Macrophage biology in development, homeostasis and disease. Nature. 496:445-55. PMID: 23619691/PMCID: PMC3725458.
30. Mills CD, Kincaid K, Alt JM, Heilman MJ, Hill AM. (2000). M-1/M-2 macrophages andthe Th1/Th2 paradigm. J Immunol. 164(12):6166-73. PMID: 10843666.
31. Mantovani A, Sozzani S, Locati M, Allavena P, Sica A. (2002). Macrophage polarization:
tumor-associated macrophages as a paradigm for polarized M2 mononuclear phagocytes. Trends in immunology. 23(11):549-55. PMID: 12401408.
32. Entenberg D, Wyckoff J, Gligorijevic B, Roussos ET, Verkhusha VV, Pollard JW,
Condeelis J. (2011). Setup and use of a two-laser multiphoton microscope for multichannel intravital fluorescence imaging. Nat Protoc. 6(10):1500-20. PMID: 21959234.
33. Wyckoff JB, Wang Y, Lin EY, Li JF, Gosj63 wami S, Stanley ER, Segall JE, Pollard JW,
Condeelis J. (2007). Direct visualization of macrophage-assisted tumor cell intravasation in
mammary tumors. Cancer Res. 67(6):2649-56. PMID: 17363585.
34. Colegio OR, Chu NQ, Szabo AL, Chu T, Rhebergen AM, Jairam V, Cyrus N, Brokowski
CE, Eisenbarth SC, Phillips GM, Cline GW, Phillips AJ, Medzhitov R. (2014). Functionalpolarization of tumour-associated macrophages by tumour-derived lactic acid. Nature.
513(7519):559-63. PMID: 25043024/PMCID: PMCPMC4301845.

Jeffrey W. Pollard, PhD, FRSE

1993-present

2000-2012

2002-2012
2008-present

Present

Professor, Department of Developmental and Molecular
Biology, Albert Einstein College of Medicine
Director, Center for the Study of Reproductive Biology
and Women’s Health, Albert Einstein College ofMedicine
Deputy Director, Albert Einstein Cancer Center
Louis Goldstein Swan Chair in Women’s Cancer
Research, Albert Einstein College of Medicine
Director, MRC University of Edinburgh Centre for
Reproductive Health, College of Medicine andVeterinary Medicine, University of Edinburgh
Wellcome Senior Investigator, Professor of Resilience
Biology, College of Medicine and Veterinary Medicine,
University of Edinburgh

Speciality and Present Interest:
Macrophages in Tumour Progression and Malignancy, Metastasis, Angiogenesis

99

CELL COMPETITION AND WARBURG EFFECT

Yasuyuki Fujita

Division of Molecular Oncology
Institute for Genetic Medicine, Hokkaido University
Kita 15, Nishi 7, Kita-ku, Sapporo, Hokkaido 060-0815, Japan(yasu@igm.hokudai.ac.jp)

At the initial step of carcinogenesis, transformation occurs in a single cell within anepithelial sheet, and the emerging transformed cells grow while being surrounded bynormal epithelial cells. However, it was not clear what happens at the boundary between
normal and transformed cells. Using newly established cell culture and mouse model

systems, we have shown that various phenomena can occur at the interface between
normal and transformed epithelial cells1,2). For example, when Ras- or Src-transformed cells

are surrounded by normal epithelial cells, various signaling pathways are activated in thetransformed cells and they are often eliminated from the apical surface of the epithelialmonolayer (Figure 1)3,4). In addition, when tumor suppressor protein Scribble- or Mahjongknockdown cells are surrounded by normal epithelial cells, the transformed cells undergo

apoptosis and are removed from the epithelium (a summary of these processes are shown

Figure 1

Apical extrusion of RasV12-transformed cells from a monolayer of normal epithelial cells.
Arrows indicate RasV12-transformed cells that are apically extruded.

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Y. Fujita

in Figure 2). These phenomena are not observed when transformed cells alone are present,

suggesting that the presence of surrounding normal cells affects the signaling pathways

and fate of transformed cells. Furthermore, we have recently demonstrated that normal

epithelial cells can recognize and actively eliminate neighboring transformed cells,

implying a notion that the normal epithelium has anti-tumor activity that does not involve

immune systems; thus we have named this process EDAC (Epithelial Defense Against

Cancer) (Figure 3)5). Cell competition is a process by which different types of cells competewith each other for survival, and was originally identified in Drosophila. But, our data

Figure 2

Cell competition between normal and transformed epithelial cells in mammals.
My lab has shown that various phenomena can occur at the interface between normal andtransformed epithelial cells.
When these transformedcells alone are present, neither apical extrusion nor apoptosis
occur, indicating that the presence of surrounding normal cells affects the signalling pathways and fate of neighbouring transformed cells. Then, what are the molecular mechanisms?

Figure 3

Schematics for EDAC ( Epithelial Defense Against Cancer)

Metabolic Aspects of Cell-Cell Interaction

101

indicate that cell competition can also occur in mammals, though the molecular mechanism

underlying this important biological process remains largely unknown.

Recently, we have found that mitochondrial activity is substantially decreased in

RasV12-transformed cells when they are surrounded by normal epithelial cells. Increased

expression of a member of PDK family is responsible for the down-regulation of

mitochondrial activity. Addition of DCA (Dichloroacetate), an inhibitor of PDKs,

significantly suppresses the apical extrusion of RasV12-transformed cells, suggesting that

PDK-mediated mitochondrial down-regulation plays a positive role in the elimination oftransformed cells. Furthermore, expression of LDH is enhanced in RasV12-transformed

cells surrounded by normal cells, and suppression of LDH activity leads to formation ofbasal protrusions. These data suggest that the Warburg effect-like phenotype can occur at

the initial stage of carcinogenesis, which plays a tumor-suppressive role by promoting
elimination of transformed cells from epithelial tissues. By further developing this new

research field, we would like to establish a novel type of cancer treatment: eradication oftransformed cells by enhancing a defensive force of neighbouring normal epithelial cells.
References

1. Hogan, C., Kajita, M., Lawrenson, K., and Fujita, Y. Interactions between normal andtransformed epithelial cells: their contributions to tumourigenesis. Int J Biochem. Cell Biol.
2011; 43: 496-503.
2. Kajita, M. and Fujita, Y. EDAC: Epithelial defence against cancer-cell competition
between normal and transformed epithelial cells in mammals. Journal of Biochemistry.
2015; 158(1): 15-23.
3. Hogan, C., Dupré-Crochet, S., Norman, M., Kajita, M., Zimmermann, C., Pelling, A.E.,
Piddini, E., Baena-López, L.A., Vincent, J. P., Hosoya, H., Itoh, Y., Pichaud, F. andFujita, Y. Characterization of the interface between normal and transformed epithelial
cells. Nature Cell Biology. 2009; 11 (4): 460-467.
4. Kajita, M., Hogan, C., Harris, A. R., Dupre-Crochet, S., Itasaki, N., Kawakami, K.,
Charras, G., Tada, M., and Fujita, Y. Interaction with surrounding normal cells influences signaling pathways and behavior of Src-transformed cells. Journal of Cell Science.
2010; 123 (Pt2): 171-80.
5. Kajita M., Sugimura, K., Ohoka, A., Burden, J., Suganuma, H., Ikegawa, M., Shimada, T.,
Kitamura, T., Shindoh, M., Ishikawa, S., Yamamoto, S., Saitoh, S., Yako, Y., Takahashi, R.,
Okajima, T., Kikuta, J., Maijima, Y., Ishii, M., Tada, M., and Fujita, Y. Filamin acts as a key
regulator in epithelial defence against transformed cells. Nature Communications. 2015;
5:4428

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Y. Fujita

Yasuyuki Fujita, MD, PhD

2002-20112010-presentGroup leader, MRC, LMCB, University College
LondonProfessor, Hokkaido University, Institute for Genetic
Medicine, Division of Molecular Oncology

Specialty and Present Interest:
Cell Competition, Carcinogenesis

103

INNOVATIVE MEDICINE FOR METABOLICREPROGRAMMING OF INTRACTABLE CANCER STEM CELLS

Hideshi Ishii

Cancer Profiling Discovery, Osaka University Graduate School of Medicine
2-2 Yamadaoka, Suita, Osaka 565-0871, Japan(hishii@gesurg.med.osaka-u.ac.jp)

The most important discovery in the field of cancer research in the past several decades

was detecting cancer as a genetic disorder. Although cancer cures are earnestly needed,cancer deaths are increasing and were reported as the most common fatality in 2010, being

only second to heart diseases. Cancer treatment remains challenging because of thediversity and heterogeneity of tumors. Cancer heterogeneity is reflected by the co-existence of cells with varying differentiation levels due to accumulation of genetic

mutations and significant association with epigenetic alterations. Recent studies suggestthat small cell populations, such as cancer stem cells, mimic normal adult stem cells in thedormant phase of the cell cycle and play a role in the biological behavior of tumors.

Considering the importance of stem cell biology in cancer, we have recently studied theaforementioned issues and have discussed their possible rationale in this study (Figure 1).

By utilizing small compounds, such as synthesized RNAs, including micro (mi) RNAs, we
examined the effects of cellular reprogramming in epigenetics and metabolomics, which

may be useful in suppressing and eradicating human malignancies, including

gastrointestinal cancers (Figures 2-4). Our data indicated that it is possible to reprogrammouse and human cells to pluripotency by direct transfection of mature double-stranded
miRNAs, which is similar to the introduction of induced pluripotent stem (iPS) genes.

Because this reprogramming method does not require vector-based gene transfer, it holds

significant potential for biomedical research and regenerative medicine. The efficiency ofthis reprogramming method needs to be improved. In this seminal study, we discuss theunderlying mechanism of miRNA-based reprogramming and its possible application incancer cell reprogramming as a novel therapeutic approach1-3) (Figures 5,6). It is naturally

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H. Ishii

Figure 1

Whole schema of the application of regenerative medicine for cancer research.
In the process of research, the epigenetic and metabolic issues have emerged for the next-generation drug discovery. The scheme indicates that cancer is derived from normally differentiated cells, in which metabolic control plays a critical role under the epigenetic control forgene expression. Cancer exhibits phenotypic appearance largely, in which multi-omics,
such as genomes, methylomes and transcriptomes, and metabolomes are involved.

Figure 2

Computational analysis of mouse chromosome 12F, a critical genomic region, which determines the maintenance and susceptibility to cellular reprogramming.
The comparison between human and mouse cells indicated that micro (mi) RNA-369, a
subclass of non-coding RNA, is conserved, whereas the other genes, such as mouse Rian andMirg, are not. This suggests the unique role of miRNA-3693).

Metabolic Aspects of Cell-Cell Interaction

105

Figure 3

Screening for micro (mi) RNAs could induce cellular reprogramming.
NANOG-green fluorescent protein (GFP) reporter mouse cells, which were isolated from subcutaneous differentiated adipocytes in NANOG promoter driven GFP transgenic mice,
were transfected by chemically synthesized miRNAs. These miRNAs were selected from an
expression study by Array, which showed substantial expression in embryonic stem (ES) cells
and induced pluripotent stem (iPS) cells but not differentiated adipocytes. The study
allowed the identification of a set of miRNAs, composed of miR-200c, miR-302, and miR-369.
These miRNAs are expressed exclusively in ES cells and iPS cells. The schema is modified(Cell Stem Cell 8(6):633-638, 2011).

Figure 4

The effect of miRNAs on tumor cells, isolated from the expression study of embryonicstem cells and induced pluripotent stem cells.
The introduction of these miRNAs resulted in the inhibition of gastrointestinal cancer cell
growth in vitro and the suppression of tumorigenicity in immune-deficient NOD-SCID
mice in vivo. The mice experiment was performed by the intravenous administration ofchemically synthesized miRNAs with a drug delivery system against pre-inoculated andformed tumors in NOD-SCID mice4).

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H. Ishii

Figure 5

miR-369-dependent alternative splicing elicits the switching of pyruvate kinase (PK).
To end the biological effect of miR-369, we studied PK knock-in mice (Pkm1-KI and Pkm2-KI)
in collaboration with Dr Numata and Dr Shima at the Miyagi Cancer Institute, Japan. The
study of Pkm1-KI and Pkm2-KI provided information on the biological effect of miR-369, atleast partially as a phenocopy3).

Figure 6

The protein-protein association analysis indicated three hab networks in a miR-369
dependent manner during cell reprograming.
Computational analysis revealed three networks: splicing factors (hnRNPa2b1, SFPQ etc.),
redox control (GSTP, PKM, etc.), and proteasome group (UBC, etc.). The computationally predicted networks were confirmed by a wet experiment3).

Metabolic Aspects of Cell-Cell Interaction

107

expected that the pharmacogenomics of targeting iPS genes may sensitize cancer cell

populations that were largely refractory to conventional chemotherapy2-4). Taken together,
these novel approaches are beneficial for cancer research and may provide an avenue totreat gastrointestinal cancers that are currently difficult to cure (Figure 7).

Figure 7

The innovative drug target of nucleotide medicine involves transomic feedback.
We propose a schema using an innovative drug target for cell-targeting therapy of cancer andregenerative medicine. Based on the study of mouse chromosome 12F, we identified several
sets of reprogramming factors inducing micro (mi) RNAs, including miR-369. SuchmiRNAs work as a unique complex and they involve Ago, hnRNPa2b1 and Fxr. The
hnRNPa2b1 exerts a unique function downstream, which dictates the metabolic process,where PKM is involved. The PKM regulates further epigenetic alterations of histone modifications via S-adenosylmethionine (SAM) and unsaturated fatty acids (USFA). In thereverse way, we showed that histone modifications control the epigenetics of miR-369 atchromosome 12F. Thus, the novel pin-point drug discovery targeting transomics (transcriptome, epigenome, and metabolome) would provide an avenue for innovative cancer
medicine in the near future1-4).

References
1. Hamabe A, Konno M, Tanuma N, Shima H, Tsunekuni K, et al. Role of pyruvate kinase
M2 in transcriptional regulation leading to epithelial-mesenchymal transition. Proc.
Natl. Acad. Sci. USA, 111(43):15526-15531, 2014.
2. Noguchi K, Eguchi H, Konno M, Kawamoto K, Nishida N, et al. Susceptibility of pancreatic cancer stem cells to reprogramming. Cancer Science, 106(9):1182-1187, 2015.

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3. Konno M, Koseki J, Kawamoto K, Nishidam N, Matsui H, et al. Embryonic microRNA369 controls metabolic splicing factors and urges cellular reprograming. PLOS ONE,10(7):e0132789, 2015.
4. Ogawa H, Wu X, Kawamoto K, Nishida N, Konno M, et al. MicroRNAs induce epigenetic
reprogramming and suppress malignant phenotypes of human colon cancer cells.
PLOS ONE, 10(5):e0127119, 2015.

Hideshi Ishii, MD, PhD

1983-1988
1988-1989
1989-1992
1992-1995

1995-1996

1996-2000
2000-2001
2001-2002
2002-2008
2008-20112011-20132014-present

Graduated from Chiba University School of Medicine
Resident, Chiba Municipal Hospital
Resident, Chiba Hospital of Kawasaki Steel Corporation
Research Resident, National Cancer Center ResearchInstitute
Staff Scientist, National Institute of Radiological
Sciences
Postdoctoral Fellow, Thomas Jefferson University
Research Instructor, Kimmel Cancer Center
Assistant Professor, Kimmel Cancer Center
Lecturer, Jichi Medical University
Associate Professor, Kyusyu University
Endowed Chair Professor, Cancer Frontier Science,
Osaka University
Specially Appointed Professor, Cancer Profiling
Discovery, Osaka University

Specialty and Present Interest:
microRNA (miRNA), Metabolic and Epigenetic Reprogramming, Gastrointestinal Cancer

109

PI KINASES AND CANCER METABOLISM

Lewis C. Cantley

Sandra and Edward Meyer Cancer Center at Weill Cornell Medical College
413 East 69th St., 13th Floor, Box 50, New York, NY 10021, USA(lcantley@med.cornell.edu)

Phosphoinositide 3-Kinase (PI3K) is a central enzyme in a signaling pathway thatmediates cellular responses to growth factors. This enzyme phosphorylates the 3 positionof phosphatidylinositol-4,5-bisphosphate to produce phosphatidylinositol-3,4,5-

trisphosphate (PIP3) at the plasma membrane. A number of signaling proteins, including

the Ser/Thr protein kinases, AKT and PDK1, contain pleckstrin homology domains thatbind specifically to PIP3. Thus, the generation of PIP3 at the plasma membrane in responseto activation of PI3K by growth factors results in the initiation of downstream Ser/Thr
phosphorylation cascades that control a variety of cellular responses. The signaling
pathway downstream of PI3K is highly conserved from worms and flies to humans andgenetic analysis of the pathway has revealed a conserved role in regulating glucose

metabolism and cell growth. Based on deletion of genes encoding the catalytic or
regulatory subunits of PI3K in the mouse, PI3K mediates insulin dependent regulation of

glucose metabolism, and defects in activation of this pathway result in insulin resistance.
In contrast, mutational events that lead to hyperactivation of the PI3K pathway result incancers. Activating mutations in PIK3CA, encoding the p110alpha catalytic subunit of

PI3K or inactivating mutations in PTEN, a phosphoinositide 3-phosphatases that reverses
the effects of PI3K, are among the most common events in solid tumors.

We have generated mouse models to interrogate the role of PI3Ks in metabolic control

and in the generation of cancers. These studies indicate that inhibitors of PI3Ks could be
effective in treating cancers in specific mutational backgrounds. Our first genetic knockoutof the gene encoding the regulatory subunit of class Ia PI3K, PIK3R1 in hematopoietic cells
revealed that class Ia PI3Ks are critical for activation of B cell lineage lymphocytes1). This

110

L. C. Cantleyobservation and subsequent studies revealing the importance of the class Ia catalytic

subunit, PIK3CD for B cell activation led to exploration of inhibitors specific to the geneproduct of PIK3CD. One such inhibitor, idelalisib was approved for treating chronic
lymphocytic leukemia, a B cell lymphoma, in 20142).

More than 20 PI3K inhibitors that target the gene product of PIK3CA have enteredclinical trials for treating solid cancers. Although these inhibitors look promising, a variety

of toxicities have created challenges. Some of the toxicities, such as hyperglycemia, are on

target since inhibition of the PIK3CA gene product is expected to cause insulin resistance.

However, other toxicities, such as severe rashes and diarrhea are unlikely to be aconsequence of inhibition of PIK3CA. These results suggest that more specific inhibitors

and inhibitors with specificity for the mutant form of PIK3CA could show better
efficacy/toxicity profiles. In any event, there are ongoing approval trials for several PI3K

inhibitors, including a pan-PI3K inhibitor from Novartis (BKM120) and an alpha-specific
PI3K inhibitor from Novartis (BYL719) that will be discussed below.

Since the gene product of PIK3CA mediates glucose uptake and metabolism

downstream of insulin and other growth factors, it is expected that PI3K inhibitors thattarget the PIK3CA gene product should cause inhibition of glucose uptake and impair

anabolic processes downstream of glucose in tumors that have mutations in PIK3CA or

where growth factor stimulation of PIK3CA is driving glycolysis. Consistent with this idea,

lung adenocarcinomas that develop in the context of PIK3CA activating mutations havehigh rates of glucose uptake as judged by imaging of 18F-flurodeoxyglucose positronemission tomography (FDG-PET)3). Importantly, inhibition of PI3K activity causes a

dramatic decline in FDG-PET signal and causes tumor regression in this model.

In order to examine whether PI3K inhibitors affect FDG-PET in human patients andwhether an acute decline in FDG-PET correlates with clinical responses, we performed
quantitative FDG-PET measurements in ER-positive breast cancer patients prior to and two

weeks after treatment with the PI3K inhibitor, BKM120. This study showed that BKM120

caused a decline in FDG-PET in a significant subset of patients. Importantly the subset ofpatients whose FDG-PET showed a decline were ultimately found to show clinical benefit

as judged by stable disease or tumor shrinkage, while those patients whose tumors did notshow an acute drop in FDG-PET also failed to show clinical benefit4). Interestingly thechange in FDG-PET and the clinical benefit did not always correlate with the presence of

PIK3CA mutations, suggesting that tumors without PIK3CA mutations may still bedependent on wild type PIK3CA.

In order to better understand how PI3K inhibitors affect glucose metabolism, we haverecently evaluated the effect of these inhibitors on flux through glycolysis as well as on thesteady state levels of intermediates in glycolysis 5) . These studies have revealed theNakahara Memorial Lecture111

surprising results that PI3K inhibitors have a major effect on aldolase activity. This affecton aldolase is not a consequence of inhibition of AKT. Inhibition of AKT impairs glucose

uptake and phosphorylation, as expected, however the PI3K-dependent activation ofaldolase is mediated by Rac-dependent mobilization of actin. In quiescent cells, much of

the aldolase is bound to F-actin in an inactive state and the mobilization of actin causes

release and activation of aldolase.

References
1. Fruman, D. A., Snapper, S. B, Yballe, C., Davidson, L., Yu, J. Y., Alt, F. W. and Cantley, L.
C. (1999) Impaired B cell development and proliferation in absence of phosphoinositide 3kinase p85a. Science 283, 393-397.
2. Fruman DA, Cantley LC. Idelalisib--a PI3Kd inhibitor for B-cell cancers. N Engl J Med.
2014 Mar 13;370(11):1061-2. doi: 10.1056/NEJMe1400055. PMID: 24620870
3. Engelman JA, Chen L, Tan X, Crosby K, Guimaraes AR, Upadhyay R, Maira M,
McNamara K, Perera SA, Song Y, Chirieac LR, Kaur R, Lightbown A, Simendinger J, Li T,
Padera RF, García-Echeverría C, Weissleder R, Mahmood U, Cantley LC, Wong KK.
Effective use of PI3K and MEK inhibitors to treat mutant Kras G12D and PIK3CA
H1047R murine lung cancers. Nat Med. 2008 Dec;14(12):1351-6. Epub 2008 Nov 30.
4. Mayer IA, Abramson VG, Isakoff SJ, Forero A, Balko JM, Kuba MG, Sanders ME, Yap JT,
Van den Abbeele AD, Li Y, Cantley LC, Winer E, Arteaga CL. Stand up to cancer phase
Ib study of pan-phosphoinositide-3-kinase inhibitor buparlisib with letrozole in estrogen
receptor-positive/human epidermal growth factor receptor 2-negative metastatic breast
cancer. J Clin Oncol. 2014 Apr 20;32(12):1202-9. doi: 10.1200/JCO.2013.54.0518. Epub
2014 Mar 24. PMID: 24663045
5. Hu H, Juvekar A, Lyssiotis CA. Lien EC, Albeck JG, Oh D, Varma G, Hung YP, Ullas S,
Lauring J, Seth P, Lundquist MR, Tolan DR, Grant AK, Needleman DJ, Asara JM.
Cantley LC, Wulf GM. Phosphoinositide 3-Kinase regulates glycolysis through mobilization of Aldolase from the Actin cytoskeleton. Cell. 2016 Jan 28;164(3):433-46. doi 10.
1016/j. cell. 2015.12.042. PMID: 26824656

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L. C. Cantley

Lewis C. Cantley, PhD

1978-1981

1981-1985

1985-1992

1992-2003
1992-2012

2003-20122007-20122012-present
2012-present

Assistant Professor, Dept. of Biochemistry andMolecular Biology, Harvard Univ.
Associate Professor, Dept. of Biochemistry andMolecular Biology, Harvard Univ.
Professor of Physiology, Tufts University School of
Medicine
Professor of Cell Biology, Harvard Medical School
Chief, Division of Signal Transduction, Beth Israel
Deaconess Medical Center
Professor of Systems Biology, Harvard Medical School
Director, Beth Israel Deaconess Cancer Center
Professor of Medicine, Weill Cornell Medical College
Director, Sandra and Edward Meyer Cancer Center at
Weill Cornell Medical College/ Ronald P. Stanton
Clinical Cancer Program at NewYork-Presbyterian

Specialty and Present Interest:
PI3K and Cancer Metabolism

113

TARGETING CANCER MICROENVIRONMENT-SPECIFICMETABOLISM REORGANIZES TUMOR VASCULATURE

Hiroyasu Esumi1, Takanori Kawashima2, Kenta Murata2 andMasayuki Yamaguchi3
Research Institute for Biomedical Sciences, Tokyo University of Science2669 Yamazaki, Noda, Chiba 278-0022, Japan(hesumi@rs.tus.ac.jp)
2
Kampo Research Laboratories, Kracie Pharma, Ltd.
3
NCC-EPOC, National Cancer Center Hospital East

1

The heterogeneity present in tumor cells and in the tumor microenvironment is ahallmark of many advanced cancers. This hallmark is especially true for hypovasculartumors, such as pancreatic cancer. We have examined molecular and biochemicalmechanism of cancer cells adapting to severe hypoxic and nutrient-deprived condition

present in pancreatic cancer. Our results showed that tumor cells alter cell cycle regulationand cell death programs through both the PI3K-Akt pathway and AMPK and its
downstream kinase pathways. We also employed biochemical analyses to study themechanism of cancer cell survival under these conditions and found that the cells use
anaerobic respiration and fumarate respiration1-3).

Hypoxia and nutrient starvation affect the biological behavior of cancer cells and canalter the outcomes of most of chemotherapy treatments4). Thus, we investigated novel

agents with cytotoxicity in hypoxia and nutrient-deprived conditions. We successfully

identified several candidate compounds that reduced cancer cell tolerance to nutrient
starvation. The candidate compounds include kigamicin D, pyrvinium pamoate,angelmarin, and arctigenin5). Several of these candidate compounds actually showed

antitumor activity in xenograft models. Based on these results, we conducted aninvestigator-initiated clinical trial using arctigenin-rich extracts from fruite of Arctinum

lappa Linne in patients with gemcitabine-refractory pancreatic cancer.

Arctigenin shows clear antitumor activity in various xenograft models. There is also an

additive or synergistic effect with anticancer drugs including gemcitabine, irinotecan, cis-

platinum, and nab-paclitaxel (our unpublished data). We hypothesized that arctigenin

might remodel the tumor microenvironment and improve blood supply because there was

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H. Esumi et al.

preferential arctigenin toxicity in poorly perfused hypoxic tissue. In order to examine thispossibility, we utilized perfusion analysis on MRI with Gadolinium-DTPA as a contrastingmaterial, bioluminescence analysis using a hypoxia-resposive luciferase construct
integrated tumor cells and pimonidazole staining. Microvasculature was analyzed byimmunohistochemical analysis using antibodies against CD31 and a-smooth muscle actin

and electron microscopic analysis.

When a human pancreatic cancer cell line xenograft using Suite-2 integrating 5X

HRE-

Luc was examined MRI perfusion with and without arctigenin administration for 14 days,

MRI signal in the tumor was markedly reduced in mice treated with arctigenin than incontrol mice. Furthermore, hypoxia imaging using HIF-driven luciferase illuminated on
IVIS showed remarkable decrease in hypoxia-induced luminescence as shown in Fig. 1.

These results clearly showed the increased tissue perfusion in tumor following arctigenin

treatment. Same results were obtained with xenograft model using human pancreaticcancer cell MiaPaca-2 integrating the same hypoxia-inducible luciferase construct.
Reduction of hypoxia in tumor tissue has a critically important impact on cancer cell

behavior including sensitivity to treatment we further examined the degree of hypoxia intumor tissue by pimonidazole staining. Pimonidazole was given to mice before sacrifice.

A representative example is shown in Fig. 2. The results clearly confirmed the reduction oftumor hypoxic region by arctigenin treatment. We also conducted morphological analyses

using CD31 staining and found that microvessel density in tumor in mice treated witharctigenin was almost same to that in control mice. However, pericyte coverage wasanalyzed by staining with a-sma antibody and analysis of co-localization of CD31 and a-sma showed pericyte coverage of tumor microvessel increased in tumors in mice treatedwith arctigenin than in control mice. Electron-microscopic analysis of tumor vasculature

revealed that arctigenin treatment result in less branching and more ordered and straight

network of vasculature. All these results clearly showed that arctigenin treatment

remodeled and normalized tumor vasculature. Present “vascular normalization” describedby bevacizumab treatment6) may explain the mechanisms for the combinational effects ofarctigenin and other anticancer drugs by improving drug delivery and sensitivity to drugs.

Arctigenin also shows preferential toxicity to the “cancer initiating cell population,”

which is similar to metformin and phenformin in vitro and in vivo (our unpublished data).

Cumulatively, our data suggest that arctigenin, and potentially antiausterity agents, could
be a novel class of anticancer treatments.

115

Targeting Metabolism: Clinical and Preclinical Challenge

150000116542

100000

62456

50000
24505
16854

0
#1

#2

#3

AG

#4
Cont

Figure 1

Suit2/5HRE-Luc cells,supplied by Harada, Kondoh, Hiraoka (2006) were transplanted into
nude mice and luciferase activity was measured by IVIS after 14 days treatment with arctigenin or control diets.

Figure 2

Pimonidazole staining of xenograft tissues of MiaPaca 2 cell containing 5X HRE-Luc construct
after 4-week treatment as indicated.

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H. Esumi et al.

References
1. Izuishi,K., Kato,K., Ogura, T., Kinoshita,T., Esumi, H. Remarkable Tolerance of Tumor
Cells to Nutrient Deprivation : Possible New Biochemical Target for Cancer Therapy.
Cancer Res 60:6201-6207, 2000
2. Esumi H., Izuishi K., Kato K., Hashimoto K., Kurashima Y., Kishimoto A., Ogura T.,
and Ozawa T. Hypoxia and Nitric Oxide Treatment Confer Tolerance to Glucose
Starvation in a 5’ -AMP-activated Protein Kinase-dependent Manner. J Biol Chem
36:32791-32798, 2002
3. Esumi H. "S'ils n'ont pas de pain, qu'ils mangent de la brioche." Focus on "anaerobic
respiration sustains mitochondrial membrane potential in a prolyl hydroxylase pathway-activated cancer cell line in a hypoxic microenvironment". Am J Physiol Cell
Physiol. 306;3211, 2014
4. Onozuka H, Tsuchihara K, Esumi H. Hypoglycemic/hypoxic condition in vitro mimicking the tumor microenvironment markedly reduced the efficacy of anticancer drugs.
Cancer Sci. 02:975-82. 2011
5. Awale S., Lu J., Kalauni SK, Kurashima Y., Tezuka Y., Kadota S. and Esumi H.
Identification of arctigenin as an anti-tumor agent having the ability to eliminate thetolerance of cancer cells to nutrient starvation. Cancer Res. 66:1751-1757, 2006
6. Jain RK. Normalization of tumor vasculature: an emerging concept in antiangiogenic
therapy. Science. 307:58-62, 2005

Hiroyasu Esumi, MD

1972-1975
1975-1983

1983-1988
1988-1992
1992-1993
2006
2007
2012-present

SurgeonResearch Associate, Biochemistry Division, National
Cancer Center (NCC)
Dermatology, Saitama Medical University
Chief, Biochemistry Division, NCC
Deputy Director, Research Institute, NCC
Director, Research Center for Innovative Oncology
Director, Hospitsl East, NCC
Professor, Tokyo University of Science

Specialty and Present Interest:
Biochemistry. Tumor Microenvironment and Cancer Cell Metabolism

117

INHIBITORS OF MUTATED ISOCITRATE DEHYDROGENASE
AS A POTENTIAL NEW THERAPY FOR AML:FROM THE BENCH TO THE CLINIC

David P. Schenkein

Agios Pharmaceuticals, Inc.
88 Sidney Street, Cambridge, MA 02139, USA(david.schenkein@agios.com)

Somatic mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) have been identified ina spectrum of solid and hematologic malignancies following their initial discovery inglioma1,2) and acute myeloid leukemia (AML)3). Our understanding of IDH1/2 mutation

prevalence continues to evolve with further genomic analysis of different tumors.

Currently, 68-74% of low grade gliomas and secondary glioblastomas, 40-52% ofchondrosarcomas, 11-24% of intrahepatic cholangiocarcinomas (IHCCs), 6-10% of AMLs,

and 3% of myelodysplastic/myeloproliferative neoplasms (MDS/MPNs) are thought tohave IDH1 mutations; 9-13% of AMLs, 3-6% of MDS/MPNs, and 2-6% of IHCCs arethought to have IDH2 mutations.

Wild-type IDH1 and IDH2 are metabolic enzymes found in the cytoplasm andmitochondria, respectively, which function as homodimers to convert isocitrate to alpha-ketoglutarate (a-KG) with the concomitant generation of NADPH. The recurrent

mutations identified in cancer all map to one of three key residues in the active site (R132 in

IDH1, and R172 and R140 in IDH2) (Figure 1). Since the cancer-associated mutant enzymes

are deficient in a-KG production, an initial hypothesis suggested that IDH1/2 were tumor
suppressors in which mutations led to a loss of function. However, in IDH1/2 mutant

cancers the wild-type allele is still expressed, and the heterozygous nature of thesemutations suggested a gain-of-function mechanism of oncogenesis. An Agios study

combining non-targeted metabolomics and structural biology identified this gain offunction, demonstrating that mutant IDH1 generates the oncometabolite, R-2hydroxyglutarate (2-HG), from a-KG and NADPH4). This neomorphic enzymatic activity isshared by mutant IDH25). 2-HG is usually produced at low levels (<300 mM) through errors

118

D. P. Schenkein

Mitochondrion

Cytoplasm
CitrateCitrateNAD+Isocitrate

IDH3
NADH
CO2

Figure 1

NADP+

IDH2

DKG

R132

Isocitrate

IDH1 ATT GGC AGG CAC GCC
NADP+

IDH2 ATA GGT CGT CAT GCT

IDH1

NADPH
CO2

DKG

R172

NADPH
CO2

Isocitrate dehydrogenase (IDH) 1/2 normal enzyme function, and site of mutations found incancerIncreased levels of 2-HG affect epigenetic control of the cell
Me

IDH

Isocitrate

Me

mIDH

Epigenetic“Re-Wiring”

2HG
CancerMe

D-KG
DIFFERENTIATION

Stem
Cell

Progenitor
BLOCKED

Inhibiting mutant IDH1 and IDH2 promotes cell maturation

IDH

Isocitrate

Epigenetic“Re-Wiring”
reversed

Inhibitor

mIDH

Normal cell death/
Homeostasis

D-KG

Figure 2

Stem
Cell

Progenitor

DIFFERENTIATION

Maturecell

IDH mutations lead to a block in normal cellular differentiation

Targeting Metabolism: Clinical and Preclinical Challenge

119

of metabolism, and is cleared by dehydrogenase housekeeping enzymes. The very highlevels of 2-HG (in the mM range) produced by mutant IDH1/2 overwhelm the clearance

mechanism, leading to the significant 2-HG accumulation seen in IDH1/2 mutant cells andtumor samples from patients with IDH1/2-mutant cancers4,5). 2-HG accumulation can also

be measured in patient blood samples6), and can be detected by imaging of inaccessible
solid tumors 7) , although further research is required to fully interpret its role as apharmacodynamic marker of mutant IDH1/2 activity.

IDH1/2 mutations are thought to initiate and drive oncogenesis in cooperation withother mutations, and there is significant evidence to support the role of 2-HG as afunctional oncometabolite. Due to its structural similarity to a-KG, 2-HG is a competitive

inhibitor of several a-KG-dependent histone and DNA demethylases, and high levels of 2HG thus result in DNA and histone hypermethylation8). These epigenetic changes lead to a

block in cellular differentiation. Multiple pre-clinical studies have demonstrated that

inhibition of mutant IDH1/2 reverses the hypermethylation and restores normal cellulardifferentiation9-11) (Figure 2).

Such promising pre-clinical data provided the rationale for the clinical development ofinhibitors of mutant IDH1/2. AG-221 and AG-120 (Agios) are first-in-class, oral, potent,

reversible, selective, small molecule inhibitors of the IDH2 and IDH1 mutant enzymes,

respectively. Both molecules can restore cellular differentiation, as demonstrated in IDH-

mutant patient AML samples ex vivo12) and in a mouse xenograft model13). In a primary

human IDH2-mutant AML xenograft mouse model, AG-221 treatment reduced 2-HG levels
and conferred a dose-dependent, statistically significant survival benefit versus vehicle
treatment14).

The clinical development of AG-221 and AG-120 encompasses both hematologic andsolid IDH-mutant malignancies. Approximately 6-10% of AMLs have IDH1 mutations,and 9-13% have IDH2 mutations. AML has a poor prognosis, with a 5-year survival rate of

26%15). The median age at diagnosis is 67 years15), consequently many patients cannot
tolerate standard of care chemotherapy and transplant treatment strategies. There havebeen few significant advances in AML treatment for decades, the notable exception being

for one particular AML subtype, acute promyelocytic leukemia (APL). Here, use of thedifferentiation agent, all-trans retinoic acid, in combination with arsenic trioxide has beeneffective in achieving high remission and overall survival rates, even without additionaluse of cytotoxic chemotherapy.

Phase 1/2 clinical trials for both AG-221 and AG-120 are ongoing in patients with IDH2

or IDH1 mutation-positive advanced hematologic malignancies, respectively(ClinicalTrials.gov NCT01915498 and NCT02074839). Both are single-arm, open-label, dose
escalation and expansion studies, in which either AG-221 or AG-120 is given orally as a

120

D. P. Schenkein

single agent. Along with safety endpoints, objective responses are assessed byinvestigators using International Working Group (IWG) AML and MDS criteria. In bothstudies patients enrolled are heavily pre-treated, and the majority have relapsed/refractory

AML. To date, both AG-221 and AG-120 have been well tolerated, and display favorable

pharmacokinetic/pharmacodynamic profiles; for both drugs, after multiple doses, plasma

2-HG levels were reduced from the elevated baseline levels by up to 98%[16, 17). Preliminary

overall response rates (including complete response [CR], partial response [PR], andplatelet and marrow responses) were ~30-40%. However, traditional IWG criteria may
overlook certain aspects of clinical benefit in patients who do not achieve CR. For example,

complete recovery of platelet and neutrophil counts is required to achieve a PR. In somepatients, the PRs appear to be as durable as complete remissions. Some patients with stabledisease are also demonstrating an improvement in neutrophil and/or platelet counts. Such

PRs may convert to CRs over time, or remain as the best response but still confer significant

clinical benefit for the patient. These observations might reflect the differentiation

mechanism of action of mutant IDH1/2 inhibitors, which is fundamentally different tocytotoxic chemotherapy, and may occur over a longer timeframe. Evidence fordifferentiation of cancer cells has been demonstrated in patients receiving AG-221 or AG120. Further data will be required to fully characterize the response patterns to, and clinicalbenefit of, targeted differentiation agents such as mutant IDH inhibitors. A phase 3, open-

label, randomized trial comparing AG-221 with the standard of care in IDH2-mutant

relapsed/refractory AML (IDHENTIFY, NCT02577406), and a phase 1, open-label trial of

AG-881 (an inhibitor of both mutant IDH1 and IDH2) in advanced IDH1 or IDH2-mutant

hematologic malignancies (NCT02492737), have recently opened. Further clinical studies

combining AG-221 or AG-120 with standard chemotherapy are also planned (Figure 3).

Relapsed

Newly Diagnosed (Untreated) IDH-mutant AML

2nd+ Relapse

Intensive

Non-Intensive

Phase 1
AG-221 Expansion

Phase 1
Induction (7+3)+
AG-221 or AG-120

Phase 1Æ 2
VIDAZA®
+
AG-221 or AG-120

Ongoing
PlannedPhase 1
AG-120 Expansion

Figure 3

Phase 3
IDHENTIFY
AG-221 vs SOC

IDH development program targets multiple lines of treatment from relapsed/refractory tofrontline AML

Targeting Metabolism: Clinical and Preclinical Challenge

121

In IDH-mutant low-grade glioma, secondary glioblastomas, IHCCs andchondrosarcomas, IDH1 mutations are more frequently observed than IDH2 mutations.

These tumors are currently difficult to treat and have low survival rates. A phase 1 dose

escalation and expansion trial of AG-120 in patients with advanced IDH1 mutant solid
malignancies, including glioma, chondrosarcoma and IHCC, is ongoing (NCT02073994),

and preliminary results suggest that treatment is well tolerated, with treatment durationsof up to 12 months18). Reductions in 2-HG levels were observed, and magnetic resonanceimaging/spectroscopy is being utilized to non-invasively measure 2-HG levels in gliomas,

along with tumor volume changes, pre- and during AG-120 therapy. A phase 1 study of

AG-881, which has improved blood-brain barrier penetration, in patients with IDH1 or
IDH2-mutant advanced solid tumors is also underway (NCT02481154). Results from thesestudies are awaited and will inform the continued development of mutant IDH inhibitors.

Christine Tomlins, PhD, of Excel Scientific Solutions, Horsham, UK, provided editorial

Acknowledgements:
support.

References
1. Parsons DW, Jones S, Zhang X, Lin JC, Leary RJ, Angenendt P, Mankoo P, Carter H, Siu
IM, Gallia GL, Olivi A, McLendon R, Rasheed BA, Keir S, Nikolskaya T, Nikolsky Y,
Busam DA, Tekleab H, Diaz LA, Jr., Hartigan J, Smith DR, Strausberg RL, Marie SK,
Shinjo SM, Yan H, Riggins GJ, Bigner DD, Karchin R, Papadopoulos N, Parmigiani G,
Vogelstein B, Velculescu VE, Kinzler KW. An integrated genomic analysis of humanglioblastoma multiforme. Science. 2008;321:1807-12.
2. Yan H, Parsons DW, Jin G, McLendon R, Rasheed BA, Yuan W, Kos I, Batinic-Haberle I,
Jones S, Riggins GJ, Friedman H, Friedman A, Reardon D, Herndon J, Kinzler KW,
Velculescu VE, Vogelstein B, Bigner DD. IDH1 and IDH2 mutations in gliomas. N Engl J
Med. 2009;360:765-73.
3. Mardis ER, Ding L, Dooling DJ, Larson DE, McLellan MD, Chen K, Koboldt DC, Fulton
RS, Delehaunty KD, McGrath SD, Fulton LA, Locke DP, Magrini VJ, Abbott RM,
Vickery TL, Reed JS, Robinson JS, Wylie T, Smith SM, Carmichael L, Eldred JM, Harris
CC, Walker J, Peck JB, Du F, Dukes AF, Sanderson GE, Brummett AM, Clark E,
McMichael JF, Meyer RJ, Schindler JK, Pohl CS, Wallis JW, Shi X, Lin L, Schmidt H,
Tang Y, Haipek C, Wiechert ME, Ivy JV, Kalicki J, Elliott G, Ries RE, Payton JE,
Westervelt P, Tomasson MH, Watson MA, Baty J, Heath S, Shannon WD, Nagarajan R,
Link DC, Walter MJ, Graubert TA, DiPersio JF, Wilson RK, Ley TJ. Recurring mutations
found by sequencing an acute myeloid leukemia genome. N Engl J Med. 2009;361:1058-66.

122

D. P. Schenkein

4. Dang L, White DW, Gross S, Bennett BD, Bittinger MA, Driggers EM, Fantin VR, Jang HG,
Jin S, Keenan MC, Marks KM, Prins RM, Ward PS, Yen KE, Liau LM, Rabinowitz JD,
Cantley LC, Thompson CB, Vander Heiden MG, Su SM. Cancer-associated IDH1 mutations produce 2-hydroxyglutarate. Nature. 2009;462:739-44.
5. Ward PS, Patel J, Wise DR, Abdel-Wahab O, Bennett BD, Coller HA, Cross JR, Fantin VR,
Hedvat CV, Perl AE, Rabinowitz JD, Carroll M, Su SM, Sharp KA, Levine RL,
Thompson CB. The common feature of leukemia-associated IDH1 and IDH2 mutations is
a neomorphic enzyme activity converting alpha-ketoglutarate to 2-hydroxyglutarate.
Cancer Cell. 2010;17:225-34.
6. Borger DR, Goyal L, Yau T, Poon RT, Ancukiewicz M, Deshpande V, Christiani DC,
Liebman HM, Yang H, Kim H, Yen K, Faris JE, Iafrate AJ, Kwak EL, Clark JW, Allen JN,
Blaszkowsky LS, Murphy JE, Saha SK, Hong TS, Wo JY, Ferrone CR, Tanabe KK,
Bardeesy N, Straley KS, Agresta S, Schenkein DP, Ellisen LW, Ryan DP, Zhu AX.
Circulating oncometabolite 2-hydroxyglutarate is a potential surrogate biomarker inpatients with isocitrate dehydrogenase-mutant intrahepatic cholangiocarcinoma. Clin
Cancer Res. 2014;20:1884-90.
7. Pope WB, Prins RM, Albert Thomas M, Nagarajan R, Yen KE, Bittinger MA, Salamon N,
Chou AP, Yong WH, Soto H, Wilson N, Driggers E, Jang HG, Su SM, Schenkein DP, Lai
A, Cloughesy TF, Kornblum HI, Wu H, Fantin VR, Liau LM. Non-invasive detection of 2hydroxyglutarate and other metabolites in IDH1 mutant glioma patients using magnetic
resonance spectroscopy. J Neurooncol. 2012;107:197-205.
8. Losman JA, Kaelin WG, Jr. What a difference a hydroxyl makes: mutant IDH, (R)-2hydroxyglutarate, and cancer. Genes Dev. 2013;27:836-52.
9. Kernytsky A, Wang F, Hansen E, Schalm S, Straley K, Gliser C, Yang H, Travins J,
Murray S, Dorsch M, Agresta S, Schenkein DP, Biller SA, Su SM, Liu W, Yen KE. IDH2
mutation-induced histone and DNA hypermethylation is progressively reversed bysmall-molecule inhibition. Blood. 2015;125:296-303.
10. Rohle D, Popovici-Muller J, Palaskas N, Turcan S, Grommes C, Campos C, Tsoi J, Clark O,
Oldrini B, Komisopoulou E, Kunii K, Pedraza A, Schalm S, Silverman L, Miller A,
Wang F, Yang H, Chen Y, Kernytsky A, Rosenblum MK, Liu W, Biller SA, Su SM,
Brennan CW, Chan TA, Graeber TG, Yen KE, Mellinghoff IK. An inhibitor of mutant
IDH1 delays growth and promotes differentiation of glioma cells. Science. 2013;340:626-30.
11. Wang F, Travins J, DeLaBarre B, Penard-Lacronique V, Schalm S, Hansen E, Straley K,
Kernytsky A, Liu W, Gliser C, Yang H, Gross S, Artin E, Saada V, Mylonas E, Quivoron C,
Popovici-Muller J, Saunders JO, Salituro FG, Yan S, Murray S, Wei W, Gao Y, Dang L,
Dorsch M, Agresta S, Schenkein DP, Biller SA, Su SM, de Botton S, Yen KE. Targeted inhibition of mutant IDH2 in leukemia cells induces cellular differentiation. Science.
2013;340:622-6.

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123

12. Hansen E, Quivoron C, Straley K, Lemieux R, Popovici-Muller J, Sadrzadeh H, Fathi A,
Gliser C, David M, Saada V, Micol J-B, Bernard O, Dorsch M, Yang H, Su SM, Agresta S,
De Botton S, Penard-Lacronique V, Yen KE. AG-120, an oral, selective, first-in-class,potent inhibitor of mutant IDH1, reduces intracellular 2HG and induces cellular differentiation in TF-1 R132H cells and primary human IDH1 mutant AML patient samples
treated ex vivo. 56th Annual American Society of Hematology Meeting. 2014;124(21):Abs 3734
13. Quivoron C, David M, Straley K, Travins J, Kim H, Chen Y, Zhu D, Saada V, Bawa O,
Opolon P, Polrot M, Micol J-B, Willekens C, Bernard O, Yang H, Agresta S, De Botton S,
Yen KE, Penard-Lacronique V. AG-221, an oral, selective, first-in-class, potent IDH2R140Q mutant inhibitor, induces differentiation in a xenotransplant model. 56th
Annual American Society of Hematology Meeting. 2014;124(21):Abs 3735
14. Yen KE, Wang F, Travins J, Chen Y, Yang H, Straley K, Choe S, Dorsch M, Schenkein DP,
Agresta S, Biller SA, Su SM. AG-221 offers a survival advantage in a primary human
IDH2 mutant AML xenograft model. 55th Annual American Society of Hematology
Meeting. 2013;122(21):Abs 240
15. National Cancer Institute. Surveillance, Epidemiology, and End Results Program. Cited
Dec 2015, from http://seer.cancer.gov/statfacts/html/amyl.html.
16. DiNardo C, Stein E, Altman J, Collins R, DeAngelo D, Fathi A, Flinn I, Frankel A,
Levine RL, Medeiros B, Patel M, Pollyea A, Roboz G, Stone R, Swords R, Tallman MS,
Almon C, Fan B, Goldwasser M, Yen KE, Attar E, De Botton S. AG-221, an oral, selective,
first-in-class, potent inhibitor of the IDH2 mutant enzyme, induced durable responses ina phase 1 study of IDH2-mutation positive advanced hematologic malignancies.
European Hematologic Association Annual Meeting. 2015;Abs P569
17. De Botton S, Pollyea A, Stein E, DiNardo C, Fathi A, Roboz G, Collins R, Swords R,
Flinn I, Altman J, Tallman MS, Kantarjian H, Colby K, Fan B, Goldwasser M, Prahl M,
Agresta S, Stone R. Clinical safety and activity of AG-120, a first-in-class, potent
inhibitor of the IDH1 mutant protein, in a phase 1 study of patients with advanced
IDH1-mutant hematologic malignancies. European Hematologic Association Annual
Meeting. 2015;Abs P563
18. Burris H, Mellinghoff IK, Maher E, Wen P, Beeram M, Touat M, Faris J, Azad N,
Cloughesy T, Gore L, Trent J, Von Hoff D, Goldwasser M, Fan B, Liu H, Agresta S. The
first reported results of AG-120, a first-in-class, potent inhibitor of the IDH1 mutant
protein, in a Phase I study of patients with advanced IDH1-mutant solid tumors,
including gliomas. AACR-NCI-EORTC Molecular Targets Conference. 2015;Abs PL04-05

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D. P. Schenkein

David P. Schenkein, MD

2006-20092009-present
2012-present

Senior Vice President of Clinical Hematology andOncology at Genentech, Inc.
CEO, Agios Pharmaceuticals, Inc.
Adjunct Attending Physician in Hematology at Tufts
Medical Center and a Member of the Board of
Directors for the Biotechnology Industry Organization

Specialty and Present Interest:
Drug Development, Hematologic Malignancies

125

LARGE-SCALE POOLED SHRNA SCREENS AND
THE DISCOVERY OF NOVEL CANCER TARGETS FORDRUG DISCOVERY

William R. Sellers

VP/Global Head of Oncology, Novartis Institutes for Biomedical Research
4th Floor, 250 Massachusetts Avenue, Cambridge, MA 02139, USA(william.sellers@novartis.com)

Cancer is driven by the accumulation of somatic genetic alterations that later the activityof tumor suppressor and oncogenes. Together these activities lead to the dysregulation ofcell proliferation, cell survival and metabolic pathways, among many other, that culminate

in the acquisition of the transformed phenotype characteristic of malignant tumors.

Advances in Next-Generation Sequencing and projects like the Cancer Genome Anatomy

project and the International Cancer Genome Consortium have given us a detailed pictureof the genetic alterations across a broad spectrum of cancer. This however, does not

immediately lead us to a detailed functional understanding of those genes whose protein

products are necessary for the maintenance of cancer viability. In order to more deeply

understand the genes and pathways critical to the survival of cancer cells we have taken on

the functional annotation of the Cancer Cell Line Encyclopedia (Nature 2012;483:603-7)

through the use of deep pooled shRNA libraries. The resulting effort termed Project

DRIVE (Deep RNAi-screening for Viablity Effects in cancer) has led to the generation ofdrop-out shRNA data for 7,500 genes targeted by 20 shRNAs per gene across 300 cancer

cell lines. The data from this effort are highly robust identifying all known oncogenes
mutated in the set of interrogated cell lines, along with multiple new cancer dependentfeatures not previously described. These data and several exemplary targets will be
discussed in further detail in this lecture.

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W. R. Sellers

William R. Sellers

1999-2004

2005-present

Assistant Professor of Medicine, Harvard Medical
School and Dana-Farber Cancer InstituteVice President and Global Head of Oncology, Novartis
Institutes for Biomedical ResearchSpecialty and Present Interest:
Cancer Drug Discovery

127

WHAT CAUSES ALTERED METABOLISM
IN COLON CANCER CELLS?

Tomoyoshi Soga

Institute for Advanced Biosciences, Keio University
246-2 Mizukami, Kakuganji, Tsuruoka, Yamagata 997-0052, Japan(soga@sfc.keio.ac.jp)

Metabolic reprogramming is a common hallmark of cancer. Most cancer cells produce

ATP and the precursors of biomass (e.g., nucleotides, amino acids and lipids) by shiftingfrom oxidative phosphorylation to aerobic glycolysis, commonly known as the “Warburg

effect”. Recently these cancer specific metabolic pathways have been considered as
attractive targets for cancer therapy. However, the mechanisms underlying the control ofcancer cell metabolism is poorly understood.

To address underlying mechanisms that induce metabolic reprogramming of cancer

metabolism, we applied capillary electrophoresis mass spectrometry (CE-MS) metabolic

profiling1-3) to paired normal tissues and tumor tissues obtained from 275 patients withcolon cancer. Significant changes in the levels of many metabolites were observed between
normal and tumor tissues. Unexpectedly metabolite levels altered quite early andremained unchanged among cancer stages. S-adenosylmethionine (SAM), was the mostincreased metabolite. Glucose was the second most decreased metabolites in tumor tissues,

whereas lactate, the final product of glycolysis, was increased, which indicates activation of
the glycolysis, termed the Warburg effect.

We then applied transcriptome analysis to paired normal-cancer colon tissues andrevealed that alterations in mRNA levels also occurred at the adenoma stage. Regarding

energy metabolism, expression of mRNAs encoding pentose phosphate pathway, someglycolysis and fatty acid synthesis were increased in tumor tissues. On the other hand,

expression of mRNAs encoding most of gluconeogenesis, fatty acid oxidation andtricarboxylic acid (TCA) cycle were reduced.

Colon cancer is associated with mutations in oncogenes and tumor suppressor genes

128

T. Sogasuch as adenomatous polyposis coli (APC), Kirsten-ras (KRAS) and TP53. We applied next

generation sequencing technologies for the detection of somatic mutations to the tumor

tissues and found mutation frequencies in APC of 78%, TP53 of 68% and KRAS of 48%.

Similarly to metabolome and transcriptome data, these mutations were umbiased amongcancer stages. We investigated whether APC, KRAS and TP53 mutations affected

metabolism. However, regardless of presence or absence of these mutations, the levels of

most of metabolites were largely-unaltered in tumor tissues.

Next, we investigated the structural properties of normal and tumor tissues obtained

from patients with various cancer stages using transmission electron microscopy and foundmitochondrial swelling, cristae disappearance and matrix clearout in tumor tissues. These

results indicate that mitochondrial abnormality may be associated with colon cancer

development as well as metabolic reprogramming of colon cancer metabolism.
References

1. Soga T, Ohashi Y, Ueno Y, Naraoka H, Tomita M, et al., Quantitative Metabolome
Analysis Using Capillary Electrophoresis Mass Spectrometry. J Proteome Res. 2: 488-494
2003.
2. Soga T, Baran R, Suematsu M, Ueno Y, Ikeda S, Sakurakawa T, et al., Differential
Metabolomics Reveals Ophthalmic Acid As An Oxidative Stress Biomarker Indicating
Hepatic Glutathione Consumption. J Biol Chem. 281: 16768-16776 2006.
3. Hirayama A, Kami K, Sugimoto M, Sugawara M, Toki N, Onozuka H, et al.,Quantitative Metabolome Profiling of Colon and Stomach Cancer Microenvironment
by Capillary Electrophoresis Time-of-flight Mass Spectrometry. Cancer Res. 69: 49184925 2009.

Tomoyoshi Soga, PhD

1984-1992
1993-2001
2001-2006

2003-20102006-present

2008-present
2010-present

Yokogawa Corp.
Yokogawa Analytical Systems Inc.
Associate Professor, Institute for Advanced
Biosciences, Keio Univ.
Founder & Director, Human Metabolome Technologies
Inc.
Professor, Institute for Advanced Biosciences Studies,
Keio Univ.
Professor, School of Medicine, Keio Univ.
Technical Advisory Board, Human Metabolome
Technologies Inc.

Specialty and Present Interest:
Metabolomics, Cancer Metabolism, Cancer Epigenetics

129

NUTRIENT SENSING AND METABOLIC REPROGRAMING
IN THE TUMOR STROMA

Jorge Moscat

Sanford Burnham Prebys Medical Discovery Institute10901 N. Torrey Pines Road, La Jolla, CA 92037, USA(jmoscat@sbpdiscovery.org)

Cancer therapeutic targets have been extensively and almost exclusively focused on

oncogenes and tumor suppressor genes, as well as on their mechanisms of action andregulation. An emerging alternative strategy involves the identification of proteins that

although not being encoded by oncogenes or tumor suppressors, however, are required toaccommodate cancer-driven nutrient and metabolic stresses.

It is becoming more apparent that the ability of cells to respond to changes in nutrient

availability is essential for an adequate control of metabolic homeostasis. In this regard,
mTORC1 is central to these processes. The autophagy substrate and signaling adaptor p62

is located in the lysosomes and participates in both the activation of mTORC1 in responseto amino acids and the shuttling of autophagy-targeted proteins to the autophagosome1).

We have shown that p62 binds Raptor and the Rag proteins, as well as TRAF6, which isalso required for mTORC1 translocation to the lysosome and its subsequent activation.
This involves the TRAF6-catalyzed K63 ubiquitination of mTOR that is critical for its

activation by amino acids. More recently we have identified new components of the p62interacting complex that translate nutrient sensing events into mTORC1 activation2). Morerecently, studies in our laboratory have identified a new kinase cascade critically involvedin the activation of the mTORC1 complex in response to the nutritional environment of thetumor cell, specifically the levels of amino acids3). That is, our new data demonstrate that

p62 is phosphorylated at two specific residues T269/S272 and that this is mediated via anovel cascade that includes MEK3/6 and p38d. This new pathway is orchestrated by the

PB1-containing kinase MEKK3 through a PB1-PB1 selective interaction that links MEKK3 to

p62 on the lysosome surface. This phosphorylation results in the recruitment of TRAF6 to130

J. Moscat

p62 on the lysosomes, which triggers the K63-mediated polyubiquitination. This event has

recently been shown to be important for the full activation of mTORC12). Importantly, we

have also shown that both MEKK3 and p38d, like p62, regulate autophagy throughmTORC1 and are required for the proliferation of various types of cancer cells in vitro andin vivo. Furthermore, genetic inactivation of MEKK3 or p38d mimics that of p62 in that it

leads to inhibited growth of PTEN-deficient prostate organoids. Of great relevance fromthe point of view of human disease, analysis of human prostate cancer samples showed

upregulation of these three components of the pathway (namely, p62, MEKK3, and p38d),

which correlated with enhanced mTORC1 activation in human cancer. In order todetermine the in vivo relevance of this pathway in an endogenous model of hepatocellular

carcinoma (HCC), in collaboration with Dr. Karin (UCSD) we have generated mice inwhich the tumor and mTORC1 suppressor, TSC1, was genetically inactivated inhepatocytes using an Alb-Cre mouse line. This was done in a wild-type background or inmice in which p62 has been knocked out specifically in the hepatocyte fraction.
Interestingly, whereas the inactivation of TSC1 in hepatocytes (that results is sustained

activation of mTORC1) results in HCC induction, the simultaneous inactivation of p62

severely abrogates tumorigenesis induced by TSC1 deficiency. Consistent with our model,

mTORC1 activation by TSC1 deficiency was totally inhibited in a p62-deficient

background. These results demonstrate that p62 is a novel and bona fide positive regulatorof mTORC1, and is critically involved in various types of cancer and in the control of

autophagy.

The role of the tumor stroma is also an important aspect of the carcinogenic process andits potential resistance to therapy. We have recently shown that at least one of the criticalmechanisms by which the microenvironment influences the tumor epithelium involves
p62-deficient stromal cells. That is, p62 levels are reduced in the stroma of several tumors

and its loss in stromal fibroblasts resulted in increased tumorigenesis of epithelial prostatecancer cells. The mechanism involves the regulation of cellular redox through an

mTORC1/c-Myc pathway of stromal amino acid metabolism, resulting in increased

stromal IL-6 production4). Our newest data establishes how tumor-derived signals promote

p62 downregulation in the stroma, as well as how the loss of p62 in that cell fraction
contributes to resistance to anti-tumor therapy. To firmly establish this notion, we havegenerated a mouse line in which p62 was selectively inactivated in fibroblasts by crossing

p62-floxed mice with FSP1-Cre mice. Surprisingly, these mice develop spontaneous

prostate tumors, which even reach the high-grade PIN phenotype. Therefore, p62, althougha tumor promoter in the epithelium, is a powerful tumor suppressor in the stroma throughits ability to regulate mTORC1 in both cellular compartments. These results raised thequestion of whether mTORC1 inhibitors efficacy could be improved if mTORC1 isselectively targeted in the epithelium without affecting the stroma.

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131

References
1. Duran, A., Amanchy, R., Linares, J. F., Joshi, J., Abu-Baker, S., Porollo, A., Hansen, M.,
Moscat, J., and Diaz-Meco, M. T. (2011) p62 Is a Key Regulator of Nutrient Sensing in themTORC1 Pathway. Mol Cell 44, 134-146
2. Linares, J. F., Duran, A., Yajima, T., Pasparakis, M., Moscat, J., and Diaz-Meco, M. T.
(2013) K63 polyubiquitination and activation of mTOR by the p62-TRAF6 complex innutrient-activated cells. Mol Cell 51, 283-296
3. Linares, J. F., Duran, A., Reina-Campos, M., Aza-Blanc, P., Campos, A., Moscat, J., andDiaz-Meco, M. T. (2015) Amino Acid Activation of mTORC1 by a PB1-Domain-Driven
Kinase Complex Cascade. Cell reports 12, 1339-1352
4. Valencia, T., Kim, J. Y., Abu-Baker, S., Moscat-Pardos, J., Ahn, C. S., Reina-Campos, M.,
Duran, A., Castilla, E. A., Metallo, C. M., Diaz-Meco, M. T., and Moscat, J. (2014)
Metabolic reprogramming of stromal fibroblasts through p62-mTORC1 signaling promotes inflammation and tumorigenesis. Cancer Cell 26, 121-135

Jorge Moscat, PhD

2008-20112011-present

2013-present

2015-present

Professor and Chair, Department of Cancer
and Cell Biology, University of Cincinnati
College of Medicine
Professor, Sanford-Burnham Medical
Research Institute (SBMRI)
Director, Cell Death and Survival Program,
NCI-Cancer Center, SBMRI
Associate Director of Basic Research,
NCI-Cancer Center, SBMRI

Specialty and Present Interest:
Nutrient Sensing and Cancer Metabolism in the Tumor Microenvironment

132

THE MICROBIOME AND MICROBIAL-DERIVEDMETABOLITES IN CARCINOGENESIS:THE FIBER-MICROBIOTA-BUTYRATE AXIS IN HISTONEACETYLATION AND
TUMOR SUPPRESSION AS A CASE STUDY
Scott J. Bultman

Department of Genetics and Lineberger Comprehensive Cancer Center
University of North Carolina
Chapel Hill, NC, 27599-7264, USA(Scott_Bultman@med.unc.edu)

It is highly controversial whether dietary fiber protects against colorectal cancer becauseof conflicting results from human epidemiologic studies. These studies have been
complicated by the participants’ genetic heterogeneity as well as differences in thecomposition of their gut microbiota and the utilization of different fiber sources. To

eliminate these confounding variables, we analyzed a gnotobiotic mouse model ofcolorectal cancer1). Our experiments investigated the function of butyrate because it is a
short-chain fatty acid produced by bacterial fermentation of fiber in the colon at high (mM)levels and has potent energetic and epigenetic properties in colonocytes2-4). Here, we reportthat fiber does have a strong tumor-suppressive effect but in a butyrate-dependent mannerthat is mediated through the microbiota. The incidence, number, size, and histopathologic

progression of AOM-induced colorectal tumors were significantly diminished in BALB/cmice provided a high-fiber diet only when they were colonized with defined microbiota

that included a butyrate-producing bacterium. The tumor-suppressive effect was

attenuated when mice were colonized with the same microbiota except with the wild-typebutyrate producer replaced by a strain harboring a 0.8-kb deletion in the butyryl-CoA
synthesis operon. To confirm that butyrate is a causal factor, the tumor-suppressive effect
was recapitulated in mice lacking any butyrate-producing bacteria when they were

provided a butyrate-fortified diet. Our data support a general mechanism that includes

bacterial fermentation rather than fiber exclusively bulking luminal contents and speedingcolonic transit to minimize exposure of colonocytes to ingested carcinogens. Our data also
support a molecular mechanism that is metaboloepigenetic. Due to the Warburg effect5),

cancerous colonocytes rely on glucose so butyrate was metabolized less and accumulated

Close Encounters of Metabolism, The Third Kind?

133

as an HDAC inhibitor that increased global histone acetylation levels, increased apoptosis,

and decreased cell proliferation. Fas and p16 are hyperacetylated and overexpressed target

genes that may account for these effects. To support the relevance of this model to humancancer, we demonstrate that butyrate accumulates in human colorectal adenocarcinomas,

and this is associated with higher global histone acetylation levels. These results link dietand microbiota to an abundant metabolite that influences epigenetics and cancer

predisposition.

References
1. Donohoe, D.R., Montgomery, S.A., Collins, L.B., Curry, K.P., Holley, D., Renner, S.W.,
Tillotson, C., Ryan, E.P., Godfrey, V., Han, A., Swenberg, J.A., Threadgill, D.S.,
Threadgill, D.W., and Bultman, S.J.. A gnotobiotic mouse model demonstrates that
dietary fiber protects against colorectal tumorigenesis in a microbiota- and butyratedependent manner. Cancer Discovery 4, 1387-1397 (2014).
2. Hamer, H. M. et al. The role of butyrate on colonic function. Alimentary Pharmacology &Therapeutics 27, 104-119 (2008).
3. Donohoe, D. R. et al. The microbiome and butyrate regulate energy metabolism andautophagy in the mammalian colon. Cell Metabolism 13, 517-526 (2011).
4. Donohoe, D. R. et al. The Warburg effect dictates the mechanism of butyrate-mediatedhistone acetylation and cell proliferation. Molecular Cell 48, 612-626 (2012).
5. Vander Heiden, M. G., Cantley, L. C. & Thompson, C. B. Understanding the Warburg
effect: the metabolic requirements of cell proliferation. Science 324, 1029-1033 (2009).

Scott J. Bultman, PhD

1995-2000
2000-2006

2006-2015

Postdoctoral Fellow, Case Western Reserve University
Research Assistant Professor, Department of Genetics,
University of North Carolina (UNC)
Assistant Professor, Department of Genetics, Lineberger
Comprehensive Cancer Center, UNC

Speciality and Present Interest:
Mouse Models of Disease, Epigenetics, Microbiome

134

BEYOND GLUCOSE AND GLUTAMINE: SOME CANCERCELLS UTILIZE EXTRACELLULAR MACROMOLECULES TO
FUEL CELL GROWTH

Wilhelm Palm1, Jiangbin Ye1, Bryan King1, Youngkyu Park2,
Kevin Wright2, Natalya N. Pavlova1, David A. Tuveson2 andCraig B. Thompson1

Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center
New York, NY 10065, USA
2
Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA(thompsonc@mskcc.org)

1

To grow and divide, cells require a continuous supply of nutrients that support energy

production and macromolecular synthesis1). Despite being surrounded by a diverse supplyof bioenergetic substrates, mammalian cells preferentially metabolize low molecular weightnutrients such as glucose and amino acids. However, proteins are the most abundantorganic constituents in body fluids and thus have the potential to function as importantalternative nutrients, if accessible to cells. Consistent with this idea, it was recently shownthat Ras-induced macropinocytosis could promote the uptake of extracellular proteins andlipids2-4).

Lysosomal degradation of extracellular macromolecules can sustain cell survival andinduce activation of the amino acid sensor, mechanistic target of rapamycin complex 1(mTORC1)5). However, catabolism of endocytosed proteins as an essential amino acidsource fails to elicit significant cell accumulation. Unlike its growth-promoting activity

under amino acid-replete conditions, we discovered that mTORC1 activation suppresses

proliferation when cells rely on extracellular proteins as an amino acid source (Figure 1).

Inhibiting mTORC1 results in increased catabolism of endocytosed proteins and enhancescell proliferation during nutrient-depleted conditions in vitro and within vascularlycompromised tumors in vivo. Thus, by preventing nutritional consumption of extracellular

proteins, mTORC1 couples growth to availability of free amino acids. These results may
have important implications for the use of mTOR inhibitors as therapeutics.

Besides mTORC1, mammalian cells possess a second amino acid-sensing kinase: generalcontrol non-derepressible 2 (GCN2). The combined effects of these two kinases allow cells
to adapt to varying levels of essential and non-essential amino acids, but how their

Concluding Keynote Lecture

Figure 1

Figure 2

135

136

C. B. Thompson et al.

activities are coordinated remains poorly understood. We recently found an important link

between GCN2 and mTORC1 signaling 6) . Upon deprivation of various amino acids,

activated GCN2 up-regulates ATF4 to induce expression of the stress response protein
Sestrin2. The upregulation of Sestrin2 is required to block lysosomal localization ofmTORC1 and therefore to inhibit its kinase activity (Figure 2). The GCN2/ATF4-dependent

induction of Sestrin2 occurs in response to deprivation of a variety of essential amino acids.

In addition, Sestrin2 induction is necessary for cell survival during glutamine deprivation,

indicating Sestrin2 is a critical effector of the GCN2-ATF4 pathway that regulates bothessential and nonessential amino acid homeostasis through mTORC1 suppression.
References
1. Thompson CB (2011). Rethinking the regulation of cellular metabolism. Cold Spring
Harb Symp Quant Biol 76, 23-29.
2. Commisso C, Davidson SM, Soydaner-Azeloglu RG, Parker SJ, Kamphorst JJ, Hackett S,
Grabocka E, Nofal M, Drebin JA, Thompson CB, Rabinowitz JD, Metallo CM, Vander
Heiden MG, Bar-Sagi D (2013). Macropinocytosis of protein is an amino acid supply
route in Ras-transformed cells. Nature 497: 633-637.
3. Kamphorst JJ, Cross JR, Fan J, de Stanchina E, Mathew R, White EP, Thompson CB,
Rabinowitz JD (2013). Hypoxic and Ras-transformed cells support growth by scavenging
unsaturated fatty acids from lysophospholipids. Proc Natl Acad Sci USA 110: 88828887.
4. Kamphorst JJ, Nofal M, Commisso C, Hackett SR, Lu W, Grabocka E, Vander Heiden MG,
Miller G, Drebin JA, Bar-Sagi D, Thompson CB, Rabinowitz JD (2015). Human pancreatic cancer tumors are nutrient poor and tumor cells actively scavenge extracellular protein.
Cancer Research 75: 544-553.
5. Palm W, Park Y, Wright K, Pavlova NN, Tuveson DA, Thompson CB (2015). The utilization of extracellular proteins as nutrients is suppressed by mTORC1. Cell. 162: 259-270.
6. Ye J, Palm W, Peng M, King B, Lindsten T, Li MO, Koumenis C, Thompson CB (2015).
GCN2 sustains mTORC1 suppression upon amino acid deprivation by inducing sestrin2. Genes Dev. 29:2331-6.

Concluding Keynote Lecture

Craig B. Thompson, MD

1999-2007
2006-20102010-present

137

Chair, Dept of Cancer Biology; Scientific Director,
Abramson Family Cancer Research Institute, Universityof Pennsylvania
Director, Abramson Cancer Center, University ofPennsylvania
Professor of Cell & Developmental Biology, Weill
Cornell Graduate School of Medical Sciences
Professor of Immunology & Microbial Pathogenesis,
Weill Cornell Graduate School of Medical Sciences
President and Chief Executive Officer of Memorial
Sloan Kettering Cancer Center (MSKCC)

Specialty and Present Interest:
The Regulation of Cellular Metabolism During Cell Growth/Differentiation and on theRole that Metabolic Changes Play in the Origin and Progression of Cancer

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CONCLUDING REMARKS

Hiroyasu Esumi

Research Institute for Biomedical Sciences, Tokyo University of Science2669 Yamazaki, Noda, Chiba 278-0022, Japan(hesumi@rs.tus.ac.jp)

The most remarkable characteristics of cancer is unregulated and disorganizedunlimited proliferation and tremendous progress in understanding of underlying
molecular mechanisms of this unlimited proliferation yielded many promising ways ofdiagnosis and treatment of cancer for last three decades. Molecular target therapy
accomplished remarkable achievement in several types of cancers but unexpectedly highdiversity of genetic mechanisms and unexpectedly early emergence of resistance to a

specific inhibitor are somehow frustrating. Diversity of cancer and emergence of resistancemight have a same root, plasticity and diversification occurs by intrinsic andmicroenvironment-induced mechanisms. Considering development of cancer treatment,
this plasticity of cancer is an extremely difficult obstacle. A longstanding well accepted

metabolic hallmark of cancer is Warburg effect. This metabolic characteristic has beenmostly cancer specific and can be applicable for wide range of cancers. Much attention has

been paid for cancer metabolism recently partly because widely applicable therapeutics

might be possible when based on cancer metabolism. Another reason is discoveries inmechanistic links between genetic alteration of cancer and metabolic processes.

Elucidation of link between p53 function and mitochondrial respiration and K-ras function

and glucose and glutamine metabolism are representative ones. In addition, discovery ingenetic alteration in genes coding for enzymes involved in TCA cycle and associatedmetabolism and discovery in the biological activity of the resultant metabolic intermediate

and endproducts, large amount of 2-hydroxyglutarate and fumarate were another turning
point.

The 46th symposium concentrated on the mechanisms of metabolic regulation of centralcarbon metabolism and amino acid metabolisms in particularly cancer specific context,

epigenetic modulation by metabolic intermediates, microenvironment metabolic

interactions, and diagnostic and therapeutic application of metabolism. Reactive oxygen,

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nitrogen and sulfur species are another interesting topics discussed in this symposium,
especially in connection to cancer cell biology. Through wide range of discussions andtopics on cancer metabolism, cancer metabolism turned out to be not so simple as Warburg

stated about 100 years ago. Oxygen supply to cancer tissue is quite limited. Therefore
tumor tissues are mostly facing to severe hypoxia that simulates anaerobic glycolysis as anenvironmental factor through HIF-1 and HIF-2 pathways, which is often activated bygenetic alteration in tumor cells or even by change in metabolism of cancer. But some
genetic alterations in cancer cells sometimes more directly activates anaerobic glycolysis byinactivating oxidative phosphorylation machinery such as cytochrome C oxidase by p53
loss of function mutations. However, as discussed in the present symposium, tocompensate loss of efficient ATP generation system oxidative phosphorylation, cancer cells

utilize many ATP generation systems other than anaerobic glycolysis as pointed out by

Warburg. Alteration in glutamine metabolism is a representative one but understanding of

the role of glutaminolysis is changing from previously proposed one, anaplerosis. Someinterlink between energy metabolism and nucleic acid precursor synthesis is critical forcontinuous cancer cells proliferation. Understanding of the importance of glycolysis inmaintaining cancer cells metabolism is also changing. Glycolysis is not simply importantfor ATP generation but is important for synthesis of precursor of purine and reducing

equivalent for regeneration of NADPH, which is important for lipid synthesis andrecycling reducing equivalent that is critically important for defense mechanism against

oxidative stress. Protection of cancer cells from oxy-stress through oxidation response
pathway, sulfur containing compounds and even sulfur containing gas are criticallyimportant for cancer cells survival, therefore could be targets for anticancer strategy.

Cancer specific metabolites are critically important not only for biomarker of diagnosis

but also for understanding cancer biology, which is directly related to cancer treatment. 2-hydroxyglutarate is the representative onco-metabolite which is generate in large amountby mutant isocitrate dehydrogenase I and II (IDH-I and II). The biological or pathologicalrole of 2-hydroxyglutarate is found to be inhibition of demethylase. Specific inhibitor

against mutant IDH I and II were invented and evaluated in preclinical and clinical studies.

However, efficacy of this inhibitor was not perfect and some proportion of the patients

respond well. If the role of the 2-hydroxyglutarate is modulation of epigenetic regulationin leukemogenesis or carcinogenesis by IDH-1 and 2 mutation, why the efficacy of theinhibitor is not 100%? Genetic and epigenetic background other than IDH mutation might
influence the outcome and microenvironment might be another determinant.

As illustrated in the present symposium, cancer metabolism is obviously a new clue tounderstand cancer but obviously we need more extensive study on cancer metabolisms inorder to understand cancer and to develop novel methods of treatment.