bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract A23: Differentiation of PI3K/Akt/mTOR inhibition in cancer models using dual dissociative TORC1/TORC2 (P529), single dissociative TORC1 (rapalogs) and catalytic inhibitors (PI3K/Akt, PI3K/mTOR)
Authors: Sherris David , Dennis Philip A. , Wilson Willie , Kawabata Shigeru , Yu Chunrong , Gravina Giovanni Luca , Mancini Andrea , Festuccia Claudio
Published: October 2015

P529 is a first-in-class, allosteric, small molecule, dual dissociative inhibitor of the TORC1 and TORC2 protein complexes within the PI3K/Akt/mTOR pathway. P529 has been previously shown to cause the rapid dissociation of the TORC1 and TORC2 complexes both in vitro and in vivo tumor xenograft models. Other PI3K inhibitors include rapalogs which cause the dissociation of the TORC1 complex and, PI3K/mTOR and PI3K/Akt inhibitors aimed at protein kinase catalytic site inhibition. Although these inhibitors have shown some utility in inhibiting cancer growth, they have deficiencies inherent to their mechanism of action. Rapalogs have been shown to stimulate the PI3K pathway through the induction of the IRS-1 negative feedback loop. Although mTOR resides in both TORC1 and TORC2, catalytic inhibition of mTOR does not dissociate both complexes allowing other proteins within the complexes to retain and exert signalling activity. Hence they are not true dual TORC1/TORC2 inhibitors, but only mTOR inhibitors. Here we show comparison of P529 with rapalogs and the catalytic inhibitors PI3K/mTOR and PI3K/Akt delineating activity in prostate, lung, glioblastoma and hepatic tumor evaluating cell survival, phosphoprotein PI3K signaling (IC50), CT scanning, tumor volume and growth arrest in cellular models and murine xenografts, showing superiority of allosteric dual TORC1/TORC2 dissociative inhibition. Furthermore, in addition to evaluation in cancer treatment models, data will be presented showing activity in cancer prevention using the mutant EGFR (L858R/T790M) transgene lung expression model.

Citation Format: David Sherris, Philip A. Dennis, Willie Wilson, III, Shigeru Kawabata, Chunrong Yu, Giovanni Luca Gravina, Andrea Mancini, Claudio Festuccia. Differentiation of PI3K/Akt/mTOR inhibition in cancer models using dual dissociative TORC1/TORC2 (P529), single dissociative TORC1 (rapalogs) and catalytic inhibitors (PI3K/Akt, PI3K/mTOR). [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr A23.
Abstract A24: Comprehensive PI3K pathway inhibition through combination of the PI3Kβ/δ inhibitor AZD8186 and the mTORC1/2 inhibitor AZD2014 drives tumor regression in vivo
Authors: Hancox Urs , Polanska Urszula , Hanson Lyndsay , Ellston Rebecca , Maynard Julia , Kraus Manfred , Curwen jon , Klinowska Teresa , Ward Lara , Cruzalegui Francisco , Green Stephen , Symeonides Stefan , Cronin Kathryn , Barry Simon
Published: October 2015

AZD8186 inhibits the PI3K isoforms PI3Kβ and δ In solid tumors when the tumor suppressor PTEN is deleted, mutated or downregulated PI3Kβ becomes a key driver of tumor cell growths. In hematological tumors such as DLBCL PTEN is down-regulated, moreover PI3Kδ is important in signaling from the B-cell receptor, creating potential for targeted treatment of hematological malignancies. AZD8186 has single agent activity in a range of pre-clinical models representative of different tumor types. However efficacy of agents in the PI3K pathway are anticipated to be limited by compensatory PI3K isoform activation or relief of feedback loops. As a result it is likely that maximal benefit will be seen when combining different agents that target the PI3K pathway. Unlike other inhibitors of the PI3K pathway AZD8186 does not affect peripheral glucose levels. However AZD8186 specifically modulates tumor FDG uptake in the PTEN null tumor model 786-0. This is associated with tumor specific modulation of the PI3K pathway biomarkers. Therefore in PTEN null tumors AZD8186 can be combined with other PI3K pathway inhibitors to give increased pathway suppression without increasing normal tissue toxicity. To test this AZD8186 has been combined with the mTORC1/2 inhibitor AZD2014 in a number of different PTEN null tumors. The combination increased reduction in tumor growth, and even induced tumor regression. This is associated with increase depth or duration of pathway suppression. Interestingly tumor regressions can be achieved with intermittent of both AZD8186 and AZD2014, implying that acute complete pathway suppression is sufficient to drive the anti-tumor effect. This data establishes the potential for AZD8186 to be used in combination with a mTOR inhibitors with the ability to customize dose and schedule to optimize both tolerability as well as anti-tumor effects. Further exploration of the combination opportunities for AZD8186 with other molecular targeted agents would inform on the potential for inhibitors of PI3Kβ and δ to give benefit in different tumor types.

Citation Format: Urs Hancox, Urszula Polanska, Lyndsay Hanson, Rebecca Ellston, Julia Maynard, Manfred Kraus, jon Curwen, Teresa Klinowska, Lara Ward, Francisco Cruzalegui, Stephen Green, Stefan Symeonides, Kathryn Cronin, Simon Barry. Comprehensive PI3K pathway inhibition through combination of the PI3Kβ/δ inhibitor AZD8186 and the mTORC1/2 inhibitor AZD2014 drives tumor regression in vivo. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr A24.
Abstract A42: Increased Akt3 expression as a resistance mechanism to targeted therapy
Authors: Chin Y Rebecca , Toker Alex
Published: October 2015

Resistance to targeted therapy poses a major challenge for successful treatment of cancers. Despite a number of drugs targeting the PI 3-K/Akt pathway are in clinical development, mechanisms of resistance to drugs targeting this pathway have not been studied extensively. To determine how breast cancer cells develop resistance to Akt inhibitors (Akti), we generated Akti-resistant lines using dose escalation regimens with pan-Akt inhibitors (ATP-competitive: GDC0068, GSK690693; Allosteric: MK2206). Interestingly, in multiple Akti-resistant breast cancer lines, we observed a robust increase in the protein expression of Akt3, but not Akt1 or Akt2. We were intrigued by this observation, as we have recently demonstrated that Akt3 has a predominant function in regulating growth of triple negative breast cancer cells in three-dimensional spheroids and in xenograft models. The increased expression of Akt3 in Akti-resistant cells occurs via a genetic event, since the levels of Akt3 remain elevated after 3 weeks of Akt inhibitor removal. Moreover, upon Akt inhibitor removal in resistant lines, cells remain resistant to Akt inhibition. The enhanced Akt3 expression is observed in both luminal and basal-like triple-negative breast cancer resistant lines. Importantly, knockdown of Akt3, but not Akt1 or Akt2, results in re-sensitization of resistant cells to Akti. Mechanistically, using anti-phosphotyrosine receptor antibody array (RTK array), we observed increased phosphorylation of several RTKs in resistant cells, including EGFR, Her2, and ROR1. Akt3 knockdown in resistant cells results in reduction of pEGFR, pHer3 and pHer2, suggesting that Akt3 plays a role in the regulation of multiple RTKs in resistant cells. Epithelial-mesenchymal transition (EMT) is implicated in therapeutic resistance of cancers. Interestingly, several of our resistant lines display EMT properties including downregulation of E-Cadherin, and upregulation of N-Cadherin as well as Vimentin. We propose that enhanced Akt3 expression is a general mechanism by which breast cancer cells acquire resistance to Akti targeted therapy. Current efforts are aimed at investigating the mechanistic basis for Akt3-mediated resistance, and demonstrating the physiological relevance of these findings in clinical samples as well as in mouse models. The role of Akt3 in regulating EMT during establishment of resistance will also be explored. These studies will provide insights into molecular mechanisms of therapeutic resistance to drugs targeting the PI 3-K/Akt pathway, as well as information for designing combination strategies and next generation therapy.

Citation Format: Y Rebecca Chin, Alex Toker. Increased Akt3 expression as a resistance mechanism to targeted therapy. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr A42.
Abstract A56: A dual PI3K-mTOR inhibitor induced cardiac hypertrophy in mice: Role of the insulin signaling
Authors: Onufrak Jillian , Yang Yongayo , Lee Juyong , Fuseler John , Sun Maoyun , Sasi Sharath , McDonald J. Tyson , Hlatky Lynn , Price Robert L. , Carrozza Joseph , Goukassian David , Borg Thomas K. , Morgan James P. , Yan Xinhua
Published: October 2015

Studies from us and others showed that mice with cardiomyocyte-specific overexpression of a dominant negative PI3Kp110α had smaller hearts but maintained normal cardiac function. Yet, these mice developed systolic heart failure in the presence of cardiac stress, such as chemotherapy drug doxorubicin and aortic stenosis. Currently, drugs targeting the PI3K pathway are being tested in clinical trials for cancer therapy. Therefore, it is important to assess the cardiac effects of pharmacological inhibition of this pathway. Here, we report that a dual PI3K-mTOR inhibitor, BEZ235 (BEZ), induced cardiac hypertrophy and subsequent heart failure in mice when it was used alone or in combination with doxorubicin. This effect of BEZ was associated with increased hepatic gluconeogenesis, hyperglycemia, hyperinsulinemia and increased activation of the IGFR/insulin receptor (IR) in the heart. In isolated cardiomyocyte culture, BEZ induced feedback activation of IGFR/IR, but this effect was negligible compared to the IGFR/IR activation in the presence of insulin. Injections of insulin in mice lowered blood glucose, improved glucose and pyruvate tolerance but further increased the activation of IGFR/IR in the heart and aggravated BEZ-induced cardiac dysfunction; whereas OSI-906, an IGFR/IR inhibitor, alleviated this cardiac dysfunction. In addition, low dose Metformin significantly prolonged survival and improved cardiac function in BEZ-treated mice. Taken together, these results identified hyperinsulinemia, a systematic effect of the drug, as one of the mechanisms of BEZ-induced cardiac hypertrophy, arguing animal studies are indispensable for evaluating drug toxicity. They also suggest that Metformin but not insulin should be considered for controlling hyperglycemia and preventing the cardiac side effects that may be caused by PI3K pathway inhibitors in clinic.

Citation Format: Jillian Onufrak, Yongayo Yang, Juyong Lee, John Fuseler, Maoyun Sun, Sharath Sasi, J. Tyson McDonald, Lynn Hlatky, Robert L. Price, Joseph Carrozza, David Goukassian, Thomas K. Borg, James P. Morgan, Xinhua Yan. A dual PI3K-mTOR inhibitor induced cardiac hypertrophy in mice: Role of the insulin signaling. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr A56.
Abstract B08: Kicking the addictions: Therapeutic strategies for NSCLC
Authors: Solitro Abigail R. , MacKeigan Jeffrey P.
Published: October 2015

The discovery of cancer cells' dependency on the self-cannibalization program autophagy has spawned the investigation of its efficacy as a therapeutic target. However, the use of lysomotropic agents to inhibit autophagy as single-agent therapeutics has proven less than optimal. Recent investigations into the design of sequential treatment strategies targeting pathways preferentially used by cancer cells have shown promise. This novel spin on personalized medicine seeks to exploit the kinetics and mechanisms by which cancer cells become dependent on proliferation and survival pathways. Oncogenic KRAS-driven non-small cell lung cancer (NSCLC) is known to depend on mTORC1-driven growth and autophagy for survival; such autophagy addiction is beneficial to cancer cells, as it eliminates the need for exogenous nutrient supplies. We aim to elucidate the mechanism and kinetics of autophagy addiction in NSCLC, in order to design sequential treatment strategies against this disease.

Citation Format: Abigail R. Solitro, Jeffrey P. MacKeigan. Kicking the addictions: Therapeutic strategies for NSCLC. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr B08.
Abstract B11: PI3-kinase inhibition forestalls the development of drug resistance in BRAFV600E/PTENNull melanoma
Authors: Deuker Marian M. , Durban Victoria Marsh , Phillips Wayne , McMahon Martin
Published: October 2015

The phosphatidylinositol 3′-kinase (PI3K) signaling pathway is frequently dysregulated in BRAF mutated melanomas and serves to promote the growth of tumors initiated by expression of BRAFV600E. The most prevalent mechanism of PI3K pathway activation occurs via silencing expression of the phosphatase and tensin homolog (PTEN) tumor suppressor, but mutational activation of PIK3CA, encoding the 110kDa catalytic subunit of PI3□-kinase-α, has also been observed at low frequency. However, the PI3K catalytic isoform dependence of BRAF mutated melanoma remains poorly understood, as does the differential isoform dependence imbued by discrete nodes of PI3K pathway dysregulation. To characterize the PI3K isoform dependence of BRAFV600E/PTENNull as well as BRAFV600E/PIK3CAH1047R melanoma, we administered a panel of isoform-selective PI3K inhibitors to cultured melanoma cells and to genetically engineered mouse (GEM) models of melanoma of both genotypes. These studies revealed that, while BRAFV600E/PIK3CAH1047R melanoma was sensitive to p110α-selective inhibition, inhibition of BRAFV600E/PTENNull melanoma cell proliferation required combined blockade of p110α (PIK3CA), p110δ (PIK3CD) and p110γ (PIK3CG). Moreover, in GEM models of BRAFV600E/PIK3CAH1047R or BRAFV600E/PTENNull melanoma, isoform-selective PI3K inhibition elicited only a cytostatic effect as monotherapy, but enhanced melanoma regression in response to BRAFV600E→MEK→ ERK pathway-targeted inhibition. Most interestingly, treatment with GDC0032, a p110β-sparing PI3K inhibitor, forestalled the onset of MEK inhibitor (GDC-973, cobimetinib) resistant disease in the BRAFV600E/PTENNull GEM model of melanoma. Together, these data suggest that PIK3CA mutational status may be used to select for melanoma patients that will respond to a p110α-selective inhibitor. By contrast, PTENNull melanoma patients will require a p110β sparing pan-class I PI3K inhibitor. Moreover, although monotherapy PI3K inhibition did not elicit dramatic tumor regression, blockade of this pathway may be a useful strategy to extend the duration of response to BRAFV600E pathway-targeted therapy.

Citation Format: Marian M. Deuker, Victoria Marsh Durban, Wayne Phillips, Martin McMahon. PI3-kinase inhibition forestalls the development of drug resistance in BRAFV600E/PTENNull melanoma. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr B11.
Abstract B13: Regulation of Akt activity, cell proliferation, and viability in ovarian cancer cells by calcium/calmodulin-dependent protein kinase kinase 2
Authors: Gocher Angela M. , Franke Thomas F. , Azabdaftari Gissou , Karacosta Loukia G. , Edelman Arthur M.
Published: October 2015

Calcium/Calmodulin-dependent protein kinase kinase 2 (CaMKK2) has been implicated in the regulation of G1/S phase cell cycle progression in prostate and breast cancer but has not been studied in ovarian cancer (OvCa). We report here the following. CaMKK2 is abundantly expressed in high grade serous papillary cystadenocarcinoma. Knockdown (KD) of CaMKK2 expression using RNAi in OVCAR-3 OvCa cells resulted in slowing of cell growth, decreased cyclin D1 protein and mRNA, decreased phosphorylation of the tumor suppressor protein Rb at Ser807/811 and decreased DNA synthesis, indicating an arrest at the G1/S interface. CaMKK2 KD decreased cell viability, increased PARP cleavage and increased Caspase-3/7 activity suggesting apoptosis induction by CaMKK2 loss. CaMKK2 KD decreased p-Akt at Thr308 to a similar extent (59.3%) as that of PDK1 KD (50.4%) in OVCAR-3 cells. Combined CaMKK2 and PDK1 KDs led to an additive (80.7%) decrease in p-Akt suggesting that CaMKK2 regulates Akt activating phosphorylation independently of the canonical-PDK1 pathway. Additive effects of combined PDK1/CaMKK2 KD on p-Akt Thr308 in SKOV-3 OvCa cells were also obtained. The ability of baculovirus expressed and purified Akt to phosphorylate a specific peptide substrate was enhanced at least 9-fold by purified CaMKK2, whereas kinase activity of Akt T308A/S473A was not increased by CaMKK2. Direct phosphorylation of baculovirus expressed Akt at Thr308 by CaMKK2 was confirmed by Western blotting using Akt phospho-specific antibodies. Altogether these data show that CaMKK2 regulates cell growth and viability in OvCa cells and directly phosphorylates Akt to promote its activity via a non-PDK1-dependent pathway. These data indicate that CaMKK2 may be a novel therapeutic target for OvCa in combination with a PI3K inhibitor.

Citation Format: Angela M. Gocher, Thomas F. Franke, Gissou Azabdaftari, Loukia G. Karacosta, Arthur M. Edelman. Regulation of Akt activity, cell proliferation, and viability in ovarian cancer cells by calcium/calmodulin-dependent protein kinase kinase 2. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr B13.
Abstract B20: Lysosomal interaction of Akt with Phafin2: A critical step in the induction of autophagy
Authors: Noguchi Masayuki , Suizu Futoshi , Lennikov-Matsuda Mami , Jyoti Bala , Ohba Yusuke , Hirata Noriyuki
Published: October 2015

Autophagy is an evolutionarily conserved mechanism for the gross disposal of intracellular proteins in mammalian cells and dysfunction in this pathway has been associated with human disease. Although the serine threonine kinase Akt is suggested to play a role in this process, little is known about the molecular mechanisms by which Akt induces autophagy. Using a yeast two-hybrid screen, Phafin2 (EAPF or PLEKHF2), a lysosomal protein with a unique structure of N-terminal PH (pleckstrin homology) domain and C-terminal FYVE (Fab 1, YOTB, Vac 1, and EEA1) domain was found to interact with Akt. A sucrose gradient fractionation experiment revealed that both Akt and Phafin2 co-existed in the same lysosome enriched fraction after autophagy induction. Confocal microscopic analysis and BiFC analysis demonstrated that both Akt and Phafin2 accumulate in the lysosome after induction of autophagy. BiFC analysis using PtdIns (3)P interaction defective mutant of Phafin2 demonstrated that lysosomal accumulation of the Akt-Phafin2 complex and subsequent induction of autophagy were lysosomal PtdIns (3)P dependent events. Furthermore, in murine macrophages, both Akt and Phafin2 were required for digestion of fluorescent bacteria and/or LPS-induced autophagy. Furthermore, overexpression of wild-type Phafin2, but not PtdIns (3)P interaction defective mutant Phafin2, suppressed oncogenic potency in Ras (or Myr-Akt, or TCL1)-induced colony transformation assays. Taken together, these findings establish that lysosomal accumulation of Akt and Phafin2 is a critical step in the induction of autophagy via an interaction with PtdIns (3)P.

Citation Format: Masayuki Noguchi, Futoshi Suizu, Mami Lennikov-Matsuda, Bala Jyoti, Yusuke Ohba, Noriyuki Hirata. Lysosomal interaction of Akt with Phafin2: A critical step in the induction of autophagy. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr B20.
Abstract B31: Targeting PI3K and RAS pathways in a novel preclinical model of prostate cancer
Authors: Pearson Helen B. , Humbert Patrick O. , Phillips Wayne A.
Published: October 2015

Oncogenic activation of the PI3K cascade increases cell proliferation, survival, and migration to facilitate tumor growth in many human cancers, including prostate cancer. Prostate cancer is the second major cause of cancer-related deaths in men globally, reflecting resistance to standard clinical regimens (i.e. androgen ablation, radiation, and chemotherapy). To better understand the role of the PI3K pathway in the initiation, progression and treatment of prostate cancer we have developed a novel mouse model of prostate cancer by expressing a single activating mutation in Pik3ca, the gene coding for the p110α catalytic subunit of PI3K, in the prostate.

To generate the model we have used a mouse with a conditional (Cre recombinase-inducible) knock-in of the common activating mutation, Pik3caH1047R, expressed from the endogenous locus and targeted the mutation to the prostate gland by crossing this mouse to mice expressing Cre under the control of prostate-specific promoter PB. Histological analysis of mice expressing Pik3caH1047R in their prostate revealed flat and cribriform hyperplastic lesions at 200 d, which progressed to locally invasive carcinoma at 400 d. This data demonstrates that a heterozygous activating mutation in Pik3ca is sufficient to cause invasive prostate cancer. Furthermore, we show that mice co-expressing heterozygous Pik3ca and KRas oncogenic mutations in the prostate display accelerated progression to invasive prostate carcinoma (100% incidence) compared to single mutants, demonstrating a synergistic relationship between these two oncogenic events.

We then used this model to perform a preclinical trial to assess the therapeutic efficacy of TORC1, panPI3K/mTORC1 and MEK inhibitors, either alone or in combination, in prostate cancer. Our results show that the combination of MEK and PI3K/mTOR inhibitors has superior efficacy compared to all other combinations or single agents assessed and with no sign of toxicity.

Our data provide strong evidence that simultaneously targeting both the PI3K and RAS pathways in advanced prostate cancer patients may prove to hold enhanced therapeutic outcome.

Citation Format: Helen B. Pearson, Patrick O. Humbert, Wayne A. Phillips. Targeting PI3K and RAS pathways in a novel preclinical model of prostate cancer. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr B31.
Abstract B38: p90RSK mediates resistance to PI3K-pathway inhibitors in breast cancer
Authors: Eichhorn Pieter J.A. , Serra Violeta , Garcia-Garcia Celina , Ibrahim Yasir H. , Prudkin Lumilla , Parra Josep-Lluis , Prat Aleix , Hahn William C. , Kim So Young , Baselga Jose
Published: October 2015

The PI3K signaling pathway regulates diverse cellular processes including proliferation, survival and metabolism and is aberrantly activated in human cancer. As such, numerous compounds targeting the PI3K pathway are currently being clinically evaluated for the treatment of cancer and several have shown some early indications of efficacy in breast cancer. However, resistance against these agents, both de novo and acquired, may ultimately limit the efficacy of these compounds. Here, we have taken a systematic functional approach to uncover potential mechanisms of resistance to PI3K inhibitors and have identified several genes whose expression promotes survival under conditions of PI3K/mTOR blockade, including the ribosomal S6 kinases RPS6KA2 (RSK3) and RPS6KA6 (RSK4). We demonstrate that overexpression of RSK3 or RSK4 supports proliferation upon PI3K inhibition both in vitro and in vivo, in part through the attenuation of apoptotic response and upregulation of protein translation. Notably, the addition of MEK- or RSK-specific inhibitors can overcome these resistance phenotypes, both in breast cancer cell lines and patient derived xenograft models with elevated levels of RSK activity. These observations provide a strong rationale for the combined use of RSK and PI3K pathway inhibitors to elicit favorable responses in breast cancer patients with activated RSK.

Citation Format: Pieter J.A. Eichhorn, Violeta Serra, Celina Garcia-Garcia, Yasir H. Ibrahim, Lumilla Prudkin, Josep-Lluis Parra, Aleix Prat, William C. Hahn, So Young Kim, Jose Baselga. p90RSK mediates resistance to PI3K-pathway inhibitors in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr B38.
Abstract IA02: G-proteins regulating PI3Ks and PI4KIIIβ regulating a G-protein
Authors: Burke John E. , Inglis Alison J. , Vadas Oscar , Perisic Olga , Masson Glenn R. , McLaughlin Stephen H. , Williams Roger L.
Published: October 2015

Dysregulation of phosphoinositide levels is associated with many cancer phenotypes. A variety of mechanisms activate phosphoinositide 3-kinases (PI3Ks), including association with G-proteins. Increased activity of PI 4-kinases (PI4Ks) also has been associated with cancer phenotypes. Although PI4Ks associate with G proteins, the relationship with enzyme activity is more complex. To understand regulation of these enzymes, we have used hydrogen/deuterium exchange mass spectrometry (HDX-MS) in conjunction with X-ray crystallography. The method enables us to explore not only three-dimensional structures but also the dynamics of the activation process and to map key regulatory interactions with G-proteins.

PI3Ks and PI4Ks, along with PI3K-related protein kinases such as TOR are members of a closely related family that all have a common core structure, consisting of a helical domain closely associated with a kinase domain. The helical domains have important roles both in stabilizing the structures and forming regulatory interactions. For example, PI3Kγ is activated by associating with Gβγ heterodimers released upon activation of GPCRs, and HDX-MS shows how the helical domain of the enzyme interacts with the p101 regulatory subunit and with Gβγ heterodimers. When the p110γ/p101 complex interacts with lipid membranes a conformational change takes place that prepares the helical domain for association with prenylated Gβγ heterodimers on membranes.

Type IIIβ PI4K (PI4KB) synthesizes PI4P on Golgi membranes. The enzyme is important for processes that require rapid expansion and remodeling of PI4P-containing membranes, including replication of a range of viruses. Its activity has also been associated with decreased cell-cell adhesion in invasive cancer cell lines. Using both HDX-MS and X-ray crystallography we have shown how the helical domain of PI4KB interacts with the Ras superfamily G protein Rab11. In this unique interaction, PI4KB grasps the Rab11 but leaves the switch regions of the G protein unencumbered and free to interact with Rab11 effectors. Consistent with this type of interaction, PI4K can interact with complexes of Rab11 bound to both GDP and GTP. Furthermore, the structure of the complex of the Rab11 effector FIP3 bound to the Rab11-PI4KB complex shows that PI4KB does not perturb Rab11 effector binding and suggests that PI4KB may be able to recruit both Rab11 and its downstream effectors to Golgi membranes.

Citation Format: John E. Burke, Alison J. Inglis, Oscar Vadas, Olga Perisic, Glenn R. Masson, Stephen H. McLaughlin, Roger L. Williams. G-proteins regulating PI3Ks and PI4KIIIβ regulating a G-protein. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr IA02.
Abstract IA10: Nuclear PTEN in regulation of genome integrity
Authors: Stambolic Vuk
Published: October 2015

Loss of PTEN function has been implicated in the etiology of numerous human malignancies, including sporadic brain, prostate, thyroid, breast, uterine and kidney tumors. PTEN germline have also been found in patients with PHTS, an autosomal-dominant cancer predisposition syndrome. Despite a thorough understanding of the inhibitory role for PTEN in the PI3K pathway, acting as a 3′PI phosphatase and directly antagonizing PI3'K, relatively little is known about PTEN's functions in the nucleus, a cellular compartment where it also resides. Our recent work has uncovered a nuclear function of PTEN in tumor suppression, which is independent from its 3′PI-phosphatase activity, but relies on PTEN protein phosphatase activity. Nuclear PTEN, which is SUMOylated, plays a prominent role in homologous recombination-based DNA repair, acting downstream of the protein kinase ATM. PTEN-deficient cells and tumors are impaired in their response to DNA damage and display heightened sensitivity to genotoxic agents, which may have significant implications for the treatment of PTEN-deficient cancers.

Citation Format: Vuk Stambolic. Nuclear PTEN in regulation of genome integrity. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr IA10.
Abstract IA11: Targeting PI3K-mTOR signaling in glioblastoma: A central role for mTORC2 in drug resistance and metabolic reprogramming
Authors: Mischel Paul
Published: October 2015

Metabolic reprogramming is a central hallmark of cancer, enabling tumor cells to obtain the macromolecular precursors and energy needed for rapid tumor growth. mTOR plays a central role in this process. Understanding how mTOR coordinates metabolic reprogramming may yield new insights into tumor pathogenesis, and provide a landscape of promising drug targets, while yielding important clues into mechanisms of resistance to the signal transduction inhibitors currently in use. This talk will focus on the emerging central regulatory role for mTOR complex 2 (mTORC2) in cancer metabolic reprogramming and targeted therapy resistance highlighting a recently identified, novel, Akt-independent, c-Myc-dependent pathway.

Citation Format: Paul Mischel. Targeting PI3K-mTOR signaling in glioblastoma: A central role for mTORC2 in drug resistance and metabolic reprogramming. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr IA11.
Abstract IA17: The therapeutic potential of PI(3) inhibition in lymphoma
Authors: Staudt Louis M.
Published: October 2015

The PI(3) kinase is engaged by multiple genetic, epigenetic and signaling abnormalities that are unique to particular molecular subtypes of lymphoma. Central to our understanding of lymphomas is signaling from the B cell receptor (BCR), which can occur by different mechanisms in each lymphoma subtype. A natural consequence of BCR signaling is engagement, which activates the SYK tyrosine kinase and consequently PI(3) kinase. The ABC subtype of diffuse large B cell lymphoma has chronic active BCR signaling in which PI(3) kinase is engaged along with NF-κB, both of which promote the survival of the malignant cells. In Burkitt lymphoma, a different mode of BCR signaling, known as tonic signaling, is responsible for cell survival and this is entirely mediated by the PI(3) kinase, with little or no involvement of NF-κB. A small subset of lymphomas belonging to the GCB subtype of DLBCL also appears to utilize tonic BCR signaling for survival. However, over half of GCB DLBCL tumors activate PI(3) kinase signaling due to genetic and epigenetic mechanisms, including PTEN inactivation and amplification of the mIR-17-92 cluster of microRNAs, which negatively regulate PTEN. Normal and malignant B cells express the PI(3) kinase delta catalytic subunit, which is not expressed highly outside of the lymphoid lineage. Selective PI(3) kinase delta inhibition with idelalisib has produced objective responses in several lymphoma malignancies. An open question, however, is whether other PI(3) kinase isoforms also contribute to signaling in lymphoma. Successful treatment of lymphomas with PI(3) kinase inhibitors will require drug combinations that inactivate parallel survival mechanisms. For example, in ABC DLBCL, PI(3) kinase inhibition synergizes strongly with inhibition of NF-κB using the BTK inhibitor ibrutinib.

Citation Format: Louis M. Staudt. The therapeutic potential of PI(3) inhibition in lymphoma. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr IA17.
Abstract IA22: Targeting mTOR for breast cancer therapy
Authors: Meric-Bernstam Funda
Published: October 2015

The Phosphatidylinositol 3-kinase (PI3K)/Akt/ mammalian target of rapamycin (mTOR) pathway plays a central role in cell metabolism, growth, proliferation, and survival. Activation of PI3K/Akt/mTOR signaling contributes to the oncogenesis of many tumor types, and thus the pathway is being actively pursued as a promising therapeutic target. mTOR, an important component of this pathway, exists in two multiprotein complexes: mTOR complex 1 and mTOR complex 2 (mTORC1 and mTORC2). mTORC1 controls cell growth, survival, and protein translation by regulating the phosphorylation of two major substrates: S6 kinase (S6K) and 4E-BP1. mTORC2 phosphorylates Akt at Serine 473 (S473), enhancing the catalytic activity of Akt, that has already been phosphorylated at Threonine 308 (T308). Thus, the mTOR complexes play an important role both upstream and downstream of Akt. Rapamycin and its analogs are allosteric mTOR inhibitors that bind FKBP12 and mTOR, and predominantly inhibit mTORC1. The rapamycin analog temsirolimus is approved by the Food and Drug Administration for the treatment of advanced renal cell carcinoma and the rapamycin analog everolimus is FDA approved for the treatment of pancreatic neuroendocrine tumors, renal cell carcinoma, sub-ependymal giant cell astrocytoma associated with tuberous sclerosis, as single agent therapy, and for the treatment of hormone-receptor positive breast cancer as combination therapy with exemestane. In spite of proven clinical benefit in breast cancer, objective responses remain rare. Thus there remains a need to identify predictors of response, optimal strategies for mTOR inhibition and rational combination therapies. There is emerging data on predictors of response to rapalog. Although preclinical studies suggest PIK3CA and PTEN alterations may confer sensitivity to rapalogs, the predictive value of these markers in the clinic has been controversial. Rare alterations such as mTOR activating mutations and TSC1 have been reported to confer sensitivity, while mTOR mutations in FKBP domain may confer rapalog resistance. The optimal strategy to target mTOR signaling is unknown. Preclinically, mTOR catalytic site inhibitors are more effective in cell lines with intrinsic or acquired rapamycin-resistance. The relative efficacy of other PI3K/Akt/mTOR inhibitors in breast cancer compared to rapalogs is not yet understood. Combinations with chemotherapy and other targeted agents are ongoing.

Citation Format: Funda Meric-Bernstam. Targeting mTOR for breast cancer therapy. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr IA22.
Abstract PR03: The Hippo pathway effector YAP1 contributes to escape from proliferation arrest under chronic PI3K/mTOR inhibition
Authors: Muranen Taru , Selfors Laura M. , Thoreen Carson C. , Gallegos Lisa L. , Coloff Jonathan L. , Mills Gordon B. , Brugge Joan S.
Published: October 2015

The PI3K/mTOR pathway is one of the most frequently mutated pathways in cancer. This pathway controls cell proliferation and cell survival, and represents an attractive target for cancer intervention. In animal tumor models, chronic treatment with PI3K/mTOR inhibitors initially induces tumor cytostasis. However, over time, tumor cells escape from drug-mediated proliferative suppression, resulting in relapse and tumor growth even in the presence of continued PI3K/mTOR inhibition. In this study we investigated the underlying molecular mechanisms that allow outgrowth of drug resistant tumor cells (“PI3K escapers”) after long-term treatment of tumor cell spheroids with PI3K/mTOR inhibitors. We identified several signaling pathways that were distinguishably altered in tumor cells that escaped proliferative suppression after chronic drug treatment compared to tumor cells acutely treated with inhibitor. Both MYC, which has been previously implicated in resistance to PI3K/mTOR inhibitors, and the Hippo pathway effector, YAP, were significantly upregulated in the “PI3K escapers” as well as multiple tumor cells lines that are resistant to PI3K/mTOR inhibition. Downregulation of either MYC or YAP abrogated the drug resistance. Although over-expression of MYC alone was sufficient to induce drug resistance, YAP was required for the MYC-induced resistant state, as knock-down of endogenous YAP resulted in loss of MYC-mediated resistance. Interestingly this resistance phenotype was associated with a KRAS or BRAF mutation, suggesting that MYC and YAP confer resistance in tumor cells in the context of ERK pathway activation. These data suggest that escape from proliferation suppression after chronic treatment with PI3K/mTOR in the context of activation of the KRAS pathway can be mediated by upregulation of MYC and YAP, which coordinately confer resistance.

This abstract is also being presented as Poster B41.

Citation Format: Taru Muranen, Laura M. Selfors, Carson C. Thoreen, Lisa L. Gallegos, Jonathan L. Coloff, Gordon B. Mills, Joan S. Brugge. The Hippo pathway effector YAP1 contributes to escape from proliferation arrest under chronic PI3K/mTOR inhibition. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr PR03.
Abstract A16: Molecular tracking of metformin chemoprevention in a 300-person ALLIANCE randomized placebo controlled trial in women at high risk for triple-negative breast cancer
Authors: Seewaldt Victoria , Marshall James
Published: October 2015

Background: Metformin (1,1-dimethylbiguanide hydrochloride) is a well-tolerated biguanide currently undergoing testing for secondary breast cancer prevention in women with prior breast cancer. Recently published population studies suggest that metformin decreases the incidence of cancer and cancer-related mortality in diabetic patients. While metformin is one of the most extensively studied drugs in vitro and in vivo, many pre-clinical studies use concentrations of metformin that are >1,000 times what is clinically achievable in humans and would be rapidly fatal if achieved. As a result, the molecular action of metformin in the breast tissue of women at high-risk who take metformin chemoprevention is poorly understood.

Methods/Results: Here we use combined proteomic profiling, breast tissue cytokine analysis, and in vivo analysis of live mammary epithelial cells for mitochondrial membrane and glucose uptake to track the molecular action of metformin chemoprevention in overweight and obese women at high-risk for triple-negative breast cancer. Women are enrolled in a 300 person ALLIANCE randomized, placebo controlled chemoprevention trial. Our studies to date have shown that overweight and obese women have activation of Akt/mTOR and IL6 /Stat3 epithelial activation, epithelial glucose uptake and increased mitochondrial membrane potential, and increased mammary tissue IL6/insulin.

Conclusion: These studies show our ability to directly track the molecular response to metformin chemoprevention.

Note: This abstract was not presented at the conference.

Citation Format: Victoria Seewaldt, James Marshall. Molecular tracking of metformin chemoprevention in a 300-person ALLIANCE randomized placebo controlled trial in women at high risk for triple-negative breast cancer. [abstract]. In: Proceedings of the Thirteenth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2014 Sep 27-Oct 1; New Orleans, LA. Philadelphia (PA): AACR; Can Prev Res 2015;8(10 Suppl): Abstract nr A16.
Abstract A54: Possible targets of metformin in colorectal cancer
Authors: Sehdev Amikar , Khramtsova Galina , Joseph Nora , Polite Blase P. , Bissonnette Marc B. , Olufunmilayo Olopade I.
Published: October 2015

Background: Growing evidence suggests metformin improves survival in diabetic colorectal cancer (CRC) patients. However, the mechanism of action remains unclear. In order to explore the mechanistic relationship between metformin and colon cancer, we examined the expression levels of several proteins implicated in metformin's action.

Methods: We collected clinical-pathological and outcomes data on all CRC patients treated at the University of Chicago (UC) from 2006-2010. The data was collected through UC cancer registry and chart review. Diabetic CRC patients and matched controls (matched on race and stage) were selected for immunostaining (IHC). Tissues blocks (FFPE) were retrieved and tissue microarray (TMA) were constructed. IHC was performed for pAMPK, pmTOR, p70S6K, 4E-BP1, pAKT, IGF1R, beta-Catenin and Ki-67. Total score was calculated from the intensity and percentage scoring. The results were analyzed in two groups: diabetic CRC patients on metformin (group 1) and diabetic or non-diabetic CRC patients not on metformin (group 2). Chi-square test was done for estimating statistical significance between groups keeping a level of significance at 10% (alpha < 0.10).

Results: The mean age of CRC patients was 62.7 years, 49% were males. Out of a total of 700 patients, 79 (11.21%) were diabetic. Metformin was prescribed to 40.5% of diabetic patients. Only 52 diabetics have FFPE block available so TMA was constructed with 104 patients in total (52 cases and 52 controls). Immunostaining was analyzable for a total of 84 patients. There were 18 and 66 patients in group 1 and 2, respectively. We found a lower expression of Ki-67 in the metformin group (group 1; 39%) as compared to non-metformin group (group 2; 64%) which was statistically significant (p = 0.04). IGF1R and beta-Catenin showed a higher expression in non-metformin group (89% and 95%, respectively) as compared to metformin group (72% and 83%, respectively) which was close to significance (p=0.12 and p=0.13, respectively). There was no significant difference between the expression of pAMPK, pmTOR, p70S6K, 4E-BP1 and pAKT in the two groups.

Conclusions: Metformin appears to improve the survival of CRC patients by decreasing proliferation. The results need to be validated in a larger study.

Citation Format: Amikar Sehdev, Galina Khramtsova, Nora Joseph, Blase P. Polite, Marc B. Bissonnette, Olopade I. Olufunmilayo. Possible targets of metformin in colorectal cancer. [abstract]. In: Proceedings of the Thirteenth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2014 Sep 27-Oct 1; New Orleans, LA. Philadelphia (PA): AACR; Can Prev Res 2015;8(10 Suppl): Abstract nr A54.
Abstract B21: The effect of mutant Kras associated changes in redox signaling in pancreatic neoplasia.
Authors: Schultz Michelle A. , Shah Rital , DeCant Brian , Mascarinas Windel E. , Bentrem David , Grippo Paul
Published: October 2015

Recent studies have suggested that mutant Kras associated activation of the Nrf2 pathway may have a significant role in pancreatic cancer (PaCa) tumorigenesis. Nrf2 regulates a battery of antioxidant genes, including peroxiredoxin-1 (Prdx-1), that are responsible for maintaining redox homeostasis in the cell. While ROS detoxification may have significant roles in cancer, several non-classical mechanisms of redox signaling associated with protein oxidation and phosphorylation are also important in tumorigenesis. Prdx-1 can regulate transcription as a cofactor in multiple oxidation states, represses tumorigenesis in its reduced state, and can act as a nuclear chaperone in elevated states of oxidation and when phosphorylated. We are therefore investigating the role of Prdx-1 and its oxidation states in oncogenic signaling pathways in mutant Kras models of pancreatic tumorigenesis and neoplasia. Our data suggests that throughout tumor progression, Prdx-1 is differentially regulated and that its localization changes. In vitro, neoplastic and tumorigenic pancreatic cell lines have more total prdx-1 expression and oxidized Prdx-1 expression than non-tumorigenic cell lines. Moreover, cells that have mutant Kras were associated with higher levels of protein oxidation than those with wildtype Kras expression. Interestingly, mutant Kras was also associated with differences in pERK interaction with Prdx-1, which was also differentially regulated by NOX inhibitors and fatty acids that control pancreatic neoplasia. In addition, AKT, ERK , and FOXO3a phosphorylation were also differntially modified by DPI treatment and fatty acid treatment in wildtype and mutant Kras normal, neoplastic, and tumorigenic cell lines. We therefore conclude that mutant Kras is associated with elevated levels of Prdx-1 oxidation and that Kras mediated activation of ERK could be associated with changes in Prdx-1 signaling in pancreatic neoplasia.

Citation Format: Michelle A. Schultz, Rital Shah, Brian DeCant, Windel E. Mascarinas, David Bentrem, Paul Grippo. The effect of mutant Kras associated changes in redox signaling in pancreatic neoplasia.. [abstract]. In: Proceedings of the Thirteenth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2014 Sep 27-Oct 1; New Orleans, LA. Philadelphia (PA): AACR; Can Prev Res 2015;8(10 Suppl): Abstract nr B21.