bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4540: Development of a biochemical High Throughput Screening (HTS) assay for chemical inhibitors of MALT1, a target for lymphoma therapeutics.
Authors: Zhai Dayong , Shu Chih-Wen , Diaz Paul , Reed John C.
Published: August 2014

MALT1 plays critical role in antigen receptor-mediated lymphocyte activation and lymphomagenesis. MALT1 acts both as a scaffold protein to recruit upstream signal transducers and as an intracellular cysteine protease with proteolytic activity that cleaves substrate proteins involved in NF-kB induction. Dysregulation of MALT1 activity occurs in lymphoma as a result of chromosomal translocations that fuse the catalytic domain of MALT1 with portions of BirC3 (c-IAP2). Previous studies have shown that MALT1’s proteolytic activity is required for its participation in NF-kB induction and for promoting cell viability of certain types of lymphomas, particularly the activated B-cell (ABC) type of diffuse large B-cell lymphoma.

To lay a foundation for discovery of drug-like chemical inhibitors of MALT1, we developed a fluorescence-based, biochemical assay for measuring protease activity of MALT1 in vitro in 384 and 1,536 well formats. The assay components included recombinant purified full-length MALT1 protein, the MALT1-binding protein, Bcl-10, and the fluorigenic substrate Ac-LRSR-AMC (Ac-Leu-Arg-Ser-Arg-7-amino-4-methylcoumarin). Experiments showed that addition of Bcl-10 increases MALT1‘s protease activity up to 20-40 fold. The high throughput screening (HTS) assay was validated with excellent Z’ factors (>0.7) in both 384 and 1,536 well formats, with performance remaining robust for more than 6 hours, and thus suitable for screening of large chemical libraries. The HTS assay was validated by performing pilot screens with two small collections of compounds enriched in bioactive molecules (n = 1280 for LopacTM and 2000 for SpectrumTM library), yielding confirmed hit rates of 0.2 % and 0.7%, respectively. Counter-screening assays were also generated using other types of intracellular cysteine proteases, specifically Caspase-3 and Autophagin-1 (ATG4B), for eliminating non-specific inhibitors. Various supporting cell-based assays have also been generated to support hit characterization. In conclusion, we have generated a robust biochemical HTS assays for detection of chemical inhibitors of the MALT1 protease. We plan to utilize the HTS assay for screening large compound libraries as a starting point for discovery of novel therapeutics for treatment of lymphoma, autoimmunity, and allograft rejection.

Citation Format: Dayong Zhai, Chih-Wen Shu, Paul Diaz, John C. Reed. Development of a biochemical High Throughput Screening (HTS) assay for chemical inhibitors of MALT1, a target for lymphoma therapeutics. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4540. doi:10.1158/1538-7445.AM2013-4540
Abstract 4548: Identify novel molecular structures targeting X-Linked Inhibitor of Apoptosis Protein by ligand-based virtual screening.
Authors: Wang Jin , Duke Charles B. , Li Wei
Published: August 2014

Resistance to apoptosis is believed as one of the mechanism underlying a number of human malignancies notorious resistances to current chemotherapeutics. The X-linked inhibitor of apoptosis protein (XIAP) inhibits apoptosis by directly binding and deactivating caspase-9, caspase-3/7. Elevated XIAP expression in tumor tissue is closely associated with metastatic progression. Mimicking the XIAP-binding motif of second mitochondria-derived activator of caspase (SMAC) is the most widely used strategy to discover or design therapeutic agents targeting XIAP. Here we screened a large database of 362K compounds (the University of Cincinnati's Drug Discovery Center Library) in silico to find molecules with novel structures that can strongly interact with the third BIR domain of XIAP. We chose AVPI tetrapeptide from the SMAC as the lead structure and run the shape similarity search using program Canvas, version 1.4 (Schrodinger, LLC, New York, USA). Then top 10,000 structures of the similarity rank were docked into SMAC binding sites in two different SMAC-BIR3 domain complexes crystal structure (PDB ID: 1TW1 and 1G73) separately. The highest docking score of the screened structures is up to -9.0 in both of complexes while the SMAC AVPI tetrapeptide showed docking score at -9.4. Considering the docking scores in both complexes and the flexibility of chemical scaffolds, we manually selected 72 compounds with docking scores varied from -8.4 to -5.5 for in vitro anti-proliferation and caspase functional testing. Four compounds which haven't been reported with any anti-cancer activity, inhibited the cell growth by over 70% in two human melanoma cell lines (A375 and MDA-MB-435) and two human prostate cell lines (PC-3 and LNCap) at the concentration of 10uM after 48hr incubation. IC50 values of those compounds were at least 4 folds smaller than that of Embelin, a well-recognized XIAP inhibitor, in all four cell lines tested. The best of them, compound 402112, have IC50 values of 650nM to 2.5uM against the four tested cancer cell lines. Caspase functional assay showed that compound 402112 activates capase-9 activity at 4 μM, significantly better than Embelin did at a 10-fold higher concentration (40 μM) after 48hr treatment in PC-3 and A375 cells. Taken together, our study identified novel molecule structures which are amicable to further extensive modification for the development of high potent XIAP inhibitors.

Citation Format: Jin Wang, Charles B. Duke, Wei Li. Identify novel molecular structures targeting X-Linked Inhibitor of Apoptosis Protein by ligand-based virtual screening. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4548. doi:10.1158/1538-7445.AM2013-4548
Abstract 4554: A docking domain directed inhibitor targeting ERK regulated F-site substrates.
Authors: Zhang Jun , Oashi Taiji , Samadani Ramin , Nevels Kerrick , Burkhard Kimberly , Priyakumar Deva , Raman Prabhu , Fletcher Steven , Pozharsky Edvin , Shapiro Paul , MacKerell Alexander
Published: August 2014

Extracellular signal-regulated kinases 1 and 2 (ERK1/2) are ubiquitously expressed in almost all tissues and cell types. Accordingly, ERK1/2 regulate a diversity of cellular processes by phosphorylating more than 150 substrate proteins. With its constitutive activation being frequently implicated in human malignancies, ERK has been an appealing target in anti-cancer drug development. However, currently available ERK1/2 inhibitors are ATP-competitive. Since these ERK inhibitors block all ERK functions, the potential for side effects in normal tissues and cells may limit their potential of being developed into therapeutic agents. To overcome this limitation, we used a combination of computational and experimental methods to develop small molecular weight inhibitors that target ERK-substrate docking domains. Here we report the identification, characterization and optimization of a compound with a thienyl benzenesulfonate scaffold that specifically inhibits substrates containing an F-site or DEF (docking site for ERK, FXF) motif. Our X-ray crystallography data showed that this compound bound to a pocket in the vicinity of the F-site docking domain on ERK. Biological analysis further demonstrated that this compound and its analogs could preferentially inhibit phosphorylation and function of F-site-containing ERK substrates, and that these compounds preferentially inhibited growth of melanoma cells harboring a V600E B-Raf mutant. Additionally, this compound was shown to inhibit the phosphorylation of specific PKC isoforms via an ERK dependent mechanism. These findings represent the first identification and validation of chemicals that selectively inhibit ERK substrates and signaling events through ATP-independent mechanisms. These compounds have potential utility for elucidating the complex biological roles of ERK1/2 and development into novel anti-cancer agents.

Citation Format: Jun Zhang, Taiji Oashi, Ramin Samadani, Kerrick Nevels, Kimberly Burkhard, Deva Priyakumar, Prabhu Raman, Steven Fletcher, Edvin Pozharsky, Paul Shapiro, Alexander MacKerell. A docking domain directed inhibitor targeting ERK regulated F-site substrates. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4554. doi:10.1158/1538-7445.AM2013-4554
Abstract 4565: Whole exome sequencing links MAPK1 mutation to exquisite sensitivity to brief erlotinib monotherapy in head and neck cancer.
Authors: Lui Vivian WY , Van Allen Eliezer , Li Hua , Zhang Fan , Zeng Yan , Johnson Jonas T. , Garraway Levi A. , Mills Gordon B. , Grandis Jennifer Rubin
Published: August 2014

Activating mutations of EGFR, primarily found in a subgroup of lung cancers, serve as predictive biomarkers for EGFR tyrosine kinase inhibitor (TKI) sensitivity. Clinical responses to EGFR TKIs in other cancers are only moderate and predictive biomarkers are lacking. Here, we report a head and neck cancer patient with no EGFR genetic alterations, who experienced a complete histologic clinical response after 14 days of erlotinib monotherapy on a window-of-opportunity presurgical trial. To determine the somatic genetic events underlying the extraordinary and unexpected response to erlotinib alone, whole exome sequencing was performed. A bi-allelic missense mutation of MAPK1(E322K) was detected in the tumor in the absence of other mutations reported to be correlated with EGFR TKI sensitivity in preclinical models or cancer patients. Combined bioinformatics analyses of genomics-drug databases revealed, for the first time, that loss-of-function of MAPK1 or MAPK1 mutation correlates with erlotinib sensitivity. Functional studies confirmed heightened sensitivity to erlotinib by loss-of-function of MAPK1 in HNSCC cells. Our preliminary findings suggest that this MAPK1 mutation, which is also found at low frequencies in HNSCC and cervical cancers, may identify a subgroup of cancer patients who may be cured with EGFR TKI monotherapy.

Citation Format: Vivian WY Lui, Eliezer Van Allen, Hua Li, Fan Zhang, Yan Zeng, Jonas T. Johnson, Levi A. Garraway, Gordon B. Mills, Jennifer Rubin Grandis. Whole exome sequencing links MAPK1 mutation to exquisite sensitivity to brief erlotinib monotherapy in head and neck cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4565. doi:10.1158/1538-7445.AM2013-4565
Abstract 457: Matrix metalloproteinase-2 modulates antitumor immunity.
Authors: Godefroy Emmanuelle , Gallois Anne , Manches Olivier , Ensanyat Shaheen , Jotereau Francine , Bhardwaj Nina
Published: August 2014

Background: Cancer cells, including melanoma, can be highly resistant to traditional treatments such as chemotherapy and radiotherapy. As a result, a lot of effort has been put into developing immune therapies to treat cancer patients. The main barrier to design effective immunotherapies is the immunosuppression induced by the tumor. Matrix metalloproteinase-2 (MMP-2) is over-expressed in most cancers including melanoma, and its expression is associated with increased dissemination and poorer survival/prognosis. Here, we not only uncovered an immunosuppressive role of MMP-2 in inducing ineffective/detrimental TH2 immune responses, but also the underlying mechanisms.

Methods: Human dendritic cells (DCs) were cultured in the presence of MMP-2 and/or various TLR agonists. DCs responses were monitored using immunostaining, ELISA or cytometric bead array methods. Autologous CD4+ T cells were stimulated using these conditioned tumor-associated antigen (TAA)-pulsed DCs. TAA-specific CD4+ T cells were cloned and characterized using mainly the same techniques as for DCs.

Results: Here we showed that MMP-2-conditioned human DCs primed naïve CD4+ T cells to differentiate into an inflammatory TH2 phenotype, i.e. mainly secreting TNFα, IL-4 and IL-13 and expressing GATA3. Accordingly, we detected MMP-2-specific CD4+ T cells displaying the same inflammatory TH2 profile in tumor-infiltrating lymphocytes from melanoma patients. We revealed the underlying mechanisms: on the one hand, MMP-2 was found to degrade the type-I IFN receptor IFNAR1, thereby preventing STAT1 phosphorylation, which is necessary for IL-12 production. On the other hand, MMP-2 triggers the Toll-like receptor (TLR)-2 on DCs, which leads to NF-κB activation and OX40L expression. Both lack of IL-12 and over-expression of OX40L were found responsible for skewing T cell responses towards a detrimental TH2 phenotype.

Conclusions: MMP-2, therefore, acts as an endogenous TH2 "conditioner" and may underlie the prevalence of detrimental TH2 responses in cancer. Our findings also described the responsible MMP-2-dependent mechanisms, which open the way to novel therapeutic strategies and/or targets to skew anti-tumor CD4+ T cell responses towards a more efficient anti-tumor TH1 phenotype to treat cancer patients.

Citation Format: Emmanuelle Godefroy, Anne Gallois, Olivier Manches, Shaheen Ensanyat, Francine Jotereau, Nina Bhardwaj. Matrix metalloproteinase-2 modulates antitumor immunity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 457. doi:10.1158/1538-7445.AM2013-457
Abstract 4573: STINGVAX - A novel tumor vaccine with cyclic dinucleotides - can induce potent anti-tumor responses in vivo.
Authors: Dubensky Thomas W. , Leong Meredith L. , Kanne David B. , Lemmens Edward E. , Metchette Ken , Liu Weiqun , Fasso Marcella , Fu Juan , Woodward Joshua J. , Pardoll Drew , Portnoy Daniel A. , Kim Young J.
Published: August 2014

Cyclic dinucleotides (CDN) are ubiquitous bacterial intracellular messengers, and they have recently been shown to also function as a pathogen associated molecular pattern (PAMP) molecules that are sensed by eukaryotic host Stimulator of Interferon Genes (STING) that can activate the TBK-1/IRF-3 signaling pathway to induce type I interferon and other co-regulated genes. CDN was found to induce STING dependent augmentation of T-cell priming in multiple vaccination models, potentially by activating dendritic cells. We formulated CDN with a GM-CSF secreting tumor cell vaccine (STINGVAX) to mobilize and as well as activate dendritic cells both in vitro and in vivo. STINGVAX was tested in a stringent B16 melanoma treatment model, and we demonstrated significant reduction of tumor growth rate in vivo. STINGVAX's anti-tumor response was correlated with increased tumor infiltrating CD8 T-cells as well as increased number of p15E tumor-specific cytotoxic T-cells. STINGVAX's in vivo anti-tumor response was T-cell dependent as well as STING dependent. When we combined STINGVAX with multiple TLR 4/7/8 agonists signaling through the MyD88/TRIF pathway that is distinct from CDN/STING/TBK-1 signaling treatment of palpable B16 tumor resulted in regression of 30-60% of these non-immunogenic tumors. When the STINGVAX treated tumor microenvironment was examined, both IFNγ+CD8+ and PD-L1 was upregulated, potentially demonstrating an adaptive immune resistance mechanism which would render STINGVAX an excellent candidate to be combined with anti-PD-1 blockade. Cumulatively, STINGVAX is a novel tumor vaccine with a high potential for translation in clinical oncology.

Citation Format: Thomas W. Dubensky, Meredith L. Leong, David B. Kanne, Edward E. Lemmens, Ken Metchette, Weiqun Liu, Marcella Fasso, Juan Fu, Joshua J. Woodward, Drew Pardoll, Daniel A. Portnoy, Young J. Kim. STINGVAX - A novel tumor vaccine with cyclic dinucleotides - can induce potent anti-tumor responses in vivo. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4573. doi:10.1158/1538-7445.AM2013-4573
Abstract 4576: Immunization with CPG in combination with MHC class I and class II peptides stimulates rapid and strong tumor antigen-specific CTL responses in melanoma patients.
Authors: Fourcade Julien , Sun Zhaojun , Pagliano Ornella , Chauvin Joe-Marc , Sander Cindy , Moschos Stergios , Tarhini Ahmad , Tawbi Hussein , Kirkwood John M. , Zarour Hassane M.
Published: August 2014

There is now ample evidence that the toll-like receptor 9 ligand CPG/PF-3512676 represents a potent adjuvant for peptide/protein-based cancer vaccines capable of stimulating ex vivo detectable tumor antigen (TA)-specific CD8+ T cell responses in patients with advanced melanoma. Although CD4+ T cells appeared to promote the expansion and functions of CD8+ T cells in experimental models, melanoma-associated helper peptides have been shown to paradoxically decrease CD8+ T cell responses to a multipeptide melanoma vaccine emulsified in Montanide, possibly by inducing antigen-specific Tregs. To further investigate this question, we have performed one Phase I study of immunization where patients with advanced melanoma were immunized with CPG, Montanide and the HLA-A2-restricted analog peptide NY-ESO-1 157-165V either alone (Arm 1, n=5) or in combination with the pan-DR CD4 helper peptide NY-ESO-1 119-143 (Arm 2, n=7). Patients in both arms exhibited a rapid increase of NY-ESO-1-specific CD8+ T cells that were detectable ex vivo by flow cytometry. Strikingly, the mean frequency of vaccine-induced NY-ESO-1-specific CD8+ T cells after 6 biweekly immunizations was five times higher in patients who also received the helper peptide. Melanoma patients immunized with the helper peptide (Arm 2) developed a rapid increase of Foxp3− NY-ESO-1-specific CD4+ T cells that produced Th-1 type cytokines and IL-21 ex vivo. As compared to Arm 1, melanoma patients included in Arm 2 developed vaccine-induced NY-ESO-1-specific CD8+ T cells that were more differentiated, produced more IFN-γ and exhibited higher cytolytic functions in ex vivo assays. Collectively, our data support the inclusion of well-defined MHC class II peptides in combination with CPG/PF-3512676 and MHC class I peptides in multipeptide melanoma vaccines to better augment the expansion, differentiation, IFN-γ production and lytic function of TA-specific CTLs in patients with advanced melanoma.

This work was supported by the National Institutes of Health/National Cancer Institute grants R01CA90360 and R01CA157467 (to H.M. Zarour).

Citation Format: Julien Fourcade, Zhaojun Sun, Ornella Pagliano, Joe-Marc Chauvin, Cindy Sander, Stergios Moschos, Ahmad Tarhini, Hussein Tawbi, John M. Kirkwood, Hassane M. Zarour. Immunization with CPG in combination with MHC class I and class II peptides stimulates rapid and strong tumor antigen-specific CTL responses in melanoma patients. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4576. doi:10.1158/1538-7445.AM2013-4576
Abstract 4577: Identification of twenty three novel prostate cancer susceptibility loci using a custom assay (the icogs) in an international consortium: PRACTICAL.
Authors: Eeles Rosalind A. , Kote-jarai Zsofia , Consortium The PRACTICAL
Published: August 2014

Identification Prostate cancer (PrCa) is the most frequently diagnosed male cancer in developed countries. To identify common PrCa susceptibility alleles, we genotyped 211,155 SNPs on a custom chip (iCOGS) in blood DNA from 25,074 PrCa cases and 24,272 controls from 32 studies in the international PRACTICAL consortium. The chip was composed of GWAS hits from a meta analysis of 4 GWAS [11,085 cases and 11,463 controls from UK,Australia, Cancer Genetic Markers of Susceptibility (CGEMS); Cancer of the Prostate in Sweden (CAPS) and the Breast and Prostate Cancer Cohort Consortium (BPC3)], fine mapping of known regions and genes in candidate pathways. Results from the GWAS component are reported here. Twenty-three new PrCa susceptibility loci were identified at genome-wide significance (P<5x10-8). Neighbouring genes include MDM4, NOTCH4, MMP7, RAD51L1, SPOP and HOXB13. There was no strong evidence for heterogeneity in the per-allele OR among studies. All alleles are common (MAF 8-49%) with per allele ORs from 1.06 to 1.15. All but two exhibit a pattern of association consistent with a log-additive model, as observed for most common cancer susceptibility alleles. Aggressive disease was defined as Gleason score > 8, PSA >100, disease stage of distant (i.e. outside the pelvis) or death from PrCa. The majority of SNPs showed clear association when analysis was restricted to aggressive disease (13 SNPs significant at P<.01) and for 22 of the 23 SNPs the estimated OR was in the same direction for aggressive and non-aggressive disease. Two SNPs were associated with PSA level in controls. Six SNPs showed a trend in OR with age at diagnosis, with a higher OR at younger ages. GeneGo pathway enrichment analysis showed the most strongly associated pathways were cell adhesion (P=1.31 x10−6), extracellular matrix (ECM) remodelling (P=3.6x10−9), and transcriptional regulation by androgen receptor (P= 3.5x10−8). Comparison of the direction of effects in the GWAS and COGS replication stages indicate that more than 1800 loci on the array are associated with this disease. More than 70 PrCa susceptibility loci, explaining 30% of the familial risk in this disease, have now been identified. Based on the combined risks conferred by these loci, the top 1% of the risk distribution has a 4.7fold increased risk compared with the average of the population. These results will facilitate population risk stratification for clinical studies.

Citation Format: Rosalind A. Eeles, Zsofia Kote-jarai, The PRACTICAL Consortium. Identification of twenty three novel prostate cancer susceptibility loci using a custom assay (the icogs) in an international consortium: PRACTICAL. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4577. doi:10.1158/1538-7445.AM2013-4577
Abstract 4584: Bone marrow-derived Gr1+ cells can generate a metastasis-resistant microenvironment via induced secretion of thrombospondin-1.
Authors: Catena Raul , Bhattacharya Nandita , El Rayes Tina , Choi Hyejin , Wang Suming , Gao Dingcheng , Joshi Natasha , Ryu Seongho , Bielenberg Diane , Lee Sharrell , Haukaas Svein , Gravdal Karsten , Halvorsen Ole , Akslen Lars , Watnick Randolph , Mittal Vivek
Published: August 2014

Previous studies have shown that metastatic tumors establish premetastatic niches comprised of recruited bone marrow (BM)-derived cells in potential distant target metastatic organs. These premetatatic niches serve as permissive hubs for supporting future metastases. However, little is known about whether tumors that lack metastatic potential establish such niches, or generate niches that inhibit metastasis. Here, we show that metastasis-incompetent tumors are also capable of systemically generating BM-derived niches in the metastatic organs. However, by secreting the glycoprotein prosaposin these tumors systemically stimulate expression of antitumorigenic factor thrombospondin-1 (Tsp-1) in the Gr1+ myeloid cells converting them it into a metastasis-refractory niche. Genetic deletion of Tsp-1 abolished the inhibition of metastasis, which was restored by BM transplant from Tsp-1+ donors.

We developed a 5-amino acid peptide from prosaposin as a pharmacological inducer of Tsp-1 in Gr1+ BM cells. Strikingly, the peptide suppresses metastasis. These results provide mechanistic insights into why certain tumors are deficient in metastatic potential and implicate recruited Gr1+ myeloid cells as the main source of Tsp-1, contrary to previous notion. These results also underscore the plasticity of Gr1+ cells, which, depending on the context and the nature of paracrine factor/s can either promote or inhibit metastasis. Clinically, examination of a tumor tissue microarray (TMA) compiled from 103 prostate cancer patients with long and complete follow up, revealed that prosaposin expression positively correlated with increased overall survival, suggesting that the prosaposin peptide may be a potential therapeutic agent against metastatic cancer.

Citation Format: Raul Catena, Nandita Bhattacharya, Tina El Rayes, Hyejin Choi, Suming Wang, Dingcheng Gao, Natasha Joshi, Seongho Ryu, Diane Bielenberg, Sharrell Lee, Svein Haukaas, Karsten Gravdal, Ole Halvorsen, Lars Akslen, Randolph Watnick, Vivek Mittal. Bone marrow-derived Gr1+ cells can generate a metastasis-resistant microenvironment via induced secretion of thrombospondin-1. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4584. doi:10.1158/1538-7445.AM2013-4584
Abstract 4585: Breaking metastatic dormancy during surgical resection of a primary tumor and implications for treatment strategies.
Authors: Granitto Selena R. , Giles Amber , Lavotshkin Simon , Rutigliano Daniel , Lyden David , Kaplan Rosandra N.
Published: August 2014

At the time of a cancer diagnosis, most patients have localized tumors. Despite elaborate staging schemas for each cancer type in an attempt to stratify patients, the vast majority of patients that die will do so from metastatic disease. We hypothesized that surgery augments the already ongoing activation and mobilization of bone marrow-derived progenitor cells that are critical to colonizing tumor cells at distant sites. These bone marrow-derived cells, by inducing a local inflamed tumor microenvironment, provide survival signals to these seeding tumor cells. Our data show increased metastatic burden in the lung after surgical resection of the primary tumor using two murine cancer models, B16 melanoma and E0771 breast carcinoma. In these models, we also show a surge in hematopoietic and endothelial progenitor cells in the hours and days immediately following resection of the primary tumor, which is not similarly observed in control mice, where surgery was performed in the absence of the primary tumor. We also confirmed that a factor specific to the plasma of the tumor-bearing mice is responsible for this mobilization by using in vitro migration assays, whereby plasma from tumor-bearing mice and surgically resected mice induced an increased migration of lineage negative bone marrow cells compared to the plasma of wild type mice. We confirmed increased levels of MCP-1 and MCSF, both known to mobilize progenitor cells, in the plasma of mice with surgical resection. Additionally, targeting these bone marrow derived hematopoietic and endothelial progenitor cells with Pazopanib prevents the surge in bone marrow-derived cells into the circulation, abolishes the enhanced metastatic spread in mice undergoing surgical resection of the primary tumor, and provides a significant prolongation of survival. Finally, we correlated these data to a cohort of breast cancer patients where circulating levels of progenitor cells were analyzed at time points before and after surgery, which confirmed the mobilization of progenitor cells with surgery. Together, these results provide evidence for the increased risk of metastatic spread after surgical resection of the primary tumor and suggest that blocking progenitor cell mobilization by adjuvant treatment during or immediately following surgery, the incidence of metastatic recurrence may be reduced.

Citation Format: Selena R. Granitto, Amber Giles, Simon Lavotshkin, Daniel Rutigliano, David Lyden, Rosandra N. Kaplan. Breaking metastatic dormancy during surgical resection of a primary tumor and implications for treatment strategies. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4585. doi:10.1158/1538-7445.AM2013-4585
Abstract 4598: Identification of driver genes in hepatocellular carcinoma by exome sequencing.
Authors: Cleary Sean P. , Jeck William R. , Zhao Xiaobei , Selitsky Sara R. , Savich Gleb L. , Tan Ting-Xu , Wu Michael C. , Getz Gad , Lawrence Michael S. , Parker Joel S. , Li Jinyu , Powers Scott , Kim Hyeja , Fischer Sandra E. , Guindi Maha , Ghanekar Anand , Chiang Derek Y.
Published: August 2014

Genetic alterations in specific driver genes lead to disruption of cellular pathways and are critical events in the instigation and progression of hepatocellular carcinoma. As a prerequisite for individualized cancer treatment, we sought to characterize the landscape of recurrent somatic mutations in hepatocellular carcinoma. We performed whole exome sequencing on 87 hepatocellular carcinomas and matched normal adjacent tissues to an average coverage of 59x. The overall mutation rate was roughly 2.5 mutations per Mb, with a median of 45 non-synonymous mutations that altered the amino acid sequence (range 2 to 381). We found recurrent mutations in several genes with high transcript levels: TP53 (20%), CTNNB1 (15%), IGSF3 (9%) and KEAP1 (8%). Significantly affected gene families include the nucleotide-binding domain and leucine rich repeat containing family, calcium channel subunits, and histone methyltransferases. In particular, the MLL family of methyltransferases for histone H3 lysine 4 were mutated in 20% of tumors. Clinical data demonstrated significantly decreased disease-free survival in tumors with p53 mutations (p=0.006) and a trend was observed towards higher rates of recurrence and shorter disease free survival among MLL mutation carriers. The NFE2L2-KEAP1 and MLL pathways are recurrently mutated in multiple cohorts of hepatocellular carcinoma. The elucidation of genetic changes in HCC will enhance our understanding of hepatocarcinogenesis and influence disease prognosis and therapy.

Citation Format: Sean P. Cleary, William R. Jeck, Xiaobei Zhao, Sara R. Selitsky, Gleb L. Savich, Ting-Xu Tan, Michael C. Wu, Gad Getz, Michael S. Lawrence, Joel S. Parker, Jinyu Li, Scott Powers, Hyeja Kim, Sandra E. Fischer, Maha Guindi, Anand Ghanekar, Derek Y. Chiang. Identification of driver genes in hepatocellular carcinoma by exome sequencing. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4598. doi:10.1158/1538-7445.AM2013-4598
Abstract 4605: CDIP-BAP31 complex bridges endoplasmic reticulum and mitochondria during ER-stress mediated apoptosis.
Authors: Namba Takushi , Tian Fang , Chu Kiki , Hwang So-Young , Lee Sam W.
Published: August 2014

Resolved ER stress response is fundamentally essential for intracellular homeostatic balance, but unsettled ER stress can lead to apoptosis. In addition, imbalances in homeostasis would result in improper cell functioning that may become an important pathogenic factor in a number of prevalent diseases, including neurodegenerative diseases, metabolic disorders, and cancers. However, the molecular mechanisms relating ER stress to apoptosis still remain largely unfamiliar. Here, we show that a proapoptotic p53 target, CDIP, acts as a key signal transducer of ER stress response. Applying a biochemical/proteomics approach, we characterize BAP31, B-cell receptor-associated protein 31 that is an integral protein of ER and known to crosstalk with mitochondrial proteins during the apoptosis process, as an interacting partner of CDIP. Upon ER stress, CDIP is induced and enhances an association with BAP31 at the ER membrane. We found that the CDIP-BAP31 complex formation is essential for ER stress-mediated apoptotic process. CDIP-/- or CDIP knock-down cells are strongly resistant to ER-stress-induced apoptosis. Moreover, we show that CDIP up-regulation and binding to BAP31 is required for BAP31 cleavage via caspase-8 activation in response to ER stress and also responsible for BAP31-Bcl-2 association, then resulting in Bcl-2 sequestration from BAX. The recruitment of Bcl-2 with BAP31-CDIP complex triggers BAX oligomerization/activation that causes its translocation to the mitochondria, followed by cyctochrome c release. Genetic knockout of CDIP in mice also leads to impaired response to ER-stress-mediated apoptosis, likely through reduced BAX activation. Together, our data demonstrate that the CDIP-BAP31→Bcl-2/BAX regulatory circuit represents a novel mechanism for establishing an ER-mitochondrial cross-talk for ER stress-mediated apoptosis signaling.

Citation Format: Takushi Namba, Fang Tian, Kiki Chu, So-Young Hwang, Sam W. Lee. CDIP-BAP31 complex bridges endoplasmic reticulum and mitochondria during ER-stress mediated apoptosis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4605. doi:10.1158/1538-7445.AM2013-4605
Abstract 4608: Autophagy is required for prostate cancer progression.
Authors: De Velasco Marco A. , Kura Yurie , Ando Naomi , Fukushima Emiko , Hatanaka Yuji , Yamamoto Yutaka , Shimizu Nobutaka , Yoshimura Kazuhiro , Nozawa Masahiro , Yoshikawa Kazuhiro , Nishio Kazuto , Uemura Hirotsugu
Published: August 2014

Autophagy (also referred to as macroautophagy) is a self-catabolic process that maintains intracellular homeostasis and prolongs cell survival by the autophagosomic-lysosomal degradation of cytoplasmic contents during metabolic stress or nutrient starvation. Inactivation of PTEN tumor suppressor function is a common characteristic of human prostate cancer that leads to the upregulation of the PI3K/Akt/mTOR signaling pathway, and has emerged as promising candidate for targeted therapies. mTOR is one of the upstream regulators of autophagy, therefore, inhibiting the PI3K/Akt/mTOR axis putatively stimulates autophagy. However, the role of autophagy in cancer is complex, acting both as a tumor suppressor and promoter, and its role in prostate tumorigenesis remains to be defined. Here we demonstrate that prostate cancer is dependent on autophagy for its growth and progression through the use genetically modified mouse models of prostate cancer.

We had previously reported a conditional knockout mouse model in which homozygous deletion of PTEN lead to the stage-specific development of invasive adenocarcinoma. Characterization of tumors from the mice showed that although autophagy was suppressed early in tumor development, tumors eventually restored autophagic function despite increased PI3K/Akt/mTOR signaling. To determine the effect of defective autophagy on prostate cancer progression, we generated prostate-specific conditional double knockout mice harboring both inactivated PTEN and ATG7, a key regulator for the formation of autophagosomes. Prostate-specific inactivation of ATG7 function alone did not result in changes to the development or function of the prostate gland. Prostate-specific inactivation of both PTEN and ATG7 led to the early development of prostate adenocarcinoma similar to homozygous PTEN-mutant mice. However, prostate tumors failed to progress and showed regression. Our data suggests that impaired autophagy can be induced independent of mTOR and defective autophagy contributes to suppress tumor progression. Thus, our findings support the role of autophagy as a therapeutic target for advance prostate cancer.

Citation Format: Marco A. De Velasco, Yurie Kura, Naomi Ando, Emiko Fukushima, Yuji Hatanaka, Yutaka Yamamoto, Nobutaka Shimizu, Kazuhiro Yoshimura, Masahiro Nozawa, Kazuhiro Yoshikawa, Kazuto Nishio, Hirotsugu Uemura. Autophagy is required for prostate cancer progression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4608. doi:10.1158/1538-7445.AM2013-4608
Abstract 4612: Phosphoglycerate mutase 1 coordinates glycolysis and biosynthesis to promote tumor growth.
Authors: Hitosugi Taro , Zhou Lu , Arellano Martha , Khoury Hanna , Boggon Titus , Kang Sumin , He Chuan , Chen Jing
Published: August 2014

The current understanding of the Warburg effect consists of an increase in aerobic glycolysis in cancer cells. The connection between glycolysis and PPP/biosynthesis is based upon a model in which glycolytic intermediates can be diverted into PPP and biosynthesis pathways as precursors. However, it remains unclear how cancer cells coordinate glycolysis and biosynthesis to fulfill the request of rapidly growing tumors. We found that glycolytic enzyme phosphoglycerate mutase 1 (PGAM1), commonly upregulated in human cancers due to loss of TP53, regulates anabolic biosynthesis by controlling intracellular levels of its substrate 3-phosphoglycerate (3-PG) and product 2-phosphoglycerate (2-PG). Our co-crystal structure based analysis revealed that 3-PG binds to and inhibits 6-phosphogluconate dehydrogenase in the oxidative pentose phosphate pathway (PPP) as a competitive inhibitor, while 2-PG activates 3-phosphoglycerate dehydrogenase (PHGDH) to provide feedback control of 3-PG levels. Inhibition of PGAM1 by shRNA results in increased 3-PG and decreased 2-PG levels in cancer cells, leading to significantly decreased glycolysis, PPP flux and biosynthesis, as well as attenuated cell proliferation and tumor growth. These findings uncover that PGAM1 controls the intracellular 3-PG and 2-PG levels to serve as a novel link between glycolysis, biosynthesis, cancer cell proliferation and tumor development.

Moreover, we screened and developed novel small molecule PGAM1 inhibitor (PGMI)-004A, which effectively inhibits PGAM1 enzyme activity, resulting in aberrant cancer cell metabolism with reduced glycolysis and PPP/anabolic biosynthesis, as well as attenuated cell proliferation in diverse human cancer cell lines, but not control normal human proliferating cells including human dermal fibroblasts and human foreskin fibroblasts. PGMI-004A treatment attenuates tumor growth in xenograft nude mice with minimal toxicity in vivo. Furthermore, PGMI-004A inhibits cell proliferation of primary leukemia cells from human AML, CML and B-ALL patients, but not control CD34+ cells isolated from bone marrow samples or mononucleocytes isolated from peripheral blood samples from healthy donors, suggesting minimal toxicity of PGMI-004A in human cells.These results together provide “proof of principle” for the development of PGAM1 inhibitors as anti-cancer agents.

Citation Format: Taro Hitosugi, Lu Zhou, Martha Arellano, Hanna Khoury, Titus Boggon, Sumin Kang, Chuan He, Jing Chen. Phosphoglycerate mutase 1 coordinates glycolysis and biosynthesis to promote tumor growth. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4612. doi:10.1158/1538-7445.AM2013-4612
Abstract 463: Differential cellular localization of programmed death ligand 1 (PD-L1) and enhanced expression of programmed death-1 (PD-1) in melanoma progression.
Authors: Chakravarti Nitin , Bassett Roland , Hwu Wen-Jen , Ivan Doina , Curry Jonathan L. , Prieto Victor G.
Published: August 2014

Melanoma is an aggressive malignancy with a relatively high metastatic rate, responsible for almost 60% of lethal skin tumors. Although many human cancers such as melanoma express tumor antigens recognized by T cells, host immune responses often fail to control tumor growth for mostly unexplained reasons. Escaping immune destruction is one of the tenets of tumorigenesis; there have been several studies suggesting an important role of PD-1/PD-L1 (B7-H1) interactions in inhibiting the effector functions of cytotoxic T-lymphocyte in tumor. Ghebeh et al., reported that doxorubicin treatment caused decrease in cytosolic B7-H1 and increased nuclear translocalization in breast carcinoma (Breast Cancer Res. 2010;12(4):R48). It is unknown whether differential cellular expression of PD-L1 contributes to the pathogenesis of melanoma. To address the role of PD-L1 and its receptor PD-1 in different stages of malignant melanoma, we have performed semiquantitative immunohistochemical analysis of PD-L1 and PD-1 in benign nevi (n=59), primary melanomas without metastasis (n=22) or with metastasis (n=17), and melanoma metastases (n=32 cases). Benign nevi had significantly higher levels of nuclear PD-L1 immunopositivity (p=0.004) and low levels of PD-1 protein expression (p=0.002). With progression from benign nevi to primary melanoma there was an increase in cytoplasmic positivity for PD-L1 (p=0.03). Also, a significant positive correlation was observed between the percentage of positive cells (p=0.0007) and intensity (p=0.0009) for PD-1 in primary melanomas. Although there was no significant difference in PD-1 levels in between primaries with or without metastasis, a trend was observed for PD-L1 expression: primaries with metastasis having a larger number of tumor cells with cytosolic localization than primaries without metastasis (p=0.07). Interestingly, primary melanomas without metastasis showed significantly larger number of tumor infiltrating lymphocytes (TILs) cells having intense immunoreactivity for PD-L1 protein than did metastatic melanomas (p=0.03). In conclusion, differential cellular localization of PD-L1 protein and high levels of PD-1 protein correlates with disease progression and high-risk histologic features in melanoma and might partially explain the resistance of melanoma cells against the immune system.

Citation Format: Nitin Chakravarti, Roland Bassett, Wen-Jen Hwu, Doina Ivan, Jonathan L. Curry, Victor G. Prieto. Differential cellular localization of programmed death ligand 1 (PD-L1) and enhanced expression of programmed death-1 (PD-1) in melanoma progression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 463. doi:10.1158/1538-7445.AM2013-463
Abstract 4635: The avidity hypothesis: comparing bispecific and monospecific antibodies in preclinical oncology models.
Authors: Sachsenmeier Kris F. , Dimasi Nazzareno , Huang Qihui , Sult Erin , Bezabeh Binyam , Fleming Ryan , Hay Carl , Zhong Helen , Breen Shannon , Gao Changshou , Agoram Balaji , Jermutus Lutz , Hollingsworth Robert
Published: August 2014

Bi-specific antibodies are suggested to possess unique advantages when compared with mono-specific antibodies as anti-cancer therapeutics. These advantages include greater efficacy, superior combination with standard of care agents, and evasion of resistance mechanisms. Data are presented supporting the hypothesis that the increased avidity of a tetravalent (non BiTE-like) bi-specific antibody mediates such advantages when compared with conventional bi-valent mono-specific antibodies. Experiments include functional comparisons of antibodies binding the Epidermal Growth Factor (EGFR) and the Insulin-like Growth Factor 1 Receptor (IGF1R) at the biochemical, cellular and tumor xenograft levels. These data support the ongoing use of multi-specific antibodies in oncology research and describe a mechanism that may contribute to superior activity compared to mono-specific antibodies and drug combinations. It is noted that advantages of bi-specific antibody formats remain to be seen clinically in terms of both efficacy and safety.

Citation Format: Kris F. Sachsenmeier, Nazzareno Dimasi, Qihui Huang, Erin Sult, Binyam Bezabeh, Ryan Fleming, Carl Hay, Helen Zhong, Shannon Breen, Changshou Gao, Balaji Agoram, Lutz Jermutus, Robert Hollingsworth. The avidity hypothesis: comparing bispecific and monospecific antibodies in preclinical oncology models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4635. doi:10.1158/1538-7445.AM2013-4635
Abstract 4638: Anti-tumor activity of pimasertib in combination with SAR245409 or SAR245408 in human primary colorectal cancer xenograft models bearing PI3K/KRas and KRas mutations.
Authors: Sidhu Sukhvinder S. , Egile Coumaran , Malfilatre Marc , Lefranc Celine , Ruffin Yvette , Ma Jianguo , Hsu Karl , Lager Joanne , Marzabal Stephane , Ogden Janet A. , Vincent Loic
Published: August 2014

The PI3K/AKT/mTOR and Ras/Raf/MEK/ERK are interlinked growth and survival signaling pathways that are often constitutively activated in human tumors as a result of amplifications and/or mutations in PIK3CA, RAS and BRAF genes. In the present work, we have investigated the anti-tumor activity and documented the steady state pharmacodynamic impact of combinations between 2 selective pan PI3K Class I inhibitors, SAR245408 (XL147) and SAR245409 (XL765) with the selective allosteric inhibitor of MEK1/2, pimasertib (AS703026; MSC1936369B) against several different patient derived colorectal cancer (CRC) xenograft models harboring either KRAS mutations, dual KRAS and BRAF mutations or dual KRAS and PIK3CA mutations.

In the BRAF driven CRC model, effects of the combinations were not significantly different from that of single agent treatment of pimasertib alone. In the G12V KRAS or G12D KRAS mutant CRC models, both combinations led to significantly greater anti-tumor activity than that of single agent treatment. In the dual PIK3CA/KRAS mutant tumors, combination therapies lead to a potent inhibition of both TORC1 and TORC2 pathways, in addition to inhibition of the MAPK pathway in contrast to single agent treatments. Overall, pimasertib combined with either SAR245409 or SAR245408 showed a significantly greater anti-tumor activity as compared to single agent treatment alone in primary models bearing KRAS mutations or dual PIK3CA/KRAS mutations. The combination of pimasertib and SAR245409 is currently being investigated in patients with PIK3CA/KRAS mutant colorectal cancer as part of an ongoing Phase Ib dose escalation trial of oral combination therapy with pimasertib (MSC1936369B) and SAR245409 in subjects with locally advanced or metastatic solid tumors.

Citation Format: Sukhvinder S. Sidhu, Coumaran Egile, Marc Malfilatre, Celine Lefranc, Yvette Ruffin, Jianguo Ma, Karl Hsu, Joanne Lager, Stephane Marzabal, Janet A. Ogden, Loic Vincent. Anti-tumor activity of pimasertib in combination with SAR245409 or SAR245408 in human primary colorectal cancer xenograft models bearing PI3K/KRas and KRas mutations. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4638. doi:10.1158/1538-7445.AM2013-4638