Diverse Tumor Types Research Articles

ErbB-targeted CAR T-cell immunotherapy of cancer.

Chimeric antigen receptor (CAR) based immunotherapy has been under development for the last 25 years and is now a promising new treatment modality in the field of cancer immunotherapy. The approach involves genetically engineering T cells to target malignant cells through expression of a bespoke fusion receptor that couples an HLA-independent antigen recognition domain to one or more intracellular T-cell activating modules. Multiple clinical trials are now underway in several centers to investigate CAR T-cell immunotherapy of diverse hematologic and solid tumor types. The most successful results have been achieved in the treatment of patients with B-cell malignancies, in whom several complete and durable responses have been achieved. This review focuses on the preclinical and clinical development of CAR T-cell immunotherapy of solid cancers, targeted against members of the ErbB family.

Arachidonoyl-ethanolamide activates endoplasmic reticulum stress-apoptosis in tumorigenic keratinocytes: Role of cyclooxygenase-2 and novel J-series prostamides.

Non-melanoma skin cancer and other epithelial tumors overexpress cyclooxygenase-2 (COX-2), differentiating them from normal cells. COX-2 metabolizes arachidonic acid to prostaglandins including, the J-series prostaglandins, which induce apoptosis by mechanisms including endoplasmic reticulum (ER) stress. Arachidonoyl-ethanolamide (AEA) is a cannabinoid that causes apoptosis in diverse tumor types. Previous studies from our group demonstrated that AEA was metabolized by COX-2 to J-series prostaglandins. Thus, the current study examines the role of COX-2, J-series prostaglandins, and ER stress in AEA-induced apoptosis. In tumorigenic keratinocytes that overexpress COX-2, AEA activated the PKR-like ER kinase (PERK), inositol requiring kinase-1 (IRE1), and activating transcription factor-6 (ATF6) ER stress pathways and the ER stress apoptosis-associated proteins, C/EBP homologous protein-10 (CHOP10), caspase-12, and caspase-3. Using an ER stress inhibitor, it was determined that ER stress was required for AEA-induced apoptosis. To evaluate the role of COX-2 in ER stress-apoptosis, HaCaT keratinocytes with low endogenous COX-2 expression were transfected with COX-2 cDNA or an empty vector and AEA-induced ER stress-apoptosis occurred only in the presence of COX-2. Moreover, LC-MS analysis showed that the novel prostaglandins, 15-deoxyΔ(12,14) PGJ2 -EA and Δ(12) PGJ2 /PGJ2-EA, were synthesized from AEA. These findings suggest that AEA will be selectively toxic in tumor cells that overexpress COX-2 due to the metabolism of AEA by COX-2 to J-series prostaglandin-ethanolamides (prostamides). Hence, AEA may be an ideal topical agent for the elimination of malignancies that overexpress COX-2.

DAP1 high expression increases risk of lymph node metastases in squamous cell carcinoma of the oral cavity.

Death-associated protein 1 (DAP1) is a member of the DAP family. Its expression is associated with cell growth and normal death of the neoplastic cells, regulated by the mammalian target of the rapamycin protein. Activated DAP1 negatively regulates autophagy, which has been associated with the development and progression of several diseases, such as cancer, and with prognosis and survival of diverse tumor types. Therefore, in this study we analyzed DAP1 expression in 54 oral squamous cell carcinoma tumor samples and in 20 non-tumoral margins by immunohistochemistry. The results showed that DAP1 is more frequently expressed in tumor tissues compared with marginal non-tumoral cells. Additionally, high DAP1 expression is associated with a 4-fold increase in the risk of lymph node metastases. Our results suggest that the DAP1 protein can be used as a potential marker of lymph node metastases predisposition, helping define the best therapy for each patient to minimize risk of developing metastases.

Inhibition of intracerebral glioblastoma growth by targeting the insulin-like growth factor 1 receptor involves different context-dependent mechanisms.

Signaling by insulin-like growth factor 1 receptor (IGF-1R) can contribute to the formation and progression of many diverse tumor types, including glioblastoma. We investigated the effect of the IGF-1R blocking antibody IMC-A12 on glioblastoma growth in different in vivo models. U87 cells were chosen to establish rapidly growing, angiogenesis-dependent tumors in the brains of nude mice, and the GS-12 cell line was used to generate highly invasive tumors. IMC-A12 was administered using convection-enhanced local delivery. Tumor parameters were quantified histologically, and the functional relevance of IGF-1R activation was analyzed in vitro. IMC-A12 treatment inhibited the growth of U87 and GS-12 tumors by 75% and 50%, respectively. In GS-12 tumors, the invasive tumor extension and proliferation rate were significantly reduced by IMC-A12 treatment, while apoptosis was increased. In IMC-A12-treated U87 tumors, intratumoral vascularization was markedly decreased, and tumor cell proliferation was moderately reduced. Flow cytometry showed that <2% of U87 cells but >85% of GS-12 cells expressed IGF-1R. Activation of IGF-1R by IGF-1 and IGF-2 in GS-12 cells was blocked by IMC-A12. Both ligands stimulated GS-12 cell proliferation, and IGF-2 also stimulated migration. IMC-A12 inhibited these stimulatory effects and increased apoptosis. In U87 cells, stimulation with either ligand had no functional effect. IGF-1R blockade can inhibit glioblastoma growth by different mechanisms, including direct effects on the tumor cells as well as indirect anti-angiogenic effects. Hence, blocking IGF-1R may be useful to target both the highly proliferative, angiogenesis-dependent glioblastoma core component as well as the infiltrative periphery.

Biomarkers of Response to the Glutaminase Inhibitor CB-839 in Multiple Myeloma Cells

CB-839 is a potent, orally bioavailable small-molecule inhibitor of the tumor metabolism target glutaminase (GLS) that is currently in Phase 1 clinical trials for the treatment of solid and hematological malignancies. GLS is a mitochondrial enzyme that converts Gln to glutamate (Glu) to support several metabolic processes including amino acid synthesis, maintenance of cellular redox homeostasis, and the replacement of TCA cycle intermediates. CB-839 has in vitro antiproliferative activity across a sub-set of cell lines from diverse hematological tumor types including multiple myeloma (MM), acute lymphocytic leukemia, and non–Hodgkin’s lymphoma [Parlati et al. Blood 2013 122:4226]. To identify biomarkers that would predict sensitivity to CB-839 in MM, we profiled cellular metabolites, mRNA transcripts, and oncogenic signaling pathways in eight MM cell lines representing four CB-839-sensitive cell lines (RPMI8226, MM.1S, KMS-11, and IM-9) and four CB-839-resistant cell lines (AMO-1, L-363, KMS-28PE, and OPM-2).CB-839 treatment for 4 hours significantly decreased the levels of amino acids (Glu, aspartate, proline) as well as TCA cycle intermediates (fumarate, malate, succinate) across all cell lines. However, prior to treatment, CB-839-sensitive cells had significantly lower baseline levels of pyruvate-, fumarate-, and succinyl-CoA-derived amino acids compared to CB-839-resistant cells. In addition, both the adenylate and guanylate energy charges, a measure of cellular metabolic activity [Atkinson and Walton, J. Biol. Chem. 1967 242: 3239-41], were significantly lower in CB-839 sensitive cells. These observations suggest that cells with low levels of amino acids and/or low cellular energy charge are more susceptible to the pharmacological effects of CB-839.Reverse phase protein array and immunoblot analysis were used to evaluate the impact of CB-839 on signaling pathways across the panel of cell lines. Consistent with the observed decreases in amino acid pools, CB-839 treatment for 4 hours led to an acute inhibition of the amino-acid sensing kinase mTORC1 across all cell lines, as evidenced by decreased phosphorylation of p70S6K and S6. CB-839 treatment also reduced the levels of c-Myc across all cell lines, consistent with a block in five-prime cap-dependent translation as a likely consequence of mTORC1 inhibition. However, untreated sensitive cells had lower baseline levels of both 4E-BP1 and phospho-4E-BP1 in comparison to resistant cells, and at 24 hours following treatment, the mTORC1-dependent activation of CAD, a key enzyme in pyrimidine biosynthesis (Ben-Sahra et al., Science 2013 15: 1323-8), was reduced in CB-839-sensitive but not in resistant cells. These results suggest that CB-839 inhibits mTORC1 signaling in MM cells and that sensitive cells may lack compensatory mechanisms to overcome this inhibition.Consistent with differential regulation of mTORC1 in CB-839-sensitive versus resistant cells, RNAseq analysis showed greater down-regulation of several mTORC1-regulated transcripts encoding glycolytic enzymes (such as ALDOA and TPI1, Düvel et al., 2010 Mol. Cell 39: 171-83) in sensitive cells versus resistant cells following CB-839 treatment. RNAseq analysis also revealed elevated levels of pyruvate carboxylase (PC) in CB-839 resistant cells as compared to sensitive cells. Immunoblot analysis of twenty four MM cell lines supported this observation; PC protein levels were inversely correlated with response to CB-839 (r = 0.64, p = 0.001) and PC protein and mRNA transcript levels were highly correlated (r = 0.94, p = 0.0004). PC is an anaplerotic enzyme that converts pyruvate to oxaloacetate. Therefore, sufficiently high levels of glycolytic enzymes and PC could lessen the dependence of a MM cell line on Gln metabolism for replenishment of TCA intermediates.The studies described herein elucidate differences in metabolic and signaling pathways among MM cell lines. These differences in metabolites, mTORC1 signaling, glycolytic enzymes and PC may account for differential sensitivity to CB-839. We are exploring the potential utility of PC expression as a biomarker in our ongoing clinical studies of CB-839. DisclosuresMackinnon:Calithera Biosciences: Employment, Equity Ownership. Bennett:Calithera Biosciences: Employment, Equity Ownership. Rodriguez:Calithera Biosciences: Employment, Equity Ownership. Parlati:Calithera Biosciences: Employment, Equity Ownership.

A chimeric antibody targeting CD147 inhibits hepatocellular carcinoma cell motility via FAK-PI3K-Akt-Girdin signaling pathway.

CD147 is expressed at low levels in normal tissues but frequently highly expressed in a wide range of tumor types such as lung, breast, and liver and therefore it is a potentially unique therapeutic target for these diverse tumor types. We previously generated a murine antibody HAb18 which suppresses matrix met al.loproteinase-2 and matrix metalloproteinase-9 secretion, attenuates cell invasion by blocking the CD147 molecule in tumor cells. Here, we generated a chimeric antibody containing the variable heavy and variable light chains of murine HAb18 and the constant regions of human IgG1γ1 and human κ chain as a potential therapeutic agent (designated cHAb18). Quantitative measurement of cHAb18 antibody affinity for antigen CD147 with surface plasmon resonance showed the equilibrium dissociation constant KD was 2.66 × 10(-10) mol/L, similar to that of KD 2.73 × 10(-10) mol/L for murine HAb18. cHAb18 induced antibody-dependent cell-mediated cytotoxicity in two hepatocellular carcinoma cell lines, SMMC-7721 and Huh-7 cells. It inhibited cancer invasion and migration in hepatocellular carcinoma cells by specifically blocking CD147. Except for the depression of matrix metalloproteinase-2 and matrix metalloproteinase-9 expressions, cHAb18 antibody suppressed cell motility by rearrangement of actin cytoskeleton, which was probably induced by decreasing the phosphorylation of focal adhesion kinase, phosphatidylinositide-3 kinase (PI3K), Akt, and Girdin in the integrin signaling pathway. In an orthotopic model of hepatocellular carcinoma in BALB/c nude mice, cHAb18 treatment effectively reduced the tumor metastasis in liver and prolonged the survival. These findings reveal new therapeutic potential for cHAb18 antibody targeting CD147 on tumor therapy.

Potential microRNA-mediated oncogenic intercellular communication revealed by pan-cancer analysis.

Carcinogenesis consists of oncogenesis and metastasis, and intriguingly microRNAs (miRNAs) are involved in both processes. Although aberrant miRNA activities are prevalent in diverse tumor types, the exact mechanisms for how they regulate cancerous processes are not always clear. To this end, we performed a large-scale pan-cancer analysis via a novel probabilistic approach to infer recurrent miRNA-target interactions implicated in 12 cancer types using data from The Cancer Genome Atlas. We discovered ~20,000 recurrent miRNA regulations, which are enriched for cancer-related miRNAs/genes. Notably, miRNA 200 family (miR-200/141/429) is among the most prominent miRNA regulators, which is known to be involved in metastasis. Importantly, the recurrent miRNA regulatory network is not only enriched for cancer pathways but also for extracellular matrix (ECM) organization and ECM-receptor interactions. The results suggest an intriguing cancer mechanism involving miRNA-mediated cell-to-cell communication, which possibly involves delivery of tumorigenic miRNA messengers to adjacent cells via exosomes. Finally, survival analysis revealed 414 recurrent-prognostic associations, where both gene and miRNA involved in each interaction conferred significant prognostic power in one or more cancer types. Together, our comprehensive pan-cancer analysis provided not only biological insights into metastasis but also brought to bear the clinical relevance of the proposed recurrent miRNA-gene associations.

Generation of cytotoxic T lymphocytes specific for native or modified peptides derived from the epidermal growth factor receptor pathway substrate 8 antigen.

The ideal tumor antigen for the development of a cancer immunotherapy is one that is expressed only in tumor cells. The epidermal growth factor receptor pathway substrate 8 gene (Eps8) might be an effective antigen for cancer immunotherapy as it is overexpressed in a variety of cancer cells but not in normal tissues. In this study, the potential utility of an Eps8-derived immunotherapy was tested in vitro and in vivo. Three computer-based algorithms were used to design eight Eps8 native epitopes with potentially high binding affinity to the HLA-A2.1 molecule, which is found at a high frequency in the Chinese population. Of these eight, three peptides with a moderate affinity to the HLA-A2.1 molecule were modified at anchor residue positions to achieve stronger immunogenicity. These four modified peptides displayed stronger binding affinity to HLA-A2.1 molecules on T2 cells and a lower dissociation rate. In functional assays with human PBMCs in vitro and in HLA-A2.1/K(b) transgenic mice in vivo, CTLs primed by each native and modified peptide secreted IFN-γ and were toxic to cancer cells from a variety of tissue types in an HLA-A2.1-restricted and Eps8-specific manner. p101-109-2L and p276-284-1Y9V were superior to other modified and native epitopes both in vitro and in vivo. These results indicate that employing the native and modified epitopes identified here in Eps8-based immunotherapy for HLA-A2.1 positive cancer patients may result in efficient anticancer immune responses for diverse tumor types.

Abstract A23: Comprehensive next-generation sequencing-based genomic profiling identifies actionable genomic alterations in diverse pediatric tumor types: The Foundation Medicine (FMI) experience

Abstract Background: Solid tumor oncology is amidst a paradigm shift with the advent and increasingly successful utilization of targeted therapies that inhibit specific genomic alterations driving an individual patient's disease. Unfortunately, many pediatric tumors lack approved targeted therapies, and routine genomic profiling of pediatric tumors has yet to be applied in a widespread manner. More comprehensive testing platforms are required to determine the landscape of genomic alterations in pediatric solid tumors and thereby broaden targeted treatment options. We have developed a solid tumor next-generation sequencing (NGS)-based diagnostic test, optimized for routine clinical FFPE specimens including needle biopsies and malignant effusions, and report here on 193 pediatric patients' tumors analyzed to date in our CLIA-certified and CAP-accredited laboratory. Methods: Hybridization capture of 3,320 exons from 182 cancer-related genes and 37 introns of 14 genes commonly rearranged in cancer (previous version of the test) or 3,769 exons from 236 cancer-related genes and 47 introns of 19 genes commonly rearranged in cancer (current version of the test) was applied to ≥ 50ng of DNA extracted from over 190 pediatric FFPE tumor specimens and sequenced to high, uniform coverage. Genomic alterations (base substitutions, small indels, rearrangements, copy number alterations) were determined and then reported for these patient samples. Results: Successful profiles were generated from 193 pediatric patient specimens from individuals ≤21 years old at the time of biopsy. A total of 361 genomic alterations were identified, with 145/193 (75%, 95% CI 68%-81%) of cases harboring at least one genomic alteration, for an average of 2.5 genomic alterations per case (range 0-14). The number of genomic alterations varied widely depending on tumor type (average alterations per sample): 4 colon cancer samples (9.0), 11 bone sarcoma samples (3.7), 13 leukemia samples (3.1), 23 brain cancer samples (2.4), 27 soft tissue sarcoma samples (2.2), 6 liver samples (2.0), 26 neuroblastoma samples (1.7), 6 lung cancer samples (1.2), 5 kidney cancer samples (1.2), and 24 other tumor types. These alterations included 146 base substitutions, 54 indels, 133 copy number alterations, and 28 rearrangements. This corresponded to alterations within 102 genes with recurrent alterations observed in 61 genes. We identified 249 actionable alterations with 109/193 (56%) of these pediatric patients found to have at least one actionable alteration. Seventy one percent (176/249) of the actionable genomic alterations would not be detected by available tumor type specific tests or hotspot panels. This approach has led to novel insights into pediatric cancers including: novel potentially druggable kinase gene fusions (e.g. ALK, RET) and non-fusion alterations in known drug targets (e.g. EGFR, BRAF, ALK, MET, PTCH1, PIK3CA, KIT, PDGFRA, FGFR1). Conclusions: Comprehensive NGS-based genomic profiling identified alterations in 75% of 193 unselected pediatric cancer clinical cases; and 56% of these patients (N=109) were found to have at least one therapeutically actionable alteration. Widespread deployment of this approach may provide treatment options for pediatric cancer patients. Citation Format: Matthew J. Hawryluk, Kai Wang, Juliann Chmielecki, Siraj M. Ali, Gary Palmer, Lazaro Garcia, Emily White, Roman Yelensky, Philip J. Stephens, Jeffrey S. Ross, Vincent A. Miller. Comprehensive next-generation sequencing-based genomic profiling identifies actionable genomic alterations in diverse pediatric tumor types: The Foundation Medicine (FMI) experience. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A23.

Abstract 5338: Inferring evolutionary models of tumor progression from single-cell heterogeneity data

Abstract We describe computational methods to compute likely evolutionary histories from tumor single-cell copy number data and next generation sequencing data and apply the methods to data collected from diverse types of tumors. Experimental techniques for assessing heterogeneity in tumor cell populations have undergone great advances, but these improvements have created a great need for more sophisticated computer algorithms capable of making sense of these data sources in terms of coherent models of tumor evolution. We have addressed this problem by developing computer algorithms for building phylogenetic trees describing evolution of individual tumors based on copy numbers of fluorescence in situ hybridization (FISH) probes from single cells in these tumors. These algorithms reconstruct evolutionary trees for observed cell populations so as to heuristically minimize the number of mutational events needed to explain the observed combinations of probe counts by evolution from a common diploid ancestral cell. We have extended this work from initial simple evolutionary models of evolution by single copy number changes to account for distinct mechanisms of evolution at the gene, chromosome, or whole-genome scale, with potentially different rates of evolution by mutation type. We have applied these algorithms to several FISH data sets, including cervical cancers probed for four genes (LAMP3, PROX1, PRKAA1 and CCND1) measured for up to 250 cells of paired primary and metastatic samples from 16 patients, head-and-neck cancers probed for four genes (TERC, CCND1, EGFR and TP53) measured on up to 250 cells per patient for 65 patients at four tumor stages, prostate cancers probed for six genes (TBL1XR1, CTTNBP2, MYC, PTEN, MEN1 and PDGFB) measured for up to 407 cells in 6 non-progressive and 7 progressive carcinomas, and breast cancers probed for eight genes (COX-2, MYC, CCND1, HER-2, ZNF217, DBC2, CDH1 and TP53) measured on up to 220 cells of paired of ductal carcinoma in situ and invasive ductal carcinoma samples from 13 patients. We have then applied statistical and machine learning analysis to examine the ability of these trees to classify tumors by stage or potential for progression. The evolutionary tree models reveal robust features of evolutionary processes distinguishing progression stages and predicting future progression that lead to improved classification accuracy relative to predictions from cellular heterogeneity data alone. Our software is freely available at ftp://ftp.ncbi.nlm.nih.gov/pub/FISHtrees. In continuing work, we are exploring extension of these approaches to better modeling and analysis of tumor evolution using single-cell sequencing data and to more detailed models of tumor evolution. Citation Format: Salim A. Chowdhury, Ayshwarya Subramanian, Alejandro A. Schäffer, Stanley E. Shackney, Darawalee Wangsa, Kerstin Heselmeyer-Haddad, Thomas Ried, Russell Schwartz. Inferring evolutionary models of tumor progression from single-cell heterogeneity data. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5338. doi:10.1158/1538-7445.AM2014-5338

Abstract 3625: Tumor-specific human monoclonal antibodies isolated from cancer patients

Abstract Introduction: New cancer-specific antigens are needed to fully realize the potential of antibody-based cancer therapies. Cancer cells express novel antigens that are characteristic of the transformed state and may engender an antibody response in patients. We hypothesize that some of these antibodies detect neo-antigens shared by diverse tumor types. We optimized a hybridoma method for cloning human IgG monoclonal antibodies (mAbs) by screening for binding to heterologous cell lines. Here, we describe four mAbs, isolated from cancer patients, that bind to cancer cells. Methods: We developed immunofluorescence binding assays with the A431, MCF-7, and MDA-MB-231 cell lines, detecting cell-reactive human IgG with the Operetta (Perkin Elmer). The assays were adapted for extracellular binding as well as internalization using specific culture conditions. We tested sera from 59 lung cancer patients with diverse clinical features. 24 of the samples contained IgG reactivity with A431 cells at &amp;gt; 1:100 titer, and 17 had evidence of IgG internalization. We generated hybridomas using peripheral blood B-cells isolated from two of these lung cancer patients, as well as B-cells from a lymph node of a stage I, ER+PR+ breast cancer patient. We characterized the binding of four mAbs using immunohistochemistry (IHC), microscopy, flow cytometry and immunoblotting. One mAb was used to identify its cognate antigen by immunoprecipitation and mass spectrometry. Results: In a panel of 59 lung cancer patient sera, 24 had IgG that bound the A431 cell line, and 17 of these had evidence of internalization. We isolated three human mAbs from two lung cancer patients and one mAb from the breast cancer lymph node. We performed IHC with three of the mAbs in formalin fixed, paraffin embedded normal and tumor tissues and found primarily tumor cell binding to a variety of tumor types. The 5A6 (lung) bound 3 of 4 non-small cell lung, 3 of 4 colon, 2 of 4 ovarian and 1 of 4 small cell lung cancers. 19F10 (lung) bound 1 of 4 colon and 2 of 4 small cell lung. 2G4 (breast) bound 4 of 4 non-small cell lung, 3 of 4 prostate, 3 of 4 ovarian, and 2 of 4 colon cancers. Some normal tissue binding was observed, such as small intestine and distal renal tubule (2G4 and 19F10), pancreatic islet (19F10), and prostate (2G4). The 6A10 mAb (lung) bound to the constitutive heat shock protein, HSC70, which was localized in the plasma membrane of cancer cell lines. Conclusion: We have cloned 4 human mAbs from cancer patients. This supports the hypothesis that patients with active malignancies make antibodies capable of binding cancer-specific antigens. We identified HSC70, a protein that has an altered cell localization in cancer cells, as one such antigen. The three mAbs tested by IHC bound to diverse tumor types that often differed from the tumors of the patients where the B-cells originated. The human anti-cancer antibody repertoire holds promise as a tool to identify new targets for mAb-based cancer diagnostics and therapeutics. Citation Format: Huiwu Zhao, Jiping Zhang, Ramdev Puligedda, Cezary Swider, Paul Simon, Baron Heimbach, Sharad Adekar, Maureen Murphy, Hossein Borghaei, Scott Dessain. Tumor-specific human monoclonal antibodies isolated from cancer patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3625. doi:10.1158/1538-7445.AM2014-3625

Abstract 1815: Efficacy of FASN-selective small molecule inhibitors in preclinical tumor models

Abstract Fatty acid synthase (FASN) expression increases with tumor progression and is associated with impaired response to chemotherapy and diminished patient survival in many solid and hematopoietic tumor types. Palmitate, the product of FASN enzymatic activity, functions in vital cellular processes including energy metabolism and cell growth, cellular membrane biosynthesis and architecture, and protein localization and function. 3-V Biosciences has discovered and developed a series of potent, selective, orally available, and reversible FASN inhibitors with excellent pharmaceutical properties. Single agent FASN inhibition has been shown to cause anti-tumor activities in preclinical models of human cancer, including inhibition of AKT and mTOR signal transduction, induction of tumor cell apoptosis, and tumor growth inhibition and regression in pancreatic, ovarian, and lung cancer xenograft tumor models of human cancer. Current studies are supporting the active clinical development of TVB-2640, 3-V Biosciences' first-in-class FASN inhibitor, as an oncology therapeutic. These studies have multiple objectives: (1) characterizing a wide range of tumor types for responsiveness to FASN inhibition using tumor cell line and Champions TumorGraftTM efficacy models; (2) investigating the benefit of FASN combination therapy with chemotherapeutic or targeted anti-cancer agents; (3) developing biomarkers that characterize efficacy response; and (4) further elucidating the mechanisms of action that emanate from the inhibition of palmitate synthesis and lead to tumor cell apoptosis and in vivo anti-tumor efficacy. The results of these investigations show that FASN inhibition causes tumor growth inhibition or regression in diverse tumor types; and moreover, that tumor growth inhibition additivity or synergy is observed when FASN inhibition is combined with chemotherapy agents. Genomics and directed analysis of mRNA, proteins, and lipids following FASN inhibition have identified biomarker candidates and provided insights into the tumor growth inhibition mechanisms of action. For example, genome-wide gene expression analysis shows FASN inhibition to significantly increase the expression of many genes in biosynthetic (sterol, lipid, etc.) and apoptosis pathways while significantly reducing the expression of many genes in cell growth and proliferation pathways (DNA replication, cell cycle progression, mitosis, etc.). These data strongly support the clinical development of TVB-2640 as a first-in-class single and combination oncology therapeutic and are advancing the discovery and validation of biomarkers to inform clinical utility and response. Citation Format: Timothy S. Heuer, Richard Ventura, Joanna Waszczuk, Kasia Mordec, Julie Lai, Russell Johnson, Lilly Hu, Haiying Cai, Allan Wagman, Douglas Buckley, Stanley T. Parish, Elizabeth Bruckheimer, George Kemble. Efficacy of FASN-selective small molecule inhibitors in preclinical tumor models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1815. doi:10.1158/1538-7445.AM2014-1815

Abstract 2094: Regulation of site-specific liver metastasis by collagen IV-conveyed signals

Abstract The liver is a primary site of metastasis for some of the most common human malignancies. At present, surgical resection is the most effective curative option for liver metastases, but most patients with liver metastases still succumb to their disease. A better understanding of the underlying biology is essential for design of more effective therapy. We previously identified basement membrane type IV collagen α1/α2 expression levels as determinants of the liver colonizing potential in a murine lung carcinoma model (1) and observed high collagen IV expression levels in surgical specimens of liver metastases from diverse tumor types. The aim of this study was to elucidate the functional relevance of increased collagen IV expression to liver metastases formation. We reported that collagen IV α1/α2 overexpression in non-metastatic lung carcinoma M-27 (M27colIV) cells increased specifically their liver (but not lung) metastasizing potential. A transcriptome analysis was therefore performed on these cells in order to identify changes to gene expression that could account for the newly acquired metastatic potential. This analysis revealed that type IV collagen levels regulated the expression of multiple genes. Prominent among them were genes encoding for chemokines such as CCL-5 and CCL-7 and for growth factors such as amphiregulin (AREG) that were all upregulated by ≥ 3 fold. These changes were validated at the RNA and protein levels using qPCR and Western blotting, respectively. When the expression of these genes in M27colIV cells was subsequently silenced using shRNA, a significant decrease (5-10 folds) was observed in their ability to generate experimental liver metastases, as compared to wild type or mock-transfected cells and they failed to develop metastases in 40-50 % of injected mice. A similar correlation between CCL-5 and CCL-7 expression levels and liver metastases formation was also noted in human colon carcinoma cell lines. The results identify a type IV collagen-regulated gene expression signature that promotes liver metastasis and suggest that collagen IV-induced changes in chemokine and growth factor production levels mediate this effect. They provide a rationale for further exploration of the clinical utility of CCL-5, CCL-7 and AREG as targets in the management of liver metastases. Supported by a grant from the Canadian Institute for Health Research (to PB) and a Henry R. Shibata Cedars Cancer Fellowship (to RR). REFERENCES 1. Burnier JV, Wang N, Michel RP, Hassanain M, Li S, Lu Y, et al. Type IV collagen-initiated signals provide survival and growth cues required for liver metastasis. Oncogene. 2011;30:3766-83. Citation Format: Roni F. Rayes, Ni Wang, Julia V. Burnier, France Bourdeau, Pnina Brodt. Regulation of site-specific liver metastasis by collagen IV-conveyed signals. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2094. doi:10.1158/1538-7445.AM2014-2094

Abstract 3728: A discovery platform for allosteric modulators of c-Abl kinase function

Abstract The c-Abl protein-tyrosine kinase regulates intracellular signaling pathways controlling cell growth, adhesion, and responses to genotoxic stress. The kinase activity of c-Abl is repressed by intramolecular interactions involving its non-catalytic SH3 and SH2 domains. The SH3 domain interacts with a polyproline type II helix formed by the SH2-kinase linker, while the SH2 domain interacts with the C-lobe of kinase domain away from the active site. In addition, the myristoylated N-terminal cap region (Ncap) binds a hydrophobic pocket in the C-lobe of the kinase domain, clamping the SH3 and SH2 domains against the back of the kinase domain. Small molecules that allosterically regulate c-Abl kinase activity through its non-catalytic domains may represent selective probes of c-Abl function in diverse tumor types. In this study, we developed a chemical library screening assay designed to identify chemical modulators of c-Abl kinase activity that either disrupt or stabilize the regulatory interaction of the SH3 domain with the SH2-kinase linker. This fluorescence polarization (FP) assay is based on a recombinant purified protein consisting of the c-Abl Ncap, SH3 and SH2 domains plus the linker (N32L protein) and a short fluorescein-labeled probe peptide that binds to the SH3 domain. In assay development experiments, we found that the probe peptide binds to the recombinant c-Abl N32L protein in vitro, producing a robust FP signal that can be competed with an excess of unlabeled peptide. The FP signal is not observed with control N32L proteins bearing either an inactivating mutation in the SH3 domain or enhanced SH3:linker interaction. The assay was optimized with respect to probe peptide sequence, probe and N32L protein concentrations, DMSO tolerance, and FP signal stability over time. Pilot library screens were then performed with the NCI Diversity Set III and an FDA-approved drug library (2800 compounds total). Seven compounds were identified that specifically reduced the FP signal by at least three standard deviations from the untreated controls. Secondary assays showed that one of these hit compounds enhances c-Abl kinase activity in vitro, suggesting that it disrupts regulation of kinase activity through the non-catalytic region. These results show that screening assays based on the non-catalytic domains of c-Abl can identify small molecule regulators of kinase function. Citation Format: Prerna Grover, Thomas E. Smithgall. A discovery platform for allosteric modulators of c-Abl kinase function. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3728. doi:10.1158/1538-7445.AM2014-3728

Abstract 2811: Allogeneic cell cased vaccine development by shared antigen screening

Abstract The fundamental premise of allogeneic vaccines for cancer relies upon the presence of common antigens that are shared between established cell lines and a large proportion of patients with histologically related tumors. Recent results from The Cancer Genome Atlas have clearly demonstrated that somatic mutations in exon region DNA, while potentially immunogenic, do not result in the translation of antigenic proteins that are similar from patient to patient. Instead, these data reinforce the concept that dysregulation of common pathways contributes to the general property of de-differentiation in tumor cells and the acquisition of immature phenotypes as compared to untransformed precursor cells. This phenomenon leads to expression of mucinous and embryonal antigens, collectively known as cancer testis antigens, that are otherwise rare in mature tissues and to which central tolerance remains incomplete. We have developed allogeneic vaccines based on parallel screening of non-small cell lung, bladder, triple-negative breast, ovarian and melanoma tumor cell lines and patient tumor samples. The baseline expression of certain antigens, including SSX2, SSX4, CT10, MAGE-A1, MAGE-A10, LAGE-1 and NY-ESO-1 was found to vary widely both between tumor samples and non-transformed tissue controls. Among all antigens screened, MAGE-A3 was most commonly upregulated across transformed tissue samples, however other antigens including BAGE-1, XAGE-1b and MAGE-A10 were found to be most dramatically upregulated. This analysis provides a screening tool with which to appropriately profile and prioritize individual cell lines for vaccine development in histologically diverse tumor types. Citation Format: Vadim V. Deyev, Neal Schilling, Taylor H. Schreiber. Allogeneic cell cased vaccine development by shared antigen screening. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2811. doi:10.1158/1538-7445.AM2014-2811

Abstract 1040: An integrated analysis of EMT across diverse cancer types identifies new potential therapeutic targets

Abstract Background: Epithelial-to-mesenchymal transition (EMT) is a marker of resistance to specific targeted drugs in non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSC), and other epithelial cancer and may be associated with poor patient outcomes. Here we performed an integrated molecular analysis in 1934 tumors representing 11 different cancer types in the Cancer Genome Atlas (TCGA) and 1264 representative cell lines to investigate new potential therapeutic targets (PTTs) in mesenchymal tumors. Methods: For each tumor, we correlated the EMT score (based on expression of an EMT gene signature) with mutation status, copy number alterations (CNA), mRNA, miRNA, and protein expression. PTTs expressed in mesenchymal tumors were investigated in vitro using publically available drug sensitivity datasets (DSDs) by correlating IC50s with EMT score. Results: Most cancer types exhibited a range of EMT scores that included both epithelial and mesenchymal tumors, with the exception of kidney clear cell carcinoma (KIRC) (all mesenchymal) and ovarian serous cystadenocarcinoma (OV) (primarily epithelial). Patients with mesenchymal OV and HNSC experienced shorter overall survival (p=0.029 and 0.076, respectively). BAP1 and PP2R1A mutations were more frequent in mesenchymal tumors, independent of primary site (adjusted p value &amp;lt;0.05). Conversely, NSD1 and KEAP1 mutations were more frequent in tumors with low EMT scores (epithelial) (p&amp;lt;0.05). Overall mutation burden and CNAs were not associated with mesenchymal status. A striking association between miR expression and EMT score was observed across most tumor types, including low miR 200 family and high miR 199a and 214 expression in mesenchymal cancers (r&amp;gt;0.3). PTTs commonly expressed in mesenchymal tumors included AXL, PDGFRA/B, MMP2 and DDR2 (r&amp;gt;0.6). Of these, investigation of the DSDs supported in vitro sensitivity of mesenchymal cell lines overall to PDGFR and GSK3 inhibitors, but resistance to drugs targeting ErbB family members, including the dual VEGF/EGFR inhibitor vandetanib (p&amp;lt;0.001) and drugs targeting Her2 and/or EGFR (p&amp;lt;0.001). In contrast, other EMT-associated drug resistance was tissue specific, such as resistance of mesenchymal breast cancer lines to mTOR inhibition (p 0.03). Conclusions: Application of the EMT signature to diverse tumor types identifies highly conserved patterns of gene and miRs expression, including high miR-199a expression in mesenchymal cancers. While mutational landscape and CNAs were most often driven by tumor type, protein expression and drug sensitivity were influenced by a combination of tumor type and EMT status. New associations between drug response and EMT were identified, such as the sensitivity of mesenchymal cancers to PDGFR and GSK3 inhibitors, that warrant further investigation. Citation Format: Pan Tong, Milena P. Mak, Lixia Diao, Jing Wang, Patrick Kwok-Shing Ng, You-hong Fan, William N. William, John V. Heymach, Kevin R. Coombes, Lauren Averett Byers. An integrated analysis of EMT across diverse cancer types identifies new potential therapeutic targets. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1040. doi:10.1158/1538-7445.AM2014-1040

Stereotactic Body Radiation Therapy for Oligometastatic Pulmonary Lesions

Chemotherapy is standard therapy for metastatic disease. However, aggressive local therapies may improve outcomes for selected patients (pts) with oligometastatic disease. SBRT in early stage non-small cell lung cancer (NSCLC) has high rates of local control (LC), but little data exist assessing SBRT to treat lung metastases. We assess safety and efficacy of SBRT in pts with 1-5 pulmonary metastases and limited extrathoracic disease, and we compare outcomes by disease. We hypothesized SBRT to lung metastases could achieve high LC across diverse primary tumor types, and overall survival (OS) would vary by disease site of origin and time from stage IV diagnosis.

467P - Fgfr Inhibitor and Chemotherapy in Gastric Cancer (Facing): Phase I Results from an Ecmc Combinations Alliance Phase I/II Trial of Azd4547 in Combination with Cisplatin and Capecitabine (Cx)

ABSTRACT Aim: Fibroblast growth factors (FGFs) and their receptors (FGFRs) control a wide range of biological functions, and are implicated in the pathogenesis of diverse tumour types including gastro-oesophageal adenocarcinoma (OGA). AZD4547 is a potent, selective, small molecule inhibitor of FGFR 1, 2, & 3 tyrosine kinases. The aims of the phase I component of this study were to determine the safety, MTD, & PK profile of AZD4547 when administered with cisplatin (C) and capecitabine (X). Methods: Eligible patients (pts) with refractory advanced solid tumors, adequate performance status (PS), haematologic, renal, hepatic function received 1 of 3 escalating doses of AZD4547 in a “rolling six” dose escalation design. AZD4547 was administered orally (po) bid days 1-14 in combination with C (80 mg/m2 i/v) and X (1000 mg/m2 po bid days 1–14) in 3-weekly cycles. DLTs were based on cycle 1 toxicities which were assessed weekly (clinical, haematologic, biochemical) with ophthalmic assessment and echocardiogram at 4 weeks. Plasma AZD4547 was measured in blood over 24 hrs (day 1). Disease (CT scan) was assessed 9-weekly. Results: 19 pts (11 M, 8F), median age 57 (range: 40-78) were enrolled; 16 with PS 0 (9) or 1 (7) were evaluable for toxicity at 40mg BD (4 pts), 60mg BD (6), or 80mg BD (6) dose levels. Median duration of treatment was 3 cycles (range: 1-6). 4 DLTs occurred in 3 pts: 3 DLTs at 80mg BD (grade 3/4 neutropenia with fever; & > 2 week dose interruption for cardiac toxicity; - 1 pt; grade 3 LFT elevation – 1 pt); 1 DLT at 60mg BD (grade 3 oral mucositis). Cumulative (worst grade, all cycles) grade 3/4 toxicities (excluding DLTs) included hypomagnesaemia (5 pts), electrolyte disturbance (4) neutropenia (3), pulmonary embolism (3), elevated AST / ALT (3), nausea (2), febrile neutropenia (1). 1 partial response (basaloid cervical cancer) was observed at 40mg BD. Stable disease was observed in 7 pts at 9 weeks and in 3 pts at 18 weeks. Conclusions: The recommended dose of AZD4547 is 60mg BD when combined with CX. A randomised phase IIa study of CX + AZD4547 or placebo is on-going in patients with advanced OGA and polysomy or amplification of FGFRs. Disclosure: D. Landers: Employee of AstraZeneca UK Limited, involved in development of the reported drug. All other authors have declared no conflicts of interest.

Patterns of DNA damage response in intracranial germ cell tumors versus glioblastomas reflect cell of origin rather than brain environment: Implications for the anti-tumor barrier concept and treatment

The DNA damage response (DDR) machinery becomes commonly activated in response to oncogenes and during early stages of development of solid malignancies, with an exception of testicular germ cell tumors (TGCTs). The active DDR signaling evokes cell death or senescence but this anti-tumor barrier can be breached by defects in DDR factors, such as the ATM-Chk2-p53 pathway, thereby allowing tumor progression. The DDR barrier is strongly activated in brain tumors, particularly gliomas, due to oxidative damage and replication stress. Here, we took advantage of rare human primary intracranial germ cell tumors (PIGCTs), to address the roles of cell-intrinsic factors including cell of origin, versus local tissue environment, in the constitutive DDR activation in vivo. Immunohistochemical analysis of 7 biomarkers on a series of 21 PIGCTs (germinomas and other subtypes), 20 normal brain specimens and 20 glioblastomas, revealed the following: i) The overall DDR signaling (γH2AX) and activation of the ATM-Chk2-p53 pathway were very low among the PIGCTs, reminiscent of TGCTs, and contrasting sharply with strong DDR activation in glioblastomas; ii) Except for one case of embryonal carcinoma, there were no clear aberrations in the ATM-Chk2-p53 pathway components among the PIGCT cohort; iii) Subsets of PIGCTs showed unusual cytosolic localization of Chk2 and/or ATM. Collectively, these results show that PIGCTs mimic the DDR activation patterns of their gonadal germ cell tumor counterparts, rather than the brain tumors with which they share the tissue environment. Hence cell-intrinsic factors and cell of origin dictate the extent of DDR barrier activation and also the ensuing pressure to select for DDR defects. Our data provide conceptually important insights into the role of DNA damage checkpoints in intracranial tumorigenesis, with implications for the differential biological responses of diverse tumor types to endogenous stress as well as to genotoxic treatments such as ionizing radiation or chemotherapy.

DIFFERENT MECHANISMS OF TUMOR GROWTH INHIBITION BY BLOCKADE OF IGF-IR IN U87-INDUCED AND STEM-LIKE CELL-INDUCED GLIOBLASTOMAS

BACKGROUND: Insulin-like growth factor receptor I (IGF-IR) signaling can contribute to the formation, maintenance, and/or progression of many diverse tumor types, including glioblastomas. We investigated the effect of an IGF-IR blocking antibody (IMC-A12) on tumors derived either from U87 cells or from glioblastoma stem-like (GS) cells in vitro and in vivo. METHODS: Two different xenograft models were established in nude mice. U87 cells were used to generate solid tumors, and the glioblastoma stem-like cell line GS-12 was used to generate diffusely invasive tumors. In both models, IMC-A12 was administered intratumorally via osmotic mini-pumps with catheters for 3-4 weeks. Tumor size, proliferation, apoptosis and angiogenesis were evaluated histologically and immunohistochemically. Receptor expression and activation were analyzed by flow cytometry and immunoprecipitation. Proliferation, apoptosis and migration were studied in vitro. RESULTS: Growth of U87 and of GS-12 tumors was inhibited by 75% and by 50%, respectively. In the solid, non-invasively growing U87 tumors, the intratumoral blood vessel density was reduced significantly, whereas the tumor cell proliferation and apoptosis rates did not differ from controls. In contrast, in the highly invasive GS-12 model, IMC-A12 treatment had no effect on tumor neovascularization, while the proportion of proliferating cells was decreased by 30% compared to controls. We therefore hypothesized that IMC-A12 interfered directly with the stem-like GS-12 glioblastoma cells but not with U87 cells. FACS analyses revealed that only 6% of U87 cells expressed IGF-IR, whereas 87% of GS-12 cells expressed the receptor. IGF-I and IGF-II-induced receptor activation was blocked by IMC-A12 in vitro, and the antibody reduced proliferation of GS-12 cells by 40% and migration by 27%. In contrast, IMC-A12 had no effect on the proliferation or migration of U87 cells. CONCLUSIONS: IGF-IR blockade can inhibit glioblastoma growth in vivo by different mechanisms. In the highly invasive GS-12 model it inhibits proliferation and migration directly. In contrast, in highly angiogenic tumors derived from U87 cells, which barely express IGF-IR, it acts by blocking neovascularization. These findings suggest that the IGF-IR signaling pathway might be useful to target both the highly proliferative angiogenesis-dependent solid tumor component as well as the non-angiogenic infiltrative tumors cells at the invasive edge in glioblastomas. SECONDARY CATEGORY: Tumor Biology.