Petit, Joachim L.

PURPOSE This investigation sought to evaluate the prognostic value of pretreatment of circulating tumor DNA (ctDNA) in metastatic biliary tract cancers (BTCs) treated with platinum-based first-line chemotherapy treatment. MATERIALS AND METHODS We performed a retrospective analysis of 67 patients who underwent ctDNA testing before platinum-based chemotherapy for first-line treatment for metastatic BTC. For analysis, we considered the detected gene with highest variant allele frequency as the dominant clone allele frequency (DCAF). Results of ctDNA analysis were correlated with patients' demographics, progression-free survival (PFS), and overall survival (OS). RESULTS The median age of patients was 67 (27-90) years. Fifty-four (80.6%) of 67 patients evaluated had intrahepatic cholangiocarcinoma; seven had extrahepatic cholangiocarcinoma, and six gallbladder cancers. Forty-six (68.6%) of the patients were treated with cisplatin plus gemcitabine, and 16.4% of patients received gemcitabine and other platinum (carboplatin or oxaliplatin) combinations, whereas 15% of patients were treated on a clinical trial with gemcitabine and cisplatin plus additional agents (CX4945, PEGPH20, or nab-paclitaxel). TP53, KRAS, FGFR2, ARID1A, STK11, and IDH1 were the genes with highest frequency as DCAF. The median DCAF was 3% (0%-97%). DCAF > 3% was associated with worse OS (median OS: 10.8 v 18.8 months, P = .032). Stratifying DCAF in quartiles, DCAF > 10% was significantly related to worse PFS (median PFS: 3 months, P = .014) and worse OS (median OS: 7.0 months, P = .001). Each 1% increase in ctDNA was associated with a hazard ratio of 13.1 in OS when adjusting for subtypes, metastatic sites, size of largest tumor, age, sex, and CA19-9. CONCLUSION DCAF at diagnosis of advanced BTC can stratify patients who have worse outcomes when treated with upfront platinum-based chemotherapy. Each increase in %ctDNA decreases survival probabilities.

176 Background: Despite androgen deprivation therapy, prostate cancer will develop resistance known as castrate resistant prostate cancer (CRPC). Androgen signaling is associated with pathways that influence cell proliferation and survival via cell cycle regulation. AR signaling results in inactivation of Rb tumor suppressor by way of AKT and reduction in intrinsic CDK4/6 inhibitors. Since PTEN loss is found in 40% of mCRPC and AKT pathway is a mechanism of CDK4/6 inhibitor resistance, we hypothesize adding an AKT inhibitor to a CDK4/6 inhibitor will have synergistic activity while delaying CDK4/6 inhibitor resistance. Preclinical xenografts have been shown to recapitulate patient response, therefore, can be used to guide clinical trials using novel combinations. Methods: We have established patient derived tumor xenograft models (GUR-017M, TMA-027 and PRJ-88T) from patients with mCRPC with each having a unique genomic alteration profile. In the clinical setting, GUR-017M was resistant to abiraterone, enzalutamide, cabazitaxel and had exposure to docetaxel. TMA-027 and PRJ-88T were resistant to enzalutamide. We combined a CDK4/6 inhibitor with an AKT inhibitor and treated each of these patient tumors in NOD-SCID mice for a total duration of 40 days. Mice were implanted and separated into 4 groups (placebo, CDK4/6 inhibitor, AKT inhibitor and CDK4/6 + AKT inhibitor) with 10-13 mice in each group. Tumor volumes were measured twice a week. For GUR-017M, abemaciclib (50 mg/kg) was combined with MK2206 (120 mg/kg) and was compared to each placebo and each single agent alone. For TMA-027, palbociclib (50 mg/kg) was combined with ipatasertib (50 mg/kg). After 40 days, tumors were harvested for immunohistochemistry analysis. PRJ-88T was grown as 3D spheroids in ultra-low attachment plates and treated with palbociclib + ipatasertib (10 uM-100uM) to evaluate for synergy. Results: GUR-017M and TMA-027 showed significant delayed tumor growth compared to placebo or either single agent alone. In the GUR-017M group, the final mean tumor volume for the combination group was 149 mm3, compared to 377 mm3 (abemaciclib), 358 mm3 (MK2206) and 527 mm3 placebo group, representing a percent decrease compared to control by 72%, 29%, and 32%, respectively. For TMA-027, the final mean tumor volume for the combination group was 102 mm3, compared to 259 mm3 (palbociclib), 416 mm3 (ipatasertib), and 402 mm3 placebo group, representing a percent decrease compared to control by 75%, 36%, and 4%, respectively. PRJ-88T 3D spheroids showed synergy with palbociclib + ipatasertib. In vivo experiments using PRJ-88T are in progress. Conclusions: The preclinical results show combining CDK4/6 inhibitors with AKT inhibitors is a promising treatment strategy and provides the basis for a clinical trial in patients with mCRPC.

Introduction Despite continuous improvement of clinical outcome in multiple myeloma (MM), disease relapse remains a major challenge, leading to progressively shorter remissions and fewer treatment options. Strategies attempting to counteract this challenge include recent efforts resulting in an increase in the availability of novel promising anti-MM agents and targeting specific genetic profiles of the disease. In this context, we aim to develop predictive models of sensitivity and resistance to novel compounds by connecting an ex vivo high-throughput drug screen with genetic, transcriptomics, FISH, and clinical features. Methods Twenty compounds (afatinib, afuresertib, belinostat, buparlisib, cobimetinib, CPI-0610, crenolanib, dinaciclib, dovitinib, JQ1, LGH447, osimertinib, OTX015, panobinostat, romidepsin, selinexor, sunitinib, trametinib, venetoclax, and vorinostat) were selected based on overall promising anti-MM activity from an ex vivo high throughput drug screen with a panel of 79 single agents incubated for 24 hours. The area under the curve (AUC) was used to rank order the ex vivo responses for each compound and the lowest and highest quartile samples were identified for further analysis. Clinical data and FISH data, including t(11;14), t(4;14), t(14;16), del(17p), +1q, monosomy 13, and MYC rearrangement, were collected. Targeted DNA sequencing was performed using a 2.3 Mb custom capture panel covering 139 MM-relevant genes. mRNA-sequencing was performed and differential gene expression analysis in the highest and lowest quartile identified subsets of markers positively and negatively associated with the AUC response for a given compound. An additional unbiased selection of markers using lasso techniques was performed, resulting in predictive generalized linear models (GLM) for each agent. Responses from the remaining intermediate samples were estimated with the predictive models, with overall predictive ability assessed by correlating predicted AUCs with their actual counterparts. Results Our integrative analysis was performed on 50 primary patient samples (36% untreated and 64% relapsed MM). Venetoclax, dinaciclib, romidepsin, panobinostat, osimertinib, belinostat and selinexor were the most active compounds in the cohort. Interestingly, LGH447, dovitinib, selinexor, JQ1, OTX-015, cobimetinib, and trametinib showed increased activity in relapsed MM when compared to untreated samples (Wilcoxon Test; p<0.05). We generated GLMs using an average of 92 markers (range 64-107) per compound, combining mRNA-sequencing expression with FISH and mutation data. The analysis proposed in the present study was validated through the unbiased selection of BCL2 among the subset of markers included in the GLM predicting sensitivity to venetoclax, a first-in-class orally bioavailable selective BCL2 inhibitor. Expression level of critical NF-kB and cell cycle genes, such as BIRC3, CKS1B, PAX5, NFKB2, and CCND2, were included in 60% of our predictive models. Mutations of DNA repair genes (ATM,TP53) were included in the GLMs of three epigenetic therapies, one histone deacetylase inhibitor and two BET inhibitors, associated to ex vivo resistance to the drugs. The presence of monosomy 13 was also a marker for ex vivo resistance for five epigenetic therapies, four HDAC inhibitors and one BET inhibitor. The three BET inhibitors, JQ1, CPI-0610, and OTX015, were among the compounds most accurately predicted by our integrative approach, with Spearman correlation values between 0.773-0.858. Overall, our models accurately predicted the ex vivo response for 16 (80%) of the compounds (r>0.7). Five (25%) of these compounds displayed a remarkably accurate prediction model in both training (highest and lowest quartiles) and validation (intermediate quartiles) samples (r>0.8). Conclusions The GLM data integration approach enabled the establishment of effective predictive models, identifying FISH, transcriptomics, and mutations of putative driver genes important in anti-MM agent responsiveness. In addition, the resulting dataset is promising for future research focusing on the discovery of novel mechanisms of action and establishing markers of sensitivity and resistance to novel compounds. We are currently increasing our dataset and seek to create an omnibus approach that predicts responses to multiple anti-MM agents simultaneously. Disclosures Bergsagel: Celgene: Consultancy; Ionis Pharmaceuticals: Consultancy; Janssen Pharmaceuticals: Consultancy. Stewart:Amgen: Consultancy, Research Funding; Bristol Myers-Squibb: Consultancy; Celgene: Consultancy, Research Funding; Ionis: Consultancy; Janssen: Consultancy, Research Funding; Oncopeptides: Consultancy; Ono: Consultancy; Roche: Consultancy; Seattle Genetics: Consultancy; Takeda: Consultancy.

Abstract Quiescin sulfhydryl oxidase 1 (QSOX1) is an enzyme overexpressed by many different tumor types. QSOX1 catalyzes the formation of disulfide bonds in proteins. Because short hairpin knockdowns (KD) of QSOX1 have been shown to suppress tumor growth and invasion in vitro and in vivo, we hypothesized that chemical compounds inhibiting QSOX1 enzymatic activity would also suppress tumor growth, invasion, and metastasis. High throughput screening using a QSOX1-based enzymatic assay revealed multiple potential QSOX1 inhibitors. One of the inhibitors, known as “SBI-183,” suppresses tumor cell growth in a Matrigel-based spheroid assay and inhibits invasion in a modified Boyden chamber, but does not affect viability of nonmalignant cells. Oral administration of SBI-183 inhibits tumor growth in 2 independent human xenograft mouse models of renal cell carcinoma. We conclude that SBI-183 warrants further exploration as a useful tool for understanding QSOX1 biology and as a potential novel anticancer agent in tumors that overexpress QSOX1.

Abstract Introduction: QSOX1 is a flavin-dependent sulfhydryl oxidase and the only known human enzyme with both disulfide generating and transferring activities. Using tissue microarrays, we previously demonstrated that QSOX1 is over-expressed in multiple tumor types including breast, pancreas, prostate and myeloma. Loss of QSOX1 leads to a significant decrease in tumor cell proliferation and invasion. The over-expression of QSOX1 in tumor cells compared to non-malignant cells combined with its role in tumor cell proliferation and invasion make it an attractive target for potential anti-neoplastic agents. Methods: Using a high throughput screening assay to detect sulfhydryl oxidase activity, we screened a library of compounds for inhibition of the QSOX1 enzyme. We used ROS-GloTM, a luciferase-based assay that detects H2O2, to determine if any compounds in the library inhibited QSOX1-mediated disulfide bond formation using reduced RNAse A as a substrate. QSOX1 generates H2O2 as a by-product of disulfide bond formation. To determine if compounds that showed activity in the enzymatic assay were also active against tumor cells, we treated pancreatic, breast, and myeloma cells with the compounds in a dose-dependent fashion, using non-malignant cells as controls. We assayed growth/viability by MTT and CellTiter-Glo assays. Invasive activity of tumor cells was assessed using matrigel-coated inserts in a modified Boyden chamber assay. Results: High throughput library screening identified 4 compounds that specifically inhibited QSOX1 activity (designated A-D) compared to a glucose oxidase counter-screen. Growth/viability assays showed that compound D suppressed the growth of tumor cells with IC50s between 300nM-750nM, depending on tumor cell line. In non-malignant cells the IC50s for compound D were more than 10-fold higher than in tumor cells. Compounds A and D also inhibited invasion of breast and pancreatic tumor cells through Matrigel. Conclusion: This is the first report of chemical compounds that inhibit QSOX1 enzymatic activity. The discovery of QSOX1 inhibitors with cellular efficacy establishes the chemical tractability of the target. Our data demonstrate that these First-In-Class compounds have potential as anti-neoplastic therapeutics via inhibition of QSOX1. Citation Format: Paul D. Hanavan, Douglas O. Faigel, Chen-Ting Ma, Eduard Sergienko, Nathalie Meurice, Joachim L. Petit, Yvette W. Ruiz, Benjamin A. Katchman, Douglas F. Lake. Small molecule inhibitors of QSOX1 suppress tumor cell growth and invasion. [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 1790. doi:10.1158/1538-7445.AM2014-1790

Abstract Deregulation of the TWEAK-Fn14 signaling pathway is observed in many diseases including inflammation, autoimmune diseases, and cancer, notably glioblastoma. Activation of Fn14 signaling by TWEAK binding triggers glioma cell invasion and survival and therefore represents an attractive pathway for therapeutic intervention. Based on structural studies of the TWEAK-binding cysteine rich domain of Fn14, several homology models of TWEAK were built to investigate plausible modes of TWEAK-Fn14 interaction. Two promising models, centered on different anchoring residues of TWEAK [Tyrosine 176 (Y176) and Tryptophan 231 (W231)], were prioritized using a data-driven strategy. Site-directed mutagenesis of TWEAK at Y176, but not W231, resulted in the loss of TWEAK binding to Fn14 substantiating Y176 as the anchoring residue. Importantly, mutation of TWEAK at Y176 did not disrupt TWEAK trimerization, but failed to induce Fn14 mediated nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling. The validated structural models were utilized in a virtual screen to design a targeted library of small molecules predicted to disrupt the TWEAK-Fn14 interaction. 129 small molecules were screened iteratively, with identification of molecules producing up to 37% inhibition of TWEAK-Fn14 binding. One specific compound, L524-0366 binds specifically to the Fn14 receptor and disrupt TWEAK-Fn14 interaction. Treatment of L524-0366 suppresses TWEAK-induced cell migration and survival in glioblastoma cells. In summary, we present a data-driven in silico study revealing key structural elements of the TWEAK-Fn14 interaction, followed by experimental validation, serving as a guide for the design of small molecule inhibitors of the TWEAK-Fn14 ligand-receptor interaction. Our results provide the foundation for further exploration utilizing L524-0366 as a small molecule inhibitor to the TWEAK-Fn14 signaling axis. Citation Format: Harshil D. Dhruv, Joseph C. Loftus, Pooja Narang, Joachim L. Petit, Donald Chow, Holly Yin, Michael Berens, Nathalie Meurice, Nhan L. Tran. Identification and characterization of L524-0366 as a small molecule inhibitor that disrupt TWEAK-Fn14 signaling in glioblastoma. [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 1780. doi:10.1158/1538-7445.AM2014-1780