Ataxia-telangiectasia mutated (ATM) plays a central role in the cellular response to DNA damage and ATM alterations are common in several tumor types including bladder cancer. However, the specific impact of ATM alterations on therapy response in bladder cancer is uncertain. Here, we combine preclinical modeling and clinical analyses to comprehensively define the impact of ATM alterations on bladder cancer. We show that ATM loss is sufficient to increase sensitivity to DNA-damaging agents including cisplatin and radiation. Furthermore, ATM loss drives sensitivity to DNA repair–targeted agents including poly(ADP-ribose) polymerase (PARP) and Ataxia telangiectasia and Rad3 related (ATR) inhibitors. ATM loss alters the immune microenvironment and improves anti-PD1 response in preclinical bladder models but is not associated with improved anti-PD1/PD-L1 response in clinical cohorts. Last, we show that ATM expression by immunohistochemistry is strongly correlated with response to chemoradiotherapy. Together, these data define a potential role for ATM as a predictive biomarker in bladder cancer.

Abstract Introduction: Upper Gastrointestinal Cancers (UGCs) exhibit resistance to conventional chemotherapy due to variable P53 status and constitutive overactivity of EGFR, ERBB2/HER-2, Aurora kinases, and JAK2 oncogenes. UGC is a leading cause of cancer related deaths worldwide and development of multi-kinase targeting inhibitors could pave the way for improved chemotherapy and better therapeutic outcomes. In this study, we investigated a series of N4-phenylsubstituted-7H-pyrrolo[2,3-d]pyrimidin-4-amines and selected a promising novel investigational derivative (RS-41) that targets multiple kinases and exhibits significant anticancer activity in P53 mutant and wild type models of UGC. Methods: Target kinase inhibition and selectivity screening (T-KISS) and MTT-cell viability assays were performed to determine potency and selectivity of kinase inhibition and anticancer efficacy of primary derivatives, respectively. RS-41 was identified as the most promising agent following analysis via T-KISS and MTT-cell viability assay. Next, clonogenic cell survival assay, cell cycle analyses, and western blot analyses were done to evaluate the effect of RS-41 treatment on cell survival, cell cycle progression, and expression of apoptotic markers in P53 mutant (FLO-1) and P53 wild type (AGS) UGC cells, respectively. The initial T-KISS and cell-viability data aided in the design of second-generation derivatives of RS-41. Results: The initial T-KISS and cell viability data aided in identification of structural moieties that were modified to make the second generation of derivatives with improved multi-kinase inhibition. Evaluation with T-KISS showed that RS-41 derivative selectively inhibits AURKA (IC50-0.96±0.03µM), JAK2 (IC50-1.21±0.17µM), and EGFR (IC50-5.92±0.75µM) kinases, respectively. The cell viability and clonogenic cell survival data indicate that treatment with RS-41 mediates significant (P≤0.05) inhibition of FLO-1 and AGS UGC cell viability and survival. The cell cycle data and western blotting data also exhibited a marked increase (P≤0.05) in the percentage of sub-G1-phase (cell death) and expression of various apoptotic markers in FLO-1 and AGS cells following treatment with RS-41 for 24 and 72 hrs. Conclusions: Our T-KISS data indicate that multi-targeted inhibition of EGFR, AURKA and JAK2 can be achieved with the pyrrolo[2,3-d]pyrimidine scaffold. In addition, we demonstrate that inhibition of various oncogenic kinases with RS-41 is an effective therapeutic strategy for inducing apoptosis in both P53 mutant and wild type UGC cells. Citation Format: Amruta Samant, Tanvi Visal, Priya Pancholi, Dhvanir Kansara, Tanmay Dichwalkar, Robert Senones, Sonali Kurup, Vikas Sehdev. Design, synthesis, selection, and evaluation of a potent multi-kinase targeting N4-phenylsubstituted-7H-pyrrolo[2,3-d]pyrimidin-4-amine derivative with significant anticancer activity in P53 mutant and wild type models of upper gastrointestinal cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2800.

Abstract Background: Upper Gastrointestinal Cancers (UGCs) respond poorly to conventional chemotherapy due to overactive intrinsic mechanisms that mediate cellular proliferation and drug resistance. Dysregulated cell division due to increased activity of cyclin-dependent kinases 4/6 (CDK4/6) is one such mechanism that drives progression of UGCs, making them an important therapeutic target. Palbociclib is a second generation cdk4/6 specific inhibitor approved for treatment of ER+ breast cancers. Since Cisplatin (CDDP) is a first line therapeutic agent frequently used for treatment of UGCs we hypothesized that Palbociclib would complement the DNA-damaging activity of CDDP. To test this hypothesis, we investigated the potential therapeutic benefit of Palbociclib alone and in combination with CDDP in P53 mutant model of UGCs. Methods: In this study, we evaluated the effect of Palbociclib treatment alone and/or in combination with CDDP on FLO-1 (P53 mutant) UGC cell viability, survival, and cell cycle progression, respectviely. The MTT cell viability assay and Compusyn mediated median effect plot analysis (MEPA)(Chou and Talaly) were used to determine synergistic drug combinations of Palbociclib and CDDP in FLO-1 UGC cells. Results: The cell viability data and MEPA indicated that Palbociclib and CDDP show significant synergistic anticancer activity in FLO-1 UGC cells at a ratio of 1:2 and 1:3, respectively. The clonogenic cell survival assay data showed that in comparison to treatment with Palbociclib (0.5µM) or CDDP (1.0 and 1.5µM) alone, the combination treatments (Palbociclib:CDDP) at a ratio of 1:2 and 1:3 exhibited significantly elevated (p<0.05) inhibition of cellular survival in FLO-1 UGC cell lines. In addition, the Combination Index (CI) values of 0.66 and 0.71 for the aforementioned combination ratios suggests a strong synergism between the two drugs. Furthermore, the cell cycle data exhibited a marked increase (p<0.05) in the percentage of FLO-1 cells in the sub-G1-phase (cell death) after treatment with Palbocicb and CDDP combinations for 72 hrs., respectively . Conclusions: Our in vitro data indicate that Palbociclib, as a single agent, is an effective therapeutic strategy for inhibiting UGC cells. Additionally, Palbociclib in combination with CDDP synergistically enhances the anti-tumor activity of CDDP against P53 mutant UGC cells[MM1] . The observed synergism can potentially translate into reduced individual drug dosages and toxicity for Palbociclib and/or CDDP when used in combination. Citation Format: Dhvanir Kansara, Amruta Samant, Priya Pancholi, Tanvi Visal, Shraddha Patel, Vikas Sehdev. Palbociclib synergistically enhances the anticancer activity of cisplatin in P53 mutant model of upper gastrointestinal cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5805.

Background: The overexpression of cyclin-dependent kinases 4/6 (CDK4/6) is known to cause cell cycle dysregulation in certain cancer types, making these cell cycle kinases attractive targets for pharmacological inhibition. The effectiveness of first-generation non-selective cyclin-dependent kinases, such as roscovitine and flavopiridol, was hampered by toxicities, leading to the development of second-generation compounds like IBRANCE®/Palbociclib that specifically inhibit CDK4 and 6. ON 123300 is a third-generation potent CDK4/6 inhibitor that also inhibits ARK5 with low nanomolar potency and has the potential to improve upon second-generation compounds. Previous studies have demonstrated the inhibitory effect of single-agent ON 123300 in various pre-clinical cancer models of MM and leukemia. In this study, we investigated the comparative therapeutic potential of ON 123300 as an oral anticancer agent and a second-generation inhibitor, Palbociclib, in xenografted Rb+ve mouse models. Methods: MDA-MB-435S xenografted mice were treated once a day for 21 days with ON 123300 (125mg/kg) or Palbociclib (125mg/kg). Tumor volumes were measured and peripheral blood was gathered to evaluate the effects on hematological parameters. Separately, Western blot analyses were performed to determine the effect of CDK4/6 inhibition on p-Rb following intra-tumoral treatment with ON 123300 (2.5µM) or Palbociclib (2.5µM). Results: ON 123300 and Palbociclib reduced tumor growth with an equivalent magnitude during the 21-day treatment period, suggesting that the two compounds were equally effective in this model. Both compounds decreased RBC and platelet counts, however Palbociclib had a more prominent and statistically significant (P≤0.05) inhibitory effect on neutrophil counts when compared to ON 123300 (30.70 ± 3.55 vs. 45.10 ± 2.04). Western blot analysis of tumor tissues demonstrated equivalent effects on p-Rb for both compounds. Conclusions: Xenograft data indicates that a third-generation CDK4/6 inhibitor, ON 123300, is as effective as Palbociclib in an Rb+ve xenograft model. Moreover, this study also suggests that ON 123300 may have the added advantage of reduced neutropenia compared to Palbociclib. Prior preclinical data suggest that ON 123300 may be efficacious in Rb-ve tumors, where second-generation compounds have diminished single-agent activity, and our ongoing studies are aimed at further characterizing the in vivo activity of ON 123300 in this setting. Citation Format: Shraddha Patel, Priya Pancholi, Tanvi Visal, Amruta Samant, Dhvanir Kansara, V J. Rajadhyaksha, Benjamin S. Hoffman, Manoj Maniar, Vikas Sehdev. ON 123300, an orally administered novel CDK4/6 + ARK5 inhibitor, exhibits potent antitumor activity in vivo: comparative studies with Palbociclib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2172. doi:10.1158/1538-7445.AM2017-2172

Introduction: Upper Gastrointestinal Cancers (UGCs) are a leading cause of cancer-related mortality and account for approximately 1.1 million deaths worldwide. UGCs respond poorly to conventional chemotherapy due to constitutive over activity of multiple oncogenic signaling mechanisms, including the epidermal growth factor receptor (EGFR), ERBB2/HER-2, Aurora kinases, and JAK-STAT pathways. In addition, presence of mutant P53 further imparts resistance to conventional chemotherapeutic agents. Therefore, novel small molecule inhibitors that target multiple kinases associated with oncogenic progression could pave the way for improved chemotherapy and better therapeutic outcomes. In this study, we characterized the anticancer activity of RS-41, an investigational 4-phenylbenzamidopyrrolo[2,3-d]-pyrimidin-4-amine multi-kinase inhibitor, in P53 mutant and wild type models of UGC. Methods: Target kinase inhibition and selectivity screening assays were performed to determine potency and selectivity of kinase inhibition for RS-41. In addition, MTT-cell viability assay, clonogenic cell survival assay, cell cycle analyses, and western blot analyses were done to evaluated the effect of RS-41 treatment on cell viability, survival, cell cycle progression, and expression of apoptotic markers in P53 mutant (FLO-1) and P53 wild type (AGS) UGC cells, respectively. Results: The kinase selectivity screening assay characterized RS-41 for its selectivity against a panel of 90 human kinases. The kinase screening analyses showed that RS-41 selectively inhibits AURKA (IC50-0.96±0.03µM), JAK2 (IC50-1.21±0.17µM), and EGFR (IC50-5.92±0.75µM) kinases, respectively. The cell viability data indicate that treatment with RS-41 mediates significant (P≤0.05) inhibition of FLO-1 and AGS UGC cell viability. The clonogenic cell survival data showed that treatment with RS-41 for 24 hrs. suppresses subsequent formation of colonies in both FLO-1 and AGS UGC cells. The cell cycle data exhibited a marked increase (P≤0.05) in the percentage of FLO-1 and AGS cells in the sub-G1-phase (cell death) after treatment with RS-41 for 24 and 72 hrs., respectively. The western blotting data further confirmed induction of apoptosis in FLO-1 and AGS cells as evidenced by an increase in expression of various markers of apoptosis (P73/P53, cleaved PARP, and/or cleaved caspase 3) following treatment with RS-41 for 24 and 72 hrs. Conclusions: Our in vitro data indicate that inhibition of various oncogenic kinases with RS-41 is an effective therapeutic strategy for inducing apoptosis in both P53 mutant and wild type UGC cells. Our study suggests that RS-41 is a promising multi-kinase inhibitor with a potential to further enhance chemotherapeutic options for treatment of UGC. Citation Format: Tanvi Visal, Shraddha Patel, Priya Pancholi, Samhita Bapat, Amruta Samant, Dhvanir Kansara, Sonali Kurup, Vikas Sehdev. RS-41, a multi-targeted kinase inhibitor, induces cell cycle arrest and apoptosis in p53 mutant and wild type models of upper gastrointestinal cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4180. doi:10.1158/1538-7445.AM2017-4180