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Ataxia-telangiectasia mutated and Rad3-related kinase (ATR) is one of the key players in the DNA damage response (DDR) to replication stress. M6620 is a potent and selective ATR inhibitor which has been shown to sensitize cancer cells to the lethal effects of DNA-damaging chemotherapeutic agents. We previously demonstrated that M6620 significantly enhances the anti-tumor efficacy of dual combinations of platinum-based chemotherapy and avelumab, an FDA-approved anti-PD-L1 monoclonal antibody, in syngeneic tumor models in vivo. The purpose of this study was to further elucidate the potential mechanisms of action that drive the therapeutic benefit of this triplet combination therapy. Treatment with certain chemotherapeutic agents has been reported to promote immunogenic cell death (ICD) in tumor cells. We utilized in vitro assays to test the expression of markers of immunogenic cell death in murine MC38 colorectal carcinoma cells. After 72 hours of treatment, carboplatin and M6620 dual combination treatment significantly increased the levels of Calreticulin, HMGB1, and ATP relative to treatment with either single agent. In addition, gene expression profiling was used to determine pathways differentially induced by the drug treatments. RNA-sequencing (RNA-seq) analysis was performed on MC38 cells treated in vitro with carboplatin, M6620, or dual combination therapy. Comparison of gene expression between treatment groups at 48 hours post-treatment revealed that the dual combination therapy significantly increased expression of genes regulated by type I/II interferons (IFN), including CCl5, CXCL10, CXCL11 and ISG15. RNA-seq gene expression analysis was also performed on MC38 tumor samples from mice treated with carboplatin, M6620, avelumab, carboplatin/avelumab dual therapy, or carboplatin/M6620/avelumab triple combination therapy. Triple combination therapy induced a more robust upregulation of IFN-inducible genes at day 3 compared with monotherapies or carboplatin/avelumab dual combination, consistent with in vitro observations that M6620 and carboplatin dual combination therapy enhanced expression of genes regulated by IFNs. Taken together, these data suggest that potentiation of immunogenic cell death and induction of pro-inflammatory responses by M6620, in the context of chemotherapy-induced DNA damage, may contribute to the enhanced anti-tumor efficacy observed with carboplatin, M6620, and avelumab triple combination therapy. Citation Format: Marat Alimzhanov, Ashwin George, Parantu Shah, Astrid Zimmerman, Andreas Schroeder, Martin Falk, Christiane Amendt, Joern-Peter Halle, Andree Blaukat, Frank Zenke, Patricia Soulard. Induction of immunogenic cell death and interferon signaling by carboplatin and the ATR inhibitor M6620 may contribute to anti-tumor activity of M6620-carboplatin-avelumab triplet combination in MC38 tumor model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 964.

Background: There is increasing interest in using combination immunotherapy in the neoadjuvant setting in prostate cancer, however, endpoints for such studies remain elusive. We have conducted a clinical trial evaluating immunotherapy with ADT in patients with high risk prostate cancer. Patients were assessed for immune responses and changes in endorectal (er) MRI which can be used to assess intraprostatic tumors. Methods: Treatment-naïve high-risk (Gleason 8-10, PSA>20, or stage T3) prostate cancer patients (pts) were randomized to standard ADT+Radiation + an immunotherapy targeting MUC1 (tecemotide, aka L-BLP25) in this trial (NCT01496131). ADT consisted of gonadotropin-releasing hormone therapy. Immunotherapy included low dose (300 mg/m2, maximum 600 mg) pre-treatment cyclophosphamide for regulatory T-cell depletion. erMRI was done at baseline and after 2 months of immunotherapy including multiparametric MRI evaluation of apparent diffusion coefficient (ADC) maps from diffusion-weighted MRI. Monthly peripheral blood assessments analyzed immune cell subsets using flow cytometry and intracellular cytokine (ICC) staining for MUC-1 specific responses. Results: 28 pts with high risk prostate cancer were enrolled (n=14/arm). As expected, PSA declined in all pts 2 months after ADT. erMRI after 2 months of treatment suggested greater improvements in ADC values in pts receiving immunotherapy+ADT vs. ADT alone. Improved ADC on MRI indicates increased intratumoral diffusion and has been associated with decreased tumor density. The improvements in ADC were seen when one dominant tumor per patient was evaluated (p=0.17) but were more pronounced when up to 3 lesions were evaluated per pt (n=44 lesions; p=0.031). Compared to baseline, there were trends to increases in CTLA4+ CD8+ T-cells consistent with immune activation and decreases in myeloid derived suppressor cells (MDSCs) in pts receiving immunotherapy+ADT coinciding with the erMRI changes. These immune findings were not seen in the ADT alone group. 3 of 14 pts had MUC1 specific immune response by ICC. 2 of these patients had the greatest changes in ADC noted on erMRI over a 2-year period. Conclusions: Based on assessments by erMRI, pts who received ADT+immunotherapy had greater improvements in ADC than pts receiving ADT alone. Given that ADC improvements are associated with decreased tumor density, this suggests a possible greater anti-tumor effect of the ADT-immunotherapy combination vs. ADT alone. These findings were associated with trends to increased activated CD8+ T-cells and decreased MDSCs in pts receiving immunotherapy+ADT, with 3/14 pts having MUC1 specific immune responses. Further studies are required to confirm the potential to use ADC on erMRI as a potential (bio)marker of anti-tumor effect of immune combinations including ADT. Citation Format: Ravi A. Madan, Baris Turkbey, Lauren M. Lepone, Renee N. Donahue, Italia Grenga, Samuel Samuel Borofsky, Peter A. Pinto, Deborah Citrin, Aradhana Kaushal, Andra Krauze, Sheri McMahon, Myrna Rauchhorst, Anna Couvillon, Martin H. Falk, S Peter Eggleton, Stephen C. Greco, Peter L. Choyke, William L. Dahut, Jeffrey Schlom, James L. Gulley. Neoadjuvant immunotherapy with androgen deprivation therapy (ADT) prior to radiation in prostate cancer: Impact on multiparametric prostate MRI and immune responses [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 LB-059. doi:10.1158/1538-7445.AM2017-LB-059