ATR inhibitor–induced CDK1-SPOP axis destabilizes PD-L1 and enhances cytotoxicity in prostate cancer

We show that treatment of prostate cancer cells with potent, specific ATR inhibitors (ATRi) leads to robust activation of the cGAS-STING signaling pathway, growth inhibition, and cell death. We also demonstrate that ATRis repress ATR-Chk1 replication stress signaling, which results in activation of a CDK1-SPOP axis, PD-L1 degradation, and derepression of a cGAS-STING-IFN-ß-IFNAR1–driven, autocrine, cytotoxic signaling pathway. ATRi and anti–PD-L1 combination treatment resulted in synergistic, anti-tumor activity in a syngeneic mouse androgen-indifferent, aggressive prostate cancer model. scRNA-seq analysis of tumor tissue samples from this model demonstrated increased activation of cGAS-STING, IFN-ß, and apoptotic signaling in combination versus ATRi single agent treatment. Our findings provide a molecular mechanistic rationale for combining ATR-targeted agents with immune checkpoint blockade, leading to the development of active early-phase clinical trials and reveal mechanism-based opportunities for improving outcomes for men with advanced prostate cancer by combining immunotherapy and agents that induce mitotic catastrophe. ABSTRACT Purpose: Despite significant benefit for other cancer subtypes, immune checkpoint blockade (ICB) therapy has not yet been shown to significantly improve outcomes for men with castration-resistant prostate cancer (CRPC). Prior data have shown that DNA damage response (DDR) deficiency, via genetic alteration and/or pharmacologic induction using DDR inhibitors (DDRi), may improve ICB response in solid tumors in part due to induction of mitotic catastrophe and innate immune activation. Discerning the underlying mechanisms of this DDRi-ICB interaction in a prostate-cancer specific manner is vital to guide novel clinical trials and provide durable clinical responses for men with CRPC. Experimental Design: We treated prostate cancer cell lines with potent, specific inhibitors of ATR kinase, as well as with PARP inhibitor, olaparib. We performed analyses of cGAS-STING and DDR signaling in treated cells, and treated a syngeneic androgen-indifferent, prostate cancer model with combined ATR inhibition and anti-PDL1, and performed scRNA-seq analysis in treated tumors. Results: ATRi (BAY1895433) directly repressed ATR-CHK1 signaling, activated CDK1-SPOP axis, leading to destabilization of PD-L1 protein. These effects of ATRi are distinct from those of olaparib, and resulted in a cGAS-STING–initiated, IFN-ß–mediated, autocrine, apoptotic response in CRPC. The combination of ATRi with anti-PDL1 therapy resulted in robust innate immune activation and a synergistic, T-cell dependent therapeutic response in our syngeneic mouse model. Conclusions: This work provides a molecular mechanistic rationale for combining ATR-targeted agents with immune checkpoint blockade for patients with CRPC. Multiple early phase clinical trials of this combination are underway. 5 that compare and contrast these agents with regard to their mechanisms of action, impact on immune activation, and therapeutic opportunities for combination with ICB in immunocompetent models of prostate cancer. Previous reports have demonstrated that PARPis activate cGAS-STING signaling and induce PD-L1 protein expression through IRF3 activity (11,17-19), yet the impact of ATRi on immune pathway activation is not clear. Furthermore, a recent report showed that ATRi treatment led to downregulation of PD-L1 in select cancer cell lines, although the underlying mechanism of this activity was not identified and synergy with anti-PD-1/L1 therapy not explored (20,21). Because of these knowledge gaps, we sought to determine the resulting impact and the mechanism of action of ATR signaling inhibition on cytotoxicity, immune activation, PD-L1 protein regulation, and its capacity for safely improving the efficacy of ICB in prostate cancer, which can directly inform biomarker-driven ICB treatment strategies for patients with CRPC. signaling, the effects (JAK1-STAT3 signal transduction to phosphorylate STAT3 at Y705) were largely blunted compared to ATRi treatment. These results are similar to those from a recent study which demonstrated a cancer cell intrinsic IFNAR1-JAK1-STAT3(p-Y705)-caspase 7– dependent cytotoxic pathway induced by deletion of a specific intracytoplasmic domain of PDL1 (42). In addition, multiple studies previously showed that caspase 7 mediates IFN-ß–induced apoptosis in cancer cells (42-44). RT-qPCR analysis of cGAS mRNA levels indicated that ATRi (BAY1895344) or olaparib treatment produced a dose-dependent increase in cGAS mRNA Gene set enrichment analysis in the epithelial cell cluster demonstrated enrichment of IFN-γ signaling, IFN-ß signaling, STING signaling, JAK-STAT signaling pathways in ATRi and anti-PD-L1–treated cells compared to ATRi- or anti-PD-L1–