Supplementary Table S1 shows the origin and selected gene alteration status of C4-2b, PC-3, RM-9, RM-1 and RM-1-BM prostate cancer cell line models. Supplementary Table S2 shows details of the antibodies used in the experiments. Supplementary Table S3 shows primer sequences for RT-qPCR analysis. Supplementary Table S4 shows siRNA sequences used in the experiments. Supplementary Table S5 shows quantification of cell types among treatments derived from single cell RNA sequencing data.
ARTICLE ABSTRACT
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.
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–programmed death ligand 1 (anti–PD-L1), and performed single-cell RNA sequencing analysis in treated tumors.
ATR inhibitor (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-β–mediated, autocrine, apoptotic response in CRPC. The combination of ATRi with anti–PD-L1 therapy resulted in robust innate immune activation and a synergistic, T-cell–dependent therapeutic response in our syngeneic mouse model.
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.
