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Background Ataxia telangiectasia and Rad3-related (ATR) is a key mediator of DNA damage response and cancer cell survival. ATR inhibitors have been shown to suppress cancer cell viability and potentiate conventional anticancer therapy and immunotherapy. This study investigates the immunomodulatory potential of the ATRi tuvusertib to improve natural killer (NK) cell targeting of prostate cancer (PCa).Methods Human PCa cells (DU145 and 22Rv1) were exposed to tuvusertib for 48 hours prior to downstream experiments. Impedance-based real-time cell analysis was used to assess the lysis of tuvusertib-treated PCa cells upon co-incubation with PBMC-derived NK cells in the presence or absence of anti-PD-L1 avelumab, PD-L1 targeting high-affinity NK cell line (PD-L1 t-hANK) cells, or TRAIL ligand. In some assays, NK cells were treated with the interleukin-15 (IL-15) superagonist N-803 (also known as nogapendekin alfa inbakicept, NAI) overnight prior to co-incubation with tumor cells. Flow cytometric and RNA analyses of tuvusertib-treated cells were used to assess immunomodulatory effects. A DU145 PCa xenograft model in athymic nude mice was utilized to evaluate the efficacy of tuvusertib and N-803 combination therapy in vivo. Tuvusertib and avelumab were provided by the healthcare business of Merck KGaA, Darmstadt, Germany (CrossRef Funder ID: 10.13039/100009945). PD-L1 t-haNK and N-803 were provided by ImmunityBio, Culver City, CA, USA.Results Tuvusertib induced immunogenic modulation in PCa cells, including upregulated surface expression of TRAIL-R2, ULBP-1, and PD-L1, and differential gene expression of p21 (elevated) and Bcl-xL (reduced). Tuvusertib sensitized DU145 and 22Rv1 cells to NK-mediated lysis. Additionally, tuvusertib increased the susceptibility of the PCa cells to PD-L1-directed therapies such as avelumab-mediated antibody-dependent cellular cytotoxicity (ADCC) and PD-L1 t-hANK cells. In vitro lysis of tuvusertib-exposed cells was further enhanced when the effector NK cells were treated with N-803. TRAIL signaling blockade mitigated the NK cell lysis of tuvusertib-treated cells. In vivo, tuvusertib and N-803 combination therapy resulted in the significant tumor growth control of the DU145 xenograft (p<0.0001) and the prolonged survival of the animals (p<0.05).Conclusions This study supports the combination of the ATR inhibitor tuvusertib with the anti-PD-L1 avelumab and/or IL-15 superagonist N-803 for prostate carcinoma.Acknowledgements This work was funded by the Intramural Research Program of the Center for Cancer Research, National Cancer Institute (NCI), National Institutes of Health and via Cooperative Research and Development Agreements (CRADAs) between the NCI and the healthcare business of Merck KGaA, Darmstadt, Germany (CrossRef Funder ID: 10.13039/100009945) and the NCI and ImmunityBio, Culver City, CA, USA.Ethics Approval All animal studies were approved and conducted in accordance with the National Cancer Institute Animal Care and Use Committee approved animal protocols CIO-002 and LTIB-057.