Abstract B061: The oxidative phosphorylation inhibitor, atovaquone, elevates PD-L1 expression through ATM/ATR signaling

Abstract Ovarian cancer is typically diagnosed at an advanced stage when standard treatments are less effective and hence there is an unmet need to develop new treatment strategies. Previously our lab has shown that atovaquone, an FDA-approved anti-malarial drug, reduces ovarian cancer proliferation under in vitro and in vivo conditions. Atovaquone is a potent inhibitor of oxidative phosphorylation (OXPHOS) and as a result causes a significant increase in intracellular oxygen radicals. The oxidative stress mediated by atovaquone causes double strand DNA breaks leading to cancer cell death. When experiencing oxidative damage, cancer cells also promote cellular processes that protect them from cell death and immune recognition. One such mechanism is the expression of PD-L1 allowing the stressed cells to evade immune responses. Here, we examine if the DNA damage by atovaquone leads to activation of the ATM/ATR pathway and, consequently, upregulates PD-L1. Abnormal nuclei were observed in 75% of the atovaquone (10-25 μM) treated OVCAR-5 cells. Increase in expression of 8-OHdG confirmed DNA damage caused due to oxidative stress induced in atovaquone-treated cells. Exposure to atovaquone for 72 h resulted in significant upregulation of phosphorylated ATM, ATR, CHK1, CHK2 and STAT1 proteins. Treatment with atovaquone resulted in 55-65% increase in the number of OVCAR-5 and p53negID8 cells expressing PD-L1. Inhibition of ATM/ATR signaling by pretreatment with AZD1056 or AZD6738 reversed PD-L1 increase in atovaquone treated cells. PD-L1 expression was unaffected by inhibition of HIF-1α and p53. On the contrary, knockdown of IRF-1, resulted in further increase in PD-L1. These studies suggest an intricate interplay between transcription factors that regulate PD-L1 expression. Our studies are also examining if PD-L1 from intracellular pools is recruited to the cell surface under oxidative stress conditions. Immunohistology of p53negID8 tumors obtained from mice receiving daily dose of atovaquone (200 mg/kg body weight) for 1 week showed a clear increase in PD-L1 expression. Experiments are underway to investigate combination of atovaquone with anti-PD-L1 can result in more efficient control of p53negID8 tumor growth in vivo. Overall, our studies demonstrate PD-L1 expression in atovaquone treated ovarian cancer cells. Expression of this immune checkpoint likely compromises efficacy of atovaquone as immunologic elimination of the cancer cells is not fully realized. Our observations provide support for combining atovaquone with anti-PD-L1 therapy for effective control of ovarian tumor growth. Citation Format: Sejal Sharma, Meghana Roy Peddoddi, Maria Virumbrales Munoz, Lisa Barroilhet, Manish S. Patankar. The oxidative phosphorylation inhibitor, atovaquone, elevates PD-L1 expression through ATM/ATR signaling abstract. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Ovarian Cancer Research; 2025 Sep 19-21; Denver, CO. Philadelphia (PA): AACR; Cancer Res 2025;85 (18Suppl): Abstract nr B061.