Abstract 527: Atovaquone upregulates PD-L1 expression via activation of the ATM/ATR DNA damage response pathway

Abstract Background: Ovarian cancer is one of the deadliest gynecological malignancies, typically diagnosed at an advanced stage complicating standard treatments and shows a need for new treatment options. Previously our lab has shown that atovaquone, a known 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. Here, we examine if the DNA damage by atovaquone leads to activation of the ATM/ATR pathway and, consequently, upregulate PD-L1. Increased PD-L1 expression in ovarian cancer patients after treatment with atovaquone will provide a rationale in combining atovaquone with anti -PDL1 therapy to enhance anti-tumor response and improve overall treatment outcome. Methods: For all experiments, ovarian cancer cells (OVCAR-5 and ID8) were treated with atovaquone at IC50 concentrations (10-30 µM). DNA damage was assessed by staining with picogreen and monitoring the cells by fluorescent microscopy as well as 8-OHdG (marker of DNA damage due to oxidative stress) flow cytometry. Activation of the ATM/ATR pathway was evaluated by western blotting using cell lysates for the expression of phosphorylated ATM, ATR, CHK1, CHK2, JAK1, STAT1 and IRF1 proteins. Flow cytometry was used to monitor expression of PD-L1 on the tumor cells. In some experiments, the ATM/ATR inhibitor, AZD1056, was added to the media at 2.5 µM-5 µM concentration for 5 h prior to determining the effect of this treatment on PD-L1 expression. Results: A 75% increase in abnormal nuclei with DNA damage was observed after 72 h treatment of OVCAR-5 and ID8 with atovaquone. Mild dysfunction in nuclear morphology was visible at the 24 h. Increase in expression of 8-OHdG was observed after atovaquone treatment confirming DNA damage. Exposure to atovaquone for 72 h resulted in significant upregulation of phosphorylated ATM, ATR, CHK1, CHK2, JAK1 and STAT1 proteins. Flow cytometry data demonstrated a (20-55%) increase in the level of PDL1 expression after 48h to 72 h atovaquone treatment in OVCAR 5 and ID8 cells. A (55-65%) increase in the number of both OVCAR 5 and ID8 cells expressing PDL1 expression was also observed. Inhibition of ATM/ATR signaling by pretreatment with AZD1056 reversed the level of increase in PDL1 expression in atovaquone treated OVCAR-5 and ID8 cells. Conclusion: These findings underscores ATM/ATR pathway as a novel molecular mechanism of PDL1 upregulation in atovaquone-treated ovarian tumors. Thus, our next step is to investigate if combining atovaquone with anti-PD1/PDL1 therapy will be effective in in vivo models of ovarian cancer. Citation Format: Sejal Tarun Sharma, Maria Virumbrales-Munoz, Mayra Betancourt Ponce, Lisa Barroilhet, Manish Patankar. Atovaquone upregulates PD-L1 expression via activation of the ATM/ATR DNA damage response pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 527.