The oxidative phosphorylation inhibitor, atovaquone, upregulates PD-L1 via activation of the ATM/ATR DNA damage response pathway

Oxidative phosphorylation (OXPHOS), a major metabolic pathway in normal/differentiated cells is also active in tumors and a target for cancer drug development. Atovaquone, an FDA-approved antiprotozoal and OXPHOS inhibitor, blocks electron transport at mitochondrial Complex III resulting in an oxygen radical surge that triggers cancer cell death. Here, we examine mechanisms that attenuate the efficacy of atovaquone as an anti-cancer agent. First, we demonstrate that exposure to atovaquone causes DNA damage and loss of nuclear integrity in cancer cells. DNA damage by atovaquone does not activate cGAS-STING signaling, likely due to repressed cGAS expression in the cell lines tested. Instead, ATM/ATR signaling is activated in response to atovaquone. Recently, we demonstrated that oxidative and endoplasmic reticulum stress in atovaquone-treated cancer cells was associated with elevation in danger associated molecular patterns (DAMPs) corresponding to increased lysis by natural killer cells. Contrary to this immune activating effect, we now report that cancer cells also employ an immunosuppressive mechanism upon exposure to atovaquone. Specifically, we observed ATM/ATR-dependent increase in expression of PD-L1 on the cancer cells. Increase in PD-L1 required STAT1 signaling but was not regulated by IRF1, HIF1α or p53. Increase in PD-L1 was confirmed on peritoneal p53-/- ID8-F3 tumors growing in mice receiving atovaquone therapy. Combining atovaquone with anti-PD-L1 resulted in significant delay in tumor growth. Data from this study provides a mechanistic basis for PD-L1 elevation in tumors treated with atovaquone. Our studies support further development of atovaquone-anti-PD-L1 combination for the treatment of ovarian and other malignancies.