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Abstract Immunomodulatory agents represent an important recent advance in cancer therapy, but utility is often limited by tumor immune evasion mechanisms. Strategic therapeutic activation of intracellular antiviral immune responses offers an opportunity to reverse immune evasion mechanisms and improve treatment outcomes. Anti-cancer agents such as, DNA methyltransferase inhibitors (DNMTis) induce re-expression of endogenous retroviral elements (ERVs), leading to cytosolic double-stranded RNA (dsRNA) accumulation that activates interferon/inflammasome signaling. Moreover, poly (ADP ribose) polymerase inhibitors (PARPi) increase cytosolic dsDNA leading to activation of stimulator of interferon genes (STING). We previously reported in triple negative breast cancer (TNBC) and ovarian cancer (OC) that DNMTis in combination with PARPi induce STING-dependent interferon/inflammasome signaling in a process termed pathogen mimicry response (PMR). Mitochondria (mt) are an important gateway for antiviral inflammasome signaling, but upstream activating events in cancer are not understood. We now show NFX1-type zinc finger–containing 1 (ZNFX1), a little-studied innate immune gene, acts as a master nucleic acid (dsRNA/DNA) sensor for this mt gateway function. Importantly, in primary ovarian tumors from TCGA and clinical trial RNAseq datasets, increased ZNFX1 expression tracks with tumor stage and grade but inversely correlates with a mt dysfunction signature. In studies of high grade serous (TYK-nu, OVCAR4) as well as endometrial ovarian (A2780) cancer cells, transfection of dsRNA/DNA mimics or DNMTi azacytidine (AZA) and PARPi (talazoparib) treatments induce increased ZNFX1 expression and binding to mt antiviral protein (MAVs) localized on the mt outer membrane, using immunofluorescence-based proximity ligation assays. In studies of mt dysfunction, we further show that dsRNA/DNA as well as above-described anti-cancer drugs increase mt reactive oxygen species (ROS), using flow cytometry of ROS dye mitoSOX. DNMTi and PARPi treatments also increase fragmented mtDNA and oxidative mtDNA base damage, as measured long range PCR and 8-oxoguanine (8-oxoG) ELISA assays, respectively. Importantly, these drug treatment increase release of mtDNA into the cytosol, resulting in STING-dependent inflammasome signaling and cytokine release. ZNFX1 knockout robustly attenuates these dynamics, thus defining this immune gene as essential for interferon/inflammasome signaling induced by mtDNA damage. Our data indicates a master role for the little-studied dsRNA/DNA sensor ZNFX1 in initiating a pathogen mimicry response to DNMTi and PARPi combination treatment, and further highlights the critical role of the mt in cellular antiviral signaling mechanisms. Importantly, this work suggests a novel avenue for manipulation of inflammasome signaling to improve cancer therapy responses and disease outcomes. Citation Format: Lora Stojanovic, Rachel Abbotts, Kaushlendra Tripathi, Collin M. Coon, Sheng Liu, Jun Wan, Michael J. Topper, Kenneth P. Nephew, Stephen B. Baylin, Feyruz V. Rassool. ZNFX1 is a master regulator for epigenetic reprograming of mitochondrial inflammasome signaling and pathogen mimicry in cancer cells abstract. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84 (5 Suppl₂): Abstract nr PR-005.
Stojanovic et al. (Mon,) studied this question.