Abstract PR001: ZNFX1 is a master regulator and tumor suppressor in epigenetically-induced pathogen mimicry, mitochondrial dysfunction and STING-dependent signaling in cancer
Authors: Rassool Feyruz V , Stojanovic Lora , Abbotts Rachel , Tripathi Kaushlendra , Rajendran Saranya , Coon Colin M , Abbasi Elnaz , Liu Sheng , Wan Jun , Topper Michael J , Baylin Stephen B , Nephew Kenneth P 
Published: February 2025

Abstract
Immunomodulatory agents are 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 retroviruses (ERVs), leading to cytosolic double-stranded RNA (dsRNA) accumulation that activates interferon/inflammasome signaling. Moreover, poly (ADP ribose) polymerase inhibitors (PARPi) increase cytosolic dsDNA, activating stimulator of interferon (IFN) genes (STING). We reported that DNMTis in combination with PARPis induce STING-dependent signaling in a process termed pathogen mimicry response (PMR). Although we and others have demonstrated that mitochondria (mt) are an important gateway for antiviral inflammasome signaling, the underlying mechanism in cancer remains to be fully elucidated. In this regard, our studies of the little-known gene, NFX1-type zinc finger–containing 1 (ZNFX1), show that ZNFX1 acts as a master nucleic acid (dsRNA/DNA) sensor for mt gateway function. Bioinformatics analysis in primary ovarian tumors from TCGA and clinical trial RNAseq datasets shows that increased ZNFX1 expression tracks with tumor stage and grade but inversely correlates with a mt dysfunction signature. In a panel of ovarian cancer (OC) cell lines, transfection of dsRNA/DNA mimics or DNMTi azacytidine (AZA) and PARPi (talazoparib [TAL]) treatments induce increased ZNFX1 expression and binding to mt antiviral protein (MAVs) localized on the mt outer membrane. Functional studies of mt dysfunction in OC cells further show that dsRNA/DNA as well as above viral mimicry drugs increase mt reactive oxygen species (ROS) as measured by mitosox flow cytometry. AZA and TAL treatments also increase fragmented mtDNA and oxidative mtDNA base damage, as measured by long range PCR and 8-oxoguanine (8-oxoG) ELISA assays. These drug treatments also increase release of mtDNA into the cytosol, resulting in STING-dependent inflammasome signaling and cytokine release. Notably, ZNFX1 knockout (KO) attenuates these dynamics, further supported by bioinformatics analysis of RNA seq data, thus defining ZNFX1 as a gene as essential for IFN/inflammasome signaling induced by mtDNA damage. Pathway analysis of ZNFX1 KO vs wild-type RNAseq data, combined with in vitro and in vivo validation assays, demonstrate increased proliferation and epithelial to mesenchymal transition, suggesting that not only is ZNFX1 a master regular of the above mt dynamics but also a new tumor suppressor in OC. Abrogating ZNFX1 expression in OC cells triggers increased cell proliferation, invasive capacity and, increased tumor growth in vivo. Taken together, this work then suggests novel approaches to improve immune therapy responses in OC through manipulating ZNFX1.
Citation Format: Feyruz V Rassool, Lora Stojanovic, Rachel Abbotts, Kaushlendra Tripathi, Saranya Rajendran, Colin M Coon, Elnaz Abbasi, Sheng Liu, Jun Wan, Michael J Topper, Stephen B Baylin, Kenneth P Nephew. ZNFX1 is a master regulator and tumor suppressor in epigenetically-induced pathogen mimicry, mitochondrial dysfunction and STING-dependent signaling in cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Methylation, Clonal Hematopoiesis, and Cancer; 2025 Feb 1-4; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2025;85(3 Suppl):Abstract nr PR001.