Abstract 5359: M6A regulated NNMT mediates resistance to platinum in ovarian cancer

Abstract

Abstract Background: Ovarian cancer (OC) remains one of the deadliest malignancies. Development of resistance to platinum (Pt) is a major clinical problem and understanding its underpinnings will help find new approaches to overcome it. Fat mass and obesity associated protein (FTO) is a N6-methyladenosine (m6A) demethylase and plays an important role regulating how the messenger RNA is processed translating into functional proteins. We recently showed that m6A modifications induced by FTO play a suppressive role in tumorigenicity and survival of OC stem cells. Here we hypothesized that RNA modifications caused by FTO regulate the response of OC cells to Pt. Methods: To study the mechanisms related to FTO implicated in response to Pt; we used OC cells in which FTO was knocked down (KD) via shRNA or overexpressed (OE). Additionally, Pt-resistant (Pt-R) OC cells were obtained through repeated (3-4) exposures to Pt. Cell viability assay determined the IC50 (half maximal inhibitory concentration) to Pt. Pt response in vivo was assessed in FTO expressing vs. FTO KD xenografts. Induction of DNA damage was assessed by immunofluorescence (IF) for γ-H2AX. Apoptosis was evaluated by Annexin V staining in the IncuCyte system. To identify potential targets of FTO-mediated m6A modifications in Pt induced response, RNA-seq and MeRIP-seq were integrated. Results: FTO was significantly downregulated in Pt-R vs. sensitive OC cells. Forced expression of FTO increased sensitivity to Pt in vitro and in vivo, while FTO KD increased Pt resistance (p<0.05). A catalytic mutant FTO did not appreciably alter responsiveness to Pt. Increased γ-H2AX foci and increased apoptosis were observed after exposure to Pt in FTO OE vs. control cells. Through integrated RNA-seq and MeRIP-seq, we identified and validated several potential targets involved in response to Pt including IER5, IER5, ST3Gal, and the enzyme nicotinamide N-methyltransferase (NNMT). NNMT was upregulated and significantly hypomethylated in FTO OE cells and was downregulated in Pt resistant cells. Treatment with an NNMT inhibitor rescued the FTO induced sensitivity to Pt in OC cells demonstrating that its function is necessary in the response to Pt. Conclusions: We identified a new function of FTO-dependent m6A RNA modifications in regulating response to Pt through NNMT, a novel RNA methylated target. Activating FTO could improve response to Pt in OC. Citation Format: Hao Huang, Guangyuan Zhao, Andres Felipe Valdivia, Horacio Cardenas, Yinu Wang, Daniela Daniela Matei. M6A regulated NNMT mediates resistance to platinum in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5359.