Abstract 5797: NTX301, a novel DNA hypomethylating agent, targets lipid metabolism in ovarian cancer
Authors: Wang Yinu , Situ Xiaolei , Cardenas Horacio , Matei Daniela , Siu Ellie , Alhunayan Sayedabdulrazzaq A. 
Published: April 2023

Abstract
Background: DNA methylation causes the silencing of tumor suppressor and differentiation-associated genes being linked to chemoresistance and stemness. Previous studies demonstrated that hypomethylating agents (HMA) re-sensitize ovarian cancer (OC) cells to chemotherapy, however, translation to the clinic has been slow. NTX 301 (PinotBio) is a novel, highly potent, and orally bioavailable HMA, in early clinical development.
Methods: We assessed the anti-tumor effects of NTX301 in OC models and studied the underlying molecular mechanisms by using cell proliferation, stemness, and ferroptosis assays, RNA sequencing, and validation.
Results: OC cells (SKOV3, IC50=5.089nM; OVCAR5 IC50=3.664nM) were highly sensitive to NTX301 (p&lt;0.05) compared to immortalized fallopian tube epithelial cells (FT-190) (IC50=103.3nM). Treatment with NTX301 induced significant downregulation of the DNA methyltransferases (DNMTs) 1-3 expression in SKOV3 and OVCAR5 cells compared with decitabine (p&lt;0.05). Treatment with low dose NTX301 (100nM) induced significant transcriptomic reprogramming with 15,000 differentially expressed genes (DEGs) compared to DMSO (p&lt;0.05). Among the NTX301 down-regulated DEGs, Gene Ontology (GO) enrichment analysis identified pathways related to the regulation of alcohol, cholesterol, and fatty acid biosynthetic process and molecular functions related to aldehyde dehydrogenase (ALDH) and oxidoreductase activity, known features of cancer stem cells (CSCs). Indeed, treatment with low dose NTX301 (100nM) reduced the ALDH(+) cell population and expression of stemness-associated transcription factors (Oct4 and Sox2). Additionally, stearoyl-Coenzyme A desaturase 1 (SCD), a key enzyme that regulates unsaturated fatty acid homeostasis in the lipogenesis process was found among the top DEGs downregulated in response to NTX3 (fold change=9.36, FDR&lt;0.05). As blockade of SCD had been shown to induce ferroptosis, oxidized lipids levels were measured by C11-BODIPY staining. NTX301 treatment increased the levels of oxidized lipids compared to DMSO and this was blunted by deferoxamine, indicating that NTX301 promoted cell death via ferroptosis. NTX301-induced ferroptosis was successfully rescued by the addition of oleic acid (200μM) into the culture medium, supporting that it was mediated through SCD depletion. In vivo, monotherapy with NTX301 (0.5mg/kg), significantly inhibited subcutaneous OC xenograft tumor growth (p&lt;0.05). Decreased SCD levels and increased oxidized lipids were recorded in NTX301-treated xenografts.
Conclusions: NTX301 is a potent HMA active in OC models. Our findings point to a new mechanism by which epigenetic blockade disrupts lipid homeostasis and promotes cancer cell death.
Citation Format: Yinu Wang, Xiaolei Situ, Horacio Cardenas, Daniela Matei, Ellie Siu, Sayedabdulrazzaq A. Alhunayan. NTX301, a novel DNA hypomethylating agent, targets lipid metabolism in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5797.