Purpose: DNA methylation causes silencing of tumor suppressor and differentiation-associated genes, being linked to chemoresistance. Previous studies demonstrated that hypomethylating agents (HMA) re-sensitize ovarian cancer (OC) to chemotherapy. NTX-301 is a highly potent and orally bioavailable HMA, in early clinical development. Experimental Design: The anti-tumor effects of NTX-301 were studied in OC models by using cell viability, stemness and ferroptosis assays, RNA sequencing, lipidomic analyses and stimulated Raman spectroscopy. Results: OC cells (SKOV3, IC50=5.08nM; OVCAR5 IC50=3.66nM) were highly sensitive to NTX-301 compared to fallopian tube epithelial cells. NTX-301 downregulated expression of DNA methyltransferases (DNMTs)1-3 and induced transcriptomic reprogramming with 15,000 differentially expressed genes (DEGs, p<0.05). Among them, Gene Ontology enrichment analysis identified regulation of fatty acid biosynthesis and molecular functions related to aldehyde dehydrogenase (ALDH) and oxidoreductase, known features of cancer stem cells. Low dose NTX-301 reduced the ALDH(+) cell population and expression of stemness associated transcription factors. Stearoyl-Coenzyme A desaturase 1 (SCD), which regulates production of unsaturated fatty acids (UFAs), was among the top DEGs downregulated by NTX-301. NTX-301 decreased levels of UFAs and increased oxidized lipids and this was blunted by deferoxamine, indicating cell death via ferroptosis. NTX-301 induced ferroptosis was rescued by oleic acid. In vivo, monotherapy with NTX-301 significantly inhibited OC and patient derived xenograft (PDX) growth (p<0.05). Decreased SCD levels and increased oxidized lipids were detected in NTX-301-treated xenografts. Conclusions: NTX-301 is active in OC models. Our findings point to a new mechanism by which epigenetic blockade disrupts lipid homeostasis and promotes cancer cell death.
