Abstract Pluripotency transcription factor NANOG represses mitochondrial oxidative phosphorylation (OXPHOS) genes, but activates fatty acid oxidation (FAO) to support self-renewal and drug resistance of tumor-initiating stem-like cells (TICs). Hepatoceullular carcinomas (HCCs: 39%) associated with HCV and alcohol have mutations in ARID1A, a component of the chromatin remodeling complex SWI/SNF. We hypothesized that NANOG binds polycomb repressive complex 2 (PRC2) to reprogram metabolism to promote slow cycling phenotype. Here, the Genome-scale CRISPR Knock-Out (GeCKO) screening with ARID1A mutation identified PRC2 complex genes (EZH2, SUZ12 and EED) in patient-derived TICs. NANOG together with the PRC2 complex inhibits OXPHOS to generate TICs with label-retaining ability and therapy resistance. NANOG binds PRC2 complex. ARID1A gene loss promotes sorafenib-resistance phenotype and self-renewal through PRC2 complex genes. ARID1A mutation promoted HCC development in humanized FRG mice with HCCs. The studies established a novel therapy to overcome the chemoresistance of TICs for HCC treatment. Citation Format: Da-Wei Yeh, Juan Carlos Hernandez, Keigo Machida. NANOG binds polycomb repressive complex 2 to reprogram metabolism to promote slow cycling phenotype in experimental mouse and clinical carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1455.