Abstract Potential cell surface markers specifically enriching the leukemia stem cell (LSC) subfraction based on stemness genes expression have not yet been identified. Previously, we reported ABCG2, a drug efflux pump, enriches a subpopulation of cells exhibiting a very high level of stemness genes expression in ES cells. These ABCG2+ ES cells exhibited high HIF-2α, which in cooperation with MYC-regulated Nanog and Sox-2. ABCG2+ cells demonstrated highly cytoprotective altruistic behavior by secreting high levels of glutathione. In the present study, we evaluated whether ABCG2+ subfraction of human LSCs exhibits high level of stemness genes and altruistic behavior. We isolated the ABCG2+ cell subfraction from cervical lymph node and blood of human T-ALL(n=5) and CML(n=7) patients exhibiting high levels of stemness genes in addition to MYC and HIF-2α. We investigated the altruistic stemness phenotype of the ABCG2+ cells by studying the molecular signature of MYC binding to HIF-2α. siRNA HIF-2α treatment led to ABCG2+ cell loss of proliferative capacity and reduced GSH levels, suggesting reduction of cytoprotective altruistic behavior in vitro. Treatment of ABCG2+ cell harboring mice with FM19G11 led to marked loss in the secondary engraftment in NOD/SCID mice, indicating loss of ABCG2+ cell self-renewal capacity. We found siRNA MYC inhibition led to loss of HIF-2α expression, suggesting that MYC might be regulating HIF-2α mediated altruistic stemness program in the ABCG2+ cells. Overall, we found that ABCG2 can be a probable marker to enrich the LSC subfraction having high expression of stemness genes and altruistic stemness phenotype. Citation Format: Shirsajit Mitra, Lekhika Pathak, Partha Jyoti Saikia, Bidisha Pal, Bikul Das. MYC mediated HIF-2α modulates the altruistic stemness reprogramming in leukemia stem cells [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 1266.