Abstract Leukemia is hierarchically organized into two distinct population of cancer cells, cancer stem cells (CSCs) and non-stem cancer cells (NSCC). Numerous studies suggest that CSCs may express embryonic stem (ES) cells related stemness genes including Nanog, Sox-2, and Oct-4. However, potential cell surface markers that could specifically enrich the leukemia stem cell (LSC) subfraction based on the expression of these stemness genes have not yet been identified. Previously, we reported that ABCG2, a drug efflux pump, could enrich a subpopulation of cells exhibiting very high level of Nanog, Sox-2, and Oct-4 in ES cells. Additionally, these ABCG2+ ES cells also exhibited high HIF-2α, a transcription factor, which in cooperation with MYC-regulated both Nanog and Sox-2. In addition to these features, 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 Nanog, Sox2, and Oct4. We also investigated the altruistic behavior of the LSCs, a potential novel mechanism of drug resistance and disease relapse. Methods: ABCG2+ cells from cervical lymph node and peripheral blood of T- cell acute lymphoblastic lymphoma/leukemia (T-ALL) and chronic myeloid leukemia (CML) patients (n=12) were enriched using immunomagnetic sorting. These cells were then expanded and used for different experiments including flow cytometry, ChiP on ChiP assay, in vivo transplantation assay, and siRNA inhibition treatment, to demonstrate the role of HIF-2α and MYC in regulating the altruistic stemness program in human LSCs. Results: We were able to enrich ABCG2+ cell subfraction from human T-ALL (n=5) and CML (n=7) patients exhibiting high levels of stemness genes such as Nanog, Sox-2, and Oct-4 in addition to MYC and HIF-2α. Higher engraftment potential was observed in ABCG2+ cells in comparison to ABCG2- cells as indicated by in vivo transplantation assay in NOD/SCID mice. The inhibition of stemness genes Nanog and Sox2 by siRNA gene silencing led to the loss of expression of MYC and HIF-2α, indicating that these genes are regulated MYC and HIF-2α. We then investigated the altruistic stemness phenotype of the ABCG2+ cells by studying the distinct molecular signature of MYC binding to HIF-2α in these cells. siRNA HIF-2α treatment led to ABCG2+ cell loss of proliferative capacity and reduced GSH levels, suggesting reduction of cytoprotective altruistic behavior in vitro. Additionally, siRNA HIF-2α treated ABCG2+ cells, when injected in mice, exhibited increased survival. Importantly, treatment of ABCG2+ cell harboring mice with FM19G11, a HIF inhibitor, led to marked loss in the secondary engraftment in NOD/SCID mice, thus indicating loss of ABCG2+ cell self-renewal capacity. Then we found that 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. Conclusion: To summarize, we found that ABCG2 is an excellent marker to enrich the LSC subfraction having high expression of stemness genes. The ABCG2+ LSCs exhibit altruistic stemness phenotype that includes high secretion of GSH and the distinct binding of MYC to HIF-2a, Sox2, and Nanog. Thus, we now report that a subfraction of LSC could also be enriched such that it exhibits altruistic stemness phenotype which was previously reported by us in ES cells. Our findings may open up new understanding of the LSC-mediated cancer relapse and drug resistance. Citation Format: Bidisha Pal, Anupam Sarma, Joyeeta Talukdar, Seema Bhuyan, Sora Sandhya, Sukanya Gayan, Gayatri Gogoi, Debabrat Baishya, Amal Chandra Kataki, Dean W. Felsher, Bikul Das. MYC through HIF-2α regulates the altruistic stemness program in human leukemia stem cells [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr 42.