Bcl-2 Inhibitor ABT-263 Targets Oxidative Phosphorylation and Selectively Eradicates Quiescent Human Leukemia Stem Cells

Abstract 206

Targeting leukemia stem cells (LSCs) is a priority for the development of improved therapeutic regimens. However, the intrinsic heterogeneity of malignant populations in acute myelogenous leukemia (AML) has made it challenging to identify biological properties appropriately conserved amongst primitive cell types. To better characterize physiological features of LSCs related to growth and survival, we previously investigated oxidative state and demonstrated that the majority of functionally-defined LSCs are characterized by relatively low levels of reactive oxygen species (termed “ROS-low”)(Lagadinou et al, abstract 639 ASH 2011). Based on these findings herein we have used primary AML specimens and flow cytometric sorting for endogenous ROS levels so as to enrich for ROS-low LSCs, and we have characterized mechanisms controlling LSC energy production and redox state. We report here that LSC-enriched ROS-low cells are metabolically dormant tumor populations characterized by low levels of oxygen–dependent mitochondrial respiration (OXPHOS), low rates of anaerobic glycolysis, and a low overall cellular ATP content. These properties are unique for LSCs, as bulk leukemic cells and non-tumorigenic ROS-high cells were found to be significantly more metabolically active with regard to both aerobic and anaerobic types of energy production. Intriguingly, we further demonstrate that in contrast to bulk leukemic cells, ROS-low subsets are deficient in their ability to utilize glycolysis when mitochondrial respiration is pharmacologically blocked, indicating a paradoxical dependence of LSCs on mitochondrial energy production. To investigate the mechanisms that underlie the distinct metabolic properties of ROS-low cells, we performed gene expression studies using RNA-seq based methods. In agreement with an important role of mitochondrial metabolism in LSCs we found several mitochondrial-related genes up-regulated in ROS-low cells. Importantly, we found that ROS-low cells express significantly higher levels of bcl-2 both at the mRNA and protein level. To determine if bcl-2 up-regulation relates to the metabolic status of ROS-low cells, we evaluated the bio-energetic profile of bulk AML cells and isolated ROS-low subsets +/− in vitro treatment with the bcl-2 pharmacologic inhibitor ABT-263 and the closely related compound ABT-737. We found that functional inhibition of bcl-2 by this class of drugs results in severe OXPHOS blockage both in total AML cells and ROS-low subsets, indicating a novel non-canonical activity of bcl-2 in promoting AML cell mitochondrial bioenergetics. In unfractionated total AML cells, the bcl-2-inhibitor initiated impairment of OXPHOS was associated with a robust induction of glycolysis and variable toxicity, indicating glycolysis as a compensatory protective response of leukemic cells in this class of drugs. In contrast, bcl-2 inhibition and OXPHOS impairment in ROS-low cells was not compensated by glycolysis, and resulted in depletion of cellular ATP levels, elimination of cellular glutathione pool, oxidation and profound toxicity in the LSC-enriched ROS-low compartment in vitro. Taken together, these studies indicated ABT-263 as an approach to eradicate LSCs by impairing fundamental aspects of LSC metabolism. To more directly investigate this issue we performed xenograft analyses. We first treated ROS-low AML populations in vitro with ABT-263 concentrations equal to the IC₅₀ concentration of the total AML cells for each sample, and then transplanted treated vs. vehicle control cells into immune deficient NSG mice. We found that ABT-263 reduced LSC potential in all AML specimens evaluated by this functional assay. Next, we treated mice engrafted with primary human AML cells with ABT-737 in vivo (50mg/kg IP for 15d), and then performed serial transplantation analyses with the engrafted cells from treated and control mice. These functional studies showed that ABT-737 clearly reduced leukemia burden in the treated primary recipients, and also significantly reduced the capacity of engrafted leukemia cells to establish AML in secondary recipients. Based on these findings, our studies propose a model wherein the unique physiology of ROS-low LSCs provides an opportunity for selective targeting via disruption of Bcl-2-dependent oxidative phosphorylation.