Reactive Oxygen Species (ROS) Levels Define Functional Heterogeneity in Human Leukemia Stem Cells and Represent a Critical Parameter for Therapeutic Targeting

Abstract 639

Failure to eliminate leukemia stem cells (LSC) appears to be a major limitation of standard regimens and a significant challenge in the development of improved therapies. To effectively target LSC, it is essential to unravel the unique functional properties of these cells with respect to bulk leukemic and normal cells. Reactive oxygen species (ROS) are oxygen-containing chemicals continuously generated in cells through various biochemical pathways. Normal stem cells appear to tightly control intracellular ROS at low levels so as to maintain long-term self-renewal and survival. Although cancer cells are generally considered to have increased ROS levels due to oncogene activation and acquisition of a tumor-specific metabolic profile, the redox regulation in cancer stem cells remains largely unknown. We hypothesized that, similar to normal stem cells, that redox state is important for LSC survival. By employing primary human acute myeloid leukemia (AML) samples and several redox-sensitive dyes that provide information on different aspects of intracellular oxidative state, we show that leukemic cells display a range of intracellular ROS, with functionally defined LSC being distributed across the ROS gradient. However, leukemic cells prospectively isolated by flow cytometry from the lowest end of the ROS gradient contain the highest concentration of leukemic stem cell activity, as defined by their preferentially quiescent cell cycle profile, enhanced potential in colony forming assays, increased engraftment in immune deficient NSG mice, and capacity to re-establish in the xenograft model an oxidative state heterogeneity similar to that observed in the primary tumor. Further, in serial transplantation assays, which evaluate the long-term maintenance of self-renewal capacity, the ROS-low leukemic population is preferentially able to engraft NSG mice in most cases. In contrast, leukemic cells residing in higher oxidative levels (ROS-high) are more actively cycling; show reduced in vivo self-renewal potential in NSG mice, and evidence of increased DNA damage. Upon therapeutic challenge, prospectively isolated ROS-low primary leukemic cells are less responsive to in vitro drug treatment with both conventional (daunorubicin) and experimental (parthenolide, temsirolimus) anti-leukemic agents, and are better able to resist induction towards an increased oxidative state after pro-oxidant treatment (hydrogen peroxide, BSO). To further investigate the relative sensitivity of ROS-low vs high primitive populations to anti-leukemic therapies, we treated sorted primary AML subsets with anti-leukemic drugs for 18hr and then transplanted into NSG mice. In this functional assay, only the ROS-low LSC fraction retains its self-renewal capacity after therapy, thereby indicating that ROS-low cells may be a critical target for therapy. To better understand mechanisms controlling redox heterogeneity in leukemia, we performed biochemical analyses on ROS-low vs high subsets. Our studies indicate that ROS-low cells adopt unique metabolic and redox regulation properties. Whereas ROS-high cells exhibit robust activation of pathways implicated in cell cycle promotion, metabolism, inflammation and senescence, the respective gene and protein expression in ROS-low cells is either markedly decreased or diminished, and ROS-low cells also show a dramatically reduced dependence on mitochondrial oxidative phosphorylation, as determined by bio-energetic analyses. Further, ROS-low cells seem to contain increased levels of glutathione, a critical cellular antioxidant. Taken together, these findings suggest that ROS-low LSC represent an important target for therapy, but as a consequence of their unique biological properties may be very difficult to eradicate. To this end, we have begun to characterize agents with the ability to enhance leukemia selective targeting of the ROS-low population. Intriguingly, our initial studies show that the naturally occurring compound rocaglamide is a potent inducer of cell death in the ROS-low compartment, as determined by both in vitro culture and xenograft analyses. Importantly, rocaglamide is much less toxic to normal stem and progenitor, indicating that this approach may have a significant therapeutic index. Thus, we propose that regimens designed to include compounds of this class may result in more effective elimination of human LSCs.