Developing effective strategies to target human LSCs has proven to be a challenging task. While a number of strategies have been proposed based on studies in preclinical models, the clinical utility of such methods remains unclear. An added difficulty that has come to light in recent years is the likely complexity and heterogeneity of the LSC population(Sarry et al., 2011
). Evidence from human leukemia studies, as well as related efforts in other cancers, have suggested that the properties defining “stemness” may vary amongst individual AML patients, as well as within the same patients during the course of disease pathogenesis (Eppert et al., 2011
; Sarry et al., 2011
). Consequently, it has become increasingly important to determine whether any biological properties are relatively consistent in LSC populations, irrespective of disease stage or the nuances of any individual tumor population. Indeed, fundamental LSC-specific characteristics remain a highly desirable focal point for the development of improved therapies.
In the present study, we examined the properties of LSCs with respect to oxidative status and energy production. In agreement with previous reports on normal stem cells and breast CSCs (Diehn et al., 2009
; Jang and Sharkis, 2007
; Smith et al., 2000
), we observe substantial heterogeneity in LSC oxidative state, an underlying measure of energy metabolism. However, we show that similar to normal stem cell populations (Jang and Sharkis, 2007
; Smith et al., 2000
), functionally defined LSCs preferentially reside in a reduced state (ROS-low). Our findings in LSC-enriched populations, and previous studies in breast CSCs indicate a low oxidative state as a potentially frequent CSC property (Diehn et al., 2009
). Further, we show here that LSC-enriched populations are characterized by quiescent cell cycle status and low energy production. This relatively dormant condition likely enables LSCs to persist, even under restrictive conditions such as low nutrient or oxygen levels. Intriguingly though, in contrast to our findings in normal CD34+ and previous findings in murine HSCs, both showing that normal stem cells efficiently utilize glycolysis for energy homeostasis (Simsek et al., 2010
), ROS-low LSCs appear to be deficient in their ability to employ glycolysis, and are highly reliant on oxidative phosphorylation. Glioma stem cells were also shown to be more reliant on oxidative respiration for energy generation, suggesting that lack of glycolytic activity in stem cells may be a broader characteristic of cancers (Vlashi et al., 2011
). If true, then therapeutic strategies based on the assumption that tumors are preferentially reliant on glycolysis may need to be reconsidered.
Why ROS-low self-renewing LSC populations, in contrast to the bulk tumor and normal HSC (Simsek et al., 2010
) are less efficient in employing glycolysis represents an important avenue of future investigation. We consider it unlikely that the LSC metabolic profile identified in our study is an artifact of ex vivo
cell manipulation, since if this were the case we would expect all leukemic cells to exhibit this phenotype, instead of just the ROS-low subset. Intriguingly, it is recently proposed that cancer cell subsets with different dependencies in energy generating pathways co-exist within tumors in a symbiotic manner(Nakajima and Van Houten, 2012
). In particular, cancer populations that depend on glycolysis for energy generation secrete lactate as a byproduct that is subsequently imported and used as a fuel for oxidative respiration by cancer subsets dependent on oxygen metabolism. Evaluating if this symbiotic model has relevance to the CSC model represents an intriguing line of future investigation. Further, it is possible that the paradoxical OXPHOS dependence of the ROS-low LSC-enriched subset is part of a wider metabolic adaptation of these cells, as they successfully maintain survival despite a dramatically reduced overall metabolic rate. Since oxidative respiration is a slower but significantly more efficient mechanism of energy generation, quiescent LSC subsets may benefit in the long term by using OXPHOS in the context of the tumor microenvironment, as they can more efficiently utilize the limited available nutrients.
By performing gene expression analyses and molecular studies, we show that BCL-2 is preferentially elevated in LSC enriched ROS-low cells. Although BCL-2 is commonly up-regulated in cancer, a differential role for BCL-2 in cancer stem cells has not been widely considered. Notably though, one study by Konopleva et al previously demonstrated the activity of BCL-2 inhibitor ABT-737 towards leukemia cells types, including primary AML CD34+/CD38− cells(Konopleva et al., 2006
). These initial studies first established the concept of using BCL-2 inhibition as a strategy for targeting primitive leukemia cells. Our studies extend the work by Konopleva et al and provide novel insights on the role of BCL-2 in promoting LSC metabolic homeostasis. Further, by employing xenograft analyses we demonstrate here that both ABT-737 and its optimized orally bioavailable analogue ABT-263, not only decrease bulk leukemia burden, but also target functionally defined LSCs.
From a mechanistic standpoint the high levels of BCL-2 expression we observe in ROS-low cells may promote LSC survival in response to stressful stimuli such as chemotherapy and irradiation by inhibiting the mitochondrial pro-apoptotic pathway (Del Poeta et al., 2003
). In addition, we demonstrate that BCL-2 mediates oxidative respiration, which our findings indicate as essential for LSC energy homeostasis (). The newly identified role of BCL-2 in promoting mitochondrial bioenergetics offers a unique opportunity for drug targeting in the LSC-specific context, as LSC not only express high levels of BCL-2 but also have a selective dependency on oxidative respiration. In this vein, we demonstrate that the BCL-2 inhibitors ABT-737 and ABT-263 inhibit LSC oxidative respiration, which is accompanied by selective eradication of ROS-low cells. Importantly, we, and others have shown that this class of drugs does not affect normal HSCs(Konopleva et al., 2006
). The identification of BCL-2 inhibitors as LSC targeting agents together with the absence of significant toxicity to normal cells is intriguing, since it provides a basis for clinical investigation of those drugs at disease stages where targeting residual LSCs is essential, as for instance in consolidation therapy or maintenance treatment during remission.
Model for selective targeting of LSCs by Bcl-2 inhibition
In conclusion, the concept of targeting cancer cell metabolism has emerged as an intriguing approach to the development of improved therapeutic regimens. We demonstrate here that it is feasible to eradicate resistant LSC populations by targeting their unique metabolic dependencies. Understanding further the metabolic regulation of leukemia stem cells may yield improved therapeutic strategies to eradicate this highly drug resistant tumor population.