BCL-2 Inhibition Targets Oxidative Phosphorylation and Selectively Eradicates Quiescent Human Leukemia Stem Cells

Most forms of chemotherapy employ mechanisms involving induction of oxidative stress, a strategy that can be effective due to the elevated oxidative state commonly observed in cancer cells. However, recent studies have shown that relative redox levels in primary tumors can be heterogeneous, suggesting that regimens dependent on differential oxidative state may not be uniformly effective. To investigate this issue in hematological malignancies, we evaluated mechanisms controlling oxidative state in primary specimens derived from acute myelogenous leukemia (AML) patients. Our studies demonstrate three striking findings. First, the majority of functionally defined leukemia stem cells (LSCs) are characterized by relatively low levels of reactive oxygen species (termed “ROS-low”). Second, ROS-low LSCs aberrantly overexpress BCL-2. Third, BCL-2 inhibition reduced oxidative phosphorylation and selectively eradicated quiescent LSCs. Based on these findings, we 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. Most forms of chemotherapy employ mechanisms involving induction of oxidative stress, a strategy that can be effective due to the elevated oxidative state commonly observed in cancer cells. However, recent studies have shown that relative redox levels in primary tumors can be heterogeneous, suggesting that regimens dependent on differential oxidative state may not be uniformly effective. To investigate this issue in hematological malignancies, we evaluated mechanisms controlling oxidative state in primary specimens derived from acute myelogenous leukemia (AML) patients. Our studies demonstrate three striking findings. First, the majority of functionally defined leukemia stem cells (LSCs) are characterized by relatively low levels of reactive oxygen species (termed “ROS-low”). Second, ROS-low LSCs aberrantly overexpress BCL-2. Third, BCL-2 inhibition reduced oxidative phosphorylation and selectively eradicated quiescent LSCs. Based on these findings, we 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. LSC are prospectively isolated from the bulk tumor on the basis of low ROS levels Metabolic dependencies discriminating LSCs, bulk tumor, and normal HSCs are described BCL-2 is identified as a regulator of LSC mitochondrial respiration BCL-2 pharmacologic inhibitors demonstrate LSC-specific targeting Biological analyses of leukemia stem cells (LSCs) has been the focus of numerous studies over the past decade, with reports describing molecular, cellular, biochemical, and genetic properties in both human and mouse systems. It has been assumed to at least some extent that the biology of LSCs mirrors that of normal tissues, where malignant stem cells reside at the apex of a hierarchical developmental structure. Indeed, key stem cell properties such as self-renewal are readily evident in stem cells from multiple tumor types (Magee et al., 2012Magee J.A. Piskounova E. Morrison S.J. Cancer stem cells: impact, heterogeneity, and uncertainty.Cancer Cell. 2012; 21: 283-296Abstract Full Text Full Text PDF PubMed Scopus (889) Google Scholar). In addition, quiescent cell cycle status, a very well described feature of normal hematopoietic stem cells (HSCs), also appears to be a central property of functionally defined acute myeloid leukemia (AML) stem cells (Saito et al., 2010Saito Y. Uchida N. Tanaka S. Suzuki N. Tomizawa-Murasawa M. Sone A. Najima Y. Takagi S. Aoki Y. Wake A. et al.Induction of cell cycle entry eliminates human leukemia stem cells in a mouse model of AML.Nat. Biotechnol. 2010; 28: 275-280PubMed Google Scholar). Further, several molecular mechanisms that regulate normal stem cell properties are also frequently active (and/or aberrant) in cancer cell types as well (Magee et al., 2012Magee J.A. Piskounova E. Morrison S.J. Cancer stem cells: impact, heterogeneity, and uncertainty.Cancer Cell. 2012; 21: 283-296Abstract Full Text Full Text PDF PubMed Scopus (889) Google Scholar). These observations have led investigators to attempt targeting of LSC populations based on properties that are thought to be shared among normal and tumor primitive cells and are conserved from patient to patient. Despite the similarities noted above, it has become increasingly clear that primitive cancer cell types possess important differences from normal tissues, some of which run counter to conventional views of stem cell biology. For example, recent studies have shown that cell surface immunophenotype can vary widely among specimens derived from primary AML patients, and that commonly used stem cell antigens such as CD34 and CD38 do not show consistent levels of expression on LSCs (Eppert et al., 2011Eppert K. Takenaka K. Lechman E.R. Waldron L. Nilsson B. van Galen P. Metzeler K.H. Poeppl A. Ling V. Beyene J. et al.Stem cell gene expression programs influence clinical outcome in human leukemia.Nat. Med. 2011; 17: 1086-1093Crossref PubMed Scopus (728) Google Scholar; Sarry et al., 2011Sarry J.E. Murphy K. Perry R. Sanchez P.V. Secreto A. Keefer C. Swider C.R. Strzelecki A.C. Cavelier C. Récher C. et al.Human acute myelogenous leukemia stem cells are rare and heterogeneous when assayed in NOD/SCID/IL2Rγc-deficient mice.J. Clin. Invest. 2011; 121: 384-395Crossref PubMed Scopus (290) Google Scholar). Thus, unlike normal hematopoietic tissues, which show substantial interspecimen similarities, some of the biological properties of independent LSCs may be highly disparate and/or unstable. Given the observations outlined above, we sought to better define fundamental aspects of LSC biology, which are conserved irrespective of interpatient or intrapatient heterogeneity. To this end, we investigated energy metabolism, a basic requirement of any cell type. Cancer cells appear to commonly acquire aberrancies in energy metabolism and cellular redox state (Cairns et al., 2011Cairns R.A. Harris I.S. Mak T.W. Regulation of cancer cell metabolism.Nat. Rev. Cancer. 2011; 11: 85-95Crossref PubMed Scopus (3564) Google Scholar). In contrast to their normal counterparts, cancer cells tend in the presence of oxygen to employ glycolysis instead of aerobic mitochondrial respiration to generate energy (Warburg, 1956Warburg O. On the origin of cancer cells.Science. 1956; 123: 309-314Crossref PubMed Scopus (9557) Google Scholar). Notably, in at least some instances, cancer cells generate increased levels of reactive oxygen species (ROS) (Trachootham et al., 2008Trachootham D. Zhang H. Zhang W. Feng L. Du M. Zhou Y. Chen Z. Pelicano H. Plunkett W. Wierda W.G. et al.Effective elimination of fludarabine-resistant CLL cells by PEITC through a redox-mediated mechanism.Blood. 2008; 112: 1912-1922Crossref PubMed Scopus (164) Google Scholar). This notion has provided the impetus to explore redox modulation as a therapeutic target in cancer, with the rationale that pro-oxidants may eradicate tumor cells by pushing them beyond the limits of viability (Trachootham et al., 2009Trachootham D. Alexandre J. Huang P. Targeting cancer cells by ROS-mediated mechanisms: a radical therapeutic approach?.Nat. Rev. Drug Discov. 2009; 8: 579-591Crossref PubMed Scopus (3793) Google Scholar). However, while such tumor-related metabolic adaptations characterize bulk tumor tissues, it is unclear whether cancer stem cells (CSCs) also exhibit an increased oxidative state. Indeed, previous studies by Diehn et al., 2009Diehn M. Cho R.W. Lobo N.A. Kalisky T. Dorie M.J. Kulp A.N. Qian D. Lam J.S. Ailles L.E. Wong M. et al.Association of reactive oxygen species levels and radioresistance in cancer stem cells.Nature. 2009; 458: 780-783Crossref PubMed Scopus (1860) Google Scholar demonstrated that breast cancer cells enriched for the breast CSC-related CD24−/CD44+ phenotypic profile showed lower levels of ROS than the “non CD24−/CD44+” cells. Gaining further insights into the metabolic properties of CSCs may reveal physiological dependencies that can be targeted for therapy. In the present study we used primary human AML specimens and a stringent xenograft assay to investigate the oxidative state and the bioenergetic properties of LSCs. We show that in contrast to the nontumorigenic cells, the majority of self-renewing and chemotherapy-resistant primitive leukemic populations are quiescent tumor subsets characterized by a low rate of energy metabolism and a low cellular oxidative status (termed “ROS-low”). Surprisingly, ROS-low cells are unable to utilize glycolysis when mitochondrial respiration is inhibited. Thus, we show that the maintenance of mitochondrial function is essential for LSC survival. Finally, we demonstrate a unique role for BCL-2 in ROS-low cell mitochondrial respiration, and show that small molecule BCL-2 inhibitors can effectively target chemotherapy-resistant LSCs by impairing their energy generation capacity and redox control. Our findings indicate it is feasible to eradicate therapy-resistant LSC populations by targeting their unique metabolic dependencies. To characterize leukemia cells with differing oxidative state, we employed a method previously reported by Jang and Sharkis, 2007Jang Y.Y. Sharkis S.J. A low level of reactive oxygen species selects for primitive hematopoietic stem cells that may reside in the low-oxygenic niche.Blood. 2007; 110: 3056-3063Crossref PubMed Scopus (652) Google Scholar. In this study, the authors used cell sorting based on the relative fluorescence of redox-sensitive probes to isolate HSCs with differing endogenous ROS levels. Since a number of recent studies have highlighted that LSCs may display disparate phenotypic patterns besides CD34+/CD38−/CD123+ (Eppert et al., 2011Eppert K. Takenaka K. Lechman E.R. Waldron L. Nilsson B. van Galen P. Metzeler K.H. Poeppl A. Ling V. Beyene J. et al.Stem cell gene expression programs influence clinical outcome in human leukemia.Nat. Med. 2011; 17: 1086-1093Crossref PubMed Scopus (728) Google Scholar; Sarry et al., 2011Sarry J.E. Murphy K. Perry R. Sanchez P.V. Secreto A. Keefer C. Swider C.R. Strzelecki A.C. Cavelier C. Récher C. et al.Human acute myelogenous leukemia stem cells are rare and heterogeneous when assayed in NOD/SCID/IL2Rγc-deficient mice.J. Clin. Invest. 2011; 121: 384-395Crossref PubMed Scopus (290) Google Scholar), in performing our initial analyses of ROS levels in primary AML samples, we did not rely on standard phenotypic profiles, but rather examined the entire leukemic population. By employing several redox-sensitive probes commonly used to measure intracellular ROS (CM-H2DCFDA, CellROX, MitoSox, Mitotracker, and DHE) we consistently observed substantial heterogeneity in the redox staining profile of AML specimens (Figure 1A and Figures S1A and S1B available online). Since the fluorescent dyes CM-H2DCFDA and CellROX provide a broader measure of intracellular redox levels, we used those two dyes to sort AML specimens into subsets with low and high endogenous ROS (defined as the 15% dimmest and brightest dye fluorescence distribution as shown in Figure 1A) and then analyzed the two populations for stem cell properties. A critical feature of both normal and leukemia primitive populations is quiescence, as increased proliferation can lead to exhaustion of stem cell function (Ito et al., 2006Ito K. Hirao A. Arai F. Takubo K. Matsuoka S. Miyamoto K. Ohmura M. Naka K. Hosokawa K. Ikeda Y. Suda T. Reactive oxygen species act through p38 MAPK to limit the lifespan of hematopoietic stem cells.Nat. Med. 2006; 12: 446-451Crossref PubMed Scopus (1072) Google Scholar; Tothova et al., 2007Tothova Z. Kollipara R. Huntly B.J. Lee B.H. Castrillon D.H. Cullen D.E. McDowell E.P. Lazo-Kallanian S. Williams I.R. Sears C. et al.FoxOs are critical mediators of hematopoietic stem cell resistance to physiologic oxidative stress.Cell. 2007; 128: 325-339Abstract Full Text Full Text PDF PubMed Scopus (1241) Google Scholar). We hypothesized that quiescence may reduce LSC metabolic activity and thus lower ROS production, so slowly proliferating LSCs should be enriched in the redox low compartment. To investigate this issue, we colabeled sorted ROS-low and ROS-high fractions with anti-Ki67 and 7AAD to evaluate cell cycle status. As shown in Figures 1B and 1C, leukemic ROS-low cells are preferentially enriched for G0 quiescent cells. This finding was corroborated by analyses of cell cycle components, which demonstrated reduced expression of CDK1; cyclins A2, B2, D3 and E2; and increased expression of the cell cycle inhibitor p27 in ROS-low cells (Figures S1C and S1D). ROS-low cells also showed reduced expression of the MAPK kinase phospho-p38, which is associated with redox state regulation and cell cycle activity in normal hematopoiesis (Figure S1D) (Ito et al., 2006Ito K. Hirao A. Arai F. Takubo K. Matsuoka S. Miyamoto K. Ohmura M. Naka K. Hosokawa K. Ikeda Y. Suda T. Reactive oxygen species act through p38 MAPK to limit the lifespan of hematopoietic stem cells.Nat. Med. 2006; 12: 446-451Crossref PubMed Scopus (1072) Google Scholar). Interestingly, ROS-low subsets isolated from the M9-ENL1 AML cell line and from normal marrow CD34+ cells were also more quiescent as compared to corresponding ROS-high cells (Figure S1E), indicating that the association between lower ROS and quiescence is conserved in both cell lines and normal HSCs. To assess whether AML cells with lower endogenous ROS are enriched for primitive leukemia cells, we first performed in vitro colony assays to determine colony-forming units (CFUs). In four evaluable AML samples, the leukemic cells with lower ROS gave growth to more CFUs in comparison to ROS-high or total AML cells (Figure 1D), suggesting enrichment of primitive cells in the ROS-low compartment. To functionally evaluate LSC potential, xenograft studies were performed using transplantation of primary human cells into immune deficient NOD/SCID-IL2Rγ−/− (NSG) mice. As shown in Figure 1E, LSC activity (i.e., successful engraftment of NSG mice) is readily evident in all ROS-low AML subsets evaluated (five of five independent specimens). Notably, in two of five specimens we also detected some LSC activity (albeit at lower levels) with ROS-high AML cells, suggesting that functionally defined primitive leukemia populations can exist across a relatively broad ROS gradient in at least some cases. However, the leukemic subsets isolated from the lowest end of the ROS range were consistently more enriched for LSC activity as compared to AML cells containing high levels of ROS (Figure 1E and Figure S1F). Indeed, in transplantation experiments (Figure 1E), despite the fact that AML ROS-low cells were injected in NSG mice at a 5-fold lower dose than unfractionated total AML cells (Table S2), the ROS-low population engrafted at levels comparable to the total AML cells (AML ROS-low = 44.6 ± 38 versus total AML = 51 ± 38, p = 0.122 > 0.05, n = 5). Further, to address whether ROS-low cells fulfill the stem cell criterion of long-term self-renewal, we performed serial transplantation studies in secondary mouse recipients. ROS-low leukemic cells reestablished AML in the secondary recipients in all cases, thus confirming their leukemic stem cell potential (Table S3). Interestingly, in two out of three AML specimens where ROS-high AML cells engrafted at levels that allowed comparative serial transplantation analyses, we observed long-term self-renewal potential in secondary recipients, suggesting that LSCs are significantly fewer but evident within the redox high leukemic compartment (Table S3). Since normal HSCs have been suggested to reside in a reduced oxidized state, we next tested if the increased engraftment potential of the ROS-low AML subset resulted from contributing HSCs (Tothova et al., 2007Tothova Z. Kollipara R. Huntly B.J. Lee B.H. Castrillon D.H. Cullen D.E. McDowell E.P. Lazo-Kallanian S. Williams I.R. Sears C. et al.FoxOs are critical mediators of hematopoietic stem cell resistance to physiologic oxidative stress.Cell. 2007; 128: 325-339Abstract Full Text Full Text PDF PubMed Scopus (1241) Google Scholar). To this end we analyzed the human cells from mice engrafted with ROS-low or ROS-high AML cells (patient sample #1) for expression of mutant nucleophosmin (NPM), which is a frequently observed leukemia marker (Falini et al., 2007Falini B. Nicoletti I. Martelli M.F. Mecucci C. Acute myeloid leukemia carrying cytoplasmic/mutated nucleophosmin (NPMc+ AML): biologic and clinical features.Blood. 2007; 109: 874-885Crossref PubMed Scopus (429) Google Scholar). Engrafted AML cells from both leukemic subsets were positive for this marker (Figure S1G), thus confirming the leukemic origin of engrafted cells in both cases. We next asked if AML cells containing lower levels of ROS are enriched for the CD34+/CD38− phenotypic profile, which is commonly associated with stem cells in both normal and leukemic hematopoiesis. We found an increased percentage of CD34+/CD38− cells in the ROS-low subset as compared to those in the ROS-high, but no significant enrichment of this phenotypic profile when the ROS-low cells where compared to the total AML population (Figure S1H). Further, analysis of AML specimens with an additional collection of antibodies associated with primitive cell types (CD123, CD33, CD117, CD90, and CD44) did not reveal any clear association between surface expression profile and a lower oxidative state (data not shown). Taken together, these findings indicate that endogenous ROS levels can be used as a tool to prospectively isolate LSC-enriched populations. To identify properties of LSCs that can be targeted for therapy, we therefore used endogenous ROS sorting to enrich for LSCs, and studied mechanisms controlling energy metabolism in LSC-enriched populations. To gain insights into the metabolic regulation of LSCs, we performed bioenergetic analyses in ROS-low and ROS-high primary AML specimens. We first investigated the relative dependence of the ROS-low AML cells to the two major energy-generating pathways: mitochondrial oxidative phosphorylation (OXPHOS) and glycolysis. To this end, we used the Seahorse XF24 extracellular flux analyzer to measure the oxygen consumption rate (OCR) indicative of OXPHOS, and the lactate production indicative of glycolysis (extracellular acidification rate; ECAR). As shown in Figure 2A, LSC-enriched ROS-low cells showed a significantly lower OCR rate, indicative of lower oxidative metabolism, as compared to the total and ROS-high AML cells. While oxidative processes occur mainly in mitochondria, oxidative activity can also be found in the cytoplasmic membrane due to the presence of the NADPH oxidases (Hole et al., 2010Hole P.S. Pearn L. Tonks A.J. James P.E. Burnett A.K. Darley R.L. Tonks A. Ras-induced reactive oxygen species promote growth factor-independent proliferation in human CD34+ hematopoietic progenitor cells.Blood. 2010; 115: 1238-1246Crossref PubMed Scopus (94) Google Scholar). Therefore, we evaluated the protein expression of NADPH oxidase subunit NOX2 (gp91phox) by flow cytometry. As shown in Figure 2B, NOX2 levels were found to be quite low in ROS-low cells, indicating mitochondria as the primary site of oxidative metabolism in LSC-enriched populations. AML ROS-low cells isolated based on the fluorescence of the redox dye CM-H2DCFDA also showed reduced labeling with the mitochondrial-specific redox probe MitoSox-Red, indicating a lower content of mitochondrial-derived ROS (Figure S2). We next examined relative rates of glycolysis and observed that ROS-low cells are substantially less active with respect to this anaerobic form of energy production as well (Figure 2C). While the basal rate of glycolysis in ROS-low cells is low, it is possible that under certain conditions (i.e., in hypoxic niches in vivo) LSCs might compensate for low OXPHOS by upregulating glycolysis. To investigate this issue we measured lactate generation indicative of the cellular glycolytic rate after treatment of cells with the mitochondrial respiration inhibitors oligomycin (OLI) and FCCP. OLI and FCCP inhibit mitochondrial respiration and force cells to the glycolytic pathway to maintain energy supply, so treatment with these agents is indicative of the reserved cellular glycolytic potential. Surprisingly, LSC-enriched ROS-low AML cells showed a significantly decreased potential to upregulate the glycolytic machinery and thus sustain energy generation when mitochondrial energy production was inhibited (Figures 2D and 2E). In agreement with our bioenergetic data, ROS-low cells showed significantly decreased ATP levels in all samples evaluated (Figure 2F). To determine whether the metabolic status of ROS-low AML cells represents an aberrant condition, we performed comparative bioenergetic analyses of normal bone marrow CD34+ progenitors isolated from healthy volunteers. A recent study showed that murine hematopoietic stem populations are predominantly glycolytic (Simsek et al., 2010Simsek T. Kocabas F. Zheng J. Deberardinis R.J. Mahmoud A.I. Olson E.N. Schneider J.W. Zhang C.C. Sadek H.A. The distinct metabolic profile of hematopoietic stem cells reflects their location in a hypoxic niche.Cell Stem Cell. 2010; 7: 380-390Abstract Full Text Full Text PDF PubMed Scopus (761) Google Scholar), but to our knowledge the bioenergetic profile of human marrow progenitors has not been previously investigated. We found that, in contrast to AML ROS-low cells, normal CD34+ cells are metabolically active cells with baseline glycolysis and OXPHOS rates similar to CD34− cells (Figures 2G and 2I). Further, normal CD34+ progenitors preferentially (in comparison with CD34− cells) upregulate glycolysis when mitochondrial OXPHOS is blocked (Figures 2G and 2H). Taken together, our metabolic analyses indicate that LSC-enriched subsets are metabolically dormant tumor populations that reside in a substantially decreased energetic state. Importantly, our findings further suggest that in contrast to normal progenitor cells, LSC-enriched subsets may paradoxically depend more on mitochondrial respiration rather than glycolysis to meet their energy demands and maintain their survival. To investigate mechanisms that control oxidative state and metabolic processes in ROS-low and ROS-high populations, we performed gene expression studies using RNA-seq-based methods. Since our findings indicated LSC-enriched populations contained lower levels of ROS, we investigated if upregulation of antioxidant genes represents a prominent feature of ROS-low AML cells. We found only a small number of genes related to antioxidant defenses to be significantly altered in ROS-low AML cells, none of which were consistently upregulated in the ROS-low population (Figure S3A). We next investigated the relative expression of genes related to mitochondria and energy metabolism. In agreement with an important role of mitochondrial metabolism in LSCs, we found several mitochondrial-related genes upregulated in ROS-low cells. These included the SLC25A42 and SLC25A38 mitochondrial carriers that transport metabolites and amino acids across the mitochondrial membrane, and the gene FIS1, which is critical for mitochondrial dynamics and fission (Figure 3A) (Westermann, 2010Westermann B. Mitochondrial fusion and fission in cell life and death.Nat. Rev. Mol. Cell Biol. 2010; 11: 872-884Crossref PubMed Scopus (1349) Google Scholar). Intriguingly, we found that the LSC-enriched ROS-low population expressed significantly higher levels of BCL-2 (Figure 3A). We validated this initial observation using quantitative PCR in a larger AML sample cohort (Figure 3B). BCL-2 mRNA expression correlated with protein expression, as ROS-low AML cells expressed higher levels of BCL-2 protein (Figure 3C), whereas they showed no significant upregulation of other antiapoptotic BCL-2 family members including BCL-XL and MCL-1 (Figures S3B and S3C). Parallel analyses of BCL-2 gene expression in normal marrow specimens showed, in agreement with previous observations (Delia et al., 1992Delia D. Aiello A. Soligo D. Fontanella E. Melani C. Pezzella F. Pierotti M.A. Della Porta G. bcl-2 proto-oncogene expression in normal and neoplastic human myeloid cells.Blood. 1992; 79: 1291-1298Crossref PubMed Google Scholar), that normal CD34+ progenitors express significantly higher levels of BCL-2 as compared to more differentiated CD34− cells (Figures S3D–S3F). To investigate the extent to which normal CD34+ progenitors share the BCL-2 expression profile observed in leukemic cells, we further examined BCL-2 levels in normal CD34+ ROS-low versus ROS-high populations. To this end, we enriched for CD34+ progenitors using an immunomagnetic affinity column and then isolated CD34+ ROS-low and ROS-high populations by flow cytometry. CD34+ ROS-low cells showed a modest increase in BCL-2 mRNA expression as compared to CD34+ ROS-high, but we found no difference in the BCL-2 protein levels among the two populations in three independent experiments (Figures S3G–S3H). Based on these data, we conclude there is no differential activity of BCL-2 between normal ROS-low and ROS-high compartments. The upregulation of BCL-2 in the LSC is potentially important, since (1) BCL-2 has an established role as an inhibitor of the mitochondrial-initiated proapoptotic pathway and thus can represent an important contributor in the chemoresistance properties of LSCs (Del Poeta et al., 2003Del Poeta G. Venditti A. Del Principe M.I. Maurillo L. Buccisano F. Tamburini A. Cox M.C. Franchi A. Bruno A. Mazzone C. et al.Amount of spontaneous apoptosis detected by Bax/Bcl-2 ratio predicts outcome in acute myeloid leukemia (AML).Blood. 2003; 101: 2125-2131Crossref PubMed Scopus (288) Google Scholar), and (2) recent evidence points to a noncanonical activity of BCL-2 in regulating oxidative state and mitochondrial metabolism (Chen and Pervaiz, 2007Chen Z.X. Pervaiz S. Bcl-2 induces pro-oxidant state by engaging mitochondrial respiration in tumor cells.Cell Death Differ. 2007; 14: 1617-1627Crossref PubMed Scopus (128) Google Scholar). Thus, elevated BCL-2 could contribute to key functional properties of LSCs. To investigate the role of BCL-2 in the metabolic homeostasis of primitive leukemia cells, we determined the bioenergetic properties of primary AML cells treated with BCL-2 pharmacologic inhibitors. We found that within minutes of treatment, the BCL-2 inhibitor ABT-263 induces severe impairment of OXPHOS in primary unfractionated AML cells (Figure 4A and Figure S4A). Similarly, a second chemically distinct BCL-2 inhibitor, obatoclax, also inhibited oxidative respiration in leukemia cells (Figure S4B), albeit to a lesser extent than ABT-263. This effect is accompanied by a robust glycolytic response (Figure 4D), indicating that glycolysis is a compensatory mechanism activated upon inhibition of OXPHOS by BCL-2 inhibitors. ABT-263 similarly impaired OXPHOS in the LSC-enriched ROS-low cells (Figure 4B). However, in agreement with our previous findings showing that ROS-low AML cells have reduced reserved glycolytic capacity (Figures 2D and 2E), ROS-low cells were not able to induce glycolysis (Figure 4D). These findings suggested that ABT-263 should selectively inhibit energy generation in the ROS-low population. Indeed, ABT-263 rapidly depleted cellular ATP in the ROS-low AML subset (Figure 4E, left panel), whereas it had no effect in the ATP content of the ROS-high population (Figure 4E, middle panel). To investigate the role of BCL-2 in normal populations, we tested the effect of ABT-263 treatment in CD34+ cells from healthy donors. As shown in Figure 4C, OCR was impaired, albeit to a lesser extent than that observed in leukemic cells. In agreement with our data showing that normal CD34+ cells possess reserve glycolytic capacity (Figure 2H), we detected a clear upregulation of glycolysis in normal CD34+ subsets in response to ABT-263-induced OXPHOS inhibition (Figure 4D). Importantly, evaluation of the effect of ABT-263 on the ATP content of normal CD34+ progenitors revealed no significant impact, unless ABT-263 was combined with the glycolysis inhibitor 2-deoxyglucose (2-DG) (Figure 4E, right panel). The combination of ABT-263 and 2-DG induced a decrease in the ATP content of normal CD34+ cells to levels similar to those observed in ROS-low AML cells treated with ABT-263 alone. These data indicate that glycolytic capacity represents an important biological property that distinguishes ROS-low AML cells from normal CD34+ cells. To more directly address the role of BCL-2 in the oxidative respiration of leukemia cells, we employed an shRNA strategy to genetically reduce gene expression. Using lentiviral-mediated gene transfer, we expressed BCL-2 shRNAs in primary AML cells and the U937 leukemia cell line (Figure S4C). Primary leukemic and U937 cells transduced with BCL-2 shRNA showed a significantly reduced basal oxygen consumption rate as compared to cells transduced with a control vector (Figure 4F and Figure S4D), indicating that BCL-2 is a positive regulator of OXPHOS in leukemia cells. Further, the capacity of ABT-263 to suppress oxidative respiration was markedly decreased in BCL-2 shRNA-transduced primary AML cells as compared to vector control cells (Figure 4G and Figure S4E). Knocking down BCL-XL (the other known target of ABT-263) (van Delft et al., 2006van Delft M.F. Wei A.H. Mason K.D. Vandenberg C.J. Chen L. Czabotar P.E. Willis S.N. Scott C.L. Day C.L. Cory S. et al.The BH3 mimetic ABT-737 targets selective Bcl-2 proteins and efficiently induces apoptosis via Bak/Bax if Mcl-1 is neutralized.Cancer Cell. 2006; 10: 389-399Abstract Full Text Full Text PDF PubMed Scopus (1057) Google Scholar) by specific shRNA did not have a significant effect on the OXPHOS of