Chronic Myelogenous Leukemia Progenitor Cells Research Articles

Metabolic Adaptation to Tyrosine Kinase Inhibition in Chronic Myelogenous Leukemia Stem Cells

Objectives: BCR-ABL tyrosine kinase inhibitors (TKI) are highly effective in treatment of chronic myelogenous leukemia (CML) but fail to eliminate leukemia stem cells, which persist as a source of disease recurrence. Previous studies have suggested that primitive CML cells rely on upregulated oxidative metabolism for survival. However, there is limited knowledge about the effects of TKI treatment on CML stem cell metabolism and the role of metabolic adaptations in stem cell persistence after TKI treatment. Here we evaluated the metabolic response to TKI treatment in CML stem and progenitor cells using a representative SCL-tTA/BCR-ABL mouse model of CML. Methods: CML mice were treated with the TKI Nilotinib or vehicle for 2 days, to evaluate initial effects of treatment, and for 2 weeks, to evaluate response to continued treatment. Bioenergetics, metabolite profiling, and stable isotope metabolic labeling was performed on BM c-Kit+ progenitor cells obtained from vehicle and TKI treated mice. The effects of TKI treatment on metabolism-related gene expression in CML hematopoietic stem cells (HSC) was studied using single cell RNA-Seq (scRNA) analysis of BM Lin- c-Kit+ Sca-1+ (LSK) cells from CML mice treated with vehicle or TKI for 2 days or 2 weeks. Results: Extracellular flux analysis revealed that TKI treatment initially inhibited oxygen consumption rate (OCR), representing mitochondrial respiration, and extracellular acidification rate (ECAR), representing glycolysis, in CML progenitor cells, but that these were restored with continued TKI treatment. Metabolite profiling using high-resolution mass spectrometry (HRMS) identified significant initial reduction in glycolytic, glutaminolytic and tricarboxylic acid cycle (TCA) intermediates after 2 days of TKI treatment. However, metabolite levels were restored to levels similar to controls with continued TKI treatment. Steady-state metabolic labeling with [U-13C6]-glucose or [U-13C5]-Glutamine showed reduced glycolytic flux, glutaminolysis and TCA intermediates after treatment with TKI for 2 days, but restoration of glycolytic activity and increased glucose-mediated lactate generation, and increased glutaminolytic and TCA cycle activity with continued treatment. The above studies indicate initial inhibition of energy metabolism in CML progenitors with TKI treatment, but metabolic adaptation to continued TKI exposure. scRNA analysis of CML HSC populations showed that TKI exposure led to selective enrichment of primitive HSC subsets characterized by reduced OXPHOS, glycolysis, nucleotide metabolism, and MYC gene signatures. Treatment with TKI for 2 days further reduced metabolic gene signatures, but continued treatment led to restoration of MYC and OXPHOS gene signature expression coupled with enrichment of HSC and quiescence signatures in persistent HSC. TKI-treated stem cells showed increased levels of Hif1a protein and enhanced activity of HIF-1, a key transcriptional regulator of energy metabolism. Treatment of CML mice with a HIF-1 inhibitor, Echinomycin, in combination with TKI treatment, resulted in significant depletion of CML stem cells compared to TKI alone. The combination of HIF-1 inhibitor with TKI treatment also depleted human CML stem cells in xenografted mice. Conclusions: Our results demonstrate metabolic adaptation in CML and progenitor stem cells following TKI treatment, and identify HIF-1 activation as a targetable mechanism to enhance elimination of CML stem cell that persist after TKI treatment.

Dual inhibition of Bcr-Abl and Hsp90 by C086 potently inhibits the proliferation of imatinib-resistant CML cells.

Although tyrosine kinase inhibitors (TKI) such as imatinib provide an effective treatment against Bcr-Abl kinase activity in the mature cells of patients with chronic myelogenous leukemia (CML), TKIs probably cannot eradicate the leukemia stem cell (LSC) population. Therefore, alternative therapies are required to target both mature CML cells with wild-type (WT) or mutant Bcr-Abl and LSCs. To investigate the effect of C086, a derivative of curcumin, on imatinib-resistant cells, we explored its underlying mechanisms of Bcr-Abl kinase and heat shock protein 90 (Hsp90) function inhibition. Biochemical assays were used to test ABL kinase activity; fluorescence measurements using recombinant NHsp90, Hsp90 ATPase assay, immunoprecipitation, and immunoblotting were applied to examine Hsp90 function. Colony-forming unit, long-term culture-initiating cells (LTC-IC), and flow cytometry were used to test CML progenitor and stem cells. Biochemical assays with purified recombinant Abl kinase confirmed that C086 can directly inhibit the kinase activity of Abl, including WT and the Q252H, Y253F, and T315I mutations. Furthermore, we identified C086 as a novel Hsp90 inhibitor with the capacity to disrupt the Hsp90 chaperone function in CML cells. Consequently, it inhibited the growth of both imatinib-sensitive and -resistant CML cells. Interestingly, C086 has the capacity to inhibit LTC-ICs and to induce apoptosis in both CD34(+)CD38(+) and CD34(+)CD38(-) cells in vitro. Moreover, C086 could decrease the number of CD45(+), CD45(+)CD34(+)CD38(+), and CD45(+)CD34(+)CD38(-) cells in CML NOD-SCID mice. Dual suppression of Abl kinase activity and Hsp90 chaperone function by C086 provides a new therapeutic strategy for treating Bcr-Abl-induced leukemia resistant to TKIs.

MiR-300 Acts As a Tumor Supressor in Ph+ Progenitors By Modulating the JAK2-SET/PP2A/β-Catenin Interplay

The persistence of tyrosine kinase inhibitor (TKI)-resistant malignant Philadelphia-positive (Ph+) hematopoietic stem cells (HSC) in chronic myelogenous leukemia (CML) TKI-treated patients in complete molecular remission, and the dismal prognosis of blast crisis CML indicate that the molecular mechanisms underlying its emergence, maintenance and progression are not totally dependent on the unrestrained kinase activity of BCR-ABL1 and might rely on other cell autonomous and/or microenvironmental signal capable of sustaining survival and self-renewal of the chronic phase and blast crisis Ph+ HSC compartment(s).We recently demonstrated that the Jak2/SET-PP2A/β-catenin pathway is essential for survival and self-renewal of quiescent TKI-resistant CD34+CD38- Ph+ HSC and that activation of such oncogenic signals requires the expression but not the activity of BCR-ABL1. Because microRNAs (miRNAs) are likely to control in a canonical and/or decoy manner the expression of different components of the Jak2 signalosome, this makes them an attractive target for further understanding the mechanisms of leukemogenesis and, perhaps, for developing new alternative therapies that selectively eradicate quiescent leukemic HSCs.In silico analysis revealed that a specific miR subset shares multiple targets of the BCR-ABL1/Jak2/SET-PP2A signalosome. Nanostring Array analysis performed on primary bone marrow cells from normal individuals and chronic phase or blast crisis CML patients revealed that expression of some of these miRNA is altered in CML. For example, expression of miR-300 and miR-101, which are predicted to simultaneously modulate directly Jak2, hnRNP-A1 and β-catenin and, indirectly, other molecules of the BCR-ABL1/Jak2/SET-PP2A/β-catenin pathway, is significantly inhibited in chronic phase CML and further decreases in advanced CML samples. Additionally, miR-300 expression is several folds lower in dividing (div. 1) compared to quiescent (CFSEMAX) CD34+ CML cells.Lentiviral-transduction of miR-300 in human BCR-ABL+ cell lines resulted in marked downmodulation of JAK2, β-Catenin hnRNPA1 and SET and, as expected, in increased PP2A activity. Moreover, ectopic miR-300 expression decreased reduced clonogenic potential and proliferation, and increased susceptibility to TKI (e.g. Imatinib) induced apoptosis. Interestingly, it appears that forced BCR-ABL1 expression in TF-1 leukemic cells decreases miR-300, consistent with the reported activation in these cells of the Jak2-SET-PP2A-β-catenin pathway.Altogether our results suggest that miR-300 expression and, potentially, that of other deregulated non-coding RNAs might be dispensable or deleterious for the phenotype of Ph+ progenitors and/or indispensable for that of Ph+ HSCs. Thus, experiments in BCR-ABL1 cell lines as well as primary stem and progenitor cell fractions are currently ongoing to assess the role of this and other miRNAs in survival, self-renewal/proliferation of CML stem and progenitor cells. DisclosuresNo relevant conflicts of interest to declare.

KIT Signaling Governs Differential Sensitivity of Mature and Primitive CML Progenitors to Tyrosine Kinase Inhibitors

Imatinib and other BCR-ABL1 inhibitors are effective therapies for chronic myelogenous leukemia (CML), but these inhibitors target additional kinases including KIT, raising the question of whether off-target effects contribute to clinical efficacy. On the basis of its involvement in CML pathogenesis, we hypothesized that KIT may govern responses of CML cells to imatinib. To test this, we assessed the growth of primary CML progenitor cells under conditions of sole BCR-ABL1, sole KIT, and dual BCR-ABL1/KIT inhibition. Sole BCR-ABL1 inhibition suppressed mature CML progenitor cells, but these effects were largely abolished by stem cell factor (SCF) and maximal suppression required dual BCR-ABL1/KIT inhibition. In contrast, KIT inhibition did not add to the effects of BCR-ABL1 inhibition in primitive progenitors, represented by CD34(+)38(-) cells. Long-term culture-initiating cell assays on murine stroma revealed profound depletion of primitive CML cells by sole BCR-ABL1 inhibition despite the presence of SCF, suggesting that primitive CML cells are unable to use SCF as a survival factor upon BCR-ABL1 inhibition. In CD34(+)38(+) cells, SCF strongly induced pAKT(S473) in a phosphoinositide 3-kinase (PI3K)-dependent manner, which was further enhanced by inhibition of BCR-ABL1 and associated with increased colony survival. In contrast, pAKT(S473) levels remained low in CD34(+)38(-) cells cultured under the same conditions. Consistent with reduced response to SCF, KIT surface expression was significantly lower on CD34(+)38(-) compared with CD34(+)38(+) CML cells, suggesting a possible mechanism for the differential effects of SCF on mature and primitive CML progenitor cells.

Enhanced ABL-inhibitor-induced MAPK-activation in T315I-BCR-ABL-expressing cells: a potential mechanism of altered leukemogenicity

Targeted treatment of chronic myelogenous leukemia using imatinib has dramatically improved patient outcome. However, residual disease can be detected in the majority of patients treated with imatinib. Compensatory activation of MAP kinases (MAPK1/2) in response to BCR-ABL-inhibitors has been reported as a potential cytokine-dependent resistance mechanism leading to the rescue of leukemic progenitor cells. Differential MAPK-modulating activity of clinically approved tyrosine kinase inhibitors was assessed in vitro using BCR-ABL-transformed cells. CD34+-enriched progenitors of newly diagnosed chronic myelogenous leukemia patients were exposed to tyrosine kinase inhibitors. MAPK-signaling was studied by Western blot technique. Proliferation assays were used to analyze response to antileukemic treatment. The ABL-inhibitors imatinib and nilotinib activate MAPKs in CD34+ chronic myelogenous leukemia progenitor cells, whereas treatment with the SRC/ABL-inhibitor dasatinib does not affect MAPK-activation at clinically relevant concentrations. Similar results are seen in BCR-ABL-transformed cells in the presence of interleukin-3 (IL-3). Experiments using BCR-ABL-mutant T315I, a resistance mutation not amenable to tyrosine kinase inhibitor binding, demonstrate that ABL-inhibitor-induced MAPK-activation does not depend on BCR-ABL-inhibition and cannot be prevented by selective SRC-inhibition. However, BCR-ABL-T315I enhances MAPK-activation, suggesting a T315I-dependent positive feedback of MAPK-activation. An autocrine IL-3-loop as trigger for aberrant T315I-dependent MAPK-activation was excluded. Aberrant MAPK-activation triggered by ABL-inhibitors and positively regulated by BCR-ABL kinase mutation T315I might be an experimental explanation for the clinical observation that patients carrying high-resistance mutations show a highly aggressive course of their disease when tyrosine kinase inhibitor treatment is not discontinued in time.

Small GTPase RAB45-mediated p38 activation in apoptosis of chronic myeloid leukemia progenitor cells

Chronic myelogenous leukemia (CML) is characterized by a reciprocal chromosomal translocation (9;22) that generates the Bcr-Abl fusion gene. BCR-ABL transforming activity is mediated by critical downstream signaling pathways that are aberrantly activated by tyrosine kinases. However, the mechanisms of BCR-ABL anti-apoptotic effects and the signaling pathways by which BCR-ABL influences apoptosis in BCR-ABL-expressing cells are poorly defined. In this study, we found that treatment with ABL kinase inhibitors or depletion of BCR-ABL induced the expression of RAB45 messenger RNA and protein and induced apoptosis via reduction of mitochondrial membrane potential and p38 activation in CML cell lines and BCR-ABL(+) progenitor cells from CML patients. Overexpressed RAB45 induced the activation of caspases-3 and -9 and reduced the expression of Survivin, XIAP, c-IAP1 and c-IAP2 in CML cells. Moreover, in colony-forming cells derived from CML-aldehyde dehydrogenase(hi)/CD34(+) cells, treatment with ABL kinase inhibitors induced RAB45 expression and reduced mitochondrial membrane potential, resulting in inhibited colony formation of Bcr-Abl(+) progenitor cells. The overexpression of RAB45 significantly decreased colony numbers and induced apoptosis through the activation of caspases-3 and -9. Furthermore, the overexpression of RAB45 increased the phosphorylation levels of p38, resulting in the induction of apoptosis and inhibition of proliferation of CML progenitor cells. Our results identify a new signaling molecule involved in BCR-ABL modulation of apoptosis and suggest that RAB45 induction strategies may have therapeutic utility in patients with CML.

Nilotinib: A Novel, Selective Tyrosine Kinase Inhibitor

The development of tyrosine kinase inhibitors (TKIs) for the treatment of chronic myelogenous leukemia (CML) was based on the discovery that CML stem and progenitor cells overexpress the abnormal fusion protein kinase BCR-ABL. The prototype TKI, imatinib, selectively inhibits BCR-ABL, as well as several other kinases, including stem cell factor receptor (KIT), discoidin domain receptor (DDR), platelet-derived growth factor receptor (PDGFR), and colony-stimulating factor receptor-1 (CSF-1R). Although the management of CML improved dramatically with the introduction of imatinib, not all patients benefit from treatment because of resistance or intolerance. Consequently, research efforts have focused on developing more potent TKIs with the ability to circumvent imatinib resistance. Nilotinib, a second-generation oral TKI, was rationally designed based on the crystal structure of imatinib to be highly active against a wide range of imatinib-resistant BCR-ABL mutants and is approved for the treatment of newly diagnosed or imatinib-resistant or -intolerant CML, and has shown superiority over imatinib in first-line treatment for newly diagnosed CML. Furthermore, the activity of nilotinib against KIT and PDGFRα has led to its evaluation in advanced gastrointestinal stromal tumors (GIST). The purpose of this review is to describe the development of nilotinib, providing a structural explanation for the differential activity of nilotinib and imatinib in GIST. Activity of nilotinib against KIT and PDGFR and emerging evidence of differences in cellular uptake between nilotinib and imatinib are discussed.

Oxidative Stress in CML Stem and Progenitor Cells Correlates with Response to Imatinib

AbstractAbstract 4473 Introduction:The BCR/ABL kinase alters the oxidative environment in chronic myelogenous leukemia (CML) cells, but the consequences of the increased reactive oxygen species (ROS) levels on signaling pathways remain unknown. Increased intracellular peroxides in progenitors cells have been linked to DNA damage, and promote self-mutation which subsequently render imatinib to resistance and failure to eliminate all leukemia cells. Material and Method:A total of 8 patients and 3 health controls were included in this study. 1 patient only at diagnosis, 3 patients at diagnosis and 3 months after treatment with Imatinib, 1 case after 3 and 6 month with Imatinib, 2 cases are long follow-up resistant patients and 1 with long follow-up and good response. Oxidative stress biomarkers were studied by flow cytometry in the bone marrow progenitor cells. Result:Not only levels of BCR-ABR and the number of CML stem cells were lower with the treatment, the oxidative stress biomarkers were lower too in normal controls and in CML cells in patients with response to imatinib but in the resistant ones oxidative stress biomarkers were higher in all stem and progenitor cells. Conclusion:The levels of oxidative stress are significantly higher in CML cells than in healthy subjects and correlate with tumor burden as well as the response to imatinib. This data demonstrate a role of oxidative stress in restore normal hematopoiesis in CML patients and probably in imatinib resistance. Disclosures:García:Celgene: Research Funding.

Effective Targeting of Quiescent Chronic Myelogenous Leukemia Stem Cells by Histone Deacetylase Inhibitors in Combination with Imatinib Mesylate

Imatinib mesylate (IM) induces remission in chronic myelogenous leukemia (CML) patients but does not eliminate leukemia stem cells (LSCs), which remain a potential source of relapse. Here we investigated the ability of HDAC inhibitors (HDACis) to target CML stem cells. Treatment with HDACis combined with IM effectively induced apoptosis in quiescent CML progenitors resistant to elimination by IM alone, and eliminated CML stem cells capable of engrafting immunodeficient mice. In vivo administration of HDACis with IM markedly diminished LSCs in a transgenic mouse model of CML. The interaction of IM and HDACis inhibited genes regulating hematopoietic stem cell maintenance and survival. HDACi treatment represents an effective strategy to target LSCs in CML patients receiving tyrosine kinase inhibitors.

Efficacious Immune Therapy in Chronic Myelogenous Leukemia (CML) Recognizes Antigens That Are Expressed on CML Progenitor Cells

Curative effects of graft-versus-leukemia-based therapies such as donor lymphocyte infusion (DLI) for chronic myelogenous leukemia (CML) may result from immunologic ablation of self-renewing CML progenitor cells. Patients who achieved durable remissions after DLI developed a significant B-cell lymphocytosis after treatment, which did not occur in patients who were unresponsive to DLI. In this study, we identified antigen targets of this B-cell response by probing two immunoproteomic platforms with plasma immunoglobulins from seven CML patients with clinically apparent graft-versus-leukemia responses after DLI. In total, 62 antigens elicited greater reactivity from post-DLI versus pre-DLI plasma. Microarray analysis revealed that >70% of the antigens were expressed in CML CD34(+) cells, suggesting that expression in malignant progenitor cells is a feature common to antibody targets of DLI. We confirmed elevated expression of three target antigens (RAB38, TBCE, and DUSP12) in CML that together consistently elicited antibody responses in 18 of 21 of an additional cohort of CML patients with therapeutic responses, but not in normal donors and rarely in non-CML patients. In summary, immunologic targets of curative DLI responses include multiple antigens on CML progenitor cells, identifying them as potential immunogens for vaccination and/or monitoring of immunotherapeutics designed to eliminate myeloid leukemia stem cells.

Physiologic Hypoxia Protects Chronic Myelogenous Leukemia Progenitors From Elimination by Imatinib Mesylate.

Abstract 2181Poster Board II-158The use of Abl tyrosine kinase inhibitors (TKI), such as imatinib mesylate (IM), has seen a major advance in the control of chronic myelogenous leukemia (CML). However, since TKIs do not seem to eliminate CML stem cells, the use of TKIs may not represent a curative approach. Recent reports have shown that the bone marrow (BM) microenvironment, which facilitates the proliferation and renewal of resident hematopoietic stem cells (HSC), is hypoxic. The average BM O2 tension has been measured at 6-7% O2 in humans (physiologic hypoxia), including in patients with leukemia (Fiegl et al. Blood, 2009), while levels in the HSC niche are estimated to be even lower (<1% O2). In light of the above, it is likely that CML stem cells also reside in the HSC niche, and are similarly adapted for survival and self-renewal in this environment. Furthermore, although hypoxia is known to be associated with both radio- and chemo-resistance in solid tumors, its role in leukemia has has not been thoroughly investigated, particularly in the context of resistance to targeted therapies. Accordingly, we hypothesized that hypoxia protects CML progenitor cells from elimination by IM, a phenomenon which may contribute to disease persistence. To test the above, we examined if hypoxia modified the response of CML progenitor cells toward IM. Primary chronic phase (CP) CML samples “from either BM or peripheral blood (PB)” were obtained at the time of diagnosis from seven individuals with clinically-defined IM-sensitive (IM-S) or IM-resistant (IM-R) disease. Cells were then incubated with IM (0, 0.25, 1 or 5μM) under hypoxic (0.5% O2) or normoxic (21% O2) conditions for 96 hours. The treated cells were then harvested and plated in methylcellulose for 14 days (under 21% O2), after which the number of colony-forming cells (CFCs) were counted by two independent observers. In the absence of IM, four of the seven samples had a significant increase in CFCs (1.5-2.4 fold) when cultured in 0.5% O2 vs 21% O2, suggesting that primary CML CFCs may be better maintained in hypoxia. Furthermore, when treated with IM, six out of seven samples cultured under 0.5% O2 demonstrated dramatic increases in CFCs compared to those treated under 21% O2: a 2.3 to 9.0-fold increase at 1μM, and a 4.4 to 35.0-fold increase at 5μM IM. We also found that the protective effect of hypoxia was independent of the original source of the CFCs (BM or PB). In addition, we found that progenitors from both IM-S and IM-R patients were protected from IM under 0.5% O2, suggesting that hypoxia-induced protection is a general feature of CML progenitors. In order to establish a model for further study, we also tested if hypoxia elicits similar responses in four human CML cell lines (K562, AR230, LAMA84, and BV173). In contrast to primary CML cells, we found that hypoxia actually impaired the ability of all four cell lines to form colonies, and also did not confer protection from IM. These results show that CML cell lines have adapted to conditions of normoxia, and are thus inappropriate models to study the hypoxic response in CML. Additional data will be presented describing the mechanisms that may underlie the protective effect of hypoxia, as well as compounds that can counteract such effects. Ongoing experiments using the LT-CIC assay will also determine the response of primitive CML progenitors to growth under conditions of physiologic hypoxia vs normoxia. In conclusion, our results show that physiologic hypoxia protects CML progenitors from IM, and suggest that blocking of hypoxia-induced survival pathway(s) in CML progenitor cells may facilitate the elimination of residual CML progenitors. Disclosures:No relevant conflicts of interest to declare.

ROS-Induced DNA Damage Causing Genomic Instability in CML Stem and/or Progenitor Cells and in Quiescent and/or Proliferating Cells: Role of Mitochondrial Respiratory Chain Complex III.

Abstract 3268Poster Board III-1▪▪This icon denotes an abstract that is clinically relevant.BCR/ABL kinase transforms hematopoietic stem cells to induce chronic myelogenous leukemia (CML). CML in chronic phase (CML-CP) is a leukemia stem cell (LSC)-derived but leukemia progenitor cell (LPC)-driven disease, which is, in most cases, sensitive to ABL tyrosine kinase inhibitors (TKIs) monotherapy. TKIs do not eradicate the leukemia but instead usually render the disease ‘inactive', since the residual quiescent LSCs are intrinsically insensitive to BCR-ABL inhibition and, in a significant cohort of CML patients, LPCs are also refractory or acquire resistance to TKIs due to mutations in BCR/ABL kinase. In the post-imatinib era, these cells may eventually undergo transformation and initiate fatal CML blast crisis (CML-BC). The malignant progression is usually associated with enhanced expression of BCR/ABL and accumulation of additional genetic aberrations, such as TKI-resistant mutations and chromosomal aberrations. In CML-CP, LSCs and LPCs reside in the CD34+CD38- and CD34+CD38+ populations, respectively, whereas in CML-BC, LSCs are also found in the CD34+CD38+ population. In addition, LSCs and LPCs usually belong to quiescent (CFSEmax) and proliferative (CFSElow) populations, respectively. However, the origin of CML-BC clone and the role of BCR/ABL “dosage” are not known. Since genomic instability usually results from DNA damage, we investigated the mechanisms responsible for enhanced DNA damage in CML cells. Much endogenous DNA damage arises from free radicals such as reactive oxygen species (ROS). Here we show that LSCs-enriched CD34+CD38- and quiescent (CFSEmax) CML cells and LPCs-enriched CD34+CD38+ cells contain higher levels of ROS (superoxide anion, hydrogen peroxide, and hydroxyl radical) than corresponding cells from normal donors (CML-BC>CML-CP>Normal). Interestingly, CFSEmax and CFSElow CML cells displayed similar elevation of ROS indicating that the presence of BCR/ABL and not the proliferative status enhances ROS. In addition, total cellular ROS and mitochondrial ROS levels were proportional to the expression of BCR/ABL kinase implicating the role of BCR/ABL kinase “dosage”. Higher levels of ROS caused more oxidative DNA lesions, such as 8-oxoG and DNA double-strand breaks (DSBs) in CD34+ and also in CD34+CD38- CML cells than in normal counterparts (CML-BC>CML-CP>Normal). Inhibition of BCR/ABL kinase with imatinib partially reduced ROS and oxidative DNA damage in CD34+ CML-CP cells, implicating BCR/ABL-dependent and -;independent mechanisms. Our previous studies showed that elevated levels of oxidative DNA damage in BCR/ABL-transformed cells were responsible for accumulation of TKI-resistant BCR/ABL mutants and chromosomal aberrations (Blood, 2006; Leukemia, 2008), highlighting the importance of identification of the sources of ROS in CML. Mitochondrial respiratory chain (MRC) is a major site of ATP production via oxidative phosphorylation, which is associated with electron flux through MRC. Some of the electrons may escape and react with molecular oxygen to form ROS. To shut down MRC, cells were depleted of mitochondrial DNA (mtDNA) by long-term exposure to ethidium bromide in the presence of uridine and pyruvate as confirmed by RT-PCR showing the absence/reduction of mtDNA-coded Cox II gene transcript. The absence of functional MRC reduced the level of ROS by 40% and 20% in CD34+ CML-CP cells and normal counterparts, respectively, suggesting that MRC is an important source of ROS in leukemia cells. Using selective inhibitors of various MRC complexes we identified complex III as major producer of ROS in LSCs and LPCs in CML-CP. The role of complex III in CML-BC cells is somehow diminished in concordance with the observation that prolonged exposure of MRC to elevated levels of ROS results in “mitochondrial injury” and reduction of MRC activity in advanced stages of cancer. In summary, we postulate that BCR/ABL kinase generates ROS and oxidative DNA damage in a dose-dependent manner not only in LPCs-enriched CD34+CD38+ and CFSElow cells, but also in LSCs-enriched CD34+CD38- and CFSEmax cells, and that MRC complex III generates significant amount of ROS in CML-CP cells. Thus, genomic instability causing TKI resistance and progression to CML-BC may originate in LSCs as well as in LPCs. Disclosures:No relevant conflicts of interest to declare.

Triptolide induces cell death independent of cellular responses to imatinib in blast crisis chronic myelogenous leukemia cells including quiescent CD34+ primitive progenitor cells

The advent of Bcr-Abl tyrosine kinase inhibitors (TKI) has revolutionized the treatment of chronic myelogenous leukemia (CML). However, resistance evolves due to BCR-ABL mutations and other mechanisms. Furthermore, patients with blast crisis CML are less responsive and quiescent CML stem cells are insensitive to these inhibitors. We found that triptolide, a diterpenoid, at nanomolar concentrations, promoted equally significant death of KBM5 cells, a cell line derived from a Bcr-Abl-bearing blast crisis CML patient and KBM5STI571 cells, an imatinib-resistant KBM5 subline bearing the T315I mutation. Similarly, Ba/F3 cells harboring mutated BCR-ABL were as sensitive as Ba/F3Bcr-Abl(p210wt) cells to triptolide. Importantly, triptolide induced apoptosis in primary samples from blast crisis CML patients, who showed resistance to Bcr-Abl TKIs in vivo, with less toxicity to normal cells. Triptolide decreased X-linked inhibitor of apoptosis protein, Mcl-1, and Bcr-Abl protein levels in K562, KBM5, and KBM5STI571 cells and in cells from blast crisis CML patients. It sensitized KBM5, but not KBM5STI571, cells to imatinib. More importantly, triptolide also induced death of quiescent CD34(+) CML progenitor cells, a major problem in the therapy of CML with TKIs. Collectively, these results suggest that triptolide potently induces blast crisis CML cell death independent of the cellular responses to Bcr-Abl TKIs, suggesting that triptolide could eradicate residual quiescent CML progenitor cells in TKI-treated patients and benefit TKI-resistant blast crisis CML patients.

Depletion of Pleckstrin Homology Domain Leucine-rich Repeat Protein Phosphatases 1 and 2 by Bcr-Abl Promotes Chronic Myelogenous Leukemia Cell Proliferation through Continuous Phosphorylation of Akt Isoforms

The constitutive activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway commonly occurs in cancers and is a crucial event in tumorigenesis. Chronic myelogenous leukemia (CML) is characterized by a reciprocal chromosomal translocation (9;22) that generates the Bcr-Abl fusion gene. The PI3K/Akt pathway is activated by Bcr-Abl chimera protein and mediates the leukemogenesis in CML. However, the mechanism by which Bcr-Abl activates the PI3K/Akt pathway is not completely understood. In the present study, we found that pleckstrin homology domain leucine-rich repeat protein phosphatases 1 and 2 (PHLPP1 and PHLPP2) were depleted in CML cells. We investigated the interaction between PHLPPs and Bcr-Abl in CML cell lines and Bcr-Abl+ progenitor cells from CML patients. The Abl kinase inhibitors and depletion of Bcr-Abl induced the expression of PHLPP1 and PHLPP2, which dephosphorylated Ser-473 on Akt1, -2, and -3, resulting in inhibited proliferation of CML cells. The reduction of PHLPP1 and PHLPP2 expression by short interfering RNA in CML cells weakened the Abl kinase inhibitor-mediated inhibition of proliferation. In colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte; colony-forming unit-granulocyte, macrophage; and burst-forming unit-erythroid, treatment with the Abl kinase inhibitors and depletion of Bcr-Abl induced PHLPP1 and PHLPP2 expression and inhibited colony formation of Bcr-Abl+ progenitor cells, whereas depletion of PHLPP1 and PHLPP2 weakened the inhibition of colony formation activity by the Abl kinase inhibitors in Bcr-Abl+ progenitor cells. Thus, Bcr-Abl represses the expression of PHLPP1 and PHLPP2 and continuously activates Akt1, -2, and -3 via phosphorylation on Ser-473, resulting in the proliferation of CML cells.

Aurora-A kinase: a novel target of cellular immunotherapy for leukemia

AbstractAurora-A kinase (Aur-A) is a member of the serine/threonine kinase family that regulates the cell division process, and has recently been implicated in tumorigenesis. In this study, we identified an antigenic 9–amino-acid epitope (Aur-A207-215: YLILEYAPL) derived from Aur-A capable of generating leukemia-reactive cytotoxic T lymphocytes (CTLs) in the context of HLA-A*0201. The synthetic peptide of this epitope appeared to be capable of binding to HLA-A*2402 as well as HLA-A*0201 molecules. Leukemia cell lines and freshly isolated leukemia cells, particularly chronic myelogenous leukemia (CML) cells, appeared to express Aur-A abundantly. Aur-A–specific CTLs were able to lyse human leukemia cell lines and freshly isolated leukemia cells, but not normal cells, in an HLA-A*0201–restricted manner. Importantly, Aur-A–specific CTLs were able to lyse CD34+ CML progenitor cells but did not show any cytotoxicity against normal CD34+ hematopoietic stem cells. The tetramer assay revealed that the Aur-A207-215 epitope–specific CTL precursors are present in peripheral blood of HLA-A*0201–positive and HLA-A*2402–positive patients with leukemia, but not in healthy individuals. Our results indicate that cellular immunotherapy targeting Aur-A is a promising strategy for treatment of leukemia.

The Polyamide-Chlorambucil Conjugate 1R-Chl Effectively Inhibits Proliferation and Induces Apoptosis in CML Progenitor Cells

Imatinib mesylate (IM) is highly effective in the treatment of chronic myelogenous leukemia (CML). However, resistance of malignant progenitors to elimination by IM remains a clinical challenge and new strategies to improve elimination of residual primitive CML cells are required. The polyamide-chlorambucil conjugate 1R-Chl has been developed with the rationale that coupling to a polyamide will increase the DNA sequence specificity of the alkylating agent, and decrease unwanted side effects while retaining the ability to kill cancer cells. In previous studies 1R-Chl was shown to inhibit growth of K562 CML cells in vitro and in vivo (Mol Cancer Ther. 2008; 7:769). Histone H4c, which is highly expressed in a wide range of cancer cells, was identified as a gene target of 1R-Chl. H4c downregulation was associated with chromatin decondensation, possibly exposing otherwise hidden 1R-Chl binding sites. The purpose of the current studies was to investigate whether 1R-Chl could effectively induce apoptosis and inhibit growth of primary CML hematopoietic progenitors. CD34+ progenitors obtained from untreated CML patients and from healthy donors were cultured for 96 hours in growth factor supplemented medium in a range of concentrations of 1R-Chl (0–1μM), its inactive stereoisomer 1S-Chl (0–1μM), and with 5μM IM for comparison. Cells were labeled with CFSE prior to culture and with Annexin-V at culmination of culture to allow flow cytometry assessment of the effects of drug exposure on cell proliferation and apoptosis. In addition CD34+ cells incubated under the same conditions were plated in methylcellulose progenitor culture to assess effects on colony forming cell (CFC) growth. 1R-Chl treatment resulted in significantly increased apoptosis of CML CD34+ cells (from 5.0±1.2% [control] to 41.2±8.0% ([1μM 1R-Chl], n=6, p=001). In contrast, 1S-Chl (1μM) or IM (5μM) treatment did not significantly increase CML CD34+ cell apoptosis. Importantly 1R-Chl, 1S-Chl and IM treatment did not significantly increase apoptosis in normal CD34+ cells (n=3). Combined treatment with 1R-Chl and IM did not result in increased apoptosis of CML CD34+ cells compared to 1R-Chl alone (n=3). Analysis of CFSE fluorescence indicated that 1R-Chl treatment induced dose dependent reduction in proliferation of CML CD34+ progenitors (proliferation relative to untreated cells of 0.39±0.1 with 1μM 1R-Chl, n=4–6, p=.01) with an IC50 value of 0.4μM. The antiproliferative effects of 1R-Chl were significantly greater than those of 1S-Chl (0.6±0.2, n=4–6, p=.02) and IM (0.6±0.1, n=4–6, p=.03). Minor antiproliferative effects were observed in normal controls (0.8±0.1, n=3, p=.0005 [1μM 1R-Chl]; 0.9±0.1, n=3, p=.008 [1μM 1S-Chl] and 0.98±0.1, n=3, p=NS [5μM IM]). The combination of IM and 1R-Chl did not result in further reduction in proliferation of CML CD34+ cells compared to 1R-Chl alone (n=3). A trend towards an increased proportion of undivided cells was observed in response to 1R-Chl, 1S-Chl and IM treatment (from 1.5±0.5% [control] to 19.1±8.7%, 4.4±1.9%, and 11.3±6.7%, n=4–6, respectively). CML CFC growth was suppressed by 96.0±1.5% by 1μM 1R-Chl (n=4–5, p<.001) with an IC50 value of 0.25μM. In comparison, 1S-Chl did not significantly inhibit CML CFC growth and IM suppressed CFC CML growth by 76.1±8.9% (n=4–5, p<.001). 1R-Chl (1μM) suppressed normal CFC growth by 59.3±13.9% (n=3, p=.005, IC50=0.88μM), whereas 1S-Chl and IM did not significantly inhibit normal CFC growth. In summary we observed that the alkylating agent-polyamide conjugate 1R-Chl exerted significant and selective pro-apoptotic and antiproliferative effects on CML CD34+ progenitors. The pro-apoptotic effects of 1R-Chl were more potent than those observed with IM, although as with IM treatment, non-dividing CML progenitors were not eliminated. We conclude that 1R-Chl demonstrates considerable activity against primary CML progenitors and that further studies of its potential role in CML therapy are warranted.

CXCR4 up-regulation by imatinib induces chronic myelogenous leukemia (CML) cell migration to bone marrow stroma and promotes survival of quiescent CML cells

Chronic myelogenous leukemia (CML) is driven by constitutively activated Bcr-Abl tyrosine kinase, which causes the defective adhesion of CML cells to bone marrow stroma. The overexpression of p210Bcr-Abl was reported to down-regulate CXCR4 expression, and this is associated with the cell migration defects in CML. We proposed that tyrosine kinase inhibitors, imatinib or INNO-406, may restore CXCR4 expression and cause the migration of CML cells to bone marrow microenvironment niches, which in turn results in acquisition of stroma-mediated chemoresistance of CML progenitor cells. In KBM5 and K562 cells, imatinib, INNO-406, or IFN-alpha increased CXCR4 expression and migration. This increase in CXCR4 levels on CML progenitor cells was likewise found in samples from CML patients treated with imatinib or IFN-alpha. Imatinib induced G0-G1 cell cycle block in CML cells, which was further enhanced in a mesenchymal stem cell (MSC) coculture system. MSC coculture protected KBM-5 cells from imatinib-induced cell death. These antiapoptotic effects were abrogated by the CXCR4 antagonist AMD3465 or by inhibitor of integrin-linked kinase QLT0267. Altogether, these findings suggest that the up-regulation of CXCR4 by imatinib promotes migration of CML cells to bone marrow stroma, causing the G0-G1 cell cycle arrest and hence ensuring the survival of quiescent CML progenitor cells.

Imatinib Inhibits Both CD4+ T Regulatory Cells and CD8+ T Lymphocytes Specifically Directed Against the Leukemia-Associated Antigen RHAMM/CD168.

Imatinib mesylate (IM, STI571, Gleevec®) is highly effective in the treatment of patients with chronic myelogenous leukemia (CML). In patients with residual disease or relapse after allogeneic stem cell transplantation (allo-SCT), the optimal strategy is a matter of debate: donor lymphocyte infusions (DLIs) alone, IM alone or a combination of both have been administered to CML patients in this clinical situation. As an impairment of anti-viral CD8+ T lymphocyte function by IM has been described, we questioned whether IM also affects specific anti-leukemic CD8+ T lymphocytes. Moreover, we asked to which extent CD4+CD25hi T regulatory cells might be affected by IM thus changing the balance between cytotoxic and tolerogenic T cells. After mixed lymphocyte peptide culture (MLPC), we assessed CD8+ T cells from healthy donors and patients with CML after allo-SCT by tetramer staining/multi-color flow cytometry and enzyme linked immunosorbent spot (ELISPOT) assays, as well as CD4+CD25hicells after 3 days of culture with anti-CD3 and anti-CD28. The release of interferon gamma and granzyme B by CD8+ T lymphocytes specific for R3 (ILSLELMKL), a T cell epitope peptide derived from the leukemia-associated antigen designated RHAMM/CD168 (receptor for hyaluronic acid mediated motility) was inhibited by IM in a clearly dose-dependent fashion in a range from 1 to 25 μM. These T cells were further characterized as CD8+ HLA-A2/R3_tetramer+ WT1_tetra-CD45RA+CD27-CD28-CCR7- effector T cells with the ability to lyse R3 peptide labeled T2 cells and CD34+ CML progenitor cells. The inhibition of CD8+ T lymphocytes was a function of the time of exposure to IM and reversible after removal of IM from the MLPC. On the other hand, the proliferation and function of CD4+CD25hiFoxP3+GITR+TGFß1+ CD69+CD152+ T regulatory cells were also significantly inhibited by IM in a dose-related fashion. These findings suggest that the clinical impact IM must not necessarily result in a reduced efficacy of the graft-versus-leukemia effect observed after allo-SCT and/or DLIs but might depend rather on the ratio of CD8+ cytotoxic T cells and CD4+CD25hi T regulatory cells.

Distinct molecular phenotype of malignant CD34+ hematopoietic stem and progenitor cells in chronic myelogenous leukemia

Chronic myelogenous leukemia (CML) is a malignant disorder of the hematopoietic stem cell characterized by the BCR-ABL oncogene. We examined gene expression profiles of highly enriched CD34(+) hematopoietic stem and progenitor cells from patients with CML in chronic phase using cDNA arrays covering 1.185 genes. Comparing CML CD34(+) cells with normal CD34(+) cells, we found 158 genes which were significantly differentially expressed. Gene expression patterns reflected BCR-ABL-induced functional alterations such as increased cell-cycle and proteasome activity. Detoxification enzymes and DNA repair proteins were downregulated in CML CD34(+) cells, which might contribute to genetic instability. Decreased expression of junction plakoglobulin and CXC chemokine receptor 4 (CXCR-4) might facilitate the release of immature precursors from bone marrow in CML. GATA-2 was upregulated in CML CD34(+) cells, suggesting an increased self-renewal in comparison with normal CD34(+) cells. Moreover, we found upregulation of the proto-oncogene SKI and of receptors for neuromediators such as opioid mu1 receptor, GABA B receptor, adenosine A1 receptor, orexin 1 and 2 receptors and corticotropine-releasing hormone receptor. Treatment of CML progenitor cells with the selective adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) resulted in a dose-dependent significant inhibition of clonogenic growth by 40% at a concentration of 10(-5) M, which could be reversed by the equimolar addition of the receptor agonist 2-chloro-N6-cyclopentyladenosine (P<0.05). The incubation of normal progenitor cells with DPCPX resulted in an inhibition of clonogenic growth to a significantly lesser extent in comparison with CML cells (P<0.05), suggesting that the adenosine A1 receptor is of functional relevance in CML hematopoietic progenitor cells.

Identification of human chronic myelogenous leukemia progenitor cells with hemangioblastic characteristics

AbstractOverwhelming evidence from leukemia research has shown that the clonal population of neoplastic cells exhibits marked heterogeneity with respect to proliferation and differentiation. There are rare stem cells within the leukemic population that possess extensive proliferation and self-renewal capacity not found in the majority of the leukemic cells. These leukemic stem cells are necessary and sufficient to maintain the leukemia. Interestingly, the BCR/ABL fusion gene, which is present in chronic myelogenous leukemia (CML), was also detected in the endothelial cells of patients with CML, suggesting that CML might originate from hemangioblastic progenitor cells that can give rise to both blood cells and endothelial cells. Here we isolated fetal liver kinase-1–positive (Flk1+) cells carrying the BCR/ABL fusion gene from the bone marrow of 17 Philadelphia chromosome–positive (Ph+) patients with CML and found that these cells could differentiate into malignant blood cells and phenotypically defined endothelial cells at the single-cell level. These findings provide direct evidence for the first time that rearrangement of the BCR/ABL gene might happen at or even before the level of hemangioblastic progenitor cells, thus resulting in detection of the BCR/ABL fusion gene in both blood and endothelial cells.