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Chronic myeloid leukemia is a stem cell-initiated but progenitor-driven disease induced by the BCR-ABL oncogene. Tyrosine kinase inhibitors (TKIs) were introduced in the late 1990s and have revolutionized the management of chronic myeloid leukemia in chronic phase. The majority of patients can now expect to live a normal life as long as they continue to comply with TKI treatment. However, in a significant proportion of cases TKI resistance develops over time, requiring a switch of therapy. The most frequent mechanism for drug resistance is the development of kinase domain mutations that reduce or completely ablate drug efficacy. Fortunately, the last 10 years have seen an impressive array of new drugs, some modeled on the mechanism of action of imatinib, others employing more novel approaches, for these patients. [ABSTRACT FROM AUTHOR]

Summary:A blinded prospective study was performed to determine whether screening of whole blood using a real-time, panfungal polymerase chain reaction (PCR) technique could predict the development of invasive fungal infection (IFI) in immunocompromised haemato-oncology patients. In all, 78 patients (125 treatment episodes) were screened twice weekly by real-time panfungal PCR using LightCycler™technology. IFI was documented in 19 treatment episodes (five proven, three probable and 11 possible), and in 12, PCR was sequentially positive. PCR positivity occurred in: 4/5 proven; 2/3 probable; 6/11 possible; and 29/106 with no IFI. In 8/12 with IFI and sequentially positive PCR results, PCR positivity occurred before (median 19.5 days) and in 4/12 (median 10.5 days) after the initiation of empirical antifungal therapy. Based on sequential positive results for proven/probable IFI sensitivity, specificity, positive predictive value and negative predictive value were 75, 70, 15 and 98%, respectively. Real-time panfungal PCR is a sensitive tool for the early diagnosis of IFI in immunocompromised haemato-oncology patients. It may be most useful as a screening method in high-risk patients, either to direct early pre-emptive antifungal therapy or to determine when empirical antifungal therapy can be withheld in patients with antibiotic--resistant neutropenic fever. However, these strategies require further assessment in comparative clinical trials.Bone Marrow Transplantation (2005) 35, 389-395. doi:10.1038/sj.bmt.1704768 Published online 10 January 2005 [ABSTRACT FROM AUTHOR]

Background : CML progenitor-cell studies would be greatly facilitated if samples could be repeatedly accessed from a source of well-characterized cells. The present study was designed to develop a simple, inexpensive Ab cocktail that would provide subpopulations of cells enriched for CD34 + cells and simultaneously depleted of CD15 + mature myeloid cells. Methods : Cells from leukapheresis products from CML patients at diagnosis were incubated with each of two Ab cocktails. The standard cocktail (debulking, DB), containing 11 Abs, is recommended for obtaining a highly enriched population of CD34 + cells. The efficacy of an alternative, simpler cocktail (CML custom, CC), containing only four Abs was tested. The recoveries of CD34 + cells, CD15 + cells, colony-forming unit granulocyte-macrophage, and LTCIC were monitored. The samples were then cryopreserved, thawed, and the recoveries remeasured. Results : The purity of CD34 + cells was significantly superior using the DB cocktail than with the CC cocktail. Conversely, using the CC cocktail, the yield of CD34 + cells was significantly higher compared to the DB cocktail. These results were maintained even when the amount of Ab was reduced 10-fold. Both Ab cocktails consistently removed > 99% of the CD15 + cells. Consistent with the CD34 + cell-enrichment data, higher colony-forming cell (CFC) frequencies were obtained with the DB cocktail, although superior yields of CFC were obtained with the CC cocktail. After cryopreservation and thawing the yield of CD34 + cells remained high, and a further reduction in the number of CD15 + cells was obtained. Discussion : A method is described that allows the rapid and efficient debulking of large CML samples. This strategy will provide a source of well-characterized CML stem/progenitor cells that can be repeatedly accessed. [ABSTRACT FROM AUTHOR]

Background : Traditionally, autologous BM or PBSC have been stored as a secondary source ('back-up') of hematopoietic stem cells (HSC) prior to allogeneic and autologous HSC transplantation. Method : We conducted an audit of a single transplant center practice for providing back-up HSC and compared this practice with other European centers. Laboratory records relating to the collection and re-infusion of consecutive HSC harvests were reviewed for 515 transplants (300 autologous and 215 allogeneic HSC transplants). Results : In our experience, 2.3% (five of 215) of allogeneic HSC transplants required secondary HSC rescue for failure to engraft or graft failure (MUD, n = 2; un-manipulated sibling BMT, n = 1; T-cell depleted sibling BMT, n = 2). For autologous transplants, 4.7% (14 of 300) required rescue due to failure to engraft or late graft failure (ABMT for AML, n = 8; CD34 + cell selection/ex vivo expanded, n = 4; ABMT/PBSCT, n = 2). Among the European centers, 69.7% replied to a postal questionnaire, demonstrating that 81.4% and 45.6% of centers stored a secondary HSC source for manipulated and unmanipulated MUD BMT, respectively; 50% and 11.6% of centers stored a secondary source of HSC for manipulated and unmanipulated matched sibling BMT, respectively; 36.4% and 12.7% of centers stored HSC for manipulated and unmanipulated matched sibling PBSCT, respectively. In the autologous setting, 15.2% and 62.1% of centers stored back-up for unmanipulated and manipulated BMT, respectively and 19.5% and 68.5% stored back-up for unmanipulated and manipulated PBSCT, respectively, when myeloablative conditioning regimens were used. Discussion : These data suggest that a small minority of patients require a secondary source of HSC rescue, most commonly in transplants with higher risk of graft failure. This is reflected in the practice across Europe of storing 'back-up' HSC. Guidelines should accommodate the need for storage of a secondary source of HSC only in those transplants associated with a higher risk of graft failure, especially in relation to graft engineering. [ABSTRACT FROM AUTHOR]

In chronic-phase chronic myeloid leukaemia (CP-CML), residual BCR-ABL1+ leukaemia stem cells are responsible for disease persistence despite TKI. Based on in vitro data, CHOICES (CHlorOquine and Imatinib Combination to Eliminate Stem cells) was an international, randomised phase II trial designed to study the safety and efficacy of imatinib (IM) and hydroxychloroquine (HCQ) compared with IM alone in CP-CML patients in major cytogenetic remission with residual disease detectable by qPCR. Sixty-two patients were randomly assigned to either arm. Treatment 'successes' was the primary end point, defined as ≥0.5 log reduction in 12-month qPCR level from trial entry. Selected secondary study end points were 24-month treatment 'successes', molecular response and progression at 12 and 24 months, comparison of IM levels, and achievement of blood HCQ levels >2000 ng/ml. At 12 months, there was no difference in 'success' rate (p = 0.58); MMR was achieved in 80% (IM) vs 92% (IM/HCQ) (p = 0.21). At 24 months, the 'success' rate was 20.8% higher with IM/HCQ (p = 0.059). No patients progressed. Seventeen serious adverse events, including four serious adverse reactions, were reported; diarrhoea occurred more frequently with combination. IM/HCQ is tolerable in CP-CML, with modest improvement in qPCR levels at 12 and 24 months, suggesting autophagy inhibition maybe of clinical value in CP-CML.

The introduction of BCR-ABL tyrosine kinase inhibitors has revolutionized the treatment of chronic myeloid leukemia (CML). A major clinical aim remains the identification and elimination of low-level disease persistence, termed "minimal residual disease". The phenomenon of disease persistence suggests that despite targeted therapeutic approaches, BCR-ABL-independent mechanisms exist which sustain the survival of leukemic stem cells (LSCs). Although other markers of a primitive CML LSC population have been identified in the preclinical setting, only CD26 appears to offer clinical utility. Here we demonstrate consistent and selective expression of CD93 on a lin - CD34 + CD38 - CD90 + CML LSC population and show in vitro and in vivo data to suggest increased stem cell characteristics, as well as robust engraftment in patient-derived xenograft models in comparison with a CD93 - CML stem/progenitor cell population, which fails to engraft. Through bulk and single-cell analyses of selected stem cell and cell survival-specific genes, we confirmed the quiescent character and demonstrate their persistence in a population of CML patient samples who demonstrate molecular relapse on TKI withdrawal. Taken together, our results identify that CD93 is consistently and selectively expressed on a lin - CD34 + CD38 - CD90 + CML LSC population with stem cell characteristics and may be an important indicator in determining poor TKI responders.

In chronic myeloid leukemia (CML), tyrosine kinase inhibitor (TKI) treatment induces autophagy that promotes survival and TKI-resistance in leukemic stem cells (LSCs). In clinical studies hydroxychloroquine (HCQ), the only clinically approved autophagy inhibitor, does not consistently inhibit autophagy in cancer patients, so more potent autophagy inhibitors are needed. We generated a murine model of CML in which autophagic flux can be measured in bone marrow-located LSCs. In parallel, we use cell division tracing, phenotyping of primary CML cells, and a robust xenotransplantation model of human CML, to investigate the effect of Lys05, a highly potent lysosomotropic agent, and PIK-III, a selective inhibitor of VPS34, on the survival and function of LSCs. We demonstrate that long-term haematopoietic stem cells (LT-HSCs: Lin - Sca-1 + c-kit + CD48 - CD150 + ) isolated from leukemic mice have higher basal autophagy levels compared with non-leukemic LT-HSCs and more mature leukemic cells. Additionally, we present that while HCQ is ineffective, Lys05-mediated autophagy inhibition reduces LSCs quiescence and drives myeloid cell expansion. Furthermore, Lys05 and PIK-III reduced the number of primary CML LSCs and target xenografted LSCs when used in combination with TKI treatment, providing a strong rationale for clinical use of second generation autophagy inhibitors as a novel treatment for CML patients with LSC persistence.

Background: Imatinib and second-generation tyrosine kinase inhibitors (TKIs) nilotinib and dasatinib have statistically significantly improved the life expectancy of chronic myeloid leukemia (CML) patients; however, resistance to TKIs remains a major clinical challenge. Although ponatinib, a third-generation TKI, improves outcomes for patients with BCR-ABL-dependent mechanisms of resistance, including the T315I mutation, a proportion of patients may have or develop BCR-ABL-independent resistance and fail ponatinib treatment. By modeling ponatinib resistance and testing samples from these CML patients, it is hoped that an alternative drug target can be identified and inhibited with a novel compound., Methods: Two CML cell lines with acquired BCR-ABL-independent resistance were generated following culture in ponatinib. RNA sequencing and gene ontology (GO) enrichment were used to detect aberrant transcriptional response in ponatinib-resistant cells. A validated oncogene drug library was used to identify US Food and Drug Administration-approved drugs with activity against TKI-resistant cells. Validation was performed using bone marrow (BM)-derived cells from TKI-resistant patients (n = 4) and a human xenograft mouse model (n = 4-6 mice per group). All statistical tests were two-sided., Results: We show that ponatinib-resistant CML cells can acquire BCR-ABL-independent resistance mediated through alternative activation of mTOR. Following transcriptomic analysis and drug screening, we highlight mTOR inhibition as an alternative therapeutic approach in TKI-resistant CML cells. Additionally, we show that catalytic mTOR inhibitors induce autophagy and demonstrate that genetic or pharmacological inhibition of autophagy sensitizes ponatinib-resistant CML cells to death induced by mTOR inhibition in vitro (% number of colonies of control[SD], NVP-BEZ235 vs NVP-BEZ235+HCQ: 45.0[17.9]% vs 24.0[8.4]%, P = .002) and in vivo (median survival of NVP-BEZ235- vs NVP-BEZ235+HCQ-treated mice: 38.5 days vs 47.0 days, P = .04)., Conclusion: Combined mTOR and autophagy inhibition may provide an attractive approach to target BCR-ABL-independent mechanism of resistance.

Leukemia stem cells (LSCs) in individuals with chronic myelogenous leukemia (CML) (hereafter referred to as CML LSCs) are responsible for initiating and maintaining clonal hematopoiesis. These cells persist in the bone marrow (BM) despite effective inhibition of BCR-ABL kinase activity by tyrosine kinase inhibitors (TKIs). Here we show that although the microRNA (miRNA) miR-126 supported the quiescence, self-renewal and engraftment capacity of CML LSCs, miR-126 levels were lower in CML LSCs than in long-term hematopoietic stem cells (LT-HSCs) from healthy individuals. Downregulation of miR-126 levels in CML LSCs was due to phosphorylation of Sprouty-related EVH1-domain-containing 1 (SPRED1) by BCR-ABL, which led to inhibition of the RAN-exportin-5-RCC1 complex that mediates miRNA maturation. Endothelial cells (ECs) in the BM supply miR-126 to CML LSCs to support quiescence and leukemia growth, as shown using mouse models of CML in which Mir126a (encoding miR-126) was conditionally knocked out in ECs and/or LSCs. Inhibition of BCR-ABL by TKI treatment caused an undesired increase in endogenous miR-126 levels, which enhanced LSC quiescence and persistence. Mir126a knockout in LSCs and/or ECs, or treatment with a miR-126 inhibitor that targets miR-126 expression in both LSCs and ECs, enhanced the in vivo anti-leukemic effects of TKI treatment and strongly diminished LSC leukemia-initiating capacity, providing a new strategy for the elimination of LSCs in individuals with CML.

Chronic myeloid leukemia (CML) stem/progenitor cells (SPCs) express a transcriptional program characteristic of proliferation, yet can achieve and maintain quiescence. Understanding the mechanisms by which leukemic SPCs maintain quiescence will help to clarify how they persist during long-term targeted treatment. We have identified a novel BCR-ABL1 protein kinase-dependent pathway mediated by the upregulation of hsa-mir183 , the downregulation of its direct target early growth response 1 (EGR1), and, as a consequence, upregulation of E2F1. We show here that inhibition of hsa-mir183 reduced proliferation and impaired colony formation of CML SPCs. Downstream of this, inhibition of E2F1 also reduced proliferation of CML SPCs, leading to p53-mediated apoptosis. In addition, we demonstrate that E2F1 plays a pivotal role in regulating CML SPC proliferation status. Thus, for the first time, we highlight the mechanism of hsa-mir183 /EGR1-mediated E2F1 regulation and demonstrate this axis as a novel, critical factor for CML SPC survival, offering new insights into leukemic stem cell eradication., (© 2018 by The American Society of Hematology.)

Treatment of chronic myeloid leukemia (CML) with imatinib mesylate and other second- and/or third-generation c-Abl-specific tyrosine kinase inhibitors (TKIs) has substantially extended patient survival. However, TKIs primarily target differentiated cells and do not eliminate leukemic stem cells (LSCs). Therefore, targeting minimal residual disease to prevent acquired resistance and/or disease relapse requires identification of new LSC-selective target(s) that can be exploited therapeutically. Considering that malignant transformation involves cellular metabolic changes, which may in turn render the transformed cells susceptible to specific assaults in a selective manner, we searched for such vulnerabilities in CML LSCs. We performed metabolic analyses on both stem cell-enriched (CD34 + and CD34 + CD38 - ) and differentiated (CD34 - ) cells derived from individuals with CML, and we compared the signature of these cells with that of their normal counterparts. Through combination of stable isotope-assisted metabolomics with functional assays, we demonstrate that primitive CML cells rely on upregulated oxidative metabolism for their survival. We also show that combination treatment with imatinib and tigecycline, an antibiotic that inhibits mitochondrial protein translation, selectively eradicates CML LSCs both in vitro and in a xenotransplantation model of human CML. Our findings provide a strong rationale for investigation of the use of TKIs in combination with tigecycline to treat patients with CML with minimal residual disease.

Background: Protein tyrosine phosphatase receptor gamma (PTPRG) is a ubiquitously expressed member of the protein tyrosine phosphatase family known to act as a tumor suppressor gene in many different neoplasms with mechanisms of inactivation including mutations and methylation of CpG islands in the promoter region. Although a critical role in human hematopoiesis and an oncosuppressor role in chronic myeloid leukemia (CML) have been reported, only one polyclonal antibody (named chPTPRG) has been described as capable of recognizing the native antigen of this phosphatase by flow cytometry. Protein biomarkers of CML have not yet found applications in the clinic, and in this study, we have analyzed a group of newly diagnosed CML patients before and after treatment. The aim of this work was to characterize and exploit a newly developed murine monoclonal antibody specific for the PTPRG extracellular domain (named TPγ B9-2) to better define PTPRG protein downregulation in CML patients., Methods: TPγ B9-2 specifically recognizes PTPRG (both human and murine) by flow cytometry, western blotting, immunoprecipitation, and immunohistochemistry., Results: Co-localization experiments performed with both anti-PTPRG antibodies identified the presence of isoforms and confirmed protein downregulation at diagnosis in the Philadelphia-positive myeloid lineage (including CD34 + /CD38 bright/dim cells). After effective tyrosine kinase inhibitor (TKI) treatment, its expression recovered in tandem with the return of Philadelphia-negative hematopoiesis. Of note, PTPRG mRNA levels remain unchanged in tyrosine kinase inhibitors (TKI) non-responder patients, confirming that downregulation selectively occurs in primary CML cells., Conclusions: The availability of this unique antibody permits its evaluation for clinical application including the support for diagnosis and follow-up of these disorders. Evaluation of PTPRG as a potential therapeutic target is also facilitated by the availability of a specific reagent capable to specifically detect its target in various experimental conditions.

Purpose: BCR-ABL kinase inhibitors are employed successfully for chronic myeloid leukemia (CML) treatment. However, resistant disease and persistence of BCR-ABL1-independent leukemia stem and progenitor cells (LSPC) remain clinical challenges. The receptor tyrosine kinase Axl can mediate survival and therapy resistance of different cancer cells. We investigated the therapeutic potential of Axl inhibition in CML. Experimental Design: We used primary cells from patients with CML and TKI-sensitive and -resistant BCR-ABL1 + CML cell lines and a novel ponatinib-resistant cell line KCL-22 PonR. We analyzed the effects of genetic and pharmacologic Axl blockade by the small-molecule Axl inhibitor BGB324 in vitro and in vivo In BCR-ABL1-unmutated cells, we also investigated BGB324 in combination with imatinib. Results: We demonstrate overexpression of Axl receptor tyrosine kinase in primary cells of patients with CML compared with healthy individuals and a further increase of Axl expression in BCR-ABL TKI-resistant patients. We show that Axl blockage decreased growth of BCR-ABL TKI-sensitive CML cells including CD34 + cells and exerts additive effects with imatinib via inhibition of Stat5 activation. BGB324 also inhibits BCR-ABL TKI-resistant cells, including T315I-mutated and ponatinib-resistant primary cells. BGB324 exerted therapeutic effects in BCR-ABL1 T315I-mutated and ponatinib-resistant preclinical mouse models. Notably, BGB324 does not inhibit BCR-ABL1 and consequently inhibits CML independent of BCR-ABL1 mutational status. Conclusions: Our data show that Axl inhibition has therapeutic potential in BCR-ABL TKI-sensitive as well as -resistant CML and support the need for clinical trials. Clin Cancer Res; 23(9); 2289-300. ©2016 AACR ., (©2016 American Association for Cancer Research.)

Chronic myeloid leukemia (CML) is caused by the acquisition of the tyrosine kinase BCR-ABL1 in a hemopoietic stem cell, transforming it into a leukemic stem cell (LSC) that self-renews, proliferates, and differentiates to give rise to a myeloproliferative disease. Although tyrosine kinase inhibitors (TKIs) that target the kinase activity of BCR-ABL1 have transformed CML from a once-fatal disease to a manageable one for the vast majority of patients, only ∼10% of those who present in chronic phase (CP) can discontinue TKI treatment and maintain a therapy-free remission. Strong evidence now shows that CML LSCs are resistant to the effects of TKIs and persist in all patients on long-term therapy, where they may promote acquired TKI resistance, drive relapse or disease progression, and inevitably represent a bottleneck to cure. Since their discovery in patients almost 2 decades ago, CML LSCs have become a well-recognized exemplar of the cancer stem cell and have been characterized extensively, with the aim of developing new curative therapeutic approaches based on LSC eradication. This review summarizes our current understanding of many of the pathways and mechanisms that promote the survival of the CP CML LSCs and how they can be a source of new gene coding mutations that impact in the clinic. We also review recent preclinical approaches that show promise to eradicate the LSC, and future challenges on the path to cure., (© 2017 by The American Society of Hematology.)

Advances in the design of targeted therapies for the treatment of chronic myeloid leukemia (CML) have transformed the prognosis for patients diagnosed with this disease. However, leukemic stem cell persistence, drug intolerance, drug resistance, and advanced-phase disease represent unmet clinical needs demanding the attention of CML investigators worldwide. The availability of appropriate preclinical models is essential to efficiently translate findings from the bench to the clinic. Here we review the current approaches taken to preclinical work in the CML field, including examples of commonly used in vivo models and recent successes from systems biology-based methodologies., (Copyright © 2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.)

Targeting the fusion oncoprotein BCR-ABL with tyrosine kinase inhibitors has significantly affected chronic myeloid leukemia (CML) treatment, transforming the life expectancy of patients; however the risk for relapse remains, due to persistence of leukemic stem cells (LSCs). Therefore it is imperative to explore the mechanisms that result in LSC survival and develop new therapeutic approaches. We now show that major histocompatibility complex (MHC)-II and its master regulator class II transactivator (CIITA) are downregulated in CML compared with non-CML stem/progenitor cells in a BCR-ABL kinase-independent manner. Interferon γ (IFN-γ) stimulation resulted in an upregulation of CIITA and MHC-II in CML stem/progenitor cells; however, the extent of IFN-γ-induced MHC-II upregulation was significantly lower than when compared with non-CML CD34 + cells. Interestingly, the expression levels of CIITA and MHC-II significantly increased when CML stem/progenitor cells were treated with the JAK1/2 inhibitor ruxolitinib (RUX). Moreover, mixed lymphocyte reactions revealed that exposure of CD34 + CML cells to IFN-γ or RUX significantly enhanced proliferation of the responder CD4 + CD69 + T cells. Taken together, these data suggest that cytokine-driven JAK-mediated signals, provided by CML cells and/or the microenvironment, antagonize MHC-II expression, highlighting the potential for developing novel immunomodulatory-based therapies to enable host-mediated immunity to assist in the detection and eradication of CML stem/progenitor cells., (© 2017 by The American Society of Hematology.)

The introduction of tyrosine kinase inhibitors in chronic myeloid leukaemia (CML) has revolutionised disease outcome. However, despite this, progression to blast phase disease is high in those that do not achieve complete cytogenetic and major molecular response on standard therapy. As well as BCR-ABL-dependent mechanisms, disease persistence has been shown to play a key role. Disease persistence suggests that, despite a targeted therapeutic approach, BCR-ABL-independent mechanisms are being exploited to sustain the survival of a small population of cells termed leukaemic stem cells (LSCs). Increasing evidence highlights the importance of self-renewal and survival pathways in this process. This review will focus on the role of stem-cell restricted self-renewal pathways, namely Hedgehog, Notch, and Bone Morphogenic Pathway (BMP). Wingless-Int/β-Catenin (Wnt/β-Catenin) signalling will be discussed within a further review in this series in view of its regulatory role in GSK3β. Further to this, we will highlight the role of key transcriptional regulators, namely p53 and c- MYC, in targeting wider deregulated networks., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Treatment of chronic myelogenous leukemia (CML) with BCR-ABL tyrosine kinase inhibitors (TKI) fails to eliminate leukemia stem cells (LSC). Patients remain at risk for relapse, and additional approaches to deplete CML LSC are needed to enhance the possibility of discontinuing TKI treatment. We have previously reported that expression of the pivotal proinflammatory cytokine interleukin-1 (IL-1) is increased in CML bone marrow. We show here that CML LSC demonstrated increased expression of the IL-1 receptors, IL-1 receptor accessory protein and IL-1 receptor type 1 (IL-1R1), and enhanced sensitivity to IL-1-induced NF-κB signaling compared with normal stem cells. Treatment with recombinant IL-1 receptor antagonist (IL-1RA) inhibited IL-1 signaling in CML LSC and inhibited growth of CML LSC. Importantly, the combination of IL-1RA with TKI resulted in significantly greater inhibition of CML LSC compared with TKI alone. Our studies also suggest that IL-1 signaling contributes to overexpression of inflammatory mediators in CML LSC, suggesting that blocking IL-1 signaling could modulate the inflammatory milieu. We conclude that IL-1 signaling contributes to maintenance of CML LSC following TKI treatment and that IL-1 blockade with IL-1RA enhances elimination of TKI-treated CML LSC. These results provide a strong rationale for further exploration of anti-IL-1 strategies to enhance LSC elimination in CML., (© 2016 by The American Society of Hematology.)

A major obstacle to curing chronic myeloid leukemia (CML) is residual disease maintained by tyrosine kinase inhibitor (TKI)-persistent leukemic stem cells (LSC). These are BCR-ABL1 kinase independent, refractory to apoptosis, and serve as a reservoir to drive relapse or TKI resistance. We demonstrate that Polycomb Repressive Complex 2 is misregulated in chronic phase CML LSCs. This is associated with extensive reprogramming of H3K27me3 targets in LSCs, thus sensitizing them to apoptosis upon treatment with an EZH2-specific inhibitor (EZH2i). EZH2i does not impair normal hematopoietic stem cell survival. Strikingly, treatment of primary CML cells with either EZH2i or TKI alone caused significant upregulation of H3K27me3 targets, and combined treatment further potentiated these effects and resulted in significant loss of LSCs compared to TKI alone, in vitro, and in long-term bone marrow murine xenografts. Our findings point to a promising epigenetic-based therapeutic strategy to more effectively target LSCs in patients with CML receiving TKIs., Significance: In CML, TKI-persistent LSCs remain an obstacle to cure, and approaches to eradicate them remain a significant unmet clinical need. We demonstrate that EZH2 and H3K27me3 reprogramming is important for LSC survival, but renders LSCs sensitive to the combined effects of EZH2i and TKI. This represents a novel approach to more effectively target LSCs in patients receiving TKI treatment. Cancer Discov; 6(11); 1248-57. ©2016 AACR.See related article by Xie et al., p. 1237This article is highlighted in the In This Issue feature, p. 1197., (©2016 American Association for Cancer Research.)

Effective differentiation therapy for acute myeloid leukemia (AML) has been restricted to a small subset of patients with one defined genetic abnormality. Using an unbiased small molecule screen, Sykes et al. now identify a mechanism of de-repression of differentiation in several models of AML driven by distinct genetic drivers., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The use of tyrosine kinase inhibitors (TKI), including nilotinib, has revolutionized the treatment of chronic myeloid leukemia (CML). However current unmet clinical needs include combating activation of additional survival signaling pathways in persistent leukemia stem cells after long-term TKI therapy. A ubiquitous signaling alteration in cancer, including CML, is activation of reactive oxygen species (ROS) signaling, which may potentiate stem cell activity and mediate resistance to both conventional chemotherapy and targeted inhibitors. We have developed a novel nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor, imipramine blue (IB) that targets ROS generation. ROS levels are known to be elevated in CML with respect to normal hematopoietic stem/progenitor cells and not corrected by TKI. We demonstrate that IB has additive benefit with nilotinib in inhibiting proliferation, viability, and clonogenic function of TKI-insensitive quiescent CD34+ CML chronic phase (CP) cells while normal CD34+ cells retained their clonogenic capacity in response to this combination therapy in vitro. Mechanistically, the pro-apoptotic activity of IB likely resides in part through its dual ability to block NF-κB and re-activate the tumor suppressor protein phosphatase 2A (PP2A). Combining BCR-ABL1 kinase inhibition with NADPH oxidase blockade may be beneficial in eradication of CML and worthy of further investigation., Competing Interests: J.L.A. is the inventor of imipramine blue (US Patent 8435979) which is owned by Emory University. Imipramine blue has been licensed by Emory to ABBY Therapeutics of which JLA is a cofounder.

The regulation of hematopoietic stem cell (HSC) survival and self-renewal within the bone marrow (BM) niche is not well understood. We therefore investigated global transcriptomic profiling of normal human HSC/hematopoietic progenitor cells [HPCs], revealing that several chemokine ligands (CXCL1-4, CXCL6, CXCL10, CXCL11, and CXCL13) were upregulated in human quiescent CD34(+)Hoescht(-)Pyronin Y(-) and primitive CD34(+)38(-), as compared with proliferating CD34(+)Hoechst(+)Pyronin Y(+) and CD34(+)38(+) stem/progenitor cells. This suggested that chemokines might play an important role in the homeostasis of HSCs. In human CD34(+) hematopoietic cells, knockdown of CXCL4 or pharmacologic inhibition of the chemokine receptor CXCR2, significantly decreased cell viability and colony forming cell (CFC) potential. Studies on Cxcr2(-/-) mice demonstrated enhanced BM and spleen cellularity, with significantly increased numbers of HSCs, hematopoietic progenitor cell-1 (HPC-1), HPC-2, and Lin(-)Sca-1(+)c-Kit(+) subpopulations. Cxcr2(-/-) stem/progenitor cells showed reduced self-renewal capacity as measured in serial transplantation assays. Parallel studies on Cxcl4 demonstrated reduced numbers of CFC in primary and secondary assays following knockdown in murine c-Kit(+) cells, and Cxcl4(-/-) mice showed a decrease in HSC and reduced self-renewal capacity after secondary transplantation. These data demonstrate that the CXCR2 network and CXCL4 play a role in the maintenance of normal HSC/HPC cell fates, including survival and self-renewal., (© 2016 by The American Society of Hematology.)

Chronic myeloid leukaemia (CML) arises after transformation of a haemopoietic stem cell (HSC) by the protein-tyrosine kinase BCR-ABL. Direct inhibition of BCR-ABL kinase has revolutionized disease management, but fails to eradicate leukaemic stem cells (LSCs), which maintain CML. LSCs are independent of BCR-ABL for survival, providing a rationale for identifying and targeting kinase-independent pathways. Here we show--using proteomics, transcriptomics and network analyses--that in human LSCs, aberrantly expressed proteins, in both imatinib-responder and non-responder patients, are modulated in concert with p53 (also known as TP53) and c-MYC regulation. Perturbation of both p53 and c-MYC, and not BCR-ABL itself, leads to synergistic cell kill, differentiation, and near elimination of transplantable human LSCs in mice, while sparing normal HSCs. This unbiased systems approach targeting connected nodes exemplifies a novel precision medicine strategy providing evidence that LSCs can be eradicated., Competing Interests: Competing Interest Declaration–The work presented in Fig. 6 was in part supported by funding from Constellation Pharmaceuticals and Roche.

The hematopoietic stem cell (HSC) pool is maintained under hypoxic conditions within the bone marrow microenvironment. Cellular responses to hypoxia are largely mediated by the hypoxia-inducible factors, Hif-1 and Hif-2. The oxygen-regulated α subunits of Hif-1 and Hif-2 (namely, Hif-1α and Hif-2α) form dimers with their stably expressed β subunits and control the transcription of downstream hypoxia-responsive genes to facilitate adaptation to low oxygen tension. An initial study concluded that Hif-1α is essential for HSC maintenance, whereby Hif-1α-deficient HSCs lost their ability to self-renew in serial transplantation assays. In another study, we demonstrated that Hif-2α is dispensable for cell-autonomous HSC maintenance, both under steady-state conditions and following transplantation. Given these unexpected findings, we set out to revisit the role of Hif-1α in cell-autonomous HSC functions. Here we demonstrate that inducible acute deletion of Hif-1α has no impact on HSC survival. Notably, unstressed HSCs lacking Hif-1α efficiently self-renew and sustain long-term multilineage hematopoiesis upon serial transplantation. Finally, Hif-1α-deficient HSCs recover normally after hematopoietic injury induced by serial administration of 5-fluorouracil. We therefore conclude that despite the hypoxic nature of the bone marrow microenvironment, Hif-1α is dispensable for cell-autonomous HSC maintenance., (© 2016 by The American Society of Hematology.)

A major drawback of tyrosine kinase inhibitor (TKI) treatment in chronic myeloid leukemia (CML) is that primitive CML cells are able to survive TKI-mediated BCR-ABL inhibition, leading to disease persistence in patients. Investigation of strategies aiming to inhibit alternative survival pathways in CML is therefore critical. We have previously shown that a nonspecific pharmacological inhibition of autophagy potentiates TKI-induced death in Philadelphia chromosome-positive cells. Here we provide further understanding of how specific and pharmacological autophagy inhibition affects nonmitochondrial and mitochondrial energy metabolism and reactive oxygen species (ROS)-mediated differentiation of CML cells and highlight ATG7 (a critical component of the LC3 conjugation system) as a potential specific therapeutic target. By combining extra- and intracellular steady state metabolite measurements by liquid chromatography-mass spectrometry with metabolic flux assays using labeled glucose and functional assays, we demonstrate that knockdown of ATG7 results in decreased glycolysis and increased flux of labeled carbons through the mitochondrial tricarboxylic acid cycle. This leads to increased oxidative phosphorylation and mitochondrial ROS accumulation. Furthermore, following ROS accumulation, CML cells, including primary CML CD34(+) progenitor cells, differentiate toward the erythroid lineage. Finally, ATG7 knockdown sensitizes CML progenitor cells to TKI-induced death, without affecting survival of normal cells, suggesting that specific inhibitors of ATG7 in combination with TKI would provide a novel therapeutic approach for CML patients exhibiting persistent disease.

Targeting the Hedgehog (Hh) pathway represents a potential leukaemia stem cell (LSC)-directed therapy which may compliment tyrosine kinase inhibitors (TKIs) to eradicate LSC in chronic phase (CP) chronic myeloid leukaemia (CML). We set out to elucidate the role of Hh signaling in CP-CML and determine if inhibition of Hh signaling, through inhibition of smoothened (SMO), was an effective strategy to target CP-CML LSC. Assessment of Hh pathway gene and protein expression demonstrated that the Hh pathway is activated in CD34(+) CP-CML stem/progenitor cells. LDE225 (Sonidegib), a small molecule, clinically investigated SMO inhibitor, used alone and in combination with nilotinib, inhibited the Hh pathway in CD34(+) CP-CML cells, reducing the number and self-renewal capacity of CML LSC in vitro. The combination had no effect on normal haemopoietic stem cells. When combined, LDE225 + nilotinib reduced CD34(+) CP-CML cell engraftment in NSG mice and, upon administration to EGFP(+) /SCLtTA/TRE-BCR-ABL mice, the combination enhanced survival with reduced leukaemia development in secondary transplant recipients. In conclusion, the Hh pathway is deregulated in CML stem and progenitor cells. We identify Hh pathway inhibition, in combination with nilotinib, as a potentially effective therapeutic strategy to improve responses in CP-CML by targeting both stem and progenitor cells.

Purpose: In chronic myelogenous leukemia (CML), leukemic stem cells (LSC) represent a critical target of therapy. However, little is known about markers and targets expressed by LSCs. The aim of this project was to identify novel relevant markers of CML LSCs., Experimental Design: CML LSCs were examined by flow cytometry, qPCR, and various bioassays. In addition, we examined the multipotent CD25(+)CML cell line KU812., Results: In contrast to normal hematopoietic stem cells, CD34(+)/CD38(-)CML LSCs expressed the IL-2 receptor alpha chain, IL-2RA (CD25). STAT5 was found to induce expression of CD25 in Lin(-)/Sca-1(+)/Kit(+)stem cells in C57Bl/6 mice. Correspondingly, shRNA-induced STAT5 depletion resulted in decreased CD25 expression in KU812 cells. Moreover, the BCR/ABL1 inhibitors nilotinib and ponatinib were found to decrease STAT5 activity and CD25 expression in KU812 cells and primary CML LSCs. A CD25-targeting shRNA was found to augment proliferation of KU812 cellsin vitroand their engraftmentin vivoin NOD/SCID-IL-2Rγ(-/-)mice. In drug-screening experiments, the PI3K/mTOR blocker BEZ235 promoted the expression of STAT5 and CD25 in CML cells. Finally, we found that BEZ235 produces synergistic antineoplastic effects on CML cells when applied in combination with nilotinib or ponatinib., Conclusions: CD25 is a novel STAT5-dependent marker of CML LSCs and may be useful for LSC detection and LSC isolation in clinical practice and basic science. Moreover, CD25 serves as a growth regulator of CML LSCs, which may have biologic and clinical implications and may pave the way for the development of new more effective LSC-eradicating treatment strategies in CML., (©2015 American Association for Cancer Research.)

Chronic myeloid leukemia (CML) is driven by malignant stem cells that can persist despite therapy. We have identified Metastasis suppressor 1 (Mtss1/MIM) to be downregulated in hematopoietic stem and progenitor cells from leukemic transgenic SCLtTA/Bcr-Abl mice and in patients with CML at diagnosis, and Mtss1 was restored when patients achieved complete remission. Forced expression of Mtss1 decreased clonogenic capacity and motility of murine myeloid progenitor cells and reduced tumor growth. Viral transduction of Mtss1 into lineage-depleted SCLtTA/Bcr-Abl bone marrow cells decreased leukemic cell burden in recipients, and leukemogenesis was reduced upon injection of Mtss1-overexpressing murine myeloid 32D cells. Tyrosine kinase inhibitor (TKI) therapy and reversion of Bcr-Abl expression increased Mtss1 expression but failed to restore it to control levels. CML patient samples revealed higher DNA methylation of specific Mtss1 promoter CpG sites that contain binding sites for Kaiso and Rest transcription factors. In summary, we identified a novel tumor suppressor in CML stem cells that is downregulated by both Bcr-Abl kinase-dependent and -independent mechanisms. Restored Mtss1 expression markedly inhibits primitive leukemic cell biology in vivo, providing a therapeutic rationale for the Bcr-Abl-Mtss1 axis to target TKI-resistant CML stem cells in patients.

Leukemogenesis occurs under hypoxic conditions within the bone marrow (BM). Knockdown of key mediators of cellular responses to hypoxia with shRNA, namely hypoxia-inducible factor-1α (HIF-1α) or HIF-2α, in human acute myeloid leukemia (AML) samples results in their apoptosis and inability to engraft, implicating HIF-1α or HIF-2α as therapeutic targets. However, genetic deletion of Hif-1α has no effect on mouse AML maintenance and may accelerate disease development. Here, we report the impact of conditional genetic deletion of Hif-2α or both Hif-1α and Hif-2α at different stages of leukemogenesis in mice. Deletion of Hif-2α accelerates development of leukemic stem cells (LSCs) and shortens AML latency initiated by Mll-AF9 and its downstream effectors Meis1 and Hoxa9. Notably, the accelerated initiation of AML caused by Hif-2α deletion is further potentiated by Hif-1α codeletion. However, established LSCs lacking Hif-2α or both Hif-1α and Hif-2α propagate AML with the same latency as wild-type LSCs. Furthermore, pharmacological inhibition of the HIF pathway or HIF-2α knockout using the lentiviral CRISPR-Cas9 system in human established leukemic cells with MLL-AF9 translocation have no impact on their functions. We therefore conclude that although Hif-1α and Hif-2α synergize to suppress the development of AML, they are not required for LSC maintenance., (© 2015 Vukovic et al.)

This protocol describes a highly reproducible antibody-based method that provides protein level and phosphorylation status information from nanogram quantities of protein cell lysate. Nanocapillary isoelectric focusing (cIEF) combines with UV-activated linking chemistry to detect changes in phosphorylation status. As an example application, we describe how to detect changes in response to tyrosine kinase inhibitors (TKIs) in the phosphorylation status of the adaptor protein CrkL, a major substrate of the oncogenic tyrosine kinase BCR-ABL in chronic myeloid leukemia (CML), using highly enriched CML stem cells and mature cell populations in vitro. This protocol provides a 2.5 pg/nl limit of protein detection (<0.2% of a stem cell sample containing <10(4) cells). Additional assays are described for phosphorylated tyrosine 207 (pTyr207)-CrkL and the protein tyrosine phosphatase PTPRC/CD45; these assays were developed using this protocol and applied to CML patient samples. This method is of high throughput, and it can act as a screen for in vitro cancer stem cell response to drugs and novel agents.

The introduction of protein tyrosine kinase inhibitors (TKIs) in 1998 transformed the management of chronic myeloid leukemia (CML), leading to significantly reduced mortality and improved 5 year survival rates. However, the CML community is faced with several clinical issues that need to be addressed. Ten to 15% of CML patients are diagnosed in advanced phase, and small numbers of chronic phase (CP) cases experience disease progression each year during treatment. For these patients, TKIs induce only transient responses and alternative treatment strategies are urgently required. Depending on choice of first line TKI, approximately 30% of CML CP cases show suboptimal responses, due to a combination of poor compliance, drug intolerance, and drug resistance, with approximately 50% of TKI-resistance caused by kinase domain mutations and the remainder due to unknown mechanisms. Finally, the chance of successful treatment discontinuation is on the order of only 10-20% related to disease persistence. Disease persistence is a poorly understood phenomenon; all CML patients have functional Philadelphia positive (Ph+) stem and progenitor cells in their bone marrows and continue to express BCR-ABL1 by DNA PCR, even when in very deep remission and following treatment discontinuation. What controls the maintenance of these persisting cells, whether it is necessary to fully eradicate the malignant clone to achieve cure, and how that might be approached therapeutically are open questions., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Approximately 40% of patients with chronic myeloid leukemia (CML) receiving imatinib fail treatment. There is an increased risk of CML in subjects with (i) deletions of genes encoding glutathione-S-transferase (GST)-θ1 (GSTT1) and -μ1, (GSTM1) and (ii) the GST-π1 (GSTP1) single-nucleotide polymorphism (SNP) Ile105Val (GSTP1*B; rs1695); however, their effects on imatinib treatment outcome are not known. Here, we assess the role of these GSTs in relation to imatinib treatment outcome in 193 CML patients. Deletion of GSTT1 alone, or in combination with deletion of the GSTM1 gene, significantly increased the likelihood of imatinib failure (P = 0.021 and P < 0.001, respectively). The GSTP1*B SNP was not associated with time to imatinib failure. Losses of the GSTT1 and GSTM1 genes are therefore important determinants of imatinib failure in CML. Screening for GSTT1 and GSTM1 gene deletions during diagnosis may identify patients who may be better treated using an alternative therapy.

Chronic myeloid leukemia (CML) is initiated and maintained by the tyrosine kinase BCR-ABL which activates a number of signal transduction pathways, including PI3K/AKT signaling and consequently inactivates FOXO transcription factors. ABL-specific tyrosine kinase inhibitors (TKIs) induce minimal apoptosis in CML progenitor cells, yet exert potent antiproliferative effects, through as yet poorly understood mechanisms. Here, we demonstrate that in CD34+ CML cells, FOXO1 and 3a are inactivated and relocalized to the cytoplasm by BCR-ABL activity. TKIs caused a decrease in phosphorylation of FOXOs, leading to their relocalization from cytoplasm (inactive) to nucleus (active), where they modulated the expression of key FOXO target genes, such as Cyclin D1, ATM, CDKN1C, and BCL6 and induced G1 arrest. Activation of FOXO1 and 3a and a decreased expression of their target gene Cyclin D1 were also observed after 6 days of in vivo treatment with dasatinib in a CML transgenic mouse model. The over-expression of FOXO3a in CML cells combined with TKIs to reduce proliferation, with similar results seen for inhibitors of PI3K/AKT/mTOR signaling. While stable expression of an active FOXO3a mutant induced a similar level of quiescence to TKIs alone, shRNA-mediated knockdown of FOXO3a drove CML cells into cell cycle and potentiated TKI-induced apoptosis. These data demonstrate that TKI-induced G1 arrest in CML cells is mediated through inhibition of the PI3K/AKT pathway and reactivation of FOXOs. This enhanced understanding of TKI activity and induced progenitor cell quiescence suggests that new therapeutic strategies for CML should focus on manipulation of this signaling network., (© 2014 The Authors. STEM CELLS Published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.)

Cancer stem cells (CSCs) are responsible for the initiation and maintenance of some types of cancer, suggesting that inhibition of these cells may limit disease progression and relapse. Unfortunately, few CSC-specific genes have been identified. Here, we determined that the gene encoding arachidonate 15-lipoxygenase (Alox15/15-LO) is essential for the survival of leukemia stem cells (LSCs) in a murine model of BCR-ABL-induced chronic myeloid leukemia (CML). In the absence of Alox15, BCR-ABL was unable to induce CML in mice. Furthermore, Alox15 deletion impaired LSC function by affecting cell division and apoptosis, leading to an eventual depletion of LSCs. Moreover, chemical inhibition of 15-LO function impaired LSC function and attenuated CML in mice. The defective CML phenotype in Alox15-deficient animals was rescued by depleting the gene encoding P-selectin, which is upregulated in Alox15-deficient animals. Both deletion and overexpression of P-selectin affected the survival of LSCs. In human CML cell lines and CD34+ cells, knockdown of Alox15 or inhibition of 15-LO dramatically reduced survival. Loss of Alox15 altered expression of PTEN, PI3K/AKT, and the transcription factor ICSBP, which are known mediators of cancer pathogenesis. These results suggest that ALOX15 has potential as a therapeutic target for eradicating LSCs in CML.

Chronic myeloid leukemia (CML) stem cell survival is not dependent on BCR-ABL protein kinase and treatment with ABL tyrosine kinase inhibitors cures only a minority of CML patients, thus highlighting the need for novel therapeutic targets. The Janus kinase (JAK)2/signal transducer and activator of transcription (STAT)5 pathway has recently been explored for providing putative survival signals to CML stem/progenitor cells (SPCs) with contradictory results. We investigated the role of this pathway using the JAK2 inhibitor, ruxolitinib (RUX). We demonstrated that the combination of RUX, at clinically achievable concentrations, with the specific and potent tyrosine kinase inhibitor nilotinib, reduced the activity of the JAK2/STAT5 pathway in vitro relative to either single agent alone. These effects correlated with increased apoptosis of CML SPCs in vitro and a reduction in primitive quiescent CML stem cells, including NOD.Cg-Prkdc(scid) IL2rg(tm1Wjl) /SzJ mice repopulating cells, induced by combination treatment. A degree of toxicity toward normal SPCs was observed with the combination treatment, although this related to mature B-cell engraftment in NOD.Cg-Prkdc(scid) IL2rg(tm1Wjl) /SzJ mice with minimal effects on primitive CD34(+) cells. These results support the JAK2/STAT5 pathway as a relevant therapeutic target in CML SPCs and endorse the current use of nilotinib in combination with RUX in clinical trials to eradicate persistent disease in CML patients., (© 2014 by The American Society of Hematology.)

Chronic myeloid leukemia (CML) is a stem cell (SC) neoplasm characterized by the BCR/ABL1 oncogene. Although mechanisms of BCR/ABL1-induced transformation are well-defined, little is known about effector-molecules contributing to malignant expansion and the extramedullary spread of leukemic SC (LSC) in CML. We have identified the cytokine-targeting surface enzyme dipeptidylpeptidase-IV (DPPIV/CD26) as a novel, specific and pathogenetically relevant biomarker of CD34(+)/CD38(─) CML LSC. In functional assays, CD26 was identified as target enzyme disrupting the SDF-1-CXCR4-axis by cleaving SDF-1, a chemotaxin recruiting CXCR4(+) SC. CD26 was not detected on normal SC or LSC in other hematopoietic malignancies. Correspondingly, CD26(+) LSC decreased to low or undetectable levels during successful treatment with imatinib. CD26(+) CML LSC engrafted NOD-SCID-IL-2Rγ(-/-) (NSG) mice with BCR/ABL1(+) cells, whereas CD26(─) SC from the same patients produced multilineage BCR/ABL1(-) engraftment. Finally, targeting of CD26 by gliptins suppressed the expansion of BCR/ABL1(+) cells. Together, CD26 is a new biomarker and target of CML LSC. CD26 expression may explain the abnormal extramedullary spread of CML LSC, and inhibition of CD26 may revert abnormal LSC function and support curative treatment approaches in this malignancy., (© 2014 by The American Society of Hematology.)

Oncogene addiction describes the dependence of some cancers on one or a few genes for their survival. Inhibition of the corresponding oncoproteins can lead to dramatic responses. However, in some cases, such as chronic myeloid leukemia (CML), a disease characterized by the presence of the abnormal fusion tyrosine kinase BCR-ABL, cancer stem cells may never acquire addiction to the oncogene that drives disease development. The suggested mechanism(s) for treatment failure include a quiescent stem cell population capable of reinstating disease, high levels of oncoprotein expression, or acquired mutations in the oncogene. In this review, we discuss the evidence for oncogene addiction in several solid tumors and their potential escape mechanism(s) with a particular focus on CML stem cells., (© 2014 AlphaMed Press.)

The tyrosine kinase inhibitor (TKI) imatinib has transformed the treatment and outlook of chronic myeloid leukemia (CML); however, the development of drug resistance and the persistence of TKI-resistant stem cells remain obstacles to eradicating the disease. Inhibition of proteasome activity with bortezomib has been shown to effectively induce apoptosis in TKI-resistant cells. In this study, we show that exposure to the next generation proteasome inhibitor carfilzomib is associated with a decrease in ERK signaling and increased expression of Abelson interactor proteins 1 and 2 (ABI-1/2). We also investigate the effect of carfilzomib in models of imatinib-sensitive and -resistant CML and demonstrate a potent reduction in proliferation and induction of apoptosis in a variety of models of imatinib-resistant CML, including primitive CML stem cells. Carfilzomib acts synergistically with the TKIs imatinib and nilotinib, even in imatinib-resistant cell lines. In addition, we found that the presence of immunoproteasome subunits is associated with an increased sensitivity to carfilzomib. The present findings provide a rational basis to examine the potential of carfilzomib in combination with TKIs as a potential therapy for CML, particularly in imatinib-resistant disease.