Abstract

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.

CML: the classic stem cell disease

Chronic myeloid leukemia (CML) is a classic example of a stem cell cancer and arises when the t9;22 translocation (the Philadelphia chromosome [Ph+])1-3  occurs in a hemopoietic stem cell (HSC). This event results in the constitutive expression of the fusion tyrosine kinase BCR-ABL1, transforming the HSC into the CML stem cell (referred to here as the leukemic stem cell or LSC), which then gives rise to a clonal myeloproliferative disease. Early evidence regarding the HSC origins of CML came from observations that transfusion of peripheral blood cells from CML patients into severely neutropenic recipients resulted in temporary homologous bone marrow (BM) engraftment and Ph+ progeny in the blood.4  This was later explained by the presence of high numbers of mobilized LSCs in the peripheral blood of chronic phase (CP) CML patients.5  A possible hemopoietic progenitor origin of CML was ruled out when BCR-ABL1 expression in murine hemopoietic progenitors failed to confer self-renewal capabilities to BCR-ABL1+ cells, and these cells failed to induce leukemia in mice.6  Until very recently, BCR-ABL1 expression was considered sufficient to cause a CML-like disease in mouse models using retrovirus transduction or transgene insertional mutagenesis to express the oncogene in LSC.7-9  However, issues with BCR-ABL1 copy number, high oncogene expression, and/or secondary mutations arising by retroviral or transgene insertional mutagenesis or genomic instability could theoretically contribute to leukemogenesis. In a recent knock-in model, a single copy of BCR-ABL1 expressed from the endogenous BCR locus was able to confer enhanced BM engraftment; however, this model was unable to induce leukemia.10 

Although the cell of origin of CML is generally accepted to be the HSC, several studies implicate an HSC precursor cell—the multipotent hemangioblast that gives rise to both hemopoietic and endothelial cells. The BCR-ABL1 fusion can be detected in endothelial cells obtained from BM and peripheral blood of CML patients at varying frequencies.11,12  These cells show altered intracellular signaling and protein expression that may affect crosstalk between LSC and the BM microenvironment (BMM), alter immune-modulation and LSC exit from quiescence into proliferation.13,14  Collectively, these data suggest that the acquisition of BCR-ABL1 in the hemangioblast may contribute to both malignant hemopoiesis and endotheliopoiesis.

The natural history of CML

CML is a rare stem cell disease with an annual incidence of 1 to 2 cases per 100 000 individuals, peaking in the sixth and seventh decades of life.15  Data derived from atomic bomb survivors16  suggest that following a latent period of ∼7 years, the natural history of CML is for 85% to 90% of cases to present in CP, but to progress to accelerated phase and then to either myeloid or lymphoid blast crisis over a 5-year time frame.17  However, the mechanism of disease progression is complex and disease behavior is highly variable for individual patients, with some progressing within a few months and others remaining in stable CP for up to 20 years. This heterogeneity between patients may relate to the mutations subsequently acquired in the BCR-ABL1 clone,18  variations in gene expression patterns between patients,19  or the subtype of HSC in which BCR-ABL1 is first expressed—with recent evidence delineating multiple HSC subsets defined by variably fixed lineage potentialities, transcriptional profiles, and phenotypes.20-23  Furthermore, intriguing work on preleukemia24,25  and the detection of BCR-ABL1 in blood cells of normal individuals26,27  present the possibility that heterogeneity could also be driven by mutations acquired before or after BCR-ABL1, or other factors such as deregulation and skewing of lineage specification, clonal hemopoiesis, DNA damage, activation of inflammatory responses, and epigenetic alterations, all of which occur in hemopoiesis during aging.28,29  Some, or all, of these factors may also be required for, or contribute to, disease development in mouse models of CML.

LSC persistence: a bottleneck to cure

The introduction of a potent BCR-ABL1 tyrosine kinase inhibitor (TKI), imatinib, almost 2 decades ago, followed by subsequent generations of TKI (dasatinib, nilotinib, bosutinib, ponatinib) has transformed the management of CML.30,31  What was once a universally fatal disorder, unless treated with an allogeneic transplant, is now well-controlled in the outpatient setting, and overall survival has improved significantly (http://seer.cancer.gov/statfacts/html/cmyl.html), with the majority of patients requiring life-long TKI. In keeping with disease heterogeneity, patient responses to TKI are also variable. The majority of cases (50% to 70%) achieve major molecular response (MMR) in which BCR-ABL1 levels detectable by quantitative polymerase chain reaction in the blood show a 3 log10 fold reduction (ie, 0.1%, compared with a standardized baseline, reviewed elsewhere32 ). However, patient-to-patient variation in leukemic cell blood counts at diagnosis and variations in BCR-ABL1 expression between early and late stages of cell differentiation can often confound these interpretations. Approximately 10% to 20% of all patients develop even deeper molecular responses triggering dose deescalation and discontinuation/stopping trials (Stop Imatinib [STIM], Phase II Study of Withdrawal of Imatinib Therapy in Adult Patients With Chronic Phase Chronic Myeloid Leukaemia in Stable Molecular Remission [TWISTER], Dasatinib Discontinuation [DADI]), in which 50% of patients relapse within 12 months.33,34  When CP relapse occurs, the doubling time (∼9 days) for increasing disease burden mirrors the CML disease at diagnosis.35  One-quarter of CP patients fail TKI therapy,36  and approximately one-half of these cases can be explained by BCR-ABL1 kinase domain mutations,32,37  but the reason for failure in the remaining patients is unclear.

Ironically, the earliest evidence of CML LSC38  predated the introduction of TKI; this was followed by definitive evidence of a deeply, but reversibly, quiescent subpopulation of leukemic cells in patients with CML.39  In the subsequent years, the consensus view has emerged that virtually all CP patients on TKI therapy and in MMR are not cured of CML and show signs of residual disease burden from the presence of LSC in the BM (termed “LSC persistence”). In a typical cohort of 100 CP CML patients who undertake TKI therapy over a 5-year period, almost two-thirds will have this “LSC persistence” phenotype (Figure 1). Researchers have consistently detected BCR-ABL1+ primitive cells in the BM of TKI-treated patients in MMR, which are capable of growth in colony-forming cell and long-term culture initiation cell assays, even in patients in deep molecular response with no detectable BCR-ABL1 transcripts by quantitative polymerase chain reaction.40-43  The most recent of these studies has shown that, although LSCs are not always detectable in cases of very deep molecular response, most likely from technical limitations, some patients with no detectable LSCs can subsequently relapse after TKI discontinuation.43  Others have shown that the LSCs that persist in patients in MMR express BCR-ABL1 at lower levels than the LSCs at the point of diagnosis. Furthermore, murine BM cells engineered to express low levels of BCR-ABL1 levels were far less sensitive to imatinib, whereas those expressing higher levels were prone to de novo mutations.44  These findings point to LSC persistence as a “low mutator” phenotype, perhaps explaining why the majority of these patients do not develop drug resistance or progress to BC. The eradication of the LSC remains a challenge in the majority of CML patients, a significant bottleneck to cure, and an area of intensive research.

Figure 1.

The CP CML patients’ journey through TKI therapy for 5 years. The schematic shows the clinical outcome for a typical 100 CP CML patients with respect to response to imatinib (IM) over the course of 5 years and the decision-tree leading to discontinuing TKI or switching to second- or third-generation TKIs for various reasons. Outcomes were compiled based on data obtained from various sources.31,143-146  By the end of year 5: 12 (green segment of the pie chart) of these 100 patients will typically be off TKI and in therapy-free remission (TFR), more than one-quarter (26) (red segment of the pie chart) will have failed TKI therapy (even after drug switching or through disease progression to accelerated phase or blast crisis), and the majority (62) (amber segment of the pie chart) will remain on long-term TKI therapy but have residual disease resulting from LSC persistence in their BM.

Figure 1.

The CP CML patients’ journey through TKI therapy for 5 years. The schematic shows the clinical outcome for a typical 100 CP CML patients with respect to response to imatinib (IM) over the course of 5 years and the decision-tree leading to discontinuing TKI or switching to second- or third-generation TKIs for various reasons. Outcomes were compiled based on data obtained from various sources.31,143-146  By the end of year 5: 12 (green segment of the pie chart) of these 100 patients will typically be off TKI and in therapy-free remission (TFR), more than one-quarter (26) (red segment of the pie chart) will have failed TKI therapy (even after drug switching or through disease progression to accelerated phase or blast crisis), and the majority (62) (amber segment of the pie chart) will remain on long-term TKI therapy but have residual disease resulting from LSC persistence in their BM.

General features of the LSC

At the time of CP diagnosis, BCR-ABL1 cells coexist with BCR-ABL1+ cells and enriched CD34+ populations require dual-fluorescent in situ hybridization to determine the proportion of cells that carry Ph+ (usually >90% BCR-ABL1+). The more primitive LSC fraction can be purified by fluorescence-activated cell sorting (FACS) in a variety of ways, giving rise to overlapping, primitive, quiescent populations (Figure 2). Phenotypically and functionally, we define CP CML LSCs as those primitive stem/progenitor cells that show a higher capacity to engraft in immunocompromised mice than bulk CD34+ cells,45  have stem cell properties (self-renewal), are resistant to apoptosis,46,47  are prone to genomic instability,48,49  and have impaired DNA damage responses.50-52  Because BCR-ABL1 drives survival and proliferation, it is somewhat of a paradox that CML LSCs express BCR-ABL1 but can also be quiescent39 —a feature that may enable them to become refractory to TKI-induced apoptosis. However, TKIs also exert a potent antiproliferative effect on CML CD34+ cells and LSCs to induce quiescence46,53 ; and subsequent evidence has shown that TKIs exert additional effects to subvert a number of pathways to promote survival (see the following section).

Figure 2.

General features and critical pathways that contribute to CP CML LSCs being quiescent, refractory to apoptosis, and prone to DNA damage. Typically LSCs represent 1% to 5% of the bulk CML CD34+ cells, are enriched by FACS as CD34+CD38, and show more variable levels of Ph+ cells than bulk CML CD34+ cells. Some researchers also include Lin/CD90+/CD45RA cells as part of the CD34+CD38 LSC definition.112  Other FACS approaches can also be used to isolate LSCs by using Hoechst, Pyronin Y, and carboxyfluorescein succinimidyl ester (CFSE) intracellular staining in combination with CD34 to identify quiescent/undivided cells.39,46,47,147  CD34+CD38 CML cells from patients at diagnosis that retain high levels of CFSE (CFSEmax) or are CD34+ and both Hoechstlo and Pyronin Ylo, and survive exposure to TKI, are often considered surrogate in vitro models for the TKI-resistant cells found in patients with LSC persistence. The schematic diagram of the LSC shows key (but not exhaustive) pathways and components and whether the published evidence points to TKI-dependent (blue) or independent (olive green) mechanisms of regulation. Dotted lines denote translocation of components from the cytoplasm (light red) to the nucleus (white). TK, tyrosine kinase. Activation and repression are denoted according to convention. Specific details of each pathway are described in the text.

Figure 2.

General features and critical pathways that contribute to CP CML LSCs being quiescent, refractory to apoptosis, and prone to DNA damage. Typically LSCs represent 1% to 5% of the bulk CML CD34+ cells, are enriched by FACS as CD34+CD38, and show more variable levels of Ph+ cells than bulk CML CD34+ cells. Some researchers also include Lin/CD90+/CD45RA cells as part of the CD34+CD38 LSC definition.112  Other FACS approaches can also be used to isolate LSCs by using Hoechst, Pyronin Y, and carboxyfluorescein succinimidyl ester (CFSE) intracellular staining in combination with CD34 to identify quiescent/undivided cells.39,46,47,147  CD34+CD38 CML cells from patients at diagnosis that retain high levels of CFSE (CFSEmax) or are CD34+ and both Hoechstlo and Pyronin Ylo, and survive exposure to TKI, are often considered surrogate in vitro models for the TKI-resistant cells found in patients with LSC persistence. The schematic diagram of the LSC shows key (but not exhaustive) pathways and components and whether the published evidence points to TKI-dependent (blue) or independent (olive green) mechanisms of regulation. Dotted lines denote translocation of components from the cytoplasm (light red) to the nucleus (white). TK, tyrosine kinase. Activation and repression are denoted according to convention. Specific details of each pathway are described in the text.

BCR-ABL1 kinase-independent survival

To understand why LSCs were refractory to the effects of TKI, we exposed CML CD34+ cells to high concentrations of dasatinib for 12 days, and subjected them, in parallel, to BCR-ABL1 knockdown. These in vitro studies were complemented in vivo using the inducible transgenic SCL-tTA/BCR-ABL model.9  BCR-ABL1 expression was induced in mice to lead to the development of CML-like disease, then switched off to determine whether the LSC population required BCR-ABL1 for survival, and then induced for a second time to see whether the LSCs were still functional and could again drive the development of CML-like disease. In the in vitro studies, functional BCR-ABL1+ LSCs persisted in culture despite evidence for complete kinase inhibition and significant BCR-ABL1 knockdown. In the mouse model, CML-like disease reoccurred following the second induction of BCR-ABL1. This work demonstrated that LSC survival is not dependent on BCR-ABL1 kinase activity54  and suggested that BCR-ABL1 may have nonkinase-mediated functions that modulate signaling pathways to promote LSC survival. These conclusions were further supported by others who used imatinib to fully inhibit BCR-ABL1 kinase activity in both LSCs and quiescent cells.55  Taken together, these studies concluded that CML LSCs were not “oncogene-addicted” and that targeting of BCR-ABL1 kinase activity alone would not eliminate them. Furthermore, this work has led investigators worldwide to search for LSC-selective, BCR-ABL1 kinase-independent targets and pathways that might offer potential for improved targeting of LSCs in CML. To date, several mechanisms, pathways, and drug-able targets have been proposed to contribute to the TKI-resistant LSC phenotype (Figures 2 and 3; Tables 1 and 2).

Figure 3.

LSC survival signaling in the CP CML BMM. The schematic diagram of the BMM shows key (but not exhaustive) pathway components that mediate signaling between the LSC (light red) and other BMM cell types. HSC is shown in blue. OB, osteoblast cells (tan). Ligands involved in various signaling pathways are shown as small, colored spheres. IL-1/IL-1RAP regulates NFKβ signaling in LSCs and can be blocked using a monoclonal antibody to IL-1RAP.135  MPL, the thrombopoietin (TPO) receptor, regulates JAK/STAT signaling and CML patients with high MPL expression on their LSCs have reduced sensitivity to BCR-ABL1 kinase inhibition with TKI, but a higher sensitivity to JAK inhibitors.139  Leukemic progenitor expansion is driven by exposure of LSC, overexpressing BMPR1B, to BMP2 and BMP4.127 The CML BMM is also thought to overexpress the NOTCH ligand JAGGED-1, implicating NOTCH signaling in LSC quiescence.138  LSCs stimulate the production of placental growth factor (PIGF) by BM stromal cells that work in a positive feedback loop to increase angiogenesis of the BM and promote CML cell proliferation through FLT1 (VEGFR1) signaling.140  Stimulation of BM osteoblasts with parathyroid hormone (PTH) resulted in bone remodelling and production of TGF-β1, eradicated LSCs by stimulating TGF-β signaling142  (the opposite effect to other reports of TGF-β signaling in LSCs58,122 ). Similarly, others have shown that expansion of the osteoblast layer of the CML BMM can contribute to creating a hostile environment for HSCs; these effects are mediated by TPO, CCL3, and direct cell–cell interactions that alter TGF-β, NOTCH, and pro-inflammatory signaling in the remodelled osteoblasts.148  Other abbreviations are as described in the text. Other features are as described in Figures 2 and 3.

Figure 3.

LSC survival signaling in the CP CML BMM. The schematic diagram of the BMM shows key (but not exhaustive) pathway components that mediate signaling between the LSC (light red) and other BMM cell types. HSC is shown in blue. OB, osteoblast cells (tan). Ligands involved in various signaling pathways are shown as small, colored spheres. IL-1/IL-1RAP regulates NFKβ signaling in LSCs and can be blocked using a monoclonal antibody to IL-1RAP.135  MPL, the thrombopoietin (TPO) receptor, regulates JAK/STAT signaling and CML patients with high MPL expression on their LSCs have reduced sensitivity to BCR-ABL1 kinase inhibition with TKI, but a higher sensitivity to JAK inhibitors.139  Leukemic progenitor expansion is driven by exposure of LSC, overexpressing BMPR1B, to BMP2 and BMP4.127 The CML BMM is also thought to overexpress the NOTCH ligand JAGGED-1, implicating NOTCH signaling in LSC quiescence.138  LSCs stimulate the production of placental growth factor (PIGF) by BM stromal cells that work in a positive feedback loop to increase angiogenesis of the BM and promote CML cell proliferation through FLT1 (VEGFR1) signaling.140  Stimulation of BM osteoblasts with parathyroid hormone (PTH) resulted in bone remodelling and production of TGF-β1, eradicated LSCs by stimulating TGF-β signaling142  (the opposite effect to other reports of TGF-β signaling in LSCs58,122 ). Similarly, others have shown that expansion of the osteoblast layer of the CML BMM can contribute to creating a hostile environment for HSCs; these effects are mediated by TPO, CCL3, and direct cell–cell interactions that alter TGF-β, NOTCH, and pro-inflammatory signaling in the remodelled osteoblasts.148  Other abbreviations are as described in the text. Other features are as described in Figures 2 and 3.

Table 1.

Candidate therapeutic targets in CP CML LSCs

Target or survival factor Pathway Exemplar inhibitors/activators Reference CML clinical trial 
ALOX15 β-catenin; PI3K/AKT signaling PD146176 106  No 
ALOX5 Wnt/β-catenin signaling Zileuton 69  Yes 
Autophagy Autophagy Chloroquine; bafilomycin A1 113  Yes 
As2O3 
 114  
 115  
BCL6 PI3K/AKT/FOX3a/BCL6 signaling RI-BPI 56  No 
BLK MYC/PAX5/BLK/CDKN1B signaling NA 116  No 
CCN Wnt/Ca2+/NFAT signaling Cyclosporin A 68  No 
CD25 JAK/STAT signaling BEZ235 111  No 
CD70/CD27 Wnt/β-catenin signaling αCD70 mAb 67  No 
c-MYC and TP53 apoptosis CPI-203; RITA/RG7388 98  No 
EZH2 Histone H3 K27 trimethylation GSK 343; GSK126 101  No 
EPZ-6438 (tazemetostat) 100  
Farnesyl transferases RAS signaling; protein farnesylation BMS-214662 117  Yes 
FOX03A TGF-β/AKT/FOXO3a/BCL6 signaling NA 57  No 
58  
GSK3β Wnt/β-catenin signaling SB216763 118  No 
HDACs Histone acetylation LBH589 119  Yes 
JAK2 JAK/STAT signaling Ruxolitinib 74  Yes 
PML Apoptosis; mTOR repression As2O3 120  Yes 
PP2A JAK/STAT/β-catenin signaling 1,9-dideoxy-forskolin; FTY720 76  No 
77  
PPARγ STAT5/HIF2α/CITED2 Pioglitazone 102  Yes 
103  
RAD52 DNA repair RAD52 F79 peptide aptamer 82  No 
SIRT1 Deacetylation of p53 Tenovin-6 121  No 
SMO Hedgehog signaling Cyclopamine; LDE225 60  Yes 
61  
62  
STAT3 JAK/STAT signaling BP-5087 80  No 
TGF-β RI, ALK5 TGF-β/AKT/FOXO3a/BCL6 signaling Ly364947; EW-7197 58  No 
122  
Target or survival factor Pathway Exemplar inhibitors/activators Reference CML clinical trial 
ALOX15 β-catenin; PI3K/AKT signaling PD146176 106  No 
ALOX5 Wnt/β-catenin signaling Zileuton 69  Yes 
Autophagy Autophagy Chloroquine; bafilomycin A1 113  Yes 
As2O3 
 114  
 115  
BCL6 PI3K/AKT/FOX3a/BCL6 signaling RI-BPI 56  No 
BLK MYC/PAX5/BLK/CDKN1B signaling NA 116  No 
CCN Wnt/Ca2+/NFAT signaling Cyclosporin A 68  No 
CD25 JAK/STAT signaling BEZ235 111  No 
CD70/CD27 Wnt/β-catenin signaling αCD70 mAb 67  No 
c-MYC and TP53 apoptosis CPI-203; RITA/RG7388 98  No 
EZH2 Histone H3 K27 trimethylation GSK 343; GSK126 101  No 
EPZ-6438 (tazemetostat) 100  
Farnesyl transferases RAS signaling; protein farnesylation BMS-214662 117  Yes 
FOX03A TGF-β/AKT/FOXO3a/BCL6 signaling NA 57  No 
58  
GSK3β Wnt/β-catenin signaling SB216763 118  No 
HDACs Histone acetylation LBH589 119  Yes 
JAK2 JAK/STAT signaling Ruxolitinib 74  Yes 
PML Apoptosis; mTOR repression As2O3 120  Yes 
PP2A JAK/STAT/β-catenin signaling 1,9-dideoxy-forskolin; FTY720 76  No 
77  
PPARγ STAT5/HIF2α/CITED2 Pioglitazone 102  Yes 
103  
RAD52 DNA repair RAD52 F79 peptide aptamer 82  No 
SIRT1 Deacetylation of p53 Tenovin-6 121  No 
SMO Hedgehog signaling Cyclopamine; LDE225 60  Yes 
61  
62  
STAT3 JAK/STAT signaling BP-5087 80  No 
TGF-β RI, ALK5 TGF-β/AKT/FOXO3a/BCL6 signaling Ly364947; EW-7197 58  No 
122  

Column 1 indicates key survival factors/drug targets intrinsic to the LSC. Column 2 lists the roles of these factors/targets in cellular pathways (if known or as proposed by investigators). Column 3 shows exemplar compounds that have been identified as having potential therapeutic value for each factor/target or pathway (if known). Column 4 provides the references in which the factors/targets are described. Column 5 indicates whether factors/targets or compounds have been evaluated in clinical trials.

As2O3, arsenic trioxide; NA, not available.

Table 2.

Candidate therapeutic targets implicated in CP CML LSC/BMM interactions

Target or survival factor Pathway Exemplar inhibitors/activators Reference CML clinical trial 
β1-integrins Cell adhesion; proliferation IFN-α 87  Yes 
86  
CD26 CXCL12/CXCR4 signaling Gliptins 90  No 
CD44 E-selectin ligands; homing, engraftment NA 83  No 
CXCR4 CXCL12/CXCR4 axis AMD3465; AMD3100; plerixafor 123  Yes 
124  
125  
126  
BMP2/4 BMP signaling NA 127  No 
Galectin-3 BM lodgement NA 85  No 
HIF1α Hypoxia response NA 128, 129  No 
IFN-α Immune surveillance; cytokine-mediated proliferation Interferon alfa-2a 130  Yes 
131  
132  
133  
134  
IL-1RAP/IL-1 NFKβ/AKT signaling IL-1RAP mAb (mAb81.2) 110  Yes 
135  
136  
IL-6 IL-6Rα signaling from the microenvironment αIL-6 mAb 92  No 
LYN CXCR4/CXCL12 signaling in lipid rafts Methyl-β-cyclodextrin; PP2 89  No 
MHC-II/CIITA JAK/STAT signaling Ruxolitinib, IFN-γ 97  Yes 
miR-126 Exosomal transfer 2-O-Me-miR-126 137  No 
N-cadherins N-cadherin/Wnt/β-catenin signaling ICG001 88  No 
JAGGED-1 NOTCH signaling NA 138  No 
MPL JAK/STAT signaling; engraftment NA 139  No 
PD-1/PD-L1 Immune surveillance αPD-L1 mAb 95, 96  Yes 
αPD-1 mAb  
PIGF VEGF signaling: BM angiogenesis; proliferation; metabolism 5D11D4 140  No 
Selectins BM homing; engraftment GMI-1271 84, 141  No 
TGF-β1 Osteoblast → LSC TGF-β signaling Parathyroid hormone 142  No 
Target or survival factor Pathway Exemplar inhibitors/activators Reference CML clinical trial 
β1-integrins Cell adhesion; proliferation IFN-α 87  Yes 
86  
CD26 CXCL12/CXCR4 signaling Gliptins 90  No 
CD44 E-selectin ligands; homing, engraftment NA 83  No 
CXCR4 CXCL12/CXCR4 axis AMD3465; AMD3100; plerixafor 123  Yes 
124  
125  
126  
BMP2/4 BMP signaling NA 127  No 
Galectin-3 BM lodgement NA 85  No 
HIF1α Hypoxia response NA 128, 129  No 
IFN-α Immune surveillance; cytokine-mediated proliferation Interferon alfa-2a 130  Yes 
131  
132  
133  
134  
IL-1RAP/IL-1 NFKβ/AKT signaling IL-1RAP mAb (mAb81.2) 110  Yes 
135  
136  
IL-6 IL-6Rα signaling from the microenvironment αIL-6 mAb 92  No 
LYN CXCR4/CXCL12 signaling in lipid rafts Methyl-β-cyclodextrin; PP2 89  No 
MHC-II/CIITA JAK/STAT signaling Ruxolitinib, IFN-γ 97  Yes 
miR-126 Exosomal transfer 2-O-Me-miR-126 137  No 
N-cadherins N-cadherin/Wnt/β-catenin signaling ICG001 88  No 
JAGGED-1 NOTCH signaling NA 138  No 
MPL JAK/STAT signaling; engraftment NA 139  No 
PD-1/PD-L1 Immune surveillance αPD-L1 mAb 95, 96  Yes 
αPD-1 mAb  
PIGF VEGF signaling: BM angiogenesis; proliferation; metabolism 5D11D4 140  No 
Selectins BM homing; engraftment GMI-1271 84, 141  No 
TGF-β1 Osteoblast → LSC TGF-β signaling Parathyroid hormone 142  No 

Column 1 indicates key survival factors that act through LSC/BMM interactions. Column 2 shows the roles of these survival factors in cellular pathways (if known or as proposed by investigators). Column 3 lists exemplar compounds that have been identified as having potential therapeutic value for each target (if known). Column 4 provides the references in which the targets were described. Column 5 indicates whether targets or compounds have been evaluated in clinical trials.

miR, microRNA; NA, not applicable; VEGF, vascular endothelial growth factor.

PI3K/AKT/FOXO signaling

BCR-ABL1 has been shown to upregulate phosphatidylinositol 3-kinase (PI3K)/AKT signaling, and AKT-mediated phosphorylation of FOXO transcription factors results in their cytoplasmic localization, where they are inactive (Figure 2). One important consequence of TKI exposure is inhibition of BCR-ABL1 and downregulation of PI3K/AKT signaling in the LSC (kinase-dependent), leading in turn to relocalization of FOXO1 and FOXO3a from the cytoplasm to the nucleus, where they modulate expression of CCND1, ATM, CDKN1C, and BCL6, causing a G1 arrest56,57  and may fuel an antiapoptotic phenotype. The transcriptional repressor BCL6, a FOXO3A target, likely plays an important role in this process by repressing the tumor suppressors p53 and ARF. In this respect, TKI exposure permits FOXO3A-mediated upregulation of BCL6, resulting in a protective, pro-survival effect. Others have shown that the PI3K signaling axis in LSC is also under the control of transforming growth factor β (TGF-β) signaling (kinase-independent) and that blocking this pathway reversed the effects of FOXO nuclear translocation.58,59  However, the precise mechanism of how this occurs is not fully understood and may not be completely cell-autonomous.

Hedgehog signaling

Several studies have implicated the hedgehog pathway in the maintenance (self-renewal) and proliferation of the LSC,60-62  where Smoothened (SMO) is a critical mediator. Hedgehog binding to Patched activates SMO that in turn activates the transcription factor GLI1. This leads to reductions of NUMB expression and increased MDM2-mediated degradation of the p53 protein (Figure 2). This has the effect of suppressing apoptotic responses and/or cell-cycle arrest through repression of p53 targets. SMO deletion or pharmacological inhibition in mouse models of CML blocked this pathway and led to loss of LSCs.60,61  However, TKI treatment alone was unable to block this pathway, suggesting that hedgehog signaling was kinase-independent. More recently, similar results were obtained using SMO inhibitors in human CML samples in vitro and in vivo using xenografts in immunocompromised nonobese diabetic severe combined immunodeficiency γ mice.62 

Canonical and noncanonical Wnt signaling

β-catenin is a central mediator of both canonical and non-Wnt signaling and has a dual role in regulating cell-to-cell contact through tight junctions and acting as a transcriptional regulator when translocated to the nucleus (Figure 2). In the absence of Wnt signaling, cytoplasmic β-catenin is ultimately phosphorylated by GSK3β and targeted for degradation by an axin-mediated multimeric complex. Nuclear β-catenin is required for self-renewal and survival of normal HSCs63 ; therefore, it is not surprising that it has also been shown to be a key mediator of LSC survival. Loss of β-catenin in a murine model of CML impaired the development of the disease by inhibiting LSC self-renewal,64  and genetic and pharmacological inhibition of β-catenin activity synergized with TKI to target the loss of LSC.65  Several alternative Wnt-regulated pathways have been implicated in CML LSC. TKI exposure induced the upregulation of CD70 ligand-induced CD27 signaling66,67  resulting in β-catenin nuclear translocation and activation of Wnt target genes, including NOTCH, and c-MYC (kinase-dependent). TKI exposure also induces a noncanonical Wnt signaling mediated through NFAT signaling, which reduces levels of the pro-survival cytokine interleukin-4 (IL-4)68  (kinase-dependent). Fatty acid metabolism was demonstrated to be important in LSC when arachidonate 5-lipoxygenase, encoded by ALOX5, was shown to be upregulated in LSCs in a kinase-independent manner,69  where it is thought to regulate β-catenin levels. Inhibition of ALOX5, through genetic deletion or by pharmacological inhibition in mouse models, targeted the loss of LSCs, implicating this component as an important mediator of LSC survival.

JAK/STAT signaling

The Janus kinases family of intracellular nonreceptor kinases play important roles in regulating cytokine-mediated signal transduction via the JAK/STAT pathway (Figure 2). Activation of STAT5 was demonstrated in primary CML and CML cell lines 20 years ago70  and involves its phosphorylation and translocation to the nucleus where it regulates transcription. Subsequent evidence has also shown that a single null mutation in the STAT5a isoform can attenuate CML-like disease in mouse models71  and knockdown can impair Ph+ myeloid colony formation from CML patient samples.72  Modulating JAK2 activity in human and mouse cell lines reduces BCR-ABL1 and STAT5 signaling,73  and pharmacological inhibition using ruxolitinib resulted in the loss of LSCs both in vitro and in vivo,74  implicating JAK2 as an upstream mediator of a CML JAK/STAT signaling cascade in LSCs. However, BCR-ABL1 has also been implicated in the direct activation of STAT575  (kinase-dependent), suggesting that JAK2 may not be necessary for CML disease maintenance. Furthermore, the reactivation of the tumor suppressor and serine-threonine phosphatase PP2A, through either knockdown or pharmacological inhibition of its repressor SET, has been shown to inhibit BCR-ABL1 and STAT5 activation in CML blast crisis.76  The scenario, however, is different in LSCs, where BCR-ABL1 exerts kinase-independent roles to recruit JAK2 to modulate JAK/STAT signaling77,78  (see also the following section). Activation of STAT3 has also been implicated in the JAK/STAT cascade, where it exerts a protective effect on CML cells upon exposure to TKI.79  Inhibition of STAT3 in combination with TKI-induced synthetic lethality to target the loss of LSC.80 

Genomic instability, DNA damage, and repair

Whether TKI-induced quiescence contributes to LSC persistence in patients is still an open question. Two possible beneficial consequences of TKI treatment would be to reduce the turnover and expansion of LSCs in patients and enhance a “low mutator” phenotype.44  However, a more cautionary interpretation of these possible benefits has come from examining the mechanisms and pathways that contribute to genomic instability in LSCs. BCR-ABL1 kinase activity leads to increased levels of reactive oxygen species (ROS),48,49,81  including H2O2, and these lead to oxidative DNA damage, including point mutations and double-stranded breaks. In this regard, the RAC2 GTPase has been shown to alter the function of the mitochondrial respiratory chain complex to generate ROS and DNA damage in LSCs, as evidenced by the accumulation of chromosomal aberrations and clinically relevant BRC-ABL1 kinase domain mutations.49  This effect was also observed under hypoxia, the conditions that LSCs are exposed to in the BMM, and during exposure to TKIs in which RAC2 levels were unaffected, thus demonstrating a kinase-independent pathway. Inhibition of RAC2 or disruption of the mitochondrial respiratory chain complex reduced the level of genomic instability. Similarly, high ROS levels and associated genomic damage were recapitulated using the transgenic SCL-tTA/BCR-ABL model,48  in which both BCR-ABL1 kinase domain mutations and various base pair additions/deletions in genes linked to progression to blast crisis were identified in LSCs in both TKI-naïve and TKI-treated mice. Evidence as to why such DNA damage is tolerated in LSCs has also emerged. BCR-ABL1 can inhibit mismatch repair to protect cells from apoptosis50  and can stimulate single-strand annealing, homologous recombination repair, and nonhomologous end-joining, all of which are error-prone in BCR-ABL1–expressing cells.52,81  Furthermore, LSCs are dependent on the alternative RAD52-RAD51 pathway of homologous recombination repair to deal with double-stranded breaks rather than BRCA1/2-RAD51 because of the kinase-independent downregulation of BRCA1.82  Although we are unable to reconcile these data with a “low mutator” phenotype,44  they point to the LSC as a potent source of clinically relevant mutations and argue that CML is constantly evolving at the molecular level even in CP, countering the clinical view that it is a disease of 3 distinct phases.

The LSC BMM

Although the pathways described here have ostensibly been studied as primarily cell-intrinsic or cell-autonomous, it is likely that some, if not all, are regulated through interactions between the CML LSCs and the BMM—and several of these interactions have been identified (Figure 3; Table 2), some of which mediate TKI resistance.

LSC adhesion within the BMM is likely to contribute to homing and lodgement—critical steps in LSC engraftment subsequent to transplantation. CD44, expressed on LSCs, is a ligand for e-selectins; lack of CD44 reduced homing and engraftment of LSCs.83  Similarly, a critical role for selectins and their ligands in engraftment has also been shown,84  and e-selectins can be blocked pharmacologically to reduce the number of LSCs. The lectin galectin-3 mediates resistance to TKIs through binding β-galactosides on stromal cells and overexpression-activated AKT signaling and increased lodgement of LSCs in the BM.85  β1-integrins mediate adhesion of LSCs to BM stromal cells, a process likely to be regulated by interferon-α (IFN-α).86,87  TKI-induced upregulation of N-cadherin in LSCs, and adhesion to mesenchymal stem cells led to increased canonical Wnt signaling and protection of the LSC from apoptosis.88  The CXCL12 ligand and its receptor CXCR4 has been linked to intracellular LYN signaling in LSCs,89  and the CXCL12/CXCR4 axis is regulated through CXCL12 cleavage by CD26.90  Reduced homing capacity of LSCs has also been attributed to alterations of the CXCL12/CXCR4 signaling pathway as a result of increased granulocyte colony-stimulating factor levels that conferred a selective growth disadvantage to normal HSCs.91  LSCs also exert other molecular and phenotypic effects on HSCs through extrinsic IL-6 signaling in the CML BMM.92,93  Indeed, a variety of ligand receptor–mediated signaling pathways regulate CML LSCs in the BMM (Figure 3; Table 2).

It is likely that LSCs also avoid eradication by modulation of host immune surveillance in the BMM (reviewed in detail elsewhere94 ). In this respect, cytotoxic T lymphocytes (CTLs) are unable to elicit an appropriate immune response against CML cells through CTL exhaustion; and this is believed to be mediated by the interaction of the PD-1 receptor expressed on CTLs with its inhibitory ligand PD-L1 expressed on CML cells. PD-L1 is expressed on patient-derived CML cells95  and on LSCs in mouse models of CML.96  Blockade of the PD-1/PD-L1 interaction in combination with T-cell immunotherapy was able to trigger the loss of LSCs and prevent development of CML-like disease.96  Our recent work has demonstrated that cytokine-mediated downregulation of MHC-II expression may be an alternative way that LSCs evade immune surveillance; treatment with ruxolitinib or IFN-γ can reverse this effect in vitro and enhance proliferation of responder CD4+CD69+ T cells in mixed lymphocyte reactions.97  These examples represent exciting areas of research that could lead to new immune therapy-based therapeutic approaches.

New therapies to target LSC: recent approaches

The many examples summarized here illustrate the scope of potentially drug-able targets that have been identified in CML to eradicate LSC (Tables 1 and 2). Disappointingly, drugs against these targets have yet to be implemented in the clinic as standard of care. In the past 3 to 4 years, additional drug-able targets and pathway have been identified, whereas others previously identified have been further elaborated in preclinical studies (Figure 4). Our analysis of global proteomics and transcriptomics in drug-naïve primary patient material (bulk CD34+ cells and LSCs) pointed toward a dependency of CML cells on a p53 and c-MYC regulated network.98  This provided a rationale to use a combination of MDM2 and BET inhibitors (MDM2i and BETi, respectively) to target the synergistic eradication of LSCs through upregulation of the p53 apoptotic pathway and downregulation of c-MYC by both drugs (Figure 4A). Given that BETi acts generally as a transcriptional repressor, how its effects lead to upregulation of apoptosis in CML LSCs is not fully understood, although this appears to be a common phenomenon of BETi in preclinical cancer studies.99  We have also used global epigenetic and transcriptomic analysis of drug-naïve primary patient material to reveal that misregulation of the PRC2 complex (including kinase-independent downregulation of EZH1 in LSCs) results in the functional dependency of LSCs on EZH2 and its biochemical readout H3K27me3. Using murine models, others have also reported that CML LSCs are dependent on EZH2.100  Combining an EZH2 inhibitor (EZH2i) with TKI was highly effective at eradicating the LSC population.101  Our data support a model whereby apoptosis is induced in CML LSCs through upregulation of EZH2 targets upstream of p53 (such as ARF), which could lead to increased p53 levels, or through upregulation of p53 target genes directly, which are normally repressed by EZH2 activity (Figure 4A).

Figure 4.

Recent therapeutic approaches to target the eradication of CP CML LSC. (A) Dual targeting of c-MYC and TP53 (p53) or combined treatment with TKI and EZH2 inhibitor (EZH2i).98,100,101  Both approaches converge on upregulating p53-mediated apoptosis through different mechanisms. BETi and MDM2i lead to synergistic repression of c-MYC transcription and upregulation of p53 target genes. A dependency on EZH2 for LSC survival is accompanied by a TKI-independent downregulation of EZH1. (B) Inhibition of STAT5 upstream of the HIF2α-CITED2 pathway that governs LSC quiescence. Combining a PPARγ activator (PPARγa) with TKI102,103  inhibits STAT5 transcription and STAT5 phosphorylation, respectively, and downregulates HIF2α-CITED2 leading to LSC exit from quiescence. (C) Inhibition of noncanonical Wnt/β-catenin signaling mediated by CD70/CD27. TKI upregulates the Wnt/β-catenin pathway by inhibiting miR-29 expression, facilitating both increased CD70 expression and CD70/CD27 receptor/ligand interaction. Treatment with a monoclonal antibody that blocks the CD70/CD27 interaction (αCD70) in a TKI background blocks the pathway.66,67  (D) Activation of PP2A to inhibit a novel CML network driven by JAK2-β-catenin signaling. PP2A activating drugs (PADs) disrupt the PP2A-SET interaction, thereby allowing PP2A reactivation, which inhibits BCR-ABL1 recruitment of JAK2 (TKI-independent) and impairs β-catenin signaling through GSK-3β activation.77  (E) Inhibition of ALOX15 to inhibit β-catenin and PI3K/AKT signaling. Knockdown of ALOX15 or treatment with a 15-LO inhibitor (15-LOi), which blocks ALOX15 enzymatic activity, reduced LSC survival in association with reduced PI3K/AKT and β-catenin levels. This “kill” phenotype was rescued by loss of p-selectin (SELP), which is thought to negatively regulate LSC self-renewal and survival.106  Activation and repression are denoted according to convention. Drug treatments are shown in yellow. Further details are described in the text.

Figure 4.

Recent therapeutic approaches to target the eradication of CP CML LSC. (A) Dual targeting of c-MYC and TP53 (p53) or combined treatment with TKI and EZH2 inhibitor (EZH2i).98,100,101  Both approaches converge on upregulating p53-mediated apoptosis through different mechanisms. BETi and MDM2i lead to synergistic repression of c-MYC transcription and upregulation of p53 target genes. A dependency on EZH2 for LSC survival is accompanied by a TKI-independent downregulation of EZH1. (B) Inhibition of STAT5 upstream of the HIF2α-CITED2 pathway that governs LSC quiescence. Combining a PPARγ activator (PPARγa) with TKI102,103  inhibits STAT5 transcription and STAT5 phosphorylation, respectively, and downregulates HIF2α-CITED2 leading to LSC exit from quiescence. (C) Inhibition of noncanonical Wnt/β-catenin signaling mediated by CD70/CD27. TKI upregulates the Wnt/β-catenin pathway by inhibiting miR-29 expression, facilitating both increased CD70 expression and CD70/CD27 receptor/ligand interaction. Treatment with a monoclonal antibody that blocks the CD70/CD27 interaction (αCD70) in a TKI background blocks the pathway.66,67  (D) Activation of PP2A to inhibit a novel CML network driven by JAK2-β-catenin signaling. PP2A activating drugs (PADs) disrupt the PP2A-SET interaction, thereby allowing PP2A reactivation, which inhibits BCR-ABL1 recruitment of JAK2 (TKI-independent) and impairs β-catenin signaling through GSK-3β activation.77  (E) Inhibition of ALOX15 to inhibit β-catenin and PI3K/AKT signaling. Knockdown of ALOX15 or treatment with a 15-LO inhibitor (15-LOi), which blocks ALOX15 enzymatic activity, reduced LSC survival in association with reduced PI3K/AKT and β-catenin levels. This “kill” phenotype was rescued by loss of p-selectin (SELP), which is thought to negatively regulate LSC self-renewal and survival.106  Activation and repression are denoted according to convention. Drug treatments are shown in yellow. Further details are described in the text.

Two groups have shown that activators of the peroxisome proliferator-activated receptor γ (PPARγ) have increased antileukemic activities in combination with TKI.102,103  Quiescence of LSCs is regulated by a pathway involving the receptor PPARγ, STAT5, HIF2α, and CITED2, a master regulator of blood stem cell quiescence (Figure 4B). Activators of PPARγ result in transcriptional downregulation of STAT5, whereas TKIs block phosphorylation of STAT5, with the combined effects of both drugs significantly down-regulating this pathway and causing LSCs to exit quiescence where they were eradicated by TKIs.104  Recently, EZH2 has been shown to be activated by STAT5 in CML cells105  suggesting possible crosstalk between the effects of PPARy activators and those of EZH2i.

The TKI-mediated upregulation of CD70 has been further examined to provide a clear rationale for inhibiting noncanonical Wnt/β-catenin signaling in LSCs.67  Upon exposure to TKIs, the microRNA miR-29 is downregulated, the consequence of which is upregulation of CD70 through the opposing roles of miR-29 on SP1 and DNMT1a regulation (Figure 4C). Thus, antibody-based blockade of the interaction between CD70 and CD27 resulted in a potent loss of LSCs in the presence of TKIs.67  Two other routes for inhibiting β-catenin signaling in LSCs have also recently been deduced. In the first, BCR-ABL1 interacts directly with JAK2 in a kinase-independent manner to activate a JAK2/β-catenin survival/self-renewal pathway that results in inhibition of PP2A and activation of β-catenin (Figure 4D). Use of PP2A-activating drugs reversed these effects, resulting in GSKβ-dependent degradation of β-catenin and eradication of LSCs.77  In the second, another enzyme in fatty acid metabolism arachidonate 15-lipoxygenase (15-LO encoded by ALOX15) has been implicated in the kinase-independent upregulation of β-catenin, although the exact mechanism is unclear. However, pharmacological inhibition of 15-LO in combination with nilotinib on human LSCs in vitro appeared synergistic.106  In addition, the p-selectin SELP appears to be a key downstream target of 15-LO, which is normally repressed to promote LSC survival. Further preclinical studies and mechanistic studies are required to provide a clearer rationale for taking 15-LO inhibitors into clinical trials, as has been done with zileuton, which inhibits 5-LO.69 

Future challenges

We know little about how TKI-resistant LSC clones evolve in patients in MMR and the degree of intra- and interpatient heterogeneity that is likely to exist—not only at the DNA level, but also with respect to the many pathways we have identified by studying diagnostic drug-naïve LSCs for many years. This is because (1) the TKI-resistant LSCs are extremely rare in the BM of these CML patients and (2) the LSCs cannot be selectively isolated from the normal HSCs that reconstitute normal hemopoiesis in the BM subsequent to TKI therapy. Surrogate in vivo analysis has also been problematic because the majority of CML primary samples do not engraft well in commonly used immunodeficient mice strains. These issues most likely underpin the failure of many promising new drugs to deliver results in clinical trials. However, recent advances in tracking individual normal and malignant clones in xenograft models using bar-coding,21,107  the development of humanized xenograft models,108  an explosion of single-cell technologies,20,109  and the identification of a number of leukemia-specific cell surface markers, make the analysis of individual LSCs or LSC clones much more accessible. Furthermore, several groups have identified markers that discriminate LSCs from HSCs (CD26,90  IL-1RAP,110  CD25,111  and CD93112 ), but how these will perform in samples from patients in MMR has yet to be determined. For those of us intent on curing CML, this new era of game-changing technologies provides some tantalizing prospects that will enable us to finally stem the tide on drug-resistant LSC.

Authorship

Contribution: T.L.H. and D.V. reviewed the literature and wrote the manuscript.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Tessa Holyoake, R314 Level 3 Paul O'Gorman Leukaemia Research Centre, Institute of Cancer Sciences, University of Glasgow, Gartnavel General Hospital, Glasgow, United Kingdom G12 0YN; e-mail: tessa.holyoake@glasgow.ac.uk; and David Vetrie, Room 311 Wolfson Wohl Cancer Research Centre, Institute of Cancer Sciences, University of Glasgow, Garscube Estate, Glasgow, United Kingdom G61 1QH; e-mail: david.vetrie@glasgow.ac.uk.

References

References
1.
Nowell
PC
,
Hungerford
DA
.
Chromosome studies in human leukemia. II. Chronic granulocytic leukemia
.
J Natl Cancer Inst
.
1961
;
27
:
1013
-
1035
.
2.
Rowley
JD
.
Letter: a new consistent chromosomal abnormality in chronic myelogenous leukaemia identified by quinacrine fluorescence and Giemsa staining
.
Nature
.
1973
;
243
(
5405
):
290
-
293
.
3.
Heisterkamp
N
,
Stephenson
JR
,
Groffen
J
, et al
.
Localization of the c-ab1 oncogene adjacent to a translocation break point in chronic myelocytic leukaemia
.
Nature
.
1983
;
306
(
5940
):
239
-
242
.
4.
Levin
RH
,
Whang
J
,
Tjio
JH
,
Carbone
PP
,
Frei
E
III
,
Freireich
EJ
.
Persistent mitosis of transfused homologous leukocytes in children receiving antileukemic therapy
.
Science
.
1963
;
142
(
3597
):
1305
-
1311
.
5.
Petzer
AL
,
Eaves
CJ
,
Lansdorp
PM
,
Ponchio
L
,
Barnett
MJ
,
Eaves
AC
.
Characterization of primitive subpopulations of normal and leukemic cells present in the blood of patients with newly diagnosed as well as established chronic myeloid leukemia
.
Blood
.
1996
;
88
(
6
):
2162
-
2171
.
6.
Huntly
BJ
,
Shigematsu
H
,
Deguchi
K
, et al
.
MOZ-TIF2, but not BCR-ABL, confers properties of leukemic stem cells to committed murine hematopoietic progenitors
.
Cancer Cell
.
2004
;
6
(
6
):
587
-
596
.
7.
Daley
GQ
,
Van Etten
RA
,
Baltimore
D
.
Induction of chronic myelogenous leukemia in mice by the P210bcr/abl gene of the Philadelphia chromosome
.
Science
.
1990
;
247
(
4944
):
824
-
830
.
8.
Huettner
CS
,
Koschmieder
S
,
Iwasaki
H
, et al
.
Inducible expression of BCR/ABL using human CD34 regulatory elements results in a megakaryocytic myeloproliferative syndrome
.
Blood
.
2003
;
102
(
9
):
3363
-
3370
.
9.
Koschmieder
S
,
Göttgens
B
,
Zhang
P
, et al
.
Inducible chronic phase of myeloid leukemia with expansion of hematopoietic stem cells in a transgenic model of BCR-ABL leukemogenesis
.
Blood
.
2005
;
105
(
1
):
324
-
334
.
10.
Foley
SB
,
Hildenbrand
ZL
,
Soyombo
AA
, et al
.
Expression of BCR/ABL p210 from a knockin allele enhances bone marrow engraftment without inducing neoplasia
.
Cell Reports
.
2013
;
5
(
1
):
51
-
60
.
11.
Gunsilius
E
,
Duba
HC
,
Petzer
AL
, et al
.
Evidence from a leukaemia model for maintenance of vascular endothelium by bone-marrow-derived endothelial cells
.
Lancet
.
2000
;
355
(
9216
):
1688
-
1691
.
12.
Fang
B
,
Zheng
C
,
Liao
L
, et al
.
Identification of human chronic myelogenous leukemia progenitor cells with hemangioblastic characteristics
.
Blood
.
2005
;
105
(
7
):
2733
-
2740
.
13.
Zhu
X
,
Wang
L
,
Zhang
B
,
Li
J
,
Dou
X
,
Zhao
RC
.
TGF-beta1-induced PI3K/Akt/NF-kappaB/MMP9 signalling pathway is activated in Philadelphia chromosome-positive chronic myeloid leukaemia hemangioblasts
.
J Biochem
.
2011
;
149
(
4
):
405
-
414
.
14.
Li
Q
,
Wu
Y
,
Fang
S
, et al
.
BCR/ABL oncogene-induced PI3K signaling pathway leads to chronic myeloid leukemia pathogenesis by impairing immuno-modulatory function of hemangioblasts
.
Cancer Gene Ther
.
2015
;
22
(
5
):
227
-
237
.
15.
Rohrbacher
M
,
Hasford
J
.
Epidemiology of chronic myeloid leukaemia (CML)
.
Best Pract Res Clin Haematol
.
2009
;
22
(
3
):
295
-
302
.
16.
Ichimaru
M
,
Tomonaga
M
,
Amenomori
T
,
Matsuo
T
.
Atomic bomb and leukemia
.
J Radiat Res (Tokyo)
.
1991
;
32
(
Suppl 2
):
14
-
19
.
17.
Giralt
S
,
Kantarjian
H
,
Talpaz
M
.
The natural history of chronic myelogenous leukemia in the interferon era
.
Semin Hematol
.
1995
;
32
(
2
):
152
-
158
.
18.
Calabretta
B
,
Perrotti
D
.
The biology of CML blast crisis
.
Blood
.
2004
;
103
(
11
):
4010
-
4022
.
19.
Yong
AS
,
Szydlo
RM
,
Goldman
JM
,
Apperley
JF
,
Melo
JV
.
Molecular profiling of CD34+ cells identifies low expression of CD7, along with high expression of proteinase 3 or elastase, as predictors of longer survival in patients with CML
.
Blood
.
2006
;
107
(
1
):
205
-
212
.
20.
Paul
F
,
Arkin
Y
,
Giladi
A
, et al
.
Transcriptional heterogeneity and lineage commitment in myeloid progenitors [published correction appears in Cell. 2016;164(1-2):325]
.
Cell
.
2015
;
163
(
7
):
1663
-
1677
.
21.
Cheung
AM
,
Nguyen
LV
,
Carles
A
, et al
.
Analysis of the clonal growth and differentiation dynamics of primitive barcoded human cord blood cells in NSG mice
.
Blood
.
2013
;
122
(
18
):
3129
-
3137
.
22.
Grover
A
,
Sanjuan-Pla
A
,
Thongjuea
S
, et al
.
Single-cell RNA sequencing reveals molecular and functional platelet bias of aged haematopoietic stem cells
.
Nat Commun
.
2016
;
7
:
11075
.
23.
Notta
F
,
Zandi
S
,
Takayama
N
, et al
.
Distinct routes of lineage development reshape the human blood hierarchy across ontogeny
.
Science
.
2016
;
351
(
6269
):
aab2116
.
24.
Shlush
LI
,
Zandi
S
,
Mitchell
A
, et al. 
;
HALT Pan-Leukemia Gene Panel Consortium
.
Identification of pre-leukaemic haematopoietic stem cells in acute leukaemia [published correction appears in Nature. 2014 Apr 17;508(7496):420]
.
Nature
.
2014
;
506
(
7488
):
328
-
333
.
25.
Schmidt
M
,
Rinke
J
,
Schäfer
V
, et al
.
Molecular-defined clonal evolution in patients with chronic myeloid leukemia independent of the BCR-ABL status
.
Leukemia
.
2014
;
28
(
12
):
2292
-
2299
.
26.
Biernaux
C
,
Loos
M
,
Sels
A
,
Huez
G
,
Stryckmans
P
.
Detection of major bcr-abl gene expression at a very low level in blood cells of some healthy individuals
.
Blood
.
1995
;
86
(
8
):
3118
-
3122
.
27.
Bose
S
,
Deininger
M
,
Gora-Tybor
J
,
Goldman
JM
,
Melo
JV
.
The presence of typical and atypical BCR-ABL fusion genes in leukocytes of normal individuals: biologic significance and implications for the assessment of minimal residual disease
.
Blood
.
1998
;
92
(
9
):
3362
-
3367
.
28.
Rossi
DJ
,
Bryder
D
,
Zahn
JM
, et al
.
Cell intrinsic alterations underlie hematopoietic stem cell aging
.
Proc Natl Acad Sci USA
.
2005
;
102
(
26
):
9194
-
9199
.
29.
Beerman
I
,
Bock
C
,
Garrison
BS
, et al
.
Proliferation-dependent alterations of the DNA methylation landscape underlie hematopoietic stem cell aging
.
Cell Stem Cell
.
2013
;
12
(
4
):
413
-
425
.
30.
Druker
BJ
,
Tamura
S
,
Buchdunger
E
, et al
.
Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl positive cells
.
Nat Med
.
1996
;
2
(
5
):
561
-
566
.
31.
O’Brien
SG
,
Guilhot
F
,
Larson
RA
, et al. 
;
IRIS Investigators
.
Imatinib compared with interferon and low-dose cytarabine for newly diagnosed chronic-phase chronic myeloid leukemia
.
N Engl J Med
.
2003
;
348
(
11
):
994
-
1004
.
32.
O’Hare
T
,
Zabriskie
MS
,
Eiring
AM
,
Deininger
MW
.
Pushing the limits of targeted therapy in chronic myeloid leukaemia
.
Nat Rev Cancer
.
2012
;
12
(
8
):
513
-
526
.
33.
Mahon
FX
,
Réa
D
,
Guilhot
J
, et al. 
;
Intergroupe Français des Leucémies Myéloïdes Chroniques
.
Discontinuation of imatinib in patients with chronic myeloid leukaemia who have maintained complete molecular remission for at least 2 years: the prospective, multicentre Stop Imatinib (STIM) trial
.
Lancet Oncol
.
2010
;
11
(
11
):
1029
-
1035
.
34.
Ross
DM
,
Branford
S
,
Seymour
JF
, et al
.
Safety and efficacy of imatinib cessation for CML patients with stable undetectable minimal residual disease: results from the TWISTER study
.
Blood
.
2013
;
122
(
4
):
515
-
522
.
35.
Branford
S
,
Yeung
DT
,
Prime
JA
, et al
.
BCR-ABL1 doubling times more reliably assess the dynamics of CML relapse compared with the BCR-ABL1 fold rise: implications for monitoring and management
.
Blood
.
2012
;
119
(
18
):
4264
-
4271
.
36.
Baccarani
M
,
Deininger
MW
,
Rosti
G
, et al
.
European LeukemiaNet recommendations for the management of chronic myeloid leukemia: 2013
.
Blood
.
2013
;
122
(
6
):
872
-
884
.
37.
Soverini
S
,
Hochhaus
A
,
Nicolini
FE
, et al
.
BCR-ABL kinase domain mutation analysis in chronic myeloid leukemia patients treated with tyrosine kinase inhibitors: recommendations from an expert panel on behalf of European LeukemiaNet
.
Blood
.
2011
;
118
(
5
):
1208
-
1215
.
38.
Udomsakdi
C
,
Eaves
CJ
,
Swolin
B
,
Reid
DS
,
Barnett
MJ
,
Eaves
AC
.
Rapid decline of chronic myeloid leukemic cells in long-term culture due to a defect at the leukemic stem cell level
.
Proc Natl Acad Sci USA
.
1992
;
89
(
13
):
6192
-
6196
.
39.
Holyoake
T
,
Jiang
X
,
Eaves
C
,
Eaves
A
.
Isolation of a highly quiescent subpopulation of primitive leukemic cells in chronic myeloid leukemia
.
Blood
.
1999
;
94
(
6
):
2056
-
2064
.
40.
Bhatia
R
,
Holtz
M
,
Niu
N
, et al
.
Persistence of malignant hematopoietic progenitors in chronic myelogenous leukemia patients in complete cytogenetic remission following imatinib mesylate treatment
.
Blood
.
2003
;
101
(
12
):
4701
-
4707
.
41.
Chu
S
,
McDonald
T
,
Lin
A
, et al
.
Persistence of leukemia stem cells in chronic myelogenous leukemia patients in prolonged remission with imatinib treatment
.
Blood
.
2011
;
118
(
20
):
5565
-
5572
.
42.
Chomel
JC
,
Bonnet
ML
,
Sorel
N
, et al
.
Leukemic stem cell persistence in chronic myeloid leukemia patients with sustained undetectable molecular residual disease
.
Blood
.
2011
;
118
(
13
):
3657
-
3660
.
43.
Chomel
JC
,
Bonnet
ML
,
Sorel
N
, et al
.
Leukemic stem cell persistence in chronic myeloid leukemia patients in deep molecular response induced by tyrosine kinase inhibitors and the impact of therapy discontinuation
.
Oncotarget
.
2016
;
7
(
23
):
35293
-
35301
.
44.
Kumari
A
,
Brendel
C
,
Hochhaus
A
,
Neubauer
A
,
Burchert
A
.
Low BCR-ABL expression levels in hematopoietic precursor cells enable persistence of chronic myeloid leukemia under imatinib
.
Blood
.
2012
;
119
(
2
):
530
-
539
.
45.
Gerber
JM
,
Qin
L
,
Kowalski
J
, et al
.
Characterization of chronic myeloid leukemia stem cells
.
Am J Hematol
.
2011
;
86
(
1
):
31
-
37
.
46.
Graham
SM
,
Jørgensen
HG
,
Allan
E
, et al
.
Primitive, quiescent, Philadelphia-positive stem cells from patients with chronic myeloid leukemia are insensitive to STI571 in vitro
.
Blood
.
2002
;
99
(
1
):
319
-
325
.
47.
Copland
M
,
Hamilton
A
,
Elrick
LJ
, et al
.
Dasatinib (BMS-354825) targets an earlier progenitor population than imatinib in primary CML but does not eliminate the quiescent fraction
.
Blood
.
2006
;
107
(
11
):
4532
-
4539
.
48.
Bolton-Gillespie
E
,
Schemionek
M
,
Klein
HU
, et al
.
Genomic instability may originate from imatinib-refractory chronic myeloid leukemia stem cells
.
Blood
.
2013
;
121
(
20
):
4175
-
4183
.
49.
Nieborowska-Skorska
M
,
Kopinski
PK
,
Ray
R
, et al
.
Rac2-MRC-cIII-generated ROS cause genomic instability in chronic myeloid leukemia stem cells and primitive progenitors
.
Blood
.
2012
;
119
(
18
):
4253
-
4263
.
50.
Stoklosa
T
,
Poplawski
T
,
Koptyra
M
, et al
.
BCR/ABL inhibits mismatch repair to protect from apoptosis and induce point mutations
.
Cancer Res
.
2008
;
68
(
8
):
2576
-
2580
.
51.
Skorski
T
.
BCR/ABL, DNA damage and DNA repair: implications for new treatment concepts
.
Leuk Lymphoma
.
2008
;
49
(
4
):
610
-
614
.
52.
Cramer
K
,
Nieborowska-Skorska
M
,
Koptyra
M
, et al
.
BCR/ABL and other kinases from chronic myeloproliferative disorders stimulate single-strand annealing, an unfaithful DNA double-strand break repair
.
Cancer Res
.
2008
;
68
(
17
):
6884
-
6888
.
53.
Jørgensen
HG
,
Allan
EK
,
Jordanides
NE
,
Mountford
JC
,
Holyoake
TL
.
Nilotinib exerts equipotent antiproliferative effects to imatinib and does not induce apoptosis in CD34+ CML cells
.
Blood
.
2007
;
109
(
9
):
4016
-
4019
.
54.
Hamilton
A
,
Helgason
GV
,
Schemionek
M
, et al
.
Chronic myeloid leukemia stem cells are not dependent on Bcr-Abl kinase activity for their survival
.
Blood
.
2012
;
119
(
6
):
1501
-
1510
.
55.
Corbin
AS
,
Agarwal
A
,
Loriaux
M
,
Cortes
J
,
Deininger
MW
,
Druker
BJ
.
Human chronic myeloid leukemia stem cells are insensitive to imatinib despite inhibition of BCR-ABL activity
.
J Clin Invest
.
2011
;
121
(
1
):
396
-
409
.
56.
Hurtz
C
,
Hatzi
K
,
Cerchietti
L
, et al
.
BCL6-mediated repression of p53 is critical for leukemia stem cell survival in chronic myeloid leukemia
.
J Exp Med
.
2011
;
208
(
11
):
2163
-
2174
.
57.
Pellicano
F
,
Scott
MT
,
Helgason
GV
, et al
.
The antiproliferative activity of kinase inhibitors in chronic myeloid leukemia cells is mediated by FOXO transcription factors
.
Stem Cells
.
2014
;
32
(
9
):
2324
-
2337
.
58.
Naka
K
,
Hoshii
T
,
Muraguchi
T
, et al
.
TGF-beta-FOXO signalling maintains leukaemia-initiating cells in chronic myeloid leukaemia
.
Nature
.
2010
;
463
(
7281
):
676
-
680
.
59.
Pellicano
F
,
Holyoake
TL
.
Assembling defenses against therapy-resistant leukemic stem cells: Bcl6 joins the ranks
.
J Exp Med
.
2011
;
208
(
11
):
2155
-
2158
.
60.
Zhao
C
,
Chen
A
,
Jamieson
CH
, et al
.
Hedgehog signalling is essential for maintenance of cancer stem cells in myeloid leukaemia
.
Nature
.
2009
;
458
(
7239
):
776
-
779
.
61.
Dierks
C
,
Beigi
R
,
Guo
GR
, et al
.
Expansion of Bcr-Abl-positive leukemic stem cells is dependent on Hedgehog pathway activation
.
Cancer Cell
.
2008
;
14
(
3
):
238
-
249
.
62.
Irvine
DA
,
Zhang
B
,
Kinstrie
R
, et al
.
Deregulated hedgehog pathway signaling is inhibited by the smoothened antagonist LDE225 (Sonidegib) in chronic phase chronic myeloid leukaemia
.
Sci Rep
.
2016
;
6
:
25476
.
63.
Reya
T
,
Duncan
AW
,
Ailles
L
, et al
.
A role for Wnt signalling in self-renewal of haematopoietic stem cells
.
Nature
.
2003
;
423
(
6938
):
409
-
414
.
64.
Zhao
C
,
Blum
J
,
Chen
A
, et al
.
Loss of beta-catenin impairs the renewal of normal and CML stem cells in vivo
.
Cancer Cell
.
2007
;
12
(
6
):
528
-
541
.
65.
Heidel
FH
,
Bullinger
L
,
Feng
Z
, et al
.
Genetic and pharmacologic inhibition of β-catenin targets imatinib-resistant leukemia stem cells in CML
.
Cell Stem Cell
.
2012
;
10
(
4
):
412
-
424
.
66.
Schürch
C
,
Riether
C
,
Matter
MS
,
Tzankov
A
,
Ochsenbein
AF
.
CD27 signaling on chronic myelogenous leukemia stem cells activates Wnt target genes and promotes disease progression
.
J Clin Invest
.
2012
;
122
(
2
):
624
-
638
.
67.
Riether
C
,
Schürch
CM
,
Flury
C
, et al
.
Tyrosine kinase inhibitor-induced CD70 expression mediates drug resistance in leukemia stem cells by activating Wnt signaling
.
Sci Transl Med
.
2015
;
7
(
298
):
298ra119
.
68.
Gregory
MA
,
Phang
TL
,
Neviani
P
, et al
.
Wnt/Ca2+/NFAT signaling maintains survival of Ph+ leukemia cells upon inhibition of Bcr-Abl
.
Cancer Cell
.
2010
;
18
(
1
):
74
-
87
.
69.
Chen
Y
,
Hu
Y
,
Zhang
H
,
Peng
C
,
Li
S
.
Loss of the Alox5 gene impairs leukemia stem cells and prevents chronic myeloid leukemia
.
Nat Genet
.
2009
;
41
(
7
):
783
-
792
.
70.
Ilaria
RL
Jr
,
Van Etten
RA
.
P210 and P190(BCR/ABL) induce the tyrosine phosphorylation and DNA binding activity of multiple specific STAT family members
.
J Biol Chem
.
1996
;
271
(
49
):
31704
-
31710
.
71.
Ye
D
,
Wolff
N
,
Li
L
,
Zhang
S
,
Ilaria
RL
Jr
.
STAT5 signaling is required for the efficient induction and maintenance of CML in mice
.
Blood
.
2006
;
107
(
12
):
4917
-
4925
.
72.
Scherr
M
,
Chaturvedi
A
,
Battmer
K
, et al
.
Enhanced sensitivity to inhibition of SHP2, STAT5, and Gab2 expression in chronic myeloid leukemia (CML)
.
Blood
.
2006
;
107
(
8
):
3279
-
3287
.
73.
Samanta
A
,
Perazzona
B
,
Chakraborty
S
, et al
.
Janus kinase 2 regulates Bcr-Abl signaling in chronic myeloid leukemia
.
Leukemia
.
2011
;
25
(
3
):
463
-
472
.
74.
Gallipoli
P
,
Cook
A
,
Rhodes
S
, et al
.
JAK2/STAT5 inhibition by nilotinib with ruxolitinib contributes to the elimination of CML CD34+ cells in vitro and in vivo
.
Blood
.
2014
;
124
(
9
):
1492
-
1501
.
75.
Hantschel
O
,
Warsch
W
,
Eckelhart
E
, et al
.
BCR-ABL uncouples canonical JAK2-STAT5 signaling in chronic myeloid leukemia
.
Nat Chem Biol
.
2012
;
8
(
3
):
285
-
293
.
76.
Neviani
P
,
Santhanam
R
,
Trotta
R
, et al
.
The tumor suppressor PP2A is functionally inactivated in blast crisis CML through the inhibitory activity of the BCR/ABL-regulated SET protein
.
Cancer Cell
.
2005
;
8
(
5
):
355
-
368
.
77.
Neviani
P
,
Harb
JG
,
Oaks
JJ
, et al
.
PP2A-activating drugs selectively eradicate TKI-resistant chronic myeloid leukemic stem cells
.
J Clin Invest
.
2013
;
123
(
10
):
4144
-
4157
.
78.
Chen
M
,
Gallipoli
P
,
DeGeer
D
, et al
.
Targeting primitive chronic myeloid leukemia cells by effective inhibition of a new AHI-1-BCR-ABL-JAK2 complex
.
J Natl Cancer Inst
.
2013
;
105
(
6
):
405
-
423
.
79.
Traer
E
,
MacKenzie
R
,
Snead
J
, et al
.
Blockade of JAK2-mediated extrinsic survival signals restores sensitivity of CML cells to ABL inhibitors
.
Leukemia
.
2012
;
26
(
5
):
1140
-
1143
.
80.
Eiring
AM
,
Page
BD
,
Kraft
IL
, et al
.
Combined STAT3 and BCR-ABL1 inhibition induces synthetic lethality in therapy-resistant chronic myeloid leukemia
.
Leukemia
.
2015
;
29
(
3
):
586
-
597
.
81.
Nowicki
MO
,
Falinski
R
,
Koptyra
M
, et al
.
BCR/ABL oncogenic kinase promotes unfaithful repair of the reactive oxygen species-dependent DNA double-strand breaks
.
Blood
.
2004
;
104
(
12
):
3746
-
3753
.
82.
Cramer-Morales
K
,
Nieborowska-Skorska
M
,
Scheibner
K
, et al
.
Personalized synthetic lethality induced by targeting RAD52 in leukemias identified by gene mutation and expression profile
.
Blood
.
2013
;
122
(
7
):
1293
-
1304
.
83.
Krause
DS
,
Lazarides
K
,
von Andrian
UH
,
Van Etten
RA
.
Requirement for CD44 in homing and engraftment of BCR-ABL-expressing leukemic stem cells
.
Nat Med
.
2006
;
12
(
10
):
1175
-
1180
.
84.
Krause
DS
,
Lazarides
K
,
Lewis
JB
,
von Andrian
UH
,
Van Etten
RA
.
Selectins and their ligands are required for homing and engraftment of BCR-ABL1+ leukemic stem cells in the bone marrow niche
.
Blood
.
2014
;
123
(
9
):
1361
-
1371
.
85.
Yamamoto-Sugitani
M
,
Kuroda
J
,
Ashihara
E
, et al
.
Galectin-3 (Gal-3) induced by leukemia microenvironment promotes drug resistance and bone marrow lodgment in chronic myelogenous leukemia
.
Proc Natl Acad Sci USA
.
2011
;
108
(
42
):
17468
-
17473
.
86.
Bhatia
R
,
McCarthy
JB
,
Verfaillie
CM
.
Interferon-alpha restores normal beta 1 integrin-mediated inhibition of hematopoietic progenitor proliferation by the marrow microenvironment in chronic myelogenous leukemia
.
Blood
.
1996
;
87
(
9
):
3883
-
3891
.
87.
Bhatia
R
,
Wayner
EA
,
McGlave
PB
,
Verfaillie
CM
.
Interferon-alpha restores normal adhesion of chronic myelogenous leukemia hematopoietic progenitors to bone marrow stroma by correcting impaired beta 1 integrin receptor function
.
J Clin Invest
.
1994
;
94
(
1
):
384
-
391
.
88.
Zhang
B
,
Li
M
,
McDonald
T
, et al
.
Microenvironmental protection of CML stem and progenitor cells from tyrosine kinase inhibitors through N-cadherin and Wnt-β-catenin signaling
.
Blood
.
2013
;
121
(
10
):
1824
-
1838
.
89.
Tabe
Y
,
Jin
L
,
Iwabuchi
K
, et al
.
Role of stromal microenvironment in nonpharmacological resistance of CML to imatinib through Lyn/CXCR4 interactions in lipid rafts
.
Leukemia
.
2012
;
26
(
5
):
883
-
892
.
90.
Herrmann
H
,
Sadovnik
I
,
Cerny-Reiterer
S
, et al
.
Dipeptidylpeptidase IV (CD26) defines leukemic stem cells (LSC) in chronic myeloid leukemia
.
Blood
.
2014
;
123
(
25
):
3951
-
3962
.
91.
Zhang
B
,
Ho
YW
,
Huang
Q
, et al
.
Altered microenvironmental regulation of leukemic and normal stem cells in chronic myelogenous leukemia
.
Cancer Cell
.
2012
;
21
(
4
):
577
-
592
.
92.
Welner
RS
,
Amabile
G
,
Bararia
D
, et al
.
Treatment of chronic myelogenous leukemia by blocking cytokine alterations found in normal stem and progenitor cells
.
Cancer Cell
.
2015
;
27
(
5
):
671
-
681
.
93.
Reynaud
D
,
Pietras
E
,
Barry-Holson
K
, et al
.
IL-6 controls leukemic multipotent progenitor cell fate and contributes to chronic myelogenous leukemia development
.
Cancer Cell
.
2011
;
20
(
5
):
661
-
673
.
94.
Ilander
M
,
Hekim
C
,
Mustjoki
S
.
Immunology and immunotherapy of chronic myeloid leukemia
.
Curr Hematol Malig Rep
.
2014
;
9
(
1
):
17
-
23
.
95.
Mumprecht
S
,
Schürch
C
,
Schwaller
J
,
Solenthaler
M
,
Ochsenbein
AF
.
Programmed death 1 signaling on chronic myeloid leukemia-specific T cells results in T-cell exhaustion and disease progression
.
Blood
.
2009
;
114
(
8
):
1528
-
1536
.
96.
Riether
C
,
Gschwend
T
,
Huguenin
AL
,
Schürch
CM
,
Ochsenbein
AF
.
Blocking programmed cell death 1 in combination with adoptive cytotoxic T-cell transfer eradicates chronic myelogenous leukemia stem cells
.
Leukemia
.
2015
;
29
(
8
):
1781
-
1785
.
97.
Tarafdar
A
,
Hopcroft
LE
,
Gallipoli
P
, et al
.
CML cells actively evade host immune surveillance through cytokine-mediated downregulation of MHC-II expression
.
Blood
.
2017
;
129
(
2
):
199
-
208
.
98.
Abraham
SA
,
Hopcroft
LE
,
Carrick
E
, et al
.
Dual targeting of p53 and c-MYC selectively eliminates leukaemic stem cells
.
Nature
.
2016
;
534
(
7607
):
341
-
346
.
99.
Fu
LL
,
Tian
M
,
Li
X
, et al
.
Inhibition of BET bromodomains as a therapeutic strategy for cancer drug discovery
.
Oncotarget
.
2015
;
6
(
8
):
5501
-
5516
.
100.
Xie
H
,
Peng
C
,
Huang
J
, et al
.
Chronic myelogenous leukemia-initiating cells require Polycomb group protein EZH2
.
Cancer Discov
.
2016
;
6
(
11
):
1237
-
1247
.
101.
Scott
MT
,
Korfi
K
,
Saffrey
P
, et al
.
Epigenetic reprogramming sensitizes CML stem cells to combined EZH2 and tyrosine kinase inhibition
.
Cancer Discov
.
2016
;
6
(
11
):
1248
-
1257
.
102.
Prost
S
,
Relouzat
F
,
Spentchian
M
, et al
.
Erosion of the chronic myeloid leukaemia stem cell pool by PPARγ agonists
.
Nature
.
2015
;
525
(
7569
):
380
-
383
.
103.
Glodkowska-Mrowka
E
,
Manda-Handzlik
A
,
Stelmaszczyk-Emmel
A
, et al
.
PPARγ ligands increase antileukemic activity of second- and third-generation tyrosine kinase inhibitors in chronic myeloid leukemia cells
.
Blood Cancer J
.
2016
;
6
:
e377
.
104.
Holyoake
T
,
Vetrie
D
.
Cancer: Repositioned to kill stem cells
.
Nature
.
2015
;
525
(
7569
):
328
-
329
.
105.
Nishioka
C
,
Ikezoe
T
,
Yang
J
,
Yokoyama
A
.
BCR/ABL increases EZH2 levels which regulates XIAP expression via miRNA-219 in chronic myeloid leukemia cells
.
Leuk Res
.
2016
;
45
:
24
-
32
.
106.
Chen
Y
,
Peng
C
,
Abraham
SA
, et al
.
Arachidonate 15-lipoxygenase is required for chronic myeloid leukemia stem cell survival
.
J Clin Invest
.
2014
;
124
(
9
):
3847
-
3862
.
107.
Nguyen
LV
,
Pellacani
D
,
Lefort
S
, et al
.
Barcoding reveals complex clonal dynamics of de novo transformed human mammary cells
.
Nature
.
2015
;
528
(
7581
):
267
-
271
.
108.
Goyama
S
,
Wunderlich
M
,
Mulloy
JC
.
Xenograft models for normal and malignant stem cells
.
Blood
.
2015
;
125
(
17
):
2630
-
2640
.
109.
Wilson
NK
,
Kent
DG
,
Buettner
F
, et al
.
Combined single-cell functional and gene expression analysis resolves heterogeneity within stem cell populations
.
Cell Stem Cell
.
2015
;
16
(
6
):
712
-
724
.
110.
Järås
M
,
Johnels
P
,
Hansen
N
, et al
.
Isolation and killing of candidate chronic myeloid leukemia stem cells by antibody targeting of IL-1 receptor accessory protein
.
Proc Natl Acad Sci USA
.
2010
;
107
(
37
):
16280
-
16285
.
111.
Sadovnik
I
,
Hoelbl-Kovacic
A
,
Herrmann
H
, et al
.
Identification of CD25 as STAT5-dependent growth regulator of leukemic stem cells in Ph+ CML
.
Clin Cancer Res
.
2016
;
22
(
8
):
2051
-
2061
.
112.
Kinstrie
R
,
Horne
GA
,
Morrison
H
, et al
.
CD93 is a novel biomarker of leukemia stem cells in chronic myeloid leukemia
.
Blood
.
2015
;
126
:
49
.
113.
Bellodi
C
,
Lidonnici
MR
,
Hamilton
A
, et al
.
Targeting autophagy potentiates tyrosine kinase inhibitor-induced cell death in Philadelphia chromosome-positive cells, including primary CML stem cells
.
J Clin Invest
.
2009
;
119
(
5
):
1109
-
1123
.
114.
Goussetis
DJ
,
Gounaris
E
,
Wu
EJ
, et al
.
Autophagic degradation of the BCR-ABL oncoprotein and generation of antileukemic responses by arsenic trioxide
.
Blood
.
2012
;
120
(
17
):
3555
-
3562
.
115.
Karvela
M
,
Baquero
P
,
Kuntz
EM
, et al
.
ATG7 regulates energy metabolism, differentiation and survival of Philadelphia-chromosome-positive cells
.
Autophagy
.
2016
;
12
(
6
):
936
-
948
.
116.
Zhang
H
,
Peng
C
,
Hu
Y
, et al
.
The Blk pathway functions as a tumor suppressor in chronic myeloid leukemia stem cells
.
Nat Genet
.
2012
;
44
(
8
):
861
-
871
.
117.
Copland
M
,
Pellicano
F
,
Richmond
L
, et al
.
BMS-214662 potently induces apoptosis of chronic myeloid leukemia stem and progenitor cells and synergizes with tyrosine kinase inhibitors
.
Blood
.
2008
;
111
(
5
):
2843
-
2853
.
118.
Reddiconto
G
,
Toto
C
,
Palamà
I
, et al
.
Targeting of GSK3β promotes imatinib-mediated apoptosis in quiescent CD34+ chronic myeloid leukemia progenitors, preserving normal stem cells
.
Blood
.
2012
;
119
(
10
):
2335
-
2345
.
119.
Zhang
B
,
Strauss
AC
,
Chu
S
, et al
.
Effective targeting of quiescent chronic myelogenous leukemia stem cells by histone deacetylase inhibitors in combination with imatinib mesylate
.
Cancer Cell
.
2010
;
17
(
5
):
427
-
442
.
120.
Ito
K
,
Bernardi
R
,
Morotti
A
, et al
.
PML targeting eradicates quiescent leukaemia-initiating cells
.
Nature
.
2008
;
453
(
7198
):
1072
-
1078
.
121.
Li
L
,
Wang
L
,
Li
L
, et al
.
Activation of p53 by SIRT1 inhibition enhances elimination of CML leukemia stem cells in combination with imatinib
.
Cancer Cell
.
2012
;
21
(
2
):
266
-
281
.
122.
Naka
K
,
Ishihara
K
,
Jomen
Y
, et al
.
Novel oral transforming growth factor-β signaling inhibitor EW-7197 eradicates CML-initiating cells
.
Cancer Sci
.
2016
;
107
(
2
):
140
-
148
.
123.
Jin
L
,
Tabe
Y
,
Konoplev
S
, et al
.
CXCR4 up-regulation by imatinib induces chronic myelogenous leukemia (CML) cell migration to bone marrow stroma and promotes survival of quiescent CML cells
.
Mol Cancer Ther
.
2008
;
7
(
1
):
48
-
58
.
124.
Weisberg
E
,
Azab
AK
,
Manley
PW
, et al
.
Inhibition of CXCR4 in CML cells disrupts their interaction with the bone marrow microenvironment and sensitizes them to nilotinib
.
Leukemia
.
2012
;
26
(
5
):
985
-
990
.
125.
Dillmann
F
,
Veldwijk
MR
,
Laufs
S
, et al
.
Plerixafor inhibits chemotaxis toward SDF-1 and CXCR4-mediated stroma contact in a dose-dependent manner resulting in increased susceptibility of BCR-ABL+ cell to Imatinib and Nilotinib
.
Leuk Lymphoma
.
2009
;
50
(
10
):
1676
-
1686
.
126.
Vianello
F
,
Villanova
F
,
Tisato
V
, et al
.
Bone marrow mesenchymal stromal cells non-selectively protect chronic myeloid leukemia cells from imatinib-induced apoptosis via the CXCR4/CXCL12 axis
.
Haematologica
.
2010
;
95
(
7
):
1081
-
1089
.
127.
Laperrousaz
B
,
Jeanpierre
S
,
Sagorny
K
, et al
.
Primitive CML cell expansion relies on abnormal levels of BMPs provided by the niche and on BMPRIb overexpression
.
Blood
.
2013
;
122
(
23
):
3767
-
3777
.
128.
Zhang
H
,
Li
H
,
Xi
HS
,
Li
S
.
HIF1α is required for survival maintenance of chronic myeloid leukemia stem cells
.
Blood
.
2012
;
119
(
11
):
2595
-
2607
.
129.
Ng
KP
,
Manjeri
A
,
Lee
KL
, et al
.
Physiologic hypoxia promotes maintenance of CML stem cells despite effective BCR-ABL1 inhibition
.
Blood
.
2014
;
123
(
21
):
3316
-
3326
.
130.
Preudhomme
C
,
Guilhot
J
,
Nicolini
FE
, et al. 
;
SPIRIT Investigators; France Intergroupe des Leucémies Myéloïdes Chroniques (Fi-LMC)
.
Imatinib plus peginterferon alfa-2a in chronic myeloid leukemia
.
N Engl J Med
.
2010
;
363
(
26
):
2511
-
2521
.
131.
Burchert
A
,
Müller
MC
,
Kostrewa
P
, et al
.
Sustained molecular response with interferon alfa maintenance after induction therapy with imatinib plus interferon alfa in patients with chronic myeloid leukemia
.
J Clin Oncol
.
2010
;
28
(
8
):
1429
-
1435
.
132.
Simonsson
B
,
Gedde-Dahl
T
,
Markevärn
B
, et al. 
;
Nordic CML Study Group
.
Combination of pegylated IFN-α2b with imatinib increases molecular response rates in patients with low- or intermediate-risk chronic myeloid leukemia
.
Blood
.
2011
;
118
(
12
):
3228
-
3235
.
133.
Nicolini
FE
,
Etienne
G
,
Dubruille
V
, et al
.
Nilotinib and peginterferon alfa-2a for newly diagnosed chronic-phase chronic myeloid leukaemia (NiloPeg): a multicentre, non-randomised, open-label phase 2 study
.
Lancet Haematol
.
2015
;
2
(
1
):
e37
-
e46
.
134.
Hjorth-Hansen
H
,
Stentoft
J
,
Richter
J
, et al
.
Safety and efficacy of the combination of pegylated interferon-α2b and dasatinib in newly diagnosed chronic-phase chronic myeloid leukemia patients
.
Leukemia
.
2016
;
30
(
9
):
1853
-
1860
.
135.
Ågerstam
H
,
Hansen
N
,
von Palffy
S
, et al
.
IL1RAP antibodies block IL-1-induced expansion of candidate CML stem cells and mediate cell killing in xenograft models
.
Blood
.
2016
;
128
(
23
):
2683
-
2693
.
136.
Zhang
B
,
Chu
S
,
Agarwal
P
, et al
.
Inhibition of interleukin-1 signaling enhances elimination of tyrosine kinase inhibitor-treated CML stem cells
.
Blood
.
2016
;
128
(
23
):
2671
-
2682
.
137.
Taverna
S
,
Amodeo
V
,
Saieva
L
, et al
.
Exosomal shuttling of miR-126 in endothelial cells modulates adhesive and migratory abilities of chronic myelogenous leukemia cells
.
Mol Cancer
.
2014
;
13
:
169
.
138.
Bowers
M
,
Zhang
B
,
Ho
Y
,
Agarwal
P
,
Chen
CC
,
Bhatia
R
.
Osteoblast ablation reduces normal long-term hematopoietic stem cell self-renewal but accelerates leukemia development
.
Blood
.
2015
;
125
(
17
):
2678
-
2688
.
139.
Zhang
B
,
Li
L
,
Ho
Y
, et al
.
Heterogeneity of leukemia-initiating capacity of chronic myelogenous leukemia stem cells
.
J Clin Invest
.
2016
;
126
(
3
):
975
-
991
.
140.
Schmidt
T
,
Kharabi Masouleh
B
,
Loges
S
, et al
.
Loss or inhibition of stromal-derived PlGF prolongs survival of mice with imatinib-resistant Bcr-Abl1(+) leukemia
.
Cancer Cell
.
2011
;
19
(
6
):
740
-
753
.
141.
Aggoune
D
,
Wessenberger
E
,
Magnani
JL
,
Van Etten
RA
,
Krause
DS
.
The vascular niche is involved in regulating leukemic stem cells in murine chronic myelogenous leukemia
[Abstract].
Blood
.
2014
;
124
(
21
). Abstract 516.
142.
Krause
DS
,
Fulzele
K
,
Catic
A
, et al
.
Differential regulation of myeloid leukemias by the bone marrow microenvironment
.
Nat Med
.
2013
;
19
(
11
):
1513
-
1517
.
143.
Apperley
JF
.
TWIST it but don’t spin it
.
Blood
.
2013
;
122
(
4
):
470
-
471
.
144.
de Lavallade
H
,
Apperley
JF
,
Khorashad
JS
, et al
.
Imatinib for newly diagnosed patients with chronic myeloid leukemia: incidence of sustained responses in an intention-to-treat analysis
.
J Clin Oncol
.
2008
;
26
(
20
):
3358
-
3363
.
145.
Lucas
CM
,
Wang
L
,
Austin
GM
, et al
.
A population study of imatinib in chronic myeloid leukaemia demonstrates lower efficacy than in clinical trials
.
Leukemia
.
2008
;
22
(
10
):
1963
-
1966
.
146.
Gallipoli
P
,
Shepherd
P
,
Irvine
D
,
Drummond
M
,
Holyoake
T
.
Restricted access to second generation tyrosine kinase inhibitors in the UK could result in suboptimal treatment for almost half of chronic myeloid leukaemia patients: results from a West of Scotland and Lothian population study
.
Br J Haematol
.
2011
;
155
(
1
):
128
-
130
.
147.
Graham
SM
,
Vass
JK
,
Holyoake
TL
,
Graham
GJ
.
Transcriptional analysis of quiescent and proliferating CD34+ human hemopoietic cells from normal and chronic myeloid leukemia sources
.
Stem Cells
.
2007
;
25
(
12
):
3111
-
3120
.
148.
Schepers
K
,
Pietras
EM
,
Reynaud
D
, et al
.
Myeloproliferative neoplasia remodels the endosteal bone marrow niche into a self-reinforcing leukemic niche
.
Cell Stem Cell
.
2013
;
13
(
3
):
285
-
299
.