How does the novel T315L mutation of breakpoint cluster region-abelson (BCR-ABL) kinase confer resistance to ponatinib: a comparative molecular dynamics simulation study

Abstract

Acute lymphocytic leukemia (ALL) is one of the most dangerous types of leukemia, and about 40% of them is Philadelphia chromosome-positive acute lymphocytic leukemia (Ph?+?ALL). Ph?+?ALL is caused by the fusion of the breakpoint cluster region (BCR) and the Ableson (ABL) genes, named the BCR-ABL fused gene that codes for an autonomously active tyrosine kinase. Tyrosine kinase inhibitors (TKIs) are among the first-line therapeutic agents for the treatment of Ph?+?ALL. Drug resistance are the major obstacle, limiting their clinical utility. The latest third-generation TKIs, ponatinib, can tackle most abnormal BCR-ABL kinases, including the T315I mutant that is resistant to first- and second-generations TKIs such as imatinib. However, drug resistance still emerges with the novel T315L mutation and the underlying mechanisms remain elusive. Here, using molecular dynamics (MD) simulations, we explored into the detailed interactions between ponatinib and BCR-ABL in the wild-type (WT), T315I, and T315L systems. The simulations revealed the significant conformational changes of ponatinib in its binding site due to the T315L mutation and the underlying structural mechanisms. Binding free energy analysis unveiled that the affinity of ponatinib to BCR-ABL decreased upon T315L mutation, which resulted in its unfavorable binding and drug resistance. Key residues responsible for the unfavored unbinding were also identified. This study elucidates the detailed mechanisms for the resistance of ponatinib in Ph?+?ALL triggered by the T315L mutation and will provide insights for future drug development and optimization.     

Regulatory Effects of Sestrin 3 (SESN3) in BCR-ABL Expressing Cells

Chronic myeloid leukemia (CML) and Ph+ acute lymphoblastic leukemia (ALL) are characterized by the presence of the BCR-ABL oncoprotein, which leads to activation of a plethora of pro-mitogenic and pro-survival pathways, including the mTOR signaling cascade. We provide evidence that in BCR-ABL expressing cells, treatment with tyrosine kinase inhibitors (TKIs) results in upregulation of mRNA levels and protein expression of sestrin3 (SESN3), a unique cellular inhibitor of mTOR complex 1 (mTORC1). Such upregulation appears to be mediated by regulatory effects on mTOR, as catalytic inhibition of the mTOR kinase also induces SESN3. Catalytic mTOR inhibition also results in upregulation of SESN3 expression in cells harboring the TKI-insensitive T315I-BCR-ABL mutant, which is resistant to imatinib mesylate. Overexpression of SESN3 results in inhibitory effects on different Ph+ leukemic cell lines including KT-1-derived leukemic precursors, indicating that SESN3 mediates anti-leukemic responses in Ph+ cells. Altogether, our findings suggest the existence of a novel mechanism for the generation of antileukemic responses in CML cells, involving upregulation of SESN3 expression.     

针对T315I 突变的Bcr-Abl 酪氨酸激酶抑制剂研究进展

BCR-ABL 是一种由bcr 基因和c-abl 原癌基因融合产生的致癌基因。该基因表达的Bcr–Abl 癌蛋白是慢性粒细胞白血病的病理学基础。因此研发选择性的Bcr–Abl 酪氨酸激酶抑制剂成为治疗慢性粒细胞白血病的一种有效策略。目前已有数个Bcr–Abl 酪氨酸激酶抑制剂获准上市。然而,Abl 激酶结构域的突变或其他原因导致肿瘤耐药性的出现,其中T315I 突变是最重要的突变之一,引发的耐药性更是难以克服。重点介绍了针对T315I 突变的Bcr–Abl 酪氨酸激酶抑制剂的研究进展。     

ATRA-Induced Cellular Differentiation and CD38 Expression Inhibits Acquisition of BCR-ABL Mutations for CML Acquired Resistance

Acquired resistance through genetic mutations is a major obstacle in targeted cancer therapy, but the underlying mechanisms are poorly understood. Here we studied mechanisms of acquired resistance of chronic myeloid leukemia (CML) to tyrosine kinase inhibitors (TKIs) by examining genome-wide gene expression changes in KCL-22 CML cells versus their resistant KCL-22M cells that acquire T315I BCR-ABL mutation following TKI exposure. Although T315I BCR-ABL is sufficient to confer resistance to TKIs in CML cells, surprisingly we found that multiple drug resistance pathways were activated in KCL-22M cells along with reduced expression of a set of myeloid differentiation genes. Forced myeloid differentiation by all-trans-retinoic acid (ATRA) effectively blocked acquisition of BCR-ABL mutations and resistance to the TKIs imatinib, nilotinib or dasatinib in our previously described in vitro models of acquired TKI resistance. ATRA induced robust expression of CD38, a cell surface marker and cellular NADase. High levels of CD38 reduced intracellular nicotinamide adenine dinucleotide (NAD⁺) levels and blocked acquired resistance by inhibiting the activity of the NAD⁺-dependent SIRT1 deacetylase that we have previously shown to promote resistance in CML cells by facilitating error-prone DNA damage repair. Consequently, ATRA treatment decreased DNA damage repair and suppressed acquisition of BCR-ABL mutations. This study sheds novel insight into mechanisms underlying acquired resistance in CML, and suggests potential benefit of combining ATRA with TKIs in treating CML, particularly in advanced phases.     

Stem cell and kinase activity-independent pathway in resistance of leukaemia to BCR-ABL kinase inhibitors

BCR-ABL tyrosine kinase inhibitors, such as imatinib (Gleevec) are highly effective in treating human Philadelphia chromosome-positive (Ph+) chronic myeloid leukaemia (CML) in chronic phase but not in terminal acute phase; acquired drug resistance caused mainly by the development of BCR-ABL kinase domain mutations prevents cure of the leukaemia. In addition, imatinib is ineffective in treating Ph+ B-cell acute lymphoblastic leukaemia (B-ALL) and CML blast crisis, even in the absence of the kinase domain mutations. This type of drug resistance that is unrelated to BCR-ABL kinase domain mutations is caused by the insensitivity of leukaemic stem cells to kinase inhibitors such as imatinib and dasatinib, and by activation of a newly-identified signalling pathway involving SRC kinases that are independent of BCR-ABL kinase activity for activation. This SRC pathway is essential for leukaemic cells to survive imatinib treatment and for CML transition to lymphoid blast crisis. Apart from BCR-ABL and SRC kinases, stem cell pathways must also be targeted for curative therapy of Ph+ leukaemia.     

The novel Aurora A kinase inhibitor MLN8237 is active in resistant chronic myeloid leukaemia and significantly increases the efficacy of nilotinib

Novel therapies are urgently needed to prevent and treat tyrosine kinase inhibitor resistance in chronic myeloid leukaemia (CML). MLN8237 is a novel Aurora A kinase inhibitor under investigation in multiple phase I and II studies. Here we report that MLN8237 possessed equipotent activity against Ba/F3 cells and primary CML cells expressing unmutated and mutated forms of breakpoint cluster region-Abelson kinase (BCR-ABL). Notably, this agent retained high activity against the T315I and E255K BCR-ABL mutations, which confer the greatest degree of resistance to standard therapy. MLN8237 treatment disrupted cell cycle kinetics, induced apoptosis, caused a dose-dependent reduction in the expression of the large inhibitor of apoptosis protein Apollon, and produced a morphological phenotype consistent with Aurora A kinase inhibition. In contrast to other Aurora kinase inhibitors, MLN8237 did not significantly affect BCR-ABL activity. Moreover, inhibition of Aurora A with MLN8237 significantly increased the in vitro and in vivo efficacy of nilotinib. Targeted knockdown of Apollon sensitized CML cells to nilotinib-induced apoptosis, indicating that this is an important factor underlying MLN8237's ability to increase the efficacy of nilotinib. Our collective data demonstrate that this combination strategy represents a novel therapeutic approach for refractory CML that has the potential to suppress the emergence of T315I mutated CML clones.     

Molecular and cellular bases of chronic myeloid leukemia

Chronic myeloid leukemia (CML) is a myeloproliferative disease characterized by the overproduction of granulocytes, which leads to high white blood cell counts and splenomegaly in patients. Based on clinical symptoms and laboratory findings, CML is classified into three clinical phases, often starting with a chronic phase, progressing to an accelerated phase and ultimately ending in a terminal phase called blast crisis. Blast crisis phase of CML is clinically similar to an acute leukemia; in particular, B-cell acute lymphoblastic leukemia (B-ALL) is a severe form of acute leukemia in blast crisis, and there is no effective therapy for it yet. CML is induced by the BCR-ABL oncogene, whose gene product is a BCR-ABL tyrosine kinase. Currently, inhibition of BCR-ABL kinase activity by its kinase inhibitor such as imatinib mesylate (Gleevec) is a major therapeutic strategy for CML. However, the inability of BCR-ABL kinase inhibitors to completely kill leukemia stem cells (LSCs) indicates that these kinase inhibitors are unlikely to cure CML. In addition, drug resistance due to the development of BCR-ABL mutations occurs before and during treatment of CML with kinase inhibitors. A critical issue to resolve this problem is to fully understand the biology of LSCs, and to identify key genes that play significant roles in survival and self-renewal of LSCs. In this review, we will focus on LSCs in CML by summarizing and discussing available experimental results, including the original studies from our own laboratory.     

Overcoming kinase resistance in chronic myeloid leukemia

Imatinib is a small-molecule inhibitor of BCR-ABL tyrosine kinase activity, with proven efficacy and tolerability. Despite imatinib's activity, the development of resistance, whether BCR-ABL dependent or independent, is a concern. BCR-ABL-dependent resistance is commonly a result of mutations in the BCR-ABL gene, which can induce a structural predisposition towards the active conformation of the protein, resulting in a shift in the equilibrium of BCR-ABL from inactive, which imatinib binds, to active, which imatinib is unable to bind. BCR-ABL gene amplification may play a role in the development of imatinib resistance in patients with CML. There are a number of BCR-ABL-independent mechanisms of imatinib resistance, including the efflux protein multidrug resistance protein-1, of which imatinib is a substrate. Another mechanism may be the development of alternative pathways of disease progression, leading to less reliance on BCR-ABL; indeed, the SRC family tyrosine kinases LYN and HCK have been frequently implicated in treatment resistance and progression of CML. Clearly, imatinib resistance requires the development of other treatment options. Dasatinib, with increased binding potency (325-fold greater potency than imatinib for wild-type BCR-ABL), inhibition of both the active and inactive formation of BCR-ABL, and targeting of SRC family kinases, is the only agent approved for the treatment of patients with imatinib-resistant or -intolerant CML and Ph+ ALL. Dasatinib is highly active in all phases of these diseases, and is active in the majority of imatinib-resistant mutations, with the exception of T315I. The development of agents that effectively inhibit T315I mutations suggests that future treatment options will include combination therapy.     

Isolation of specific and biologically active peptides that bind cells from patients with acute myeloid leukemia (AML)

Imatinib was the first BCR-ABL-targeted agent approved for the treatment of patients with chronic myeloid leukemia (CML) and confers significant benefit for most patients; however, a substantial number of patients are either initially refractory or develop resistance. Point mutations within the ABL kinase domain of the BCR-ABL fusion protein are a major underlying cause of resistance. Of the known imatinib-resistant mutations, the most frequently occurring involve the ATP-binding loop (P-loop). In vitro evidence has suggested that these mutations are more oncogenic with respect to other mutations and wild type BCR-ABL. Dasatinib and nilotinib have been approved for second-line treatment of patients with CML who demonstrate resistance (or intolerance) to imatinib. Both agents have marked activity in patients resistant to imatinib; however, they have differential activity against certain mutations, including those of the P-loop. Data from clinical trials suggest that dasatinib may be more effective vs. nilotinib for treating patients harboring P-loop mutations. Other mutations that are differentially sensitive to the second-line tyrosine kinase inhibitors (TKIs) include F317L and F359I/V, which are more sensitive to nilotinib and dasatinib, respectively. P-loop status in patients with CML and the potency of TKIs against P-loop mutations are key determinants for prognosis and response to treatment. This communication reviews the clinical importance of P-loop mutations and the efficacy of the currently available TKIs against them.     

Inhibition of PI3K/mTOR Overcomes Nilotinib Resistance in BCR-ABL1 Positive Leukemia Cells through Translational Down-Regulation of MDM2

Chronic myeloid leukemia (CML) is a cytogenetic disorder resulting from formation of the Philadelphia chromosome (Ph), that is, the t(9;22) chromosomal translocation and the formation of the BCR-ABL1 fusion protein. Tyrosine kinase inhibitors (TKI), such as imatinib and nilotinib, have emerged as leading compounds with which to treat CML. t(9;22) is not restricted to CML, 20-30% of acute lymphoblastic leukemia (ALL) cases also carry the Ph. However, TKIs are not as effective in the treatment of Ph+ ALL as in CML. In this study, the Ph+ cell lines JURL-MK2 and SUP-B15 were used to investigate TKI resistance mechanisms and the sensitization of Ph+ tumor cells to TKI treatment. The annexin V/PI (propidium iodide) assay revealed that nilotinib induced apoptosis in JURL-MK2 cells, but not in SUP-B15 cells. Since there was no mutation in the tyrosine kinase domain of BCR-ABL1 in cell line SUP-B15, the cells were not generally unresponsive to TKI, as evidenced by dephosphorylation of the BCR-ABL1 downstream targets, Crk-like protein (CrkL) and Grb-associated binder-2 (GAB2). Resistance to apoptosis after nilotinib treatment was accompanied by the constitutive and nilotinib unresponsive activation of the phosphoinositide 3-kinase (PI3K) pathway. Treatment of SUP-B15 cells with the dual PI3K/mammalian target of rapamycin (mTOR) inhibitor BEZ235 alone induced apoptosis in a low percentage of cells, while combining nilotinib and BEZ235 led to a synergistic effect. The main role of PI3K/mTOR inhibitor BEZ235 and the reason for apoptosis in the nilotinib-resistant cells was the block of the translational machinery, leading to the rapid downregulation of the anti-apoptotic protein MDM2 (human homolog of the murine double minute-2). These findings highlight MDM2 as a potential therapeutic target to increase TKI-mediated apoptosis and imply that the combination of PI3K/mTOR inhibitor and TKI might form a novel strategy to combat TKI-resistant BCR-ABL1 positive leukemia.     

Dihydroorotate dehydrogenase inhibition reveals metabolic vulnerability in chronic myeloid leukemia

The development of different generations of BCR-ABL1 tyrosine kinase inhibitors (TKIs) has led to the high overall survival of chronic myeloid leukemia (CML) patients. However, there are CML patients who show resistance to TKI therapy and are prone to progress to more advanced phases of the disease. So, implementing an alternative approach for targeting TKIs insensitive cells would be of the essence. Dihydroorotate dehydrogenase (DHODH) is an enzyme in the de novo pyrimidine biosynthesis pathway that is located in the inner membrane of mitochondria. Here, we found that CML cells are vulnerable to DHODH inhibition mediated by Meds433, a new and potent DHODH inhibitor recently developed by our group. Meds433 significantly activates the apoptotic pathway and leads to the reduction of amino acids and induction of huge metabolic stress in CML CD34+ cells. Altogether, our study shows that DHODH inhibition is a promising approach for targeting CML stem/progenitor cells and may help more patients discontinue the therapy.Subject terms: Cancer metabolism, Apoptosis     

An overview of chronic myeloid leukemia and its animal models

Chronic myeloid leukemia(CML) is a form of leukemia characterized by the presence of clonal bone marrow stem cells with the proliferation of mature granulocytes(neutrophils, eosinophils, and basophils) and their precursors. CML is a type of myeloproliferative disease associated with a characteristic chromosomal translocation called the Philadelphia(Ph) chromosome or t(9;22) translocation(BCR-ABL). CML is now usually treated with targeted drugs called tyrosine kinase inhibitors(TKIs). The mechanism and natural history of CML is still unclear. Here, we summarize the present CML animal disease models and compare them with each other. Meanwhile, we propose that it is a very wise choice to establish zebrafish(Danio rerio) CML model mimics clinical CML. This model could be used to learn more about the mechanism of CML, and to aid in the development of new drugs to treat CML.     

Molecular mechanisms for survival regulation of chronic myeloid leukemia stem cells

Studies on chronic myeloid leukemia (CML) have served as a paradigm for cancer research and therapy. These studies involve the identifi cation of the fi rst cancer-associated chromosomal abnormality and the subsequent development of tyrosine kinase inhibitors (TKIs) that inhibit BCR-ABL kinase activity in CML. It becomes clear that leukemia stem cells (LSCs) in CML which are resistant to TKIs, and eradication of LSCs appears to be extremely diffi cult. Therefore, one of the major issues in current CML biology is to understand the biology of LSCs and to investigate why LSCs are insensitive to TKI monotherapy for developing curative therapeutic strategies. Studies from our group and others have revealed that CML LSCs form a hierarchy similar to that seen in normal hematopoiesis, in which a rare stem cell population with limitless selfrenewal potential gives rise to progenies that lack such potential. LSCs also possess biological features that are different from those of normal hematopoietic stem cells (HSCs) and are critical for their malignant characteristics. In this review, we summarize the latest progress in CML field, and attempt to understand the molecular mechanisms of survival regulation of LSCs.     

BCR-ABL uncouples canonical JAK2-STAT5 signaling in chronic myeloid leukemia

Constitutive activation of STAT5 is critical for the maintenance of chronic myeloid leukemia (CML) characterized by the BCR-ABL oncoprotein. Tyrosine kinase inhibitors (TKIs) for the STAT5-activating kinase JAK2 have been discussed as a treatment option for CML patients. Using murine leukemia models combined with inducible ablation of JAK2, we show JAK2 dependence for initial lymphoid transformation, which is lost once leukemia is established. In contrast, initial myeloid transformation and leukemia maintenance were independent of JAK2. Nevertheless, several JAK2 TKIs induced apoptosis in BCR-ABL(+) cells irrespective of the presence of JAK2. This is caused by the previously unknown direct 'off-target' inhibition of BCR-ABL. Cellular and enzymatic analyses suggest that BCR-ABL phosphorylates STAT5 directly. Our findings suggest uncoupling of the canonical JAK2-STAT5 module upon BCR-ABL expression, thereby making JAK2 targeting dispensable. Thus, attempts to pharmacologically target STAT5 in BCR-ABL(+) diseases need to focus on STAT5 itself.     

Targeted therapies and autophagy: new insights from chronic myeloid leukemia

Patients who develop chronic myeloid leukemia (CML) are currently treated with tyrosine kinase inhibitors (TKIs), which inhibit the function of the oncogene BCR/Abl. Most CML cells undergo apoptosis when BCR/Abl tyrosine kinase activity is suppressed by TKIs. Cells surviving drug treatment are either stem cells (CML in early phase) or cells with BCR/Abl-dependent or -independent mechanisms of drug resistance (CML in advanced phase). Since survival of these cells is thought to be responsible for disease recurrence, it is critical to find ways to fully eradicate CML stem cells. We have recently shown that when CML cells, including stem cells, are exposed to TKI they activate an autophagic program, which relies on intracellular calcium and is not inhibited by Bcl-2. Pharmacological or RNAi-mediated inhibition of autophagy potentiates the effect of TKI in inducing death of CML cells, including the stem cells. These data strongly suggest that inhibition of autophagy may improve the therapeutic effects of TKIs in the treatment of CML. In addition, they give credence to the idea that in cancer cells autophagy is part of a stereotypic response to stress and specifically to abrogation of their main oncogenic signal(s).     

Combination of tyrosine kinase inhibitors and the MCL1 inhibitor S63845 exerts synergistic antitumorigenic effects on CML cells

Tyrosine kinase inhibitor (TKI) treatment has dramatically improved the survival of chronic myeloid leukemia (CML) patients, but measurable residual disease typically persists. To more effectively eradicate leukemia cells, simultaneous targeting of BCR-ABL1 and additional CML-related survival proteins has been proposed. Notably, several highly specific myeloid cell leukemia 1 (MCL1) inhibitors have recently entered clinical trials for various hematologic malignancies, although not for CML, reflecting the insensitivity of CML cell lines to single MCL1 inhibition. Here, we show that combining TKI (imatinib, nilotinib, dasatinib, or asciminib) treatment with the small-molecule MCL1 inhibitor {"type":"entrez-nucleotide","attrs":{"text":"S63845","term_id":"400540","term_text":"S63845"}}S63845 exerted strong synergistic antiviability and proapoptotic effects on CML lines and CD34+ stem/progenitor cells isolated from untreated CML patients in chronic phase. Using wild-type BCR-ABL1-harboring CML lines and their T315I-mutated sublines (generated by CRISPR/Cas9-mediated homologous recombination), we prove that the synergistic proapoptotic effect of the drug combination depended on TKI-mediated BCR-ABL1 inhibition, but not on TKI-related off-target mechanisms. Moreover, we demonstrate that colony formation of CML but not normal hematopoietic stem/progenitor cells became markedly reduced upon combination treatment compared to imatinib monotherapy. Our results suggest that dual targeting of MCL1 and BCR-ABL1 activity may efficiently eradicate residual CML cells without affecting normal hematopoietic stem/progenitors.Subject terms: Cancer stem cells, Targeted therapies, Preclinical research     

Abcg2 overexpression represents a novel mechanism for acquired resistance to the multi-kinase inhibitor Danusertib in BCR-ABL-positive cells in vitro

The success of Imatinib (IM) therapy in chronic myeloid leukemia (CML) is compromised by the development of IM resistance and by a limited IM effect on hematopoietic stem cells. Danusertib (formerly PHA-739358) is a potent pan-aurora and ABL kinase inhibitor with activity against known BCR-ABL mutations, including T315I. Here, the individual contribution of both signaling pathways to the therapeutic effect of Danusertib as well as mechanisms underlying the development of resistance and, as a consequence, strategies to overcome resistance to Danusertib were investigated. Starting at low concentrations, a dose-dependent inhibition of BCR-ABL activity was observed, whereas inhibition of aurora kinase activity required higher concentrations, pointing to a therapeutic window between the two effects. Interestingly, the emergence of resistant clones during Danusertib exposure in vitro occurred considerably less frequently than with comparable concentrations of IM. In addition, Danusertib-resistant clones had no mutations in BCR-ABL or aurora kinase domains and remained IM-sensitive. Overexpression of Abcg2 efflux transporter was identified and functionally validated as the predominant mechanism of acquired Danusertib resistance in vitro. Finally, the combined treatment with IM and Danusertib significantly reduced the emergence of drug resistance in vitro, raising hope that this drug combination may also achieve more durable disease control in vivo.     

Leukemia stem cells: the root of chronic myeloid leukemia

Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder characterized by a chromosome translocation that generates the Bcr-Abl oncogene encoding a constitutive kinase activity. Despite remarkable success in controlling CML at chronic phase by Bcr-Abl tyrosine kinase inhibitors (TKIs), a significant proportion of CML patients treated with TKIs develop drug resistance due to the inability of TKIs to kill leukemia stem cells (LSCs) that are responsible for initiation, drug resistance, and relapse of CML. Therefore, there is an urgent need for more potent and safer therapies against leukemia stem cells for curing CML. A number of LSCassociated targets and corresponding signaling pathways, including CaMKII-γ, a critical molecular switch for co-activating multiple LSC-associated signaling pathways, have been identified over the past decades and various small inhibitors targeting LSC are also under development. Increasing evidence shows that leukemia stem cells are the root of CML and targeting LSC may offer a curable treatment option for CML patients. This review summarizes the molecular biology of LSC and itsassociated targets, and the potential clinical application in chronic myeloid leukemia.     

Variable Behavior of iPSCs Derived from CML Patients for Response to TKI and Hematopoietic Differentiation

Chronic myeloid leukemia disease (CML) found effective therapy by treating patients with tyrosine kinase inhibitors (TKI), which suppress the BCR-ABL1 oncogene activity. However, the majority of patients achieving remission with TKI still have molecular evidences of disease persistence. Various mechanisms have been proposed to explain the disease persistence and recurrence. One of the hypotheses is that the primitive leukemic stem cells (LSCs) can survive in the presence of TKI. Understanding the mechanisms leading to TKI resistance of the LSCs in CML is a critical issue but is limited by availability of cells from patients. We generated induced pluripotent stem cells (iPSCs) derived from CD34⁺ blood cells isolated from CML patients (CML-iPSCs) as a model for studying LSCs survival in the presence of TKI and the mechanisms supporting TKI resistance. Interestingly, CML-iPSCs resisted to TKI treatment and their survival did not depend on BCR-ABL1, as for primitive LSCs. Induction of hematopoietic differentiation of CML-iPSC clones was reduced compared to normal clones. Hematopoietic progenitors obtained from iPSCs partially recovered TKI sensitivity. Notably, different CML-iPSCs obtained from the same CML patients were heterogeneous, in terms of BCR-ABL1 level and proliferation. Thus, several clones of CML-iPSCs are a powerful model to decipher all the mechanisms leading to LSC survival following TKI therapy and are a promising tool for testing new therapeutic agents.