Molecular mechanisms of BCR::ABL1 in chronic myeloid leukaemia: From treatment-free remission to curative therapy

Abstract

Chronic myeloid leukaemia (CML) is a clonal malignancy defined by the dysregulated proliferation of granulocytic cells in the bone marrow. The aetiology is attributed to the chimeric oncogene BCR::ABL1 which is formed by the reciprocal translocation between chromosome 9 and 22, i.e., (t9;22)(q34;q11). The oncoprotein product of the gene modulates leukaemogenesis through its intrinsic tyrosine kinase activity. The management of CML has improved significantly due to the discovery of tyrosine kinase inhibitors (TKIs), making treatment-free remission possible in selected patients with durable deep molecular remission (DMR). However, the therapeutic impact of TKIs has been impaired by the occurrence of mutations within the BCR::ABL1 kinase domain which confer resistance to TKIs. Also, leukaemic stem cells (LSCs) persist in a quiescent state within the bone marrow niche. Resistance mechanisms that are not related to BCR::ABL1 include drug efflux, microenvironmental influences, and epigenetic changes. Next‑generation sequencing and digital polymerase chain reaction (dPCR) enhance the detection of mutations and minimal residual disease, although real-time quantitative polymerase chain reaction remains the validated standard for routine monitoring. Kinase domain mutations are treated with second-generation TKIs (e.g., nilotinib), third-generation TKIs (ponatinib) or allosteric inhibitors (asciminib). Emerging therapeutic strategies including combination regimens, immunotherapy, and precision medicine based on mutation profiling are currently being investigated. Most LSC- targeted and immunotherapeutic strategies remain in preclinical or early phase clinical trials. This review explores the molecular biology of BCR::ABL1, evaluates resistance mechanisms, and establishes a conceptual framework which distinguishes functional remission and from biological cure.

Key Words: Chronic myeloid leukaemia; BCR::ABL1; Tyrosine kinase inhibitor; Drug resistance; Treatment-free remission; Leukaemia stem cells; Cure

Core Tip: Chronic myeloid leukaemia is a clonal haematological malignancy with a well-defined aetiology: The Philadelphia chromosome. The backbone of therapy are the tyrosine kinase inhibitors (TKIs) which have transformed the disease from a fatal cancer into a manageable, chronic disease. The TKIs produced remarkable cytogenetic and molecular remission but they do not produce cure in about half of the patients due to the non-eradication of leukaemia stem cells. This review evaluates the molecular mechanisms underlying BCR::ABL1 signalling and leukaemia stem cell survival, providing a framework that distinguishes guideline-supported operational remission from investigational “double-hit” therapies aimed at leukaemic clone eradication.

INTRODUCTION

Chronic myelocytic leukaemia (CML) is a clonal haematological malignancy characterised by the dysregulated proliferation of granulocytic cells in the bone marrow, typically manifesting as peripheral blood leucocytosis[1]. It is driven by an acquired cytogenetic mutation: A reciprocal translocation between the long arms of chromosomes 9 and 22 designated as t(9;22)(q34;q11)[2]. The Abelson murine leukaemia viral homolog 1 (ABL1) gene is translocated from chromosome 9 to fuse with the breakpoint cluster region (BCR) gene on chromosome 22. A mutant chimeric gene BCR::ABL1 is formed, which resides on a shortened chromosome 22, famously known as the Philadelphia chromosome[2-4]. This landmark finding, which was discovered in 1960 by Peter Nowell and David Hungerford, established CML as the first human cancer directly linked to a specific cytogenetic abnormality[4,5].

There are several structural variants of the BCR::ABL1 gene due to differences in the recombination points between BCR and ABL1. These encode oncoproteins of varying molecular weights. The most prevalent variant is the p210^BCR::ABL1, which has two forms, either the e13a2 or e14a2 mRNA transcript and serves as the hallmark of CML[3,6]. In contrast, the p190 is found in acute lymphoblastic leukaemia (ALL), while the p230 variant is mainly associated with chronic neutrophilic leukaemia[6].

The BCR::ABL1 onco-protein is a constitutively active, dysregulated tyrosine kinase. Its sustained intrinsic activity triggers aberrant autophosphorylation of its catalytic domain leading to the activation of critical, downstream signalling targets like signal transducer and activator of transcription 5 (STAT5) and CRKL; and adaptor proteins including SHC and GRB2-associated binding protein 2 (GAB2). This drives uncontrolled myeloid proliferation, impairs normal apoptosis, prolongs cell survival, disrupts normal haematopoiesis and ultimately forces massive bone marrow expansion[7,8].

Tyrosine kinase inhibitors (TKIs) directly target the dysregulated oncoprotein and have dramatically transformed CML from a fatal myeloproliferative neoplasm into a manageable chronic condition with near-normal life expectancy[9]. Yet, most CML patients require lifelong therapy because standard TKIs do not eradicate the underlying leukaemia stem cells (LSCs), which remain quiescent within the protective bone marrow niche[10,11]. Some patients achieve sustained deep molecular remission (DMR) and can safely discontinue TKI therapy, a phase referred to as treatment-free remission (TFR). However, about 40% to 50% of such patients experience molecular relapse depending on trial eligibility, specific TKI used, transcript variant, and duration of follow-up, necessitating the recommencement of therapy[12]. This review adopts a holistic approach, covering the basic molecular structure of BCR::ABL1 through to global implementation of immunotherapies, which is essential for understanding and addressing the barriers in CML management. Although TKI selection[13], clinical history[14], and LSC heterogeneity[15] have been reviewed independently, a broad synthesis connecting molecular structural biology directly to LSC persistence and global therapeutic feasibility remains highly essential. This review addresses three main questions: (1) How do structural kinase domain mutations and kinase-independent pathways limit catalytic TKIs, and how are allosteric agents positioned to overcome them; (2) What distinct biological mechanisms allow quiescent LSCs to survive TKI therapy, and what is the direct clinical evidence for “double-hit” combinations; and (3) How do standard diagnostic tools compare with experimental predictive biomarkers, and what infrastructure barriers hinder the global delivery of advanced CML care?

NARRATIVE REVIEW METHODS AND LITERATURE SELECTION

This review was conducted according to the Scale for the Assessment of Narrative Review Articles guidelines. A literature search was performed across PubMed/MEDLINE, Google Scholar and Scopus covering records published from the inception of the database through August 2026.

Search strategy and selection criteria

Search terms were developed using Medical Subject Headings (MeSH) and included “chronic myeloid leukaemia”, “BCR::ABL1”, “leukaemia stem cells”, “drug resistance”, “asciminib”, and “immunotherapy”. Additional studies were identified by manual screening of the reference lists of retrieved publications.

Literature selection criteria

Inclusion criteria: Peer-reviewed original research articles, prospective randomised clinical trials, clinical practice guidelines such as European LeukemiaNet (ELN), and authoritative molecular studies published in English language.

Exclusion criteria: Publications in other languages, unverified preprints and non-peer-reviewed online commentaries.

Evidence hierarchy: High priority was assigned to randomised phase 3 clinical trials, meta-analyses, and practice guidelines for clinical claims. Preclinical cell-line data, animal xenografts, phase 1/2 exploratory studies and conference abstracts were identified as preliminary or hypothesis-generating evidence.

EPIDEMIOLOGY

The incidence of CML is about 1-2 per 100000 population, accounting for about 15% of all adult leukaemia[16]. It is more common in older individuals but can affect any age group. The median age at diagnosis in Western countries is about 64 years in the United States and 52-56 years across Europe[17-19]. Lower baseline median ages were observed in Nigeria (38 years), Kenya (39.8 years), China (46.5 years) and India (41.1 years)[3,20,21]. It is slightly more common in males with a male to female ratio of about 1.2:1[3,18].

CLINICAL FEATURES

About half of CML patients are asymptomatic at the time of diagnosis. The main features include pallor, fatigue, splenomegaly and/or hepatomegaly. The blood count reveals anaemia and leucocytosis; the platelets could be decreased, increased or normal[3]. Examination of the peripheral blood and bone marrow shows an expansion of the myelocytic lineage showing all spectrums of maturation. There are three phases of CML, namely, chronic phase, accelerated phase and blastic phase[3]. However, the World Health Organisation removed the accelerated phase from its 2022 classification for CML[22].

STRUCTURE OF THE BCR::ABL1 ONCOPROTEIN

Under physiological conditions, the wild-type ABL1 protein is a highly regulated non-receptor tyrosine kinase. It cycles between the nucleus and the cytoplasm to coordinate DNA repair, actin remodelling and apoptosis. It is maintained in a strictly-locked, auto-inhibited conformation by the myristoylation of its N-terminal cap. When the myristoyl group inserts into the myristoyl pocket, a hydrophobic pocket within the C-loop, the SRC homology domains 3 (SH3) and 2 (SH2) clamp down on the catalytic domain (SH1), effectively blocking adenosine triphosphate (ATP) from accessing its substrate binding site (Figure 1)[23,24].

Figure 1 Different conformations of the ABL1 kinase domain. A: The active conformation of the ABL1 kinase domain showing the activation loop (A-loop) which is open, the phosphate-binding loop (P-loop) and the αC helix; B: The inactive conformation of the ABL1 kinase domain when bound to TKI showing the A-loop closed; C: The autoinhibited conformation of the ABL1 kinase domain showing the engagement of the N-terminal myristoyl group within the myristoylation pocket, clamped by the SH2 and SH3 domains. Citation: Wu A, Liu X, Fruhstorfer C, Jiang X. Clinical Insights into Structure, Regulation, and Targeting of ABL Kinases in Human Leukemia. Int J Mol Sci 2024; 25: 3307 [PMID: 38542279 DOI: 10.3390/ijms25063307].

In contrast, in the mutated BCR::ABL1, the N-terminal myristoyl cap of ABL1 is deleted and replaced by structural segments of the BCR protein. The coiled-coil domain of BCR induces direct oligomerisation, homodimerisation and reciprocal autophosphorylation of adjacent BCR::ABL1 molecules. This structural disruption permanently locks the kinase domain in an open, hyperactive state[25].

MOLECULAR MECHANISMS OF BCR::ABL1 LEUKAEMOGENESIS

The uninhibited kinase activity of BCR::ABL1 creates a complex network of intracellular signalling. It bypasses the standard cellular cytokine-dependent checkpoints by directly phosphorylating key adaptor proteins and major transcription factors. This drives leukaemic transformation[26]. These pathways operate in parallel, so blocking a single aspect of the network is often ineffective, as it can be circumvented by alternative pathways.

The Janus kinase/signal transducer and activator of transcription pathway: Typical activation of the signal transducer and activator of transcription (STAT) pathway requires upstream Janus kinase (JAK) but the BCR::ABL1 oncoprotein can directly phosphorylate STAT5. Once phosphorylated, STAT5 homodimerises and moves into the nucleus where it upregulates cyclin D2 and MCL1. This renders the cell resistant to apoptosis and accelerates cell cycle progression[26].

The RAS/MAPK/ERK pathway: Autophosphorylation of BCR::ABL1 tyrosine residues, mainly the Y177 on the BCR component, creates a high affinity binding site for growth factor receptor-bound protein 2 (GRB2). This leads to the phosphorylation of GAB2, allowing the guanine-nucleotide exchange factor to convert GDP to GTP on RAS. Activated RAS recruits RAF to the cell membrane and initiates a downstream kinase cascade through MEK1/2 and ERK1/2. Translocated ERK1/2 activates nuclear transcription factors like c-FOS and C-MYC leading to cytokine-independent proliferation[27].

The PI3/AKT/mammalian target of rapamycin pathway: Phosphorylated tyrosine motifs within BCR recruit phosphoinositide 3-kinase (PI3K) through intermediary adapters like GRB2 and GAB2. This leads to the activation of serine/threonine kinase (AKT) and mammalian target of rapamycin (mTOR) pathway. The PI3K/AKT/mTOR pathway actively suppresses apoptosis by inactivating apoptotic proteins like BAD while simultaneously upregulating anti-apoptotic proteins like BCL-xL[28].

Oxidative stress and genomic instability: Oxygen free radicals are generated in large quantities during cellular processes in CML cells. This chronic oxidative stress causes extensive DNA damage and suppresses endogenous DNA repair mechanisms. The resultant genomic instability predisposes the clone to secondary mutations, favouring disease progression from the chronic phase to the accelerated and blastic phases[29].

Disruption of cell adhesion: Normal haematopoietic stem and progenitor cells (HSPCs) are confined to the bone marrow through integrin- and fibronectin-mediated adhesion to the bone marrow stroma. In CML, this tethering is disrupted. The BCR::ABL1 kinase phosphorylates critical focal adhesion molecules like paxillin, p130Cas, β-catenin and FAK while actively downregulating key integrins like CXCR4 and α4β1/VLA-4. The loss of adhesion molecules leads to the premature release of immature leukaemic progenitor cells into the peripheral circulation[30,31].

HISTORICAL EVOLUTION OF CHRONIC MYELOID LEUKAEMIA THERAPY

The treatment of CML can be divided into three major chronological eras: The pre-TKI era, the TKI era and the post-TKI era.

The Pre-TKI era (Prior to 2001): Early management relied on non-specific cytoreductive agents such as hydroxyurea, interferon-alpha (IFN-α) and busulfan[32,33]. These were mainly palliative with no effect on the cytogenetic mutation or its molecular pathways. Complete cytogenetic remission (CCR) was rare. Majority of patients invariably progressed with a bleak median survival of about 3 years to 6 years. Allogeneic stem cell transplantation (ASCT) was the only curative therapy, mainly reserved for young, eligible patients or those who have already progressed to blast crisis. The major limitations to ASCT were treatment-related mortality and donor availability[34].

The TKI era (2001-2021): This was the era of targeted therapy, heralded by the introduction of imatinib (STI571), a first generation TKI that competitively blocks the ATP-binding site of the ABL kinase domain (Table 1)[35]. Imatinib revolutionised the prognosis of CML, delivering sustained cytogenetic responses far superior to IFN-α or hydroxyurea with a significantly better safety profile and quality of life[36,37]. The landmark International Randomised Study of Interferon vs STI571 trial demonstrated a staggering CCR rate of 87% with imatinib compared to just 35% with interferon[38].

Table 1 Classification of BCR::ABL1 inhibitors.

Classification	Example and Ref.	Mechanism of action	Resistance mutations	Toxicities
First generation TKI	Imatinib1: Corbin et al[35], 2002; Druker et al[36], 2001; O'Brien et al[38], 2003 - IRIS	Competitive ATP inhibition	E255V, Y253F, G250E, T315I	Fluid retention, hepatotoxicity, muscle cramps
Second generation TKI	Nilotinib1: Kantarjian et al[39], 2006	High affinity ATP inhibition	E255K/V, Y253H, T315I	QTc prolongation, arterial occlusion
	Dasatinib1: Osman and Deininger[34], 2021	Dual SRC/ABL inhibition	V299L, T315I	Pleural effusion, pulmonary hypertension
	Bosutinib1: Osman and Deininger[34], 2021	Dual SRC/ABL1 inhibition	V299L, T315I	Diarrhoea, hepatotoxicity
Third generation TKI	Ponatinib1: Schenk et al[40], 2026 PONDEROSA real-world observational cohort study; Huang et al[48], 2010	ATP binding inhibition with ethynyl linkage	T315I+Y253H, T315I+E255V/K, T315I+F359V/C	Cardiovascular toxicity, pancreatitis
	Olverembatinib2: Haque et al[43], 2024; Wen et al[44], 2025	Competitive ATP inhibition. Multi-kinase activity including FLT3, KIT, FGFR1	Possibly parallel pathways e.g., RUNX1, STAT5. BCR::ABL1 independent e.g., Hedgehog, JAK/STAT	Cytopenias, proteinuria
STAMP inhibitor	Asciminib1: Cortes et al[41], 2026 (ASC4FIRST trial); Hughes et al[42], 2019; Wylie et al[68], 2017	Allosteric inhibition, binds to the myristoyl pocket	P465S, A337V	Asymptomatic hyperlipidaemia, cytopenias

¹Approved.

²Investigational.

ATP: Adenosine triphosphate; FGFR1: Fibroblast growth factor receptor 1; FLT3: FMS-like tyrosine kinase 3; JAK: Janus kinase; RUNX1: Runt-related transcription factor 1; STAMP: Specifically targeting the ABL myristoyl pocket; STAT5: Signal transducer and activator of transcription 5; TKI: Tyrosine kinase inhibitor.

Resistance to imatinib led to the development of second-generation (2G) TKIs like nilotinib, dasatinib and bosutinib (Table 1)[39]. Ponatinib, a third-generation TKI is indicated in CML patients harbouring the T315I mutation or were resistant to multiple 2G-TKIs[40]. Notably, the concept of TFR emerged as a therapeutic goal in the TKI era. The Stop Imatinib trial proved that stopping imatinib was feasible for patients with sustained DMR for at least two years. After 12 months, 41% of the study cohort maintained a durable DMR while none of the patients who relapsed progressed to blastic phase[12].

The post-TKI era (2021-present): This is the current era, focusing on new structural targets beyond the classic ATP binding pocket. The main priorities are the eradication of LSCs and avoidance of ponatinib-related cardiovascular toxicity. Asciminib and olverembatinib are the prototype drugs of the post-TKI era. Asciminib, approved in 2021, is a first-in-class Specifically Targeting the ABL Myristoyl Pocket inhibitor. It is an allosteric BCR::ABL1 inhibitor (Table 1)[41,42]. Olverembatinib, developed as a highly selective ATP-competitive TKI offers another powerful option for resistant CML and relapsed or refractory Ph-positive ALL (Table 1)[43,44].

MECHANISMS OF RESISTANCE TO TYROSINE KINASE INHIBITORS

There are two major mechanisms of resistance to TKIs based on whether the resistance is related to mutations within the BCR::ABL1 kinase domain or not (Table 2).

Table 2 Comparison of resistance mechanisms in chronic myeloid leukaemia.

Mechanism	BCR::ABL1 kinase-dependent resistance[47]2,[49]1	BCR::ABL1 kinase independent resistance[47]2
Pathophysiology	Intrinsic changes to BCR::ABL1 gene or protein e.g., point mutations in the tyrosine kinase domain e.g., T315I[68]1. BCR::ABL1 gene amplification or over expression. Compound mutations on the same allele causing resistance to multiple TKIs	Alternative pathways or protective niches e.g., activation of parallel oncogenic pathways e.g., STAT3, PI3/AKT, Wnt/β-catenin[47]. Quiescence of leukaemia stem cell. Altered pharmacokinetics e.g., through the overexpression of efflux transporters (ABCB1) or downregulation of influx transporters (OCT1)
Effect of dose escalating or TKI switch	Very effective	Limited efficacy because BCR::ABL1 is already suppressed
Clinical presentation	Loss of major molecular remission	Primary TKI resistance. Persistence of minimal residual disease. Failure of treatment-free remission
Diagnosis	RT-qPCR for BCR::ABL1. Sanger sequencing	Cytogenetics. Transcriptomic profiling
Management	Mutation-guided TKI switching. Allosteric binding combinations e.g., competitive TKI + asciminib. Allogeneic Haematopoietic stem cell transplantation	Combination targeted therapies e.g., TKI + BCL-2 inhibitor like venetoclax. Leukaemia stem cell eradication strategies. Allogeneic haematopoietic stem cell transplantation

¹Preclinical study.

²Review.

ABCB1: ATP-binding cassette sub-family B member 1; AKT: Serine/threonine kinase; OCT1: Organic cation transporter 1; PI3: Phosphoinositide 3; STAT3: Signal transducer and activator of transcription 3; TKI: Tyrosine kinase inhibitor.

BCR::ABL1 dependent resistance: This is mainly due to point mutations within the ABL1 tyrosine kinase domain (TKD) that disrupt TKI binding or prevent the kinase from adopting its inactive conformation. There are many clinically relevant TKD mutations such as E255K, E255V, H396R, G250E, Y253H, F359V, M351T and F317[45,46]. The most challenging is the T315I gatekeeper mutation which substitutes a threonine residue with a bulky isoleucine side chain. This creates a steric clash that confers resistance to imatinib and all 2G-TKIs[47]. Ponatinib circumvents the steric clash by incorporating a unique ethynyl-CCC triple bond that projects beyond the bulky isoleucine residue[48]. Other resistance mechanisms include genomic amplification of the BCR::ABL1 locus and the occurrence of complex compound mutations on a single BCR::ABL1 allele[49].

BCR::ABL1 independent resistance: Resistance develops through the activation of parallel signal transduction pathways like JAK, MAPK and PI3K that bypass BCR::ABL1 inhibition[47]. Overexpression of SRC kinases like HCK, also drive independent cell survival pathways[50]. Furthermore, cells can limit intracellular drug accumulation by upregulating ATP-binding cassette efflux transporters like ABCB1 (P-glycoprotein) or downregulating influx proteins like the organic cation transporter 1 (OCT1/SLC22A1). Alterations in drug metabolism like cytochrome p450 variations also affect TKI efficacy[47].

TREATMENT-FREE REMISSION

Treatment-free remission (TFR) is defined as the safe discontinuation of TKI treatment without loss of major molecular remission i.e., BCR::ABL1 ≤ 0.1%. It has become a major goal in CML management.

Selection criteria for TFR: The ELN 2020 guidelines recommend that TFR should be restricted to CML patients in chronic phase who[51,52]: (1) Are on first-line TKI or second-line therapy if switched only for intolerance; (2) Have been on TKI therapy for at least five years or minimum of 4 years for 2G-TKI; (3) Have maintained stable DMR, i.e. MR^4.0 [BCR::ABL1 < 0.01% on the international scale (IS)] or MR^4.5 (BCR::ABL1 ≤ 0.0032% IS) for more than two consecutive years; and (4) Have access to standardised rapid real-time quantitative polymerase chain reaction (RT-qPCR) monitoring with results available within four to six weeks. Regular molecular monitoring is very important after TKI discontinuation. This entails monthly RT-qPCR tests for the first six months, bimonthly for months 7-12 and quarterly thereafter[52].

Molecular relapse during TFR: Molecular relapse is defined as a loss of major molecular response (MMR), BCR::ABL1 > 0.1% IS. This occurs in about 40%-50% of patients who attempt TFR, with majority of relapses occurring within the first six months. Recommencement of therapy leads to restoration of MMR in over 95% of such patients, reflecting the safety of TFR when combined with stringent monitoring[12].

INDICATORS OF SUCCESSFUL TREATMENT-FREE REMISSION

Advanced molecular techniques like digital polymerase chain reaction (dPCR), next-generation sequencing and immunological biomarkers like CD8⁺ or natural killer (NK) cells enhance the detection of mutations and minimal residual disease. These are more sensitive than conventional polymerase chain reaction (PCR) or clinical variables like TKI duration and depth of response in predicting TFR outcomes. However, their broad application in routine clinical practice is limited by cost, availability and the need for standardised clinical thresholds. Thus, RT-qPCR remains the standard for routine clinical monitoring[53].

Digital PCR (dPCR): Conventional RT-qPCR is less sensitive in detecting ultra-low BCR::ABL1: levels, as the result could be “undetectable” in the presence of significant disease burden. Droplet digital PCR (ddPCR) provides greater precision and allows absolute BCR::ABL1 quantification without external standard curves. A study by Kockerols et al[54] showed that ddPCR transcript levels below 0.0023% IS prior to attempting TFR is associated with a significantly lower risk of molecular relapse.

Transcript variant: The structure of the BCR::ABL1 transcript variant influences TFR success. The e14a2 variant is associated with deeper, more stable molecular responses and higher rates of successful TFR than the e13a2 variant. The difference may be attributed to higher kinase activity and distinct immunogenicity of the e13a2 variant[55].

Immune profiling: The maintenance of TFR depends on active immuno-surveillance and tumour burden. Multiparametric flow cytometric studies indicate that higher TFR success rates are associated with higher counts of circulating NK cells and effector-memory CD8⁺ T-lymphocytes at the time of TKI discontinuation[56,57]. In contrast, a higher number of regulatory T-cells is a strong predictor of rapid molecular relapse[58].

The role of artificial intelligence in monitoring TFR: Artificial intelligence (AI) provides a predictive framework to optimise TFR monitoring. The use of AI enhances the precision of patient selection for TFR. Machine learning algorithms can predict the probability of successful TFR based on the depth and kinetics of molecular response[59]. Artificial intelligence can also be utilised to rank biomarkers based on single cell RNA sequencing data. This will provide information about NK cell function and transcription factor profiles (e.g., RUNX3) which correlate with durable TFR or early molecular relapse[60]. Residual leukaemic clones can be detected directly in the peripheral blood without fluorophore staining by ghost cytometry which involves the use of AI-driven optofluidic technologies[61].

SURVIVAL MECHANISMS OF LEUKAEMIA STEM CELLS

Leukaemia stem cells express the BCR::ABL1 mutation, but its kinase activity is suppressed in non-dividing stem cells. Rather, the survival of LSCs depend on intrinsic cellular developmental pathways and protective extrinsic signals within the bone marrow to survive. Conventional TKIs mainly target cells that are actively driven by the kinase; thus, they clear the bulk of proliferating progenitor cells but the primitive LSC pool often persists[62]. The pathways of LSCs persistence include.

Microenvironmental quiescence: The LSCs exist in a quiescent state within hypoxic bone marrow niches. This quiescence is mediated by adhesion molecules such as integrins and homing receptors like CXCR4 which anchor LSCs to the bone marrow stroma. The survival of quiescent LSCs depend more on niche-mediated signalling rather than BCR::ABL1 kinase activity. Pro-inflammatory cytokines inside the bone marrow also enhance LSC survival[63,64].

The Wnt/beta-catenin pathway: Beta-catenin is abnormally stabilised within granulocyte-macrophage progenitors. This enhances their self-renewal capacity, allowing them to persist as functional LSCs even when their BCR::ABL1 kinase activity is completely suppressed by TKIs[65].

The hedgehog pathway: The CML LSCs show marked amplification of hedgehog signalling. The activation of smoothened (SMO) initiates a signalling cascade that translocates GLI transcription factors into the nucleus. This pathway drives the expression of survival factors and cyclin-dependent kinase inhibitors that sustain LSC dormancy and TKI resistance[66].

Autophagy: The exposure of LSCs to TKI activates protective autophagy, a process where they digest and recycle damaged organelles. As a result, LSCs maintain metabolic homeostasis and sidestep apoptosis[67].

INNOVATIVE STRATEGIES FOR CURATIVE THERAPY

The achievement of definitive cure for CML requires that functional remission must be distinguished from biological eradication (Table 3).

Table 3 Summary of approved monotherapies and investigational combination therapies in chronic myeloid leukaemia.

Strategy	Target	Intervention	Status	Mechanism/use	Ref.
Monotherapy	BCR::ABL1 kinase domain (competitive binding at ATP site)	First and second generation TKIs e.g., Imatinib, Nilotinib, Dasatinib, Bosutinib	Approved standard	First- and second-line therapy. Inhibits proliferation of leukaemic clones	Druker et al[36], 2001
Monotherapy	BCR::ABL1 kinase domain (ATP site)	Ponatinib	Approved. Standard. Real world study	T315I and compound mutations	Schenk et al[40], 2026
Monotherapy	BCR::ABL1 (allosteric binding at the myristoyl pocket i.e. STAMP)	Asciminib1	Approved Standard	Allosteric inhibitor	Hughes et al[42], 2019
Monotherapy	Multi-kinase domain/T315I mutation	Olverembatinib	Investigational real-world clinical study	multi-TKI resistant CML	Wen et al[44], 2025
Combination	Myristoyl pocket + Competitive ATP binding	Asciminib1 + TKI	Investigational	Prevents escape mutation and enhances DMR	Ernst et al[71], 2024
Combination	Inhibition of BCL-2 + BCR::ABL1	Venetoclax + TKI	Investigational	Triggers mitochondrial apoptosis in LSCs	Ko et al[76], 2014
Combination	Autophagy + BCR::ABL1	Hydroxychloroquine + TKI	Investigational, preclinical	Inhibits survival of LSCs	Bellodi et al[73], 2009
Combination	Inhibition of hedgehog pathway + BCR::ABL1	Sonidegib + TKI	Investigational	SMO inhibition blocks LSC self-renewal	Irvine et al[72], 2016
Combination	Niche disruptor (CXXR4 inhibitor) + BCR::ABL1	Plerixafor + TKI	Investigational, preclinical	Forces LSCs into cell cycle	Vianello et al[77], 2010; Weisberg et al[78], 2012
Combination	Immunotherapy + BCR::ABL1	Pegylated interferon + TKI	Investigational. Observational study[80], phase 3 trials[81,82]	Immunological eradication of LSCs	Puzzolo et al, 2022[80]; Nicolini et al[81], 2026; Burchert et al[82], 2026

¹Asciminib monotherapy is approved by the United States Food and Drug Administration for frontline treatment in chronic phase chronic myeloid leukaemia (CML) and in heavily-pretreated CML patients but asciminib combination strategies are still investigational.

ATP: Adenosine triphosphate; BCL-2: B-cell lymphoma 2; DMR: Deep molecular remission; LSC: Leukaemia stem cell; TKI: Tyrosine kinase inhibitor.

Functional cure: TFR represents a state of functional cure characterised by stable control of leukaemic clones below the detection threshold (BCR::ABL1 ≤ 0.1% IS) without TKI administration[47]. The disease is suppressed despite the presence of dormant LSCs in the bone marrow by host immunosurveillance and microenvironment factors like endosteal hypoxia, secretion of Transforming growth factor-β, and adhesion molecules which anchor LSCs[47].

Biological cure: Total eradication of all LSC reservoirs which has historically been achieved only through ASCT but not with standard TKI therapy[47,49].

Combination therapy with catalytic and allosteric inhibition: This involves the combination of asciminib with a conventional catalytic TKI like imatinib or dasatinib to target two distinct sites on the kinase domain. The traditional TKI binds the ATP-binding pocket while asciminib binds to the myristoyl pocket on the opposite end of the kinase domain which induces a structural conformation that disrupt BCR::ABL1 homodimers[68]. Preclinical and early phase clinical trials suggest that this dual targeting prevents the emergence of compound mutations and enhances the depth of molecular remission[69-71] (Table 4).

Table 4 Some preclinical studies and clinical trials on double-hit combination therapies in chronic myeloid leukaemia.

S/N	Trial/study	Trial type, phase and strategy	Combination drugs	Mechanism	Experimental or clinical model	Findings
1	NCT02081378 Wylie et al[68], 2017 (Nature)	Preclinical and early phase 1: BCR::ABL1 (TKI) + STAMP	Imatinib or nilotinib or ponatinib + asciminib	Dual targeting competitive ATP binding and allosteric inhibitor	Mouse xenograft models of human leukaemia (Ba/F3 cell lines with single or compound mutations and patient-derived cells)	Complete and sustained regression in mouse xenografts
2	Ko et al[76], 2014 (Oncotarget)	Preclinical: TKI + BCL-2	Imatinib + venetoclax	BCL-2 inhibitor induces apoptosis of LSCs	CML cell lines. Primary human CML progenitor cells and healthy normal cord blood progenitor cells	Selective elimination of stem cells. Venetoclax enhances imatinib activity against CML progenitors
3	Bellodi et al[73], 2009 (J Clin Invest)	Preclinical: TKI + autophagy inhibitor	Imatinib + chloroquine or bafilomycinA1	Autophagy inhibitors block LSC self-renewal	CML cell lines, primary human CML stem/progenitor cells xenograft mice carrying Ph+ leukaemic cells	Imatinib induces autophagy. Autophagy inhibitors selectively eradicate LSCs and improve sensitivity of mutant cells to imatinib-induced apoptosis
4	NCT02081378 Cortes et al[92], 2025 (Leukaemia)	Phase 1 trial: TKI + STAMP	TKI + asciminib	Dual targeting competitive ATP binding and allosteric inhibitor	Chronic phase or accelerated phase CML without T315I mutation	High rates of durable MMR, high rates of adverse events
5	NCT02689440 Jabbour et al[75], 2024 (Cancer)	Phase 2 trial: TKI + BCL-2 inhibitor	dasatinib + venetoclax	Dual targeting competitive ATP binding and anti-apoptotic	Newly diagnosed chronic phase chronic myeloid leukaemia (CML-CP) or accelerated phase with clonal evolution	80% major molecular remission rate, 96% 4-year event-free survival, 100% overall survival
6	NCT04188405 Short et al[93], 2024 (Lancet Haematol)	Phase 2: Target leukemic stem cell survival	Ponatinib + decitabine + venetoclax	Triple combination: TKI + hypomethylating agent + BCL-2 inhibitor	Advanced phase CML and Ph+ AML	50% complete remission, poor overall survival (11.1 months), poor survival rates: 41% at 1 year and 34% at 2 years
7	NCT02201459 PETALs trial, Nicolini et al[81], 2026	Phase 3 randomised clinical trial	Nilotinib vs Nilotinib + Peg-IFN-alpha-2a	Comparative	Newly-diagnosed CML-CP patients	Higher treatment discontinuation rate due to haematological and psychiatric adverse events
8	NCT03117816 trial Burchert et al[82], 2026	Phase 3 randomised clinical trial	Ropeginterferon alfa-2b vs Observation after TKI discontinuation	Comparative	CML patients in DMR preparing for TFR	No significant improvement in 3-year relapse-free survival vs observation

AML: Acute myeloblastic leukaemia; ATP: Adenosine triphosphate; BCL-2: B-cell lymphoma 2; CML: Chronic myeloid leukaemia; CML-CP: Chronic myeloid leukaemia-chronic phase; DMR: Deep molecular remission; LSC: Leukaemia stem cell; MMR: Major molecular remission; STAMP: Specifically targeting the ABL myristoyl pocket; TFR: Treatment-free remission; TKI: Tyrosine kinase inhibitor; IFN: Interferon.

Direct therapeutic targeting of leukaemia stem cell signalling: Standard TKIs are also being combined with drugs that inhibit LSC self-renewal or anti-apoptotic pathways.

Hedgehog and autophagy inhibitors: SMO inhibitors e.g., sonidegib, block Hedgehog signalling to force dormant LSCs out of quiescence and increase their susceptibility to TKI-induced apoptosis[72]. Hydroxychloroquine also inhibited autophagy and increased the susceptibility of LSCs to TKIs in a preclinical model[73].

Disruption of LSC self-renewal: Beta-catenin antagonists disrupt the interaction between beta-catenin and its nuclear transcriptional co-activators, thereby inhibiting LSC self-renewal[74].

Anti-apoptotic pathway inhibition: The combination of venetoclax with TKIs targets mitochondrial dependency in resistant progenitor cells and advanced-phase CML[75,76].

Disruption of bone marrow niche adhesion: The CXCR4 antagonist, plerixafor disrupts the interaction between LSCs and bone marrow stroma, and mobilises dormant LSCs into the peripheral circulation where they are exposed to TKIs[77,78].

Immunotherapy: The immune system can be harnessed to eliminate TKI-resistant LSCs regardless of their mutational profile.

The resurgence of interferon-alpha: Pegylated IFN-α is being reintroduced as part of combination protocols for its broad immunotherapeutic benefits. It acts through multiple mechanisms: Enhancing host immune surveillance, driving dormant LSCs back into the cell cycle and selectively suppressing of leukaemic clones. Combination of low-dose pegylated IFN-α with 2G-TKIs have shown higher rates of DMR and superior TFR outcomes[79,80].

Clinical trials of IFN therapy produced variable results. The Pegylated Interferon Alpha with Nilotinib in Chronic Myeloid Leukaemia trial (NCT02201459) showed deeper molecular responses for pegylated IFN-alpha-2a and nilotinib combination compared to nilotinib alone at 12 months (24% vs 15%) but increased toxicity, including haematological, psychiatric, and vascular events limited tolerability[81]. In contrast, the (CML-IX) trial (NCT03117816; EUDRA-CT 2016-001030-94) found no significant improvement in relapse-free survival with ropeginterferon compared with observation after TKI discontinuation[82].

Immune checkpoint blockade: Persistent LSCs usually protect themselves by upregulating programmed death ligand-1 which drives programmed death 1 (PD-1) expression on cytotoxic T cells and exhausts the immune response. A phase 2 clinical trial showed that combining anti-PD-1 (pembrolizumab) or anti CTLA-4 monoclonal antibodies with TKIs can re-energise the immune microenvironment to recognise and eradicate residual LSCs[83,84].

BCR::ABL1 vaccines and adoptive T-cell therapies: Next-generation immunotherapy includes vaccines targeted against specific BCR::ABL1 junctional peptides. A 10-year follow up study of 109 imatinib-treated CML patients with residual disease who received GM-CSF-adjuvanted b3a2 and b2a2 peptide vaccines showed excellent long-term safety with complete absence of off-target toxicity. Overall survival was 89%, while 80% of the participants had specific CD4⁺ T-cell responses and 16.5% achieved durable TFR. The study presented the results of two concurrent multicentre phase II trials conducted by the “Gruppo Italiano Malattie Emmatologiche dell’Adulto” (GIMEMA) study group i.e. GIMEMA CML0206 and SI0207 involving the use of CMLVAX100 and CMLVAXb2a2-25 respectively[85]. Similar encouraging results and the eradication of TKD mutations were observed in blastic phase CML following treatment with chimeric-antigen receptor T-cells engineered against LSC-specific antigens like CD38[86,87].

CRITICAL EVALUATION OF LEUKAEMIC STEM CELL TARGETED THERAPY

Experimental regimens demonstrated LSC clearance in cell culture and xenograft models but the following challenges limit their translation into clinical practice.

Cardiovascular toxicities: The risk of adverse events are higher with potent TKIs and combination strategies. Nilotinib is associated with cardiovascular and metabolic risks[39]. Ponatinib increases the risk of arterial occlusive events and hypertension[40]. Asciminib causes asymptomatic elevations and cytopenias[41,42]. Interferon combinations are associated with increased rates of haematological and psychiatric toxicities[81].

Preclinical to clinical translation: Complete clearance in xenografts does not guarantee biological eradication in humans. High molecular response rates in single-arm phase 1/2 trials require validation in phase 3 studies before updating standard practice guidelines.

Diagnostic heterogeneity: Experimental biomarkers—such as dPCR cutoff thresholds, Treg frequencies, single-cell RNA sequencing profiles, and label-free ghost cytometry—require standardization across international reference laboratories[53,59,61].

BARRIERS TO THE GLOBAL ADOPTION OF ADVANCED THERAPIES IN CHRONIC MYELOID LEUKAEMIA

Geographical disparities in health infrastructure impairs equitable CML management. This constitutes a significant challenge to the global adoption of immunotherapy and other advanced therapies[88].

Patient selection: A critical hurdle is the lack of clinical guidelines to optimise patient selection for CML immunotherapy. Immunotherapies depend on recognition of cellular antigens and a dynamic host immune system in contrast to small molecule TKIs which target a well-defined, standard oncoprotein. Thus, it is challenging to identify suitable immunotherapy targets on LSCs without off-target toxicity to normal HSPCs[88,89]. Similarly, biomarkers for vaccine or checkpoint blockade efficacy are also not fully characterised such that responses vary depending on human leukocyte antigen polymorphisms, bone marrow suppression, and NK cell functionality[88,90].

Manufacturing: Cellular immunotherapies require autologous and personalised manufacturing process comprising ex vivo gene transfer, expansion and stringent quality control prior to re-infusion, unlike TKIs which are produced through uniform chemical synthesis, with stable shelf-life and cost-effective oral formulations. Prolonged exposure to TKIs leads to T-cell exhaustion and senescence which reduce the yield and functional quality of harvested T lymphocytes[89]. The huge financial requirements for establishing facilities and cold-chain transport may make immunotherapy less appealing than TKIs.

Adverse reactions: TKIs offer outpatient convenience and manageable side effects. However, immunotherapy is associated with increased risk of cytokine release syndrome, immune effector cell-associated neurotoxicity syndrome or severe autoimmunity. The management of these adverse reactions requires the availability of specialised intensive care facilities, highly-trained personnel and access to biological therapy which limit the widespread adoption of immunotherapy in low-resource settings[91].

CONCLUSION

The evolution of CML from a fatal cytogenetic disorder to a prototype for targeted therapy illustrates the success of translational research. Yet, the persistence of LSCs constitutes the final barrier to definitive cure. Biology-driven “double-hit” combinations of high-potency TKIs with allosteric inhibitors or selective BCL-2 inhibitors, LSC pathway inhibitors and immunotherapies show promise in preclinical and early phase clinical trials. However, their potential to deliver universal biological cure remains investigational. Clinicians must maintain a distinction between guideline-supported operational TFR and experimental LSC-eradication strategies as research continues.