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International Journal of Molecular Sciences 25 (6): 3307 (2024)
BC Cancer Research Centre. Terry Fox Laboratory
Wu, Andrew
Liu, Xiaohu
Fruhstorfer, Clark
Jiang, Xiaoyan
Chronic myeloid leukemia is a multistep, multi-lineage myeloproliferative disease that originates from a translocation event between chromosome 9 and chromosome 22 within the hematopoietic stem cell compartment. The resultant fusion protein BCR::ABL1 is a constitutively active tyrosine kinase that can phosphorylate multiple downstream signaling molecules to promote cellular survival and inhibit apoptosis. Currently, tyrosine kinase inhibitors (TKIs), which impair ABL1 kinase activity by preventing ATP entry, are widely used as a successful therapeutic in CML treatment. However, disease relapses and the emergence of resistant clones have become a critical issue for CML therapeutics. Two main reasons behind the persisting obstacles to treatment are the acquired mutations in the ABL1 kinase domain and the presence of quiescent CML leukemia stem cells (LSCs) in the bone marrow, both of which can confer resistance to TKI therapy. In this article, we systemically review the structural and molecular properties of the critical domains of BCR::ABL1 and how understanding the essential role of BCR::ABL1 kinase activity has provided a solid foundation for the successful development of molecularly targeted therapy in CML. Comparison of responses and resistance to multiple BCR::ABL1 TKIs in clinical studies and current combination treatment strategies are also extensively discussed in this article.
Citation: Wu, A.; Liu, X.; Fruhstorfer,C.; Jiang, X. Clinical Insights intoStructure, Regulation, and Targetingof ABL Kinases in Human Leukemia.Int. J. Mol. Sci. 2024, 25, 3307.https://doi.org/10.3390/ijms25063307Academic Editor: Myunggon KoReceived: 21 February 2024Revised: 12 March 2024Accepted: 13 March 2024Published: 14 March 2024Copyright: © 2024 by the authors.Licensee MDPI, Basel, Switzerland.This article is an open access articledistributed under the terms andconditions of the Creative CommonsAttribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). International Journal of Molecular SciencesReviewClinical Insights into Structure, Regulation, and Targeting ofABL Kinases in Human LeukemiaAndrew Wu 1,2, Xiaohu Liu 1,2, Clark Fruhstorfer 1 and Xiaoyan Jiang 1,2,3,*1 Collings Stevens Chronic Leukemia Research Laboratory, Terry Fox Laboratory, British Columbia CancerResearch Institute, Vancouver, BC V5Z 1L3, Canada; awu@bccrc.ca (A.W.); wliu@virogin.com (X.L.)2 Department of Medicine, University of British Columbia, Vancouver, BC V6T 1Z4, Canada3 Department of Medical Genetics, University of British Columbia, Vancouver, BC V6T 1Z4, Canada* Correspondence: xjiang@bccrc.ca; Tel.: +604-675-8141Abstract: Chronic myeloid leukemia is a multistep, multi-lineage myeloproliferative disease that origi-nates from a translocation event between chromosome 9 and chromosome 22 within the hematopoieticstem cell compartment. The resultant fusion protein BCR::ABL1 is a constitutively active tyrosinekinase that can phosphorylate multiple downstream signaling molecules to promote cellular survivaland inhibit apoptosis. Currently, tyrosine kinase inhibitors (TKIs), which impair ABL1 kinase activityby preventing ATP entry, are widely used as a successful therapeutic in CML treatment. However,disease relapses and the emergence of resistant clones have become a critical issue for CML therapeu-tics. Two main reasons behind the persisting obstacles to treatment are the acquired mutations inthe ABL1 kinase domain and the presence of quiescent CML leukemia stem cells (LSCs) in the bonemarrow, both of which can confer resistance to TKI therapy. In this article, we systemically reviewthe structural and molecular properties of the critical domains of BCR::ABL1 and how understandingthe essential role of BCR::ABL1 kinase activity has provided a solid foundation for the successfuldevelopment of molecularly targeted therapy in CML. Comparison of responses and resistance tomultiple BCR::ABL1 TKIs in clinical studies and current combination treatment strategies are alsoextensively discussed in this article.Keywords: BCR::ABL1; ABL regulation; X-ray crystallographic structures; chronic myeloid leukemia;leukemia stem cell; small-molecule inhibitors; tyrosine kinase inhibitors; TKI resistance;BCR::ABL1 mutations1. IntroductionChronic myeloid leukemia (CML) is characterized by the unregulated proliferationof myeloid granulocytic cells originating from leukemic stem cells (LSCs) and myeloidprogenitors in the bone marrow. As of 2023, the disease accounts for about 15% of newlydiagnosed leukemias with a median age of 60 years [1]. The primary defining featureof CML for diagnosis is the presence of the BCR::ABL1 chimeric fusion oncogene, orPhiladelphia chromosome (Ph), which arises from a t(9;22)(q34;q11) translocation betweenchromosomes 9 and 22 [2]. Based on the European LeukemiaNet (ELN) and MD Anderson(MDACC) criteria, CML progresses through three stages: chronic (CP), accelerated phase(AP), and blast crisis (BC), with each phase defined by increasing blast percentages, accu-mulation of additional cytogenic abnormalities, and other hematologic parameters [3,4].However, since 2022, the World Health Organization (WHO) has removed the CML-APdesignation and instead stratifies CML into CML-CP and CML-BP only, based on thepresence of high-risk features [5]. The precise classification of CML continues to be a matterof debate.The p210 isoform of the BCR::ABL1 oncogene is the main driver of CML pathogenesis in themajority of CML patients [6]. On the other hand, the p190 isoform is predominantly expressedin Ph+ acute lymphoblastic leukemia (ALL) and is associated with a worse prognosis [7].Int. J. Mol. Sci. 2024, 25, 3307. https://doi.org/10.3390/ijms25063307 https://www.mdpi.com/journal/ijmsInt. J. Mol. Sci. 2024, 25, 3307 2 of 23Interestingly, the p230 isoform was found to comprise a distinct clinical entity called neutrophilicCML, with a more benign clinical course than that associated with the p210 and p190 isoforms [8].After translocation, the coiled-coil domain of BCR::ABL1 and the removal of specific regulatorystructures like the N-terminal myristoyl and Cap region allow for constitutive kinase activity.These isoforms have been known to promote various oncogenic signaling pathways whichincrease cell proliferation, survival, and erroneous DNA repair mechanisms. Because CMLpathogenesis is heavily dependent on the p210 isoform of the BCR::ABL1, this protein alsoserves as an attractive target for therapies such as tyrosine kinase inhibitors (TKI). Indeed, TKIshave demonstrated remarkable efficacy in managing the disease and even extending the lifeexpectancy of CML patients to near-normal levels, and the goals for managing CML have shiftedfrom survival to treatment-free remission (TFR) [9]. Despite the success of TKIs, they are rarelycurative, and patients are often required to adhere to lifelong treatment which accumulateseconomic burden. Although TFR and TKI discontinuation is achievable for CML patients, thereis a lack of consistency in TFR treatment policies, and in many cases, treatment discontinuationcan lead to eventual disease relapse due to the persistence of CML cells with BCR::ABL1mutations or LSCs that harbor various BCR::ABL1-independent resistance mechanisms [10–17].In this review, we provide an up-to-date structural characterization of the ABL1 kinasesand further describe their complexes with past, present, and future inhibitors. Specifically,we focus on the ABL1B isoform, referred to as ABL1, and compare its domain structureto that of BCR::ABL1. We also review several elegant clinical studies investigating theclinical response and resistance of CML patients to six FDA-approved TKIs, TKI doseoptimization strategies, and the efficacy of combination treatment strategies as comparedto TKI monotherapies to improve treatment outcomes and overcome drug resistance.2. Structural and Molecular Description of ABL1 RegulationIn contrast to the highly regulated wild-type ABL1 kinase, BCR::ABL1 escapes spatialand temporal control to be constitutively activated (Figure 1) [18]. To better understandABL1 regulation, we discuss the different conformations of the ABL1 kinase domain, themediation of ABL1 autoinhibition via regulatory domains, and the consequences of losingautoinhibition after BCR fusion.Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 3 of 26 Figure 1. Generation of the BCR::ABL1 Fusion Protein. The BCR::ABL1 chimeric gene is formed when the ABL1 gene on chromosome 9 is translocated to the BCR gene on chromosome 22. This results in a constitutively active fusion protein that phosphorylates several downstream oncogenic signaling pathways that contribute to leukemia pathogenesis. Created with BioRender.com. 2.1. ABL1 Kinase Domain The ABL1 kinase domain is a bi-lobal structure with a myristoylated NH2-terminal (N-lobe) and the COOH-terminal (C-lobe, Figures 1 and 2) [18,19]. The N-lobe includes the αC helix and phosphate binding loop (P-loop), whereas the C-lobe houses the peptide substrate-binding site [20]. In between the N-lobe and the C-lobe is the active site which comprises the ATP-binding pocket and activation loop (A-loop, Figures 1 and 2A) [20,21]. Figure 1. Generation of the BCR::ABL1 Fusion Protein. The BCR::ABL1 chimeric gene is formedwhen the ABL1 gene on chromosome 9 is translocated to the BCR gene on chromosome 22. Thisresults in a constitutively active fusion protein that phosphorylates several downstream oncogenicsignaling pathways that contribute to leukemia pathogenesis. Created with BioRender.com.Int. J. Mol. Sci. 2024, 25, 3307 3 of 232.1. ABL1 Kinase DomainThe ABL1 kinase domain is a bi-lobal structure with a myristoylated NH2-terminal(N-lobe) and the COOH-terminal (C-lobe, Figures 1 and 2) [18,19]. The N-lobe includesthe αC helix and phosphate binding loop (P-loop), whereas the C-lobe houses the peptidesubstrate-binding site [20]. In between the N-lobe and the C-lobe is the active site whichcomprises the ATP-binding pocket and activation loop (A-loop, Figures 1 and 2A) [20,21].Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 4 of 26 Figure 2. The different conformations of the ABL1 kinase domain and the ABL1 conformational changes between active and inactive states. (A) The ABL1 active conformation with the P-loop is represented in yellow, the A-loop in red, and the αC helix in green. (B) The ABL1 inactive confor-mation with the DFG side chains of D381 and F382 are presented in red. (C) The Src-like inactive conformation. (D) The ABL1 assembled conformation. The SH2 domain, the connector, and the SH2-kinase linker are colored in pink. The N-terminal myristoylation group and the Cap region are col-ored in cyan. (E) Tetramerization of BCR::ABL1 via the coiled-coil (CC) domain leads to proximity-induced trans-phosphorylation, depicted in yellow. Created with BioRender.com. Like other kinases, ABL1 switches between active and inactive conformations. In the active state, the A-loop is in an open conformation and provides a platform for substrate binding (Figure 2A) [22,23]. The phosphorylation of Y393 in the A-loop stabilizes the ac-tive conformation by hydrogen bonding with R386 and forming salt bridges with R362 and R384 [24–26]. One of the hallmark structural features of the active state is the adoption of the “DFG-in” motif (D381-F382-G383) at the N-terminal base of the A-loop [26]. In this activated state, D381 points towards the ATP-binding site and coordinates a Mg2+ ion for catalysis, while another residue, F382, is buried in a hydrophobic pocket (Figure 2B) [26]. Concurrently, the substrate-binding site at the C-terminus accommodates substrate phos-phorylation and downstream signaling [26]. In contrast, the inactive form of ABL1 has a “DFG-out” motif where the activation loop folds back into the ATP-binding site to block Mg2+ from initiating catalysis (Figure 2B) [26]. This causes the unphosphorylated Y393 residue to replace kinase-targeting sub-strates in the active site and further frees the R386 residue to move toward E282 under the αC-helix [26]. Meanwhile, the DFG motif undergoes a drastic rotation such that the F382 residue occupies the ATP-binding site and contacts other residues (Figure 2B) [26]. This stabilizes the inactive conformation and creates a specificity pocket behind the gatekeeper residue T315 which increases the binding specificity of several TKIs [27,28]. Figure 2. The different conformations of the ABL1 kinase domain and the ABL1 conformationalchanges between active and inactive states. (A) The ABL1 active conformation with the P-loopis represented in yellow, the A-loop in red, and the αC helix in green. (B) The ABL1 inactiveconformation with the DFG side chains of D381 and F382 are presented in red. (C) The Src-likeinactive conformation. (D) The ABL1 assembled conformation. The SH2 domain, the connector,and the SH2-kinase linker are colored in pink. The N-terminal myristoylation group and the Capregion are colored in cyan. (E) Tetra erization of BCR::ABL1 via the coiled-coil (CC) domain leads toproximity-i duced trans-phos horylation, depicted in yell w. Created with BioR nder.com.Like other kinases, ABL1 switches between active and inactive confor ations. In theactive state, the A-loop is in an open conformation and provides a platform for substratebinding (Figure 2A) [22,23]. The phosp orylation of Y393 in the A-loop stabilizes the activeconformation by hydr gen bonding with R386 nd forming salt bridges with R362 andR384 [24–26]. One of the hallmark structural features of the active state is the adoption ofthe “DFG-in” motif (D381-F382-G383) at the N-terminal base of the A-loop [26]. In thisactivated state, D381 points towards the ATP-binding site and coordinates a Mg2+ ion forcatalysis, while another residue, F382, is buried in a hydrophobic pocket (Figure 2B) [26].Concurrently, the substrate-binding site at the C-terminus accommodates substrate phos-phorylation and downstream signaling [26].In contrast, the inactive form of ABL1 has a “DFG-out” motif where the activation loopfolds back into the ATP-binding site to block Mg2+ from initiating catalysis (Figure 2B) [26].This causes the unphosphorylated Y393 residue to replace kinase-targeting substrates in theactive site and further frees the R386 residue to move toward E282 under the αC-helix [26].Meanwhile, the DFG motif undergoes a drastic rotation such that the F382 residue occupiesInt. J. Mol. Sci. 2024, 25, 3307 4 of 23the ATP-binding site and contacts other residues (Figure 2B) [26]. This stabilizes the inactiveconformation and creates a specificity pocket behind the gatekeeper residue T315 whichincreases the binding specificity of several TKIs [27,28].In addition, there is a third intermediate conformation of the ABL1 kinase domain whichresembles c-Src in the inactive state, hence its name ‘Src-like inactive ABL1’ (Figure 2C) [29].Like the c-Src inactive conformation, the A-loop is present in a compacted form that blocks thesubstrate binding site [28–30]. However, unlike the regular inactive ABL1, the Src-like inactiveABL1 exists with the DFG-in motif [29]. Furthermore, to relieve the strong steric hinderance inthe active site, the αC-helix is swung out of the active site, creating an enlarged ATP-bindingpocket that is believed to facilitate the flipping of the DFG motif during the inactive/activeconformation switch [29]. The major structural change from adopting the Src-like inactivestate may accommodate kinase domain mutations that promote ABL1 kinase activity whilefacilitating escape from TKI targeting [29]. The discovery of this intermediate state has thusencouraged the development of alternative strategies to design new ABL1 inhibitors that canovercome this structural change in the kinase domain [29].2.2. ABL SH2 and SH3 DomainsThe SH2 and SH3 domains of ABL1 play a vital role in ABL autoinhibition. In theinhibited form of ABL1, the SH2 and SH3 domains function as a clamp to restrict the confor-mation of the kinase domain to remain in the inactive form (Figure 2D) [19,31]. Specifically,the SH2 domain physically docks onto the distal side of the C-lobe through an extensivenetwork of hydrophobic interactions and hydrogen bonding such that the movement ofthe kinase C-lobe is severely restrained (Figure 2D) [31,32]. In contrast, the SH3 domainforms interactions with the kinase N-lobe, which also maintains the ABL1 autoinhibition(Figure 2D) [31,32]. The SH3 domain also forms many intermolecular interactions with theSH2-kinase adjoining linker region, which functions as the cognate ligand for SH3 domains.In addition, the connector between the SH2 and SH3 domains adopts a rigid structure thatfurther stabilizes these domains into the kinase domain, hence reinforcing the inhibitoryeffect (Figure 2D) [19,31,32]. Thus, for ABL1 to be activated, the rigid SH2-SH3 clamp mustbe removed (Figure 2D). In fact, biochemical and crystallographic studies have shownthat upon activation, the SH2 domain dissociates from the C-lobe and repositions ontothe N-lobe, thereby diminishing autoinhibition and allowing ABL1 to adopt an elongatedconformation [31]. This activated conformation is proposed to result from two possibilities.Firstly, the phosphorylation of the SH2-kinase linker located Tyr-245 residue disrupts thebinding of the SH3 domain [19]. Another possibility is for the SH2 domain to bind to thephosphorylated tyrosine of ABL1 substrates, resulting in the dissociation of the SH2 domainfrom the C-lobe [19]. In both cases, the rigid SH2-SH3 clamp is removed from ABL1 whichallows for SH2- or SH3- processive phosphorylation of substrates.2.3. ABL1 N-Terminal Myristoylation Group and Cap RegionThe N-terminal myristoylation group and Cap region play a crucial role in ABL1 reg-ulation, as they act as another autoinhibitory mechanism by binding to the myristoylpocket (Figure 2D) [33]. Instead of functioning as an anchor for membrane binding, theN-terminally located myristoylation group is bound in a deep hydrophobic pocket at thebase of the C-lobe of the ABL1 kinase domain [19]. Insertion of this myristoylation group re-sults in a 90◦ bend in the middle region of the C-terminal-located α1 helix, which otherwiseexists in an extended form and occludes the SH2 domain from adopting onto the C-lobe forinhibitory machinery assembly [32]. The Cap region, which connects the myristoylationgroup and SH3 domain, positions the myristoylation group for binding to the C-lobe.Moreover, it facilitates SH2-SH3 unit fastening to the rear of the ABL1 kinase domain bymaking extensive contact with the SH2 domain, the SH2-SH3 connector, and possibly theSH3 domain [31]. In the BCR::ABL1 fusion protein, the N-terminal myristoylation groupand Cap region are removed after translocation, resulting in an open myristoyl bindingInt. J. Mol. Sci. 2024, 25, 3307 5 of 23pocket and uncontrolled ABL kinase activity. Thus, allosteric inhibitors that target themyristoyl binding pocket have also been successful in the treatment of CML [34–36].2.4. BCR::ABL1 Coiled-Coil DomainIn addition, the BCR::ABL1 translocation also induces the gain of the coiled-coil do-main at the BCR N-terminal, severely abrogates the ABL1 autoregulation, and changes thekinase dynamics, resulting in subsequent phosphorylation of numerous substrates, includ-ing GRB2/GAB2, CRKL, JAK/STAT, MAPK, and PI3K/AKT pathways (Figure 2E) [36–39].In particular, this coiled-coil domain is responsible for BCR::ABL1 tetramerization, which in-creases the chances of ABL activation by proximity-induced trans-phosphorylation of Y393in the A-loop and constitutive activation [37]. Hence, the oligomeric state of BCR::ABL1 isessential for its high kinase activity and transforming ability. Moreover, it has been shownthat coiled-coil-deleted BCR::ABL1 fails to induce CML-like disease in mouse models,indicating the vital role of this domain in BCR::ABL1 transformation [40].3. Structural Characterization of BCR::ABL1 Kinase InhibitorsUnderstanding molecular and X-ray crystallographic structures of BCR::ABL1 oncoproteinhas led to the development of several potent TKIs for the treatment of CML in the clinical [36].Currently, six BCR::ABL1-targeting TKIs have been approved for CML frontline therapy. Theseinclude the first-generation TKI Imatinib (IM), second-generation TKIs Nilotinib (NL), Dasatinib(DA), and Bosutinib (BOS), the third-generation TKIs Ponatinib (PON), and most recently,the allosteric TKI Asciminib (ASC). These TKIs have been extensively characterized and aredistinguished by their binding mechanism to BCR::ABL1 (Figure 3).Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 6 of 26 that target the myristoyl binding pocket have also been successful in the treatment of CML [34–36]. 2.4. BCR::ABL1 Coiled-Coil Domain In addition, the BCR::ABL1 translocation also induces the gain of the coiled-coil do-main at the BCR N-terminal, severely abrogates the ABL1 autoregulation, and changes the kinase dynamics, resulting in subsequent phosphorylation of numerous substrates, including GRB2/GAB2, CRKL, JAK/STAT, MAPK, and PI3K/AKT pathways (Figure 2E) [36–39]. In particular, this coiled-coil domain is responsible for BCR::ABL1 tetrameriza-tion, which increases the chances of ABL activation by proximity-induced trans-phos-phorylation of Y393 in the A-loop and constitutive activation [37]. Hence, the oligomeric state of BCR::ABL1 is essential for its high kinase activity and transforming ability. More-over, it has been shown that coiled-coil-deleted BCR::ABL1 fails to induce CML-like dis-ease in mouse models, indicating the vital role of this domain in BCR::ABL1 transfor-mation [40]. 3. Structural Characterization of BCR::ABL1 Kinase Inhibitors Understanding molecular and X-ray crystallographic structures of BCR::ABL1 onco-protein has led to the development of several potent TKIs for the treatment of CML in the clinical [36]. Currently, six BCR::ABL1-targeting TKIs have been approved for CML fr nt-line therapy. Th s include the first-generation TKI Imatinib (IM), second-generation TKIs Niloti ib (NL), Dasatinib (DA), and Bosutini (BOS), the third-generation TKIs Ponatinib (PON), and most recently, the allosteric TKI Asciminib (ASC). These TKIs have been ex-tensively charac er zed and ar distin uished by their bi ding mechanism to BCR::ABL1 (Figure 3). (A) (B) (C) Figure 3. BCR::ABL1 conformation changes between active and inactive states targeted by TKIs. IM, NL, and PON target the inactive state (A) with the DFG motif out of the catalytic cleft, whereas DA and BOS target the active (DFG-in) state (B). The T315I mutation (C) prevents conventional TKIs from binding. Created with BioRender.com. 3.1. BCR::ABL1 DFG-Out TKIs 3.1.1. Imatinib Briefly, IM, NL, and PON are TKIs that bind specifically to the ABL1 kinase domain in the DFG-out (inactive) conformation (Figure 3A). In the ABL1-IM complex, IM locks ABL1 in the inactive conformation by inserting into the central region of the kinase do-main between the αC helix and A-loop (Figure 4A) [41]. The interaction is stabilized by six hydrogen bonds and numerous van der Waals interactions between IM with the sur-rounding residues of the active site. Consequently, the association of IM and the ABL1 Figure 3. BCR::ABL1 conformation changes between active and inactive states targeted by TKIs. IM,NL, and PON target the inactive state (A) with the DFG motif out of the catalytic cleft, whereas DAand BOS target the active (DFG-in) state (B). The T315I mutation (C) prevents conventional TKIs frombinding. Created with BioRender.com.3.1. BCR::ABL1 DFG-Out TKIs3.1.1. ImatinibBriefl , , and PON are TKIs that bind specifi ally to the ABL1 kinase domain in theDFG-out (inac ive) conformation (Figure 3A). In the ABL1-IM complex, IM locks ABL1 in theinactive conformation by inserting into the central region of the kinase domain betwe n theαC helix and A-loop (Figure 4A) [41]. The interaction is stabiliz d by six hydrogen bonds andnumerous van der Waals interactions betw en IM with the surrounding residues of the activesite. Consequently, the association of IM a d the ABL1 kinase domain relies on the adoption ofABL1 into the inactive conformation and the integrity of the total interaction network. Hence,kinase domain mutations, which cause loss of drug interaction contacts, destabilization of theinactive conformation (disruption of the kinked P-loop conformation), or an active-conformationInt. J. Mol. Sci. 2024, 25, 3307 6 of 23favoring equilibrium shift, result in decreased or even total loss of binding between IM andthe ABL1 kinase [42]. The well-documented T315I mutation provides an excellent example ofhow a single mutation greatly impairs IM binding (Figure 3C). The substitution of isoleucineleads to the loss of hydrogen bonding between the T315 residue and IM, which increases activesite steric hindrance, thereby restricting access to the ATP-binding site. Moreover, this eventreinforces the “activating” hydrophobic spine formation, which results in inefficient IM bindingand loss of its therapeutic effects [42,43].Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 7 of 26 kinase domain relies on the adoption of ABL1 into the inactive conformation and the in-tegrity of the total interaction network. Hence, kinase domain mutations, which cause loss of drug interaction contacts, destabilization of the inactive conformation (disruption of the kinked P-loop conformation), or an active-conformation favoring equilibrium shift, result in decreased or even total loss of binding between IM and the ABL1 kinase [42]. The well-documented T315I mutation provides an excellent example of how a single mutation gr atly impairs IM binding (Figure 3C). The substitution of isoleuci e leads to the loss ydroge bonding between the T315 residue and IM, which increases active site steric hindrance, thereby restricting access to the ATP-binding site. Moreover, this vent re n-forces the “activating” hydrophobic spine formation, which results in ineffici nt IM bind-ing and loss of its therapeutic effects [42,43]. Figure 4. BCR::ABL1 and TKIs binding mechanisms. IM (A), NL (B), and PON (C) bind BCR::ABL1 in the DFG-out (inactive) state. IM relies on hydrogen bonding and van der Waals interactions, NL exhibits increased potency through extensive van der Waals contacts, and PON employs an ethynyl linker, successfully overcoming the challenging T315I mutation. DA (A) and BOS (D) bind BCR::ABL1 in the DFG-in (active) state. DA forms numerous van der Waals interactions with the hinge region of the ABL1 kinase and hydrogen bonds with the T315 residue making it susceptible to the T315I mutation. BOS binds similarly to DA but forms crucial van der Waals interactions with the pivotal T315 residue rather than hydrogen bonds. ASC (B) inhibits BCR::ABL1 through allosteric inhibition by targeting the myristoyl pocket. Axitinib (E) and rebastinib (F) both specifically target the BCR::ABL1 T315I mutation. Axitinib binds BCR::ABL1 in an active formation whereas rebastinib (F) is a non-competitive conformational switch inhibitor. Structures were obtained from the Protein Databank [44]. Created with BioRender.com. 3.1.2. Nilotinib NL is 20–50-fold more potent than IM, due to more extensive van der Waals interac-tions between NL and the ABL1 kinase domain which increases binding affinity (Figures 3A and 4B) [34]. This creates a better fit in the binding cleft and limits solvent exposure such that the energy required for desolvation is conserved, unlike for the IM binding [45]. While studies have shown that NL can overcome several IM-resistant BCR::ABL1 :: I ( ), ( ), ( ) i ::in the F -out (inactive) state. IM relies on hydrogen bonding and van der Waals interactions,NL exhibits increased potency through extensive van der Waals contacts, and PON employs anethynyl linker, successfully overcoming the challenging T315I mutation. DA (A) and BOS (D) bindBCR::ABL1 in the DFG-in (active) state. DA forms numerous van der Waals interactions with thehinge region of the ABL1 kinase and hydrogen bonds with the T315 residue making it susceptible tothe T315I mutation. BOS binds similarly to DA but forms crucial van der Waals interactions with thepivotal T315 residue rather than hydrogen bonds. ASC (B) inhibits BCR::ABL1 through allostericinhibition by targeting the myristoyl pocket. Axitinib (E) a d rebastinib (F) b th sp cifically targetthe BCR::ABL1 T315I mutati n. Axiti ib binds BCR::ABL1 in an active formatio whereas r bastin b(F) is a non-competitive conformational switch inhibitor. Structures were obtained from the ProteinDatabank [44]. Created with BioRender.com.3.1.2. NilotinibNL is 20–50-fold more potent than IM, due to more extensive van der Waals interactions be-tween NL and the ABL1 kinase domain which increases binding affinity (Figures 3A and 4B) [34].This creates a better fit in the binding cleft and limits solvent exposure such that the energyrequired for desolvation is conserved, unlike for the IM binding [45]. While studies have shownthat NL can overcome several IM-resistant BCR::ABL1 mutations, NL is still susceptible to theT315I mutation as well as other compound mutations [46,47].3.1.3. PonatinibPON is a third-generation TKI that is 500 times more potent than IM and shows efficacyagainst the T315I mutation. Although the PON-ABL complex resembles that of IM and NL,Int. J. Mol. Sci. 2024, 25, 3307 7 of 23there are several key differences (Figures 3A and 4C) [48]. Firstly, the key structural attributeof PON is the ethynyl linker that accommodates the T315/T315I mutation with favorablevan der Walls interactions instead of hydrogen bonding which effectively eliminates sterichinderance issues [49]. Secondly, a functional group of PON binds to the pocket vacatedby F382 in the DFG-out mode to further increase the binding affinity [49]. Despite highselectivity against nearly all kinase mutations, in vitro binding assays showed that PONis still vulnerable to BCR::ABL1 P-loop mutations, such as E255V, which appears to be acommon issue for TKIs that bind to the inactive ABL1 conformation as the integrity of theP-loop is essential for maintaining the inactive ABL1 conformation and binding affinity ofDFG-out TKIs [48,49].3.2. BCR::ABL1 DFG-In TKIs3.2.1. DasatinibDA is an oral multityrosine BCR::ABL1 and SRC family tyrosine inhibitor that is 300-foldmore potent than IM and can overcome most of the IM-resistant mutations except T315I [50,51].Unlike the previous TKIs discussed, DA binds to the ABL DFG-in (active) conformation andhas fewer interactions with the ABL1 kinase domain (Figures 3B and 4A) [24,41]. Firstly, thefunctional group on the right end of DA does not protrude into the specific pocket, butrather occupies a hydrophobic pocket created by T315, V299, and three other surroundingresidues; hence, the DFG motif no longer needs to be fixed to any certain conformation tofacilitate the inhibitor binding (Figure 4A). This allows the A-loop to adopt the extendedform in the activated state [24]. Secondly, instead of lying deep in the binding cleft, DAextends in the opposite direction such that the leftmost group forms two hydrogen bondsand numerous Van Der Waals interactions with the hinge region [24]. In addition, in theDA-ABL complex, the P-loop seems to possess mobility, as it is not required to be in aspecial conformation (Figure 4A) [41]. This suggests that a stringent P-loop conformationis not necessary for the DA binding. Therefore, the flexibility of the DA-binding modemight grant DA the ability to target both the active and inactive conformations of the ABL1kinase, which could account for the high affinity and efficacy of the drug [24]. However,this idea was challenged by an NMR study, which showed DA primarily interacts withABL1 in its active state [52]. Additionally, the high degree of P-loop freedom may indicatethat no critical contacts are made between DA and the P-loop. This may partially explainthe high activity of DA towards the P-loop mutations. Despite the increased sensitivity toa broad spectrum of ABL1 mutants, DA exhibits much less inhibitory effects against themutations around two essential binding sites: the hinge region and hydrophobic pocket.The hydrogen bonding and van der Waals contact between T315 and DA make the T315residue indispensable for DA binding [24]. Thus, even the less common T315A mutantshows much higher resistance to DA than NL or PON. Compared to DA, NL and PONform more contacts with the ABL inactive conformation such that the loss of some contactscan be compensated by others [53]. Two clinically resistant mutations against DA—F317Lin the hinge region and V299L—exemplify this reasoning well.3.2.2. BosutinibLike DA, BOS is also a dual ABL1-Src kinase inhibitor that binds to the ABL1 kinasedomain in its active conformation (Figures 3B and 4D) [54]. However, in the BOS-ABL1 com-plex, the DFG motif adopts a DFG-flip conformation [23,54]. Specifically, the D381 andF382 residues in the DFG-flip conformation occupy the space where F382 and D381 residein the DFG-in conformation, respectively. Other than this feature, BOS and DA bind toABL1 in a very similar pattern, and both share similar drug potency and mutation selec-tivity (Figure 3). One notable structural distinction of BOS is that it forms multiple crucialvan der Waals interactions with the T315 residue in contrast to DA, which usually formshydrogen bonding with the T315 residue [54]. However, the disruption of this interactionresulting from a T315I mutation can still result in the BOS dissociation from ABL1. Anotherclinically relevant mutation is V299L, which is located in the hydrophobic binding pocketInt. J. Mol. Sci. 2024, 25, 3307 8 of 23around T315. The leucine substitute results in steric clashes with BOS, thereby reducing thedrug-binding affinity [46,54].3.3. Allosteric Inhibitors of the ABL1 Kinase DomainAsciminibDespite the efficacy of competitive TKIs described above, kinase mutations (e.g., T315I)that hinder drug binding remain major obstacles to overcome. This has led to the devel-opment of non-competitive allosteric inhibitors of BCR::ABL1, such as ASC, that specif-ically target the ABL myristoyl pocket (STAMP) [34,55,56]. ASC mimics the N-terminalmyristoylation group that is lost as a result of BCR::ABL1 fusion and inserts in the myris-tate binding pocket which redocks the SH2 domain to the kinase domain through theα1 helix bending and induces a conformational change to the BCR::ABL1 inactive state(Figures 3 and 4B) [34,57]. Unlike conventional TKIs like DA and BOS that can target thekinase domain of other tyrosine kinases such as SRC, ASC has been shown to have feweroff-target effects as there are fewer tyrosine kinases with myristoyl binding sites. How-ever, point mutations in BCR::ABL1 affecting the myristoyl pocket such as the A337V,P465S, V468F, and C464W sites have been reported to generate resistance that hindersASC binding [58]. Additionally, ASC resistance has been found to arise from upregulatedABCG2-mediated drug efflux mechanisms [58]. To overcome these ASC resistance mecha-nisms, studies are evaluating the combination of ASC with conventional TKIs like PON,which can exert inhibitory effects on ABCG2, and demonstrated success in eliminatingT315I mutated CML primary patient cells [59,60].3.4. Other BCR::ABL1 Inhibitors3.4.1. AxitinibTwo other TKIs, axitinib and rebastinib, were also considered for CML therapiesas they both could target the T315I mutation. Axitinib is a vascular endothelial growthfactor receptor (VEGFR) inhibitor that was found to selectively inhibit the T315I-mutatedBCR::ABL1 in CML and ALL through structural profiling and ex vivo characterizationof drug sensitivity and resistance in patient cells [61]. Mechanistically, axitinib bindsT315I-mutated BCR::ABL1 in the A-loop DFG-in (active) conformation and prevents kinaseautophosphorylation (Figure 4E) [61]. While axitinib was able to demonstrate significantanti-leukemic activity against T315I-mutated cells in vitro, certain drawbacks prevented itsadoption in the clinic [61,62]. These included the observations that axitinib was unable totarget wild-type BCR::ABL1 and was ineffective against cells with compound mutations [63].Furthermore, doses required for the efficacy of axitinib monotherapy were not tolerablein patients and did not display added benefit even when combined with other TKIs likeASC [63]. A clinical trial that aimed to compare the efficacy of axitinib vs. BOS by theM.D. Anderson Cancer Center was also terminated due to low accrual of patients due tocompeting studies.3.4.2. RebastinibRebastinib is a non-competitive BCR::ABL1 conformation switch control inhibitorthat also demonstrated efficacy in targeting T315I-mutated CML cells [20]. When bound,hydrophobic interactions between rebastinib and BCR::ABL1 force the DFG motif awayfrom the catalytic site, which prevents BCR::ABL1 autophosphorylation (Figure 4F) [20,64].In both in vitro studies and a phase I clinical trial, rebastinib performed well in exertinganti-leukemic effects and inhibiting downstream substrates of BCR::ABL1 [65,66]. However,the clinical benefit observed was deemed to be insufficient for further development ofrebastinib in the CML therapeutic pipeline [66]. Despite both axitinib and rebastinib nolonger being actively developed, the characterization of these inhibitors provides value inthe discovery of future BCR::ABL1 inhibitors.Int. J. Mol. Sci. 2024, 25, 3307 9 of 234. TKI Treatment Strategies in the ClinicSince the advent of IM, the different generations of TKIs have been successful inmanaging chronic phase CML and extending the life expectancy of patients to near-normallevels. Thus, the focus in CML therapy has now shifted to emphasize the achievement oftreatment discontinuation and treatment-free remission (TFR) in patients [67]. However,some patients in the accelerated phase or blast crisis still fail treatment and can progress intosudden blast crisis [68–70]. Thus, there is a continuing need to develop curative treatmentsand to predict patient responses to specific modalities, as well as to anticipate diseaserelapse and/or progression. These concerns have renewed interest in developing newtreatment strategies to overcome TKI tolerance and resistance.4.1. Long-Term Clinical Efficacy of TKIs and TKI ToleranceTo be considered for treatment discontinuation, patients must first experience a sus-tained deep molecular response (DMR, ≤0.01 BCR::ABL1IS), and it has been shown thatthose who discontinue treatment after a longer duration of DMR have a higher TFRrate [67,71–73]. Overall, TFR rates are estimated to range between 25 and 55%, and differenttreatment strategies such as rotating TKIs are often employed to chase a TFR status forpatients who do not experience a DMR [9,67,71–73]. However, the extent of success withthese strategies remains debatable as some patients who switch TKIs do not experience anymeaningful outcomes [1]. Historically, patients have also undergone TKI switching whenexperiencing TKI toxicity when it is now clear that lower dose scheduling is preferred. Forexample, DA at 50 mg QD was found to be just as effective and safer than the original100 mg QD regimen [74–76]. Lower dosing of TKIs is not only less toxic but also moreaffordable for patients.Lack of TKI affordability is a major contributor to treatment non-compliance andtreatment failure. Patients who do not end up achieving TFR are often required to remainon lifelong TKI therapy due to the high incidence of relapse upon treatment discontinuation,which increases the economic burden on patients, and thus, despite the efficacy of next-generation TKIs, they are often inaccessible to patients due to lack of affordability. Forcomparison, the cost of patented TKIs can be over 250,000 USD per year whereas IM, whichis the only TKI currently available as a generic formulation, is drastically more affordableranging from US $300 to $3000 per year [1,9]. As a result, CML patients are limited to TKIsthat provide the most optimal cost-benefit.Currently, treatment strategies start with the selection of a front-line TKI, which is basedon several factors such as efficacy, cost, and toxicity [9]. Furthermore, increasing studies areuncovering endogenous biomarkers that can indicate TKI resistance and can predict treatmentresponse which may also influence front-line TKI selection. In particular, there is a growinginterest in identifying unique biomarkers that can predict TKI response at diagnosis, whichwould allow clinicians to proactively optimize treatment plans for patients. For example, ithas been reported that white blood count (WBC) can predict deep molecular responses toTKI in newly diagnosed CML patients, since patients with low WBC at diagnosis were morelikely to be TKI responders as opposed to patients with high initial WBC who likely went onto be TKI-nonresponders [77,78]. It has also been reported that CML patients who expressedlower levels of BCR::ABL1 transcripts 3 or/and 6 months post-treatment were significantlyassociated with TKI response [78–80]. In addition, differential gene expression panels andmicroRNA (miRNA) expression patterns are also useful predictive biomarkers [81–85]. In tworecent studies, it was found that expression profiles of certain miRNAs combined with in vitrocolony-forming cell assays could predict response to IM and NL [78,86]. While these are earlystudies, screening of endogenous biomarkers may be a valuable tool in facilitating optimal TKIselection in the future.After the application of an appropriate front-line TKI, patients are monitored fortolerance and response. In the event of TKI toxicity to front-line IM therapy, it is recom-mended that the dose is first reduced before resorting to switching to a second-generationTKI [1,9]. For patients who start with second-generation TKIs that experience toxicity, it isInt. J. Mol. Sci. 2024, 25, 3307 10 of 23recommended that the second-generation TKI is rotated between NL, DA, and BOS whileconsidering the patient comorbidities [1,9]. Other options include treatment with IM orswitching to a third-generation TKI at lower doses [1,9]. On the other hand, patients whodevelop resistance to front-line IM are recommended to switch to second-generation TKIsdepending on the type of ABL1 kinase resistance mutation acquired [1,9]. If resistancecontinues with second-generation TKIs, then third-generation TKIs like PON and ASCare considered especially for patients harboring the T315I mutation. Although there arecurrently no clinical studies directly comparing PON with ASC, PON is more favoredbased on the results of two separate clinical studies on PON and ASC with similar studyconditions [87–89]. Currently, several ongoing clinical trials have been applied to usesecond- or third-generation TKIs in combination with BCL-2 inhibitors or other agents toimprove the efficacy of treatment to overcome TKI resistance.4.2. Clinical Trials of Developing TKI Treatment Strategies for CMLThe approval of several major TKIs phase III clinical trials, including the comparisonof efficacy of different TKIs in the treatment of CML patients, have demonstrated promis-ing clinical results, and these clinical trials include the IRIS (IM), DASISON (DA vs. IM),ENESTnd (NL vs. IM), BFORE (BOS vs. IM), EPIC (PON vs. IM), and ASCEMBL (ASC vs. BOS)trials. Additionally, several non-BCR::ABL1 targeting compounds which include histone deacety-lase inhibitors and JAK/STAT, Hedgehog, Aurora kinase, and PPAR-γ signaling pathwayinhibitors have been observed to be effective against CML in phase I/II clinical trials [90].The efficacy of these compounds are attributed to the dependency of BCR::ABL1-independentsignaling mechanisms, particularly for advanced-stage CML and LSCs, and identification ofnovel targetable factors in CML that confer BCR::ABL1-independent resistance continues toremain a priority [47,91]. As the results of these clinical trials have already been summarizedin previous publications, the rest of this review will instead highlight updates to existing andemerging phase II/III clinical trials of TKI treatment strategies [1,87,90,92].4.2.1. ASC4FIRSTCurrently, ASC is approved as a third-line therapy for CML. The ASC4FIRST(NCT4971226) is an ongoing phase III multi-center clinical trial comparing ASC against aninvestigator-selected approved TKI as first-line therapies in newly diagnosedBCR::ABL1+ CML-CP adult patients (Table 1) [93,94]. Approximately 404 randomized pa-tients will either receive ASC 80 mg once daily (QD) or one of IM (400 mg QD), NL (300 mgtwice per day), DA (100 mg QD), or BOS (400 mg QD) (Table 1) [93,94]. The ASC4FIRSTstudy is estimated to be completed in 2028 and will aim to assess major molecular response(MMR, BCR::ABL1/ABL ratio of ≤0.1% BCR::ABL1IS on the international scale), adverseevents, pharmacokinetics, and patient-reported outcomes of ASC vs. investigator-selectedTKIs at week 48 and week 96 [93,94]. Ultimately, the study is expected to provide valuableinsight into further development of first-line treatment strategies in CML.Int. J. Mol. Sci. 2024, 25, 3307 11 of 23Table 1. Summary of emerging TKI clinical trials for the treatment of CML. MHR, major hematologic response includes complete hematologic response (CHR) and noevidence of leukemia. CR, complete remission; CRi, CR with incomplete hematologic recovery; MLFS, morphologic leukemia-free state. MCyR, major cytogenic response;CCyR, complete cytogenic response; MR2 (≤1% BCR::ABL1IS); MR3/MMR (≤0.1% BCR::ABL1IS); MR4 (≤0.01% BCR::ABL1IS); MR4.5 (≤0.0032% BCR::ABL1IS). All responsecriteria are defined according to the European Leukemia-net (ELN) guidelines [3,95].Trial Name(Clinical TrialNumber)Compound(s) Combination Rationale Phase Number of Patients Outcome StatusASC4FIRST(NCT04971226)Asciminib(80 mg QD) MonotherapyComparison of efficacy of ASC as a first-line therapy againstother first-line TKI:III TBDActiveNot recruitingIM 400 mg QD 404NL 300 mg BIDDA 100 mg QD Newly diagnosed CML-CPBOS 400 mg QDASC2ESCALATE(NCT05384587)Asciminib Monotherapy Safety and efficacy of ASC dose escalation II92TBD Recruiting(80 mg, 200 mg QD, 200 mg BID) CML-CP with prior TKI failurePACE(NCT01207440)Ponatinib(45 mg QD) Monotherapy Safety and efficacy of PON to overcome the T315I mutation II449CML-CP (267 patients):Completed60% MCyR54% CCyRCML-CP, AP, BP 40% MMRPh+ ALL 24% MR4.5Resistant to DA, NL or have theT315I mutation CML-AP (83 patients):61% MHR49% MCyR31% CCyR22% MMRCML-BP (62 patients):31% MHR23% MCyR38% CCyR13% MMRPh+ ALL (32):47% MCyR38% CCyROPTIC(NCT02467270)Ponatinib(45 mg, 30 mg, 15 mg QD) Monotherapy Safety and efficacy of 3 different starting doses of PON II283Patients with T315I MR2ActiveNot recruiting64% (45 mg)25% (30 mg)16% (15 mg)Patients without T315I MR2CML-CP with prior TKI failureor have the T315I mutation59% (45 mg)44% (30 mg)46% (15 mg)DAC-VEN-PON(NCT04188405)Ponatinib(45 mg QD)+Decitabine (20 mg/m2 QD)+Venetoclax(400 mg QD)Safety and efficacy of DAC-VEN-PON combinationAssess BCL-2 dependency in response to treatment regimen II14 11 patients responded ActiveNot recruitingCML-AP, BP with prior TKI orchemotherapy exposure40% CR/CRi33% MLFSInt. J. Mol. Sci. 2024, 25, 3307 12 of 23Table 1. Cont.Trial Name(Clinical TrialNumber)Compound(s) Combination Rationale Phase Number of Patients Outcome StatusNilo Post-STIM(NCT01774630)Nilotinib(300 mg BID) MonotherapySafety and efficacy of NL to achieve 2nd TFR after priorIM discontinuation II317 patients discontinuedtherapy after experiencingadverse eventsCompletedCML patients with molecularrelapse after IMdiscontinuation attempt22 patients achieved TFRrates of:• 59.1% (12 months 95%CI: 41.7–83.7%)• 42.1% (24 months 95%CI: 25–71%)DAstop2(NCT03573596)Dasatinib(100 mg, 70 mg QD) MonotherapySafety and efficacy of DA to achieve 2nd TFR after priorfailed TKI discontinuation attempt II9462 patients attempted 2ndTFR attempt with TFR ratesof:RecruitingCML-CP patients who relapsedafter 3 years of TKI therapy andachieved deep molecularresponse (EURO-SKI)• 61% (6 months)• 56% (12 months)• 46% (24 months)HQP1351 vs. BAT(NCT04126681)Olverembatinib(40 mg QD) MonotherapySafety and efficacy of olverembatinib compared to bestavailable therapy (BAT) II14497 patients discontinuedtreatment due to adverseeventsActiveNot recruitingOlverembatinib arm:CML-CP patients resistantand/or intolerant to IM, DA,and NL85% CHR48% MCyR36% CCyR27% MMRBAT arm:35% CR30% MCyR16% CCyR8% MMRHQP1351-CP(NCT03883087)Olverembatinib(40 mg QD) Monotherapy Safety and efficacy of olverembatinib against CML-CP I/II12779% MCyR (95% CI: 70–85%)ActiveNot recruiting69% CCyR (95% CI: 60–77%)CML-CP patients with theT315I mutation56% MMR (95% CI: 47–64%)44% MR4.0 (95% CI: 35–52%)39% MR4.5 (95% CI: 47–64%)HQP1351-AP(NCT03883100)Olverembatinib(40 mg QD) Monotherapy Safety and efficacy of olverembatinib against CML-AP II3847% MCyR (95% CI: 31–62%)ActiveNot recruiting47% CCyR (95% CI: 31–62%)CML-AP patients with theT315I mutation45% MMR (95% CI: 28–60%)39% MR4.0 (95% CI: 22–56%)32% MR4.5 (95% CI: 18–48%)Int. J. Mol. Sci. 2024, 25, 3307 13 of 23Table 1. Cont.Trial Name(Clinical TrialNumber)Compound(s) Combination Rationale Phase Number of Patients Outcome StatusHQP1351-CML/Ph+ ALL(NCT04260022)Olverembatinib(30 mg, 40 mg, 50 mg QD)Monotherapy+BlinatumomabSafety and efficacy of olverembatinib against CML-CP,AP and BP or Ph+ ALL in patients resistant to PON andASCI57 CML-CP19 Ph+ ALL12 CML-CP and 7 Ph+ ALLpatients discontinuedtreatment due to adverseeventsRecruitingCML-CP:57% CCyR43% MMRCML-CP patients with ≥4TKI failures:57% CCyR42% MMRCML-CP with T315I:Previously treated and resistantto PON and/or ASC60% CCyR44% MMRCML-CP with prior PONfailure:53% CCyR38% MMRCML-CP with prior ASCfailure:43% CCyR38% MMRCML-CP with prior PON andASC failure:25% MMRPh+ ALL:23% MMRHQP1351-VEN(NCT05594784)Olverembatinib(40 mg QD)+Venetoclax(100 mg d1, 200 mg d2, 400 mgd3–d28)+Chemotherapy (10 mgmethotrexate, 50 mg cytarabine,10 mg dexamethasone)Safety and efficacy of olverembatinib combined withvenetoclax and chemotherapy against Ph+ ALL II31Ph+ ALL patients with relapsedor refractory disease32% (10) MMR6.5% (2) < MMR61% (19) MR4.5RecruitingInt. J. Mol. Sci. 2024, 25, 3307 14 of 234.2.2. ASC2ESCALATEAnother ongoing clinical trial with ASC is the ASC2ESCALATE (NCT05384587), aphase II multi-center, single-arm, dose-escalation study that aims to evaluate the efficacyand safety of ASC dose-escalation in CML-CP patients as a second-line therapy (Table 1) [96].The patients recruited for this study include a cohort of 92 adult CML-CP patients with-out the T315I mutation who displayed resistance or intolerance to first-line treatment(Table 1) [96]. Additionally, a cohort of 60–90 newly diagnosed CML-CP patients will alsobe included in this study to compare the effects of ASC as a first-line therapy [96]. Patientswill receive ASC 80 mg QD, and the study will measure the proportion of patients that willachieve MMR at 12 months (Table 1) [96]. For those that do not meet response milestones,the study will also assess whether dose escalation to 200 mg QD and 200 mg BID willfacilitate the achievement of MMR [96].4.2.3. PACE and OPTICThe PACE (NCT01207440) clinical trial was a phase II trial evaluating the efficacy of single-dose PON at 45 mg QD in 449 adult CML or Ph+ ALL patients who were resistant or intolerantto DA or NL or harbored the T315I mutation. The results from this trial found that PON helped60% of patients achieve a major cytogenic response (MCyR), 40% achieve MMR, and 24%achieve a 4.5-log molecular response (≤0.0032% BCR::ABL1IS) (Table 1) [97]. Most strikingly,the majority of these patients sustained these responses even after 40 months following dosereduction and demonstrated a 5-year overall survival rate of 73% [97]. However, significantadverse events such as cardiovascular toxicities were associated with PON treatment which ledto its temporary withdrawal from the market in 2012 [97].To adjust against the adverse effects of PON on CML patients, the OPTIC (NCT02467270)clinical trial was performed. OPTIC is an ongoing phase II randomized, dose-optimizationstudy of PON designed to evaluate the efficacy of three different starting doses of PON inCML-CP patients who experienced resistance to more than two previous TKIs or who possessthe T315I mutation [98,99]. In this study, 283 patients received either 45 mg, 30 mg, or 15 mg QDof PON and were assessed for a primary endpoint of MR2 (≤1% BCR::ABL1IS) at 12 months(Table 1) [98,99]. Primary analysis showed that 44% of patients receiving 45 mg, 29% of patientsreceiving 30 mg, and 23% of patients receiving 15 mg achieved MR2 at 12 months (Table 1) [98].A three-year follow-up analysis of the OPTIC trial showed that the percentage of patients whoachieved the primary endpoint rose to 60% in the 45 mg cohort and 40% in both the 30 mgand 15 mg cohorts (Table 1) [100]. At both 12-month and 36-month time points, the 45 mg QDdose strategy with a reduction to 15 mg QD upon reaching MR2 displayed the most optimalbenefit-to-risk ratio [98,100]. Overall, a comparison study found that patients from the OPTICtrial had lower incidences of adverse effects compared to patients from the PACE trial andhighlighted the benefits of dose optimization strategies for TKIs [101].4.2.4. Decitabine, Venetoclax, and Ponatinib for the Treatment of PhiladelphiaChromosome-Positive Acute Myeloid Leukemia or Myeloid Blast Phase or AcceleratedPhase CML (M.D. Anderson Cancer Center)It has previously been reported that in a phase I/II clinical study, DA could be combinedwith the hypomethylating agent, decitabine, to effectively treat advanced phase CML (CML-AP,CML-BP) [102]. Furthermore, it has been shown that the anti-apoptotic BCL-2 family proteinsare upregulated in CML blast crisis cells and LSCs via BCR::ABL1 signaling, and preclinicalstudies combining TKIs with the BCL-2 inhibitor venetoclax revealed synergistic anti-leukemiceffects in BCR::ABL1+ myeloid leukemias [103,104]. Following up on these results, a phase IIclinical trial (NCT04188405) combining PON (45 mg QD) with decitabine (20 mg/m2 QD), andvenetoclax (400 mg QD) is currently underway to determine the efficacy of this combination innewly diagnosed or relapsed/refractory CML-AP or CML-BP (Table 1). While the trial continuesto accumulate patients, recent updates have shown that out of 14 advanced-phase CML patientstreated so far, 11 patients responded well with 3 experiencing continued response without stemcell therapy (Table 1) [105].Int. J. Mol. Sci. 2024, 25, 3307 15 of 234.2.5. TFR and TKI Discontinuation TrialsAs discussed earlier, TFR is the desired outcome for CML, and TKI discontinuationis eligible for patients who experience a sustained DMR. The first TKI discontinuationtrial was the STIM1 trial which assessed outcomes in 100 CML patients with DMR forat least 2 years who discontinued IM therapy [106]. Long-term follow up of the STIM1trial found that 38% of patients maintained molecular remission after a median follow-upof 77 months after TKI discontinuation and that no patients experienced disease progres-sion [106]. Another large TKI discontinuation trial called EURO-SKI followed 755 patients,which agreed with the findings from the STIM1 trial and helped establish the criteria thatpatient candidates for TKI stopping should have experienced at least 3 years of TKI therapyand at least 1 year of sustained DMR [107].However, about 50% of patients who discontinue therapy experience relapse thoughthey are able to regain DMR status with TKI re-initiation [108–110]. Thus, there are clinicaltrials that aim to determine the feasibility of patients to achieve a second TFR. The firststudy to investigate this was the French Nilo Post-STIM (NCT01774630) study whichstudied 31 patients who had relapsed after attempting TFR following IM discontinuation(Table 1) [111]. In this study, patients were treated with NL at 300 mg twice per day over twoyears, and it was found that TFR rates after NL discontinuation were 59% at 24 months and42% at 48 months which were similar to the rates after the first TFR attempt (Table 1) [111].Despite these promising results, the investigators conceded that switching from IM toNL for a second TFR was not recommended due to a relatively small sample size and arelatively high 23% rate of NL-associated adverse events [111].Another TKI discontinuation trial is the DAstop2 (NCT03573596) clinical trial whichis an ongoing study that aims to investigate TFR rates following a 2nd TFR attemptafter DA treatment in patients who previously failed TFR with any TKI [112]. A totalof 94 patients were enrolled in the trial and treated with DA 100 mg or 70 mg QD fortwo years. These patients were cleared for a second TFR attempt if patients were ableto re-achieve a sustained 4-log molecular response (≤0.01% BCR::ABL1IS) (Table 1) [112].Although the trial is ongoing, interim results as of 2024 showed that 66% of patientshad attempted a second TFR attempt with TFR rates of 56% and 46% at 12 months and24 months, respectively (Table 1) [112]. Additionally, 87% of patients re-achieved a 4-logmolecular response within 3 months of TKI re-commencement [112]. Thus, attempting asecond TFR using second-generation TKIs appears to show promising results and mayinform future strategies to achieve TFR in patients.4.2.6. OlverembatinibOlverembatinib is a new third-generation TKI that was developed to fill a need incountries where PON and ASC are not available and was approved in China for adultCML-CP or CML-AP patients who are TKI-resistant and have the T315I mutation [9]. Mech-anistically, olverembatinib can bind to the ATP-binding site of both phosphorylated andunphosphorylated forms of wild-type and mutated BCR::ABL1 including the T315I mutantthrough extensive hydrogen bond networks [113]. Compared to the other TKIs discussedpreviously, olverembatinib exhibits impressive anti-leukemic activity against virtually allABL1 kinase domain mutations and other ABL1 region compound mutations that causeTKI resistance [9].In a multi-center, randomized phase II study (NCT04126681) in China conducted onCML-CP patients intolerant to IM, DA, and NL, the olverembatinib treatment arm signif-icantly increased event-free survival and response rates compared to the best availabletherapy arm (Table 1) [114]. Two other phase II clinical trials on olverembatinib monother-apy for CML-CP (NCT03883087) and CML-AP (NCT03883100) patients in China with theT315I mutation also demonstrated favorable response rates (Table 1) [115]. Although notyet approved by the FDA for treatment in the U.S., the FDA designates olverembatinib asan orphan drug, as the drug has undergone several emerging clinical trials which highlightits strong potential as a CML therapeutic option [116].Int. J. Mol. Sci. 2024, 25, 3307 16 of 23Clinical studies with olverembatinib showed that the maximum tolerated dose was 50mg QD and that the drug consistently achieved ≥70% complete cytogenic response (CCyR,≤1% BCR::ABL1IS) and≥50% MMR with minimal adverse effects in patients with the T315Imutation as well as those with resistance to other TKIs such as PON [117–120]. Results froma recent phase I clinical trial (NCT04260022) on 76 CML-CP and Ph+ ALL patients outsideof China treated with olverembatinib also demonstrated promising results comparableto those acquired from Chinese patients (Table 1). In this trial, olverembatinib was ableto induce overall CCyR and MMR rates of 57% and 43%, respectively in all CML-CPpatients (Table 1) [121]. Strikingly, olverembatinib was also able to induce impressive CCyRand MMR rates in CML-CP patients with the T315I mutation as well as in patients whohad previously failed PON and/or ASC treatment (Table 1). CCyR and MMR rates inPON-resistant patients were 53% and 38%, respectively (Table 1) [121]. For ASC-resistantpatients, CCyR was 43% and MMR was 38% (Table 1) [121]. For patients that experiencedprior failure to both ASC and PON, 25% were able to achieve MMR with olverembatinib(Table 1) [121]. Although this study is continuing to recruit patients, these updated resultssuggest that olverembatinib monotherapy was able to overcome the T315I mutation andcould benefit patients who were resistant to PON and ASC [121].A phase II study (NCT05594784) on 31 Ph+ ALL has also demonstrated that olverembat-inib combined with venetoclax can deliver high response rates in patients with relapsed orrefractory Ph+ ALL where the p190 BCR::ABL1 isoform is the most dominant (Table 1) [122].Aside from targeting BCR::ABL1 in CML and Ph+ ALL, olverembatinib has also shownefficacy against FLT3-ITD AML and IM-resistant gastrointestinal stromal tumors by beingable to inhibit several other kinases such as KIT and FLT3 among others [123]. Giventhat olverembatinib can overcome a broad range of TKI-resistant mutants including T315I,olverembatinib is anticipated to be an effective third-line treatment option. Regimens forusing olverembatinib as a frontline treatment are also currently being investigated.4.2.7. Preclinical Developments of CML Therapeutic StrategiesThere have also been promising developments in therapeutic strategies being exploredon the preclinical front. Several non-BCR::ABL1 targeted therapies are known to be effectivein the treatment of CML, especially when combined with TKIs, and have already beenreviewed [90,124]. More recently, it has been shown that the induction of nitric oxidesynthase (NOS) expression in leukemic cells leads to apoptosis and pyroptosis throughvarious mechanisms including caspase activation, mitochondrial dysfunction, and reactivenitrogen species (RONS)-mediated modulation of the PI3K/Akt, p38MAPK/Erk1/2, andJNK pathways [125]. Exploiting NOS vulnerability in CML may thus be beneficial fortherapeutic purposes and warrants further study, particularly with clinical samples.Given that TKI therapies are rarely curative, there has also been a growing numberof preclinical studies on the application of CRISPR/Cas9 in the context of CML in thelast few years. Conventionally, allogenic hematopoietic stem cell (HSC) transplantationis considered the only curative treatment for CML but is associated with major issuessuch as graft-versus-host-disease and donor compatibility. However, these risks may beaddressed with autologous transplantation of gene-edited HSCs. The CRISPR/Cas9 systemhas been demonstrated to effectively disrupt BCR::ABL1 at the genomic level leadingto leukemia cell death in vitro and in vivo with no off-target effects [126–129]. Despitehigh gene-editing efficiency using the CRISPR/Cas9 system, a major barrier to curativegene therapy in CML is the presence of unedited cells that have the potential to initiaterelapse after transplantation [127]. To overcome this, a proof-of-concept study describedthe efficacy of a CRISPR-gene trapping strategy that would allow for CRISPR-edited cellsto be selected before transplantation [127]. In this study, a donor gene trap cassette carryinga fluorescent reporter gene was inserted into the BCR::ABL1 fusion site in K562 cells [127].The selected cells were indeed shown to have abrogated BCR::ABL1 expression and werealso shown to have reduced proliferation capacity and increased apoptosis in vitro andin vivo xenograft mouse models [127]. Integration of CRISPR/Cas9 with artificial intelli-Int. J. Mol. Sci. 2024, 25, 3307 17 of 23gence design algorithms is also being explored and may lead to more efficient developmentof robust gene therapies against BCR::ABL1-dependent and BCR::ABL1-independent resis-tance mechanisms in the future [130,131]. While further studies are needed to assess theefficacy and safety of CRISPR/Cas9 gene therapy for CML in the clinical setting, the recentFDA approval of gene therapies for sickle cell anemia indicates that this is a promisingavenue for the future of CML treatments.5. ConclusionsThe discovery of IM and the development of next-generation TKIs have revolutionizedCML treatments, turning a previously fatal disease into a manageable chronic condition.The groundbreaking success in CML treatment serves as a paradigm for targeted therapiesand promotes the investigation of small-molecule inhibitors in other types of malignancies.However, despite the outstanding clinical outcomes in CML patients, the disease relapses,and the emergence of resistance seems to be inevitable. Although second-generation TKIsare more potent and can target most of the IM-resistant mutants, several mutations such asthe notorious T351I are still immune to most treatments and confer a poor prognosis forsuch patients. While approved third-generation TKIs like PON and ASC have demonstratedefficacy against the T315I mutant, some patients continue to respond sub-optimally due totheir inability to overcome compound mutations. Additionally, the different p190, p210, andp230 isoforms of BCR::ABL1 may also respond differently to therapies. Therefore, targetingBCR::ABL1 alone may not be sufficient. Further deciphering the molecular and cellularnetwork of CML pathogenesis by advanced global sequencing, proteomics, bioinformaticsanalysis, and artificial intelligence technology may identify key molecular targets that areplaying important roles in drug resistance, disease maintenance, and progression, whichwill lead to the development of new combination therapies. Lastly, exploring new clinicalstrategies that aim to optimize TKI dose and treatment regimens may offer a fresh outlookon achieving TFR with existing TKIs that can minimize adverse events, improve quality oflife, and reduce healthcare costs.Author Contributions: A.W., X.L. and C.F. wrote the manuscript, and X.J. edited and finalized themanuscript. All authors have read and agreed to the published version of the manuscript.Funding: X.J. is generously supported by the Canadian Institutes of Health Research, the CanadianCancer Society, the Leukemia & Lymphoma Society of Canada, and the Collings Stevens ChronicLeukemia Research Fund. A.W. received a MITACS Accelerate Fellowship.Acknowledgments: We also acknowledge many colleagues whose relevant research we were notable to cite due to space limitations.Conflicts of Interest: The authors declare no conflicts of interest.References1. Jabbour, E.; Kantarjian, H. Chronic Myeloid Leukemia: 2022 Update on Diagnosis, Therapy, and Monitoring. Am. J. Hematol.2022, 97, 1236–1256. [CrossRef]2. Pane, F.; Intrieri, M.; Quintarelli, C.; Izzo, B.; Muccioli, G.C.; Salvatore, F. BCR/ABL Genes and Leukemic Phenotype: FromMolecular Mechanisms to Clinical Correlations. Oncogene 2002, 21, 8652–8667. [CrossRef]3. 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ABL regulation
X-ray crystallographic structures
chronic myeloid leukemia
leukemia stem cell
small-molecule inhibitors
tyrosine kinase inhibitors
TKI resistance
BCR::ABL1 mutations
Clinical Insights into Structure, Regulation, and Targeting of ABL Kinases in Human Leukemia
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