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Imatinib resistance in chronic myeloid leukaemia caused by Bcr-Abl kinase domain and non-Bcr-Abl mutations: a comparison and review.

Chronic myeloid leukaemia (CML) is a myeloproliferative disorder caused by BCR-ABL1 igureusion encoding for a tyrosine kinase oncoprotein. Since the introduction of the tyrosine kinase inhibitor (TKI), imatinib, in 2000, CML survival rates have increased, to the point where life expectancy is equal to that of the general population. One obstacle patients face is imatinib resistance. Literature about resistance has mainly focussed on mutations in the Bcr-Abl kinase domain (KD), which have been well described. Areas that have not been as well established include the origin of KD mutations and resistance from mechanisms outside of KD mutations. This review focuses on how KD mutations arise and their mechanisms of resistance and the roles of BCR-ABL1 gene amplification, Erk1, and Lyn kinase in creating resistance outside of the KD. Experimental therapies to combat imatinib resistance are also mentioned. Using database searches to obtain the current literature, this review attempts to determine the current consensus on these topics and highlight areas where research could be beneficial. While the origin of KD-mutations and non-KD resistance is not entirely clear, the many possible causes that have been elucidated thus far have already paved the way for new therapies.

 

Introduction

Chronic myeloid leukaemia (CML) was the first cancer where the pathological chromosomal abnormality was identified, and is one of the most understood and well-managed cancers [1,2]. CML is a clonal disorder of pluripotent stem cells that results in over-proliferation of mature myeloid cells [3]. Constitutive and aberrant tyrosine kinase activity is responsible for pathological cell proliferation in CML [4].
Before the advent of tyrosine kinase inhibitors (TKIs), 5-year survival rate for patients aged 20-44 was 40%, and less than 20% for patients over 65 years. For patients aged 15-44 diagnosed in 2000, this jumped to 71.6%, increasing to 86.4%, if diagnosed in 2005 [5]. Responsible for these leaps in survival was imatinib mesylate, a TKI approved in 2001 [6]. Imatinib antagonises tyrosine kinase activity by competing with ATP binding to the Bcr-Abl protein, reducing unchecked cell-cycle progression [3]. Imatinib resistance undermines therapy, putting patients at risk, and occurs in approximately 25% of patients [1]. Hence, it is important for doctors and medical students alike to understand that resistance occurs, some of the mechanisms behind resistance and how new pharmacotherapies can combat these. This review summarises the pathophysiology of CML and synthesises the literature around competing theories of imatinib resistance.

 

Pathophysiology of CML

CML is a myeloproliferative disease caused by a reciprocal translocation between chromosome 9 and 22 (9;22)(q34;q11.2)[1,7]. This creates an abnormal chromosome 22 called the Philadelphia (Ph) chromosome, named after the city it was discovered in in 1960 [2,8]. The oncogenic effects of this translocation are caused by 5’ exons of the BCR (breakpoint cluster region) gene fusing to the 3’ exons of ABL1 (Abelson tyrosine protein kinase 1) [3]. This creates the BCR-ABL1 oncogene on the Ph chromosome encoding for Bcr-Abl tyrosine kinase, or p210BCR/ABL [9-11].  Retroviral insertion of p210BCR-ABL in murine models induces a myeloproliferative disorder similar to CML [11]. However, in less than 10% of cases, gene fusion occurs in different exons creating kinases p190 or p230 depending on where the fusion occurs [12,13]. Bcr-Abl has constitutive tyrosine kinase activity, causing modulated gene transcription, proliferation, and enforced survival of myeloid progenitor cells [14]. Unregulated cells grow and enter the S-Phase of the cell cycle independently of physiological growth factors and avoid apoptosis [8,14,15]. Abl and Bcr-Abl are non-receptor tyrosine kinases that travel between the nucleus and the cytoplasm and phosphorylate proteins via SH2 and SH3 domains [16].

Figure 1. Intracellular pathways influenced by Bcr-Abl Kinase [24,25,26].
A synthesis and simplification of selected pathways (JAK/STAT, Gab2, Lyn kinase, IGF-1 and β-1 integrin) showing the leukaemogenic downstream effects of Bcr-Abl signalling. Imatinib is shown solely inhibiting Bcr-Abl, however, research shows imatinib therapy also affects Lyn kinase expression and activity.
Targets of increased phosphorylation that have proliferative effects include insulin-like growth factor receptor 1 (IGF-1R) [17], Ras [16], p27Kip [18], and others. This review will focus on a selection of the most well-known pathways (Figure 1). Bcr-Abl regulates IGF-1R expression via Stat5, which enhances IGF-1R gene expression [19,20]. IGF-1R tyrosine kinase stimulates haematopoietic stem cells and CML patients show higher than normal levels of IGF-1R mRNA [17]. When CML cells were treated with an inhibitor of tyrosine kinase phosphorylation of IGF-1R, the cells underwent apoptosis, cell-cycle arrest, and decreased cell proliferation, illustrating how IGF-1R and CML are linked [17]. One protein both IGF-1R and Bcr-Abl stimulate is Ras [21]. Ras is a well-studied oncoprotein that regulates several downstream pathways that increase cell proliferation including PI3/AKT and JAK/STAT [16]. The importance of Ras in CML pathophysiology was confirmed when association of Bcr-Abl and Ras was blocked and subsequent attempts to induce a CML-like disorder failed [22]. P27Kip is a cyclin dependent kinase (cdk) inhibitor and decreases cdk2 activity, thus inhibiting G1/S-phase progression. In CML, while the amount of P27Kip is unchanged, 80% of P27Kip is relocated to the cytoplasm, where it cannot interact with nucleic cdk2, allowing unregulated cell-cycle progression [18].  This is caused by Bcr-Abl interfering with cytoskeletal proteins such as 1-integrins [23]. These three pathways highlight that Bcr-Abl is central to CML pathology[11]. As a consequence, pharmacotherapy targeting Bcr-Abl has been developed in the form of imatinib.

 

Imatinib

Imatinib is a TKI hailed as a conceptual breakthrough in targeted chemotherapy, and is the first line treatment in CML, while also being used in Ph+ acute lymphocytic leukaemia and some gastric cancers [27]. In early evaluation studies, it was found to specifically inhibit cellular proliferation and tumour formation of Bcr-Abl expressing cells by 92%-98% [28]. Imatinib is effective when compared to other early treatments for CML, increasing the likelihood of survival at ten years by 20% when compared to the closest alternative therapy (Figure 2) [29]. The current parameters to measure successful treatment outcomes are listed with definitions in Table 1. In the International Randomized Study of Interferon and STI571, it was found that complete haematological response (CHR), complete cytogenetic response (CCyR), and major molecular response (MMR) scores were superior in imatinib-treated patients compared with interferon-treated patients [30]. Additionally, in the original trials for imatinib approval, three phase II studies involving 1027 patients showed over 90% of patient leukocyte counts returned to normal, and when combined with interferon, 100% of evaluable patients achieved CHR [31].

Figure 2. Survival with CML over time [29].
This figure from the German CML-Study group shows patient survival probability as a function of time after diagnosis in five consecutive randomised treatment optimisation studies.

 

 

Table 1. Measurements of CML therapy outcome [1,32]

Mechanism of action

Bcr-Abl has an activation loop, a P-loop with an ATP binding site (in the kinase domain) and substrate anchoring SH2 and SH3 domains [33,34,35]. Imatinib binds specifically to the ATP-binding sites of Bcr-Abl, c-kit, and platelet-derived growth factor receptors, and inhibits their tyrosine kinase activity by both preventing ATP binding and stabilising the activation loop in an inactive conformation [34,36,37]. When Bcr-Abl tyrosine kinase activity is inhibited, there is no downstream signalling and treatment is successful in 77% of all patients [1] and virtually 100% of patients treated in the early stages of the disease [34]. The European LeukemiaNet (ELN) 2013 guidelines use MMR as the marker to indicate success of TKI therapy [32], however, Thompson et al. [1]. have argued that MMR overestimates the number of patients with treatment failure.

 

Treatment considerations and mechanisms of Imatinib resistance in CML

Failure of TKI therapy is caused by a number of factors, including inappropriate drug choice, patient non-adherence, and drug resistance. There are a number of opinions as to what constitutes treatment failure, but the definition used by this paper is the one put forward by the ELN and European Society for Medical Oncology. Treatment failure depends on which measurement is used; using haematological parameters (testing peripheral leukocyte counts), treatment failure is defined as no haematological response by three months, or any loss of CHR. Using cytogenetic response, however, treatment failure is no cytogenetic response within six months, no CCyr by 18 months, or any loss of CCyr, as detected by CBA [38]. Finally, a sub-optimal molecular response is defined as no MMR by 18 months or loss of MMR at any point using PCR for BCR-ABL1 transcripts [38]. While this review focuses on imatinib (the gold standard in CML therapy) [30], second generation TKIs such as nilotinib, ponatinib, and dasatinib are now also used, both as first line agents and for use in imatinib resistant patients [1,27].  Which TKI to use and at what dose depends on a number of factors, including imatinib sensitivity and which disease phase the patient is in. CML is staged into chronic phase (CP), accelerated phase (AP), and blast phase (BP) (Table 2) [39]. For instance, a patient in AP who has never had a TKI is still treated with imatinib, but if they have taken imatinib and then progressed to AP, a second generation TKI is used [1]. Using imatinib inappropriately could cause treatment failure, while immediately using a second generation TKI or increasing TKI dosages has been found to limit adherence, as well as side effects (Table 2), especially for patients who require more potent TKIs and higher doses [40].

Table 2. Stages of CML with respective treatment options and side effects[1,27,41,42].
The most serious complication in CML treatment is resistance to therapy. This occurs most frequently in the AP and BP stages of the disease22.  Traditionally, resistance is thought to occur from point mutations in the Bcr-Abl kinase domain (KD) [1,15,34,43]. There are several mutations that can cause mutations in this setting (Table 3). Bcr-Abl KD mutations can be intrinsic (primary) or acquired (secondary) and interfere with TKI therapy in a number of ways [1]. The most common mechanisms are by directly interfering with TKI binding via amino acid substitutes in the ATP binding site or by preventing the activation loop from adopting the inactive conformational state [34]. Although Abl KD mutations causing TKI resistance were first induced in laboratory cell lines in 2000 [44], and detected in vivo clinically in 2001 [45], novel mutations are continually being discovered, with two new mutations conferring resistance sequenced in 2014 [46]. The discovery and sequencing of mutations has led to individualised therapy and a more accurate prognosis for specific mutations, for instance the T315I mutation. In this common mutation, threonine-315 is replaced with isoleucine, effectively removing a hydrogen-bonding site required for TKI binding resulting in enormous treatment difficulty [34]. Only one TKI has any activity in T315I+ patients and that is the highly potent ponatinib. However, this drug is not used as a front line therapy as it has a higher rate of arterial thrombosis and pancreatitis when compared to imatanimb [1]. A novel drug that binds to a nearby site not affected by the mutation, HS-438, is being investigated for use in T315I+ CML and has shown success in pre-clinical trials [47]. Another drug, ABL001, also acts at another molecular site and thus avoids the resistance caused by KD mutations. ABL001 mimics the autoregulating region of ABL1 that is lost upon fusion of BCR, restoring negative regulation, and has been shown to remain effective against clinically significant mutations in an in vivo model [48]. This example highlights how sequencing mutations can provide valuable, individualised prognostic information and guide future research.

Table 3. Sample of p210BCR/ABL kinase domain mutations known to cause resistance [1,34,49-51].  *Incidence based off Ursan et al.’s meta-analysis of a total of 1,698 patients. Overall mutation rate for imatinib was 9.7% [50].
How these mutations arise is not clear; in some patients with secondary resistance pre-therapeutic samples revealed the same KD mutation detected at relapse, consistent with selection of pre-existing resistant clones during therapy giving an evolutionary advantage [52,53]. There is some contention as to whether these stem cell mutations can cause primary resistance or whether KD point mutations can only cause secondary resistance and relapse. Previous studies have concluded that Bcr-Abl KD mutations are a rare cause for primary resistance, however, more recent research found KD mutations in 56% of patients with primary resistance [54,55]. This discrepancy may be explained by the more recent study utilising more sensitive technology and patients who had CML for a longer period of time, which has been linked to mutagenicity [28]. KD resistance in Bcr-Abl1 CML differs from other diseases in that unlike traditional drug resistance, where treatment resistance arises via positive selection of tumour cells with mechanisms to avoid DNA damage, in CML resistant cells there is a tendency to accumulate more rather than less DNA damage [56]. The mechanism behind this is unknown, but it is an example of how the malignancy directly causes resistance. Nevertheless, the evidence suggests that pre-existing KD mutations in cancer stem cells are more likely to be responsible for secondary resistance.

Other research suggests that BCR-ABL1 has the ability to cause self-mutagenesis.  Mutation rate and advanced disease phase were correlated, consistent with mutations being related to exposure time to Bcr-Abl activity [34]. One suggested mechanism is production of reactive oxygen species causing genomic instability, shown in vitro and in murine models, but beyond the original studies, no further research has been undertaken [34,57]. While KD mutations are a highly researched area in CML therapy (over 60 unique point mutations have been identified), there remains an information deficit, for example, the prevalence of mutations in specific populations, or randomised controlled trials for TKI choice following imatinib failure [1,58].

 

Non Bcr-Abl kinase domain mediated resistance

Recent research adds complexity by suggesting there are a number of mutations and events occurring outside of Bcr-Abl KD that impact drug resistance [43].This includes mutations of Bcr-Abl1 outside of the kinase domain, such as BCR-ABL1 amplification, and causes outside of the Bcr-Abl1 protein altogether, such as mesenchymal cells, drug transporters and bypass molecular pathways. Increased BCR-ABL1 expression via gene amplification is found in most TKI resistant cells, whether the mutations are primary, secondary, KD, or non-KD, implying a link between increased expression and resistance [59]. However, increased expression is not the sole cause of non-KD mediated resistance as studies have shown that increasing imatinib concentration in non-mutated, sensitive cells with induced BCR-ABL1 amplification still reduces Bcr-Abl activity, whereas some resistant cells without KD mutations remain resistant at any dose [60].

Extracellular Signal-Regulated Kinase 2 (Erk2) is a Mitogen Activated Protein Kinase (MAPK) and has been implicated in both primary and secondary resistance (i.e. immediate resistance to therapy and resistance that builds over time)[60,61]. In a study of non-KD mutated resistant cells treated with imatinib, Erk2 was found in the nucleus of resistant cells only, and inhibiting Erk2 caused damage to resistant cells [60]. Mechanisms for how Erk2 could cause primary resistance were then elucidated. To achieve this, mutated Ras (which activates Erk2), was virally transduced into sensitive cells that were cultured and treated with imatinib. Using proliferation assays to determine cell survival, it was discovered activating Erk2 gave previously sensitive cells resistance without any prior exposure to imatinib. Erk2 is a key regulator of the pro-apoptotic molecule Bim and it is proposed interactions between Erk2 and Bcr-Abl over-stimulate Erk2 and reduce CML cell apoptosis [62]. Research in 2016 by Wong et al. [63] extended these results to create a pharmacotherapy inhibiting Erk2, showing areas outside of the KD can cause primary and secondary resistance and can be targeted.

 

Lyn kinase

Lyn kinase is a non-receptor tyrosine kinase regulated by Bcr-Abl. Imatinib resistant but Bcr-Abl KD-mutation negative cells were found to overexpress Lyn kinase following treatment with imatinib [64]. In cell lines from these patients, while imatinib effectively inhibited Bcr-Abl activity, Lyn kinase phosphorylation continued, allowing proliferation to continue. Interestingly, prior to imatinib therapy, there was no consistent difference in Lyn expression between sensitive and resistant cells, but afterward there were consistent distinctions in their control of phosphorylation. This implies imatinib treatment uncouples Lyn expression from Bcr-Abl, leading to resistance. Lyn overexpression can induce a three to fourfold resistance, equal to some KD mutations, yet the mechanisms of its overexpression and how it worsens CML are not yet known [65]. One theory is that because silencing of Lyn kinase induces apoptosis in CML cells, overexpression causes cell survival, signalled through via Gab2 [66,67]. The fact that this effect is not seen in imatinib-naïve CML patient cells supports the idea that Lyn kinase only causes acquired resistance, leaving the mechanisms behind primary resistance a mystery.

 

Drug transporters

Alterations in drug transporters are yet another mechanism by which medication resistance can occur and will be mentioned briefly. A drug must both reach the target organ in sufficient amounts and be present at an effective therapeutic concentration for it to exert and effect, and both influx and efflux transporters can interfere with these pharmacokinetics [27]. Radiolabelled imatinib assays have determined that the level of kinase inhibition is dependent on the level of uptake and retention of imatinib achieved [68]. Imatinib enters the circulation from the gastrointestinal tract by a member of the organic cation transporter (OCT) family, OCT-1, thus mutations in OCT-1 are thought to contribute to treatment failure [68]. Conversely, imatinib leaves the cell via the p-glycoprotein multidrug resistance protein-1 (MDR1 or ABCB1) [69]. In other drugs, MDR1 overexpression has been confirmed to cause drug resistance by increasing efflux before a therapeutic concentration can be reached, and this is a relationship currently under investigation in CML.

Non-Bcr-Abl KD resistance is not a well-studied area and much research is yet to be undertaken. Two recent CML mutation reviews by Jabbour et al. and O’Hare et al. only provide a brief mention of non Bcr-Abl mutations causing resistance, even though these mutations cause from 10%-40% of TKI resistance [3,70]. In addition, there was much disagreement among researchers concerning molecular pathways to resistance. Erk2 is part of a super-family of MAPKs, other members such as Erk1, Erk5, and P38MAPK, have been considered in imatinib resistance [71,72]. Aceves-Luquero et al. carried out knock-out studies of MAPKs, which identified only Erk2 as having a resistance-inducing effect [52]. Extremely resistant patients require potent TKIs or stem-cell transplantation, both of which greatly affect quality of life, which could be avoided if mechanisms behind resistance were uncovered and targeted treatment developed1.

 

Limitations

While p210BCR-ABL accounts for 90% of cases of CML, other Bcr-Abl variants were not examined despite their different treatment responses, limiting the applicability of this review. Furthermore, the diagram in Figure 1 is a simplified representation of the pathways associated with Bcr-Abl, especially in the case of JAK/STAT. Only pathways that have been clearly implicated in CML and imatinib resistance by research literature were included. Systematic database searches were used to carry out this review. Spelling and terminology variations that influence search results, for example, “myeloid” and “myelogenous”, were a limiting factor.

 

Clinical implications

This article holds a number of clinical implications for all medical students, not just the aspiring oncologist. For instance, the prevailing view in oncology is that mutations that confer imatinib resistance occur in the kinase domain. With the explosion of advances in genome sequencing, it is becoming possible to prospectively genetically screen patients to determine whether resistance will occur. If the current wisdom regarding CML resistance prevails, then mutations outside the kinase domain (that have been reported to cause between 10% – 40% of resistance) could be ignored, potentially putting patients at risk of ineffective treatment which could cost them their lives [2]. By investigating and becoming aware of the role of non-KD mutations, doctors could also give more accurate prognoses to patients with these mutations and begin studies looking at the best treatment for these cases (for example, randomised controlled trials comparing current therapy to higher doses of imatinib, or new pharmacological agents altogether). This review also provides a general overview into CML pathophysiology, imatinib pharmacology and chemotherapy resistance, topics every medical practitioner should be very familiar with.

 

Conclusion

Imatinib is a TKI that revolutionised leukaemia treatment and increased the length and quality of life of CML patients. While it has been known for many years that primary and secondary resistance to imatinib exist, the mechanisms have not been fully explained. While mutations in the Bcr-Abl KD account for the majority of resistance and are well known, what remains unclear is the origin of these mutations, and how resistance occurs without KD mutations.  Stem cell mutations and self-mutagenesis are possible explanations for how KD mutation occurs, and gene amplification, Lyn kinase and Erk2 for resistance occurring outside of the KD. Further research identifying key events in downstream pathways will offer new approaches for overcoming all forms of imatinib resistance.

 

Acknowledgements

The author would like to thank and acknowledge A/Prof Peter Johnson and A/Prof Bill Warren of James Cook University School of Medicine and Dentistry who provided comments and feedback on this paper. The author would also like to thank Dr. Donna Rigano and Miss Shalisa Maisrikrod for their assistance and editing help.

 

Conflicts of interest

None declared

 

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