BCR-ABL Kinase Domain Research Articles

Impact Of Baseline (BL) Mutations, Including Low-Level and Compound Mutations, On Ponatinib Response and End Of Treatment (EOT) Mutation Analysis In Patients (Pts) With Chronic Phase Chronic Myeloid Leukemia (CP-CML)

Impact Of Baseline (BL) Mutations, Including Low-Level and Compound Mutations, On Ponatinib Response and End Of Treatment (EOT) Mutation Analysis In Patients (Pts) With Chronic Phase Chronic Myeloid Leukemia (CP-CML)

Dual Specific Inhibitors Of The BCR-ABL and MNK Kinases As Potential Therapeutics For Blast Crisis Chronic Myeloid Leukemia

Pharmacologic inhibition of BCR-ABL by tyrosine kinase inhibitors (TKI) provides effective therapy for chronic phase but not blast crisis (BC) CML. The inability of TKIs to control BC CML is due to the reactivation of BCR-ABL through TKI resistance-conferring mutations, as well as the activation of alternative oncogenic pathways that mediate acquired leukemia stem cell (LSC) function and differentiation block in leukemic progenitors. Accordingly, the development of effective BC therapeutics will require reversing multiple contributors to the BC phenotype, and indicates that targeting BCR-ABL alone will not be sufficient to control BC CML. Indeed, despite the ability of third-generation TKIs to effectively inhibit the majority of clinically-relevant BCR-ABL kinase domain mutations, most patients with BC treated with these agents continue to relapse despite initial responses. Because our recent work has highlighted the importance of the MNK kinase-eIF4E axis in b-catenin-associated BC self-renewal (Lim et al., PNAS18;110(25):E2298-307, 2013), we set out to develop compounds capable of targeting BCR-ABL, TKI-resistant BCR-ABL mutants, as well as the MNK kinases simultaneously. Here, we report the discovery and characterization of two lead compounds ETC-219 and ETC-027 that inhibit clinically relevant BCR-ABL isoforms (E255K, T315I) as well as the MNK1/2 kinases at nanomolar potency (Table 1).Table 1In vitro kinase assays for ETC-219 and ETC-027.Compound IDIC50 MNK1 (µM)IC50 MNK2 (µM)IC50 ABL (µM)IC50 ABL (T315I) (µM)IC50 ABL (E255K) (µM)ETC-2190.2540.0150.0140.0130.148ETC-0275.0150.0240.2140.0913.19Using a panel of 104 kinases, we showed that ETC-219 and ETC-027 inhibited 90% of the activity of 15 and 12 kinases respectively when tested at 1uM. ETC-219 and ETC-027 interact with only 27 and 20 of off-target kinases respectively, and offer significant selectivity over kinases outside of the MNK and BCR-ABL families.ETC-219 and ETC-027 have favorable pharmacokinetic properties, and include the following ADME characteristics (Table 2).Table 2ADME characteristics of ETC-219 and ETC-027.ETC-219ETC-027ParametersUnitIVPOIVPODosemg/kg1050550C0ng/ml21241714Cmaxng/ml12122490Tmaxh12T1/2h22.21.82.1CLL/h/kg39.72.83.5VzL/kg8.831.37.410.8VssL/kg7.25.8AUC0-t(last)ng.h/ml32905128179514337AUC0-infng.h/ml33025133179814349Extrapolated area for AUC0-inf%0.37000.11000.16000.0800F%3180Regarding targeting of BCR-ABL-dependent cellular proliferation and survival, we found that ETC-219 and ETC-027 exhibited potent anti-proliferative activity against a panel of BC CML cell lines (K562, BV-173, EM-2, KCL-22, JURL-MK1), as well as Ba/F3-BCR-ABL cells containing the T315I mutation. In an in vivo xenograft model, employing K562-eIF4E cell lines in NOD-SCID mice, we found that both compounds inhibited tumor growth in a dose-dependent manner, and a dose-dependent decrease in eIF4E phosphorylation in tumor explants were also observed. Tumor regression was observed at 100 mg/kg for ETC-219. Importantly, mice given ETC-219 and ETC-027 at 100 mg/kg and 200 mg/kg doses did not exhibit significant toxicity.With respect to MNK-eIF4E-b-catenin axis inhibition, ETC-219 and ETC-027 inhibited eIF4E phosphorylation at IC50s of 0.186 nM and 0.28 nM respectively in HeLa cells. In parallel, both compounds inhibited Wnt/b-catenin reporter activity in BC CML cell lines, as well as the expression of a panel of Wnt-regulated genes. Using the serial replating assay as a readout for LSC function and self-renewal capacity, we found that ETC-219 and ETC-027 were effective at 1nM at preventing serial replating using primary CD34+ cells from patients in BC. Furthermore, ETC-219 and ETC-027 inhibited the serial replating efficacy of BC cells from patients with clinical resistance to the third generation TKI, ponatinib. ConclusionOur results demonstrate that: i) small molecule compounds with favorable ADME properties can be developed to potently and specifically inhibit the BCR-ABL and MNK kinases simultaneously; ii) Dual specific MNK and BCR-ABL inhibitors are effective at abrogating BCR-ABL-driven growth and proliferation, as well as the MNK-eIF4E-dependent self-renewal function of BC LSCs. We conclude that dual specific BCR-ABL and MNK inhibitors may warrant testing in patients with BC CML. Disclosures:Chuah:Novartis: Honoraria; BMS: Honoraria.

Phasing Analysis Of TKI Resistance Mutations In The BCR-ABL1 Kinase Domain and Neighboring Domains Using Next-Generation Sequencing

BackgroundMany CML patients treated with tyrosine kinase inhibitors (TKIs) eventually develop resistance as a result of ABL1 kinase domain (KD) mutations, and sequential treatment with different TKIs may select for multiple BCR-ABL1 mutations. Whether multiple mutations arise in distinct clones (in trans, or polyclonal mutations) or instead are present within the same BCR-ABL1 molecule (in cis, or compound mutations), has been shown to have important implications with respect to TKI sensitivities (Eide, C.A. et al., Blood 2011). Distinguishing between polyclonal and compound mutations, or mutation phasing, for the ABL1 KD has not been clinically practical with standard mutation detection methods. Here we have developed a highly sensitive next-generation sequencing (NGS) assay on the Ion Torrent PGM along with a proprietary data analysis pipeline that together enable deep sequencing of the BCR-ABL1 KD and neighboring domains with a 1% limit of detection and quantitative reporting of mutation phasing. MethodsRT and long range PCR was performed to amplify BCR-ABL1 e1a2/3, e13a2/3, and e14a2/3 fusion transcripts and the PCR products were enzymatically randomly fragmented and ligated with Ion Torrent sequencing adaptors. Size-selected libraries were quantified, pooled, amplified with the OneTouch system and sequenced with the Ion Torrent PGM using 400 bp sequencing chemistry. Sequencing data were analyzed with Torrent Suite 3.4.2 with variant frequency cutoff adjusted to 1%. Variants were further annotated with a proprietary analysis pipeline and variant report was produced after manually reviewing variants by Integrative Genomics Viewer. If more than one non-synonymous variant was reported in a sample, a proprietary phasing analysis pipeline was applied to report the mutation spectrum of all of the combinations of multiple mutations in the sample. ResultsTo validate the accuracy of the sequencing method which employs 400 bp sequencing chemistry, we compared this assay with our previously validated BCR-ABL1 NGS assay based on Ion Torrent 200 bp sequencing chemistry for a set of clinical specimens from CML patients previously treated with TKI. Results were highly concordant and similarly sensitive, with 11/11 variants (frequencies ranging from 2% to 100%) identified with comparable frequencies by both methods.To evaluate the specificity of the phasing analysis, an artificial sample was created by mixing two samples (b2_10 and b2_6) with 6 distinct variants present at ratio of 1:19. Because variants are unique in each sample, any compound mutation composed of b2_10 variant and b2_6 variant identified would be false positive. The false positive error rate (percentage of b2_6 variant as compound mutation with b2_10 variant and vice versa) ranged from 0 – 0.6%, which was consistent with sequencing error rate. We conservatively define a compound mutation as true if it is present in at least 5% of any one of the component variants in the compound mutation. Mutation detection and phasing analysis were reproducible on different chips (314 v2 and 318 v2) and different library preps from the same long range PCR product of BCR-ABL1.Table 1 shows the mutation spectrum from sample b2_6. Of the four variants detected, L248V and G250E were mutually exclusive (in trans), while T315I and M351T were present as compound mutations with each other and, separately, with either L248V or G250E. Notably, >86% of the molecules harbored single mutations, and no compound mutations containing more than 2 variants were observed.Table 1Phasing analysis of sample b2_6L248G250T315M351% of moleculesL248VWTT315IWT2.38%L248VWTWTM351T0.84%L248VWTWTWT32.78%WTG250ET315IWT2.80%WTG250EWTM351T1.20%WTG250EWTWT43.00%WTWTT315IM351T0.16%WTWTT315IWT8.7%WTWTWTM351T1.80%WTWTWTWT6.34% ConclusionsWe have developed and validated a sensitive NGS assay that enables deep sequencing of the BCR-ABL1 KD and neighboring domains along with quantitative mutational phasing. This method has been applied in evaluating >250 clinical specimens for a clinical trial of a third-generation TKI(results reported separately). The ability to easily determine the mutation phasing of a CML patients' mutation profile using this assay will allow for investigations into compound mutation-based resistance mechanisms and may be used to better guide treatment decisions. Disclosures:Li:MolecularMD: Employment. Yan:MolecularMD: Employment. Darwanto:MolecularMD: Employment. Fang:MolecularMD: Employment. Liu:MolecularMD: Employment. Drafahl:MolecularMD: Employment. Toplin:MolecularMD: Employment. Spittle:MolecularMD: Employment. Galderisi:MolecularMD: Employment.

Ponatinib Is a Pan-BCR-ABL Kinase Inhibitor: MD Simulations and SIE Study

BCR-ABL kinase domain inhibition can be used to treat chronic myeloid leukemia. The inhibitors such as imatinib, dasatinib and nilotinib are effective drugs but are resistant to some BCR-ABL mutations. The pan-BCR-ABL kinase inhibitor ponatinib exhibits potent activity against native, T315I, and all other clinically relevant mutants, and showed better inhibition than the previously known inhibitors. We have studied the molecular dynamics simulations and calculated solvated interaction energies of native and fourteen mutant BCR-ABL kinases (M244V, G250E, Q252H, Y253F, Y253H, E255K, E255V, T315A, T315I, F317L, F317V, M351T, F359V and H396P) complexed with ponatinib. These studies revealed that the interactions between ponatinib and individual residues in BCR-ABL kinase are also affected due to the remote residue mutations. We report that some residues, Met244, Lys245, Gln252, Gly254, Leu370 and Leu298 do not undergo any conformational changes, while the fluctuations in residues from P-loop, β3-, β5- strands and αC- helix are mainly responsible for ponatinib binding to native and all mutant BCR-ABL kinases. Our work provides the molecular mechanisms of native and mutant BCR-ABL kinases inhibition by ponatinib at atomic level that has not been studied before.

A Phase 2 Trial of Ponatinib in Philadelphia Chromosome–Positive Leukemias

BackgroundPonatinib is a potent oral tyrosine kinase inhibitor of unmutated and mutated BCR-ABL, including BCR-ABL with the tyrosine kinase inhibitor–refractory threonine-to-isoleucine mutation at position 315 (T315I). We conducted a phase 2 trial of ponatinib in patients with chronic myeloid leukemia (CML) or Philadelphia chromosome–positive acute lymphoblastic leukemia (Ph-positive ALL).MethodsWe enrolled 449 heavily pretreated patients who had CML or Ph-positive ALL with resistance to or unacceptable side effects from dasatinib or nilotinib or who had the BCR-ABL T315I mutation. Ponatinib was administered at an initial dose of 45 mg once daily. The median follow-up was 15 months.ResultsAmong 267 patients with chronic-phase CML, 56% had a major cytogenetic response (51% of patients with resistance to or unacceptable side effects from dasatinib or nilotinib and 70% of patients with the T315I mutation), 46% had a complete cytogenetic response (40% and 66% in the two subgroups, respectively), and 34% had a major molecular response (27% and 56% in the two subgroups, respectively). Responses were observed regardless of the baseline BCR-ABL kinase domain mutation status and were durable; the estimated rate of a sustained major cytogenetic response of at least 12 months was 91%. No single BCR-ABL mutation conferring resistance to ponatinib was detected. Among 83 patients with accelerated-phase CML, 55% had a major hematologic response and 39% had a major cytogenetic response. Among 62 patients with blast-phase CML, 31% had a major hematologic response and 23% had a major cytogenetic response. Among 32 patients with Ph-positive ALL, 41% had a major hematologic response and 47% had a major cytogenetic response. Common adverse events were thrombocytopenia (in 37% of patients), rash (in 34%), dry skin (in 32%), and abdominal pain (in 22%). Serious arterial thrombotic events were observed in 9% of patients; these events were considered to be treatment-related in 3%. A total of 12% of patients discontinued treatment because of an adverse event.ConclusionsPonatinib had significant antileukemic activity across categories of disease stage and mutation status. (Funded by Ariad Pharmaceuticals and others; PACE ClinicalTrials.gov number, NCT01207440.)

A Single Nucleotide Polymorphism in cBIM Is Associated with a Slower Achievement of Major Molecular Response in Chronic Myeloid Leukaemia Treated with Imatinib

PurposeBIM is essential for the response to tyrosine-kinase inhibitors (TKI) in chronic myeloid leukaemia (CML) patients. Recently, a deletion polymorphism in intron 2 of the BIM gene was demonstrated to confer an intrinsic TKI resistance in Asian patients. The present study aimed at identifying mutations in the BIM sequence that could lead to imatinib resistance independently of BCR-ABL mutations.Experimental DesignBIM coding sequence analysis was performed in 72 imatinib-treated CML patients from a French population of our centre and in 29 healthy controls (reference population) as a case-control study. Real-time quantitative PCR (RT qPCR) was performed to assess Bim expression in our reference population.ResultsNo mutation with amino-acid change was found in the BIM coding sequence. However, we observed a silent single nucleotide polymorphism (SNP) c465C>T (rs724710). A strong statistical link was found between the presence of the T allele and the high Sokal risk group (p = 0.0065). T allele frequency was higher in non responsive patients than in the reference population (p = 0.0049). Similarly, this T allele was associated with the mutation frequency on the tyrosine kinase domain of BCR-ABL (p<0.001) and the presence of the T allele significantly lengthened the time to achieve a major molecular response (MMR). Finally, the presence of the T allele was related to a decreased basal expression of the Bim mRNA in the circulating mononuclear cells of healthy controls.ConclusionThese results suggest that the analysis of the c465C>T SNP of BIM could be useful for predicting the outcome of imatinib-treated CML patients.

Molecular analysis of the BCR-ABL1 kinase domain in chronic-phase chronic myelogenous leukemia treated with tyrosine kinase inhibitors in practice: Study by the Nagasaki CML Study Group

An appropriate trigger for BCR-ABL1 mutation analysis has not yet been established in unselected cohorts of chronic-phase chronic myelogenous leukemia patients. We examined 92 patients after 12 months of tyrosine kinase inhibitor (TKI) treatment in Nagasaki Prefecture, Japan. Univariate analysis revealed that significant factors associated with not attaining a major molecular response (MMR) were the presence of the minor BCR-ABL1 fusion gene, a low daily dose of TKI, and the emergence of BCR-ABL1 kinase domain mutations conferring resistance to imatinib. Factors associated with the loss of sustained MMR were a low daily dose of TKI and the emergence of alternatively spliced BCR-ABL1 mRNA with a 35-nucleotide insertion. Taken together, our results suggest that the search for BCR-ABL1 mutations should be initiated if patients have not achieved MMR following 12 months of TKI treatment.

Abstract B277: Polymutant BCR-ABL1 proteins during chronic myeloid leukemia therapy: Novel mechanisms of resitance from clinical, in vitro, and in silico evidence.

Abstract The acquisition of mutations within the BCR-ABL1 kinase domain is frequently associated to imatinib failure among patients with chronic myeloid leukemia (CML). Typically, patients failing imatinib undergo sequential therapy with second line tyrosine kinase inhibitors (TKIs) such as nilotinib or dasatinib. To investigate the prevalence of BCR-ABL1 mutations and the mechanisms of acquired TKI resistance during sequential therapy, we used a sensitive cloning technique in a cohort of chronic-phase patients failing imatinib therapy that subsequently received dasatinib. The analysis revealed that most patients failing imatinib carry BCR-ABL1 mutations, many with polymutant BCR-ABL1 alleles, and that there is progressive exhaustion of the pool of unmutated BCR-ABL1 alleles over the course of treatment. Detailed modeling of the structural effects on the ABL1 kinase domain of the various mutations and of clinically available/experimental TKIs against some of the most frequent polymutants found in our patient cohort indicates that the accumulation of more than one BCR-ABL1 mutation within the same allele promotes a higher than expected level of TKI resistance compared to that of each single mutation alone. Some polymutants exhibited levels of resistance to imatinib/dasatinib remarkably higher than those of the highly resistant T315I mutation, suggesting that the generation of polymutant BCR-ABL1 alleles represents a powerful mechanism of escape for CML cells exposed to TKI-induced selection pressure. Notably, the complexity of the polymutants is limited, suggesting that, in addition to increasing TKI resistance, the successive accumulation of single mutations within the same clone may compromise its fitness, the latter being a function of the number and type of single mutants involved in any given polymutant. Strategies to limit the generation of complex mutants and clinical resistance may require the use of frontline therapy with TKI combinations or with novel TKIs that cover a broad range of mutations and therefore may prevent the emergence of resistance. In addition to providing an understanding for the molecular basis of clinical TKI failure in our patient cohort, we modeled the activity of the novel TKI ponatinib against the most frequently encountered polymutants. Although all the polymutants were predicted to be highly resistant to imatinib and dasatinib, ponatinib was predicted to maintain a notable affinity for most clinically relevant BCR-ABL1 single mutants and some of the polymutants detected in our patient cohort. Ponatinib was able to accommodate the structural changes induced by these complex mutants, which suggest the possibility of polymutant kinase inhibition at doses frequently within a clinically achievable range. These findings were validated in cell-based biochemical assays, thus supporting the clinical applicability of in silico modeling as a means to predict the TKI approach with the highest probability of clinical success. It is worth noting, however, that some polymutants examined required ponatinib concentrations unlikely to be reached in humans without excessive toxicity, thus anticipating potential mechanisms of escape to ponatinib therapy through selection and expansion of clones carrying highly TKI resistant complex BCR-ABL1 mutant proteins. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B277. Citation Format: Sabrina Pricl, Don L. Gibbons, Paola Posocco, Erik Laurini, Barbara Giabbai, Paola Storici, Maurizio Fermeglia, Hunshi Sun, Jorge Cortes, Moshe Talpaz, Nicholas Donato, Alfonso Quintas-Cardama. Polymutant BCR-ABL1 proteins during chronic myeloid leukemia therapy: Novel mechanisms of resitance from clinical, in vitro, and in silico evidence. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B277.

Detection of BCR-ABL kinase domain mutations in patients with chronic myeloid leukemia on imatinib

BCR-ABL tyrosine kinase domain mutations are the most important factor contributing to imatinib-resistance in patients with chronic myeloid leukemia. We used a semi-nested reverse transcriptase-polymerase chain reaction followed by bidirectional sequencing to detect mutations in a cohort of 110 chronic myeloid leukemia patients. In total, 34 mutations in 19 distinct codons were identified in 32 patients, of which D276N and E279A were novel. The most commonly mutated region was drug-binding site (29%) followed by P-loop region (26%) and most patients bearing them were in accelerated phase and blastic phase. This report expands the spectrum of BCR-ABL mutations and stresses the use of mutation testing in imatinib-resistant patients for continuation of treatment procedure.

Ponatinib—A Step Forward in Overcoming Resistance in Chronic Myeloid Leukemia

With the current therapy, the improvement in survival of patient with early chronic phase chronic myelogenous leukemia (CML) is unrivaled by that of any other leukemia. In fact, extrapolation of the survival curves may suggest that life expectancy of patients who achieve and maintain predetermined milestones may not differ from that of the age-matched healthy adults. The main reasons for such success are the presence of a well-defined molecular target, the BCR-ABL oncogene, necessary and sufficient for the initiation and propagation of CML, and the powerful and selective agents that inhibit it. Five U.S. Food and Drug Administration (FDA)-approved tyrosine kinase inhibitors (TKI), each with unique activities and toxicity profiles, allow for individualized patient care. Despite the remarkable responses of most patients, a small but significant fraction of patients develops clinical resistance to the TKIs, some of which is attributed to the BCR-ABL kinase domain mutations affecting TKI binding and activity. The recently approved third-generation TKI ponatinib showed remarkable activity in the patients with multi-TKI-resistant disease. Particularly impressive was its efficacy in patients with T315I mutation that is resistant to all other TKIs. In lieu of the current emphasis on achieving earlier and more profound responses and excellent activity of ponatinib in the refractory setting, its optimal position among the available armamentarium of agents is being established.

Implications of BCR-ABL1 kinase domain-mediated resistance in chronic myeloid leukemia

Patients with chronic myeloid leukemia develop resistance to both first-generation and second-generation tyrosine kinase inhibitors (TKIs) as a result of mutations in the kinase domain (KD) of BCR-ABL1. A wide range of BCR-ABL1 KD mutations that confer resistance to TKIs have been identified, and the T315I mutant has proven particularly difficult to target. This review summarizes the prevalence, impact, and prognostic implications of BCR-ABL1 KD mutations in patients with chronic myeloid leukemia who are treated with current TKIs and provides an overview of recent treatment guidelines and future trends for the detection of mutations.

Combined effects of PI3K and SRC kinase inhibitors with imatinib on intracellular calcium levels, autophagy, and apoptosis in CML-PBL cells

Imatinib induces a complete cytogenetic regression in a large percentage of patients affected by chronic myeloid leukemia (CML) until mutations in the kinase domain of BCR-ABL appear. Alternative strategies for CML patients include the inhibition of phosphatidylinositol 3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) pathway, which is constitutively activated in leukemia cells and seems important for the regulation of cell proliferation, viability, and autophagy. In this study, we verified the effect of imatinib mesylate (IM), alone or in association with LY294002 (LY) (a specific PI3K protein tyrosine kinase inhibitor) or 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]-pyrimidine (PP1) (a Src tyrosine kinase inhibitor), on viability, intracellular calcium mobilization, apoptosis, and autophagy, in order to verify possible mechanisms of interaction. Our data demonstrated that PP1 and LY interact synergistically with IM by inducing apoptosis and autophagy in Bcr/Abl+ leukemia cells and this mechanism is related to the stress of the endoplasmic reticulum (ER). Our findings suggest a reasonable relationship between apoptotic and autophagic activity of tyrosine kinase inhibitors (TKIs) and the functionality of smooth ER Ca2+-ATPase and inositol triphosphate receptors, independently of intracellular calcium levels. Therapeutic strategies combining imatinib with PI3K and/or Src kinase inhibitors warrant further investigations in Bcr/Abl+ malignancies, particularly in the cases of imatinib mesylate-resistant disease.

Unraveling the complexity of tyrosine kinase inhibitor–resistant populations by ultra-deep sequencing of the BCR-ABL kinase domain

AbstractIn chronic myeloid leukemia and Philadelphia chromosome–positive acute lymphoblastic leukemia, tyrosine kinase inhibitor (TKI) therapy may select for drug-resistant BCR-ABL mutants. We used an ultra-deep sequencing (UDS) approach to resolve qualitatively and quantitatively the complexity of mutated populations surviving TKIs and to investigate their clonal structure and evolution over time in relation to therapeutic intervention. To this purpose, we performed a longitudinal analysis of 106 samples from 33 patients who had received sequential treatment with multiple TKIs and had experienced sequential relapses accompanied by selection of 1 or more TKI-resistant mutations. We found that conventional Sanger sequencing had misclassified or underestimated BCR-ABL mutation status in 55% of the samples, where mutations with 1% to 15% abundance were detected. A complex clonal texture was uncovered by clonal analysis of samples harboring multiple mutations and up to 13 different mutated populations were identified. The landscape of these mutated populations was found to be highly dynamic. The high degree of complexity uncovered by UDS indicates that conventional Sanger sequencing might be an inadequate tool to assess BCR-ABL kinase domain mutation status, which currently represents an important component of the therapeutic decision algorithms. Further evaluation of the clinical usefulness of UDS-based approaches is warranted.

Targeting survival pathways in chronic myeloid leukaemia stem cells

Chronic myeloid leukaemia (CML) is a clonal myeloproliferative disorder characterized by the presence of a fusion oncogene BCR-ABL, which encodes a protein with constitutive TK activity. The implementation of tyrosine kinase inhibitors (TKIs) marked a major advance in CML therapy; however, there are problems with current treatment. For example, relapse occurs when these drugs are discontinued in the majority of patients who have achieved a complete molecular response on TKI and these agents are less effective in patients with mutations in the BCR-ABL kinase domain. Importantly, TKI can effectively target proliferating mature cells, but do not eradicate quiescent leukaemic stem cells (LSCs), therefore allowing disease persistence despite treatment. It is essential that alternative strategies are used to target the LSC population. BCR-ABL activation is responsible for the modulation of different signalling pathways, which allows the LSC fraction to evade cell death. Several pathways have been shown to be modulated by BCR-ABL, including PI3K/AKT/mTOR, JAK-STAT and autophagy signalling pathways. Targeting components of these survival pathways, alone or in combination with TKI, therefore represents an attractive potential therapeutic approach for targeting the LSC. However, many pathways are also active in normal stem cells. Therefore, potential targets must be validated to effectively eradicate CML stem cells while sparing normal counterparts. This review summarizes the main pathways modulated in CML stem cells, the recent developments and the use of novel drugs to target components in these pathways which may be used to target the LSC population. This article is part of a themed section on Emerging Therapeutic Aspects in Oncology. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2013.169.issue-8.

Endoplasmic reticulum stress-mediated apoptosis in imatinib-resistant leukemic K562-r cells triggered by AMN107 combined with arsenic trioxide

The first tyrosine kinase inhibitor (TKI) imatinib mesylate (imatinib) targets the kinase domain of BCR-ABL and induces apoptosis in newly diagnosed chronic myeloid leukaemia (CML). However, resistant and relapse are common problems in imatinib-treated patients. Although second-generation TKI such as AMN107 appears to improve the treatment of CML, TKI resistance and relapse are also frequently occurred in the patients. To test whether arsenic trioxide (ATO) could potentiate the efficacy of AMN107 in imatinib-resistant cells, we conducted a series of assays in TKI-resistant K562-r cells treated with AMN107 and ATO. Based on a time-course cDNA microarray analysis, we found many genes typically involved in the endoplasmic reticulum (ER) stress signalling were significantly up-regulated, implicating the occurrence of ER stress-mediated apoptosis in K562-r cells treated with the combination of ATO and AMN107. Such implication was also supported by the data showing the activation of members in the JNK pathway, which are known to be characteristic markers bridging ER-stress and apoptosis. Partial knock-down of the JNK activities alleviated the effects of apoptosis (p < 0.05) triggered by combining AMN107 with ATO. In conclusion, this study for the first time demonstrates a synergistic effect of AMN107 with ATO, allowing insights into the possible mechanisms underlying imatinib-induced resistance in CML. Our data also suggest that combination of AMN107 with ATO may represent a new strategy for the treatment of imatinib-resistant CML patients.

Imatinib mesylate resistance and mutations: An Indian experience

The treatment of chronic myeloid leukemia (CML) has been revolutionized by the small molecule selective kinase inhibitor imatinib mesylate. Imanitib was the first BCR-ABL targeted agent approved for the treatment of CML patients and confers significant response in most patients; however, a substantial number of patients are initially refractory to the drug or may develop resistance during the course of treatment. Point mutations in the kinase domain (KD) of BCR-ABL that impact drug binding have been identified as one of the major mechanisms of resistance. We present here an overview of the current practice in monitoring for such mutations, including the methods used, criteria for investigating and guidelines for reporting the mutations. We further present and discuss the experience of our own laboratory in studying the KD mutations in Indian CML patients on imatinib treatment.

Dasatinib for the treatment of chronic phase chronic myeloid leukemia

SUMMARY Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder driven by the oncogenic tyrosine kinase BCR-ABL. The discovery of the tyrosine kinase inhibitor imatinib revolutionized the management of patients with CML. However, imatinib can have significant side effects, and is not effective in all patients. The development of second-generation (dasatinib and nilotinib) and third-generation (ponatinib) tyrosine kinase inhibitors sought to address the issue of imatinib-resistance and -intolerance and furthermore, to determine if these agents are superior to imatinib. Dasatinib, a multitargeted inhibitor, is effective against most BCR-ABL kinase domain mutations resistant to imatinib and capable of inducing faster and deeper molecular responses. This article describes the development of dasatinib and documents its current clinical use in chronic phase CML.

BCR-ABL1 kinase domain mutations may persist at very low levels for many years and lead to subsequent TKI resistance

Background:BCR-ABL1 mutation analysis is recommended for chronic myeloid leukaemia patients. However, mutations may become undetectable after changing therapy, and it is unknown whether they have been eradicated.Methods:We examined longitudinal data of patients with imatinib-resistant mutations, which became undetectable by Sanger sequencing to determine whether mutations could reappear, and the related circumstances.Results:Identical imatinib- and nilotinib-resistant mutations reappeared following further therapy changes in five patients, and was associated with subsequent nilotinib resistance in four.Conclusion:The data suggest that some BCR-ABL1 mutations may persist at undetectable levels for many years after changing therapy, and can be reselected and confer resistance to subsequent inhibitors.

Molecular signatures of chronic myeloid leukemia stem cells

BCR-ABL tyrosine kinase inhibitors (TKIs) are effective in controlling Philadelphia-positive (Ph+) chronic myeloid leukemia (CML) are unlikely to cure the disease because TKIs are unable to eradicate leukemia stem cells (LSCs) responsible for the disease relapse even after tyrosine kinase inhibition. In addition, the TKI resistance of LSCs is not associated with the BCR-ABL kinase domain mutations. These observations indicate that TKI-insensitive LSCs and TKI-sensitive leukemic progenitor cells are biologically different, which leads us to believe that LSCs and more differentiated leukemic cells have different genetic mechanisms. Further study of LSCs to identify the novel gene signatures and mechanisms that control the function and molecular phenotype of LSCs is critical. In this mini-review, we will discuss our current understanding of the biology of LSCs and novel genes that could serve as a molecular signature of LSCs in CML. These novel genes could also serve as potential targets for eradicating LSCs in CML.

Suppression of Survivin Induced by a BCR-ABL/JAK2/STAT3 Pathway Sensitizes Imatinib-Resistant CML Cells to Different Cytotoxic Drugs

The BCR-ABL oncoprotein of chronic myelogenous leukemia (CML) displays exclusive cytoplasmic localization and constitutive tyrosine kinase activity leading to the activation of different pathways that favor cell proliferation and survival. BCR-ABL induces survivin expression at both the mRNA and protein level, thus inhibiting the apoptotic machinery of CML cells and contributing to the expansion of the leukemic clone. We report that, in human CML cell lines, BCR-ABL-mediated upregulation of survivin involves the JAK2/STAT3 pathway since silencing of either protein caused a consistent reduction in survivin expression. Cell lines unresponsive to imatinib mesylate (IM) because of BCR-ABL gene amplification were not resensitized to the drug after survivin downregulation. However, cells insensitive to IM because of point mutations in the BCR-ABL kinase domain were highly responsive to hydroxyurea (HU) after survivin silencing. To address the possible clinical applications of our results, we used shepherdin, a cell-permeable peptidomimetic compound that downregulates survivin expression by preventing its interaction with Hsp90. Incubation with shepherdin of immortalized cell lines both sensitive and resistant to IM enhanced cell death induced by HU and doxorubicin. Similarly, the combination of shepherdin with first- and second-generation tyrosine kinase inhibitors reduced the colony-forming potential of human progenitors derived from both patients with IM-sensitive and IM-resistant CML. These results suggest that strategies aimed at reducing survivin levels may represent a potential therapeutic option for patients with CML unresponsive to IM.