Detection of BCR-ABL kinase domain mutations in CD34+ cells from newly diagnosed chronic phase CML patients and their association with imatinib resistance

Abstract Imatinib mesylate is a selective Bcr-Abl kinase inhibitor, effective in the treatment of chronic myelogenous leukemia. Most patients in chronic phase maintain durable responses; however, many in blast crisis fail to respond, or relapse quickly. Kinase domain mutations are the most commonly identified mechanism associated with relapse. Many of these mutations decrease the sensitivity of the Abl kinase to imatinib, thus accounting for resistance to imatinib. The role of other mutations in the emergence of resistance has not been established. Using biochemical and cellular assays, we analyzed the sensitivity of several mutants (Met244Val, Phe311Leu, Phe317Leu, Glu355Gly, Phe359Val, Val379Ile, Leu387Met, and His396Pro/Arg) to imatinib mesylate to better understand their role in mediating resistance.While some Abl mutations lead to imatinib resistance, many others are significantly, and some fully, inhibited. This study highlights the need for biochemical and biologic characterization, before a resistant phenotype can be ascribed to a mutant.

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Properties of CD34+ CML Cells That Predict Clinical Response to Tyrosine Kinase Inhibitors.

Blood ◽

10.1182/blood.v110.11.324.324 ◽

2007 ◽

Vol 110 (11) ◽

pp. 324-324

Author(s):

Xiaoyan Jiang ◽

Donna Forrest ◽

Franck Nicolini ◽

Karen Lambie ◽

Kyi Min Saw ◽

...

Keyword(s):

Stem Cells ◽

Tyrosine Kinase ◽

Blast Crisis ◽

Chronic Phase ◽

Kinase Domain ◽

Abl Kinase ◽

Cd34 Cells ◽

Clinical Responses ◽

Kinase Domain Mutations

Abstract Imatinib (IM) treatment causes remission in a majority of patients with chronic myeloid leukemia (CML) but relapses remain a problem. The frequent presence in relapsing cells of BCR-ABL kinase domain mutations suggests that their prior but undetected acquisition by rare CML stem cells may be a major contributor to IM treatment failures. We have recently demonstrated that enriched populations of CML stem cells (lin−CD34+CD38− cells) are relatively insensitive to IM and possess multiple unique features that would be expected to promote both innate and acquired mechanisms of resistance to BCR-ABL-targeted therapeutics. These include elevated BCR-ABL expression and tyrosine kinase activity, increased expression of ABCB1/MDR1 and ABCG2, decreased expression of OCT1, and a high degree of genetic instability, as demonstrated by a rapid accumulation of BCR-ABL mutations in vitro. To determine whether these parameters may be predictive of clinical responses to IM, immunomagnetically selected CD34+ stem/progenitor cells from 18 chronic phase CML patients’ samples obtained prior to IM therapy were evaluated and the results compared with subsequent clinical responses. Direct sequencing of transcripts cloned from extracts of freshly isolated CD34+ cells (10 clones/sample) detected a high frequency of pre-existing BCR-ABL kinase mutations in the CD34+ cells from 12 of 12 patients regardless of their subsequent IM responses (20–80%). Interestingly, a higher incidence of BCR-ABL kinase domain mutations was found in 5 IM-nonresponders (33–80% of transcripts showed ≥1 BCR-ABL kinase domain mutation) as compared to 5 IM-responders (values of 20-30%, P<0.02). A higher frequency of BCR-ABL kinase domain mutations was also detected in extracts of colonies generated from assays of cells harvested from 3-week suspension cultures initiated with the same starting CD34+ CML cells (21–68% vs 10–43%). A high incidence of BCR-ABL kinase domain mutations was also documented in freshly isolated or cultured CD34+ cells from 2 patients who developed sudden blast crisis (50–63% and 17–83%). Overall, 38 different mutations were identified from freshly isolated CD34+ CML cells and >50 additional mutations were identified in the progeny of CD34+ CML cells cultured ± IM. These included 15 point mutations frequently associated with clinical IM resistance (including G250, Q252, E255, T315, M351, F359 and H396) and >40 mutations not previously described. Furthermore, freshly isolated CD34+ cells from IM-nonresponders (including the 2 patients who developed blast crisis, n=10) showed a greater resistance to IM in vitro (∼2 fold, P< 0.001 with 5 μM and P<0.02 with 10 μM IM) as compared to CD34+ cells from IM-responders (n=8) in the presence of 5 and 10 μM IM, as determined by colony-forming cell (CFC) assays. Although more IM-resistant CFCs were obtained in the presence of IM from 3-week cultures initiated with CD34+ cells from the same IM-nonresponders than from IM responders, these latter differences were not significantly different (P= 0.28). These results suggest that the CD34+ leukemic cells from individual chronic phase CML patients harbor differences in their biologic properties that are predictive of how they will respond to IM therapy and that assessment of these differences may form the basis of rapid, practical and quantitative tests to assist in optimized patient management.

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Low Level BCR-ABL Mutations Below the Detection Limit of Current Standard Screening Techniques Occur Predominantly in the CD34+ Progenitor Cell Compartment in Chronic Phase CML Patients At Diagnosis. A Substudy of the ENEST1st Trial

Blood ◽

10.1182/blood.v120.21.1671.1671 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1671-1671

Author(s):

Jacqueline Maier ◽

Karoline Schubert ◽

Michael Cross ◽

Sabine Leiblein ◽

Kathrin Wildenberger ◽

...

Keyword(s):

Detection Limit ◽

Research Funding ◽

Progenitor Cell ◽

Chronic Phase ◽

Kinase Domain ◽

Cell Compartment ◽

Screening Techniques ◽

Abl Kinase ◽

Cd34 Cells ◽

Kinase Domain Mutations

Abstract Abstract 1671 The presence of BCR-ABL kinase domain mutations below the detection limit of conventional screening techniques (low level mutations, LLM) predicts outcome of subsequent therapy in patients with imatinib resistance (Parker et. al JCO 2011 and Blood 2012). We have further evaluated LLM in the context of the ENEST1st trial, which addresses the frequency of complete molecular responses after 18 months on nilotinib 300mg BID (NI) in newly diagnosed patients with chronic myeloid leukemia (CML) in chronic phase (CP). Here, we have investigated the incidence of detectable LLM in the CD34+ progenitor cell compartment in comparison to total white cells (TWBC). Sixty nine ENEST1st study patients with CP CML provided 10ml of peripheral blood or 2ml bone marrow after written informed consent. CD34+ selection was carried out by MACS® (Miltenyi Biotec) and the CD34+ purity was subsequently determined by fluorescent activated cell sorting (FACS). The results were compared to those derived from stored TWBC from 23 of the same patients and a further 16 patients at diagnosis. Aliquots of 105 CD34+ or at least 106 TWBC were used for RNA extraction, cDNA synthesis and BCR-ABL amplification followed by Ligation PCR (L-PCR) for mutations T315I, Y253H, E255K/V, and F359V. This method has previously been shown to achieve a dynamic detection range of 100% to <0.1% mutant allele (3–3.5 log). No patients showed BCR-ABL kinase domain mutations detected by Sanger sequencing spanning ABL exons 4–9. Forty five of 69 patients (65%) with 105 CD34+ cells and a documented CD34+ purity of >50% were available for BCR-ABL amplification. Amplification was successful from 36 (52%) of these CD34+ samples and from 38 of the 39 (97%) TWBC samples. A total of 180 L-PCR assays of CD34+ cells identified 29 (16%) mutations (T315Ix12, Y253Hx7, E255Kx8/Vx1 and F359Vx1) in CD34+ cells from 21/36 patients (58%). In comparison, 190 assays of TWBC identified 10 (5%) mutations (T315Ix3, Y253Hx6, E255Vx1, p=0.0005) in 8/38 patients (21%, p=0.001 Fishers exact test). Significantly more T315I (33%) and E255K (22%) mutations were observed in CD34+ cells than in TWBC (8%, p=0.007 and 0% p= 0.003 respectively). The quantitative levels of all mutant alleles were median 0.135 (range 0.06–0.535) and 0.1 (range 0.04-0, 25) BCR-ABLmutant/ BCR-ABLunmutated for mutations in CD34+ cells and TWBC, respectively and were not significantly different. Where both CD34+ and TWBC were available from the same patient (n=23), 11 patients showed a total of 18 mutations in the CD34+ fraction but only one of these mutations was confirmed in TWBC. One additional mutation was detectable in the TWBC. The remaining 12 patients with no detectable mutation in the CD34+ fraction showed 3 mutations (2x Y253H, T315I) in 2 patients in TWBC only. In conclusion, LLM with either no (T315I) or intermediate (Y253H, E255K/V, F359V) sensitivity to nilotinib are detectable in CP CML patients at a frequency of 21% in the TWBC but with a significantly higher frequency of 58% in the enriched CD34+ progenitor cell compartment. Longterm patient follow up on the ENEST1st and ENESTobserve studies will allow analysis of the relationship between LLM and clinical outcomes on nilotinib. Disclosures: Hochhaus: Novartis, BMS, MSD, Ariad, Pfizer: Consultancy Other, Honoraria, Research Funding. Frank:Novartis: Employment. Lange:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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Predictors of Primary Imatinib Resistance in Chronic Myelogenous Leukemia Are Distinct From Those in Secondary Imatinib Resistance

Journal of Clinical Oncology ◽

10.1200/jco.2008.19.4076 ◽

2009 ◽

Vol 27 (22) ◽

pp. 3642-3649 ◽

Cited By ~ 62

Author(s):

Wenyong W. Zhang ◽

Jorge E. Cortes ◽

Hui Yao ◽

Li Zhang ◽

Neelima G. Reddy ◽

...

Keyword(s):

Gene Expression ◽

Expression Patterns ◽

Chronic Phase ◽

Kinase Domain ◽

Myelogenous Leukemia ◽

Newly Diagnosed ◽

Blast Phase ◽

Imatinib Resistance ◽

Secondary Resistance ◽

Kinase Domain Mutations

Purpose A subset of patients with chronic myelogenous leukemia (CML) do not respond to the tyrosine kinase inhibitor (TKI) imatinib mesylate. Such primary imatinib resistance is distinguished from secondary resistance which reemerges after attainment of cytogenetic remission. Patients and Methods We studied gene expression patterns in total WBCs using a panel of 21 genes previously implicated in TKI handling, resistance, or progression comparing patients who had newly diagnosed TKI-naive CML that had optimal (n = 41), or suboptimal (n = 7) responses to imatinib, or primary resistance (n = 20). Expression patterns were compared to those in secondary TKI-resistant chronic phase CML without ABL1 kinase domain mutations (n = 29), and to lymphoid (n = 15) or myeloid blast phase disease (n = 12). Results Fifteen genes in the panel distinguished blast phase from chronic phase disease, and 12 genes distinguished newly diagnosed CML from TKI-resistant CML without ABL1 kinase domain mutations, but only a single gene, prostaglandin-endoperoxide synthase 1/cyclooxgenase 1 (PTGS1/COX1; P = .005), differentiated imatinib-responsive from primary imatinib-resistant CML. The association of primary imatinib resistance with higher transcript levels of the drug metabolism gene PTGS1 was confirmed in a separate data set of 68 newly diagnosed, imatinib-treated CML (P = .008). In contrast, up to 11 different genes were identified in a multivariate model that optimally discriminated secondary imatinib resistance lacking ABL1 kinase domain mutation from imatinib-responsive cases, likely related to the more complex pathogenesis of secondary resistance. Conclusion Gene expression profiling of CML at diagnosis for PTGS1 may be useful in predicting imatinib response and in selecting alternate therapy.

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Low Level BCR-ABL Mutations and Response to Treatment: A Substudy of the ENEST1st Trial

Blood ◽

10.1182/blood.v124.21.3143.3143 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3143-3143

Author(s):

Georg-Nikolaus Franke ◽

Jacqueline Maier ◽

Karoline Schubert ◽

Michael Cross ◽

Sabine Leiblein ◽

...

Keyword(s):

Detection Limit ◽

Research Funding ◽

Sanger Sequencing ◽

Kinase Domain ◽

Imatinib Resistance ◽

Detection Range ◽

Low Level ◽

Abl Kinase ◽

Cd34 Cells ◽

Kinase Domain Mutations

Abstract The presence of BCR-ABL kinase domain mutations below the detection limit of sanger sequencing (low level mutations, LLM) predicts the outcome of subsequent therapy in patients with imatinib resistance (Parker et al. JCO 2011, Blood 2012). We have evaluated LLM in the context of the ENEST1st trial, which addresses the frequency of deep molecular response (MR4) after 18 months on nilotinib 300mg BID (NI) in newly diagnosed patients with chronic myeloid leukemia (CML) in chronic phase (CP). We have investigated the incidence of detectable LLM in the CD34+ progenitor cell compartment and correlated the mutation status (LLM vs. no LLM) with the primary endpoint of the ENEST1st trial, MR4at 18 months. Additionally, we evaluated MMR at 18 months. Seventy eight ENEST1st study patients (pts) provided 10ml of peripheral blood or 2ml bone marrow after written informed consent. CD34+ selection was carried out by MACS® (Miltenyi Biotec) and the CD34+purity was subsequently determined by fluorescent activated cell sorting (FACS). Aliquots of 105 CD34+ were used for RNA extraction, cDNA synthesis and BCR-ABL amplification followed by Ligation PCR (L-PCR) for mutations T315I, Y253H, E255K/V, and F359V. This method has previously been shown to achieve a dynamic detection range of 100% to <0.1% mutant allele (3–3.5 log). No patient showed BCR-ABL kinase domain mutations detected by Sanger sequencing spanning ABL exons 4–9. Fifty one of 78 pts (65%) with 105 CD34+ cells and a documented CD34+ purity of >50% were available for BCR-ABL amplification. Amplification was successful from 41 (52%) of these CD34+samples. A total of 205 L-PCR assays of CD34+ cells identified 29 (14%) mutations (T315Ix12, Y253Hx7, E255Kx8/Vx1 and F359Vx1) from 21/41 pts (51%). The median quantitative level of mutant alleles in CD34+ cells was 0.13 (range 0.06–0.54) BCR-ABLmutant/ BCR-ABLunmutated. One pt was taken off the study because of adverse events before the 3 months evaluation, 2 pts between 3 and 6 months and 1 after the 6 months evaluation (9.7%). Two pts were screened for the trial and did not fulfil the inclusion criteria (4.9%). One pt lost complete hematological response after 6 months and was switched to a different TKI and one pt had a persistent suboptimal response and switched TKI after 12 months on NI (4.9%). These two pts were considered as non-optimal responders at all time points after the change of treatment. All patients except one had typical transcripts (b2/a2 or b3/a2). One pt had an atypical transcript and was not available for MR4 assessment. At 18 months, 31 of 41 patients were evaluable for BCR-ABL levels. Thirteen out of 31 (42%) pts had MR4 or higher at 18 months (6 pts in the LLM group). Twenty six (84%) pts had MMR (10 in the LLM group), while five pts did not reach MMR at 18 months (4 pts with LLM). No statistically significant differences in the proportion of patients achieving MR4 or MMR could be detected between the two groups. In conclusion, T315I, Y253H, E255K or F359V mutations in CD34 selected cells below the detection limit of sanger sequencing do not predict the achievement of MR4 or higher in patients on nilotinib in the ENEST1st study. Therefore, screening for low level BCR-ABL mutations at diagnosis of CML is currently not recommended. Disclosures Giles: Novartis: Honoraria, Research Funding. Hochhaus:Novartis: Research Funding; BMS: Research Funding; MSD: Research Funding; Ariad: Research Funding; Pfizer: Research Funding. Frank:Novartis: Employment. Lange:Novartis: Consultancy, Honoraria, Research Funding.

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Leukemic Stem Cells of Chronic Phase CML Patients Possess Uniquely Elevated BCR-ABL Kinase Activity and Acquire Spontaneous BCR-ABL Kinase Domain Mutations at a High Frequency.

Blood ◽

10.1182/blood.v106.11.438.438 ◽

2005 ◽

Vol 106 (11) ◽

pp. 438-438 ◽

Cited By ~ 1

Author(s):

Xiaoyan Jiang ◽

Kyi Min Saw ◽

Allen Eaves ◽

Connie Eaves

Keyword(s):

Stem Cells ◽

Tyrosine Kinase ◽

Kinase Activity ◽

Point Mutations ◽

Chronic Phase ◽

Kinase Domain ◽

Tyrosine Kinase Activity ◽

Abl Kinase ◽

Kinase Domain Mutations

Abstract Growing evidence indicates that the therapeutic potential of imatinib mesylate (IM) for the treatment of CML may be limited initially by a relative innate resistance of the leukemic stem cells and eventually by an accumulation of cells with BCR-ABL tyrosine kinase domain mutations. We now show that the amount and tyrosine kinase activity of p210-BCR-ABL in the most primitive and relatively IM-unresponsive lin−CD34+CD38− CML cells is 3 to 10-fold higher than in the majority of the lin−CD34+CD38+ CML progenitors (n=3). These results confirm previous BCR-ABL transcript data and identify elevated p210-BCR-ABL expression to be a likely important factor in the characteristic IM-insensitivity of very primitive CML cells. To determine whether in vivo, CML stem cells also accumulate gene mutations affecting the BCR-ABL kinase domain, cDNAs were prepared from RNA extracts of purified lin−CD34+CD38− cells isolated from 3 chronic phase patients that had not received IM therapy. Bidirectional sequencing of individually cloned cDNAs from these samples revealed BCR-ABL kinase domain mutations in 2 of the 3 patients at frequencies of 10% (1/10), 20% (2*/10,*identical mutations). Incubation of these lin−CD34+CD38− cells in vitro for 2–3 wk ± a high concentration of IM (up to 10 μM, which was sufficient to reduce the tyrosine kinase activity in the input cells by 70±12% and in their 2 wk progeny by 10±5%) selected a subpopulation of more differentiated and completely IM-resistant cells. This was shown in Western blots by the inability of 10 μM IM to reduce either their p210-BCR-ABL tyrosine kinase activity or CrkL phosphorylation and in methylcellulose assays ±5 μM IM. As predicted, IM-selected cells showed a higher frequency of kinase domain mutations (13–20% vs 0–20% of cDNA clones analyzed from 3 wk cells cultured ±IM). Analysis of individual colonies produced from CFCs in the cultured cells showed all (21/21) colonies from IM-selected cells had mutations vs 50% (5/10) in those cultured without IM. The total frequency of mutant cDNAs detected was also increased in the IM-resistant cells (35–55% vs 10–25% mutant cDNAs in selected vs control cells). Interestingly, in most cases, both wild-type and mutant cDNAs were identified in the same colony, indicating de novo generation of mutations in vitro. Overall, >50 different mutations were identified. These included 10 point mutations previously associated with clinical IM resistance (including G250 and T315), another 13 point mutations previously identified in a comprehensive mutational screen, and >20 previously undescribed mutations. Several of the latter affect the critical region of the P loop, the c-helix and the activation loop and would be predicted to confer significant IM resistance. To investigate the possibility that the observed genomic instability of very primitive CML cells might be related to their elevated innate p210-BCR-ABL activity, BCR-ABL transcript levels in individual IM-selected, fully resistant and control (similarly treated but no IM exposure) colonies were compared. This showed that BCR-ABL transcripts were ~20-fold higher (P<0.05) in the resistant colonies (30 assessed from 3 patients). These findings suggest that the increased BCR-ABL expression and activity that uniquely characterizes the most primitive CML cells may contribute not only to their innate insensitivity to IM but also to a deregulation of genomic stability leading to the emergence of IM-resistant mutants and other subclones associated with disease progression.

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Correlation of Clinical Response to Nilotinib with BCR-ABL Mutation Status in Advanced Phase Chronic Myelogenous Leukemia (CML-AP) Patients with Imatinib-Resistance or Intolerance.

Blood ◽

10.1182/blood.v110.11.1940.1940 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1940-1940 ◽

Cited By ~ 2

Author(s):

Giuseppe Saglio ◽

Dong-Wook Kim ◽

Andreas Hochhaus ◽

Simona Soverini ◽

P. Erben ◽

...

Keyword(s):

Disease Progression ◽

Cytogenetic Response ◽

Kinase Domain ◽

Mutant Clone ◽

Imatinib Resistance ◽

Mutation Status ◽

Abl Kinase ◽

Imatinib Resistant ◽

Kinase Domain Mutations

Abstract The 2nd-generation bcr-abl inhibitor nilotinib is more potent than imatinib (IC50 <30 nM) against unmutated bcr-abl and active against 32/33 imatinib-resistant BCR-ABL mutants in vitro. We investigated the in vivo activity of nilotinib stratified by the baseline BCR-ABL mutation status in 127 imatinib-resistant or -intolerant CML-AP patients (pts) enrolled in an open-label phase II trial of nilotinib. Eighty-five pts (85/127, 67%) were screened prior to nilotinib therapy for BCR-ABL kinase domain mutations by direct sequencing. Of the 85 pts, 75 (88%) were resistant to imatinib and 10 (12%) were intolerant using standard published criteria. Twenty-two different baseline mutations involving 19 amino acids were identified in 50 (59%) pts analyzed. Other 35 (41%) pts did not have a baseline mutation. The most frequent mutation types identified included M351T (8 pts), G250E (7 pts), Y253H (6 pts), M244V (5 pts), F359V (5 pts) and T315I (5 pts). Twenty-two percent of pts with baseline mutations (11/50) showed more than one mutation (9 with two, 1 with three, and 1 with four mutations). All baseline mutations occurred in imatinib-resistant pts but none in intolerant pts. After 12 months of therapy, confirmed (confirmed in two consecutive analyses 4 week apart) hematologic response (HR) was achieved in 48% (21/50), major cytogenetic response (MCR) in 20% (10/50), and complete cytogenetic response (CCR) in 16% (8/50) of imatinib-resistant pts with baseline mutation versus 44% (12/25), 40% (10/25), and 20% (2/25) of imatinib-resistant pts without baseline mutation, respectively. Responses appeared to be affected by the in vitro sensitivity of the mutant clone against nilotinib. Pts with less sensitive mutation (cellular IC50 of >200nM: Y253H, E255K, E255V, F359C) representing 13% (11/85) of all patients assessed for baseline mutation, showed 13% (1/11) HR and 13% (1/11) MCyR compared to 74% (17/28) and 18% (5/28) respectively in the mutant group with IC50 of ≤200 nM. The nilotinib resistant T315I mutation occurred in 5 pts. Only one of these 5 pts who had T315I and G250E dual mutation achieved HR conceivably reflecting the sensitivity of G250E or non-mutant clone to nilotinib. At the time of data analyses, 50% of pts with baseline mutation were free of disease progression versus 62% of pts without baseline mutation. Rate of progression was 64% (7/11) in the group with less sensitive mutations and 60% (3/5) in pts. with T315I. However, the mutants most frequently associated with progression were F359V and M244V both having 4/5 pts (80%) progressed. In summary, BCR-ABL kinase domain mutations were identified at baseline in 59% of all pts in this cohort and in 67% of pts with imatinib resistance. Responses were observed across a broad spectrum of mutant genotypes. The rate of responses and disease progression may be affected by the baseline mutation types, although a larger data set with longer follow up is needed to further establish the correlation.

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Optimal Treatment for Patients after Dasatinib or Nilotinib: Comparison of Preclinical Activities of Approved and Promising Investigational Kinase Inhibitors Against BCR-ABL Kinase Domain Mutations That Confer Clinical Resistance to Dasatinib or Nilotinib.

Blood ◽

10.1182/blood.v110.11.4589.4589 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4589-4589

Author(s):

Corynn Kasap ◽

Christopher Weier ◽

Neil P. Shah

Keyword(s):

Second Generation ◽

Kinase Inhibitors ◽

Kinase Inhibitor ◽

Kinase Domain ◽

Inhibitor Therapy ◽

Drug Resistant ◽

Imatinib Resistance ◽

Abl Kinase ◽

Clinical Resistance ◽

Kinase Domain Mutations

Abstract The optimal management of patients with chronic myeloid leukemia (CML) is increasingly reliant upon molecular studies. Loss of response to imatinib in CML is most commonly associated with selection for a limited number of BCR-ABL kinase domain mutations that impair the ability of imatinib to effectively bind to BCR-ABL Molecular understanding of imatinib resistance mechanisms has led to the development of effective “second generation” BCR-ABL kinase inhibitors, such as dasatinib and nilotinib, which have clinical activity against most, but not all, drug-resistant mutations. Analysis of the BCR-ABL kinase domain in patients who develop resistance to second-generation inhibitors has implicated further selection of drug-resistant BCR-ABL kinase domain mutants in nearly all cases reported to date. Encouragingly, the number of resistant mutations capable of conferring clinical resistance to the most clinically-advanced second-generation agents, dasatinib (approved by the US FDA and EMEA) and nilotinib (approved in Mexico and Switzerland), appears to be restricted to a relatively small number of amino acid substitutions. As clinical experience with dasatinib and nilotinib grows, an understanding of the relative sensitivities of dasatinib- and nilotinib-resistant BCR-ABL mutants to other kinase inhibitors, both approved and investigational, is critical to optimize clinical outcomes in patients with resistance to dasatinib or nilotinib. At the present time, kinase inhibitor therapy options for patients with resistance to one of these agents include the investigational options bosutinib and MK-0457 (VX-680), as well as dasatinib and nilotinib (for patients not yet exposed to one of these agents) and re-exposure imatinib. It is likely that the success of therapeutic intervention in these cases can be predicted based upon the preclinical sensitivity of the mutation(s) involved with the agent chosen. We have therefore conducted a thorough biochemical and biological cross-analysis of the activities of each of these clinically-useful kinase inhibitors against mutations that confer clinical resistance to dasatinib or nilotinib. These studies provide clinicians with a useful reference for choosing an appropriate kinase inhibitor based upon the identity of the resistant BCR-ABL kinase domain mutation(s) detected at the time of relapse when faced with a patient who has lost response to dasatinib or nilotinib. It is hoped that the application of such “personalized medicine” strategies to the clinical management of CML cases will further improve outcomes in patients treated with kinase inhibitor therapy.

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A Novel β-catenin Inhibitor, AV65 Suppresses the Growth of CML Cell Lines Which Acquire Imatinib-Resistance Because of Abl Kinase Domain Mutations Including T315I and Hypoxia-Adaptation.

Blood ◽

10.1182/blood.v112.11.1081.1081 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1081-1081 ◽

Cited By ~ 2

Author(s):

Rina Nagao ◽

Shinya Kimura ◽

Eishi Ashihara ◽

Miki Takeuchi ◽

Ruriko Tanaka ◽

...

Keyword(s):

Stem Cells ◽

Cell Lines ◽

Second Generation ◽

Kinase Domain ◽

Parental Cell ◽

Imatinib Resistance ◽

Abl Kinase ◽

P Glycoprotein ◽

Imatinib Resistant ◽

Kinase Domain Mutations

Abstract Imatinib and the second-generation ABL tyrosine kinase inhibitors (TKIs) such as dasatinib and nilotinib have dramatically changed the management of CML. However, these agents are effective neither for CML cells harboring T315I mutation nor for CML stem cells. Genetic analysis of blasts from CML patients in blast crisis has identified numerous members of the Wnt/b-catenin pathway as being activated (Radich et al, PNAS 2006) and loss of b-catenin has been reported to impair the renewal of CML stem cells (Chao et al, Cancer Cell 2007). Thus, b-catenin signaling inhibition may be useful for CML treatment. We herein describe the effects of a novel b-catenin inhibitor, AV65 on various imatinib-sensitive and –resistant CML cell lines (Table 1). Eight imatinib-sensitive human CML cell lines and one normal cell line derived from hepatocyte were exposed to AV65 for 72 hours and its anti-proliferative effects were examined by MTT assay. AV65 induced apoptosis in these CML cell lines time- and does-dependent manners and inhibited the growth of all eight CML cell lines with IC50 at ranging from 9.8 to 33.1nM. While that for hepatocyte was 204.8nM. Interestingly, AV65 induced polyploidies in K562 but not in BV173. In addition, AV65 augmented the anti-proliferative effects of imatinib additively against K562 and BV173. These findings suggested that AV65 alone or the combination with imatinib was effective for imatinib-sensitive CML cells. To investigated the effects of AV65 against imatinib-resistant CML cells, four imatinib-resistant CML cell lines such as K562/IMR (bcr-abl amplification), MYL-R1 (LYN overexpression), KBM5/ STIR (harboring T315I) and K562/D1-9 (P-glycoprotein overexpression), and BaF3 cells expressing the wild type BCR-ABL or its ten different mutant BCR-ABL forms including T315I, were used. There was little difference on the induction of apoptosis and anti-proliferative effects of AV65 except K562/D1-9 between each parental cell line and its resistant clone and AV65 inhibited the growth of all examined BaF3 cells harboring various mutations including T315I with IC50 at ranging from 21.6 to 46.5nM. IC50 values of AV65 for K562 and K562/D1-9 were 11.0 and 60.1nM, respectively. These findings suggested that the effects of AV65 were independent either to BCR-ABL expression level, ABL mutations or LYN overexpression, but might be affected by P-glycoprotein. Next, the effects of AV65 against the hypoxia-adapted CML cell lines such as K562/ HA and KCL22/HA were also investigated. These cell lines were resistant to imatinib, dasatinib, INNO-406 (another second-generation ABL TKI; Kimura et al, Blood 2005) and alkylating agents via the up-regulation of glyoxalase-I, a detoxification enzyme for the cytotoxic byproducts of glycolysis. Intriguingly, AV65 inhibited the growth of hypoxiaadapted CML cell lines at almost same concentration compared with their parental cell lines. Although CML stem cell niche has not been definitely identified, it might located in hypoxia because the inoculated human leukemia cells to immunodeficient mice preferably localized on the surface of osteoblasts in the epiphysis (Ninomiya et al, Leukemia 2007) where could be hypoxic. Taken together, AV65 may be effective for CML stem cells in hypoxia. In conclusion, AV65 inhibited the growth of CML cell lines which acquire imatinib-resistance because of Abl kinase domain mutations including T315I and hypoxiaadaptation. Therefore, AV65 may become a promising agent for CML treatment including both imatinib and the second-generation Abl TKIs-resistant patients. Figure

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