BCR-ABL Tyrosine Kinase Domain Research Articles

Bcr-Abl Tyrosine Kinase Domain Mutations Both Pre-Exist and Develop during Imatinib Treatment and Are Responsible for Resistance in Patients with Philadelphia-Chromosome Positive Acute Lymphoblastic Leukemia (Ph+ALL).

Background: Point mutations in the tyrosine kinase domain (TKD) of BCR-ABL are an important cause of resistance to imatinib (IM) in pts. with CML and Ph+ ALL. The significantly inferior response to IM in Ph+ALL pts. who failed prior chemotherapy compared to those with de novo Ph+ALL suggests that treatment with cytotoxic drugs may promote the development of TKD mutations. However, it is not known whether the frequency and pattern of TKD mutations at the time of treatment initiation with TK inhibitors are related to disease stage or prior anti-leukemic therapy. Moreover, the potential of combined treatment with IM and multi-agent chemotherapy to influence the development of mutational resistance, as compared to IM alone, has not been determined.Patients and methods: 51 pts. with newly diagnosed Ph+ALL (>55 yrs.) enrolled in a GMALL study of combined IM and chemotherapy, and 68 Ph+ALL pts. who had failed prior chemotherapy and received single-agent IM as salvage therapy were analysed for the occurrence of point mutations within the TKD. Bone marrow samples collected pre-treatment, during therapy and at relapse were examined by denaturing high-performance liquid chromatography (D-HPLC) and cDNA sequencing.Results: The frequency of TKD mutations pre-IM was 44% in newly diagnosed Ph+ALL and 53% (34/64) in pts. with advanced Ph+ALL. At relapse after combination therapy (n=19), the frequency of de novo ALL pts. harbouring a TKD mutation had increased to 89% (P-loop 47%, T315I 29%, A-loop 24%), 2 pts. (11%) showed wild-type BCR-ABL. The frequency of TKD mutations in pts. with advanced disease who relapsed after IM was 55% (P-loop 73%, T315I 23%, A-loop 4 %).In both patient groups, the D-HPLC pattern showed concordance between the mutation detected in pre-therapeutic specimens and the dominant mutation detected at relapse.The CR rate in de novo pts. receiving IM induction was 90 % irrespective of detectable mutations pre-study. Bcr-abl transcripts became undetectable during the course of therapy in 40% of pts. with and 37% of pts. without a mutation. Median remission duration in pts. with a T315I mutation (n=4) was 130 d (range: 53–319d), in contrast to 526 d (range: 504–549d) with activation loop and 411 d (range: 106–745d) with P-loop mutations. To date, 7 pts. with an initially detected mutation remain in CR after median FU of 12.8 mo (range 2.4–24.5 mo.).Conclusions: Bcr-abl TKD mutations are detectable prior to first imatinib exposure in approximately 50% of Ph+ALL patients. Clinical imatinib resistance is in most cases associated with the identical mutation detected pre-IM, which is not eradicated by the combination of chemotherapy and IM. Identification and elimination of TKD mutations during early stages of treatment is essential to improve treatment.

Chronic Myeloid Leukemia Stem Cells Possess Unique Properties That Predict Intrinsic and Acquired Resistance to Imatinib Mesylate.

Chronic myeloid leukemia (CML) is sustained by a clonally amplified, but still rare population of BCR-ABL+ hematopoietic stem cells (HSCs). Understanding their properties is assuming increasing importance in light of recent evidence that they are innately resistant to BCR-ABL−targeted therapies and that, over time, the CML clone acquires BCR-ABL tyrosine kinase domain mutations. The relative resistance of CML HSCs (lin−CD34+CD38− cells) to imatinib mesylate (IM) may be explained at least in part by their elevated expression of BCR-ABL and higher tyrosine kinase activity than is seen in the more prevalent lin−CD34+CD38+ leukemic cells. We have now obtained formal support for this concept from experiments with a BCR-ABL−transduced BaF3 cell line in which p210BCR-ABL expression can be regulated by doxycycline (dox)-mediated activation of an upstream tet-regulated repressor. Treatment of these cells for 48 hr with 0.1–5 μM IM in the absence of IL-3 and in the presence of 0.1–1 μg dox/ml confirmed the expected dox-control of intracellular phospho-CrkL levels and demonstrated a corresponding p210BCR-ABL kinase activity-dependent effect on IM sensitivity. To investigate the possibility that CML HSCs possess other unique properties that contribute to their relative resistance to many therapies, we compared the expression of 3 transporter genes (OCT1, ABCB1 and ABCG2) in CML cells at different stages of differentiation. Interestingly, we found that transcript levels for OCT1 (which regulates IM uptake) were very low in the most primitive (lin−CD34+CD38−) normal bone marrow cells and progressively increased (>100-fold) as these cells differentiate into mature (lin+CD34−) cells (n=4). Importantly, this difference in OCT1 expression was exaggerated in CML samples where OCT1 transcripts were even lower in the most primitive populations (below the level of detection in 2 of the 4 CML HSC isolates analyzed). Conversely, transcript levels for ABCB1 and ABCG2 (which regulate the efflux of many drugs) were highest in the normal lin−CD34+CD38− (HSC) population and lowest (>6-fold) in the most mature lin+CD34− normal cells, and again this difference was enhanced in the CML samples. The combination of a very low expression of OCT1 (low IM uptake) and highly elevated expression of ABCB1 and ABCB2 (high drug efflux) and BCR-ABL (elevated kinase activity) in CML HSCs identifies multiple mechanisms that would predict their broad insensitivity to IM and other therapeutics. In addition, we have found both by direct sequencing of cloned transcripts and allele-specific RT-PCR analyses, that a significant fraction of BCR-ABL transcripts in CML HSCs from chronic phase patients who have never received BCR-ABL-targeted therapy contain readily detectable kinase domain mutations (20–33%, n=3). A high incidence of mutations was also found at 2 additional sites in the BCR-ABL gene 5′ to the kinase domain and extending into the region encoded by BCR. After primitive CML cells had been cultured for 3 weeks (± 5 μM IM), we frequently found new BCR-ABL mutants in their clonal progeny. These in vivo and in vitro findings suggest that primary CML HSCs are also characterized by a high degree of genetic instability. We have thus identified multiple unique features of chronic phase CML HSCs that underscore the importance of evaluating these critical cells when considering new therapeutic approaches.

Hematologic and Cytogenetic Response Dynamics to Nilotinib (AMN107) Depend on the Type of BCR-ABL Mutations in Patients with Chronic Myelogeneous Leukemia (CML) after Imatinib Failure.

Nilotinib (AMN107) is a novel, orally active aminopyrimidine-derivative, which selectively inhibits the BCR-ABL tyrosine kinase with greater potency compared with imatinib (Im). In preclinical models, nilotinib was 20–50 times more potent than imatinib in Im-sensitive CML cell lines, and 3–7 times more potent in Im-resistant cell lines. Activity of nilotinib was also demonstrated in 32/33 Im-resistant mutant cell lines. We sought to investigate the efficacy of nilotinib in vivo according to the type of preexisting BCR-ABL mutations. We have investigated peripheral blood samples from 101 CML patients (pts, 63m, 38 f) with a median age of 61 yrs (range, 23–85) who had been enrolled in an international phase II study investigating the efficacy and safety of 400mg nilotinib bid after Im failure (chronic phase, CP, n=64; accelerated phase, AP, n=22; blast crisis, BC, n=15). Screening for BCR-ABL mutations was performed by D-HPLC combined with DNA sequencing. The analysis covered amino acids 207–517 of the BCR-ABL tyrosine kinase domain. The proportion of the mutated clone was estimated from sequencing plots by comparison to plots from serial standard dilutions of Ba/F3 cell lines harboring various concentrations of mutated BCR-ABL. Hematologic and cytogenetic response data are available for a median of 4 mo (range, 1–10) after start of nilotinib therapy. Prior to nilotinib, 28 different BCR-ABL mutations involving 22 amino acids were detected in 61/101 pts (60%). Nine pts showed two, three pts three, and one patient four mutations. Mutations were observed in 37 pts in CP (49%), 15 pts in AP (68%), and 9 pts in BC (60%). In pts with mutations, the overall rate of hematologic response was 70% (78% in CP, 75% in AP, 25% in BC) compared with 88% in pts without mutations. In CP CML, complete cytogenetic response was achieved within 3–6 mo in pts with mutations with high in vitro sensitivity to nilotinib according to O'Hare et al., Cancer Res 2005 and Weisberg et al., Br J Cancer 2006 (M244V, 38nM, n=2; M351T, 15nM; H396R, 41nM; D276G+M351T, 69 and 15nM; E355G+L387F, 47 and 46nM). 4 pts with mutations resulting in relatively higher cellular IC50 to nilotinib had progressed (Y253F+E255K, 125 and 200nM; L248V, 102nM; Y253H, 450nM; F359V, 175nM). 2 pts with small clones (ratio mutated BCR-ABL/ABL 0.14% and 8.6%) harboring the BCR-ABL mutation T315I being highly resistant to Im and nilotinib in vitro did not show signs of relapse after one and 11 mo of nilotinib therapy, respectively. During therapy, novel BCR-ABL mutations (Y253F, E255V, T315I, and F359V) appeared in 4 pts. In conclusion, preliminary data suggest that nilotinib may overcome most of the mutation-associated resistance to Im, and may have an important therapeutic role in Im resistance and in frontline CML therapy to prevent emergence of resistant clones. Response dynamics depend on the individual type of the mutation which may be the basis for individualized dosage of nilotinib according to the mutation pattern. The molecular analysis of the total phase II study population will be reported at the meeting.

Novel tyrosine kinase inhibitors in chronic myelogenous leukemia

The successful introduction of the tyrosine kinase inhibitors has initiated a new era in the management of chronic myeloid leukemia. Imatinib therapy has significantly improved prognosis of chronic myeloid leukemia. A minority of patients with chronic-phase disease (4% annually) and considerably more in advanced stages develop resistance. This is attributed, in 40-50% of cases, to the development of BCR-ABL (breakpoint cluster region/Abelson oncogene) tyrosine kinase domain mutations that impair imatinib binding. This has led to the development of more potent novel tyrosine kinase inhibitors that can overcome both BCR-ABL-dependent and BCR-ABL-independent mechanisms of resistance. Preliminary results of phase I and II trials with dasatinib and nilotinib have provided promising data that may reduce disease progression and potentially prevent acquired resistance to the tyrosine kinase inhibitors. Novel tyrosine kinase inhibitors with more potent and selective Bcr-Abl inhibition and with multitargeted inhibition of Bcr-Abl and Src family kinases are promising and may further improve prognosis in chronic myeloid leukemia.

AMN107 (nilotinib): a novel and selective inhibitor of BCR-ABL.

Chronic myelogenous leukaemia (CML) and Philadelphia chromosome positive (Ph+) acute lymphoblastic leukaemia (ALL) are caused by the BCR-ABL oncogene. Imatinib inhibits the tyrosine kinase activity of the BCR-ABL protein and is an effective, frontline therapy for chronic-phase CML. However, accelerated or blast-crisis phase CML patients and Ph+ ALL patients often relapse due to drug resistance resulting from the emergence of imatinib-resistant point mutations within the BCR-ABL tyrosine kinase domain. This has stimulated the development of new kinase inhibitors that are able to over-ride resistance to imatinib. The novel, selective BCR-ABL inhibitor, AMN107, was designed to fit into the ATP-binding site of the BCR-ABL protein with higher affinity than imatinib. In addition to being more potent than imatinib (IC50<30 nM) against wild-type BCR-ABL, AMN107 is also significantly active against 32/33 imatinib-resistant BCR-ABL mutants. In preclinical studies, AMN107 demonstrated activity in vitro and in vivo against wild-type and imatinib-resistant BCR-ABL-expressing cells. In phase I/II clinical trials, AMN107 has produced haematological and cytogenetic responses in CML patients, who either did not initially respond to imatinib or developed imatinib resistance. Dasatinib (BMS-354825), which inhibits Abl and Src family kinases, is another promising new clinical candidate for CML that has shown good efficacy in CML patients. In this review, the early characterisation and development of AMN107 is discussed, as is the current status of AMN107 in clinical trials for imatinib-resistant CML and Ph+ ALL. Future trends investigating prediction of mechanisms of resistance to AMN107, and how and where AMN107 is expected to fit into the overall picture for treatment of early-phase CML and imatinib-refractory and late-stage disease are discussed.

Overcoming Resistance to Imatinib Mesylate in a CML Cell Line by Downregulation of P-gp with siRNAs.

Imatinib Mesylate (Gleevec, formely STI571 from NOVARTIS) is a highly selective drug, capable of inhibiting the BCR-ABL tyrosine kinase which is responsible for CML. However, resistance to this drug has been documented, mainly due to an overexpression of the BCR-ABL protein, to mutations in the tyrosine-kinase domain of BCR-ABL itself or to an overexpression of the multidrug resistance protein 1 (P-gp). In the current study we investigated if downregulation of P-gp expression with siRNAs, in a cell line resistant to Imatinib which overexpressed P-gp (K562Dox), would allow to overcome resistance to this drug. In order to optimise transfection of the siRNAs, we used a fluorochrome labelled control-siRNA and fluorescence microscopy to evaluate the % of uptake of the siRNA under different transfection conditions. The optimised conditions (200 nM siRNA using the jetSi™ transfection reagent) allowed us to obtain uptake of siRNAs in 53% of cells. In order to confirm if the siRNAs were capable of downregulating the expression of P-gp we carried out, 24h after transfection, Real-Time Quantitative PCR studies and Western Blots of cells: i) treated with medium only (Blank), ii) transfected with the siRNAs suspension buffer, iii) transfected with a control siRNA (CRNAi), iv) transfected with two different siRNAs for P-gp (MDR-FE or MDR-CR). We verified that in cells treated with the MDR-CR siRNA there was a reduction in the P-gp mRNA expression of 26%, when compared to the CRNAi. Furthermore, when cells were treated with the MDR-FE or MDR-CR siRNAs, protein levels were also reduced by 33% and 57% respectively, when compared to the CRNAi. It was then verified if this downregulation of MDR expression caused sensitization of cells to Imatinib, in comparison to control cells, using both viability (Trypan Blue) and apoptosis (TUNEL) assays. For this purpose, Imatinib Mesylate was added to the cells previously transfected with siRNAs (for P-gp or control siRNAs) and to cells in exponential growth (Blank cells). Results showed that there was an enhancement of the effect of Imatinib in cells previously transfected with both P-gp siRNAs (MDR-FE or MDR-CR). This increase in the effect of Imatinib was due to an increase in cellular apoptosis. Indeed, the % of apoptotic cells increased from 3 % in cells treated with solvent to 7 % in cells treated with 1 μM Imatinib. It was concluded that downregulation of P-gp protein expression by RNAi technology permitted sensitisation of CML cells to Imatinib, suggesting that P-gp inhibition may be considered as an adjuvant therapy in cases of Imatinib resistance mediated by this mechanism.

A Randomized Phase II Study of Single-Agent Imatinib Versus Chemotherapy Therapy in Elderly Patients with Philadelphia-Positive Acute Lymphoblastic Leukemia (Ph+ALL) Including Analysis of Resistance Patterns.

Background: Elderly pts. with Ph+ALL have a very poor prognosis with a low CR rate, high induction mortality and short remission duration. Single-agent Imatinib (IM) has shown rapid anti-leukemic activity and a favorable toxicity in advanced Ph+ALL.Study design: To determine the value of single-agent IM as induction (ind.) therapy, we designed a prospective, randomized multicenter trial comparing a 4 week course of IM at 600 mg/d (IndIM) with age-adapted multi-agent chemotherapy (Indchemo) as front-line treatment of elderly Ph+ALL (>55 yrs.) patients. In addition, we investigated the tolerability and efficacy of an IM-based consolidation therapy in which responding pts. received successive chemotherapy cycles parallel to IM, given continuously for one year.Methods: As mutations of the BCR-ABL tyrosine kinase domain (KD) constitute the leading cause of resistance to IM, we evaluated 17/21 responding pts. at the time of hematologic relapse for the presence of these mutations using direct sequencing and denaturing HPLC.Results: To date, 52 pts. with newly diagnosed Ph+ALL (49) or lyBC (n=3) and a median age of 68 (58–79) yrs. were randomly assigned to receive IndIM (n=25) or Indchemo (n=27). Three pts. not entered in the study but treated according to the Indchemo regimen are included in the analysis. Twenty-four of 25 pts. assigned to IndIM received a CR (96%) and 1 pt. a PR (4%). In comparison, 14 of 23 evaluable pts. allocated to Indchemo achieved a CR (61%) and 2 pts. a PR (9%), while 7 pts. failed treatment (30%) (p=0.004). Two deaths occurred during Indchemo and none during IndIM. Severe infections during ind. developed in 13 of 23 evaluable pts. (57%) assigned to Indchemo and in 3 of 25 pts.(12%) receiving IndIM (p=0.002). After a median follow-up of 8.2 (0.6–35.4+) mo. 17 pts. are in ongoing CR and 21 pts. have relapsed (10 pts. during study, 7 pts. after end of study and 4 pts. after discontinuation). Ten pts. died in CR a med. of 4.1 (0.4–11.7) mo. after starting treatment and 1 pt. died after allogeneic SCT. Estimated DFS and OS at 18 mo. is 27±8% and 43±8%, respectively. Seventeen of 45 pts.(38%) with serially analysed MRD-levels achieved a molecular remission during treatment. Med. DFS was 15,7 mo. in molecular responders compared to 8.3 mo. in pts. remaining MRD positive (p=0.03). At the time of relapse 10 different BCR-ABL tyrosine KD mutations were detected in 13 of 17 evaluated pts. (76.5%). P-loop mutations were detected in 77% (10 of 13 pts.), activation loop and the T315I mutation each in 15% (2 of 13) of pts. In 3 pts. (23%) two different mutations were detected. In both pts. harboring the T315I mutation, it was already present at the time of diagnosis conferring an inferior remission duration of 1.8 and 5.2 mo.Conclusion: Single-agent IM is highly effective as ind. therapy in elderly pts. with Ph+ALL and is superior to standard chemotherapy induction with respect to tolerability, induction mortality and CR-rate. Monitoring of resistance patterns will help to identify candidates at risk of relapse and can guide treatment with 2nd generation tyrosine kinase inhibitors.

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.

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.

Early Detection of Imminent Hematologic Relapse Due to BCR-ABL Kinase Domain Mutations in CML Patients on Imatinib Therapy by D-HPLC.

Mutations of the BCR-ABL tyrosine kinase domain constitute the major cause of resistance in chronic myeloid leukemia (CML) patients (pts) on imatinib monotherapy. Sensitivity of conventional sequencing may allow the detection of a proportion of 10% mutated cells. We sought to improve the diagnostic armamentarium to screen for mutations prior to frank relapse. A total of 95 pts (chronic phase, CP, n=47; accelerated phase, AP, n=27; myeloid blast crisis, BC, n=19; lymphoid BC, n=2) who relapsed during imatinib therapy were screened for BCR-ABL kinase domain mutations applying denaturing high performance liquid chromatography (D-HPLC) and direct sequencing. Mutations were detectable in 47/79 (59%) pts with hematologic relapse and in 8/16 (50%) pts with cytogenetic relapse. 22 different point mutations affecting 18 amino acids and a novel deletion of 81bp of ABL exon 4 mapping to the P-loop region of the kinase domain were observed. To investigate the dynamics of the mutated clones D-HPLC was applied to 453 cDNA samples tracking back from relapse towards start of imatinib therapy. D-HPLC was optimized to detect 0.1–0.5% BaF3BCR-ABL cells harboring various mutations in a background of unmutated cells. Hematologic relapse occurred after a median of 12.9 mo (range 0.9–44.2) of imatinib therapy. However, BCR-ABL mutations became first detectable by D-HPLC at a median of 5.8 mo (range 0.0–30.5) after commencing imatinib (p<0.0001). Nine pts (18%; CP, n=4; AP, n=3, myeloid and lymphoid BC, n=1 each) showed evidence for BCR-ABL mutations even prior to imatinib therapy (T315I, n=4; M351T, n=3; M244V, Y253H, n=1 each). The median interval between first detection of the mutation and relapse was 5.6 mo in CP, 8.1 mo in AP, and 2.4 mo in myeloid BC. P-loop mutations were revealed at a median of 2.8 mo, activation loop mutations 2.9 mo, and T315I 6.3 mo prior to relapse. Cytogenetic relapse occured at a median of 19.2 mo (range 10.3–36.3) after start of imatinib therapy. BCR-ABL mutations became first detectable at a median time of 15.8 mo (range 0.0–26.4) after commencing imatinib (difference n.s., p=0.061). In two pts BCR-ABL mutations (M244V in AP, L324Q in CP) were observed in a small clone prior to imatinib therapy. Twenty-five CP pts in continuous complete cytogenetic remission were screened for BCR-ABL mutations to determine the predictive value of minor clones harboring mutations for consecutive relapse. No mutation was found in any of these pts. We conclude that (i) D-HPLC is a reliable and sensitive method to screen for BCR-ABL mutations before and during therapy with tyrosine kinase inhibitors. (ii) The observation of BCR-ABL mutations during imatinib therapy is predictive for relapse. (iii) Mutations may be detectable several months before hematologic relapse, and (iv) the sensitive detection of small mutated clones could provide clinical benefit by triggering early therapeutic interventions, which should be demonstrated in prospective clinical trials.

Double-Gradient–Denaturing-Gradient Gel Electrophoresis for Mutation Screening of the BCR-ABL Tyrosine Kinase Domain in Chronic Myeloid Leukemia Patients

Chronic myeloid leukemia (CML) is a rare hematopoietic stem cell disorder characterized by the t(9;22) translocation. This somatic event leads to the formation of the BCR-ABL fusion gene, which is translated in a functional protein (1). The Bcr-Abl oncoprotein differs from the endogenous c-Abl protein in both its subcellular localization and its tyrosine kinase (TK) activity. The deregulated Abl TK activity of the Bcr-Abl protein initiates the oncogenic process and represents a target to TK inhibitors. Among a series of compounds exhibiting TK inhibition, STI 571 (imatinib) was found to be highly effective against Abl and its derivative Bcr-Abl (2)(3). This molecule targets the inactive conformation of the kinase, which leads to stabilization of the protein in its inactive form and impairs ATP binding (4). Approved for the treatment of CML, imatinib induces hematologic and cytogenetic remissions in the chronic phase as well as in the blast phase (5)(6)(7). A significant proportion of patients, however, become resistant to the treatment. Different mechanisms of imatinib resistance have been described, in particular point mutations within the sequence of the BCR-ABL gene coding for the TK domain (8)(9)(10). Approximately 30 missense mutations have been identified in CML patients. Within the Bcr-Abl TK domain, these mutations are located in the nucleotide binding loop (P-loop), in the active site (imatinib contact site), or in the activation loop (A-loop; Fig. 1A⇓ ). Among these mutations, some prevent the binding of imatinib, and others make this binding more difficult. In the latter case, a dose increase can overcome the resistance. Recently, a novel TK inhibitor was described (11). In vitro, this molecule remains active for the majority of Bcr-Abl mutants, with the exception of T315I. Molecular monitoring of CML patients is based on the quantification …

Molecular Long-Term Surveillance of CML Patients on Imatinib Therapy. Follow-Up of German Patients Treated within the IRIS Trial.

High rates of complete cytogenetic response (CCR), the availability of sensitive methods to detect residual disease, and direct therapeutic consequences are leading motives to integrate regular molecular monitoring into the standards for the management of patients (pts) with chronic myeloid leukemia (CML). We sought to determine long-term dynamics of BCR-ABL mRNA expression levels in 132 CML pts (75 m, 57 f, median age 51, range 20–71 yrs) recruited into the IRIS study in 17 German centers. Pts were randomized to receive imatinib (n=69) or interferon α+Ara-C (IFN, n=63). Due to intolerance or lack of response 41 pts crossed over from IFN to imatinib. Response to therapy was sequentially monitored by conventional cytogenetics from bone marrow metaphases (n=806). BCR-ABL transcripts were determined in 1414 peripheral blood samples by quantitative real time RT-PCR (RQ-PCR) using the LightCycler technology. In case of low level (<10 transcripts/2μl cDNA) or neg RQ-PCR, nested PCR was performed. Total ABL transcripts were quantified as internal controls. A single series of BCR-ABL plasmid dilutions served as standard for both BCR-ABL and ABL transcripts. In pts on 1st-line imatinib therapy median ratios BCR-ABL/ABL gradually decreased: 4.8% at mo 3, 0.88% at mo 6, 0.22% at mo 12, 0.17% at mo 18, 0.058% at mo 24, 0.066% at mo 30, and 0.023% at mo 36. After crossover to imatinib results were not significantly different: 15.5% at mo 3, 1.6% at mo 6, 0.28% at mo 12, 0.068% at mo 18, 0.045% at mo 24, and 0.041% at mo 30. After a median follow-up of 40 mo (1–47) 31/69 pts (45%) on 1st-line imatinib were still RQ-PCR pos, 20 pts (29%) were RQ-PCR neg and nested PCR pos, and in 4 pts (5.8%) BCR-ABL became undetectable by RQ- and nested PCR. After a median time of 25 mo (3–43) on 2nd-line imatinib therapy 19/41 pts (46%) were RQ-PCR pos, 9 pts (22%) were RQ-PCR neg and nested PCR pos, and in 5 pts (12%) BCR-ABL was undetectable by RQ- and nested PCR. Considering adequate RNA quality BCR-ABL became repeatedly undetectable in 4 pts after 18–33 mo of 1st-line imatinib therapy and in 5 pts 9–33 mo after crossover from IFN to imatinib. In one patient, BCR-ABL remained undetectable after a treatment free interval of 4 weeks. After achieving CCR, 5 pts (7.2%) on 1st-line and 2 pts (4.9%) on 2nd-line imatinib therapy experienced cytogenetic relapse after a median time of 10 mo (4–21). In none of these pts mutations of the tyrosine kinase domain of BCR-ABL were detected. BCR-ABL/ABL ratios after 12 mo of imatinib therapy were significantly lower in pts in continuous CCR vs pts with subsequent relapse (0.18 vs 0.60%, respectively, p=0.04). None of the relapsing patients had achieved a ratio BCR-ABL/ABL <0.12% after 12 mo, which represents a 3-log reduction from baseline. During total follow-up ratios BCR-ABL/ABL <0.12% have been achieved in 51 pts (74%) on 1st-line and in 21 pts (51%) on 2nd-line imatinib therapy. We conclude that (i) treatment with imatinib in newly diagnosed CML pts is associated with a rapid and steady decrease of BCR-ABL transcript levels, (ii) a short trial of IFN does not jeopardize molecular response to subsequent imatinib therapy, (iii) an increasing minority of pts achieve complete molecular remission, and (iv) ratios of BCR-ABL/ABL <0.12% after 12 mo of therapy predict for long-term response.

Imatinib (STI571) resistance in chronic myelogenous leukemia: molecular basis of the underlying mechanisms and potential strategies for treatment.

Following the paradigm set by STI571, protein tyrosine kinase inhibitors are emerging as a promising class of drugs, capable of modulating intracellular signaling and demonstrating therapeutic potential for the treatment of proliferative diseases. Although the majority of chronic phase CML patients treated with STI571 respond, some patients, especially those with more advanced disease, relapse. This article reviews the reasons for relapse and, in particular, analyses resistance resulting from Bcr-Abl tyrosine kinase domain mutations at the molecular level. Arguments are based upon the structure of the STI571-Abl complex, which is compared to the crystal structures of PD173955-Abl and PD180970-Abl, which bind to the kinase differently. Strategies to potentially circumvent or overcome resistance are discussed.

Comparative gene expression profile of chronic myeloid leukemia cells innately resistant to imatinib mesylate

Objective Resistance to imatinib mesylate monotherapy is clearly a barrier to successful treatment of chronic myeloid leukemia (CML) patients. In some patients, resistance arises due to powerful selective pressure on rare cells that carry amplified copies of the BCR-ABL fusion oncogene or point mutations in the Bcr-Abl tyrosine kinase domain that affect binding of the drug to the oncoprotein. However, in a proportion of patients neither mechanism operates, and resistance appears to be a priori, existing prior to exposure to the drug. These mechanisms of imatinib resistance are poorly understood and may be heterogeneous. Materials and methods We have previously described such innate resistance to imatinib in subclones of a myeloid leukemia cell line, KCL22, in which imatinib exposure inhibits the activity of Bcr-Abl and yet fails to induce apoptosis. We describe here whole-genome expression analysis of imatinib-sensitive and -resistant cells derived from the original KCL22 line, using Affymetrix microarray analysis. Results We detected differential expression of 39 genes that correlate with the imatinib-resistant phenotype. The resistant cells overexpress several genes associated with the suppression of apoptosis or with conferral of a transformed phenotype. Conclusion Amongst the differentially-expressed genes correlating with imatinib resistance, several suggest the activation of alternative pathway(s) that maintain viability and growth independently of Bcr-Abl kinase activity. Given the high rate of primary imatinib resistance in blast crisis, the potential of activating such alternative pathways appears to correlate with disease progression.