Dasatinib Resistance Research Articles

Therapeutic implications of Src independent calcium mobilization in diffuse large B-cell lymphoma

We report that 38% of primary large B-cell lymphoma (DLBCL) tested expressed active Src family kinases, which are targeted by dasatinib. The expression of active Src family of kinases (SFK) in primary DLBCL tumors correlated with unfavorable prognostic markers such as Ki67 and Mum1. Using four DLBCL cell lines we found that: (1) sensitivity to dasatinib (but not imatinib) varied 400-fold; (2) dasatinib resistance was associated with distinct signaling profiles downstream of BCR activation. In particular, although Src family kinase phosphorylation was inhibited by 100–150 nM dasatinib in all cell lines, this failed to inhibit BCR-mediated Blnk phosphorylation, calcium signaling and proliferation in a dasatinib resistant cell line.

Primary del 17 chronic lymphocytic leukaemia lymphocytes are hypersensitive to dasatinib in vitro

Primary del 17 chronic lymphocytic leukaemia lymphocytes are hypersensitive to dasatinib <i>in vitro</i>

Outcome of Patients with Chronic Myeloid Leukemia with T315I BCR-ABL Mutation

The most frequent causes of imatinib resistance are BCR-ABL mutations, usually selected during treatment. Most of them destabilize the inactive conformation necessary for imatinib binding. The T315I mutation, located at the ATP binding pocket is resistant to imatinib and other available kinase inhibitors and is associated with a poor outcome. The aim of this study was to evaluate the frequency of T315I mutation in patients with CML resistant to imatinib and to describe their outcome. Mutation analysis was performed in two institutions, through direct automated sequencing. Total RNA was extracted from peripheral blood or bone marrow and amplification of the kinase domain of ABL from BCR/ABL was performed, using a semi-nested RT-PCR, to cover amino acids 244–486. PCR product was submitted to direct automated sequencing and compared with normal sequences of BCR-ABL gene (M14752, GenBank). A total of 173 resistant imatinib resistant patients were analyzed. BCR-ABL mutations were detected in 47 out of 173 (27%) patients. Among these patients, 14 (30%) presented T315I mutation. The mutation was found in ten patients during imatinib treatment, in one before start of imatinib, in three after dasatinib (2) and nilotinib (1) failure. Three patients have more than one mutation analysis, performed during different time-points of treatment: imatinib resistance, dasatinib resistance (3) and bone marrow transplantation relapse (1). Overall survival from diagnosis was 39,5% and 17,5% from mutation detection, in a median time of 69 and 13 months respectively. After imatinib resistance, eight patients were treated with dasatinib, one with nilotinib and had no hematological response; tree were treated with Hydrea and two were submitted to allogeneic bone marrow transplantation. Five patients died due to disease progression in median time of 19 months after mutation detection. One patient died after bone marrow transplantation, due to GVHD and infection. Eight patients are alive, three in CP, two in BC, two in hematological response and one in complete cytogenetic response after bone marrow transplantation. In conclusion, in this study we confirmed the high frequency of T315I mutation in imatinib resistant patients and their poor prognosis. Most of them started imatinib in a late chronic or advanced phase, with exception of two patients which started imatinib 800mg in early chronic phase, suggesting that there was inhibition of Ph positive sensitive cells and selection of resistant clones.

Bcr-Abl-Independent Activation of Src Kinases Associated with Development of Dasatinib Resistance in a CML Patient.

Activation of Src family of kinases (SFK) is associated with chronic myeloid leukemia (CML) disease progression and resistance to imatinib (IM) therapy. Activation of SFK can be either Bcr-Abl-dependent or Bcr-Abl-independent in IM-resistant CML patients and clinical importance of this phenomenon is not completely understood. Second generation of tyrosine kinase inhibitors (TKIs) such as dasatinib, targeting both Bcr-Abl kinase as well as SFK, could represent a logical alternative for treatment of patients with acquired IM resistance due to SFK activation. Recent studies raised question whether overexpression of SFK could lead to an acquired resistance to dasatinib. Sensitivity of individual patients' leukemia cells to TKIs can be carried out by assessment of inhibition of phosphorylation of Crkl and SFK after incubation of patient's leukocytes with the drug in vitro. We used 10 μM IM or 250 nM dasatinib in vitro and detection with western blot analysis with anti-Crkl 32H4 monoclonal antibody (Cell SignalingTechnology, Beverly, MA) and Phospho-Src Family (Tyr416) Antibody (Cell Signaling Technology Inc., Danvers, MA). By this functional approach we identified several IM-resistant CML cases, where IM failed to inhibit phospho-Crkl and phospho-SFK in vitro; these patients were either in blast crisis or carried a BCR-ABL mutation. In most cases, however, phospho-SFK were inhibited after incubation of the patients' leukocytes with dasatinib. The exception was a patient with hematological resistance to IM and acquired resistance to dasatinib therapy due to Bcr-Abl-independent activation of SFK. This patient was a 56-year-old female diagnosed with CML in chronic phase. The cytogenetic examination at the time of diagnosis revealed Ph chromosome and an additional aberration involving derivative chromosomes 2, 3 and 5. After 10 months of IM therapy the BCR-ABL-positive clone was eradicated, but the clinical response to the treatment was unsatisfactory. At the same time the number of cells with the complex additional aberration remained high (>70%). Western blot analysis of the patient's leukocytes revealed overexpression of phospho-SFK suggesting activation of an alternative signaling pathway not inhibited by IM and independent of Bcr-Abl. In vitro sensitivity to dasatinib provided a rationale for switch to dasatinib therapy. Therefore, dasatinib was administered to the patient (2×70 mg daily). However, a rapid development of resistance to dasatinib followed, which corresponded with a loss of inhibition of SFK in in vitro phosphorylation assay. Interestingly, the phenotype of the patient's disease resembles a mouse model of v-src gene induced myeloproliferative disease, characterized by splenomegaly, anemia, and increased numbers of immature erythroid cells (Keller G and Wagner EF, Genes Dev. 1989; 3: 827–37). Our data suggest that the oncogene driving myeloproliferative disease in this patient belongs to SFK. We demonstrate that the detection of activated SFK by immunoblot could lead to recognition of Bcr-Abl-independent and SFK-dependent resistance to dasatinib.

Dasatinib sensitizes primary chronic lymphocytic leukaemia lymphocytes to chlorambucil and fludarabine in vitro

The dual c-abl/Src kinase inhibitor, dasatinib, utilized to treat chronic myeloid leukaemia (CML) when used at clinically attainable sublethal concentrations, synergistically sensitized primary chronic lymphocytic leukaemia (CLL) lymphocytes to chlorambucil and fludarabine. In contrast, dasatinib alone demonstrated toxicity to CLL lymphocytes at concentrations that are generally not clinically attainable. Dasatinib resistance and poorer dasatinib-mediated sensitization to chlorambucil and fludarabine was associated with higher expression of c-abl protein levels. In contrast, chlorambucil and fludarabine resistance correlated with basal p53 protein levels. Moreover, Western blot analysis after in vitro treatment of primary CLL lymphocytes with dasatinib, chlorambucil and/or fludarabine, showed that dasatinib: (i) inhibited c-abl function (e.g. downregulation of c-abl protein levels and decreased the phosphorylation of a c-abl downstream target, Dok2), (ii) decreased chlorambucil/fludarabine induced accumulation of p53 protein levels, (iii) altered the response to chlorambucil/fludarabine induced DNA-damage as evidenced by an increase in chlorambucil/fludarabine-induced H2AX phosphorylation, and (iv) accentuated the c-abl downregulation induced by chlorambucil/fludarabine. Our results suggest that dasatinib in combination with chlorambucil or fludarabine may improve the therapy of CLL.

Dasatinib Resistance in Patients with Chronic Myelogenous Leukemia: Identification of a Novel bcr-abl Kinase Domain Mutation

BackgroundDasatinib is a multi–tyrosine kinase–specific inhibitor with potent inhibitory activity against bcrabl kinase. It has been increasingly used in the treatment of imatinib-resistant chronic myelogenous leukemia (CML) and Philadelphia chromosome–positive acute lymphoblastic leukemia. However, despite its excellent activity, a proportion of patients present evidence of dasatinib resistance. Patients and MethodsTo assess the potential mechanisms of resistance, we have analyzed the bcr-abl kinase domain from 22 patients with CML who demonstrated clinical evidence of dasatinib failure. ResultsMutations were present in 16 of 22 patients, and the majority were mapped to the adenosine triphosphate binding pocket. Of these, 7 had panresistant T315I, and 4 had F317L, with 1 F317I mutation. In addition, 3 of 16 patients evaluated had a V299L mutation. All of these mutations were previously reported in association with dasatinib resistance. We also observed that 1 of 7 patients in the chronic phase and 5 of 15 patients in the advanced phase had > 1 mutation in separate clones, as well as compound mutations (1 mRNA had ≥ 2 mutant codons). Interestingly, 4 of 15 patients with documented mutations were found to have a novel V304D mutation, which is located outside of the dasatinib binding site in the β 4-5 loop structure. ConclusionIt appears that bcr-abl mutations in advanced-phase CML are more common but occur at lower frequencies in patients with chronic-phase disease (93% vs. 62%, respectively). Our results suggest that selection for T315I and F317L mutations occurs frequently in dasatinib-resistant patients, and both mutations were previously shown to affect dasatinib binding. Two additional mutations (V299L and V304D) have been identified with incidences that exceed random occurrence. The significance of these mutations is being evaluated.

Characteristics of Dasatinib- and Imatinib-Resistant Chronic Myelogenous Leukemia Cells

Although dual src-family kinase/BCR/ABL inhibitor, dasatinib (BMS-354825), provides therapeutic advantages to imatinib-resistant cells, the mechanism of dasatinib resistance was not fully known. We used TF-1 BCR/ABL cells, by introducing the BCR/ABL gene into a leukemia cell line, TF-1 and K562, and established dasatinib- (BMS-R) and imatinib-resistant (IM-R) cells. We characterized chronic myelogenous leukemia drug-resistant cells and examined intracellular signaling. The IC(50) of dasatinib was 0.75 nmol/L (TF-1 BCR/ABL), 1 nmol/L (K562), 7.5 nmol/L (TF-1 BCR/ABL IM-R), 10 nmol/L (K562 IM-R), 15 micromol/L (TF-1 BCR/ABL BMS-R), and 25 micromol/L (K562 BMS-R). The number of BCR/ABL copies in resistant cell lines was the same as the parental cell line by fluorescence in situ hybridization analysis. There was no mutation in Abl kinase. We found that protein levels of BCR/ABL were reduced in dasatinib-resistant cell lines. BCR/ABL protein was increased by treatment of an ubiquitin inhibitor. The Src kinase, Lck, as well as mitogen-activated protein kinase and Akt were activated, but p21(WAF), phosphatase and tensin homologue was reduced in K562 BMS-R cells. Removal of dasatinib from the culture medium of K562 BMS-R cells led to apoptosis, and activated caspase 3 and poly (ADP-ribose) polymerase. These results suggest that the expression and protein activation signatures identified in this study provide insight into the mechanism of resistance to dasatinib and imatinib and may be of therapeutic chronic myelogenous leukemia value clinically.

Dasatinib Resistance in CML Patients. Identification of Novel BCR-ABL Kinase Domain Mutation.

Dasatinib is a dual specific SRC-ABL kinase inhibitor which has been increasingly used in treatment of CML and Ph + ALL patients. Although it has excellent activity, a proportion of patients, particularly those with advanced disease had developed evidence of dasatinib resistance. In order to assess the reasons for resistance we have analyzed BCR-ABL kinase domain utilizing single step PCR reaction followed by amplification of ABL kinase domain with subsequent subcloning (≥10 clones) from 23 CML patients who demonstrated clinical evidence of dasatinib failure. We have found mutations in the BCR-ABL domain in 19 out of 23 patients. 14 were mapped to ATP “binding pocket”, of those, seven had pan-resistant T315I and five had F317L with one F317I mutation. In addition, three of 19 patients had V299L mutation. All of these mutations were previously reported in association with dasatinib resistance. We also observed that one of 8 patients in chronic phase and three of 15 patients in advanced phase had two different mutations in separate clones§. Interestingly, four of 19 patients were found to have novel V304D mutation, which is located outside of dasatinib binding site in β 4–5 loop structure and may not be directly involved in dasatinib resistance. It appears that BCR-ABL mutations in advanced phase CML are more common, but occur at lower frequency in dasatinib resistant patients in chronic phase, 93% vs. 62%. In conclusion, our clinical sample suggests that selection for T315I and F317L mutations occurs frequently in dasatinib resistant patients which was predicted from preclinical study. Both mutations were proposed to directly and indirectly affect dasatinib binding. Two additional mutations have been identified with incidence that exceeds random occurrence. The significance of these mutations (V299L, D304D) will be further studied.Table 1Mutations status at different phases of CMLchronic phaseadvanced phasemutationsyes 5 §no 3yes 14§no 1T315I27F317L14F317I01V299L12V304D131 patient in chronic phase group have subcloning results pending.

SGX70393 Inhibits Bcr-AblT315I In Vitro and In Vivo and Completely Suppresses Resistance When Combined with Nilotinib or Dasatinib.

Overview: Bcr-AblT315I is detected in the majority of CML patients who relapse after dasatinib- or nilotinib-based second-line Bcr-Abl kinase inhibitor therapy. SGX70393, an azapyridine-based Abl kinase inhibitor, is effective against Bcr-Abl and Bcr-AblT315I at low nanomolar concentrations in vitro and in cell lines. Here, we comprehensively profiled SGX70393 against native and mutant Bcr-Abl in vitro and in vivo. We also used a cell-based mutagenesis screen to evaluate the resistance profile of SGX70393 alone and in combination with imatinib, nilotinib, or dasatinib.Methods: We assessed colony formation in the presence of SGX70393 by murine bone marrow infected with retroviruses for expression of Bcr-Abl, Bcr-AblT315I, or a variety of other mutants. Toxicity was tested in clonogenic assays of normal bone marrow. SGX70393 effects on cellular tyrosine phosphorylation were measured by immunoblot and FACS in primary Bcr-AblT315I cells isolated from patients with CML or Ph+ B-ALL. In vivo activity was evaluated in a xenograft model using Ba/F3 cells expressing Bcr-AblT315I. Lastly, the resistance profile of SGX70393 was evaluated alone and in dual combinations with imatinib, nilotinib, or dasatinib in a cell-based mutagenesis assay.Results: Colony formation by murine bone marrow cells expressing Bcr-AblT315I (IC50: 180 nM) was reduced by SGX70393 in a dose dependent manner, while no toxicity was observed in colony forming assays of normal human or murine mononuclear cells at concentrations up to 2 μM. Ex vivo exposure of human Bcr-AblT315I mononuclear cells to SGX70393 decreased CrkL phosphorylation, while imatinib, nilotinib, or dasatinib had no effect. SGX70393 inhibited Bcr-AblT315I-driven tumor growth in mice and this was correlated with reduced levels of pCrkL in tumor tissue, while imatinib was ineffective. A cell-based mutagenesis screen revealed a profile of resistant clones confined to four p-loop residues and position 317. SGX70393 in combination with imatinib contracted the spectrum of resistant mutations relative to either single agent, though outgrowth could not be completely suppressed. Combining SGX70393 with low concentrations of nilotinib or dasatinib narrowed the resistance profile still further (residues 248 and 255 for nilotinib; 317 for dasatinib) and, with clinically achievable doses of either second drug, completely abrogated emergence of resistant subclones.Conclusions:SGX70393, a potent inhibitor of Bcr-AblT315I, exhibits a resistance profile centered around the p-loop and residue 317 of Bcr-Abl. Remarkably, in combination with nilotinib or dasatinib, outgrowth of resistant clones is completely suppressed. Single-agent therapy with an effective T315I inhibitor may provide a viable option for patients who relapse with Bcr-AblT315I. However, as a broader spectrum of mutations accounts for imatinib resistance, patients with acquired dasatinib or nilotinib resistance may continue to harbor residual mutant clones other than T315I. Thus, the full clinical potential of SGX70393 may be realized in combinations with a second Abl kinase inhibitor. Our findings provide the first demonstration that Abl kinase inhibitor combinations that include a T315I-targeted component such as SGX70393 have the potential to pre-empt Bcr-Abl-dependent resistance.

BCR-ABL mutant kinetics in CML patients treated with dasatinib

Dasatinib is efficient in vitro against most of CML cells harboring ABL kinase domain mutations and induces high rates of response in imatinib-resistant CML patients. Here, we monitored the mutated BCR -ABL transcripts during the follow-up of 12 CML patients treated with dasatinib. We identified four groups of patients based on their sensitivity to dasatinib. Clinical responses were correlated to the in vitro sensitivity of BCR-ABL mutants to dasatinib, however, some discrepancies were observed in a subfraction of CML patients, suggesting subtle differences between in vitro observations and clinical entities and/or the onset of other mechanisms responsible for dasatinib resistance.

Mechanism of Drug Resistance to Dasatinib (BMS-354825) and Imatinib in Chronic Myelogenous Leukemia Cells.

The selective tyrosine kinase inhibitor, imatinib has shown remarkable clinical activity in patients with chronic myelogenous leukemia (CML), however, imatinib does not completely eradicate BCR/ABL-expressing cells, and cells resistant to imatinib develop. It has been reported that mutations in the kinase domain (KD) of BCR/ABL impairs binding of imatinib and causes resistance to it. So, additional therapy is necessary to overcome imatinib resistance in patients with an acute form of CML. The novel BCR/ABL inhibitor, dasatinib (BMS-354825), which A dual inhibitor of both Src and Abl kinases, suppresses the activity of these kinases at subnanomolar concentrations in imatinib resistance cells. Dasatinib is presently being evaluated in a clinical trial in CML patients with imatinib resistance, but drug resistance to dasatinib and imatinib are not fully evaluated. In this study, we used TF-1 BCR/ABL cell lines, which were transfected with the BCR/ABL gene, as well as parental TF-1 cells and K562 cell lines and established dasatinib resistant TF-1 BCR-ABL BMS-R and K562 BMS-R cells and imatinib resistant TF-1 BCR-ABL IM-R and K562 IM-R cells. We show here that dasatinib potently induced apoptosis of TF-1 BCR-ABL and K562 cells in 72 hours treatment. IC50 of dasatinib was 0.75nM (TF-1 BCR/ABL) and 1nM (K562) and IC50 of imatinib was 500nM (TF-1 BCR/ABL) and 750nM (K562). In the resistant cell lines, IC50 of dasatinib was 15μM (TF-1 BCR/ABL BMS-R) and 25μM (K562 BMS-R). TF-1 BCR/ABL BMS-R and K562 BMS-R cells were also resistant to imatinib (IC50: more than 10μM). TF-1 BCR-ABL IM-R and K562 IM-R cells were also resistant to dasatinib (IC50: 7.5 nM (TF-1 BCR/ABL IM-R) and the value was more than 10nM for K562 IM-R). There was no mutation in Abl kinase in these resistant cell lines suggesting that BCR/ABL mutation-independent mechanism is involved in resistance to imatinib and dasatinib. Because the correlation of parameters with defined protein expression patterns and protein signatures would predict disease progression and drug resistant, we investigated the protein expression pattern by using antibody microarray system (Antibody Microarray: Lab Vision Corporation). We found that dasatinib resistant cells had reduced protein levels of BCR/ABL. In TF-1 BCR/ABL BMS-R cells, Zap-70 protein was increased and activated. In contrast, the src kinase, lck, is increased and activated in K562 BMS-R cells. Moreover, mitogen-activated protein kinase (MAPK) and Akt were also activated in K562 BMS-R cells. We also found that src kinases, especially Lyn, as well as MAPK and Akt were activated in K562 IM-R cells. The phosphatase SHP-2 was decreased in dasatinib resistant cell lines. Signal-transducing activators of transcription 5 (STAT5) phosphorylation was reduced in K562 BMS-R cells. 24 hour removal of dasatinib from culture medium of K562 BMS-R led to apoptosis of the cells and activated caspase 3 and poly (ADP-ribose) polymerase (PARP). We also found that the phosphatase SHP-1 was increased after removal of dasatinib. The expression and protein activation signatures identified in this study provide insight into the mechanism of resistance to dasatinib and imatinib. Our study demonstrates development of dasatinib resistance and suggests that this information may be of therapeutic relevance.

Response to Dasatinib after Imatinib Failure According to Type of Preexisting BCR-ABL Mutations.

Dasatinib (BMS-354825) is a novel, oral, multi-targeted kinase inhibitor of BCR-ABL and SRC kinases with preclinical activity against 20/21 imatinib resistant BCR-ABL mutations and clinical phase I/II efficacy in patients with chronic myelogenous leukemia (CML) and BCR-ABL positive acute lymphoblastic leukemia (ALL). We sought to establish a relationship between type of preexisting BCR-ABL mutations associated with imatinib resistance and efficacy of dasatinib in patients (pts) with CML and ALL. We have investigated 872 peripheral blood samples from 394 pts (53% male, median age 60 yrs, range 17–85) who had been enrolled in international phase II studies investigating the activity of 70mg dasatinib BID after imatinib failure (chronic phase, CP, n=198; accelerated phase, AP, n=78; myeloid blast crisis, MyBC, n=53; lymphoid blast crisis, LyBC, or ALL, n=65).Screening for BCR-ABL mutations was performed by D-HPLC combined with DNA sequencing. During follow up, pts were monitored in 3-monthly intervals by RQ-PCR for BCR-ABL mRNA transcripts and by mutation analysis to determine the quantitative course of the preexisting mutation or the emergence of new mutations. Hematologic and cytogenetic response data have been collected sequentially for a median of 8 months (range, 2–11) after start of therapy. Prior to dasatinib, 46 different BCR-ABL mutations involving 36 amino acids were detected in 202/394 pts (51%). 162 pts showed one, 33 pts two, 6 pts three, and 1 pt four mutations. Mutations were observed in 84 pts in CP (42%), 47 pts in AP (60%), 23 pts in MyBC (43%), and 48 pts in LyBC and ALL (74%). In patients with mutations, hematologic response was 91% in CP, 62% in AP, 41% in MyBC, and 34% in LyBC/ALL (p<0.01), major and complete cytogenetic response did not differ significantly (47% and 34% in CP, 35% and 27% in AP, 33% and 28% in MyBC, 53% and 51% in LyBC/ALL). Major cytogenetic response rates were comparable in pts bearing no mutations (44%), mutations within the P-Loop (43%), SH2 domain (47%), activation loop (56%), or other sites (49%), but not for T315I (0%, p<0.001). Sorting individual pts by the underlying mutation and cellular IC50 values of dasatinib revealed clearly higher hematologic and cytogenetic response rates in those with lower IC50 values. In line with the virtual insensitivity to dasatinib in vitro, none of the 17 pts carrying a T315I mutation showed any hematologic response. Two distinct patterns of response were observed:a parallel decrease of the BCR-ABL load and the mutated clone ora decrease of the BCR-ABL load followed by a decrease of the mutated clone after a delay of up to 4–6 months.Up until now 5 pts developed new mutations associated with dasatinib resistance (T315I, n=2, AP and MyBC pts; F317L+F486S, n=2, LyBC and MyBC pts; E507G, n=1, CP pt). We conclude that dasatinib is capable of inducing hematologic and cytogenetic remissions in a significant proportion of imatinib resistant pts harboring BCR-ABL mutations, except T315I. Response dynamics depend on the individual type of mutation which may be a basis for individual dose adaptation according to the mutation pattern.

Monitoring Imatinib Resistance with a “Polony” Assay: Towards Tailored Therapy of Chronic Myelogenous Leukemia (CML).

Although tyrosine kinase inhibitor therapy with imatinib in CML patients leads to a high rate of long-term disease control, the development of resistance largely due to bcr-abl point mutations has become a therapeutic challenge. Dasatinib, a recently approved kinase inhibitor, and other new agents invariably have a unique pattern of activity against particular drug-resistant forms of BCR-ABL. It will therefore be helpful to determine the specific mutations that mediate resistance in order to employ the optimal alternative agents in relapsed or refractory patients. As such, there is a need for a rapid, sensitive, and quantitative assay to identify and monitor drug resistance mutations. We have adapted a method for genotyping single nucleotide polymorphisms to mutation detection in clinical samples of CML patients. This “polony” technique entails solid phase PCR amplification of single DNA molecules in a polyacrylamide gel (forming a PCR-colony, or “polony”), followed by discrimination of wild type and mutated nucleotides by a fluorescent single nucleotide base extension assay. The technique is versatile and sensitive (detection limit: 1 mutant in 10−4 wild type cells); it allows us to quantify the BCR-ABL to ABL ratio and to haplotype and screen for several mutations in the same cell population. We have used this assay to screen for the most common BCR-ABL mutations in CML to discern the dynamics of specific mutations following treatment with BCR-ABL kinase inhibitors. One imatinib-treated patient sustained a cytogenetic relapse and was switched to dasatinib. This patient was found to have a T315I mutation that caused subsequent dasatinib resistance. The polony assay detected this mutation from a rare presence to the dramatic takeover in concert with the patient's rapid disease progression. In another imatinib-treated patient who sustained a cytogenetic relapse on imatinib the polony assay detected an M244V mutation, modestly resistant to imatinib (IC50 3.1μM). Doubling the imatinib dose the patient regained cytogenetic response, which was documented by the polony assay. A third patient who exhibited primary resistance to imatinib was switched to nilotinib after progression to accelerated phase, but without success. The polony assay identified an E255K mutation, known to be quite resistant to imatinib (IC50 5.2μM), relatively resistant to nilotinib (IC50 200nM) but sensitive to dasatinib (IC50 5.6nM). Dasatinib therapy resulted in a rapid hematologic response, with a drop in WBC from 60,000 to 5000, and a complete clearance of blasts. The polony assay showed a rapid decrease of the E255K mutation from a frequency of almost 100 percent to about 20 percent. These patients and others in our clinical surveys illustrate the importance of having a reliable means of detecting and quantifying the mutational burden of patients in order to make informed treatment decisions. A prospective clinical trial testing the role of mutational monitoring in guiding the choice of novel agents is required to prove the likely clinical utility of the polony assay in the current era of burgeoning target-directed therapy in CML.

The Most Common Dasatinib-Resistant BCR-ABL Kinase Domain Mutations in Patients with Chronic Myeloid Leukemia Are Sensitive to VX-680: Rationale for Early Combination Kinase Inhibitor Therapy.

Selection for CML cells with BCR-ABL kinase domain mutations represents the predominant molecular mechanism responsible for loss of response to imatinib. Similarly, we have found acquired resistance to dasatinib to be associated with kinase domain mutations in 100% of cases (n=15). However, unlike the multitude of imatinib resistant mutations, which to a large extent occur at non-contact residues and destabilize the inactive confirmation to which imatinib binds, only two mutations appear to be responsible for nearly all cases of dasatinib resistance, T315I and V299L (Shah et al, submitted, ASH 2006). Both of these mutations occur at critical contact residues between the ABL kinase domain and dasatinib. Successful treatment of dasatinib-resistant cases will therefore require strategies to successfully eliminate cells that harbor these mutations.Use of a combination of kinase inhibitors with the ability to collectively suppress all BCR-ABL kinase domain mutants would be predicted to lead to profoundly minimize disease resistance and relapse on targeted therapy. Although the combination of imatinib and dasatinib may prevent selection of the dasatinib-resistant V299L mutation, these agents share many targets, and their combination may therefore result in substantial toxicity. Moreover, the combination of imatinib and dasatinib is not predicted to effectively inhibit the growth of cells harboring BCR-ABL/T315I.We previously have shown that the Aurora kinase inhibitor VX-680 can bind to the ABL kinase domain and inhibit the kinase activity of the T315I mutation at low micromolar concentration. VX-680 is showing early signs of efficacy in CML cases associated with the T315I mutation. Interestingly, the co-crystal structure of VX-680 reveals that V299 is one of 14 contact residues within the ABL kinase domain. Substitution of leucine at this residue might therefore be expected to diminish the potential affinity of VX-680 for BCR-ABL/V299L. However, analysis of the amino acid sequence of Aurora-A revealed divergence from native BCR-ABL at this the corresponding amino acid position due to the presence of a leucine in Aurora-A. BCR-ABL/V299L is therefore a mimetic of Aurora-A, and as a result, predicted to retain sensitivity to VX-680.We therefore assessed VX-680 for its ability to inhibit the kinase activity of BCR-ABL/V299L in Ba/F3 cells and found effective inhibition of the kinase activity at low micromolar concentration. Consistent with predictions based upon structural considerations, the V299L mutation is somewhat more sensitive to VX-680 than BCR-ABL/T315I. We confirmed these results in an analysis of primary human PBMCs obtained from a dasatinib-resistant patient who had evolved the V299L mutation on therapy.Our findings suggest that early combination therapy with two kinase inhibitors, dasatinib and VX-680, may successfully suppress resistant disease by collectively eliminating BCR-ABL kinase domain mutation as a mechanism of resistance, and thereby achieve effective long-term disease control in the vast majority of patients.

Molecular Analysis of Dasatinib Resistance Mechanisms in CML Patients Identifies Novel BCR-ABL Mutations Predicted To Retain Sensitivity to Imatinib: Rationale for Combination Tyrosine Kinase Inhibitor Therapy.

Point mutations within the BCR-ABL kinase domain represent the most common mechanism of resistance to imatinib in patients with CML. Preclinical studies have shown that dasatinib (BMS-354825) is effective at inhibiting the kinase activity of imatinib-resistant BCR-ABL mutants with the notable exception of the T315I mutation, which remains highly resistant to imatinib, dasatinib, and AMN107 (Gorre et al, Science 2001; Shah et al, Science 2004; Weisberg et al, Cancer Cell, 2005). Clinical data from Phase I and II studies of dasatinib in CML confirms the in vitro findings. Each of three imatinib-resistant patients bearing the T315I mutation (CP=1; AP=2) did not achieve objective hematologic or cytogenetic responses during treatment with dasatanib on a Phase I study. Additionally, each of two phase II patients with the T315I mutation (CP=1; LBC=1) treated at UCLA showed no evidence of objective response. We have also detected the T315I mutation in each of two cases of acquired resistance in a phase II (LBC =2) study, and in seven of nine patients with acquired resistance to dasatinib in phase I and II studies (CP=1; MBC=3; LBC=2; Ph+ ALL=1).Notably, we detected a novel BCR-ABL mutation, T315A, in one of the two patients who relapsed without a detectable T315I mutation. The patient is a 53 year-old female whose chronic phase CML had progressed to myeloid blast phase while being treated with imatinib. The imatinib-resistant mutation M244V was identified prior to dasatinib treatment. The patient achieved a major hematologic response (<5% blasts with partial recovery of peripheral blood counts) on dasatinib 90 mg orally given twice daily, but relapsed with MBC after six months. Sequence analysis of the BCR-ABL kinase domain at the time of relapse revealed the presence of the imatinib-resistant mutation M244V as well as the novel mutation T315A. This finding is of particular interest because T315A and several other novel BCR-ABL mutations were recently recovered in a saturation mutagenesis study designed to define potential mechanisms of dasatinib resistance. Remarkably, many of these mutations retain sensitivity to imatinib in vitro (Burgess et al, PNAS, 2005). Through periodic molecular monitoring of other dasatinib-treated patients, we have identified a second novel BCR-ABL mutant, F317I, that developed in an imatinib-resistant CP patient after 9 months of treatment. Similar to T315A, F317I was isolated in the saturation mutagenesis screen for dasatinib resistance and is predicted to retain sensitivity to imatinib. Taken together, our findings implicate the T315I mutation as the principle mechanism of resistance to dasatinib, but more importantly, strongly support the use for combination kinase inhibitor therapy in CML to prevent emergence of drug resistant clones. A phase I trial to assess the safety of combining imatinib with dasatinib is planned.