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 ◽  
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.

Sequential Kinase Inhibitor Therapy in CML Patients Can Select for Cells Harboring Compound BCR-ABL Kinase Domain Mutations with Increased Oncogenic Potency: Rationale for Early Combination Therapy of ABL Kinase Inhibitors.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 751-751 ◽  
Author(s):  
Neil P. Shah ◽  
Brian Skaggs ◽  
Susan Branford ◽  
Timothy P. Hughes ◽  
John M. Nicoll ◽  
...  
Keyword(s):  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Kinase Domain ◽  
Inhibitor Therapy ◽  
Significant Finding ◽  
Abl Kinase ◽  
Imatinib Resistant ◽  
Clinical Resistance ◽  
T315i Mutation ◽  
Kinase Domain Mutations

Abstract A critical question in the targeted therapy era relates to whether treatment outcomes will be optimized by sequential or combinatorial use of targeted agents. Selection for CML cells with BCR-ABL kinase domain mutations is the main mechanism responsible for loss of response to imatinib. Dasatinib is an ABL tyrosine kinase inhibitor that has activity against nearly all imatinib-resistant mutations and is approved for the treatment of imatinib-resistant and -intolerant BCR-ABL-associated leukemias. Acquired clinical resistance to sequential use of dasatinib following imatinib failure has been observed. We analyzed the BCR-ABL kinase domain at the time of relapse in 15 patients who lost an initial response to dasatinib, and found evolution of a total of three new mutations at the time of relapse in all cases. The highly resistant BCR-ABL/T315I mutation was detected in 11 cases. The four remaining cases were associated with the evolution of novel mutations (V299L, 3 cases; T315A, 1 case). V299L was also detected in a fourth case that had also evolved T315I. These three dasatinib-resistant mutations were part of a small number of amino acid substitutions previously isolated in a preclinical mutagenesis screen for dasatinib resistance-conferring BCR-ABL mutations. While the T315I mutation is highly resistant to imatinib, V299L and T315A retain sensitivity to imatinib in vitro and have not been previously described in imatinib-resistant cases, raising the potential utility of imatinib rechallenge in select dasaitinib-resistant cases. A significant finding of our studies is the evolution of five unique “compound” mutations (i.e. greater than one mutation on a DNA strand) in the BCR-ABL kinase domain of patients treated sequentially with imatinib and dasatinib. It is noteworthy that although the imatinib-sensitive V299L and T315A mutations evolved in five cases, they were detected in the context of a pre-existing imatinib-resistant mutation in three of these cases, and these cases are therefore unlikely to respond to rechallenge with IM. The T315A mutation was detected in the context of 2 pre-existing IM-resistant mutations (M244V/L364I). Interestingly, in bone marrow transformation assays, the clinically-identified dasatinib-resistant M244V/L364I/T315A mutation was more potently oncogenic than non-mutated BCR-ABL, in contrast to the baseline imatinib resistant M244V/L364I, which like T315A in isolation, was less potent than native BCR-ABL Our studies of CML cases resistant to sequential kinase inhibitor therapy reinforce BCR-ABL kinase domain mutation as the predominant mechanism of resistance to kinase inhibitor therapy, and provide evidence that compound mutations acquired as a result of sequential therapy can not only limit further therapeutic options, but also create more biologically aggressive isoforms of BCR-ABL. Together, these findings provide a strong rationale for early treatment of CML with combinations of kinase inhibitors that have the capacity to collectively prevent selection of resistant kinase domain mutations.

Sorafenib and Nilotinib Are Candidates to Overcome Imatinib Resistance in Myeloproliferation with FIP1L1-PDGFRA.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2912-2912
Author(s):  
Nikolas von Bubnoff ◽  
Sivahari P Gorantla ◽  
Richard A. Engh ◽  
Taiana de Oliveira ◽  
Silvia Thoene ◽  
...  
Keyword(s):  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Kinase Domain ◽  
Cloning And Expression ◽  
Cross Resistance ◽  
Response Analysis ◽  
Resistance Mutations ◽  
Imatinib Resistance ◽  
Clinical Resistance ◽  
Kinase Domain Mutations

Abstract Abstract 2912 Poster Board II-888 Constitutively activated variants of PDGFRA, PDGFRB can be found in a subset of patients with myeloid neoplasms associated with eosinophilia. The most common is FIP1L1-PDGFRA (FP). Patients with PDGFR-A and -B rearranged myeloproliferation respond to treatment with imatinib. However, single cases of clinical resistance due to a secondary FP/T674I mutation have been reported. In CML, more than 40 different exchanges have been described that confer imatinib resistance, and sequential treatment with imatinib and novel Abl kinase inhibitors has become reality. Nilotinib and sorafinib are potent alternative inhibitors of PDGFR-A and -B. We therefore hypothesized that available PDGFR kinase inhibitors might produce specific profiles of secondary FP kinase domain mutations mediating inhibitor resistance. To this aim, we selected clones of FP expressing Ba/F3 cells resistant to rising concentrations of imatinib, nilotinib, and sorafinib. In these, we identified 27 different PDGFRA kinase domain mutations. Imatinib, nilotinib and sorafenib produced distinct profiles of resistance mutations. During selection with imatinib, FP/T674I predominated with rising concentrations. FP/T674I corresponds to Bcr-Abl/T315I, which is frequently found in imatinib resistant CML. In contrast to imatinib, nilotinib and sorafenib produced a significantly lower frequency of resistant cell clones. Also, T674I disappeared at therapeutic nilotinib concentrations in favour of T674I+T874I and D842V. Sorafinib displayed a distinct profile of mutations including D842V, but not T674I. Following cloning and expression of all FP variants, dose-response analysis indicated that full cross-resistance to all three inhibitors was limited to D842V, whereas the gatekeeper T674I exchange retained sensitivity to sorafenib and nilotinib, and T674I+T874I was responsive to sorafenib only. In silico structure modelling indicated that differences in inhibitor response observed in distinct clusters of FP mutations identified in drug-resistant clones are based on differences of sorafenib versus imatinib and nilotinib in key drug - protein target interactions in PDGFR family kinases. Besides direct inhibitor binding effects, we propose that the identified exchanges shift an inactive-active conformation equilibrium and thereby affect binding of type II inhibitors like imatinib, nilotinib and sorafenib. Our results predict PDGFR variants that might come up in patients with myeloproliferation positive for PDGFR-A or -B fusions treated with imatinib, nilotinib or sorafenib. These findings will help in selection of an appropriate second line PDGFR kinase inhibitor when resistance to imatinib emerges, and will guide drug design. Moreover, our data can be translated to other neoplasms driven by activated forms of PDGFR-A or -B including GIST, CMML, and dermatofibrosarcoma protuberans. Disclosures: Off Label Use: Use of sorafenib and nilotinib in myeloproliferation with prominent eosinophilia.

Several Bcr-Abl kinase domain mutants associated with imatinib mesylate resistance remain sensitive to imatinib

Blood ◽  
2003 ◽  
Vol 101 (11) ◽  
pp. 4611-4614 ◽  
Author(s):  
Amie S. Corbin ◽  
Paul La Rosée ◽  
Eric P. Stoffregen ◽  
Brian J. Druker ◽  
Michael W. Deininger
Keyword(s):  
Imatinib Mesylate ◽  
Kinase Inhibitor ◽  
Blast Crisis ◽  
Chronic Phase ◽  
Kinase Domain ◽  
Myelogenous Leukemia ◽  
Imatinib Resistance ◽  
Abl Kinase ◽  
Cellular Assays ◽  
Kinase Domain Mutations

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.

Overcoming Resistance in Chronic Myelogenous Leukemia

2013 ◽  
pp. 306-312
Author(s):  
Michael J. Mauro
Keyword(s):  
Chronic Myelogenous Leukemia ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Point Mutations ◽  
Kinase Domain ◽  
Drug Binding ◽  
Myelogenous Leukemia ◽  
Novel Therapy ◽  
Abl Kinase ◽  
Clinical Resistance

Resistance in chronic myelogenous leukemia is an issue that has developed in parallel to the availability of rationally designed small molecule tyrosine kinase inhibitors to treat the disease. A significant fraction of patients with clinical resistance are recognized to harbor point mutations/substitutions in the Abl kinase domain, which limit or preclude drug binding and activity. Recent data suggest that compound mutations may develop as well. Proper identification of clinical resistance and prudent screening for all causes of resistance, ranging from adherence to therapy to Abl kinase mutations, is crucial to success with kinase inhibitor therapy. There is currently an array of Abl kinase inhibitors with unique toxicity and activity profiles available, allowing for individualizing therapy beginning with initial choice at diagnosis and as well informed choice of subsequent therapy in the face of toxicity or resistance, with or without Abl kinase domain mutations. Recent studies continue to highlight the merits of increasingly aggressive initial therapy to subvert resistance and importance of early response to identify need for change in therapy. Proper knowledge and navigation amongst novel therapy options and consideration of drug toxicities, individual patient characteristics, disease response, and vigilance for development of resistance are necessary elements of optimized care for the patient with chronic myelogenous leukemia.

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 ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1081-1081 ◽  
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

Mutation Mediated Resistance to the Tyrosine Kinase Inhibitors Imatinib, Dasatinib & Nilotinib in Philadelphia Positive Leukaemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4245-4245 ◽  
Author(s):  
Lisa M O’ Connor ◽  
Stephen Langabeer ◽  
Shaun R. McCann ◽  
Eibhlin Conneally
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitors ◽  
Molecular Mechanisms ◽  
Second Generation ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Kinase Domain ◽  
Patient Specific ◽  
Abl Kinase ◽  
Clinically Significant

Abstract The Philadelphia chromosome is formed as a result of a reciprocal translocation between chromosomes 9 and 22 and results in the formation of the hybrid oncoprotein BCR-ABL. It is observed in over 95% of Chronic Myeloid Leukaemia (CML) and approximately 30% of adult Acute Lymphoblastic Leukaemia (ALL) cases. Imatinib Mesylate (IM), a tyrosine kinase inhibitor that specifically binds BCR-ABL in its inactive conformation has revolutionized therapy for CML and Ph+ ALL. However, resistance develops in a significant proportion of patients and is predominantly mediated by single base-pair substitutions within the BCR-ABL kinase domain leading to changes in the amino acid composition that inhibit IM binding whilst retaining BCR-ABL phosphorylation capacity. Second generation tyrosine kinase inhibitors such as Dasatinib and Nilotinib retain activity in IM-resistant patients due to less stringent binding requirements and represent viable alternatives for IM-resistant patients with a suitable molecular profile. In this study, we undertook to examine the molecular mechanisms underlying IM resistance. A cohort of 40 patients with either primary or acquired resistance or intolerance to IM was identified by persistent high or increasing levels of BCR-ABL transcripts determined by real-time quantitative PCR. An allele-specific PCR screen was used to sensitively detect the clinically significant T315I mutation, which renders patients insensitive to currently available tyrosine kinase inhibitors: five (12.5%) IM resistant/intolerant patients were T315I positive. To further elucidate the molecular mechanisms of mutation induced resistance, the BCR-ABL kinase domain was screened for the presence of a mutation using a sensitive denaturing high performance liquid chromatography (dHPLC) approach. Samples showing evidence of mutation were examined by direct sequencing to identify the mutation(s) present. Kinase domain mutations have been identified in 20 of the 40 (50%) patients examined to date and these include p-loop mutations (M244V, G250E, Q252H), IM-binding domain mutations (T315I), catalytic domain mutations (M351T), an activation-loop mutation (L387M). Three previously unreported mutations were identified in patients with indications of IM resistance (T267A, E275Q) and Nilotinib resistance (L273M). The L273 residue lies adjacent to a region of the BCR-ABL kinase domain bound by Nilotinib. Three patients were found to harbour mutations at two distinct kinase domain residues while one patient harboured mutations at three distinct residues, supporting the theory that patients who develop mutation-mediated resistance to one kinase inhibitor may become resistant to subsequent inhibitors by a similar mechanism. The identification of clinically significant mutations facilitates selection of alternative approaches to therapy such as dose escalation of IM, second generation tyrosine kinase inhibitors or allogeneic stem cell transplant, if eligible, at an early stage in a patient’s disease, tailoring patient specific approaches to therapy.

Dynamics of BCR-ABL Kinase Domain Mutations in Patients with Chronic Myeloid Leukemia (CML) after Treatment with One, Two or Three Tyrosine Kinase Inhibitors (TKI).

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 750-750 ◽  
Author(s):  
Elias Jabbour ◽  
Dan Jones ◽  
Hagop Kantarjian ◽  
Susan O’Brien ◽  
Guillermo Garcia-Manero ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitors ◽  
Kinase Inhibitors ◽  
Kinase Domain ◽  
Imatinib Resistance ◽  
Mutation Status ◽  
Abl Kinase ◽  
Kinase Domain Mutations ◽  
New Mutations

Abstract Dasatinib (D) and nilotinib (N) are potent tyrosine kinase inhibitors (TKIs) with activity against many imatinib (IM) resistant BCR-ABL kinase domain mutants, except T315I. In vitro mutant models have selected specific mutations occurring after incubation with IM, D and N. Therapy with these new TKI may select for patients with T315I or other mutations relatively insensitive to them. We assessed the change in mutation status of the bcr-abl kinase domain (codons 220 to 500) in 113 patients (pts) with CML who received therapy with D and/or N after imatinib failure. Median age was 60 years (range, 21 to 82 years). Seventy-one (63%) pts received prior interferon (IFN). Median time on imatinib was 28 months (range, 2 to 78 months). At the time of imatinib failure, mutations were detected in 46 of 85 (54%) pts who had DNA sequencing. The evolution of mutations after a second TKI was as follows (Table 1). Twenty pts received a third TKI after failing IM and a second TKI. The evolution of mutations in this cohort was as follows (Table 2). Overall, 19 of 101 evaluable pts (19%), cases had new mutations emerge following TKI switch 17 after a 2nd TKI (12 nilotinib, 5 dasatinib), and 2 after a 3rd TKI (2 dasatinib). We analyzed whether these N- and D-associated new mutations were at sites that have been detected following D and N treatment in vitro (Burgess et al, PNAS 2005; Bradeen et al, Blood 2006; Von Bubnoff et al, Blood 2006). Only 14/46 (30%) kinase domain mutations that developed after D (7) or N (7) corresponded with an in vitro-identified site. Only 5 of 134 (4%) mutations identified were T315I (3 after dasatinib, 2 after nilotinib), but the mutation status of these patients was unknown after IM. We conclude that the spectrum of mutations that develops in vivo after TKI switch is broader and includes common imatinib-resistance sites as well. There appears to no marked increase in the incidence of T315I mutation after TKI switch. Table 1. Dynamics of mutations after 2nd TKI Post IM mutation No. Post-2nd TKI Mutation (New + Same + Lost) *1 pt acquired new mutation with persistence of pre-existing mutation, 1 lost 3 mutations and acquired 1, and 1 pt lost 2 mutations. Nilotinib Dasatinib Absent 39 8+NA+NA/21 3+NA+NA/18 Present 46 3+20+3/26 2+16+2*/20 Unknown 28 8/9 13/19 Table 2. Dynamics of mutations after 3rd TKI Post IM mutation No. Post-3nd TKI Mutation (compared to status after 2nd TKI) (New + Same + Lost) Nilotinib Dasatinib Absent 5 0/1 1+NA+NA/4 Present 12 0+1+0/1 2+6+3/11 Unknown 3 1/3 NA

Bcr-Abl resistance screening predicts a limited spectrum of point mutations to be associated with clinical resistance to the Abl kinase inhibitor nilotinib (AMN107)

Blood ◽  
2006 ◽  
Vol 108 (4) ◽  
pp. 1328-1333 ◽  
Author(s):  
Nikolas von Bubnoff ◽  
Paul W. Manley ◽  
Jurgen Mestan ◽  
Jana Sanger ◽  
Christian Peschel ◽  
...  
Keyword(s):  
Imatinib Mesylate ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Point Mutations ◽  
Treatment Strategies ◽  
Kinase Domain ◽  
Abl Kinase ◽  
Advanced Phase ◽  
Clinical Resistance ◽  
Limited Spectrum

Abstract In advanced-phase chronic myeloid leukemia (CML), resistance to imatinib mesylate is associated with point mutations in the BCR-ABL kinase domain. A new generation of potent ABL kinase inhibitors is undergoing clinical evaluation. It is important to generate specific resistance profiles for each of these compounds, which could translate into combinatorial and sequential treatment strategies. Having characterized nilotinib (AMN107) against a large panel of imatinib mesylate–resistant Bcr-Abl mutants, we investigated which mutants might arise under nilotinib therapy using a cell-based resistance screen. In contrast to imatinib mesylate, resistance to nilotinib was associated with a limited spectrum of Bcr-Abl kinase mutations. Among these were mutations affecting the P-loop and T315I. Rarely emerging resistant colonies at a concentration of 400 nM nilotinib exclusively expressed the T315I mutation. With the exception of T315I, all of the mutations that were identified were effectively suppressed when the nilotinib concentration was increased to 2000 nM, which falls within the peak-trough range in plasma levels (3.6-1.7 μM) measured in patients treated with 400 mg twice daily. Our findings suggest that nilotinib might be superior to imatinib mesylate in terms of the development of resistance. However, our study indicates that clinical resistance to nilotinib may be associated with the predominant emergence of T315I.

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.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2175-2175 ◽  
Author(s):  
Neil P. Shah ◽  
Brian Skaggs ◽  
Susan Branford ◽  
Timothy P. Hughes ◽  
John M. Nicoll ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Kinase Domain ◽  
Aurora A ◽  
Abl Kinase ◽  
Micromolar Concentration ◽  
T315i Mutation ◽  
Contact Residues ◽  
Kinase Domain Mutations

Abstract 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.

Beyond the Gatekeeper: Imatinib- and Dasatinib-Resistant BCR-ABL/F317 Mutations Confer Cross-Resistance to VX-680 but Are Sensitive to a Structural Derivative of VX-680

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 725-725
Author(s):  
Corynn Kasap ◽  
Nicholas Hertz ◽  
Debora Makino ◽  
Kevan Shokat ◽  
John Kuriyan ◽  
...  
Keyword(s):  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Aurora Kinase ◽  
Hematologic Malignancies ◽  
Kinase Domain ◽  
Blast Phase ◽  
Abl Kinase ◽  
Aurora Kinase Inhibitors ◽  
Kinase Domain Mutations ◽  
High Degree

Abstract The management of chronic phase CML has been revolutionized by selective ABL tyrosine kinase inhibitor (TKI) therapy. Despite the effectiveness of these targeted agents, long-term control of blast phase CML and Ph+ ALL has been elusive, where the majority of patients relapse within 6–12 months. For blast phase CML and Ph+ ALL, two TKIs are currently approved: imatinib and dasatinib. While head-to-head comparisons of these agents have not been performed, it is generally believed that dasatinib is the more active agent for these phases of disease. In most cases, loss of response to these agents is driven by BCR-ABL kinase domain mutations. While more than 70 mutations have been associated with clinical resistance to imatinib, dasatinib appears vulnerable primarily to five mutations: V299L, T315A, T315I, F317I, and F317L. Of these, T315I and F317L are cross-resistant to imatinib. For the achievement of long-term remissions in blast phase CML and Ph+ ALL, a combination of TKIs that can collectively suppress all resistant BCR-ABL kinase domain mutations holds therapeutic promise. The BCR-ABL/T315I mutation, which confers a high degree of resistance to all approved BCR-ABL TKIs, has been referred to as a “molecular gatekeeper”, as it restricts access to a deeper hydrophobic pocket within the ABL kinase domain and makes an important stabilizing H-bond with imatinib, dasatinib and nilotinib. The Aurora kinase inhibitor VX-680 was the first compound to have activity against BCR-ABL/T315I in vitro, as well as clinically. To determine the promise of a kinase inhibitor combination of dasatinib and VX-680, we assessed the activity of VX-680 against the five dasatinib-resistant mutations using a cell-based flow cytometric assay of BCR-ABL kinase activity. While three mutants are sensitive, mutations at F317 demonstrated a high degree of resistance. We tested a number of other Aurora kinase inhibitors of different chemotypes and found that each of these had similar difficulty at inhibiting the kinase activity of BCR-ABL/F317 mutants. Based upon the co-crystal structure of VX-680 complexed with ABL, we have performed structure-activity relationship studies of 12 VX-680 scaffold derivatives, and have successfully identified structural modifications that increase kinase inhibitory activity against F317 mutants. Moreover, one of these derivatives increases the selectivity for ABL relative to Aurora kinases, which may help reduce the likelihood of suppressing normal hematopoiesis, a dose-limiting toxicity of Aurora kinase inhibitors that may substantially limit their effectiveness for the management of hematologic malignancies such as blast phase CML and Ph+ ALL. Lastly, we have performed structural studies of ABL/F317 mutants complexed with select VX-680 derivatives in an effort to understand how F317 mutations confer resistance to a broad range of ABL and Aurora kinase inhibitors. Interestingly, a recent study reported the successful selection of Aurora kinase inhibitor-resistant clones derived from a human colon cancer cell line (Girdler et al, 2008). While no resistance-conferring mutations were isolated at L154, the Aurora kinase gatekeeper residue, mutations were detected at Y156 in Aurora B, which corresponds to F317 in ABL. Aurora B Y156 mutations were found to confer resistance to a number of Aurora kinase inhibitors, including VX-680. As Aurora kinase inhibitors are being studied in a variety of non-hematologic malignancies, there is an increasing need to understand and overcome the mechanisms whereby mutations at this residue confer resistance to these agents. It is hoped that our studies will lead not only to the development of an effective adjunctive kinase inhibitor for the treatment of blast phase CML and Ph+ ALL, but will also shed light on the growing problem of resistance conferred by mutations at residues that correspond to BCR-ABL/F317 in other kinases.

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