Treatment with Nilotinib for Patients with Chronic Myeloid Leukemia (CML) Who Failed Prior Therapy with Imatinib and Dasatinib.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2171-2171
Author(s):  
Elias Jabbour ◽  
Hagop Kantarjian ◽  
Francis Giles ◽  
Susan O’Brien ◽  
Jorge Cortes
Keyword(s):  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Cross Resistance ◽  
New Agents ◽  
Prior Therapy ◽  
Abl Kinase ◽  
Imatinib Resistant ◽  
Blastic Phase ◽  
Complete Hematologic Response

Nilotinib (AMN107) is a selective Bcr-Abl kinase inhibitor which is aproximately 30-fold more potent than imatinib. Phase I and II studies confirmed the efficacy of nilotinib in imatinib-resistant CML. Dasatinib (BMS-354825) is a dual Src-Abl kinase inhibitor recently approved for the treatment of CML in all stages of the disease after imatinib failure. With the availability of more new tyrosine kinase inhibitors, one important question is the existence of cross-resistance between these new agents. To help answer this question, we analyzed the outcome of patients with CML who receive therapy with nilotinib after having failed therapy with dasatinib. Seven patients were treated: 6 had previously shown hematologic resistance to imatinib and one had intolerance to imatinib. Dasatinib failure was due to hematologic resistance in all 7 patients after a median of 16 weeks (range 4 to 32) on therapy. Median age was 50 years (range, 15 to 78 years); median follow-up on nilotinib was 5 months (range, 1 to 14 months). At the start of therapy with nilotinib, 1 patient was in chronic (CP), 1 in accelerated (AP), and 5 in blastic phase (BP). Mutations were found in 2 of 4 patients assessed before nilotinib therapy (F317L and E355G, respectively). All patients received nilotinib 400 mg BID (n=5) or 800 mg single daily dose (n=2). Of the 7 evaluable patients, one patient (in AP) responded (complete hematologic response). This patient had no mutations identified at baseline. At the last follow-up, 5 patients are alive: 3 with active disease, one in CHR, and one in complete molecular remission post allogeneic stem cell transplantation. We conclude that nilotinib has modest activity in patients with CML resistant to both imatinib and dasatinib.

Dasatinib (BMS354825), a dual Src-Abl inhibitor, is active in Philadelphia chromosome-positive chronic myelogenous leukemia (Ph+CML) following treatment failure with imatinib mesylate and AMN107

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 6530-6530 ◽  
Author(s):  
Z. Estrov ◽  
S. O’Brien ◽  
F. Giles ◽  
G. Garcia-Manero ◽  
G. Borthakur ◽  
...  
Keyword(s):  
Treatment Failure ◽  
Kinase Inhibitor ◽  
Philadelphia Chromosome ◽  
Therapy Response ◽  
Myelogenous Leukemia ◽  
Prior Therapy ◽  
Abl Kinase ◽  
Resistant Disease ◽  
Daily Dose

6530 Background: Dasatinib is a dual Src-Abl kinase inhibitor which is 300 fold more potent than imatinib and 20-fold more potent than AMN107 (a selective potent Bcr-Abl kinase inhibitor). Dasatinib also inhibits Src which may overcome CML resistance. Methods: Study Aims: To report the experience with dasatinib in patients who develop CML failure post AMN107. Patients and Methods: All patients on the ongoing dasatinib studies at our institution were reviewed for prior therapy/response to AMN107 and outcome on imatinib. Results: 16 patients with Ph+CML following treatment failure with AMN107 received dasatinib. All patients had previously shown hematologic resistance to imatinib. AMN107 failure was due to hematologic resistance in all 16 pts. Median age was 60 years (range 22 to 76 years); median follow-up on dasatinib is 3 months (range <1 to 9 months). CML phase was chronic in 2, accelerated in 6, blastic in 6, and second chronic/accelerated in 2. Abl mutations were found in 7/16 patients before dasatinib therapy: G250E+F359L, G250E, Y253F, Y253H, H396R (1 patient each), T315I (2 patients). All patients received dasatinib 70 mg orally BID (n = 10), 140 mg single daily dose (n = 5), or 120 mg orally BID (n = 1). Responses were observed in 5 of 7 patients with mutations including 4/4 with P loop mutations and 1/2 with other mutations; the 2 patients with T315I had resistant disease. Conclusions: Dasatinib is active in patients with CML resistant to both imatinib and AMN107. [Table: see text] [Table: see text]

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.

Inhibition of T315I Bcr-Abl and Other Imatinib-Resistant Bcr-Abl Mutants by the Selective Abl Kinase Inhibitor SGX70393.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1373-1373 ◽  
Author(s):  
Thomas O’Hare ◽  
Christopher A. Eide ◽  
Jeffrey W. Tyner ◽  
Matthew J. Wong ◽  
Caitlyn A. Smith ◽  
...  
Keyword(s):  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Kinase Domain ◽  
Imatinib Resistance ◽  
Abl Kinase ◽  
Imatinib Resistant ◽  
Escape Mutants ◽  
A Cell ◽  
Almost All ◽  
Proliferation Assays

Abstract Imatinib effectively inhibits the tyrosine kinase activity of Bcr-Abl, the molecular driver of CML. Emergence of imatinib resistance due to mutations within the Bcr-Abl kinase domain (KD) has prompted the development of new Abl kinase inhibitors. A particularly important target is Bcr-Abl(T315I), which accounts for 15–20% of patients with resistance. To address this unresolved need, we profiled the novel Abl kinase inhibitor SGX70393 against native and mutant Bcr-Abl. Methods: We assessed the efficacy of SGX70393 in cellular and biochemical assays against a panel of KD mutants. Cell proliferation assays and Bcr-Abl tyrosine phosphorylation immunoblot analyses were performed for parental Ba/F3 cells, Ba/F3 cells expressing unmutated Bcr-Abl, or Ba/F3 cells expressing a single Bcr-Abl KD mutation (M244V, G250E, Q252H, Y253F, Y253H, E255K, E255V, F311L, T315I, F317L, M351T, F359V, V379I, L387M, H396P, or H396R). The resistance profile of SGX70393 was also evaluated using a recently developed accelerated, cell-based mutagenesis assay (Bradeen, et al. Blood, June 2006; doi:10.1182). Results: SGX70393 inhibited growth of cells expressing Bcr-Abl(T315I) (IC50: 7.3 nM) or unmutated Bcr-Abl (IC50: 12 nM). Sensitivity of Bcr-Abl mutants to SGX70393 partitioned into three categories: high (IC50&lt;25 nM: M244V, T315I, F359V, V379I, L387M, H396P, and H396R), medium (IC50&lt;300 nM: Q252H, Y253H, E255K, and F311L), and low (IC50&gt;500 nM: G250E, Y253F, E255V, and F317L). A cell-based mutagenesis screen for Bcr-Abl kinase domain escape mutants emerging in the presence of SGX70393 revealed a concentration-dependent reduction in surviving clones, with five previously reported Bcr-Abl mutations (L248M; G250E; Y253F; E255V; F317V) accounting for almost all resistance. Conclusions: (a) SGX70393 is a potent inhibitor of native and T315I mutant Bcr-Abl. (b) SGX70393 coverage extends to most clinically relevant mutants except mutations of the p-loop and F317.

Long-Term Mutation Follow-up of Philadelphia-Chromosome Positive Leukemia Patients Treated with Second-Generation Tyrosine Kinase Inhibitors after Imatinib Failure Shows That Newly Acquired Bcr-Abl Kinase Domain Mutations Leading to Relapse Are Mainly Detected during the First Year.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2118-2118 ◽  
Author(s):  
Simona Soverini ◽  
Alessandra Gnani ◽  
Sabrina Colarossi ◽  
Fausto Castagnetti ◽  
Francesca Palandri ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Median Time ◽  
Kinase Inhibitors ◽  
Initial Response ◽  
Kinase Domain ◽  
Abl Kinase ◽  
Imatinib Resistant ◽  
Tki Treatment ◽  
Selection Of

Abstract Resistance to imatinib in Philadelphia-positive (Ph+) leukemia patients is often associated with selection of point mutations in the Bcr-Abl kinase domain (KD). Dasatinib and nilotinib are second-generation tyrosine kinase inhibitors (TKIs) with different binding modes with respect to imatinib, that have been shown to confer in vitro and in vivo activity against many Bcr-Abl mutated forms. However, both dasatinib and nilotinib have been shown to retain some ‘Achilles heels’, and they include both imatinib-resistant mutations (e.g., T315I) and some novel, inhibitor-specific ones. Selection of either type of KD mutations has frequently been observed in patients (pts) who relapse after an initial response to dasatinib or nilotinib and represents one of the major hurdles on the road to successful treatment of imatinib-resistant pts. We have monitored Abl KD mutation status in a total of 121 pts who received dasatinib (n= 78) or nilotinib (n=43) as 2nd TKI after imatinib failure since February 2005. Fifty-eight (48%) pts had chronic phase (CP) chronic myelogenous leukemia (CML), 63 pts (52%) had accelerated phase (AP) or blast crisis (BC) CML or Ph+ acute lymphoblastic leukemia (ALL). Median age was 55 years (range, 18–76); median time from diagnosis was 49 months (range, 4–181); median time on imatinib was 32 months (range, 4–66). Median follow-up of all pts who received a 2nd TKI is 7 months (range, 1–38). Median follow-up of pts who are still on 2nd TKI treatment is 32 months (range, 28–38). Relapses after an initial response have so far been observed in 46/121 pts. Thirty-eight out of these 46 pts had AP/BC CML or Ph+ ALL at the time 2nd TKI was started. Forty-one out of 121 (34%) pts have experienced relapse after an initial response during the first 12 months of 2nd TKI treatment (median time to relapse, 6,5 months; range 4–12 months), while only five of the 45 (11%) pts who were still on 2nd TKI treatment after &gt;12 months have relapsed (at 13, 15, 18, 20 and 33 months, respectively). Interestingly, none of these 5 pts had never achieved more than a minor cytogenetic response (CgR), and 4/5 pts were receiving a reduced TKI dose because of toxicity. In 36/46 (78%) cases, relapse was associated with newly acquired Abl KD mutations. In particular 26/30 (87%) pts who relapsed on dasatinib and 10/16 (63%) pts who relapsed on nilotinib had evidence of a newly acquired KD mutation presumably responsible for treatment failure. Newly acquired mutations in pts who relapsed on dasatinib as 2nd TKI were T315I (n= 12 pts) F317L (n= 8 pts) T315A (n=3 pts); V299L (n=3 pts); F317I (n=2 pts); 2 pts had multiple mutations. Newly acquired mutations in pts who relapsed on nilotinib as 2nd TKI were E255K (n=3); E255V (n=2); Y253H (n=2); T315I (n=1); F359V (n=1); F359C (n=1). Sixteen pts (but none of those harboring the T315I) switched to dasatinib or nilotinib or high-dose imatinib as 3rd TKI and this rescued hematologic or even cytogenetic responses in a proportion of cases. Our observations suggest that: newly acquired mutations leading to relapse in Ph+ leukemia pts receiving dasatinib or nilotinib as 2nd TKI usually arise rapidly; the likelihood of mutation selection consistently decreases over time, and seems mainly confined to advanced phase pts and to pts with no or minor CgR; almost all (87%) cases who developed resistance to dasatinib had newly acquired KD mutations - suggesting that the higher potency with respect to imatinib can overcome Bcr-Abl gene amplification and that Src kinase inhibition may turn off Bcr- Abl-independent resistance mechanisms; a lower incidence (63%) of newly acquired KD mutations was observed in pts who developed resistance to nilotinib; with the exception of T315I, there is little if no overlap between dasatinib and nilotinib-resistant mutants, which may allow to regain responses by switching TKIs.

Lyn Kinase Alters Gab2 Phosphorylation and c-Cbl Protein Levels To Regulate Imatinib Sensitivity and Survival of Chronic Myelogenous Leukemia Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2132-2132
Author(s):  
Ji Wu ◽  
Feng Meng ◽  
Moshe Talpaz ◽  
Nicholas J. Donato
Keyword(s):  
Tyrosine Phosphorylation ◽  
Kinase Inhibitor ◽  
K562 Cells ◽  
Imatinib Resistance ◽  
Kinase Inhibition ◽  
Protein Levels ◽  
Abl Kinase ◽  
Lyn Kinase ◽  
Imatinib Resistant ◽  
Cbl Protein

Abstract The tyrosine kinase inhibitor imatinib mesylate (Gleevec) is effective in controlling BCR-ABL expressing leukemias but resistance occurs in some early phase patients while it is more common in advanced disease. Resistance has been generally associated with mutations in the BCR-ABL kinase that effect drug affinity. However patients are also increasingly reported to fail imatinib therapy while retaining wild-type BCR-ABL expression. Our previous studies suggested a role for Lyn, a Src-related kinase, in imatinib resistance. K562 cells selected for imatinib resistance (K562R) overexpress Lyn kinase and its targeted silencing overcomes imatinib resistance and engages apoptosis. Overexpression of Lyn in K562 cells reduces imatinib sensitivity (3-fold) and patients that fail imatinib therapy in the absence of BCR-ABL mutations express a highly activated Lyn kinase that is not suppressed by imatinib. Silencing Lyn expression in patient specimens induces changes in cell survival that are proportional to the level of Lyn protein reduction. To understand the role of Lyn kinase in imatinib resistance and apoptosis we examined proteins associated with this kinase in imatinib resistant cell lines, leukemic cells overexpressing Lyn and specimens derived from imatinib resistant patients. Lyn overexpression blocked complete suppression of BCR-ABL tyrosine phosphorylation by imatinib and affected BCR-ABL signaling adaptors. Although BCR-ABL forms a stable complex with the leukemogenic-critical adaptor protein Gab2 in imatinib sensitive cells, Lyn overexpression resulted in the formation of Lyn:Gab2 complexed in resistant cells. BCR-ABL kinase inhibition failed to reduce tyrosine phosphorylation of Gab2 in these cells while Lyn silencing or kinase inhibition (with dasatinib) completely suppressed Gab2 tyrosine phosphorylation and correlated with the induction of apoptosis. Lyn silencing in K562R cells also lead to a reciprocal increase in the tyrosine phosphorylation and association with a protein of ~120kDa, identified as the E3 ligase, c-Cbl. Lyn overexpression in K562 cells reduced their imatinib sensitivity and reduced c-Cbl protein levels. Kinase inhibitor and co-transfection studies demonstrated that tyrosine phosphorylation of c-Cbl at a critical signaling site (Y774) is primarily controlled by BCR-ABL and deletion or mutation of the c-Cbl RING domain altered its BCR-ABL phosphorylation. These results suggest that c-Cbl complexes are regulated at both the protein and phosphorylation level by Lyn and BCR-ABL kinase activities, respectively. Overexpression and/or activation of Lyn may disrupt the balance and regulation of critical regulators of leukemogenic signaling (Gab2) or protein trafficking and stability (c-Cbl), resulting in increased cell survival and reduced responsiveness to BCR-ABL kinase inhibition. We conclude that Lyn alters the level and function of critical signaling adaptor proteins in CML cells.

MAPK-Activation in CD34+ CML Stem Cells Induced by Selective Abl-Inhibitors Is Not Observed Using the Combined Src/Abl-Inhibitor Dasatinib.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 652-652 ◽  
Author(s):  
Paul La Rosee ◽  
Nicolai Haertel ◽  
Thomas Klag ◽  
Heiko Konig ◽  
Ruediger Hehlmann ◽  
...  
Keyword(s):  
Stem Cell ◽  
Time Course ◽  
Kinase Inhibitors ◽  
Mononuclear Cells ◽  
Kinase Inhibitor ◽  
Single Agent ◽  
Cell Activity ◽  
Myelogenous Leukemia ◽  
Mapk Activation ◽  
Abl Kinase

Abstract The BCR-ABL oncoprotein promotes growth and survival by activating Ras-dependent MAPK-signalling, which can be blocked by the Abl-kinase inhibitor imatinib, the current standard treatment for chronic myelogenous leukemia (CML). However, paradoxical MAPK-activation is observed in CD34+ progenitor cells of CML patients when exposed to imatinib in the presence of growth factors. Cytokine-dependent MAPK-activation may underlie primary resistance of the leukemic stem cell to imatinib. Second generation BCR-ABL inhibitors have been developed, but their potential to modulate the MAPK has not been evaluated. Here we demonstrate differential MAPK-modulating activity of currently available ABL-inhibitors and report a cell line model mimicking paradoxical, cytokine dependent MAPK-activation in response to BCR-ABL-inhibition. CD34+ enriched mononuclear cells (MNC) derived from CML patients prior to treatment (n=5) were cultured in serum-free media in the presence of standard growth factor mix (GF). Overnight exposure of single agent or combined inhibitors (nilotinib, dasatinib), and subsequent analysis of MAPK-activation was performed. BCR-ABL-transformed Baf3 and 32D cells were grown in the presence of increasing concentrations of tyrosine kinase inhibitors up to 24 hrs. Cultures were kept either with or without Interleukin 3 (IL3). Signaling was studied after SDS-page of whole cell lysates and subsequent Western blot analysis. Inhibitor isodoses were determined using tetrazolium based proliferation assays. Results: CD34+ CML MNC show significant and dose dependent activation of MAPK1/2 in response to nilotinib ([fold change of control]; 0.2μM: 2.7±1.1, p<0.05; 1.0μM: 3.2±1.2, p<0.05). Co-treatment with imatinib tends to enhance the MAPK-activation seen with nilotinib alone. In contrast, exposure of patient cells (n=5) to dasatinib results in a significant MAPK-inhibition (12nM: 0.4±0.3, p<0.05; 60nM: 0.5±0.3, p<0.05). However, combined application of nilotinib with dasatinib overrides the MAPK-inhibitory activity of dasatinib (dasatinib: 0.4±0.3, p<0.05 vs dasatinib+nilotinib: 2.3±2; n.s.). The myeloid 32Dp210 cells when grown in the presence of IL3 reflect the results seen in primary cells with activation of MAPK1/2 in the presence of nilotinib. Time course experiments show peak-activation at 6hrs after start of treatment (0.04μM: 2.8±1.6, n.s.; 0.2μM: 2.8±1.9; n.s.). Without IL3, MAPK’s are significantly inhibited confirming the cytokine-dependence of MAPK-activation (p<0.05). This contrasts data obtained with dasatinib, which leads to MAPK-inhibition even in the presence of IL3 (12nM: 0.5±0.2, n.s., 60nM: 0.5±0.1, n.s.). In lymphoid Bafp210, nilotinib and dasatinib both effectively inhibit MAPK1/2 even in the presence of IL3, demonstrating cell context dependent MAPK-modulation in response to Abl-kinase-inhibitors. Conclusions: Nilotinib induces significant MAPK1/2-activation in CD34+ CML progenitors similar to previously reported results with imatinib. Effective MAPK-inhibition in CD34+ cells by dasatinib may underlie a possible higher stem cell activity of dasatinib. Reversal of MAPK-inhibition in combination experiments indicates that nilotinib acts through mechanisms that cannot be influenced by dasatinib. 32Dp210 cells may prove useful to study the exact mechanisms of paradoxical MAPK-activation.

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.

Dissecting the Complexity of Philadelphia-Positive Mutated Populations by Ultra-Deep Sequencing of the Bcr-Abl Kinase Domain: Biological and Clinical Implications

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 692-692 ◽  
Author(s):  
Simona Soverini ◽  
Caterina De Benedittis ◽  
Katerina Machova Polakova ◽  
Adela Brouckova ◽  
Fausto Castagnetti ◽  
...  
Keyword(s):  
Deep Sequencing ◽  
Research Funding ◽  
Kinase Inhibitor ◽  
Lymphoblastic Leukemia ◽  
Kinase Domain ◽  
Mutation Status ◽  
Abl Kinase ◽  
Selection Of ◽  
Higher Sensitivity

Abstract Abstract 692 Background and Aims: In chronic myeloid leukemia (CML) and Philadelphia-positive acute lymphoblastic leukemia (ALL), tyrosine kinase inhibitor (TKI) therapy may select for drug-resistant Bcr-Abl mutants. Mutation status of resistant patients is usually investigated by Sanger sequencing (SS) of the Bcr-Abl kinase domain (KD). Novel ultra-deep sequencing (UDS) technologies allow to conjugate higher sensitivity with the unprecedented possibility to perform instant cloning of thousands of DNA molecules. We thus decided to take advantage of an UDS-based approach in order to: Methods: We retrospectively performed a longitudinal analysis of a total of 111 samples from 35 CML or Ph+ ALL patients who had received sequential treatment with multiple TKIs (two to four TKIs among imatinib, dasatinib, nilotinib, ponatinib) and had experienced sequential relapses accompanied by selection of TKI-resistant mutations. All samples had already been scored by SS; 74/111 (67%) were positive for one (n=33) or multiple (n=41) mutations. UDS of the Bcr-Abl KD was done using Roche 454 technology. UDS allowed to achieve a lower detection limit of at least 0.1% – as compared to 20% of SS. Results: Bcr-Abl KD mutation status was found to be more complex than SS had previously shown in 85/111 (77%) samples (representative examples are detailed in Table 1). In 33/74 (44%) samples known to harbour one or more mutations by SS, UDS revealed that up to four ‘minor’ mutations with 1–20% abundance were present in addition to the ‘dominant’ one(s). The type of mutations could easily be accounted for by TKI exposure history, since the majority were known to be poorly sensitive either to the current or to the previous TKI received. The higher degree of complexity was evident also when the clonal relationships of multiple mutations were reconstructed (Table 1). This revealed that identical mutations may be acquired in parallel by independent populations (e.g., one wild-type and one already harboring a mutation), via the same or different nucleotide changes leading to the same amino acid substitution (convergent evolution). In addition, longitudinal quantitative follow-up of mutated populations revealed that: Conclusions: Disclosures: Soverini: ARIAD: Consultancy; Bristol-Myers Squibb: Consultancy; Novartis: Consultancy. Castagnetti:Novartis: Honoraria; Bristol Myers Squibb: Honoraria. Luppi:CELGENE CORPORATION: Research Funding. Rosti:Bristol Myers Squibb: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding. Baccarani:ARIAD, Novartis, Bristol Myers-Squibb, and Pfizer: Consultancy, Honoraria, Speakers Bureau. Martinelli:NOVARTIS: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.

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.

Alternatively spliced truncated BCR-ABL1 protein in CML patients with resistance to kinase inhibitors

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. 7026-7026
Author(s):  
J. Bruey ◽  
H. Kantarjian ◽  
W. Ma ◽  
C. Yeh ◽  
R. Peralta ◽  
...  
Keyword(s):  
Structural Changes ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Residual Disease ◽  
Chronic Phase ◽  
K562 Cells ◽  
Aurora Kinase ◽  
Imatinib Resistant ◽  
Alternatively Spliced

7026 Background: We have reported that some patients with imatinib-resistant chronic myeloid leukemia (CML) express an alternatively spliced BCR-ABL mRNA with a 35-bp insertion (BCR-ABL135INS), resulting in the addition of 10 residues and truncation of 653 residues. Molecular dynamic simulation suggested that this truncation and insertion of new 10 AA results in structural changes similar to those seen in BCR-ABL with T315I mutation. Here we evaluate the prevalence of BCR-ABL135INS in imatinib-resistant CML, examine the effect of this mutation on resistance to compared the efficiency of various kinase inhibitors in vitro, and suggest a model for persistent CML and a possible strategy to eradicate residual disease. Methods: Using a sensitive PCR method, we determined the prevalence of the alternatively spliced BCR-ABL135INS mRNA in 288 patients with chronic-phase CML resistant to imatinib. Expression of truncated protein was confirmed by Western blot. We then tested the effectiveness of various kinase inhibitors on human K562 CML cells expressing different levels of BCR-ABL135INS along with wild-type BCR-ABL1. Results: BCR-ABL135INS mRNA was detected in 210 (73%) of the 288 patients. Only 25% of BCR-ABL135INS positive cases showed coexistance of ABL1 kinase point mutation. Immunoprecipitation studies demonstrated that expression of the predicted 143-kD BCR-ABL135INS protein at levels proportional to those predicted by mRNA. Expression of BCR-ABL135INS in K562 cells was sufficient to conferred resistance to imatinib, dasatinib, and nilotinib in a dose-dependant fashion. However, no resistance was detected using aurora kinase inhibitor (MK 0457) or homoharringtonine (HHT). BCR-ABL135INS suppressed imatinib, nilotinib, and dasatinib-mediated dephosphorylation of CRKL, LYN, SRC, and STAT5, but had no effect on MK 0457-mediated dephosphorylation. The combination of imatinib with nilotinib or HHT showed strong synergy, overcoming BCR-ABL135INS-induced resistance in vitro. Conclusions: These findings emphasize the importance of the overlooked alternatively spliced BCR-ABL135INS protein and may provide a strategy to treat resistant disease and eradicate residual CML. No significant financial relationships to disclose.

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