Tyrosine kinases are aberrantly activated in numerous malignancies, including acute myeloid leukemia (AML). To identify tyrosine kinases activated in AML, we developed a screening strategy that rapidly identifies tyrosine-phosphorylated proteins using mass spectrometry. This allowed the identification of an activating mutation (A572V) in the JAK3 pseudokinase domain in the acute megakaryoblastic leukemia (AMKL) cell line CMK. Subsequent analysis identified two additional JAK3 alleles, V722I and P132T, in AMKL patients. JAK3(A572V), JAK3(V722I), and JAK3(P132T) each transform Ba/F3 cells to factor-independent growth, and JAK3(A572V) confers features of megakaryoblastic leukemia in a murine model. These findings illustrate the biological importance of gain-of-function JAK3 mutations in leukemogenesis and demonstrate the utility of proteomic approaches to identifying clinically relevant mutations.
Members of the fibroblast growth factor receptor family of kinases (FGFR1-4) are dysregulated in multiple cancers. Ponatinib (AP24534) is an oral multitargeted tyrosine kinase inhibitor being explored in a pivotal phase II trial in patients with chronic myelogenous leukemia due to its potent activity against BCR-ABL. Ponatinib has also been shown to inhibit the in vitro kinase activity of all four FGFRs, prompting us to examine its potential as an FGFR inhibitor. In Ba/F3 cells engineered to express activated FGFR1-4, ponatinib potently inhibited FGFR-mediated signaling and viability with IC 50 values <40 nmol/L, with substantial selectivity over parental Ba/F3 cells. In a panel of 14 cell lines representing multiple tumor types (endometrial, bladder, gastric, breast, lung, and colon) and containing FGFRs dysregulated by a variety of mechanisms, ponatinib inhibited FGFR-mediated signaling with IC 50 values <40 nmol/L and inhibited cell growth with GI 50 (concentration needed to reduce the growth of treated cells to half that of untreated cells) values of 7 to 181 nmol/L. Daily oral dosing of ponatinib (10-30 mg/kg) to mice reduced tumor growth and inhibited signaling in all three tumor models examined. Importantly, the potency of ponatinib in these models is similar to that previously observed in BCR-ABL-driven models and plasma levels of ponatinib that exceed the IC 50 values for FGFR1-4 inhibition can be sustained in patients. These results show that ponatinib is a potent pan-FGFR inhibitor and provide strong rationale for its evaluation in patients with FGFR-driven cancers.
Ponatinib (AP24534) is a novel multitargeted kinase inhibitor that potently inhibits native and mutant BCR-ABL at clinically achievable drug levels. Ponatinib also has in vitro inhibitory activity against a discrete set of kinases implicated in the pathogenesis of other hematologic malignancies, including FLT3, KIT, fibroblast growth factor receptor 1 (FGFR1), and platelet derived growth factor receptor α (PDGFRα). Here, using leukemic cell lines containing activated forms of each of these receptors, we show that ponatinib potently inhibits receptor phosphorylation and cellular proliferation with IC50 values comparable to those required for inhibition of BCR-ABL (0.3 to 20 nmol/L). The activity of ponatinib against the FLT3-ITD mutant, found in up to 30% of acute myeloid leukemia (AML) patients, was particularly notable. In MV4-11 (FLT3-ITD+/+) but not RS4;11 (FLT3-ITD−/−) AML cells, ponatinib inhibited FLT3 signaling and induced apoptosis at concentrations of less than 10 nmol/L. In an MV4-11 mouse xenograft model, once daily oral dosing of ponatinib led to a dose-dependent inhibition of signaling and tumor regression. Ponatinib inhibited viability of primary leukemic blasts from a FLT3-ITD positive AML patient (IC50 4 nmol/L) but not those isolated from 3 patients with AML expressing native FLT3. Overall, these results support the investigation of ponatinib in patients with FLT3-ITD–driven AML and other hematologic malignancies driven by KIT, FGFR1, or PDGFRα.
Imatinib inhibitsgatekeeper mutation ͉ imatinib resistance ͉ kinase inhibitor
Background: Imatinib mesylate (IM) induces complete cytogenetic remission (CCR) in 82% of newly diagnosed patients with CML in chronic phase. Most of these patients display minimal residual disease by nested RT-PCR for BCR-ABL. A previous study suggested that kinase domain (KD) mutations in BCR-ABL may be responsible for disease persistence in a significant percentage (9/13) of CCR patients (Chu et al. Blood, 2005). As the relapse rate in this small cohort was high, we decided to investigate the incidence of KD mutants in a more representative group of patients with stable CCR. Methods: We performed nested RT-PCR for BCR-ABL in 72 CCR patients, using 2 sets of primers spanning the breakpoint and entire kinase domain, with ABL as a control gene. Mutation analysis was performed with direct sequencing and denaturing-HPLC of BCR-ABL amplicons. Median time on IM in these patients was 32 months (range, 8–65). In case of wild-type sequence, PCR products were subcloned and a median of 16 individual clones (range, 11–20) sequenced. Results: Amplification was successful in 42/72 patients (58%). We detected 11 different point mutations in 9/42 (21%), of which 3 were novel (C305S, T212R, N231D). The remaining 8 mutants are known to be IM-resistant. C305S was found only by analysis of subclones, while all other mutations were detected upon initial analysis. 5/9 patients with a mutation had a corresponding rise in BCR-ABL transcripts by qRT-PCR. In the other 4, low or undetectable levels of BCR-ABL were maintained. For 2 of these, follow-up mutation analysis detected wild-type BCR-ABL, indicating a decrease of the mutant clone below the detection threshold. For patient 5, with G250E, an initial rise in BCR-ABL was followed by a continual decrease and stable CCR. Although most of the mutants detected were expected to lead to hematologic relapse, at a median follow-up of 12 months, this occurred in only one case. Conclusions: KD mutations were detected in a modest proportion of CCR patients (12% of all patients and 21% of BCR-ABL-positive patients). The relatively high rate of PCR negativity may be due to the long amplicons (1.4kb). Our data imply that, in the majority of patients, disease persistence must be mediated by mechanisms other than KD mutations. In addition, some mutants failed to persist upon follow-up analysis, suggesting that the mutations occurred in transiently amplifying cells that are not capable of prolonged survival. Characteristics of CCR patients with a KD mutation. Patient No. Age at Study (years) Time on IM (months) CD34+ Mutation Detected CD34-/MNC Mutation Detected Time to Last Follow-up (months) Current Cytogenetic Status Follow-up Sample Mutation Status No Amp: No PCR amplification. NA: No sample available. Mutations detected by nested RT-PCR and sequencing (1), or D-HPLC (2), or both in parallel (3). Subcloning results are shown in parentases. 3 84 40 NA T212R3(15,15) 11 5% Ph+ In Progress 5 27 28 No Amp G250E3(17/17) 14 CCR G250E 19 52 18 N231D2 WT3(C305S:3/20) 5 CCR NA 20 43 43 Y253F, M244V2 M244V3(20/20) NA NA NA 21 24 63 WT3 (16/16) G321E, E355G2 NA NA NA 28 80 31 NA T315I1 (15/15) 12 CCR In Progress 30 51 39 NA Y253H1 (16/16) 10 CCR WT 31 39 43 NA T315I2 17 CCR No Amp 33 69 32 NA F359V2 5 100% Ph+ NA
Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN) is a rare and aggressive malignancy that usually presents with diffuse cutaneous lesions. While a favorable response to therapy occurs in a majority of cases, a sustained long-term response is uncommon. Most patients subsequently relapse within a year. In the following report, we present the case of a 41-year-old woman who has not displayed many of the clinical features traditionally associated with BPDCN. The patient received sporadic chemotherapy treatment over the course of 2 years, before undergoing an allogeneic stem cell transplant. Although she ultimately relapsed following her transplant, her disease has repeatedly returned into remission after donor lymphocyte infusion (DLI). Currently, the patient is in remission following her fourth DLI. We believe that allogeneic transplantation should be considered as front-line therapy for the treatment of this rare malignancy.
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<25 nM: M244V, T315I, F359V, V379I, L387M, H396P, and H396R), medium (IC50<300 nM: Q252H, Y253H, E255K, and F311L), and low (IC50>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.
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