RESUMEN
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.
Asunto(s)
Imidazoles/farmacología , Neoplasias/tratamiento farmacológico , Piridazinas/farmacología , Receptores de Factores de Crecimiento de Fibroblastos/antagonistas & inhibidores , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Apoptosis/efectos de los fármacos , Línea Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Femenino , Amplificación de Genes , Humanos , Immunoblotting , Ratones , Ratones SCID , Mutación , Neoplasias/metabolismo , Neoplasias/patología , Fosforilación/efectos de los fármacos , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/genética , Receptores de Factores de Crecimiento de Fibroblastos/metabolismoRESUMEN
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.
Asunto(s)
Neoplasias Hematológicas/tratamiento farmacológico , Imidazoles/farmacología , Leucemia Mieloide Aguda/tratamiento farmacológico , Piridazinas/farmacología , Proteínas Tirosina Quinasas Receptoras/metabolismo , Tirosina Quinasa 3 Similar a fms/metabolismo , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Femenino , Neoplasias Hematológicas/enzimología , Neoplasias Hematológicas/patología , Humanos , Leucemia Mieloide Aguda/enzimología , Leucemia Mieloide Aguda/patología , Ratones , Ratones SCID , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-kit/metabolismo , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Transducción de Señal/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Imatinib inhibits Bcr-Abl, the oncogenic tyrosine kinase that causes chronic myeloid leukemia. The second-line inhibitors nilotinib and dasatinib are effective in patients with imatinib resistance resulting from Bcr-Abl kinase domain mutations. Bcr-Abl(T315I), however, is resistant to all Abl kinase inhibitors in clinical use and is emerging as the most frequent cause of salvage therapy failure. SGX393 is a potent inhibitor of native and T315I-mutant Bcr-Abl kinase that blocks the growth of leukemia cell lines and primary hematopoietic cells expressing Bcr-Abl(T315I), with minimal toxicity against Bcr-Abl-negative cell lines or normal bone marrow. A screen for Bcr-Abl mutants emerging in the presence of SGX393 revealed concentration-dependent reduction in the number and range of mutations. Combining SGX393 with nilotinib or dasatinib preempted emergence of resistant subclones, including Bcr-Abl(T315I). These findings suggest that combination of a T315I inhibitor with the current clinically used inhibitors may be useful for reduction of Bcr-Abl mutants in Philadelphia chromosome-positive leukemia.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Proteínas de Fusión bcr-abl/antagonistas & inhibidores , Proteínas de Fusión bcr-abl/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Mutación Missense , Inhibidores de Proteínas Quinasas/farmacología , Línea Celular Tumoral , Dasatinib , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Humanos , Pirimidinas/farmacología , Tiazoles/farmacologíaRESUMEN
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.