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1.
Cancer Cell ; 39(9): 1214-1226.e10, 2021 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-34375612

RESUMO

PARP7 is a monoPARP that catalyzes the transfer of single units of ADP-ribose onto substrates to change their function. Here, we identify PARP7 as a negative regulator of nucleic acid sensing in tumor cells. Inhibition of PARP7 restores type I interferon (IFN) signaling responses to nucleic acids in tumor models. Restored signaling can directly inhibit cell proliferation and activate the immune system, both of which contribute to tumor regression. Oral dosing of the PARP7 small-molecule inhibitor, RBN-2397, results in complete tumor regression in a lung cancer xenograft and induces tumor-specific adaptive immune memory in an immunocompetent mouse cancer model, dependent on inducing type I IFN signaling in tumor cells. PARP7 is a therapeutic target whose inhibition induces both cancer cell-autonomous and immune stimulatory effects via enhanced IFN signaling. These data support the targeting of a monoPARP in cancer and introduce a potent and selective PARP7 inhibitor to enter clinical development.


Assuntos
Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Interferon Tipo I/metabolismo , Neoplasias/tratamento farmacológico , Proteínas de Transporte de Nucleosídeos/genética , Proteínas de Transporte de Nucleosídeos/metabolismo , Bibliotecas de Moléculas Pequenas/administração & dosagem , Imunidade Adaptativa/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células HEK293 , Células HeLa , Humanos , Camundongos , Neoplasias/genética , Neoplasias/metabolismo , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Evasão Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
2.
Oncotarget ; 9(51): 29654-29664, 2018 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-30038711

RESUMO

Genomic studies are revolutionizing clinical oncology, but bridging the lab and the bedside requires the ability to efficiently interrogate rare genetic lesions in unexpected pathological settings using preclinical models. Oncogenes can exhibit intrinsic drug resistance to targeted therapy in different cells of origin, adding complexity to clinical interpretations of genomic findings. Here, we capitalize on the flexibility of engineered cell systems to rapidly profile known multi-kinase inhibitors that harbor rearranged during transfection (RET) kinase activity across multiple RET fusions. Identifying ponatinib as the most potent RET inhibitor tested, we used ponatinib to gauge therapeutic responsiveness in RET fusion-positive patient-derived xenograft (PDX) models. Using a genomics guided outlier approach, we identified 4 RET fusion PDX models with 3 different fusion partners (KIF5B, CCDC6, and NCOA4) in both non-small cell lung cancer and colorectal cancer. By comparing ponatinib activity in RET fusion-positive and RET fusion-negative PDX models alongside a standard of care chemotherapeutic agent, we show that RET fusions in colorectal tumors are therapeutically responsive to RET inhibition. Finally, we suggest that coupling engineered cell systems and genomics guided PDX model selection provides a rapid workflow to triage rare genomics findings.

3.
Clin Cancer Res ; 20(22): 5745-5755, 2014 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-25239608

RESUMO

PURPOSE: KIT is the major oncogenic driver of gastrointestinal stromal tumors (GIST). Imatinib, sunitinib, and regorafenib are approved therapies; however, efficacy is often limited by the acquisition of polyclonal secondary resistance mutations in KIT, with those located in the activation (A) loop (exons 17/18) being particularly problematic. Here, we explore the KIT-inhibitory activity of ponatinib in preclinical models and describe initial characterization of its activity in patients with GIST. EXPERIMENTAL DESIGN: The cellular and in vivo activities of ponatinib, imatinib, sunitinib, and regorafenib against mutant KIT were evaluated using an accelerated mutagenesis assay and a panel of engineered and GIST-derived cell lines. The ponatinib-KIT costructure was also determined. The clinical activity of ponatinib was examined in three patients with GIST previously treated with all three FDA-approved agents. RESULTS: In engineered and GIST-derived cell lines, ponatinib potently inhibited KIT exon 11 primary mutants and a range of secondary mutants, including those within the A-loop. Ponatinib also induced regression in engineered and GIST-derived tumor models containing these secondary mutations. In a mutagenesis screen, 40 nmol/L ponatinib was sufficient to suppress outgrowth of all secondary mutants except V654A, which was suppressed at 80 nmol/L. This inhibitory profile could be rationalized on the basis of structural analyses. Ponatinib (30 mg daily) displayed encouraging clinical activity in two of three patients with GIST. CONCLUSION: Ponatinib possesses potent activity against most major clinically relevant KIT mutants and has demonstrated preliminary evidence of activity in patients with refractory GIST. These data strongly support further evaluation of ponatinib in patients with GIST.


Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Tumores do Estroma Gastrointestinal/genética , Imidazóis/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-kit/genética , Piridazinas/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Benzamidas/farmacologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Éxons , Feminino , Tumores do Estroma Gastrointestinal/diagnóstico , Tumores do Estroma Gastrointestinal/tratamento farmacológico , Tumores do Estroma Gastrointestinal/patologia , Humanos , Mesilato de Imatinib , Imidazóis/química , Imidazóis/uso terapêutico , Indóis/farmacologia , Concentração Inibidora 50 , Modelos Moleculares , Conformação Molecular , Mutação , Recidiva Local de Neoplasia , Piperazinas/farmacologia , Ligação Proteica , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-kit/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-kit/química , Piridazinas/química , Piridazinas/uso terapêutico , Pirimidinas/farmacologia , Pirróis/farmacologia , Sunitinibe , Tomografia Computadorizada por Raios X , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/genética , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Mol Cancer Res ; 12(10): 1460-9, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24966347

RESUMO

UNLABELLED: Malignant pleural mesothelioma (MPM) is associated with asbestos exposure and is a cancer that has not been significantly affected by small molecule-based targeted therapeutics. Previously, we demonstrated the existence of functional subsets of lung cancer and head and neck squamous cell carcinoma (HNSCC) cell lines in which fibroblast growth factor receptor (FGFR) autocrine signaling functions as a nonmutated growth pathway. In a panel of pleural mesothelioma cell lines, FGFR1 and FGF2 were coexpressed in three of seven cell lines and were significantly associated with sensitivity to the FGFR-active tyrosine kinase inhibitor (TKI), ponatinib, both in vitro and in vivo using orthotopically propagated xenografts. Furthermore, RNAi-mediated silencing confirmed the requirement for FGFR1 in specific mesothelioma cells and sensitivity to the FGF ligand trap, FP-1039, validated the requirement for autocrine FGFs. None of the FGFR1-dependent mesothelioma cells exhibited increased FGFR1 gene copy number, based on a FISH assay, indicating that increased FGFR1 transcript and protein expression were not mediated by gene amplification. Elevated FGFR1 mRNA was detected in a subset of primary MPM clinical specimens and like MPM cells; none harbored increased FGFR1 gene copy number. These results indicate that autocrine signaling through FGFR1 represents a targetable therapeutic pathway in MPM and that biomarkers distinct from increased FGFR1 gene copy number such as FGFR1 mRNA would be required to identify patients with MPM bearing tumors driven by FGFR1 activity. IMPLICATIONS: FGFR1 is a viable therapeutic target in a subset of MPMs, but FGFR TKI-responsive tumors will need to be selected by a biomarker distinct from increased FGFR1 gene copy number, possibly FGFR1 mRNA or protein levels.


Assuntos
Amplificação de Genes , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Mesotelioma/genética , Mesotelioma/patologia , Neoplasias Pleurais/genética , Neoplasias Pleurais/patologia , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Animais , Comunicação Autócrina/efeitos dos fármacos , Comunicação Autócrina/genética , Linhagem Celular Tumoral , Proliferação de Células , Células Clonais , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Fator 2 de Crescimento de Fibroblastos/metabolismo , Humanos , Imidazóis/farmacologia , Mesotelioma Maligno , Camundongos Nus , Piridazinas/farmacologia , Interferência de RNA , Transdução de Sinais
5.
Clin Cancer Res ; 20(12): 3299-309, 2014 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-24771645

RESUMO

PURPOSE: FGFR1 gene copy number (GCN) is being evaluated as a biomarker for FGFR tyrosine kinase inhibitor (TKI) response in squamous cell lung cancers (SCC). The exclusive use of FGFR1 GCN for predicting FGFR TKI sensitivity assumes increased GCN is the only mechanism for biologically relevant increases in FGFR1 signaling. Herein, we tested whether FGFR1 mRNA and protein expression may serve as better biomarkers of FGFR TKI sensitivity in lung cancer. EXPERIMENTAL DESIGN: Histologically diverse lung cancer cell lines were submitted to assays for ponatinib sensitivity, a potent FGFR TKI. A tissue microarray composed of resected lung tumors was submitted to FGFR1 GCN, and mRNA analyses and the results were validated with The Cancer Genome Atlas (TCGA) lung cancer data. RESULTS: Among 58 cell lines, 14 exhibited ponatinib sensitivity (IC50 values ≤ 50 nmol/L) that correlated with FGFR1 mRNA and protein expression, but not with FGFR1 GCN or histology. Moreover, ponatinib sensitivity associated with mRNA expression of the ligands, FGF2 and FGF9. In resected tumors, 22% of adenocarcinomas and 28% of SCCs expressed high FGFR1 mRNA. Importantly, only 46% of SCCs with increased FGFR1 GCN expressed high mRNA. Lung cancer TCGA data validated these findings and unveiled overlap of FGFR1 mRNA positivity with KRAS and PIK3CA mutations. CONCLUSIONS: FGFR1 dependency is frequent across various lung cancer histologies, and FGFR1 mRNA may serve as a better biomarker of FGFR TKI response in lung cancer than FGFR1 GCN. The study provides important and timely insight into clinical testing of FGFR TKIs in lung cancer and other solid tumor types.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Dosagem de Genes , Inibidores de Proteínas Quinases/farmacologia , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Western Blotting , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Proliferação de Células , Estudos de Coortes , Seguimentos , Amplificação de Genes , Humanos , Imidazóis/farmacologia , Técnicas Imunoenzimáticas , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Estadiamento de Neoplasias , Piridazinas/farmacologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Células Tumorais Cultivadas
6.
J Cell Physiol ; 229(9): 1160-9, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24443386

RESUMO

S-phase kinase-associated protein 2 (SKP2) is an important cell cycle regulator, targeting the cyclin-dependent kinase (CDK) inhibitor p27 for degradation, and is frequently overexpressed in breast cancer. p27 regulates G1 /S transition by abrogating the activity of cyclin/CDK complexes. p27 can undergo phosphorylation at serine 10 (pSer10p27). This phosphorylation event is associated with increased cell proliferation and poor prognosis in patients with glioma. The relationship between SKP2 and pSer10p27 in breast cancer has not been previously investigated. Immunohistochemistry (IHC) of SKP2, p27, pSer10p27, and other genes involved in this pathway, was analyzed in 188 breast tumors and 50 benign reduction mammoplasty samples. IHC showed SKP2 to be more highly expressed in estrogen receptor α (ERα)-negative breast cancers and demonstrated that triple-negative tumors were more likely to have high expression of SKP2 than were non-triple negative, ERα-negative tumors. A significant positive relationship was discovered for SKP2 and pSer10p27. High levels of SKP2 and pSer10p27 were observed significantly more often in ERα-negative and triple-negative than in ERα-positive breast cancers. Use of the triple-negative TMX2-28 breast cancer cell line to address the role of SKP2 in cell cycle progression confirmed that SKP2 contributes to a more rapid cell cycle progression and may regulates pSer10p27 levels. Together, the results indicate that presence of high SKP2 plus high pSer10p27 levels in triple-negative breast cancers is associated with aggressive growth, and highlight the validity of using SKP2 inhibitors as a therapeutic approach for treating this subset of breast cancers.


Assuntos
Biomarcadores Tumorais/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Proteínas Quinases Associadas a Fase S/metabolismo , Neoplasias de Mama Triplo Negativas/enzimologia , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Ciclo Celular , Proliferação de Células , Ciclina D1/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/genética , Receptor alfa de Estrogênio/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Células MCF-7 , Pessoa de Meia-Idade , Gradação de Tumores , Invasividade Neoplásica , Fosforilação , Interferência de RNA , Proteínas Quinases Associadas a Fase S/genética , Serina , Transdução de Sinais , Transfecção , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Regulação para Cima
8.
Cancer Chemother Pharmacol ; 71(5): 1315-23, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23468082

RESUMO

PURPOSE: Activating mutations in FGFR2 have been identified as potential therapeutic targets in endometrial cancer, typically occurring alongside genetic alterations that disrupt the mTOR pathway, such as PTEN loss. These observations suggest that the mTOR pathway may act in concert with oncogenic FGFR2 to drive endometrial cancer growth in a subset of patients. The aim of this study was to examine the therapeutic potential of a rational drug combination based on the simultaneous targeting of mutant-FGFR2 and mTOR-driven signaling pathways in endometrial cancer cells. METHODS: Ponatinib is an oral multitargeted kinase inhibitor that potently inhibits all 4 members of the FGFR family. Ridaforolimus is a selective inhibitor of mTOR that has demonstrated positive clinical activity in endometrial cancer. The combinatorial effects of ponatinib and ridaforolimus on growth of endometrial cancer models, and their modes of action, were evaluated in vitro and in vivo. RESULTS: The combination of ponatinib and ridaforolimus had a synergistic effect on the in vitro growth of endometrial lines bearing an activating FGFR2 mutation, irrespective of PTEN status. Concomitant inhibition of both FGFR2 and mTOR signaling pathways was observed, with simultaneous blockade resulting in enhanced cell cycle arrest. Ponatinib and ridaforolimus each demonstrated inhibition of tumor growth in vivo, but dual inhibition by the combination of agents resulted in superior efficacy and induced tumor regression in an endometrial xenograft. CONCLUSIONS: These encouraging preclinical findings suggest the inhibition of both FGFR2 and mTOR by the ponatinib-ridaforolimus combination may provide a new therapeutic strategy to treat advanced endometrial cancers with dual pathway dysregulation.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias do Endométrio/tratamento farmacológico , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Serina-Treonina Quinases TOR/antagonistas & inibidores , Animais , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sinergismo Farmacológico , Neoplasias do Endométrio/patologia , Feminino , Humanos , Imidazóis/administração & dosagem , Camundongos , Camundongos Nus , Terapia de Alvo Molecular , Mutação , Inibidores de Proteínas Quinases/farmacologia , Piridazinas/administração & dosagem , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Transdução de Sinais/efeitos dos fármacos , Sirolimo/administração & dosagem , Sirolimo/análogos & derivados , Ensaios Antitumorais Modelo de Xenoenxerto
9.
J Clin Endocrinol Metab ; 98(5): E811-9, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23526464

RESUMO

CONTEXT: The RET tyrosine kinase encoding gene acts as a dominantly transforming oncogene in thyroid carcinoma and other malignancies. Ponatinib (AP24534) is an oral ATP-competitive tyrosine kinase inhibitor that is in advanced clinical experimentation in leukemia. OBJECTIVE: We tested whether ponatinib inhibited RET kinase and oncogenic activity. METHODS: Ponatinib activity was studied by an in vitro RET immunocomplex kinase assay and immunoblotting. The effects of ponatinib on proliferation of human TT, MZ-CRC-1, and TPC-1 thyroid carcinoma cells, which harbor endogenous oncogenic RET alleles, and of NIH3T3 fibroblasts transfected with oncogenic RET mutants were determined. Ponatinib activity on TT cell xenografted tumors in athymic mice was measured. RESULTS: Ponatinib inhibited immunopurified RET kinase at the IC50 of 25.8 nM (95% confidence interval [CI] = 23.15-28.77 nM). It also inhibited (IC50 = 33.9 nM; 95% CI = 26.41-43.58 nM) kinase activity of RET/V804M, a RET mutant displaying resistance to other tyrosine kinase inhibitor. Ponatinib blunted phosphorylation of point-mutant and rearranged RET-derived oncoproteins and inhibited proliferation of RET-transformed fibroblasts and RET mutant thyroid carcinoma cells. Finally, after 3 weeks of treatment with ponatinib (30 mg/kg/d), the volume of TT cell (medullary thyroid carcinoma) xenografts was reduced from 133 mm³ to an unmeasurable size (difference = 133 mm³, 95% CI = -83 to 349 mm³) (P < .001). Ponatinib-treated TT cell tumors displayed a reduction in the mitotic index, RET phosphorylation, and signaling. CONCLUSIONS: Ponatinib is a potent inhibitor of RET kinase and has promising preclinical activity in models of RET-driven medullary thyroid carcinoma.


Assuntos
Antineoplásicos/uso terapêutico , Carcinoma/tratamento farmacológico , Imidazóis/uso terapêutico , Proteínas Mutantes/antagonistas & inibidores , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-ret/antagonistas & inibidores , Piridazinas/uso terapêutico , Neoplasias da Glândula Tireoide/tratamento farmacológico , Animais , Antineoplásicos/farmacologia , Carcinoma/genética , Carcinoma/metabolismo , Carcinoma/patologia , Carcinoma Neuroendócrino , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Imidazóis/farmacologia , Camundongos , Camundongos Nus , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-ret/genética , Proteínas Proto-Oncogênicas c-ret/metabolismo , Piridazinas/farmacologia , Distribuição Aleatória , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/metabolismo , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/patologia , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
10.
Cancer Cell Int ; 12(1): 17, 2012 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-22574838

RESUMO

BACKGROUND: Paralemmin-1 is a phosphoprotein lipid-anchored to the cytoplasmic face of membranes where it functions in membrane dynamics, maintenance of cell shape, and process formation. Expression of paralemmin-1 and its major splice variant (Δ exon 8) as well as the extent of posttranslational modifications are tissue- and development-specific. Paralemmin-1 expression in normal breast and breast cancer tissue has not been described previously. RESULTS: Paralemmin-1 mRNA and protein expression was evaluated in ten breast cell lines, 26 primary tumors, and 10 reduction mammoplasty (RM) tissues using real time RT-PCR. Paralemmin-1 splice variants were assessed in tumor and RM tissues using a series of primers and RT-PCR. Paralemmin-1 protein expression was examined in cell lines using Western Blots and in 31 ductal carcinomas in situ, 65 infiltrating ductal carcinomas, and 40 RM tissues using immunohistochemistry. Paralemmin-1 mRNA levels were higher in breast cancers than in RM tissue and estrogen receptor (ER)-positive tumors had higher transcript levels than ER-negative tumors. The Δ exon 8 splice variant was detected more frequently in tumor than in RM tissues. Protein expression was consistent with mRNA results showing higher paralemmin-1 expression in ER-positive tumors. CONCLUSIONS: The differential expression of paralemmin-1 in a subset of breast cancers suggests the existence of variation in membrane dynamics that may be exploited to improve diagnosis or provide a therapeutic target.

11.
Mol Cancer Ther ; 11(3): 690-9, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22238366

RESUMO

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.


Assuntos
Imidazóis/farmacologia , Neoplasias/tratamento farmacológico , Piridazinas/farmacologia , Receptores de Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Amplificação de Genes , Humanos , Immunoblotting , Camundongos , Camundongos SCID , Mutação , Neoplasias/metabolismo , Neoplasias/patologia , Fosforilação/efeitos dos fármacos , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/genética , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo
12.
Mol Cancer Ther ; 10(6): 1028-35, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21482694

RESUMO

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.


Assuntos
Neoplasias Hematológicas/tratamento farmacológico , Imidazóis/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Piridazinas/farmacologia , Receptores Proteína Tirosina Quinases/metabolismo , Tirosina Quinase 3 Semelhante a fms/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Neoplasias Hematológicas/enzimologia , Neoplasias Hematológicas/patologia , Humanos , Leucemia Mieloide Aguda/enzimologia , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos SCID , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-kit/metabolismo , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Cancer Cell ; 16(6): 487-97, 2009 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-19962667

RESUMO

Persistent activation of Stat3 is oncogenic and is prevalent in a wide variety of human cancers. Chronic cytokine stimulation is associated with Stat3 activation in some tumors, implicating cytokine receptor-associated Jak family kinases. Using Jak2 inhibitors, we demonstrate a central role of Jaks in modulating basal and cytokine-induced Stat3 activation in human solid tumor cell lines. Inhibition of Jak2 activity is associated with abrogation of Stat3 nuclear translocation and tumorigenesis. The Jak2 inhibitor AZD1480 suppresses the growth of human solid tumor xenografts harboring persistent Stat3 activity. We demonstrate the essential role of Stat3 downstream of Jaks by inhibition of tumor growth using short hairpin RNA targeting Stat3. Our data support a key role of Jak kinase activity in Stat3-dependent tumorigenesis.


Assuntos
Janus Quinase 2/antagonistas & inibidores , Neoplasias da Próstata/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , Fator de Transcrição STAT3/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Masculino , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/patologia , Fator de Transcrição STAT3/metabolismo
14.
J Biol Chem ; 283(47): 32334-43, 2008 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-18775810

RESUMO

The Janus-associated kinase 2 (JAK2) V617F mutation is believed to play a critical role in the pathogenesis of polycythemia vera, essential thrombocythemia, and idiopathic myelofibrosis. We have characterized a novel small molecule JAK2 inhibitor, AZ960, and used it as a tool to investigate the consequences of JAK2 V617F inhibition in the SET-2 cell line. AZ960 inhibits JAK2 kinase with a K(i) of 0.00045 microm in vitro and treatment of TEL-JAK2 driven Ba/F3 cells with AZ960 blocked STAT5 phosphorylation and potently inhibited cell proliferation (GI(50)=0.025 microm). AZ960 demonstrated selectivity for TEL-JAK2-driven STAT5 phosphorylation and cell proliferation when compared with cell lines driven by similar fusions of the other JAK kinase family members. In the SET-2 human megakaryoblastic cell line, heterozygous for the JAK2 V617F allele, inhibition of JAK2 resulted in decreased STAT3/5 phosphorylation and inhibition of cell proliferation (GI(50)=0.033 microm) predominately through the induction of mitochondrial-mediated apoptosis. We provide evidence that JAK2 inhibition induces apoptosis by direct and indirect regulation of the anti-apoptotic protein BCL-xL. Inhibition of JAK2 blocked BCL-XL mRNA expression resulting in a reduction of BCL-xL protein levels. Additionally, inhibition of JAK2 resulted in decreased PIM1 and PIM2 mRNA expression. Decreased PIM1 mRNA corresponded with a decrease in Pim1 protein levels and inhibition of BAD phosphorylation at Ser(112). Finally, small interfering RNA-mediated suppression of BCL-xL resulted in apoptotic cell death similar to the phenotype observed following JAK2 inhibition. These results suggest a model in which JAK2 promotes cell survival by signaling through the Pim/BAD/BCL-xL pathway.


Assuntos
Aminopiridinas/farmacologia , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica , Janus Quinase 2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Pirazóis/farmacologia , Proteína de Morte Celular Associada a bcl/metabolismo , Proteína bcl-X/metabolismo , Apoptose , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular , Humanos , Fenótipo , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Transdução de Sinais
15.
Mol Cancer Res ; 4(12): 905-13, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17189381

RESUMO

An estrogen receptor-negative variant of the MCF-7 breast cancer cell line, TMX2-28, was used as a model in which to study breast cancer cell invasion. Using a reconstituted basement membrane (Matrigel) assay to evaluate cell invasion, we determined that TMX2-28 cells are more invasive than MCF-7 cells and that the invasiveness of TMX2-28 is similar to that of the aggressive MDA-MB-231 breast cancer cell line. TMX2-28 cells displayed a rounded, epithelial cell-like morphology, suggesting an amoeboid mode of cell invasion, in contrast to the mesenchymal mode of invasion characteristic of spindle-shaped, fibroblast-like MDA-MB-231 cells. Using real-time reverse transcription-PCR, we found that mitogen-inducible gene 2 (MIG2) is expressed at a 17-fold higher level in TMX2-28 cells than in nonaggressive MCF-7 cells and that MIG2 mRNA levels are low in the nontumorigenic human mammary epithelial cell line, 184. We determined that MIG2 plays a role in cell invasion by using small interfering RNA (siRNA) to suppress the expression of MIG2 mRNA levels in TMX2-28 cells. TMX2-28 cell invasion was reduced by 48% when the cells were transfected with siRNAs targeting MIG2, relative to cells transfected with siRNAs against glyceraldehyde-3-phosphate dehydrogenase. Finally, MIG2 expression was evaluated in reductive mammoplasty and breast tumor tissue. Although all 21 normal tissues from reduction mammoplasty showed immunoreactivity for MIG2, ranging from weak (62%) to strong (24%), only half of the 34 formalin-fixed breast tumors showed immunoreactivity for MIG2. Of these 17 positive cases, 10 were considered to overexpress MIG2 (moderate to strong staining). Examination of 30 frozen breast tumors supported the finding that MIG2 is overexpressed in a subset of breast cancers. We suggest that MIG2's normal regulation and function are disrupted in breast cancer.


Assuntos
Neoplasias da Mama/patologia , Invasividade Neoplásica/fisiopatologia , Receptores de Estrogênio/fisiologia , Neoplasias da Mama/metabolismo , Moléculas de Adesão Celular/metabolismo , Linhagem Celular Tumoral , Proteínas do Citoesqueleto/metabolismo , Feminino , Humanos , Reação em Cadeia da Polimerase
16.
Toxicol Appl Pharmacol ; 196(1): 58-67, 2004 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-15050408

RESUMO

Polycyclic aromatic hydrocarbons (PAHs) are common environmental pollutants that have been extensively studied for multiple toxicological endpoints in both laboratory animals and humans. The purpose of this study was to investigate the estrogenicity of PAHs in the human breast cancer cell line MCF-7. We investigated 14 PAHs for their ability to bind either the estrogen receptor (ER) or the aryl hydrocarbon receptor (AhR) and to activate target gene expression. PAHs were tested in a human recombinant estrogen receptor (hrER) competitive binding assay, and in both an estrogen response element (ERE)- and xenobiotic response element (XRE)-mediated reporter gene assay. We used quantitative RT-PCR to examine selected PAHs that showed activity in the ERE reporter gene assay for their ability to upregulate estrogen-responsive genes HEM45, progesterone receptor, and pS2, and the aryl hydrocarbon-responsive CYP1A1 gene. None of the 14 PAHs bound the hrER, but five of the PAHs (anthracene, B[a]A, chrysene, B[b]F, and B[a]P) induced ER-reporter activity. This activity was dependent on the metabolism of PAHs in MCF-7 cells via the AhR pathway, which resulted in the formation of metabolites that bound the ER. None of the five PAHs that induced the ER-reporter were found to upregulate estrogen-responsive genes, yet four of the five PAHs induced AhR-dependent CYP1A1 gene expression. In contrast, a metabolite of B[a]P, 3'OH-B[a]P, and a PCB metabolite, 4'OH-2,4,6-BP, did weakly upregulate all three estrogen-responsive genes. Data from these studies indicate that induction of ER-reporter activity alone does not necessarily parallel endogenous gene transcription, and that the reporter gene assay may detect interactions that are not functional in vivo.


Assuntos
Expressão Gênica/efeitos dos fármacos , Genes Reporter/genética , Hidrocarbonetos Policíclicos Aromáticos/toxicidade , Receptores de Estrogênio/metabolismo , Ligação Competitiva , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Humanos , Hidrocarbonetos Policíclicos Aromáticos/química , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Receptores de Estrogênio/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Elementos de Resposta/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção
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