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1.
Nature ; 535(7610): 148-52, 2016 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-27362227

RESUMO

The non-receptor protein tyrosine phosphatase SHP2, encoded by PTPN11, has an important role in signal transduction downstream of growth factor receptor signalling and was the first reported oncogenic tyrosine phosphatase. Activating mutations of SHP2 have been associated with developmental pathologies such as Noonan syndrome and are found in multiple cancer types, including leukaemia, lung and breast cancer and neuroblastoma. SHP2 is ubiquitously expressed and regulates cell survival and proliferation primarily through activation of the RAS­ERK signalling pathway. It is also a key mediator of the programmed cell death 1 (PD-1) and B- and T-lymphocyte attenuator (BTLA) immune checkpoint pathways. Reduction of SHP2 activity suppresses tumour cell growth and is a potential target of cancer therapy. Here we report the discovery of a highly potent (IC50 = 0.071 µM), selective and orally bioavailable small-molecule SHP2 inhibitor, SHP099, that stabilizes SHP2 in an auto-inhibited conformation. SHP099 concurrently binds to the interface of the N-terminal SH2, C-terminal SH2, and protein tyrosine phosphatase domains, thus inhibiting SHP2 activity through an allosteric mechanism. SHP099 suppresses RAS­ERK signalling to inhibit the proliferation of receptor-tyrosine-kinase-driven human cancer cells in vitro and is efficacious in mouse tumour xenograft models. Together, these data demonstrate that pharmacological inhibition of SHP2 is a valid therapeutic approach for the treatment of cancers.


Assuntos
Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Piperidinas/farmacologia , Proteína Tirosina Fosfatase não Receptora Tipo 11/antagonistas & inibidores , Pirimidinas/farmacologia , Receptores Proteína Tirosina Quinases/metabolismo , Regulação Alostérica/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Humanos , Concentração Inibidora 50 , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Camundongos Nus , Modelos Moleculares , Neoplasias/patologia , Proteína Oncogênica p21(ras)/metabolismo , Piperidinas/química , Piperidinas/uso terapêutico , Inibidores de Proteínas Quinases/farmacologia , Estabilidade Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína/efeitos dos fármacos , Proteína Tirosina Fosfatase não Receptora Tipo 11/química , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Pirimidinas/química , Pirimidinas/uso terapêutico , Reprodutibilidade dos Testes , Ensaios Antitumorais Modelo de Xenoenxerto
2.
Proc Natl Acad Sci U S A ; 108(38): 16002-7, 2011 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-21911392

RESUMO

Increased numbers of S100A4(+) cells are associated with poor prognosis in patients who have cancer. Although the metastatic capabilities of S100A4(+) cancer cells have been examined, the functional role of S100A4(+) stromal cells in metastasis is largely unknown. To study the contribution of S100A4(+) stromal cells in metastasis, we used transgenic mice that express viral thymidine kinase under control of the S100A4 promoter to specifically ablate S100A4(+) stromal cells. Depletion of S100A4(+) stromal cells significantly reduced metastatic colonization without affecting primary tumor growth. Multiple bone marrow transplantation studies demonstrated that these effects of S100A4(+) stromal cells are attributable to local non-bone marrow-derived S100A4(+) cells, which are likely fibroblasts in this setting. Reduction in metastasis due to the loss of S100A4(+) fibroblasts correlated with a concomitant decrease in the expression of several ECM molecules and growth factors, particularly Tenascin-C and VEGF-A. The functional importance of stromal Tenascin-C and S100A4(+) fibroblast-derived VEGF-A in metastasis was established by examining Tenascin-C null mice and transgenic mice expressing Cre recombinase under control of the S100A4 promoter crossed with mice carrying VEGF-A alleles flanked by loxP sites, which exhibited a significant decrease in metastatic colonization without effects on primary tumor growth. In particular, S100A4(+) fibroblast-derived VEGF-A plays an important role in the establishment of an angiogenic microenvironment at the metastatic site to facilitate colonization, whereas stromal Tenascin-C may provide protection from apoptosis. Our study demonstrates a crucial role for local S100A4(+) fibroblasts in providing the permissive "soil" for metastatic colonization, a challenging step in the metastatic cascade.


Assuntos
Proteínas S100/metabolismo , Células Estromais/metabolismo , Tenascina/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Linhagem Celular Tumoral , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Ganciclovir/farmacologia , Perfilação da Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Metástase Neoplásica , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Proteína A4 de Ligação a Cálcio da Família S100 , Proteínas S100/genética , Células Estromais/efeitos dos fármacos , Tenascina/genética , Timidina Quinase/genética , Timidina Quinase/metabolismo , Microambiente Tumoral/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/genética
3.
J Med Chem ; 67(2): 1447-1459, 2024 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-38198520

RESUMO

Uveal melanoma (UM) is the most common primary intraocular malignancy in the adult eye. Despite the aggressive local management of primary UM, the development of metastases is common with no effective treatment options for metastatic disease. Genetic analysis of UM samples reveals the presence of mutually exclusive activating mutations in the Gq alpha subunits GNAQ and GNA11. One of the key downstream targets of the constitutively active Gq alpha subunits is the protein kinase C (PKC) signaling pathway. Herein, we describe the discovery of darovasertib (NVP-LXS196), a potent pan-PKC inhibitor with high whole kinome selectivity. The lead series was optimized for kinase and off target selectivity to afford a compound that is rapidly absorbed and well tolerated in preclinical species. LXS196 is being investigated in the clinic as a monotherapy and in combination with other agents for the treatment of uveal melanoma (UM), including primary UM and metastatic uveal melanoma (MUM).


Assuntos
Melanoma , Neoplasias Uveais , Adulto , Humanos , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Melanoma/tratamento farmacológico , Melanoma/patologia , Neoplasias Uveais/tratamento farmacológico , Neoplasias Uveais/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Mutação
4.
Proc Natl Acad Sci U S A ; 105(39): 15040-5, 2008 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-18818312

RESUMO

Tumstatin is an angiogenesis inhibitor that binds to alphavbeta3 integrin and suppresses tumor growth. Previous deletion mutagenesis studies identified a 25-aa fragment of tumstatin (tumstatin peptide) with in vitro antiangiogenic activity. Here, we demonstrate that systemic administration of this tumstatin peptide inhibits tumor growth and angiogenesis. Site-directed mutagenesis identified amino acids L, V, and D as essential for the antiangiogenic activity of tumstatin. The tumstatin peptide binds to alphavbeta3 integrin on proliferating endothelial cells and localizes to select tumor endothelium in vivo. Using 3D molecular modeling, we identify a putative interaction interface for tumstatin peptide on alphavbeta3 integrin. The antitumor activity of the tumstatin peptide, in combination with bevacizumab (anti-VEGF antibody), displays significant improvement in efficacy against human renal cell carcinoma xenografts when compared with the single-agent use. Collectively, our results demonstrate that tumstatin peptide binds specifically to the tumor endothelium, and its antiangiogenic action is mediated by alphavbeta3 integrin, and, in combination with an anti-VEGF antibody it exhibits enhanced tumor suppression of renal cell carcinoma.


Assuntos
Inibidores da Angiogênese/farmacologia , Autoantígenos/farmacologia , Carcinoma de Células Renais/tratamento farmacológico , Colágeno Tipo IV/farmacologia , Neoplasias Renais/tratamento farmacológico , Neovascularização Patológica/tratamento farmacológico , Substituição de Aminoácidos/genética , Inibidores da Angiogênese/genética , Inibidores da Angiogênese/metabolismo , Inibidores da Angiogênese/uso terapêutico , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados , Protocolos de Quimioterapia Combinada Antineoplásica , Ácido Aspártico/genética , Ácido Aspártico/metabolismo , Autoantígenos/genética , Autoantígenos/metabolismo , Autoantígenos/uso terapêutico , Bevacizumab , Carcinoma de Células Renais/irrigação sanguínea , Proliferação de Células , Células Cultivadas , Colágeno Tipo IV/genética , Colágeno Tipo IV/metabolismo , Colágeno Tipo IV/uso terapêutico , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Humanos , Integrina alfaVbeta3/metabolismo , Neoplasias Renais/irrigação sanguínea , Leucina/genética , Leucina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese Sítio-Dirigida , Transplante de Neoplasias , Conformação Proteica , Valina/genética , Valina/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Clin Cancer Res ; 27(7): 2061-2073, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33355204

RESUMO

PURPOSE: Targeting RAF for antitumor therapy in RAS-mutant tumors holds promise. Herein, we describe in detail novel properties of the type II RAF inhibitor, LXH254. EXPERIMENTAL DESIGN: LXH254 was profiled in biochemical, in vitro, and in vivo assays, including examining the activities of the drug in a large panel of cancer-derived cell lines and a comprehensive set of in vivo models. In addition, activity of LXH254 was assessed in cells where different sets of RAF paralogs were ablated, or that expressed kinase-impaired and dimer-deficient variants of ARAF. RESULTS: We describe an unexpected paralog selectivity of LXH254, which is able to potently inhibit BRAF and CRAF, but has less activity against ARAF. LXH254 was active in models harboring BRAF alterations, including atypical BRAF alterations coexpressed with mutant K/NRAS, and NRAS mutants, but had only modest activity in KRAS mutants. In RAS-mutant lines, loss of ARAF, but not BRAF or CRAF, sensitized cells to LXH254. ARAF-mediated resistance to LXH254 required both kinase function and dimerization. Higher concentrations of LXH254 were required to inhibit signaling in RAS-mutant cells expressing only ARAF relative to BRAF or CRAF. Moreover, specifically in cells expressing only ARAF, LXH254 caused paradoxical activation of MAPK signaling in a manner similar to dabrafenib. Finally, in vivo, LXH254 drove complete regressions of isogenic variants of RAS-mutant cells lacking ARAF expression, while parental lines were only modestly sensitive. CONCLUSIONS: LXH254 is a novel RAF inhibitor, which is able to inhibit dimerized BRAF and CRAF, as well as monomeric BRAF, while largely sparing ARAF.


Assuntos
Sistema de Sinalização das MAP Quinases/fisiologia , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-raf/antagonistas & inibidores , Animais , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Células HCT116 , Humanos , Camundongos , Mutação , Neoplasias/genética , Inibidores de Proteínas Quinases/farmacologia , Multimerização Proteica , Proteínas Proto-Oncogênicas B-raf/química , Proteínas Proto-Oncogênicas c-raf/química , Proteínas Proto-Oncogênicas p21(ras)/genética
6.
J Med Chem ; 63(5): 2013-2027, 2020 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-31059256

RESUMO

Direct pharmacological inhibition of RAS has remained elusive, and efforts to target CRAF have been challenging due to the complex nature of RAF signaling, downstream of activated RAS, and the poor overall kinase selectivity of putative RAF inhibitors. Herein, we describe 15 (LXH254, Aversa, R.; et al. Int. Patent WO2014151616A1, 2014), a selective B/C RAF inhibitor, which was developed by focusing on drug-like properties and selectivity. Our previous tool compound, 3 (RAF709; Nishiguchi, G. A.; et al. J. Med. Chem. 2017, 60, 4969), was potent, selective, efficacious, and well tolerated in preclinical models, but the high human intrinsic clearance precluded further development and prompted further investigation of close analogues. A structure-based approach led to a pyridine series with an alcohol side chain that could interact with the DFG loop and significantly improved cell potency. Further mitigation of human intrinsic clearance and time-dependent inhibition led to the discovery of 15. Due to its excellent properties, it was progressed through toxicology studies and is being tested in phase 1 clinical trials.


Assuntos
Antineoplásicos/química , Descoberta de Drogas/métodos , Mutação/genética , Inibidores de Proteínas Quinases/química , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/genética , Animais , Antineoplásicos/farmacologia , Desenho de Fármacos , Descoberta de Drogas/tendências , Humanos , Simulação de Acoplamento Molecular/métodos , Simulação de Acoplamento Molecular/tendências , Mutação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
7.
Dev Biol ; 313(1): 246-55, 2008 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-18022613

RESUMO

Junctional adhesion molecules (JAMs) that are expressed in endothelial and epithelial cells and function in tight junction assembly, also perform important roles in testis where the closely-related JAM-A, JAM-B, and JAM-C are found. Disruption of murine Jam-B and Jam-C has varying effects on sperm development and function; however, deletion of Jam-A has not yet been studied. Here we show for the first time that in addition to expression in the Sertoli-Sertoli tight junctions in the seminiferous tubules, the approximately 32 kDa murine JAM-A is present in elongated spermatids and in the plasma membrane of the head and flagellum of sperm. Deletion of Jam-A, using the gene trap technology, results in flagellar defects at the ultrastructural level. In Jam-A-deficient mice, which have reduced litter size, both progressive and hyperactive motility are significantly affected (P<0.0001) before and, more severely, after capacitation. The findings show that JAM-A is involved in sperm tail formation and is essential for normal motility, which may occur via its signal transduction and protein phosphorylation properties. Detection of JAM-A in human sperm proteins indicates that its role may be conserved in sperm motility and that JAM-A may be a candidate gene for the analysis of idiopathic sperm motility defects resulting in male subfertility in the human population.


Assuntos
Moléculas de Adesão Celular/fisiologia , Imunoglobulinas/fisiologia , Receptores de Superfície Celular/fisiologia , Motilidade dos Espermatozoides , Espermatozoides/química , Animais , Moléculas de Adesão Celular/análise , Epididimo/fisiologia , Humanos , Imunoglobulinas/análise , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Superfície Celular/análise
8.
Cancer Res ; 78(6): 1537-1548, 2018 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-29343524

RESUMO

Resistance to the RAF inhibitor vemurafenib arises commonly in melanomas driven by the activated BRAF oncogene. Here, we report antitumor properties of RAF709, a novel ATP-competitive kinase inhibitor with high potency and selectivity against RAF kinases. RAF709 exhibited a mode of RAF inhibition distinct from RAF monomer inhibitors such as vemurafenib, showing equal activity against both RAF monomers and dimers. As a result, RAF709 inhibited MAPK signaling activity in tumor models harboring either BRAFV600 alterations or mutant N- and KRAS-driven signaling, with minimal paradoxical activation of wild-type RAF. In cell lines and murine xenograft models, RAF709 demonstrated selective antitumor activity in tumor cells harboring BRAF or RAS mutations compared with cells with wild-type BRAF and RAS genes. RAF709 demonstrated a direct pharmacokinetic/pharmacodynamic relationship in in vivo tumor models harboring KRAS mutation. Furthermore, RAF709 elicited regression of primary human tumor-derived xenograft models with BRAF, NRAS, or KRAS mutations with excellent tolerability. Our results support further development of inhibitors like RAF709, which represents a next-generation RAF inhibitor with unique biochemical and cellular properties that enables antitumor activities in RAS-mutant tumors.Significance: In an effort to develop RAF inhibitors with the appropriate pharmacological properties to treat RAS mutant tumors, RAF709, a compound with potency, selectivity, and in vivo properties, was developed that will allow preclinical therapeutic hypothesis testing, but also provide an excellent probe to further unravel the complexities of RAF kinase signaling. Cancer Res; 78(6); 1537-48. ©2018 AACR.


Assuntos
2,2'-Dipiridil/análogos & derivados , Antineoplásicos/farmacologia , Benzamidas/farmacologia , Proteínas Proto-Oncogênicas B-raf/genética , Quinases raf/antagonistas & inibidores , Proteínas ras/genética , 2,2'-Dipiridil/farmacologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Humanos , MAP Quinase Quinase Quinases/antagonistas & inibidores , Camundongos Nus , Mutação , Inibidores de Proteínas Quinases/farmacologia , Multimerização Proteica , Ensaios Antitumorais Modelo de Xenoenxerto , Quinases raf/metabolismo
9.
Int J Biochem Cell Biol ; 39(3): 576-85, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17118692

RESUMO

Junctional adhesion molecule-A (JAM-A, JAM-1, F11R) is an Ig domain containing transmembrane protein that has been proposed to function in diverse processes including platelet activation and adhesion, leukocyte transmigration, angiogenesis, epithelial cell shape and endothelial cell migration although its function in vivo is less well established. In the mouse eye, JAM-A protein expression is first detected at 12.5 dpc in the blood vessels of the tunica vasculosa, while it is first detected in both the corneal epithelium and lens between 13.5 and 14.5 dpc. In the corneal epithelium, JAM-A levels remain appreciable throughout life, while JAM-A immunostaining becomes stronger in the lens as the animals age. Both the cornea and lens of mice lacking an intact JAM-A gene are transparent until at least a year of age, although the cells of the JAM-A null corneal epithelium are irregularly shaped. In wild-type mice, JAM-A protein is found at the leading edge of repairing corneal epithelial wounds, however, corneal epithelial wound repair was qualitatively normal in JAM-A null animals. In summary, JAM-A is expressed in the corneal epithelium where it appears to regulate cell shape.


Assuntos
Moléculas de Adesão Celular/deficiência , Epitélio Corneano/anormalidades , Receptores de Superfície Celular/deficiência , Animais , Sequência de Bases , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Forma Celular/genética , Forma Celular/fisiologia , Primers do DNA/genética , Epitélio Corneano/citologia , Epitélio Corneano/embriologia , Epitélio Corneano/metabolismo , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Heterozigoto , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados/deficiência , Fatores de Transcrição Box Pareados/genética , Fatores de Transcrição Box Pareados/metabolismo , Fenótipo , Gravidez , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Cicatrização/fisiologia
10.
Arterioscler Thromb Vasc Biol ; 26(9): 2005-11, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16809549

RESUMO

OBJECTIVE: We have previously shown that JAM-A regulates fibroblast growth factor-2 (FGF-2)-induced endothelial cell morphology, proliferation, and migration. Whether JAM-A is involved in FGF-2-induced angiogenesis in vivo is not known. We used JAM-A null mice to conclusively determine the role of JAM-A in FGF-2-induced neovascularization. METHODS AND RESULTS: We generated JAM-A null (JAM-A(-/-)) mice using gene trap technology. These mice, although viable and fertile, exhibited distorted Mendelian and sex ratios, suggesting partial embryonic lethality. Retinal fluorescein angiogram did not reveal any significant morphological differences in the vasculature of JAM-A(-/-) mice compared with wild-type (JAMA-A(+/+)) littermates. To evaluate the role of JAM-A in angiogenesis, we performed an aortic ring assay. FGF-2-induced microvessel growth was evident in aortic rings from JAM-A(+/+) mice, but FGF-2 failed to induce microvessel sproutings in aortic rings from JAM-A(-/-) mice. In a Matrigel plug assay, a known in vivo model for angiogenesis, we found that FGF-2 induced a robust vessel growth in JAM-A(+/+) mice, whereas FGF-2 failed to induce blood vessel formation in plugs from JAM-A(-/-) mice. CONCLUSIONS: Our results using JAM-A(-/-) mice presented here conclusively establish an essential role for JAM-A in FGF-2-induced angiogenesis.


Assuntos
Moléculas de Adesão Celular/deficiência , Moléculas de Adesão Celular/fisiologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Neovascularização Fisiológica/efeitos dos fármacos , Neovascularização Fisiológica/fisiologia , Receptores de Superfície Celular/deficiência , Animais , Aorta Torácica/fisiologia , Vasos Sanguíneos/embriologia , Perda do Embrião/etiologia , Feminino , Técnicas In Vitro , Moléculas de Adesão Juncional , Tamanho da Ninhada de Vivíparos , Masculino , Camundongos , Camundongos Knockout , Isoformas de Proteínas/deficiência , Isoformas de Proteínas/fisiologia , Vasos Retinianos/fisiologia , Razão de Masculinidade
11.
J Med Chem ; 60(12): 4869-4881, 2017 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-28557458

RESUMO

RAS oncogenes have been implicated in >30% of human cancers, all representing high unmet medical need. The exquisite dependency on CRAF kinase in KRAS mutant tumors has been established in genetically engineered mouse models and human tumor cells. To date, many small molecule approaches are under investigation to target CRAF, yet kinase-selective and cellular potent inhibitors remain challenging to identify. Herein, we describe 14 (RAF709) [ Aversa , Biaryl amide compounds as kinase inhibitors and their preparation . WO 2014151616, 2014 ], a selective B/C RAF inhibitor, which was developed through a hypothesis-driven approach focusing on drug-like properties. A key challenge encountered in the medicinal chemistry campaign was maintaining a balance between good solubility and potent cellular activity (suppression of pMEK and proliferation) in KRAS mutant tumor cell lines. We investigated the small molecule crystal structure of lead molecule 7 and hypothesized that disruption of the crystal packing would improve solubility, which led to a change from N-methylpyridone to a tetrahydropyranyl oxy-pyridine derivative. 14 proved to be soluble, kinase selective, and efficacious in a KRAS mutant xenograft model.


Assuntos
2,2'-Dipiridil/análogos & derivados , Antineoplásicos/farmacologia , Benzamidas/farmacologia , Quinases raf/antagonistas & inibidores , Proteínas ras/genética , 2,2'-Dipiridil/química , 2,2'-Dipiridil/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/farmacocinética , Benzamidas/química , Cristalografia por Raios X , Cães , Desenho de Fármacos , Descoberta de Drogas , Estabilidade de Medicamentos , Humanos , Concentração Inibidora 50 , Camundongos , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Neoplasias/genética , Proteínas Proto-Oncogênicas B-raf/química , Proteínas Proto-Oncogênicas p21(ras)/genética , Ratos , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Cancer Cell ; 29(4): 574-586, 2016 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-27070704

RESUMO

More than 90% of drugs with preclinical activity fail in human trials, largely due to insufficient efficacy. We hypothesized that adequately powered trials of patient-derived xenografts (PDX) in mice could efficiently define therapeutic activity across heterogeneous tumors. To address this hypothesis, we established a large, publicly available repository of well-characterized leukemia and lymphoma PDXs that undergo orthotopic engraftment, called the Public Repository of Xenografts (PRoXe). PRoXe includes all de-identified information relevant to the primary specimens and the PDXs derived from them. Using this repository, we demonstrate that large studies of acute leukemia PDXs that mimic human randomized clinical trials can characterize drug efficacy and generate transcriptional, functional, and proteomic biomarkers in both treatment-naive and relapsed/refractory disease.


Assuntos
Xenoenxertos , Leucemia/patologia , Linfoma/patologia , Bancos de Tecidos , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais , Linhagem da Célula , Feminino , Perfilação da Expressão Gênica , Genes p53 , Humanos , Internet , Isoquinolinas/farmacologia , Isoquinolinas/uso terapêutico , Leucemia/metabolismo , Leucemia Experimental/tratamento farmacológico , Linfoma/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos NOD , Terapia de Alvo Molecular , Proteínas de Neoplasias/antagonistas & inibidores , Transplante de Neoplasias , Fenótipo , Piperazinas/farmacologia , Piperazinas/uso terapêutico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Proteoma , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Distribuição Aleatória , Ensaios Clínicos Controlados Aleatórios como Assunto/métodos , Projetos de Pesquisa , Transcriptoma
13.
Cell Rep ; 10(7): 1066-81, 2015 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-25704811

RESUMO

Strategies to target angiogenesis include inhibition of the vessel-stabilizing properties of vascular pericytes. Pericyte depletion in early-stage non-hypoxic tumors suppressed nascent angiogenesis, tumor growth, and lung metastasis. In contrast, pericyte depletion in advanced-stage hypoxic tumors with pre-established vasculature resulted in enhanced intra-tumoral hypoxia, decreased tumor growth, and increased lung metastasis. Furthermore, depletion of pericytes in post-natal retinal blood vessels resulted in abnormal and leaky vasculature. Tumor transcriptome profiling and biological validation revealed that angiopoietin signaling is a key regulatory pathway associated with pericyte targeting. Indeed, pericyte targeting in established mouse tumors increased angiopoietin-2 (ANG2/Angpt2) expression. Depletion of pericytes, coupled with targeting of ANG2 signaling, restored vascular stability in multiple model systems and decreased tumor growth and metastasis. Importantly, ANGPT2 expression correlated with poor outcome in patients with breast cancer. These results emphasize the potential utility of therapeutic regimens that target pericytes and ANG2 signaling in metastatic breast cancer.


Assuntos
Angiopoietina-2/metabolismo , Neoplasias da Mama/patologia , Neoplasias Pulmonares/secundário , Angiopoietina-2/imunologia , Animais , Anticorpos/imunologia , Anticorpos/farmacologia , Antígenos/genética , Antígenos/metabolismo , Hipóxia Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Transição Epitelial-Mesenquimal , Feminino , Mesilato de Imatinib/farmacologia , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Neovascularização Patológica , Pericitos/citologia , Pericitos/efeitos dos fármacos , Pericitos/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteoglicanas/deficiência , Proteoglicanas/genética , Proteoglicanas/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/deficiência , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Retina/fisiologia , Transdução de Sinais/efeitos dos fármacos
14.
Mol Cancer Ther ; 14(10): 2249-59, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26206331

RESUMO

The tumor suppressor p53 is a key regulator of apoptosis and functions upstream in the apoptotic cascade by both indirectly and directly regulating Bcl-2 family proteins. In cells expressing wild-type (WT) p53, the HDM2 protein binds to p53 and blocks its activity. Inhibition of HDM2:p53 interaction activates p53 and causes apoptosis or cell-cycle arrest. Here, we investigated the ability of the novel HDM2 inhibitor CGM097 to potently and selectively kill WT p53-expressing AML cells. The antileukemic effects of CGM097 were studied using cell-based proliferation assays (human AML cell lines, primary AML patient cells, and normal bone marrow samples), apoptosis, and cell-cycle assays, ELISA, immunoblotting, and an AML patient-derived in vivo mouse model. CGM097 potently and selectively inhibited the proliferation of human AML cell lines and the majority of primary AML cells expressing WT p53, but not mutant p53, in a target-specific manner. Several patient samples that harbored mutant p53 were comparatively unresponsive to CGM097. Synergy was observed when CGM097 was combined with FLT3 inhibition against oncogenic FLT3-expressing cells cultured both in the absence as well as the presence of cytoprotective stromal-secreted cytokines, as well as when combined with MEK inhibition in cells with activated MAPK signaling. Finally, CGM097 was effective in reducing leukemia burden in vivo. These data suggest that CGM097 is a promising treatment for AML characterized as harboring WT p53 as a single agent, as well as in combination with other therapies targeting oncogene-activated pathways that drive AML.


Assuntos
Antineoplásicos/farmacologia , Isoquinolinas/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteína Supressora de Tumor p53/metabolismo , Animais , Apoptose/efeitos dos fármacos , Benzotiazóis/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sinergismo Farmacológico , Feminino , Expressão Gênica , Humanos , Concentração Inibidora 50 , Camundongos Endogâmicos NOD , Camundongos SCID , Compostos de Fenilureia/farmacologia , Estaurosporina/análogos & derivados , Estaurosporina/farmacologia , Carga Tumoral/efeitos dos fármacos , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética , Ensaios Antitumorais Modelo de Xenoenxerto
15.
Nat Med ; 21(5): 440-8, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25849130

RESUMO

Resistance to cancer therapies presents a significant clinical challenge. Recent studies have revealed intratumoral heterogeneity as a source of therapeutic resistance. However, it is unclear whether resistance is driven predominantly by pre-existing or de novo alterations, in part because of the resolution limits of next-generation sequencing. To address this, we developed a high-complexity barcode library, ClonTracer, which enables the high-resolution tracking of more than 1 million cancer cells under drug treatment. In two clinically relevant models, ClonTracer studies showed that the majority of resistant clones were part of small, pre-existing subpopulations that selectively escaped under therapeutic challenge. Moreover, the ClonTracer approach enabled quantitative assessment of the ability of combination treatments to suppress resistant clones. These findings suggest that resistant clones are present before treatment, which would make up-front therapeutic combinations that target non-overlapping resistance a preferred approach. Thus, ClonTracer barcoding may be a valuable tool for optimizing therapeutic regimens with the goal of curative combination therapies for cancer.


Assuntos
Código de Barras de DNA Taxonômico/métodos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Diferenciação Celular , Linhagem Celular Tumoral , Crizotinibe , DNA/química , DNA Complementar/metabolismo , Transição Epitelial-Mesenquimal , Cloridrato de Erlotinib , Proteínas de Fusão bcr-abl/genética , Dosagem de Genes , Biblioteca Gênica , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Modelos Teóricos , Oligonucleotídeos/genética , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas c-abl/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-met/metabolismo , Pirazóis/administração & dosagem , Piridinas/administração & dosagem , Quinazolinas/administração & dosagem , Análise de Sequência de RNA
16.
Cancer Res ; 74(12): 3294-305, 2014 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-24747911

RESUMO

Tankyrases (TNKS) play roles in Wnt signaling, telomere homeostasis, and mitosis, offering attractive targets for anticancer treatment. Using unbiased combination screening in a large panel of cancer cell lines, we have identified a strong synergy between TNKS and MEK inhibitors (MEKi) in KRAS-mutant cancer cells. Our study uncovers a novel function of TNKS in the relief of a feedback loop induced by MEK inhibition on FGFR2 signaling pathway. Moreover, dual inhibition of TNKS and MEK leads to more robust apoptosis and antitumor activity both in vitro and in vivo than effects observed by previously reported MEKi combinations. Altogether, our results show how a novel combination of TNKS and MEK inhibitors can be highly effective in targeting KRAS-mutant cancers by suppressing a newly discovered resistance mechanism.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Proteínas Proto-Oncogênicas/genética , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Tanquirases/metabolismo , Proteínas ras/genética , Acetamidas/administração & dosagem , Aminopiridinas/administração & dosagem , Compostos de Anilina/administração & dosagem , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linhagem Celular Tumoral , Sinergismo Farmacológico , Cloridrato de Erlotinib , Retroalimentação Fisiológica , Feminino , Humanos , MAP Quinase Quinase Quinases/antagonistas & inibidores , MAP Quinase Quinase Quinases/metabolismo , Camundongos , Camundongos Nus , Morfolinas/administração & dosagem , Mutação , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas p21(ras) , Pirimidinonas/administração & dosagem , Quinazolinas/administração & dosagem , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Transdução de Sinais , Sulfonamidas/administração & dosagem , Tanquirases/antagonistas & inibidores , Tiazóis/administração & dosagem , Ensaios Antitumorais Modelo de Xenoenxerto
17.
Nat Med ; 20(1): 87-92, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24362935

RESUMO

Mantle cell lymphoma (MCL) is an aggressive malignancy that is characterized by poor prognosis. Large-scale pharmacological profiling across more than 100 hematological cell line models identified a subset of MCL cell lines that are highly sensitive to the B cell receptor (BCR) signaling inhibitors ibrutinib and sotrastaurin. Sensitive MCL models exhibited chronic activation of the BCR-driven classical nuclear factor-κB (NF-κB) pathway, whereas insensitive cell lines displayed activation of the alternative NF-κB pathway. Transcriptome sequencing revealed genetic lesions in alternative NF-κB pathway signaling components in ibrutinib-insensitive cell lines, and sequencing of 165 samples from patients with MCL identified recurrent mutations in TRAF2 or BIRC3 in 15% of these individuals. Although they are associated with insensitivity to ibrutinib, lesions in the alternative NF-κB pathway conferred dependence on the protein kinase NIK (also called mitogen-activated protein 3 kinase 14 or MAP3K14) both in vitro and in vivo. Thus, NIK is a new therapeutic target for MCL treatment, particularly for lymphomas that are refractory to BCR pathway inhibitors. Our findings reveal a pattern of mutually exclusive activation of the BCR-NF-κB or NIK-NF-κB pathways in MCL and provide critical insights into patient stratification strategies for NF-κB pathway-targeted agents.


Assuntos
Linfoma de Célula do Manto/tratamento farmacológico , NF-kappa B/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Pirróis/farmacologia , Quinazolinas/farmacologia , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Adenina/análogos & derivados , Proteína 3 com Repetições IAP de Baculovírus , Sequência de Bases , Western Blotting , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Linhagem Celular , Sobrevivência Celular , Primers do DNA/genética , Guanilato Ciclase/metabolismo , Humanos , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Medições Luminescentes , Análise em Microsséries , Dados de Sequência Molecular , Piperidinas , Proteínas Serina-Treonina Quinases/genética , Pirazóis/farmacologia , Pirimidinas/farmacologia , Interferência de RNA , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Antígenos de Linfócitos B/antagonistas & inibidores , Análise de Sequência de RNA , Fator 2 Associado a Receptor de TNF/genética , Fator 2 Associado a Receptor de TNF/metabolismo , Fator 3 Associado a Receptor de TNF/metabolismo , Azul Tripano , Ubiquitina-Proteína Ligases , Quinase Induzida por NF-kappaB
18.
Nat Med ; 19(8): 1047-53, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23817022

RESUMO

Myofibroblasts are associated with organ fibrosis, but their precise origin and functional role remain unknown. We used multiple genetically engineered mice to track, fate map and ablate cells to determine the source and function of myofibroblasts in kidney fibrosis. Through this comprehensive analysis, we identified that the total pool of myofibroblasts is split, with 50% arising from local resident fibroblasts through proliferation. The nonproliferating myofibroblasts derive through differentiation from bone marrow (35%), the endothelial-to-mesenchymal transition program (10%) and the epithelial-to-mesenchymal transition program (5%). Specific deletion of Tgfbr2 in α-smooth muscle actin (αSMA)(+) cells revealed the importance of this pathway in the recruitment of myofibroblasts through differentiation. Using genetic mouse models and a fate-mapping strategy, we determined that vascular pericytes probably do not contribute to the emergence of myofibroblasts or fibrosis. Our data suggest that targeting diverse pathways is required to substantially inhibit the composite accumulation of myofibroblasts in kidney fibrosis.


Assuntos
Nefropatias/metabolismo , Nefropatias/patologia , Rim/metabolismo , Rim/patologia , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Actinas/metabolismo , Animais , Antígenos/metabolismo , Células da Medula Óssea/patologia , Diferenciação Celular , Proliferação de Células , Transição Epitelial-Mesenquimal , Feminino , Fibrose , Masculino , Células-Tronco Mesenquimais/metabolismo , Camundongos , Pericitos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteoglicanas/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transdução de Sinais
19.
Cancer Discov ; 3(9): 1030-43, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23842682

RESUMO

UNLABELLED: Castration-resistant prostate cancer (CRPC) is the most aggressive, incurable form of prostate cancer. MDV3100 (enzalutamide), an antagonist of the androgen receptor (AR), was approved for clinical use in men with metastatic CRPC. Although this compound showed clinical efficacy, many initial responders later developed resistance. To uncover relevant resistant mechanisms, we developed a model of spontaneous resistance to MDV3100 in LNCaP prostate cancer cells. Detailed characterization revealed that emergence of an F876L mutation in AR correlated with blunted AR response to MDV3100 and sustained proliferation during treatment. Functional studies confirmed that AR(F876L) confers an antagonist-to-agonist switch that drives phenotypic resistance. Finally, treatment with distinct antiandrogens or cyclin-dependent kinase (CDK)4/6 inhibitors effectively antagonized AR(F876L) function. Together, these findings suggest that emergence of F876L may (i) serve as a novel biomarker for prediction of drug sensitivity, (ii) predict a "withdrawal" response to MDV3100, and (iii) be suitably targeted with other antiandrogens or CDK4/6 inhibitors. SIGNIFICANCE: We uncovered an F876L agonist-switch mutation in AR that confers genetic and phenotypic resistance to the antiandrogen drug MDV3100. On the basis of this fi nding, we propose new therapeutic strategies to treat patients with prostate cancer presenting with this AR mutation.


Assuntos
Antagonistas de Receptores de Andrógenos/farmacologia , Feniltioidantoína/análogos & derivados , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Receptores Androgênicos/genética , Antagonistas de Androgênios/farmacologia , Sequência de Bases , Benzamidas , Linhagem Celular Tumoral , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Masculino , Mutação , Nitrilas , Feniltioidantoína/farmacologia , Análise de Sequência de DNA
20.
Nat Genet ; 45(11): 1386-91, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24076604

RESUMO

Epigenetic dysregulation is an emerging hallmark of cancers. We developed a high-information-content mass spectrometry approach to profile global histone modifications in human cancers. When applied to 115 lines from the Cancer Cell Line Encyclopedia, this approach identified distinct molecular chromatin signatures. One signature was characterized by increased histone 3 lysine 36 (H3K36) dimethylation, exhibited by several lines harboring translocations in NSD2, which encodes a methyltransferase. A previously unknown NSD2 p.Glu1099Lys (p.E1099K) variant was identified in nontranslocated acute lymphoblastic leukemia (ALL) cell lines sharing this signature. Ectopic expression of the variant induced a chromatin signature characteristic of NSD2 hyperactivation and promoted transformation. NSD2 knockdown selectively inhibited the proliferation of NSD2-mutant lines and impaired the in vivo growth of an NSD2-mutant ALL xenograft. Sequencing analysis of >1,000 pediatric cancer genomes identified the NSD2 p.E1099K alteration in 14% of t(12;21) ETV6-RUNX1-containing ALLs. These findings identify NSD2 as a potential therapeutic target for pediatric ALL and provide a general framework for the functional annotation of cancer epigenomes.


Assuntos
Cromatina/genética , Histona-Lisina N-Metiltransferase/genética , Histonas/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteínas Repressoras/genética , Animais , Sequência de Bases , Linhagem Celular Tumoral , Criança , Feminino , Predisposição Genética para Doença , Variação Genética , Humanos , Camundongos , Camundongos SCID , Células NIH 3T3 , Transplante de Neoplasias , Análise de Sequência de DNA , Ensaios Antitumorais Modelo de Xenoenxerto
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