RESUMO
Gastrointestinal stromal tumors (GISTs) predominantly harbor activating mutations in the receptor tyrosine kinase KIT. To genetically dissect in vivo the requirement of different signal transduction pathways emanating from KIT for tumorigenesis, the oncogenic KitV558Δ mutation was combined with point mutations abrogating specific phosphorylation sites on KIT. Compared with single-mutant KitV558Δ/+ mice, double-mutant KitV558Δ;Y567F/Y567F knock-in mice lacking the SRC family kinase-binding site on KIT (pY567) exhibited attenuated MAPK signaling and tumor growth. Surprisingly, abrogation of the PI3K-binding site (pY719) in KitV558Δ;Y719F/Y719F mice prevented GIST development, although the interstitial cells of Cajal (ICC), the cells of origin of GIST, were normal. Pharmacologic inhibition of the PI3K pathway in tumor-bearing KitV558Δ/+ mice with the dual PI3K/mTOR inhibitor voxtalisib, the pan-PI3K inhibitor pilaralisib, and the PI3K-alpha-restricted inhibitor alpelisib each diminished tumor proliferation. The addition of the MEK inhibitor PD-325901 or binimetinib further decreased downstream KIT signaling. Moreover, combining PI3K and MEK inhibition was effective against imatinib-resistant KitV558Δ;T669I/+ tumors.
Assuntos
Carcinogênese/patologia , Resistencia a Medicamentos Antineoplásicos/genética , Tumores do Estroma Gastrointestinal/patologia , Mesilato de Imatinib/farmacologia , Mutação , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Animais , Carcinogênese/genética , Carcinogênese/metabolismo , Feminino , Neoplasias Gastrointestinais/genética , Neoplasias Gastrointestinais/metabolismo , Neoplasias Gastrointestinais/patologia , Tumores do Estroma Gastrointestinal/genética , Tumores do Estroma Gastrointestinal/metabolismo , Humanos , Masculino , Camundongos , Fosfatidilinositol 3-Quinases/genética , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-kit/genética , Transdução de Sinais , Células Tumorais CultivadasRESUMO
Most gastrointestinal stromal tumors (GISTs) harbor a gain-of-function mutation in the Kit receptor. GIST patients treated with the tyrosine kinase inhibitor imatinib frequently develop imatinib resistance as a result of second-site Kit mutations. To investigate the consequences of second-site Kit mutations on GIST development and imatinib sensitivity, we engineered a mouse model carrying in the endogenous Kit locus both the Kit(V558Δ) mutation found in a familial case of GIST and the Kit(T669I) (human KIT(T670I)) "gatekeeper" mutation found in imatinib-resistant GIST patients. Similar to Kit(V558/+) mice, Kit(V558;T669I/+) mice developed gastric and colonic interstitial cell of Cajal hyperplasia as well as cecal GIST. In contrast to the single-mutant Kit(V558/+) control mice, treatment of the Kit(V558;T669I/+) mice with either imatinib or dasatinib failed to inhibit oncogenic Kit signaling and GIST growth. However, this resistance could be overcome by treatment of Kit(V558;T669I/+) mice with sunitinib or sorafenib. Although tumor lesions were smaller in Kit(V558;T669I/+) mice than in single-mutant mice, both interstitial cell of Cajal hyperplasia and mast cell hyperplasia were exacerbated in Kit(V558;T669I/+) mice. Strikingly, the Kit(V558;T669I/+) mice developed a pronounced polycythemia vera-like erythrocytosis in conjunction with microcytosis. This mouse model should be useful for preclinical studies of drug candidates designed to overcome imatinib resistance in GIST and to investigate the consequences of oncogenic KIT signaling in hematopoietic as well as other cell lineages.
Assuntos
Eritrócitos/citologia , Tumores do Estroma Gastrointestinal/genética , Mutação , Piperazinas/farmacologia , Policitemia/genética , Proteínas Proto-Oncogênicas c-kit/genética , Pirimidinas/farmacologia , Animais , Antineoplásicos/farmacologia , Benzamidas , Linhagem da Célula , Dasatinibe , Modelos Animais de Doenças , Resistência a Medicamentos , Resistencia a Medicamentos Antineoplásicos/genética , Éxons , Tumores do Estroma Gastrointestinal/tratamento farmacológico , Mesilato de Imatinib , Camundongos , Fenótipo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Tiazóis/farmacologiaRESUMO
The KIT receptor tyrosine kinase has important roles in hematopoiesis. We have recently produced a mouse model for imatinib resistant gastrointestinal stromal tumor (GIST) carrying the Kit(V558Δ) and Kit(T669I) (human KIT(T670I) ) mutations found in imatinib-resistant GIST. The Kit(V558Δ;T669I/+) mice developed microcytic erythrocytosis with an increase in erythroid progenitor numbers, a phenotype previously seen only in mouse models of polycythemia vera with alterations in Epo or Jak2. Significantly, the increased hematocrit observed in Kit(V558Δ;T669I/+) mice normalized upon splenectomy. In accordance with increased erythroid progenitors, myeloerythroid progenitor numbers were also elevated in the Kit(V558Δ;T669I/+) mice. Hematopoietic stem cell (HSC) numbers in the bone marrow (BM) of Kit(V558Δ;T669I/+) mice were unchanged in comparison to wild-type mice. However, increased HSC numbers were observed in fetal livers and the spleen and peripheral blood of adult Kit(V558Δ;T669I/+) mice. Importantly, HSC from Kit(V558Δ;T669I/+) BM had a competitive advantage over wild-type HSC. In response to 5-fluorouracil treatment, elevated numbers of dividing Lin(-) Sca(+) cells were found in the Kit(V558Δ;T669I/+) BM compared to wild type. Our study demonstrates that signaling from the Kit(V558Δ;T669I/+) receptor has important consequences in hematopoiesis enhancing HSC self-renewal and resulting in increased erythropoiesis.
Assuntos
Hematopoese/fisiologia , Células-Tronco Hematopoéticas/citologia , Proteínas Proto-Oncogênicas c-kit/metabolismo , Animais , Processos de Crescimento Celular/fisiologia , Modelos Animais de Doenças , Células Eritroides/citologia , Células Eritroides/metabolismo , Feminino , Fluoruracila/farmacologia , Células-Tronco Hematopoéticas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas c-kit/genética , Transdução de SinaisRESUMO
Although imatinib mesylate has been a major breakthrough in the treatment of advanced gastrointestinal stromal tumors (GIST), complete responses are rare and most patients eventually develop resistance to the drug. Thus, the possibility of an imatinib-insensitive cell subpopulation within GIST tumors, harboring stem cell characteristics, may be responsible for the clinical failures. However, the existence of a cancer stem cell component in GIST has not been yet established. This study was aimed to determine whether expression of commonly used stem cell markers in other malignancies, that is, CD133 and CD44, might identify cells with characteristics of cancer stem/progenitor cells in human GIST. CD133 and CD44 expression in GIST explants was analyzed by flow cytometry, immunofluorescence, and gene expression. Their transcription levels were correlated with clinical and molecular factors in a large, well-annotated cohort of GIST patients. FACS sorted GIST cells based on CD133 and CD44 expression were isolated and used to assess phenotypic characteristics, ability to maintain their surface expression, sensitivity to imatinib, and expression signature. The enrichment in CD133/CD44 cells in the side population (SP) assay was also investigated. CD133 expression was consistently found in GIST. CD133(-) cells formed more colonies, were more invasive in a matrigel assay, and showed enrichment in the SP cells, compared to CD133(+) cells. CD133 expression was also detected in the two imatinib-sensitive GIST cell lines, while was absent in the imatinib-resistant lines. Our results show that CD133 and CD44 are universally expressed in GIST, and may represent a lineage rather than a cancer stem cell marker.
Assuntos
Antígenos CD/metabolismo , Biomarcadores Tumorais/metabolismo , Neoplasias Gastrointestinais/metabolismo , Tumores do Estroma Gastrointestinal/metabolismo , Glicoproteínas/metabolismo , Receptores de Hialuronatos/metabolismo , Células-Tronco Neoplásicas/metabolismo , Peptídeos/metabolismo , Antígeno AC133 , Adolescente , Adulto , Antígenos CD/genética , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Proliferação de Células , Criança , Pré-Escolar , Neoplasias Gastrointestinais/genética , Neoplasias Gastrointestinais/patologia , Tumores do Estroma Gastrointestinal/genética , Tumores do Estroma Gastrointestinal/patologia , Expressão Gênica , Perfilação da Expressão Gênica , Glicoproteínas/genética , Humanos , Receptores de Hialuronatos/genética , Lactente , Peptídeos/genética , Sarcoma/genética , Sarcoma/metabolismo , Células da Side Population/metabolismo , Regulação para Cima , Adulto JovemRESUMO
BACKGROUND: A subset of KIT/PDGFRA wild-type gastrointestinal stromal tumors (WT GIST) have been associated with alteration of the succinate dehydrogenase (SDH) complex II function. A recent report identified four non-syndromic, KIT/PDGFRA WT GIST harboring compound heterozygous or homozygous mutations in SDHA encoding the main subunit of the SDH complex II. METHODS: Next generation sequencing was applied on five pediatric and one young adult WT GIST, by whole exome capture and SOLiD 3-plus system sequencing. The putative mutations were first confirmed by Sanger sequencing and then screened on a larger panel of 11 pediatric and young adult WT GIST, including 5 in the context of Carney triad. RESULTS: A germline p.Arg31X nonsense SDHA mutation was identified in one of the six cases tested by SOLiD platform. An additional p.D38V missense mutation in SDHA exon 2 was identified by Sanger sequencing in the extended KIT/PDGFRA WT GIST patients cohort. Western blotting showed loss of SDHA expression in the two cases harboring SDHA mutations, while expression being retained in the other WT GIST tumors. Results were further confirmed by immunohistochemistry for both SDHA and SDHB, which showed a concurrent loss of expression of both proteins in SDHA-mutant lesions, while the remaining WT tumors showed only loss of SDHB expression. CONCLUSIONS: Germline and/or somatic aberrations of SDHA occur in a small subset of KIT/PDGFRA WT GISTs, outside the Carney's triad and are associated with loss of both SDHA and SDHB protein expression. Mutations of the SDH complex II are more particularly associated with KIT/PDGFRA WT GIST occurring in young adults. Although pediatric GIST consistently display alterations of SDHB protein expression, further molecular studies are needed to identify the crucial genes involved in their tumorigenesis.
Assuntos
Complexo II de Transporte de Elétrons/genética , Neoplasias Gastrointestinais/genética , Tumores do Estroma Gastrointestinal/genética , Mutação , Adolescente , Adulto , Criança , Feminino , Humanos , Masculino , Proteínas Proto-Oncogênicas c-kit/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Análise de Sequência de DNA , Succinato Desidrogenase/genéticaRESUMO
Juxtamembrane signaling via the membrane growth factor KitL is critical for Kit mediated functions. KitL has a conserved cytoplasmic domain and has been shown to possess a monomeric leucine-dependent basolateral targeting signal. To investigate the consequences in vivo of impaired basolateral KitL targeting in polarized epithelial cells, we have mutated this critical leucine to alanine using a knock-in strategy. KitL(L263A/L263A) mutant mice are pigmented normally and steady-state hematopoiesis is unaffected although peritoneal and skin mast cell numbers are significantly increased. KitL localization is affected in the Sertoli cells of the KitL(L263A/L263A) testis and testis size is reduced in these mice due to aberrant spermatogonial proliferation. Furthermore, the effect of the KitL L263A mutation on the testicular phenotype is dosage dependent. The tubules of hemizygous KitL(L263A/Sl) mice completely lack germ cells in contrast to the weaker testicular phenotype of KitL(L263A/L263A) mice. The onset of the testis phenotype coincides with the formation of tight junctions between Sertoli cells during postnatal development. Thus, the altered sorting of KitL is dispensable for hematopoietic and melanogenic lineages, yet is crucial in the testicular environment, where the basal membranes of adjacent polarized Sertoli cells form a niche for the proliferating spermatogonia.
Assuntos
Polaridade Celular , Hematopoese/fisiologia , Espermatogênese/fisiologia , Fator de Células-Tronco/química , Fator de Células-Tronco/metabolismo , Sequência de Aminoácidos , Animais , Apoptose , Contagem de Células , Proliferação de Células , Éxons/genética , Marcação de Genes , Linfopoese , Masculino , Mastócitos/citologia , Camundongos , Dados de Sequência Molecular , Mutação/genética , Estrutura Terciária de Proteína , Transporte Proteico , Deleção de Sequência , Relação Estrutura-Atividade , Testículo/metabolismo , Testículo/patologiaRESUMO
In mammals, primordial follicles are generated early in life and remain dormant for prolonged intervals. Their growth resumes via a process known as primordial follicle activation. Recent genetic studies have demonstrated that phosphoinositide 3-kinase (PI3K) is the essential signaling pathway controlling this process throughout life, acting via Akt to regulate nucleocytoplasmic shuttling of Foxo3, which functions as a downstream molecular switch. The receptor tyrosine kinase Kit has been implicated by numerous studies as the critical upstream regulator of primordial follicle activation via PI3K/Akt. Here we present a genetic analysis of the contribution of Kit in regulating primordial follicle activation and early follicle growth, employing a knock-in mutation (Kit(Y719F)) that completely abrogates signaling via PI3K. Surprisingly, homozygous Kit(Y719F) female mice undergo primordial follicle activation and are fertile, demonstrating that Kit signaling via PI3K is dispensable for this process. However, other abnormalities were identified in Kit(Y719F) ovaries, including accelerated primordial follicle depletion and accumulation of morphologically abnormal primary/secondary follicles with persistent nuclear Foxo3 localization. These findings reveal specific roles of Kit in the maintenance of the primordial follicle reserve and in the primary to secondary follicle transition, but argue that Kit is dispensable in primordial follicle activation.
Assuntos
Folículo Ovariano/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-kit/fisiologia , Transdução de Sinais/fisiologia , Animais , Animais Recém-Nascidos , Feminino , Técnicas de Introdução de Genes , Camundongos , Mutação , Folículo Ovariano/citologia , Ovário/citologia , Ovário/crescimento & desenvolvimento , Ovário/fisiologia , Proteínas Proto-Oncogênicas c-kit/genéticaRESUMO
The Kit receptor functions in hematopoiesis, lymphocyte development, gastrointestinal tract motility, melanogenesis, and gametogenesis. To investigate the roles of different Kit signaling pathways in vivo, we have generated knock-in mice in which docking sites for PI 3-kinase (KitY719) or Src kinase (KitY567) have been mutated. Whereas steady-state hematopoiesis is normal in KitY719F/Y719F and KitY567F/Y567F mice, lymphopoiesis is affected differentially. The KitY567F mutation, but not the KitY719F mutation, blocks pro T cell and pro B cell development in an age-dependent manner. Thus, the Src family kinase, but not the PI 3-kinase docking site in Kit, mediates a critical signal for lymphocyte development. In agreement with these results, treatment of normal mice with the Kit tyrosine kinase inhibitor imatinib (Gleevec) leads to deficits in pro T and pro B cell development, similar to those seen in KitY567F/Y567F and KitW/W mice. The two mutations do not affect embryonic gametogenesis but the KitY719F mutation blocks spermatogenesis at the spermatogonial stages and in contrast the KitY567F mutation does not affect this process. Therefore, Kit-mediated PI 3-kinase signaling and Src kinase family signaling is highly specific for different cellular contexts in vivo.
Assuntos
Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Transdução de Sinais/fisiologia , Quinases da Família src/metabolismo , Fatores Etários , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/fisiologia , Benzamidas , Western Blotting , Primers do DNA , Citometria de Fluxo , Técnicas Histológicas , Mesilato de Imatinib , Linfopoese/efeitos dos fármacos , Linfopoese/genética , Masculino , Mastócitos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutagênese Insercional , Mutagênese Sítio-Dirigida , Mutação/genética , Fosfatidilinositol 3-Quinases/genética , Inibidores de Fosfoinositídeo-3 Quinase , Piperazinas/farmacologia , Reação em Cadeia da Polimerase , Testes de Precipitina , Pirimidinas/farmacologia , Espermatogênese/genética , Linfócitos T/efeitos dos fármacos , Linfócitos T/fisiologia , Testículo/anatomia & histologia , Quinases da Família src/genéticaRESUMO
BACKGROUND & AIMS: Gastrointestinal stromal tumors (GISTs) express the receptor tyrosine kinase c-kit. Approximately 90% of GISTs have gain-of-function mutations in the Kit gene, which leads to its constitutive activation and drives malignant behavior of GISTs. Interstitial cells of Cajal (ICC) express c-kit; however, it is unknown whether uncontrolled hyperplasia of ICC is responsible for GISTs. Here, we sought to determine whether gain-of-function mutations in Kit lead to hyperplasia of all classes of ICC, whether ICC hyperplasia begins before birth, and whether functional defects occur in ICC hyperplasia or the development of GISTs. METHODS: Heterozygous mutant Kit(V558Delta)/+ mice that develop symptoms of human familial GISTs and prematurely die from pathology of the gastrointestinal tract were utilized and compared with wild-type controls. C-kit-immunohistochemistry and intracellular electrical recording of spontaneous and nerve-evoked activity were applied to examine the density and functionality of ICC in these mutants. RESULTS: There was considerable hyperplasia in all classes of ICC throughout the GI tract of Kit(V558Delta)/+ mice, except for ICC in the deep muscular plexus of the intestine. Spontaneous electrical activity and postjunctional neural responses in hyperplastic ICC tissues appeared normal but were up-regulated in the cecum, where GISTs were commonly found. CONCLUSIONS: Kit gain-of-function leads to hyperplasia of most classes of ICC throughout the GI tract. ICC retain normal pacemaker function and enteric neural responses well after development of hyperplasia.
Assuntos
Tumores do Estroma Gastrointestinal/patologia , Tumores do Estroma Gastrointestinal/fisiopatologia , Trato Gastrointestinal/patologia , Trato Gastrointestinal/fisiopatologia , Músculo Liso/patologia , Músculo Liso/fisiopatologia , Animais , Ceco/metabolismo , Ceco/patologia , Ceco/fisiopatologia , Colo/metabolismo , Colo/patologia , Colo/fisiopatologia , Modelos Animais de Doenças , Fenômenos Eletrofisiológicos , Sistema Nervoso Entérico/fisiopatologia , Feto/metabolismo , Feto/patologia , Feto/fisiopatologia , Fundo Gástrico/metabolismo , Fundo Gástrico/patologia , Fundo Gástrico/fisiopatologia , Tumores do Estroma Gastrointestinal/metabolismo , Trato Gastrointestinal/metabolismo , Hiperplasia/metabolismo , Hiperplasia/patologia , Hiperplasia/fisiopatologia , Íleo/metabolismo , Íleo/patologia , Íleo/fisiopatologia , Jejuno/metabolismo , Jejuno/patologia , Jejuno/fisiopatologia , Masculino , Camundongos , Camundongos Mutantes , Músculo Liso/metabolismo , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Antro Pilórico/metabolismo , Antro Pilórico/patologia , Antro Pilórico/fisiopatologiaRESUMO
Gastrointestinal stromal tumor (GIST) is the most common human sarcoma and arises in the gastrointestinal tract. Most GISTs are caused by activating mutations in the KIT receptor tyrosine kinase, such as the exon 11 KIT V559Δ mutation. The small molecule imatinib inhibits KIT and has been a mainstay of therapy in GIST. Unfortunately, imatinib-treated patients typically relapse, most often due to clonal emergence of the resistance-associated KIT V654A mutation. To determine the biologic impact of this second-site mutation in vivo, we created a mouse model with the corresponding V558Δ;V653A Kit double mutation restricted (a) spatially to ETV1+ cells, which include the interstitial cells of Cajal (ICCs) from which GISTs presumably originate, and (b) temporally through tamoxifen treatment after birth. This resulted in the first in vivo model of the most common second-site mutation associated with imatinib resistance in GIST and the first in vivo demonstration that cell-autonomous expression of mutant KIT in the ICC lineage leads to GIST. GISTs driven by the V558Δ;V653A Kit double mutation were resistant to imatinib, while cabozantinib was more effective in overcoming resistance than sunitinib. Compared to control mice with a single V558Δ Kit mutation, mice with a double V558Δ; V653A Kit mutation had increased tumor oncogenesis and associated KIT-dependent STAT activation. Our findings demonstrate that the biologic consequences of a second-site mutation in an oncogenic driver may include not only a mechanism for drug resistance, but changes in tumor oncogenic potential and differential activation of signaling pathways.
Assuntos
Carcinogênese/genética , Neoplasias Gastrointestinais/genética , Tumores do Estroma Gastrointestinal/genética , Mutação , Proteínas Proto-Oncogênicas c-kit/genética , Fatores de Transcrição STAT/metabolismo , Animais , Modelos Animais de Doenças , Neoplasias Gastrointestinais/patologia , Tumores do Estroma Gastrointestinal/patologia , Humanos , Camundongos , Proteínas Proto-Oncogênicas c-kit/metabolismo , Transdução de Sinais/genética , Microambiente Tumoral/genéticaRESUMO
The Kit receptor tyrosine kinase functions in hematopoiesis, melanogenesis, and gametogenesis and in interstitial cells of Cajal. We previously identified two upstream hypersensitive site (HS) clusters in mast cells and melanocytes. Here we investigated the roles of these 5' HS sequences in Kit expression using transgenic mice carrying Kit-GFP reporter constructs. In these mice there is close correspondence between Kit-GFP reporter and endogenous Kit gene expression in most tissues analyzed. Deletion analysis defined the 5' upstream HS cluster region as critical for Kit expression in mast cells. Furthermore, chromatin immunoprecipitation analysis in mast cells showed that H3 and H4 histone hyperacetylation and RNA polymerase II recruitment within the Kit promoter and in the 5' HS region were associated with Kit expression. Therefore, the 5' upstream hypersensitivity sites appear to be critical components of locus control region-mediated Kit gene activation in mast cells.
Assuntos
Regulação da Expressão Gênica , Região de Controle de Locus Gênico , Mastócitos/fisiologia , Proteínas Proto-Oncogênicas c-kit/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Células Cultivadas , Cerebelo/citologia , Cerebelo/metabolismo , Cromatina/química , Cromatina/metabolismo , Feminino , Genes Reporter , Histonas/metabolismo , Masculino , Mastócitos/citologia , Camundongos , Camundongos Transgênicos , Conformação de Ácido Nucleico , Ovário/citologia , Ovário/metabolismo , Proteínas Proto-Oncogênicas c-kit/genética , RNA Polimerase II/metabolismo , Proteínas Recombinantes de Fusão/genética , Testículo/citologia , Testículo/metabolismo , Ativação TranscricionalRESUMO
PURPOSE: Pediatric gastrointestinal stromal tumors (GIST) are rare and occur preferentially in females as multifocal gastric tumors, typically lacking mutations in KIT and PDGFRA. As KIT oncoprotein is consistently overexpressed in pediatric GIST, we sought to investigate the activation of KIT downstream targets and alterations of KIT/PDGFRA gene copy number, mine novel therapeutic targets by gene expression, and test tyrosine kinase receptor activation by proteomic profiling. EXPERIMENTAL DESIGN: Seventeen pediatric GISTs were investigated for KIT/PDGFRA genotype and biochemical activation of KIT downstream targets. The transcriptional profile of 13 nodules from 8 pediatric patients was compared with 8 adult wild-type (WT) GISTs, including 3 young adults. The drug sensitivity of second-generation kinase inhibitors was tested in murine Ba/F3 cells expressing human WT KIT, as well as in short-term culture of explants of WT GIST cells. RESULTS: A KIT/PDGFRA WT genotype was identified in all 12 female patients, whereas two of five males had either a KIT exon 11 or PDGFRA exon 18 mutation. KIT downstream targets were consistently activated. Pediatric GISTs showed a distinct transcriptional signature, with overexpression of BAALC, PLAG1, IGF1R, FGF4, and NELL1. In vitro studies showed that nilotinib, sunitinib, dasatinib, and sorafenib are more effective than imatinib against WT KIT. CONCLUSIONS: Rare cases of pediatric GIST may occur in male patients and harbor activating KIT/PDGFRA mutations. Pediatric GISTs show distinct transcriptional signature, suggesting a different biology than WT GIST in adults. In vitro drug screening showed that second-generation kinase inhibitors may provide greater clinical benefit in pediatric GIST.
Assuntos
Tumores do Estroma Gastrointestinal/genética , Perfilação da Expressão Gênica , Proteínas Proto-Oncogênicas c-kit/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Adolescente , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Western Blotting , Proliferação de Células/efeitos dos fármacos , Criança , Análise Mutacional de DNA , Feminino , Tumores do Estroma Gastrointestinal/patologia , Dosagem de Genes , Expressão Gênica , Humanos , Imunoprecipitação , Hibridização in Situ Fluorescente , Masculino , Mutação , Inibidores de Proteínas Quinases/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores SexuaisRESUMO
BRAF and NRAS are commonly mutated in cancer and represent the most frequent genetic events in malignant melanoma. More recently, a subset of melanomas was shown to overexpress KIT and harbor KIT mutations. Although most gastrointestinal stromal tumors (GISTs) exhibit activating mutations in either KIT or PDGFRA, about 10% of the cases lack mutations in these genes. It is our hypothesis following the melanoma model that mutations in BRAF or NRAS may play a role in wild-type GIST pathogenesis. Alterations in RAS/MEK/ERK pathway may also be involved in development of imatinib resistance in GIST, particularly in tumors lacking secondary KIT or PDGFRA mutations. Imatinib-naive wild-type GISTs from 61 patients, including 15 children and 28 imatinib-resistant tumors without secondary KIT mutations were analyzed. Screening for hot spots mutations in BRAF (exons 11 and 15) and NRAS (exons 2 and 3) was performed. A BRAF exon 15 V600E was identified in 3 of 61 GIST patients, who shared similar clinical features, being 49- to 55-years-old females and having their tumors located in the small bowel. The tumors were strongly KIT immunoreactive and had a high risk of malignancy. An identical V600E BRAF mutation was also identified in one of 28 imatinib resistant GIST lacking a defined mechanism of drug resistance. In conclusion, we identified a primary BRAF V600E mutations in 7% of adult GIST patients, lacking KIT/PDGFRA mutations. The BRAF-mutated GISTs show predilection for small bowel location and high risk of malignancy. A secondary V600E BRAF mutation could represent an alternative mechanism of imatinib resistance. Kinase inhibitors targeting BRAF may be effective therapeutic options in this molecular GIST subset.
Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Tumores do Estroma Gastrointestinal/tratamento farmacológico , Tumores do Estroma Gastrointestinal/genética , Mutação/genética , Piperazinas/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas B-raf/genética , Pirimidinas/uso terapêutico , Adulto , Idoso , Benzamidas , Criança , DNA de Neoplasias/genética , Feminino , Genes ras/genética , Genótipo , Humanos , Mesilato de Imatinib , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Prognóstico , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-kit/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genéticaRESUMO
Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract and are caused by activating mutations of the KIT or platelet-derived growth factor receptor alpha (PDGFRA) tyrosine kinases. GISTs can be successfully treated with imatinib mesylate, a selective small-molecule protein kinase inhibitor that was first clinically approved to target the oncogenic BCR-ABL fusion protein kinase in chronic myelogenous leukemia, but which also potently inhibits KIT and PDGFR family members. The mechanistic events by which KIT/PDGFRA kinase inhibition leads to clinical responses in GIST patients are not known in detail. We report here that imatinib triggers GIST cell apoptosis in part through the up-regulation of soluble histone H2AX, a core histone H2A variant. We found that untreated GIST cells down-regulate H2AX in a pathway that involves KIT, phosphoinositide-3-kinase, and the ubiquitin/proteasome machinery, and that the imatinib-mediated H2AX up-regulation correlates with imatinib sensitivity. Depletion of H2AX attenuated the apoptotic response of GIST cells to imatinib. Soluble H2AX was found to sensitize GIST cells to apoptosis by aberrant chromatin aggregation and a transcriptional block. Our results underscore the importance of H2AX as a human tumor suppressor protein, provide mechanistic insights into imatinib-induced tumor cell apoptosis and establish H2AX as a novel target in cancer therapy.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Tumores do Estroma Gastrointestinal/tratamento farmacológico , Tumores do Estroma Gastrointestinal/patologia , Histonas/biossíntese , Piperazinas/farmacologia , Pirimidinas/farmacologia , Animais , Apoptose/fisiologia , Benzamidas , Modelos Animais de Doenças , Regulação para Baixo , Tumores do Estroma Gastrointestinal/genética , Tumores do Estroma Gastrointestinal/metabolismo , Regulação Neoplásica da Expressão Gênica , Células HeLa , Histonas/genética , Humanos , Mesilato de Imatinib , Camundongos , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Transcrição Gênica/efeitos dos fármacos , Regulação para CimaRESUMO
PURPOSE: Gastrointestinal stromal tumor (GIST) is the most common sarcoma of the intestinal tract. Nearly all tumors express KIT protein, and most have an activating mutation in either KIT or PDGFRA. Therapy with selective tyrosine kinase inhibitors achieves a partial response or stable disease in approximately 80% of patients with advanced GIST. However, after an initial clinical response, some patients develop imatinib resistance. Our goal was to investigate the spectrum of pathologic response and molecular alterations in a group of GIST patients, clinically defined as having imatinib-stable/imatinib-responsive lesions, who underwent surgical resection. EXPERIMENTAL DESIGN: Forty-three tumor nodules from 28 patients were available for pathologic and molecular analysis, which included genotyping for primary and secondary KIT/PDGFRA-mutations, cell cycle alterations, and biochemical activation status of KIT and downstream targets. The transcriptional changes of a subset of these tumors were compared with a group of imatinib-naive GISTs on a U133A Affymetrix expression platform. RESULTS: The histologic response did not correlate with imatinib therapy duration or with proliferative activity. Second-site KIT mutation was identified in only one tumor nodule. Activation of KIT and downstream targets was consistent in all tumors analyzed. Ultrastructurally, a subset of tumors showed a smooth muscle phenotype, which correlated with overexpression of genes involved in muscle differentiation and function. CONCLUSIONS: The histologic response to imatinib is heterogeneous and does not correlate well with clinical response. Second-site KIT mutations are rare in imatinib-responsive GISTs compared with imatinib-resistant tumors. The gene signature of imatinib-response in GISTs showed alterations of cell cycle control as well as up-regulation of genes involved in muscle differentiation and function.
Assuntos
Tumores do Estroma Gastrointestinal/tratamento farmacológico , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-kit/genética , Pirimidinas/farmacologia , Benzamidas , Proliferação de Células , Análise por Conglomerados , Análise Mutacional de DNA , Resistencia a Medicamentos Antineoplásicos , Éxons , Genótipo , Humanos , Mesilato de Imatinib , Microscopia Eletrônica , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Tirosina Quinases/antagonistas & inibidores , Análise de Sequência de DNA , Resultado do TratamentoRESUMO
PURPOSE: Resistance is commonly acquired in patients with metastatic gastrointestinal stromal tumor who are treated with imatinib mesylate, often due to the development of secondary mutations in the KIT kinase domain. We sought to investigate the efficacy of second-line tyrosine kinase inhibitors, such as sorafenib, dasatinib, and nilotinib, against the commonly observed imatinib-resistant KIT mutations (KIT(V654A), KIT(T670I), KIT(D820Y), and KIT(N822K)) expressed in the Ba/F3 cellular system. EXPERIMENTAL DESIGN: In vitro drug screening of stable Ba/F3 KIT mutants recapitulating the genotype of imatinib-resistant patients harboring primary and secondary KIT mutations was investigated. Comparison was made to imatinib-sensitive Ba/F3 KIT mutant cells as well as Ba/F3 cells expressing only secondary KIT mutations. The efficacy of drug treatment was evaluated by proliferation and apoptosis assays, in addition to biochemical inhibition of KIT activation. RESULTS: Sorafenib was potent against all imatinib-resistant Ba/F3 KIT double mutants tested, including the gatekeeper secondary mutation KIT(WK557-8del/T670I), which was resistant to other kinase inhibitors. Although all three drugs tested decreased cell proliferation and inhibited KIT activation against exon 13 (KIT(V560del/V654A)) and exon 17 (KIT(V559D/D820Y)) double mutants, nilotinib did so at lower concentrations. CONCLUSIONS: Our results emphasize the need for tailored salvage therapy in imatinib-refractory gastrointestinal stromal tumors according to individual molecular mechanisms of resistance. The Ba/F3 KIT(WK557-8del/T670I) cells were sensitive only to sorafenib inhibition, whereas nilotinib was more potent on imatinib-resistant KIT(V560del/V654A) and KIT(V559D/D820Y) mutant cells than dasatinib and sorafenib.
Assuntos
Antineoplásicos/farmacologia , Benzenossulfonatos/farmacologia , Tumores do Estroma Gastrointestinal/tratamento farmacológico , Mutação , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-kit/genética , Piridinas/farmacologia , Pirimidinas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Benzamidas , Proliferação de Células/efeitos dos fármacos , Dasatinibe , Resistencia a Medicamentos Antineoplásicos , Tumores do Estroma Gastrointestinal/genética , Humanos , Mesilato de Imatinib , Camundongos , Niacinamida/análogos & derivados , Compostos de Fenilureia , Fosforilação , Sorafenibe , Tiazóis/farmacologiaRESUMO
PURPOSE: Tyrosine kinase inhibitors are effective in gastrointestinal stromal tumors (GISTs) but often are of transient benefit as resistance commonly develops. Immunotherapy, particularly blockade of the inhibitory receptor programmed death 1 (PD-1) or the ligand programmed death ligand 1 (PD-L1), has shown effectiveness in a variety of cancers. The functional effects of PD-1/PD-L1 blockade are unknown in GISTs. EXPERIMENTAL DESIGN: We analyzed tumor and matched blood samples from 85 patients with GISTs and determined the expression of immune checkpoint molecules using flow cytometry. We investigated the combination of imatinib with PD-1/PD-L1 blockade in KitV558Δ/+ mice that develop GISTs. RESULTS: The inhibitory receptors PD-1, lymphocyte activation gene 3, and T-cell immunoglobulin mucin-3 were upregulated on tumor-infiltrating T cells compared with T cells from matched blood. PD-1 expression on T cells was highest in imatinib-treated human GISTs. Meanwhile, intratumoral PD-L1 expression was variable. In human GIST cell lines, treatment with imatinib abrogated the IFNγ-induced upregulation of PD-L1 via STAT1 inhibition. In KitV558Δ/+ mice, imatinib downregulated IFNγ-related genes and reduced PD-L1 expression on tumor cells. PD-1 and PD-L1 blockade in vivo each had no efficacy alone but enhanced the antitumor effects of imatinib by increasing T-cell effector function in the presence of KIT and IDO inhibition. CONCLUSIONS: PD-1/PD-L1 blockade is a promising strategy to improve the effects of targeted therapy in GISTs. Collectively, our results provide the rationale to combine these agents in human GISTs. Clin Cancer Res; 23(2); 454-65. ©2016 AACR.
Assuntos
Antígeno B7-H1/imunologia , Tumores do Estroma Gastrointestinal/terapia , Receptor de Morte Celular Programada 1/imunologia , Fator de Transcrição STAT1/genética , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/imunologia , Antígeno B7-H1/antagonistas & inibidores , Linhagem Celular Tumoral , Tumores do Estroma Gastrointestinal/genética , Tumores do Estroma Gastrointestinal/imunologia , Tumores do Estroma Gastrointestinal/patologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Mesilato de Imatinib/administração & dosagem , Imunoterapia , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Terapia de Alvo Molecular , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Fator de Transcrição STAT1/antagonistas & inibidores , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Gastrointestinal stromal tumor (GIST) is the most common type of sarcoma and usually harbors either a KIT or PDGFRA mutation. However, the molecular basis for tumor malignancy is not well defined. Although the Wnt/ß-catenin signaling pathway is important in a variety of cancers, its role in GIST is uncertain. Through analysis of nearly 150 human GIST specimens, we found that some human GISTs expressed ß-catenin and contained active, dephosphorylated nuclear ß-catenin. Furthermore, advanced human GISTs expressed reduced levels of the Wnt antagonist DKK4. Accordingly, in human GIST T1 cells, Wnt stimulation increased ß-catenin-mediated transcriptional activity in a reporter assay as well as transcription of the downstream target genes Axin2 and CCND1 In contrast, DKK4 overexpression in GIST T1 cells reduced Wnt/ß-catenin signaling. In addition, we showed that nuclear ß-catenin stability was partially regulated by the E3 ligase COP1, as demonstrated with coimmunoprecipitation and COP1 knockdown. Three molecular inhibitors of the Wnt/ß-catenin pathway demonstrated antitumor efficacy in various GIST models, both in vitro and in vivo Notably, the tankyrase inhibitor G007-LK alone had substantial activity against tumors of genetically engineered KitV558Δ/+ mice, and the effect was increased by the addition of the Kit inhibitor imatinib mesylate. Collectively, our findings demonstrate that Wnt/ß-catenin signaling is a novel therapeutic target for selected untreated or imatinib-resistant GISTs. Mol Cancer Ther; 16(9); 1954-66. ©2017 AACR.
Assuntos
Tumores do Estroma Gastrointestinal/metabolismo , Tumores do Estroma Gastrointestinal/patologia , Via de Sinalização Wnt , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Tumores do Estroma Gastrointestinal/tratamento farmacológico , Humanos , Mesilato de Imatinib/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Camundongos , Camundongos Transgênicos , Terapia de Alvo Molecular , Gradação de Tumores , Estadiamento de Neoplasias , Ubiquitina-Proteína Ligases/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , Proteína Wnt3A/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , beta Catenina/metabolismoRESUMO
Most gastrointestinal stromal tumors (GIST) have an activating mutation in either KIT or PDGFRA. Imatinib is a selective tyrosine kinase inhibitor and achieves a partial response or stable disease in about 80% of patients with metastatic GIST. It is now clear that some patients with GIST develop resistance to imatinib during chronic therapy. To identify the mechanism of resistance, we studied 31 patients with GIST who were treated with imatinib and then underwent surgical resection. There were 13 patients who were nonresistant to imatinib, 3 with primary resistance, and 15 with acquired resistance after initial benefit from the drug. There were no secondary mutations in KIT or PDGFRA in the nonresistant or primary resistance groups. In contrast, secondary mutations were found in 7 of 15 (46%) patients with acquired resistance, each of whom had a primary mutation in KIT exon 11. Most secondary mutations were located in KIT exon 17. KIT phosphorylation was heterogeneous and did not correlate with clinical response to imatinib or mutation status. That acquired resistance to imatinib in GIST commonly occurs via secondary gene mutation in the KIT kinase domain has implications for strategies to delay or prevent imatinib resistance and to employ newer targeted therapies.
Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Tumores do Estroma Gastrointestinal/tratamento farmacológico , Mutação , Piperazinas/uso terapêutico , Pirimidinas/uso terapêutico , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequência de Aminoácidos , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Benzamidas , Western Blotting , Análise Mutacional de DNA , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Tumores do Estroma Gastrointestinal/genética , Tumores do Estroma Gastrointestinal/patologia , Genótipo , Humanos , Mesilato de Imatinib , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Piperazinas/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Pirimidinas/farmacologia , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismoRESUMO
GISTs are the most common mesenchymal neoplasms of the digestive tract and are thought to originate from or differentiate toward the interstitial cell of Cajal lineage. Almost all GISTs express KIT protein and the majority show activating mutations in either KIT or PDGFRA proto-oncogenes. Ultrastructurally, these tumors have been shown to have either a smooth muscle, neuronal, dual, or null phenotype. The objective of this study was to investigate the relationship between ultrastructural features and genotype in a large series of 125 histologically confirmed and CD117 positive GISTs. PCR analysis for the presence of KIT exon 9, 11, 13, and 17 and PDGFRA exon 12 and 18 mutations was performed. There were 62 (50%) tumors located in the stomach and 45 (36%) in the small bowel. Overall, KIT mutations were detected in 93 (75%) patients: 86 (69%) in exon 11, and 7 (6%) in exon 9. A PDGFRA mutation was detected in 7 (6%) cases and 25 (19%) cases had no mutation. Ultrastructurally, skeinoid fibers were seen in 55 (44%) cases and were more common in small bowel than stomach GISTs, and occurred in only in 1 of 16 patients with an ITD (KIT) exon 11 or PDGFRA mutation. Focal actin microfilaments were identified in 82 (65%) cases and did not correlate with location or mutation type. Rare neurosecretory-type granules (NS-G) were seen in 34 (27%) of cases, but were seen in most of the cells in only 5 (4%) cases. GISTs showing both NS-G and microtubules were associated with KIT exon 11 genotype and spindle cell morphology. PDGFRA mutated cases were associated with gastric location, predominantly epithelioid morphology and lacked NS-G.