RESUMO
The Raf/MEK/ERK and PI3K/Akt pathways are prominent effectors of oncogenic Ras. These pathways negatively regulate each other, but the mechanism involved is incompletely understood. We now identify MEK1 as an essential regulator of lipid/protein phosphatase PTEN, through which it controls phosphatidylinositol-3-phosphate accumulation and AKT signaling. MEK1 ablation stabilizes AKT activation and, in vivo, causes a lupus-like autoimmune disease and myeloproliferation. Mechanistically, MEK1 is necessary for PTEN membrane recruitment as part of a ternary complex containing the multidomain adaptor MAGI1. Complex formation is independent of MEK1 kinase activity but requires phosphorylation of T292 on MEK1 by activated ERK. Thus, inhibiting the ERK pathway reduces PTEN membrane recruitment, increasing phosphatidylinositol-3-phosphate accumulation and AKT activation. Our data offer a conceptual framework for the observation that activation of the PI3K pathway frequently mediate resistance to MEK inhibitors and for the promising results obtained by combined MEK/PI3K inhibition in preclinical cancer models.
Assuntos
Membrana Celular/enzimologia , Fibroblastos/enzimologia , Linfócitos/enzimologia , MAP Quinase Quinase 1/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Tolerância a Antígenos Próprios , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Doenças Autoimunes/enzimologia , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Células COS , Moléculas de Adesão Celular , Morte Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/imunologia , Membrana Celular/patologia , Chlorocebus aethiops , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/imunologia , Fibroblastos/patologia , Genótipo , Guanilato Quinases , Ativação Linfocitária , Linfócitos/imunologia , Linfócitos/patologia , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 1/deficiência , MAP Quinase Quinase 1/genética , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Complexos Multienzimáticos , Transtornos Mieloproliferativos/enzimologia , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/imunologia , Fenótipo , Fosfatos de Fosfatidilinositol/metabolismo , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Transporte Proteico , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Interferência de RNA , Transdução de Sinais , Fatores de Tempo , Transfecção , TirosinaRESUMO
PURPOSE: GNAQ mutations have been identified in port wine stains (both syndromic and nonsyndromic) and melanocytic ocular neoplasms. This study investigates the presence of GNAQ mutations in diffuse (those associated with Sturge-Weber syndrome [SWS]) and solitary choroidal hemangiomas. PARTICIPANTS: Tissue from 11 patients with the following diagnoses: port wine stain (n = 3), diffuse choroidal hemangioma (n = 1), solitary choroidal hemangioma (n = 6), and choroidal nevus (n = 1). METHODS: Ten specimens were interrogated with Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets, a hybridization capture-based next-generation sequencing assay for targeted deep sequencing of all exons and selected introns of 468 key cancer genes in formalin-fixed, paraffin-embedded tumors. Digital polymerase chain reaction was used to detect GNAQ Q209 mutation in 1 specimen. MAIN OUTCOME MEASURES: Detection of GNAQ codon-specific mutation. RESULTS: Activating somatic GNAQ mutations (c.547C > T; p.Arg183Cys) were found in 100% (3 of 3) of the port wine stain and in the diffuse choroidal hemangioma. Somatic GNAQ mutations (c.626A > T; p.Gln209Leu) were found in 100% (6 of 6) of the solitary choroidal hemangiomas and (c.626A > C; p.Gln209Pro) in the choroidal nevus. CONCLUSIONS: GNAQ mutations occur in both diffuse and solitary hemangiomas, although at distinct codons. An R183 codon is mutant in diffuse choroidal hemangiomas, consistent with other Sturge-Weber vascular malformations. By contrast, solitary choroidal hemangiomas have mutations in the Q209 codon, similar to other intraocular melanocytic neoplasms.
Assuntos
Neoplasias da Coroide/genética , DNA de Neoplasias/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Hemangioma/genética , Mutação , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Corioide/patologia , Neoplasias da Coroide/diagnóstico , Neoplasias da Coroide/metabolismo , Análise Mutacional de DNA , Feminino , Seguimentos , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Hemangioma/diagnóstico , Hemangioma/metabolismo , Humanos , Masculino , Estudos RetrospectivosRESUMO
PURPOSE: To investigate the clinical and morphologic characteristics of serous retinal disturbances in patients taking mitogen-activated protein kinase kinase (MEK) inhibitors. PARTICIPANTS: A total of 313 fluid foci in 50 eyes of 25 patients receiving MEK inhibitors for treatment of their metastatic cancer, who had evidence of serous retinal detachments confirmed by optical coherence tomography (OCT). DESIGN: Single-center, retrospective cohort study. METHODS: Clinical examination and OCT were used to evaluate MEK inhibitor-associated subretinal fluid. The morphology, distribution, and location of fluid foci were serially evaluated for each eye. Choroidal thickness was measured at each time point (baseline, fluid accumulation, and fluid resolution). Two independent observers performed all measurements. Statistical analysis was used to correlate interobserver findings and compare choroidal thickness and visual acuity at each time point. MAIN OUTCOME MEASURES: Comparison of OCT characteristics of retinal abnormalities at baseline to fluid accumulation. RESULTS: The majority of patients had fluid foci that were bilateral (92%) and multifocal (77%) and at least 1 focus involving the fovea (83.3%). All fluid foci occurred between the interdigitation zone and an intact retinal pigment epithelium. The 313 fluid foci were classified into 4 morphologies, as follows: 231 (73.8%) dome, 36 (11.5%) caterpillar, 31 (9.9%) wavy, and 15 (4.8%) splitting. Best-corrected visual acuity at fluid resolution was not statistically different from baseline; and no eye lost more than 2 Snellen lines from baseline at the time of fluid accumulation. There was no statistical difference in the choroidal thickness between the different time points (baseline, fluid accumulation, and fluid resolution). A strong positive interobserver correlation was obtained for choroidal thickness measurements (r = 0.97, P < 0.0001) and grading of foci morphology (r = 0.97, P < 0.0001). CONCLUSION: The subretinal fluid foci associated with MEK inhibitors have unique clinical and morphologic characteristics, which can be distinguished from the findings of central serous chorioretinopathy. In this series, MEK inhibitors did not cause irreversible loss of vision or serious eye damage.
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Antineoplásicos/efeitos adversos , Coriorretinopatia Serosa Central/diagnóstico , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Inibidores de Proteínas Quinases/efeitos adversos , Descolamento Retiniano/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Azetidinas/efeitos adversos , Benzimidazóis/efeitos adversos , Coriorretinopatia Serosa Central/induzido quimicamente , Feminino , Angiofluoresceinografia , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias/tratamento farmacológico , Piperidinas/efeitos adversos , Piridonas/efeitos adversos , Pirimidinonas/efeitos adversos , Descolamento Retiniano/induzido quimicamente , Estudos Retrospectivos , Líquido Sub-Retiniano , Tomografia de Coerência Óptica , Acuidade Visual/fisiologia , Adulto JovemRESUMO
BACKGROUND: RAF inhibitors are an effective therapy for patients with BRAF-mutant melanoma and brain metastasis. Efficacy data are derived from clinical studies enriched with physiologically fit patients; therefore, it is of interest to assess the real-world experience of vemurafenib in this population. Tumor-specific genetic variants that influence sensitivity to RAF kinase inhibitors also require investigation. METHODS: Records of patients with BRAF-mutant melanoma and brain metastases who were treated with vemurafenib were reviewed. Clinical data were extracted to determine extracranial and intracranial objective response rates, progression-free survival (PFS), overall survival (OS), and safety. A bait-capture, next-generation sequencing assay was used to identify mutations in pretreatment tumors that could explain primary resistance to vemurafenib. RESULTS: Among patients with intracranial disease treated with vemurafenib, 27 were included in survival analyses and 22 patients were assessable for response. The extracranial and intracranial objective response rates were 71% and 50%, respectively. Discordant responses were observed between extracranial and intracranial metastatic sites in 4 of 19 evaluable patients. Median PFS was 4.1 months (95% confidence interval [CI]: 2.6-7.9); median intracranial PFS was 4.6 months (95% CI: 2.7-7.9), median OS was 7.5 months (95% CI: 4.3-not reached), with a 30.4% 1-year OS rate. Outcomes were influenced by performance status. Vemurafenib was tolerable, although radiation-induced dermatitis occurred in some patients who received whole-brain radiotherapy. Adequate samples for next-generation sequencing analysis were available for seven patients. Melanomas categorized as "poorly sensitive" (≥20% tumor growth, new lesions, or ≤50% shrinkage for <4 months) harbored co-occurring mutations in genes predicted to activate the phosphatidylinositol 3-kinase-AKT (PI3K-AKT) pathway. CONCLUSION: Vemurafenib is highly active in BRAF-mutant melanoma brain metastases but has limited activity in patients with poor performance status. The safety and efficacy of concurrent radiotherapy and RAF inhibition requires careful clinical evaluation. Combination strategies blocking the MAPK and PI3K-AKT pathway may be warranted in a subset of patients. IMPLICATIONS FOR PRACTICE: Vemurafenib is active for BRAF-mutant intracranial melanoma metastases in an unselected patient population typical of routine oncologic practice. Patients with poor performance status appear to have poor outcomes despite vemurafenib therapy. Preliminary data indicate that co-occurring or secondary alterations in the phosphatidylinositol 3-kinase-AKT (PI3K-AKT) pathway are involved in resistance to RAF inhibition, thus providing a rationale for dual MAPK and PI3K-AKT pathway inhibition in this patient population.
Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Indóis/uso terapêutico , Melanoma/tratamento farmacológico , Proteínas Proto-Oncogênicas B-raf/genética , Sulfonamidas/uso terapêutico , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/efeitos adversos , Antineoplásicos/uso terapêutico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/mortalidade , Neoplasias Encefálicas/secundário , Feminino , Humanos , Indóis/efeitos adversos , Estimativa de Kaplan-Meier , Masculino , Melanoma/genética , Melanoma/mortalidade , Melanoma/patologia , Pessoa de Meia-Idade , Mutação , Estudos Retrospectivos , Sulfonamidas/efeitos adversos , Resultado do Tratamento , VemurafenibRESUMO
BACKGROUND: Metastases to the liver are often the first finding in patients with uveal melanoma with extraocular disease, but little has been published on the utility of staging MRI at initial diagnosis. We aimed to evaluate the proportion of abnormal hepatic findings on baseline MRI and accuracy of MRI in patients with newly diagnosed uveal melanoma. METHODS: This is a single-centre, retrospective, institutional review board-approved study of 145 consecutive patients diagnosed with uveal melanoma, at Memorial Sloan Kettering Cancer Center between 2004 and 2016, who had staging MRI within 1 month of diagnosis. Scans were classified as normal or abnormal, and further distinguished as abnormal non-metastatic, uncharacterisable lesions and suspicious for metastasis. Where available, follow-up MRI (at ~1 year) or biopsies were reviewed. RESULTS: MRI in 145 patients revealed 62% (90) with abnormal hepatic findings; out of these 87% (78) had non-metastatic benign findings, 6.7% (6) had unclassifiable lesions and 6.7% (6) were suspicious for metastasis (6). Abnormal non-metastatic findings included 72 focal (36 solitary and 36 multiple) and 12 diffuse lesions. Lesions suspicious for metastases were found in 6 of 145 patients (4%), despite normal liver function tests. Of these, five had confirmed liver metastases and one patient had a stable, presumed non-metastatic lesion on follow-up. In this study, the sensitivity and specificity of staging MRI for all findings were 83.3% (95% CI 35.9 to 99.6) and 99.0% (95% CI 94.3 to 99.9), respectively. CONCLUSION: Staging MRI of patients with newly diagnosed uveal melanoma accurately identified early metastases. Furthermore, imaging revealed hepatic abnormalities in the majority of patients, although as expected few of these represented metastatic disease.
Assuntos
Neoplasias Hepáticas/secundário , Melanoma/patologia , Neoplasias Uveais/patologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Análise de Variância , Feminino , Humanos , Neoplasias Hepáticas/diagnóstico por imagem , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Adulto JovemRESUMO
BACKGROUND/AIMS: Ophthalmic artery chemosurgery (OAC) has changed the face of retinoblastoma treatment and led to a higher rate of globe salvage. The introduction of intravitreal chemotherapy (IVitC) has further enhanced globe salvage with increased success in treatment of intravitreal seeds. Our group has seen success at treating non-vitreous disease that is refractory to OAC using IVitC. This study was undertaken to quantify and report on this success. METHODS: A retrospective review was used to identify patients treated with IVitC for indications other than vitreous seeds from two centres. The indication, prior and concurrent treatment, response time and duration of treatment were documented. Kaplan-Meier estimates were used to evaluate ocular and recurrence-free survival. Ocular toxicity was evaluated using the 30 Hz flicker electroretinogram (ERG). Continuous and categorical variables were compared with Student's t-test and χ2 test, respectively. RESULTS: Fifty-six eyes from 52 retinoblastoma patients were identified. There were no disease-related or treatment-related deaths. One patient developed a second primary malignancy (pinealoblastoma) and subsequent leptomeningeal spread. Ninety-eight per cent of the eyes showed clinical regression. Recurrence was seen in 14.3%. Of the recurrences, five occurred in retinal tumours and three in subretinal seeds. The Kaplan-Meier estimated risk of recurrence in all patients treated was 83.5% (95% CI 7.9 to 14.1) at 10 months. The mean change in ERG over treatment course was -17.7 µV. CONCLUSIONS: Intravitreal chemotherapy is successful for the treatment of subretinal seeds and recurrent retinal tumours and could be considered as adjunctive therapy in globe-sparing treatment of retinoblastoma.
Assuntos
Melfalan/administração & dosagem , Neoplasias da Retina/tratamento farmacológico , Retinoblastoma/tratamento farmacológico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos Alquilantes/administração & dosagem , Criança , Implantes de Medicamento , Eletrorretinografia , Feminino , Seguimentos , Humanos , Incidência , Injeções Intravítreas , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/epidemiologia , Oftalmoscopia , Neoplasias da Retina/diagnóstico , Retinoblastoma/diagnóstico , Estudos Retrospectivos , Microscopia com Lâmpada de Fenda , Resultado do Tratamento , Estados Unidos/epidemiologia , Adulto JovemRESUMO
BACKGROUND: There has been no effective method for treating newly visible ("new") subretinal seeding in retinoblastoma except enucleation. The objective of this report is to determine whether intravitreal chemotherapy combined with 810 nm indirect laser can successfully treat retinoblastoma eyes with "new" subretinal seeding which appeared after intra-arterial chemotherapy (ophthalmic arterial chemosurgery: OAC). MATERIAL AND METHODS: Single center retrospective study from a tertiary cancer hospital of a case series of 14 eyes treated with combined intravitreal chemotherapy and laser from 2012 to 2017. Ocular salvage, patient survival, recurrence-free ocular survival, metastases, and extraocular extension were assessed. RESULTS: A total of 14 eyes in 13 unilateral or bilateral retinoblastoma patients with "new" subretinal seeding after initial eye salvage therapy were treated with combined intravitreal injection of melphalan (30 ug) or melphalan (30 ug) and topotecan (20 ug) and with 810 nm indirect continuous wave laser. All eyes were salvaged. Only two eyes (14%) recurred again for subretinal seeds after 6 and 8 months, respectively, and required additional cycles of intravitreal injections and laser. Combined intravitreal injection of melphalan or melphalan plus topotecan with 810 nm indirect continuous wave laser was not associated with any metastatic events, patient deaths, extraocular extension, or need for enucleation. CONCLUSION: There has been no effective treatment for "new" subretinal seeding after OAC except enucleation or second course OAC. Combined intravitreal chemotherapy with 810 nm indirect laser may be an effective and safe alternative to enucleation.
Assuntos
Antineoplásicos/uso terapêutico , Terapia a Laser , Recidiva Local de Neoplasia/terapia , Neoplasias da Retina/terapia , Retinoblastoma/terapia , Terapia de Salvação , Corpo Vítreo/patologia , Adolescente , Adulto , Criança , Terapia Combinada , Feminino , Humanos , Injeções Intravítreas , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/patologia , Inoculação de Neoplasia , Prognóstico , Neoplasias da Retina/patologia , Retinoblastoma/patologia , Estudos Retrospectivos , Adulto JovemRESUMO
Although studies from pediatric cancers (largely acute lymphoblastic leukemia) have shown that patients undergoing systemic chemotherapy may experience decreased growth velocity during the treatment phase, no such data exist for retinoblastoma patients treated with systemic chemotherapy or ophthalmic artery chemosurgery (OAC). The purpose of this study is to report growth patterns of our retinoblastoma (Rb) population who were treated with OAC in a retrospective, single center (Memorial Sloan Kettering Cancer Center) review of 341 patients treated between 2006 and 2016. Children who only received OAC were classified as naive; those who were treated initially with systemic chemotherapy and subsequently presented to our center for OAC were termed secondary; and a small group of patients who received single-agent systemic chemotherapy prior to OAC were labeled bridge. For all patients, height and weight were recorded at monthly intervals during OAC (short-term) and then annually during a follow-up period (long-term) up to 3 years after treatment. Excluded from this study were children who received external radiation therapy and those with genetic syndromes, which are independently associated with growth derangements. During OAC, there was no significant difference in growth velocity between the naïve and secondary groups. In either group, number of treatments also did not affect growth rate. Three years after the end of OAC, naïve patients were in the 68th percentile by height (95% CI 61.30, 74.63) compared to secondary patients in the 61st percentile (95% CI 51.1, 71.47). Both groups were in the same weight percentiles during the first two years of follow-up but at the three-year follow-up period, naïve patients were in the 63rd percentile (95% CI 57.4, 69.4) and secondary patients were in the 60th percentile (95% CI 50.4, 69.7). OAC for retinoblastoma does not appear to impact short-term growth velocity, weight gain during the treatment period or after three years.
Assuntos
Sobreviventes de Câncer , Artéria Oftálmica/cirurgia , Retinoblastoma/tratamento farmacológico , Retinoblastoma/cirurgia , Adolescente , Carboplatina/administração & dosagem , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Infusões Intra-Arteriais , Masculino , Melfalan/administração & dosagem , Artéria Oftálmica/efeitos dos fármacos , Retinoblastoma/epidemiologia , Retinoblastoma/patologia , Estudos Retrospectivos , Resultado do TratamentoRESUMO
PURPOSE: The clinical use of BRAF inhibitors in patients with melanoma is limited by intrinsic and acquired resistance. We asked whether next-generation sequencing of pretreatment tumors could identify coaltered genes that predict for intrinsic resistance to BRAF inhibitor therapy in patients with melanoma as a prelude to rational combination strategies. PATIENTS AND METHODS: We analyzed 66 tumors from patients with metastatic BRAF-mutant melanoma collected before treatment with BRAF inhibitors. Tumors were analyzed for > 250 cancer-associated genes using a capture-based next-generation sequencing platform. Antitumor responses were correlated with clinical features and genomic profiles with the goal of identifying a molecular signature predictive of intrinsic resistance to RAF pathway inhibition. RESULTS: Among the 66 patients analyzed, 11 received a combination of BRAF and MEK inhibitors for the treatment of melanoma. Among the 55 patients treated with BRAF inhibitor monotherapy, objective responses, as assessed by Response Evaluation Criteria in Solid Tumors (RECIST), were observed in 30 patients (55%), with five (9%) achieving a complete response. We identified a significant association between alterations in PTEN that would be predicted to result in loss of function and reduced progression-free survival, overall survival, and response grade, a metric that combines tumor regression and duration of treatment response. Patients with melanoma who achieved an excellent response grade were more likely to have an elevated BRAF-mutant allele fraction. CONCLUSION: These results provide a rationale for cotargeting BRAF and the PI3K/AKT pathway in patients with BRAF-mutant melanoma when tumors have concurrent loss-of-function mutations in PTEN. Future studies should explore whether gain of the mutant BRAF allele and/or loss of the wild-type allele is a predictive marker of BRAFi sensitivity.
RESUMO
PURPOSE: Test the hypothesis that in BRAF-mutated melanomas, clinical responses to selumetinib, a MEK inhibitor, will be restricted to tumors in which the PI3K/AKT pathway is not activated. EXPERIMENTAL DESIGN: We conducted a phase II trial in patients with melanoma whose tumors harbored a BRAF mutation. Patients were stratified by phosphorylated-AKT (pAKT) expression (high vs. low) and treated with selumetinib 75 mg per os twice daily. Pretreatment tumors were also analyzed for genetic changes in 230 genes of interest using an exon-capture approach. RESULTS: The high pAKT cohort was closed after no responses were seen in the first 10 patients. The incidence of low pAKT melanoma tumors was low (â¼25% of melanomas tested) and this cohort was eventually closed because of poor accrual. However, among the five patients with melanoma accrued in the low pAKT cohort, there was one partial response (PR). Two other patients had near PRs before undergoing surgical resection of residual disease (one patient) or discontinuation of treatment due to toxicity (one patient). Among the two nonresponding, low pAKT patients with melanoma, co-mutations in MAP2K1, NF1, and/or EGFR were detected. CONCLUSIONS: Tumor regression was seen in three of five patients with BRAF-mutated, low pAKT melanomas; no responses were seen in the high pAKT cohort. These results provide rationale for co-targeting MEK and PI3K/AKT in patients with BRAF mutant melanoma whose tumors express high pAKT. However, the complexity of genetic changes in melanoma indicates that additional genetic information will be needed for optimal selection of patients likely to respond to MEK inhibitors.
Assuntos
Benzimidazóis/uso terapêutico , Melanoma/tratamento farmacológico , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Mutação , Proteínas Proto-Oncogênicas B-raf/genética , Administração Oral , Adulto , Idoso , Benzimidazóis/administração & dosagem , Benzimidazóis/efeitos adversos , Estudos de Coortes , Intervalo Livre de Doença , Esquema de Medicação , Exantema/induzido quimicamente , Feminino , Humanos , Linfopenia/induzido quimicamente , Masculino , Melanoma/genética , Melanoma/metabolismo , Pessoa de Meia-Idade , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Resultado do Tratamento , Adulto JovemRESUMO
The RAF inhibitor vemurafenib has unprecedented activity in BRAF-mutant melanomas, but resistance invariably develops. As Hsp90 is required for the stability of several of the oncoproteins that mediate RAF inhibitor resistance, inhibitors of this cellular chaperone may be effective in patients with intrinsic or acquired resistance to RAF inhibitors.
Assuntos
Compostos Azabicíclicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Melanoma/tratamento farmacológico , Ácidos Ftálicos/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Animais , HumanosRESUMO
Mek1 and Mek2 (also known as Map2k1 and Map2k2, respectively) are evolutionarily conserved, dual-specificity kinases that mediate Erk1 and Erk2 activation during adhesion and growth factor signaling. Here we describe a previously uncharacterized, unexpected role of Mek1 in downregulating Mek2-dependent Erk signaling. Mek1 mediates the regulation of Mek2 in the context of a previously undiscovered Mek1-Mek2 complex. The Mek heterodimer is negatively regulated by Erk-mediated phosphorylation of Mek1 on Thr292, a residue missing in Mek2. Disabling this Erk-proximal negative-feedback step stabilizes the phosphorylation of both Mek2 and Erk in cultured cells and in vivo in Mek1 knockout embryos and mice. Thus, in disagreement with the current perception of the pathway, the role of Mek1 and Mek2 in growth factor-induced Erk phosphorylation is not interchangeable. Our data establish Mek1 as the crucial modulator of Mek and Erk signaling and have potential implications for the role of Mek1 and Mek2 in tumorigenesis.
Assuntos
MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Animais , Dimerização , Camundongos , Modelos Moleculares , Fosforilação , Ligação Proteica , Estrutura Quaternária de ProteínaRESUMO
Mutations in the extracellular signal-regulated kinase (ERK) pathway, particularly in the mitogen-activated protein kinase/ERK kinase (MEK) activator B-Raf, are associated with human tumorigenesis and genetic disorders. Hence, B-Raf is a prime target for molecule-based therapies, and understanding its essential biological functions is crucial for their success. B-Raf is expressed preferentially in cells of neuronal origin. Here, we show that in mice, conditional ablation of B-Raf in neuronal precursors leads to severe dysmyelination, defective oligodendrocyte differentiation, and reduced ERK activation in brain. Both B-Raf ablation and chemical inhibition of MEK impair oligodendrocyte differentiation in vitro. In glial cell cultures, we find B-Raf in a complex with MEK, Raf-1, and kinase suppressor of Ras. In B-Raf-deficient cells, more Raf-1 is recruited to MEK, yet MEK/ERK phosphorylation is impaired. These data define B-Raf as the rate-limiting MEK/ERK activator in oligodendrocyte differentiation and myelination and have implications for the design and use of Raf inhibitors.
Assuntos
Diferenciação Celular/genética , Sistema Nervoso Central/crescimento & desenvolvimento , Sistema Nervoso Central/metabolismo , Bainha de Mielina/metabolismo , Oligodendroglia/metabolismo , Proteínas Proto-Oncogênicas B-raf/fisiologia , Animais , Células Cultivadas , Sistema Nervoso Central/citologia , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Inibidores Enzimáticos/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 1/metabolismo , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Camundongos Transgênicos , Bainha de Mielina/ultraestrutura , Oligodendroglia/citologia , Fosforilação , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas Proto-Oncogênicas c-raf/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismoRESUMO
Polymyxin B is a lipopolysaccharide binding antibiotic used to inactivate potential lipopolysaccharide contaminations when evaluating the activity of different agents on innate immune cells. We report that polymyxin B is able to induce directly in monocyte-derived human dendritic cells (DCs) several functional and molecular modifications characteristic of DCs undergoing a maturation process. DCs incubated with polymyxin B up-regulate the expression of HLA class I and II, the co-stimulatory CD86 molecule, and show an increase in the fraction of adherent cells at short time, which persist at 48 h of incubation. Adhesion to the plate was required for the polymyxin B-induced DCs maturation. A transient activation of IkappaB-alpha/NF-kappaB and ERK1/2 pathways at short time and a further ERK1/2 activation at long term were also detected. Neither up-regulation of the maturation marker CD83 nor activation of p38 nor induction of cytokines secretion was observed in DCs treated with polymyxin B. We demonstrated that inhibition of IkappaB-alpha/NF-kappaB pathway abolishes polymyxin B effects. ERK1/2 inhibition instead allowed DCs treated with polymyxin B to progress in their maturation process as revealed by the increased up-regulation of the CD83 co-stimulatory molecules, the activation of p38, and the reduced adhesion to culture plates at 48 h of incubation. Our results indicate that polymyxin B induces a partial maturation of human DCs through increased adhesion to a substrate and activation of the IkappaB-alpha/NF-kappaB pathway. The increased ERK1/2 activation observed, even though correlating with the initial phases of the maturation process, actually inhibits the occurrence of full maturation.
Assuntos
Antibacterianos/farmacologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/fisiologia , Polimixina B/farmacologia , Transdução de Sinais/fisiologia , Antígenos CD/genética , Antígenos CD/metabolismo , Antígeno B7-2 , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Células Cultivadas , Citocinas/metabolismo , Células Dendríticas/citologia , Células Dendríticas/imunologia , Ativação Enzimática , Inibidores Enzimáticos/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Flavonoides/metabolismo , Genes MHC Classe I , Genes MHC da Classe II , Antígenos HLA/genética , Antígenos HLA/metabolismo , Humanos , Proteínas I-kappa B/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Inibidor de NF-kappaB alfa , NF-kappa B/metabolismo , Tosilfenilalanil Clorometil Cetona/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Immune responses against pathogens require that microbial components promote the activation of antigen-presenting cells (APCs). Autoimmune diseases and graft rejections occur in the absence of pathogens; in these conditions, endogenous molecules, the so-called 'innate adjuvants', activate APCs. Necrotic cells contain and release innate adjuvants; necrotic cells also release high-mobility group B1 protein (HMGB1), an abundant and conserved constituent of vertebrate nuclei. Here, we show that necrotic HMGB1(-/-) cells have a reduced ability to activate APCs, and HMGB1 blockade reduces the activation induced by necrotic wild-type cell supernatants. In vivo, HMGB1 enhances the primary antibody responses to soluble antigens and transforms poorly immunogenic apoptotic lymphoma cells into efficient vaccines.