RESUMO
To further our understanding of the somatic genetic basis of uveal melanoma, we sequenced the protein-coding regions of 52 primary tumors and 3 liver metastases together with paired normal DNA. Known recurrent mutations were identified in GNAQ, GNA11, BAP1, EIF1AX, and SF3B1. The role of mutated EIF1AX was tested using loss of function approaches including viability and translational efficiency assays. Knockdown of both wild type and mutant EIF1AX was lethal to uveal melanoma cells. We probed the function of N-terminal tail EIF1AX mutations by performing RNA sequencing of polysome-associated transcripts in cells expressing endogenous wild type or mutant EIF1AX. Ribosome occupancy of the global translational apparatus was sensitive to suppression of wild type but not mutant EIF1AX. Together, these studies suggest that cells expressing mutant EIF1AX may exhibit aberrant translational regulation, which may provide clonal selective advantage in the subset of uveal melanoma that harbors this mutation.
Assuntos
Genoma Humano , Melanoma/genética , Biossíntese de Proteínas/genética , Neoplasias Uveais/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Fator de Iniciação 1 em Eucariotos/genética , Feminino , Humanos , Masculino , Melanoma/patologia , Pessoa de Meia-Idade , Mutação , Neoplasias Uveais/patologia , Adulto JovemRESUMO
PURPOSE: Implementing cancer precision medicine in the clinic requires assessing the therapeutic relevance of genomic alterations. A main challenge is the systematic interpretation of whole-exome sequencing (WES) data for clinical care. METHODS: One hundred sixty-five adults with metastatic colorectal and lung adenocarcinomas were prospectively enrolled in the CanSeq study. WES was performed on DNA extracted from formalin-fixed paraffin-embedded tumor biopsy samples and matched blood samples. Somatic and germ-line alterations were ranked according to therapeutic or clinical relevance. Results were interpreted using an integrated somatic and germ-line framework and returned in accordance with patient preferences. RESULTS: At the time of this analysis, WES had been performed and results returned to the clinical team for 165 participants. Of 768 curated somatic alterations, only 31% were associated with clinical evidence and 69% with preclinical or inferential evidence. Of 806 curated germ-line variants, 5% were clinically relevant and 56% were classified as variants of unknown significance. The variant review and decision-making processes were effective when the process was changed from that of a Molecular Tumor Board to a protocol-based approach. CONCLUSION: The development of novel interpretive and decision-support tools that draw from scientific and clinical evidence will be crucial for the success of cancer precision medicine in WES studies.Genet Med advance online publication 26 January 2017.
Assuntos
Sequenciamento do Exoma/métodos , Exoma/genética , Medicina de Precisão/métodos , Adenocarcinoma/genética , Adenocarcinoma de Pulmão , Adulto , Neoplasias Colorretais/genética , Bases de Dados Genéticas , Genômica/métodos , Mutação em Linhagem Germinativa/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Neoplasias Pulmonares/genética , Mutação/genética , Estudos Prospectivos , Análise de Sequência de DNA/métodosRESUMO
PIK3CA (which encodes the PI3K alpha isoform) is the most frequently mutated oncogene in breast cancer. Small-molecule PI3K inhibitors have shown promise in clinical trials; however, intrinsic and acquired resistance limits their utility. We used a systematic gain-of-function approach to identify genes whose upregulation confers resistance to the PI3K inhibitor BYL719 in breast cancer cells. Among the validated resistance genes, Proviral Insertion site in Murine leukemia virus (PIM) kinases conferred resistance by maintaining downstream PI3K effector activation in an AKT-independent manner. Concurrent pharmacologic inhibition of PIM and PI3K overcame this resistance mechanism. We also observed increased PIM expression and activity in a subset of breast cancer biopsies with clinical resistance to PI3K inhibitors. PIM1 overexpression was mutually exclusive with PIK3CA mutation in treatment-naïve breast cancers, suggesting downstream functional redundancy. Together, these results offer new insights into resistance to PI3K inhibitors and support clinical studies of combined PIM/PI3K inhibition in a subset of PIK3CA-mutant cancers. SIGNIFICANCE: PIM kinase overexpression confers resistance to small-molecule PI3K inhibitors. Combined inhibition of PIM and PI3K may therefore be warranted in a subset of breast cancers. Cancer Discov; 6(10); 1134-47. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 1069.
Assuntos
Neoplasias da Mama/genética , Resistencia a Medicamentos Antineoplásicos , Proteínas Proto-Oncogênicas c-pim-1/genética , Regulação para Cima , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Classe I de Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Classe I de Fosfatidilinositol 3-Quinases/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Células MCF-7 , Inibidores de Proteínas Quinases/farmacologia , Tiazóis/farmacologiaRESUMO
To explore the distinct genotypic and phenotypic states of melanoma tumors, we applied single-cell RNA sequencing (RNA-seq) to 4645 single cells isolated from 19 patients, profiling malignant, immune, stromal, and endothelial cells. Malignant cells within the same tumor displayed transcriptional heterogeneity associated with the cell cycle, spatial context, and a drug-resistance program. In particular, all tumors harbored malignant cells from two distinct transcriptional cell states, such that tumors characterized by high levels of the MITF transcription factor also contained cells with low MITF and elevated levels of the AXL kinase. Single-cell analyses suggested distinct tumor microenvironmental patterns, including cell-to-cell interactions. Analysis of tumor-infiltrating T cells revealed exhaustion programs, their connection to T cell activation and clonal expansion, and their variability across patients. Overall, we begin to unravel the cellular ecosystem of tumors and how single-cell genomics offers insights with implications for both targeted and immune therapies.
Assuntos
Melanoma/genética , Melanoma/secundário , Neoplasias Cutâneas/patologia , Microambiente Tumoral , Sequência de Bases , Comunicação Celular , Ciclo Celular , Resistencia a Medicamentos Antineoplásicos/genética , Células Endoteliais/patologia , Genômica , Humanos , Imunoterapia , Ativação Linfocitária , Melanoma/terapia , Fator de Transcrição Associado à Microftalmia/metabolismo , Metástase Neoplásica , RNA/genética , Análise de Sequência de RNA , Análise de Célula Única , Células Estromais/patologia , Linfócitos T/imunologia , Linfócitos T/patologia , TranscriptomaRESUMO
Whole-exome sequencing of metastatic castration-resistant prostate cancer (mCRPC) reveals that 5% to 7% of tumors harbor promyelocytic leukemia zinc finger (PLZF) protein homozygous deletions. PLZF is a canonical androgen-regulated putative tumor suppressor gene whose expression is inhibited by androgen deprivation therapy (ADT). Here, we demonstrate that knockdown of PLZF expression promotes a CRPC and enzalutamide-resistant phenotype in prostate cancer cells. Reintroduction of PLZF expression is sufficient to reverse androgen-independent growth mediated by PLZF depletion. PLZF loss enhances CRPC tumor growth in a xenograft model. Bioinformatic analysis of the PLZF cistrome shows that PLZF negatively regulates multiple pathways, including the MAPK pathway. Accordingly, our data support an oncogenic program activated by ADT. This acquired mechanism together with the finding of genetic loss in CRPC implicates PLZF inactivation as a mechanism promoting ADT resistance and the CRPC phenotype.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Fatores de Transcrição Kruppel-Like/genética , Neoplasias de Próstata Resistentes à Castração/genética , Animais , Antineoplásicos Hormonais/farmacologia , Benzamidas , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Masculino , Camundongos Nus , Nitrilas , Feniltioidantoína/análogos & derivados , Feniltioidantoína/farmacologia , Proteína com Dedos de Zinco da Leucemia Promielocítica , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/patologia , Transcriptoma , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The use of targeted therapeutics directed against BRAF(V600)-mutant metastatic melanoma improves progression-free survival in many patients; however, acquired drug resistance remains a major medical challenge. By far, the most common clinical resistance mechanism involves reactivation of the MAPK (RAF/MEK/ERK) pathway by a variety of mechanisms. Thus, targeting ERK itself has emerged as an attractive therapeutic concept, and several ERK inhibitors have entered clinical trials. We sought to preemptively determine mutations in ERK1/2 that confer resistance to either ERK inhibitors or combined RAF/MEK inhibition in BRAF(V600)-mutant melanoma. Using a random mutagenesis screen, we identified multiple point mutations in ERK1 (MAPK3) and ERK2 (MAPK1) that could confer resistance to ERK or RAF/MEK inhibitors. ERK inhibitor-resistant alleles were sensitive to RAF/MEK inhibitors and vice versa, suggesting that the future development of alternating RAF/MEK and ERK inhibitor regimens might help circumvent resistance to these agents.
Assuntos
Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Melanoma/tratamento farmacológico , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/genética , Mutação , Inibidores de Proteínas Quinases/farmacologia , Linhagem Celular , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Células HEK293 , Humanos , Melanoma/enzimologia , Melanoma/genética , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteínas Proto-Oncogênicas B-raf/genéticaRESUMO
Most patients with BRAF(V600)-mutant metastatic melanoma develop resistance to selective RAF kinase inhibitors. The spectrum of clinical genetic resistance mechanisms to RAF inhibitors and options for salvage therapy are incompletely understood. We performed whole-exome sequencing on formalin-fixed, paraffin-embedded tumors from 45 patients with BRAF(V600)-mutant metastatic melanoma who received vemurafenib or dabrafenib monotherapy. Genetic alterations in known or putative RAF inhibitor resistance genes were observed in 23 of 45 patients (51%). Besides previously characterized alterations, we discovered a "long tail" of new mitogen-activated protein kinase (MAPK) pathway alterations (MAP2K2, MITF) that confer RAF inhibitor resistance. In three cases, multiple resistance gene alterations were observed within the same tumor biopsy. Overall, RAF inhibitor therapy leads to diverse clinical genetic resistance mechanisms, mostly involving MAPK pathway reactivation. Novel therapeutic combinations may be needed to achieve durable clinical control of BRAF(V600)-mutant melanoma. Integrating clinical genomics with preclinical screens may model subsequent resistance studies.
Assuntos
Antineoplásicos/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/genética , Melanoma/genética , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Neoplasias Cutâneas/genética , Linhagem Celular Tumoral , Exoma , Feminino , Células HEK293 , Humanos , Imidazóis/uso terapêutico , Indóis/uso terapêutico , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 2/genética , Masculino , Melanoma/tratamento farmacológico , Pessoa de Meia-Idade , Mutação , Metástase Neoplásica , Oximas/uso terapêutico , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas B-raf/genética , Análise de Sequência de DNA , Neoplasias Cutâneas/tratamento farmacológico , Sulfonamidas/uso terapêutico , VemurafenibRESUMO
Treatment of BRAF-mutant melanoma with combined dabrafenib and trametinib, which target RAF and the downstream MAP-ERK kinase (MEK)1 and MEK2 kinases, respectively, improves progression-free survival and response rates compared with dabrafenib monotherapy. Mechanisms of clinical resistance to combined RAF/MEK inhibition are unknown. We performed whole-exome sequencing (WES) and whole-transcriptome sequencing (RNA-seq) on pretreatment and drug-resistant tumors from five patients with acquired resistance to dabrafenib/trametinib. In three of these patients, we identified additional mitogen-activated protein kinase (MAPK) pathway alterations in the resistant tumor that were not detected in the pretreatment tumor, including a novel activating mutation in MEK2 (MEK2(Q60P)). MEK2(Q60P) conferred resistance to combined RAF/MEK inhibition in vitro, but remained sensitive to inhibition of the downstream kinase extracellular signal-regulated kinase (ERK). The continued MAPK signaling-based resistance identified in these patients suggests that alternative dosing of current agents, more potent RAF/MEK inhibitors, and/or inhibition of the downstream kinase ERK may be needed for durable control of BRAF-mutant melanoma.