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While whole genome sequencing (WGS) of cell-free DNA (cfDNA) holds enormous promise for detection of molecular residual disease (MRD), its performance is limited by WGS error rate. Here we introduce AccuScan, an efficient cfDNA WGS technology that enables genome-wide error correction at single read-level, achieving an error rate of 4.2 × 10-7, which is about two orders of magnitude lower than a read-centric de-noising method. The application of AccuScan to MRD demonstrated analytical sensitivity down to 10-6 circulating variant allele frequency at 99% sample-level specificity. AccuScan showed 90% landmark sensitivity (within 6 weeks after surgery) and 100% specificity for predicting relapse in colorectal cancer. It also showed 67% sensitivity and 100% specificity in esophageal cancer using samples collected within one week after surgery. When AccuScan was applied to monitor immunotherapy in melanoma patients, the circulating tumor DNA (ctDNA) levels and dynamic profiles were consistent with clinical outcomes. Overall, AccuScan provides a highly accurate WGS solution for MRD detection, empowering ctDNA detection at parts per million range without requiring high sample input or personalized reagents.
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Despite the remarkable success of anti-cancer immunotherapy, its effectiveness remains confined to a subset of patients-emphasizing the importance of predictive biomarkers in clinical decision-making and further mechanistic understanding of treatment response. Current biomarkers, however, lack the power required to accurately stratify patients. Here, we identify interferon-stimulated, Ly6Ehi neutrophils as a blood-borne biomarker of anti-PD1 response in mice at baseline. Ly6Ehi neutrophils are induced by tumor-intrinsic activation of the STING (stimulator of interferon genes) signaling pathway and possess the ability to directly sensitize otherwise non-responsive tumors to anti-PD1 therapy, in part through IL12b-dependent activation of cytotoxic T cells. By translating our pre-clinical findings to a cohort of patients with non-small cell lung cancer and melanoma (n = 109), and to public data (n = 1440), we demonstrate the ability of Ly6Ehi neutrophils to predict immunotherapy response in humans with high accuracy (average AUC ≈ 0.9). Overall, our study identifies a functionally active biomarker for use in both mice and humans.
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Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Camundongos , Animais , Interferons , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neutrófilos/patologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Biomarcadores , ImunoterapiaRESUMO
While whole genome sequencing (WGS) of cell-free DNA (cfDNA) holds enormous promise for molecular residual disease (MRD) detection, its performance is limited by WGS error rate. Here we introduce AccuScan, an efficient cfDNA WGS technology that enables genome-wide error correction at single read level, achieving an error rate of 4.2×10 -7 , which is about two orders of magnitude lower than a read-centric de-noising method. When applied to MRD detection, AccuScan demonstrated analytical sensitivity down to 10 -6 circulating tumor allele fraction at 99% sample level specificity. In colorectal cancer, AccuScan showed 90% landmark sensitivity for predicting relapse. It also showed robust MRD performance with esophageal cancer using samples collected as early as 1 week after surgery, and predictive value for immunotherapy monitoring with melanoma patients. Overall, AccuScan provides a highly accurate WGS solution for MRD, empowering circulating tumor DNA detection at parts per million range without high sample input nor personalized reagents. One Sentence Summary: AccuScan showed remarkable ultra-low limit of detection with a short turnaround time, low sample requirement and a simple workflow for MRD detection.
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The conserved WD40-repeat protein WDR5 interacts with multiple proteins both inside and outside the nucleus. However, it is currently unclear whether and how the distribution of WDR5 between complexes is regulated. Here, we show that an unannotated microprotein EMBOW (endogenous microprotein binder of WDR5) dually encoded in the human SCRIB gene interacts with WDR5 and regulates its binding to multiple interaction partners, including KMT2A and KIF2A. EMBOW is cell cycle regulated, with two expression maxima at late G1 phase and G2/M phase. Loss of EMBOW decreases WDR5 interaction with KIF2A, aberrantly shortens mitotic spindle length, prolongs G2/M phase, and delays cell proliferation. In contrast, loss of EMBOW increases WDR5 interaction with KMT2A, leading to WDR5 binding to off-target genes, erroneously increasing H3K4me3 levels, and activating transcription of these genes. Together, these results implicate EMBOW as a regulator of WDR5 that regulates its interactions and prevents its off-target binding in multiple contexts.
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Cromatina , Peptídeos e Proteínas de Sinalização Intracelular , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proliferação de Células , Fuso Acromático , Cinesinas/genética , MicropeptídeosRESUMO
Immune-related toxicities, otherwise known as immune-related adverse events (irAEs), occur in a substantial fraction of cancer patients treated with immune checkpoint inhibitors (ICIs). Ranging from asymptomatic to life-threatening, ICI-induced irAEs can result in hospital admission, high-dose corticosteroid treatment, ICI discontinuation, and in some cases, death. A deeper understanding of the factors underpinning severe irAE development will be essential for improved irAE prediction and prevention, toward maximizing the benefits and safety profiles of ICIs. In recent work, we applied mass cytometry, single-cell RNA sequencing, single-cell V(D)J sequencing, bulk RNA sequencing, and bulk T-cell receptor (TCR) sequencing to identify pretreatment determinants of severe irAE development in patients with advanced melanoma. Across 71 patients separated into three cohorts, we found that two baseline features in circulation-elevated activated CD4 effector memory T-cell abundance and TCR diversity-are associated with severe irAE development, independent of the affected organ system within 3 months of ICI treatment initiation. Here, we provide an extended perspective on this work, synthesize and discuss related literature, and summarize practical considerations for clinical translation. Collectively, these findings lay a foundation for data-driven and mechanistic insights into irAE development, with the potential to reduce ICI morbidity and mortality in the future.
Assuntos
Antineoplásicos Imunológicos , Neoplasias , Humanos , Inibidores de Checkpoint Imunológico/efeitos adversos , Antineoplásicos Imunológicos/efeitos adversos , Linfócitos T CD4-Positivos , Neoplasias/tratamento farmacológicoRESUMO
Immune checkpoint PD-1 and its ligand PD-L1 lead to T cell exhaustion, and a high level of circulating soluble PD-L1 at baseline indicates a poor prognosis in melanoma and other solid tumor types. Here we show that the dynamic changes of circulating soluble PD-1 and PD-L1 across the course of immune checkpoint blockades (ICBs) and their changes associate with patient survival in melanoma in a retrospective study. A high change of soluble PD-L1 level at a time-point but not PD-1 significantly increased the mortality, whereas a high change of soluble PD-1/PD-L1 ratio significantly reduced the mortality. After the initial immunotherapy, both soluble PD-1 and PD-L1 increased. However, the change pattern of soluble PD-L1 level was particularly dependent on patients' survival status. These findings indicate that the magnitudes of circulating soluble PD-L1 and PD-1/PD-L1 ratio changes over the time may reflect the patients' response to ICBs or the progression of the disease and predict the survival in melanoma patients treated with ICBs.
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Inibidores de Checkpoint Imunológico , Melanoma , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Antígeno B7-H1/metabolismo , Estudos Retrospectivos , ImunoterapiaRESUMO
Acral melanoma, the most common melanoma subtype among non-White individuals, is associated with poor prognosis. However, its key molecular drivers remain obscure. Here, we perform integrative genomic and clinical profiling of acral melanomas from 104 patients treated in North America (n = 37) or China (n = 67). We find that recurrent, late-arising focal amplifications of cytoband 22q11.21 are a leading determinant of inferior survival, strongly associated with metastasis, and linked to downregulation of immunomodulatory genes associated with response to immune checkpoint blockade. Unexpectedly, LZTR1 - a known tumor suppressor in other cancers - is a key candidate oncogene in this cytoband. Silencing of LZTR1 in melanoma cell lines causes apoptotic cell death independent of major hotspot mutations or melanoma subtypes. Conversely, overexpression of LZTR1 in normal human melanocytes initiates processes associated with metastasis, including anchorage-independent growth, formation of spheroids, and an increase in MAPK and SRC activities. Our results provide insights into the etiology of acral melanoma and implicate LZTR1 as a key tumor promoter and therapeutic target.
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Melanoma , Neoplasias Cutâneas , Genômica , Humanos , Melanoma/patologia , Oncogenes , Neoplasias Cutâneas/patologia , Fatores de Transcrição/genética , Melanoma Maligno CutâneoRESUMO
The major drawback of melanoma therapy with BRAF and MAPK inhibitors is the innate and acquired drug resistance. We therefore explored alternative targets and developed a new compound, SAB298, that is a SRC-family kinase (SFK) inhibitor. The drug is cytotoxic to patient-derived melanoma cells regardless of oncogene expression and inhibits tumor growth in vivo. As expected, it inhibited SRC and PI3K activity, and had the additional property of ERBB2 inhibition, that lead to inactivation of the two ERK phosphatases PP2A and SHP2. In 57% of the melanoma cell lines tested, the consequent increase in ERK activity lead to proteolytic degradation of its substrate, the lineage specific transcription factor MITF, likely contributing to growth arrest. Treatment with a combination of SAB298 and AZD6244 (selumetinib), induced a synergistic growth inhibition, suggesting that the new compound could be used in the clinic as a substitute for, or in combination with MAPK inhibitors.
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Combination checkpoint blockade (CCB) targeting inhibitory CTLA4 and PD1 receptors holds promise for cancer therapy. Immune-related adverse events (IRAEs) remain a major obstacle for the optimal application of CCB in cancer. Here, we analyzed B cell changes in patients with melanoma following treatment with either anti-CTLA4 or anti-PD1, or in combination. CCB therapy led to changes in circulating B cells that were detectable after the first cycle of therapy and characterized by a decline in circulating B cells and an increase in CD21lo B cells and plasmablasts. PD1 expression was higher in the CD21lo B cells, and B cell receptor sequencing of these cells demonstrated greater clonality and a higher frequency of clones compared with CD21hi cells. CCB induced proliferation in the CD21lo compartment, and single-cell RNA sequencing identified B cell activation in cells with genomic profiles of CD21lo B cells in vivo. Increased clonality of circulating B cells following CCB occurred in some patients. Treatment-induced changes in B cells preceded and correlated with both the frequency and timing of IRAEs. Patients with early B cell changes experienced higher rates of grade 3 or higher IRAEs 6 months after CCB. Thus, early changes in B cells following CCB may identify patients who are at increased risk of IRAEs, and preemptive strategies targeting B cells may reduce toxicities in these patients.
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Autoimunidade , Linfócitos B/citologia , Antígeno CTLA-4/antagonistas & inibidores , Melanoma/tratamento farmacológico , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Idoso , Linfócitos B/efeitos dos fármacos , Antígeno CTLA-4/metabolismo , Feminino , Humanos , Sistema Imunitário , Imunoterapia , Estimativa de Kaplan-Meier , Leucócitos Mononucleares/citologia , Masculino , Melanoma/sangue , Pessoa de Meia-Idade , Fenótipo , Receptor de Morte Celular Programada 1/metabolismo , Risco , Neoplasias Cutâneas/sangue , Neoplasias Cutâneas/tratamento farmacológicoRESUMO
Heterogeneity of tumor cells and their microenvironment can affect outcome in cancer. Blockade of immune checkpoints (ICPs) expressed only on a subset of immune cells leads to durable responses in advanced melanoma. Tissue-resident memory T (TRM) cells have recently emerged as a distinct subset of memory T cells in nonlymphoid tissues. Here, we show that functional properties and expression of ICPs within tumor-infiltrating lymphocytes (TILs) differ from those of blood T cells. TILs secrete less IL-2, IFN-γ, and TNF-α compared with circulating counterparts, and expression of VEGF correlated with reduced TIL infiltration. Within tumors, ICPs are particularly enriched within T cells with phenotype and genomic features of TRM cells and the CD16+ subset of myeloid cells. Concurrent T cell receptor (TCR) and tumor exome sequencing of individual metastases in the same patient revealed that interlesional diversity of TCRs exceeded differences in mutation/neoantigen load in tumor cells. These findings suggest that the TRM subset of TILs may be the major target of ICP blockade and illustrate interlesional diversity of tissue-resident TCRs within individual metastases, which did not equilibrate between metastases and may differentially affect the outcome of immune therapy at each site.
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Vascular tumors are among the most common neoplasms in infants and children; 5%-10% of newborns present with or develop lesions within the first 3 months of life. Most are benign infantile hemangiomas that typically regress by 5 years of age; other vascular tumors include congenital tufted angiomas (TAs), kaposiform hemangioendotheliomas (KHEs), and childhood lobular capillary hemangiomas (LCHs). Some of these lesions can become locally invasive and unresponsive to pharmacologic intervention, leading to significant complications. Recent investigation has revealed that activating mutations in HRAS, KRAS, NRAS, GNAQ, and GNA11 can cause certain types of rare childhood vascular tumors, and we have now identified causal recurrent somatic activating mutations in GNA14 by whole-exome and targeted sequencing. We found somatic activating GNA14 c.614A>T (p.Gln205Leu) mutations in one KHE, one TA, and one LCH and a GNA11 c.547C>T (p.Arg183Cys) mutation in two LCH lesions. We examined mutation pathobiology via expression of mutant GNA14 or GNA11 in primary human endothelial cells and melanocytes. GNA14 and GNA11 mutations induced changes in cellular morphology and rendered cells growth-factor independent by upregulating the MAPK pathway. Our findings identify GNA14 mutations as a cause of childhood vascular tumors, offer insight into mechanisms of oncogenic transformation by mutations affecting Gaq family members, and identify potential targets for therapeutic intervention.
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Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Sistema de Sinalização das MAP Quinases , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mutação/genética , Neoplasias Vasculares/congênito , Neoplasias Vasculares/genética , Células Cultivadas , Pré-Escolar , Ativação Enzimática , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Lactente , Recém-Nascido , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Masculino , Melanócitos/metabolismo , Melanócitos/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Neoplasias Vasculares/enzimologia , Neoplasias Vasculares/patologiaRESUMO
We report on whole-exome sequencing (WES) of 213 melanomas. Our analysis established NF1, encoding a negative regulator of RAS, as the third most frequently mutated gene in melanoma, after BRAF and NRAS. Inactivating NF1 mutations were present in 46% of melanomas expressing wild-type BRAF and RAS, occurred in older patients and showed a distinct pattern of co-mutation with other RASopathy genes, particularly RASA2. Functional studies showed that NF1 suppression led to increased RAS activation in most, but not all, melanoma cases. In addition, loss of NF1 did not predict sensitivity to MEK or ERK inhibitors. The rebound pathway, as seen by the induction of phosphorylated MEK, occurred in cells both sensitive and resistant to the studied drugs. We conclude that NF1 is a key tumor suppressor lost in melanomas, and that concurrent RASopathy gene mutations may enhance its role in melanomagenesis.
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Exoma , Melanoma/genética , Neurofibromina 1/genética , Neoplasias Cutâneas/genética , Antineoplásicos/farmacologia , Benzimidazóis/farmacologia , Análise Mutacional de DNA , Resistencia a Medicamentos Antineoplásicos , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Concentração Inibidora 50 , Estimativa de Kaplan-Meier , Perda de Heterozigosidade , Masculino , Melanoma/tratamento farmacológico , Melanoma/etiologia , Mutação de Sentido Incorreto , Análise de Sequência de RNA , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/etiologia , Luz Solar/efeitos adversos , Células Tumorais Cultivadas , Proteínas ras/genéticaRESUMO
Mutations in sunlight-induced melanoma arise from cyclobutane pyrimidine dimers (CPDs), DNA photoproducts that are typically created picoseconds after an ultraviolet (UV) photon is absorbed at thymine or cytosine. We found that in melanocytes, CPDs are generated for >3 hours after exposure to UVA, a major component of the radiation in sunlight and in tanning beds. These "dark CPDs" constitute the majority of CPDs and include the cytosine-containing CPDs that initiate UV-signature CâT mutations. Dark CPDs arise when UV-induced reactive oxygen and nitrogen species combine to excite an electron in fragments of the pigment melanin. This creates a quantum triplet state that has the energy of a UV photon but induces CPDs by energy transfer to DNA in a radiation-independent manner. Melanin may thus be carcinogenic as well as protective against cancer. These findings also validate the long-standing suggestion that chemically generated excited electronic states are relevant to mammalian biology.
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Dano ao DNA/genética , DNA/efeitos da radiação , Melaninas/metabolismo , Melanócitos/efeitos da radiação , Melanoma/genética , Neoplasias Induzidas por Radiação/genética , Dímeros de Pirimidina/metabolismo , Neoplasias Cutâneas/genética , Animais , Células Cultivadas , Citosina/metabolismo , DNA/química , DNA/genética , Transferência de Energia , Humanos , Melaninas/química , Melanócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese , Mutação , Fótons , Receptor Tipo 1 de Melanocortina/genética , Luz Solar/efeitos adversos , Timina/metabolismo , Raios UltravioletaRESUMO
BRAF inhibitors improve melanoma patient survival, but resistance invariably develops. Here we report the discovery of a novel BRAF mutation that confers resistance to PLX4032 employing whole-exome sequencing of drug-resistant BRAF(V600K) melanoma cells. We further describe a new screening approach, a genome-wide piggyBac mutagenesis screen that revealed clinically relevant aberrations (N-terminal BRAF truncations and CRAF overexpression). The novel BRAF mutation, a Leu505 to His substitution (BRAF(L505H) ), is the first resistance-conferring second-site mutation identified in BRAF mutant cells. The mutation replaces a small nonpolar amino acid at the BRAF-PLX4032 interface with a larger polar residue. Moreover, we show that BRAF(L505H) , found in human prostate cancer, is itself a MAPK-activating, PLX4032-resistant oncogenic mutation. Lastly, we demonstrate that the PLX4032-resistant melanoma cells are sensitive to novel, next-generation BRAF inhibitors, especially the 'paradox-blocker' PLX8394, supporting its use in clinical trials for treatment of melanoma patients with BRAF-mutations.
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Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Indóis/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Sulfonamidas/farmacologia , Sequência de Aminoácidos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Elementos de DNA Transponíveis/genética , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Melanoma/enzimologia , Melanoma/patologia , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Insercional/genética , Proteínas Mutantes/metabolismo , Mutação/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , VemurafenibAssuntos
Matriz Extracelular/metabolismo , Melanócitos/citologia , Melanócitos/metabolismo , Animais , Moléculas de Adesão Celular/farmacologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sistema Livre de Células , Células Clonais , Matriz Extracelular/efeitos dos fármacos , Humanos , Melanócitos/efeitos dos fármacos , Camundongos , Ratos , CalininaRESUMO
The receptor tyrosine kinase AXL regulates melanoma cell proliferation and migration. We now demonstrate that AXL and the related kinase MERTK are alternately expressed in melanoma and are associated with different transcriptional signatures. MERTK-positive melanoma cells are more proliferative and less migratory than AXL-positive melanoma cells and overexpression of AXL increases cell motility relative to MERTK. MERTK is expressed in up to 50% of melanoma cells and shRNA-mediated knockdown of MERTK reduces colony formation and cell migration in a CDC42-dependent fashion. Targeting MERTK also decreases cell survival and proliferation in an AKT-dependent manner. Finally, we identify a novel mutation in the kinase domain of MERTK, MERTK(P) (802S) , that increases the motility of melanoma cells relative to wild-type MERTK. Together, these data demonstrate that MERTK is a possible therapeutic target in melanoma, that AXL and MERTK are associated with differential cell behaviors, and that mutations in MERTK may contribute to melanoma pathogenesis.
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Regulação Neoplásica da Expressão Gênica , Melanoma/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/fisiologia , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Proteína Tirosina Quinases/fisiologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Citofotometria , Perfilação da Expressão Gênica , Células HEK293 , Humanos , Metástase Neoplásica , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Transdução de Sinais , Neoplasias Cutâneas/metabolismo , c-Mer Tirosina Quinase , Proteína cdc42 de Ligação ao GTP/metabolismo , Receptor Tirosina Quinase AxlRESUMO
UNLABELLED: Resistance and partial responses to targeted monotherapy are major obstacles in cancer treatment. Systematic approaches to identify efficacious drug combinations for cancer are not well established, especially in the context of genotype. To address this, we have tested pairwise combinations of an array of small-molecule inhibitors on early-passage melanoma cultures using combinatorial drug screening. Results reveal several inhibitor combinations effective for melanomas with activating RAS or BRAF mutations, including mutant BRAF melanomas with intrinsic or acquired resistance to vemurafenib. Inhibition of both EGF receptor and AKT sensitized treatment-resistant BRAF mutant melanoma cultures to vemurafenib. Melanomas with RAS mutations were more resistant to combination therapies relative to BRAF mutants, but were sensitive to combinations of statins and cyclin-dependent kinase inhibitors in vitro and in vivo. These results show the use of combinatorial drug screening for discovering unique treatment regimens that overcome resistance phenotypes of mutant BRAF- and RAS-driven melanomas. SIGNIFICANCE: We have used drug combinatorial screening to identify effective combinations for mutant BRAF melanomas, including those resistant to vemurafenib, and mutant RAS melanomas that are resistant to many therapies. Mechanisms governing the interactions of the drug combinations are proposed, and in vivo xenografts show the enhanced benefit and tolerability of a mutant RAS -selective combination, which is currently lacking in the clinic.
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Antineoplásicos/administração & dosagem , Melanoma/tratamento farmacológico , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas ras/genética , Animais , Linhagem Celular Tumoral , Interações Medicamentosas , Resistencia a Medicamentos Antineoplásicos , Quimioterapia Combinada , Genes ras/genética , Ensaios de Triagem em Larga Escala , Humanos , Melanoma/genética , Camundongos , Camundongos Nus , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The heterogeneity of tumor samples is a major challenge in the analysis of high-throughput profiling of tumor biopsies and cell lines. The measured aggregate signals of multigenerational progenies often represent an average of several tumor subclones with varying genomic aberrations and different gene expression levels. The goal of the present study was to integrate copy number analyses from SNP-arrays and karyotyping, gene expression profiling, and pathway analyses to detect heterogeneity, identify driver mutations, and explore possible mechanisms of tumor evolution. We showed the heterogeneity of the studied samples, characterized the global copy number alteration profiles, and identified genes whose copy number status and expression levels were aberrant. In particular, we identified a recurrent association between two BRAF(V600E) and BRAF(V600K) mutations and changes in DKK1 gene expression levels, which might indicate an association between the BRAF and WNT pathways. These findings show that the integrated approaches used in the present study can robustly address the challenging issue of tumor heterogeneity in high-throughput profiling.
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Variações do Número de Cópias de DNA , Perfilação da Expressão Gênica/métodos , Melanoma/genética , Linhagem Celular Tumoral , Mapeamento Cromossômico , Cromossomos Humanos/genética , Evolução Molecular , Regulação Neoplásica da Expressão Gênica , Genes Neoplásicos , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Cariotipagem , Melanoma/metabolismo , Mutação , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Polimorfismo de Nucleotídeo Único , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismoRESUMO
We characterized the mutational landscape of melanoma, the form of skin cancer with the highest mortality rate, by sequencing the exomes of 147 melanomas. Sun-exposed melanomas had markedly more ultraviolet (UV)-like C>T somatic mutations compared to sun-shielded acral, mucosal and uveal melanomas. Among the newly identified cancer genes was PPP6C, encoding a serine/threonine phosphatase, which harbored mutations that clustered in the active site in 12% of sun-exposed melanomas, exclusively in tumors with mutations in BRAF or NRAS. Notably, we identified a recurrent UV-signature, an activating mutation in RAC1 in 9.2% of sun-exposed melanomas. This activating mutation, the third most frequent in our cohort of sun-exposed melanoma after those of BRAF and NRAS, changes Pro29 to serine (RAC1(P29S)) in the highly conserved switch I domain. Crystal structures, and biochemical and functional studies of RAC1(P29S) showed that the alteration releases the conformational restraint conferred by the conserved proline, causes an increased binding of the protein to downstream effectors, and promotes melanocyte proliferation and migration. These findings raise the possibility that pharmacological inhibition of downstream effectors of RAC1 signaling could be of therapeutic benefit.