RESUMEN
BACKGROUND: Ewing sarcoma (EwS) is a rare, aggressive solid tumor of childhood, adolescence and young adulthood associated with pathognomonic EWSR1-ETS fusion oncoproteins altering transcriptional regulation. Genome-wide association studies (GWAS) have identified 6 common germline susceptibility loci but have not investigated low-frequency inherited variants with minor allele frequencies below 5% due to limited genotyped cases of this rare tumor. METHODS: We investigated the contribution of rare and low-frequency variation to EwS susceptibility in the largest EwS genome-wide association study to date (733 EwS cases and 1,346 unaffected controls of European ancestry). RESULTS: We identified two low-frequency variants, rs112837127 and rs2296730, on chromosome 20 that were associated with EwS risk (OR = 0.186 and 2.038, respectively; P-value < 5×10-8) and located near previously reported common susceptibility loci. After adjusting for the most associated common variant at the locus, only rs112837127 remained a statistically significant independent signal (OR = 0.200, P-value = 5.84×10-8). CONCLUSIONS: These findings suggest rare variation residing on common haplotypes are important contributors to EwS risk. IMPACT: Motivate future targeted sequencing studies for a comprehensive evaluation of low-frequency and rare variation around common EwS susceptibility loci.
Asunto(s)
Sitios Genéticos , Predisposición Genética a la Enfermedad , Variación Genética , Células Germinativas/metabolismo , Sarcoma de Ewing/genética , Estudio de Asociación del Genoma Completo , Humanos , Desequilibrio de Ligamiento/genética , Oportunidad Relativa , Polimorfismo de Nucleótido Simple/genéticaRESUMEN
Rhabdoid tumors (RTs) are genomically simple pediatric cancers driven by the biallelic inactivation of SMARCB1, leading to SWI/SNF chromatin remodeler complex deficiency. Comprehensive evaluation of the immune infiltrates of human and mice RTs, including immunohistochemistry, bulk RNA sequencing and DNA methylation profiling studies showed a high rate of tumors infiltrated by T and myeloid cells. Single-cell RNA (scRNA) and T cell receptor sequencing highlighted the heterogeneity of these cells and revealed therapeutically targetable exhausted effector and clonally expanded tissue resident memory CD8+ T subpopulations, likely representing tumor-specific cells. Checkpoint blockade therapy in an experimental RT model induced the regression of established tumors and durable immune responses. Finally, we show that one mechanism mediating RTs immunogenicity involves SMARCB1-dependent re-expression of endogenous retroviruses and interferon-signaling activation.
Asunto(s)
Ensamble y Desensamble de Cromatina/inmunología , Tumor Rabdoide/genética , Tumor Rabdoide/inmunología , Linfocitos T/inmunología , Animales , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/inmunología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/inmunología , Humanos , Inmunohistoquímica/métodos , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación/genética , Proteínas Nucleares/genética , Factores de Transcripción/genética , Factores de Transcripción/inmunologíaRESUMEN
Ewing sarcoma (EWS) is a pediatric cancer characterized by the EWSR1-FLI1 fusion. We performed a genome-wide association study of 733 EWS cases and 1346 unaffected individuals of European ancestry. Our study replicates previously reported susceptibility loci at 1p36.22, 10q21.3 and 15q15.1, and identifies new loci at 6p25.1, 20p11.22 and 20p11.23. Effect estimates exhibit odds ratios in excess of 1.7, which is high for cancer GWAS, and striking in light of the rarity of EWS cases in familial cancer syndromes. Expression quantitative trait locus (eQTL) analyses identify candidate genes at 6p25.1 (RREB1) and 20p11.23 (KIZ). The 20p11.22 locus is near NKX2-2, a highly overexpressed gene in EWS. Interestingly, most loci reside near GGAA repeat sequences and may disrupt binding of the EWSR1-FLI1 fusion protein. The high locus to case discovery ratio from 733 EWS cases suggests a genetic architecture in which moderate risk SNPs constitute a significant fraction of risk.
Asunto(s)
Perfilación de la Expresión Génica , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Sarcoma de Ewing/genética , Alelos , Proteínas de Ciclo Celular/genética , Proliferación Celular/genética , Proteínas de Unión al ADN/genética , Regulación Neoplásica de la Expresión Génica , Genotipo , Proteína Homeobox Nkx-2.2 , Proteínas de Homeodominio/genética , Humanos , Proteínas Nucleares , Proteínas de Fusión Oncogénica/genética , Polimorfismo de Nucleótido Simple , Proteína Proto-Oncogénica c-fli-1/genética , Control de Calidad , Sitios de Carácter Cuantitativo , Proteína EWS de Unión a ARN/genética , Riesgo , Sarcoma de Ewing/etnología , Factores de Transcripción/genética , Población Blanca , Proteínas de Pez CebraRESUMEN
Activating mutations of the ALK receptor occur in a subset of neuroblastoma tumors. We previously demonstrated that Alk mutations cooperate with MYCN overexpression to induce neuroblastoma in mice and identified Ret as being strongly upregulated in MYCN/Alkmut tumors. By a genetic approach in vivo, we now document an oncogenic cooperation between activated Ret and MYCN overexpression in neuroblastoma formation. We show that MYCN/RetM919T tumors exhibit histological features and expression profiles close to MYCN/Alkmut tumors. We show that RET transcript levels decrease precedes RET protein levels decrease upon ALK inhibition in neuroblastoma cell lines. Etv5 was identified as a candidate transcription factor regulating Ret expression from murine MYCN/Alkmut tumor transcriptomic data. We demonstrate that ETV5 is regulated both at the protein and mRNA levels upon ALK activation or inhibition in neuroblastoma cell lines and that this regulation precedes RET modulation. We document that ALK activation induces ETV5 protein upregulation through stabilization in a MEK/ERK-dependent manner. We show that RNAi-mediated inhibition of ETV5 decreases RET expression. Reporter assays indicate that ETV5 is able to drive RET gene transcription. ChIP-seq analysis confirmed ETV5 binding on the RET promoter and identified an enhancer upstream of the promoter. Finally, we demonstrate that combining RET and ALK inhibitors reduces tumor growth more efficiently than each single agent in MYCN and AlkF1178L-driven murine neuroblastoma. Altogether, these results define the ERK-ETV5-RET pathway as a critical axis driving neuroblastoma oncogenesis downstream of activated ALK.
Asunto(s)
Quinasa de Linfoma Anaplásico/genética , Carcinogénesis/genética , Mutación con Ganancia de Función , Neuroblastoma/genética , Quinasa de Linfoma Anaplásico/metabolismo , Animales , Carcinogénesis/patología , Células Cultivadas , Proteínas de Unión al ADN/fisiología , Femenino , Mutación con Ganancia de Función/fisiología , Células HEK293 , Humanos , Sistema de Señalización de MAP Quinasas/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Ratones Transgénicos , Neuroblastoma/patología , Proteínas Proto-Oncogénicas c-ret/fisiología , Transducción de Señal/genética , Factores de Transcripción/fisiología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Neuroblastoma is a tumor of the peripheral sympathetic nervous system, derived from multipotent neural crest cells (NCCs). To define core regulatory circuitries (CRCs) controlling the gene expression program of neuroblastoma, we established and analyzed the neuroblastoma super-enhancer landscape. We discovered three types of identity in neuroblastoma cell lines: a sympathetic noradrenergic identity, defined by a CRC module including the PHOX2B, HAND2 and GATA3 transcription factors (TFs); an NCC-like identity, driven by a CRC module containing AP-1 TFs; and a mixed type, further deconvoluted at the single-cell level. Treatment of the mixed type with chemotherapeutic agents resulted in enrichment of NCC-like cells. The noradrenergic module was validated by ChIP-seq. Functional studies demonstrated dependency of neuroblastoma with noradrenergic identity on PHOX2B, evocative of lineage addiction. Most neuroblastoma primary tumors express TFs from the noradrenergic and NCC-like modules. Our data demonstrate a previously unknown aspect of tumor heterogeneity relevant for neuroblastoma treatment strategies.
Asunto(s)
Linaje de la Célula/genética , Regulación Neoplásica de la Expresión Génica/genética , Neuroblastoma/genética , Factores de Transcripción/genética , Animales , Western Blotting , Línea Celular Tumoral/clasificación , Linaje de la Célula/efectos de los fármacos , Doxiciclina/farmacología , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Heterogeneidad Genética , Células HEK293 , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/metabolismo , Interferencia de ARN , Tratamiento con ARN de Interferencia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de la Célula Individual , Factores de Transcripción/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
UNLABELLED: Ewing sarcoma is a primary bone tumor initiated by EWSR1-ETS gene fusions. To identify secondary genetic lesions that contribute to tumor progression, we performed whole-genome sequencing of 112 Ewing sarcoma samples and matched germline DNA. Overall, Ewing sarcoma tumors had relatively few single-nucleotide variants, indels, structural variants, and copy-number alterations. Apart from whole chromosome arm copy-number changes, the most common somatic mutations were detected in STAG2 (17%), CDKN2A (12%), TP53 (7%), EZH2, BCOR, and ZMYM3 (2.7% each). Strikingly, STAG2 mutations and CDKN2A deletions were mutually exclusive, as confirmed in Ewing sarcoma cell lines. In an expanded cohort of 299 patients with clinical data, we discovered that STAG2 and TP53 mutations are often concurrent and are associated with poor outcome. Finally, we detected subclonal STAG2 mutations in diagnostic tumors and expansion of STAG2-immunonegative cells in relapsed tumors as compared with matched diagnostic samples. SIGNIFICANCE: Whole-genome sequencing reveals that the somatic mutation rate in Ewing sarcoma is low. Tumors that harbor STAG2 and TP53 mutations have a particularly dismal prognosis with current treatments and require alternative therapies. Novel drugs that target epigenetic regulators may constitute viable therapeutic strategies in a subset of patients with mutations in chromatin modifiers.