RESUMO
Many cancers are characterized by gene fusions encoding oncogenic chimeric transcription factors (TFs) such as EWS::FLI1 in Ewing sarcoma (EwS). Here, we find that EWS::FLI1 induces the robust expression of a specific set of novel spliced and polyadenylated transcripts within otherwise transcriptionally silent regions of the genome. These neogenes (NGs) are virtually undetectable in large collections of normal tissues or non-EwS tumors and can be silenced by CRISPR interference at regulatory EWS::FLI1-bound microsatellites. Ribosome profiling and proteomics further show that some NGs are translated into highly EwS-specific peptides. More generally, we show that hundreds of NGs can be detected in diverse cancers characterized by chimeric TFs. Altogether, this study identifies the transcription, processing, and translation of novel, specific, highly expressed multi-exonic transcripts from otherwise silent regions of the genome as a new activity of aberrant TFs in cancer.
Assuntos
Carcinogênese , Regulação Neoplásica da Expressão Gênica , Proteínas de Fusão Oncogênica , Proteína Proto-Oncogênica c-fli-1 , Fatores de Transcrição , Carcinogênese/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/genética , Inativação Gênica , Genoma/genética , Genômica , Humanos , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Oncogenes/genética , Proteína Proto-Oncogênica c-fli-1/genética , Proteína Proto-Oncogênica c-fli-1/metabolismo , Sarcoma de Ewing/genética , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/patologia , Fatores de Transcrição/genética , Transcrição Gênica/genéticaRESUMO
Ewing sarcoma (EwS) is a rare bone and soft tissue malignancy driven by chromosomal translocations encoding chimeric transcription factors, such as EWSR1-FLI1, that bind GGAA motifs forming novel enhancers that alter nearby expression. We propose that germline microsatellite variation at the 6p25.1 EwS susceptibility locus could impact downstream gene expression and EwS biology. We performed targeted long-read sequencing of EwS blood DNA to characterize variation and genomic features important for EWSR1-FLI1 binding. We identified 50 microsatellite alleles at 6p25.1 and observed that EwS-affected individuals had longer alleles (>135 bp) with more GGAA repeats. The 6p25.1 GGAA microsatellite showed chromatin features of an EWSR1-FLI1 enhancer and regulated expression of RREB1, a transcription factor associated with RAS/MAPK signaling. RREB1 knockdown reduced proliferation and clonogenic potential and reduced expression of cell cycle and DNA replication genes. Our integrative analysis at 6p25.1 details increased binding of longer GGAA microsatellite alleles with acquired EWSR-FLI1 to promote Ewing sarcomagenesis by RREB1-mediated proliferation.
Assuntos
Neoplasias Ósseas , Sarcoma de Ewing , Humanos , Alelos , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Proteína Proto-Oncogênica c-fli-1/genética , Proteína Proto-Oncogênica c-fli-1/metabolismo , Proteína EWS de Ligação a RNA/genética , Proteína EWS de Ligação a RNA/metabolismo , Sarcoma de Ewing/genética , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/patologiaRESUMO
ERG family proteins (ERG, FLI1 and FEV) are a subfamily of ETS transcription factors with key roles in physiology and development. In Ewing sarcoma, the oncogenic fusion protein EWS-FLI1 regulates both transcription and alternative splicing of pre-messenger RNAs. However, whether wild-type ERG family proteins might regulate splicing is unknown. Here, we show that wild-type ERG proteins associate with spliceosomal components, are found on nascent RNAs, and induce alternative splicing when recruited onto a reporter minigene. Transcriptomic analysis revealed that ERG and FLI1 regulate large numbers of alternative spliced exons (ASEs) enriched with RBFOX2 motifs and co-regulated by this splicing factor. ERG and FLI1 are associated with RBFOX2 via their conserved carboxy-terminal domain, which is present in EWS-FLI1. Accordingly, EWS-FLI1 is also associated with RBFOX2 and regulates ASEs enriched in RBFOX2 motifs. However, in contrast to wild-type ERG and FLI1, EWS-FLI1 often antagonizes RBFOX2 effects on exon inclusion. In particular, EWS-FLI1 reduces RBFOX2 binding to the ADD3 pre-mRNA, thus increasing its long isoform, which represses the mesenchymal phenotype of Ewing sarcoma cells. Our findings reveal a RBFOX2-mediated splicing regulatory function of wild-type ERG family proteins, that is altered in EWS-FLI1 and contributes to the Ewing sarcoma cell phenotype.
Assuntos
Processamento Alternativo , Proteínas de Fusão Oncogênica/metabolismo , Proteína Proto-Oncogênica c-fli-1/metabolismo , Fatores de Processamento de RNA/metabolismo , Proteína EWS de Ligação a RNA/metabolismo , Proteínas Repressoras/metabolismo , Proteínas de Ligação a Calmodulina/genética , Proteínas de Ligação a Calmodulina/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Células HeLa , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Domínios Proteicos , Sarcoma de Ewing/genética , Sarcoma de Ewing/metabolismo , Regulador Transcricional ERG/química , Regulador Transcricional ERG/metabolismoRESUMO
The gene CXXC5, encoding a Retinoid-Inducible Nuclear Factor (RINF), is located within a region at 5q31.2 commonly deleted in myelodysplastic syndrome (MDS) and adult acute myeloid leukemia (AML). RINF may act as an epigenetic regulator and has been proposed as a tumor suppressor in hematopoietic malignancies. However, functional studies in normal hematopoiesis are lacking, and its mechanism of action is unknow. Here, we evaluated the consequences of RINF silencing on cytokineinduced erythroid differentiation of human primary CD34+ progenitors. We found that RINF is expressed in immature erythroid cells and that RINF-knockdown accelerated erythropoietin-driven maturation, leading to a significant reduction (~45%) in the number of red blood cells (RBCs), without affecting cell viability. The phenotype induced by RINF-silencing was TGFß-dependent and mediated by SMAD7, a TGFß- signaling inhibitor. RINF upregulates SMAD7 expression by direct binding to its promoter and we found a close correlation between RINF and SMAD7 mRNA levels both in CD34+ cells isolated from bone marrow of healthy donors and MDS patients with del(5q). Importantly, RINF knockdown attenuated SMAD7 expression in primary cells and ectopic SMAD7 expression was sufficient to prevent the RINF knockdowndependent erythroid phenotype. Finally, RINF silencing affects 5'-hydroxymethylation of human erythroblasts, in agreement with its recently described role as a Tet2- anchoring platform in mouse. Altogether, our data bring insight into how the epigenetic factor RINF, as a transcriptional regulator of SMAD7, may fine-tune cell sensitivity to TGFß superfamily cytokines and thus play an important role in both normal and pathological erythropoiesis.
Assuntos
Proteínas de Ligação a DNA , Leucemia Mieloide Aguda , Síndromes Mielodisplásicas , Proteína Smad7 , Fatores de Transcrição , Adulto , Animais , Ciclo Celular , Epigênese Genética , Humanos , Leucemia Mieloide Aguda/genética , Camundongos , Síndromes Mielodisplásicas/genética , RNA Mensageiro , Proteína Smad7/genéticaRESUMO
Outcomes for metastatic Ewing sarcoma and osteosarcoma are dismal and have not changed for decades. Oxidative stress attenuates melanoma metastasis, and melanoma cells must reduce oxidative stress to metastasize. We explored this in sarcomas by screening for oxidative stress sensitizers, which identified the class I HDAC inhibitor MS-275 as enhancing vulnerability to reactive oxygen species (ROS) in sarcoma cells. Mechanistically, MS-275 inhibits YB-1 deacetylation, decreasing its binding to 5'-UTRs of NFE2L2 encoding the antioxidant factor NRF2, thereby reducing NFE2L2 translation and synthesis of NRF2 to increase cellular ROS. By global acetylomics, MS-275 promotes rapid acetylation of the YB-1 RNA-binding protein at lysine-81, blocking binding and translational activation of NFE2L2, as well as known YB-1 mRNA targets, HIF1A, and the stress granule nucleator, G3BP1. MS-275 dramatically reduces sarcoma metastasis in vivo, but an MS-275-resistant YB-1K81-to-alanine mutant restores metastatic capacity and NRF2, HIF1α, and G3BP1 synthesis in MS-275-treated mice. These studies describe a novel function for MS-275 through enhanced YB-1 acetylation, thus inhibiting YB-1 translational control of key cytoprotective factors and its pro-metastatic activity.
Assuntos
Antineoplásicos/uso terapêutico , Benzamidas/uso terapêutico , Neoplasias Ósseas/tratamento farmacológico , Inibidores de Histona Desacetilases/uso terapêutico , Piridinas/uso terapêutico , Sarcoma de Ewing/tratamento farmacológico , Fatores de Transcrição/metabolismo , Acetilação , Animais , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Células Cultivadas , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Camundongos , Fator 2 Relacionado a NF-E2/metabolismo , Metástase Neoplásica , Estresse Oxidativo , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/patologiaRESUMO
Clinical responses to anticancer therapies are often restricted to a subset of patients. In some cases, mutated cancer genes are potent biomarkers for responses to targeted agents. Here, to uncover new biomarkers of sensitivity and resistance to cancer therapeutics, we screened a panel of several hundred cancer cell lines--which represent much of the tissue-type and genetic diversity of human cancers--with 130 drugs under clinical and preclinical investigation. In aggregate, we found that mutated cancer genes were associated with cellular response to most currently available cancer drugs. Classic oncogene addiction paradigms were modified by additional tissue-specific or expression biomarkers, and some frequently mutated genes were associated with sensitivity to a broad range of therapeutic agents. Unexpected relationships were revealed, including the marked sensitivity of Ewing's sarcoma cells harbouring the EWS (also known as EWSR1)-FLI1 gene translocation to poly(ADP-ribose) polymerase (PARP) inhibitors. By linking drug activity to the functional complexity of cancer genomes, systematic pharmacogenomic profiling in cancer cell lines provides a powerful biomarker discovery platform to guide rational cancer therapeutic strategies.
Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Ensaios de Seleção de Medicamentos Antitumorais , Genes Neoplásicos/genética , Marcadores Genéticos/genética , Genoma Humano/genética , Neoplasias/tratamento farmacológico , Neoplasias/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Genômica , Humanos , Indóis/farmacologia , Neoplasias/patologia , Proteínas de Fusão Oncogênica/genética , Farmacogenética , Ftalazinas/farmacologia , Piperazinas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases , Proteína Proto-Oncogênica c-fli-1/genética , Proteína EWS de Ligação a RNA/genética , Sarcoma de Ewing/tratamento farmacológico , Sarcoma de Ewing/genética , Sarcoma de Ewing/patologiaRESUMO
Ewing sarcoma is the second most frequent pediatric bone tumor. In most of the patients, a chromosomal translocation leads to the expression of the EWS-FLI1 chimeric transcription factor that is the major oncogene in this pathology. Relative genetic simplicity of Ewing sarcoma makes it particularly attractive for studying cancer in a systemic manner. Silencing EWS-FLI1 induces cell cycle alteration and ultimately leads to apoptosis, but the exact molecular mechanisms underlying this phenotype are unclear. In this study, a network linking EWS-FLI1 to cell cycle and apoptosis phenotypes was constructed through an original method of network reconstruction. Transcriptome time-series after EWS-FLI1 silencing were used to identify core modulated genes by an original scoring method based on fitting expression profile dynamics curves. Literature data mining was then used to connect these modulated genes into a network. The validity of a subpart of this network was assessed by siRNA/RT-QPCR experiments on four additional Ewing cell lines and confirmed most of the links. Based on the network and the transcriptome data, CUL1 was identified as a new potential target of EWS-FLI1. Altogether, using an original methodology of data integration, we provide the first version of EWS-FLI1 network model of cell cycle and apoptosis regulation.
Assuntos
Apoptose/genética , Neoplasias Ósseas/genética , Proliferação de Células , Redes Reguladoras de Genes , Proteínas de Fusão Oncogênica/metabolismo , Proteína Proto-Oncogênica c-fli-1/metabolismo , Proteína EWS de Ligação a RNA/metabolismo , Sarcoma de Ewing/genética , Neoplasias Ósseas/metabolismo , Linhagem Celular Tumoral , Mineração de Dados , Regulação Neoplásica da Expressão Gênica , Humanos , Modelos Genéticos , Proteínas de Fusão Oncogênica/antagonistas & inibidores , Proteínas de Fusão Oncogênica/genética , Proteína Proto-Oncogênica c-fli-1/antagonistas & inibidores , Proteína Proto-Oncogênica c-fli-1/genética , RNA Interferente Pequeno/metabolismo , Proteína EWS de Ligação a RNA/antagonistas & inibidores , Proteína EWS de Ligação a RNA/genética , Sarcoma de Ewing/metabolismo , Transdução de Sinais , Biologia de Sistemas , TranscriptomaRESUMO
Sarcomas comprise a heterogeneous group of malignant tumors of mesenchymal origin. More than 80 entities are associated with different mesenchymal lineages. Sarcomas with fibroblastic, muscle, bone, vascular, adipocytic, and other characteristics are distinguished. Nearly half of all entities contain specific chromosomal translocations that give rise to fusion proteins. These are mostly pathognomonic, and their detection by various molecular techniques supports histopathologic classification. Moreover, the fusion proteins act as oncogenic drivers, and their blockade represents a promising therapeutic approach. This review summarizes the current knowledge on fusion proteins in sarcoma. We categorize the different fusion proteins into functional classes, including kinases, epigenetic regulators, and transcription factors, and describe their mechanisms of action. Interestingly, while fusion proteins acting as transcription factors are found in all mesenchymal lineages, the others have a more restricted pattern. Most kinase-driven sarcomas belong to the fibroblastic/myofibroblastic lineage. Fusion proteins with an epigenetic function are mainly associated with sarcomas of unclear differentiation, suggesting that epigenetic dysregulation leads to a major change in cell identity. Comparison of mechanisms of action reveals recurrent functional modes, including antagonism of Polycomb activity by fusion proteins with epigenetic activity and recruitment of histone acetyltransferases by fusion transcription factors of the myogenic lineage. Finally, based on their biology, we describe potential approaches to block the activity of fusion proteins for therapeutic intervention. Overall, our work highlights differences as well as similarities in the biology of fusion proteins from different sarcomas and provides the basis for a functional classification.
RESUMO
Rhabdomyosarcoma (RMS) is a highly aggressive pediatric cancer with features of skeletal muscle differentiation. More than 80% of the high-risk patients ultimately fail to respond to chemotherapy treatment, leading to limited therapeutic options and dismal prognostic rates. The lack of response and subsequent tumor recurrence is driven in part by stem cell-like cells, the tumor subpopulation that is enriched after treatment, and characterized by expression of the AXL receptor tyrosine kinase (AXL). AXL mediates survival, migration, and therapy resistance in several cancer types; however, its function in RMS remains unclear. In this study, we investigated the role of AXL in RMS tumorigenesis, migration, and chemotherapy response, and whether targeting of AXL with small-molecule inhibitors could potentiate the efficacy of chemotherapy. We show that AXL is expressed in a heterogeneous manner in patient-derived xenografts (PDX), primary cultures and cell line models of RMS, consistent with its stem cell-state selectivity. By generating a CRISPR/Cas9 AXL knock-out and overexpressing models, we show that AXL contributes to the migratory phenotype of RMS, but not to chemotherapy resistance. Instead, pharmacologic blockade with the AXL inhibitors bemcentinib (BGB324), cabozantinib and NPS-1034 rapidly killed RMS cells in an AXL-independent manner and augmented the efficacy of the chemotherapeutics vincristine and cyclophosphamide. In vivo administration of the combination of bemcentinib and vincristine exerted strong antitumoral activity in a rapidly progressing PDX mouse model, significantly reducing tumor burden compared with single-agent treatment. Collectively, our data identify bemcentinib as a promising drug to improve chemotherapy efficacy in patients with RMS.
Assuntos
Receptor Tirosina Quinase Axl , Benzocicloeptenos , Proteínas Proto-Oncogênicas , Receptores Proteína Tirosina Quinases , Rabdomiossarcoma , Humanos , Rabdomiossarcoma/tratamento farmacológico , Rabdomiossarcoma/patologia , Rabdomiossarcoma/genética , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/metabolismo , Animais , Camundongos , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/genética , Benzocicloeptenos/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Linhagem Celular Tumoral , Criança , Proliferação de Células/efeitos dos fármacos , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Movimento Celular/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , TriazóisRESUMO
Osteosarcoma and Ewing sarcoma are bone tumors mostly diagnosed in children, adolescents, and young adults. Despite multimodal therapy, morbidity is high and survival rates remain low, especially in the metastatic disease setting. Trials investigating targeted therapies and immunotherapies have not been groundbreaking. Better understanding of biological subgroups, the role of the tumor immune microenvironment, factors that promote metastasis, and clinical biomarkers of prognosis and drug response are required to make progress. A prerequisite to achieve desired success is a thorough, systematic, and clinically linked biological analysis of patient samples, but disease rarity and tissue processing challenges such as logistics and infrastructure have contributed to a lack of relevant samples for clinical care and research. There is a need for a Europe-wide framework to be implemented for the adequate and minimal sampling, processing, storage, and analysis of patient samples. Two international panels of scientists, clinicians, and patient and parent advocates have formed the Fight Osteosarcoma Through European Research consortium and the Euro Ewing Consortium. The consortia shared their expertise and institutional practices to formulate new guidelines. We report new reference standards for adequate and minimally required sampling (time points, diagnostic samples, and liquid biopsy tubes), handling, and biobanking to enable advanced biological studies in bone sarcoma. We describe standards for analysis and annotation to drive collaboration and data harmonization with practical, legal, and ethical considerations. This position paper provides comprehensive guidelines that should become the new standards of care that will accelerate scientific progress, promote collaboration, and improve outcomes.
Assuntos
Neoplasias Ósseas , Osteossarcoma , Sarcoma de Ewing , Manejo de Espécimes , Humanos , Osteossarcoma/terapia , Osteossarcoma/patologia , Osteossarcoma/diagnóstico , Sarcoma de Ewing/terapia , Sarcoma de Ewing/patologia , Sarcoma de Ewing/diagnóstico , Europa (Continente) , Neoplasias Ósseas/terapia , Neoplasias Ósseas/patologia , Manejo de Espécimes/métodos , Manejo de Espécimes/normas , Biomarcadores Tumorais , Bancos de Espécimes BiológicosRESUMO
3D in vitro biological systems are progressively replacing 2D systems to increase the physiological relevance of cellular studies. Microfluidics-based approaches can be powerful tools towards such biomimetic systems, but often require high-end complicated and expensive processes and equipment for microfabrication. Herein, a drug screening platform is proposed, minimizing technicality and manufacturing steps. It provides an alternate way of spheroid generation in droplets in tubes. Droplet microfluidics then elicit multiple droplets merging events at programmable times, to submit sequentially the spheroids to chemotherapy and to reagents for cytotoxicity screening. After a comprehensive study of tumorogenesis within the droplets, the system is validated for drug screening (IC50) with chemotherapies in cancer cell lines as well as cells from a patient-derived-xenografts (PDX). As compared to microtiter plates methods, our system reduces the initial number of cells up to 10 times and opens new avenues towards primary tumors drug screening approaches.
Assuntos
Microfluídica , Neoplasias , Humanos , Microfluídica/métodos , Avaliação Pré-Clínica de Medicamentos , Detecção Precoce de Câncer , Esferoides Celulares , Linhagem Celular Tumoral , Neoplasias/tratamento farmacológicoRESUMO
Clinical sequencing efforts are rapidly identifying sarcoma gene fusions that lack functional validation. An example is the fusion of transcriptional coactivators, VGLL2-NCOA2, found in infantile rhabdomyosarcoma. To delineate VGLL2-NCOA2 tumorigenic mechanisms and identify therapeutic vulnerabilities, we implement a cross-species comparative oncology approach with zebrafish, mouse allograft, and patient samples. We find that VGLL2-NCOA2 is sufficient to generate mesenchymal tumors that display features of immature skeletal muscle and recapitulate the human disease. A subset of VGLL2-NCOA2 zebrafish tumors transcriptionally cluster with embryonic somitogenesis and identify VGLL2-NCOA2 developmental programs, including a RAS family GTPase, ARF6. In VGLL2-NCOA2 zebrafish, mouse, and patient tumors, ARF6 is highly expressed. ARF6 knockout suppresses VGLL2-NCOA2 oncogenic activity in cell culture, and, more broadly, ARF6 is overexpressed in adult and pediatric sarcomas. Our data indicate that VGLL2-NCOA2 is an oncogene that leverages developmental programs for tumorigenesis and that reactivation or persistence of ARF6 could represent a therapeutic opportunity.
Assuntos
Rabdomiossarcoma , Sarcoma , Criança , Adulto , Humanos , Animais , Camundongos , Peixe-Zebra/metabolismo , Fatores de Transcrição/genética , Rabdomiossarcoma/genética , Rabdomiossarcoma/patologia , Fusão Gênica , Coativador 2 de Receptor Nuclear/genética , Proteínas Musculares/genéticaRESUMO
Ewing sarcoma is a pediatric bone and soft tissue cancer with an urgent need for new therapies to improve disease outcome. To identify effective drugs, phenotypic drug screening has proven to be a powerful method, but achievable throughput in mouse xenografts, the preclinical Ewing sarcoma standard model, is limited. Here, we explored the use of xenografts in zebrafish for high-throughput drug screening to discover new combination therapies for Ewing sarcoma. We subjected xenografts in zebrafish larvae to high-content imaging and subsequent automated tumor size analysis to screen single agents and compound combinations. We identified three drug combinations effective against Ewing sarcoma cells: Irinotecan combined with either an MCL-1 or an BCL-XL inhibitor and in particular dual inhibition of the anti-apoptotic proteins MCL-1 and BCL-XL, which efficiently eradicated tumor cells in zebrafish xenografts. We confirmed enhanced efficacy of dual MCL-1/BCL-XL inhibition compared to single agents in a mouse PDX model. In conclusion, high-content screening of small compounds on Ewing sarcoma zebrafish xenografts identified dual MCL-1/BCL-XL targeting as a specific vulnerability and promising therapeutic strategy for Ewing sarcoma, which warrants further investigation towards clinical application.
Assuntos
Sarcoma de Ewing , Humanos , Animais , Camundongos , Sarcoma de Ewing/tratamento farmacológico , Sarcoma de Ewing/genética , Sarcoma de Ewing/metabolismo , Peixe-Zebra/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Avaliação Pré-Clínica de Medicamentos , Xenoenxertos , Apoptose , Proteína bcl-X/genética , Proteína bcl-X/metabolismo , Linhagem Celular TumoralRESUMO
Rhabdomyosarcoma (RMS) is a group of pediatric cancers with features of developing skeletal muscle. The cellular hierarchy and mechanisms leading to developmental arrest remain elusive. Here, we combined single-cell RNA sequencing, mass cytometry, and high-content imaging to resolve intratumoral heterogeneity of patient-derived primary RMS cultures. We show that the aggressive alveolar RMS (aRMS) subtype contains plastic muscle stem-like cells and cycling progenitors that drive tumor growth, and a subpopulation of differentiated cells that lost its proliferative potential and correlates with better outcomes. While chemotherapy eliminates cycling progenitors, it enriches aRMS for muscle stem-like cells. We screened for drugs hijacking aRMS toward clinically favorable subpopulations and identified a combination of RAF and MEK inhibitors that potently induces myogenic differentiation and inhibits tumor growth. Overall, our work provides insights into the developmental states underlying aRMS aggressiveness, chemoresistance, and progression and identifies the RAS pathway as a promising therapeutic target.
Assuntos
Antineoplásicos , Rabdomiossarcoma Alveolar , Rabdomiossarcoma , Criança , Humanos , Rabdomiossarcoma Alveolar/tratamento farmacológico , Rabdomiossarcoma Alveolar/genética , Rabdomiossarcoma Alveolar/patologia , Rabdomiossarcoma/tratamento farmacológico , Rabdomiossarcoma/genética , Rabdomiossarcoma/patologia , Músculo Esquelético/metabolismo , Diferenciação Celular , Antineoplásicos/uso terapêutico , Linhagem Celular TumoralRESUMO
Activation of oncogenic gene expression from long-range enhancers is initiated by the assembly of DNA-binding transcription factors (TF), leading to recruitment of co-activators such as CBP/p300 to modify the local genomic context and facilitate RNA-Polymerase 2 (Pol2) binding. Yet, most TF-to-coactivator recruitment relationships remain unmapped. Here, studying the oncogenic fusion TF PAX3-FOXO1 (P3F) from alveolar rhabdomyosarcoma (aRMS), we show that a single cysteine in the activation domain (AD) of P3F is important for a small alpha helical coil that recruits CBP/p300 to chromatin. P3F driven transcription requires both this single cysteine and CBP/p300. Mutants of the cysteine reduce aRMS cell proliferation and induce cellular differentiation. Furthermore, we discover a profound dependence on CBP/p300 for clustering of Pol2 loops that connect P3F to its target genes. In the absence of CBP/p300, Pol2 long range enhancer loops collapse, Pol2 accumulates in CpG islands and fails to exit the gene body. These results reveal a potential novel axis for therapeutic interference with P3F in aRMS and clarify the molecular relationship of P3F and CBP/p300 in sustaining active Pol2 clusters essential for oncogenic transcription.
Assuntos
RNA Polimerase II , Rabdomiossarcoma Alveolar , Humanos , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Cisteína/metabolismo , Fatores de Transcrição/metabolismo , Fator de Transcrição PAX3/genética , Rabdomiossarcoma Alveolar/genética , RNA/metabolismo , Ativação Transcricional , Ligação Proteica , Proteína Forkhead Box O1/metabolismoRESUMO
Renal medullary carcinoma (RMC) is an aggressive tumour driven by bi-allelic loss of SMARCB1 and tightly associated with sickle cell trait. However, the cell-of-origin and oncogenic mechanism remain poorly understood. Using single-cell sequencing of human RMC, we defined transformation of thick ascending limb (TAL) cells into an epithelial-mesenchymal gradient of RMC cells associated with loss of renal epithelial transcription factors TFCP2L1, HOXB9 and MITF and gain of MYC and NFE2L2-associated oncogenic and ferroptosis resistance programs. We describe the molecular basis for this transcriptional switch that is reversed by SMARCB1 re-expression repressing the oncogenic and ferroptosis resistance programs leading to ferroptotic cell death. Ferroptosis resistance links TAL cell survival with the high extracellular medullar iron concentrations associated with sickle cell trait, an environment propitious to the mutagenic events associated with RMC development. This unique environment may explain why RMC is the only SMARCB1-deficient tumour arising from epithelial cells, differentiating RMC from rhabdoid tumours arising from neural crest cells.
Assuntos
Carcinoma Medular , Carcinoma de Células Renais , Ferroptose , Neoplasias Renais , Traço Falciforme , Humanos , Neoplasias Renais/patologia , Carcinoma Medular/metabolismo , Carcinoma de Células Renais/patologia , Proteína SMARCB1/genética , Proteína SMARCB1/metabolismo , Proteínas Repressoras , Proteínas de HomeodomínioRESUMO
Noradrenergic and mesenchymal identities have been characterized in neuroblastoma cell lines according to their epigenetic landscapes and core regulatory circuitries. However, their relationship and relative contribution in patient tumors remain poorly defined. We now document spontaneous and reversible plasticity between the two identities, associated with epigenetic reprogramming, in several neuroblastoma models. Interestingly, xenografts with cells from each identity eventually harbor a noradrenergic phenotype suggesting that the microenvironment provides a powerful pressure towards this phenotype. Accordingly, such a noradrenergic cell identity is systematically observed in single-cell RNA-seq of 18 tumor biopsies and 15 PDX models. Yet, a subpopulation of these noradrenergic tumor cells presents with mesenchymal features that are shared with plasticity models, indicating that the plasticity described in these models has relevance in neuroblastoma patients. This work therefore emphasizes that intrinsic plasticity properties of neuroblastoma cells are dependent upon external cues of the environment to drive cell identity.
Assuntos
Plasticidade Celular , Neuroblastoma , Humanos , Neuroblastoma/metabolismo , Linhagem Celular Tumoral , Microambiente Tumoral/genéticaRESUMO
PURPOSE: ALK-activating mutations are identified in approximately 10% of newly diagnosed neuroblastomas and ALK amplifications in a further 1%-2% of cases. Lorlatinib, a third-generation anaplastic lymphoma kinase (ALK) inhibitor, will soon be given alongside induction chemotherapy for children with ALK-aberrant neuroblastoma. However, resistance to single-agent treatment has been reported and therapies that improve the response duration are urgently required. We studied the preclinical combination of lorlatinib with chemotherapy, or with the MDM2 inhibitor, idasanutlin, as recent data have suggested that ALK inhibitor resistance can be overcome through activation of the p53-MDM2 pathway. EXPERIMENTAL DESIGN: We compared different ALK inhibitors in preclinical models prior to evaluating lorlatinib in combination with chemotherapy or idasanutlin. We developed a triple chemotherapy (CAV: cyclophosphamide, doxorubicin, and vincristine) in vivo dosing schedule and applied this to both neuroblastoma genetically engineered mouse models (GEMM) and patient-derived xenografts (PDX). RESULTS: Lorlatinib in combination with chemotherapy was synergistic in immunocompetent neuroblastoma GEMM. Significant growth inhibition in response to lorlatinib was only observed in the ALK-amplified PDX model with high ALK expression. In this PDX, lorlatinib combined with idasanutlin resulted in complete tumor regression and significantly delayed tumor regrowth. CONCLUSIONS: In our preclinical neuroblastoma models, high ALK expression was associated with lorlatinib response alone or in combination with either chemotherapy or idasanutlin. The synergy between MDM2 and ALK inhibition warrants further evaluation of this combination as a potential clinical approach for children with neuroblastoma.
Assuntos
Neoplasias Pulmonares , Neuroblastoma , Camundongos , Animais , Humanos , Quinase do Linfoma Anaplásico/genética , Aminopiridinas/uso terapêutico , Lactamas Macrocíclicas/farmacologia , Lactamas Macrocíclicas/uso terapêutico , Neuroblastoma/tratamento farmacológico , Neuroblastoma/genética , Neuroblastoma/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológicoRESUMO
Pediatric patients with recurrent and refractory cancers are in most need for new treatments. This study developed patient-derived-xenograft (PDX) models within the European MAPPYACTS cancer precision medicine trial (NCT02613962). To date, 131 PDX models were established following heterotopical and/or orthotopical implantation in immunocompromised mice: 76 sarcomas, 25 other solid tumors, 12 central nervous system tumors, 15 acute leukemias, and 3 lymphomas. PDX establishment rate was 43%. Histology, whole exome and RNA sequencing revealed a high concordance with the primary patient's tumor profile, human leukocyte-antigen characteristics and specific metabolic pathway signatures. A detailed patient molecular characterization, including specific mutations prioritized in the clinical molecular tumor boards are provided. Ninety models were shared with the IMI2 ITCC Pediatric Preclinical Proof-of-concept Platform (IMI2 ITCC-P4) for further exploitation. This PDX biobank of unique recurrent childhood cancers provides an essential support for basic and translational research and treatments development in advanced pediatric malignancies.
Assuntos
Leucemia , Neoplasias , Animais , Criança , Humanos , Camundongos , Bancos de Espécimes Biológicos , Modelos Animais de Doenças , Xenoenxertos , Neoplasias/genética , Medicina de Precisão , Ensaios Clínicos como AssuntoRESUMO
Atypical teratoid rhabdoid tumors (ATRT) are divided into MYC, TYR and SHH subgroups, suggesting diverse lineages of origin. Here, we investigate the imaging of human ATRT at diagnosis and the precise anatomic origin of brain tumors in the Rosa26-CreERT2::Smarcb1flox/flox model. This cross-species analysis points to an extra-cerebral origin for MYC tumors. Additionally, we clearly distinguish SHH ATRT emerging from the cerebellar anterior lobe (CAL) from those emerging from the basal ganglia (BG) and intra-ventricular (IV) regions. Molecular characteristics point to the midbrain-hindbrain boundary as the origin of CAL SHH ATRT, and to the ganglionic eminence as the origin of BG/IV SHH ATRT. Single-cell RNA sequencing on SHH ATRT supports these hypotheses. Trajectory analyses suggest that SMARCB1 loss induces a de-differentiation process mediated by repressors of the neuronal program such as REST, ID and the NOTCH pathway.