RESUMO
Polarized vesicular trafficking directs specific receptors and ion channels to cilia, but the underlying mechanisms are poorly understood. Here we describe a role for DLG1, a core component of the Scribble polarity complex, in regulating ciliary protein trafficking in kidney epithelial cells. Conditional knockout of Dlg1 in mouse kidney causes ciliary elongation and cystogenesis, and cell-based proximity labeling proteomics and fluorescence microscopy show alterations in the ciliary proteome upon loss of DLG1. Specifically, the retromer-associated protein SDCCAG3, IFT20, and polycystin-2 (PC2) are reduced in the cilia of DLG1-deficient cells compared to control cells. This phenotype is recapitulated in vivo and rescuable by re-expression of wild-type DLG1, but not a Congenital Anomalies of the Kidney and Urinary Tract (CAKUT)-associated DLG1 variant, p.T489R. Finally, biochemical approaches and Alpha Fold modelling suggest that SDCCAG3 and IFT20 form a complex that associates, at least indirectly, with DLG1. Our work identifies a key role for DLG1 in regulating ciliary protein composition and suggests that ciliary dysfunction of the p.T489R DLG1 variant may contribute to CAKUT.
Assuntos
Proteínas de Transporte , Cílios , Proteína 1 Homóloga a Discs-Large , Canais de Cátion TRPP , Animais , Cílios/metabolismo , Canais de Cátion TRPP/metabolismo , Canais de Cátion TRPP/genética , Camundongos , Proteína 1 Homóloga a Discs-Large/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Transporte/genética , Humanos , Transporte Proteico , Camundongos Knockout , Rim/metabolismo , Células Epiteliais/metabolismo , Ligação Proteica , Refluxo Vesicoureteral/metabolismo , Refluxo Vesicoureteral/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Anormalidades UrogenitaisRESUMO
Linking clinical multi-omics with mechanistic studies may improve the understanding of rare cancers. We leverage two precision oncology programs to investigate rhabdomyosarcoma with FUS/EWSR1-TFCP2 fusions, an orphan malignancy without effective therapies. All tumors exhibit outlier ALK expression, partly accompanied by intragenic deletions and aberrant splicing resulting in ALK variants that are oncogenic and sensitive to ALK inhibitors. Additionally, recurrent CKDN2A/MTAP co-deletions provide a rationale for PRMT5-targeted therapies. Functional studies show that FUS-TFCP2 blocks myogenic differentiation, induces transcription of ALK and truncated TERT, and inhibits DNA repair. Unlike other fusion-driven sarcomas, TFCP2-rearranged tumors exhibit genomic instability and signs of defective homologous recombination. DNA methylation profiling demonstrates a close relationship with undifferentiated sarcomas. In two patients, sarcoma was preceded by benign lesions carrying FUS-TFCP2, indicating stepwise sarcomagenesis. This study illustrates the potential of linking precision oncology with preclinical research to gain insight into the classification, pathogenesis, and therapeutic vulnerabilities of rare cancers.
Assuntos
Sarcoma , Neoplasias de Tecidos Moles , Humanos , Multiômica , Medicina de Precisão , Fatores de Transcrição/genética , Sarcoma/genética , Sarcoma/terapia , Sarcoma/diagnóstico , Proteína EWS de Ligação a RNA/genética , Neoplasias de Tecidos Moles/genética , Neoplasias de Tecidos Moles/terapia , Receptores Proteína Tirosina Quinases , Biomarcadores Tumorais/genética , Proteínas de Fusão Oncogênica/genética , Proteína-Arginina N-Metiltransferases , Proteínas de Ligação a DNA/genéticaRESUMO
Polarized vesicular trafficking directs specific receptors and ion channels to cilia, but the underlying mechanisms are poorly understood. Here we describe a role for DLG1, a core component of the Scribble polarity complex, in regulating ciliary protein trafficking in kidney epithelial cells. Conditional knockout of Dlg1 in mouse kidney caused ciliary elongation and cystogenesis, and cell-based proximity labelling proteomics and fluorescence microscopy showed alterations in the ciliary proteome upon loss of DLG1. Specifically, the retromer-associated protein SDCCAG3, IFT20 and polycystin-2 (PC2) were reduced in cilia of DLG1 deficient cells compared to control cells. This phenotype was recapitulated in vivo and rescuable by re-expression of wildtype DLG1, but not a Congenital Anomalies of the Kidney and Urinary Tract (CAKUT)-associated DLG1 variant, p.T489R. Finally, biochemical approaches and Alpha Fold modelling suggested that SDCCAG3 and IFT20 form a complex that associates, at least indirectly, with DLG1. Our work identifies a key role for DLG1 in regulating ciliary protein composition and suggests that ciliary dysfunction of the p.T489R DLG1 variant may contribute to CAKUT.
RESUMO
While some follicular lymphoma (FL) patients do not require treatment or experience prolonged responses, others relapse early, and little is known about genetic alterations specific to patients with a particular clinical behavior. We selected 56 grade 1-3A FL patients according to their need of treatment or timing of relapse: never treated (n = 7), non-relapsed (19), late relapse (14), early relapse or POD24 (11), and primary refractory (5). We analyzed 56 diagnostic and 12 paired relapse lymphoid tissue biopsies and performed copy number alteration (CNA) analysis and next generation sequencing (NGS). We identified six focal driver losses (1p36.32, 6p21.32, 6q14.1, 6q23.3, 9p21.3, 10q23.33) and 1p36.33 copy-neutral loss of heterozygosity (CN-LOH). By integrating CNA and NGS results, the most frequently altered genes/regions were KMT2D (79%), CREBBP (67%), TNFRSF14 (46%) and BCL2 (40%). Although we found that mutations in PIM1, FOXO1 and TMEM30A were associated with an adverse clinical behavior, definitive conclusions cannot be drawn, due to the small sample size. We identified common precursor cells harboring early oncogenic alterations of the KMT2D, CREBBP, TNFRSF14 and EP300 genes and 16p13.3-p13.2 CN-LOH. Finally, we established the functional consequences of mutations by means of protein modeling (CD79B, PLCG2, PIM1, MCL1 and IRF8). These data expand the knowledge on the genomics behind the heterogeneous FL population and, upon replication in larger cohorts, could contribute to risk stratification and the development of targeted therapies.
Assuntos
Linfoma Folicular , Humanos , Linfoma Folicular/patologia , Recidiva Local de Neoplasia , Mutação , Genômica , RecidivaRESUMO
Histone methylation-modifiers, such as EZH2 and KMT2D, are recurrently altered in B-cell lymphomas. To comprehensively describe the landscape of alterations affecting genes encoding histone methylation-modifiers in lymphomagenesis we investigated whole genome and transcriptome data of 186 mature B-cell lymphomas sequenced in the ICGC MMML-Seq project. Besides confirming common alterations of KMT2D (47% of cases), EZH2 (17%), SETD1B (5%), PRDM9 (4%), KMT2C (4%), and SETD2 (4%), also identified by prior exome or RNA-sequencing studies, we here found recurrent alterations to KDM4C in chromosome 9p24, encoding a histone demethylase. Focal structural variation was the main mechanism of KDM4C alterations, and was independent from 9p24 amplification. We also identified KDM4C alterations in lymphoma cell lines including a focal homozygous deletion in a classical Hodgkin lymphoma cell line. By integrating RNA-sequencing and genome sequencing data we predict that KDM4C structural variants result in loss-offunction. By functional reconstitution studies in cell lines, we provide evidence that KDM4C can act as a tumor suppressor. Thus, we show that identification of structural variants in whole genome sequencing data adds to the comprehensive description of the mutational landscape of lymphomas and, moreover, establish KDM4C as a putative tumor suppressive gene recurrently altered in subsets of B-cell derived lymphomas.
Assuntos
Linfoma de Células B , Linfoma , Humanos , Histonas/metabolismo , Histona Desmetilases/genética , Homozigoto , Deleção de Sequência , Linfoma/genética , Linfoma de Células B/genética , Sequenciamento Completo do Genoma , RNA , Histona Desmetilases com o Domínio Jumonji/genética , Histona Desmetilases com o Domínio Jumonji/química , Histona Desmetilases com o Domínio Jumonji/metabolismo , Histona-Lisina N-Metiltransferase/genéticaRESUMO
Cilia are ubiquitous eukaryotic organelles impotant for cellular motility, signaling, and sensory reception. Cilium formation requires intraflagellar transport of structural and signaling components and involves 22 different proteins organized into intraflagellar transport (IFT) complexes IFT-A and IFT-B that are transported by molecular motors. The IFT-B complex constitutes the backbone of polymeric IFT trains carrying cargo between the cilium and the cell body. Currently, high-resolution structures are only available for smaller IFT-B subcomplexes leaving > 50% structurally uncharacterized. Here, we used Alphafold to structurally model the 15-subunit IFT-B complex. The model was validated using cross-linking/mass-spectrometry data on reconstituted IFT-B complexes, X-ray scattering in solution, diffraction from crystals as well as site-directed mutagenesis and protein-binding assays. The IFT-B structure reveals an elongated and highly flexible complex consistent with cryo-electron tomographic reconstructions of IFT trains. The IFT-B complex organizes into IFT-B1 and IFT-B2 parts with binding sites for ciliary cargo and the inactive IFT dynein motor, respectively. Interestingly, our results are consistent with two different binding sites for IFT81/74 on IFT88/70/52/46 suggesting the possibility of different structural architectures for the IFT-B1 complex. Our data present a structural framework to understand IFT-B complex assembly, function, and ciliopathy variants.
Assuntos
Cílios , Dineínas , Cílios/metabolismo , Dineínas/metabolismo , Transporte Biológico , Sítios de Ligação , Modelos Estruturais , Flagelos/metabolismoRESUMO
Protein S-palmitoylation, the addition of a long-chain fatty acid to target proteins, is among the most frequent reversible protein modifications in Metazoa, affecting subcellular protein localization, trafficking and protein-protein interactions. S-palmitoylated proteins are abundant in the neuronal system and are associated with neuronal diseases and cancer. Despite the importance of this post-translational modification, it has not been thoroughly studied in the model organism Drosophila melanogaster. Here we present the palmitoylome of Drosophila S2R+ cells, comprising 198 proteins, an estimated 3.5% of expressed genes in these cells. Comparison of orthologs between mammals and Drosophila suggests that S-palmitoylated proteins are more conserved between these distant phyla than non-S-palmitoylated proteins. To identify putative client proteins and interaction partners of the DHHC family of protein acyl-transferases (PATs) we established DHHC-BioID, a proximity biotinylation-based method. In S2R+ cells, ectopic expression of the DHHC-PAT dHip14-BioID in combination with Snap24 or an interaction-deficient Snap24-mutant as a negative control, resulted in biotinylation of Snap24 but not the Snap24-mutant. DHHC-BioID in S2R+ cells using 10 different DHHC-PATs as bait identified 520 putative DHHC-PAT interaction partners of which 48 were S-palmitoylated and are therefore putative DHHC-PAT client proteins. Comparison of putative client protein/DHHC-PAT combinations indicates that CG8314, CG5196, CG5880 and Patsas have a preference for transmembrane proteins, while S-palmitoylated proteins with the Hip14-interaction motif are most enriched by DHHC-BioID variants of approximated and dHip14. Finally, we show that BioID is active in larval and adult Drosophila and that dHip14-BioID rescues dHip14 mutant flies, indicating that DHHC-BioID is non-toxic. In summary we provide the first systematic analysis of a Drosophila palmitoylome. We show that DHHC-BioID is sensitive and specific enough to identify DHHC-PAT client proteins and provide DHHC-PAT assignment for ca. 25% of the S2R+ cell palmitoylome, providing a valuable resource. In addition, we establish DHHC-BioID as a useful concept for the identification of tissue-specific DHHC-PAT interactomes in Drosophila.
Assuntos
Aciltransferases , Drosophila melanogaster , Aciltransferases/genética , Animais , Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Lipoilação/fisiologia , Mamíferos/metabolismo , Processamento de Proteína Pós-TraducionalRESUMO
Advances in DNA sequencing and proteomics mean that researchers must now regularly interrogate thousands of positional gene/protein changes in order to find those relevant for potential clinical application or biological insights. The abundance of already known information on protein interactions, mechanism, and tertiary structure provides the possible means to understand these changes rapidly, though a careful and systematic integration of these diverse datasets is first needed. For this purpose, we developed Mechnetor, a tool that allows users to quickly explore and visualize integrated mechanistic data for proteins or interactions of interest. Central to the system is a careful cataloguing of diverse sources of protein interaction mechanism, and an efficient means to visualize interactions between relevant and/or known protein regions. The result is a finer resolution interaction network that provides more immediate clues as to points of intervention or mechanistic understanding. Users can import protein, interactions, genetic variants or post-translational modifications and see these data in the best known mechanistic context. We demonstrate the tool with topical examples in human genetic diseases and cancer genomics. The tool is freely available at: mechnetor.russelllab.org.
Assuntos
Variação Genética , Mapeamento de Interação de Proteínas , Software , Animais , Doenças Genéticas Inatas/genética , Humanos , Internet , Camundongos , Neoplasias/genética , Domínios e Motivos de Interação entre Proteínas , Processamento de Proteína Pós-Traducional , Proteínas/genéticaRESUMO
Cdc42-GTP is required for apical domain formation in epithelial cells, where it recruits and activates the Par-6-aPKC polarity complex, but how the activity of Cdc42 itself is restricted apically is unclear. We used sequence analysis and 3D structural modeling to determine which Drosophila GTPase-activating proteins (GAPs) are likely to interact with Cdc42 and identified RhoGAP19D as the only high-probability Cdc42GAP required for polarity in the follicular epithelium. RhoGAP19D is recruited by α-catenin to lateral E-cadherin adhesion complexes, resulting in exclusion of active Cdc42 from the lateral domain. rhogap19d mutants therefore lead to lateral Cdc42 activity, which expands the apical domain through increased Par-6/aPKC activity and stimulates lateral contractility through the myosin light chain kinase, Genghis khan (MRCK). This causes buckling of the epithelium and invasion into the adjacent tissue, a phenotype resembling that of precancerous breast lesions. Thus, RhoGAP19D couples lateral cadherin adhesion to the apical localization of active Cdc42, thereby suppressing epithelial invasion.
Assuntos
Forma Celular , Proteínas de Drosophila/metabolismo , Células Epiteliais/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster , Células Epiteliais/citologia , Proteínas de Ligação ao GTP/genética , Proteínas Ativadoras de GTPase/genética , Domínios Proteicos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
Background: Considering protein mutations in their biological context is essential for understanding their functional impact, interpretation of high-dimensional datasets and development of effective targeted therapies in personalized medicine. Methods: We combined the curated knowledge of biochemical reactions from Reactome with the analysis of interaction-mediating 3D interfaces from Mechismo. In addition, we provided a software tool for users to explore and browse the analysis results in a multi-scale perspective starting from pathways and reactions to protein-protein interactions and protein 3D structures. Results: We analyzed somatic mutations from TCGA, revealing several significantly impacted reactions and pathways in specific cancer types. We found examples of genes not yet listed as oncodrivers, whose rare mutations were predicted to affect cancer processes similarly to known oncodrivers. Some identified processes lack any known oncodrivers, which suggests potentially new cancer-related processes (e.g. complement cascade reactions). Furthermore, we found that mutations perturbing certain processes are significantly associated with distinct phenotypes (i.e. survival time) in specific cancer types (e.g. PIK3CA centered pathways in LGG and UCEC cancer types), suggesting the translational potential of our approach for patient stratification. Our analysis also uncovered several druggable processes (e.g. GPCR signalling pathways) containing enriched reactions, providing support for new off-label therapeutic options. Conclusions: In summary, we have established a multi-scale approach to study genetic variants based on protein-protein interaction 3D structures. Our approach is different from previously published studies in its focus on biochemical reactions and can be applied to other data types (e.g. post-translational modifications) collected for many types of disease.
Assuntos
Neoplasias , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Mutação , Transdução de Sinais/genética , Proteínas/genética , ProteômicaRESUMO
PURPOSE: Pathogenic variants in neuroblastoma-amplified sequence (NBAS) cause an autosomal recessive disorder with a wide range of symptoms affecting liver, skeletal system, and brain, among others. There is a continuously growing number of patients but a lack of systematic and quantitative analysis. METHODS: Individuals with biallelic variants in NBAS were recruited within an international, multicenter study, including novel and previously published patients. Clinical variables were analyzed with log-linear models and visualized by mosaic plots; facial profiles were investigated via DeepGestalt. The structure of the NBAS protein was predicted using computational methods. RESULTS: One hundred ten individuals from 97 families with biallelic pathogenic NBAS variants were identified, including 26 novel patients with 19 previously unreported variants, giving a total number of 86 variants. Protein modeling redefined the ß-propeller domain of NBAS. Based on the localization of missense variants and in-frame deletions, three clinical subgroups arise that differ significantly regarding main clinical features and are directly related to the affected region of the NBAS protein: ß-propeller (combined phenotype), Sec39 (infantile liver failure syndrome type 2/ILFS2), and C-terminal (short stature, optic atrophy, and Pelger-Huët anomaly/SOPH). CONCLUSION: We define clinical subgroups of NBAS-associated disease that can guide patient management and point to domain-specific functions of NBAS.
Assuntos
Doenças Genéticas Inatas/genética , Predisposição Genética para Doença , Proteínas de Neoplasias/genética , Alelos , Encéfalo/patologia , Criança , Pré-Escolar , Feminino , Doenças Genéticas Inatas/patologia , Humanos , Lactente , Fígado/patologia , Transplante de Fígado/efeitos adversos , Masculino , Músculo Esquelético/patologia , Mutação de Sentido Incorreto/genética , FenótipoRESUMO
MYC is the most altered oncogene in human cancer, and belongs to a large family of genes, including MYCN and MYCL. Recently, while assessing the degree of correlation between MYC gene rearrangement and MYC protein expression in aggressive B-cell lymphomas, we observed few Burkitt lymphoma (BL) cases lacking MYC protein expression despite the translocation involving the MYC gene. Therefore, in the present study we aimed to better characterize such cases. Our results identified two sub-groups of MYC protein negative BL: one lacking detectable MYC protein expression but presenting MYCN mRNA and protein expression; the second characterized by the lack of both MYC and MYCN proteins but showing MYC mRNA. Interestingly, the two sub-groups presented a different pattern of SNVs affecting MYC gene family members that may induce the switch from MYC to MYCN. Particulary, MYCN-expressing cases show MYCN SNVs at interaction interface that stabilize the protein associated with loss-of-function of MYC. This finding highlights MYCN as a reliable diagnostic marker in such cases. Nevertheless, due to the overlapping clinic, morphology and immunohistochemistry (apart for MYC versus MYCN protein expression) of both sub-groups, the described cases represent bona fide BL according to the current criteria of the World Health Organization.
Assuntos
Linfoma de Burkitt/genética , Linfoma de Burkitt/metabolismo , Regulação Neoplásica da Expressão Gênica , Genes de Troca , Genes myc , Adolescente , Adulto , Idoso , Linfoma de Burkitt/epidemiologia , Linfoma de Burkitt/patologia , Criança , Feminino , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imuno-Histoquímica , Imunofenotipagem , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Mutação , Conformação Proteica , RNA Mensageiro/genética , Relação Estrutura-Atividade , Translocação Genética , Adulto JovemRESUMO
Oncodriver genes are usually identified when mutations recur in multiple tumours. Different drivers often converge in the activation or repression of key cancer-relevant pathways. However, as many pathways contain multiple members of the same gene family, individual mutations might be overlooked, as each family member would necessarily have a lower mutation frequency and thus not identified as significant in any one-gene-at-a-time analysis. Here, we looked for mutated, functional sequence positions in gene families that were mutually exclusive (in patients) with another gene in the same pathway, which identified both known and new candidate oncodrivers. For instance, many inactivating mutations in multiple G-protein (particularly Gi/o) coupled receptors, are mutually exclusive with Gαs oncogenic activating mutations, both of which ultimately enhance cAMP signalling. By integrating transcriptomics and interaction data, we show that the Gs pathway is upregulated in multiple cancer types, even those lacking known GNAS activating mutations. This suggests that cancer cells may develop alternative strategies to activate adenylate cyclase signalling in multiple cancer types. Our study provides a mechanistic interpretation for several rare somatic mutations in multi-gene oncodrivers, and offers possible explanations for known and potential off-label cancer treatments, suggesting new therapeutic opportunities.
Assuntos
Mutação , Neoplasias/genética , Oncogenes/genética , Receptores Acoplados a Proteínas G/genética , Cromograninas/genética , Biologia Computacional , Epistasia Genética , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Frequência do Gene , Redes Reguladoras de Genes/fisiologia , Genes Supressores de Tumor , Células HEK293 , Humanos , Modelos Moleculares , Família Multigênica/genética , Mutação/fisiologia , Neoplasias/mortalidade , Neoplasias/patologia , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/genética , Análise de Sobrevida , Fatores de Transcrição/genéticaRESUMO
Heterotrimetic G proteins consist of four subfamilies (Gs, Gi/o, Gq/11, and G12/13) that mediate signaling via G-protein-coupled receptors (GPCRs), principally by receptors binding Gα C termini. G-protein-coupling profiles govern GPCR-induced cellular responses, yet receptor sequence selectivity determinants remain elusive. Here, we systematically quantified ligand-induced interactions between 148 GPCRs and all 11 unique Gα subunit C termini. For each receptor, we probed chimeric Gα subunit activation via a transforming growth factor-α (TGF-α) shedding response in HEK293 cells lacking endogenous Gq/11 and G12/13 proteins, and complemented G-protein-coupling profiles through a NanoBiT-G-protein dissociation assay. Interrogation of the dataset identified sequence-based coupling specificity features, inside and outside the transmembrane domain, which we used to develop a coupling predictor that outperforms previous methods. We used the predictor to engineer designer GPCRs selectively coupled to G12. This dataset of fine-tuned signaling mechanisms for diverse GPCRs is a valuable resource for research in GPCR signaling.
Assuntos
Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Modelos Biológicos , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Feminino , Células HEK293 , Proteínas Heterotriméricas de Ligação ao GTP/genética , Humanos , Masculino , Células PC-3 , Receptores Acoplados a Proteínas G/genéticaRESUMO
G protein-coupled receptors (GPCRs) are the largest gene family of cell membrane-associated molecules mediating signal transmission, and their involvement in key physiological functions is well-established. The ability of GPCRs to regulate a vast array of fundamental biological processes, such as cardiovascular functions, immune responses, hormone and enzyme release from endocrine and exocrine glands, neurotransmission, and sensory perception (e.g. vision, odor, and taste), is largely due to the diversity of these receptors and the layers of their downstream signaling circuits. Dysregulated expression and aberrant functions of GPCRs have been linked to some of the most prevalent human diseases, which renders GPCRs one of the top targets for pharmaceutical drug development. However, the study of the role of GPCRs in tumor biology has only just begun to make headway. Recent studies have shown that GPCRs can contribute to the many facets of tumorigenesis, including proliferation, survival, angiogenesis, invasion, metastasis, therapy resistance, and immune evasion. Indeed, GPCRs are widely dysregulated in cancer and yet are underexploited in oncology. We present here a comprehensive analysis of GPCR gene expression, copy number variation, and mutational signatures in 33 cancer types. We also highlight the emerging role of GPCRs as part of oncocrine networks promoting tumor growth, dissemination, and immune evasion, and we stress the potential benefits of targeting GPCRs and their signaling circuits in the new era of precision medicine and cancer immunotherapies.
Assuntos
Imunoterapia , Neoplasias/terapia , Receptores Acoplados a Proteínas G/metabolismo , Animais , Variações do Número de Cópias de DNA , Humanos , Mutação , Neoplasias/genética , Neoplasias/fisiopatologia , Transdução de SinaisRESUMO
Chordomas are rare bone tumors with few therapeutic options. Here we show, using whole-exome and genome sequencing within a precision oncology program, that advanced chordomas (n = 11) may be characterized by genomic patterns indicative of defective homologous recombination (HR) DNA repair and alterations affecting HR-related genes, including, for example, deletions and pathogenic germline variants of BRCA2, NBN, and CHEK2. A mutational signature associated with HR deficiency was significantly enriched in 72.7% of samples and co-occurred with genomic instability. The poly(ADP-ribose) polymerase (PARP) inhibitor olaparib, which is preferentially toxic to HR-incompetent cells, led to prolonged clinical benefit in a patient with refractory chordoma, and whole-genome analysis at progression revealed a PARP1 p.T910A mutation predicted to disrupt the autoinhibitory PARP1 helical domain. These findings uncover a therapeutic opportunity in chordoma that warrants further exploration, and provide insight into the mechanisms underlying PARP inhibitor resistance.
Assuntos
Cordoma/tratamento farmacológico , Ftalazinas/uso terapêutico , Piperazinas/uso terapêutico , Poli(ADP-Ribose) Polimerase-1/genética , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Reparo de DNA por Recombinação/genética , Adulto , Idoso , Cordoma/genética , Cordoma/patologia , Mapeamento Cromossômico , Quebras de DNA de Cadeia Dupla , Análise Mutacional de DNA , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Instabilidade Genômica , Humanos , Masculino , Pessoa de Meia-Idade , Ftalazinas/farmacologia , Piperazinas/farmacologia , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Medicina de Precisão/métodos , Domínios Proteicos/genética , Resultado do Tratamento , Sequenciamento do ExomaRESUMO
BACKGROUND: Posterior fossa A (PFA) ependymomas are one of 9 molecular groups of ependymoma. PFA tumors are mainly diagnosed in infants and young children, show a poor prognosis, and are characterized by a lack of the repressive histone H3 lysine 27 trimethylation (H3K27me3) mark. Recently, we reported overexpression of chromosome X open reading frame 67 (CXorf67) as a hallmark of PFA ependymoma and showed that CXorf67 can interact with enhancer of zeste homolog 2 (EZH2), thereby inhibiting polycomb repressive complex 2 (PRC2), but the mechanism of action remained unclear. METHODS: We performed mass spectrometry and peptide modeling analyses to identify the functional domain of CXorf67 responsible for binding and inhibition of EZH2. Our findings were validated by immunocytochemistry, western blot, and methyltransferase assays. RESULTS: We find that the inhibitory mechanism of CXorf67 is similar to diffuse midline gliomas harboring H3K27M mutations. A small, highly conserved peptide sequence located in the C-terminal region of CXorf67 mimics the sequence of K27M mutated histones and binds to the SET domain (Su(var)3-9/enhancer-of-zeste/trithorax) of EZH2. This interaction blocks EZH2 methyltransferase activity and inhibits PRC2 function, causing de-repression of PRC2 target genes, including genes involved in neurodevelopment. CONCLUSIONS: Expression of CXorf67 is an oncogenic mechanism that drives H3K27 hypomethylation in PFA tumors by mimicking K27M mutated histones. Disrupting the interaction between CXorf67 and EZH2 may serve as a novel targeted therapy for PFA tumors but also for other tumors that overexpress CXorf67. Based on its function, we have renamed CXorf67 as "EZH Inhibitory Protein" (EZHIP).
Assuntos
Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Ependimoma/patologia , Histonas/genética , Neoplasias Infratentoriais/patologia , Mutação , Proteínas Oncogênicas/metabolismo , Complexo Repressor Polycomb 2/antagonistas & inibidores , Carcinogênese , Metilação de DNA , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Ependimoma/genética , Ependimoma/metabolismo , Humanos , Neoplasias Infratentoriais/genética , Neoplasias Infratentoriais/metabolismo , Proteínas Oncogênicas/genética , Complexo Repressor Polycomb 2/metabolismoRESUMO
Burkitt lymphoma (BL) is the most common B-cell lymphoma in children. Within the International Cancer Genome Consortium (ICGC), we performed whole genome and transcriptome sequencing of 39 sporadic BL. Here, we unravel interaction of structural, mutational, and transcriptional changes, which contribute to MYC oncogene dysregulation together with the pathognomonic IG-MYC translocation. Moreover, by mapping IGH translocation breakpoints, we provide evidence that the precursor of at least a subset of BL is a B-cell poised to express IGHA. We describe the landscape of mutations, structural variants, and mutational processes, and identified a series of driver genes in the pathogenesis of BL, which can be targeted by various mechanisms, including IG-non MYC translocations, germline and somatic mutations, fusion transcripts, and alternative splicing.
Assuntos
Linfoma de Burkitt/genética , Genoma Humano , Transcriptoma/genética , Adolescente , Processamento Alternativo/genética , Sequência de Aminoácidos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Criança , Pré-Escolar , Pontos de Quebra do Cromossomo , Estudos de Coortes , Metilação de DNA/genética , Análise Mutacional de DNA , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Mutação INDEL/genética , Masculino , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Fases de Leitura Aberta/genética , Polimorfismo de Nucleotídeo Único/genética , Proteínas Proto-Oncogênicas c-myc/genética , Translocação Genética , Sequenciamento Completo do GenomaRESUMO
The new recently described provisional lymphoma category Burkitt-like lymphoma with 11q aberration comprises cases similar to Burkitt lymphoma (BL) on morphological, immunophenotypic and gene-expression levels but lacking the IG-MYC translocation. They are characterized by a peculiar imbalance pattern on chromosome 11, but the landscape of mutations is not yet described. Thus, we investigated 15 MYC-negative Burkitt-like lymphoma with 11q aberration (mnBLL,11q,) cases by copy-number analysis and whole-exome sequencing. We refined the regions of 11q imbalance and identified the INO80 complex-associated gene NFRKB as a positional candidate in 11q24.3. Next to recurrent gains in 12q13.11-q24.32 and 7q34-qter as well as losses in 13q32.3-q34, we identified 47 genes recurrently affected by protein-changing mutations (each ≥3 of 15 cases). Strikingly, we did not detect recurrent mutations in genes of the ID3-TCF3 axis or the SWI/SNF complex that are frequently altered in BL, or in genes frequently mutated in germinal center-derived B-cell lymphomas like KMT2D or CREBBP An exception is GNA13, which was mutated in 7 of 15 cases. We conclude that the genomic landscape of mnBLL,11q, differs from that of BL both at the chromosomal and mutational levels. Our findings implicate that mnBLL,11q, is a lymphoma category distinct from BL at the molecular level.
Assuntos
Biomarcadores Tumorais/genética , Linfoma de Burkitt/classificação , Linfoma de Burkitt/genética , Aberrações Cromossômicas , Cromossomos Humanos Par 11/genética , Mutação , ATPases Associadas a Diversas Atividades Celulares , Adolescente , Adulto , Linfoma de Burkitt/patologia , Criança , Pré-Escolar , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Proteínas Proto-Oncogênicas c-myc/genética , Estudos Retrospectivos , Adulto JovemRESUMO
Increasingly available genomic sequencing data are exploited to identify genes and variants contributing to diseases, particularly cancer. Traditionally, methods to find such variants have relied heavily on allele frequency and/or familial history, often neglecting to consider any mechanistic understanding of their functional consequences. Thus, while the set of known cancer-related genes has increased, for many, their mechanistic role in the disease is not completely understood. This issue highlights a wide gap between the disciplines of genetics, which largely aims to correlate genetic events with phenotype, and molecular biology, which ultimately aims at a mechanistic understanding of biological processes. Fortunately, new methods and several systematic studies have proved illuminating for many disease genes and variants by integrating sequencing with mechanistic data, including biomolecular structures and interactions. These have provided new interpretations for known mutations and suggested new disease-relevant variants and genes. Here, we review these approaches and discuss particular examples where these have had a profound impact on the understanding of human cancers.