RESUMO
Mutations in the RNA splicing factor gene SF3B1 are common across hematologic and solid cancers and result in widespread alterations in splicing, yet there is currently no therapeutic means to correct this mis-splicing. Here, we utilize synthetic introns uniquely responsive to mutant SF3B1 to identify trans factors required for aberrant mutant SF3B1 splicing activity. This revealed the G-patch domain-containing protein GPATCH8 as required for mutant SF3B1-induced splicing alterations and impaired hematopoiesis. GPATCH8 is involved in quality control of branchpoint selection, interacts with the RNA helicase DHX15, and functionally opposes SURP and G-patch domain containing 1 (SUGP1), a G-patch protein recently implicated in SF3B1-mutant diseases. Silencing of GPATCH8 corrected one-third of mutant SF3B1-dependent splicing defects and was sufficient to improve dysfunctional hematopoiesis in SF3B1-mutant mice and primary human progenitors. These data identify GPATCH8 as a novel splicing factor required for mis-splicing by mutant SF3B1 and highlight the therapeutic impact of correcting aberrant splicing in SF3B1-mutant cancers.
Assuntos
Neoplasias Hematológicas , Mutação , Fosfoproteínas , Fatores de Processamento de RNA , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , Humanos , Animais , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/patologia , Neoplasias Hematológicas/metabolismo , Camundongos , Splicing de RNA , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Hematopoese/genética , Células HEK293 , Íntrons , RNA Helicases/genética , RNA Helicases/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
Transcription and splicing of pre-messenger RNA are closely coordinated, but how this functional coupling is disrupted in human diseases remains unexplored. Using isogenic cell lines, patient samples, and a mutant mouse model, we investigated how cancer-associated mutations in SF3B1 alter transcription. We found that these mutations reduce the elongation rate of RNA polymerase II (RNAPII) along gene bodies and its density at promoters. The elongation defect results from disrupted pre-spliceosome assembly due to impaired protein-protein interactions of mutant SF3B1. The decreased promoter-proximal RNAPII density reduces both chromatin accessibility and H3K4me3 marks at promoters. Through an unbiased screen, we identified epigenetic factors in the Sin3/HDAC/H3K4me pathway, which, when modulated, reverse both transcription and chromatin changes. Our findings reveal how splicing factor mutant states behave functionally as epigenetic disorders through impaired transcription-related changes to the chromatin landscape. We also present a rationale for targeting the Sin3/HDAC complex as a therapeutic strategy.
Assuntos
Cromatina , Neoplasias , Animais , Humanos , Camundongos , Cromatina/genética , Mutação , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Splicing de RNA/genética , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismoRESUMO
SF3B1 is the most mutated splicing factor (SF) in myelodysplastic syndromes (MDSs), which are clonal hematopoietic disorders with variable risk of leukemic transformation. Although tumorigenic SF3B1 mutations have been extensively characterized, the role of "non-mutated" wild-type SF3B1 in cancer remains largely unresolved. Here, we identify a conserved epitranscriptomic program that steers SF3B1 levels to counteract leukemogenesis. Our analysis of human and murine pre-leukemic MDS cells reveals dynamic regulation of SF3B1 protein abundance, which affects MDS-to-leukemia progression in vivo. Mechanistically, ALKBH5-driven 5' UTR m6A demethylation fine-tunes SF3B1 translation directing splicing of central DNA repair and epigenetic regulators during transformation. This impacts genome stability and leukemia progression in vivo, supporting an integrative analysis in humans that SF3B1 molecular signatures may predict mutational variability and poor prognosis. These findings highlight a post-transcriptional gene expression nexus that unveils unanticipated SF3B1-dependent cancer vulnerabilities.
Assuntos
Leucemia , Síndromes Mielodisplásicas , Fosfoproteínas , Fatores de Processamento de RNA , Animais , Humanos , Camundongos , Carcinogênese/genética , Leucemia/genética , Mutação , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Splicing de RNA/genética , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismoRESUMO
Benbarche, Pineda, Galvis, et al. delineate an essential role for the G-patch motif-containing protein GPATCH8 in mis-splicing associated with cancer-driving mutations of the splicing factor SF3B1. GPATCH8 cooperates with SF3B1 mutants, affecting the splicing machinery. Targeting GPATCH8 reveals therapeutic opportunities for SF3B1 mutant cancers and other splicing-related diseases.
Assuntos
Neoplasias , Fatores de Processamento de RNA , Splicing de RNA , Humanos , Mutação , Neoplasias/genética , Neoplasias/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fatores de Processamento de RNA/metabolismo , Fatores de Processamento de RNA/genéticaRESUMO
CDK7 associates with the 10-subunit TFIIH complex and regulates transcription by phosphorylating the C-terminal domain (CTD) of RNA polymerase II (RNAPII). Few additional CDK7 substrates are known. Here, using the covalent inhibitor SY-351 and quantitative phosphoproteomics, we identified CDK7 kinase substrates in human cells. Among hundreds of high-confidence targets, the vast majority are unique to CDK7 (i.e., distinct from other transcription-associated kinases), with a subset that suggest novel cellular functions. Transcription-associated factors were predominant CDK7 substrates, including SF3B1, U2AF2, and other splicing components. Accordingly, widespread and diverse splicing defects, such as alternative exon inclusion and intron retention, were characterized in CDK7-inhibited cells. Combined with biochemical assays, we establish that CDK7 directly activates other transcription-associated kinases CDK9, CDK12, and CDK13, invoking a "master regulator" role in transcription. We further demonstrate that TFIIH restricts CDK7 kinase function to the RNAPII CTD, whereas other substrates (e.g., SPT5 and SF3B1) are phosphorylated by the three-subunit CDK-activating kinase (CAK; CCNH, MAT1, and CDK7). These results suggest new models for CDK7 function in transcription and implicate CAK dissociation from TFIIH as essential for kinase activation. This straightforward regulatory strategy ensures CDK7 activation is spatially and temporally linked to transcription, and may apply toward other transcription-associated kinases.
Assuntos
Quinases Ciclina-Dependentes/metabolismo , Modelos Biológicos , Fator de Transcrição TFIIH/metabolismo , Transcrição Gênica/genética , Processamento Alternativo/genética , Sobrevivência Celular/efeitos dos fármacos , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/genética , Ativação Enzimática/genética , Células HL-60 , Humanos , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
Intron branchpoint (BP) recognition by the U2 snRNP is a critical step of splicing, vulnerable to recurrent cancer mutations and bacterial natural product inhibitors. The BP binds a conserved pocket in the SF3B1 (human) or Hsh155 (yeast) U2 snRNP protein. Amino acids that line this pocket affect the binding of splicing inhibitors like Pladienolide-B (Plad-B), such that organisms differ in their sensitivity. To study the mechanism of splicing inhibitor action in a simplified system, we modified the naturally Plad-B resistant yeast Saccharomyces cerevisiae by changing 14 amino acids in the Hsh155 BP pocket to those from human. This humanized yeast grows normally, and splicing is largely unaffected by the mutation. Splicing is inhibited within minutes after the addition of Plad-B, and different introns appear inhibited to different extents. Intron-specific inhibition differences are also observed during cotranscriptional splicing in Plad-B using single-molecule intron tracking to minimize gene-specific transcription and decay rates that cloud estimates of inhibition by standard RNA-seq. Comparison of Plad-B intron sensitivities to those of the structurally distinct inhibitor Thailanstatin-A reveals intron-specific differences in sensitivity to different compounds. This work exposes a complex relationship between the binding of different members of this class of inhibitors to the spliceosome and intron-specific rates of BP recognition and catalysis. Introns with variant BP sequences seem particularly sensitive, echoing observations from mammalian cells, where monitoring individual introns is complicated by multi-intron gene architecture and alternative splicing. The compact yeast system may hasten the characterization of splicing inhibitors, accelerating improvements in selectivity and therapeutic efficacy.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Humanos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Íntrons/genética , Ribonucleoproteína Nuclear Pequena U2/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Splicing de RNA , Spliceossomos/genética , Aminoácidos/genética , Precursores de RNA/genéticaRESUMO
Many eukaryotic genes generate linear mRNAs and circular RNAs, but it is largely unknown how the ratio of linear to circular RNA is controlled or modulated. Using RNAi screening in Drosophila cells, we identify many core spliceosome and transcription termination factors that control the RNA outputs of reporter and endogenous genes. When spliceosome components were depleted or inhibited pharmacologically, the steady-state levels of circular RNAs increased while expression of their associated linear mRNAs concomitantly decreased. Upon inhibiting RNA polymerase II termination via depletion of the cleavage/polyadenylation machinery, circular RNA levels were similarly increased. This is because readthrough transcripts now extend into downstream genes and are subjected to backsplicing. In total, these results demonstrate that inhibition or slowing of canonical pre-mRNA processing events shifts the steady-state output of protein-coding genes toward circular RNAs. This is in part because nascent RNAs become directed into alternative pathways that lead to circular RNA production.
Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Precursores de RNA/biossíntese , Splicing de RNA , RNA Mensageiro/biossíntese , RNA/biossíntese , Spliceossomos/genética , Transcrição Gênica , Animais , Linhagem Celular , Proteínas de Drosophila/biossíntese , Drosophila melanogaster/metabolismo , Lacase/biossíntese , Lacase/genética , RNA/genética , Interferência de RNA , RNA Polimerase II/metabolismo , Precursores de RNA/genética , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , Estabilidade de RNA , RNA Circular , RNA Mensageiro/genética , Ribonucleoproteínas Nucleolares Pequenas/genética , Ribonucleoproteínas Nucleolares Pequenas/metabolismo , Spliceossomos/metabolismo , Terminação da Transcrição Genética , TransfecçãoRESUMO
Among 210 patients with myelodysplastic syndromes (MDSs) with del(5q), molecular information was available at diagnosis or at least 3 months before leukaemic transformation in 146 cases. Multivariate analysis identified therapy-related setting (p = 0.02; HR 2.3) and TP53 variant allele frequency (VAF) ≥22% (p < 0.01; HR 2.8), but not SF3B1 mutation (p = 0.65), as independent risk factors for survival. Median survival was 11.7 versus 4 years (5/10-year survival 73%/52% vs. 42%/14%) in the absence (N = 112) versus presence (N = 34) of ≥1 risk factors; leukaemia-free survival was affected by TP53 VAF ≥22% (p < 0.01). Such information might inform treatment decision-making in MDS-del(5q) regarding allogeneic stem cell transplant.
Assuntos
Síndromes Mielodisplásicas , Humanos , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/terapia , Síndromes Mielodisplásicas/diagnóstico , Frequência do Gene , Mutação , Prognóstico , Deleção Cromossômica , Cromossomos Humanos Par 5/genética , Proteína Supressora de Tumor p53/genéticaRESUMO
The criteria of myelodysplastic syndromes (MDS) with mutated SFB31 (MDS-SFB31) proposed by the 5th edition of the WHO classification (WHO 2022) and the International Consensus Classification (ICC) need validation. We analysed 125 consecutive MDS cases with SFB31 mutation or ring sideroblasts (RS) ≥15% without excess blasts. We found that SFB31-negative MDS with RS had significantly different clinical features and worse prognosis. According to WHO 2022, the detection of ≥15% RS may substitute for SF3B1 mutation and our analyses support this proposal for similar prognosis of two groups after excluding high-risk genetic features referred by WHO 2022. Patients with variant allele frequency (VAF) <10% SFB31 tend to have briefer survival, supporting the VAF 10% threshold of ICC. Patients with multilineage dysplasia (MLD) had significantly shorter OS than those with single lineage dysplasia. MLD is still a powerful morphological marker of worse outcome in WHO 2022 and ICC-defined MDS-SF3B1.
RESUMO
SF3b is a heptameric protein complex of the U2 small nuclear ribonucleoprotein (snRNP) that is essential for pre-mRNA splicing. Mutations in the largest SF3b subunit, SF3B1/SF3b155, are linked to cancer and lead to alternative branch site (BS) selection. Here we report the crystal structure of a human SF3b core complex, revealing how the distinctive conformation of SF3b155's HEAT domain is maintained by multiple contacts with SF3b130, SF3b10, and SF3b14b. Protein-protein crosslinking enabled the localization of the BS-binding proteins p14 and U2AF65 within SF3b155's HEAT-repeat superhelix, which together with SF3b14b forms a composite RNA-binding platform. SF3b155 residues, the mutation of which leads to cancer, contribute to the tertiary structure of the HEAT superhelix and its surface properties in the proximity of p14 and U2AF65. The molecular architecture of SF3b reveals the spatial organization of cancer-related SF3b155 mutations and advances our understanding of their effects on SF3b structure and function.
Assuntos
Mutação , Proteínas de Neoplasias/química , Proteínas Oncogênicas/química , Fosfoproteínas/química , Fatores de Processamento de RNA/química , Spliceossomos/química , Fator de Processamento U2AF/química , Sequência de Aminoácidos , Animais , Baculoviridae/genética , Baculoviridae/metabolismo , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , Expressão Gênica , Genes Supressores de Tumor , Células HeLa , Humanos , Modelos Moleculares , Mariposas , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Splicing de RNA , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Spliceossomos/metabolismo , Spliceossomos/ultraestrutura , Fator de Processamento U2AF/genética , Fator de Processamento U2AF/metabolismoRESUMO
Genomic analyses of the myeloid malignancies and clonal disorders of hematopoiesis that may give rise to these disorders have identified that mutations in genes encoding core spliceosomal proteins and accessory regulatory splicing factors are among the most common targets of somatic mutations. These spliceosomal mutations often occur in a mutually exclusive manner with one another and, in aggregate, account for the most frequent class of mutations in patients with myelodysplastic syndromes (MDSs) in particular. Although substantial progress has been made in understanding the effects of several of these mutations on splicing and splice site recognition, functional connections linking the mechanistic changes in splicing induced by these mutations to the phenotypic consequences of clonal and aberrant hematopoiesis are not yet well defined. This review describes our current understanding of the mechanistic and biological effects of spliceosomal gene mutations in MDSs as well as the regulation of splicing throughout normal hematopoiesis.
Assuntos
Hematopoese/genética , Síndromes Mielodisplásicas/genética , Spliceossomos/genética , Animais , Epigênese Genética , Humanos , Modelos Animais , Mutação/genética , Splicing de RNARESUMO
Mutations in the U2 snRNP component SF3B1 are prominent in myelodysplastic syndromes (MDSs) and other cancers and have been shown recently to alter branch site (BS) or 3' splice site selection in splicing. However, the molecular mechanism of altered splicing is not known. We show here that hsh155 mutant alleles in Saccharomyces cerevisiae, counterparts of SF3B1 mutations frequently found in cancers, specifically change splicing of suboptimal BS pre-mRNA substrates. We found that Hsh155p interacts directly with Prp5p, the first ATPase that acts during spliceosome assembly, and localized the interacting regions to HEAT (Huntingtin, EF3, PP2A, and TOR1) motifs in SF3B1 associated with disease mutations. Furthermore, we show that mutations in these motifs from both human disease and yeast genetic screens alter the physical interaction with Prp5p, alter branch region specification, and phenocopy mutations in Prp5p. These and other data demonstrate that mutations in Hsh155p and Prp5p alter splicing because they change the direct physical interaction between Hsh155p and Prp5p. This altered physical interaction results in altered loading (i.e., "fidelity") of the BS-U2 duplex into the SF3B complex during prespliceosome formation. These results provide a mechanistic framework to explain the consequences of intron recognition and splicing of SF3B1 mutations found in disease.
Assuntos
RNA Helicases DEAD-box/metabolismo , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , Ribonucleoproteína Nuclear Pequena U2/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Motivos de Aminoácidos/genética , RNA Helicases DEAD-box/genética , Humanos , Íntrons/genética , Mutação , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Ligação Proteica/genética , Precursores de RNA/metabolismo , Splicing de RNA/genética , Ribonucleoproteína Nuclear Pequena U2/genética , Proteínas de Saccharomyces cerevisiae/genética , Spliceossomos/genéticaRESUMO
The discovery of new genes/pathways improves our knowledge of cancer pathogenesis and presents novel potential therapeutic options. For instance, splicing factor 3b subunit 1 (SF3B1) and NOTCH1 genetic alterations have been identified at a high frequency in hematological malignancies, such as leukemia, and may be related to the prognosis of involved patients because they change the nature of malignancies in different ways like mediating therapeutic resistance; therefore, studying these gene/pathways is essential. This review aims to discuss SF3B1 and NOTCH1 roles in the pathogenesis of various types of leukemia and the therapeutic potential of targeting these genes or their mutations to provide a foundation for leukemia treatment.
Assuntos
Leucemia , Fatores de Transcrição , Humanos , Leucemia/fisiopatologia , Mutação , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Clonal hematopoiesis (CH) consists in an abnormal expansion of a hematopoietic stem cell bearing an advantageous somatic variant. A survey of known recurrent somatic missense variants in DNMT3A, SF3B1, SRSF2, and TP53, some of the most prominent genes underlying CH of indeterminate potential (CHIP), in gnomAD noncancer database shows the presence of 73 variants. Many of them reach frequencies higher than 0.01% in various populations and, in many cases, are enriched in specific populations. Consistent with a potential involvement in CHIP, we found that the age distribution of the carriers is shifted towards old ages. Moreover, the variant allele frequencies are on average lower than 50%, expected for germline heterozygous variants. The pervasive presence of some of such variants in blood DNA from elder individuals is compatible with CHIP of somatic origin. On practical grounds, CHIP can lead to misclassification of somatic variants in cancer-predisposition genes as inherited, which bear consequences for the affected individuals and their families.
Assuntos
Hematopoiese Clonal , Hematopoese , Humanos , Idoso , Mutação , Hematopoese/genética , Células-Tronco Hematopoéticas , Mutação em Linhagem GerminativaRESUMO
Myelodysplastic syndromes (MDS) are acquired bone marrow malignant disorders characterized by ineffective hematopoiesis, resulting from a complex interaction between genetic and epigenetic mutations, alterations of the marrow microenvironment, and the immune system. In 2001, the World Health Organization (WHO) proposed a classification that integrates morphologic and genetic information, considering the MDS with ring sideroblasts (MDS-RS) as a distinct entity. Considering the strong association between MDS-RS and SF3B1 mutation and its importance in the development of MDS, the last WHO classification replaced the prior entity of MDS-RS with MDS with SF3B1 mutation. Several studies were performed to explore this genotype-phenotype correlation. Mutant SF3B1 protein deregulates the expression of genes implicated in developing hematopoietic stem and progenitor cells. Of paramount importance are PPOX and ABCB7 involved in iron metabolism. Another essential role in hemopoiesis is played by the transforming growth factor-beta (TGF-ß) receptor. This gene exerts its effects on SMAD pathways, regulating hematopoiesis through effects on balancing proliferation and apoptosis cell inactivity, differentiation, and migration. Luspatercept (ACE-536) is a soluble fusion protein that inhibits molecules in the TGF-ß superfamily. Since its structure resembles the TGF-ß family receptor, it catches TGF-ß superfamily ligands before binding to the receptor, resulting in reduced activation of SMAD signaling, thus enabling erythroid maturation. Luspatercept was investigated in the phase III trial MEDALIST, showing promising efficacy in treating anemia compared to placebo. Nowadays, further studies are needed to explore the real potential of luspatercept, investigating the biological features likely associated with treatment response, the potential use in combination treatments, and its role in the treatment of naïve MDS.
Assuntos
Anemia , Síndromes Mielodisplásicas , Humanos , Fatores de Processamento de RNA/genética , Síndromes Mielodisplásicas/tratamento farmacológico , Medula Óssea/patologia , Mutação , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/uso terapêutico , Fosfoproteínas/genética , Fosfoproteínas/uso terapêutico , Flavoproteínas/genética , Flavoproteínas/uso terapêutico , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/uso terapêutico , Protoporfirinogênio Oxidase/genéticaRESUMO
Patients with myelodysplastic syndromes and ring sideroblasts (MDS RS) are clinically characterized by severe anemia and transfusion need. Erythropoiesis-stimulating agents (ESAs), which stimulate hemoglobin production and early maturation of erythroid precursors, are effective only in a portion of patients and for limited time. Luspatercept, an inhibitor of the TGF-beta pathway, is beneficial in unblocking late-stage erythropoiesis and has been approved for MDS RS patients failing or not-candidate to ESAs. ESAs and/or luspatercept failure represents an unmet clinical need and most patients become life-long transfusion dependent. Here, we describe the clinical combination of luspatercept with ESAs (subcutaneous epoetin alpha 40-80 000 IU/week) in seven MDS RS patients. Two patients had ESAs as pre-existing therapy, while five were re-challenged with ESAs as add-on treatment due to luspatercept failure. Three patients achieved hematologic improvement, and one became transfusion independent. No adverse events were noted. This is the first clinical evidence that stimulating both early and late-stage erythropoiesis may offer a further option for this challenging patient population.
Assuntos
Eritropoetina , Síndromes Mielodisplásicas , Humanos , Eritropoese , Síndromes Mielodisplásicas/complicações , Síndromes Mielodisplásicas/diagnóstico , Síndromes Mielodisplásicas/tratamento farmacológico , Proteínas Recombinantes de Fusão/uso terapêutico , Proteínas Recombinantes/uso terapêutico , Eritropoetina/uso terapêuticoRESUMO
Precursor mRNA (pre-mRNA) splicing is an essential step in human gene expression and is carried out by a large macromolecular machine called the spliceosome. Given the spliceosome's role in shaping the cellular transcriptome, it is not surprising that mutations in the splicing machinery can result in a range of human diseases and disorders (spliceosomopathies). This review serves as an introduction into the main features of the pre-mRNA splicing machinery in humans and how changes in the function of its components can lead to diseases ranging from blindness to cancers. Recently, several drugs have been developed that interact directly with this machinery to change splicing outcomes at either the single gene or transcriptome-scale. We discuss the mechanism of action of several drugs that perturb splicing in unique ways. Finally, we speculate on what the future may hold in the emerging area of spliceosomopathies and spliceosome-targeted treatments.
Assuntos
Neoplasias , Precursores de RNA , Humanos , Precursores de RNA/genética , Precursores de RNA/metabolismo , Splicing de RNA , Spliceossomos/genética , Spliceossomos/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/genéticaRESUMO
The gene encoding the core spliceosomal protein SF3B1 is the most frequently mutated gene encoding a splicing factor in a variety of hematologic malignancies and solid tumors. SF3B1 mutations induce use of cryptic 3' splice sites (3'ss), and these splicing errors contribute to tumorigenesis. However, it is unclear how widespread this type of cryptic 3'ss usage is in cancers and what is the full spectrum of genetic mutations that cause such missplicing. To address this issue, we performed an unbiased pan-cancer analysis to identify genetic alterations that lead to the same aberrant splicing as observed with SF3B1 mutations. This analysis identified multiple mutations in another spliceosomal gene, SUGP1, that correlated with significant usage of cryptic 3'ss known to be utilized in mutant SF3B1 expressing cells. Remarkably, this is consistent with recent biochemical studies that identified a defective interaction between mutant SF3B1 and SUGP1 as the molecular defect responsible for cryptic 3'ss usage. Experimental validation revealed that five different SUGP1 mutations completely or partially recapitulated the 3'ss defects. Our analysis suggests that SUGP1 mutations in cancers can induce missplicing identical or similar to that observed in mutant SF3B1 cancers.
Assuntos
Biologia Computacional/métodos , Mutação , Neoplasias/genética , Fosfoproteínas/genética , Sítios de Splice de RNA , Fatores de Processamento de RNA/genética , Splicing de RNA , Análise Mutacional de DNA , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias/patologia , SpliceossomosRESUMO
BACKGROUND: Uveal melanoma (UM) is the eye's most common primary malignancy and there are no effective therapies for disseminated disease. It is important to try to know the patient's prognosis. The aim of this study was to reflect genetic variants, studied using NGS, of a series of 69 cases of UM and its correlation with histopathology and clinical progression. METHODS: We performed targeted NGS using a 519-gene panel. RESULTS: There were selected 28 different mutated genes, showing a total of 231 genetic variants that affected the function of the protein. The most common secondary mutations occurred in SF3B1 (in 26%), followed by BAP1 (in 23%), LRP1B (22%) and FGFR4 (20%). BAP1 mutation was associated with a greater likelihood of metastases and with greater presence of epithelioid cells. LRP1B was also associated with presence of epithelioid cells SF3B1 mutation was significantly associated with a spindle morphology. We found variants in the RAD51B, TOP2A, PTPRD, TSC2, DHX9, PDK1 and MTOR that have not been previously reported in consulted databases. The presence of a mutation in: CHEK2, DHX9 and PDK1 was associated with metastases. CONCLUSIONS: BAP1 is the most solid biomarker of a poor prognosis in UM and mutations can be detected using NGS. SF3B1 is associated with the spindle cell subtype of UM, which gives it probably a favourable prognostic value. Our study suggests that mutations in DHX9 and PDK1 can have prognostic value. These potential biomarkers are related to the PI3K/AKT/mTOR pathway and makes them candidates for developing new directed therapies.
Assuntos
Fosfatidilinositol 3-Quinases , Neoplasias Uveais , Humanos , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Prognóstico , Proteínas Supressoras de Tumor/genética , Análise Mutacional de DNA , Mutação , Neoplasias Uveais/genética , Sequenciamento de Nucleotídeos em Larga EscalaRESUMO
Chronic lymphocytic leukemia (CLL) is a genetically and clinically heterogeneous malignancy affecting older individuals. There are a number of current treatment options for CLL, including monoclonal antibodies, targeted drugs, chemotherapy, and different combinations of these. However, for those patients who are intrinsically treatment resistant, or relapse following initial responses, novel targeted therapies are still needed. Targeting the mouse double-minute-2 human homolog (MDM2), a primary negative regulator of p53, is an appealing therapeutic strategy for non-genotoxic reactivation of p53, since the TP53 gene is in its wild-type state at diagnosis in approximately 90% of patients. Mutated SF3B1 and TP53 are both associated with more aggressive disease, resistance to therapies and poorer overall survival for CLL. In this study, we performed a screen for SF3B1 and TP53 mutations and tested RG7388 (idasanutlin), a second-generation MDM2 inhibitor, in a cohort of CLL primary patient samples. SF3B1 mutations were detected in 24 of 195 cases (12.3%) and found associated with poor overall survival (hazard ratio [HR] 2.12, p = 0.032) and high CD38 expression (median CD38 (%) 32 vs. 5; p = 0.0087). The novel striking finding of this study was an independent link between SF3B1 mutational status and poor response to RG7388. Overall, SF3B1 mutations in CLL patient samples were associated with resistance to treatment with RG7388 ex vivo, and patients with the wild type for both SF3B1 and TP53 are more likely to benefit from treatment with MDM2 inhibitors.