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1.
Cancer Res ; 82(5): 819-830, 2022 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-35027467

RESUMO

Mutations in SF3B1 have been identified across several cancer types. This key spliceosome component promotes the efficient mRNA splicing of thousands of genes including those with crucial roles in the cellular response to DNA damage. Here, we demonstrate that depletion of SF3B1 specifically compromises homologous recombination (HR) and is epistatic with loss of BRCA1. More importantly, the most prevalent cancer-associated mutation in SF3B1, K700E, also affects HR efficiency and as a consequence, increases the cellular sensitivity to ionizing radiation and a variety of chemotherapeutic agents, including PARP inhibitors. In addition, the SF3B1 K700E mutation induced unscheduled R-loop formation, replication fork stalling, increased fork degradation, and defective replication fork restart. Taken together, these data suggest that tumor-associated mutations in SF3B1 induce a BRCA-like cellular phenotype that confers synthetic lethality to DNA-damaging agents and PARP inhibitors, which can be exploited therapeutically. SIGNIFICANCE: The cancer-associated SF3B1K700E mutation induces DNA damage via generation of genotoxic R-loops and stalled replication forks, defective homologous recombination, and increased replication fork degradation, which can be targeted with PARP inhibitors.


Assuntos
Neoplasias , Fosfoproteínas , Inibidores de Poli(ADP-Ribose) Polimerases , Fatores de Processamento de RNA , Replicação do DNA , Humanos , Mutação , Neoplasias/tratamento farmacológico , Neoplasias/genética , Fenótipo , Fosfoproteínas/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Fatores de Processamento de RNA/genética , Mutações Sintéticas Letais
2.
Nat Struct Mol Biol ; 28(2): 190-201, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33479542

RESUMO

NUP98 fusion proteins cause leukemia via unknown molecular mechanisms. All NUP98 fusion proteins share an intrinsically disordered region (IDR) in the NUP98 N terminus, featuring repeats of phenylalanine-glycine (FG), and C-terminal fusion partners often function in gene control. We investigated whether mechanisms of oncogenic transformation by NUP98 fusion proteins are hardwired in their protein interactomes. Affinity purification coupled to mass spectrometry (MS) and confocal imaging of five NUP98 fusion proteins expressed in human leukemia cells revealed that shared interactors were enriched for proteins involved in biomolecular condensation and that they colocalized with NUP98 fusion proteins in nuclear puncta. We developed biotinylated isoxazole-mediated condensome MS (biCon-MS) to show that NUP98 fusion proteins alter the global composition of biomolecular condensates. An artificial FG-repeat-containing fusion protein phenocopied the nuclear localization patterns of NUP98 fusion proteins and their capability to drive oncogenic gene expression programs. Thus, we propose that IDR-containing fusion proteins combine biomolecular condensation with transcriptional control to induce cancer.


Assuntos
Núcleo Celular/metabolismo , Proteínas de Homeodomínio , Leucemia , Complexo de Proteínas Formadoras de Poros Nucleares , Proteínas de Fusão Oncogênica , Animais , Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Células HEK293 , Células HL-60 , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/fisiologia , Humanos , Leucemia/metabolismo , Leucemia/patologia , Camundongos , Células NIH 3T3 , Complexo de Proteínas Formadoras de Poros Nucleares/química , Complexo de Proteínas Formadoras de Poros Nucleares/fisiologia , Proteínas de Fusão Oncogênica/química , Proteínas de Fusão Oncogênica/fisiologia
3.
J Transl Med ; 18(1): 339, 2020 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-32883299

RESUMO

BACKGROUND: The cohesin complex plays a major role in folding the human genome into 3D structural domains. Mutations in members of the cohesin complex are known early drivers of myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML), with STAG2 the most frequently mutated complex member. METHODS: Here we use functional genomics (RNA-seq, ChIP-seq and HiChIP) to investigate the impact of chronic STAG2 loss on three-dimensional genome structure and transcriptional programming in a clinically relevant model of chronic STAG2 loss. RESULTS: The chronic loss of STAG2 led to loss of smaller loop domains and the maintenance/formation of large domains that, in turn, led to altered genome compartmentalisation. These changes in genome structure resulted in altered gene expression, including deregulation of the HOXA locus and the MAPK signalling pathway, resulting in increased sensitivity to MEK inhibition. CONCLUSIONS: The altered genomic architecture driven by the chronic loss of STAG2 results in altered gene expression that may contribute to leukaemogenesis and may be therapeutically targeted.


Assuntos
Leucemia Mieloide Aguda , Síndromes Mielodisplásicas , Proteínas de Ciclo Celular/genética , Cromatina/genética , Humanos , Leucemia Mieloide Aguda/genética , Mutação
4.
Nucleic Acids Res ; 45(22): 12816-12833, 2017 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-29112714

RESUMO

mRNA splicing and export plays a key role in the regulation of gene expression, with recent evidence suggesting an additional layer of regulation of gene expression and cellular function through the selective splicing and export of genes within specific pathways. Here we describe a role for the RNA processing factors THRAP3 and BCLAF1 in the regulation of the cellular DNA damage response (DDR) pathway, a key pathway involved in the maintenance of genomic stability and the prevention of oncogenic transformation. We show that loss of THRAP3 and/or BCLAF1 leads to sensitivity to DNA damaging agents, defective DNA repair and genomic instability. Additionally, we demonstrate that this phenotype can be at least partially explained by the role of THRAP3 and BCLAF1 in the selective mRNA splicing and export of transcripts encoding key DDR proteins, including the ATM kinase. Moreover, we show that cancer associated mutations within THRAP3 result in deregulated processing of THRAP3/BCLAF1-regulated transcripts and consequently defective DNA repair. Taken together, these results suggest that THRAP3 and BCLAF1 mutant tumors may be promising targets for DNA damaging chemotherapy.


Assuntos
Transporte Ativo do Núcleo Celular/genética , Dano ao DNA , Proteínas de Ligação a DNA/genética , Splicing de RNA , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Perfilação da Expressão Gênica/métodos , Células HEK293 , Humanos , Hibridização in Situ Fluorescente , Microscopia de Fluorescência , Mutação , Interferência de RNA , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo
5.
Methods Mol Biol ; 1196: 349-70, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25151174

RESUMO

Drug discovery and development are often hampered by lack of target identification and clinical tractability. Repurposing of approved drugs to life-threatening diseases such as leukemia is emerging as a promising alternative approach. Connectivity mapping systems link approved drugs with disease-related gene signatures. Relevant preclinical models provide essential tools for system validation and proof-of-concept studies. Herein we describe procedures aimed at generating disease-based gene signatures and applying them to established cross-referencing databases of potential candidate drugs. As a proof of principle, we present the identification of Entinostat as a candidate drug for the treatment of HOX-TALE-related leukemia.


Assuntos
Antineoplásicos/farmacologia , Descoberta de Drogas , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Proteínas de Homeodomínio/genética , Leucemia/genética , Animais , Antineoplásicos/administração & dosagem , Benzamidas/administração & dosagem , Benzamidas/farmacologia , Linhagem Celular Tumoral , Bases de Dados Genéticas , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Perfilação da Expressão Gênica , Inibidores de Histona Desacetilases/administração & dosagem , Inibidores de Histona Desacetilases/farmacologia , Proteínas de Homeodomínio/metabolismo , Humanos , Leucemia/tratamento farmacológico , Leucemia/metabolismo , Leucemia/patologia , Camundongos , Piridinas/administração & dosagem , Piridinas/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Haematologica ; 98(8): 1216-25, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23539541

RESUMO

The cytogenetically normal subtype of acute myeloid leukemia is associated with an intermediate risk which complicates therapeutic options. Lower overall HOX/TALE expression appears to correlate with more favorable prognosis/better response to treatment in some leukemias and solid cancer. The functional significance of the associated gene expression and response to chemotherapy is not known. Three independent microarray datasets obtained from large cohorts of patients along with quantitative polymerase chain reaction validation were used to identify a four-gene HOXA/TALE signature capable of prognostic stratification. Biochemical analysis was used to identify interactions between the four encoded proteins and targeted knockdown used to examine the functional importance of sustained expression of the signature in leukemia maintenance and response to chemotherapy. An 11 HOXA/TALE code identified in an intermediate-risk group of patients (n=315) compared to a group with a favorable risk (n=105) was reduced to a four-gene signature of HOXA6, HOXA9, PBX3 and MEIS1 by iterative analysis of independent platforms. This signature maintained the favorable/intermediate risk partition and where applicable, correlated with overall survival in cytogenetically normal acute myeloid leukemia. We further showed that cell growth and function are dependent on maintained levels of these core genes and that direct targeting of HOXA/PBX3 sensitizes cytogenetically normal acute myeloid leukemia cells to standard chemotherapy. Together the data support a key role for HOXA/TALE in cytogenetically normal acute myeloid leukemia and demonstrate that targeting of clinically significant HOXA/PBX3 elements may provide therapeutic benefit to patients with this subtype of leukemia.


Assuntos
Antineoplásicos/uso terapêutico , Análise Citogenética/métodos , Proteínas de Homeodomínio/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Proteínas Proto-Oncogênicas/genética , Antineoplásicos/farmacologia , Proliferação de Células , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Técnicas de Silenciamento de Genes/métodos , Proteínas de Homeodomínio/antagonistas & inibidores , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Proteína Meis1 , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Células U937
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