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1.
Mol Cell ; 84(10): 1886-1903.e10, 2024 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-38688280

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/metabolismo
2.
Science ; 383(6682): eadi5798, 2024 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-38301010

RESUMO

Increasing use of covalent and noncovalent inhibitors of Bruton's tyrosine kinase (BTK) has elucidated a series of acquired drug-resistant BTK mutations in patients with B cell malignancies. Here we identify inhibitor resistance mutations in BTK with distinct enzymatic activities, including some that impair BTK enzymatic activity while imparting novel protein-protein interactions that sustain B cell receptor (BCR) signaling. Furthermore, we describe a clinical-stage BTK and IKZF1/3 degrader, NX-2127, that can bind and proteasomally degrade each mutant BTK proteoform, resulting in potent blockade of BCR signaling. Treatment of chronic lymphocytic leukemia with NX-2127 achieves >80% degradation of BTK in patients and demonstrates proof-of-concept therapeutic benefit. These data reveal an oncogenic scaffold function of mutant BTK that confers resistance across clinically approved BTK inhibitors but is overcome by BTK degradation in patients.


Assuntos
Tirosina Quinase da Agamaglobulinemia , Resistencia a Medicamentos Antineoplásicos , Fator de Transcrição Ikaros , Leucemia Linfocítica Crônica de Células B , Inibidores de Proteínas Quinases , Proteólise , Humanos , Tirosina Quinase da Agamaglobulinemia/genética , Tirosina Quinase da Agamaglobulinemia/metabolismo , Fator de Transcrição Ikaros/metabolismo , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/genética , Mutação , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Transdução de Sinais , Proteólise/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos
3.
bioRxiv ; 2023 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-37034664

RESUMO

Ongoing, first-in-human clinical trials illustrate the feasibility and translational potential of human pluripotent stem cell (hPSC)-based cell therapies in Parkinson's disease (PD). However, a major unresolved challenge in the field is the extensive cell death following transplantation with <10% of grafted dopamine neurons surviving. Here, we performed a pooled CRISPR/Cas9 screen to enhance survival of postmitotic dopamine neurons in vivo . We identified p53-mediated apoptotic cell death as major contributor to dopamine neuron loss and uncovered a causal link of TNFa-NFκB signaling in limiting cell survival. As a translationally applicable strategy to purify postmitotic dopamine neurons, we performed a cell surface marker screen that enabled purification without the need for genetic reporters. Combining cell sorting with adalimumab pretreatment, a clinically approved and widely used TNFa inhibitor, enabled efficient engraftment of postmitotic dopamine neurons leading to extensive re-innervation and functional recovery in a preclinical PD mouse model. Thus, transient TNFa inhibition presents a clinically relevant strategy to enhance survival and enable engraftment of postmitotic human PSC-derived dopamine neurons in PD. Highlights: In vivo CRISPR-Cas9 screen identifies p53 limiting survival of grafted human dopamine neurons. TNFα-NFκB pathway mediates p53-dependent human dopamine neuron deathCell surface marker screen to enrich human dopamine neurons for translational use. FDA approved TNF-alpha inhibitor rescues in vivo dopamine neuron survival with in vivo function.

4.
Nat Commun ; 13(1): 5676, 2022 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-36167829

RESUMO

To identify drivers of sensitivity and resistance to Protein Arginine Methyltransferase 5 (PRMT5) inhibition, we perform a genome-wide CRISPR/Cas9 screen. We identify TP53 and RNA-binding protein MUSASHI2 (MSI2) as the top-ranked sensitizer and driver of resistance to specific PRMT5i, GSK-591, respectively. TP53 deletion and TP53R248W mutation are biomarkers of resistance to GSK-591. PRMT5 expression correlates with MSI2 expression in lymphoma patients. MSI2 depletion and pharmacological inhibition using Ro 08-2750 (Ro) both synergize with GSK-591 to reduce cell growth. Ro reduces MSI2 binding to its global targets and dual treatment of Ro and PRMT5 inhibitors result in synergistic gene expression changes including cell cycle, P53 and MYC signatures. Dual MSI2 and PRMT5 inhibition further blocks c-MYC and BCL-2 translation. BCL-2 depletion or inhibition with venetoclax synergizes with a PRMT5 inhibitor by inducing reduced cell growth and apoptosis. Thus, we propose a therapeutic strategy in lymphoma that combines PRMT5 with MSI2 or BCL-2 inhibition.


Assuntos
Linfoma de Células B , Linfoma , Linhagem Celular Tumoral , Humanos , Linfoma/genética , Mutação , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteína Supressora de Tumor p53/genética
5.
Nat Genet ; 53(5): 707-718, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33846634

RESUMO

Most eukaryotes harbor two distinct pre-mRNA splicing machineries: the major spliceosome, which removes >99% of introns, and the minor spliceosome, which removes rare, evolutionarily conserved introns. Although hypothesized to serve important regulatory functions, physiologic roles of the minor spliceosome are not well understood. For example, the minor spliceosome component ZRSR2 is subject to recurrent, leukemia-associated mutations, yet functional connections among minor introns, hematopoiesis and cancers are unclear. Here, we identify that impaired minor intron excision via ZRSR2 loss enhances hematopoietic stem cell self-renewal. CRISPR screens mimicking nonsense-mediated decay of minor intron-containing mRNA species converged on LZTR1, a regulator of RAS-related GTPases. LZTR1 minor intron retention was also discovered in the RASopathy Noonan syndrome, due to intronic mutations disrupting splicing and diverse solid tumors. These data uncover minor intron recognition as a regulator of hematopoiesis, noncoding mutations within minor introns as potential cancer drivers and links among ZRSR2 mutations, LZTR1 regulation and leukemias.


Assuntos
Predisposição Genética para Doença , Doenças Hematológicas/genética , Íntrons/genética , Neoplasias/genética , Animais , Sequência de Bases , Sistemas CRISPR-Cas/genética , Autorrenovação Celular , Transformação Celular Neoplásica/patologia , Células Clonais , Feminino , Genoma Humano , Doenças Hematológicas/patologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Masculino , Camundongos Knockout , Síndrome de Noonan/genética , Linhagem , RNA/metabolismo , Splicing de RNA/genética , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Baço/patologia , Fatores de Transcrição/genética
6.
Biochim Biophys Acta ; 1631(3): 246-54, 2003 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-12668176

RESUMO

The activation of phospholipase D (PLD) in PC12/PC2 pheochromocytoma cells involves a tyrosine kinase. However, it is not clear whether this is due to direct phosphorylation of the enzyme or some other intermediary protein. In this manuscript, we examined this issue by two methods: (1) immunoprecipitation of phosphotyrosine containing proteins and assay of phospholipase D; (2) overexpression of HA-phospholipase D2 and susbsequent immunoprecipitation. The only agent that caused phosphorylation of phospholipase D on tyrosine residues was the phosphatase inhibitor, peroxyvanadate. Other agents that activate phospholipase D, including bradykinin, ionomycin, and phorbol dibutyrate did not cause phosphorylation of the enzyme. In addition, there was a lack of correlation between the peroxyvanadate-mediated phosphorylation and activation of phospholipase D, both in terms of time course and concentration dependence. These data demonstrate that phospholipase D is directly phosphorylated on tyrosine residues. However, phosphorylation of tyrosine residues does not correlate with activation of the enzyme.


Assuntos
Fosfolipase D/metabolismo , Tirosina/química , Animais , Ativação Enzimática , Inibidores Enzimáticos , Isoenzimas/metabolismo , Células PC12 , Fosfolipase D/análise , Fosfolipase D/química , Fosforilação/efeitos dos fármacos , Fosfotirosina/análise , Testes de Precipitina , Ratos , Vanadatos
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