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1.
Gastroenterology ; 161(3): 814-826.e7, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33992635

RESUMO

BACKGROUND & AIMS: Next-generation sequencing (NGS) was recently approved by the United States Food and Drug Administration to detect microsatellite instability (MSI) arising from defective mismatch repair (dMMR) in patients with metastatic colorectal cancer (mCRC) before treatment with immune checkpoint inhibitors (ICI). In this study, we aimed to evaluate and improve the performance of NGS to identify MSI in CRC, especially dMMR mCRC treated with ICI. METHODS: CRC samples used in this post hoc study were reassessed centrally for MSI and dMMR status using the reference methods of pentaplex polymerase chain reaction and immunohistochemistry. Whole-exome sequencing (WES) was used to evaluate MSISensor, the Food and Drug Administration-approved and NGS-based method for assessment of MSI. This was performed in (1) a prospective, multicenter cohort of 102 patients with mCRC (C1; 25 dMMR/MSI, 24 treated with ICI) from clinical trials NCT02840604 and NCT033501260, (2) an independent retrospective, multicenter cohort of 113 patients (C2; 25 mCRC, 88 non-mCRC, all dMMR/MSI untreated with ICI), and (3) a publicly available series of 118 patients with CRC from The Cancer Genome Atlas (C3; 51 dMMR/MSI). A new NGS-based algorithm, namely MSICare, was developed. Its performance for assessment of MSI was compared with MSISensor in C1, C2, and C3 at the exome level or after downsampling sequencing data to the MSK-IMPACT gene panel. MSICare was validated in an additional retrospective, multicenter cohort (C4) of 152 patients with new CRC (137 dMMR/MSI) enriched in tumors deficient in MSH6 (n = 35) and PMS2 (n = 9) after targeted sequencing of samples with an optimized set of microsatellite markers (MSIDIAG). RESULTS: At the exome level, MSISensor was highly specific but failed to diagnose MSI in 16% of MSI/dMMR mCRC from C1 (4 of 25; sensitivity, 84%; 95% confidence interval [CI], 63.9%-95.5%), 32% of mCRC (8 of 25; sensitivity, 68%; 95% CI, 46.5%-85.1%), and 9.1% of non-mCRC from C2 (8 of 88; sensitivity, 90.9%; 95% CI, 82.9%-96%), and 9.8% of CRC from C3 (5 of 51; sensitivity, 90.2%; 95% CI, 78.6%-96.7%). Misdiagnosis included 4 mCRCs treated with ICI, of which 3 showed an overall response rate without progression at this date. At the exome level, reevaluation of the MSI genomic signal using MSICare detected 100% of cases with true MSI status among C1 and C2. Further validation of MSICare was obtained in CRC tumors from C3, with 96.1% concordance for MSI status. Whereas misdiagnosis with MSISensor even increased when analyzing downsampled WES data from C1 and C2 with microsatellite markers restricted to the MSK-IMPACT gene panel (sensitivity, 72.5%; 95% CI, 64.2%-79.7%), particularly in the MSH6-deficient setting, MSICare sensitivity and specificity remained optimal (100%). Similar results were obtained with MSICare after targeted NGS of tumors from C4 with the optimized microsatellite panel MSIDIAG (sensitivity, 99.3%; 95% CI, 96%-100%; specificity, 100%). CONCLUSIONS: In contrast to MSISensor, the new MSICare test we propose performs at least as efficiently as the reference method, MSI polymerase chain reaction, to detect MSI in CRC regardless of the defective MMR protein under both WES and targeted NGS conditions. We suggest MSICare may rapidly become a reference method for NGS-based testing of MSI in CRC, especially in mCRC, where accurate MSI status is required before the prescription of ICI.


Assuntos
Algoritmos , Biomarcadores Tumorais/genética , Neoplasias Colorretais/genética , Reparo de Erro de Pareamento de DNA , Sequenciamento do Exoma , Sequenciamento de Nucleotídeos em Larga Escala , Instabilidade de Microssatélites , Tomada de Decisão Clínica , Ensaios Clínicos como Assunto , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/imunologia , Bases de Dados Genéticas , França , Humanos , Inibidores de Checkpoint Imunológico/uso terapêutico , Imuno-Histoquímica , Reação em Cadeia da Polimerase Multiplex , Valor Preditivo dos Testes , Estudos Prospectivos , Reprodutibilidade dos Testes , Estudos Retrospectivos
2.
Genome Biol ; 25(1): 210, 2024 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-39107855

RESUMO

BACKGROUND: Microsatellite instability (MSI) due to mismatch repair deficiency (dMMR) is common in colorectal cancer (CRC). These cancers are associated with somatic coding events, but the noncoding pathophysiological impact of this genomic instability is yet poorly understood. Here, we perform an analysis of coding and noncoding MSI events at the different steps of colorectal tumorigenesis using whole exome sequencing and search for associated splicing events via RNA sequencing at the bulk-tumor and single-cell levels. RESULTS: Our results demonstrate that MSI leads to hundreds of noncoding DNA mutations, notably at polypyrimidine U2AF RNA-binding sites which are endowed with cis-activity in splicing, while higher frequency of exon skipping events are observed in the mRNAs of MSI compared to non-MSI CRC. At the DNA level, these noncoding MSI mutations occur very early prior to cell transformation in the dMMR colonic crypt, accounting for only a fraction of the exon skipping in MSI CRC. At the RNA level, the aberrant exon skipping signature is likely to impair colonic cell differentiation in MSI CRC affecting the expression of alternative exons encoding protein isoforms governing cell fate, while also targeting constitutive exons, making dMMR cells immunogenic in early stage before the onset of coding mutations. This signature is characterized by its similarity to the oncogenic U2AF1-S34F splicing mutation observed in several other non-MSI cancer. CONCLUSIONS: Overall, these findings provide evidence that a very early RNA splicing signature partly driven by MSI impairs cell differentiation and promotes MSI CRC initiation, far before coding mutations which accumulate later during MSI tumorigenesis.


Assuntos
Processamento Alternativo , Neoplasias Colorretais , Instabilidade de Microssatélites , Fator de Processamento U2AF , Neoplasias Colorretais/genética , Humanos , Fator de Processamento U2AF/genética , Fator de Processamento U2AF/metabolismo , Mutação , Sítios de Ligação , Éxons
3.
J Clin Invest ; 132(14)2022 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-35587378

RESUMO

Acute megakaryoblastic leukemia of Down syndrome (DS-AMKL) is a model of clonal evolution from a preleukemic transient myeloproliferative disorder requiring both a trisomy 21 (T21) and a GATA1s mutation to a leukemia driven by additional driver mutations. We modeled the megakaryocyte differentiation defect through stepwise gene editing of GATA1s, SMC3+/-, and MPLW515K, providing 20 different T21 or disomy 21 (D21) induced pluripotent stem cell (iPSC) clones. GATA1s profoundly reshaped iPSC-derived hematopoietic architecture with gradual myeloid-to-megakaryocyte shift and megakaryocyte differentiation alteration upon addition of SMC3 and MPL mutations. Transcriptional, chromatin accessibility, and GATA1-binding data showed alteration of essential megakaryocyte differentiation genes, including NFE2 downregulation that was associated with loss of GATA1s binding and functionally involved in megakaryocyte differentiation blockage. T21 enhanced the proliferative phenotype, reproducing the cellular and molecular abnormalities of DS-AMKL. Our study provides an array of human cell-based models revealing individual contributions of different mutations to DS-AMKL differentiation blockage, a major determinant of leukemic progression.


Assuntos
Síndrome de Down , Leucemia Megacarioblástica Aguda , Proteínas de Ciclo Celular/genética , Criança , Proteoglicanas de Sulfatos de Condroitina/genética , Proteínas Cromossômicas não Histona/genética , Síndrome de Down/genética , Fator de Transcrição GATA1/genética , Hematopoese , Humanos , Leucemia Megacarioblástica Aguda/complicações , Leucemia Megacarioblástica Aguda/genética , Leucemia Megacarioblástica Aguda/metabolismo , Megacariócitos/metabolismo , Mutação , Trissomia
4.
J Clin Invest ; 131(1)2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33393494

RESUMO

DYRK1A is a serine/threonine kinase encoded on human chromosome 21 (HSA21) that has been implicated in several pathologies of Down syndrome (DS), including cognitive deficits and Alzheimer's disease. Although children with DS are predisposed to developing leukemia, especially B cell acute lymphoblastic leukemia (B-ALL), the HSA21 genes that contribute to malignancies remain largely undefined. Here, we report that DYRK1A is overexpressed and required for B-ALL. Genetic and pharmacologic inhibition of DYRK1A decreased leukemic cell expansion and suppressed B-ALL development in vitro and in vivo. Furthermore, we found that FOXO1 and STAT3, transcription factors that are indispensable for B cell development, are critical substrates of DYRK1A. Loss of DYRK1A-mediated FOXO1 and STAT3 signaling disrupted DNA damage and ROS regulation, respectively, leading to preferential cell death in leukemic B cells. Thus, we reveal a DYRK1A/FOXO1/STAT3 axis that facilitates the development and maintenance of B-ALL.


Assuntos
Proteína Forkhead Box O1/metabolismo , Proteínas de Neoplasias/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Fator de Transcrição STAT3/metabolismo , Animais , Feminino , Proteína Forkhead Box O1/genética , Masculino , Camundongos , Camundongos Transgênicos , Proteínas de Neoplasias/genética , Fosforilação/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Fator de Transcrição STAT3/genética , Quinases Dyrk
5.
Clin Cancer Res ; 26(13): 3307-3318, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32220889

RESUMO

PURPOSE: Children with Down syndrome (constitutive trisomy 21) that develop acute lymphoblastic leukemia (DS-ALL) have a 3-fold increased likelihood of treatment-related mortality coupled with a higher cumulative incidence of relapse, compared with other children with B-cell acute lymphoblastic leukemia (B-ALL). This highlights the lack of suitable treatment for Down syndrome children with B-ALL. EXPERIMENTAL DESIGN: To facilitate the translation of new therapeutic agents into clinical trials, we built the first preclinical cohort of patient-derived xenograft (PDX) models of DS-ALL, comprehensively characterized at the genetic and transcriptomic levels, and have proven its suitability for preclinical studies by assessing the efficacy of drug combination between the MEK inhibitor trametinib and conventional chemotherapy agents. RESULTS: Whole-exome and RNA-sequencing experiments revealed a high incidence of somatic alterations leading to RAS/MAPK pathway activation in our cohort of DS-ALL, as well as in other pediatric B-ALL presenting somatic gain of the chromosome 21 (B-ALL+21). In murine and human B-cell precursors, activated KRASG12D functionally cooperates with trisomy 21 to deregulate transcriptional networks that promote increased proliferation and self renewal, as well as B-cell differentiation blockade. Moreover, we revealed that inhibition of RAS/MAPK pathway activation using the MEK1/2 inhibitor trametinib decreased leukemia burden in several PDX models of B-ALL+21, and enhanced survival of DS-ALL PDX in combination with conventional chemotherapy agents such as vincristine. CONCLUSIONS: Altogether, using novel and suitable PDX models, this study indicates that RAS/MAPK pathway inhibition represents a promising strategy to improve the outcome of Down syndrome children with B-cell precursor leukemia.


Assuntos
Síndrome de Down/complicações , Síndrome de Down/genética , Síndrome de Down/metabolismo , Leucemia de Células B/diagnóstico , Leucemia de Células B/etiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Transdução de Sinais , Proteínas ras/metabolismo , Animais , Biologia Computacional/métodos , Modelos Animais de Doenças , Suscetibilidade a Doenças , Perfilação da Expressão Gênica , Humanos , Imunofenotipagem , Leucemia de Células B/terapia , Camundongos , Camundongos Transgênicos , Oncogenes , Inibidores de Proteínas Quinases/farmacologia , Piridonas/farmacologia , Pirimidinonas/farmacologia , Transdução de Sinais/efeitos dos fármacos
6.
Blood Adv ; 2(13): 1616-1627, 2018 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-29986854

RESUMO

JAK3-activating mutations are commonly seen in chronic or acute hematologic malignancies affecting the myeloid, megakaryocytic, lymphoid, and natural killer (NK) cell compartment. Overexpression models of mutant JAK3 or pharmacologic inhibition of its kinase activity have highlighted the role that these constitutively activated mutants play in the T-cell, NK cell, and megakaryocytic lineages, but to date, the functional impact of JAK3 mutations at an endogenous level remains unknown. Here, we report a JAK3A572V knockin mouse model and demonstrate that activated JAK3 leads to a progressive and dose-dependent expansion of CD8+ T cells in the periphery before colonization of the bone marrow. This phenotype is dependent on the γc chain of cytokine receptors and presents several features of the human leukemic form of cutaneous T-cell lymphoma (L-CTCL), including skin involvements. We also showed that the JAK3A572V-positive malignant cells are transplantable and phenotypically heterogeneous in bone marrow transplantation assays. Interestingly, we revealed that activated JAK3 functionally cooperates with partial trisomy 21 in vivo to enhance the L-CTCL phenotype, ultimately leading to a lethal and fully penetrant disorder. Finally, we assessed the efficacy of JAK3 inhibition and showed that CTCL JAK3A572V-positive T cells are sensitive to tofacitinib, which provides additional preclinical insights into the use of JAK3 inhibitors in these disorders. Altogether, this JAK3A572V knockin model is a relevant new tool for testing the efficacy of JAK inhibitors in JAK3-related hematopoietic malignancies.


Assuntos
Cromossomos de Mamíferos/metabolismo , Neoplasias Hematológicas/metabolismo , Janus Quinase 3/metabolismo , Linfoma Cutâneo de Células T/metabolismo , Mutação de Sentido Incorreto , Neoplasias Experimentais/metabolismo , Trissomia , Substituição de Aminoácidos , Animais , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/patologia , Cromossomos de Mamíferos/genética , Técnicas de Introdução de Genes , Neoplasias Hematológicas/tratamento farmacológico , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/patologia , Janus Quinase 3/genética , Linfoma Cutâneo de Células T/tratamento farmacológico , Linfoma Cutâneo de Células T/genética , Linfoma Cutâneo de Células T/patologia , Camundongos , Camundongos Transgênicos , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia
7.
Cancer Cell ; 31(3): 452-465, 2017 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-28292442

RESUMO

Chimeric transcription factors are a hallmark of human leukemia, but the molecular mechanisms by which they block differentiation and promote aberrant self-renewal remain unclear. Here, we demonstrate that the ETO2-GLIS2 fusion oncoprotein, which is found in aggressive acute megakaryoblastic leukemia, confers megakaryocytic identity via the GLIS2 moiety while both ETO2 and GLIS2 domains are required to drive increased self-renewal properties. ETO2-GLIS2 directly binds DNA to control transcription of associated genes by upregulation of expression and interaction with the ETS-related ERG protein at enhancer elements. Importantly, specific interference with ETO2-GLIS2 oligomerization reverses the transcriptional activation at enhancers and promotes megakaryocytic differentiation, providing a relevant interface to target in this poor-prognosis pediatric leukemia.


Assuntos
Leucemia Megacarioblástica Aguda/patologia , Proteínas de Fusão Oncogênica/fisiologia , Ativação Transcricional , Animais , Diferenciação Celular , Criança , Elementos Facilitadores Genéticos , Fator de Transcrição GATA1/genética , Humanos , Camundongos , Proteínas de Fusão Oncogênica/química , Regulador Transcricional ERG/fisiologia
8.
PLoS One ; 6(8): e23676, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21886810

RESUMO

BACKGROUND: Besides regulation of actin cytoskeleton-dependent functions, Rho GTPase pathways are essential to cell cycle progression and cell division. Rho, Rac and Cdc42 regulate G1 to S phase progression and are involved in cytokinesis. RhoA GDP/GTP cycling is required for normal cytokinesis and recent reports have shown that the exchange factor Ect2 and the GTPase activating protein MgcRacGAP regulate RhoA activity during mitosis. We previously showed that the transcription factors E2F1 and CUX1 regulate expression of MgcRacGAP and Ect2 as cells enter S-phase. METHODOLOGY/PRINCIPAL FINDINGS: We now report that Ect2 is subject to proteasomal degradation after mitosis, following ubiquitination by the APC/C complex and its co-activator Cdh1. A proper nuclear localization of Ect2 is necessary for its degradation. APC-Cdh1 assembles K11-linked poly-ubiquitin chains on Ect2, depending upon a stretch of ∼25 amino acid residues that contain a bi-partite NLS, a conventional D-box and two TEK-like boxes. Site-directed mutagenesis of target sequences generated stabilized Ect2 proteins. Furthermore, such degradation-resistant mutants of Ect2 were found to activate RhoA and subsequent signalling pathways and are able to transform NIH3T3 cells. CONCLUSIONS/SIGNIFICANCE: Our results identify Ect2 as a bona fide cell cycle-regulated protein and suggest that its ubiquitination-dependent degradation may play an important role in RhoA regulation at the time of mitosis. Our findings raise the possibility that the overexpression of Ect2 that has been reported in some human tumors might result not only from deregulated transcription, but also from impaired degradation.


Assuntos
Caderinas/fisiologia , Mitose , Proteínas Proto-Oncogênicas/metabolismo , Antígenos CD , Linhagem Celular , Proteínas de Ligação a DNA , Humanos , Estabilidade Proteica , Fatores de Transcrição , Proteínas rho de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo
9.
Mol Cell Biol ; 29(2): 570-81, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19015243

RESUMO

Rho GTPases are critical for mitosis progression and completion of cytokinesis. During mitosis, the GDP/GTP cycle of Rho GTPases is regulated by the exchange factor Ect2 and the GTPase activating protein MgcRacGAP which associates with the kinesin MKLP1 in the centralspindlin complex. We report here that expression of Ect2, MgcRacGAP, and MKLP1 is tightly regulated during cell cycle progression. These three genes share similar cell cycle-related signatures within their promoter regions: (i) cell cycle gene homology region (CHR) sites located at -20 to +40 nucleotides of their transcription start sites that are required for repression in G(1), (ii) E2F binding elements, and (iii) tandem repeats of target sequences for the CUX1 transcription factor. CUX1 and E2F1 bind these three promoters upon S-phase entry, as demonstrated by chromatin immunoprecipitation, and regulate transcription of these genes, as established using promoter-luciferase reporter constructs and expression of activated or dominant negative transcription factors. Overexpression of either E2F1 or CUX1 increased the levels of the endogenous proteins whereas small interfering RNA knockdown of E2F1 or use of a dominant negative E2F1 reduced their expression levels. Thus, CUX1, E2F, and CHR elements provide the transcriptional controls that coordinate induction of Ect2, MgcRacGAP, and MKLP1 in S phase, leading to peak expression of these interacting proteins in G(2)/M, at the time they are required to regulate cytokinesis.


Assuntos
Fator de Transcrição E2F1/metabolismo , Proteínas Ativadoras de GTPase/genética , Regulação da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Repressoras/metabolismo , Fase S/fisiologia , Imunoprecipitação da Cromatina , Análise Mutacional de DNA , Fator de Transcrição E2F1/genética , Fase G1/fisiologia , Proteínas Ativadoras de GTPase/metabolismo , Proteínas de Homeodomínio/genética , Humanos , Interleucina-2/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Nucleares/genética , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Repressoras/genética , Homologia de Sequência do Ácido Nucleico , Fatores de Transcrição , Sítio de Iniciação de Transcrição
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