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1.
Blood Adv ; 5(9): 2412-2425, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33956058

RESUMO

Advances in cancer genomics have revealed genomic classes of acute myeloid leukemia (AML) characterized by class-defining mutations, such as chimeric fusion genes or in genes such as NPM1, MLL, and CEBPA. These class-defining mutations frequently synergize with internal tandem duplications in FLT3 (FLT3-ITDs) to drive leukemogenesis. However, ∼20% of FLT3-ITD-positive AMLs bare no class-defining mutations, and mechanisms of leukemic transformation in these cases are unknown. To identify pathways that drive FLT3-ITD mutant AML in the absence of class-defining mutations, we performed an insertional mutagenesis (IM) screening in Flt3-ITD mice, using Sleeping Beauty transposons. All mice developed acute leukemia (predominantly AML) after a median of 73 days. Analysis of transposon insertions in 38 samples from Flt3-ITD/IM leukemic mice identified recurrent integrations at 22 loci, including Setbp1 (20/38), Ets1 (11/38), Ash1l (8/38), Notch1 (8/38), Erg (7/38), and Runx1 (5/38). Insertions at Setbp1 led exclusively to AML and activated a transcriptional program similar, but not identical, to those of NPM1-mutant and MLL-rearranged AMLs. Guide RNA targeting of Setbp1 was highly detrimental to Flt3ITD/+/Setbp1IM+, but not to Flt3ITD/+/Npm1cA/+, AMLs. Also, analysis of RNA-sequencing data from hundreds of human AMLs revealed that SETBP1 expression is significantly higher in FLT3-ITD AMLs lacking class-defining mutations. These findings propose that SETBP1 overexpression collaborates with FLT3-ITD to drive a subtype of human AML. To identify genetic vulnerabilities of these AMLs, we performed genome-wide CRISPR-Cas9 screening in Flt3ITD/+/Setbp1IM+ AMLs and identified potential therapeutic targets, including Kdm1a, Brd3, Ezh2, and Hmgcr. Our study gives new insights into epigenetic pathways that can drive AMLs lacking class-defining mutations and proposes therapeutic approaches against such cases.


Assuntos
Leucemia Mieloide Aguda , Doença Aguda , Animais , Proteínas de Ligação a DNA , Histona-Lisina N-Metiltransferase , Leucemia Mieloide Aguda/genética , Camundongos , Mutação , Proteínas Nucleares/genética
2.
J Clin Invest ; 131(8)2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33690225

RESUMO

Melanomas commonly undergo a phenotype switch, from a proliferative to an invasive state. Such tumor cell plasticity contributes to immunotherapy resistance; however, the mechanisms are not completely understood and thus are therapeutically unexploited. Using melanoma mouse models, we demonstrated that blocking the MNK1/2-eIF4E axis inhibited melanoma phenotype switching and sensitized melanoma to anti-PD-1 immunotherapy. We showed that phospho-eIF4E-deficient murine melanomas expressed high levels of melanocytic antigens, with similar results verified in patient melanomas. Mechanistically, we identified phospho-eIF4E-mediated translational control of NGFR, a critical effector of phenotype switching. Genetic ablation of phospho-eIF4E reprogrammed the immunosuppressive microenvironment, exemplified by lowered production of inflammatory factors, decreased PD-L1 expression on dendritic cells and myeloid-derived suppressor cells, and increased CD8+ T cell infiltrates. Finally, dual blockade of the MNK1/2-eIF4E axis and the PD-1/PD-L1 immune checkpoint demonstrated efficacy in multiple melanoma models regardless of their genomic classification. An increase in the presence of intratumoral stem-like TCF1+PD-1+CD8+ T cells, a characteristic essential for durable antitumor immunity, was detected in mice given a MNK1/2 inhibitor and anti-PD-1 therapy. Using MNK1/2 inhibitors to repress phospho-eIF4E thus offers a strategy to inhibit melanoma plasticity and improve response to anti-PD-1 immunotherapy.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Fator de Iniciação 4E em Eucariotos/imunologia , Imunidade Celular , Sistema de Sinalização das MAP Quinases/imunologia , Melanoma Experimental/imunologia , Proteínas Serina-Treonina Quinases/imunologia , Animais , Antígeno B7-H1/genética , Antígeno B7-H1/imunologia , Linhagem Celular Tumoral , Fator de Iniciação 4E em Eucariotos/genética , Imunoterapia , Sistema de Sinalização das MAP Quinases/genética , Melanoma Experimental/genética , Melanoma Experimental/terapia , Camundongos , Camundongos Transgênicos , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/imunologia , Proteínas Serina-Treonina Quinases/genética , Receptor de Fator de Crescimento Neural/genética , Receptor de Fator de Crescimento Neural/imunologia
3.
Antioxidants (Basel) ; 9(12)2020 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-33287312

RESUMO

OBJECTIVE: Heme oxygenase-1 (HO-1) is a cytoprotective, proangiogenic and anti-inflammatory enzyme that is often upregulated in tumors. Overexpression of HO-1 in melanoma cells leads to enhanced tumor growth, augmented angiogenesis and resistance to anticancer treatment. The effect of HO-1 in host cells on tumor development is, however, hardly known. METHODS AND RESULTS: To clarify the effect of HO-1 expression in host cells on melanoma progression, C57BL/6xFvB mice of different HO-1 genotypes, HO-1+/+, HO-1+/-, and HO-1-/-, were injected with the syngeneic wild-type murine melanoma B16(F10) cell line. Lack of HO-1 in host cells did not significantly influence the host survival. Nevertheless, in comparison to the wild-type counterparts, the HO-1+/- and HO-1-/- males formed bigger tumors, and more numerous lung nodules; in addition, more of them had liver and spleen micrometastases. Females of all genotypes developed at least 10 times smaller tumors than males. Of importance, the growth of primary and secondary tumors was completely blocked in HO-1+/+ females. This was related to the increased infiltration of leukocytes (mainly lymphocytes T) in primary tumors. CONCLUSIONS: Although HO-1 overexpression in melanoma cells can enhance tumor progression in mice, its presence in host cells, including immune cells, can reduce growth and metastasis of melanoma.

4.
J Exp Med ; 216(4): 966-981, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30890554

RESUMO

Epigenetic regulators, such as EZH2, are frequently mutated in cancer, and loss-of-function EZH2 mutations are common in myeloid malignancies. We have examined the importance of cellular context for Ezh2 loss during the evolution of acute myeloid leukemia (AML), where we observed stage-specific and diametrically opposite functions for Ezh2 at the early and late stages of disease. During disease maintenance, WT Ezh2 exerts an oncogenic function that may be therapeutically targeted. In contrast, Ezh2 acts as a tumor suppressor during AML induction. Transcriptional analysis explains this apparent paradox, demonstrating that loss of Ezh2 derepresses different expression programs during disease induction and maintenance. During disease induction, Ezh2 loss derepresses a subset of bivalent promoters that resolve toward gene activation, inducing a feto-oncogenic program that includes genes such as Plag1, whose overexpression phenocopies Ezh2 loss to accelerate AML induction in mouse models. Our data highlight the importance of cellular context and disease phase for the function of Ezh2 and its potential therapeutic implications.


Assuntos
Progressão da Doença , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/mortalidade , Mutação com Perda de Função , Animais , Células da Medula Óssea/metabolismo , Transplante de Medula Óssea , Linhagem Celular Tumoral , Estudos de Coortes , Modelos Animais de Doenças , Frequência do Gene , Histonas/metabolismo , Humanos , Leucemia Mieloide Aguda/sangue , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Endogâmicos C57BL , Prognóstico , Taxa de Sobrevida , Transdução Genética , Transplante Homólogo
5.
Nat Genet ; 50(6): 883-894, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29736013

RESUMO

The histone H3 Lys27-specific demethylase UTX (or KDM6A) is targeted by loss-of-function mutations in multiple cancers. Here, we demonstrate that UTX suppresses myeloid leukemogenesis through noncatalytic functions, a property shared with its catalytically inactive Y-chromosome paralog, UTY (or KDM6C). In keeping with this, we demonstrate concomitant loss/mutation of KDM6A (UTX) and UTY in multiple human cancers. Mechanistically, global genomic profiling showed only minor changes in H3K27me3 but significant and bidirectional alterations in H3K27ac and chromatin accessibility; a predominant loss of H3K4me1 modifications; alterations in ETS and GATA-factor binding; and altered gene expression after Utx loss. By integrating proteomic and genomic analyses, we link these changes to UTX regulation of ATP-dependent chromatin remodeling, coordination of the COMPASS complex and enhanced pioneering activity of ETS factors during evolution to AML. Collectively, our findings identify a dual role for UTX in suppressing acute myeloid leukemia via repression of oncogenic ETS and upregulation of tumor-suppressive GATA programs.


Assuntos
Cromatina/genética , Elementos Facilitadores Genéticos , Fatores de Transcrição GATA/genética , Histona Desmetilases/genética , Leucemia Mieloide/genética , Proteínas Proto-Oncogênicas c-ets/genética , Animais , Linhagem Celular , Montagem e Desmontagem da Cromatina/genética , Regulação Leucêmica da Expressão Gênica , Células HEK293 , Histonas/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteômica/métodos , Sequências Reguladoras de Ácido Nucleico/genética , Ativação Transcricional
6.
Oncotarget ; 9(24): 16917-16931, 2018 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-29682194

RESUMO

Fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) is one of the most common genetic lesions in acute myeloid leukemia patients (AML). Although FLT3 tyrosine kinase inhibitors initially exhibit clinical activity, resistance to treatment inevitably occurs within months. PIM kinases are thought to be major drivers of the resistance phenotype and their inhibition in relapsed samples restores cell sensitivity to FLT3 inhibitors. Thus, simultaneous PIM and FLT3 inhibition represents a promising strategy in AML therapy. For such reasons, we have developed SEL24-B489 - a potent, dual PIM and FLT3-ITD inhibitor. SEL24-B489 exhibited significantly broader on-target activity in AML cell lines and primary AML blasts than selective FLT3-ITD or PIM inhibitors. SEL24-B489 also demonstrated marked activity in cells bearing FLT3 tyrosine kinase domain (TKD) mutations that lead to FLT3 inhibitor resistance. Moreover, SEL24-B489 inhibited the growth of a broad panel of AML cell lines in xenograft models with a clear pharmacodynamic-pharmacokinetic relationship. Taken together, our data highlight the unique dual activity of the SEL24-B489 that abrogates the activity of signaling circuits involved in proliferation, inhibition of apoptosis and protein translation/metabolism. These results underscore the therapeutic potential of the dual PIM/FLT3-ITD inhibitor for the treatment of AML.

7.
Cell Rep ; 17(4): 1193-1205, 2016 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-27760321

RESUMO

Acute myeloid leukemia (AML) is an aggressive cancer with a poor prognosis, for which mainstream treatments have not changed for decades. To identify additional therapeutic targets in AML, we optimize a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) screening platform and use it to identify genetic vulnerabilities in AML cells. We identify 492 AML-specific cell-essential genes, including several established therapeutic targets such as DOT1L, BCL2, and MEN1, and many other genes including clinically actionable candidates. We validate selected genes using genetic and pharmacological inhibition, and chose KAT2A as a candidate for downstream study. KAT2A inhibition demonstrated anti-AML activity by inducing myeloid differentiation and apoptosis, and suppressed the growth of primary human AMLs of diverse genotypes while sparing normal hemopoietic stem-progenitor cells. Our results propose that KAT2A inhibition should be investigated as a therapeutic strategy in AML and provide a large number of genetic vulnerabilities of this leukemia that can be pursued in downstream studies.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Testes Genéticos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Terapia de Alvo Molecular , Adulto , Apoptose , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células , Histona Acetiltransferases/antagonistas & inibidores , Histona Acetiltransferases/metabolismo , Humanos , Reprodutibilidade dos Testes
8.
Cell Cycle ; 15(16): 2108-2114, 2016 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-27399214

RESUMO

The first hematopoietic cells are generated very early in ontogeny to support the growth of the embryo and to provide the foundation to the adult hematopoietic system. There is a considerable therapeutic interest in understanding how these first blood cells are generated in order to try to reproduce this process in vitro. This would allow generating blood products, or hematopoietic cell populations from embryonic stem (ES) cells, induced pluripotent stem cells or through directed reprogramming. Recent studies have clearly established that the first hematopoietic cells originate from a hemogenic endothelium (HE) through an endothelial to hematopoietic transition (EHT). The molecular mechanisms underlining this transition remain largely unknown with the exception that the transcription factor RUNX1 is critical for this process. In this Extra Views report, we discuss our recent studies demonstrating that the transcriptional repressors GFI1 and GFI1B have a critical role in the EHT. We established that these RUNX1 transcriptional targets are actively implicated in the downregulation of the endothelial program and the loss of endothelial identity during the formation of the first blood cells. In addition, our results suggest that GFI1 expression provides an ideal novel marker to identify, isolate and study the HE cell population.


Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Hemangioblastos/metabolismo , Hematopoese , Células-Tronco Hematopoéticas/metabolismo , Fatores de Transcrição/metabolismo , Animais , Modelos Biológicos
9.
Nat Cell Biol ; 18(1): 21-32, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26619147

RESUMO

In vertebrates, the first haematopoietic stem cells (HSCs) with multi-lineage and long-term repopulating potential arise in the AGM (aorta-gonad-mesonephros) region. These HSCs are generated from a rare and transient subset of endothelial cells, called haemogenic endothelium (HE), through an endothelial-to-haematopoietic transition (EHT). Here, we establish the absolute requirement of the transcriptional repressors GFI1 and GFI1B (growth factor independence 1 and 1B) in this unique trans-differentiation process. We first demonstrate that Gfi1 expression specifically defines the rare population of HE that generates emerging HSCs. We further establish that in the absence of GFI1 proteins, HSCs and haematopoietic progenitor cells are not produced in the AGM, revealing the critical requirement for GFI1 proteins in intra-embryonic EHT. Finally, we demonstrate that GFI1 proteins recruit the chromatin-modifying protein LSD1, a member of the CoREST repressive complex, to epigenetically silence the endothelial program in HE and allow the emergence of blood cells.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Embrião de Mamíferos/metabolismo , Hemangioblastos/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Histona Desmetilases/metabolismo , Fatores de Transcrição/metabolismo , Animais , Aorta/citologia , Aorta/embriologia , Diferenciação Celular/fisiologia , Embrião de Mamíferos/citologia , Hemangioblastos/citologia , Células-Tronco Hematopoéticas/citologia , Camundongos
10.
Blood ; 120(2): 314-22, 2012 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-22668850

RESUMO

Recent studies have established that during embryonic development, hematopoietic progenitors and stem cells are generated from hemogenic endothelium precursors through a process termed endothelial to hematopoietic transition (EHT). The transcription factor RUNX1 is essential for this process, but its main downstream effectors remain largely unknown. Here, we report the identification of Gfi1 and Gfi1b as direct targets of RUNX1 and critical regulators of EHT. GFI1 and GFI1B are able to trigger, in the absence of RUNX1, the down-regulation of endothelial markers and the formation of round cells, a morphologic change characteristic of EHT. Conversely, blood progenitors in Gfi1- and Gfi1b-deficient embryos maintain the expression of endothelial genes. Moreover, those cells are not released from the yolk sac and disseminated into embryonic tissues. Taken together, our findings demonstrate a critical and specific role of the GFI1 transcription factors in the first steps of the process leading to the generation of hematopoietic progenitors from hemogenic endothelium.


Assuntos
Proteínas de Ligação a DNA/fisiologia , Hemangioblastos/citologia , Hemangioblastos/fisiologia , Hematopoese/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Repressoras/fisiologia , Fatores de Transcrição/fisiologia , Animais , Diferenciação Celular , Linhagem Celular , Subunidade alfa 2 de Fator de Ligação ao Core/deficiência , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Subunidade alfa 2 de Fator de Ligação ao Core/fisiologia , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/fisiologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos , Camundongos Knockout , Modelos Biológicos , Neovascularização Fisiológica , Gravidez , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , Proteínas Repressoras/deficiência , Proteínas Repressoras/genética , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Saco Vitelino/citologia , Saco Vitelino/embriologia , Saco Vitelino/fisiologia
11.
Antioxid Redox Signal ; 16(2): 113-27, 2012 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-21827279

RESUMO

AIMS: Heme oxygenase-1 (HMOX1) is a cytoprotective enzyme degrading heme to biliverdin, iron ions, and carbon monoxide, whose expression is induced in response to oxidative stress. Its overexpression has been suggested as a strategy improving survival of transplanted muscle precursors. RESULTS: Here we demonstrated that HMOX1 inhibits differentiation of myoblasts and modulates miRNA processing: downregulates Lin28 and DGCR8, lowers the total pool of cellular miRNAs, and specifically blocks induction of myomirs. Genetic or pharmacological activation of HMOX1 in C2C12 cells reduces the abundance of miR-1, miR-133a, miR-133b, and miR-206, which is accompanied by augmented production of SDF-1 and miR-146a, decreased expression of MyoD, myogenin, and myosin, and disturbed formation of myotubes. Similar relationships between HMOX1 and myomirs were demonstrated in murine primary satellite cells isolated from skeletal muscles of HMOX1(+/+), HMOX1(+/-), and HMOX1(-/-) mice or in human rhabdomyosarcoma cell lines. Inhibition of myogenic development is independent of antioxidative properties of HMOX1. Instead it is mediated by CO-dependent inhibition of c/EBPδ binding to myoD promoter, can be imitated by SDF-1, and partially reversed by enforced expression of miR-133b and miR-206. Control C2C12 myoblasts injected to gastrocnemius muscles of NOD-SCID mice contribute to formation of muscle fibers. In contrast, HMOX1 overexpressing C2C12 myoblasts form fast growing, hyperplastic tumors, infiltrating the surrounding tissues, and disseminating to the lungs. INNOVATION: We evidenced for the first time that HMOX1 inhibits differentiation of myoblasts, affects the miRNA processing enzymes, and modulates the miRNA transcriptome. CONCLUSION: HMOX1 improves the survival of myoblasts, but concurrently through regulation of myomirs, may act similarly to oncogenes, increasing the risk of hyperplastic growth of myogenic precursors.


Assuntos
Diferenciação Celular/fisiologia , Heme Oxigenase-1/fisiologia , Mioblastos/citologia , Animais , Linhagem Celular , Humanos , Camundongos , Camundongos Knockout , MicroRNAs/genética , MicroRNAs/metabolismo , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , Transcriptoma
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