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1.
Nat Immunol ; 25(6): 1007-1019, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38816617

RESUMO

Rare multipotent stem cells replenish millions of blood cells per second through a time-consuming process, passing through multiple stages of increasingly lineage-restricted progenitors. Although insults to the blood-forming system highlight the need for more rapid blood replenishment from stem cells, established models of hematopoiesis implicate only one mandatory differentiation pathway for each blood cell lineage. Here, we establish a nonhierarchical relationship between distinct stem cells that replenish all blood cell lineages and stem cells that replenish almost exclusively platelets, a lineage essential for hemostasis and with important roles in both the innate and adaptive immune systems. These distinct stem cells use cellularly, molecularly and functionally separate pathways for the replenishment of molecularly distinct megakaryocyte-restricted progenitors: a slower steady-state multipotent pathway and a fast-track emergency-activated platelet-restricted pathway. These findings provide a framework for enhancing platelet replenishment in settings in which slow recovery of platelets remains a major clinical challenge.


Assuntos
Plaquetas , Diferenciação Celular , Células-Tronco Hematopoéticas , Megacariócitos , Plaquetas/imunologia , Plaquetas/metabolismo , Animais , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Camundongos , Diferenciação Celular/imunologia , Megacariócitos/citologia , Linhagem da Célula , Camundongos Endogâmicos C57BL , Hematopoese , Trombopoese , Camundongos Knockout , Humanos , Células-Tronco Multipotentes/citologia , Células-Tronco Multipotentes/metabolismo , Células-Tronco Multipotentes/imunologia
2.
Nature ; 560(7717): 253-257, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-30069049

RESUMO

Acetylation of histones by lysine acetyltransferases (KATs) is essential for chromatin organization and function1. Among the genes coding for the MYST family of KATs (KAT5-KAT8) are the oncogenes KAT6A (also known as MOZ) and KAT6B (also known as MORF and QKF)2,3. KAT6A has essential roles in normal haematopoietic stem cells4-6 and is the target of recurrent chromosomal translocations, causing acute myeloid leukaemia7,8. Similarly, chromosomal translocations in KAT6B have been identified in diverse cancers8. KAT6A suppresses cellular senescence through the regulation of suppressors of the CDKN2A locus9,10, a function that requires its KAT activity10. Loss of one allele of KAT6A extends the median survival of mice with MYC-induced lymphoma from 105 to 413 days11. These findings suggest that inhibition of KAT6A and KAT6B may provide a therapeutic benefit in cancer. Here we present highly potent, selective inhibitors of KAT6A and KAT6B, denoted WM-8014 and WM-1119. Biochemical and structural studies demonstrate that these compounds are reversible competitors of acetyl coenzyme A and inhibit MYST-catalysed histone acetylation. WM-8014 and WM-1119 induce cell cycle exit and cellular senescence without causing DNA damage. Senescence is INK4A/ARF-dependent and is accompanied by changes in gene expression that are typical of loss of KAT6A function. WM-8014 potentiates oncogene-induced senescence in vitro and in a zebrafish model of hepatocellular carcinoma. WM-1119, which has increased bioavailability, arrests the progression of lymphoma in mice. We anticipate that this class of inhibitors will help to accelerate the development of therapeutics that target gene transcription regulated by histone acetylation.


Assuntos
Benzenossulfonatos/farmacologia , Senescência Celular/efeitos dos fármacos , Histona Acetiltransferases/antagonistas & inibidores , Hidrazinas/farmacologia , Linfoma/tratamento farmacológico , Linfoma/patologia , Sulfonamidas/farmacologia , Acetilação/efeitos dos fármacos , Animais , Benzenossulfonatos/uso terapêutico , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Desenvolvimento de Medicamentos , Fibroblastos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Histona Acetiltransferases/deficiência , Histona Acetiltransferases/genética , Histonas/química , Histonas/metabolismo , Hidrazinas/uso terapêutico , Linfoma/enzimologia , Linfoma/genética , Lisina/química , Lisina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Sulfonamidas/uso terapêutico
3.
Nature ; 538(7626): 477-482, 2016 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-27760111

RESUMO

Avoidance of apoptosis is critical for the development and sustained growth of tumours. The pro-survival protein myeloid cell leukemia 1 (MCL1) is overexpressed in many cancers, but the development of small molecules targeting this protein that are amenable for clinical testing has been challenging. Here we describe S63845, a small molecule that specifically binds with high affinity to the BH3-binding groove of MCL1. Our mechanistic studies demonstrate that S63845 potently kills MCL1-dependent cancer cells, including multiple myeloma, leukaemia and lymphoma cells, by activating the BAX/BAK-dependent mitochondrial apoptotic pathway. In vivo, S63845 shows potent anti-tumour activity with an acceptable safety margin as a single agent in several cancers. Moreover, MCL1 inhibition, either alone or in combination with other anti-cancer drugs, proved effective against several solid cancer-derived cell lines. These results point towards MCL1 as a target for the treatment of a wide range of tumours.


Assuntos
Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Modelos Biológicos , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Tiofenos/farmacologia , Tiofenos/uso terapêutico , Animais , Antineoplásicos/administração & dosagem , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Feminino , Humanos , Leucemia/tratamento farmacológico , Leucemia/metabolismo , Leucemia/patologia , Linfoma/tratamento farmacológico , Linfoma/metabolismo , Linfoma/patologia , Masculino , Camundongos , Modelos Moleculares , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/química , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Neoplasias/metabolismo , Pirimidinas/administração & dosagem , Tiofenos/administração & dosagem , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo
4.
Blood ; 132(15): 1573-1583, 2018 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-30139826

RESUMO

Myeloid cell leukemia-1 (MCL-1) is a prosurvival B-cell lymphoma 2 (BCL-2) family member required for the sustained growth of many cancers. Recently, a highly specific MCL-1 inhibitor, S63845, showing sixfold higher affinity to human compared with mouse MCL-1, has been described. To accurately test efficacy and tolerability of this BH3-mimetic (BH3-only protein mimetic) drug in preclinical cancer models, we developed a humanized Mcl-1 (huMcl-1) mouse strain in which MCL-1 was replaced with its human homolog. huMcl-1 mice are phenotypically indistinguishable from wild-type mice but are more sensitive to the MCL-1 inhibitor S63845. Importantly, nontransformed cells and lymphomas from huMcl-1;Eµ-Myc mice are more sensitive to S63845 in vitro than their control counterparts. When huMcl-1;Eµ-Myc lymphoma cells were transplanted into huMcl-1 mice, treatment with S63845 alone or alongside cyclophosphamide led to long-term remission in ∼60% or almost 100% of mice, respectively. These results demonstrate the potential of our huMcl-1 mouse model for testing MCL-1 inhibitors, allowing precise predictions of efficacy and tolerability for clinical translation.


Assuntos
Antineoplásicos/uso terapêutico , Linfoma/tratamento farmacológico , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Pirimidinas/uso terapêutico , Tiofenos/uso terapêutico , Alelos , Animais , Antineoplásicos/farmacologia , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos/métodos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Feminino , Humanos , Linfoma/metabolismo , Linfoma/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Pirimidinas/farmacologia , Tiofenos/farmacologia
5.
Cell Death Differ ; 31(2): 159-169, 2024 02.
Artigo em Inglês | MEDLINE | ID: mdl-38110554

RESUMO

Transcriptional activation of target genes is essential for TP53-mediated tumour suppression, though the roles of the diverse TP53-activated target genes in tumour suppression remains poorly understood. Knockdown of ZMAT3, an RNA-binding zinc-finger protein involved in regulating alternative splicing, in haematopoietic cells by shRNA caused leukaemia only with the concomitant absence of the PUMA and p21, the critical effectors of TRP53-mediated apoptosis and cell cycle arrest respectively. We were interested to further investigate the role of ZMAT3 in tumour suppression beyond the haematopoietic system. Therefore, we generated Zmat3 knockout and compound gene knockout mice, lacking Zmat3 and p21, Zmat3 and Puma or all three genes. Puma-/-p21-/-Zmat3-/- triple knockout mice developed tumours at a significantly higher frequency compared to wild-type, Puma-/-Zmat3-/- or p21-/-Zmat3-/-deficient mice. Interestingly, we observed that the triple knockout and Puma-/-Zmat3-/- double deficient animals succumbed to lymphoma, while p21-/-Zmat3-/- animals developed mainly solid cancers. This analysis suggests that in addition to ZMAT3 loss, additional TRP53-regulated processes must be disabled simultaneously for TRP53-mediated tumour suppression to fail. Our findings reveal that the absence of different TRP53 regulated tumour suppressive processes changes the tumour spectrum, indicating that different TRP53 tumour suppressive pathways are more critical in different tissues.


Assuntos
Neoplasias , Proteína Supressora de Tumor p53 , Animais , Camundongos , Apoptose/genética , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Incidência , Camundongos Knockout , Neoplasias/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
6.
Cell Death Dis ; 11(10): 877, 2020 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-33082333

RESUMO

TP53 is a critical tumor suppressor that is mutated in approximately 50% of human cancers. Unveiling the downstream target genes of TP53 that fulfill its tumor suppressor function is an area of intense investigation. Zmat3 (also known as Wig-1 or PAG608) is one such downstream target of p53, whose loss in hemopoietic stem cells lacking the apoptosis and cell cycle regulators, Puma and p21, respectively, promotes the development of leukemia. The function of Zmat3 in tumorigenesis however remains unclear. Here, to investigate which oncogenic drivers co-operate with Zmat3 loss to promote neoplastic transformation, we utilized Zmat3 knockout mice in models of c-MYC-driven lymphomagenesis and KrasG12D-driven lung adenocarcinoma development. Interestingly, unlike loss of p53, Zmat3 germline loss had little impact on the rate of tumor development or severity of malignant disease upon either the c-MYC or KrasG12D oncogenic activation. Furthermore, loss of Zmat3 failed to rescue KrasG12D primary lung tumor cells from oncogene-induced senescence. Taken together, we conclude that in the context of c-MYC-driven lymphomagenesis or mutant KrasG12D-driven lung adenocarcinoma development, additional co-occurring mutations are required to resolve Zmat3 tumor suppressive activity.


Assuntos
Adenocarcinoma de Pulmão/genética , Carcinogênese/genética , Proteínas de Ligação a DNA/genética , Neoplasias Pulmonares/genética , Mutação/genética , Proteínas de Ligação a RNA/genética , alfa-Amilases Salivares/genética , Adenocarcinoma/genética , Adenocarcinoma de Pulmão/metabolismo , Animais , Proliferação de Células/genética , Transformação Celular Neoplásica/genética , Neoplasias Pulmonares/patologia , Camundongos Transgênicos , Proteínas Proto-Oncogênicas p21(ras)/genética , Transdução de Sinais/genética
7.
Cell Death Differ ; 26(5): 902-917, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30042493

RESUMO

BCL2A1 is an anti-apoptotic member of the BCL-2 family that contributes to chemoresistance in a subset of tumors. BCL2A1 has a short half-life due to its constitutive processing by the ubiquitin-proteasome system. This constitutes a major tumor-suppressor mechanism regulating BCL2A1 function. However, the enzymes involved in the regulation of BCL2A1 protein stability are currently unknown. Here, we provide the first insight into the regulation of BCL2A1 ubiquitination. We present evidence that TRIM28 is an E3 ubiquitin-ligase for BCL2A1. Indeed, endogenous TRIM28 and BCL2A1 bind to each other at the mitochondria and TRIM28 knock-down decreases BCL2A1 ubiquitination. We also show that TRIM17 stabilizes BCL2A1 by blocking TRIM28 from binding and ubiquitinating BCL2A1, and that GSK3 is involved in the phosphorylation-mediated inhibition of BCL2A1 degradation. BCL2A1 and its close relative MCL1 are thus regulated by common factors but with opposite outcome. Finally, overexpression of TRIM28 or knock-out of TRIM17 reduced BCLA1 protein levels and restored sensitivity of melanoma cells to BRAF-targeted therapy. Therefore, our data describe a molecular rheostat in which two proteins of the TRIM family antagonistically regulate BCL2A1 stability and modulate cell death.


Assuntos
Apoptose/genética , Antígenos de Histocompatibilidade Menor/genética , Neoplasias/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas com Motivo Tripartido/genética , Proteína 28 com Motivo Tripartido/genética , Ubiquitina-Proteína Ligases/genética , Morte Celular/genética , Linhagem Celular Tumoral , Doxiciclina/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Quinase 3 da Glicogênio Sintase/genética , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Fosforilação/genética , Complexo de Endopeptidases do Proteassoma/genética , Ligação Proteica/genética , Estabilidade Proteica , Proteólise/efeitos dos fármacos , Ubiquitinação/genética
9.
Nat Med ; 24(7): 947-953, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29892060

RESUMO

It has long been assumed that p53 suppresses tumor development through induction of apoptosis, possibly with contributions by cell cycle arrest and cell senescence1,2. However, combined deficiency in these three processes does not result in spontaneous tumor formation as observed upon loss of p53, suggesting the existence of additional mechanisms that are critical mediators of p53-dependent tumor suppression function3-5. To define such mechanisms, we performed in vivo shRNA screens targeting p53-regulated genes in sensitized genetic backgrounds. We found that knockdown of Zmat3, Ctsf and Cav1, promoted lymphoma/leukemia development only when PUMA and p21, the critical effectors of p53-driven apoptosis, cell cycle arrest and senescence, were also absent. Notably, loss of the DNA repair gene Mlh1 caused lymphoma in a wild-type background, and its enforced expression was able to delay tumor development driven by loss of p53. Further examination of direct p53 target genes implicated in DNA repair showed that knockdown of Mlh1, Msh2, Rnf144b, Cav1 and Ddit4 accelerated MYC-driven lymphoma development to a similar extent as knockdown of p53. Collectively, these findings demonstrate that extensive functional overlap of several p53-regulated processes safeguards against cancer and that coordination of DNA repair appears to be an important process by which p53 suppresses tumor development.


Assuntos
Reparo do DNA , Proteína Supressora de Tumor p53/metabolismo , Animais , Reparo do DNA/genética , Células-Tronco Hematopoéticas/metabolismo , Estimativa de Kaplan-Meier , Camundongos Endogâmicos C57BL , Proteína 1 Homóloga a MutL/metabolismo , RNA Interferente Pequeno/metabolismo , Reprodutibilidade dos Testes
11.
Cell Rep ; 10(8): 1422-32, 2015 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-25732831

RESUMO

The CRISPR/Cas9 technology enables the introduction of genomic alterations into almost any organism; however, systems for efficient and inducible gene modification have been lacking, especially for deletion of essential genes. Here, we describe a drug-inducible small guide RNA (sgRNA) vector system allowing for ubiquitous and efficient gene deletion in murine and human cells. This system mediates the efficient, temporally controlled deletion of MCL-1, both in vitro and in vivo, in human Burkitt lymphoma cell lines that require this anti-apoptotic BCL-2 protein for sustained survival and growth. Unexpectedly, repeated induction of the same sgRNA generated similar inactivating mutations in the human Mcl-1 gene due to low mutation variability exerted by the accompanying non-homologous end-joining (NHEJ) process. Finally, we were able to generate hematopoietic cell compartment-restricted Trp53-knockout mice, leading to the identification of cancer-promoting mutants of this critical tumor suppressor.


Assuntos
Genes Essenciais/genética , Genes Neoplásicos/genética , Lentivirus/genética , Neoplasias/genética , RNA Guia de Cinetoplastídeos/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Sequência de Bases , Proteína 11 Semelhante a Bcl-2 , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Reparo do DNA por Junção de Extremidades , Doxiciclina/farmacologia , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Mutação , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transplante Heterólogo , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
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