RESUMO
The twenty-three Fanconi anemia (FA) proteins cooperate in the FA/BRCA pathway to repair DNA interstrand cross-links (ICLs). The cell division cycle and apoptosis regulator 1 (CCAR1) protein is also a regulator of ICL repair, though its possible function in the FA/BRCA pathway remains unknown. Here, we demonstrate that CCAR1 plays a unique upstream role in the FA/BRCA pathway and is required for FANCA protein expression in human cells. Interestingly, CCAR1 co-immunoprecipitates with FANCA pre-mRNA and is required for FANCA mRNA processing. Loss of CCAR1 results in retention of a poison exon in the FANCA transcript, thereby leading to reduced FANCA protein expression. A unique domain of CCAR1, the EF hand domain, is required for interaction with the U2AF heterodimer of the spliceosome and for excision of the poison exon. Taken together, CCAR1 is a splicing modulator required for normal splicing of the FANCA mRNA and other mRNAs involved in various cellular pathways.
Assuntos
Proteínas Reguladoras de Apoptose , Proteínas de Ciclo Celular , Proteína do Grupo de Complementação A da Anemia de Fanconi , Anemia de Fanconi , Splicing de RNA , Fator de Processamento U2AF , Humanos , Proteína BRCA1/metabolismo , Proteína BRCA1/genética , Proteína BRCA2/metabolismo , Proteína BRCA2/genética , Reparo do DNA , Endodesoxirribonucleases , Éxons , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação A da Anemia de Fanconi/genética , Proteína do Grupo de Complementação A da Anemia de Fanconi/metabolismo , Células HEK293 , Células HeLa , Ligação Proteica , Precursores de RNA/metabolismo , Precursores de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Spliceossomos/metabolismo , Spliceossomos/genética , Fator de Processamento U2AF/metabolismo , Fator de Processamento U2AF/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismoRESUMO
Hematopoietic stem cells (HSCs) undergo progressive functional decline over time due to both internal and external stressors, leading to aging of the hematopoietic system. A comprehensive understanding of the molecular mechanisms underlying HSC aging will be valuable in developing novel therapies for HSC rejuvenation and to prevent the onset of several age-associated diseases and hematological malignancies. This review considers the general causes of HSC aging that range from cell-intrinsic factors to cell-extrinsic factors. In particular, epigenetics and inflammation have been implicated in the linkage of HSC aging, clonality, and oncogenesis. The challenges in clarifying mechanisms of HSC aging have accelerated the development of therapeutic interventions to rejuvenate HSCs, the major goal of aging research; these details are also discussed in this review.
Assuntos
Senescência Celular , Células-Tronco Hematopoéticas , Epigênese Genética , Hematopoese , Células-Tronco Hematopoéticas/fisiologiaRESUMO
Clonal hematopoiesis of indeterminate potential (CHIP) is an age-associated phenomenon characterized by clonal expansion of blood cells harboring somatic mutations in hematopoietic genes, including DNMT3A, TET2, and ASXL1. Clinical evidence suggests that CHIP is highly prevalent and associated with poor prognosis in solid-tumor patients. However, whether blood cells with CHIP mutations play a causal role in promoting the development of solid tumors remained unclear. Using conditional knock-in mice that express CHIP-associated mutant Asxl1 (Asxl1-MT), we showed that expression of Asxl1-MT in T cells, but not in myeloid cells, promoted solid-tumor progression in syngeneic transplantation models. We also demonstrated that Asxl1-MT-expressing blood cells accelerated the development of spontaneous mammary tumors induced by MMTV-PyMT. Intratumor analysis of the mammary tumors revealed the reduced T-cell infiltration at tumor sites and programmed death receptor-1 (PD-1) upregulation in CD8+ T cells in MMTV-PyMT/Asxl1-MT mice. In addition, we found that Asxl1-MT induced T-cell dysregulation, including aberrant intrathymic T-cell development, decreased CD4/CD8 ratio, and naïve-memory imbalance in peripheral T cells. These results indicate that Asxl1-MT perturbs T-cell development and function, which contributes to creating a protumor microenvironment for solid tumors. Thus, our findings raise the possibility that ASXL1-mutated blood cells exacerbate solid-tumor progression in ASXL1-CHIP carriers.
Assuntos
Hematopoiese Clonal , Neoplasias , Proteínas Repressoras , Animais , Linfócitos T CD8-Positivos/metabolismo , Hematopoiese Clonal/genética , Hematopoese/genética , Camundongos , Mutação , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição/genética , Microambiente TumoralRESUMO
Additional sex combs-like 1 (ASXL1), an epigenetic modulator, is frequently mutated in myeloid neoplasms. Recent analyses of mutant ASXL1 conditional knockin (ASXL1-MT-KI) mice suggested that ASXL1-MT alone is insufficient for myeloid transformation. In our previous study, we used retrovirus-mediated insertional mutagenesis, which exhibited the susceptibility of ASXL1-MT-KI hematopoietic cells to transform into myeloid leukemia cells. In this screening, we identified the hematopoietically expressed homeobox (HHEX) gene as one of the common retrovirus integration sites. In this study, we investigated the potential cooperation between ASXL1-MT and HHEX in myeloid leukemogenesis. Expression of HHEX enhanced proliferation of ASXL1-MT-expressing HSPCs by inhibiting apoptosis and blocking differentiation, whereas it showed only modest effect in normal HSPCs. Moreover, ASXL1-MT and HHEX accelerated the development of RUNX1-ETO9a and FLT3-ITD leukemia. Conversely, HHEX depletion profoundly attenuated the colony-forming activity and leukemogenicity of ASXL1-MT-expressing leukemia cells. Mechanistically, we identified MYB and ETV5 as downstream targets for ASXL1-MT and HHEX by using transcriptome and chromatin immunoprecipitation-next-generation sequencing analyses. Moreover, we found that expression of ASXL1-MT enhanced the binding of HHEX to the promoter loci of MYB or ETV5 via reducing H2AK119ub. Depletion of MYB or ETV5 induced apoptosis or differentiation in ASXL1-MT-expressing leukemia cells, respectively. In addition, ectopic expression of MYB or ETV5 reversed the reduced colony-forming activity of HHEX-depleted ASXL1-MT-expressing leukemia cells. These findings indicate that the HHEX-MYB/ETV5 axis promotes myeloid transformation in ASXL1-mutated preleukemia cells.
Assuntos
Transformação Celular Neoplásica/genética , Predisposição Genética para Doença , Proteínas de Homeodomínio/genética , Mutação , Células Mieloides/metabolismo , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Animais , Apoptose/genética , Biomarcadores Tumorais , Biópsia , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Ciclo Celular/genética , Diferenciação Celular/genética , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica/metabolismo , Ensaio de Unidades Formadoras de Colônias , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Estudos de Associação Genética , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Imunofenotipagem , Leucemia Mieloide/genética , Leucemia Mieloide/metabolismo , Leucemia Mieloide/mortalidade , Leucemia Mieloide/patologia , Camundongos , Células Mieloides/patologia , Prognóstico , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Recent genome-wide studies have revealed that aging or chronic inflammation can cause clonal expansion of cells in normal tissues. Clonal hematopoiesis has been the most intensively studied form of clonal expansion in the last decade. Clonal hematopoiesis of indeterminate potential (CHIP) is an age-related phenomenon observed in elderly individuals with no history of hematological malignancy. The most frequently mutated genes in CHIP are DNMT3A, TET2, and ASXL1, which are associated with initiation of leukemia. Importantly, CHIP has been the focus of a number of studies because it is an independent risk factor for myeloid malignancy, cardiovascular disease (CVD), and all-cause mortality. Animal models recapitulating human CHIP revealed that CHIP-associated mutations alter the number and function of hematopoietic stem and progenitor cells (HSPCs) and promote leukemic transformation. Moreover, chronic inflammation caused by infection or aging confers a fitness advantage to the CHIP-associated mutant HSPCs. Myeloid cells, such as macrophages with a CHIP-associated mutation, accelerate chronic inflammation and are associated with increased levels of inflammatory cytokines. This positive feedback loop between CHIP and chronic inflammation promotes development of atherosclerosis and chronic heart failure and thereby increases the risk for CVD. Notably, HSPCs with a CHIP-associated mutation may alter not only innate but also acquired immune cells. This suggests that CHIP is involved in the development of solid cancers or immune disorders, such as aplastic anemia. In this review, we provide an overview of recent findings on CHIP. We also discuss potential interventions for treating CHIP and preventing myeloid transformation and CVD progression.
Assuntos
Doenças Cardiovasculares/genética , Hematopoiese Clonal/genética , Mutação , Neoplasias/genética , Idoso , Envelhecimento/sangue , Animais , Aterosclerose/genética , Transformação Celular Neoplásica , Doença Crônica , Citocinas/sangue , DNA (Citosina-5-)-Metiltransferases/genética , DNA Metiltransferase 3A , Proteínas de Ligação a DNA/genética , Dioxigenases , Células-Tronco Hematopoéticas/fisiologia , Humanos , Inflamação/sangue , Inflamação/complicações , Inflamação/genética , Leucemia Mieloide/genética , Leucemia Mieloide/patologia , Macrófagos/patologia , Camundongos , Modelos Animais , Proteínas Proto-Oncogênicas/genética , Proteínas Repressoras/genética , Fatores de RiscoRESUMO
Recent high-throughput genome-wide sequencing studies have identified recurrent somatic mutations in myeloid neoplasms. An epigenetic regulator, Additional sex combs-like 1 (ASXL1), is one of the most frequently mutated genes in all subtypes of myeloid malignancies. ASXL1 mutations are also frequently detected in clonal hematopoiesis, which is associated with an increased risk of mortality. Therefore, it is important to understand how ASXL1 mutations contribute to clonal expansion and myeloid transformation in hematopoietic cells. Studies using ASXL1-depleted human hematopoietic cells and Asxl1 knockout mice have shown that deletion of wild-type ASXL1 protein leads to impaired hematopoiesis and accelerates myeloid malignancies via loss of interaction with polycomb repressive complex 2 proteins. On the other hand, ASXL1 mutations in myeloid neoplasms typically occur near the last exon and result in the expression of C-terminally truncated mutant ASXL1 protein. Biological studies and biochemical analyses of this variant have shed light on its dominant-negative and gain-of-function features in myeloid transformation via a variety of epigenetic changes. Based on these results, it would be possible to establish novel promising therapeutic strategies for myeloid malignancies harboring ASXL1 mutations by blocking interactions between ASXL1 and associating epigenetic regulators. Here, we summarize the clinical implications of ASXL1 mutations, the role of wild-type ASXL1 in normal hematopoiesis, and oncogenic functions of mutant ASXL1 in myeloid neoplasms.
Assuntos
Hematopoese , Mutação , Síndromes Mielodisplásicas/fisiopatologia , Proteínas Repressoras/metabolismo , Animais , Humanos , Síndromes Mielodisplásicas/metabolismo , Proteínas Repressoras/genéticaRESUMO
Mutations in ASXL1, which occur frequently in myeloid neoplasms, often confer poor prognosis. Despite their clinical importance, the precise molecular mechanisms underlying the contribution of mutant ASXL1 to cancer pathogenesis remain to be elucidated. Thus, we analyzed the roles of the hyperactive complex formed by mutant ASXL1 and the deubiquitinase BAP1 in promoting myeloid leukemogenesis. BAP1 expression resulted in the stabilization and increased monoubiquitination of mutant but not wildtype ASXL1. Monoubiquitination of mutant ASXL1 enhanced the catalytic function of BAP1, resulting in a profound reduction in H2AK119ub by counteracting the PRC1 complex. The mutant ASXL1-BAP1 hyperactive complex impaired the multi-lineage differentiation of hematopoietic progenitor cells and accelerated myeloid leukemogenesis. Mechanistically, the mutant ASXL1/BAP1 complex induced the upregulation of HOXA5, HOXA7, HOXA9, and IRF8 via a reduction in H2AK119ub. Importantly, BAP1 depletion inhibited the leukemogenicity of mutant ASXL1-expressing myeloid leukemia cells and MLL-rearranged leukemia cells by reducing the expression levels of HOXA5, HOXA7, and HOXA9. Our findings highlight the potential of BAP1 as a therapeutic target in a broad range of myeloid neoplasms.
Assuntos
Leucemia Mieloide , Transtornos Mieloproliferativos , Humanos , Proteínas Repressoras , Proteínas Supressoras de Tumor , Ubiquitina TiolesteraseRESUMO
RUNX1 is a member of RUNX transcription factors and plays important roles in hematopoiesis. Disruption of RUNX1 activity has been implicated in the development of hematopoietic neoplasms. Chromosomal translocations involving the RUNX1 gene are associated with several types of leukemia, including acute myeloid leukemia driven by a leukemogenic fusion protein RUNX1-RUNX1T1. Previous studies have shown that RUNX1 is an unstable protein and is subjected to proteolytic degradation mediated by the ubiquitin-proteasome pathway. However, the precise mechanisms of RUNX1 ubiquitination have not been fully understood. Furthermore, much less is known about the mechanisms to regulate the stability of RUNX1-RUNX1T1. In this study, we identified several RUNX1-interacting E3 ubiquitin ligases using a novel high-throughput binding assay. Among them, we found that STUB1 bound to RUNX1 and induced its ubiquitination and degradation mainly in the nucleus. Immunofluorescence analyses revealed that the STUB1-induced ubiquitination also promoted nuclear export of RUNX1, which probably contributes to the reduced transcriptional activity of RUNX1 in STUB1-overexpressing cells. STUB1 also induced ubiquitination of RUNX1-RUNX1T1 and down-regulated its expression. Importantly, STUB1 overexpression showed a substantial growth-inhibitory effect in myeloid leukemia cells that harbor RUNX1-RUNX1T1, whereas it showed only a marginal effect in other non-RUNX1-RUNX1T1 leukemia cells and normal human cord blood cells. Taken together, these data suggest that the E3 ubiquitin ligase STUB1 is a negative regulator of both RUNX1 and RUNX1-RUNX1T1. Activation of STUB1 could be a promising therapeutic strategy for RUNX1-RUNX1T1 leukemia.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Humanos , Estabilidade Proteica , Proteína 1 Parceira de Translocação de RUNX1RESUMO
JADE is a core subunit of the HBO1 acetyltransferase complex that regulates developmental and epigenetic programs and promotes gene transcription. Here we describe the mechanism by which JADE facilitates recruitment of the HBO1 complex to chromatin and mediates its enzymatic activity. Structural, genomic and complex assembly in vivo studies show that the PZP (PHD1-zinc-knuckle-PHD2) domain of JADE engages the nucleosome through binding to histone H3 and DNA and is necessary for the association with chromatin targets. Recognition of unmethylated H3K4 by PZP directs enzymatic activity of the complex toward histone H4 acetylation, whereas H3K4 hypermethylation alters histone substrate selectivity. We demonstrate that PZP contributes to leukemogenesis, augmenting transforming activity of the NUP98-JADE2 fusion. Our findings highlight biological consequences and the impact of the intact JADE subunit on genomic recruitment, enzymatic function and pathological activity of the HBO1 complex.
Assuntos
Histona Acetiltransferases , Histonas , Humanos , Histonas/metabolismo , Histona Acetiltransferases/metabolismo , Histona Acetiltransferases/genética , Animais , Cromatina/metabolismo , Acetilação , Camundongos , Nucleossomos/metabolismo , Ligação Proteica , Metilação , Modelos Moleculares , Domínios Proteicos , Proteínas de Homeodomínio , Proteínas Supressoras de TumorRESUMO
Decitabine (DAC) is clinically used to treat myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Our genome-wide CRISPR-dCas9 activation screen using MDS-derived AML cells indicates that mitotic regulation is critical for DAC resistance. DAC strongly induces abnormal mitosis (abscission failure or tripolar mitosis) in human myeloid tumors at clinical concentrations, especially in those with TP53 mutations or antecedent hematological disorders. This DAC-induced mitotic disruption and apoptosis are significantly attenuated in DNMT1-depleted cells. In contrast, overexpression of Dnmt1, but not the catalytically inactive mutant, enhances DAC-induced mitotic defects in myeloid tumors. We also demonstrate that DAC-induced mitotic disruption is enhanced by pharmacological inhibition of the ATR-CLSPN-CHK1 pathway. These data challenge the current assumption that DAC inhibits leukemogenesis through DNMT1 inhibition and subsequent DNA hypomethylation and highlight the potent activity of DAC to disrupt mitosis through aberrant DNMT1-DNA covalent bonds.
Assuntos
Azacitidina , Leucemia Mieloide Aguda , Humanos , Decitabina/farmacologia , Decitabina/uso terapêutico , Azacitidina/farmacologia , Azacitidina/uso terapêutico , Antimetabólitos Antineoplásicos/farmacologia , Leucemia Mieloide Aguda/patologia , Metilação de DNA/genética , DNA , Proteínas Adaptadoras de Transdução de Sinal/genéticaRESUMO
Immunotherapy has attracted considerable attention as a therapeutic strategy for cancers including acute myeloid leukemia (AML). In this study, we found that the development of several aggressive subtypes of AML is slower in Rag2-/- mice despite the lack of B and T lymphocytes, even compared to the immunologically normal C57BL/6 mice. Furthermore, an orally active p53-activating drug shows stronger antileukemia effect on AML in Rag2-/- mice than C57BL/6 mice. Intriguingly, Natural Killer (NK) cells in Rag2-/- mice are increased in number, highly express activation markers, and show increased cytotoxicity to leukemia cells in a coculture assay. B2m depletion that triggers missing-self recognition of NK cells impairs the growth of AML cells in vivo. In contrast, NK cell depletion accelerates AML progression in Rag2-/- mice. Interestingly, immunogenicity of AML keeps changing during tumor evolution, showing a trend that the aggressive AMLs generate through serial transplantations are susceptible to NK cell-mediated tumor suppression in Rag2-/- mice. Thus, we show the critical role of NK cells in suppressing the development of certain subtypes of AML using Rag2-/- mice, which lack functional lymphocytes but have hyperactive NK cells.
Assuntos
Células Matadoras Naturais , Leucemia Mieloide Aguda , Animais , Camundongos , Camundongos Knockout , Camundongos Endogâmicos C57BL , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Linfócitos T , Proteínas de Ligação a DNA/genéticaRESUMO
Inosine monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme in de novo guanine nucleotide synthesis pathway. Although IMPDH inhibitors are widely used as effective immunosuppressants, their antitumor effects have not been proven in the clinical setting. Here, we found that acute myeloid leukemias (AMLs) with MLL-fusions are susceptible to IMPDH inhibitors in vitro. We also showed that alternate-day administration of IMPDH inhibitors suppressed the development of MLL-AF9-driven AML in vivo without having a devastating effect on immune function. Mechanistically, IMPDH inhibition induced overactivation of Toll-like receptor (TLR)-TRAF6-NF-κB signaling and upregulation of an adhesion molecule VCAM1, which contribute to the antileukemia effect of IMPDH inhibitors. Consequently, combined treatment with IMPDH inhibitors and the TLR1/2 agonist effectively inhibited the development of MLL-fusion AML. These findings provide a rational basis for clinical testing of IMPDH inhibitors against MLL-fusion AMLs and potentially other aggressive tumors with active TLR signaling.
Assuntos
Leucemia Mieloide Aguda , Proteína de Leucina Linfoide-Mieloide , Humanos , Proteína de Leucina Linfoide-Mieloide/metabolismo , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/patologia , Inibidores Enzimáticos/farmacologia , NF-kappa B , Imunossupressores/uso terapêuticoRESUMO
Leukemia stem cells (LSCs) in chronic myeloid leukemia (CML) are quiescent, insensitive to BCR-ABL1 tyrosine kinase inhibitors (TKIs) and responsible for CML relapse. Therefore, eradicating quiescent CML LSCs is a major goal in CML therapy. Here, using a G0 marker (G0M), we narrow down CML LSCs as G0M- and CD27- double positive cells among the conventional CML LSCs. Whole transcriptome analysis reveals NF-κB activation via inflammatory signals in imatinib-insensitive quiescent CML LSCs. Blocking NF-κB signals by inhibitors of interleukin-1 receptor-associated kinase 1/4 (IRAK1/4 inhibitors) together with imatinib eliminates mouse and human CML LSCs. Intriguingly, IRAK1/4 inhibitors attenuate PD-L1 expression on CML LSCs, and blocking PD-L1 together with imatinib also effectively eliminates CML LSCs in the presence of T cell immunity. Thus, IRAK1/4 inhibitors can eliminate CML LSCs through inhibiting NF-κB activity and reducing PD-L1 expression. Collectively, the combination of TKIs and IRAK1/4 inhibitors is an attractive strategy to achieve a radical cure of CML.
Assuntos
Quinases Associadas a Receptores de Interleucina-1/efeitos dos fármacos , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mieloide/tratamento farmacológico , Células-Tronco Neoplásicas/metabolismo , Animais , Antineoplásicos/farmacologia , Doença Crônica , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Proteínas de Fusão bcr-abl/metabolismo , Humanos , Mesilato de Imatinib/farmacologia , Inibidores de Checkpoint Imunológico/farmacologia , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Inibidores de Proteínas Quinases/farmacologiaRESUMO
Myelodysplastic syndrome (MDS) is a clonal disorder of hematopoietic stem cells (HSCs), characterized by ineffective hematopoiesis and frequent progression to leukemia. It has long remained unresolved how MDS cells, which are less proliferative, inhibit normal hematopoiesis and eventually dominate the bone marrow space. Despite several studies implicating mesenchymal stromal or stem cells (MSCs), a principal component of the HSC niche, in the inhibition of normal hematopoiesis, the molecular mechanisms underlying this process remain unclear. Here, we demonstrate that both human and mouse MDS cells perturb bone metabolism by suppressing the osteolineage differentiation of MSCs, which impairs the ability of MSCs to support normal HSCs. Enforced MSC differentiation rescues the suppressed normal hematopoiesis in both in vivo and in vitro MDS models. Intriguingly, the suppression effect is reversible and mediated by extracellular vesicles (EVs) derived from MDS cells. These findings shed light on the novel MDS EV-MSC axis in ineffective hematopoiesis.
Assuntos
Vesículas Extracelulares , Células-Tronco Mesenquimais , Síndromes Mielodisplásicas , Animais , Vesículas Extracelulares/metabolismo , Hematopoese , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Mesenquimais/metabolismo , Camundongos , Síndromes Mielodisplásicas/metabolismoRESUMO
KMT2A-rearranged infant acute lymphoblastic leukemia (ALL) represents the most refractory type of childhood leukemia. To uncover the molecular heterogeneity of this disease, we perform RNA sequencing, methylation array analysis, whole exome and targeted deep sequencing on 84 infants with KMT2A-rearranged leukemia. Our multi-omics clustering followed by single-sample and single-cell inference of hematopoietic differentiation establishes five robust integrative clusters (ICs) with different master transcription factors, fusion partners and corresponding stages of B-lymphopoietic and early hemato-endothelial development: IRX-type differentiated (IC1), IRX-type undifferentiated (IC2), HOXA-type MLLT1 (IC3), HOXA-type MLLT3 (IC4), and HOXA-type AFF1 (IC5). Importantly, our deep mutational analysis reveals that the number of RAS pathway mutations predicts prognosis and that the most refractory subgroup of IC2 possesses 100% frequency and the heaviest burden of RAS pathway mutations. Our findings highlight the previously under-appreciated intra- and inter-patient heterogeneity of KMT2A-rearranged infant ALL and provide a rationale for the future development of genomics-guided risk stratification and individualized therapy.
Assuntos
Histona-Lisina N-Metiltransferase/genética , Proteína de Leucina Linfoide-Mieloide/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras , Fusão Gênica , Humanos , Lactente , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Fatores de Transcrição/genéticaRESUMO
Cell behavior is controlled by complex gene regulatory networks. Although studies have uncovered diverse roles of individual genes, it has been challenging to record or control sequential genetic events in living cells. In this study, we designed two cellular chain reaction systems that enable sequential sgRNA activation in mammalian cells using a nickase Cas9 tethering of a cytosine nucleotide deaminase (nCas9-CDA). In these systems, thymidine (T)-to-cytosine (C) substitutions in the scaffold region of the sgRNA or the TATA box-containing loxP sequence (TATAloxP) are corrected by the nCas9-CDA, leading to activation of the next sgRNA. These reactions can occur multiple times, resulting in cellular chain reactions. As a proof of concept, we established a chain reaction by repairing sgRNA scaffold mutations in 293 T cells. Importantly, the results obtained in yeast or in vitro did not match those obtained in mammalian cells, suggesting that in vivo chain reactions need to be optimized in appropriate cellular contexts. Our system may lay the foundation for building cellular chain reaction systems that have a broad utility in the future biomedical research.
Assuntos
Sistemas CRISPR-Cas , Reparo do DNA , Edição de Genes , Mutação , RNA Guia de Cinetoplastídeos/genética , Citosina Desaminase/genética , Citosina Desaminase/metabolismo , Células HEK293 , Humanos , TATA Box/genética , Timidina/genéticaRESUMO
Paraspeckles are membraneless organelles formed through liquid-liquid phase separation and consist of multiple proteins and RNAs, including NONO, SFPQ, and NEAT1. The role of paraspeckles and the component NONO in hematopoiesis remains unknown. In this study, we show histone modifier ASXL1 is involved in paraspeckle formation. ASXL1 forms phase-separated droplets, upregulates NEAT1 expression, and increases NONO-NEAT1 interactions through the C-terminal intrinsically disordered region (IDR). In contrast, a pathogenic ASXL mutant (ASXL1-MT) lacking IDR does not support the interaction of paraspeckle components. Furthermore, paraspeckles are disrupted and Nono localization is abnormal in the cytoplasm of hematopoietic stem and progenitor cells (HSPCs) derived from ASXL1-MT knockin mice. Nono depletion and the forced expression of cytoplasmic NONO impair the repopulating potential of HSPCs, as does ASXL1-MT. Our study indicates a link between ASXL1 and paraspeckle components in the maintenance of normal hematopoiesis.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Paraspeckles/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/metabolismo , Animais , Proteínas de Ligação a DNA/genética , Feminino , Células HL-60 , Células HeLa , Hematopoese , Humanos , Camundongos , Camundongos Transgênicos , Paraspeckles/genética , Proteínas de Ligação a RNA/genética , Proteínas Repressoras/genética , Células THP-1RESUMO
Somatic mutations of ASXL1 are frequently detected in age-related clonal hematopoiesis (CH). However, how ASXL1 mutations drive CH remains elusive. Using knockin (KI) mice expressing a C-terminally truncated form of ASXL1-mutant (ASXL1-MT), we examined the influence of ASXL1-MT on physiological aging in hematopoietic stem cells (HSCs). HSCs expressing ASXL1-MT display competitive disadvantage after transplantation. Nevertheless, in genetic mosaic mouse model, they acquire clonal advantage during aging, recapitulating CH in humans. Mechanistically, ASXL1-MT cooperates with BAP1 to deubiquitinate and activate AKT. Overactive Akt/mTOR signaling induced by ASXL1-MT results in aberrant proliferation and dysfunction of HSCs associated with age-related accumulation of DNA damage. Treatment with an mTOR inhibitor rapamycin ameliorates aberrant expansion of the HSC compartment as well as dysregulated hematopoiesis in aged ASXL1-MT KI mice. Our findings suggest that ASXL1-MT provokes dysfunction of HSCs, whereas it confers clonal advantage on HSCs over time, leading to the development of CH.
Assuntos
Envelhecimento/genética , Hematopoiese Clonal/genética , Hematopoese/genética , Células-Tronco Hematopoéticas/metabolismo , Proteínas Repressoras/genética , Serina-Treonina Quinases TOR/metabolismo , Idoso , Envelhecimento/metabolismo , Envelhecimento/fisiologia , Animais , Apoptose/genética , Ciclo Celular/genética , Proliferação de Células/genética , Células Cultivadas , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Técnicas de Introdução de Genes , Hematopoese/fisiologia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/fisiologia , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/genética , Camundongos , Camundongos Transgênicos , Mutação , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA-Seq , Espécies Reativas de Oxigênio/farmacologia , Proteínas Repressoras/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Sirolimo/farmacologia , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina Tiolesterase/metabolismo , Ubiquitinação/efeitos dos fármacos , Ubiquitinação/genéticaRESUMO
An epigenetic modulator Additional sex combs-like 1 (ASXL1) is recurrently mutated in myeloid neoplasms such as myelodysplastic syndromes (MDS), acute myeloid leukemia (AML) and myeloproliferative neoplasms (MPNs). ASXL1 mutations are also frequently detected in clonal hematopoiesis with indeterminate potential (CHIP), which is the clonal expansion of premalignant hematopoietic cells without any evidence of hematological malignancies. Thus, understanding the roles of ASXL1 in hematopoiesis and myeloid neoplasms is a clinically crucial issue. ASXL1 mutations in hematological neoplasms are typically frameshift or nonsense mutations and occur near the 5' end of the last exon, thereby the transcripts would escape from nonsense-mediated decay, Indeed, we identified the C-terminally truncated mutant protein of ASXL1 in several cell lines derived from patients with myeloid leukemia. In mouse models, expression of the mutant ASXL1 results in impaired hematopoiesis and promotes development of myeloid neoplasms. In addition, recent findings from biochemical analysis have demonstrated that the mutant ASXL1 protein gains new functions including enhancing catalytic activity of BRCA1-associated protein 1 (BAP1), resulting in reduction of H2AK119ub and aberrant gene expression essential for myeloid transformation. In this review, we will focus on the pivotal roles of the mutant ASXL1 on histone modifications and myeloid transformation.
Assuntos
Código das Histonas , Leucemia Mieloide Aguda/genética , Síndromes Mielodisplásicas/genética , Transtornos Mieloproliferativos/genética , Proteínas de Neoplasias/genética , Proteínas Repressoras/genética , Animais , Transformação Celular Neoplásica/genética , Códon sem Sentido , Mutação da Fase de Leitura , Mutação com Ganho de Função , Regulação Leucêmica da Expressão Gênica , Hematopoese/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Leucemia Experimental/genética , Leucemia Experimental/fisiopatologia , Camundongos , Terapia de Alvo Molecular , Complexos Multiproteicos/fisiologia , Proteínas de Neoplasias/fisiologia , Processamento de Proteína Pós-Traducional , Proteínas Repressoras/deficiência , Proteínas Repressoras/fisiologia , Relação Estrutura-Atividade , Proteínas Supressoras de Tumor/fisiologia , Ubiquitina Tiolesterase/fisiologia , UbiquitinaçãoRESUMO
Decitabine is a DNA methyltransferase inhibitor and is considered a promising drug to treat myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) with p53 mutations. However, whether loss of p53 in fact increases the response of MDS/AML cells to decitabine remains unclear. In this study, we assessed the role of p53 in MDS and AML cells treated with decitabine using mouse models for MLL-AF9-driven AML and mutant ASXL1-driven MDS/AML. CRISPR/Cas9-mediated depletion of p53 in MDS/AML cells did not increase, but rather decreased their sensitivity to decitabine. Forced expression of a dominant-negative p53 fragment (p53DD) in these cells also decreased their responses to decitabine, confirming that acute inhibition of p53 conferred resistance to decitabine in AML and MDS/AML cells. In contrast, MLL-AF9-expressing AML cells generated from bone marrow progenitors of Trp53-deficient mice were more sensitive to decitabine in vivo than their wild-type counterparts, suggesting that long-term chronic p53 deficiency increases decitabine sensitivity in AML cells. Taken together, these data revealed a multifaceted role for p53 to regulate responses of myeloid neoplasms to decitabine treatment.