RESUMO
The emergence of drug resistance is a major obstacle for the success of targeted therapy in melanoma. Additionally, conventional chemotherapy has not been effective as drug-resistant cells escape lethal DNA damage effects by inducing growth arrest commonly referred to as cellular dormancy. We present a therapeutic strategy termed "targeted chemotherapy" by depleting protein phosphatase 2A (PP2A) or its inhibition using a small molecule inhibitor (1,10-phenanthroline-5,6-dione [phendione]) in drug-resistant melanoma. Targeted chemotherapy induces the DNA damage response without causing DNA breaks or allowing cellular dormancy. Phendione treatment reduces tumor growth of BRAFV600E-driven melanoma patient-derived xenografts (PDX) and diminishes growth of NRASQ61R-driven melanoma, a cancer with no effective therapy. Remarkably, phendione treatment inhibits the acquisition of resistance to BRAF inhibition in BRAFV600E PDX highlighting its effectiveness in combating the advent of drug resistance.
Assuntos
Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Melanoma/tratamento farmacológico , Pirazóis/farmacologia , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Humanos , Melanoma/enzimologia , Melanoma/fisiopatologia , Proteína Fosfatase 2/antagonistas & inibidoresRESUMO
Patients treated with cytotoxic therapies, including autologous stem cell transplantation, are at risk for developing therapy-related myeloid neoplasms (tMN). Preleukemic clones (ie, clonal hematopoiesis [CH]) are detectable years before the development of these aggressive malignancies, although the genomic events leading to transformation and expansion are not well defined. Here, by leveraging distinctive chemotherapy-associated mutational signatures from whole-genome sequencing data and targeted sequencing of prechemotherapy samples, we reconstructed the evolutionary life-history of 39 therapy-related myeloid malignancies. A dichotomy was revealed, in which neoplasms with evidence of chemotherapy-induced mutagenesis from platinum and melphalan were hypermutated and enriched for complex structural variants (ie, chromothripsis), whereas neoplasms with nonmutagenic chemotherapy exposures were genomically similar to de novo acute myeloid leukemia. Using chemotherapy-associated mutational signatures as temporal barcodes linked to discrete clinical exposure in each patient's life, we estimated that several complex events and genomic drivers were acquired after chemotherapy was administered. For patients with prior multiple myeloma who were treated with high-dose melphalan and autologous stem cell transplantation, we demonstrate that tMN can develop from either a reinfused CH clone that escapes melphalan exposure and is selected after reinfusion, or from TP53-mutant CH that survives direct myeloablative conditioning and acquires melphalan-induced DNA damage. Overall, we revealed a novel mode of tMN progression that is not reliant on direct mutagenesis or even exposure to chemotherapy. Conversely, for tMN that evolve under the influence of chemotherapy-induced mutagenesis, distinct chemotherapies not only select preexisting CH but also promote the acquisition of recurrent genomic drivers.
Assuntos
Antineoplásicos , Transplante de Células-Tronco Hematopoéticas , Leucemia Mieloide Aguda , Segunda Neoplasia Primária , Humanos , Melfalan , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Transplante Autólogo/efeitos adversos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Segunda Neoplasia Primária/induzido quimicamente , Segunda Neoplasia Primária/genética , Antineoplásicos/farmacologiaRESUMO
Bromodomain-containing protein 4 (BRD4) is overexpressed and functionally implicated in various myeloid malignancies. However, the role of BRD4 in normal hematopoiesis remains largely unknown. Here, utilizing an inducible Brd4 knockout mouse model, we find that deletion of Brd4 (Brd4Δ/Δ ) in the hematopoietic system impairs hematopoietic stem cell (HSC) self-renewal and differentiation, which associates with cell cycle arrest and senescence. ATAC-seq analysis shows increased chromatin accessibility in Brd4Δ/Δ hematopoietic stem/progenitor cells (HSC/HPCs). Genome-wide mapping with cleavage under target and release using nuclease (CUT&RUN) assays demonstrate that increased global enrichment of H3K122ac and H3K4me3 in Brd4Δ/Δ HSC/HPCs is associated with the upregulation of senescence-specific genes. Interestingly, Brd4 deletion increases clipped H3 (cH3) which correlates with the upregulation of senescence-specific genes and results in a higher frequency of senescent HSC/HPCs. Re-expression of BRD4 reduces cH3 levels and rescues the senescence rate in Brd4Δ/Δ HSC/HPCs. This study unveils an important role of BRD4 in HSC/HPC function by preventing H3 clipping and suppressing senescence gene expression.
Assuntos
Histonas , Fatores de Transcrição , Animais , Camundongos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Histonas/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Senescência Celular/genética , Células-Tronco Hematopoéticas/metabolismo , Diferenciação Celular , HematopoeseRESUMO
SETD2, the histone H3 lysine 36 methyltransferase, previously identified by us, plays an important role in the pathogenesis of hematologic malignancies, but its role in myelodysplastic syndromes (MDSs) has been unclear. In this study, low expression of SETD2 correlated with shortened survival in patients with MDS, and the SETD2 levels in CD34+ bone marrow cells of those patients were increased by decitabine. We knocked out Setd2 in NUP98-HOXD13 (NHD13) transgenic mice, which phenocopies human MDS, and found that loss of Setd2 accelerated the transformation of MDS into acute myeloid leukemia (AML). Loss of Setd2 enhanced the ability of NHD13+ hematopoietic stem and progenitor cells (HSPCs) to self-renew, with increased symmetric self-renewal division and decreased differentiation and cell death. The growth of MDS-associated leukemia cells was inhibited though increasing the H3K36me3 level by using epigenetic modifying drugs. Furthermore, Setd2 deficiency upregulated hematopoietic stem cell signaling and downregulated myeloid differentiation pathways in the NHD13+ HSPCs. Our RNA-seq and chromatin immunoprecipitation-seq analysis indicated that S100a9, the S100 calcium-binding protein, is a target gene of Setd2 and that the addition of recombinant S100a9 weakens the effect of Setd2 deficiency in the NHD13+ HSPCs. In contrast, downregulation of S100a9 leads to decreases of its downstream targets, including Ikba and Jnk, which influence the self-renewal and differentiation of HSPCs. Therefore, our results demonstrated that SETD2 deficiency predicts poor prognosis in MDS and promotes the transformation of MDS into AML, which provides a potential therapeutic target for MDS-associated acute leukemia.
Assuntos
Anemia Refratária com Excesso de Blastos/patologia , Calgranulina B/fisiologia , Histona-Lisina N-Metiltransferase/deficiência , Histona-Lisina N-Metiltransferase/fisiologia , Leucemia Mieloide Aguda/etiologia , Anemia Refratária com Excesso de Blastos/genética , Anemia Refratária com Excesso de Blastos/metabolismo , Animais , Calgranulina B/biossíntese , Calgranulina B/genética , Transformação Celular Neoplásica , Células Cultivadas , Decitabina/farmacologia , Regulação para Baixo , Regulação Leucêmica da Expressão Gênica , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/patologia , Código das Histonas/efeitos dos fármacos , Histona-Lisina N-Metiltransferase/biossíntese , Histona-Lisina N-Metiltransferase/genética , Proteínas de Homeodomínio/genética , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/mortalidade , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Síndromes Mielodisplásicas/patologia , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Proteínas de Fusão Oncogênica/genética , Prognóstico , Proteínas Recombinantes/uso terapêutico , Fatores de Tempo , Análise Serial de Tecidos , TranscriptomaRESUMO
Protein arginine methyltransferase 5 (PRMT5) catalyzes the symmetric di-methylation of arginine residues in histones H3 and H4, marks that are generally associated with transcriptional repression. However, we found that PRMT5 inhibition or depletion led to more genes being downregulated than upregulated, indicating that PRMT5 can also act as a transcriptional activator. Indeed, the global level of histone H3K27me3 increases in PRMT5 deficient cells. Although PRMT5 does not directly affect PRC2 enzymatic activity, methylation of histone H3 by PRMT5 abrogates its subsequent methylation by PRC2. Treating AML cells with an EZH2 inhibitor partially restored the expression of approximately 50% of the genes that are initially downregulated by PRMT5 inhibition, suggesting that the increased H3K27me3 could directly or indirectly contribute to the transcription repression of these genes. Indeed, ChIP-sequencing analysis confirmed an increase in the H3K27me3 level at the promoter region of a quarter of these genes in PRMT5-inhibited cells. Interestingly, the anti-proliferative effect of PRMT5 inhibition was also partially rescued by treatment with an EZH2 inhibitor in several leukemia cell lines. Thus, PRMT5-mediated crosstalk between histone marks contributes to its functional effects.
Assuntos
Arginina/metabolismo , Histonas/metabolismo , Proteínas do Grupo Polycomb/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Transcrição Gênica , Animais , Ciclo Celular/genética , Linhagem Celular Tumoral , Deleção de Genes , Regulação da Expressão Gênica , Células-Tronco Hematopoéticas/metabolismo , Metilação , Camundongos Knockout , Modelos Biológicos , Nucleossomos/metabolismo , Proteína-Arginina N-Metiltransferases/antagonistas & inibidoresRESUMO
The AML1-ETO fusion protein, generated by the t(8;21) chromosomal translocation, is causally involved in nearly 20% of acute myeloid leukemia (AML) cases. In leukemic cells, AML1-ETO resides in and functions through a stable protein complex, AML1-ETO-containing transcription factor complex (AETFC), that contains multiple transcription (co)factors. Among these AETFC components, HEB and E2A, two members of the ubiquitously expressed E proteins, directly interact with AML1-ETO, confer new DNA-binding capacity to AETFC, and are essential for leukemogenesis. However, the third E protein, E2-2, is specifically silenced in AML1-ETO-expressing leukemic cells, suggesting E2-2 as a negative factor of leukemogenesis. Indeed, ectopic expression of E2-2 selectively inhibits the growth of AML1-ETO-expressing leukemic cells, and this inhibition requires the bHLH DNA-binding domain. RNA-seq and ChIP-seq analyses reveal that, despite some overlap, the three E proteins differentially regulate many target genes. In particular, studies show that E2-2 both redistributes AETFC to, and activates, some genes associated with dendritic cell differentiation and represses MYC target genes. In AML patients, the expression of E2-2 is relatively lower in the t(8;21) subtype, and an E2-2 target gene, THPO, is identified as a potential predictor of relapse. In a mouse model of human t(8;21) leukemia, E2-2 suppression accelerates leukemogenesis. Taken together, these results reveal that, in contrast to HEB and E2A, which facilitate AML1-ETO-mediated leukemogenesis, E2-2 compromises the function of AETFC and negatively regulates leukemogenesis. The three E proteins thus define a heterogeneity of AETFC, which improves our understanding of the precise mechanism of leukemogenesis and assists development of diagnostic/therapeutic strategies.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Leucemia Mieloide Aguda/etiologia , Proteínas de Fusão Oncogênica/metabolismo , Proteína 1 Parceira de Translocação de RUNX1/metabolismo , Proteína 2 Semelhante ao Fator 7 de Transcrição/metabolismo , Diferenciação Celular , Linhagem Celular Tumoral , Humanos , Leucemia Mieloide Aguda/metabolismo , RecidivaRESUMO
Additional Sex Combs-Like 1 (ASXL1) is mutated at a high frequency in all forms of myeloid malignancies associated with poor prognosis. We generated a Vav1 promoter-driven Flag-Asxl1Y588X transgenic mouse model, Asxl1Y588X Tg, to express a truncated FLAG-ASXL1aa1-587 protein in the hematopoietic system. The Asxl1Y588X Tg mice had an enlarged hematopoietic stem cell (HSC) pool, shortened survival, and predisposition to a spectrum of myeloid malignancies, thereby recapitulating the characteristics of myeloid malignancy patients with ASXL1 mutations. ATAC- and RNA-sequencing analyses revealed that the ASXL1aa1-587 truncating protein expression results in more open chromatin in cKit+ cells compared with wild-type cells, accompanied by dysregulated expression of genes critical for HSC self-renewal and differentiation. Liquid chromatography-tandem mass spectrometry and coimmunoprecipitation experiments showed that ASXL1aa1-587 acquired an interaction with BRD4. An epigenetic drug screening demonstrated a hypersensitivity of Asxl1Y588X Tg bone marrow cells to BET bromodomain inhibitors. This study demonstrates that ASXL1aa1-587 plays a gain-of-function role in promoting myeloid malignancies. Our model provides a powerful platform to test therapeutic approaches of targeting the ASXL1 truncation mutations in myeloid malignancies.
Assuntos
Mutação com Ganho de Função/genética , Leucemia Mieloide/genética , Proteínas Repressoras/genética , Animais , Células da Medula Óssea/metabolismo , Diferenciação Celular/genética , Linhagem da Célula/genética , Cromatina/metabolismo , Regulação Leucêmica da Expressão Gênica , Células-Tronco Hematopoéticas/metabolismo , Leucemia Mieloide/patologia , Camundongos Transgênicos , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica , Proteínas Proto-Oncogênicas c-kit/metabolismo , Proteínas Proto-Oncogênicas c-vav/metabolismo , Fatores de Transcrição/metabolismoRESUMO
HOX gene dysregulation is a common feature of acute myeloid leukemia (AML). The molecular mechanisms underlying aberrant HOX gene expression and associated AML pathogenesis remain unclear. The nuclear protein CCCTC-binding factor (CTCF), when bound to insulator sequences, constrains temporal HOX gene-expression patterns within confined chromatin domains for normal development. Here, we used targeted pooled CRISPR-Cas9-knockout library screening to interrogate the function of CTCF boundaries in the HOX gene loci. We discovered that the CTCF binding site located between HOXA7 and HOXA9 genes (CBS7/9) is critical for establishing and maintaining aberrant HOXA9-HOXA13 gene expression in AML. Disruption of the CBS7/9 boundary resulted in spreading of repressive H3K27me3 into the posterior active HOXA chromatin domain that subsequently impaired enhancer/promoter chromatin accessibility and disrupted ectopic long-range interactions among the posterior HOXA genes. Consistent with the role of the CBS7/9 boundary in HOXA locus chromatin organization, attenuation of the CBS7/9 boundary function reduced posterior HOXA gene expression and altered myeloid-specific transcriptome profiles important for pathogenesis of myeloid malignancies. Furthermore, heterozygous deletion of the CBS7/9 chromatin boundary in the HOXA locus reduced human leukemic blast burden and enhanced survival of transplanted AML cell xenograft and patient-derived xenograft mouse models. Thus, the CTCF boundary constrains the normal gene-expression program, as well as plays a role in maintaining the oncogenic transcription program for leukemic transformation. The CTCF boundaries may serve as novel therapeutic targets for the treatment of myeloid malignancies.
Assuntos
Fator de Ligação a CCCTC/metabolismo , Montagem e Desmontagem da Cromatina , Regulação Leucêmica da Expressão Gênica , Proteínas de Homeodomínio/biossíntese , Leucemia Mieloide Aguda/metabolismo , Proteínas de Neoplasias/metabolismo , Transcrição Gênica , Animais , Fator de Ligação a CCCTC/genética , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Proteínas de Homeodomínio/genética , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Endogâmicos NOD , Proteínas de Neoplasias/genéticaRESUMO
The germinal center (GC) reaction plays an important role in generating humoral immunity and is believed to give rise to most B-cell lymphomas. GC entry and exit are tightly regulated processes, controlled by the actions of transcription factors such as BCL6. Herein, we demonstrate that protein arginine methyltransferase 5 (PRMT5), a symmetric dimethyl arginine methyltransferase, is also necessary for GC formation and affinity maturation. PRMT5 contributes to GC formation and affinity maturation at least in part through its direct interaction with and methylation of BCL6 at arginine 305 (R305), a modification necessary for the full transcriptional repressive effects of BCL6. Inhibition of PRMT5 in B-cell lymphoma lines led to significant upregulation of BCL6 target genes, and the concomitant inhibition of both BCL6 and PRMT5 exhibited synergistic killing of BCL6-expressing lymphoma cells. Our studies identify PRMT5 as a novel regulator of the GC reaction and highlight the mechanistic rationale of cotargeting PRMT5 and BCL6 in lymphoma.
Assuntos
Centro Germinativo/metabolismo , Linfoma/metabolismo , Mapas de Interação de Proteínas , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas Proto-Oncogênicas c-bcl-6/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Regulação Neoplásica da Expressão Gênica , Centro Germinativo/citologia , Centro Germinativo/patologia , Humanos , Linfoma/genética , Linfoma/patologia , Camundongos Knockout , Proteína-Arginina N-Metiltransferases/genética , Proteínas Proto-Oncogênicas c-bcl-6/genéticaRESUMO
Double plant homeodomain finger 2 (DPF2) is a highly evolutionarily conserved member of the d4 protein family that is ubiquitously expressed in human tissues and was recently shown to inhibit the myeloid differentiation of hematopoietic stem/progenitor and acute myelogenous leukemia cells. Here, we present the crystal structure of the tandem plant homeodomain finger domain of human DPF2 at 1.6-Å resolution. We show that DPF2 interacts with the acetylated tails of both histones 3 and 4 via bipartite binding pockets on the DPF2 surface. Blocking these interactions through targeted mutagenesis of DPF2 abolishes its recruitment to target chromatin regions as well as its ability to prevent myeloid differentiation in vivo. Our findings suggest that the histone binding of DPF2 plays an important regulatory role in the transcriptional program that drives myeloid differentiation.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Histonas/química , Histonas/metabolismo , Células Mieloides/citologia , Células Mieloides/metabolismo , Acetilação , Diferenciação Celular/fisiologia , Cromatina/química , Cromatina/metabolismo , Cristalografia por Raios X , Hematopoese/fisiologia , Humanos , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , Fatores de TranscriçãoRESUMO
AML1-ETO (AE), a fusion oncoprotein generated by t(8;21), can trigger acute myeloid leukemia (AML) in collaboration with mutations including c-Kit, ASXL1/2, FLT3, N-RAS, and K-RAS. Caspase-3, a key executor among its family, plays multiple roles in cellular processes, including hematopoietic development and leukemia progression. Caspase-3 was revealed to directly cleave AE in vitro, suggesting that AE may accumulate in a Caspase-3-compromised background and thereby accelerate leukemogenesis. Therefore, we developed a Caspase-3 knockout genetic mouse model of AML and found that loss of Caspase-3 actually delayed AML1-ETO9a (AE9a)-driven leukemogenesis, indicating that Caspase-3 may play distinct roles in the initiation and/or progression of AML. We report here that loss of Caspase-3 triggers a conserved, adaptive mechanism, namely autophagy (or macroautophagy), which acts to limit AE9a-driven leukemia. Furthermore, we identify ULK1 as a novel substrate of Caspase-3 and show that upregulation of ULK1 drives autophagy initiation in leukemia cells and that inhibition of ULK1 can rescue the phenotype induced by Caspase-3 deletion in vitro and in vivo. Collectively, these data highlight Caspase-3 as an important regulator of autophagy in AML and demonstrate that the balance and selectivity between its substrates can dictate the pace of disease.
Assuntos
Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Autofagia , Carcinogênese/patologia , Caspase 3/metabolismo , Leucemia/metabolismo , Leucemia/patologia , Proteínas de Fusão Oncogênica/metabolismo , Animais , Antígenos CD34/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/antagonistas & inibidores , Autorrenovação Celular , Modelos Animais de Doenças , Feto/patologia , Deleção de Genes , Técnicas de Silenciamento de Genes , Humanos , Transplante de Fígado , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Fenótipo , Especificidade por SubstratoRESUMO
Transcription factors are frequently altered in leukaemia through chromosomal translocation, mutation or aberrant expression. AML1-ETO, a fusion protein generated by the t(8;21) translocation in acute myeloid leukaemia, is a transcription factor implicated in both gene repression and activation. AML1-ETO oligomerization, mediated by the NHR2 domain, is critical for leukaemogenesis, making it important to identify co-regulatory factors that 'read' the NHR2 oligomerization and contribute to leukaemogenesis. Here we show that, in human leukaemic cells, AML1-ETO resides in and functions through a stable AML1-ETO-containing transcription factor complex (AETFC) that contains several haematopoietic transcription (co)factors. These AETFC components stabilize the complex through multivalent interactions, provide multiple DNA-binding domains for diverse target genes, co-localize genome wide, cooperatively regulate gene expression, and contribute to leukaemogenesis. Within the AETFC complex, AML1-ETO oligomerization is required for a specific interaction between the oligomerized NHR2 domain and a novel NHR2-binding (N2B) motif in E proteins. Crystallographic analysis of the NHR2-N2B complex reveals a unique interaction pattern in which an N2B peptide makes direct contact with side chains of two NHR2 domains as a dimer, providing a novel model of how dimeric/oligomeric transcription factors create a new protein-binding interface through dimerization/oligomerization. Intriguingly, disruption of this interaction by point mutations abrogates AML1-ETO-induced haematopoietic stem/progenitor cell self-renewal and leukaemogenesis. These results reveal new mechanisms of action of AML1-ETO, and provide a potential therapeutic target in t(8;21)-positive acute myeloid leukaemia.
Assuntos
Transformação Celular Neoplásica , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Complexos Multiproteicos/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Fatores de Transcrição/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Divisão Celular , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Subunidade alfa 2 de Fator de Ligação ao Core/química , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Humanos , Leucemia Mieloide Aguda/genética , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multiproteicos/química , Proteínas de Fusão Oncogênica/química , Mutação Puntual , Ligação Proteica , Multimerização Proteica , Estabilidade Proteica , Estrutura Terciária de Proteína , Proteína 1 Parceira de Translocação de RUNX1RESUMO
Inhibitor of DNA binding 1 (Id1) functions as an E protein inhibitor, and overexpression of Id1 is seen in acute myeloid leukemia (AML) patients. To define the effects of Id1 on leukemogenesis, we expressed MLL-AF9 in fetal liver (FL) cells or bone marrow (BM) cells isolated from wild-type, Id1(-/-), p21(-/-), or Id1(-/-)p21(-/-) mice, and transplanted them into syngeneic recipient mice. We found that although mice receiving MLL-AF9-transduced FL or BM cells develop AML, loss of Id1 significantly prolonged the median survival of mice receiving FL cells but accelerated leukemogenesis in recipients of BM cells. Deletion of Cdkn1a (p21), an Id1 target gene, can rescue the effect of Id1 loss in both models, suggesting that Cdkn1a is a critical target of Id1 in leukemogenesis. It has been suggested that the FL transplant model mimics human fetal-origin (infant) MLL fusion protein (FP)-driven leukemia, whereas the BM transplantation model resembles postnatal MLL leukemia; in fact, the analysis of clinical samples from patients with MLL-FP(+) leukemia showed that Id1 expression is elevated in the former and reduced in the latter type of MLL-FP(+) AML. Our findings suggest that Id1 could be a potential therapeutic target for infant MLL-AF9-driven leukemia.
Assuntos
Proteína 1 Inibidora de Diferenciação/metabolismo , Neoplasias Experimentais/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Animais , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Humanos , Proteína 1 Inibidora de Diferenciação/genética , Camundongos , Camundongos Knockout , Neoplasias Experimentais/genética , Proteínas de Fusão Oncogênica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genéticaRESUMO
The identification of somatic activating mutations in JAK2 (refs 14) and in the thrombopoietin receptor gene (MPL) in most patients with myeloproliferative neoplasm (MPN) led to the clinical development of JAK2 kinase inhibitors. JAK2 inhibitor therapy improves MPN-associated splenomegaly and systemic symptoms but does not significantly decrease or eliminate the MPN clone in most patients with MPN. We therefore sought to characterize mechanisms by which MPN cells persist despite chronic inhibition of JAK2. Here we show that JAK2 inhibitor persistence is associated with reactivation of JAKSTAT signalling and with heterodimerization between activated JAK2 and JAK1 or TYK2, consistent with activation of JAK2 in trans by other JAK kinases. Further, this phenomenon is reversible: JAK2 inhibitor withdrawal is associated with resensitization to JAK2 kinase inhibitors and with reversible changes in JAK2 expression. We saw increased JAK2 heterodimerization and sustained JAK2 activation in cell lines, in murine models and in patients treated with JAK2 inhibitors. RNA interference and pharmacological studies show that JAK2-inhibitor-persistent cells remain dependent on JAK2 protein expression. Consequently, therapies that result in JAK2 degradation retain efficacy in persistent cells and may provide additional benefit to patients with JAK2-dependent malignancies treated with JAK2 inhibitors.
Assuntos
Janus Quinase 2/antagonistas & inibidores , Janus Quinase 2/metabolismo , Transtornos Mieloproliferativos/tratamento farmacológico , Multimerização Proteica , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais , Animais , Linhagem Celular , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Granulócitos/efeitos dos fármacos , Granulócitos/enzimologia , Granulócitos/metabolismo , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Janus Quinase 1/biossíntese , Janus Quinase 1/deficiência , Janus Quinase 1/genética , Janus Quinase 1/metabolismo , Janus Quinase 2/deficiência , Janus Quinase 2/genética , Camundongos , Transtornos Mieloproliferativos/enzimologia , Transtornos Mieloproliferativos/metabolismo , Transtornos Mieloproliferativos/patologia , Fosforilação , Biossíntese de Proteínas , Interferência de RNA , Transdução de Sinais/efeitos dos fármacos , TYK2 Quinase/biossíntese , TYK2 Quinase/deficiência , TYK2 Quinase/genética , TYK2 Quinase/metabolismoRESUMO
Transcriptional regulators are recurrently altered through translocations, deletions, or aberrant expression in acute myeloid leukemia (AML). Although critically important in leukemogenesis, the underlying pathogenetic mechanisms they trigger remain largely unknown. Here, we identified that Id1 (inhibitor of DNA binding 1) plays a pivotal role in acute myeloid leukemogenesis. Using genetically modified mice, we found that loss of Id1 inhibited t(8;21) leukemia initiation and progression in vivo by abrogating protein kinase B (AKT)1 activation, and that Id1 interacted with AKT1 through its C terminus. An Id1 inhibitor impaired the in vitro growth of AML cells and, when combined with an AKT inhibitor, triggered even greater apoptosis and growth inhibition, whereas normal hematopoietic stem/progenitor cells were largely spared. We then performed in vivo experiments and found that the Id1 inhibitor significantly prolonged the survival of t(8;21)(+) leukemic mice, whereas overexpression of activated AKT1 promoted leukemogenesis. Thus, our results establish Id1/Akt1 signaling as a potential therapeutic target in t(8;21) leukemia.
Assuntos
Proteína 1 Inibidora de Diferenciação/metabolismo , Leucemia Mieloide Aguda/etiologia , Leucemia Mieloide Aguda/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Apoptose , Carcinogênese , Linhagem Celular Tumoral , Progressão da Doença , Técnicas de Silenciamento de Genes , Humanos , Proteína 1 Inibidora de Diferenciação/deficiência , Proteína 1 Inibidora de Diferenciação/genética , Proteínas Inibidoras de Diferenciação/antagonistas & inibidores , Proteínas Inibidoras de Diferenciação/genética , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Domínios e Motivos de Interação entre Proteínas , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Transdução de Sinais , Translocação GenéticaRESUMO
The RUNX family of transcription factors plays important roles in tissue-specific gene expression. Many of their functions depend on specific post-translational modifications (PTMs), and in this review, we describe how PTMs govern RUNX DNA binding, transcriptional activity, protein stability, cellular localization, and protein-protein interactions. We also report how these processes can be disrupted in disease settings. Finally, we describe how alterations of RUNX1, or the enzymes that catalyze its post-translational modifications, contribute to hematopoietic malignancies.
Assuntos
Subunidades alfa de Fatores de Ligação ao Core/genética , Subunidades alfa de Fatores de Ligação ao Core/metabolismo , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Mapas de Interação de Proteínas/genética , Processamento de Proteína Pós-Traducional/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica/genéticaRESUMO
RUNX1/CBFß (core binding factor [CBF]) is a heterodimeric transcription factor complex that is frequently involved in chromosomal translocations, point mutations, or deletions in acute leukemia. The mixed lineage leukemia (MLL) gene is also frequently involved in chromosomal translocations or partial tandem duplication in acute leukemia. The MLL protein interacts with RUNX1 and prevents RUNX1 from ubiquitin-mediated degradation. RUNX1/CBFß recruits MLL to regulate downstream target genes. However, the functional consequence of MLL fusions on RUNX1/CBFß activity has not been fully understood. In this report, we show that MLL fusion proteins and the N-terminal MLL portion of MLL fusions downregulate RUNX1 and CBFß protein expression via the MLL CXXC domain and flanking regions. We confirmed this finding in Mll-Af9 knock-in mice and human M4/M5 acute myeloid leukemia (AML) cell lines, with or without MLL translocations, showing that MLL translocations cause a hypomorph phenotype of RUNX1/CBFß. Overexpression of RUNX1 inhibits the development of AML in Mll-Af9 knock-in mice; conversely, further reducing Runx1/Cbfß levels accelerates MLL-AF9-mediated AML in bone marrow transplantation assays. These data reveal a newly defined negative regulation of RUNX1/CBFß by MLL fusion proteins and suggest that targeting RUNX1/CBFß levels may be a potential therapy for MLLs.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Subunidade beta de Fator de Ligação ao Core/metabolismo , Células-Tronco Hematopoéticas/patologia , Leucemia Mieloide Aguda/patologia , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Proteínas de Fusão Oncogênica/fisiologia , Animais , Western Blotting , Transplante de Medula Óssea , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Subunidade beta de Fator de Ligação ao Core/genética , Citometria de Fluxo , Células-Tronco Hematopoéticas/metabolismo , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas de Fusão Oncogênica/genética , Fenótipo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Translocação GenéticaRESUMO
RUNX1 (previously termed AML1) is a frequent target of human leukaemia-associated gene aberrations, and it encodes the DNA-binding subunit of the Core-Binding Factor transcription factor complex. RUNX1 expression is essential for the initiation of definitive haematopoiesis, for steady-state thrombopoiesis, and for normal lymphocytes development. Recent studies revealed that protein arginine methyltransferase 1 (PRMT1), which accounts for the majority of the type I PRMT activity in cells, methylates two arginine residues in RUNX1 (R206 and R210), and these modifications inhibit corepressor-binding to RUNX1 thereby enhancing its transcriptional activity. In order to elucidate the biological significance of these methylations, we established novel knock-in mouse lines with non-methylable, double arginine-to-lysine (RTAMR-to-KTAMK) mutations in RUNX1. Homozygous Runx1(KTAMK) (/) (KTAMK) mice are born alive and appear normal during adulthood. However, Runx1(KTAMK) (/) (KTAMK) mice showed a reduction in CD3(+) T lymphoid cells and a decrease in CD4(+) T cells in peripheral lymphoid organs, in comparison to their wild-type littermates, leading to a reduction in the CD4(+) to CD8(+) T-cell ratio. These findings suggest that arginine-methylation of RUNX1 in the RTAMR-motif is dispensable for the development of definitive haematopoiesis and for steady-state platelet production, however this modification affects the role of RUNX1 in the maintenance of the peripheral CD4(+) T-cell population.
Assuntos
Arginina/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Hematopoese , Linfócitos T/metabolismo , Alelos , Motivos de Aminoácidos , Animais , Contagem de Linfócito CD4 , Subunidade alfa 2 de Fator de Ligação ao Core/química , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Células-Tronco Embrionárias/metabolismo , Técnicas de Introdução de Genes , Ordem dos Genes , Marcação de Genes , Vetores Genéticos/genética , Genótipo , Células Germinativas/metabolismo , Hematopoese/genética , Humanos , Metilação , Camundongos , Camundongos Knockout , Mutação , Mielopoese/genética , Fenótipo , Ativação TranscricionalRESUMO
BACKGROUND: Myelodysplastic syndromes (MDS) are characterized by ineffective erythropoiesis with dysplastic bone marrow leading to peripheral cytopenia, risk of infection, and progression to acute myelogenous leukemia. Maitake mushroom beta-glucan, a dietary supplement, stimulates hematopoietic progenitor cell differentiation, granulocyte colony-stimulating factor production, and recovery of peripheral blood leukocytes after bone marrow injury. This phase II trial examined the effects of Maitake on innate immune function in MDS. METHODS: Myelodysplastic syndromes patients with International Prognostic Scoring System Low- and Intermediate-1-risk disease received oral Maitake extract at 3 mg/kg twice daily for 12 weeks. Primary endpoints included neutrophil count and function tested as endogenous or stimulated neutrophil production of reactive oxygen species (ROS) by flow cytometry compared with age-matched healthy controls (HC). ROS activators were Escherichia coli, phorbol ester, and the bacterial peptide N-formylmethionyl-leucyl-phenylalanine (fMLP). Complete blood counts, chemistry panels, iron studies, and monocyte function were evaluated. RESULTS: Of 21 patients enrolled, 18 completed the study and were evaluable. Maitake increased endogenous (basal) neutrophil (p = 0.005) and monocyte function (p = 0.021). Pre-treatment monocyte response to E. coli was reduced in MDS patients compared with HC (p = 0.002) and increased (p = 0.0004) after treatment. fMLP-stimulated ROS production response also increased (p = 0.03). Asymptomatic eosinophilia occurred in 4 patients (p = 0.014). Other changes in albumin, hemoglobin, and total protein were not clinically relevant. CONCLUSIONS: Maitake was well tolerated. Enhanced in vitro neutrophil and monocyte function following treatment demonstrate that Maitake has beneficial immunomodulatory potential in MDS. Further study is warranted.
Assuntos
Antineoplásicos/uso terapêutico , Misturas Complexas/uso terapêutico , Grifola/química , Síndromes Mielodisplásicas/tratamento farmacológico , Idoso , Antineoplásicos/administração & dosagem , Antineoplásicos/efeitos adversos , Biomarcadores/sangue , Biomarcadores/metabolismo , Medula Óssea/patologia , Células da Medula Óssea/metabolismo , Estudos de Casos e Controles , Misturas Complexas/administração & dosagem , Misturas Complexas/efeitos adversos , Feminino , Humanos , Cariótipo , Masculino , Pessoa de Meia-Idade , Monócitos/imunologia , Monócitos/metabolismo , Síndromes Mielodisplásicas/diagnóstico , Neutrófilos/imunologia , Neutrófilos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Resultado do TratamentoRESUMO
Myeloid ELF1-like factor (MEF/ELF4), a member of the ETS transcription factors, can function as an oncogene in murine cancer models and is overexpressed in various human cancers. Here, we report a mechanism by which MEF/ELF4 may be activated by a common leukemia-associated mutation in the nucleophosmin gene. By using a tandem affinity purification assay, we found that MEF/ELF4 interacts with multifactorial protein nucleophosmin (NPM1). Coimmunoprecipitation and GST pull-down experiments demonstrated that MEF/ELF4 directly forms a complex with NPM1 and also identified the region of NPM1 that is responsible for this interaction. Functional analyses showed that wild-type NPM1 inhibited the DNA binding and transcriptional activity of MEF/ELF4 on the HDM2 promoter, whereas NPM1 mutant protein (Mt-NPM1) enhanced these activities of MEF/ELF4. Induction of Mt-NPM1 into MEF/ELF4-overexpressing NIH3T3 cells facilitated malignant transformation. In addition, clinical leukemia samples with NPM1 mutations had higher human MDM2 (HDM2) mRNA expression. Our data suggest that enhanced HDM2 expression induced by mutant NPM1 may have a role in MEF/ELF4-dependent leukemogenesis.