RESUMO
With age, haematopoietic stem cells lose their ability to regenerate the blood system, and promote disease development. Autophagy is associated with health and longevity, and is critical for protecting haematopoietic stem cells from metabolic stress. Here we show that loss of autophagy in haematopoietic stem cells causes accumulation of mitochondria and an activated metabolic state, which drives accelerated myeloid differentiation mainly through epigenetic deregulations, and impairs haematopoietic stem-cell self-renewal activity and regenerative potential. Strikingly, most haematopoietic stem cells in aged mice share these altered metabolic and functional features. However, approximately one-third of aged haematopoietic stem cells exhibit high autophagy levels and maintain a low metabolic state with robust long-term regeneration potential similar to healthy young haematopoietic stem cells. Our results demonstrate that autophagy actively suppresses haematopoietic stem-cell metabolism by clearing active, healthy mitochondria to maintain quiescence and stemness, and becomes increasingly necessary with age to preserve the regenerative capacity of old haematopoietic stem cells.
Assuntos
Autofagia , Autorrenovação Celular , Senescência Celular , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/fisiologia , Animais , Autofagia/genética , Autorrenovação Celular/genética , Senescência Celular/genética , Epigênese Genética , Feminino , Hematopoese/genética , Células-Tronco Hematopoéticas/metabolismo , Masculino , Camundongos , Mitocôndrias/metabolismo , Células Mieloides/citologia , Células Mieloides/metabolismoRESUMO
Somatic mutations in genes coding for splicing factors, e.g. SF3B1, U2AF1, SRSF2, and others are found in approximately 50% of patients with Myelodysplastic Syndromes (MDS). These mutations have been predicted to frequently occur early in the mutational hierarchy of the disease therefore making them particularly attractive potential therapeutic targets. Recent studies in cell lines engineered to carry splicing factor mutations have revealed a strong association with elevated levels of DNA:RNA intermediates (R-loops) and a dependency on proper ATR function. However, data confirming this hypothesis in a representative cohort of primary MDS patient samples have so far been missing. Using CD34+ cells isolated from MDS patients with and without splicing factor mutations as well as healthy controls we show that splicing factor mutation-associated R-loops lead to elevated levels of replication stress and ATR pathway activation. Moreover, splicing factor mutated CD34+ cells are more susceptible to pharmacological inhibition of ATR resulting in elevated levels of DNA damage, cell cycle blockade, and cell death. This can be enhanced by combination treatment with low-dose splicing modulatory compound Pladienolide B. We further confirm the direct association of R-loops and ATR sensitivity with the presence of a splicing factor mutation using lentiviral overexpression of wild-type and mutant SRSF2 P95H in cord blood CD34+ cells. Collectively, our results from n=53 MDS patients identify replication stress and associated ATR signaling to be critical pathophysiological mechanisms in primary MDS CD34+ cells carrying splicing factor mutations, and provide a preclinical rationale for targeting ATR signaling in these patients.
Assuntos
Síndromes Mielodisplásicas , Fosfoproteínas , Humanos , Mutação , Síndromes Mielodisplásicas/tratamento farmacológico , Síndromes Mielodisplásicas/genética , Fosfoproteínas/genética , Splicing de RNA , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , Fatores de Processamento de Serina-Arginina/genética , Fator de Processamento U2AF/genéticaRESUMO
Non-targeted effects (NTE) of ionizing radiation may initiate myeloid neoplasms (MN). Here, protein mediators (I) in irradiated human mesenchymal stromal cells (MSC) as the NTE source, (II) in MSC conditioned supernatant and (III) in human bone marrow CD34+ cells undergoing genotoxic NTE were investigated. Healthy sublethal irradiated MSC showed significantly increased levels of reactive oxygen species. These cells responded by increasing intracellular abundance of proteins involved in proteasomal degradation, protein translation, cytoskeleton dynamics, nucleocytoplasmic shuttling, and those with antioxidant activity. Among the increased proteins were THY1 and GNA11/14, which are signaling proteins with hitherto unknown functions in the radiation response and NTE. In the corresponding MSC conditioned medium, the three chaperones GRP78, CALR, and PDIA3 were increased. Together with GPI, these were the only four altered proteins, which were associated with the observed genotoxic NTE. Healthy CD34+ cells cultured in MSC conditioned medium suffered from more than a six-fold increase in γH2AX focal staining, indicative for DNA double-strand breaks, as well as numerical and structural chromosomal aberrations within three days. At this stage, five proteins were altered, among them IQGAP1, HMGB1, and PA2G4, which are involved in malign development. In summary, our data provide novel insights into three sequential steps of genotoxic signaling from irradiated MSC to CD34+ cells, implicating that induced NTE might initiate the development of MN.
Assuntos
Células da Medula Óssea/metabolismo , Diferenciação Celular , Dano ao DNA , Células-Tronco Mesenquimais/metabolismo , Proteoma , Transdução de Sinais , Idoso , Antígenos CD34/metabolismo , Biomarcadores , Células da Medula Óssea/citologia , Diferenciação Celular/genética , Diferenciação Celular/efeitos da radiação , Sobrevivência Celular/genética , Instabilidade Cromossômica , Meios de Cultivo Condicionados/metabolismo , Chaperona BiP do Retículo Endoplasmático , Feminino , Histonas/metabolismo , Humanos , Masculino , Células-Tronco Mesenquimais/citologia , Modelos Biológicos , Proteômica/métodos , Radiação Ionizante , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos da radiaçãoRESUMO
Reciprocal RUNX1 fusions are traditionally found in up to 10% of acute myeloid leukemia (AML) patients, usually associated with a translocation (8;21)(q22;q22) corresponding to the RUNX1-RUNX1T1 fusion gene. So far, alternative RUNX1 rearrangements have been reported only rarely in AML, and the few reports so far have focused on results based on cytogenetics, fluorescence in situ hybridization, and polymerase chain reaction. Acknowledging the inherent limitations of these diagnostic techniques, the true incidence of rare RUNX1 rearrangements may be underestimated. In this report, we present two cases of adult AML, in which we detected rare RUNX1 rearrangements not by conventional cytogenetics but rather by next-generation panel sequencing. These include t(16;21)(q24;q22)/RUNX1-CBFA2T3 and t(7;21)(p22;q22)/RUNX1-USP42, respectively. In both patients the AML was therapy-related and associated with additional structural and numerical alterations thereby conferring bad prognosis. This is in line with previous reports on rare RUNX1 fusions in AML and emphasizes the clinical importance of their detection. In summary, our report not only confirms the clinical utility of NGS for diagnostics of rare reciprocal rearrangements in AML in a real-life scenario but also sheds light on the variety and complexity within AML. It further emphasizes the need for collection of additional cases for deepening insights on their clinical meaning as well as their frequency.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/genética , Rearranjo Gênico , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/genética , Translocação Genética , Idoso , Biomarcadores Tumorais , Linhagem Celular Tumoral , Cromossomos Humanos Par 16 , Cromossomos Humanos Par 21 , Estudos de Associação Genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Hibridização in Situ Fluorescente , Masculino , Proteínas de Fusão Oncogênica/genética , Proteínas Repressoras/genéticaRESUMO
The significance of clonal evolution in myelofibrosis (MF) relapse remains poorly understood. Here we performed panel sequencing in paired samples of 30 patients with MF who relapsed after undergoing allogeneic hematopoietic stem cell transplantation (alloSCT). We identified a median of 2 mutations (range, 0 to 12) in a median of 2 genes (range, 0 to 8) before allo-SCT, along with a median of 2 mutations (range, 0 to 12) in 2 genes (range, 0 to 6) at relapse. Additional whole-genome sequencing (n = 6) did not elucidate additional molecular changes. Taken together, our data provide further evidence, here on MF, that clonal evolution after alloSCT is limited and that instead, alloSCT selects specific (sub)clones.
Assuntos
Evolução Clonal , Transplante de Células-Tronco Hematopoéticas , Mielofibrose Primária , Humanos , Mutação , Mielofibrose Primária/genética , Mielofibrose Primária/terapia , RecidivaRESUMO
Relapse of acute myeloid leukemia (AML) remains a major determinant of outcome. A number of molecularly directed treatment options have recently emerged making comprehensive diagnostics an important pillar of clinical decision making at relapse. Acknowledging the high degree of individual genetic variability at AML relapse, next-generation sequencing (NGS) has opened the opportunity for assessing the unique clonal hierarchy of individual AML patients. Knowledge on the genetic makeup of AML is reflected in patient customized treatment strategies thereby providing improved outcomes. For example, the emergence of druggable mutations at relapse enable the use of novel targeted therapies, including FLT3 inhibitors or the recently approved IDH1/2 inhibitors ivosidenib and enasidenib, respectively. Consequently, some patients may undergo novel bridging approaches for reinduction before allogeneic stem cell transplantation, or the identification of an adverse prognostic marker may initiate early donor search. In this review, we summarize the current knowledge of NGS in identifying clonal stability, clonal evolution, and clonal devolution in the context of AML relapse. In light of recent improvements in AML treatment options, NGS-based molecular diagnostics emerges as the basis for molecularly directed treatment decisions in patients at relapse.
Assuntos
Antineoplásicos/uso terapêutico , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Leucemia Mieloide Aguda/tratamento farmacológico , Mutação , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Recidiva Local de Neoplasia/tratamento farmacológico , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Terapia de Alvo Molecular , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologiaRESUMO
Separase, a cysteine endopeptidase, is a key player in mitotic sister chromatid separation, replication fork dynamics, and DNA repair. Aberrant expression and/or altered separase proteolytic activity are associated with aneuploidy, tumorigenesis, and disease progression. Since genomic instability and clonal evolution are hallmarks of progressing chronic myeloid leukemia (CML), we have comparatively examined separase proteolytic activity in TKI-treated chronic phase CML. Separase proteolytic activity was analyzed on single cell level in 88 clinical samples and in 14 healthy controls by a flow cytometric assay. In parallel, BCR-ABL1 gene expression and replication fork velocity were measured by qRT-PCR and DNA fiber assays, respectively. The separase activity distribution (SAD) value indicating the occurrence of MNCs with elevated separase proteolytic activity within samples was found to positively correlate with BCR-ABL1 gene expression levels and loss of MMR (relapse) throughout routine BCR-ABL1 monitoring. Analyses of CD34+ cells and MNCs fractionized by flow cytometric cell sorting according to their separase activity levels (H- and L-fractions) revealed that CD34+ cells with elevated separase activity levels (H-fractions) displayed enhanced proliferation/viability when compared with cells with regular (L-fraction) separase activity (mean 3.3-fold, p = 0.0011). BCR-ABL1 gene expression positivity prevailed in MNC H-fractions over L-fractions (42% vs. 8%, respectively). Moreover, expanding CD34+ cells of H-fractions showed decreased replication fork velocity compared with cells of L-fractions (p < 0.0001). Our data suggests an association between high separase activity, residual BCR-ABL1 gene expression, and enhanced proliferative capacity in hematopoietic cells within the leukemic niche of TKI-treated chronic phase CML.
Assuntos
Antígenos CD34/metabolismo , Biomarcadores Tumorais/metabolismo , Proliferação de Células/efeitos dos fármacos , Leucemia Mielogênica Crônica BCR-ABL Positiva , Inibidores de Proteínas Quinases/administração & dosagem , Separase/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Proteínas de Fusão bcr-abl/metabolismo , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/enzimologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Masculino , Pessoa de Meia-IdadeRESUMO
Haematopoietic stem cells (HSCs) self-renew for life, thereby making them one of the few blood cells that truly age. Paradoxically, although HSCs numerically expand with age, their functional activity declines over time, resulting in degraded blood production and impaired engraftment following transplantation. While many drivers of HSC ageing have been proposed, the reason why HSC function degrades with age remains unknown. Here we show that cycling old HSCs in mice have heightened levels of replication stress associated with cell cycle defects and chromosome gaps or breaks, which are due to decreased expression of mini-chromosome maintenance (MCM) helicase components and altered dynamics of DNA replication forks. Nonetheless, old HSCs survive replication unless confronted with a strong replication challenge, such as transplantation. Moreover, once old HSCs re-establish quiescence, residual replication stress on ribosomal DNA (rDNA) genes leads to the formation of nucleolar-associated γH2AX signals, which persist owing to ineffective H2AX dephosphorylation by mislocalized PP4c phosphatase rather than ongoing DNA damage. Persistent nucleolar γH2AX also acts as a histone modification marking the transcriptional silencing of rDNA genes and decreased ribosome biogenesis in quiescent old HSCs. Our results identify replication stress as a potent driver of functional decline in old HSCs, and highlight the MCM DNA helicase as a potential molecular target for rejuvenation therapies.
Assuntos
Senescência Celular/fisiologia , Replicação do DNA/fisiologia , Células-Tronco Hematopoéticas/patologia , Estresse Fisiológico , Animais , Proliferação de Células , Senescência Celular/genética , Dano ao DNA/genética , DNA Ribossômico/genética , Feminino , Regulação da Expressão Gênica , Células-Tronco Hematopoéticas/citologia , Histonas/genética , Histonas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Manutenção de Minicromossomo/genéticaRESUMO
DNA damage and alterations in the DNA damage response (DDR) are critical sources of genetic instability that might be involved in BCR-ABL1 kinase-mediated blastic transformation of chronic myeloid leukemia (CML). Here, increased DNA damage is detected by γH2AX foci analysis in peripheral blood mononuclear cells (PBMCs) of de novo untreated chronic phase (CP)-CML patients (n = 5; 2.5 γH2AX foci per PBMC ± 0.5) and blast phase (BP)-CML patients (n = 3; 4.4 γH2AX foci per PBMC ± 0.7) as well as CP-CML patients with loss of major molecular response (MMR) (n = 5; 1.8 γH2AX foci per PBMC ± 0.4) when compared to DNA damage in PBMC of healthy donors (n = 8; 1.0 γH2AX foci per PBMC ± 0.1) and CP-CML patients in deep molecular response or MMR (n = 26; 1.0 γH2AX foci per PBMC ± 0.1). Progressive activation of erroneous non-homologous end joining (NHEJ) repair mechanisms during blastic transformation in CML is indicated by abundant co-localization of γH2AX/53BP1 foci, while a decline of the DDR is suggested by defective expression of (p-)ATM and (p-)CHK2. In summary, our data provide evidence for the accumulation of DNA damage in the course of CML and suggest ongoing DNA damage, erroneous NHEJ repair mechanisms, and alterations in the DDR as critical mediators of blastic transformation in CML.
Assuntos
Dano ao DNA , Reparo do DNA , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Feminino , Instabilidade Genômica , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
Blood production is ensured by rare, self-renewing haematopoietic stem cells (HSCs). How HSCs accommodate the diverse cellular stresses associated with their life-long activity remains elusive. Here we identify autophagy as an essential mechanism protecting HSCs from metabolic stress. We show that mouse HSCs, in contrast to their short-lived myeloid progeny, robustly induce autophagy after ex vivo cytokine withdrawal and in vivo calorie restriction. We demonstrate that FOXO3A is critical to maintain a gene expression program that poises HSCs for rapid induction of autophagy upon starvation. Notably, we find that old HSCs retain an intact FOXO3A-driven pro-autophagy gene program, and that ongoing autophagy is needed to mitigate an energy crisis and allow their survival. Our results demonstrate that autophagy is essential for the life-long maintenance of the HSC compartment and for supporting an old, failing blood system.
Assuntos
Autofagia/genética , Metabolismo Energético/genética , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Estresse Fisiológico/genética , Envelhecimento , Animais , Apoptose , Restrição Calórica , Sobrevivência Celular/genética , Senescência Celular , Citocinas/deficiência , Citocinas/metabolismo , Privação de Alimentos , Proteína Forkhead Box O3 , Homeostase , Camundongos , Camundongos Endogâmicos C57BLAssuntos
Transplante de Células-Tronco Hematopoéticas , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Mutação , Proteínas de Neoplasias/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Aloenxertos , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Estudos RetrospectivosRESUMO
Limited response rates and frequent relapses during standard of care with hypomethylating agents in myelodysplastic neoplasms (MN) require urgent improvement of this treatment indication. Here, by combining 5-azacytidine (5-AZA) with the pan-lysyl oxidase inhibitor PXS-5505, we demonstrate superior restoration of erythroid differentiation in hematopoietic stem and progenitor cells (HSPCs) of MN patients in 20/31 cases (65%) versus 9/31 cases (29%) treated with 5-AZA alone. This effect requires direct contact of HSPCs with bone marrow stroma components and is dependent on integrin signaling. We further confirm these results in vivo using a bone marrow niche-dependent MN xenograft model in female NSG mice, in which we additionally demonstrate an enforced reduction of dominant clones as well as significant attenuation of disease expansion and normalization of spleen sizes. Overall, these results lay out a strong pre-clinical rationale for efficacy of combination treatment of 5-AZA with PXS-5505 especially for anemic MN.
Assuntos
Síndromes Mielodisplásicas , Transtornos Mieloproliferativos , Neoplasias , Humanos , Feminino , Camundongos , Animais , Azacitidina/farmacologia , Azacitidina/uso terapêutico , Eritropoese , Proteína-Lisina 6-Oxidase , Células-Tronco Hematopoéticas , Síndromes Mielodisplásicas/tratamento farmacológico , Síndromes Mielodisplásicas/patologia , Transtornos Mieloproliferativos/patologia , Neoplasias/patologiaRESUMO
Autologous hematopoietic cell transplantation (HCT) is suitable for consolidation of favorable-/intermediate-risk AML patients in CR1. However, ~50% of AML patients relapse after autologous HCT, and efficacy of subsequent salvage strategies including allogeneic HCT remains unclear. We studied 123 consecutive patients with newly diagnosed AML undergoing high-dose chemotherapy (HDCT)/autologous HCT in CR1. In relapsing patients afterwards, we analyzed salvage treatments and outcomes focusing particularly on salvage allogeneic HCT. Of 123 patients, 64 (52%) relapsed after autologous HCT. Subsequently, 13 (21%) received palliative therapy, whereas 51 (79%) proceeded to salvage therapy with a curative intent. Of the 47 patients with a curative intent and who did not proceed directly to allogeneic HCT, 23 (49%) achieved CR2 or had ongoing hematologic CR1 despite molecular relapse. Finally, 30 patients (47%) received allogeneic HCT with estimated 3-year leukemia-free and overall survival rates of 33% and 43%. Hematologic remission at allogeneic HCT and lack of acute GvHD had a positive impact on OS and LFS (p < 0.05). Our study suggests that almost 80% of AML patients can undergo salvage therapy following relapse after front-line HDCT/autologous HCT. Allogeneic HCT can provide cure in one third of patients relapsing after front-line HDCT/autologous HCT.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Leucemia Mieloide Aguda , Estudos de Viabilidade , Humanos , Leucemia Mieloide Aguda/terapia , Recidiva Local de Neoplasia , Indução de Remissão , Estudos Retrospectivos , Terapia de Salvação , Transplante AutólogoRESUMO
Patient-derived xenograft (PDX) models have emerged as versatile preclinical platforms for investigation of functional pathomechanisms in myelodysplastic syndromes (MDS) and other myeloid neoplasms. However, despite increasingly improved methodology, engraftment efficiencies frequently remain low. Humanized three-dimensional scaffold models (ossicle xenotransplantation models) in immunocompromised mice have recently been found to enable improved engraftment rates of healthy and malignant human hematopoiesis. We therefore interrogated the feasibility of using four different three-dimensional ossicle-based PDX models for application with primary MDS samples. In a fully standardized comparison, we evaluated scaffold materials such as Gelfoam, extracellular matrix (ECM), and human or xenogenous bone substance in comparison to intrafemoral (IF) co-injection of bone marrow (BM)-derived mesenchymal stromal cells (MSCs) and CD34+ hematopoietic stem and progenitor cells (HSPCs). Our study included13 primary MDS patient samples transplanted in parallel according to these five different conditions. Engraftment of MDS samples was assessed by flow cytometry, immunohistological staining, and molecular validation. We determined that three-dimensional ossicle-based methods achieved higher relative rates of engraftment and enabled long-term retrievability of patient-derived MSCs from implanted ossicles. In summary, HSPCs and MSCs derived from MDS BM, which did not significantly engraft in NSG mice after intrafemoral injection, were able to colonize humanized scaffold models. Therefore, these models are promising new xenotransplantation techniques for addressing preclinical and functional questions of the interaction between hematopoiesis and the BM niche in MDS.
Assuntos
Células-Tronco Mesenquimais , Síndromes Mielodisplásicas , Animais , Células da Medula Óssea/patologia , Modelos Animais de Doenças , Hematopoese , Células-Tronco Hematopoéticas/patologia , Humanos , Células-Tronco Mesenquimais/patologia , Camundongos , Síndromes Mielodisplásicas/patologia , Transplante HeterólogoRESUMO
Preclinical research of myelodysplastic syndromes (MDSs) is hampered by a lack of feasible disease models. Previously, we have established a robust patient-derived xenograft (PDX) model for MDS. Here we demonstrate for the first time that this model is applicable as a preclinical platform to address pending clinical questions by interrogating the efficacy and safety of the thrombopoietin receptor agonist eltrombopag. Our preclinical study included n = 49 xenografts generated from n = 9 MDS patient samples. Substance efficacy was evidenced by FACS-based human platelet quantification and clonal bone marrow evolution was reconstructed by serial whole-exome sequencing of the PDX samples. In contrast to clinical trials in humans, this experimental setup allowed vehicle- and replicate-controlled analyses on a patient-individual level deciphering substance-specific effects from natural disease progression. We found that eltrombopag effectively stimulated thrombopoiesis in MDS PDX without adversely affecting the patients' clonal composition. In conclusion, our MDS PDX model is a useful tool for testing new therapeutic concepts in MDS preceding clinical trials.
Assuntos
Benzoatos/uso terapêutico , Hidrazinas/uso terapêutico , Síndromes Mielodisplásicas/tratamento farmacológico , Pirazóis/uso terapêutico , Idoso , Idoso de 80 Anos ou mais , Animais , Apoptose , Proliferação de Células , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/patologia , Prognóstico , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Transcription poses a threat to genomic stability through the formation of R-loops that can obstruct progression of replication forks. R-loops are three-stranded nucleic acid structures formed by an RNA-DNA hybrid with a displaced non-template DNA strand. We developed RNA-DNA Proximity Proteomics to map the R-loop proximal proteome of human cells using quantitative mass spectrometry. We implicate different cellular proteins in R-loop regulation and identify a role of the tumor suppressor DDX41 in opposing R-loop and double strand DNA break accumulation in promoters. DDX41 is enriched in promoter regions in vivo, and can unwind RNA-DNA hybrids in vitro. R-loop accumulation upon loss of DDX41 is accompanied with replication stress, an increase in the formation of double strand DNA breaks and transcriptome changes associated with the inflammatory response. Germline loss-of-function mutations in DDX41 lead to predisposition to acute myeloid leukemia in adulthood. We propose that R-loop accumulation and genomic instability-associated inflammatory response may contribute to the development of familial AML with mutated DDX41.
Assuntos
RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Instabilidade Genômica , Proteômica , Estruturas R-Loop , Transcrição Gênica , Adulto , Linhagem Celular Tumoral , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Técnicas de Silenciamento de Genes , Genes Supressores de Tumor , Células HEK293 , Humanos , Leucemia Mieloide Aguda , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , Regiões Promotoras Genéticas , Estruturas R-Loop/genética , RNA/metabolismoRESUMO
Genotoxic bystander signals released from irradiated human mesenchymal stromal cells (MSC) may induce radiation-induced bystander effects (RIBEs) in human hematopoietic stem and progenitor cells (HSPC), potentially causing leukemic transformation. Although the source of bystander signals is evident, the identification and characterization of these signals is challenging. Here, RIBEs were analyzed in human CD34+ cells cultured in distinct molecular size fractions of medium, conditioned by 2 Gy irradiated human MSC. Specifically, γH2AX foci (as a marker of DNA double-strand breaks) and chromosomal instability were evaluated in CD34+ cells grown in approximate (I) < 10 kDa, (II) 10-100 kDa and (III) > 100 kDa fractions of MSC conditioned medium and un-/fractionated control medium, respectively. Hitherto, significantly increased numbers of γH2AX foci (p = 0.0286) and aberrant metaphases (p = 0.0022) were detected in CD34+ cells grown in the (II) 10-100 kDa fraction (0.67 ± 0.10 γH2AX foci per CD34+ cell ⨠3.8 ± 0.3 aberrant metaphases per CD34+ cell sample; mean ± SEM) when compared to (I) < 10 kDa (0.19 ± 0.01 ⨠0.3 ± 0.2) or (III) > 100 kDa fractions (0.23 ± 0.04 ⨠0.4 ± 0.4) or un-/fractionated control medium (0.12 ± 0.01 ⨠0.1 ± 0.1). Furthermore, RIBEs disappeared after heat inactivation of medium at 75 °C. Taken together, our data suggest that RIBEs are mainly mediated by the heat-sensitive (II) 10-100 kDa fraction of MSC conditioned medium. We postulate proteins as RIBE mediators and in-depth proteome analyses to identify key bystander signals, which define targets for the development of next-generation anti-leukemic drugs.
Assuntos
Efeito Espectador/efeitos da radiação , Meios de Cultivo Condicionados/farmacologia , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/efeitos da radiação , Mutagênicos/toxicidade , Idoso , Idoso de 80 Anos ou mais , Antígenos CD34/metabolismo , Efeito Espectador/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Instabilidade Cromossômica/efeitos dos fármacos , Instabilidade Cromossômica/efeitos da radiação , Dano ao DNA , Feminino , Humanos , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Pessoa de Meia-Idade , Peso Molecular , Raios XRESUMO
The bone marrow (BM) stroma in myeloid neoplasms is altered and it is hypothesized that this cell compartment may also harbor clonal somatically acquired mutations. By exome sequencing of in vitro expanded mesenchymal stromal cells (MSCs) from n = 98 patients with myelodysplastic syndrome (MDS) and n = 28 healthy controls we show that these cells accumulate recurrent mutations in genes such as ZFX (n = 8/98), RANK (n = 5/98), and others. MDS derived MSCs display higher mutational burdens, increased replicative stress, senescence, inflammatory gene expression, and distinct mutational signatures as compared to healthy MSCs. However, validation experiments in serial culture passages, chronological BM aspirations and backtracking of high confidence mutations by re-sequencing primary sorted MDS MSCs indicate that the discovered mutations are secondary to in vitro expansion but not present in primary BM. Thus, we here report that there is no evidence for clonal mutations in the BM stroma of MDS patients.
Assuntos
Medula Óssea/patologia , Células-Tronco Mesenquimais/patologia , Síndromes Mielodisplásicas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Medula Óssea/metabolismo , Células Cultivadas , Exoma/genética , Feminino , Genótipo , Humanos , Masculino , Células-Tronco Mesenquimais/metabolismo , Pessoa de Meia-Idade , Mutação , Síndromes Mielodisplásicas/patologia , Fenótipo , Microambiente TumoralRESUMO
While young blood can restore many aged tissues, its effects on the aged blood system itself and old hematopoietic stem cells (HSCs) have not been determined. Here, we used transplantation, parabiosis, plasma transfer, exercise, calorie restriction, and aging mutant mice to understand the effects of age-regulated systemic factors on HSCs and their bone marrow (BM) niche. We found that neither exposure to young blood, nor long-term residence in young niches after parabiont separation, nor direct heterochronic transplantation had any observable rejuvenating effects on old HSCs. Likewise, exercise and calorie restriction did not improve old HSC function, nor old BM niches. Conversely, young HSCs were not affected by systemic pro-aging conditions, and HSC function was not impacted by mutations influencing organismal aging in established long-lived or progeroid genetic models. Therefore, the blood system that carries factors with either rejuvenating or pro-aging properties for many other tissues is itself refractory to those factors.