Gene expression profile of HSC/Ps and mature myeloid cells in Mdivi-1 treated MDS mutant clone
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ABSTRACT: MDS are characterized by bone marrow (BM) failure due to ineffective hematopoiesis. However, the underlying mechanisms of ineffective hematopoiesis, in which clonal expansion coexists with accelerated cell death, remain to be elucidated. Recently, we detected CBL exon 8/9 deletion mutation (CBLΔE8/9) in several MDS patients with RUNX1 mutations. The CBLΔE8/9/RUNX1S291fs mice (CR-mice) developed a variety of MDS phenotypes, such as pancytopenia, dysplasia, and ineffective hematopoiesis in BM. RNA-Seq of HSC/progenitor (HSC/P) and mutant mature myeloid BM cells revealed that gene signature induced in the CR-mice is similar to that in MDS patients with BM failure. Especially, inflammation and innate immune-related genes were significantly dysregulated. Mdivi-1 treatment significantly attenuated inflammaotry signaling activation and rescued MDS phenotypes.
Project description:MDS are characterized by bone marrow (BM) failure due to ineffective hematopoiesis. However, the underlying mechanisms of ineffective hematopoiesis, in which clonal expansion coexists with accelerated cell death, remain to be elucidated. Recently, we detected CBL exon 8/9 deletion mutation (CBLΔE8/9) in several MDS patients with RUNX1 mutations. The CBLΔE8/9/RUNX1S291fs mice (CR-mice) developed a variety of MDS phenotypes, such as pancytopenia, dysplasia, and ineffective hematopoiesis in BM. RNA-Seq of HSC/progenitor (HSC/P) and mutant mature myeloid BM cells revealed that gene signature induced in the CR-mice is similar to that in MDS patients with BM failure. Especially, innate immune-related genes were significantly dysregulated.
Project description:Control of oxidative stress in the bone marrow (BM) is key for maintaining the balance between self-renewal, proliferation, and differentiation of hematopoietic cells. Breakdown of this regulation can lead to diseases characterized by BM failure such as the myelodysplastic syndromes (MDS). To better understand the role of oxidative stress in MDS development, we compared protein carbonylation as an oxidative stress marker in BM of patients with MDS and control subjects, and also patients with MDS under treatment with the iron chelator deferasirox.
Project description:Myelodysplastic Syndromes (MDSs) are a heterogeneous family of clonal disorders of hematopoietic stem cells characterized by ineffective hematopoiesis and frequently leukemia progression. To exlore how MDS develop into leukemia, we performed the transcriptional profiling of lesional cells and normal lymphoid cells from the MDS patients. Bone marrow cells from the MDS patients were sorted into blastic, myeloid, erythroid and lymphoid fractions by flow cytometry and then were profiled transcriptionally by Human Human Genome U133 Plus 2.0 arrays. Differentially expressed genes between lesional cells and lymphoid cells were identified by SAM.
Project description:Myelodysplastic Syndromes (MDSs) are a heterogeneous family of clonal disorders of hematopoietic stem cells characterized by ineffective hematopoiesis and frequently leukemia progression. To exlore how MDS develop into leukemia, we performed the transcriptional profiling of lesional cells and normal lymphoid cells from the MDS patients.
Project description:Myelodysplastic syndromes (MDS) are clonal hematopoietic stem cell (HSC) malignancies characterized by ineffective hematopoiesis with increased incidence in elderly individuals. Genetic alterations do not fully explain the molecular pathogenesis of the disease, indicating that other types of lesions may play a role in its development. In this work, we analyzed the transcriptional lesions of human HSCs, demonstrating how aging and MDS are characterized by a complex transcriptional rewiring that manifests as diverse linear and non-linear transcriptional dynamisms. While aging-associated lesions seemed to predispose elderly HSCs to myeloid transformation, disease-specific alterations may be involved in triggering MDS development. Among MDS-specific lesions, we detected the overexpression of the transcription factor (TF) DDIT3. Exogenous upregulation of DDIT3 in human healthy HSCs induced an MDS-like transcriptional state, and a delay in erythropoiesis, with an accumulation of cells in early stages of erythroid differentiation, as determined by single-cell RNA-sequencing. Increased DDIT3 expression was associated with downregulation of transcription factors required for normal erythropoiesis, such as KLF1, TAL1 or SOX6, and with a failure in the activation of their erythroid transcriptional programs. Finally, DDIT3 knockdown in CD34+ cells from MDS patients was able to restore erythropoiesis, as demonstrated by immunophenotypic and transcriptional profiling. These results demonstrate that DDIT3 may be a driver of MDS transformation, and a potential therapeutic target to restore the inefficient erythropoiesis characterizing these patients.
Project description:Myelodysplastic syndromes (MDS) are clonal hematopoietic stem cell (HSC) malignancies characterized by ineffective hematopoiesis with increased incidence in elderly individuals. Genetic alterations do not fully explain the molecular pathogenesis of the disease, indicating that other types of lesions may play a role in its development. In this work, we analyzed the transcriptional lesions of human HSCs, demonstrating how aging and MDS are characterized by a complex transcriptional rewiring that manifests as diverse linear and non-linear transcriptional dynamisms. While aging-associated lesions seemed to predispose elderly HSCs to myeloid transformation, disease-specific alterations may be involved in triggering MDS development. Among MDS-specific lesions, we detected the overexpression of the transcription factor (TF) DDIT3. Exogenous upregulation of DDIT3 in human healthy HSCs induced an MDS-like transcriptional state, and a delay in erythropoiesis, with an accumulation of cells in early stages of erythroid differentiation, as determined by single-cell RNA-sequencing. Increased DDIT3 expression was associated with downregulation of transcription factors required for normal erythropoiesis, such as KLF1, TAL1 or SOX6, and with a failure in the activation of their erythroid transcriptional programs. Finally, DDIT3 knockdown in CD34+ cells from MDS patients was able to restore erythropoiesis, as demonstrated by immunophenotypic and transcriptional profiling. These results demonstrate that DDIT3 may be a driver of MDS transformation, and a potential therapeutic target to restore the inefficient erythropoiesis characterizing these patients.
Project description:Myelodysplastic syndromes (MDS) are clonal hematopoietic stem cell (HSC) malignancies characterized by ineffective hematopoiesis with increased incidence in elderly individuals. Genetic alterations do not fully explain the molecular pathogenesis of the disease, indicating that other types of lesions may play a role in its development. In this work, we analyzed the transcriptional lesions of human HSCs, demonstrating how aging and MDS are characterized by a complex transcriptional rewiring that manifests as diverse linear and non-linear transcriptional dynamisms. While aging-associated lesions seemed to predispose elderly HSCs to myeloid transformation, disease-specific alterations may be involved in triggering MDS development. Among MDS-specific lesions, we detected the overexpression of the transcription factor (TF) DDIT3. Exogenous upregulation of DDIT3 in human healthy HSCs induced an MDS-like transcriptional state, and a delay in erythropoiesis, with an accumulation of cells in early stages of erythroid differentiation, as determined by single-cell RNA-sequencing. Increased DDIT3 expression was associated with downregulation of transcription factors required for normal erythropoiesis, such as KLF1, TAL1 or SOX6, and with a failure in the activation of their erythroid transcriptional programs. Finally, DDIT3 knockdown in CD34+ cells from MDS patients was able to restore erythropoiesis, as demonstrated by immunophenotypic and transcriptional profiling. These results demonstrate that DDIT3 may be a driver of MDS transformation, and a potential therapeutic target to restore the inefficient erythropoiesis characterizing these patients.
Project description:TRAF-interacting protein with forkhead-associated domain B (TIFAB) is a haploinsufficient gene in del(5q) Myelodysplastic syndrome (MDS). Hematopoietic-specific deletion of Tifab results in progressive bone marrow (BM) and blood defects, including skewed hematopoietic stem/progenitor cells (HSPC) proportions, altered myeloid differentiation, and progressive cytopenia. A subset of mice transplanted with Tifab knockout (KO) hematopoietic cells develop a bone marrow failure (BMF)-like disease with neutrophil dysplasia and cytopenia. In competitive transplants, Tifab KO HSPC are out-competed by wild-type (WT) cells, suggesting a cell-intrinsic HSPC defect. Gene expression analysis of Tifab KO HSPC identified dysregulation of immune-related signatures, and hypersensitivity to Toll-like receptor 4 (TLR4) stimulation. TIFAB also forms a complex with TRAF6, a mediator of immune signaling, and reduces TRAF6 protein stability by a lysosome-dependent mechanism. In contrast, TIFAB loss increases TRAF6 protein and the dynamic range of TLR4 signaling in HSPC, contributing to ineffective hematopoiesis. Moreover, combined deletion of TIFAB and miR-146a, two genes associated with del(5q) MDS/AML, results in a cooperative increase in TRAF6 expression and hematopoietic dysfunction in vivo. Re-expression of TIFAB in human del(5q) leukemic cells results in attenuated TLR4 signaling and reduced cell viability. These findings underscore the importance of efficient regulation of innate immune/TRAF6 signaling within HSPC by TIFAB, and its cooperation with miR-146a as it relates to the pathogenesis of hematopoietic malignancies, such as del(5q) MDS/AML. We performed an expression analysis on sorted lineage-Sca1+cKit+ (LSK) isolated from bone marrow (BM) of 3 month old mice transplanted with Tifab+/+ (WT) or Tifab-/- (KO) BM cells (n = 3 mice/group). We selected this time point to capture the gene expression profile of Tifab-/- LSK after engraftment but prior to overt hematopoietic failure. Total RNA was extracted, purified, reverse transcribed, labeled, and hybridized onto the GeneChip MoGene 2.0 ST Array (Affymetrix). Comparison comprises mRNA expression profile of Tifab+/+ LSK vs. Tifab-/- LSK.
Project description:High ploidy large cytoplasmic megakaryocytes (LCM) are critical negative regulators of hematopoietic stem cells (HSC) and are responsible for platelet formation. Using a mouse knockout model with normal megakaryocyte numbers but essentially devoid of LCM (MK-LCM KO), we demonstrated a pronounced increase in bone marrow HSC concurrent with endogenous mobilization and extramedullary hematopoiesis. When HSC isolated from a MK-LCM KO microenvironment were transplanted in lethally irradiated mice, the absence of LCM increased HSC in BM, blood and spleen. Severe thrombocytopenia was observed in animals with diminished LCM, although there was no change in megakaryocyte ploidy distribution. In contrast, WT HSC-generated LCM regulated a normal HSC pool and prevented thrombocytopenia. The present label-free quantitative LC-MSMS data was used to determine proteins that are differentially expressed in bone marrow cells of MK-LCM WT versus MK-LCM KO mice.
Project description:Myelodysplastic syndromes(MDS) are a group of heterogeneous disease. Emerging evidence has shown the bone marrow(BM) endothelial progenitor cells(EPCs) are also heterogeneity. To uncover the underlying mechanism of heterogeneous BM EPCs in different types of MDS patients. RNA sequencing(RNA-seq) analyses were performed to analyse BM EPCs at day 7 in culture from lower-risk MDS(N=3), higher-risk MDS(N=3), acute myloid leukemia(AML) patients(N=3) and healthy donors(HDs)(N=3). We analysed the hematopoiesis- and immune-related genes and pathways.