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Acute myeloid leukemia cells functionally compromise hematopoietic stem/progenitor cells inhibiting normal hematopoiesis through the release of extracellular vesicles

ABSTRACT: Acute myeloid leukemia (AML) is an aggressive and heterogeneous clonal disorder of hematopoietic stem/progenitor cells (HSPCs). It is not well known how leukemia cells alter hematopoiesis promoting tumor growth and leukemic niche formation. In this study, we investigated how AML deregulates the hematopoietic process of HSPCs through the release of extracellular vesicles (EVs). First, we found that AML cells released a heterogeneous population of EVs (AML-EVs) containing microRNAs involved in AML pathogenesis. Notably AML-EVs were able to influence the fate of HSPCs modifying their transcriptome. In fact, a gene expression profile of AML-EV-treated HSPCs identified approximately 923 down and 630 up-regulated genes involved in hematopoiesis/differentiation, inflammatory cytokine production and cell movement. Indeed, most of the downregulated genes are targeted by AML-EV-derived miRNAs. Furthermore, we demonstrated that AML-EVs were able to affect HSPCs phenotype, modifying several biological functions, such as inhibiting cell differentiation and clonogenicity, activating inflammatory cytokine production and compromising cell movement. Indeed, a redistribution of HSPC populations was observed in AML-EV treated cells with a significant increase in the frequency of common myeloid progenitors and a reduction in granulocyte-macrophage progenitors and megakaryocyte-erythroid progenitors. This effect was accompanied by a reduction in HSPC colony formation. AML-EV treatment of HSPCs increased the levels of CCL3, IL1B and CSF2 cytokines, involved in the inflammatory process and in cell movement, and decreased CXCR4 expression associated with a reduction of SDF-1 mediated-migration. In conclusion, this study demonstrates the existence of a powerful communication between AML cells and HSPCs, mediated by EVs, which suppresses normal hematopoiesis and potentially contributes to create a leukemic niche favorable to neoplastic development.

ORGANISM(S): Homo sapiens

PROVIDER: GSE189492 | GEO | 2022/03/11

REPOSITORIES: GEO

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Project description:Hematopoietic stem cell transplantation (HSCT) is a common practice in the treatment of (malignant-) hematopoietic diseases. Umbilical Cord Blood (UCB) has become an important hematopoietic stem and progenitor cell (HSPC) source for allogeneic HSCT. However, the relatively low number of HSPCs that are present in a single UCB unit is associated with a delayed engraftment and a higher risk for graft failure. To overcome these limitations, the discovery of new therapeutics and the development of efficient ex vivo HSPC expansion conditions will help towards the successful treatment of malignant hematopoietic disease. Bone marrow derived mesenchymal stromal cells (BMSCs) are known to support the characteristic properties of HSPCs in the bone marrow microenvironment. Recently, we and others have illustrated that extracellular vesicles (EVs) have the potential to support HSPC expansion, however, the mechanism and processes responsible for the cell-cell communication of EVs are still unknown. In the current study, we investigate whether primary human BMSC EVs isolated from two different origins, fetal (fEV) and adult (aEV) tissue respectively, can increase the relative low number of HSPC found in UCB and which EV-derived components are responsible for ex vivo HSPC expansion. Interestingly, aEVs but not fEVs showed supportive ex vivo expansion capacity of UCB-HSPC. Taking advantage of the two BMSC sources with different supportive effects, we performed small RNA sequencing and proteomics analyses on the EV cargo and investigated how gene expression is modulated in HSPC after incubation with aEVs and fEVs. Proteomics analyses of the protein cargo composition of the supportive aEV versus the non-supportive fEV identified 90% of the Top100 exosome proteins present in the ExoCarta database. We identified well-defined EV markers such as ANXA1, ANXA2, ANXA5 and GAPDH as the most abundant proteins detected in both aEV and fEV. Gene Ontology (GO) analyses of the enriched proteins in aEVs and fEVs illustrated that of the proteins overrepresented in aEVs were annotated to oxidation-reduction process (eg. HADHA, HADHB), mitochondrial ATP synthesis coupled proton transport (eg. ATP5A1, ATP5B and ATP5O) or protein folding (eg. FKBP11, FKBP10, FKBP2, MESDC2). In contrast, the proteins overrepresented in fEVs were annotated to extracellular matrix organization (eg. FN1, COL12A1, COL1A1, COL6A1, CCDC80) or positive regulation of cell migration (eg. ITGA4, ITGA6, GDF15, SEMA3, APDSD6, MMP14, ICAM1, FGFR1, PDGFRA, ADAMTS1). Interestingly, we found various proteins that were overrepresented in the non-supportive fEV cargo, that are involved in transforming growth factor beta receptor (TGFBR) signaling pathway (TGFBR1, TGFBR2, TGFB1, TGFB2, LTBP1, LTBP2, BMPR1A, GDF5, GDF15, PARP1, RPS27A and COL3A1). Together this illustrates the large differences in the protein content of EV populations derived from two different origins. Small RNA sequencing identified different molecular signatures between the supportive aEVs and the non-supportive fEVs. miRNA, yRNA and piRNA were abundantly found in aEV, while fEVs contained a significant enrichment of snoRNAs. Interestingly, the microRNA cluster miR-99b/let-7e/miR-125a, previously identified to increase the number of HSPC by targeting multiple proapoptotic genes, was highly and significantly enriched in aEV in comparison to fEV. Although we identified a significant difference in EV cargo and supportive effects of aEVs and fEVs, RNAseq analyses of 24hrs treated HSPCs with aEVs or fEVs indicated that only a limited set of genes was differentially regulated when compared to cells that were only treated with cytokines. Interestingly, we identified 5 genes, (eg MTF1, PER1, HOMER1, HSPA6, ENSG00000260534) that were significantly upregulated upon aEV compared to fEV treatment of HSPC. Moreover, HSPC treated with fEVs, resulted in increased expression of genes (SKIL, SMAD7, ENG, LDLRAD4) that are involved in the negative regulation of the TGFbeta signalling pathway. Together with the TGFbeta pathway proteins that were specifically identified in the fEVs, our results suggest that HSPCs may activate a negative feedback loop upon fEV treatment that consolidate HSPC expansion. In conclusion, our study gives novel insights into the complex biological role of EVs in the bone marrow microenvironment. Systematic analyses of supportive and non-supportive human BMSC derived extracellular vesicles indicated known molecules, eg. microRNA cluster miR-99b/let-7e/miR-125a, and the presence of TGFbeta pathway components that are important regulators in the cell-cell communication via EVs and open new means for the application of EVs in the discovery of therapeutics for more efficient ex vivo HSPC expansion.

Project description:Recently, we and others have illustrated that extracellular vesicles (EVs) have the potential to support hematopoietic stem and progenitor cell (HSPC) expansion; however, the mechanism and processes responsible for the intercellular communication by EVs are still unknown. In the current study, we investigate whether primary human bone marrow derived mesenchymal stromal cells (BMSC) EVs isolated from two different origins, fetal (fEV) and adult (aEV) tissue, can increase the relative low number of HSPCs found in umbilical cord blood (UCB) and which EV-derived components are responsible for ex vivo HSPC expansion. Interestingly, aEVs and to a lesser extent fEVs, showed supportive ex vivo expansion capacity of UCB-HSPCs. Taking advantage of the two BMSC sources with different supportive effects, we analyzed the EV cargo and investigated how gene expression is modulated in HSPCs after incubation with aEVs and fEVs. Proteomics analyses of the protein cargo composition of the supportive aEV versus the less-supportive fEV identified 90% of the Top100 exosome proteins present in the ExoCarta database. Gene Ontology (GO) analyses illustrated that the proteins overrepresented in aEVs were annotated to oxidation-reduction process, mitochondrial ATP synthesis coupled proton transport, or protein folding. In contrast, the proteins overrepresented in fEVs were annotated to extracellular matrix organization positive regulation of cell migration or transforming growth factor beta receptor (TGFBR) signaling pathway. Small RNA sequencing identified different molecular signatures between aEVs and fEVs. Interestingly, the microRNA cluster miR-99b/let-7e/miR-125a, previously identified to increase the number of HSPCs by targeting multiple pro-apoptotic genes, was highly and significantly enriched in aEVs. Although we identified significant differences in the supportive effects of aEVs and fEVs, RNAseq analyses of the 24hrs treated HSPCs indicated that a limited set of genes was differentially regulated when compared to cells that were treated with cytokines only. Together, our study provides novel insights into the complex biological role of EVs and illustrates that aEVs and fEVs differentially support ex vivo expansion capacity of UCB-HSPCs. Together opening new means for the application of EVs in the discovery of therapeutics for more efficient ex vivo HSPC expansion.

Project description:Recently, we and others have illustrated that extracellular vesicles (EVs) have the potential to support hematopoietic stem and progenitor cell (HSPC) expansion; however, the mechanism and processes responsible for the intercellular communication by EVs are still unknown. In the current study, we investigate whether primary human bone marrow derived mesenchymal stromal cells (BMSC) EVs isolated from two different origins, fetal (fEV) and adult (aEV) tissue, can increase the relative low number of HSPCs found in umbilical cord blood (UCB) and which EV-derived components are responsible for ex vivo HSPC expansion. Interestingly, aEVs but and to a lesser extent fEVs, showed supportive ex vivo expansion capacity of UCB-HSPCs. Taking advantage of the two BMSC sources with different supportive effects, we analyzed the EV cargo and investigated how gene expression is modulated in HSPCs after incubation with aEVs and fEVs. Proteomics analyses of the protein cargo composition of the supportive aEV versus the less-supportive fEV identified 90% of the Top100 exosome proteins present in the ExoCarta database. Gene Ontology (GO) analyses illustrated that the proteins overrepresented in aEVs were annotated to oxidation-reduction process, mitochondrial ATP synthesis coupled proton transport, or protein folding. In contrast, the proteins overrepresented in fEVs were annotated to extracellular matrix organization positive regulation of cell migration or transforming growth factor beta receptor (TGFBR) signaling pathway. Small RNA sequencing identified different molecular signatures between aEVs and fEVs. Interestingly, the microRNA cluster miR-99b/let-7e/miR-125a, previously identified to increase the number of HSPCs by targeting multiple pro-apoptotic genes, was highly and significantly enriched in aEVs. Although we identified significant differences in the supportive effects of aEVs and fEVs, RNAseq analyses of the 24hrs treated HSPCs indicated that a limited set of genes was differentially regulated when compared to cells that were treated with cytokines only. Together, our study provides novel insights into the complex biological role of EVs and illustrates that aEVs and fEVs differentially support ex vivo expansion capacity of UCB-HSPCs. Together opening new means for the application of EVs in the discovery of therapeutics for more efficient ex vivo HSPC expansion.

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Acute myeloid leukemia cells functionally compromise hematopoietic stem/progenitor cells inhibiting normal hematopoiesis through the release of extracellular vesicles

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