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Single-cell analysis of megakaryopoiesis in peripheral CD34+ cells: insights into ETV6-related thrombocytopenia

ABSTRACT: Using single-cell RNA sequencing (scRNAseq), we investigated ETV6-variant (P214L and F417LTer4) carriers who are affected by constitutional thrombopenia. We demonstrate that ETV6-variant carriers presented transcriptomic aberrant populations of megakaryocytes (MK) and mega-erythroid progenitors (MEP) with affected pathways. We confirmed this bioinformatic analyzes by functional assays. We also demonstrate a bypass of common myeloid progenitors (CMP) during physiologic differentiation from hematopoietic stem/progenitors cells (HSPC) to MK and we identified a new population of MK-primed CMP in our culture model. We raise this analysis as a new exploratory modus operandi for constitutional diseases.

ORGANISM(S): Homo sapiens

PROVIDER: GSE206089 | GEO | 2023/06/15

REPOSITORIES: GEO

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Project description:ETV6-RUNX1 is associated with childhood B-cell acute lymphoblastic leukemia (B-ALL), but the consequence of ETV6-RUNX1 expression on cell lineage decision during B-cell leukemogenesis remains largely evasive. Clinically silent ETV6-RUNX1 preleukemic clones are frequently found in neonatal cord blood, but only a few carriers develop B-ALL as a result of the acquisition of secondary postnatal genetic hits, like KDM5C or PAX5 loss. However, understanding the mechanism involved in this transformation would advance the development of non-toxic prophylactic interventions to preleukemic carriers. Using genetic lineage tracing, we have examined the capacity of ETV6-RUNX1 in instructing a B-cell malignant phenotype. Restricted Cre-mediated activation of ETV6-RUNX1 from the endogenous Etv6 locus into B-cell precursors does not trigger B-ALL development. Similarly, concomitant transient ETV6-RUNX1 expression in hematopoietic progenitors close to restricted Cre-mediated introduction of second hit (Kdm5c loss) in B-cells fail to induce B-ALL. In contrast, targeting ETV6-RUNX1 in hematopoietic progenitors was required to induce leukemia. These mice develop either T-ALL or B-ALL, suggesting that the nature of the second hit may define tumor cell identity during ETV6-RUNX1 leukemogenesis. In order to uncover a potential exclusive effect of the second hit in establishing the leukemic phenotype, we next showed that the introduction of the second hit, either Kdm5c or Pax5 loss, to the ETV6-RUNX1 preleukemic clone confers the tumor B-cell identity without need of environmental infection exposure. The resulting B-ALLs clustered according to the second-hit by RNA sequencing (RNA-Seq) analysis. Together these results provide a novel paradigm for the generation of tumor B cells through ETV6-RUNX1 in vivo where the second hit confers the tumor cell-identity to the ETV6-RUNX1 preleukemic clone. These findings could be relevant to develop new therapeutic approaches to prevent disease development.

Project description:This SuperSeries is composed of the SubSeries listed below. ETS Variant 6 (ETV6) encodes an essential transcriptional repressor abundantly expressed in hematopoietic stem and progenitor cells (HSPCs), where it is required for adult hematopoiesis. Heterozygous pathogenic germline ETV6 variants are associated with Thrombocytopenia 5 (T5), a poorly understood genetic condition predisposing to thrombocytopenia and hematologic malignancies. To elucidate how germline ETV6 variants impact the HSPC compartment and contribute to disease, we generated a knock-in mouse model harboring an Etv6R355X loss-of-function variant, which is equivalent to the T5-associated variant ETV6R359X. Under homeostatic conditions, all HSPC subpopulations are present in the bone marrow (BM) of Etv6R355X/+ mice; however, these animals display subtle shifts in the proportions and/or numbers of specific progenitor subtypes. To examine whether the Etv6R355X/+ mutation impacts HSPC function, we carried out serial competitive transplantation and observed that Etv6R355X/+ lineage-sca1+cKit+ (LSK) cells exhibit significantly impaired reconstitution compared to Etv6+/+ LSK cells with near complete failure to repopulate irradiated-recipients by the tertiary transplant. Mechanistic studies incorporating CUT&RUN, ATAC-Seq and Hi-C identify ETV6 binding at inflammatory gene loci, including those within the TNF signaling pathway, in Etv6+/+ HSPCs, mouse BM-progenitor-derived HPC5 cells, and human CD34+ cells. Further, single-cell RNA-Seq of BM cells isolated post-competitive transplantation reveals upregulation of inflammatory genes in Etv6R355X/+ compared to Etv6+/+ progenitors. Corroborating these findings, Etv6R355X/+ HSPCs produce significantly more TNF than Etv6+/+ cells post-transplantation. From these studies, we conclude that ETV6 is required to repress inflammatory gene expression in HSPCs under conditions of hematopoietic stress and this mechanism may be critical to sustain HSPC function.

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Single-cell analysis of megakaryopoiesis in peripheral CD34+ cells: insights into ETV6-related thrombocytopenia

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