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

Abstract

Background: Germline mutations in the ETV6 transcription factor gene are responsible for familial thrombocytopenia and leukemia predisposition syndrome. Although previous studies have shown that ETV6 plays an important role in megakaryocyte (MK) maturation and platelet formation, the mechanisms by which ETV6 dysfunction promotes thrombocytopenia remain unclear.

Objectives: To decipher the transcriptional mechanisms and gene regulatory network linking ETV6 germline mutations and thrombocytopenia.

Methods: Presuming that ETV6 mutations result in selective effects at a particular cell stage, we applied single-cell RNA sequencing to understand gene expression changes during megakaryopoiesis in peripheral CD34⁺ cells from healthy controls and patients with ETV6-related thrombocytopenia.

Results: Analysis of gene expression and regulon activity revealed distinct clusters partitioned into 7 major cell stages: hematopoietic stem/progenitor cells, common-myeloid progenitors (CMPs), MK-primed CMPs, granulocyte-monocyte progenitors, MK-erythroid progenitors (MEPs), progenitor MKs/mature MKs, and platelet-like particles. We observed a differentiation trajectory in which MEPs developed directly from hematopoietic stem/progenitor cells and bypassed the CMP stage. ETV6 deficiency led to the development of aberrant cells as early as the MEP stage, which intensified at the progenitor MK/mature MK stage, with a highly deregulated core "ribosome biogenesis" pathway. Indeed, increased translation levels have been documented in patient CD34⁺-derived MKs with overexpression of ribosomal protein S6 and phosphorylated ribosomal protein S6 in both CD34⁺-derived MKs and platelets. Treatment of patient MKs with the ribosomal biogenesis inhibitor CX-5461 resulted in an increase in platelet-like particles.

Conclusion: These findings provide novel insight into both megakaryopoiesis and the link among ETV6, translation, and platelet production.

Keywords: ETV6; megakaryopoiesis; ribosomal protein S6; single-cell RNA sequencing; thrombocytopenia.