ASSESSING THE IMPACT OF A B-ALL-ASSOCIATED GERMLINE ETV6 VARIANT ON MURINE HEMATOPOIESIS AND STEM CELL FUNCTION

Abstract

The transcription factor ETV6 is required for bone marrow (BM) hematopoiesis and survival of adult hematopoietic stem cells (HSCs), but the mechanisms by which ETV6 regulates these processes remain unclear. We and others reported that germline variants in ETV6 are associated with autosomal dominant thrombocytopenia and occurrence of B-ALL. We identified likely pathogenic variants in 0.8% of 4,405 childhood ALL cases, and functional evaluation of these variants revealed impairment of transcriptional repression activity of ETV6. To investigate the impact of ETV6 variants on hematopoiesis, we generated a CRISPR-Cas9-derived mouse model (Etv6R355X/+) that mirrors an ETV6 R359X variant observed in multiple ALL cases. Flow cytometry of the BM revealed progressive changes in stem and progenitor cells, with 12-month-old ETV6R355X/+ mice showing ∼40% reductions in HSPCs and ∼40% increases in pro-B cells. Lineage-negative, cKit+, Sca1+ (LSK) cells from 3-month-old ETV6R355X/+ mice showed impaired engraftment in a competitive transplant model, as demonstrated by significantly reduced chimerism in primary, secondary, and tertiary recipients. To determine how the ETV6 R355X variant influences the hematopoietic transcriptional landscape, we performed RNA-seq and ATAC-seq from LSK and pro-B cells of 3-month-old WT or Etv6R355X/+ mice. We found 90 and 125 differentially expressed genes in LSK and pro-B cells, respectively. GSEA revealed enrichment of genes associated with hypoxia response and stem cell maintenance. ATAC-seq revealed a preferential shift toward an increase in open chromatin in Etv6R355X/+ cells within regions highly enriched for ETS DNA consensus motifs, indicating a potential loss of ETV6-mediated repression. These findings demonstrate that a pathogenic ETV6 variant can disrupt normal hematopoiesis and alter the transcriptional profile of stem and progenitor cells.