ETV6 represses Tumor Necrosis Factor During Stress Hematopoiesis to Regulate Mouse Stem Cell Function

At the root of the blood system lies a heterogenous population of hematopoietic stem and progenitor cells (HSPCs), which reside in the bone marrow (BM) and give rise to diverse blood lineages. Maintaining this pool of self-renewing HSPCs is critical to uphold hematopoiesis during period of stress such as bleeding or infection1. ETS Variant Transcription Factor 6 (ETV6) is a transcriptional repressor that is highly expressed in HSPCs2 where it is essential for development and maintenance of the adult hematopoiesis 3. In 2015, our group4 and others5-7  identified germline pathogenic ETV6 variants in families with predisposition to B-acute lymphoblastic leukemia (B-ALL) and thrombocytopenia, defining a new genetic syndrome known as Thrombocytopenia 5 (T5). Subsequently, we performed targeted germline ETV6 sequencing of remission blood samples from over 4,000 children with B-ALL and identified germline ETV6 variants in ~1% of cases7. In vitro studies revealed that ETV6 variant proteins exhibit impaired repressor activity, reduced DNA binding, and aberrant subcellular localization4,5,7,8. Overall, these studies indicated that T5-associated germline ETV6 variants negatively impact the repressor activity of ETV6. In support of this notion, transcriptional profiling of peripheral blood cells from T5 patients has revealed upregulation of interferon response genes9. Nevertheless, little remains known about the mechanisms by which ETV6 regulates the HSPC compartment and how T5-associated ETV6 variants contribute to disease.

To address these questions, we used CRISPR-Cas9 gene editing to generate a novel mouse model harboring a pathogenic heterozygous germline Etv6 variant, R355X, the murine equivalent to the human T5-associated variant R359X10. Through the comprehensive study of this model, we describe a novel role for ETV6 during aging and regenerative hematopoiesis and show that the heterozygous Etv6R355X variant impairs HSC function in vitro and in vivo. Using genomic approaches to interrogate mouse and human HSCs, we identify new ETV6 targets, including the gene encoding Tumor Necrosis Factor (TNF) and genes involved in TNF signaling. Further, we show increased TNF production and cell cycling in Etv6R355X/+ mouse HSPCs post-BM transplantation. Finally, we demonstrate that genetic ablation of Tnf restores the long-term potential of Etv6R355X/+ cells in serial replating assays in vitro. Together, these findings provide novel insights into the pathways regulated by ETV6 and demonstrate how a pathogenic variant impacts ETV6 function in the context of hematopoietic stress.

All bulk RNAseq, Cut&Run, ATACseq, and Hi-C data has been submitted to in the Gene Expression Omnibus (accession number GSE213597) and Sequence Read Archive (BioProject number PRJNA880871). Due to the journal’s limitation on submitted supplementary data  as an Excel file, all post-analysis supplemental data files are deposited in Dryad data repository to be made available with this manuscript.