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 others^5-7 identified germline pathogenic <em>ETV6</em> 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 <em>ETV6 </em>sequencing of remission blood samples from over 4,000 children with B-ALL and identified germline <em>ETV6</em> variants in ~1% of cases⁷. <em>In vitro</em> studies revealed that ETV6 variant proteins exhibit impaired repressor activity, reduced DNA binding, and aberrant subcellular localization^4,5,7,8. Overall, these studies indicated that T5-associated germline <em>ETV6</em> 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 <em>ETV6</em> 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 <em>Etv6 </em>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 <em>Etv6</em>^R355X variant impairs HSC function <em>in vitro</em> and <em>in vivo</em>. 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 <em>Etv6^R355X/+</em> mouse HSPCs post-BM transplantation. Finally, we demonstrate that genetic ablation of <em>Tnf</em> restores the long-term potential of <em>Etv6^R355X/+</em> cells in serial replating assays <em>in vitro</em>. 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&amp;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.