3036 – ETV6 REPRESSES TNF EXPRESSION IN HSCS DURING STRESS HEMATOPOIESIS AND REGULATES HSC SELF-RENEWAL

Abstract

ETV6 encodes an essential transcription factor during normal hematopoiesis. Our group and others identified heterozygous pathogenic germline ETV6 variants in families with autosomal dominant thrombocytopenia and predisposition to hematologic malignancies. To elucidate the mechanisms by which germline ETV6 variants impact hematopoiesis, we generated a knock-in mouse model harboring Etv6 R355X, the murine equivalent of a human disease variant. Except for reduced MPP4 cells, 3-month-old Etv6R355X/+ mice maintain normal numbers of hematopoietic stem and progenitor cells (HSPCs) in the bone marrow (BM). However, by 12 months, they exhibit a decrease in all HSPC compartments. RNA-sequencing of Etv6R355X/+ HSPCs at 12 months identified 402 significant differentially-expressed genes. Among these, we observe upregulation of “active stem cell genes” (e.g., Myc, Cdk6) and downregulation of “self-renewal genes” (e.g., Procr, Mpl, Jun). Moreover, we observe a significant loss of self -renewal potential when challenging Etv6R355X/+ HSPCs with repetitive stressors: First, through serial competitive transplantation, we observe a failure of mutant cells to reconstitute the bone marrow by the tertiary transplant. Next, following serial 5FU treatment, we find that Etv6R355X/+ mice exhibit inferior survival compared to Etv6+/+ controls. To determine which ETV6 target genes contribute to these phenotypes, we performed ETV6 CUT&RUN, ATACseq and HiC using the HPC5 stem cell line, as well as ETV6 CUT&RUN using primary mouse HSPCs and human CD34+ cells. Through these studies, we identify TNF as a putative ETV6 target gene. Notably, using intracellular staining and flow cytometry, we find elevated TNF expression in Etv6R355X/+ HSPCs post-transplant. Together, these data suggest that ETV6 regulates HSPC self-renewal and represses TNF signaling during stress hematopoiesis. ETV6 encodes an essential transcription factor during normal hematopoiesis. Our group and others identified heterozygous pathogenic germline ETV6 variants in families with autosomal dominant thrombocytopenia and predisposition to hematologic malignancies. To elucidate the mechanisms by which germline ETV6 variants impact hematopoiesis, we generated a knock-in mouse model harboring Etv6 R355X, the murine equivalent of a human disease variant. Except for reduced MPP4 cells, 3-month-old Etv6R355X/+ mice maintain normal numbers of hematopoietic stem and progenitor cells (HSPCs) in the bone marrow (BM). However, by 12 months, they exhibit a decrease in all HSPC compartments. RNA-sequencing of Etv6R355X/+ HSPCs at 12 months identified 402 significant differentially-expressed genes. Among these, we observe upregulation of “active stem cell genes” (e.g., Myc, Cdk6) and downregulation of “self-renewal genes” (e.g., Procr, Mpl, Jun). Moreover, we observe a significant loss of self -renewal potential when challenging Etv6R355X/+ HSPCs with repetitive stressors: First, through serial competitive transplantation, we observe a failure of mutant cells to reconstitute the bone marrow by the tertiary transplant. Next, following serial 5FU treatment, we find that Etv6R355X/+ mice exhibit inferior survival compared to Etv6+/+ controls. To determine which ETV6 target genes contribute to these phenotypes, we performed ETV6 CUT&RUN, ATACseq and HiC using the HPC5 stem cell line, as well as ETV6 CUT&RUN using primary mouse HSPCs and human CD34+ cells. Through these studies, we identify TNF as a putative ETV6 target gene. Notably, using intracellular staining and flow cytometry, we find elevated TNF expression in Etv6R355X/+ HSPCs post-transplant. Together, these data suggest that ETV6 regulates HSPC self-renewal and represses TNF signaling during stress hematopoiesis.