ETV6 Represses TNF during Stress Hematopoiesis and Regulates HSC Self Renewal

INTRO: ETS Variant Transcription Factor 6 (ETV6), formerly known as Translocation ETS Leukemia (TEL), functions primarily as a transcriptional repressor and is highly expressed in mouse and human HSPCs where it is essential for the development and maintenance of adult hematopoiesis. Nevertheless, it has remained poorly understood how ETV6 regulates the HSC compartment. In 2015, our group and others identified heterozygous pathogenic germline ETV6 variants in families with autosomal dominant thrombocytopenia and predisposition to hematologic malignancies (most often B-acute lymphoblastic leukemia), a condition now known as thrombocytopenia 5 (T5).

METHODS: To elucidate the mechanisms by which germline ETV6 variants impact hematopoiesis and promote leukemogenesis, we generated a knock-in mouse model harboring Etv6 R355X, the murine equivalent of a human T5-associated variant (R359X). We used complementary in vitro and in vivo assays to comprehensively characterize the numbers and functions of Etv6^R355X/+ and Etv6^+/+ hematopoietic progenitors.

RESULTS:Etv6^R355X/+ mice display several of the cardinal features of T5, including abnormally rounded platelets with increased area compared to platelets from wild-type Etv6^+/+ littermates. Further, Etv6^R355X/+ platelets exhibit significantly delayed and incomplete clot formation as measured through clot retraction assays. Together, these findings indicate that this novel model will serve as a viable means to study the human disease.

We observe that 3-month-old Etv6^R355X/+mice maintain normal frequencies and numbers of lineage-sca1+cKit+ (LSK) cells in the bone marrow (BM); however, at 12 months of age, they exhibit a significant decrease in frequency in this compartment. At each of these timepoints, there are variable shifts in the proportions and/or numbers of certain HSC subpopulations. To examine whether the Etv6^R355X/+mutation impacts HSC function, we carried out in vitrosEtv6^R355X/+BM cells generate more colonies than wild-type cells in the first two rounds of plating, but significantly fewer or no colonies by the fourth plating. Similarly, Etv6^R355X/+LSK cells exhibit significantly impaired engraftment when challenged via competitive transplantation, with near complete failure to reconstitute irradiated recipients by the tertiary transplant. Etv6^R355X/+LSKs home to the bone marrow comparably to Etv6^+/+cells, indicating that homing does not account for the defect in repopulation capacity.

To investigate how ETV6 functions in HSCs, we performed RNA-seq of Etv6^R355X/+LSKs and flow cytometry-purified HSPC subpopulations from 12-month-old mice, which revealed upregulation of genes associated with an active stem cell phenotype, downregulation of genes supporting stem cell quiescence, and upregulated expression of Tnf with enrichment for the Hallmark TNFa Signaling via NFKB geneset. Subsequent experiments using CUT&RUN of Etv6^+/+LSK cells revealed several known and novel ETV6 target genes, including Tnf, a cytokine implicated in regulating stress hematopoiesis. The sites bound by ETV6 at the Tnf locus aligned with ATAC-seq peaks generated from HPC5 cells, a mouse bone marrow progenitor cell line, suggesting that this is a region of open chromatin and active transcriptional regulation. Corroborating these findings, examination of Etv6^R355X/+HSPCs at 6 weeks post-transplant revealed increased cell cycling and intracellular TNF production.

CONCLUSION: From these studies, we conclude that ETV6 regulates HSC maintenance. Further, it binds to a regulatory sequence within the Tnf locus, whereby it represses TNF expression. The loss or reduction of this repression in Etv6^R355X/+HSCs is associated with increased cell cycling under conditions of hematopoietic stress and a loss of self-renewal capacity. Studies are ongoing to determine how these changes promote leukemogenesis and contribute to the other features of T5.

Weiss:Graphite Bio: Consultancy; Glaxo SmithKline: Consultancy; Dyne Therapeutics: Consultancy; Forma therapeutics: Consultancy; Cellarity Inc: Current equity holder in private company. Yang:Takeda Pharmaceutical Company: Research Funding. Nichols:Incyte: Research Funding.