Germline Mutations in ETV6 are Associated with In Vivo cytoplasmic Localization of ETV6 and Cause Transcriptional Dysregulation of Interferon Response Genes

ETV6, an ETS family transcription factor, has been characterized as a master regulator of hematopoiesis and megakaryocyte development. In this work we identified a novel role for ETV6 as a key repressor of peripheral inflammation and interferon response in humans. Flow cytometry based immunophenotyping of peripheral blood mononuclear cells (PBMC) from 5 patients carrying a germline mutation in ETV6 (p.Pro214Leu) of various ages demonstrated normal frequencies of peripheral cell populations, including B and T cells (CD3+ and CD19+, respectively), progenitor populations, including CLP (CD19- CD3- CD34+ CD10+) and MEP (CD10- CD34+ CD38+ CD135- CD45RA-), but significantly increased circulating HSCs (CD34+ CD38- CD90+) as compared to healthy related and unrelated controls. Immunofluorescence studies in these PBMC showed cytoplasmic localization of ETV6 in all affected individuals. Furthermore, 10X genomics single cell RNA sequencing of these peripheral blood cells demonstrated profound transcriptomic dysregulation as compared to PBMCs of related and unrelated healthy controls. We initially detected a total of 204 differentially expressed transcripts in monocytes, NK cells and T and B cell populations, demonstrating a cell-type specific upregulation of inflammatory genes. We then analyzed the intersection of the significantly increased transcripts across these populations, identifying a highly specific suite of 22 genes, which depending on cell type were upregulated 4 to 400-fold. These significantly upregulated 22 transcripts in the affected individuals were highly enriched for the interferon response pathway, consisting of pro-inflammatory transcripts such as CCL4, CCL4L2, CCL3, CCL3L3, IFIT2, ISG15, MX1, IFIT3, IRF1, and NFKBIA among others. Ingenuity Pathway Analysis revealed HDAC3 as a highly predicted upstream master regulator of this group of genes. We then demonstrated by protein immunoprecipitation that both wild-type and P214L ETV6 form a complex with HDAC3, suggesting that the ETV6-HDAC3 complex is being removed from the nucleus, disrupting homeostatic transcriptional repression of interferon response genes. In summary, we have determined a new role for ETV6 as a repressive regulator of interferon response genes likely via its interaction with HDAC3. These findings have potential implications for the development of myelodysplasia and hematological malignancies observed in individuals with ETV6 germline mutations. Disclosures Callaghan: Novonordisk: Consultancy, Speakers Bureau; Octapharma: Consultancy; Pfizer: Research Funding; Roche: Research Funding; Biomarin, Bioverativ, Grifols, Kedrion, Pfizer, Roche/Genentech, Shire, and Spark Therapeutics: Consultancy; Alnylum: Equity Ownership; Bayer: Consultancy, Speakers Bureau; Takeda: Consultancy, Research Funding; Sanofi: Consultancy; Global Blood Therapeutics: Consultancy; Shire/Takeda: Speakers Bureau; Roche/Genentech: Speakers Bureau.