A germline ETV6 mutation disrupts hematopoiesis via de novo creation of a nuclear export signal

Germline mutations in the transcriptional regulator ETV6 are a root cause of familial inherited thrombocytopenia and predispose carriers to myelodysplastic syndromes and acute leukemias. Here, we report that the ETV6 P214L mutation creates an XPO1-dependent nuclear export signal to cause protein mislocalization. Strategies to disrupt XPO1 nuclear export activity fully restore ETV6 P214L protein nuclear localization and transcription regulation activity, establishing XPO1-dependent mislocalization as a critical mechanism underscoring ETV6 P214L dysfunction. Mechanistic insight inspired the design of 'humanized' ETV6 mice in which the germline P214L mutation is sufficient to elicit severe defects in thrombopoiesis and hematopoietic stem cell maintenance and survival in animals. These studies define a unique mechanism by which the ETV6 P214L mutation exerts a dominant negative effect on protein function and reveal critical mutation-dependent disruptions to hematopoiesis that underlie disease phenotypes. Overall design: RNA-seq profiling of Ba/F3 cells expressing NRAS G12D and one of ZsGreen (control), ETV6-IRES-ZsGreen, ETV6 P214L-IRES-ZsGreen, or ETV6 I209S P214L-IRES-ZsGreen on day 3 after removal of IL-3 from media conditions. The purpose of this experiment is to determining if breaking the nuclear export sequence created by the ETV6 P214L mutation with a secondary I209S mutation would result in ETV6 I209S P214L samples transcriptionally phenocopying wild-type ETV6 samples.