Familial Thrombocytopenia-Associated Germline ETV6 P214L Mutation Results in XPO1-Mediated Nuclear Export

In recent years, a number of heterozygous germline mutations in ETV6 have been associated with familial thrombocytopenia and predisposition to hematologic malignancies. While most of these missense mutations occur in the DNA-binding-domain of ETV6, the recurrent P214L mutation is unique in that it occurs in the central disordered region of the protein. The ETV6 P214L mutation has previously been shown to disrupt the nuclear localization of ETV6 and the ability of ETV6 to act as a transcriptional repressor. Unexpectedly, we have found that the P214L missense mutation creates a recognition motif for the nuclear export protein XPO1. We have demonstrated in cellular and protein reconstitution assays that XPO1 directly engages the P214L but not the wild-type ETV6 construct. Consistent with this mechanism, we are able to fully rescue the nuclear localization of ETV6 P214L in mammalian cells with small molecule XPO1 inhibitors. We have also found that once localized to the nucleus, the P214L mutant construct exhibits functional consequences indistinguishable from wild-type ETV6 in Ba/F3 transformation assays. When looking at the evolutionary conservation of the ETV6 amino acid sequence involved in the XPO1 recognition motif, we observed that the synonymous P216L mutation in the mouse ETV6 ortholog does not create a nuclear export signal. In order to study XPO1-mediated export of ETV6 P214L and its impact on hematopoiesis, we have generated a mouse model in which two point mutations (P216L and S211I) have been introduced into a single endogenous ETV6 allele, which precisely matches the sequence of this region in human P214L carriers. Using CRISPR-Cas9, we generated 3 transgenic mouse lines: Etv6 P216L, Etv6 S211I, and Etv6 S211IP216L. Immunofluorescence of fixed bone marrow shows that ETV6 is only mislocalized in megakaryocytes from ETV6 S211I P216L mice. Complete blood counts revealed a significant decrease in platelet counts in ETV6 S211I P216L heterozygotes (485 x 10 3/µL) compared to ETV6 S211I heterozygotes (1140 x 10 3/µL, p