Abstract 2164 Familial Thrombocytopenia-Associated Germline ETV6 P214L Mutation Results in XPO1-Mediated Nuclear Export

Objective: Determine the mechanism by which the recurrent thrombocytopenia-associated germline ETV6 P214L mutation results in protein mislocalization and disease. Methods: We utilized a variety of in vitro biochemical (fluorescence polarization), tissue culture (immunofluoresence microscopy, growth curves, RNA sequencing), and animal model (complete blood counts, competitive bone marrow transplant) assays to study the effects of the ETV6 P214L mutation. All animal studies were approved by UT Southwestern's Institutional Animal Care and Use Committee (IACUC). Results: 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 sequence. 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: Etv6P216L, Etv6S211I, and Etv6S211I P216L. 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 103/μL) compared to ETV6 S211I heterozygotes (1140 x 103/μL, p<0.0001), ETV6 P216L heterozygotes (1065 x 103/μL, p<0.0006), and wild-type littermates (1254 x 103/μL, p<0.0001). Flow cytometry phenotyping of bone marrow from the three transgenic lines and wild-type littermates shows a significant decrease in the absolute numbers of hematopoietic stem cells (HSCs) and megakaryocyte precursors (MkPs) in ETV6 S211I P216L mice. In a competitive bone marrow transplant assay, bone marrow cells from ETV6 S211I P216L mice fail to engraft in recipient mice, further suggesting that loss of ETV6 function as a result of XPO1-mediated nuclear export leads to a defect in HSC function. Discussion: We have determined that XPO1-mediated nuclear export is the mechanistic basis for ETV6 P214L mislocalization. We developed a mouse model that recapitulates this export sequence in the endogenous ETV6 allele and found that this model demonstrates a thrombocytopenic phenotype as seen in human patients. Failure of HSCs to engraft in transplant assays and reductions in HSCs and MkPs suggest both a decreased fitness of HSCs, as well as dysregulated megakaryopoiesis in this model. We acknowledge the following institutions/organizations for funding this research: Cancer Prevention and Research Institute of Texas (CPRIT), National Institutes of Health (NIH), Welch Foundation