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1298 Familial Thrombocytopenia-Associated Germline ETV6 P214L Mutation Results in XPO1-Mediated Nuclear ExportClinically Relevant Abstract

Program: Oral and Poster Abstracts
Session: 501. Hematopoietic Stem and Progenitor Cells and Hematopoiesis: Basic and Translational: Poster I
Hematology Disease Topics & Pathways:
Research, Fundamental Science, hematopoiesis, Biological Processes, molecular biology, Technology and Procedures, gene editing
Saturday, December 9, 2023, 5:30 PM-7:30 PM

Michael McCONVILLE1*, Toby Thomas, B.S.2*, Catherine Valadez, B.S. M.S.3*, Ryan Beckner, B.S.3*, Yuh Min Chook, Ph.D.4*, Stephen Chung, M.D.2 and Glen Liszczak, Ph.D.3*

1Department of Biochemistry, University of Texas Southwestern Medical Center, Irving, TX
2Division of Hematology/Oncology, Department of Internal Medicine, Children's Research Institute, University of Texas Southwestern, Dallas, TX
3Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX
4Departments of Pharmacology and Biophysics, University of Texas Southwestern Medical Center, Dallas, TX

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: 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 summary, 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 signal in the endogenous ETV6 allele and found that this model demonstrates a thrombocytopenic phenotype as seen in human patients. Reductions in HSCs and MkPs suggest both a decreased fitness of HSCs, as well as dysregulated megakaryopoiesis in this model.

Disclosures: No relevant conflicts of interest to declare.

*signifies non-member of ASH