Studying the Role of ETV6 in Megakaryopoiesis and Thrombopoiesis Using a Novel CRISPR-Cas9 Halotag Genome Editing Strategy

Emerging data indicate that germline mutations in transcription factors involved in hematopoiesis can lead to a cascade of downstream molecular alterations that modify the function of megakaryocytes (MK) and platelets. Our group and others have found that mutations in ETV6 lead to mild thrombocytopenia with a bleeding diathesis, red cell macrocytosis, and predisposition to lymphoblastic leukemia. The mechanisms responsible for thrombocytopenia and propensity for bleeding in patients with ETV6 mutations are unknown. We described families with missense mutations in the central domain (p.Pro214Leu) and the ETS DNA binding domain (p.Arg418Gly) of ETV6 that result in aberrant cellular localization of ETV6, decreased transcriptional repression, and impaired MK maturation. Deep sequencing of the platelet transcriptome revealed significant differences in mRNA expression levels between patients with the ETV6 p.Pro214Leu mutation and non-affected family members, indicating that ETV6 is critically involved in defining the molecular phenotype and function of platelets. We hypothesize that normal regulation and function of ETV6 is essential for the transcriptional machinery that controls megakaryocyte differentiation and formation of platelets that function normally under homeostatic conditions. We have successfully generated a CRISPR-Cas9 model to edit the genome of ETV6-expressing iPSC derived megakaryocyte cell line (imMKCL) to characterize the role of wild-type ETV6 in megakaryocyte development and elucidate the molecular mechanism driving mutant ETV6 mislocalization, transcriptional dysregulation, and subsequent dysmegakaryopoiesis and thrombocytopenia. In this imMKCL model, we have genetically engineered the cells to express wild-type, P214L, and the DNA binding domain mutations R418G and R369Q ETV6 fused to HALOtag, a reporter protein that can react with ligands carrying a variety of functionalities, including fluorescent labels, affinity handles, and attachment to solid phase, making this novel reporter conducive to immunofluorescence imaging, biochemical pulldown, and ChIPSeq. This system allows us to express wild type and mutant forms of ETV6 in appropriate allele ratios in imMKCL cells and various hematopoietic-relevant cell lines. Using this approach, we detected nuclear localization of wild-type ETV6 and altered cytoplasmic localization of both P214L and R418G ETV6 mutants. We have also demonstrated dimerization between both wild-type and mutant ETV6 in this cell model. Importantly, we have used HALOtag protein immunoprecipitation to demonstrate ETV6 binding to FLI1, another ETS family member and key transcriptional regulator of megakaryocyte development, suggesting that ETV6 and FLI1 cooperate to regulate megakaryopoiesis under homeostatic conditions. Altogether, these data suggest that mutant ETV6 functions as a dominant negative, sequestering wild type ETV6 in the cytoplasm, de-regulating key transcriptional targets for homeostatic megakaryocyte development. Ongoing studies will define the full repertoire of protein interactions and transcriptional targets of wild-type and mutant ETV6. Discoveries from this novel tool will further advance our understanding of normal megakaryocyte and platelet biology, and will provide potential therapeutic targets for disorders of platelet number and function to optimize the clinical approach to these patients. Disclosures Callaghan: Bayer: Consultancy, Speakers Bureau; Alnylum: Equity Ownership; Biomarin, Bioverativ, Grifols, Kedrion, Pfizer, Roche/Genentech, Shire, and Spark Therapeutics: Consultancy; Takeda: Consultancy, Research Funding; Sanofi: Consultancy; Global Blood Therapeutics: Consultancy; Novonordisk: Consultancy, Speakers Bureau; Octapharma: Consultancy; Pfizer: Research Funding; Roche: Research Funding; Shire/Takeda: Speakers Bureau; Roche/Genentech: Speakers Bureau.

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