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Date of Award
Doctor of Philosophy (PhD)
Somatic mutations in the spliceosome gene U2AF1 are common in patients with myelodysplastic syndromes (MDS). We asked why U2AF1 mutations that code for the most common amino acid change (S34F) are always heterozygous and why the wild-type (WT) allele is retained in mutant cells. We hypothesized that mutant hematopoietic cells require the residual WT allele for survival. In order to understand the role of WT U2af1 in normal and abnormal hematopoiesis, we created a conditional U2af1 knock-out (KO) mouse (U2af1flox/flox).Homozygous embryonic deletion of U2af1 using Vav1-Cre was lethal and led to reduction of fetal liver hematopoietic stem and progenitor cells as well as impaired hemtopoietic progenitor cell colony forming ability in E14.5 embryos. The E14.5 fetal liver progenitor cells from homozygous KO embryos had aberrant splicing and alterations in gene expression compared to control cells by RNA-seq. To study the hematopoietic cell-intrinsic effects of U2af1 deletion in adult mice, we harvested bone marrow cells from Mx1-Cre/U2af1flox/flox, Mx1-Cre/U2af1flox/wt or Mx1-Cre/U2af1wt/wt mice and performed a non-competitive bone marrow transplant into lethally irradiated congenic recipient mice. Following poly I:C-induced U2af1 deletion, mice that received homozygous KO cells, but not cells from other genotypes (including heterozygous KO cells), had multilineage cytopenias, rapid bone marrow failure and depletion of hematopoietic progenitor cells. In a competitive bone marrow transplant repopulation assay, we observed a multilineage competitive disadvantage of homozygous KO cells, but not cells from other genotypes, including a rapid depletion of neutrophils. The data indicate that normal hematopoiesis is dependent on WT U2af1 expression, and that U2af1 heterozygous KO cells that retain one U2af1 allele are normal.Next, we tested whether mutant U2af1(S34F) hematopoietic cells require expression of WT U2af1 for survival. We crossed the U2af1 KO mouse to the previously published U2af1wt/S34F heterozygous knock-in (KI) mouse to generate heterozygous mutant (Mx1-Cre/U2af1wt/S34F) and hemizygous mutant (Mx1-Cre/U2af1S34F/flox) littermate mice and performed a competitive repopulation assay. There was a complete loss of neutrophil chimerism for hemizygous S34F cells within two weeks, similar to homozygous KO cells. The data confirm that S34F mutant U2af1 hematopoietic cells are dependent on expression of WT U2af1 for survival. Furthermore, we found that S34F mutant U2af1 cells are more sensitive to reduced, but not absent, levels of WT U2AF1 than control cells, suggesting that the ratio of U2af1 WT:S34F is critical for cell survival. Therefore, selectively targeting the WT U2AF1 allele in heterozygous mutant cells could be a novel therapeutic strategy.U2AF1 mutations significantly co-occur with ASXL1 mutations in MDS. We hypothesized that Asxl1 deletion will cooperate with mutant U2AF1(S34F) to induce MDS or acute myeloid leukemia (AML) development. To directly test this, we crossed available conditional Asxl1 KO mice to transgenic mutant U2AF1(S34F) mice to generate single and compound mutant mice. We characterized hematopoiesis and monitored the mice for development of MDS or AML. Our results showed that the two mutations do not cooperate to induce MDS or AML, but when Asxl1 KO is induced before mutant U2AF1(S34F) expression there is abnormal hematopoietic progenitor cell expansion, suggesting that they may cooperate to alter hematopoiesis. Future studies are needed to further explore the significance of this cooperation for disease pathogenesis.
Chair and Committee
Matthew J. Walter
Timothy Ley, Daniel Link, Eugene Oltz, Laura Schuettpelz,
Wadugu, Brian A., "The Role of U2AF1 in Normal and Abnormal Hematopoiesis" (2019). Arts & Sciences Electronic Theses and Dissertations. 2021.
Available for download on Friday, December 15, 2119