Normal Hematopoiesis Is Dependent on the Expression of U2af1, a Spliceosome Gene Commonly Mutated in MDS

Recently, somatic mutations in genes encoding factors involved in pre-mRNA splicing have been identified in up to 50% of myelodysplastic syndrome (MDS) patients. U2AF1, a spliceosome gene, has recurrent missense mutations in 11% of MDS patients. This gene is a key component of the spliceosome that recognizes the AG dinucleotide in the 3' splice acceptor site of pre-mRNA. U2AF1 mutations are always heterozygous and the wild-type allele is expressed, suggesting that cells may not tolerate biallelic mutations. Mice expressing mutant U2AF1 have altered hematopoiesis and RNA splicing, similar to mutant MDS patients but the role of wild-type U2AF1 in normal hematopoiesis has not been studied. A complete understanding of the role of wild-type U2AF1 on hematopoiesis and RNA splicing is critical to our understanding of how mutant U2AF1 contributes to abnormal hematopoiesis and MDS. In order to understand the role of wild-type U2af1 in normal hematopoiesis, we created a conditional U2af1 knock-out (KO) mouse by flanking exon 2 with LoxP sites.

We confirmed deletion at the genomic DNA and mRNA level in hematopoietic cells by crossing mice to various Cre-recombinase expressing models. We first generated U2af1^flox/flox , U2af1^flox/wt and U2af1^wt/wt littermate control mice all carrying ERT2- Cre (and U2af1^flox/flox without ERT2- Cre as a control). To study the hematopoietic cell-intrinsic effects of U2af1 deletion, we performed a non-competitive bone marrow transplant into lethally irradiated congenic recipient mice. Four weeks after engraftment, we treated mice with tamoxifen to active ERT2- Cre and monitored their survival and blood counts. Homozygous U2af1 KO mice ( ERT2- Cre/ U2af1^flox/flox ), but not other genotypes, became moribund (and were sacrificed) as early as only 6 doses of tamoxifen (Fig. 1A). The bone marrow cellularity in homozygous U2af1 KO mice was reduced (Fig. 1B). Analysis of mice at day 24 (from 1^st tamoxifen dose) revealed anemia and a reduction in blood and bone marrow neutrophils, monocytes, and B-cells ( p ≤ 0.01 ).

We also generated mice carrying the interferon inducible Mx1- Cre to verify the observed phenotypes using an independent model. After a non-competitive bone marrow transplant into lethally irradiated congenic recipient mice, deletion of U2af1 was induced by polyinosinic:polycytidylic acid (poly I:C). The homozygous U2af1 KO mice, but not other genotypes, became moribund. Analysis of peripheral blood up to 11 days post poly I:C treatment revealed anemia (decrease >1.7 fold) and a reduction in platelets (>9 fold) and white blood cell counts (>4.7 fold) in homozygous U2af1 KO mice compared to all other genotypes (p ≤ 0.001, n=5) . Peripheral blood neutrophils, monocytes, B-cells, and T-cells were reduced (p ≤ 0.001, n=5). Deletion of U2af1 also led to rapid bone marrow failure and a reduction in bone marrow neutrophils, monocytes, and B-cells as well as a significant depletion hematopoietic progenitor cells [ KL (Lin-, cKit+, Sca1-), and KLS (Lin-, cKit+, Sca1+)], (Fig. 1C). In order to study the hematopoietic cell-intrinsic effects of U2af1 deletion on stem cell function, we mixed equal numbers of whole bone marrow test cells (Ly5.2) with congenic control wild-type bone marrow competitor cells (Ly5.1/5.2) and transplanted them into lethally irradiated congenic recipient mice (Ly5.1). As early as 10 days following Cre-induction we observed a significant decrease in white blood cell count chimerism (Fig. 1D) and complete loss of homozygous U2af1 KO neutrophils and monocytes, as well as a severe reduction in B-cells and T-cells ( p ≤ 0.001 ). Heterozygous U2af1 KO bone marrow had a modest competitive disadvantage compared to controls after 120 days (Figure 1D, p ≤ 0.05 ).

Collectively, the data indicate that stem cell function, multilineage hematopoiesis and hematopoietic cell survival are dependent on U2af1 expression. In ongoing studies to understand why U2AF1 mutations are always heterozygous, we will cross the U2af1 KO mouse to existing transgenic mutant U2AF1(S34F)-expressing mice and determine whether mutant U2AF1-expression is sufficient to rescue cell survival and normal hematopoiesis when both endogenous U2af1 wild-type alleles are deleted. Proving that U2AF1 mutant cells are vulnerable to loss of the wild-type U2AF1 allele could provide a therapeutic vulnerability to mutant cells by selectively targeting the wild-type U2AF1 allele in heterozygous mutant cells.

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