Mutant U2AF1 Expression Alters Hematopoiesis In Transgenic Mice

Mutations in 8 spliceosome genes have been identified in up to half of patients with myelodysplastic syndromes (MDS), suggesting that perturbations in pre-mRNA splicing play a role in MDS pathogenesis. Our group and others discovered recurrent heterozygous mutations in U2AF1, a gene encoding a splicing factor involved in intronic 3’-splice site recognition, in 11% of MDS patients. The most common U2AF1 mutation causes a serine to phenylalanine substitution at amino acid 34 (S34F). To study the effects of U2AF1(S34F) expression on hematopoiesis, we created site-specific, single-copy, doxycycline-inducible (controlled by the reverse tetracycline-transactivator [rtTA] transgene) U2AF1(WT) and U2AF1(S34F) transgenic mice. To examine the cell-autonomous effects of mutant U2AF1(S34F), we transplanted transgenic donor bone marrow into wild type mouse recipients prior to doxycycline induction of transgene expression.

We observed a doxycycline dose-dependent increase in expression of U2AF1(S34F) and U2AF1(WT) transgenes by RT-PCR and pyrosequencing analysis, and we subsequently chose a dose that induces equivalent levels of exogenous U2AF1 transgene to endogenous mouse U2af1 transcripts. Following 4 weeks of doxycycline induction of transgene expression in vivo, U2AF1(S34F)-recipient mouse bone marrow cells showed increased alternative splicing of endogenous Fmr1 pre-mRNA, a transcript previously shown to be alternatively-spliced in U2AF1 mutant MDS patient samples, when compared to U2AF1(WT)- and rtTA only-recipient mouse bone marrow cells (p<0.02, n=9-11). This effect was seen as early as 5 days (p<0.001, n=3-7).

Following 4 weeks of transgene induction, U2AF1(S34F)-recipient mice display a decrease in WBCs in the peripheral blood compared to U2AF1(WT)- and rtTA only-recipient controls (average 4.5 vs 6.7 and 7.1 K/µl, respectively; p≤0.01). However, there are no significant differences in bone marrow cellularity or spleen size in these mice (n=9-11). U2AF1(S34F)-recipient mice have reduced frequency of peripheral blood monocytes (average 1.2 vs 3.2 and 2.6%, p<0.05), as well as bone marrow monocytes (3.9 vs 7 and 6.1%, p<0.001) and B cells (average 10.1 vs 20.7 and 20.6%, p<0.001) when compared to U2AF1(WT)- and rtTA only-recipient mice, respectively (n=9-11). Moreover, bone marrow monocytes in U2AF1(S34F)-recipient mice have an increased frequency of Annexin V+ staining compared to controls (average 11.2 vs 6.9 and 7.5%, p<0.05, n=5-6), indicating that loss of these cells may be due to apoptosis. U2AF1(S34F)-recipient mice also have an increase in the frequency of bone marrow neutrophils compared to U2AF1(WT)- and rtTA only-recipient mice (average 65 vs 53 and 50%, respectively, p<0.001, n=9-11). Taken together, these data indicate U2AF1(S34F) expression alters hematopoietic lineage distribution in vivo.

In addition, 4 weeks of U2AF1(S34F) expression results in alterations of progenitor and stem cell frequency. U2AF1(S34F)-recipient mice have approximately a 1.5-fold increase in the frequency of progenitor cells in their bone marrow measured by flow cytometry (lin^low/c-kit+/Sca-1-) (average 4.1 vs 2.6 and 2.9%; p<0.001, n=5-6) and methylcellulose progenitor colony forming assays (average 85 vs 55 and 59 colonies/plate; p<0.02, n=9-11) when compared to U2AF1(WT)- and rtTA only-recipient mice, respectively. U2AF1(S34F)-recipient mice also display a 1.5-fold increase in the frequency of common myeloid progenitors compared to U2AF1(WT) and rtTA only controls in both bone marrow and spleen (p≤0.02), along with a 2-fold increase of the granulocyte-macrophage progenitors in the spleen (p≤0.03) (n=5-6). In addition to changes in progenitors, U2AF1(S34F)-recipient mice have a 1.4-fold increase in the frequency of bone marrow KLS cells, short-term HSCs, and dormant long-term HSCs (p≤0.05), as well as a 2-fold increase in spleen dormant long-term HSCs (p≤0.03) compared to U2AF1(WT)- and rtTA only-recipient controls (n=5-6). Competitive repopulation studies are ongoing.

Collectively, these data indicate that mutant U2AF1(S34F) expression in mouse bone marrow cells results in altered pre-mRNA splicing and changes in hematopoietic cell lineage distribution, as well as increased bone marrow and spleen progenitor and stem cell populations. Furthermore, these results suggest that mutant U2AF1 may contribute to altered hematopoiesis in patients.