Mutant U2AF1 Expression Alters Hematopoiesis and Pre-mRNA Splicing in Transgenic Mice

Our group and others discovered recurrent heterozygous missense mutations in U2AF1 in 11% of patients with myelodysplastic syndromes (MDS). The U2AF1 gene encodes a splicing factor involved in intronic 3’-splice site recognition, which suggests that perturbations in pre-mRNA splicing play a role in MDS pathogenesis. To study the effects of the most common U2AF1 mutation, U2AF1(S34F), on hematopoiesis and pre-mRNA splicing in vivo, we created site-specific, single-copy, doxycycline-inducible 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 recipient mice prior to induction of transgene expression.

Following 4 weeks of transgene induction, U2AF1(S34F)-recipient mice have reduced total WBCs in the peripheral blood compared to U2AF1(WT)- and rtTA only-recipient controls (4.3 vs 7.11 and 7.13 K/µl, respectively, p≤0.01), but no significant changes in bone marrow cellularity or spleen size (n=9-11). U2AF1(S34F)-recipient mice have a perturbed mature cell lineage distribution, including reduced monocytes and B cells in both peripheral blood (p≤0.05) and bone marrow (p≤0.01) when compared to control mice (n=9-11). Reduction of bone marrow monocytes occurs as early as 5 days and is associated with increased Annexin V+ (p≤0.05) and phospho-H2AX (p≤0.05) compared to controls, suggesting loss of these cells may be due to apoptosis. In addition, U2AF1(S34F)-recipient mice have increased numbers of progenitors in both bone marrow and spleen by CFU-C methylcellulose assay and flow cytometry for c-Kit+/Lineage- cells, as well as common myeloid progenitors (CMPs), when compared to U2AF1(WT) and rtTA only controls (p≤0.05, n=5-10). U2AF1(S34F)-recipient mice also have an increase in the frequency of bone marrow hematopoietic stem cells (HSCs) measured by flow cytometry for bone marrow KLS (c-Kit+/Lineage-/Sca-1+) cells (p≤0.05). The increase in bone marrow KLS cells in U2AF1(S34F)-recipient mice is seen as early as 5 days and is associated with higher levels of intracellular Ki67 (a marker of cell proliferation) in KLS cells compared to U2AF1(WT) controls (p<0.05, n=8-13). Competitive repopulation studies show a disadvantage for bone marrow cells expressing mutant U2AF1(S34F) compared to U2AF1(WT) at ≥4 months post-transplant in both primary and secondary transplant recipient mice (p≤0.05, n=3-12), suggesting that the increase in KLS cell cycling following U2AF1(S34F) expression may lead to stem cell exhaustion. Collectively, these data indicate U2AF1(S34F) expression alters hematopoiesis in vivo.

Next, we performed unbiased RNA sequencing on sorted bone marrow CMPs following 5 days of transgene induction in U2AF1(S34F)- and U2AF1(WT)-transplanted mice (n=3 each). We identified 460 splicing junctions that were differentially expressed in U2AF1(S34F) samples compared to U2AF1(WT) controls (FDR <5%). We observed a preference of the mutant U2AF1(S34F) to skip exons (p=1.3e-05, n=72) and alternative splice sites (p=0.014, n=45) with a T in the -3 position relative to the AG splice acceptor site of differentially-spliced genes; this effect has been previously reported in AML patient samples with U2AF1 mutations. To prioritize altered junctions for further analysis, we intersected mouse CMP junction results with RNA sequencing data from AML patient samples with and without U2AF1 mutations and primary human CD34+ cells over-expressing U2AF1(S34F) or U2AF1(WT). Across species and present in all 3 datasets, we identified homologous dysregulated junctions in 2 genes known to be involved in cancer and stem cell biology: H2AFY and MED24. We validated concordant changes in both H2AFY and MED24 isoform expression by RT-PCR using MDS patient bone marrow samples that have mutant U2AF1(S34F) versus U2AF1(WT) (p<0.001, n=5-6). We are currently testing these isoform changes for their functional contribution to mutant U2AF1-associated phenotypes.

Together, these results suggest that mutant U2AF1 expression contributes to the altered hematopoiesis and pre-mRNA splicing observed in patients with U2AF1 mutations. This study also identifies changes in gene isoform expression unique to U2AF1 mutations that may have functional significance for MDS pathogenesis.