Abstract PR06: Mutant U2AF1 alters hematopoiesis and pre-mRNA splicing in transgenic mice.

Abstract

Abstract 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 patients with myelodysplastic syndromes (MDS), suggesting that perturbations in pre-mRNA splicing play a role in MDS pathogenesis. 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 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 mouse recipients 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 frequency of Annexin V+ staining (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) expression results in alterations of progenitor and stem cell frequency in bone marrow and spleen of mice. 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 - (KL) 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 KLS (c-Kit+ Lineage- Sca-1+) cells, short-term HSCs, and dormant long-term HSCs (p≤0.05), as well as an increase in spleen dormant long-term HSCs (p≤0.03) compared to U2AF1(WT)- and rtTA only-recipient controls (n=5-6). The increase in bone marrow KLS cells in U2AF1(S34F)-recipient mice at 5 days is associated with higher levels of intracellular Ki67 (a marker of cell proliferation) in KLS cells compared to controls (p<0.05, n=8-13). Competitive repopulation studies show a disadvantage for bone marrow cells expressing mutant U2AF1(S34F) compared to controls (p≤0.05, n=6-12), 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 transgene induction in U2AF1(S34F)- and U2AF1(WT)-transplanted mice. We identified 402 splicing junctions that were differentially expressed in U2AF1(S34F) samples compared to U2AF1(WT) (FDR <10%, >2 |log2| fold change). We also observed a preference of the mutant U2AF1(S34F) to skip exons (n=72) and alternative splice sites (n=45) with a T in the -3 position relative to the AG splice acceptor site. We intersected our junction results with RNA sequencing from AML patient samples with U2AF1 mutations and human CD34+ cells expressing U2AF1(S34F), and we identified homologous dysregulated junctions in all 3 datasets that are currently being tested for their functional significance. Together, these results suggest that mutant U2AF1 expression contributes to the altered pre-mRNA splicing and hematopoiesis observed in patients with U2AF1 mutations. Citation Format: Cara Lunn Shirai, James N. Ley, Brian White, Justin Tibbitts, Jin Shao, Matthew Ndonwi, Sanghyun Kim, Theresa Okeyo-Owuor, Timothy A. Graubert, Matthew J. Walter. Mutant U2AF1 alters hematopoiesis and pre-mRNA splicing in transgenic mice. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr PR06.