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ObjectiveIn epidemiological and experimental research, high folic acid intake has been demonstrated to accelerate tumor development among populations with genetic and/or molecular susceptibility to cancer. Neurofibromatosis type 1 (NF1) is a common autosomal dominant disorder predisposing affected individuals to tumorigenesis, including benign plexiform neurofibromas; however, understanding of factors associated with tumor risk in NF1 patients is limited. Therefore, we investigated whether pregestational folic acid intake modified plexiform-like peripheral nerve sheath tumor risk in a transgenic NF1 murine model.ResultsWe observed no significant differences in overall survival according to folate group. Relative to controls (180 days), median survival did not statistically differ in deficient (174 days,P=0.56) or supplemented (177 days,P=0.13) folate groups. Dietary folate intake was positively associated with RBC folate levels at weaning, (P=0.023, 0.0096, and 0.0006 for deficient vs. control, control vs. supplemented, and deficient vs. supplemented groups, respectively). Dorsal root ganglia (DRG), brachial plexi, and sciatic nerves were assessed according to folate group. Mice in the folate deficient group had significantly more enlarged DRG relative to controls (P=0.044), but no other groups statistically differed. No significant differences for brachial plexi or sciatic nerve enlargement were observed according to folate status.

Summary Heterozygous somatic mutations in the spliceosome gene U2AF1 occur in ∼11% of patients with myelodysplastic syndromes (MDS), the most common adult myeloid malignancy. It is unclear how these mutations contribute to disease. We examined in vivo hematopoietic consequences of the most common U2AF1 mutation using a doxycycline-inducible transgenic mouse model. Mice expressing mutant U2AF1(S34F) display altered hematopoiesis and changes in pre-mRNA splicing in hematopoietic progenitor cells by whole transcriptome analysis (RNA-seq). Integration with human RNA-seq datasets determined that common mutant U2AF1-induced splicing alterations are enriched in RNA processing genes, ribosomal genes, and recurrently mutated MDS and acute myeloid leukemia-associated genes. These findings support the hypothesis that mutant U2AF1 alters downstream gene isoform expression, thereby contributing to abnormal hematopoiesis in patients with MDS.

This abstract is being presented as a short talk. A full abstract is printed in the Proffered Abstracts section (PR06) of the Conference Proceedings. 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 B41.

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 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 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.