Mutant U2AF1^S34F and U2AF1^Q157P Induce Distinct RNA Splicing and Hematopoietic Phenotypes In Vivo

Myelodysplastic syndromes (MDS) are the most common myeloid malignancies among the elderly. Patients present with bone marrow (BM) failure manifested by low peripheral blood (PB) counts and are at increased risk of developing acute myeloid leukemia. Mutations of U2AF1, a gene that encodes a spliceosome protein, are identified in 11% of MDS patients. The two most common U2AF1 mutants, S34F and Q157P, alter the splicing of two distinct sets of pre-mRNA targets in vitro and each co-occur with unique gene mutations in MDS patients, suggesting these mutants may affect MDS pathogenesis differently. In mice, U2AF1^S34F expression leads to altered splicing, reduced B-cell counts, and features of MDS. Similar studies have not been performed for U2AF1^Q157P. To study the impact of U2AF1^Q157P expression on splicing and hematopoiesis in vivo, we created a doxycycline (DOX)-inducible ("Tet-On") transgenic mouse that expresses mutant U2AF1^Q157P and is isogenic to our previously reported U2AF1^S34F and U2AF1^WT transgenic mice.

First, we confirmed DOX-inducible expression of the U2AF1^Q157P transgene in BM by RT-PCR-seq. To study the hematopoietic cell-intrinsic effects of U2AF1^Q157P, we performed non-competitive BM transplants into lethally irradiated congenic recipient mice. Donor BM from U2AF1^WT or U2AF1^S34F mice was also transplanted for comparison. Six weeks after transplant, mice were maintained on DOX chow to induce U2AF1 transgene (U2AF1^WT, U2AF1^S34F, or U2AF1^Q157P) expression (n = 10 mice per genotype). After six weeks on DOX, there were no significant changes in PB counts for U2AF1^Q157P mice compared to U2AF1^WT controls. In contrast, white blood cell (WBC) and B-cell counts were significantly reduced in U2AF1^S34F mice, as reported previously. Assessment of the BM revealed increased numbers (per five leg bones) of hematopoietic stem and progenitor cells (LSK [Lin⁻ Sca-1⁺ c-kit⁺] and LK [Lin⁻ Sca-1⁻ c-kit⁺]) in U2AF1^S34F mice (1.33×10⁵ LSK and 7.13×10⁵ LK cells) compared to U2AF1^WT (1.04×10⁵ LSK and 5.69×10⁵ LK cells; p < 0.05 for LSK and LK), as reported previously. In contrast, there was no change in LSK cells (1.03×10⁵, p = 0.9668) and a non-significant increase in LK cells (6.84×10⁵, p = 0.0547) in U2AF1^Q157P mice compared to U2AF1^WT. Both U2AF1^S34F and U2AF1^Q157P mice shared a significant increase in the number of common myeloid progenitors (CMP) compared to U2AF1^WT (2.43×10⁵ and 2.39×10⁵ vs. 1.66×10⁵ cells; p < 0.001 and p < 0.01, respectively), although CFU-C interrogated by methylcellulose assay were significantly increased only for U2AF1^S34F mice.

To study the hematopoietic cell-intrinsic effects of U2AF1^Q157P on stem cell function, we mixed equal numbers of whole BM test cells (CD45.2⁺; U2AF1^Q157P or U2AF1^WT) with congenic control wild-type BM competitor cells (CD45.1⁺/CD45.2⁺) and transplanted them into lethally irradiated congenic recipient mice (CD45.1⁺/CD45.2⁺ ; n = 6 per genotype). As in non-competitive transplants, DOX chow was administered six weeks after transplant. After six weeks on DOX, we observed a relative multi-lineage competitive disadvantage by analysis of peripheral blood chimerism (%CD45.2⁺ WBC) for U2AF1^Q157P test compared to U2AF1^WT test cells (49.5% vs. 71.7%, respectively, p < 0.001). In addition, stem and progenitor cells were all significantly reduced in the BM of U2AF1^Q157P competitive transplant mice compared to U2AF1^WT after 18 weeks of DOX (LSK, 36.1% vs. 92.2%, respectively, p < 0.001; LK, 53.1% vs. 92.0%, p < 0.001).

Lastly, using a Nanostring array, we identified consensus 3' splice sites of cassette exons that were increased or decreased in RNA from c-kit enriched mutant (U2AF1^S34F or U2AF1^Q157P) BM cells relative to U2AF1^WT (FDR < 0.1). As expected, we observed altered consensus 3' splice sites at the −3 position (for U2AF1^S34F) and +1 position (for U2AF1^Q157P) of differentially spliced exons, indicating altered but different pre-mRNA splicing induced by either U2AF1 mutant.

In aggregate, hematopoietic expression of U2AF1^Q157P causes a multi-lineage competitive disadvantage of BM stem cells and expanded myeloid progenitors in the non-competitive transplant setting, like U2AF1^S34F. However, PB counts and lineage distribution are not affected, indicating that the two common U2AF1 mutants, Q157P and S34F, are associated with different hematopoietic phenotypes and alterations to splicing, and may have different roles in MDS pathogenesis.