The Role of H2AFY in U2AF1 Mutant Cells and Normal Hematopoiesis

Mutations in genes that are involved in pre-mRNA splicing (i.e., spliceosome genes; U2AF1, SF3B1, SRSF2 and ZRSR2) are particularly frequent in MDS, affecting ~50% of MDS patients. Our group showed that expression of U2AF1(S34F), the most common U2AF1 mutant, causes altered splicing in human and mouse cells and altered hematopoiesis in mice. How U2AF1(S34F) expression contributes to altered hematopoiesis is unclear. We hypothesized that U2AF1(S34F) alters splicing of target genes that affect hematopoiesis. Using three independent sets of RNA-seq data from human acute myeloid leukemia (AML) patients, human CD34+ hematopoietic cells, and mouse common myeloid progenitor cells that express either wild-type or mutant U2AF1, we identified consistent alternative splicing of H2AFY. H2AFY, also known as macroH2A1, is a histone H2A variant implicated in transcription, development, and tumorigenesis. Alternative splicing of H2AFY generates two isoforms, H2AFY1.1 and H2AFY1.2. In many solid cancers, alternative splicing of H2AFY is observed, resulting in reductions in H2AFY1.1 isoform expression. Similarly, in all three RNA-seq datasets and RT-PCR of primary MDS samples, mutant U2AF1 samples express H2AFY1.1 at 30% of the levels observed in samples without a spliceosome mutation (p<0.01). In order to determine whether expression of H2AFY1.1 could rescue a cellular phenotype in U2AF1 mutant cells, we exogenously expressed H2AFY1.1 in doxycycline-inducible U2AF1(S34F) mouse bone marrow cells ex vivo using a lentivirus co-expressing GFP. We observed an attenuation of cell death induced by U2AF1(S34F) expression (%GFP+ live cells relative to uninduced cells: U2AF1(S34F) cells-45% [control virus] vs. 89% [H2AFY1.1 virus], p<0.001; wild-type cells-94% [control virus] vs. 97% [H2AFY1.1 virus], ns). Next, we asked whether loss of H2afy1.1 expression phenocopies any of the hematopoietic alterations observed in U2AF1(S34F) mice (reductions in white blood cell counts [WBC], B-cells and monocytes, or expansion of progenitor cells). To address this question, we first investigated the role of H2afy in normal mouse hematopoiesis. H2afy^-/- mice (lacking both H2afy1.1 and H2afy1.2 isoforms) are viable and born at the expected Mendelian frequency. H2afy^-/- and H2afy^+/-mice exhibited reduced WBC compared to H2afy^+/+ littermate mice (7.7, 8.1, and 9.9 K cells/µL, respectively, p<0.01 compared to wild-type mice, N=15 each genotype, 8-12 weeks old), whereas the red blood cell and platelet counts were no different. Flow cytometric analysis of the peripheral blood cell lineages revealed reduced B-cell numbers in H2afy^-/- and H2afy^+/-mice compared to H2afy^+/+ mice (mean 4.0, 4.5, and 5.6 K cells/µL, respectively, p<=0.003). Stem (KLS and KLS-SLAM) and progenitor cells (CMP, GMP, MEP) were not different between genotypes. There was a 2-fold reduction in CFU-pre-B colonies in the H2afy^-/- bone marrow cells compared to wild-type controls (p= 0.01, N=10), but CFU-C colonies were no different. To determine whether the reduction in B-cells is hematopoietic cell-intrinsic, we transplanted mouse bone marrow cells collected from H2afy^+/+ or H2afy^-/- mice into lethally irradiated congenic recipients. At 6 weeks post-transplant, mice who received the H2afy^-/- bone marrow cells compared to mice who received H2afy^+/+ cells exhibited a marked reduction in WBC (5.0, 9.1 K cells/µL, respectively, p<0.001, N=10) and peripheral blood B-cells (1.9, 5.3 K cells/µL, respectively, p<0.001), but not in myeloid cells. This suggests that hematopoietic expression of H2afy contributes to B-cell development in mice. To assess stem/progenitor cell function in vivo, we performed a competitive repopulation assay. As early as 1 month post-transplant (and persistent to 4 months), there was a reduction in H2afy^-/- donor-derived B-cells in the peripheral blood compared to mice receiving H2afy^+/+ cells (14%, 58%, respectively, p<0.001, N=14-15). The reduction in peripheral white blood cell counts and B-cells in H2afy^-/- mice mirrors the phenotypic changes observed in U2AF1(S34F) mice, suggesting that alterations in H2afy may contribute to altered hematopoiesis in U2AF1 mutant mice. Ongoing studies are designed to address whether the B-cell phenotype in H2afy^-/- and U2AF1 mutant mice is due to a specific loss of H2afy1.1 or 1.2 isoform expression in hematopoietic cells.