Abstract 5108: The role of H2afy in normal and malignant hematopoiesis
Authors: Kim Sanghyun , Chavez Monique , Shirai Cara , Walter Matt 
Published: August 2018

Abstract
Mutations in the spliceosome gene U2AF1 are found in 11% of patients with myelodysplastic syndrome (MDS), a common hematologic malignancy, and at lower frequencies in some solid cancers. We have shown that expression of a common U2AF1 mutant, U2AF1(S34F), causes altered pre-mRNA splicing and abnormal hematopoiesis in mice, including reduced white blood cell counts (WBC) and B-cells, which are phenotypes similar to patients with MDS. We consistently find that expression of mutant U2AF1 results in reduced RNA expression of H2AFY1.1, a splice isoform of H2AFY, in patient samples and mouse hematopoietic cells. H2AFY (also known as macroH2A1) is a histone H2A variant implicated in transcriptional gene regulation, development, and tumorigenesis. This gene encodes two splice isoforms, H2AFY1.1 and H2AFY1.2. Expression of the H2AFY1.1 isoform is also reduced in several solid cancers. We hypothesize that reduced H2AFY1.1 expression contributes to abnormal hematopoiesis induced by mutant U2AF1(S34F). We first characterized hematopoiesis in adult constitutive H2afy knock-out mice (lacking both H2afy1.1 and H2afy1.2 isoforms). We transplanted bone marrow cells from H2afy+/+ or H2afy-/- mice into lethally irradiated congenic recipients to study the hematopoietic cell-intrinsic effects of H2afy loss. At 6 weeks post-transplant, recipients of H2afy-/- bone marrow cells compared to recipients of H2afy+/+ cells exhibited a marked reduction in WBC (5.0 vs. 9.1 K cells/µL, respectively, p&lt;0.001, N=10), peripheral blood B-cells (1.9 vs. 5.3 K cells/µL, respectively, p&lt;0.001, N=10), and bone marrow B-cells (6.33e6 vs. 2.5e6 cells/mouse, p=0.001, N=7). There is also a 2-fold reduction in pre-B methylcellulose colony forming units of H2afy-/- bone marrow cells compared to wild-type controls (p&lt; 0.01, N=20), indicating a role for H2afy in B-cell development. In competitive repopulation assays, we observed a reduction in H2afy-/- donor-derived B-cells in the peripheral blood compared to H2afy+/+ B-cells (14 vs. 58%, respectively, p&lt;0.001, N=14-15) and reductions in bone marrow pro-B-cells through mature B-cells, suggesting potential roles for H2afy in pre-pro- to pro-B-cell development. Viral re-expression of H2AFY1.1 in H2afy-/- bone marrow cells rescued the reduced frequency of peripheral blood B-cells (61 vs. 36%, p&lt;0.001, N=10) and bone marrow B cells (11 vs. 5%, p=0.018, N=5-6) in vivo relative to control virus-transduced H2afy-/- cells, whereas H2AFY1.2 expression did not significantly affect B-cell counts. Collectively, our data indicate that the H2afy1.1 isoform is important for B-cell development and that reductions in the H2afy1.1 isoform may contribute to U2AF1(S34F)-induced abnormal hematopoiesis in mice. Ongoing studies are designed to address how H2afy1.1 affects B-cell development and to determine if the B-cell phenotype in U2AF1(S34F) mice is due to reduced H2afy1.1 isoform expression.
Citation Format: Sanghyun Kim, Monique Chavez, Cara Shirai, Matt Walter. The role of H2afy in normal and malignant hematopoiesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5108.