Essential Role for Rbm15 (Ott1) in the Maintenance of Hematopoietic Stem Cell Function and Cell Cycle Control.

RBM15 is the 5′ fusion partner in RBM15-MKL1 (aka OTT1-MAL), a putative oncoprotein in non-Down syndrome infants and children with acute megakaryoblastic leukemia (FAB-M7) containing t(1;22). RBM15 belongs to the “spen” family, which is characterized by the presence of three RNA recognition motifs and a spen paralog and ortholog C-terminal (SPOC) domain. RBM15-homologous Drosophila proteins are involved in regulation of a variety of signaling cascades including MAPK, Wnt, Notch, cyclin E and Hox pathways, but the normal functions of mammalian RBM15 remain largely uncharacterized. We determined that Rbm15 is highly expressed in hematopoietic stem cells (HSCs) as well as T-lineage cells. To study Rbm15 specifically in the regulation of HSC function, we generated Rbm15 conditional knockout mice using the Cre-LoxP system to overcome embryonic death observed with a conventional knockout. Using an inducible Mx1-Cre transgenic line, we conditionally deleted Rbm15 (deletion efficiency ∼96–100%) in HSCs. Both the percentage and absolute number of long-term HSCs (Lin-Sca1+ckit+/Flk2-) were increased in Rbm15-deleted (Rbm15lx/lx;Mx1-Cre+) mice (0.23 ± 0.02% of total nucleated marrow cells [TNMC], 2.3-fold higher) compared to matched littermate controls (Rbm15lx/lx;Mx1-Cre-) (0.10 ± 0.01% TNMC) (P<0.0001, n=18 mice per group). By contrast, total white blood cells (WBCs) were significantly decreased in the peripheral blood (PB) of Rbm15-deleted animals compared to controls (Rbm15-deleted: 5.33 ± 0.40 × 10³/uL, Rbm15-intact: 10.26 ± 0.49 × 10³/uL; P <0.0001, n=31 per group). Functional analysis of Rbm15-deleted HSCs by competitive repopulation showed these cells to be markedly impaired in their reconstitution of hematopoiesis in lethally-irradiated recipient mice, with only 9.73 ± 2.32% donor-derived cells in the PB of transplanted animals compared to a 47.52 ± 7.26% contribution by donor cells from littermate controls (P=0.00015; n=10 mice per group; 1:1 ratio of donor:wild-type competitor marrow). The serial transplantation ability of Rbm15-deleted HSCs was also severely decreased, with a decline in their engraftment and contribution to the blood of recipient mice noted beginning with the 3rd round of transplantation, culminating in essentially complete failure to engraft in the 4^th round (wild-type donor-derived PB cells in 4^th round ∼60.95% vs. ∼2.71% Rbm15-deleted donor-derived PB cells; P=0.00003). These defects in HSC function may be due in part to altered HSC cell cycle status in the absence of Rbm15. For example, in representative experiments using Ki-67/Hoechst 33342 and BrdU/7AAD staining, 24.1% and 19.3% of Rbm15-deleted HSCs were found to be in G1 and G2/M phases, respectively - a marked increase compared to Rbm15-intact controls (8.3% and 4.3%, respectively); furthermore, Rbm15-deleted HSCs exhibited significantly less BrdU incorporation (6.8%) compared to control HSCs (27.1%) in in vivo labeling studies. Consistent with these altered cell cycle parameters, hematopoiesis in Rbm15-deleted mice recovered significantly more slowly than controls following 5-FU exposure, with only half the number of total nucleated marrow cells at day 9 and half the number of mature PB WBCs at day 12 following 5-FU as compared to Rbm15-intact littermates (p=0.003, n=3 per group). Collectively, these data demonstrate a critical role for Rbm15 in maintaining HSC integrity and suggest a yet-to-be fully elucidated function for Rbm15 in modulating HSC cell cycle kinetics.