Rbm15 Affects Notch Signaling and Myelopoiesis.

Acute Megakaryoblastic Leukemia (AMKL) presents with one of two different genotypic abnormalities; either Down Syndrome (DS) accompanied with GATA-1 mutations, or a non-DS translocation t(1;22)(p13;q13). In AMKL associated with DS, there is trisomy 21 and various partial loss of function mutations in GATA-1. In non-DS AMKL, the translocation t(1;22)(p13;q13) encodes a fusion protein, RBM15-MKL, comprised of the transcriptional co-factors RBM15 (chromosome 1) and MKL (chromosome 22). In order to elucidate the role of the RBM15-MKL fusion protein in AMKL, we must understand the normal functions of RBM15 and MKL. Herein we demonstrate a role for Rbm15 in myeloid differentiation.

Previous work in the lab showed that Rbm15 is expressed at highest levels in hematopoietic stem cells, and at more moderate levels during myelopoiesis of murine cell lines and primary murine cells. Therefore, we hypothesized that Rbm15 plays a role in myeloid differentiation. Indeed, we demonstrate that shRNA-mediated knockdown of Rbm15 enhanced the differentiation of 32DWT18 myeloid precursor cells. Recent studies have shown the Rbm15 homolog, Sharp, interacts with RBPJk, a critical transcription factor in the Notch signaling pathway; and since Notch signaling is essential for hematopoiesis, we hypothesized that Rbm15 may also modulate Notch signaling. We demonstrate that Rbm15 alters Notch-induced HES1 promoter activity in a cell-type specific manner. Rbm15 inhibits Notch-induced HES1 activity in non-hematopoietic cell lines, but stimulates this activity in hematopoietic cell lines including 32DWT18 and HEL (human erythroleukemia). In addition, we show that the N-terminus of Rbm15 co-immunoprecipitates with RBPJk and has a dominant negative effect by impairing the activation ability of HES1 promoter activity by full length-Rbm15.

Thus, Rbm15 is differentially expressed during hematopoiesis and may act to inhibit myeloid differentiation in hematopoietic cells via a mechanism that is mediated, at least in part, by stimulation of Notch signaling via interaction with RBPJk. Consistent with a potential mechanistic role of Rbm15 and Notch in myelopoiesis, we have shown that shRNA-mediated knockdown of Rbm15 in 32DWT18 cells promotes myeloid differentiation, suggesting that the RBM15 component of the RBM15-MKL fusion protein may act by blocking differentiation. Currently, we are examining the effect of RBM15 overexpression and knockdown in primary mouse bone marrow cells using in vitro and in vivo assays.