Abstract
Acute megakaryoblastic leukemia (AMKL, FAB-M7) with the t(1;22)(p13;q13) chromosome translocation is a unique subtype of AMKL, characterized by its early onset, poor response to existing therapies, rapid progression, and poor prognosis. t(1;22) is often the sole abnormality found in infants with this AMKL subtype, suggesting that the RBM15-MKL1 (also known as OTT1-MAL) fusion gene created by the translocation plays a crucial role in the pathogenesis of this leukemia. Here we have explored the normal functions of one of the t(1;22) partner genes, Rbm15, by conditionally knocking it out in the mouse (Rbm15 CKO mice) and by crossing these mice with an Mx1-Cre transgenic line to delete Rbm15 in the hematopoietic system. We found both the percentage and absolute number of long-term hematopoietic stem cells (Lin-Sca1+ckit+/Flk2-, LT-HSCs) to be 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 age- and gender-matched wild-type littermates (Rbm15lx/lx;Mx1-Cre-) (0.10 ± 0.01% TNMC) (P <0.0001, n = 18 mice per group). However, Rbm15-deleted bone marrow (BM) cells were markedly impaired in their ability to reconstitute normal hematopoiesis in competitive repopulation studies, with only 9.73 ± 2.32% donor-derived cells in the peripheral blood (PB) of recipient animals compared to the contribution by donor cells from littermate controls (47.52 ± 7.26%) (P = 0.00015; n = 10 mice per group) when transplanted at a 1:1 ratio of donor vs. wild-type competitor bone marrow. Additional analysis showed that Rbm15-deleted donor-derived cells were also significantly decreased in the recipient BM as a whole; importantly, however, the percentages of donor-derived HSC and LT-HSC populations were markedly increased (8.8-fold and 5.3-fold, respectively) in recipients transplanted with Rbm15 mutant cells compared to those transplanted with wild-type control marrow. This stem cell accumulation was more profound than that observed in the original Rbm15 mutant mice. Additional analysis that will be presented suggested an HSC activation/differentiation defect due to Rbm15 absence rather than abnormalities of HSC homing, survival or proliferation. Collectively, these alterations are highly reminiscent of those seen in c-Myc knockout (KO) mice, in which the expression levels of several adhesion molecules have been shown to be altered and an HSC-niche interaction defect posited to be the underlying mechanism of the HSC abnormalities observed. We found that similar to the c-Myc KO phenotype, the expression of both N-cadherin and beta1-integrin is increased on Rbm15-deficient HSCs and the N-cadherin expression level can be downregulated by ectopic expression of RBM15. Interestingly, in addition to the abnormalities of c-Myc-null HSCs that have been previously reported, we have identified a heretofore unrecognized significant increase of megakaryopoiesis in c-Myc knockout mice as well. We also determined that Rbm15 deficiency predisposes HSCs toward megakaryocyte lineage differentiation – the percentages of CD41-positive megakaryocytes were found to be increased both in vivo in the spleens of Rbm15 mutant mice and after the in vitro culture of Rbm15-deleted HSCs in a megakaryocyte liquid differentiation medium, as compared to wild-type littermate controls – thus uncovering another phenotypic similarity between mice lacking Rbm15 and c-Myc in their hematopoietic systems. Furthermore, c-Myc expression was decreased in Rbm15-deficient HSCs (nearly 2-fold lower compared to littermate control HSCs), as confirmed by TaqMan real-time PCR. Importantly, the megakaryocytic differentiation bias of Rbm15-deficient HSCs could be at least partially rescued by c-Myc overexpression in liquid culture conditions in vitro, suggesting a functional interaction between Rbm15 and c-Myc activity. Our data suggest that c-Myc may be a major target and effector of Rbm15 function in the regulation of HSC and megakaryocyte development. The putative oncoprotein RBM15-MKL1 has been hypothesized to be involved in the pathogenesis of AMKL in part through a dominant-negative mechanism that impairs normal RBM15 function; our data imply that such an inhibition of normal RBM15 function would in turn lead to diminished c-MYC activity, thus contributing to the aberrant megakaryocyte lineage involvement observed in t(1;22)-positive AMKL.
Clinical Evidence for the Importance of the Wild-Type PRPF31 Allele in the Phenotypic Expression of RP11
