Mutant U2AF1(S34F) Expression Alters Hematopoiesis in Mice

Abstract 553

Myelodysplastic syndromes (MDS) are stem cell disorders characterized by ineffective hematopoiesis with increased levels of hematopoietic cell apoptosis. Recent discoveries by our group and others suggest that perturbations in pre-mRNA splicing may play a role in MDS pathogenesis. Indeed, more than half of all MDS patients have a mutation in one of eight splicing factors. U2AF1 (U2AF35), a gene encoding a splicing factor involved in intronic 3'-splice site recognition, is mutated in 8.7% of MDS patients. The consequence of the highly recurrent serine to phenylalanine mutation at position 34 (S34F) of U2AF1 in hematopoiesis is unknown. Therefore, to examine the effects of mutant U2AF1(S34F) on hematopoiesis, we utilized the MSCV-IRES-GFP retroviral system to introduce mutant U2AF1(S34F) or wild type U2AF1(WT), or an empty vector control, into mouse bone marrow cells for in vitro and in vivo studies. Expression of U2AF1(S34F) results in reduced expansion of transduced bone marrow cells (marked by GFP) compared to both U2AF1(WT) or empty vector-transduced cells grown in suspension culture (2 vs 4-fold change, respectively; p<0.001, n=3). Additionally, U2AF1(S34F)-transduced cells have increased levels of apoptosis (Annexin V+/7AAD+) in culture compared to U2AF1(WT) (p=0.03) and empty vector-transduced cells (p=0.02) (n=3). We also examined the effects of the U2AF1(S34F) mutation in vivo using bone marrow transplantation. The percentage of GFP+ cells in the peripheral blood of recipient mice transplanted with MSCV-transduced bone marrow was significantly reduced at 6 months post-transplant with U2AF1(S34F) expression (average=4%) compared to U2AF1(WT) (average=44%) and empty vector (average=65%) (p<0.02, n= 6–9 mice each). Transduction efficiencies were similar within experiments. There was no consistent alteration in lineage distribution of GFP+ cells in the peripheral blood of these mice.

To overcome some of the limitations of retroviral models, we created a single-copy, doxycycline-inducible U2AF1(S34F) transgenic mouse to model the effect of U2AF1(S34F) expression on hematopoiesis. As a control for U2AF1 protein overexpression, we created an additional single-copy, doxycycline-inducible U2AF1(WT) transgenic mouse with transgene integration into the same locus as the U2AF1(S34F) mouse. Induction of U2AF1(S34F) transgene expression in bone marrow cells in culture with doxycycline treatment (200 ng/ml for 5 days) resulted in reduced cell numbers when compared to uninduced U2AF1(S34F) transgenic cells (ratio of growth of induced/uninduced cells = 0.38), while cell proliferation was not altered for U2AF1(WT) transgenic cells (ratio of growth of induced/uninduced cells = 1.13) (p<0.001, n=3). In addition, doxycycline-induced U2AF1(S34F) expression results in increased apoptosis (Annexin V+) compared to uninduced U2AF1(S34F) transgenic cells (21% vs 11%, p=0.01) and induced U2AF1(WT) transgenic cells in culture (21% vs 9.3%, p=0.008) (n=4). To examine the effects of mutant U2AF1(S34F) induction in vivo, we transplanted mutant U2AF1(S34F) or U2AF1(WT) transgenic bone marrow cells into congenic wild type recipient mice and induced transgene expression 6 weeks post-transplant using 2 mg/ml doxycycline in the drinking water for 5 days. Induction of U2AF1(S34F) expression in vivo results in reduced number of WBCs in the peripheral blood of recipient mice compared to mice with uninduced U2AF1(S34F) transgenic bone marrow (3.4k vs 5.6k, p=0.01, n=3). In addition, recipient mice with induced U2AF1(S34F) bone marrow had reduced number of bone marrow cells per femur when compared to uninduced U2AF1(S34F) recipient mice (3.9M vs 13.1M, p=0.04) and induced U2AF1(WT) recipient mice (3.9M vs 12.4M, p=0.03) (n=3). The number of neutrophils in peripheral blood (p<0.001), bone marrow (p=0.04), and spleen (p=0.04) of induced U2AF1(S34F) recipient mice were all significantly lower compared to uninduced U2AF1(S34F) mice (n=3). The total numbers of c-Kit+/lineage-/Sca+ hematopoietic progenitor cells were not affected in induced U2AF1(S34F) recipient mice compared to uninduced U2AF1(S34F) (p=0.75) or induced U2AF1(WT) recipient mice (p=0.46, n=3) after 5 days of treatment. Collectively, these results suggest that the U2AF1(S34F) mutation may contribute to abnormal hematopoiesis in vivo. Longer periods of doxycycline-induction in vivo are ongoing and will be presented.