R e s e a R c h a R t i c l e3 5 3 3 jci.orgVolume 125 Number 9 September 2015 levels decreased dramatically, suggesting important posttranscriptional regulation of PRMT5 expression in these cells. Since straight Prmt5-KO mice die before birth (12), we generated Prmt5-conditional KO mice by first crossing Prmt5 FLIP-OUT mice (obtained from the European Mutant Mouse Archive [EMMA]) with Flp recombinase-expressing transgenic (Tg) mice to generate Prmt5-floxed mice, whereby exon 7 of the Prmt5 time PCR (qPCR) ( Figure 1A) and Western blot analysis ( Figure 1B). Prmt5 mRNA and protein levels were readily detected in HSPCs, with little change in mRNA levels in the various stem and progenitor cell populations. However, when cells underwent myeloid, erythroid, or lymphoid differentiation, PRMT5 protein levels decreased to 5% to 24% of the levels seen in HSPCs. Although Prmt5 mRNA was maintained in differentiated B cells, its protein BM cells and found a complete loss of this modification 7 days after Cre induction ( Figure 1C), suggesting that PRMT5 is indeed the major type 2 PRMT in these cells. Deletion of PRMT5 during adult hematopoiesis leads to severe cytopenias. The deletion of PRMT5 in 2-month-old mice had a profound effect on hematopoiesis. Two weeks after the first poly(I:C) injection, the Mx1Cre + Prmt5 fl/fl mice developed severe pallor due to anemia, and most of these PRMT5-deleted mice were moribund 1 to 2 days later, requiring euthanasia. Analysis of the peripheral blood of these mice revealed severe pancytopenia, with a more than 10-fold decrease in wbc, a 5-fold decrease in rbc, and a 100-fold decrease in platelet counts 15 days after Cre induction ( Figure 1D). BM cellularity of the PRMT5-deleted mice was reduced by more than 50% on day 7 and by more than 95% on day 15 ( Figure 1E), consistent with the development of aplastic anemia ( Figure 1G). While PRMT5 loss had little effect on spleen weight (or cellularity) on day 15 (Supplemental Figure 2, A and B), the size and cellularity of the thymus were significantly reduced on day 15 gene was flanked by 2 LoxP sites (Supplemental Figure 1A; mice and Mx1Cre-negative littermate controls. To delete Prmt5 in hematopoietic cells, 2 i.p. injections of poly(I:C) (10 mg/kg on days 0 and 1) were given to both the Mx1Cre -and Mx1Cre + Prmt5-floxed mice. Prmt5 loss was confirmed by PCR analysis of genomic DNA (Supplemental Figure 1B), by quantitative qPCR to detect Prmt5 mRNA (Supplemental Figure 1C), and by Western blot analysis to detect PRMT5 protein (Supplemental Figure 1D). This injection strategy was used in all subsequent experiments. Interestingly, loss of PRMT5 triggered the loss of MEP50 protein (Supplemental Figure 1D), a cofactor that is required for the enzymatic activity of PRMT5 on histones, without changing Mep50 mRNA levels (data not shown). This suggests a probable posttranscriptional effect of PRMT5 on MEP50 protein levels. We also determined the overall level of symmetrically dimethylated arginine in PRMT5-deleted and Prmt5 Δ/Δ mice (n = 3). P...

Protein arginine methyltransferase 5 (PRMT5) is overexpressed in many cancer types and is a promising therapeutic target for several of them, including leukemia and lymphoma. However, we and others have reported that PRMT5 is essential for normal physiology. This dependence may become dose limiting in a therapeutic setting, warranting the search for combinatorial approaches. Here, we report that PRMT5 depletion or inhibition impairs homologous recombination (HR) DNA repair, leading to DNA-damage accumulation, p53 activation, cell-cycle arrest, and cell death. PRMT5 symmetrically dimethylates histone and non-histone substrates, including several components of the RNA splicing machinery. We find that PRMT5 depletion or inhibition induces aberrant splicing of the multifunctional histone-modifying and DNA-repair factor TIP60/KAT5, which selectively affects its lysine acetyltransferase activity and leads to impaired HR. As HR deficiency sensitizes cells to PARP inhibitors, we demonstrate here that PRMT5 and PARP inhibitors have synergistic effects on acute myeloid leukemia cells.

Constitutive activation of FLT3 by internal tandem duplication (ITD) is one of the most common molecular alterations in acute myeloid leukemia (AML