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Somatic mutations in spliceosome genes are detectable in ∼50% of patients with myelodysplastic syndromes (MDS). We hypothesize that cells harbouring spliceosome gene mutations have increased sensitivity to pharmacological perturbation of the spliceosome. We focus on mutant U2AF1 and utilize sudemycin compounds that modulate pre-mRNA splicing. We find that haematopoietic cells expressing mutant U2AF1(S34F), including primary patient cells, have an increased sensitivity to in vitro sudemycin treatment relative to controls. In vivo sudemycin treatment of U2AF1(S34F) transgenic mice alters splicing and reverts haematopoietic progenitor cell expansion induced by mutant U2AF1 expression. The splicing effects of sudemycin and U2AF1(S34F) can be cumulative in cells exposed to both perturbations—drug and mutation—compared with cells exposed to either alone. These cumulative effects may result in downstream phenotypic consequences in sudemycin-treated mutant cells. Taken together, these data suggest a potential for treating haematological cancers harbouring U2AF1 mutations with pre-mRNA splicing modulators like sudemycins. Spliceosome mutations occur in approximately 50% of patients with myelodysplastic syndromes. Here, the authors show that tumour cells harbouring the S34F mutation in the U2AF spliceosome gene is sensitive to compounds that further perturb the spliceosome.

Myelodysplastic syndromes (MDS) are the most common adult myeloid blood cancers in the US. Patients have increased apoptosis in their bone marrow cells leading to low peripheral blood counts. The full complement of gene mutations that contribute to increased apoptosis in MDS remains unknown. Up to 25% of MDS patients harbor and acquired interstitial deletion on the long arm of chromosome 5 [del(5q)], creating haploinsufficiency for a large set of genes including HSPA9. Knockdown of HSPA9 in primary human CD34+ hematopoietic progenitor cells significantly inhibits growth and increases apoptosis. We show here that HSPA9 knockdown is associated with increased TP53 expression and activity, resulting in increased expression of target genes BAX and p21. HSPA9 protein interacts with TP53 in CD34+ cells and knockdown of HSPA9 increases nuclear TP53 levels, providing a possible mechanism for regulation of TP53 by HSPA9 haploinsufficiency in hematopoietic cells. Concurrent knockdown of TP53 and HSPA9 rescued the increased apoptosis observed in CD34+ cells following knockdown of HSPA9. Reduction of HSPA9 below 50% results in severe inhibition of cell growth, suggesting that del(5q) cells may be preferentially sensitive to further reductions of HSPA9 below 50%, thus providing a genetic vulnerability to del(5q) cells. Treatment of bone marrow cells with MKT-077, an HSPA9 inhibitor, induced apoptosis in a higher percentage of cells from MDS patients with del(5q) compared to non-del(5q) MDS patients and normal donor cells. Collectively, these findings indicate that reduced levels of HSPA9 may contribute to TP53 activation and increased apoptosis observed in del(5q)-associated MDS.

Somatic mutations in the spliceosome gene U2AF1 are common in patients with myelodysplastic syndromes. U2AF1 mutations that code for the most common amino acid substitutions are always heterozygous, and the retained WT allele is expressed, suggesting that mutant hematopoietic cells may require the residual WT allele to be viable. We show that hematopoiesis and RNA splicing in U2af1 heterozygous knockout mice were similar to those in control mice, but that deletion of the WT allele in U2AF1(S34F) heterozygous mutant-expressing hematopoietic cells (i.e., hemizygous mutant) was lethal. These results confirm that U2AF1 mutant hematopoietic cells are dependent on the expression of WT U2AF1 for survival in vivo and that U2AF1 is a haplo-essential cancer gene. Mutant U2AF1(S34F)-expressing cells were also more sensitive to reduced expression of WT U2AF1 than nonmutant cells. Furthermore, mice transplanted with leukemia cells expressing mutant U2AF1 had significantly reduced tumor burden and improved survival after the WT U2af1 allele was deleted compared with when it was not deleted. These results suggest that selectively targeting the WT U2AF1 allele in heterozygous mutant cells could induce cancer cell death and be a therapeutic strategy for patients harboring U2AF1 mutations.

Roles of the γ-carboxyglutamic acid (Gla) and first epidermal growth factor-like (EGF1) domains in blood coagulation factors IX and X (FIX and FX) were investigated. FIX mutants-FIXPCGla [Gla domain of FIX replaced with that of protein C (PC)], FIXPCEGF1 (EGF1 domain replaced with that of PC), FIXPCGla/EGF1 (Gla and EGF 1 domains replaced with the respective PC domains), FIXQ50P (point mutant in EGF 1 domain deficient in Ca2+ binding), and the corresponding FX mutants-FXPCGla, FXPCEGF1, FXPCGla/EGF1 and FXQ49P, as well as FXΔEGF1 (EGF1 deletion mutant of FX) were made and compared to wild type (WT) counterparts in various reactions of the coagulations cascade. It was found that the Gla domain of FIX plays an essential role during activation by FXIa, and contributes modestly towards FX activation by FIXa. However, it is neither essential for FIX activation by FVIIa/tissue factor (TF) nor feedback activation of FVII/TF by FIXa. On the other hand, it was found that the EGF1 domain is not required for FIX activation by FXIa but is crucial in the activation by FVIIa/TF and feedback activation of FVII/TF by FIXa. Its contribution to the activation of FX by FIXa is modest. In FX, both the Gla and EGF1 domains were found to be essential for optimal activation by FVIIa/TF and feedback activation of FVII/TF. In addition, the Gla but not the EGF1 domain is required for activation by FIXa whereas both domains provided a modest contribution towards activation by Russell's viper venom protease and subsequent activation of prothrombin. The EGF1 domain was not required for FX inhibition by tissue factor pathway inhibitor (TFPI). Instead it was required for optimal inhibition of FVIIa/TF by the FXa/TFPI complex. We conclude that intact Gla and EGF1 domains in FIX and FX are required for some but not all the reactions of these factors in blood coagulation thus making these domains interesting targets for the design of novel therapeutic antithrombotics.

Somatic mutations in the spliceosome gene U2AF1 are common in patients with myelodysplastic syndromes. U2AF1 mutations that code for the most common amino acid substitutions are always heterozygous, and the retained wild-type allele is expressed, suggesting that mutant hematopoietic cells may require the residual wild-type allele to be viable and cause disease. We show that hematopoiesis and RNA splicing in U2af1 heterozygous knock-out mice was similar to control mice, but that deletion of the wild-type allele in U2AF1(S34F) heterozygous mutant expressing hematopoietic cells (i.e., hemizygous mutant) was lethal. These results confirm that U2AF1 mutant hematopoietic cells are dependent on the expression of wild-type U2AF1 for survival in vivo and that U2AF1 is a haplo-essential cancer gene. Mutant U2AF1 (S34F) expressing cells were also more sensitive to reduced, but not absent, expression of wild-type U2AF1 than non-mutant cells. Furthermore, mice transplanted with leukemia cells expressing mutant U2AF1 had significantly reduced tumor burden and improved survival after the wild-type U2af1 allele was deleted compared to when it was not deleted. These results suggest that selectively targeting the wild-type U2AF1 allele in heterozygous mutant cells could induce cancer cell death and be a therapeutic strategy for patients harboring U2AF1 mutations.