U2af1 is a haplo-essential gene required for hematopoietic cancer cell survival in mice.

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.

U2AF1 expression is a novel and independent prognostic indicator of childhood T-lineage acute lymphoblastic leukemia.

INTRODUCTION: U2AF1 gene is associated with various types of hematological malignancies in adults. However, the expression level of U2AF1 gene and its prognostic significance are unclear in pediatric ALL patients. The study aimed to study the mRNA level of U2AF1 in pediatric ALL patients and its clinical relevance with long-term survival. METHODS: We quantitatively determined U2AF1 gene expression at diagnosis in 132 children with ALL by real-time PCR. According to the patients' median U2AF1 value, the patients' samples were classified into low U2AF1 and high U2AF1 expression groups. Twenty-two bone marrow samples from 22 patients with ITP were recruited as control. The correlation between the expression level of U2AF1 and clinical treatment outcome was analyzed. RESULTS: Pediatric patients with ALL showed higher U2AF1 mRNA levels than controls (P = .034). The relapse rates of patients in low U2AF1 levels group were obviously higher than those of U2AF1 high expression group (28.8% vs 12.1%, P = .030). Patients of low U2AF1 expression presented worse 5-year EFS than those of high U2AF1 expression (60% vs 81%, P = .035). For T-ALL, patients with low U2AF1 mRNA level showed lower BM blast percentages (P = .031), worse EFS (37.8% vs 92.3%, P = .003), and CIR (62.2% vs 7.7%, P = .003) than those in high U2AF1 expression group. Multivariate analysis confirmed low U2AF1 mRNA level could be used as an independent risk indicator of poor EFS and CIR of children with T-ALL. CONCLUSION: Low U2AF1 mRNA level is related to inferior prognosis and can be served as a prognostic indicator for risk stratification in children with T-ALL.

Integrative Profiling of Alternative Splicing Induced by U2AF1 S34F Mutation in Lung Adenocarcinoma Reveals a Mechanistic Link to Mitotic Stress.

Mutations in spliceosome components have been implicated in carcinogenesis of various types of cancer. One of the most frequently found is U2AF1 S34F missense mutation. Functional analyses of this mutation have been largely limited to hematological malignancies although the mutation is also frequently seen in other cancer types including lung adenocarcinoma (LUAD). We examined the impact of knockdown (KD) of wild type (wt) U2AF1 and ectopic expression of two splice variant S34F mutant proteins in terms of alternative splicing (AS) pattern and cell cycle progression in A549 lung cancer cells. We demonstrate that induction of distinct AS events and disruption of mitosis at distinct sub-stages result from KD and ectopic expression of the mutant proteins. Importantly, when compared with the splicing pattern seen in LUAD patients with U2AF1 S34F mutation, ectopic expression of S34F mutants but not KD was shown to result in common AS events in several genes involved in cell cycle progression. Our study thus points to an active role of U2AF1 S34F mutant protein in inducing cell cycle dysregulation and mitotic stress. In addition, alternatively spliced genes which we describe here may represent novel potential markers of lung cancer development.

Wild-Type U2AF1 Antagonizes the Splicing Program Characteristic of U2AF1-Mutant Tumors and Is Required for Cell Survival.

We have asked how the common S34F mutation in the splicing factor U2AF1 regulates alternative splicing in lung cancer, and why wild-type U2AF1 is retained in cancers with this mutation. A human lung epithelial cell line was genetically modified so that U2AF1S34F is expressed from one of the two endogenous U2AF1 loci. By altering levels of mutant or wild-type U2AF1 in this cell line and by analyzing published data on human lung adenocarcinomas, we show that S34F-associated changes in alternative splicing are proportional to the ratio of S34F:wild-type gene products and not to absolute levels of either the mutant or wild-type factor. Preferential recognition of specific 3' splice sites in S34F-expressing cells is largely explained by differential in vitro RNA-binding affinities of mutant versus wild-type U2AF1 for those same 3' splice sites. Finally, we show that lung adenocarcinoma cell lines bearing U2AF1 mutations do not require the mutant protein for growth in vitro or in vivo. In contrast, wild-type U2AF1 is required for survival, regardless of whether cells carry the U2AF1S34F allele. Our results provide mechanistic explanations of the magnitude of splicing changes observed in U2AF1-mutant cells and why tumors harboring U2AF1 mutations always retain an expressed copy of the wild-type allele.