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.

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 wildtype 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 0 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 0 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. Author SummaryLarge-scale genomics studies have identified recurrent mutations in many genes that fall outside the conventional domain of proto-oncogenes. They include genes encoding factors that mediate RNA splicing; mutations affecting four of these genes are present in up to half of proliferative myeloid disorders and in a significant number of solid tumors, We show that the altered splice site preferences of mutant U2AF1 can be attributed to changes in its binding to relevant 3 0 splice sites. We also show that mutant U2AF1 is different from some oncogenes: the growth properties of lung cancer cell lines carrying the mutant allele are unaffected by loss of the mutant gene, while the wild-type allele is absolutely required for survival. These results advance our understanding of the molecular determinants of the mutant-associated splicing program, and they highlight previously unappreciated roles of wild-type U2AF1 in the presence of the recurrent U2AF1S34F mutation.

The splicing factor gene, U2AF1, is commonly mutated in myeloid neoplasms and occasionally in solid tumors such as lung adenocarcinomas (LUADs), yet the molecular and physiological consequences of U2AF1 mutations are not well understood. Through genome editing of the endogenous locus, we created a common U2AF1 mutant allele (U2AF1-S34F) in an immortalized human bronchial epithelial cell line, and we inactivated the U2AF1-S34F allele in two LUAD cell lines that naturally harbor this mutation. Cells expressing U2AF1-S34F deployed altered splicing of many transcripts, and they displayed a S34F-characteristic sequence pattern at the proximal 3’ splice site, as previously reported. Remarkably, S34F-associated alternative splicing patterns remained unaffected by changes in the amount of mutant U2AF1 protein, as long as the ratio of mutant and wild-type U2AF1 remained constant, as evidenced by RNA interference-mediated knockdown of the expression of both mutant and wild-type alleles. On the other hand, perturbing the ratio of mutant and wild-type U2AF1, either through overexpression or allelic-specific inactivation of wild-type U2AF1, greatly affected S34F-associated splicing. These results suggest that the ratio of mutant and wild-type U2AF1, rather than the absolute amount of either protein, is critical in controlling splicing outcome. Expression of U2AF1-S34F does not cause cell transformation and LUAD cells with endogenous U2AF1-S34F expression are not dependent on the U2AF1-S34F allele. Instead, wild-type U2AF1 is absolutely required for cell survival in the presence of the mutant allele. To study the effect of U2AF1-S34F in vivo, we created transgenic mice carrying a conditional mutant allele, MG-S34F, at the endogenous U2af1 locus. By crossing the MG-S34F mice with mice carrying a Mx1-Cre transgene and treating the progeny with poly IC, we activated MG-S34F by Cre-mediated recombination to express U2af1-S34F in bone marrow cells. This resulted in cytopenia in multiple blood cell lineages. Similar results were observed in recipient mice following non-competitive transplant. Bone marrow cells expressing U2af1-S34F were competitively disadvantaged in repopulating the hematopoietic system in irradiated mice, suggesting defective hematopoietic stem cell function, and we are currently assessing the long-term impact of U2af1-S34F expression on the incidence of myeloid neoplasms. Citation Format: Dennis Fei, Hayley Motowski, Sameer Prasad, Jovian Yu, Robert Bradley, Harold Varmus. Molecular and physiological effects of splicing factor mutant U2AF1 in human lung cell lines and in mice. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4163.