Abstract 4163: Molecular and physiological effects of splicing factor mutant U2AF1 in human lung cell lines and in mice

Abstract

Abstract 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.