Abstract 4654: Hotspot mutations in the core spliceosomal protein SF3B1 promote breast tumorigenesis

The core RNA splicing factor SF3B1 is targeted by recurrent hotspot mutations in leukemias and solid tumors with the K700E substitution observed in 2% of unselected breast cancer patients. These mutations promote usage of aberrant intron proximal 3’ splice sites. In breast cancer, the links between these aberrant splicing events and the transformed phenotype are not well understood. Here, we integrated findings from multiple genetically modified model systems and human tumor specimens to understand the contributions of mutant SF3B1 to breast cancer pathogenesis. To identify the aberrant splicing events in breast cancers specifically, we first performed RNA-seq analyses of MCF10A breast epithelial cells with CRISPR knock-in of the K700E mutation. Dysregulation of splicing at over 1,000 splice junctions was discovered, most of which involve the use of alternative 3’ splice sites. Gene expression analysis revealed upregulation of expression of NFκB pathway genes in the SF3B1 mutant cells. SF3B1 mutant cells showed higher phosphorylation of RelA (p65) and, accordingly, greater migration in wound-healing assays. Mis-splicing of exon 5 in MAP3K7, a gene encoding a central regulator of NFκB pathway, significantly reduced MAP3K7 protein expression in SF3B1 K700E mutant cells. Consistent with these data, pharmacologic MAP3K7 inhibition in SF3B1 wild-type breast cells increased phosphorylation of RelA. To further understand the effects of the K700E mutation on breast cancer development and progression, we generated a conditional knock-in mouse model, in which Sf3b1 K700E mutant allele was activated through MMTV promoter-controlled expression of Cre recombinase in mammary epithelium. Accelerated tumor formation was not identified in the mammary gland of these mice. However, RNA-seq analysis of mammary epithelial cells from the Sf3b1 K700E mutant mice revealed dysregulation of alternative splicing including Map3k7. As we found the majority of SF3B1 k700E mutations to co-occur with activating mutations in the PI3K pathway, we additionally introduced the Pik3ca H1047R mutant allele, by mating the MMTV-Cre Sf3b1 K700E mice with Rosa26-Pik3ca H1047R knock-in line. Markedly decreased survival was observed in double mutant animals, which developed mammary tumors faster, compared with single mutant controls. Overall, these data reveal that breast cancer-associated Sf3b1 mutations induce the NFkB pathway to promote invasion and cooperate with the PI3K pathway to accelerate mammary tumor development. Citation Format: Bo Liu, Michelle Ki, Omar Abdel-Wahab, Sarat Chandarlapaty. Hotspot mutations in the core spliceosomal protein SF3B1 promote breast tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4654.