Abstract 2932: SF3B1 mutations induce aberrant mRNA splicing in cancer and confer sensitivity to spliceosome inhibition
Authors: Buonamici Silvia , Lim Kian Huat , Feala Jacob , Park Eunice , Corson Laura , Aicher Michelle , Aird Daniel , Chan Betty , Corcoran Erik , Darman Rachel , Fekkes Peter , Keaney Gregg , Kumar Pavan , Kunii Kaiko , Lee Linda , Puyang Xiaoling , Rodrigues Jose , Selvaraj Anand , Thomas Michael , Wang John , Warmuth Markus , Yu Lihua , Zhu Ping , Smith Peter , Mizui Yoshiharu 
Published: May 2015

Abstract
Recurrent heterozygous mutations of the spliceosome protein SF3B1 have been identified in myelodysplastic syndromes, chronic lymphocytic leukemia (CLL), breast, pancreatic and skin cancers. SF3B1 is a component of the U2 snRNP complex which binds to the pre-mRNA branch point site and is involved in recognition and stabilization of the spliceosome at the 3′ splice site.
To understand the impact of SF3B1 mutations, we compared RNAseq profiles from tumor samples with SF3B1 hotspot mutations (SF3B1-MUT) or wild-type SF3B1 (SF3B1-WT) in breast cancer, melanoma and CLL. This analysis revealed significant increases in the usage of novel alternative splice junctions in SF3B1-MUT samples including selection of alternative 3′ splice sites and less frequently exon skipping. These events induce expression of alternative mRNAs that are translated into novel proteins or aberrant mRNAs that are decayed by cells. A common alternative splicing profile was shared across different hotspot mutations and lineages (e.g. ZDHHC16 and COASY); however, unique alternative splicing profiles were also observed suggesting lineage specific effects. RNAseq analysis of several cell lines with endogenous SF3B1 hotspot mutations confirmed the presence of the same spliced isoforms as observed in tumor samples. To prove that SF3B1-MUT were inducing alternative splicing, transient transfection of several SF3B1 hotspot mutations in 293FT cells induced the expression of the common alternatively spliced genes suggesting functional similarity. Selective shRNA depletion of mutant SF3B1 allele in SF3B1-MUT cells resulted in downregulation of the same splice isoforms. Furthermore, isogenic B-cell lines (NALM-6) expressing the most frequent SF3B1 mutation (K700E) were generated and profiled by RNAseq. As expected, similar alternatively spliced genes were observed in NALM-6 SF3B1-K700E cells exclusively. To investigate the role of nonsense-mediated mRNA decay (NMD) in eliminating aberrant mRNAs induced by SF3B1-MUT, we treated NALM-6 SF3B1-K700E cells with cycloheximide, a translation inhibitor known to inhibit NMD. In the treated samples, expression of several aberrant mRNAs was revealed and some of these transcripts were shown to be downregulated in patient samples. Taken together, these results confirm the association between different SF3B1 hotspot mutations and the presence of novel splice isoforms.
We demonstrated that E7107, a potent and selective inhibitor of wild-type SF3B1, also binds and inhibits SF3B1-MUT protein. In addition, E7107 represses the expression of several common aberrant splice mRNA products in SF3B1-MUT cells in vitro and in vivo. When tested in a NALM-6 mouse model, E7107 induced tumor regression and increased the overall survival of animals implanted with NALM-6 SF3B1-K700E cells. These data suggest splicing inhibitors as a promising therapeutic approach for cancer patients carrying SF3B1 mutations.
Citation Format: Silvia Buonamici, Kian Huat Lim, Jacob Feala, Eunice Park, Laura Corson, Michelle Aicher, Daniel Aird, Betty Chan, Erik Corcoran, Rachel Darman, Peter Fekkes, Gregg Keaney, Pavan Kumar, Kaiko Kunii, Linda Lee, Xiaoling Puyang, Jose Rodrigues, Anand Selvaraj, Michael Thomas, John Wang, Markus Warmuth, Lihua Yu, Ping Zhu, Peter Smith, Yoshiharu Mizui. SF3B1 mutations induce aberrant mRNA splicing in cancer and confer sensitivity to spliceosome inhibition. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2932. doi:10.1158/1538-7445.AM2014-2932