Abstract 4471: Novel SF3B1 deletion mutations result in aberrant RNA splicing in CLL patients
Authors: Agrawal Anant A. , Seiler Michael , Brinton Lindsey , Mantel Rose , Lapalombella Rosa , Woyach Jennifer A. , Johnson Amy J. , Yu Lihua , Byrd John C. , Smith Peter G. , Blachly* James S. , Buonamici* Silvia 
Published: July 2017

Abstract
Recurrent heterozygous mutations in SF3B1, a component of the U2snRNP complex involved in branchsite recognition during the first step of splicing catalysis, are found in various solid tumors and hematologic cancers including myelodysplastic syndromes (MDS) and chronic lymphocytic leukemia (CLL). To provide broader insight into the effects of SF3B1 mutations on RNA splicing in CLL, RNAseq was performed on 215 CLL patient samples with unknown SF3B1 mutation status. 37 patients (17%) demonstrated cryptic 3’ splice site usage we and others have previously reported in patients with known SF3B1 hotspot mutations. Interestingly, 3 patient samples harbored previously unreported in-frame deletions in SF3B1 around K700, the most common amino acid mutated in hematologic cancers with SF3B1 mutations. In particular, 2 patient sequences contained 6 nucleotide (nt) deletions (c.2186 to c.2191) resulting in replacement of Q698, Q699 and K700 with Q (p.Q698_K700delinsQ: “p.Q698del”) and 1 patient sequence had a 3 nt deletion (c.2192 to c.2194) resulting in replacement of K700 and V701 with N (p.K700_V701delinsN: “p.K700del”) in SF3B1. The variant allele fraction (VAF) of these deletions was 20-40%. Targeted resequencing of DNA confirmed presence of these mutations at similar VAFs. Differential splicing analysis across patients with wild-type SF3B1, mutant SF3B1 and our novel deletions revealed similar alternative 3’ splice site patterns. To study the mechanism of alternative 3’ splice site selection induced by these novel p.Q698del and p.K700del in-frame deletions we used our ZDHHC16 minigene system with modifications in the canonical and cryptic 3’ splice sites and respective branchsites. After confirming expression of these deletion mutants and their incorporation in the SF3b complex by qPCR and co-immunoprecipitation, we demonstrated that recognition of both canonical 3’ splice site and alternate branchsite are important for aberrant splicing. This observation is identical to the intronic features required for aberrant splicing that we previously reported for the p.K700E SF3B1 hotspot mutation in CLL and MDS. By comparing the recent cryo-electron microscopy and X-ray crystal structures of SF3b complexes (PDB: 5GM6 and 5IFE, respectively), it is evident that the K700 present at the edge of heat repeat domain 6 is important for pre-mRNA interactions. Both the in-frame deletions and substitution mutations at K700 perturb SF3B1 charge, likely affecting a side-chain interaction with pre-mRNA, thus altering splice-site recognition through a mechanism that is still not well understood. Together, these data indicate that small in-frame deletions in SF3B1 around K700 offer a novel mechanism to induce aberrant splicing in patients with CLL. The common mechanism of action of these mutations and deletions suggest that they should also be sensitive to splicing modulators.
Citation Format: Anant A. Agrawal, Michael Seiler, Lindsey Brinton, Rose Mantel, Rosa Lapalombella, Jennifer A. Woyach, Amy J. Johnson, Lihua Yu, John C. Byrd, Peter G. Smith, James S. Blachly*, Silvia Buonamici*. Novel SF3B1 deletion mutations result in aberrant RNA splicing in CLL patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4471. doi:10.1158/1538-7445.AM2017-4471