Abstract 2040: Mutations in SF3B1 lead to aberrant splicing through cryptic 3′ splice site selection and impair hematopoietic cell differentiation
Authors: Buonamici Silvia , Perino Samantha , Lim Kian Huat , Feala Jacob , Darman Rachel , Obeng Esther , Furman Richard R. , Bailey Suzanna , Keaney Gregg , Kumar Pavan , Mizui Yoshiharu , Park Eunice , Wang John , Warmuth Markus , Yu Lihua , Zhu Ping , Ebert Benjamin L. , Smith Peter 
Published: August 2015

Abstract
Heterozygous mutations in SF3B1, a component of the U2 complex involved in the recognition of 3′ splice sites (ss), have been reported with high frequency in refractory anemia with ring sideroblasts (RARS, a subtype of myelodysplastic syndrome, MDS) and have also been observed in chronic lymphocytic leukemia (CLL) and several solid tumors. To study the impact of SF3B1 mutations on splicing, RNAseq data obtained from breast cancer, melanoma, CLL and MDS samples with mutant (SF3B1MUT) or wild-type SF3B1 (SF3B1WT) were compared. The majority of aberrant junctions identified in the samples with mutant SF3B1 utilized an alternative 3′ss, suggesting its neomorphic function. Motif analysis of the sequences used by SF3B1MUT revealed the usage of a cryptic AG with a shorter and weaker polypyrimidine tract. Minigenes with modifications of these sites revealed the importance of both intronic and exonic sequence features for the recognition of the cryptic AG by SF3B1MUT. Of the aberrant junctions identified, several were common across all hotspot mutations and diseases; however, a unique aberrant splicing profile was found for each disease suggesting lineage and disease specific effects. The majority of splicing defects introduced a premature termination codon downstream of the cryptic AG leading to nonsense mediated decay (NMD) of aberrant transcripts and downregulation of gene expression, such as ABCB7. Gene-set enrichment analysis of aberrantly spliced and differentially expressed genes in SF3B1MUT MDS samples identified genes involved in cell differentiation and epigenetic pathways which are known to be deregulated in MDS. The impact on erythroid differentiation by SF3B1MUT was studied in transduced TF-1 cells following erythropoietin (EPO) stimulation. As expected, TF-1 SF3B1WT cells were able to differentiate normally after EPO treatment; however, expression of SF3B1K700E (the most common hotspot mutation found in RARS and CLL) induced a block in erythoid differentiation. This differentiation block was not observed with the expression of SF3B1G742D, a mutation found in CLL but not RARS, suggesting a context dependent role for SF3B1 mutations. Interestingly, the differentiation block observed in SF3B1K700E was associated with cytokine independent growth. Initial mining of RNAseq data from SF3B1MUT TF-1 cells highlighted several aberrantly spliced and NMD-downregulated genes previously implicated in MDS. Finally, a xenograft model was developed by subcutaneous implantation of transduced TF-1 cells. After several passages, an enrichment of TF-1 SF3B1K700E cells was observed, suggesting a growth advantage for SF3B1MUT cells over SF3B1WT cells. This data suggests that the K700E SF3B1 mutation can lead to a block in differentiation and competitive advantage as observed in human RARS.
Citation Format: Silvia Buonamici, Samantha Perino, Kian Huat Lim, Jacob Feala, Rachel Darman, Esther Obeng, Richard R. Furman, Suzanna Bailey, Gregg Keaney, Pavan Kumar, Yoshiharu Mizui, Eunice Park, John Wang, Markus Warmuth, Lihua Yu, Ping Zhu, Benjamin L. Ebert, Peter Smith. Mutations in SF3B1 lead to aberrant splicing through cryptic 3′ splice site selection and impair hematopoietic cell differentiation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2040. doi:10.1158/1538-7445.AM2015-2040