Abstract SF3B1 mutation, which occurs in 10-20% of chronic lymphocytic leukemia (CLL) patients, is associated with faster disease progression, shorter overall survival and fludarabine resistance. Mutant SF3B1 utilizes cryptic 3′ splice sites to generate aberrantly spliced mRNAs, half of which may undergo non-sense mediated mRNA decay (NMD), leading to downregulation of the affected genes. To identify potential tumor suppressor genes that are aberrantly spliced and downregulated by mutant SF3B1, we performed RNA-sequencing analysis of SF3B1-mutated primary CLL cells in the presence or absence of cycloheximide (CHX), a translation inhibitor known to inhibit NMD. Our analysis identified PPP2R5A, encoding one of the regulatory subunits of protein phosphatase-2A (PP2A B56α), as one of the key genes that were most consistently affected by an aberrantly spliced junction, and significantly downregulated (>2 fold) in CLL patient samples with various SF3B1 hotspot mutations. Splicing analyses by RT-PCR and Sanger sequencing confirmed that a 13-nucleotides sequence was added before the 5th exon of PPP2R5A via alternative splicing in CLL patients with the SF3B1 mutation. Due to this 13-nucleotides addition, three consecutive premature stop codons were created by frameshift at a position more than 55 bases upstream of an exon-exon junction, which is a canonical feature of the mRNAs degraded through NMD. The down-regulation of PPP2R5A was confirmed by quantitative PCR (p<0.05) and immunoblot at both mRNA and protein levels in CLL patients with SF3B1 mutation as compared to CLL patients without SF3B1 mutation and normal CD19+ B cell samples. Furthermore, overexpression of mutant SF3B1 in K562 and HEK 293 cells resulted in the same aberrant splicing pattern observed in CLL patients with SF3B1 mutation and downregulation of PPP2R5A (p<0.05). Treatment with CHX resulted in an increased abundance of alternatively spliced PPP2R5A transcript, suggesting that the aberrant PPP2R5A transcript was degraded by NMD. PP2A, when incorporated with PPP2R5A as the regulatory subunit, dephosphorylates c-Myc at S62, and results in the degradation of c-Myc. Here, we demonstrated that downregulation of PPP2R5A by mutant SF3B1 led to upregulation of c-Myc through promoting c-Myc protein stability in SF3B1K700E overexpressing cells when compared to SF3B1wildtype overexpressing cells. Knockdown PPP2R5A in MEC1 CLL cells by siRNA also significantly upregulated c-Myc at the protein level and upregulated mRNA expression of c-Myc downstream genes NCL and ODC1. Overall, our results demonstrate that SF3B1 mutation causes aberrant splicing and downregulation of tumor suppressor protein PP2A B56α in vitro and in vivo, leading to upregulation of c-Myc. Citation Format: Qimei Han, Libin Deng, Shuo Tu, Lirong Pei, Jeong-Hyeon Choi, Victor Jin, Huidong Shi. SF3B1 mutation promotes c-Myc protein stability through aberrant splicing and downregulation of PP2A B56α subunit in chronic lymphocytic leukemia [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 4493.