Abstract 1997: Inhibition of SF3B1 causes global intron retention and downregulation of the B-cell receptor pathway in chronic lymphocytic leukemia

Abstract

Abstract Splicing factor SF3B1 is frequently mutated in about 10-15% of CLL patients. SF3B1 mutations are predominately associated with faster disease progression and poor overall survival, and mutations are typically enriched in approximately 20% relapsed and chemorefractory CLL patients. Therefore, there is a growing interest in using SF3B1 as a therapeutic target for CLL. In this study, we investigated the effectiveness of Sudemycin D6 (SD6), a small molecule SF3B1 inhibitor, in reducing cell viability and promoting apoptosis in CLL cells. We then isolated RNA from both the SD6-treated and control cells at 2, 6, and 24hr post-treatment and performed RNA sequencing analysis to investigate the global impact. We demonstrated that SD6 effectively inhibits cell growth and induces apoptosis in CLL cells at a nanomolar concentration in both a time and dosage dependent manner. Furthermore, global analysis of the RNA sequencing data revealed that treatment with SD6 causes an aberrant splicing signature with significant increase in global intron retention. The increase in global intron retention peaked at 6hr then significantly diminished at 24hr post-treatment. Ontology analysis of the intron-retaining transcripts using the Ingenuity Pathway Analysis (IPA) software package suggested that B-cell receptor (BCR) signaling, protein ubiquitination, and PI3K signaling were among the top canonical pathways affected by SD6 treatment. Using RT-PCR analysis, we confirmed intron retention events in several components of the BCR pathway, such as BTK, BLNK, and PIK3CD, and an exon-skipping event in the exons encoding the kinase domain of Bruton's Tyrosine Kinase. The increase in intron retention significantly correlated with a decrease in overall mRNA and protein expression levels of the affected downstream BCR pathway proteins like AKT, PI3Kδ, and PLCγ2. Furthermore, SD6 treatment induced a time-dependent exon-skipping event in the apoptotic regulator MCL1, resulting in alternative splicing of the long, anti-apoptotic isoform of MCL1 into the short, pro-apoptotic isoform. SD6 treatment also caused downregulation of anti-apoptotic gene TRAF1 at both the mRNA and protein level, which could contribute to SD6-induced apoptosis. Cumulatively, these results strongly suggest that SF3B1 is a promising target for therapeutic treatment in CLL. Our study also provides solid rationale for future clinical development of spliceosome inhibitors and combination therapies based on spliceosome inhibitors. Citation Format: Qimei Han, Austin Young Shull, Fang Shi, Chandraiah Lagisetti, Thomas Webb, Justin Choi, Huidong Shi. Inhibition of SF3B1 causes global intron retention and downregulation of the B-cell receptor pathway in chronic lymphocytic leukemia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1997.