Abstract B125: Mutant SF3B1 downregulates proteins involved in differentiation, including ABCB7

Abstract

Abstract Refractory Anemia with Ringed Sideroblasts (RARS), a subtype of Myelodysplatic Syndrome (MDS), occurs with a high frequency of hotspot mutations in HEAT (Huntingtin, Elongation factor 3, protein phosphatase 2A, Targets of rapamycin 1 domains) domains of SF3B1. This protein component of the U2 snRNP complex of the spliceosome is essential in the proper selection and usage of 3' splice sites. RNAseq analysis of MDS and other tumor types in which SF3B1 hotspot mutations have been found show that alternative 3' splice site usage is the predominant cause of RNA transcript aberration. These modifications can result in mRNAs encoding novel peptides, or they can introduce premature termination codons into the pre-mRNA, most likely directing it to the Nonsense Mediated Decay (NMD) pathway for degradation. Using a predictive tool to determine the likelihood of a given aberrant transcript to be targeted for NMD, we determined that nearly 50% of the SF3B1-mutant-associated aberrant transcripts were candidates for degradation. We confirmed this experimentally by treating isogenic Nalm-6 cells (engineered by AAV homology to express SF3B1 K700E or K700K) with or without cycloheximide, an agent known to inhibit translation and RNA degradation by NMD. Investigation of the resulting RNAseq data showed significant rescue of gene expression only for the transcripts predicted to be NMD targets. Ingenuity Pathway Analysis indicated that many of the downregulated genes in SF3B1 mutant samples were involved in differentiation, which has been shown to be dysregulated in MDS. We tested the idea that such modifications in the transcriptome confer selective advantage or impair differentiation in SF3B1 mutant cells. We began by manipulating the expression of ABCB7, one of the genes identified in our RNAseq analysis to be downregulated by aberrant splicing and subsequent NMD. ABCB7 is a mitochondrial transporter important in cellular iron metabolism and, indirectly, in heme production. Additionally, loss of function of ABCB7 is causal in X-linked sideroblastic anemia and has been implicated in RARS MDS. We discovered in our SILAC proteomic analysis that ABCB7 protein was dramatically decreased in K700E SF3B1 Nalm-6 cells relative to K700K Nalm-6, in agreement with our RNAseq analysis. Using doxycycline-inducible shRNA expression, we knocked down ABCB7 mRNA and protein expression in TF-1 erythroblasts. These cells show significant decreases in erythropoeitin (EPO)-induced differentiation when expressing exogenous K700E SF3B1, but not K700R (a very conservative mutation) or WT SF3B1. With direct knock down of ABCB7, we observed a similar phenotype - impairment of EPO-induced differentiation in ABCB7 shRNA-induced cells by Day 7, with no overall decline in cell viability. Interestingly, knock down of SF3B1 expression with shRNA also reduces ABCB7 mRNA. However, it also promotes cell death. This is consistent with the heterozygous nature of SF3B1 hotspot mutations; severe loss of SF3B1 function is deleterious. We propose that hotspot SF3B1 mutants promote aberrant splicing of multiple genes, inducing a general “spliceosomal sickness” in addition to downregulating key genes (e.g. ABCB7) responsible for erythroid differentiation impairment, such as that observed in RARS. Citation Format: Rachel B. Darman, Samantha A. Perino, Michael Seiler, Shouyong Peng, Jacob Feala, Peter Fekkes, Gregg F. Keaney, Kaiko Kunii, Linda Lee, Kian Huat Lim, Yoshiya Oda, Khin Myint, Esther A. Obeng, Ermira Pazolli, Eun Sun Park, John Yuan Wang, Markus Warmuth, Lihua Yu, Ping Zhu, Yoshiharu Mizui, Benjamin L. Ebert, Peter G. Smith, Silvia Buonamici. Mutant SF3B1 downregulates proteins involved in differentiation, including ABCB7. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B125.