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Wojtek Drabarek, Job van Riet, Harmen van de Werken, Annelies de Klein, Emine Kilic; The effect of spliceosome inhibitor E7107 on SF3B1mut uveal melanoma cell lines. Invest. Ophthalmol. Vis. Sci. 2019;60(9):732. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Effective treatment for Uveal Melanoma (UM) patients with metastatic disease is unfortunately not available. Mutations in splicing factor genes (SF3B1, SRSF2) are observed in 20% of patients with uveal melanoma, suggesting that aberrant spliceosome function also plays a role in the pathogenesis of this tumor. Spliceosome inhibitors are promising agents that exploit the preferential sensitivity of splicing factor-mutant cells to these compounds. Therefore, we hypothesize that Mel202 cell line which harbors a SF3B1 mutation of the arginine amino acid residue 625 (R625) is more sensitive to splicing inhibition than SF3B1 WT cell lines.
UM cell lines were exposed for 24 hours to splicing inhibitor E7107 concentrations ranging from 0 to 20 nM. Linear regression was applied to compare the decrease in cell viability between cell lines and increasing concentration of E7107. After exposure cells were counted and RNA was isolated for expression analysis. DEXSeq analysis of RNA-seq data was used to define the most significant UM target genes in SF3B1mut tumors. Unsupervised clustering and stratification of the data to detect specific SF3B1 target genes were performed. The most significant target genes have been validated using specific designed PCR primers able to discriminate between the normal and alternatively spliced products.
The decrease in cell viability of Mel202 cells was significantly higher compared to the decrease of 92.1 cells(P = 0.026). From DEXseq analysis we selected ENOSF1 and ARMC9 as both highly expressed normal and aberrant transcript candidates for transcription level analysis. Using PCR we did observe a decrease in aberrant transcript levels compared to WT mRNA expression with increasing E7107 concentrations: CHMP2A expression was used for normalization.
Our cell viability experiments suggest increasing concentrations of E7107 result in decreased cell counts of Mel202 cells compared to SF3B1 WT cells. Although, transcription level analysis of both canonical and aberrantly expressed transcripts show a trend in decreased aberrant transcript levels, future RNA-sequencing experiments will be needed to quantify the effect of E7107 on the whole transcriptome.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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