RNAseq Study in CC-671 Treated Cal-51 Cells

CC-671 has been identified as an inhibitor of Cdc2-like kinase 2 (CLK2) and TTK in direct enzyme assays. CLK2 is a member of the CLK family that phosphorylates serine- and arginine-rich (SR) proteins of the spliceosomal complex as part of a regulatory mechanism for control of pre-mRNA splicing. SR proteins are a family of small nuclear ribonucleoprotein particle (snRNP) splicing factors involved in constitutive and alternative splicing. Monitoring specific phospho-biomarkers of CLK2 demonstrated that CC-671 inhibited phosphorylation of CLK2 substrates in cancer cells with mean IC50 of 549 nM in the triple negative breast cancer (TNBC) line CAL51. In this study, RNA sequencing approach was used to quantify the impact of CC-671 treatment on gene transcription and global alternative splicing in CAL51 cells. Differential exon usage analysis demonstrated that CC-671 changed alternative splicing of many genes. In addition, different sets of genes are impacted by CC-671 at both the alternative splicing and mRNA expression. Genes impacted by alternative splicing shared a set of common pathways with genes altered by mRNA expression. This result indicates that CC-671 regulates transcription via both gene expression and alternative splicing mechanisms. Overall design: Triple negative breast cancer (TNBC) line CAL51 was grown in DMEM medium containing 10% fetal bovine serum, as recommended by vendor. The growing cells were treated by CC-671 in three biological replicates at the following concentrations and time intervals. The treatment time points were 6 hour and 24 hour. Concentration of compounds used was 3 and 10 uM. Six million cells from each treatment were harvested and RNA was isolated by RNeasy kit. Poly-A selection and strand-specific RNA library construction were performed, followed by multiplexing indexed libraries and sequencing on the HiSeq 2500 with 2x100 bp read lengths. A total of 16 samples were included in this experiment, including 4 treatment groups with three biological replicates and 2 vehicle control groups with two biological replicates

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Zhu D, Xu S, Deyanat-Yazdi G, Peng SX, Barnes LA, Narla RK, Tran T, Mikolon D, Ning Y, Shi T, Jiang N, Raymon HK, Riggs JR, Boylan JF