CLK3 up-regulation promotes CCA development by reprogramming purine metabolism. (A) Heatmap of top 500 up-regulated genes in human CCA with a high abundance of CLK3. (B) Top 20 biological processes uncovered among CCA patients with high CLK3 expression using gene ontology term enrichment. (C) LC-MS/MS was used to examine the metabolites in HuCCT1 cells with or without CLK3 deficiency induced by Dox or with Dox-induced c-Myc introduction. The data are shown in the heatmap. (D) Schematic representation of the main metabolic pathways. (E) LC-MS/MS analysis was performed to measure ¹⁵N-glutamine–labeled intermediates of purine synthesis (upper panel) or metabolites labeled with ¹³C-glycine (lower panel) in HuCCT1 cells with Dox-induced CLK3 knockdown. (F) Measuring RNA and DNA with the incorporation of ¹⁴C-glycine using samples in E. (G) Quantitative RT-PCR assays were used to analyze the effects of CLK3 silencing on the genes deciding purine metabolism in HuCCT1 cells. (H) CLK3 silencing significantly decreased HuCCT1 cell proliferation, while overexpressing ATIC or adding purine markedly reverted this defect. (I) ATIC inhibitor significantly reverted the proliferation of HCCC9810 cells induced by CLK3 overexpression. *, P < 0.05; **, P < 0.01; ***, P < 0.001; #, not significant; one-way ANOVA. Data are from three independent experiments (C and E–I; mean ± SEM). GO, gene ontology; KD, knockdown; PPP, pentose phosphate pathway; AMP, adenosine monophosphate; GMP, guanosine monophosphate; IMP, inosine monophosphate.