Isocitrate dehydrogenase 1 mutation sensitizes intrahepatic cholangiocarcinoma cells to MDM2 inhibitors

Background: Mutations in isocitrate dehydrogenase 1 ( IDH1 ^mut ) occurs in 10-25% of intrahepatic cholangiocarcinoma (iCCA) cases. IDH1 ^mut produces oncometabolite D-2-hydroxyglutatrate (D-2-HG), which inhibits α-ketoglutarate-dependent dioxygenases activity, and further reprograms cell metabolism, epigenetically alters gene expression, promotes oncogenesis, etc. Several mutant IDH1 inhibitors have been developed and undergone clinical trials. Despite significantly prolonged progression-free survival, the mutant IDH1 inhibitor ivosidenib achieved low response rate in clinical trials, highlighting the need for new therapeutic options for IDH1 ^mut iCCA. Methods: We used in-silico analysis to identify mutually exclusive genetic alterations in iCCA mutation panels. RNA and protein expression of target genes were examined by qPCR and western blotting, respectively. D-2-HG levels and KDM5/JARID activity were determined by ELISA assay. Chromatin immunoprecipitation (IP)-qPCR assay was performed to evaluate the H3K4 methylation level on the interested gene. Cell cycle analysis and proliferation assay were done to evaluate the growth inhibitory effects of study compounds, either alone or in combinations on iCCA cells Results: Our in-silico analysis demonstrated that IDH1 and TP53 mutations were mutually exclusive in iCCA tumors and cell-lines, and IDH1 ^mut /TP53 ^wt iCCA cells expressed higher MDM2 levels than IDH1 ^wt /TP53 ^mut iCCA cells. Real-time quantitative polymerase chain reaction (Real-time -qPCR) and western blotting showed MDM2 up-regulation was at transcriptional level. Chromatin immunoprecipitation (ChIP)-qPCR showed enrichment of histone-3-lysine-4 tri-methylation (H3K4me3), an indicator of active gene transcription, at the MDM2 promoter in IDH1 ^mut iCCA cells, confirming the data from ENCODE histone-seq. Treatment with a mIDH1 inhibitor reduced 2-hydroxyglutarate (2-HG) levels, enhanced lysine-specific demethylase 5 (KDM5) activity, and attenuated the H3K4me3/H3K4me1 ratio at the MDM2 promoter, which was accompanied by a reduction in MDM2 expression and an increase in wild-type TP53 (wtTP53) protein levels in IDH1 ^mut /TP53 ^wt iCCA cells. The effect of mIDH1 inhibitor on MDM2 mRNA levels was reversed by treatment with KDOAM-25 citrate, a pan-KDM5 inhibitor. In addition, MDM2 inhibitors that could block MDM2-mediated wtTP53 degradation selectively induced TP53 reactivation, cell cycle arrest, and growth inhibition in IDH1 ^mut /TP53 ^wt iCCA cells. The combination of mIDH1 and MDM2 inhibitors synergistically suppressed the proliferation of IDH1 ^mut iCCA cells. Conclusions: Our study delineated a novel mIDH1-MDM2-wtTP53 axis and the potential application of MDM2 inhibitor therapy in IDH1mut/TP53wt iCCA.