Isocitrate dehydrogenase 1 mutation sensitizes intrahepatic cholangiocarcinoma cells to MDM2 inhibitors

Isocitrate dehydrogenase 1 mutation sensitizes intrahepatic cholangiocarcinoma cells to MDM2 inhibitors | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Isocitrate dehydrogenase 1 mutation sensitizes intrahepatic cholangiocarcinoma cells to MDM2 inhibitors Chien-Tsung Liu, Yung-Yeh Su, Nai-Jung Chiang, Chien-Feng Li, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6489065/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract 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. Intrahepatic cholangiocarcinoma (iCCA) Isocitrate dehydrogenase 1/2 (IDH1/2) mutation Tumor protein 53 (TP53) Mouse double minute 2 (MDM2) Histone-3-lysine-4 (H3K4) methylation Mutant isocitrate dehydrogenase 1 inhibitor Mouse double minute 2 inhibitor Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Biliary tract cancer (BTC) is a heterogeneous group of malignancies arising from epithelial cells of the biliary tract. Based on their primary location, BTCs are divided into intrahepatic, perihilar, and distal cholangiocarcinoma; gallbladder cancer; and ampulla of Vater carcinoma, which have different trends of incidence changes, geographic distribution, risk factors, clinical outcomes, and genetic alterations [1-3]. Intrahepatic cholangiocarcinoma (iCCA), next to hepatocellular carcinoma, is the second most common hepatic malignancy. Owing to the lack of specific symptoms and signs, iCCA is usually diagnosed at a late stage with local invasion and/or intrahepatic or extrahepatic disseminations. According to the Surveillance, Epidemiology, and End Results Program (SEER) database, the 5-year overall survival rate of iCCA diagnosed between 2012 and 2018 in the United States was 9%, with 23%, 9%, and 3% for patients with localized, regional, and metastatic diseases, respectively [4]. Before the era of precision medicine, gemcitabine plus cisplatin was the standard systemic treatment for patients with unresectable and metastatic iCCA, similar to other BTCs [5, 6]. The isocitrate dehydrogenase (IDH) protein family is a metabolic enzyme responsible for converting isocitrate into α-ketoglutarate (α-KG). Somatic mutations in IDH1 or 2 ( IDH1/2 mut ) are frequently identified in several cancers, including 10-25% of iCCA cases [7]. The mutant IDH1/2 (mIDH1/2) protein encoded by a gain-of-function IDH1/2 mutation converts α-KG to D-2-hydroxyglutarate (2-HG), an oncometabolite that competitively suppresses the activity of α-KG-dependent dioxygenases (α-KGDs), including DNA and histone demethylases, to promote epigenetic reprogramming and tumorigenesis [8-10]. Based on the structural differences between the catalytic pockets of wild type IDH1/2 (wtIDH1/2) and mIDH1/2, several inhibitors targeting mIDH1 and/or mIDH2 have been developed and have undergone clinical trials [11]. AG120 (ivosidenib) is the first FDA-approved agent for IDH1 -mutated iCCA. However, despite significant improvement in progression-free survival (PFS) versus placebo control, the confirmed response rate and absolute PFS increase of second-line ivosidenib were 2.4% and 0.7 months, respectively, in the pivotal, ClarIDHy trial for IDH1 -mutated, chemotherapy-refractory iCCA [12-14]. In addition, patients who received long-term ivosidenib treatment might develop acquired resistance to ivosidenib through acquired resistance mutations that impair ivosidenib binding affinity, such as the conversion of catalytic arginine substitution and secondary IDH1 mut at the dimer-interface region or isoform switch by acquired IDH2 mut [15-18]. Taken together, ivosidenib treatment could result in a 22% 12-month PFS rate but a modest tumor response rate and might acquire secondary resistance mutations after long-term use, suggesting that other therapeutic interventions beyond IDH1 inhibition are warranted for IDH1 mut iCCA patients. Herein, we report a novel mechanism by which mIDH1-derived 2-HG inhibits lysine-specific demethylase 5 (KDM5) to upregulate the expression of mouse double minute 2 homolog ( MDM2 ) via histone methylation and selectively promotes the proliferation of IDH1 mut iCCA cells. In addition, the combination of mIDH1 and MDM2 inhibitors may provide a new strategy for treating IDH1 mut iCCA patients. Methods Clinical sample gene mutation profile analysis Mutation profiling datasets were obtained using cBioPortal (https://www.cbioportal.org) [19-21]. Odds ratios were calculated using the following formula: ( IDH1 wt , TP53 wt sample number × IDH1 mut , TP53 mut sample number)/ ( IDH1 wt , TP53 mut sample number × IDH1 mut , TP53 wt sample number). The P value of the odds ratio was calculated using Fisher’s exact test. Immunohistochemistry staining The use of human iCCA tissue samples in the current study was approved by the Institutional Review Board of the National Cheng Kung University Hospital (NCKUH, IRB A-ER-111-019). Formalin-fixed paraffin-embedded iCCA samples were sectioned and stained with hematoxylin and eosin. For MDM2 IHC staining, sections were incubated with MDM2 antibody, secondary mouse antibody, and diaminobenzidine for colorimetric detection. Three independent pathologists who were unaware of the genetic alterations evaluated the IHC staining results. The histochemical scoring assessment (H-score) was based on the staining intensity of MDM2 and the proportion of stained cancer cells. Cell lines The following cell lines were obtained from the following sources: HuCCT1 and HuH28 were obtained from the Japanese Collection of Research Bioresources Cell Bank, SNU1079 was purchased from the Korean Cell Line Bank, and RBE and SSP25 were kindly provided by Dr. Chien-Feng Li (Chi Mei Medical Center, Tainan, Taiwan), and Dr. Chun-Nan Yeh (Chang Gung University, Taoyuan, Taiwan). HuCCT1, RBE, SSP25, and SNU1079 cells were cultivated in RPMI-1640 medium (Cytiva) supplemented with 10% fetal bovine serum (Cytiva) and 1% 100x penicillin-streptomycin-glutamine (Invitrogen). HuH28 cells were cultivated in MEM (Simply) supplemented with 20% fetal bovine serum (Cytiva) and 1% 100x penicillin-streptomycin-glutamine (Invitrogen). All cell lines were incubated in at 37°C humidified incubator with 5% CO 2 and passaged by trypsinization with 0.25% trypsin and protease solution (Cytiva). All cell lines were verified by short tandem repeat (STR) testing (FIRDI, Hsinchu, Taiwan), and experiments were performed on cells cultivated for less than ten passages. Intracellular 2-HG measurement To detect intracellular 2-HG concentration, a D-2-Hydroxyglutarate (D-2-HG) assay kit (Sigma-Aldrich, MAL320) was used following the manufacturer's protocol. Cells were seeded in 10 cm dishes (5x10 5 cells per dish) for 24 hours (h) and treated with either mutant IDH1 inhibitors or DMSO control for 48 h. After treatment, cells were washed with ice-cold PBS, scraped, and collected by centrifugation. The collected cells were lysed in CelLytic TM M Cell Lysis Reagent (Sigma-Aldrich), and the protein concentration was quantified. The lysate was deproteinized by perchloric acid (PCA) precipitation. After PCA precipitation, the supernatants for 2-HG detection were collected and stored at -80°C. The supernatants with D-2-HG complete reaction mixture for 1 h at 37°C. The fluorescence was measured at λ ex = 540 nm and λ em = 590 nm using a microplate reader. The intracellular D-2-HG concentrations were normalized to their corresponding protein concentrations. RNA extraction and complementary DNA synthesis RNA was extracted using the Total RNA Miniprep Purification Kit (GeneMark, TR01), following the manufacturer's protocol. Cells were first cultivated in 6-well plates (4x10 4 cells/well) for 24 h and then treated with inhibitors or DMSO control for 48 h. After treatment, cells were washed with cold PBS and lysed in an RNA lysis solution containing 1% 2-mercaptoethanol. Lysates were purified using a two-step wash with RNA wash solution I and ethanol/RNA wash solution II. The purified RNA was eluted in nuclease-free water and stored at -80°C. RNA quality was measured using a nanophotometer. Complementary DNA (cDNA) was reverse-transcribed from purified RNA using the ReverTra Ace -α-™ kit (TOYOBO, FSK-101), following the manufacturer's instructions. One microgram of total purified RNA was mixed with oligo-dT primers, RNase inhibitor, dNTP mixture, RT buffer, and RNase-free water. cDNA was amplified in a thermal cycler using the following program: 42°C for 20 minutes (m), 99°C for 5 min, and 4°C for 5 min. The cDNA was stored at -20 °C. Real-time polymerase chain reaction (PCR) For real-time PCR, we followed the manufacturer's protocol and used a GoTaq® qPCR Master Mix kit (Promega, A6001). GoTaq® qPCR master mix, forward primer, reverse primer, and nuclease-free water were mixed. The mixture was quantified on a QuantStudio 5 Real-Time PCR System (Thermo Fisher Scientific), using a standard qPCR amplification and dissociation program. The 2 ΔΔCT value was calculated and normalized to that of the corresponding DMSO control. Western blot Cells were first cultivated in 6 well (4x10 4 cells/well) for 24 h and treated with inhibitors or DMSO control for 72 h. To detect wild-type TP53 protein expression in IDH1 mut iCCA cells, cells were pre-treated with proteasome inhibitor MG-132 (MedChemExpress, HY-13259) 15 µM for 2 h before harvest. Cells were lysed in CelLytic TM M Cell Lysis Reagent containing a protease inhibitor cocktail (Abcam). The Lysates were purified by centrifugation at 16,000 × g for 15 min at 4 °C. The supernatants were collected and quantified using the Bradford protein assay (Bio-Rad). The Lysates were mixed with 4x loading dye buffer and heated at 95°C for 15 min. Protein lysates were loaded onto SDS-PAGE gels. The gels were run at 80 mV for 2 h and then transferred overnight at 30 mV. The membranes were sequentially blocked with 5% non-fat milk/TBST buffer, primary antibodies, and secondary antibodies. Chemiluminescence was activated by enhanced chemiluminescence (Perkin Elmer) and detected by exposure to X-ray film. Primary antibodies: p53 (7F5) Rabbit mAb (Cell Signaling Technology, 2527, 1:1000), Mic-1 (D2A3) Rabbit mAb (Cell Signaling Technology, 8479, 1:1000), MDM2 antibody [SMP14] (GeneTex, GTX70278, 1:1000) and Anti-Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) Antibody clone 6C5 (Millipore, MAB374, 1:10000). Secondary antibodies: Peroxidase AffiniPure™ Goat Anti-Rabbit IgG (H+L) (Jackson ImmunoResearch Inc., 111-007-003, 1:5000) and AffiniPure™ Fab Fragment Goat Anti-Mouse IgG (H+L) (Jackson ImmunoResearch Inc., 115-007-003, 1:5000). Nuclear protein extraction and KDM5 activity measurement To detect KDM5 activity, we performed nuclear extraction using a nuclear extraction kit (Abcam, ab113474) and KDM5 activity quantification assay kit (Abcam, ab113464), following the manufacturer's protocol. Cells were seeded in 10 cm dishes (5x10 5 cells per dish) for 24 h and treated with either mutant IDH1 inhibitors or DMSO control for 48 h. The cells were washed with ice-cold PBS and cell pellets were collected by scraping and centrifuging. Cell pellets were lysated in 1X Pre-Extraction Buffer containing 0.1% DTT and 0.1% 100x protease inhibitor cocktail, vortexed, and centrifuged to collect the nuclear pellets. Nuclear pellets were lysed in Extraction Buffer containing 0.1% DTT and 0.1% 100x protease inhibitor cocktail, vortexed, and centrifuged to collect nuclear protein. The Nuclear extracts were collected, quantified, and stored at -80°C until KDM5 activity measurement. For KDM5 activity measurement, 10 µg of purified nuclear extract was mixed with reaction buffer containing completed assay buffer and substrate, incubated at 37°C for 90 min, and washed with wash buffer. Capture antibody was added to the reaction wells, covered with Parafilm M, and incubated at room temperature for 1 h. After incubation, the capture antibody solution was removed, and the cells were washed with wash buffer. Detection antibody was added to the reaction wells, covered with Parafilm M, and incubated at room temperature for 30 min. After incubation, the detection antibody solution was removed, and the cells were washed with wash buffer. Fluoro developer was then added to the reaction wells and incubated at room temperature for 4 min in the dark. The fluorescence signal was measured at λ ex = 530 nm and λ em = 590 nm, using a microplate reader. Native chromatin immunoprecipitation Cells were seeded in 10 cm dishes (5x10 5 cells per dish) for 24 h and then treated with either mutant IDH1 inhibitors or DMSO control for 48 h. The cells were washed with ice-cold PBS, scraped, and centrifuged to collect cell pellets. The cell pellet was resuspended in CelLytic TM M Cell Lysis Reagent containing 1 mM CaCl2, 0.2% Triton X-100, and 50 mM Tris-HCl (pH 7.6) on ice for 15 min, homogenized by passing through a 27G needle, and sonicated to shear the chromatin. 5 µL of DNA fragment were used for agarose gel analysis to verify the sonication results. The sheared chromatin supernatants were mixed with IgG control or the indicated antibodies at 4°C overnight on a rotary mixer. Protein A Mag Sepharose Xtra (Cytiva) were then added to the complex solution and incubated at 4°C for 4 h on a rotary mixer. The complex solution was placed on a magnetic stand and the supernatant was removed. The magnetic beads were washed on a rotary mixer with the following buffers and times: RIPA buffer twice, RIPA buffer + 0.3M NaCl twice, LiCl buffer twice, TE buffer + 0.2% Triton X-100 twice, and TE buffer once. The Magnetic beads were resuspended in 10% SDS/proteinase K/TE buffer and incubated overnight at 65°C. The samples were vortexed briefly, placed on a magnetic stand, and the supernatants were collected. The magnetic beads were washed with 0.5M NaCl / TE buffer, placed on a magnetic stand, and the supernatants were collected. The collected supernatants were combined with the collected solutions and stored at -20°C until DNA purification. Primary antibodies: Mono-Methyl-Histone H3 (Lys4) (D1A9) XP® Rabbit mAb (Cell Signaling Technology, 5326, 1:50), Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb (Cell Signaling Technology, 9751, 1:50) and Rabbit (DA1E) mAb IgG XP® Isotype Control (Cell Signaling Technology, 3900, 1:50). DNA fragments purification DNA fragments were purified from the collected solutions using a GenepHlow™ Gel/PCR Kit (Geneaid, DFH300), according to the manufacturer's instructions. The collected solutions were mixed with gel/PCR buffer, transferred to collection tubes, and centrifuged at 16,000 g for 30 s. The collection tubes were washed with ethanol/wash buffer and centrifuged at 16,000 g for 30 s. The remaining ethanol was removed by centrifuging the collection tubes at 16,000 × g for 3 min. The purified DNA was eluted in 70°C-heated elution buffer and stored at -20°C until Real-time PCR. Cell cycle analysis Cells were seeded in 10 cm dishes (5 × 10 5 cells per dish) for 24 h and then treated with MDM2 inhibitors or DMSO control for one doubling time. The cells were harvested and washed with 2% fetal bovine serum/PBS. The cells were fixed in 70% ethanol at 4°C for one hour, then stored at -20°C overnight. The fixed cells were washed with cold PBS and stained with propidium iodide / RNase / TritonX-100 solution. FL-2 intensity was detected using BD FACSCalibur Flow Cytometer (BD Biosciences) and analyzed using ModFit LT (Verity Software House). Proliferation assay Cells were seeded in 24-well plates (3x10 3 cells/well). For single-drug treatment, increasing doses of mutant IDH1 inhibitors, MDM2 inhibitors, or DMSO controls were added. For combined treatment, mutant IDH1 and MDM2 inhibitors were added at different ratios. Cells were harvested at three doubling times for each cell line. The cells were stained with 0.5% methylene blue ethanol solution for 2 h, washed with flowing tap water, and air-dried overnight. Methylene blue-stained cells were dissolved in 1% sarkosyl/PBS buffer and the absorbance was read at 595 nm using a microplate reader. The data were normalized to those of DMSO controls. The combined index was calculated by adjusting the effective concentration of each drug combination. The data were analyzed and visualized using SynergyFinder 3.0 (https://synergyfinder.fimm.fi/) [22]. Reagents and chemicals The reagents and chemicals used in this study: dimethyl sulfoxide (DMSO) (Sigma-Aldrich, D2650), ivosidenib (Synonyms: AG-120) (MedChemExpress, HY-18767), IDH-305 (MedChemExpress, HY-104036), idasanutlin (Synonyms: RG7388) (MedChemExpress, HY-15676), siremadlin (Synonyms: NVP-HDM201; HDM201) (MedChemExpress, HY-18658), KDOAM-25 citrate (MedChemExpress, HY-102047B), and MG-132 (Synonyms: Z-Leu-Leu-Leu-al; MG132) (MedChemExpress, HY-13259). Statistical Analysis RStudio IDE (RStudio), R Project for Statistical Computing version 4.2, and the R packages ggplot2 and ggsignif were used to analyze and visualize the data [23-25]. Paired t-tests and Wilcoxon signed-rank tests were used to compare gene expression data. Data availability The data were generated by the first author and are available upon request from the corresponding author. The data generated in this study are publicly available in the following online datasets and studies: Mutation profiling datasets used in this study were downloaded from cBioPortal (https://www.cbioportal.org), Genomic Data Commons Data Portal (GDC) (https://portal.gdc.cancer.gov/) and published studies [19-21, 26, 27]. Datasets from cBioPortal : Cholangiocarcinoma (ICGC, Cancer Discov 2017), Cholangiocarcinoma (National Cancer Centre of Singapore, Nat Genet 2013), Intrahepatic Cholangiocarcinoma (JHU, Nat Genet 2013), Intrahepatic Cholangiocarcinoma (Mount Sinai 2015), Intrahepatic Cholangiocarcinoma (Shanghai, Nat Commun 2014) and Intrahepatic Cholangiocarcinoma (MSK, Hepatology 2021) [28-33]. Dataset from GDC: TCGA-CHOL cohort. Datasets from published studies: doi:10.1038/s41416-022-01932-1, doi:10.1038/s41598-022-22543-z, doi:10.1158/2159-8290.CD-17-0368, and doi:10.1158/2159-8290.CD-20-0766 [28, 34-36]. Results Mutations in IDH1/2 and TP53 are mutually exclusive in human iCCA tissues and cell lines To identify the other potential therapeutic targets in iCCA with IDH1/2 mutations, we first reviewed the comprehensive genomic profiling of BTC, in which IDH1/2 mutations were only identified in iCCA and mutually exclusive with other major genetic alterations, i.e. TP53 , KRAS and SMAD4 mutations, actionable FGFR2 fusion and BRAF V600E mutation, and ERBB2 and MDM2 amplifications [37, 38]. With the emergence of wtTP53 reactivation therapy, we examined the mutual exclusion of IDH1/2 mut and TP53 alterations. Of the 1031 iCCA that were included from 7 studies in cBioPortal database, IDH1/2 mutation was detected in 146 (14.1%) (Fig. 1A) [19-21]. TP53 mutation rate was 6.1% (9/146) in IDH1/2 mut and 29.9% (265/885) in IDH1/2 wt tumors, with an odds ratio (OR) of 0.15 (95% confidence interval, 0.08-0.31), p < 0.0001 (Fisher's exact test). Trends of mutual exclusiveness between IDH1/2 and TP53 mutations of individual studies with case number more than 100, three within cBioPortal and four other recent publications were shown in Fig. 1B. Interestingly, both iCCA cell lines with pathogenic IDH1 mut (SNU1079 and REB) in Cancer Cell Encyclopedia were TP53 wt further supports our observation (Supplementary Fig. 1A) [39]. Mutant IDH1 upregulates the expression of ubiquitin ligase MDM2 at transcription level To elucidate the functions and utilities of molecules and signaling pathways involved in IDH1 mut /TP53 wt iCCA, we compared the expression profiles of IDH1 mut /TP53 wt and IDH1 wt /TP53 mut iCCA cells from the Cancer Cell Line Encyclopedia (CCLE) mRNA dataset and found upregulation of the E3 ubiquitin ligase MDM2, which ubiquitinates and negatively modulates TP53 protein expression [39] (Supplementary Fig. 2A). The augmentation of MDM2 in IDH1 mut iCCA cells was confirmed in iCCA tissues and cell lines (Fig. 2A, 2B and 2C). Real time-qPCR and western blotting confirmed the increases in MDM2 mRNA and protein levels in both IDH1 mut iCCA cell lines (SNU1079 and RBE) (Fig. 2B and 2C). To validate the association between mIDH1 and MDM2 expression, IDH1 mut iCCA cells were treated with two mIDH1 specific inhibitors, AG120 and IDH305. Pharmacological inhibition of mIDH1 led to a reduction in intracellular 2-HG levels, accompanied by a decrease in MDM2 mRNA and protein expression (Fig. 2D, 2E and 2F). As expected, attenuation of MDM2 was accompanied by an increase in wtTP53 protein levels (Fig. 2F). In contrast, mIDH1 inhibitor treatment did not alter MDM2 and mTP53 expression in IDH1 wt iCCA cell lines (HuH28, HuCCT1, and SSP25) (Supplementary Fig. 2B and 2C). These data suggested that 2-HG upregulates the expression of MDM2 at the transcriptional level in IDH1 mut iCCA cells. Mutant IDH1 suppresses KDM5 activity and enhances H3K4 methylation level on the MDM2 promoter region Because 2-HG can inhibit the activity of lysine-specific histone demethylases (KDMs), a group of α-KG-dependent dioxygenases that modulate histone lysine methylation and gene transcription [40, 41]. We searched the histone chromatin immunoprecipitation (ChIP) sequencing data from ENCODE on the UCSC genome browser to explore the molecular mechanisms underlying the epigenetic modulation of MDM2 transcription in IDH1 mut iCCA cells [42-44]. The histone-3-lysine-4 tri-methylation (H3K4me3), an active histone code, was highly enriched in three different regions near the promoter region and transcription start site (TSS) of MDM2 in pan-cell types (Fig. 3A). ChIP-qPCR was performed to detect H3K4 mono-methylation (H3K4me1) and H3K4me3 in three H3K4me3-enriched promoter and TSS regions of MDM2 in IDH1 mut and IDH1 wt iCCA cells. Among them, the methylation score (H3K4me3/H3K4me1 ratio) of region 2 was the highest in the two IDH1 mut iCCA cells compared to regions 1 and 3 within the same cells or region 2 of the IDH1 wt iCCA cells (Fig. 3B). AG120 and IDH305 treatment profoundly reduced the H3K4 methylation level and H3K4me3/H3K4me1 ratio in region 2 of both IDH1 mut iCCA cells (Fig. 3C). As the KDM5 family (KDM5A–5D) specifically recognizes and demethylates H3K4me3 via α-KG-dependent oxidation, they can be antagonized by 2-HG [40]. We hypothesized that high H3K4 methylation at the MDM2 promoter in IDH1 mut iCCA cells is caused by low KDM5 activity. We measured total KDM5 activity in the nuclear extract of iCCA cell lines and found a significant decrease in IDH1 mut iCCA cells compared to IDH1 wt iCCA cells (Fig. 4A). Treatment with AG120 and IDH305 enhanced total KDM5 activity and reduced H3K4me3 level, which was accompanied by a reduction in MDM2 mRNA expression in IDH1 mut iCCA cells (Fig. 4B - 4E). Co-treatment with KDOAM-25 citrate, a pan-KDM5 inhibitor, restored H3K4me3 and MDM2 levels in AG120- and IDH305-treated IDH1 mut iCCA cells (Fig. 4C - 4E). These results suggested that mIDH1-derived 2-HG enhances H3K4me3 levels at the MDM2 promoter by suppressing KDM5 activity. MDM2 inhibitors restore wild-type TP53 function and suppress the growth of IDH1 mut iCCA cells We demonstrated that IDH1 and TP53 mutations are mutually exclusive in iCCAs and mutant IDH1 enhance MDM2 expression through the modulation of KDM5 activity by 2-HG in IDH1 mut iCCA cells. These findings suggest IDH1 mut iCCA is a potential candidate for MDM2 inhibitor treatment, which is under extensive clinical evaluation for MDM2 -amplified TP53 wt tumors, including BTCs [45, 46]. We treated iCCA cells with different concentrations of MDM2 inhibitors (RG7388 and HDM201) or mIDH1 inhibitors (AG120 and IDH305) for three doubling times and assessed the cell proliferation. Consistent with previous studies and clinical trials, mIDH1 inhibitors had little growth inhibitory effect on iCCA cell lines. However, IDH1 mut /TP 53 iCCA cells were more susceptible to MDM2 inhibitors than IDH1 wt / TP53 mut iCCA cells (Fig. 5A). To clarify the mechanism by which MDM2 inhibitors affect IDH1 mut iCCA cells, we treated SNU1079 and RBE cells with RG7388 and HDM201 for one doubling time and harvested the cells for analysis. Both inhibitors disrupted the interaction between MDM2 and TP53, inducing the accumulation of these two proteins in IDH1 mut / TP53 wt iCCA cells (Fig. 5B). In addition, the expression of macrophage inhibitory cytokine-1 (MIC-1), a TP53 target gene, was also increased [47] (Fig. 5B). This led to cell cycle arrest in the G1 phase and apoptosis in the SNU1079 and RBE cells (Fig. 5C and 5D). Conversely, treatment with the MDM2 inhibitor did not significantly inhibit the growth or accumulation of MDM2 and mTP53 proteins in IDH1 wt /TP53 mut iCCA cells under the same experimental conditions (Fig. 5A and 5B). As mIDH1 inhibitors suppressed MDM2 expression, it would be interesting to explore the effect of MDM2 and mIDH1 inhibitor combination treatment in IDH1 mut / TP53 wt iCCA cells. We co-treated iCCA cell lines with various combinations of MDM2 and mIDH1 inhibitors and calculated the combination index (CI) value. The CI estimation indicated that the MDM2 and mIDH1 inhibitors showed a synergistic effect (CI < 1) in suppressing the growth of IDH1 mut / TP53 wt iCCA cell lines (Fig. 6A and 6B). These results suggest that MDM2 is a therapeutic target in IDH1 mut / TP53 wt iCCA cells. Discussion In recent decades, large-scale next-generation sequencing has contributed to the discovery of pathogenic somatic mutations in the cancer genomes. Comprehensive mutation profiling provides a new perspective for studying interactions between gene alterations. Our earlier iCCA genomic profiling datasets analyses revealed mutual exclusion of IDH1/2 and TP53 mutations, and CCLE mRNA dataset analysis showed IDH1 mut /TP53 wt cells expressing higher level of MDM2 than IDH1 wt /TP53 mut cells. Our laboratory works further demonstrated both IDH1 mut /TP53 wt iCCA (SNU1079 ad RBE) cell lines expressing higher MDM2 protein levels than IDH1 wt /TP53 mut iCCA cells. Mutant IDH1 inhibitors treatment reduced the intracellular level of 2-HG that was accompanied with a reduction of MDM2 mRNA and protein expression, and enhancing wTP53 protein expression in both IDH1 mut /TP53 wt iCCA cells. Since MDM2 is a known TP53 negative regulator that binds TP53 to facilitate its degradation, we further explored the molecular mechanisms underlying the modulation of MDM2 transcription by 2-HG, which is a mutant IDH1/2-derived oncometabolite known to reprogram gene expression and cellular metabolism through suppressing α-KGD activity. It has been reported that KDM5 histone lysine demethylase as a target enzyme of 2-HG [25], and KDM5A as a negative regulator of TP53 expression via the modulation of protein translation genes [36,37]. Our study showed that KDM5 activity was lower and the level of H3K4me3 in part of the promoter region of MDM2 was higher in IDH1 mut iCCA cells than IDH1 wt iCCA cells (Figure). All the events, reduced KDM5 activity, increased H3K4me3 in MDM2 promoter region, enhanced MDM2 expression and reduced wtTP53 expression in IDH1 mut iCCA cells could be partially reversed by mutant IDH1 inhibitors treatment. These findings indicate wtTP53 inactivation plays a role in the carcinogenesis of IDH1/2 mut iCCA. In addition, combining the findings of mutual exclusion of IDH1/2 and TP53 mutations, and the enhanced MDM2 expression in IDH1 mut iCCA tissues and cells also provide a rationale to evaluate the potential use of MDM2 inhibitor in this subgroup of patients. MDM2 is an E3 ubiquitin ligase that directly targets wtTP53 to promote TP53 degradation through monoubiquitination, polyubiquitination, NEDD8 NEDDylation, and SUMO-1 modification [48]. These post-translational modifications govern TP53 cellular localization, activity, downstream target gene selection, and protein stability. Based on structural studies of the MDM2-TP53 binding motif (Phe19, Trp23, and Leu26), small-molecule inhibitors targeting MDM2 were designed and developed [49, 50]. Currently, targeted population for MDM2 inhibitors therapy in solid tumors mainly focused on those with frequent MDM2 amplified/TP53 wt genotype, and encouraging therapeutic efficacy has been observed in patients with advanced biliary tract cancers [51]. In this study, we used two MDM2 inhibitors that had undergone clinical trials to test the cytotoxic effects of IDH1 mut iCCA [52, 53]. These two MDM2 inhibitors showed more effective cytotoxicity and wtTP53 reactivation than mIDH1 inhibitor in IDH1 mut iCCA cells. The finding is clinical relevant because it provides a rationale to expand the potential targeted iCCA population for MDM2 inhibitor treatment. According to cBioportal dataset, MDM2 amplification and IDH1/2 mutations was detected in 3.5% (15/430) and 25.6% (109/430) of iCCA tissue, respectively. Of them, only two tumors had co-occurrence of IDH1 mutation and MDM2 amplification . Furthermore, MDM2 inhibitor may overcome the mutant IDH1 inhibitor resistance IDH1 mut iCCA with acquired IDH2 mutation [15]. Moreover, co-treatment with mIDH1 and MDM2 inhibitors synergistically suppressed the proliferation of IDH1 mut iCCA cells. Although mIDH1 inhibitors did not directly target wtTP53 or MDM2, indirect epigenetic regulation of MDM2 still provides a new therapeutic strategy for wtTP53 reactivation. Since one of the most important obstacles for the development of MDM2 in cancer therapy is dose-dependent hematological toxicity, notably thrombocytopenia [54]. The finding of synergism between IDH1 inhibitor and MDM2 inhibitors provide a potential strategy of combination therapy aiming to reduce dose-dependent adverse events of MDM2 inhibitor without compromising therapeutic efficacy in IDH1 mut /TP53 wt iCCA. Moreover, this mutually exclusive phenomenon was also observed in IDH1/2 mut acute myeloid leukemia [51], revealing that MDM2 inhibitors or other agents that reactivate wtTP53 are a potential treatment strategy for IDH1/2 mut AML (Supplementary Fig. 3A). 2-HG inhibits the activity of KDM4B, which increases the level of H3K9 trimethylation and affects global gene expression. Although aberrant H3K9 hypermethylation did not significantly affect the expression of homology-dependent repair (HDR) genes, however, its presence at loci near DNA strand breaks could block the activation signals for HDR execution and impair the recruitment of HDR factors [41, 55, 56]. The findings suggested the IDH1 mut cancers were associated with HDR deficiency and likely synthetic lethal with PARP1 inhibitor treatment [55, 57]. Conclusions In summary, we confirmed that mutant IDH1-derived 2HG enhances MDM2 transcription by decreasing KDM5 activity and enriching H3K4me3 at the MDM2 promoter region. Increased MDM2 promotes the degradation of the wild-type TP53 protein in IDH1 mut iCCA, and pharmacological inhibition of MDM2 reduces wild-type TP53 degradation and reactivates wild-type TP53-triggered growth inhibition and cell death in IDH1 mut iCCA. In conclusion, we identified a novel mIDH1-MDM2-wtTP53 axis that mIDH1 indirectly regulates wtTP53 protein expression through epigenetic upregulation of MDM2 transcription. This renders a wtTP53 reactivation therapeutic option for IDH1 mut iCCA. Abbreviations D-2-HG: D-2-hydroxyglutarate; α-KG: Alpha-ketoglutarate; α-KGDs: Alpha-KG-dependent dioxygenases; CCA: Cholangiocarcinoma; cDNA: Complementary DNA; ChIP: Chromatin immunoprecipitation; H3K4: Histone 3 lysine 4; H3K4me1: Histone 3 lysine 4 mono-methylation; H3K4me3: Histone 3 lysine 4 tri-methylation; H-score: Histochemical scoring assessment; iCCA: Intrahepatic cholangiocarcinoma; IDH1: Isocitrate dehydrogenases 1 ; IDH1 mut : IDH1 mutation ; IDH1 wt : IDH1 wild-type ; KDMs: Histone lysine demethylases; KEGG: Kyoto encyclopedia of genes and genomes; MDM2: Mouse double minute 2 homolog; mIDH1: Mutant IDH1; mTP53: Mutant TP53; OR: Odds ratio; TP53: Tumor protein 53; TSS: Transcription start site; wtIDH1: Wild-type IDH1; wtTP53: Wild-type TP53 Declarations Acknowledgments The authors would like to thank Dr. Chun-Nan Yeh, Chang Gung University, Taoyuan, Taiwan, for supporting the SSP25 cell line and Dr. Daw-Yang Hwang, National Institute of Cancer Research, National Health Research Institutes, Taiwan, for providing the M220 Focused-ultrasonicator (Covaris). Funding This work was supported by National Institute of Cancer Research, National Health Research Institutes, Taiwan (CA-111~112-PP-20 and CA-113-PP-17), and National Science and Technology Council, Executive Yuan, Taiwan, to the Center for Cancer Research, Kaohsiung Medical University, Taiwan (112-2321-B-037-004). Authors’ Contributions WCH and LTC initiated and designed this study. CTL performed the experiments, analyzed the data, and wrote the manuscript. YHH helps with the experiments design. YYS and NJC helped with the acquisition of clinical samples. YYS performed IHC staining. CFL , YCM , and KCC interpreted IHC results. YHH, YYS , WCH and LTC reviewed and revised the manuscript. WCH and LTC provided conceptual insights. Data availability The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. Ethics approval and consent to participate IRB was approved by the Institutional Review Board of the National Cheng Kung University Hospital (NCKUH, IRB A-ER-111-019). Consent for publication No applicable Competing interests The authors declare no competing interests. Author details 1 National Institute of Cancer Research, National Health Research Institutes, Tainan, Taiwan, 2 Department of Oncology, National Cheng Kung University Hospital, and Institute of Clinical Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan, 3 Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan, 4 Center for Cancer Research, Kaohsiung Medical University, Kaohsiung, Taiwan 5 Department of Oncology, Taipei Veterans General Hospital, Taipei, Taiwan, 6 School of Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan, 7 Clinical Pathology Department, Chi Mei Medical Center, Tainan, Taiwan, 8 Department of Pathology, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan, 9 Department of Pathology, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan, Taiwan, 10 Department of Biological Science and Technology, National Yang Ming Chiao Tung University, Hsinchu, Taiwan, 11 Department of Medical Research, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan, 12 Department of Biological Science and Technology, National Yang Ming Chiao Tung University, Hsinchu, Taiwan References Banales JM, Marin JJG, Lamarca A, Rodrigues PM, Khan SA, Roberts LR et al. Cholangiocarcinoma 2020: the next horizon in mechanisms and management. Nat Rev Gastroenterol Hepatol 2020; 17: 557-588. Kendall T, Verheij J, Gaudio E, Evert M, Guido M, Goeppert B et al. Anatomical, histomorphological and molecular classification of cholangiocarcinoma. Liver Int 2019; 39 Suppl 1: 7-18. Lendvai G, Szekerczes T, Illyes I, Dora R, Kontsek E, Gogl A et al. Cholangiocarcinoma: Classification, Histopathology and Molecular Carcinogenesis. Pathol Oncol Res 2020; 26: 3-15. SEER*Explorer: An interactive website for SEER cancer statistics. [cited 2024 Feb 19]. Available from: https://seer.cancer.gov/statistics-network/explorer/ Marin JJG, Prete MG, Lamarca A, Tavolari S, Landa-Magdalena A, Brandi G et al. Current and novel therapeutic opportunities for systemic therapy in biliary cancer. Br J Cancer 2020; 123: 1047-1059. Valle JW, Kelley RK, Nervi B, Oh DY, Zhu AX. Biliary tract cancer. Lancet 2021; 397: 428-444. Cao J, Hu J, Liu S, Meric-Bernstam F, Abdel-Wahab R, Xu J et al. Intrahepatic Cholangiocarcinoma: Genomic Heterogeneity Between Eastern and Western Patients. JCO Precis Oncol 2020; 4. Pirozzi CJ, Yan H. The implications of IDH mutations for cancer development and therapy. Nat Rev Clin Oncol 2021; 18: 645-661. Dang L, White DW, Gross S, Bennett BD, Bittinger MA, Driggers EM et al. Cancer-associated IDH1 mutations produce 2-hydroxyglutarate. Nature 2010; 465: 966. Ye D, Guan KL, Xiong Y. Metabolism, Activity, and Targeting of D- and L-2-Hydroxyglutarates. Trends Cancer 2018; 4: 151-165. Tian W, Zhang W, Wang Y, Jin R, Wang Y, Guo H et al. Recent advances of IDH1 mutant inhibitor in cancer therapy. Front Pharmacol 2022; 13: 982424. Fan B, Abou-Alfa GK, Zhu AX, Pandya SS, Jia H, Yin F et al. Pharmacokinetics/pharmacodynamics of ivosidenib in advanced IDH1-mutant cholangiocarcinoma: findings from the phase III ClarIDHy study. Cancer Chemother Pharmacol 2024; 93: 471-479. Zhu AX, Macarulla T, Javle MM, Kelley RK, Lubner SJ, Adeva J et al. Final Overall Survival Efficacy Results of Ivosidenib for Patients With Advanced Cholangiocarcinoma With IDH1 Mutation: The Phase 3 Randomized Clinical ClarIDHy Trial. JAMA Oncol 2021; 7: 1669-1677. Abou-Alfa GK, Macarulla T, Javle MM, Kelley RK, Lubner SJ, Adeva J et al. Ivosidenib in IDH1-mutant, chemotherapy-refractory cholangiocarcinoma (ClarIDHy): a multicentre, randomised, double-blind, placebo-controlled, phase 3 study. Lancet Oncol 2020; 21: 796-807. Cleary JM, Rouaisnel B, Daina A, Raghavan S, Roller LA, Huffman BM et al. Secondary IDH1 resistance mutations and oncogenic IDH2 mutations cause acquired resistance to ivosidenib in cholangiocarcinoma. NPJ Precis Oncol 2022; 6: 61. Harding JJ, Lowery MA, Shih AH, Schvartzman JM, Hou S, Famulare C et al. Isoform Switching as a Mechanism of Acquired Resistance to Mutant Isocitrate Dehydrogenase Inhibition. Cancer Discov 2018; 8: 1540-1547. Intlekofer AM, Shih AH, Wang B, Nazir A, Rustenburg AS, Albanese SK et al. Acquired resistance to IDH inhibition through trans or cis dimer-interface mutations. Nature 2018; 559: 125-129. Lyu J, Liu Y, Gong L, Chen M, Madanat YF, Zhang Y et al. Disabling Uncompetitive Inhibition of Oncogenic IDH Mutations Drives Acquired Resistance. Cancer Discov 2023; 13: 170-193. de Bruijn I, Kundra R, Mastrogiacomo B, Tran TN, Sikina L, Mazor T et al. Analysis and Visualization of Longitudinal Genomic and Clinical Data from the AACR Project GENIE Biopharma Collaborative in cBioPortal. Cancer Res 2023; 83: 3861-3867. Gao J, Aksoy BA, Dogrusoz U, Dresdner G, Gross B, Sumer SO et al. Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal. Sci Signal 2013; 6: pl1. Cerami E, Gao J, Dogrusoz U, Gross BE, Sumer SO, Aksoy BA et al. The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data. Cancer Discov 2012; 2: 401-404. Ianevski A, Giri AK, Aittokallio T. SynergyFinder 3.0: an interactive analysis and consensus interpretation of multi-drug synergies across multiple samples. Nucleic Acids Res 2022; 50: W739-W743. Wickham H. ggplot2: Elegant Graphics for Data Analysis. Springer-Verlag New York, 2016. Constantin A PI. ggsignif: R Package for Displaying SignificanceBrackets for ‘ggplot2’. PsyArxiv 2021. Team RC. R: A Language and Environment for Statistical Computing, 2024. Grossman RL, Heath AP, Ferretti V, Varmus HE, Lowy DR, Kibbe WA et al. Toward a Shared Vision for Cancer Genomic Data. N Engl J Med 2016; 375: 1109-1112. Cancer Genome Atlas Research N, Weinstein JN, Collisson EA, Mills GB, Shaw KR, Ozenberger BA et al. The Cancer Genome Atlas Pan-Cancer analysis project. Nat Genet 2013; 45: 1113-1120. Jusakul A, Cutcutache I, Yong CH, Lim JQ, Huang MN, Padmanabhan N et al. Whole-Genome and Epigenomic Landscapes of Etiologically Distinct Subtypes of Cholangiocarcinoma. Cancer Discov 2017; 7: 1116-1135. Chan-On W, Nairismagi ML, Ong CK, Lim WK, Dima S, Pairojkul C et al. Exome sequencing identifies distinct mutational patterns in liver fluke-related and non-infection-related bile duct cancers. Nat Genet 2013; 45: 1474-1478. Jiao Y, Pawlik TM, Anders RA, Selaru FM, Streppel MM, Lucas DJ et al. Exome sequencing identifies frequent inactivating mutations in BAP1, ARID1A and PBRM1 in intrahepatic cholangiocarcinomas. Nat Genet 2013; 45: 1470-1473. Sia D, Losic B, Moeini A, Cabellos L, Hao K, Revill K et al. Massive parallel sequencing uncovers actionable FGFR2-PPHLN1 fusion and ARAF mutations in intrahepatic cholangiocarcinoma. Nat Commun 2015; 6: 6087. Zou S, Li J, Zhou H, Frech C, Jiang X, Chu JS et al. Mutational landscape of intrahepatic cholangiocarcinoma. Nat Commun 2014; 5: 5696. Boerner T, Drill E, Pak LM, Nguyen B, Sigel CS, Doussot A et al. Genetic Determinants of Outcome in Intrahepatic Cholangiocarcinoma. Hepatology 2021; 74: 1429-1444. Tomczak A, Springfeld C, Dill MT, Chang DH, Kazdal D, Wagner U et al. Precision oncology for intrahepatic cholangiocarcinoma in clinical practice. Br J Cancer 2022; 127: 1701-1708. Rimini M, Fabregat-Franco C, Burgio V, Lonardi S, Niger M, Scartozzi M et al. Molecular profile and its clinical impact of IDH1 mutated versus IDH1 wild type intrahepatic cholangiocarcinoma. Sci Rep 2022; 12: 18775. Silverman IM, Hollebecque A, Friboulet L, Owens S, Newton RC, Zhen H et al. Clinicogenomic Analysis of FGFR2-Rearranged Cholangiocarcinoma Identifies Correlates of Response and Mechanisms of Resistance to Pemigatinib. Cancer Discov 2021; 11: 326-339. Nakamura H, Arai Y, Totoki Y, Shirota T, Elzawahry A, Kato M et al. Genomic spectra of biliary tract cancer. Nat Genet 2015; 47: 1003-1010. Kendre G, Murugesan K, Brummer T, Segatto O, Saborowski A, Vogel A. Charting co-mutation patterns associated with actionable drivers in intrahepatic cholangiocarcinoma. J Hepatol 2023; 78: 614-626. Ghandi M, Huang FW, Jane-Valbuena J, Kryukov GV, Lo CC, McDonald ER, 3rd et al. Next-generation characterization of the Cancer Cell Line Encyclopedia. Nature 2019; 569: 503-508. Gunn K, Myllykoski M, Cao JZ, Ahmed M, Huang B, Rouaisnel B et al. (R)-2-Hydroxyglutarate Inhibits KDM5 Histone Lysine Demethylases to Drive Transformation in IDH-Mutant Cancers. Cancer Discov 2023; 13: 1478-1497. Sulkowski PL, Oeck S, Dow J, Economos NG, Mirfakhraie L, Liu Y et al. Oncometabolites suppress DNA repair by disrupting local chromatin signalling. Nature 2020; 582: 586-591. Rosenbloom KR, Sloan CA, Malladi VS, Dreszer TR, Learned K, Kirkup VM et al. ENCODE data in the UCSC Genome Browser: year 5 update. Nucleic Acids Res 2013; 41: D56-63. Karolchik D, Hinrichs AS, Furey TS, Roskin KM, Sugnet CW, Haussler D et al. The UCSC Table Browser data retrieval tool. Nucleic Acids Res 2004; 32: D493-496. Raney BJ, Dreszer TR, Barber GP, Clawson H, Fujita PA, Wang T et al. Track data hubs enable visualization of user-defined genome-wide annotations on the UCSC Genome Browser. Bioinformatics 2014; 30: 1003-1005. Konopleva M, Martinelli G, Daver N, Papayannidis C, Wei A, Higgins B et al. MDM2 inhibition: an important step forward in cancer therapy. Leukemia 2020; 34: 2858-2874. Wang S, Zhao Y, Aguilar A, Bernard D, Yang CY. Targeting the MDM2-p53 Protein-Protein Interaction for New Cancer Therapy: Progress and Challenges. Cold Spring Harb Perspect Med 2017; 7. Osada M, Park HL, Park MJ, Liu JW, Wu G, Trink B et al. A p53-type response element in the GDF15 promoter confers high specificity for p53 activation. Biochem Biophys Res Commun 2007; 354: 913-918. Wen J, Wang D. Deciphering the PTM codes of the tumor suppressor p53. J Mol Cell Biol 2022; 13: 774-785. Zhu H, Gao H, Ji Y, Zhou Q, Du Z, Tian L et al. Targeting p53-MDM2 interaction by small-molecule inhibitors: learning from MDM2 inhibitors in clinical trials. J Hematol Oncol 2022; 15: 91. Wang S, Chen FE. Small-molecule MDM2 inhibitors in clinical trials for cancer therapy. Eur J Med Chem 2022; 236: 114334. Yamamoto N, Tolcher A, Hafez N, Lugowska I, Ramlau R, Macarulla T et al. Efficacy and Safety of the MDM2-p53 Antagonist Brigimadlin (BI 907828) in Patients with Advanced Biliary Tract Cancer: A Case Series. Onco Targets Ther 2024; 17: 267-280. Stein EM, DeAngelo DJ, Chromik J, Chatterjee M, Bauer S, Lin CC et al. Results from a First-in-Human Phase I Study of Siremadlin (HDM201) in Patients with Advanced Wild-Type TP53 Solid Tumors and Acute Leukemia. Clin Cancer Res 2022; 28: 870-881. Blotner S, Chen LC, Ferlini C, Zhi J. Phase 1 summary of plasma concentration-QTc analysis for idasanutlin, an MDM2 antagonist, in patients with advanced solid tumors and AML. Cancer Chemother Pharmacol 2018; 81: 597-607. Iancu-Rubin C, Mosoyan G, Glenn K, Gordon RE, Nichols GL, Hoffman R. Activation of p53 by the MDM2 inhibitor RG7112 impairs thrombopoiesis. Exp Hematol 2014; 42: 137-145 e135. Sulkowski PL, Corso CD, Robinson ND, Scanlon SE, Purshouse KR, Bai H et al. 2-Hydroxyglutarate produced by neomorphic IDH mutations suppresses homologous recombination and induces PARP inhibitor sensitivity. Sci Transl Med 2017; 9. Sun Y, Jiang X, Xu Y, Ayrapetov MK, Moreau LA, Whetstine JR et al. Histone H3 methylation links DNA damage detection to activation of the tumour suppressor Tip60. Nat Cell Biol 2009; 11: 1376-1382. Wang Y, Wild AT, Turcan S, Wu WH, Sigel C, Klimstra DS et al. Targeting therapeutic vulnerabilities with PARP inhibition and radiation in IDH-mutant gliomas and cholangiocarcinomas. Sci Adv 2020; 6: eaaz3221. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6489065","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":463998734,"identity":"ddf14e52-d8e3-451d-aee1-c47e64576ca6","order_by":0,"name":"Chien-Tsung Liu","email":"","orcid":"","institution":"National Health Research Institutes","correspondingAuthor":false,"prefix":"","firstName":"Chien-Tsung","middleName":"","lastName":"Liu","suffix":""},{"id":463998736,"identity":"0c3bcd60-dd8b-4948-8aa4-cbd7427089fd","order_by":1,"name":"Yung-Yeh Su","email":"","orcid":"","institution":"National Health Research Institutes","correspondingAuthor":false,"prefix":"","firstName":"Yung-Yeh","middleName":"","lastName":"Su","suffix":""},{"id":463998740,"identity":"d1c879ca-5004-4773-9b06-9777df5eae70","order_by":2,"name":"Nai-Jung Chiang","email":"","orcid":"","institution":"National Health Research Institutes","correspondingAuthor":false,"prefix":"","firstName":"Nai-Jung","middleName":"","lastName":"Chiang","suffix":""},{"id":463998743,"identity":"89061a63-dbb8-4f33-a26e-790c8127dc69","order_by":3,"name":"Chien-Feng Li","email":"","orcid":"","institution":"Chi Mei Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Chien-Feng","middleName":"","lastName":"Li","suffix":""},{"id":463998745,"identity":"284eb3de-508f-48d5-adab-bf78aeb7f2e6","order_by":4,"name":"Yu-Chun Ma","email":"","orcid":"","institution":"Kaohsiung Medical University Chung-Ho Memorial Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yu-Chun","middleName":"","lastName":"Ma","suffix":""},{"id":463998747,"identity":"b7bff55e-8ce0-4d57-9ff1-f9d8a5f24084","order_by":5,"name":"Kung-Chao Chang","email":"","orcid":"","institution":"National Cheng Kung University","correspondingAuthor":false,"prefix":"","firstName":"Kung-Chao","middleName":"","lastName":"Chang","suffix":""},{"id":463998748,"identity":"36dd39bd-cef7-4292-89cb-d50aef724015","order_by":6,"name":"Yu-Hsuan Hung","email":"","orcid":"","institution":"Kaohsiung Medical University Chung-Ho Memorial Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yu-Hsuan","middleName":"","lastName":"Hung","suffix":""},{"id":463998749,"identity":"8280527d-a2b4-4ebb-8532-c429a913fccd","order_by":7,"name":"Wen-Chun Hung","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4klEQVRIiWNgGAWjYBACAxCRUGEjx8/AA2QwMDA2EKXlwZk0Y8kGuBZmwloYH7YdTtxwgAcsQFiLuUTu4RcJbMzGxrd7j314wGAju+EA/zEJfFosZ+SlWSTwsMmZ3TmXPCOBIc14wwFmNrxaDG7kmBkkSPAYm93IMQb6BeRCZrYbhLUYSCRungHW8p8oLcYPEhIMEjdIgLUcIELLmTdmDAkHEowlwA4zSDaeeZjZ/AdeLcdzjD/+/Pdfjh/oMMYfFXayfccbHxvg0wIEyOEDUksgWkCA+QNhNaNgFIyCUTCiAQAiH0zF5IXQ0gAAAABJRU5ErkJggg==","orcid":"","institution":"National Health Research Institutes","correspondingAuthor":true,"prefix":"","firstName":"Wen-Chun","middleName":"","lastName":"Hung","suffix":""},{"id":463998750,"identity":"f5750274-86f1-4463-bd71-d4d228d46931","order_by":8,"name":"Li-Tzong Chen","email":"","orcid":"","institution":"Kaohsiung Medical University","correspondingAuthor":false,"prefix":"","firstName":"Li-Tzong","middleName":"","lastName":"Chen","suffix":""}],"badges":[],"createdAt":"2025-04-20 11:53:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6489065/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6489065/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":83815057,"identity":"d96ed37e-8d8f-4579-84be-60ac4a0b58b9","added_by":"auto","created_at":"2025-06-03 07:36:22","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":846451,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eIDH1/2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e mutations and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eTP53\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e mutation are mutually exclusive in iCCA samples.\u003cbr\u003e\n \u003c/strong\u003e(A) Genetic alteration of \u003cem\u003eIDH1\u003c/em\u003e, \u003cem\u003eIDH2\u003c/em\u003e, \u003cem\u003eTP53\u003c/em\u003e, \u003cem\u003eKRAS,\u003c/em\u003e \u003cem\u003eSMAD4\u003c/em\u003e, \u003cem\u003eFGFR2\u003c/em\u003e, \u003cem\u003eBRAF\u003c/em\u003e, \u003cem\u003eERBB2\u003c/em\u003e and \u003cem\u003eMDM2 \u003c/em\u003ein cBioportal iCCA datasets. Each column represented one individual mutation profiling data. Color in column represented different genetic alteration. Dark brown represented inframe mutation (putative driver), light brown represented inframe mutation (unknown significance), dark green represented missense mutation (putative driver), light green represented missense mutation (unknown significant), orange represented splice mutation (putative driver), black represented truncated mutation (putative driver), gray represented truncated mutation (unknown significance), purple represented structural variant (putative driver), red represented amplification, and blue represented deep deletion. (B) Odds ratio (OR) and 95% confidence interval (CI) of \u003cem\u003eIDH1/2\u003c/em\u003e and \u003cem\u003eTP53\u003c/em\u003emutations in seven iCCA mutation profiling datasets. \u003cem\u003eP\u003c/em\u003e value was evaluated by Fisher exact test.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6489065/v1/0c4a804ae6766f14a1e5264d.png"},{"id":83816016,"identity":"5ddca716-4d2f-4c78-b836-70bbc33281f6","added_by":"auto","created_at":"2025-06-03 07:44:22","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2280267,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMutant IDH1 upregulated \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMDM2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e mRNA and protein expression.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) iCCA tissue sections were stained with hematoxylin and eosin (H\u0026amp;E) or MDM2 IHC. The stained sections were divided into three groups: (1) \u003cem\u003eIDH1/2\u003c/em\u003e wild-type, (2) \u003cem\u003eIDH1/2 \u003c/em\u003emutation, and \u003cem\u003eIDH1/2\u003c/em\u003e wild-type, \u003cem\u003eMDM2/4\u003c/em\u003e amplification. Statistical significance was evaluated by Wilcoxon rank sum test (B) \u003cem\u003eMDM2\u003c/em\u003e mRNA expression in five iCCA cell lines. 2\u003csup\u003eΔΔCT\u003c/sup\u003e value was normalized to HuH28. (C) MDM2 and TP53 protein expression in five iCCA cell lines. GAPDH used as internal control. (D) Intracellular 2-HG level of two mutant \u003cem\u003eIDH1\u003c/em\u003e iCCA cell lines after DMSO or mutant IDH1 inhibitors treatment for 48 hours. Fluorescent values of 2-HG were normalized to DMSO control. (E) \u003cem\u003eMDM2\u003c/em\u003e mRNA expression of two mutant \u003cem\u003eIDH1\u003c/em\u003e iCCA cell lines after DMSO or mutant IDH1 inhibitors treatment for 48 hours. 2\u003csup\u003eΔΔCT\u003c/sup\u003e value was normalized to DMSO control. (F) MDM2 and TP53 protein expression in two \u003cem\u003eIDH1\u003c/em\u003e mutant iCCA cell lines after DMSO or mutant IDH1 inhibitors treatment for 72 hours. GAPDH used as internal control. MG-132 was used to stop proteasome degradation before harvest. \u003cem\u003eIDH1\u003c/em\u003e mutation iCCA cell lines, RBE and SNU1079. \u003cem\u003eIDH1\u003c/em\u003e wild-type iCCA cell lines, HuCCT1, HuH28 and SSP25. Mutant IDH1 inhibitors, AG-120 and IDH-305. All data were represented as average ± standard deviation. All experiments were performed and repeated in three independent biological replicates. Statistical significance was evaluated by paired t test: n.s. not significant.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6489065/v1/8e755946aec09f520c3dc3ea.png"},{"id":83815051,"identity":"142d1cae-b851-42b7-abb4-0a5baec0a632","added_by":"auto","created_at":"2025-06-03 07:36:22","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":827059,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMutant IDH1 enhanced H3K4 methylation level at the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMDM2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e promoter.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) H3K4 trimethylation and monomethylation signal at the \u003cem\u003eMDM2\u003c/em\u003epromoter region. Histone-seq data was obtained from ENCODE project and visualized on UCSC genome browser. (B) Ratio of monomethyl-H3K4 and trimethyl-H3K4 on the \u003cem\u003eMDM2\u003c/em\u003e promoter region in five iCCA cell lines. (C) H3K4 methylation level on the \u003cem\u003eMDM2\u003c/em\u003e promoter region after DMSO or mutant IDH1 inhibitors treatment for 48 hours in two mutant \u003cem\u003eIDH1\u003c/em\u003e iCCA cell lines. All data were represented as average ± standard deviation. All experiments were performed and repeated in three independent biological replicates. Statistical significance was evaluated by paired t test: n.s. not significant.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6489065/v1/7d48c44c17f6dd35a08eb349.png"},{"id":83815052,"identity":"ab633a77-d342-4b8e-bb3d-4ecc16c8e0c8","added_by":"auto","created_at":"2025-06-03 07:36:22","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":871592,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMutant IDH1 suppressed KDM5s activity to enhance H3K4 methylation level at the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMDM2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e promoter.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Methyl-H3K4 demethylase KDM5 activity in five iCCA cell lines. Fluorescent value was normalized to corresponding protein concentration and incubation time. (B) Methyl-H3K4 demethylase KDM5 activity in two mutant \u003cem\u003eIDH1\u003c/em\u003e iCCA cell lines after DMSO or mutant IDH1 inhibitors treatment for 48 hours. Fluorescent value was first normalized to corresponding protein concentration and incubation time. (C) Global H3K4 methylation level in two mutant \u003cem\u003eIDH1\u003c/em\u003e iCCA cell lines after DMSO, mutant IDH1 inhibitors or KDM5s inhibitor treatment for 48 hours. (D) \u003cem\u003eMDM2\u003c/em\u003e mRNA expression of two mutant \u003cem\u003eIDH1\u003c/em\u003e iCCA cell lines after DMSO, mutant IDH1 inhibitors or KDM5s inhibitor treatment for 48 hours. 2\u003csup\u003eΔΔCT\u003c/sup\u003e value was normalized to DMSO control. (E) MDM2 protein expression of two mutant \u003cem\u003eIDH1\u003c/em\u003e iCCA cell lines after DMSO, mutant IDH1 inhibitors or KDM5s inhibitor treatment for 72 hours. GAPDH used as internal control. \u003cem\u003eIDH1\u003c/em\u003e mutation iCCA cell lines, RBE and SNU1079. \u003cem\u003eIDH1\u003c/em\u003e wild-type iCCA cell lines, HuCCT1, HuH28 and SSP25. Mutant IDH1 inhibitors, AG-120 and IDH-305. Pan methyl-H3K4 demethylases inhibitors, KDOAM-25 citrate. All data were represented as average ± standard deviation. All experiments were performed and repeated in three independent biological replicates. Statistical significance was evaluated by paired t test: n.s. not significant.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6489065/v1/5ad8274cd1cb59ec2b883dd4.png"},{"id":83815055,"identity":"2a661a90-3605-41a8-859b-40fc27596462","added_by":"auto","created_at":"2025-06-03 07:36:22","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1315813,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMDM2 inhibitors reactivate wild-type in mutant \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eIDH1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e iCCA cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Cell viability after mutant IDH1 inhibitors or MDM2 inhibitors treatment for three doubling times in five iCCA cell lines. Data was normalized to corresponding DMSO control. (B) MDM2, TP53 and MIC1 protein expression in two mutant \u003cem\u003eIDH1\u003c/em\u003e and three wild-type \u003cem\u003eIDH1\u003c/em\u003e iCCA cell lines after DMSO or MDM2 inhibitors treatment for one doubling time. MIC1 used as indicator of wild-type TP53 activation. GAPDH used as internal control. (C) Cell cycle analysis of two mutant \u003cem\u003eIDH1\u003c/em\u003e iCCA cell lines after MDM2 inhibitors treatment for one doubling time. (D) TUNEL staining in two mutant \u003cem\u003eIDH1\u003c/em\u003eiCCA cell lines after MDM2 inhibitors treatment for one doubling times. Blue staining represents DPAI. Green staining represents TUNEL positive cells. \u003cem\u003eIDH1\u003c/em\u003emutation iCCA cell lines, RBE and SNU1079. \u003cem\u003eIDH1\u003c/em\u003e wild-type iCCA cell lines, HuCCT1, HuH28 and SSP25. Mutant IDH1 inhibitors, AG-120 and IDH-305. MDM2 inhibitors, RG-7388 and HDM-201. All data were represented as average ± standard deviation. All experiments were performed and repeated in three independent biological replicates.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6489065/v1/ff7fc1b3235cfa05c3900fb9.png"},{"id":83815053,"identity":"8f570873-21a4-4e93-acb6-ece0b99bf4c8","added_by":"auto","created_at":"2025-06-03 07:36:22","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1218419,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSynergistic effect of MDM2 and mutant IDH1 inhibitors combination treatment in mutant \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eIDH1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e iCCA cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Synergistic effect of MDM2 inhibitors and mutant IDH1 inhibitors combination treatment in SNU1079. Cells were treated with different combined doses of MDM2 inhibitors and mutant IDH1 inhibitors for three doubling times. Data was first normalized to DMSO control and then calculated for cytotoxicity and HSA synergy score. (B) Synergistic effect of MDM2 inhibitors and mutant IDH1 inhibitors combination treatment in RBE. Cells were treated with different combined doses of MDM2 inhibitors and mutant IDH1 inhibitors for three doubling times. Data was first normalized to DMSO control and then calculated for cytotoxicity and HSA synergy score. (C) A working model shows the mechanism by which mIDH1 activates \u003cem\u003eMDM2\u003c/em\u003e expression to promote TP53 degradation and proliferation in iCCA cells. \u003cem\u003eIDH1\u003c/em\u003e mutation iCCA cell lines, RBE and SNU1079. \u003cem\u003eIDH1\u003c/em\u003e wild-type iCCA cell lines, HuCCT1, HuH28 and SSP25. Mutant IDH1 inhibitors, AG-120 and IDH-305. MDM2 inhibitors, RG-7388 and HDM-201. All data were represented as average ± standard deviation. All experiments were performed and repeated in three independent biological replicates.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6489065/v1/88b5d3d11f85f45da795b9e5.png"},{"id":84591628,"identity":"f8e7abb2-d83d-48a2-b0c3-c7e18345ff8d","added_by":"auto","created_at":"2025-06-14 03:16:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8208587,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6489065/v1/5af0f5a4-9d4b-4f15-811c-18e5c721364f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Isocitrate dehydrogenase 1 mutation sensitizes intrahepatic cholangiocarcinoma cells to MDM2 inhibitors","fulltext":[{"header":"Background","content":"\u003cp\u003eBiliary tract cancer (BTC) is a heterogeneous group of malignancies arising from epithelial cells of the biliary tract. Based on their primary location, BTCs are divided into intrahepatic, perihilar, and distal cholangiocarcinoma; gallbladder cancer; and ampulla of Vater carcinoma, which have different trends of incidence changes, geographic distribution, risk factors, clinical outcomes, and genetic alterations [1-3]. Intrahepatic cholangiocarcinoma (iCCA), next to hepatocellular carcinoma, is the second most common hepatic malignancy. Owing to the lack of specific symptoms and signs, iCCA is usually diagnosed at a late stage with local invasion and/or intrahepatic or extrahepatic disseminations. According to the Surveillance, Epidemiology, and End Results Program (SEER) database, the 5-year overall survival rate of iCCA diagnosed between 2012 and 2018 in the United States was 9%, with 23%, 9%, and 3% for patients with localized, regional, and metastatic diseases, respectively [4].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Before the era of precision medicine, gemcitabine plus cisplatin was the standard systemic treatment for patients with unresectable and metastatic iCCA, similar to other BTCs [5, 6]. The isocitrate dehydrogenase (IDH) protein family is a metabolic enzyme responsible for converting isocitrate into \u0026alpha;-ketoglutarate (\u0026alpha;-KG). Somatic mutations in \u003cem\u003eIDH1 or 2\u003c/em\u003e (\u003cem\u003eIDH1/2\u003csup\u003emut\u003c/sup\u003e\u003c/em\u003e) are frequently identified in several cancers, including 10-25% of iCCA cases [7]. The mutant IDH1/2 (mIDH1/2) protein encoded by a gain-of-function \u003cem\u003eIDH1/2\u0026nbsp;\u003c/em\u003emutation\u003cem\u003e\u0026nbsp;\u003c/em\u003econverts \u0026alpha;-KG to D-2-hydroxyglutarate (2-HG), an oncometabolite that competitively suppresses the activity of \u0026alpha;-KG-dependent dioxygenases (\u0026alpha;-KGDs), including DNA and histone demethylases, to promote epigenetic reprogramming and tumorigenesis [8-10]. Based on the structural differences between the catalytic pockets of wild type IDH1/2 (wtIDH1/2) and mIDH1/2, several inhibitors targeting mIDH1 and/or mIDH2 have been developed and have undergone clinical trials [11]. AG120 (ivosidenib) is the first FDA-approved agent for \u003cem\u003eIDH1\u003c/em\u003e-mutated iCCA. However, despite significant improvement in progression-free survival (PFS) versus placebo control, the confirmed response rate and absolute PFS increase of second-line ivosidenib were 2.4% and 0.7 months, respectively, in the pivotal, ClarIDHy trial for \u003cem\u003eIDH1\u003c/em\u003e-mutated, chemotherapy-refractory iCCA [12-14]. In addition, patients who received long-term ivosidenib treatment might develop acquired resistance to ivosidenib through acquired resistance mutations that impair ivosidenib binding affinity, such as the conversion of catalytic arginine substitution and secondary \u003cem\u003eIDH1\u003csup\u003emut\u003c/sup\u003e\u003c/em\u003e at the dimer-interface region or isoform switch by acquired \u003cem\u003eIDH2\u003csup\u003emut\u003c/sup\u003e\u003c/em\u003e [15-18]. Taken together, ivosidenib treatment could result in a 22% 12-month PFS rate but a modest tumor response rate and might acquire secondary resistance mutations after long-term use, suggesting that other therapeutic interventions beyond \u003cem\u003eIDH1\u003c/em\u003e inhibition are warranted for \u003cem\u003eIDH1\u003csup\u003emut\u003c/sup\u003e\u003c/em\u003e iCCA patients. Herein, we report a novel mechanism by which mIDH1-derived 2-HG inhibits lysine-specific demethylase 5 (KDM5) to upregulate the expression of \u003cem\u003emouse double minute 2 homolog\u003c/em\u003e (\u003cem\u003eMDM2\u003c/em\u003e) via histone methylation and selectively promotes the proliferation of \u003cem\u003eIDH1\u003csup\u003emut\u003c/sup\u003e\u003c/em\u003e iCCA cells. In addition, the combination of mIDH1 and MDM2 inhibitors may provide a new strategy for treating \u003cem\u003eIDH1\u003csup\u003emut\u003c/sup\u003e\u003c/em\u003e iCCA patients. \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eClinical sample gene mutation profile analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMutation profiling datasets were obtained using cBioPortal (https://www.cbioportal.org) [19-21]. Odds ratios were calculated using the following formula: (\u003cem\u003eIDH1\u003csup\u003ewt\u003c/sup\u003e\u003c/em\u003e, \u003cem\u003eTP53\u003csup\u003ewt\u003c/sup\u003e\u003c/em\u003e sample number \u0026times; \u003cem\u003eIDH1\u003csup\u003emut\u003c/sup\u003e\u003c/em\u003e, \u003cem\u003eTP53\u003csup\u003emut\u003c/sup\u003e\u003c/em\u003e sample number)/ (\u003cem\u003eIDH1\u003csup\u003ewt\u003c/sup\u003e\u003c/em\u003e, \u003cem\u003eTP53\u003csup\u003emut\u003c/sup\u003e\u003c/em\u003e sample number \u0026times; \u003cem\u003eIDH1\u003csup\u003emut\u003c/sup\u003e\u003c/em\u003e, \u003cem\u003eTP53\u003csup\u003ewt\u003c/sup\u003e\u003c/em\u003e sample number). The P value of the odds ratio was calculated using Fisher\u0026rsquo;s exact test.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemistry staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe use of human iCCA tissue samples in the current study was approved by the Institutional Review Board of the National Cheng Kung University Hospital (NCKUH, IRB A-ER-111-019). Formalin-fixed paraffin-embedded iCCA samples were sectioned and stained with hematoxylin and eosin. For MDM2 IHC staining, sections were incubated with MDM2 antibody, secondary mouse antibody, and diaminobenzidine for colorimetric detection. Three independent pathologists who were unaware of the genetic alterations evaluated the IHC staining results. The histochemical scoring assessment (H-score) was based on the staining intensity of MDM2 and the proportion of stained cancer cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell lines\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe following cell lines were obtained from the following sources: HuCCT1 and HuH28 were obtained from the Japanese Collection of Research Bioresources Cell Bank, SNU1079 was purchased from the Korean Cell Line Bank, and RBE and SSP25 were kindly provided by Dr. Chien-Feng Li (Chi Mei Medical Center, Tainan, Taiwan), and Dr. Chun-Nan Yeh (Chang Gung University, Taoyuan, Taiwan). HuCCT1, RBE, SSP25, and SNU1079 cells were cultivated in RPMI-1640 medium (Cytiva) supplemented with 10% fetal bovine serum (Cytiva) and 1% 100x penicillin-streptomycin-glutamine (Invitrogen). HuH28 cells were cultivated in MEM (Simply) supplemented with 20% fetal bovine serum (Cytiva) and 1% 100x penicillin-streptomycin-glutamine (Invitrogen). All cell lines were incubated in at 37\u0026deg;C humidified incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e and passaged by trypsinization with 0.25% trypsin and protease solution (Cytiva). All cell lines were verified by short tandem repeat (STR) testing (FIRDI, Hsinchu, Taiwan), and experiments were performed on cells cultivated for less than ten passages. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIntracellular 2-HG measurement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo detect intracellular 2-HG concentration, a D-2-Hydroxyglutarate (D-2-HG) assay kit (Sigma-Aldrich, MAL320) was used following the manufacturer\u0026apos;s protocol. Cells were seeded in 10 cm dishes (5x10\u003csup\u003e5\u003c/sup\u003e cells per dish) for 24 hours (h) and treated with either mutant IDH1 inhibitors or DMSO control for 48 h. After treatment, cells were washed with ice-cold PBS, scraped, and collected by centrifugation. The collected cells were lysed in CelLytic\u003csup\u003eTM\u003c/sup\u003e M Cell Lysis Reagent (Sigma-Aldrich), and the protein concentration was quantified. The lysate was deproteinized by perchloric acid (PCA) precipitation. After PCA precipitation, the supernatants for 2-HG detection were collected and stored at -80\u0026deg;C. The supernatants with D-2-HG complete reaction mixture for 1 h at 37\u0026deg;C. The fluorescence was measured at \u0026lambda;\u003csub\u003eex\u003c/sub\u003e = 540 nm and \u0026lambda;\u003csub\u003eem\u003c/sub\u003e = 590 nm using a microplate reader. The intracellular D-2-HG concentrations were normalized to their corresponding protein concentrations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA extraction and complementary DNA synthesis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRNA was extracted using the Total RNA Miniprep Purification Kit (GeneMark, TR01), following the manufacturer\u0026apos;s protocol. Cells were first cultivated in 6-well plates (4x10\u003csup\u003e4 \u003c/sup\u003ecells/well) for 24 h and then treated with inhibitors or DMSO control for 48 h. After treatment, cells were washed with cold PBS and lysed in an RNA lysis solution containing 1% 2-mercaptoethanol. Lysates were purified using a two-step wash with RNA wash solution I and ethanol/RNA wash solution II. The purified RNA was eluted in nuclease-free water and stored at -80\u0026deg;C. RNA quality was measured using a nanophotometer. Complementary DNA (cDNA) was reverse-transcribed from purified RNA using the ReverTra Ace -\u0026alpha;-\u0026trade; kit (TOYOBO, FSK-101), following the manufacturer\u0026apos;s instructions. One microgram of total purified RNA was mixed with oligo-dT primers, RNase inhibitor, dNTP mixture, RT buffer, and RNase-free water. cDNA was amplified in a thermal cycler using the following program: 42\u0026deg;C for 20 minutes (m), 99\u0026deg;C for 5 min, and 4\u0026deg;C for 5 min. The cDNA was stored at -20 \u0026deg;C. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReal-time polymerase chain reaction (PCR)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor real-time PCR, we followed the manufacturer\u0026apos;s protocol and used a GoTaq\u0026reg; qPCR Master Mix kit (Promega, A6001). GoTaq\u0026reg; qPCR master mix, forward primer, reverse primer, and nuclease-free water were mixed. The mixture was quantified on a QuantStudio 5 Real-Time PCR System (Thermo Fisher Scientific), using a standard qPCR amplification and dissociation program. The 2\u003csup\u003e\u0026Delta;\u0026Delta;CT\u003c/sup\u003e value was calculated and normalized to that of the corresponding DMSO control.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blot\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were first cultivated in 6 well (4x10\u003csup\u003e4\u003c/sup\u003e cells/well) for 24 h and treated with inhibitors or DMSO control for 72 h. To detect wild-type TP53 protein expression in \u003cem\u003eIDH1\u003c/em\u003e\u003cem\u003e\u003csup\u003emut\u003c/sup\u003e\u003c/em\u003e iCCA cells, cells were pre-treated with proteasome inhibitor MG-132 (MedChemExpress, HY-13259) 15 \u0026micro;M for 2 h before harvest. Cells were lysed in CelLytic\u003csup\u003eTM\u003c/sup\u003e M Cell Lysis Reagent containing a protease inhibitor cocktail (Abcam). The Lysates were purified by centrifugation at 16,000 \u0026times; g for 15 min at 4 \u0026deg;C. The supernatants were collected and quantified using the Bradford protein assay (Bio-Rad). The Lysates were mixed with 4x loading dye buffer and heated at 95\u0026deg;C for 15 min. Protein lysates were loaded onto SDS-PAGE gels. The gels were run at 80 mV for 2 h and then transferred overnight at 30 mV. The membranes were sequentially blocked with 5% non-fat milk/TBST buffer, primary antibodies, and secondary antibodies. Chemiluminescence was activated by enhanced chemiluminescence (Perkin Elmer) and detected by exposure to X-ray film. Primary antibodies: p53 (7F5) Rabbit mAb (Cell Signaling Technology, 2527, 1:1000), Mic-1 (D2A3) Rabbit mAb (Cell Signaling Technology, 8479, 1:1000), MDM2 antibody [SMP14] (GeneTex, GTX70278, 1:1000) and Anti-Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) Antibody clone 6C5 (Millipore, MAB374, 1:10000). Secondary antibodies: Peroxidase AffiniPure\u0026trade; Goat Anti-Rabbit IgG (H+L) (Jackson ImmunoResearch Inc., 111-007-003, 1:5000) and AffiniPure\u0026trade; Fab Fragment Goat Anti-Mouse IgG (H+L) (Jackson ImmunoResearch Inc., 115-007-003, 1:5000).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNuclear protein extraction and KDM5 activity measurement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo detect KDM5 activity, we performed nuclear extraction using a nuclear extraction kit (Abcam, ab113474) and KDM5 activity quantification assay kit (Abcam, ab113464), following the manufacturer\u0026apos;s protocol. Cells were seeded in 10 cm dishes (5x10\u003csup\u003e5\u003c/sup\u003e cells per dish) for 24 h and treated with either mutant IDH1 inhibitors or DMSO control for 48 h. The cells were washed with ice-cold PBS and cell pellets were collected by scraping and centrifuging. Cell pellets were lysated in 1X Pre-Extraction Buffer containing 0.1% DTT and 0.1% 100x protease inhibitor cocktail, vortexed, and centrifuged to collect the nuclear pellets. Nuclear pellets were lysed in Extraction Buffer containing 0.1% DTT and 0.1% 100x protease inhibitor cocktail, vortexed, and centrifuged to collect nuclear protein. The Nuclear extracts were collected, quantified, and stored at -80\u0026deg;C until KDM5 activity measurement. For KDM5 activity measurement, 10 \u0026micro;g of purified nuclear extract was mixed with reaction buffer containing completed assay buffer and substrate, incubated at 37\u0026deg;C for 90 min, and washed with wash buffer. Capture antibody was added to the reaction wells, covered with Parafilm M, and incubated at room temperature for 1 h. After incubation, the capture antibody solution was removed, and the cells were washed with wash buffer. Detection antibody was added to the reaction wells, covered with Parafilm M, and incubated at room temperature for 30 min. After incubation, the detection antibody solution was removed, and the cells were washed with wash buffer. Fluoro developer was then added to the reaction wells and incubated at room temperature for 4 min in the dark. The fluorescence signal was measured at \u0026lambda;\u003csub\u003eex\u003c/sub\u003e = 530 nm and \u0026lambda;\u003csub\u003eem\u003c/sub\u003e = 590 nm, using a microplate reader.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNative chromatin immunoprecipitation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were seeded in 10 cm dishes (5x10\u003csup\u003e5\u003c/sup\u003e cells per dish) for 24 h and then treated with either mutant IDH1 inhibitors or DMSO control for 48 h. The cells were washed with ice-cold PBS, scraped, and centrifuged to collect cell pellets. The cell pellet was resuspended in CelLytic\u003csup\u003eTM\u003c/sup\u003e M Cell Lysis Reagent containing 1 mM CaCl2, 0.2% Triton X-100, and 50 mM Tris-HCl (pH 7.6) on ice for 15 min, homogenized by passing through a 27G needle, and sonicated to shear the chromatin. 5 \u0026micro;L of DNA fragment were used for agarose gel analysis to verify the sonication results. The sheared chromatin supernatants were mixed with IgG control or the indicated antibodies at 4\u0026deg;C overnight on a rotary mixer. Protein A Mag Sepharose Xtra (Cytiva) were then added to the complex solution and incubated at 4\u0026deg;C for 4 h on a rotary mixer. The complex solution was placed on a magnetic stand and the supernatant was removed. The magnetic beads were washed on a rotary mixer with the following buffers and times: RIPA buffer twice, RIPA buffer + 0.3M NaCl twice, LiCl buffer twice, TE buffer + 0.2% Triton X-100 twice, and TE buffer once. The Magnetic beads were resuspended in 10% SDS/proteinase K/TE buffer and incubated overnight at 65\u0026deg;C. The samples were vortexed briefly, placed on a magnetic stand, and the supernatants were collected. The magnetic beads were washed with 0.5M NaCl / TE buffer, placed on a magnetic stand, and the supernatants were collected. The collected supernatants were combined with the collected solutions and stored at -20\u0026deg;C until DNA purification. Primary antibodies: Mono-Methyl-Histone H3 (Lys4) (D1A9) XP\u0026reg; Rabbit mAb (Cell Signaling Technology, 5326, 1:50), Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb (Cell Signaling Technology, 9751, 1:50) and Rabbit (DA1E) mAb IgG XP\u0026reg; Isotype Control (Cell Signaling Technology, 3900, 1:50).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDNA fragments purification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDNA fragments were purified from the collected solutions using a GenepHlow\u0026trade; Gel/PCR Kit (Geneaid, DFH300), according to the manufacturer\u0026apos;s instructions. The collected solutions were mixed with gel/PCR buffer, transferred to collection tubes, and centrifuged at 16,000 g for 30 s. The collection tubes were washed with ethanol/wash buffer and centrifuged at 16,000 g for 30 s. The remaining ethanol was removed by centrifuging the collection tubes at 16,000 \u0026times; g for 3 min. The purified DNA was eluted in 70\u0026deg;C-heated elution buffer and stored at -20\u0026deg;C until Real-time PCR.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell cycle analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were seeded in 10 cm dishes (5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells per dish) for 24 h and then treated with MDM2 inhibitors or DMSO control for one doubling time. The cells were harvested and washed with 2% fetal bovine serum/PBS. The cells were fixed in 70% ethanol at 4\u0026deg;C for one hour, then stored at -20\u0026deg;C overnight. The fixed cells were washed with cold PBS and stained with propidium iodide / RNase / TritonX-100 solution. FL-2 intensity was detected using BD FACSCalibur Flow Cytometer (BD Biosciences) and analyzed using ModFit LT (Verity Software House).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProliferation assay \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were seeded in 24-well plates (3x10\u003csup\u003e3\u003c/sup\u003e cells/well). For single-drug treatment, increasing doses of mutant IDH1 inhibitors, MDM2 inhibitors, or DMSO controls were added. For combined treatment, mutant IDH1 and MDM2 inhibitors were added at different ratios. Cells were harvested at three doubling times for each cell line. The cells were stained with 0.5% methylene blue ethanol solution for 2 h, washed with flowing tap water, and air-dried overnight. Methylene blue-stained cells were dissolved in 1% sarkosyl/PBS buffer and the absorbance was read at 595 nm using a microplate reader. The data were normalized to those of DMSO controls. The combined index was calculated by adjusting the effective concentration of each drug combination. The data were analyzed and visualized using SynergyFinder 3.0 (https://synergyfinder.fimm.fi/) [22].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReagents and chemicals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe reagents and chemicals used in this study: dimethyl sulfoxide (DMSO) (Sigma-Aldrich, D2650), ivosidenib (Synonyms: AG-120) (MedChemExpress, HY-18767), IDH-305 (MedChemExpress, HY-104036), idasanutlin (Synonyms: RG7388) (MedChemExpress, HY-15676), siremadlin (Synonyms: NVP-HDM201; HDM201) (MedChemExpress, HY-18658), KDOAM-25 citrate (MedChemExpress, HY-102047B), and MG-132 (Synonyms: Z-Leu-Leu-Leu-al; MG132) (MedChemExpress, HY-13259).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRStudio IDE (RStudio), R Project for Statistical Computing version 4.2, and the R packages ggplot2 and ggsignif were used to analyze and visualize the data [23-25]. Paired t-tests and Wilcoxon signed-rank tests were used to compare gene expression data.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data were generated by the first author and are available upon request from the corresponding author. The data generated in this study are publicly available in the following online datasets and studies: Mutation profiling datasets used in this study were downloaded from cBioPortal (https://www.cbioportal.org), Genomic Data Commons Data Portal (GDC) (https://portal.gdc.cancer.gov/) and published studies [19-21, 26, 27]. Datasets from cBioPortal : Cholangiocarcinoma (ICGC, Cancer Discov 2017), Cholangiocarcinoma (National Cancer Centre of Singapore, Nat Genet 2013), Intrahepatic Cholangiocarcinoma (JHU, Nat Genet 2013), Intrahepatic Cholangiocarcinoma (Mount Sinai 2015), Intrahepatic Cholangiocarcinoma (Shanghai, Nat Commun 2014) and Intrahepatic Cholangiocarcinoma (MSK, Hepatology 2021) [28-33]. Dataset from GDC: TCGA-CHOL cohort. Datasets from published studies: doi:10.1038/s41416-022-01932-1, doi:10.1038/s41598-022-22543-z, doi:10.1158/2159-8290.CD-17-0368, and doi:10.1158/2159-8290.CD-20-0766 [28, 34-36].\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eMutations in \u003cem\u003eIDH1/2\u003c/em\u003e and \u003cem\u003eTP53\u003c/em\u003e are mutually exclusive in human iCCA tissues and cell lines\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo identify the other potential therapeutic targets in iCCA with \u003cem\u003eIDH1/2\u003c/em\u003e mutations, we first reviewed the comprehensive genomic profiling of BTC, in which \u003cem\u003eIDH1/2\u003c/em\u003e mutations were only identified in iCCA and mutually exclusive with other major genetic alterations, i.e. \u003cem\u003eTP53\u003c/em\u003e, \u003cem\u003eKRAS\u003c/em\u003e and \u003cem\u003eSMAD4\u003c/em\u003e mutations, actionable \u003cem\u003eFGFR2\u003c/em\u003e fusion and \u003cem\u003eBRAF\u003csup\u003eV600E\u003c/sup\u003e\u003c/em\u003e mutation, and \u003cem\u003eERBB2\u003c/em\u003e and\u0026nbsp;\u003cem\u003eMDM2\u003c/em\u003e amplifications\u0026nbsp;[37, 38]. With the emergence of wtTP53 reactivation therapy, we examined the mutual exclusion of \u003cem\u003eIDH1/2\u003csup\u003emut\u003c/sup\u003e\u003c/em\u003e and \u003cem\u003eTP53\u003c/em\u003e alterations. Of the 1031 iCCA that were included from 7 studies in cBioPortal database, \u003cem\u003eIDH1/2\u003c/em\u003e mutation was detected in 146 (14.1%) (Fig. 1A)\u0026nbsp;[19-21]. \u003cem\u003eTP53\u003c/em\u003e mutation rate was 6.1% (9/146) in \u003cem\u003eIDH1/2\u003csup\u003emut\u003c/sup\u003e\u003c/em\u003e and 29.9% (265/885) in \u003cem\u003eIDH1/2\u003csup\u003ewt\u003c/sup\u003e\u003c/em\u003e tumors, with an odds ratio (OR) of 0.15 (95% confidence interval, 0.08-0.31), p \u0026lt; 0.0001 (Fisher's exact test). Trends of mutual exclusiveness between \u003cem\u003eIDH1/2\u003c/em\u003e and \u003cem\u003eTP53\u003c/em\u003e mutations of individual studies with case number more than 100, three within cBioPortal and four other recent publications were shown in Fig. 1B. Interestingly, both iCCA cell lines with pathogenic \u003cem\u003eIDH1\u003csup\u003emut\u003c/sup\u003e\u003c/em\u003e (SNU1079 and REB) in Cancer Cell Encyclopedia were \u003cem\u003eTP53\u003csup\u003ewt\u003c/sup\u003e\u003c/em\u003e further supports our observation (Supplementary Fig. 1A)\u0026nbsp;[39]. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMutant IDH1 upregulates the expression of ubiquitin ligase MDM2 at transcription level\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo elucidate the functions and utilities of molecules and signaling pathways involved in \u003cem\u003eIDH1\u003csup\u003emut\u003c/sup\u003e/TP53\u003csup\u003ewt\u003c/sup\u003e\u003c/em\u003e iCCA, we compared the expression profiles of \u003cem\u003eIDH1\u003csup\u003emut\u003c/sup\u003e/TP53\u003csup\u003ewt\u003c/sup\u003e\u003c/em\u003e and \u003cem\u003eIDH1\u003csup\u003ewt\u003c/sup\u003e/TP53\u003csup\u003emut\u003c/sup\u003e\u003c/em\u003e iCCA cells from the Cancer Cell Line Encyclopedia (CCLE) mRNA dataset and found upregulation of the E3 ubiquitin ligase MDM2, which ubiquitinates and negatively modulates TP53 protein expression [39]\u0026nbsp;(Supplementary\u0026nbsp;Fig. 2A). The augmentation of MDM2 in \u003cem\u003eIDH1\u003csup\u003emut\u003c/sup\u003e\u003c/em\u003e iCCA cells was confirmed in iCCA tissues\u0026nbsp;and cell lines\u0026nbsp;(Fig. 2A, 2B and 2C). Real time-qPCR and western blotting confirmed the increases in MDM2 mRNA and protein\u0026nbsp;levels in both\u0026nbsp;\u003cem\u003eIDH1\u003csup\u003emut\u003c/sup\u003e\u003c/em\u003e iCCA cell lines (SNU1079 and RBE) (Fig. 2B and 2C). To validate the association between mIDH1 and MDM2 expression, \u003cem\u003eIDH1\u003csup\u003emut\u003c/sup\u003e\u003c/em\u003e iCCA cells were treated with two mIDH1 specific inhibitors, AG120 and IDH305. Pharmacological inhibition of mIDH1 led to\u0026nbsp;a reduction\u0026nbsp;in intracellular 2-HG levels,\u0026nbsp;accompanied by\u0026nbsp;a decrease\u0026nbsp;in MDM2 mRNA and protein expression (Fig. 2D, 2E and 2F).\u0026nbsp;As expected,\u0026nbsp;attenuation of MDM2 was accompanied by an increase in wtTP53 protein levels\u0026nbsp;(Fig. 2F). In contrast, mIDH1 inhibitor treatment did not alter MDM2 and mTP53 expression in \u003cem\u003eIDH1\u003csup\u003ewt\u003c/sup\u003e\u003c/em\u003e iCCA cell lines (HuH28, HuCCT1, and SSP25)\u0026nbsp;(Supplementary Fig. 2B and 2C). These data suggested\u0026nbsp;that 2-HG upregulates the expression of MDM2 at\u0026nbsp;the transcriptional level in \u003cem\u003eIDH1\u003csup\u003emut\u003c/sup\u003e\u003c/em\u003e iCCA cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMutant IDH1 suppresses KDM5 activity and enhances H3K4 methylation level on the \u003cem\u003eMDM2\u003c/em\u003e promoter region\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBecause 2-HG can inhibit the activity of lysine-specific histone demethylases (KDMs), a group of α-KG-dependent dioxygenases that modulate histone lysine methylation and gene transcription [40, 41]. We searched the histone chromatin immunoprecipitation (ChIP) sequencing data from ENCODE on the UCSC genome browser to explore the molecular mechanisms underlying the epigenetic modulation of\u0026nbsp;\u003cem\u003eMDM2\u003c/em\u003e transcription in \u003cem\u003eIDH1\u003csup\u003emut\u003c/sup\u003e\u003c/em\u003e iCCA cells\u0026nbsp;[42-44]. The histone-3-lysine-4 tri-methylation (H3K4me3), an active histone code, was highly enriched in three different regions near the promoter region and transcription start site (TSS) of \u003cem\u003eMDM2\u003c/em\u003e in pan-cell types (Fig. 3A). ChIP-qPCR was performed to detect H3K4 mono-methylation (H3K4me1) and H3K4me3 in three H3K4me3-enriched promoter and TSS regions of \u003cem\u003eMDM2\u003c/em\u003e in \u003cem\u003eIDH1\u003csup\u003emut\u003c/sup\u003e\u003c/em\u003e and \u003cem\u003eIDH1\u003csup\u003ewt\u003c/sup\u003e\u003c/em\u003e iCCA cells. Among them, the methylation score (H3K4me3/H3K4me1 ratio) of region 2 was the highest in the two \u003cem\u003eIDH1\u003csup\u003emut\u003c/sup\u003e\u003c/em\u003e iCCA cells compared to regions 1 and 3 within the same cells or region 2 of the \u003cem\u003eIDH1\u003csup\u003ewt\u003c/sup\u003e\u003c/em\u003e iCCA cells (Fig. 3B). AG120 and IDH305 treatment profoundly reduced the H3K4 methylation level and H3K4me3/H3K4me1 ratio in region 2 of both \u003cem\u003eIDH1\u003csup\u003emut\u003c/sup\u003e\u003c/em\u003e iCCA cells (Fig. 3C).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAs the KDM5 family (KDM5A–5D) specifically recognizes and demethylates H3K4me3 via α-KG-dependent oxidation, they can be antagonized by 2-HG [40]. We hypothesized that high H3K4 methylation at the \u003cem\u003eMDM2\u003c/em\u003e promoter in \u003cem\u003eIDH1\u003csup\u003emut\u003c/sup\u003e\u003c/em\u003e iCCA cells is caused by low KDM5 activity. We measured total KDM5 activity in the nuclear extract of iCCA cell lines and found a significant decrease in \u003cem\u003eIDH1\u003csup\u003emut\u003c/sup\u003e\u003c/em\u003e iCCA cells compared to \u003cem\u003eIDH1\u003csup\u003ewt\u003c/sup\u003e\u003c/em\u003e iCCA cells (Fig. 4A). Treatment with AG120 and IDH305 enhanced total KDM5 activity and reduced H3K4me3 level, which was accompanied by a reduction in \u003cem\u003eMDM2\u003c/em\u003e mRNA expression in \u003cem\u003eIDH1\u003csup\u003emut\u003c/sup\u003e\u003c/em\u003e iCCA cells (Fig. 4B - 4E). Co-treatment with KDOAM-25 citrate, a pan-KDM5 inhibitor, restored H3K4me3 and MDM2 levels in AG120- and IDH305-treated \u003cem\u003eIDH1\u003csup\u003emut\u003c/sup\u003e\u003c/em\u003e iCCA cells (Fig. 4C - 4E). These results suggested that mIDH1-derived 2-HG enhances H3K4me3 levels at the \u003cem\u003eMDM2\u003c/em\u003e promoter by suppressing KDM5 activity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMDM2 inhibitors restore wild-type TP53 function and suppress the growth of \u003cem\u003eIDH1\u003csup\u003emut\u003c/sup\u003e\u003c/em\u003e iCCA cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe demonstrated that \u003cem\u003eIDH1\u003c/em\u003e and \u003cem\u003eTP53\u003c/em\u003e mutations are mutually exclusive in iCCAs and mutant IDH1 enhance MDM2 expression through the modulation of KDM5 activity by 2-HG in\u0026nbsp;\u003cem\u003eIDH1\u003csup\u003emut\u003c/sup\u003e\u003c/em\u003e iCCA cells. These findings suggest \u003cem\u003eIDH1\u003csup\u003emut\u003c/sup\u003e\u003c/em\u003e iCCA is a potential candidate for MDM2 inhibitor treatment, which is under extensive clinical evaluation for \u003cem\u003eMDM2\u003c/em\u003e-amplified \u003cem\u003eTP53\u003csup\u003ewt\u003c/sup\u003e\u003c/em\u003e tumors, including BTCs\u0026nbsp;[45, 46]. We treated iCCA cells with different concentrations of MDM2 inhibitors (RG7388 and HDM201) or mIDH1 inhibitors (AG120 and IDH305) for three doubling times and\u0026nbsp;assessed the cell\u0026nbsp;proliferation. Consistent with previous studies and clinical trials, mIDH1 inhibitors had little growth inhibitory effect on iCCA cell lines. However, \u003cem\u003eIDH1\u003csup\u003emut\u003c/sup\u003e/TP\u003csup\u003e53\u003c/sup\u003e\u003c/em\u003e iCCA cells were more susceptible to MDM2 inhibitors than \u003cem\u003eIDH1\u003csup\u003ewt\u003c/sup\u003e\u003c/em\u003e/\u003cem\u003eTP53\u003csup\u003emut\u003c/sup\u003e\u003c/em\u003e iCCA cells (Fig. 5A). To clarify the mechanism by which MDM2 inhibitors affect \u003cem\u003eIDH1\u003csup\u003emut\u003c/sup\u003e\u003c/em\u003e iCCA cells, we treated SNU1079 and RBE cells with RG7388 and HDM201 for one doubling time\u0026nbsp;and harvested the cells for analysis.\u0026nbsp;Both inhibitors disrupted the interaction between MDM2 and TP53,\u0026nbsp;inducing the accumulation of these two proteins in \u003cem\u003eIDH1\u003csup\u003emut\u003c/sup\u003e\u003c/em\u003e/\u003cem\u003eTP53\u003csup\u003ewt\u003c/sup\u003e\u003c/em\u003e iCCA cells (Fig. 5B). In addition, the expression of macrophage inhibitory cytokine-1 (MIC-1), a TP53 target gene, was also increased\u0026nbsp;[47]\u0026nbsp;(Fig. 5B). This led to cell cycle arrest in the G1 phase and apoptosis in\u0026nbsp;the SNU1079 and RBE cells\u0026nbsp;(Fig. 5C and 5D). Conversely,\u0026nbsp;treatment with the MDM2 inhibitor\u0026nbsp;did not significantly inhibit\u0026nbsp;the growth\u0026nbsp;or accumulation of MDM2 and mTP53 proteins in \u003cem\u003eIDH1\u003csup\u003ewt\u003c/sup\u003e/TP53\u003csup\u003emut\u003c/sup\u003e\u003c/em\u003e iCCA cells under the same experimental conditions\u0026nbsp;(Fig. 5A and 5B). As mIDH1 inhibitors suppressed MDM2 expression, it would be interesting to explore the effect of MDM2 and mIDH1 inhibitor combination treatment in \u003cem\u003eIDH1\u003csup\u003emut\u003c/sup\u003e\u003c/em\u003e/\u003cem\u003eTP53\u003csup\u003ewt\u003c/sup\u003e\u003c/em\u003e iCCA cells. We co-treated\u0026nbsp;iCCA cell lines with various combinations of MDM2 and mIDH1 inhibitors\u0026nbsp;and calculated the combination index (CI) value. The CI estimation indicated that\u0026nbsp;the MDM2 and mIDH1 inhibitors showed a synergistic effect (CI \u0026lt; 1)\u0026nbsp;in suppressing the growth of \u003cem\u003eIDH1\u003csup\u003emut\u003c/sup\u003e\u003c/em\u003e/\u003cem\u003eTP53\u003csup\u003ewt\u003c/sup\u003e\u003c/em\u003e iCCA cell lines (Fig. 6A and 6B). These results suggest that MDM2 is a therapeutic target in \u003cem\u003eIDH1\u003csup\u003emut\u003c/sup\u003e\u003c/em\u003e/\u003cem\u003eTP53\u003csup\u003ewt\u003c/sup\u003e\u003c/em\u003e iCCA cells.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn recent decades, large-scale next-generation sequencing has contributed to the discovery of pathogenic somatic mutations in the cancer genomes. Comprehensive mutation profiling provides a new perspective for studying interactions between gene alterations. Our earlier iCCA genomic profiling datasets analyses revealed mutual exclusion of \u003cem\u003eIDH1/2\u003c/em\u003e and \u003cem\u003eTP53\u0026nbsp;\u003c/em\u003emutations, and CCLE mRNA dataset analysis showed \u003cem\u003eIDH1\u003csup\u003emut\u003c/sup\u003e/TP53\u003csup\u003ewt\u003c/sup\u003e\u003c/em\u003e cells expressing higher level of MDM2 than \u003cem\u003eIDH1\u003csup\u003ewt\u003c/sup\u003e/TP53\u003csup\u003emut\u003c/sup\u003e\u0026nbsp;\u003c/em\u003ecells. Our laboratory works further demonstrated both \u003cem\u003eIDH1\u003csup\u003emut\u003c/sup\u003e/TP53\u003csup\u003ewt\u003c/sup\u003e\u003c/em\u003e iCCA (SNU1079 ad RBE) cell lines expressing higher MDM2 protein levels than \u003cem\u003eIDH1\u003csup\u003ewt\u003c/sup\u003e/TP53\u003csup\u003emut\u003c/sup\u003e\u0026nbsp;\u003c/em\u003eiCCA\u003cem\u003e\u0026nbsp;\u003c/em\u003ecells. Mutant IDH1 inhibitors treatment reduced the intracellular level of 2-HG that was accompanied with a reduction of MDM2 mRNA and protein expression, and enhancing wTP53 protein expression in both \u003cem\u003eIDH1\u003csup\u003emut\u003c/sup\u003e/TP53\u003csup\u003ewt\u003c/sup\u003e\u003c/em\u003e iCCA cells.\u0026nbsp;Since\u0026nbsp;MDM2 is\u0026nbsp;a\u0026nbsp;known TP53 negative regulator that binds TP53 to facilitate its degradation, we further explored the molecular mechanisms underlying the modulation of MDM2 transcription by 2-HG, which is a mutant IDH1/2-derived oncometabolite known to reprogram gene expression and cellular metabolism through suppressing \u0026alpha;-KGD activity. It has been reported that KDM5 histone lysine demethylase as a target enzyme of 2-HG [25], and KDM5A as a negative regulator of TP53 expression via the modulation of protein translation genes [36,37]. Our study showed that KDM5 activity was lower and the level of H3K4me3 in part of the promoter region of \u003cem\u003eMDM2\u0026nbsp;\u003c/em\u003ewas higher\u003cem\u003e\u0026nbsp;\u003c/em\u003ein \u003cem\u003eIDH1\u003csup\u003emut\u003c/sup\u003e\u003c/em\u003e iCCA cells than \u003cem\u003eIDH1\u003csup\u003ewt\u003c/sup\u003e\u003c/em\u003e iCCA cells (Figure). All the events, reduced KDM5 activity, increased H3K4me3 in \u003cem\u003eMDM2\u003c/em\u003e promoter region, enhanced MDM2 expression and reduced wtTP53 expression in \u003cem\u003eIDH1\u003csup\u003emut\u003c/sup\u003e\u003c/em\u003e iCCA cells could be partially reversed by mutant IDH1 inhibitors treatment. These findings indicate wtTP53 inactivation plays a role in the carcinogenesis of \u003cem\u003eIDH1/2\u003csup\u003emut\u003c/sup\u003e\u003c/em\u003e iCCA. In addition, combining the findings of mutual exclusion of \u003cem\u003eIDH1/2\u0026nbsp;\u003c/em\u003eand \u003cem\u003eTP53\u0026nbsp;\u003c/em\u003emutations, and the enhanced MDM2 expression in \u003cem\u003eIDH1\u003csup\u003emut\u003c/sup\u003e\u003c/em\u003e iCCA tissues and cells also provide a rationale to evaluate the potential use of MDM2 inhibitor in this subgroup of patients.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMDM2 is an E3 ubiquitin ligase that directly targets wtTP53 to promote TP53 degradation through monoubiquitination, polyubiquitination, NEDD8 NEDDylation, and SUMO-1 modification [48]. These post-translational modifications govern TP53 cellular localization, activity, downstream target gene selection, and protein stability. Based on structural studies of the\u0026nbsp;MDM2-TP53 binding motif (Phe19, Trp23, and Leu26), small-molecule inhibitors targeting MDM2\u0026nbsp;were designed and developed [49, 50]. Currently, targeted population for MDM2 inhibitors therapy in solid tumors mainly focused on those with frequent \u003cem\u003eMDM2 amplified/TP53\u003csup\u003ewt\u003c/sup\u003e\u003c/em\u003e genotype, and encouraging therapeutic efficacy has been observed in patients with advanced biliary tract cancers [51].\u003cem\u003e\u0026nbsp;\u003c/em\u003eIn this study, we used two MDM2 inhibitors that had undergone clinical trials to test the cytotoxic effects of\u0026nbsp;\u003cem\u003eIDH1\u003csup\u003emut\u003c/sup\u003e\u003c/em\u003e iCCA\u0026nbsp;[52, 53]. These two MDM2 inhibitors showed more effective cytotoxicity and wtTP53 reactivation than mIDH1 inhibitor in\u0026nbsp;\u003cem\u003eIDH1\u003csup\u003emut\u003c/sup\u003e\u003c/em\u003e iCCA\u0026nbsp;cells. The finding is clinical relevant because it provides a rationale to expand the potential targeted iCCA population for MDM2 inhibitor treatment. According to cBioportal dataset, \u003cem\u003eMDM2 amplification\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;IDH1/2 mutations\u003c/em\u003e was\u003cem\u003e\u0026nbsp;\u003c/em\u003edetected in 3.5% (15/430) and 25.6% (109/430) of iCCA tissue, respectively. Of them, only two tumors had co-occurrence of \u003cem\u003eIDH1 mutation\u003c/em\u003e and \u003cem\u003eMDM2 amplification\u003c/em\u003e. Furthermore, MDM2 inhibitor may overcome the mutant IDH1 inhibitor resistance \u003cem\u003eIDH1\u003csup\u003emut\u003c/sup\u003e\u003c/em\u003e iCCA with acquired IDH2 mutation\u0026nbsp;[15].\u003c/p\u003e\n\u003cp\u003eMoreover, co-treatment with mIDH1 and MDM2 inhibitors synergistically suppressed the proliferation of\u0026nbsp;\u003cem\u003eIDH1\u003csup\u003emut\u003c/sup\u003e\u003c/em\u003e iCCA cells. Although mIDH1 inhibitors did not directly target wtTP53 or MDM2, indirect epigenetic regulation of MDM2 still provides a new therapeutic strategy for wtTP53 reactivation. Since one of the most important obstacles for the development of MDM2 in cancer therapy is dose-dependent hematological toxicity, notably thrombocytopenia [54]. The finding of synergism between IDH1 inhibitor and MDM2 inhibitors provide a potential strategy of combination therapy aiming to reduce dose-dependent adverse events of MDM2 inhibitor without compromising therapeutic efficacy in \u003cem\u003eIDH1\u003csup\u003emut\u003c/sup\u003e\u003c/em\u003e\u003cem\u003e/TP53\u003csup\u003ewt\u003c/sup\u003e\u003c/em\u003e iCCA. Moreover, this mutually exclusive phenomenon was also observed in \u003cem\u003eIDH1/2\u003csup\u003emut\u003c/sup\u003e\u003c/em\u003e acute\u0026nbsp;myeloid leukemia [51], revealing that MDM2 inhibitors or other agents that reactivate wtTP53 are a potential treatment strategy for \u003cem\u003eIDH1/2\u003csup\u003emut\u003c/sup\u003e\u003c/em\u003e AML (Supplementary Fig. 3A).\u003c/p\u003e\n\u003cp\u003e2-HG inhibits the activity of KDM4B, which increases the level of H3K9 trimethylation and affects global gene expression. Although aberrant H3K9 hypermethylation did not significantly affect the expression of\u0026nbsp;homology-dependent repair (HDR) genes, however, its presence at loci near DNA strand breaks could block the activation signals for HDR execution and impair the recruitment of HDR factors\u0026nbsp;[41, 55, 56].\u0026nbsp;The findings suggested the \u003cem\u003eIDH1\u003csup\u003emut\u003c/sup\u003e\u003c/em\u003e cancers were associated with HDR deficiency and likely synthetic lethal with PARP1 inhibitor treatment [55, 57].\u0026nbsp;\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn summary, we confirmed that mutant IDH1-derived 2HG enhances \u003cem\u003eMDM2\u0026nbsp;\u003c/em\u003etranscription by decreasing KDM5 activity and enriching H3K4me3 at the \u003cem\u003eMDM2\u0026nbsp;\u003c/em\u003epromoter region. Increased MDM2 promotes the degradation of the wild-type TP53 protein in\u0026nbsp;\u003cem\u003eIDH1\u003csup\u003emut\u003c/sup\u003e\u003c/em\u003e iCCA,\u0026nbsp;and pharmacological inhibition of MDM2 reduces wild-type TP53 degradation and reactivates wild-type TP53-triggered growth inhibition and cell death in \u003cem\u003eIDH1\u003csup\u003emut\u003c/sup\u003e\u003c/em\u003e iCCA. In conclusion, we identified a novel mIDH1-MDM2-wtTP53 axis that mIDH1 indirectly regulates wtTP53 protein expression through\u0026nbsp;epigenetic upregulation of \u003cem\u003eMDM2\u003c/em\u003e transcription. This renders a wtTP53 reactivation therapeutic option for\u0026nbsp;\u003cem\u003eIDH1\u003csup\u003emut\u003c/sup\u003e\u003c/em\u003e iCCA.\u0026nbsp;\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eD-2-HG: D-2-hydroxyglutarate; \u0026alpha;-KG: Alpha-ketoglutarate; \u0026alpha;-KGDs: Alpha-KG-dependent dioxygenases; CCA: Cholangiocarcinoma; cDNA: Complementary DNA; ChIP:\u0026nbsp;Chromatin immunoprecipitation; H3K4: Histone 3 lysine 4; H3K4me1: Histone 3 lysine 4 mono-methylation; H3K4me3: Histone 3 lysine 4 tri-methylation; H-score: Histochemical scoring assessment; iCCA: Intrahepatic cholangiocarcinoma; IDH1: Isocitrate dehydrogenases 1\u003c/p\u003e\n\u003cp\u003e; \u003cem\u003eIDH1\u003csup\u003emut\u003c/sup\u003e\u003c/em\u003e: \u003cem\u003eIDH1 mutation\u003c/em\u003e; \u003cem\u003eIDH1\u003csup\u003ewt\u003c/sup\u003e\u003c/em\u003e:\u003cem\u003e\u0026nbsp;IDH1 wild-type\u003c/em\u003e; KDMs: Histone lysine demethylases; KEGG: Kyoto encyclopedia of genes and genomes; MDM2: Mouse double minute 2 homolog; mIDH1: Mutant IDH1; mTP53: Mutant TP53; OR: Odds ratio; TP53: Tumor protein 53; TSS: Transcription start site; wtIDH1: Wild-type IDH1; wtTP53: Wild-type TP53\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank Dr. Chun-Nan Yeh, Chang Gung University, Taoyuan, Taiwan, for supporting the SSP25 cell line\u0026nbsp;and Dr. Daw-Yang Hwang,\u0026nbsp;National Institute of Cancer Research, National Health Research Institutes, Taiwan, for providing the\u0026nbsp;M220 Focused-ultrasonicator (Covaris).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by National Institute of Cancer Research, National Health Research Institutes, Taiwan (CA-111~112-PP-20\u0026nbsp;and CA-113-PP-17), and National Science and Technology Council, Executive Yuan, Taiwan, to the Center for Cancer Research, Kaohsiung Medical University, Taiwan (112-2321-B-037-004).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ Contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWCH\u003c/strong\u003e and \u003cstrong\u003eLTC\u003c/strong\u003e initiated and designed this study. \u003cstrong\u003eCTL\u003c/strong\u003e performed the experiments, analyzed the data, and wrote the manuscript. \u003cstrong\u003eYHH\u0026nbsp;\u003c/strong\u003ehelps with the experiments design.\u003cstrong\u003e\u0026nbsp;YYS\u003c/strong\u003e and \u003cstrong\u003eNJC\u003c/strong\u003e helped with the acquisition of clinical samples. \u003cstrong\u003eYYS\u0026nbsp;\u003c/strong\u003eperformed IHC staining. \u003cstrong\u003eCFL\u003c/strong\u003e, \u003cstrong\u003eYCM\u003c/strong\u003e, and\u003cstrong\u003e\u0026nbsp;KCC\u003c/strong\u003e interpreted IHC results. \u003cstrong\u003eYHH, YYS\u003c/strong\u003e, \u003cstrong\u003eWCH\u003c/strong\u003e and \u003cstrong\u003eLTC\u0026nbsp;\u003c/strong\u003ereviewed and revised the manuscript. \u003cstrong\u003eWCH\u003c/strong\u003e and \u003cstrong\u003eLTC\u003c/strong\u003e provided conceptual insights.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIRB was approved by the Institutional Review Board of the National Cheng Kung University Hospital (NCKUH, IRB A-ER-111-019).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eNational Institute of Cancer Research, National Health Research Institutes, Tainan, Taiwan, \u003csup\u003e2\u003c/sup\u003eDepartment of Oncology, National Cheng Kung University Hospital, and Institute of Clinical Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan, \u003csup\u003e3\u003c/sup\u003eDepartment of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan, \u003csup\u003e4\u003c/sup\u003eCenter for Cancer Research, Kaohsiung Medical University, Kaohsiung, Taiwan\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e5\u003c/sup\u003eDepartment of Oncology, Taipei Veterans General Hospital, Taipei, Taiwan, \u003csup\u003e6\u003c/sup\u003eSchool of Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan, \u003csup\u003e7\u003c/sup\u003eClinical Pathology Department, Chi Mei Medical Center, Tainan, Taiwan, \u003csup\u003e8\u003c/sup\u003eDepartment of Pathology, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan, \u003csup\u003e9\u003c/sup\u003eDepartment of Pathology, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan, Taiwan,\u0026nbsp;\u003csup\u003e10\u003c/sup\u003eDepartment of Biological Science and Technology, National Yang Ming Chiao Tung University, Hsinchu, Taiwan, \u003csup\u003e11\u003c/sup\u003eDepartment of Medical Research, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan, \u003csup\u003e12\u003c/sup\u003eDepartment of Biological Science and Technology, National Yang Ming Chiao Tung University, Hsinchu, Taiwan\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBanales JM, Marin JJG, Lamarca A, Rodrigues PM, Khan SA, Roberts LR et al. Cholangiocarcinoma 2020: the next horizon in mechanisms and management. Nat Rev Gastroenterol Hepatol 2020; 17: 557-588.\u003c/li\u003e\n\u003cli\u003eKendall T, Verheij J, Gaudio E, Evert M, Guido M, Goeppert B et al. Anatomical, histomorphological and molecular classification of cholangiocarcinoma. Liver Int 2019; 39 Suppl 1: 7-18.\u003c/li\u003e\n\u003cli\u003eLendvai G, Szekerczes T, Illyes I, Dora R, Kontsek E, Gogl A et al. Cholangiocarcinoma: Classification, Histopathology and Molecular Carcinogenesis. Pathol Oncol Res 2020; 26: 3-15.\u003c/li\u003e\n\u003cli\u003eSEER*Explorer: An interactive website for SEER cancer statistics. [cited 2024 Feb 19]. Available from: https://seer.cancer.gov/statistics-network/explorer/ \u003c/li\u003e\n\u003cli\u003eMarin JJG, Prete MG, Lamarca A, Tavolari S, Landa-Magdalena A, Brandi G et al. Current and novel therapeutic opportunities for systemic therapy in biliary cancer. Br J Cancer 2020; 123: 1047-1059.\u003c/li\u003e\n\u003cli\u003eValle JW, Kelley RK, Nervi B, Oh DY, Zhu AX. Biliary tract cancer. Lancet 2021; 397: 428-444.\u003c/li\u003e\n\u003cli\u003eCao J, Hu J, Liu S, Meric-Bernstam F, Abdel-Wahab R, Xu J et al. Intrahepatic Cholangiocarcinoma: Genomic Heterogeneity Between Eastern and Western Patients. JCO Precis Oncol 2020; 4.\u003c/li\u003e\n\u003cli\u003ePirozzi CJ, Yan H. The implications of IDH mutations for cancer development and therapy. Nat Rev Clin Oncol 2021; 18: 645-661.\u003c/li\u003e\n\u003cli\u003eDang L, White DW, Gross S, Bennett BD, Bittinger MA, Driggers EM et al. Cancer-associated IDH1 mutations produce 2-hydroxyglutarate. Nature 2010; 465: 966.\u003c/li\u003e\n\u003cli\u003eYe D, Guan KL, Xiong Y. Metabolism, Activity, and Targeting of D- and L-2-Hydroxyglutarates. Trends Cancer 2018; 4: 151-165.\u003c/li\u003e\n\u003cli\u003eTian W, Zhang W, Wang Y, Jin R, Wang Y, Guo H et al. Recent advances of IDH1 mutant inhibitor in cancer therapy. Front Pharmacol 2022; 13: 982424.\u003c/li\u003e\n\u003cli\u003eFan B, Abou-Alfa GK, Zhu AX, Pandya SS, Jia H, Yin F et al. Pharmacokinetics/pharmacodynamics of ivosidenib in advanced IDH1-mutant cholangiocarcinoma: findings from the phase III ClarIDHy study. Cancer Chemother Pharmacol 2024; 93: 471-479.\u003c/li\u003e\n\u003cli\u003eZhu AX, Macarulla T, Javle MM, Kelley RK, Lubner SJ, Adeva J et al. Final Overall Survival Efficacy Results of Ivosidenib for Patients With Advanced Cholangiocarcinoma With IDH1 Mutation: The Phase 3 Randomized Clinical ClarIDHy Trial. JAMA Oncol 2021; 7: 1669-1677.\u003c/li\u003e\n\u003cli\u003eAbou-Alfa GK, Macarulla T, Javle MM, Kelley RK, Lubner SJ, Adeva J et al. Ivosidenib in IDH1-mutant, chemotherapy-refractory cholangiocarcinoma (ClarIDHy): a multicentre, randomised, double-blind, placebo-controlled, phase 3 study. Lancet Oncol 2020; 21: 796-807.\u003c/li\u003e\n\u003cli\u003eCleary JM, Rouaisnel B, Daina A, Raghavan S, Roller LA, Huffman BM et al. Secondary IDH1 resistance mutations and oncogenic IDH2 mutations cause acquired resistance to ivosidenib in cholangiocarcinoma. NPJ Precis Oncol 2022; 6: 61.\u003c/li\u003e\n\u003cli\u003eHarding JJ, Lowery MA, Shih AH, Schvartzman JM, Hou S, Famulare C et al. Isoform Switching as a Mechanism of Acquired Resistance to Mutant Isocitrate Dehydrogenase Inhibition. Cancer Discov 2018; 8: 1540-1547.\u003c/li\u003e\n\u003cli\u003eIntlekofer AM, Shih AH, Wang B, Nazir A, Rustenburg AS, Albanese SK et al. Acquired resistance to IDH inhibition through trans or cis dimer-interface mutations. Nature 2018; 559: 125-129.\u003c/li\u003e\n\u003cli\u003eLyu J, Liu Y, Gong L, Chen M, Madanat YF, Zhang Y et al. Disabling Uncompetitive Inhibition of Oncogenic IDH Mutations Drives Acquired Resistance. Cancer Discov 2023; 13: 170-193.\u003c/li\u003e\n\u003cli\u003ede Bruijn I, Kundra R, Mastrogiacomo B, Tran TN, Sikina L, Mazor T et al. Analysis and Visualization of Longitudinal Genomic and Clinical Data from the AACR Project GENIE Biopharma Collaborative in cBioPortal. Cancer Res 2023; 83: 3861-3867.\u003c/li\u003e\n\u003cli\u003eGao J, Aksoy BA, Dogrusoz U, Dresdner G, Gross B, Sumer SO et al. Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal. Sci Signal 2013; 6: pl1.\u003c/li\u003e\n\u003cli\u003eCerami E, Gao J, Dogrusoz U, Gross BE, Sumer SO, Aksoy BA et al. The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data. Cancer Discov 2012; 2: 401-404.\u003c/li\u003e\n\u003cli\u003eIanevski A, Giri AK, Aittokallio T. SynergyFinder 3.0: an interactive analysis and consensus interpretation of multi-drug synergies across multiple samples. Nucleic Acids Res 2022; 50: W739-W743.\u003c/li\u003e\n\u003cli\u003eWickham H. ggplot2: Elegant Graphics for Data Analysis. Springer-Verlag New York, 2016.\u003c/li\u003e\n\u003cli\u003eConstantin A PI. ggsignif: R Package for Displaying SignificanceBrackets for \u0026lsquo;ggplot2\u0026rsquo;. PsyArxiv 2021.\u003c/li\u003e\n\u003cli\u003eTeam RC. R: A Language and Environment for Statistical Computing, 2024.\u003c/li\u003e\n\u003cli\u003eGrossman RL, Heath AP, Ferretti V, Varmus HE, Lowy DR, Kibbe WA et al. Toward a Shared Vision for Cancer Genomic Data. N Engl J Med 2016; 375: 1109-1112.\u003c/li\u003e\n\u003cli\u003eCancer Genome Atlas Research N, Weinstein JN, Collisson EA, Mills GB, Shaw KR, Ozenberger BA et al. The Cancer Genome Atlas Pan-Cancer analysis project. Nat Genet 2013; 45: 1113-1120.\u003c/li\u003e\n\u003cli\u003eJusakul A, Cutcutache I, Yong CH, Lim JQ, Huang MN, Padmanabhan N et al. Whole-Genome and Epigenomic Landscapes of Etiologically Distinct Subtypes of Cholangiocarcinoma. Cancer Discov 2017; 7: 1116-1135.\u003c/li\u003e\n\u003cli\u003eChan-On W, Nairismagi ML, Ong CK, Lim WK, Dima S, Pairojkul C et al. Exome sequencing identifies distinct mutational patterns in liver fluke-related and non-infection-related bile duct cancers. Nat Genet 2013; 45: 1474-1478.\u003c/li\u003e\n\u003cli\u003eJiao Y, Pawlik TM, Anders RA, Selaru FM, Streppel MM, Lucas DJ et al. Exome sequencing identifies frequent inactivating mutations in BAP1, ARID1A and PBRM1 in intrahepatic cholangiocarcinomas. Nat Genet 2013; 45: 1470-1473.\u003c/li\u003e\n\u003cli\u003eSia D, Losic B, Moeini A, Cabellos L, Hao K, Revill K et al. Massive parallel sequencing uncovers actionable FGFR2-PPHLN1 fusion and ARAF mutations in intrahepatic cholangiocarcinoma. Nat Commun 2015; 6: 6087.\u003c/li\u003e\n\u003cli\u003eZou S, Li J, Zhou H, Frech C, Jiang X, Chu JS et al. Mutational landscape of intrahepatic cholangiocarcinoma. Nat Commun 2014; 5: 5696.\u003c/li\u003e\n\u003cli\u003eBoerner T, Drill E, Pak LM, Nguyen B, Sigel CS, Doussot A et al. Genetic Determinants of Outcome in Intrahepatic Cholangiocarcinoma. Hepatology 2021; 74: 1429-1444.\u003c/li\u003e\n\u003cli\u003eTomczak A, Springfeld C, Dill MT, Chang DH, Kazdal D, Wagner U et al. Precision oncology for intrahepatic cholangiocarcinoma in clinical practice. Br J Cancer 2022; 127: 1701-1708.\u003c/li\u003e\n\u003cli\u003eRimini M, Fabregat-Franco C, Burgio V, Lonardi S, Niger M, Scartozzi M et al. Molecular profile and its clinical impact of IDH1 mutated versus IDH1 wild type intrahepatic cholangiocarcinoma. Sci Rep 2022; 12: 18775.\u003c/li\u003e\n\u003cli\u003eSilverman IM, Hollebecque A, Friboulet L, Owens S, Newton RC, Zhen H et al. Clinicogenomic Analysis of FGFR2-Rearranged Cholangiocarcinoma Identifies Correlates of Response and Mechanisms of Resistance to Pemigatinib. Cancer Discov 2021; 11: 326-339.\u003c/li\u003e\n\u003cli\u003eNakamura H, Arai Y, Totoki Y, Shirota T, Elzawahry A, Kato M et al. Genomic spectra of biliary tract cancer. Nat Genet 2015; 47: 1003-1010.\u003c/li\u003e\n\u003cli\u003eKendre G, Murugesan K, Brummer T, Segatto O, Saborowski A, Vogel A. Charting co-mutation patterns associated with actionable drivers in intrahepatic cholangiocarcinoma. J Hepatol 2023; 78: 614-626.\u003c/li\u003e\n\u003cli\u003eGhandi M, Huang FW, Jane-Valbuena J, Kryukov GV, Lo CC, McDonald ER, 3rd et al. Next-generation characterization of the Cancer Cell Line Encyclopedia. Nature 2019; 569: 503-508.\u003c/li\u003e\n\u003cli\u003eGunn K, Myllykoski M, Cao JZ, Ahmed M, Huang B, Rouaisnel B et al. (R)-2-Hydroxyglutarate Inhibits KDM5 Histone Lysine Demethylases to Drive Transformation in IDH-Mutant Cancers. Cancer Discov 2023; 13: 1478-1497.\u003c/li\u003e\n\u003cli\u003eSulkowski PL, Oeck S, Dow J, Economos NG, Mirfakhraie L, Liu Y et al. Oncometabolites suppress DNA repair by disrupting local chromatin signalling. Nature 2020; 582: 586-591.\u003c/li\u003e\n\u003cli\u003eRosenbloom KR, Sloan CA, Malladi VS, Dreszer TR, Learned K, Kirkup VM et al. ENCODE data in the UCSC Genome Browser: year 5 update. Nucleic Acids Res 2013; 41: D56-63.\u003c/li\u003e\n\u003cli\u003eKarolchik D, Hinrichs AS, Furey TS, Roskin KM, Sugnet CW, Haussler D et al. The UCSC Table Browser data retrieval tool. Nucleic Acids Res 2004; 32: D493-496.\u003c/li\u003e\n\u003cli\u003eRaney BJ, Dreszer TR, Barber GP, Clawson H, Fujita PA, Wang T et al. Track data hubs enable visualization of user-defined genome-wide annotations on the UCSC Genome Browser. Bioinformatics 2014; 30: 1003-1005.\u003c/li\u003e\n\u003cli\u003eKonopleva M, Martinelli G, Daver N, Papayannidis C, Wei A, Higgins B et al. MDM2 inhibition: an important step forward in cancer therapy. Leukemia 2020; 34: 2858-2874.\u003c/li\u003e\n\u003cli\u003eWang S, Zhao Y, Aguilar A, Bernard D, Yang CY. Targeting the MDM2-p53 Protein-Protein Interaction for New Cancer Therapy: Progress and Challenges. Cold Spring Harb Perspect Med 2017; 7.\u003c/li\u003e\n\u003cli\u003eOsada M, Park HL, Park MJ, Liu JW, Wu G, Trink B et al. A p53-type response element in the GDF15 promoter confers high specificity for p53 activation. Biochem Biophys Res Commun 2007; 354: 913-918.\u003c/li\u003e\n\u003cli\u003eWen J, Wang D. Deciphering the PTM codes of the tumor suppressor p53. J Mol Cell Biol 2022; 13: 774-785.\u003c/li\u003e\n\u003cli\u003eZhu H, Gao H, Ji Y, Zhou Q, Du Z, Tian L et al. Targeting p53-MDM2 interaction by small-molecule inhibitors: learning from MDM2 inhibitors in clinical trials. J Hematol Oncol 2022; 15: 91.\u003c/li\u003e\n\u003cli\u003eWang S, Chen FE. Small-molecule MDM2 inhibitors in clinical trials for cancer therapy. Eur J Med Chem 2022; 236: 114334.\u003c/li\u003e\n\u003cli\u003eYamamoto N, Tolcher A, Hafez N, Lugowska I, Ramlau R, Macarulla T et al. Efficacy and Safety of the MDM2-p53 Antagonist Brigimadlin (BI 907828) in Patients with Advanced Biliary Tract Cancer: A Case Series. Onco Targets Ther 2024; 17: 267-280.\u003c/li\u003e\n\u003cli\u003eStein EM, DeAngelo DJ, Chromik J, Chatterjee M, Bauer S, Lin CC et al. Results from a First-in-Human Phase I Study of Siremadlin (HDM201) in Patients with Advanced Wild-Type TP53 Solid Tumors and Acute Leukemia. Clin Cancer Res 2022; 28: 870-881.\u003c/li\u003e\n\u003cli\u003eBlotner S, Chen LC, Ferlini C, Zhi J. Phase 1 summary of plasma concentration-QTc analysis for idasanutlin, an MDM2 antagonist, in patients with advanced solid tumors and AML. Cancer Chemother Pharmacol 2018; 81: 597-607.\u003c/li\u003e\n\u003cli\u003eIancu-Rubin C, Mosoyan G, Glenn K, Gordon RE, Nichols GL, Hoffman R. Activation of p53 by the MDM2 inhibitor RG7112 impairs thrombopoiesis. Exp Hematol 2014; 42: 137-145 e135.\u003c/li\u003e\n\u003cli\u003eSulkowski PL, Corso CD, Robinson ND, Scanlon SE, Purshouse KR, Bai H et al. 2-Hydroxyglutarate produced by neomorphic IDH mutations suppresses homologous recombination and induces PARP inhibitor sensitivity. Sci Transl Med 2017; 9.\u003c/li\u003e\n\u003cli\u003eSun Y, Jiang X, Xu Y, Ayrapetov MK, Moreau LA, Whetstine JR et al. Histone H3 methylation links DNA damage detection to activation of the tumour suppressor Tip60. Nat Cell Biol 2009; 11: 1376-1382.\u003c/li\u003e\n\u003cli\u003eWang Y, Wild AT, Turcan S, Wu WH, Sigel C, Klimstra DS et al. Targeting therapeutic vulnerabilities with PARP inhibition and radiation in IDH-mutant gliomas and cholangiocarcinomas. Sci Adv 2020; 6: eaaz3221.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Intrahepatic cholangiocarcinoma (iCCA), Isocitrate dehydrogenase 1/2 (IDH1/2) mutation, Tumor protein 53 (TP53), Mouse double minute 2 (MDM2), Histone-3-lysine-4 (H3K4) methylation, Mutant isocitrate dehydrogenase 1 inhibitor, Mouse double minute 2 inhibitor","lastPublishedDoi":"10.21203/rs.3.rs-6489065/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6489065/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMutations in \u003cem\u003eisocitrate dehydrogenase 1\u003c/em\u003e (\u003cem\u003eIDH1\u003c/em\u003e\u003csup\u003e\u003cem\u003emut\u003c/em\u003e\u003c/sup\u003e) occurs in 10-25% of intrahepatic cholangiocarcinoma (iCCA) cases. \u003cem\u003eIDH1\u003c/em\u003e\u003csup\u003e\u003cem\u003emut\u003c/em\u003e\u003c/sup\u003e 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 \u003cem\u003eIDH1\u003c/em\u003e\u003csup\u003e\u003cem\u003emut\u003c/em\u003e\u003c/sup\u003e iCCA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe used \u003cem\u003ein-silico\u003c/em\u003e 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\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur \u003cem\u003ein-silico\u003c/em\u003e analysis demonstrated that \u003cem\u003eIDH1\u003c/em\u003e and \u003cem\u003eTP53\u003c/em\u003e mutations were mutually exclusive in iCCA tumors and cell-lines, and \u003cem\u003eIDH1\u003c/em\u003e\u003csup\u003e\u003cem\u003emut\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/TP53\u003c/em\u003e\u003csup\u003e\u003cem\u003ewt\u003c/em\u003e\u003c/sup\u003e iCCA cells expressed higher MDM2 levels than \u003cem\u003eIDH1\u003c/em\u003e\u003csup\u003e\u003cem\u003ewt\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/TP53\u003c/em\u003e\u003csup\u003e\u003cem\u003emut\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003eiCCA 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 \u003cem\u003eMDM2\u003c/em\u003e promoter in \u003cem\u003eIDH1\u003c/em\u003e\u003csup\u003e\u003cem\u003emut\u003c/em\u003e\u003c/sup\u003e 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 \u003cem\u003eMDM2\u003c/em\u003e promoter, which was accompanied by a reduction in MDM2 expression and an increase in wild-type TP53 (wtTP53) protein levels in \u003cem\u003eIDH1\u003c/em\u003e\u003csup\u003e\u003cem\u003emut\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/TP53\u003c/em\u003e\u003csup\u003e\u003cem\u003ewt\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003eiCCA cells. The effect of mIDH1 inhibitor on \u003cem\u003eMDM2\u003c/em\u003e 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\u003cem\u003e IDH1\u003c/em\u003e\u003csup\u003e\u003cem\u003emut\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/TP53\u003c/em\u003e\u003csup\u003e\u003cem\u003ewt\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003eiCCA cells. The combination of mIDH1 and MDM2 inhibitors synergistically suppressed the proliferation of \u003cem\u003eIDH1\u003c/em\u003e\u003csup\u003e\u003cem\u003emut\u003c/em\u003e\u003c/sup\u003e iCCA cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur study delineated a novel mIDH1-MDM2-wtTP53 axis and the potential application of MDM2 inhibitor therapy in IDH1mut/TP53wt iCCA.\u003c/p\u003e","manuscriptTitle":"Isocitrate dehydrogenase 1 mutation sensitizes intrahepatic cholangiocarcinoma cells to MDM2 inhibitors","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-03 07:36:17","doi":"10.21203/rs.3.rs-6489065/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f32acbdc-96b0-4f3f-9ca5-e6eb23a15dcc","owner":[],"postedDate":"June 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-06-14T03:08:10+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-03 07:36:17","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6489065","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6489065","identity":"rs-6489065","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}