Mutant IDH inhibits HNF-4α to block hepatocyte differentiation and promote biliary cancer

Abstract

Mutations in isocitrate dehydrogenase 1 (IDH1) and IDH2 are among the most common genetic alterations in intrahepatic cholangiocarcinoma (IHCC), a deadly liver cancer. Mutant IDH proteins in IHCC and other malignancies acquire an abnormal enzymatic activity allowing them to convert α-ketoglutarate (αKG) to 2-hydroxyglutarate (2HG), which inhibits the activity of multiple αKG-dependent dioxygenases, and results in alterations in cell differentiation, survival, and extracellular matrix maturation. However, the molecular pathways by which IDH mutations lead to tumour formation remain unclear. Here we show that mutant IDH blocks liver progenitor cells from undergoing hepatocyte differentiation through the production of 2HG and suppression of HNF-4α, a master regulator of hepatocyte identity and quiescence. Correspondingly, genetically engineered mouse models expressing mutant IDH in the adult liver show an aberrant response to hepatic injury, characterized by HNF-4α silencing, impaired hepatocyte differentiation, and markedly elevated levels of cell proliferation. Moreover, IDH and Kras mutations, genetic alterations that co-exist in a subset of human IHCCs, cooperate to drive the expansion of liver progenitor cells, development of premalignant biliary lesions, and progression to metastatic IHCC. These studies provide a functional link between IDH mutations, hepatic cell fate, and IHCC pathogenesis, and present a novel genetically engineered mouse model of IDH-driven malignancy.

Extended Data Figure 1. Impact of mutant IDH1 and IDH2 on HB cell differentiation

a. Primary HB cells were engineered to express the indicated human IDH alleles or empty vector (EV) under a Dox-inducible system. Lysates from HB cells cultured in the presence of increasing Dox concentrations were analyzed by immunoblot using an antibody that recognizes both murine and human IDH1 (Upper panel) or with an antibody specific to human IDH2 (lower panel); actin is the loading control. Note that 25 ng/ml Dox induces physiologic levels of IDH1 expression and was used in the experiments shown in Fig. 1 and 2 of the main text. b. Photomicrographs of cells grown for 2 days on collagen-coated plates in the presence of 25ng/mL Dox. c. HB cells cultured in the presence of increasing Dox concentrations were analyzed by LC-MS for levels of intracellular 2HG. d. Growth curve of HBs cultivated on collagen-coated dishes. e, f. HB cells from (a) were grown on collagen-coated or uncoated plates and analyzed for expression of hepatocyte markers by qRT-PCR. g. Enrichment plot showing downregulation of a hepatocyte gene set (Gene Expression Omnibus, GSE28892) in R132C-expressing cells. NES = Normalized Enrichment Score. FDR = False discovery rate. h. 2HG levels in HB cells expressing the indicated IDH alleles or EV and treated with AGI-5027 (+) or DMSO vehicle (-). i, j. WT HBs were treated with 500µm octyl-(R) or (S) enantiomers of 2HG or DMSO vehicle, and tested for hepatocyte differentiation upon transfer to uncoated plates by hepatocyte sphere formation (i) or qRT-PCR (j). k, l. HB cells expressing the indicated alleles were tested for biliary differentiation upon transfer to matrigel as assessed by qRT-PCR for the induction of biliary makers Bgp and Ggt1. Scale bars, 100µm. Error bars indicate ±s.d. between technical duplicates. * Student’s t-test P<0.05. Data are representative of at least 2 independent experiments.

Extended Data Figure 2. Mutant IDH represses the HNF4α-mediated hepatocyte differentiation program

a–c. Gene expression profiling of HB cells expressing the indicated alleles and grown on collagen. (a) Clustering analysis, (b) enrichment plot (GSEA) showing downregulation of HNF4α targets in the IDH mutant cells relative to controls, (c) GSEA plots using the collection of cis regulatory elements from the Molecular Signatures Database (C3 collection) reveals strong downregulation of genes containing consensus HNF4α and HNF1α binding sites in the IDH mutant HB cultures. d. qRT-PCR showing expression of Hnf4a and its target genes in HB cells expressing either EV or IDH2 R172K and grown on collagen-coated plates. e. HNF4α(7–9) expression (immunoblot) in HB cells grown on collagen-coated plates (quantification of HNF4α:actin is indicated). Data are representative of 2 independent experiments. f, g. Immunofluorescence (IF) staining for HNF4α (using an antibody that detects all isoforms) in HB cells expressing EV or IDH1 R132C and grown on collagen (f). The chart shows quantification of the ratio of the HNF4α and DAPI signals. The specificity of the antibody was demonstrated by the diminished staining in cells with knockdown of endogenous HNF4α (g). h. Immunoblot showing that R132C suppresses HNF4α(1–6) and HNF4α(7–9) isoforms on uncoated plates. i, j. AGI-5027 restores Hnf4a(1–6) induction in R132C-expressing HBs as shown by qRT-PCR (i) and immunoblot (j). DMSO=vehicle. k. WT HBs were treated with 500µm octyl-(R) or -(S) enantiomers of 2HG, DMSO vehicle, or media alone. Hnf4a1–6 levels were determined by qRT-PCR. l, m. Cultures of HB cells expressing the indicated alleles were grown on collagen or transferred to uncoated plates for 5 days and subjected to chromatin immunoprecipitation (ChIP) for H3K4me3 (l) or H3K27me3 (m). Enrichment for the promoter regions of Hhex (highly expressed gene in HB cells), Hoxa10 (transcriptionally silent in HB cells) and P1 Hnf4a was measured by qPCR. n, o. Analysis of WT HBs expressing shRNA control (shCTL) or targeting different HNF4a sequences (shHnf4a#1, shHnf4a#2): (n) hepatocyte marker expression (qRT-PCR) (o) HNF4a immunoblot. Error bars indicate ±s.e.m for (f) and ±s.d. for (d, i, k-n) between technical duplicates. Scale bars indicate 50 µm. Student’s t-test * P<0.05.

Extended Data Figure 3. HNF4 α is dispensable for biliary differentiation

a. WT HB cells expressing shRNA control (shCTL) or targeting HNF4α were tested for ability to undergo biliary differentiation upon transfer to matrigel as assessed by tubule formation 24h after transfer (left) and qRT-PCR for induction of the biliary markers Bgp and Ggt1 10 days after transfer to matrigel (right). b. Immunoblot showing ectopic expression of HNF4α1 in HB cells. Error bars indicate ±s.d. between technical replicates. Scale bar indicates 100µm.

Extended Data Figure 4. Analysis of GEM model with hepatocyte-specific mutant IDH expression

a. Schematic of Dox-inducible IDH2 mutant alleles (See Methods for details). Mice harboring mutant human IDH2 alleles were crossed with Alb-Cre and LSL-rtTA strains for liver-specific expression. b–d. Characterization of expression pattern of mutant IDH2 in Tet-IDH2 R140Q and Tet-R172K GEM models compared to control WT mice, after 4 weeks of doxycycline supplementation reveals hepatocyte-specific expression, consistent with previous models using this transgenic targeting system. (b) IF analysis of Tet-IDH2 R140Q and control WT livers using an antibody that detects both endogenous and transgenic IDH2 expression. Note that transgenic IDH2 R140Q is expressed in the hepatocytes which stain for HNF4α(1–6), but not in the bile ducts which are marked by CK19. (c) IF analysis of Tet-IDH2 R140Q and control WT livers using an antibody that is specific to IDH2 R140Q, showing an identical pattern of transgene expression. (d) IF analysis of Tet-IDH2 R172K and control WT livers using an antibody that is specific to IDH2 R172K. This allele is also expressed in the hepatocytes, but shows more focal expression compared to R140Q. In (b) and (c) an antibody to total HNF4α was used to label hepatocytes. Scale bars = 50µm.

Extended Data Figure 5. Characterization GEM model with hepatocyte-specific mutant IDH expression in the presence or absence of liver injury

a. Measurement of 2HG levels in liver lysates from IDH2 mutant and control mice treated with Dox for 1 month. b. Uninjured WT and IDH2 R140Q livers exhibit comparable expression of the hepatocyte markers, Hnf4a, Adh1, Alb, and Aldob and biliary markers Sprr1a and Onecut1 by qRT-PCR. c-d. Tet-R140Q mice and littermate controls (WT) receiving Dox were fed a DDC-containing diet for 5 days before being switched to normal chow for 1 week (See main text, Fig. 3a for schematic). Mutant IDH2 does not provoke liver injury as reflected by (c) comparable levels of serum AST, Tbili and ALT, and (d) absence of cleaved caspase 3 staining in Tet-R140Q compared to WT mice. Duodenum from a TNFα-treated mouse was used as a positive control for cleaved caspase-3 staining. Scale bars = 50µm.

Extended Data Figure 6. Characterization of response to liver injury in GEM model with hepatocyte-specific mutant IDH expression

Tet-R140Q or Tet-R172K mice and littermate controls (WT) receiving Dox were fed a DDC-containing diet for 5 days before being switched to normal chow for 7 or 21 days (See main text, Fig. 3a for schematic). a. Representative H&E images of livers from WT and Tet-R140Q mice analyzed at day 7 and 21. b. Quantification of Ki67-positive cells for indicated markers as shown in main text, Fig. 3e (N = 3 mice per group, from at least 5 high-powered field per mouse scored). Error bars, ±s.e.m. Data from Fig. 3f (day 21) is reproduced here for comparison. c, d.IF analysis of IDH2 mutant livers showing that Ki-67 co-localizes with HNF4α (c) and R140Q-expressing cells (d). As shown in main text, Fig. 3e, these cells express lower levels of HNF4α compared to WT hepatocytes. e, f. Tet-R172K mice and littermate controls (WT) receiving Dox were fed a DDC-containing diet for 5 days before being switched to normal chow for 1 week. IF analysis of IDH2 mutant livers shows that Ki-67 co-localizes with HNF4α and R172K-expressing cells (e) and greater numbers of Ki-67⁺,HNF4α⁺ cells in Tet-R172K compared to WT mice (f). Error bars indicate ±s.e.m. between 3 mice; Scale bars, 50µm. Student’s t-test * P<0.05.

Extended Data Figure 7. Characterization of LSL-IDH2 R172K GEMM

a. Schematic of LSL-IDH2 R172K GEMM (See Methods for details). Mice harboring the LSL-IDH2 R172K allele were crossed with the Alb-Cre strain to target expression to the liver. b. IF analysis revealed specific expression of IDH2 R172K in CK19⁺ biliary cells, whereas hepatocytes were negative. c. IF analysis showing comparable low levels of proliferation (Ki-67) in LSL-R172K and littermate control livers at 3 months of age. Scale bars = 50µm.

Extended Data Figure 8. Impact HNF4 α ablation on IHCC pathogenesis in vivo

a–c. Albumin-CreERT2; Hnf4a^fl/fl (HNF4α^fl/fl,Cre) mice were treated with diethylnitrosamine (DEN), on post-natal day 15, and subsequently administered Tamoxifen (TAM) or corn oil (CO) control at 8 months of age as described. DEN is activated to its carcinogenic form by cytochrome P450 (CYP) enzymes, including CYP2E1, specifically in hepatocytes^,. Livers were harvested two months later and analyzed by H&E, IF and IHC. In Hnf4a^fl/fl,Cre + TAM livers, HCC arises from HNF4α⁺ cells that have escaped HNF4α ablation (a), while CK19+ IHCC stains negative for HNF4α (b, right panel). Normal liver from control corn oil-treated livers is shown in (b, left panel). c. Representative IHC (top) and IF images (bottom) show marked expansion of Sox9⁺ oval cells in TAM-injected mice. Surrounding these oval cells are HNF4α⁺ hepatocytes which escaped Hnf4a ablation in TAM-injected mice. d. H&E stain confirming that the liver tumour arising in a Kras^G12D mouse is an HCC. e. Measurement of 2HG in liver tumours from Alb-Cre; LSL-R172K; Kras^G12D mice compared to normal liver from control mice (N=2). Scale bars, 50 µm. Error bars, ±s.d. * Student’s t-test P<0.05.

Extended Data Figure 9. Expression of mutant IDH2 in the LSL R172K GEM model

a, b. IF analysis to characterize expression of IDH2 in the liver of LSL-R172K; Kras^G12Dcompound mice. (a) Staining with an antibody that recognizes both endogenous WT and mutant IDH2 shows that endogenous IDH2 is expressed in bile duct in the non-diseased liver from Kras^G12D mice (top panels). In LSL-R172K; Kras^G12D mice, total IDH2 staining is at near endogenous levels in the normal bile ducts (middle panels) and in the BilIN lesions (bottom panels). (b) IF using an antibody specific to IDH2-R172K showing specific expression of the R172K transgene in CK19⁺ bile ducts (middle row) and BilINs (bottom row) in LSL-R172K, Kras^G12D animals. Scale bars = 50µm.

Extended Data Figure 10. Stem cell features in IDH mutant murine and human IHCC pathogenesis

a. IDH2 R172K expression using mutant-specific antibodies in livers from LSL-R172K and Kras^G12D, R172K compound mice showing specific expression of the R172K transgene in Sox9⁺ oval cells and IHCC. Scale bars = 50µm. b. Normalized enrichment plot of a cohort of 127 human IHCC samples genotyped for IDH status, showing that the subset of tumors with IDH1/IDH2 mutations exhibits enrichment of a gene signature that identifies IHCCs with hepatic stem cell/progenitor features.

Figure 1. IDH mutant alleles block hepatocyte differentiation

a–d. Hepatoblasts (HBs) expressing empty vector (EV) or the indicated IDH alleles at physiologic levels (see Extended Data Fig. 1a), were transferred to uncoated plates to induce hepatocyte differentiation. a, Hepatocyte sphere formation. b, proliferation, c, hepatocyte marker expression (qRT-PCR). d, Heat map of hepatocyte and biliary gene expression. e,f. HB cells treated with 2.5µM AGI-5027 or DMSO vehicle. e, hepatocyte sphere formation (upper panel), proliferation (lower panel). f, hepatocyte marker expression. g. HB cells in matrigel assessed for biliary differentiation. Tubular structures/6 cm dish ± std. are quantified. *P<0.05. Scale bars, 100 µm.

Figure 2. Mutant IDH blocks hepatocyte differentiation by silencing HNF4α

a. Heat map of GSEA showing top-ranked gene-sets distinguishing IDH1-R132C or IDH2-R172K from WT or EV control HBs (pairwise analysis; replicates for each condition; see Methods). NES = Normalized enrichment score. b,c. HB cells analyzed by (b) immunoblot, and (c) qRT-PCR. d,e. Analysis of WT HBs expressing the indicated shRNAs (uncoated plates). d, Hepatocyte sphere formation. e, Proliferation of shRNA-expressing HB cells co-expressing EV or shRNA-resistant Hnf4a1 cDNA. f–h. Control and R132C–expressing HBs co-expressing vector control (EV2) or HNF4α, grown on uncoated plates. f, hepatocyte sphere formation, g, hepatocyte gene expression, h, proliferation. *P<0.05, Scale bar, 100µm (d), 250µm (f).

Figure 3. Mutant IDH inhibits hepatocyte differentiation and quiescence of liver progenitors

a. Schematic of DDC study in Tet-R140Q (Tet-R140Q, Alb-Cre, Rosa26-LSL-rtTA) and WT littermate controls (Alb-Cre, Rosa26-LSL-rtTA). b–f.Livers at day 21. b, Immunoblot (HNF4α1–6:actin is quantified). c, qRT-PCR. d, Ki-67 staining. Chart: Ki-67⁺ cells/20 high-powered fields. e,f, IF analysis. Graph: mean fluorescence intensity of HNF4α:DAPI (125 cells/group were scored). Inset: high power views of boxed regions. f, Quantification of Ki-67⁺ cells co-staining for the indicated markers (N = 3 mice/group, 5 high-powered fields/mouse). g. IHC (top) and IF (bottom) of WT and LSL-R172K livers at 20 months. Note accumulation Sox9⁺ cells located >25µm away from bile duct or portal structures (dashed-line), which express IDH2-R172K and lack HNF4α. Inset: higher magnification. Chart: quantification. N = 3 mice/group; 4 high-powered images/mouse were scored. PV=Portal vein. Error bars, ±s.d. (c) and ±s.e.m. (d, f, g); Scale bars, 20 µm (d) and 50 µm (e, g).

Figure 4. Mutant IDH cooperates with Kras^G12D to drive liver progenitor cell expansion and multi-step IHCC pathogenesis

a. Kaplan-Meier analysis showing time until signs of illness necessitated euthanasia. All animals euthanized had liver tumours. b. Upper left: Representative Alb-Cre;LSL-R172K;Kras^G12D tumor, and peritoneal and spleen metastases (insets). Upper right: H&E staining showing IHCC histology. Lower panels: The tumour is Hep Par1- and CK19⁺ while adjacent hepatocytes stain Hep Par1⁺ and CK19^-. c.The livers of Alb-Cre;LSL-R172K;Kras^G12D animals exhibit oval cell expansion and increasing grades of BilIN, which stain Sox9⁺. CK19 levels increase with higher grade lesions. IF analysis reveals focal accumulation Sox9⁺ oval cells in Alb-Cre:LSL-R172K livers and pronounced oval cell expansion in Alb-Cre;LSL-R172K; Kras^G12D livers. d. Model for mutant IDH in cholangiocarcinoma pathogenesis. Scale bars, 1cm (b, upper left), 50µm (b-c).