Treatment with a Small Molecule Mutant IDH1 Inhibitor Suppresses Tumorigenic Activity and Decreases Production of the Oncometabolite 2-Hydroxyglutarate in Human Chondrosarcoma Cells

Chondrosarcomas are malignant bone tumors that produce cartilaginous matrix. Mutations in isocitrate dehydrogenase enzymes (IDH1/2) were recently described in several cancers including chondrosarcomas. The IDH1 inhibitor AGI-5198 abrogates the ability of mutant IDH1 to produce the oncometabolite D-2 hydroxyglutarate (D-2HG) in gliomas. We sought to determine if treatment with AGI-5198 would similarly inhibit tumorigenic activity and D-2HG production in IDH1-mutant human chondrosarcoma cells. Two human chondrosarcoma cell lines, JJ012 and HT1080 with endogenous IDH1 mutations and a human chondrocyte cell line C28 with wild type IDH1 were employed in our study. Mutation analysis of IDH was performed by PCR-based DNA sequencing, and D-2HG was detected using tandem mass spectrometry. We confirmed that JJ012 and HT1080 harbor IDH1 R132G and R132C mutation, respectively, while C28 has no mutation. D-2HG was detectable in cell pellets and media of JJ012 and HT1080 cells, as well as plasma and urine from an IDH-mutant chondrosarcoma patient, which decreased after tumor resection. AGI-5198 treatment decreased D-2HG levels in JJ012 and HT1080 cells in a dose-dependent manner, and dramatically inhibited colony formation and migration, interrupted cell cycling, and induced apoptosis. In conclusion, our study demonstrates anti-tumor activity of a mutant IDH1 inhibitor in human chondrosarcoma cell lines, and suggests that D-2HG is a potential biomarker for IDH mutations in chondrosarcoma cells. Thus, clinical trials of mutant IDH inhibitors are warranted for patients with IDH-mutant chondrosarcomas.     

Proteomic analysis of oligodendrogliomas expressing a mutant isocitrate dehydrogenase-1

Gliomas are primary tumors of the human central nervous system with unknown mechanisms of progression. Isocitrate dehydrogenase-1 (IDH1) mutation is frequent in diffuse gliomas such as oligodendrogliomas. To gain insights into the physiopathology of oligodendrogliomas that have a better prognosis than other diffuse gliomas, we combined microdissection, 2-D DIGE and MS/MS focusing on proteome alterations associated with IDH1 mutation. We first compared tumor tissues (TT) and minimally infiltrated parenchymal tissues (MIT) of four IDH1-mutated oligodendrogliomas to verify whether proteins specific to oligodendroglioma tumor cells could be identified from one patient to another. This study resulted in identification of 68 differentially expressed proteins, with functions related to growth of tumor cells in a nervous parenchyma. We then looked for proteins distinctly expressed in TT harboring either mutant (oligodendrogliomas, n=4) or wild-type IDH1 (oligodendroglial component of malignant glio-neuronal tumors, n=4). This second analysis resulted in identification of distinct proteome patterns composed of 42 proteins. Oligodendrogliomas with a mutant IDH1 had noteworthy enhanced expression of enzymes controlling aerobic glycolysis and detoxification, and anti-apoptosis proteins. In addition, the mutant IDH1 migrated differently from the wild-type IDH1 form. Comparative proteomic analysis might thus be suitable to identify proteome alterations associated with a well-defined mutation.     

Discovery and structure-activity-relationship study of novel imidazole cyclopropyl amine analogues for mutant isocitric dehydrogenase 1 (IDH1) inhibitors

The discovery and optimization of imidazole cyclopropyl amime analogues as mutant IDH1 inhibitors via structure-based rational design were reported. The optimal compounds demonstrated both potent inhibition in IDH1^R132H enzymatic activity and 2HG production in IDH1 mutant HT1080 cell line, moderate liver microsome stability and PK properties.     

Autocrine BMP4 Signaling Enhances Tumor Aggressiveness via Promoting Wnt/β-Catenin Signaling in IDH1-mutant Gliomas

The isocitrate dehydrogenase (IDH1/2) mutations are frequent genetic abnormalities in the majority of WHO grade II/III glioma and secondary GBM. IDH1-mutated (IDH1^Mut) glioma exhibits distinctive patterns in cancer biology and metabolism. In the present study, we showed that bone morphogenetic proteins (BMP4) are significantly upregulated in IDH1^Mut glioma. Further, we demonstrated that cancer-associated BMP4 is secreted to tumor microenvironment, which enhances the tumor migration and invasion through an autocrine manner. Mechanistically, BMP4 activates its receptor and concomitant SMAD1/5/8 signaling, which potentiates Wnt/β-catenin signaling by enhancing Frizzled receptor expression. LDN-193189, a selective BMP receptor inhibitor, prolonged the overall survival of mice bearing IDH1-mutated intracranial xenografts by limiting BMP/catenin signaling. These findings demonstrate the pivotal role of BMP4 on tumor aggressiveness in IDH1^Mut gliomas, suggesting a possible therapeutic strategy for this type of malignancy.     

Wild-type IDH1 inhibits the tumor growth through degrading HIF-α in renal cell carcinoma

The purpose of our study was to explore the effect and intrinsic mechanism of wild-type IDH1 and its substrate α-KG on renal cell carcinoma (RCC). IDH1 was observed lower expression in RCC cell lines. Phenotype experiment was carried out in the wild-type IDH1 and mutant IDH1^R132H plasmid treated cell line. The results showed that the wild-type IDH1 could significantly inhibit the proliferation, migration and promote the apoptosis of RCC cell lines, which were consistent with the IDH1''s substrate α-KG. The mutant IDH1^R132H was found to lose this biological function of IDH1. Moreover, we verified the proliferation inhibition of IDH1 in vivo. In addition, we verified the correlation between IDH1 and hypoxia signal-related proteins in vitro and in vivo, specifically, IDH1 overexpression could significantly reduce the expression of HIF-1α and HIF-2α proteins and its downstream proteins (VEGF, TGF-α). Furthermore, we preliminarily verified the possibility of α-KG in the RCC''s treatment by injecting α-KG into the xenograft model. α-KG significantly reduced tumor size and weight in tumor-bearing mice. This study provided a new therapeutic target and small molecule for the study of the treatment and mechanism of RCC.     

Selective Inhibition of Mutant Isocitrate Dehydrogenase 1 (IDH1) via Disruption of a Metal Binding Network by an Allosteric Small Molecule

Cancer-associated point mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) confer a neomorphic enzymatic activity: the reduction of α-ketoglutarate to d-2-hydroxyglutaric acid, which is proposed to act as an oncogenic metabolite by inducing hypermethylation of histones and DNA. Although selective inhibitors of mutant IDH1 and IDH2 have been identified and are currently under investigation as potential cancer therapeutics, the mechanistic basis for their selectivity is not yet well understood. A high throughput screen for selective inhibitors of IDH1 bearing the oncogenic mutation R132H identified compound 1, a bis-imidazole phenol that inhibits d-2-hydroxyglutaric acid production in cells. We investigated the mode of inhibition of compound 1 and a previously published IDH1 mutant inhibitor with a different chemical scaffold. Steady-state kinetics and biophysical studies show that both of these compounds selectively inhibit mutant IDH1 by binding to an allosteric site and that inhibition is competitive with respect to Mg²⁺. A crystal structure of compound 1 complexed with R132H IDH1 indicates that the inhibitor binds at the dimer interface and makes direct contact with a residue involved in binding of the catalytically essential divalent cation. These results show that targeting a divalent cation binding residue can enable selective inhibition of mutant IDH1 and suggest that differences in magnesium binding between wild-type and mutant enzymes may contribute to the inhibitors'' selectivity for the mutant enzyme.     

Design and synthesis of novel 2-arylbenzimidazoles as selective mutant isocitrate dehydrogenase 2 R140Q inhibitors

A series of novel 2-arylbenzimidazoles have been designed, synthesized and evaluated for their inhibitory activity against IDH2 R140Q mutant. The preliminary results indicated that four compounds 7b, 7c, 7m and 7r displayed the potent inhibitory activity against IDH2 R140Q mutant. Among them, compound 7c showed the highest inhibitory activity, with the IC₅₀ value of 0.26 μM, which was more active than positive control enasidenib. The exquisite selectivity of 7c for IDH2 R140Q mutant isoform was demonstrated by the poor activity against the IDH1 R132C mutant, IDH1 R132H mutant, wild-type IDH1, IDH2 R172K mutant and the wild-type IDH2.     

Metabolic Reprogramming in Mutant IDH1 Glioma Cells

BackgroundMutations in isocitrate dehydrogenase (IDH) 1 have been reported in over 70% of low-grade gliomas and secondary glioblastomas. IDH1 is the enzyme that catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate while mutant IDH1 catalyzes the conversion of α-ketoglutarate into 2-hydroxyglutarate. These mutations are associated with the accumulation of 2-hydroxyglutarate within the tumor and are believed to be one of the earliest events in the development of low-grade gliomas. The goal of this work was to determine whether the IDH1 mutation leads to additional magnetic resonance spectroscopy (MRS)–detectable changes in the cellular metabolome.MethodsTwo genetically engineered cell models were investigated, a U87-based model and an E6/E7/hTERT immortalized normal human astrocyte (NHA)-based model. For both models, wild-type IDH1 cells were generated by transduction with a lentiviral vector coding for the wild-type IDH1 gene while mutant IDH1 cells were generated by transduction with a lentiviral vector coding for the R132H IDH1 mutant gene. Metabolites were extracted from the cells using the dual-phase extraction method and analyzed by ¹H-MRS. Principal Component Analysis was used to analyze the MRS data.ResultsPrincipal Component Analysis clearly discriminated between wild-type and mutant IDH1 cells. Analysis of the loading plots revealed significant metabolic changes associated with the IDH1 mutation. Specifically, a significant drop in the concentration of glutamate, lactate and phosphocholine as well as the expected elevation in 2-hydroxyglutarate were observed in mutant IDH1 cells when compared to their wild-type counterparts.ConclusionThe IDH1 mutation leads to several, potentially translatable MRS-detectable metabolic changes beyond the production of 2-hydroxyglutarate.     

Discovery and structure-activity-relationship study of novel conformationally restricted indane analogues for mutant isocitric dehydrogenase 1 (IDH1) inhibitors

The discovery and optimization of various of indane amides as mutant IDH1 inhibitors via structure-based rational design were reported. The optimal compounds demonstrated both potent inhibition in IDH1^R132H enzymatic activity and 2HG production in IDH1 mutant HT1080 cell line, favorable PK properties and great selectivity against IDH1^wt and IDH2^R140Q.     

IDH1 mutation identified in one human melanoma metastasis, but not correlated with metastases to the brain

Isocitrate dehydrogenase 1 (IDH1) and isocitrate dehydrogenase 2 (IDH2) are enzymes which convert isocitrate to α-ketoglutarate while reducing nicotinamide adenine dinucleotide phosphate (NADP + to NADPH). IDH1/2 were recently identified as mutated in a large percentage of progressive gliomas. These mutations occur at IDH1^R132 or the homologous IDH2^R172. Melanomas share some genetic features with IDH1/2-mutated gliomas, such as frequent TP53 mutation. We sought to test whether melanoma is associated with IDH1/2 mutations. Seventy-eight human melanoma samples were analyzed for IDH1^R132 and IDH2^R172 mutation status. A somatic, heterozygous IDH1 c.C394T (p.R132C) mutation was identified in one human melanoma metastasis to the lung. Having identified this mutation in one metastasis, we sought to test the hypothesis that certain selective pressures in the brain environment may specifically favor the cell growth or survival of tumor cells with mutations in IDH1/2, regardless of primary tumor site. To address this, we analyzed IDH1^R132 and IDH2^R172 mutation status 53 metastatic brain tumors, including nine melanoma metastases. Results revealed no mutations in any samples. This lack of mutations would suggest that mutations in IDH1^R132 or IDH2^R172 may be necessary for the formation of tumors in a cell-lineage dependent manner, with a particularly strong selective pressure for mutations in progressive gliomas; this also suggests the lack of a particular selective pressure for growth in brain tissue in general. Studies on the cell-lineages of tumors with IDH1/2 mutations may help clarify the role of these mutations in the development of brain tumors.     

Design,synthesis and evaluation of 18F-labeled cationic carbonic anhydrase IX inhibitors for PET imaging

Carbonic anhydrase IX (CA-IX) is a marker for tumor hypoxia, and its expression is negatively correlated with patient survival. CA-IX represents a potential target for eliminating hypoxic cancers. We synthesized fluorinated cationic sulfonamide inhibitors 1–3 designed to target CA-IX. The binding affinity for CA-IX ranged from 0.22 to 0.96?μM. We evaluated compound 2 as a diagnostic PET imaging agent. Compound 2 was radiolabeled with ¹⁸F in 10?±?4% decay-corrected radiochemical yield with 85.1?±?70.3 GBq/μmol specific activity and >98% radiochemical purity. ¹⁸F-labeled 2 was stable in mouse plasma at 37?°C after 1?h incubation. PET/CT imaging was conducted at 1?h post-injection in a human colorectal cancer xenograft model. ¹⁸F-labeled 2 cleared through hepatobiliary and renal pathways. Tumor uptake was approximately 0.41?±?0.06% ID/g, with a tumor-to-muscle ratio of 1.99?±?0.25. Subsequently, tumor xenografts were visualized with moderate contrast. This study demonstrates the use of a cationic motif for conferring isoform selectively for CA-IX imaging agents.     

Fluorine-18 labeling of an anti-HER2 VHH using a residualizing prosthetic group via a strain-promoted click reaction: Chemistry and preliminary evaluation

In a previous study, we evaluated a HER2-specific single domain antibody fragment (sdAb) 2Rs15d labeled with ¹⁸F via conjugation of a residualizing prosthetic agent that was synthesized by copper-catalyzed azide-alkyne cycloaddition (CuAAC). In order to potentially increase overall efficiency and decrease the time required for labeling, we now investigate the use of a strain-promoted azide-alkyne cycloaddition (SPAAC) between the 2Rs15d sdAb, which had been pre-derivatized with an azide-containing residualizing moiety, and an ¹⁸F-labeled aza-dibenzocyclooctyne derivative. The HER2-targeted sdAb 2Rs15d and a nonspecific sdAb R3B23 were pre-conjugated with a moiety containing both azide- and guanidine functionalities. The thus derivatized sdAbs were radiolabeled with ¹⁸F using an ¹⁸F-labeled aza-dibenzocyclooctyne derivative ([¹⁸F]F-ADIBO) via SPAAC, generating the desired conjugate ([¹⁸F]RL-II-sdAb). For comparison, unmodified 2Rs15d was labeled with N-succinimidyl 4-guanidinomethyl-3-[¹²⁵I]iodobenzoate ([¹²⁵I]SGMIB), the prototypical residualizing agent for radioiodination. Radiochemical purity (RCP), immunoreactive fraction (IRF), HER2-binding affinity and cellular uptake of [¹⁸F]RL-II-2Rs15d were assessed in vitro. Paired label biodistribution of [¹⁸F]RL-II-2Rs15d and [¹²⁵I]SGMIB-2Rs15d, and microPET/CT imaging of [¹⁸F]RL-II-2Rs15d and the [¹⁸F]RL-II-R3B23 control sdAb were performed in nude mice bearing HER2-expressing SKOV-3 xenografts. A radiochemical yield of 23.9?±?6.9% (n?=?8) was achieved for the SPAAC reaction between [¹⁸F]F-ADIBO and azide-modified 2Rs15d and the RCP of the labeled sdAb was >95%. The affinity (K_d) and IRF for the binding of [¹⁸F]RL-II-2Rs15d to HER2 were 5.6?±?1.3?nM and 73.1?±?22.5% (n?=?3), respectively. The specific uptake of [¹⁸F]RL-II-2Rs15d by HER2-expressing BT474M1 breast carcinoma cells in vitro was 14–17% of the input dose at 1, 2, and 4?h, slightly higher than seen for co-incubated [¹²⁵I]SGMIB-2Rs15d. The uptake of [¹⁸F]RL-II-2Rs15d in SKOV-3 xenografts at 1?h and 2?h p.i. were 5.54?±?0.77% ID/g and 6.42?±?1.70% ID/g, respectively, slightly higher than those for co-administered [¹²⁵I]SGMIB-2Rs15d (4.80?±?0.78% ID/g and 4.78?±?1.39% ID/g). MicroPET/CT imaging with [¹⁸F]RL-II-2Rs15d at 1–3?h p.i. clearly delineated SKOV-3 tumors while no significant accumulation of activity in tumor was seen for [¹⁸F]RL-II-R3B23. With the exception of kidneys, normal tissue levels for [¹⁸F]RL-II-2Rs15d were low and cleared rapidly. To our knowledge, this is the first time SPAAC method has been used to label an sdAb with ¹⁸F, especially with residualizing functionality.     

IDH1/2 Mutations in Cancer Stem Cells and Their Implications for Differentiation Therapy

Isocitrate dehydrogenase 1 and 2 (IDH1/2) are enzymes recurrently mutated in various types of cancer, including glioma, cholangiocarcinoma, chondrosarcoma, and acute myeloid leukemia. Mutant IDH1/2 induce a block in differentiation and thereby contribute to the stemness and oncogenesis of their cells of origin. Recently, small-molecule inhibitors of mutant IDH1/2 have been Food and Drug Administration–approved for the treatment of IDH1/2-mutated acute myeloid leukemia. These inhibitors decrease the stemness of the targeted IDH1/2-mutated cancer cells and induce their differentiation to more mature cells. In this review, we elucidate the mechanisms by which mutant IDH1/2 induce a block in differentiation and the biological and clinical effects of the release into differentiation by mutant-IDH1/2 inhibitors. (J Histochem Cytochem 70:83–97, 2022)     

Identification and functional characterization of isocitrate dehydrogenase 1 (IDH1) mutations in thyroid cancer

Mutations in the genes for isocitrate dehydrogenase 1 (IDH1) and isocitrate dehydrogenase 2 (IDH2) have been recently identified in glioblastoma. In the present study, we investigated IDH1 and IDH2 mutations in follicular thyroid cancer (FTC) and anaplastic thyroid cancer (ATC), with the latter, like glioblastoma, having a rapidly aggressive and lethal clinical course. By direct genomic DNA sequencing, we analyzed exon 4 of the IDH1 and IDH2 genes that harbored the mutation hot spots codon 132 and 172 of the two genes in glioblastoma, respectively, in 12 thyroid cancer cell lines, 20 FTC, and 18 ATC tumor samples. A novel homozygous G367A IDH1 mutation, resulting in a G123R amino acid change in codon 123, was identified in a case of ATC. A previously described IDH1 V71I mutation was found in a case of FTC and a case of ATC and no mutations were found in the cell lines. The overall prevalence of mutations was thus 1/20 (5%) in FTC and 2/18 (11%) in ATC. We did not find mutation in the IDH2 gene in these thyroid cancer cell lines and tumor samples. Sequence alignment analysis of 16 species revealed that the novel IDH1 G123R mutation was located in a highly conserved region, raising the possibility of a serious functional consequence as could also be predicted by the occurrence of a positively charged amino acid from this mutation. To test this, we created a G123R mutant by site-directed mutagenesis and demonstrated a decreased enzymatic activity of IDH1, similar to the expected reduction in the enzymatic activity of the previously described R132H IDH1 mutant measured as a control. Thus, functionally relevant IDH1 mutations can also occur in thyroid cancer, particularly ATC, suggesting a potential tumorigenic role of the IDH1 system that could represent a new therapeutic target for thyroid cancer.     

Licochalcone A suppresses the proliferation of sarcoma HT-1080 cells,as a selective R132C mutant IDH1 inhibitor

IDH1 mutations are closely related to the development and progression of various human cancers, such as glioblastoma, sarcoma, and acute myeloid leukemia. By screening dozens of reported natural compounds using both wild-type and mutant IDH1 enzymatic assays, we discovered Licochalcone A is a selective inhibitor to the R132C-mutant IDH1 with an IC₅₀ value of 5.176 μM, and inhibits the proliferation of sarcoma HT-1080 cells with an IC₅₀ value of 10.75 μM. Suggested by the molecular docking results, Licochalcone A might occupy the allosteric pocket between the two monomers of IDH1 homodimer, and the R132H mutation was unfavorable for the binding of Licochalcone A with the IDH1 protein, as compared to the R132C mutation. Revealed by the RNA-Seq data analysis, the Cell Cycle pathway was the most over-represented pathway for HT-1080 cells treated with Licochalcone A. Consistent with these results, Licochalcone A induced apoptosis and cell cycle arrest of HT-1080 cells, while it showed minimal effect against the proliferation of normal RCTEC cells. The discovery of Licochalcone A as a mutation-selective IDH1 inhibitor can serve as a promising starting point for the development of mutation-selective anti-tumor lead compounds targeting IDH1.     

Glioma Cells with the IDH1 Mutation Modulate Metabolic Fractional Flux through Pyruvate Carboxylase

Background

Over 70% of low-grade gliomas carry a heterozygous R132H mutation in the gene coding for isocitrate dehydrogenase 1 (IDH1). This confers the enzyme with the novel ability to convert α-ketoglutarate to 2-hydroxyglutarate, ultimately leading to tumorigenesis. The major source of 2-hydroxyglutarate production is glutamine, which, in cancer, is also a source for tricarboxylic acid cycle (TCA) anaplerosis. An alternate source of anaplerosis is pyruvate flux via pyruvate carboxylase (PC), which is a common pathway in normal astrocytes. The goal of this study was to determine whether PC serves as a source of TCA anaplerosis in IDH1 mutant cells wherein glutamine is used for 2-hydroxyglutarate production.

Methods

Immortalized normal human astrocytes engineered to express heterozygous mutant IDH1 or wild-type IDH1 were investigated. Flux of pyruvate via PC and via pyruvate dehydrogenase (PDH) was determined by using magnetic resonance spectroscopy to probe the labeling of [2-¹³C]glucose-derived ¹³C-labeled glutamate and glutamine. Activity assays, RT-PCR and western blotting were used to probe the expression and activity of relevant enzymes. The Cancer Genome Atlas (TCGA) data was analyzed to assess the expression of enzymes in human glioma samples.

Results

Compared to wild-type cells, mutant IDH1 cells significantly increased fractional flux through PC. This was associated with a significant increase in PC activity and expression. Concurrently, PDH activity significantly decreased, likely mediated by significantly increased inhibitory PDH phosphorylation by PDH kinase 3. Consistent with the observation in cells, analysis of TCGA data indicated a significant increase in PC expression in mutant IDH-expressing human glioma samples compared to wild-type IDH.

Conclusions

Our findings suggest that changes in PC and PDH may be an important part of cellular adaptation to the IDH1 mutation and may serve as potential therapeutic targets.     

Novel allosteric properties produced by residue substitutions in the subunit interface of yeast NAD+-specific isocitrate dehydrogenase

Yeast NAD+-specific isocitrate dehydrogenase (IDH) is an octamer of four IDH1 and four IDH2 subunits, and the basic structural unit of the enzyme is an IDH1/IDH2 heterodimer. To investigate one aspect of the interaction between IDH1 and IDH2, residues in a hydrophobic region at the heterodimer interface (Val-216, Ser-220, and Val-224 in IDH1; Ile-221, Val-225, and Val-229 in IDH2) were replaced by alanine residues in each and in both subunits. Gel filtration and sedimentation velocity analyses demonstrated that the residue substitutions do not disrupt the octameric structure of IDH. However, these substitutions produce novel kinetic properties including, with respect to cofactor, positive allosteric regulation by AMP and cooperativity in the absence of AMP. These allosteric properties are also apparent in NAD+-binding experiments. Despite substantial measurable activity for the mutant enzyme containing residue substitutions in both subunits, expression of this enzyme produces growth phenotypes indicative of IDH dysfunction in vivo.