Abstract 3106: Generation and characterization of IDH1R132H and IDH2R140Q in vitro models for drug discovery and development

Abstract

Abstract Isocitrate dehydrogenase (IDH) is a metabolic enzyme that converts isocitrate to α-ketoglutarate. This process leads to NADPH production, and is involved in the control of oxidative cellular damage. Mutations in this enzyme have been linked to human cancers such as glioma and acute myeloid leukemia (AML). While there are three isoforms of IDH, mutations that lead to cancer have only been identified in IDH1 and IDH2, which result in simultaneous loss of normal catalytic activity, the production of α-ketoglutarate (α-KG), and gain of a new function, the production of 2-hydroxyglutarate (2-HG). 2-HG is structurally similar to α-KG, and acts as an α-KG antagonist to competitively inhibit multiple α-KG-dependent dioxygenases, including lysine histone demethylases (KDM) and DNA hydroxylases (TET), causing widespread changes in histone and DNA methylation and potentially promoting tumorigenesis. A number of pre-clinical and clinical studies from the mutant IDH inhibitors indicate that IDH is a valid target for a new class of cancer therapeutics. However, there is a lack of well-established and characterized in vitro models containing IDH mutants. The most prominent IDH1 mutation takes place at residue R132H and plays a role in the development of gliomas, while the majority of IDH2 mutations take place at residue R140Q, linked to AML. Given the prevalence of these mutations, we sought to use CRISPR/Cas9 gene editing technology to create two in vitro disease models harboring either the IDH1 or IDH2 mutations. To develop the most clinically relevant models we selected highly appropriate parental cell lines from our extensive portfolio. An IDH1R132H mutation was introduced in the malignant glioblastoma U-87 MG cell line, and an IDH2R140Q mutation was introduced in the TF-1 erythroblast cell line derived from an AML patient. The introduction of the IDH mutations in the respective cell lines was then confirmed via sequencing at the genomic and transcriptional levels. Furthermore, to validate the isogenic IDH mutations confer gain-of-function in vitro, we tested the intracellular and extracellular levels of 2-hydroxyglutarate (2-HG). Bio-functional evaluation data indicated that IDH1R132H U-87 MG cells showed an increase in cellular 2-HG and elevated level of histone methylation. In IDH2R140Q TF-1 cells, an increase in cellular 2-HG was also observed. In response to IDH2 specific inhibitors, AG-221 and AGI-6780, we demonstrated that IDH2R140Q TF-1 cells exhibited decreases in both cellular 2-HG and histone methylation levels. Taken together, these isogenic in vitro models are valuable tools for elucidating mechanisms involved in cancer-associated tumorigenesis and use in screening anti-cancer compounds for drug discovery and development. Citation Format: Diana Douglas, Lysa-Anne Volpe, Luping Chen, Metewo S. Enuameh, Fang Tian, Weiguo Shu. Generation and characterization of IDH1R132H and IDH2R140Q in vitro models for drug discovery and development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3106.