Generation and Characterization of IDH1R132H and IDH2R140Q In Vitro Models for Drug Discovery
AACR Annual Meeting 2018
Chicago, Illinois, United StatesApril 14, 2018
Isocitrate dehydrogenase (IDH) is a metabolic enzyme that converts isocitrate to α-ketoglutarate (α-KG). 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-KG, and the gain of a new function—the production of 2-hydroxyglutarate D-2HG. D-2HG 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 studies from the mutant IDH inhibitors indicate that IDH is a valid target for a new class of cancer therapeutics.
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, which is 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 that harbor either the IDH1 or IDH2 mutations. An IDH1R132H G ˃A mutation was introduced in the malignant glioblastoma U-87 MG (ATCC HTB-14) cell line, and an IDH2R140Q G˃A mutation was introduced in the TF-1 (ATCC CRL-2003) erythroblast cell line that was derived from an AML patient. To validate that the isogenic IDH mutations confer gain-of-function in vitro, we tested the intracellular and extracellular levels of D-2HG. Bio-functional evaluation data indicated that the IDH1R132H mutant U-87 MG Isogenic Cell Line (ATCC HTB-14IG) showed an increase in cellular D-2HG and elevated level of histone methylation. In the IDH2R140Q mutant TF-1 Isogenic Cell Line (ATCC CRL-2003IG), an increase in cellular D-2HG 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 D-2HG and histone methylation levels.
Taken together, these data demonstrate that 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.
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