Abstract 3741: Development and characterization of a cell based assay for the validation of mutant IDH1 inhibitors
Authors: Fer Nicole D. , Harris Erik , Fox Stephen , Zhou Ming , Simpson Catherine , Liu Jing , Korboukh Ilia , Hull-Ryde Emily A. , Janzen William P. , Frye Stephen V. , Monks Anne , Teicher Beverly , Rapisarda Annamaria 
Published: May 2015

Abstract
Recurrent isocitrate dehydrogenase (IDH) mutations have recently been identified in several cancer types. These point mutations specifically affect IDH1 and IDH2 active site arginine residues and confer the ability to reduce α-ketoglutarate to R-2-hydroxyglutarate (2HG), an oncometabolite that competitively inhibits α-ketoglutarate-dependent enzymes, such as histone and DNA demethylases. Accumulating evidence indicates that mIDH enzymes are attractive targets for the development of novel cancer therapeutics. To validate potential mIDH inhibitors in a cellular assay, we acquired several cell lines that harbor mIDH1 R132H and R132C: HCT116 mIDH1 (colon cancer, mIDH1 R132H +/-, Horizon Discovery), U87 MG mIDH1 (glioma, m IDH1 R132H +/+, Japan), HT1080 (fibrosarcoma, mIDH1 R132C +/-) and JJ012 (chondrosarcoma, mIDH1 R132C +/-, Rush University Medical Center). Cell lines were sequenced to confirm the presence of mIDH1 and 2HG production was measured in cellular supernatants by GC-MS. We observed that cells harboring mIDH1 R132C produce higher amounts of 2HG compared to lines harboring mIDH1 R132H, in agreement with recent reports. In addition, we show that accumulation of 2HG is time dependent and that its half-life in cellular supernatant is over 48 hours. Moreover, we demonstrate that 2HG is a stable analyte in media (through five sample free-thaw cycles) and that our samples are within linear range, providing a good intra-assay and inter-assay variability (CV &lt;10%). Using this assay we evaluated the impact of five reference compounds obtained from published structures (Agios Pharmaceuticals, Cambridge MA), on the accumulation of 2HG in the supernatant of HT1080. These compounds were reported to have different ranges of activity against mIDH1 R132H and R132C enzymes in in vitro assays. Four of the mIDH reference compounds significantly inhibited 2HG accumulation in HT1080 supernatants in a dose dependent fashion, with potencies that were comparable to enzyme inhibition values. In agreement with previous reports, none of compounds affected HT1080 proliferation over a 48 hours incubation. In conclusion, we have identified mIDH cellular models that produce differing concentrations of 2HG, detectable by a GC/MS assay with sensitivity sufficient to determine levels as low as 0.1μg/ml. We have tested several inhibitors of the mIDH enzyme and confirmed an association between in vitro enzyme inhibition and a decrease in 2HG accumulation; therefore this assay can be used for the cellular evaluation of novel mIDH1 inhibitors.
Funded by NCI Contract No. HHSN261200800001E. This research was supported [in part] by the Developmental Therapeutics Program in the Division of Cancer Treatment and Diagnosis of the National Cancer Institute.
Citation Format: Nicole D. Fer, Erik Harris, Stephen Fox, Ming Zhou, Catherine Simpson, Jing Liu, Ilia Korboukh, Emily A. Hull-Ryde, William P. Janzen, Stephen V. Frye, Anne Monks, Beverly Teicher, Annamaria Rapisarda. Development and characterization of a cell based assay for the validation of mutant IDH1 inhibitors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3741. doi:10.1158/1538-7445.AM2014-3741