Generation, characterization, and drug sensitivities of 12 patient-derived IDH1-mutant glioma cell cultures

Abstract

Background Mutations of the isocitrate dehydrogenase (IDH) gene occur in over 80% of low-grade gliomas and secondary glioblastomas. Despite considerable efforts, endogenous in vitro IDH-mutated glioma models remain scarce. Availability of these models is key for the development of new therapeutic interventions. Methods Cell cultures were established from fresh tumor material and expanded in serum-free culture media. D-2-Hydroxyglutarate levels were determined by mass-spectrometry. Genomic and transcriptomic profiling were carried out on the Illumina Novaseq platform, methylation profiling was performed with the Infinium MethylationEpic BeadChip array. Mitochondrial respiration was measured with the Seahorse XF24 Analyzer. Drug screens were performed with an NIH FDA-approved anti-cancer drug set and two IDH-mutant specific inhibitors. Results A set of twelve patient-derived IDHmt cell cultures was established. We confirmed high concordance in driver mutations, copy number and methylation profiles between the tumors and derived cultures. Homozygous deletion of CDKN2A/B was observed in all cultures. IDH-mutant cultures had lower mitochondrial reserve capacity. IDH-mutant specific inhibitors did not affect cell viability or global gene expression. Screening of 107 FDA-approved anti-cancer drugs identified nine compounds with potent activity against IDHmt gliomas, including three compounds with favorable pharmacokinetic characteristics for CNS penetration: teniposide, omacetaxine mepesuccinate, and marizomib. Conclusions Our twelve IDH-mutant cell cultures show high similarity to the parental tissues and offer a unique tool to study the biology and drug sensitivities of high-grade IDHmt gliomas in vitro. Our drug screening studies reveal lack of sensitivity to IDHmt inhibitors, but sensitivity to a set of nine available anti-cancer agents.

Effects of the IDH1 R132H Mutation on the Energy Metabolism: A Comparison between Tissue and Corresponding Primary Glioma Cell Cultures

“…During characterization of the glioma cultures, a similar observation was made at the RNA level. 14 In this characterization study, RNAseq results showed minimal changes between IDH1 wt and IDH1 mut cultures for the genes related to the proteins in our metabolic proteomics panel. In addition, a correlation between the RNAseq and proteomics data was observed.…”

“…This shows that even though limited significant changes were observed in the metabolic panel, the proteome as a whole is quite different between the two sample groups. A further description of the differences between the IDH1 wt and IDH1 mut primary cell cultures is published by Verheul et al 14 To confirm the IDH1 mutation in the samples, a PRM analysis was performed using several peptides from IDH1, including peptides with and without the mutation. The IDH1 mutation changes the arginine (R) on the wild-type "PIIIGR" peptide of which the equivalent in the IDH1 R132H mutated form is peptide "PIIIGHHAYGDQYR" (Figure 4).…”

“…In our laboratory, a culturing procedure has been developed to reliably obtain primary glioma cultures from glioma tissues, also with the IDH1 mut. 14 We showed that the IDH1 mutation is retained in a set of 12 patient-derived glioma cultures. These cultures can easily be used in drug screening experiments.…”

Metabolic adaptations in cancers expressing isocitrate dehydrogenase mutations

“… 101 , 102 , 106 , 177 , 178 , 179 These cell lines provide relatively stable models to study the effects of the presence of the mutIDH1/2 enzymes, but it is possible that the process of producing the model itself may have unknown metabolic consequences and that these models do not account for some genetic and, subsequently, metabolic differences between WT IDH and mutIDH1/2 gliomas. 180 , 181 , 182 A limited number of glioma cell lines that endogenously express mutIDH have been successfully cultured from grade II astrocytomas, 183 grade III gliomas, and what were formerly known as secondary GBMs. 174 , 183 , 184 , 185 , 186 PDX mouse models bearing cells with IDH1/2 mutations derived from affected individuals are potentially more physiologically relevant than cell culture using immortalized cell lines.…”

“… 180 , 181 , 182 A limited number of glioma cell lines that endogenously express mutIDH have been successfully cultured from grade II astrocytomas, 183 grade III gliomas, and what were formerly known as secondary GBMs. 174 , 183 , 184 , 185 , 186 PDX mouse models bearing cells with IDH1/2 mutations derived from affected individuals are potentially more physiologically relevant than cell culture using immortalized cell lines. 181 , 187 , 188 Several PDX-specific mutIDH1 glioma cell lines have been established, 99 , 188 , 189 but in comparison with cultured cells, these can be less practical and straightforward to work with.…”