PO-306 Identification of chromatin modifiers regulating temozolomide resistance in glioblastoma multiforme

Abstract

IntroductionGlioblastoma multiforme (GBM) is the most common of all malignant brain tumours. Unfortunately, only 5.1% of patients survive five years post diagnosis. Therapy options for GBM patients are very limited, majority of the patients receive radiation therapy and the chemotherapeutic Temozolomide (TMZ). However, most tumours recur making therapy resistance an extremely important issue. Cancers show aberrant global epigenetic alterations, yet, the epigenetic mechanisms that cause therapy resistance are not well-known. In this project, the role of chromatin modifying proteins in TMZ-resistance is studied by a chemical screen.Material and methodsWe established TMZ resistant cell lines by two different methodologies, 1) dose escalation method and 2) high dose TMZ selection. For the dose escalation method, U373 cells were treated TMZ every two days, starting from 25 µM. Every two weeks, dosage was doubled (or when cells become resistant to that dosage) up to 250 µM. For high dose TMZ resistant selection, cells were treated with 250 µM TMZ for 15 days. Both cells lines have been kept under TMZ treatment for 3 months. To identify chemical inhibitors that target resistant cells, the TMZ-resistant cells and their parental controls were treated with a chemical library consisting of 90 epigenetic drugs against chromatin modifiers and cell viability was measured after 72 hours.Results and discussionsWe successfully generated isogenic subpopulations of GBM cell lines that are resistant to TMZ. We observed that the high dosage TMZ treatment regimen caused a more sustainable resistant cell line compared to dose escalation regimen. Both resistant cell line models were stable, as they did not depend on prolonged TMZ treatment and remained resistant to TMZ after 3 months of drug holiday.We then screened for epigenetic compounds that target TMZ-resistant cells. In addition to PARP inhibitors, Olaparib and Rucaparib, we identified Histone Deacetylase (HDAC) inhibitors as TMZ-sensitising agents. We are currently delineating the epigenetic alterations between parental and TMZ-resistant cells and assess the role of HDAC-mediated changes in TMZ-response by RNA-seq.ConclusionHistone acetylation and deacetylation are interesting focus areas in drug resistance because of the central role of these modifications in many aspects of cell physiology and pathology. In our screen, we have shown that various HDAC inhibitors can sensitise GBM cells to TMZ in established cells as well as our newly generated in acquired TMZ resistance models.