Abstract 92: Acquisition of temozolomide resistance: Identification of a new drug tolerant stage in glioblastoma cells

Abstract

Abstract Glioblastoma multiforme (GBM) are the most aggressive and common brain tumors in adults. Despite surgery and combined radio-chemotherapy with temozolomide (TMZ), tumor reccurence always occurs. The median survival time for patients diagnosed with GBM is about 14 months with less than 5% survival at 5 years. Today the main marker of TMZ resistance is the methylation status of MGMT promoter. Patients with a methylated promoter usually have a better response to treatment than patients with an unmethylated promoter. Indeed MGMT is an enzyme involved in DNA repair mechanisms that abrogates TMZ effects. However, in clinical trials targeting the MGMT enzyme, median survival of patients was not improved. It is thus essential to decipher the mechanisms involved in the acquisition of TMZ resistance to identify new therapeutic targets. To achieve this goal TMZ resistant cells were generated by continuous treatment of the U251 human glioblastoma cell line. These cells are sensitive to TMZ and do not express MGMT. We performed transcriptomic analysis by RNA-Seq on U251 treated with TMZ (50µM) for different time and selected differentially expressed genes. We also evaluated target genes expression in single cells by RT-qPCR using C1-HD-Biomark technology (Fluidigm). Transcriptome profiling allowed to identify a transient phase with TMZ tolerant cells before acquisition of complete resistance. These cells are characterized by a modified morphology and a no proliferative state. In this population we identified a subset of genes with the same transient overexpression. Similar results were observed in other glioblastoma cell lines and under other stress conditions. In contrast, stable expression of MGMT appeared later with the emergence of the TMZ resistant population. Interestingly single cell qPCR showed that MGMT expression in the resistant cells could not be explained by clonal selection of MGMT positive cells. Drug screening on the TMZ tolerant cells revealed a potent killing activity of histone deacetylating agents (HDAC inhibitors). In conclusion, we have shown that glioblastoma cells become resistant after a transient state of TMZ tolerance. Identification of this singular population highlights new molecular targets and a new therapeutic window. Targeting these tolerant cells could avoid emergence of resistance and tumor recurrence, thereby patients survival could be improved. Citation Format: Marion Rabé, Hicham Janati, Solenne Dumont, Christelle Thibault-Carpentier, Jean Clairambault, François M. Vallette, Catherine Gratas. Acquisition of temozolomide resistance: Identification of a new drug tolerant stage in glioblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 92. doi:10.1158/1538-7445.AM2017-92