Evaluation of the influence of MGMT inhibition in the responses of glioblastoma cell lines to TMZ treatment combined to the PARP-1 inhibitor (NU1025)

Abstract

Glioblastoma (GBM) is an aggressive tumor with a discouraging prognosis. The main chemotherapy protocol includes the alkylating agent temozolomide (TMZ). DNA repair pathways, as the one mediated by the MGMT (O6-methylguanine-DNA methyltransferase) enzyme and the Base Excision Repair (BER), promote resistance to base lesions induced by TMZ, constituting a major obstacle to reach therapeutic efficacy. An emerging strategy is based on the intervention of DNA repair mechanisms, aiming a potential sensitization of tumor cells. A strategic target is the PARP-1 protein (Poly (ADP-Ribose) polymerase 1), participant of various DNA repair processes, especially the BER pathway. There is evidence in the literature that the inhibition of this enzyme associated with treatments used for patients with cancer lead to sensitization of tumor cells. However, the efficacy of TMZ combined to PARP inhibitors has been associated with the activity of MGMT enzyme, although its role as a marker of tumor responses remains controversial in the literature. In this research group, the current project entitled "INHIBITION OF DNA REPAIR IN GLIOBLASTOMA CELL LINES TARGETING A POTENTIAL APPLICATION AS A THERAPEUTIC STRATEGY" is the main project to which the present proposal is part, and the hypothesis to be tested is based on the role of MGMT as a predictor of the response to TMZ combined to NU1025 (PARP-1 inhibitor), in the sense that cells deficient for this enzyme are sensitive to TMZ, which induces cytotoxic lesions that accumulate in the absence of MGMT activity; on the other hand, MGMT proficient cells which are resistant to TMZ (due to their ability to repair O6-methylguanine), are sensitized by PARP-1 inhibition in association with TMZ, but this is probably due to other types of drug-induced damage, which can not be repaired (in consequence of intervention in the BER pathway), thus increasing the rates of cell death (as previously demonstrated in our group). Therefore, the aim of this project is to evaluate the influence of MGMT as a modulator of the responses of GBM cells to the combined treatment, TMZ plus NU1025, by applying a strategy of MGMT inhibition using the compound O6-BG (O6 -benzylguanine) in GBM cells that are proficient for MGMT. The parameters analyzed to characterize cellular responses are: cell viability, cell cycle kinetics, apoptosis induction, induction of DNA double strand breaks (DSB) and generation of reactive oxygen species (ROS) in mitochondria. Thus, according to our hypothesis, the inhibition of MGMT enzyme will abolish the potentiating activity of the combined treatment (NU1025 and TMZ). The project has a high potential to generate relevant information to clinical practice, which will be fundamental for the stratification of patients (according to the activity of MGMT in tumor cells), distinguishing those who have the potential to successfully respond to the combined treatment of TMZ associated with PARP inhibitors.