Mechanisms of Glioblastoma resistance to antitumoral Temozolomide in cells cultured as three-dimensional (3D) models in vitro and in vivo: The role of Translesion Synthesis polymerases

Abstract

Temozolomide (TMZ) is the most used antitumoral drug in Glioblastoma Multiforme (GBM). However, chemotherapy-resistant cells are recurrent due to many factors, and one of them is the Translesion Synthesis (TLS) mechanism, which allows tumor cells to tolerate genomic damage by bypassing DNA lesions. Specifically, TLS DNA polymerases Kappa (Polº) and Iota (Pol¹) are associated with chemotherapy resistance, but little is known about GBM resistance. To understand this mechanism, this work already analyzed the cytotoxic, antiproliferative, antimetastatic, and genotoxic effects of TMZ on GBM U251MG WTE (wild type) and CRISPR/Cas9 KO for TLS Polº and Pol¹ cells cultured as 3D tumor spheroids in vitro. The results showed that TMZ (10-200 µM) promotes antiproliferative effects, especially in TLS Polº and Pol¹ KO 3D spheroids. Besides, we observed significant cytotoxicity, cell death (Caspase 3/7), genotoxicity (53BP1), and antimetastatic (migration/invasion) effects in TLS Polº/Pol¹ KO spheroids when compared to WTE after TMZ treatments (25-200 ¼M). All previous results indicate that TLS Polº and Pol¹ are involved in TMZ chemoresistance in GBM cells, although it is necessary to understand better the mechanisms of action. Initially, we pretended to identify the mutational signatures profiles by Whole-Exome Sequencing (WES) from GBM TLS pols KO and WTE cells after TMZ treatments. Besides, such 3D models can demonstrate similar pathophysiological responses in vivo, establishing xenograft tumors from GBM cells (WTE and TLS Pols KO) and analyzing the mutational profile after TMZ treatments can clarify the biological responses between both models. Finally, as glioma stem cells (GSC) are described as responsible for TMZ resistance and TLS pols can be overexpressed in this cell type, we aimed to develop/establish for the first time the 3D tumor organoids or "tumoroids" model from GBM (U251MG) cells that expressing GSC in vitro can help to understand the TMZ resistance. This innovative 3D cell culture approach represents a significant advancement over methods previously employed in the laboratory, expanding the scope of GBM research. Using 3D tumoroids from U251MG cells (WTE and TLS Pol¹/Polº KO) rich in GSC, we intend to evaluate the effects of TMZ on diameter/morphology and, using immunofluorescence, analyze stem-cell markers (SOX2 and OCT4), genotoxicity (H2Ax) and cycle cellular (using the Fast-FUCCI reporter plasmid) The BEPE Internship is being submitted to execute all mentioned objectives in collaboration with Dr. Chunzhang Yang's research group at the Neuro-Oncology Branch (National Institute of Health - NIH /USA) for 12 months (January/2024 - December/2024). The NCI/NIH has facilities equipped with cutting-edge technology and state-of-the-art infrastructure to carry out genomic, bioinformatics, and in vivo analyses, with time efficiency and costs significantly lower than those in Brazil. Dr. Chunzhang has excellent experience in studies of gliomas and mechanisms of resistance to TMZ chemotherapy and utilizes molecular biology techniques and preclinical models to understand the molecular pathways (including DNA Repair) in brain tumors. We expect that the results will help us better understand the role of TLS pols in the resistance TMZ, indicating a potential target for treating GBM patients.