Abstract 1178: Role of error-prone polymerases on glioma cell resistance to Temozolomide

Abstract

Abstract Translesion DNA polymerases are capable of replicating damaged DNA without removing lesions, performing translesion synthesis (TLS), a mechanism known by DNA damage tolerance. Tumor cells use this mechanism in order to survive lesions caused by chemotherapy and therefore this may be a strategy that those cells use to resist treatments. Moreover, this process is error-prone and can lead to mutagenesis increasing resistance potential of tumor cells. Little is known about the role of TLS in in tumor therapy with Temozolomide (TMZ). This drug is an alkylating agent that damage DNA. Our aim is to investigate how TMZ affects TLS mutated cells, to understand how these polymerases are related to tumor cells resistance to this chemotherapeutic agent. Hence, we firstly treated POLH mutated cells and analyzed cell proliferation by flow cytometry, cell viability by a colorimetric assay (XTT) and for survival by apoptotic markers (such sub-G1) and clonogenic assays. In fact, POLH mutated cells are more sensitive to TMZ, indicating that TLS mechanisms are important to overcome DNA damage. TCGA data was also used to analyze TLS expression in glioblastoma patients. Curiously, in recurrently patients treated with TMZ, POLK expression was significantly increased. Therefore, we constructed U251-MG CRISPR/CAS9 knockout cells, for POLK and POLI TLS DNA polymerases enzymes genes. Gene KO was validated using Sanger sequencing and mutations resulted in stop codon. These POLI and POLK mutated cells had impaired viability after TMZ treatment. Moreover, genotoxic stress, as indicated by phosphorylated gH2AX, were increased in TMZ-treated cells and was higher in mutated cells compared to Wild-type cells. It is also possible to observe cell cycle arrest, mainly in G2 phase instead of S phase, after 48 h in all three mutated cells, which is not observed in control, wild type, cells. To analyze closer the DNA replication, we also performed fiber assay, surprisingly there was no difference in between KO cells treated with TMZ and WT treated cells! In summary, we can infer that TLS polymerases protect tumor cells from TMZ-induced DNA damage and thus play important roles on overcoming resistance to this drug. As perspectives for this work, we intend to observe further how replication is affected in these mutated cells treated with TMZ, since the lack of difference and the non observation of S phase arrest, may indicate that the role of translesion synthesis may not be the main role in this context. Citation Format: Marcela Teatin Latancia, André Uchimura Bastos, Natália C Moreno, Davi Jardim, Clarissa RR Rocha, Giovana Leandro, Carlos Frederico Martins Menck. Role of error-prone polymerases on glioma cell resistance to Temozolomide [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1178.