Abstract 4089: Investigating translesion synthesis DNA polymerases roles on tumor protection after temozolomide induced DNA damage

Abstract

Abstract Translesion DNA polymerases are capable of replicating damaged DNA, without removing the lesions, performing translesion synthesis (TLS), a mechanism of DNA damage tolerance. Tumor cells use this mechanism in order to survive lesions caused by chemotherapy and, therefore, this may be a strategy that tumor cells use to resist treatments. Moreover, it is an error-prone process and can lead to mutagenesis increasing resistance potential of tumor cells. Little is known about the role of TLS in Temozolomide (TMZ) tumor treatment. Our aim is to investigate how TMZ affects TLS mutated cells. Hence, we firstly treated primary fibroblasts cells lacking TLS mechanisms (XP-V) and analyzed cell proliferation by flow cytometry, cell viability by a colorimetric assay (XTT), for survival by apoptotic markers (such sub-G1) and clonogenic assays. In fact, XP-V 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. CRISPR/CAS9 knockout (KO) cells of glioblastoma cell lines, such as U251, were constructed for other TLS DNA polymerases enzymes genes, such as Pol Iota and Pol kappa. Gene KO was validated using Sanger sequencing and mutations resulted in stop codon. These pol iota and pol kappa KO cells had impaired viability after TMZ treatment. Moreover, genotoxic stress, as indicated by phosphorylated gH2AX, were increased in TMZ-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 how replication is affected in these KO cells treated with TMZ by DNA Fiber Assays and measure DNA strand breaks in those cells by comet assay. Financial support: FAPESP, CNPq and CAPES. Citation Format: Marcela Teatin Latancia, André Uchimura Bastos, Natália Cestari Moreno, Davi Jardim, Clarissa RR Rocha, Carlos FM Menck. Investigating translesion synthesis DNA polymerases roles on tumor protection after temozolomide induced DNA damage [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4089.