Ro 08-2750 increases radiation-induced cytotoxicity in DLBCL by dysregulating DNA damage response

Introduction Current research efforts attempt to increase therapy efficacy in diffuse large B-cell lymphoma (DLBCL). Here, we evaluated the effects of small molecule inhibitor Ro 08-2750 on therapy-induced cytotoxicity in DLBCL. Material and methods First, we established Ro 08-2750 treatment in DLBCL by verifying cell toxicity via cell viability and colony formation assays. In a next step, functional effects reducing cell viability, e.g. cell cycle dysregulation and apoptosis, were analyzed via flow cytometry. Then, we evaluated effects of Ro 08-2750 on cytotoxic therapy in cell viability and colony formation assays. After confirming an increase in irradiation-induced DNA damage via γH2AX assay, an immunoblot-based assay was used to detect changes in DNA damage response. Lastly, we analyzed Ro 08-2750-induced changes in gene and protein expression via RT-qPCR, RNA sequencing, western blot and proteomic analysis. Results Ro 08-2750 reduces cell viability dose-dependently in DLBCL. Functionally, the decrease in cell viability is combined with a cell cycle dysregulation, specifically an increase in S phase with reduced G2/M phase, and an increase in apoptosis. DNA synthesis is reduced during S phase. Assessing the therapeutical relevance, Ro 08-2750 sensitized DLBCL cell lines to irradiation but not chemotherapy and increased irradiation-induced double strand breaks. We found a dysregulation in CHK2 phosphorylation. Moreover, Ro 08-2750 dysregulated DNA damage response in genomic and proteomic analysis. Conclusion We identified Ro 08-2750 as a potential radiosensitizer in DLBCL. Therefore, our results indicate the potential therapeutical benefit of targeting RNA binding proteins