Abstract 1375: High mobility group box proteins modulate chemotherapeutic DNA damage repair processing in human cells

Abstract

Abstract Many anticancer chemotherapeutic agents induce DNA interstrand crosslinks (ICLs) and DNA double-strand breaks (DSBs) leading to cytotoxicity; however, the mechanisms involved in processing such damage is not completely understood. Thus, a better understanding of the processing of ICLs and DSBs will assist in the identification of novel pharmacological targets, and improved drug design, particularly in chemo-resistant populations. We discovered that the High Mobility Group Box proteins (HMGBs), a family of non-histone architectural proteins, modulate DNA lesion processing in human cancer cells and significantly alter cell survival. We have shown that one of the HMGB family members, HMGB1, binds with high affinity to ICLs targeted to specific sites using triplex-forming oligonucleotides (TFOs), and modulates the repair of ICLs as a co-factor of the nucleotide excision repair (NER) mechanism. We found that other HMGB family members, HMGB2 and HMGB3, which share sequence and structural homology with HMGB1, also modulate DNA repair. Based on our preliminary results, we hypothesize that the HMGB proteins modulate lesion processing via unique and distinct mechanisms, and promote the repair of chemotherapeutic and carcinogenic lesions. In our studies site-specific ICLs were formed by incubating DNA substrates with psoralen-conjugated TFOs followed by UVA irradiation (TFO-ICLs) and used in mutagenesis studies in human cells. Protein recruitment to the damaged site was determined by chromatin immuno-precipitation (ChIP). Non-homologous end-joining (NHEJ) and homologous recombination (HR) were studied via fluorescence activated cell sorting of fluorescent reporter genes in human cells. Cellular localization was visualized by immunofluorescence microscopy. Chromosomal aberrations were determined from metaphase spreads using light microscopy. In response to ICLs, siRNA-mediated depletion of HMGB proteins increased mutagenesis, suggesting involvement in error-free DNA damage repair. Interestingly, analysis of the mutants indicated an increase in single nucleotide mutations in the absence of HMGB1, whereas depletion of HMGB2 or HMGB3 predominantly resulted in small deletions, indicating their involvement in different repair pathways. Specifically, HMGB2 or HMGB3 depletion reduced ICL-induced single-strand annealing (a sub-pathway of HR) events. Interestingly, we found that both HMGB1 and HMGB2 forms distinct foci after damage induction. HMGB2 depletion stimulated NHEJ events, while depletion of HMGB2 or HMGB3 reduced DSB-induced HR events. Finally, depletion of HMGB proteins increased chromosomal aberrations in cancer cells treated with ICL- and DSB-inducing agents. Our novel findings suggest that the HMGB proteins are involved in processing of carcinogenic and chemotherapeutic lesions in human cells via distinct repair mechanisms. Citation Format: Anirban Mukherjee, Wade A. Reh, Karen M. Vasquez. High mobility group box proteins modulate chemotherapeutic DNA damage repair processing in human cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1375.