Abstract 348: Targeting histone chaperone FACT complex and APE1 sensitizes colon cancer cells to chemotherapy

Abstract

Abstract Apurinic/apyrimidinic (AP) sites are one of the most common types of DNA damage that occur spontaneously and following chemotherapy. Human AP-endonuclease (APE1) is primarily responsible for initiating the repair of AP sites via the highly coordinated DNA base excision repair (BER) pathway. Although the repair of AP sites by APE1 utilizing naked DNA or nucleosomal DNA substrate has been extensively investigated in vitro, till to date, how APE1 repairs AP sites in chromatin in cells via BER pathway remains largely unknown. Chromatin remodeling histone chaperone FACT (facilitates chromatin transcription) complex has been previously shown to mediate nucleosome disruption which allows DNA repair proteins to gain access to damage sites. Its involvement in nucleotide excision repair (NER) pathway has been demonstrated. However, the role of FACT in BER remains unclear. Our lab has recently found that APE1 is acetylated at AP sites damage in chromatin and acetylation enhances the DNA repair activity of APE1. Here, we show that APE1 interacts with histone chaperone complex FACT. Subsequently, we confirmed the interaction of APE1 with FACT complex by Co-IP and immunofluorescence (IF) and found that both subunit of FACT complex, SPT16 and SSRP1, interact with APE1 in the nucleus and in chromatin. By Western blot (WB), IF and co-IP, we found enhanced interaction upon induction of DNA damage. To understand the functional importance of FACT, we downregulated its levels using siRNA and noted increased cell sensitivity upon damage by using MTT and colony formation. The number of AP sites in the genome increased significantly upon FACT knockdown. FRAP data demonstrated increased APE1 turnover time in damage sites after FACT knock-down suggesting the role of FACT in nucleosome disruption in facilitating the APE1 function in BER pathway. Decreasing FACT level led to reduced acetylation level of APE1 at damage sites and impaired DNA repair as evident by comet assay. Additionally, we found elevated levels of AcAPE1 and FACT in colon cancer tissues and multiple cell lines as compared to normal adjacent tissues and cells. We used curaxin which blocks the function of FACT and found increased cell sensitivity to various chemotherapeutic agents in multiple colon cancer cell lines. In nude mice xenograft, combination of curaxin and 5-FU group slowed the growth of tumor to the largest extent as compared to 5-FU and curaxin groups. Together our study suggests that FACT is required for APE1 to gain access to AP sites in the context of chromatin. Interruption of APE1 and FACT interaction exhibits synergistic effect on cell viability and proliferation upon DNA damage with chemotherapeutic drugs. We demonstrate targeting both FACT and BER pathway together as a novel strategy for chemosensitization of colon cancer cells. Citation Format: Heyu Song, Shrabasti Roychoudhury, Pranjal Biswas, Jiping Zeng, Kishor Bhakat. Targeting histone chaperone FACT complex and APE1 sensitizes colon cancer cells to chemotherapy [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 348.