A novel topoisomerase I inhibitor DIA-001 induces DNA damage mediated cell cycle arrest and apoptosis in cancer cell.

BACKGROUND: DNA topoisomerase enzyme plays an essential role in controlling the DNA topology structure by binding to DNA and cutting the phosphate backbone of either one or both of the DNA strands. Here, we have identified a small molecule inhibitor, DIA-001, that directly binds to Topoisomerase 1 (Topo I) and promotes the Topo I-DNA adducts. METHODS: In this study, we investigated the antitumor effects of DIA-001 using MTS assay and colony formation. We examined cell cycle of tumor cells with DIA-001 treatment in vitro by flow cytometry. And we investigated DNA damage and cell cycle marker protein after treatment with DIA-001 at different concentration and time point by western blot. Immunofluorescence was performance to detect the nuclear foci. The effects of DIA-001 on Topo I and Topo II activities were examined by DNA relaxation assays. RESULTS: We demonstrate that DIA-001 inhibit DNA replication and arrest cell cycle progression at the G2/M phase by directly binds to Topo I and promotes the Topo I-DNA adducts. In addition, DIA-001 can activate the DNA damage response signaling cascade, resulting in apoptosis in treated cells. CONCLUSIONS: Our findings show a novel compound for treatment of cancer cells with the potential as a chemotherapy candidate that is less toxic to normal cells.

CHK2-FOXK axis promotes transcriptional control of autophagy programs.

Autophagy is an evolutionarily conserved catabolic process, which plays a vital role in removing misfolded proteins and clearing damaged organelles to maintain internal environment homeostasis. Here, we uncovered the checkpoint kinase 2 (CHK2)-FOXK (FOXK1 and FOXK2) axis playing an important role in DNA damage-mediated autophagy at the transcriptional regulation layer. Mechanistically, following DNA damage, CHK2 phosphorylates FOXK and creates a 14-3-3γ binding site, which, in turn, traps FOXK proteins in the cytoplasm. Because FOXK functions as the transcription suppressor of ATGs, DNA damage-mediated FOXKs' cytoplasmic trapping induces autophagy. In addition, we found that a cancer-derived FOXK mutation induces FOXK hyperphosphorylation and enhances autophagy, resulting in chemoresistance. Cotreatment with cisplatin and chloroquine overcomes the chemoresistance caused by FOXK mutation. Overall, our study highlights a mechanism whereby DNA damage triggers autophagy by increasing autophagy genes via CHK2-FOXK-mediated transcriptional control, and misregulation of this pathway contributes to chemoresistance.

USP39 regulates DNA damage response and chemo-radiation resistance by deubiquitinating and stabilizing CHK2.

The serine/threonine kinase, CHK2 (checkpoint kinase 2), is a key mediator in DNA damage response and a tumor suppressor, which is implicated in promoting cell cycle arrest, apoptosis and DNA repair. Accumulating evidence suggests that these functions are primarily exerted through phosphorylation downstream factors such as p53 and BRCA1. Recent studies have shown that ubiquitination is an important mode of regulation of CHK2. However, it remains largely unclear whether deubiquitinases participate in regulation of CHK2. Here, we report that a deubiquitinase, USP39, is a new regulator of CHK2. Mechanistically, USP39 deubiquitinates and stabilizes CHK2, which in turn enhances CHK2 stability. Short hairpin RNA (shRNA) mediated knockdown of USP39 led to deregulate CHK2, which resulted in compromising the DNA damage-induced G2/M checkpoint, decreasing apoptosis, and conferring cancer cells resistance to chemotherapy drugs and radiation treatment. Collectively, we identify USP39 as a novel regulator of CHK2 in the DNA damage response.