Background: NK314 is a novel synthetic benzo[c]phenanthridine analog under Phase I clinical trial in Japan. NK314 inhibits topoisomerase (topo) II and rapidly induces DNA double strand breaks (DSBs). The drug exhibits tumor cytotoxicity and inhibits growth of various human tumors xenografted in nude mice. In the last AACR meeting (#5525), we reported that NK314 selectively and strongly inhibited topoIIα in tumor cells. Specifically, 1) NK314 stabilizes topoIIα-DNA cleavage complexes, but not topoIIβ cleavage complexes. 2) The cytotoxic activity of NK314 is inhibited by topoIIα siRNA but not by topoIIβ siRNA. 3) NK314-induced cleavage complexes are much more stable than etoposide-induced complexes. To further examine the functional role for each topoII isoform in NK314-induced cytotoxicity and to explain the stability of NK314-induced cleavage complexes, we constructed several gene knockout cell lines and evaluated the NK314’s action on these cells. Results: 1) Involvement of topoIIβ. We generated TOP2β-/- cells by gene targeting using the human pre-B ALL cell line Nalm-6 and examined the sensitivity to topoII inhibitors including NK314. In clonogenic assays, TOP2β-/- cells were more resistant to etoposide, mitoxantrone and doxorubicin (DOX) than wild-type cells. Notably, TOP2β-/- cells exhibited almost the same sensitivity to NK314 as wild-type cells, indicating that the cytotoxic effect of NK314 is independent of topoIIβ. 2) Involvement of DSB repair pathways. Since human cells have two distinct pathways for repairing DSBs, homologous recombination (HR) and non-homologous end-joining (NHEJ), we used two mutant cell lines lacking Rad54 or DNA ligase IV (Lig4), which are key repair proteins in the HR and NHEJ pathways, respectively. RAD54-/- cells showed no increased sensitivity to NK314, while LIG4-/- cells showed 2-3 times higher sensitivity to NK314, suggesting that the NHEJ pathway is important for the repair process of NK314-induced DSBs. However, the degree of sensitization by LIG4 knockout was much higher with etoposide and DOX (10-20 times), implying that NK314-caused DSBs might be difficult for cells to cope with compared to other topoII inhibitors. Conclusions: NK314 is a specific and potent topoIIα-targeting agent. The fact that topoIIα is highly expressed in growing tumor cells, in contrast to topoIIβ that is ubiquitously expressed, may account for the tumor specific cytotoxicity of NK314. Additionally, our observation that NK314-induced DSBs seem hardly repaired in cells, may explain the strong anti-tumor activity of NK314.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA