Abstract 3948: Identification of phosphorylated NF45 (T388) as a potential pharmacodynamic biomarker for ARQ 501 (β-lapachone)

Abstract ARQ 501, a fully synthetic version of β-Lapachone, has demonstrated anti-cancer activity and entered multiple phase II trials. Although the mechanism of the cytotoxic activity of ARQ 501 towards cancer cells is not completely understood, it appears that both DNA damage and DNA checkpoint activation play a role in cell death caused by this compound. We used a proteomic approach to search for pharmacodynamic (PD) biomarkers for ARQ 501 by profiling phosphoproteins. ARQ 501 treatment strongly induced threonine phosphorylation of certain proteins around 45 kD in MIA PaCa2 cells. One of the proteins was identified by mass spectrometry analysis as NF45, also known as ILF2. Immunoprecipitation experiments using a customized antibody against pNF45 confirmed that NF45 is phosphorylated in response to ARQ 501 at threonine 388 in a TQ site (an ATM/ATR phosphorylation motif) near the carboxyl terminus. ARQ 501 increased pNF45 expression in a time- and concentration-dependent manner accompanied by activation of ATM and DNAPK. Inhibition of ATM and DNAPK suppressed ARQ 501-induced pNF45 (T388) elevation. Evidence of ARQ 501-induced DNA damage is supported by the observation of CHK2 phosphorylation and γ-H2AX elevation. We therefore examined the effects of several DNA damaging agents on NF45 phosphorylation and found that only ARQ 501 was able to increase pNF45 (T388) levels, suggesting that induction of pNF45 is not a general response to DNA damage, and that ARQ 501 may inhibit cell proliferation by an alternate mechanism. Immunofluorescent staining using pNF45 (T388) antibody showed there is a significant increase in pNF45 (T388) levels predominantly in nuclei. In order to validate pNF45 (T388) as a PD biomarker, we examined pNF45 (T388) levels in human tumor mouse xenograft models treated with ARQ 501. The ARQ 501 treated tumors treated with ARQ 501 showed increased pNF45 (T388) nuclear staining by immunohistochemistry compared to the vehicle group. Therefore, phosphorylation of NF45 at threonine 388 may serve as a PD biomarker for ARQ 501. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3948.