Companion Diagnostics and Cancer Biomarkers Activation of Nrf2 Pathways Correlates with Resistance of NSCLC Cell Lines to CBP501 In Vitro

CBP501 is an anticancer drug candidate that was investigated in two randomized phase II clinical trials for patientswith nonsquamous non–small cell lung cancer (NSCLC) andmalignant pleuralmesothelioma (MPM). CBP501 has been shown to have twomechanisms of action, namely calmodulinmodulation andG2 checkpoint abrogation. Here, we searched for a biomarker to predict sensitivity to CBP501. Twenty-eight NSCLC cell lines were classified into two subgroups, CBP501-sensitive and -insensitive, by quantitatively analyzing the cisdiamminedichloro-platinum (II) (CDDP)–enhancing activity of CBP501 through treatments with short-term (1 hour) coexposure to CDDP and CBP501 or to either alone. Microarray analysis was performed on these cell lines to identify gene expression patterns that correlated with CBP501 sensitivity. We found that multiple nuclear factor erythroid-2–related factor 2 (Nrf2) target genes showed high expression in CBP501-insensitive cell lines. Western blot and immunocytochemical analysis for Nrf2 in NSCLC cell lines also indicated higher protein level in CBP501-insensitive cell lines. Moreover, CBP501 sensitivity is modulated by silencing or sulforaphane-induced overexpression of Nrf2. These results indicate that Nrf2 transcription factor is a potential candidate as a biomarker for resistance to CBP501. This study might help to identify those subpopulations of patients who would respond well to the CBP501 and CDDP combination treatment of NSCLC. Mol Cancer Ther; 13(9); 2215–25. 2014 AACR. Introduction Establishing quantifiable markers that predict sensitivity to a particular drug is an aimof personalizedmedicine. During the last decade, several anticancer drugs were developed on the basis of known oncogenic mutations, and the presence of the mutation by itself has been found tobe auseful biomarker for thesedrugs. Examples include some tyrosine kinase inhibitors, including gefitinib and erlotinib, that target epidermal growth factor receptor (EGFR) in NSCLC and for which efficacy depends on the presence of particular EGFR mutations (1, 2); crizotinib, which is highly effective when NSCLC cells harbor the EML4–ALK fusion gene (3); and the BRAF inhibitor vemurafenib, which is highly effective when melanoma cells carry the V600E mutation of BRAF (4). CBP501 is a peptide anticancer drug candidate for which two phase II clinical studies in malignant pleural mesothelioma (MPM) and non–small cell lung cancer (NSCLC) have recently been completed. In phase I trials, the combination of cis-diamminedichloro-platinum (II) (CDDP) plus CBP501 showed hint of clinical activity in patients with platinum-resistant ovarian carcinoma and MPM (5). The primary endpoint for efficacywas achieved in the phase II study on MPM (6). G2 checkpoint abrogation had been proposed as a mechanism of action (MOA) based on the observation that CBP501 (i) inhibits multiple kinases that phosphorylate CDC25C at Ser216, (ii) binds directly to 14-3-3 protein, which forms suppressive complexes with phospho-CDC25C, (iii) attenuates phosphorylation of CDC25C at Ser216, and (iv) reduces the accumulation of cancer cells at G2–M upon lengthy combined exposure to CDDP or bleomycin (7, 8). We recently demonstrated an additional MOA for CBP501. This entails direct interaction of CBP501 with calmodulin, which leads to increased uptake of CDDP into cancer cells upon much shorter exposure to and at lower doses of CBP501 than the conditions required forG2 checkpoint abrogation (9). CDDP uptake is known to depend on multiple transporters and channels (10). Because it is still not yet clear which of the many transporters and channels aremost affectedby the calmodulin– CBP501 interaction, it is challenging to rationally predict biomarkers that might be useful for this mode of action of CBP501. Nuclear factor erythroid-2–related factor 2 (Nrf2) is a transcription factor that regulates the expression of many antioxidant genes, including those related to glutathione (GSH) synthesis (11). In tumorigenesis, the expression of CanBas Co., Ltd., Numazu, Japan. Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts. Translational Genomics Research Institute (TGen), Phoenix, Arizona. Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). Corresponding Author: Takumi Kawabe, CanBas Co., Ltd., 2-2-1 Otemachi, Numazu City, Shizuoka 410-0801, Japan. Phone: 81-55-9543666; Fax: 81-55-954-3668; E-mail: [email protected] doi: 10.1158/1535-7163.MCT-13-0808 2014 American Association for Cancer Research. Molecular Cancer Therapeutics www.aacrjournals.org 2215 on June 24, 2017. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from Published OnlineFirst July 22, 2014; DOI: 10.1158/1535-7163.MCT-13-0808