Optimization of circulating biomarkers of obatoclax-induced cell death in patients with small cell lung cancer

Abstract

Small cell lung cancer (SCLC) is an aggressive disease in which, after initial sensitivity to platinum/etoposide chemotherapy, patients frequently relapse with drug-resistant disease. Deregulation of the Bcl-2 pathway is implicated in the pathogenesis of SCLC, and early phase studies of Bcl-2 inhibitors have been initiated in SCLC. Obatoclax is a small-molecule drug designed to target the antiapoptotic Bcl-2 family members to a proapoptotic effect. Preclinical studies were conducted to clarify the kinetics of obatoclax-induced apoptosis in a panel of SCLC cell lines to assist with the interpretation of biomarker data generated during early phase clinical trials. In vitro, obatoclax was synergistic with cisplatin and etoposide, and "priming" cells with obatoclax before the cytotoxics maximized tumor cell death. Peak levels of apoptosis, reflected by cleaved cytokeratin 18 (CK18) levels (M30 ELISA) and caspase activity (SR-DEVD-FMK), occurred 24 hours after obatoclax treatment. A phase 1b-2 trial of obatoclax administered using two infusion regimens in combination with carboplatin and etoposide has been completed in previously untreated patients with extensive-stage SCLC. Circulating pharmacodynamic biomarkers of cell death, full-length and/or cleaved CK18, and oligonucleosomal DNA were studied in the phase 1b trial. All SCLC patients classified as "responders" after two cycles of treatment showed significantly increased levels of full-length and cleaved CK18 (M65 ELISA) on day 3 of study. However, the preclinical data and the absence of a peak in circulating caspase-cleaved CK18 in trial patients suggest suboptimal timing of blood sampling, which will need refinement in future trials incorporating obatoclax.

Figure 1

Obatoclax decreased the viability of SCLC cells with no clear correlation with antiapoptotic Bcl-2 family protein expression levels. (A) Concentration-effect curves for a panel of SCLC cell lines treated at varying concentrations of obatoclax for 96 hours, using the MTS assay. (B) IC₅₀ values for log phase SCLC cells, treated with obatoclax for 96 and 72 hours. (C) Immunoblot analysis of antiapoptotic Bcl-2 family members in SCLC cell lines. Untreated log-phase SCLC cells were lysed, and lysates were normalized for total protein content (15 µg per lane) and analyzed by SDS-PAGE using antibodies recognizing A1, Bcl-2, Bcl-w, Bcl-x_L, and Mcl-1. Tubulin was used as a protein loading control. Data are representative of n = 3 repeat experiments ± SEM.

Figure 2

Obatoclax induced apoptosis in SCLC cells. (A and B) Immunoblot analysis of H526 cells treated with 48 hours of obatoclax at the indicated concentrations (A) or with a fixed dose of 0.2 µM for the indicated times (B). Membranes were probed with antibodies specific for cleaved (“c” as indicated) and full-length PARP. Cleaved caspase-3 as a percentage of total caspase-3 measured using the Mesoscale Discovery assay are reported below. (C) Ratio of drug-treated and vehicle-treated H526 cells with respect to staining for active caspase-3/7 by SR-DEVD-FMK by flow cytometry after cells were treated with obatoclax at the IC₅₀ concentration for 48 hours, then 1 day and 2 days after the drug was removed and replaced by medium. Levels of cleaved CK18 (by M30 assay) in the cell culture medium after 48 hours of obatoclax at the IC₅₀ concentration at the same time points. CTRL indicates control; ETOP, etoposide 20 µM. Results are representative of n = 3 ± SEM.

Figure 3

Synergistic combination of obatoclax, cisplatin and etoposide in SCLC cells. (A) H526 cells were treated with obatoclax for 48 hours followed by the addition of cisplatin and etoposide for 48 hours. Equipotent concentrations of the single agents were selected to cover the concentration-effect curve; the x axis shows the fixed ratio. Data are representative of six SCLC cell lines. (B) SCLC cells were treated either concurrently with the three drugs for 96 hours or 48 hours with the first agent (termed cytotoxics) followed by the addition of obatoclax for 48 hours, covering the concentration-effect curve. After 96 hours, surviving fraction was determined using the MTS assay, and synergy was evaluated using CalcuSyn software.

Figure 4

Pharmacodynamic biomarker analysis from the phase 1 trial of obatoclax in combination with cisplatin and etoposide in SCLC. (A) Schematic for clinical trial drug administration for obatoclax, cisplatin, and etoposide and timing of blood samples for pharmacodynamic biomarker analysis. Patients could receive up to six cycles of chemotherapy. (B) Circulating caspase cleaved plus full-length CK18 levels (M65 assay) in “responders” (n = 17) and “nonresponders” (n = 7). Statistical analysis by Wilcoxon matched-pairs signed rank test. (C) Percentage change from pretreatment baseline values in full-length plus caspase cleaved CK18 (M65 assay) and nucleosomal DNA (nDNA assay) between day 3 circulating biomarker increases and day 8 decreases (P = .002 and P = .02, respectively), and group comparisons between patients who received the 3-hour infusion and those who received the 24-hour infusion.