European Urology Oncology Efficacy of Novel Bromodomain and Extraterminal Inhibitors in Combination with Chemotherapy for Castration-Resistant Prostate Cancer  Ramiro Vázquez, Gianluca Civenni, Aleksandra Kokanovic, Dheeraj Shinde, Jasmine Cantergiani, Martina Marchetti, Giada Zoppi, Bruce Ruggeri, Phillip C.C. Liu, Giuseppina M. Carbone, Carlo V. Catapano  European Urology Oncology  DOI: 10.1016/j.euo.2019.07.013 Copyright © 2019 The Authors Terms and Conditions

Fig. 1 Evaluation of the anticancer activity of INCB054329 and INCB057643 in prostate cancer models. (A) The antiproliferative activity of drugs was evaluated after 72-h treatments in bulk cultures and in colony- and sphere-forming assays. Sigmoidal dose response curves were fitted to the experimental results by using GraphPad Prism 5.00 for MS Windows software. Data represent mean ± SEM (n ≥ 3). (B) Mice (eight per group) with 22Rv1 tumor xenografts were treated with 50 mg/kg INCB054329, 3 mg/kg INCB057643, or vehicle (5% N,N-acetylacetamide in 0.5% methylcellulose) per os twice a day for 7 d a week. Tumor growth and body weight were monitored every 2–3 d throughout the treatment period. Direct T/C% values (T/Cd%) were calculated from tumor weight at the end of the experiment. Differences in the aforementioned variables in drug-treated animals versus vehicle-treated mice were determined by using one-way ANOVA test (p < 0.01) followed by SNK a posteriori test. Data were log-transformed to achieve homoscedasticity if necessary. Different letters indicate significant (p < 0.05) variations among the experimental groups. Dots and vertical lines represent mean and standard deviation, respectively. The dotted vertical line indicates treatment start. ANOVA = analysis of variance; SEM = standard error of the mean; SNK = Student-Newman-Keuls. European Urology Oncology DOI: (10.1016/j.euo.2019.07.013) Copyright © 2019 The Authors Terms and Conditions

Fig. 2 In vitro and in vivo evaluation of INCB057643 in combination with docetaxel. (A) The antiproliferative effects of 72-h treatments with INCB057643 and docetaxel were evaluated in DU145, 22Rv1, and LNCaP cells using the MTT method. Results were processed according with the Chou-Talalay algorithm. Circles represent individual CIs at specific concentrations of each drug in a defined range of activities (25–75% growth inhibition) determined in multiple experiments (n ≥ 3). The reported overall CI is the mean of all the individual CI values with CI95% (horizontal bars). (B) Distribution of CI values as a function of the antiproliferative effect of the docetaxel/INCB057643 combination in 22Rv1 cells. Percentages indicate the fraction of CI values in the low- and moderate/high-activity areas, respectively. (C) Variations of growth inhibitory activity and CI as a function of docetaxel (top) and INCB057643 (bottom) concentrations in 22Rv1 cells. The 22Rv1 cells were treated for 72 h with (D) INCB057643, (E) docetaxel, or (F) the combination of both drugs. Cell proliferation (left panels) was evaluated by MTT and cell cycle (right panels) was analyzed by FACS. After 72 h, aliquots of vehicle- and drug-treated cells were collected, washed with PBS, counted, and replated in bulk and colony-forming culture conditions. Bulk culture cells were quantified after 1 wk, and colonies were counted after 10 d. Significant differences in the ratio of colonies and spheres with respect to 0.1% DMSO-treated control cells were determined by Kruskal-Wallis method or one-way ANOVA (p < 0.001) followed by Tukey a posteriori test. Different letters indicate significant (p <  0.05) variation with respect to vehicle. Data represent mean ± SEM (n ≥ 3). (G) Mice (12/group) with 22Rv1 xenografts were treated with 10 mg/kg INCB057643 per os once a day for 5 d a week, vehicle (5% N,N-acetylacetamide in 0.5% methylcellulose), or 15 mg/kg docetaxel IP once a week. Tumor growth (top), T/Ci values (bottom left), and body weight (bottom right) were examined throughout the treatment period. Variations in terms of tumor and body weight among experimental groups were evaluated by one-way ANOVA tests (p < 0.01) followed by SNK a posteriori tests. If necessary, variables were log-transformed to achieve homoscedasticity. Different letters indicate significant (p < 0.05) differences between experimental groups. Dots and vertical lines represent mean and standard deviation, respectively. The dotted line shows treatment start. ANOVA = analysis of variance; CI = combination index; DMSO = dimethyl sulfoxide; Doc. = docetaxel; IP = intraperitoneally; PBS = phosphate-buffered saline; SEM = standard error of the mean; SNK = Student-Newman-Keuls. European Urology Oncology DOI: (10.1016/j.euo.2019.07.013) Copyright © 2019 The Authors Terms and Conditions

Fig. 3 Effect of INCB057643 concomitant treatments with olaparib or carboplatin. (DU145, PC3, 22Rv1, and LNCaP cells were treated in vitro with (A) INCB057643 and olaparib or (B) carboplatin for 72 h. Antiproliferative activity was determined with the MTT assay and analyzed using the Chou-Talalay algorithm. Data represent individual CI values (circles) and the mean CI with CI95% (horizontal bars) from multiple experiments (n ≥ 3). Mice bearing 22Rv1 cell line–derived tumors were treated with 10 mg/kg INCB057643 per os once a day for 5 d a week, vehicle (5% N,N-acetylacetamide in 0.5% methylcellulose), and either (C) 50 mg/kg olaparib IP once a day for 5 d a week or (D) 50 mg/kg carboplatin IP twice a week. Tumor growth, T/Ci values, and body weight were monitored throughout the experiment. Tumor and body weight variations among experimental groups were evaluated by one-way ANOVA (p < 0.01) followed by SNK a posteriori tests. Variables were accordingly log-transformed to achieve homoscedasticity if necessary. Different letters indicate significant (p < 0.05) differences between experimental groups. Dots and vertical lines represent mean and standard deviation, respectively. Dotted lines indicate treatment start. Twelve (Fig. 3C) and 10 (Fig. 3D) mice/group were used. ANOVA = analysis of variance; CI = combination index; IP = intraperitoneally; SNK = Student-Newman-Keuls. European Urology Oncology DOI: (10.1016/j.euo.2019.07.013) Copyright © 2019 The Authors Terms and Conditions

Fig. 4 Comparative evaluation of treatment efficacy. (A) T/Ci% values of all treatments in 22Rv1 tumor xenografts were pooled and fitted to exponential decay or linear curves by employing GraphPad Prism 5.00 for MS Windows software. (B) Experimental tumor size data were fitted to exponential growth equation to determine the doubling time (DT). The relative DT (rDT) was calculated as the DTtreated/DTvehicle for each individual experiment. DT and rDT are shown in a table format. (C) Variations of body weight with respect to control for each drug throughout treatments were fitted to exponential decay or linear curves. (D) Efficacy scores calculated from antitumor activity and toxicity parameters for each treatment regimen. European Urology Oncology DOI: (10.1016/j.euo.2019.07.013) Copyright © 2019 The Authors Terms and Conditions