Fig. 4: In vivo efficacy study validates HTDBP as an approach to identify efficacious hits in MPM.

Malignant pleural mesothelioma PDX model CPDM_0011x, was implanted subcutaneously in the right flank of immunocompromised mice and when tumors reached 150–250 mm³, randomized into 5 treatment groups: vehicle (red, n = 8 mice), 100 mg/kg/qd navitoclax (blue, n = 7 mice), 16 mg/kg/qd AZD8055 (green, n = 7 mice), 100 mg/kg/qd navitoclax plus 16 mg/kg/qd AZD8055 (purple, n = 9 mice), or 100 mg/kg/qd venetoclax (black, n = 7 mice). Mice dosed for 21 days and sacrificed when tumors reached the endpoint of 5 times the initial tumor volume (5xITV) (a–d). Mice bearing CPDM_0011x was administered with one dose of indicated drug treatment and 24 h later tumors were harvested and dissociated and used for western blotting analysis (e) or iBH3 profiling to measure mitochondrial priming (f). a Graph showing mean tumor growth over 21 days of dosing. Error bars represent ± standard deviation for 7–9 mice per treatment group. P value is <0.0001 for navitoclax plus AZD8055 combination treatment compared to all other treatment groups using a 2-way ANOVA multiple comparisons test. b Kaplan–Meier survival curve, mice were sacrificed at 5xITV endpoint. Navitoclax plus AZD8055 combination treatment is statistically significant compared to every other treatment group (p value is <0.0001) based on a Log-rank (Mantle–Cox) test. c Graph showing the individual tumor volume for each mouse for each treatment group over time. Orange line indicates the dosing period (21 days). d Bar graph showing relative tumor burden calculated for each treatment group relative to the mean tumor volume of the vehicle-control group on day 7 and day 14 respectively. Data represents the mean and shows corresponding data points with error bars ± standard deviation. Significance calculated using 2-way ANOVA multiple comparisons. P values stated on the graph. e Representative immunoblots (n = 3) of cell lysates of CPDM_0011x tumor cells after 24-h treatment in vivo for MCL-1, S6, phosphoSer235/236-S6 (pS6), PARP, cleaved PARP (Cl.PARP), cleaved caspase 3 (CC3) and β-Actin (loading control). f Intracellular BH3 profiling was performed on CPDM_0011x tumor cells after 24 h treatment in vivo. Cells analyzed by flow cytometry for pan-cytokeratin/vimentin positive cells (parent population) and cytochrome c positive cells %. Graphs represent the mean of three mice ± standard deviation. Table represent BIM area under curve (AUC) ± 95% confidence intervals (CI) and significance is determined according to one-tailed unpaired t test comparing vehicle control to treatment. *P < 0.05 was considered statistically significant.