Fig. 6

Activation of DDIT3 expression synergizes with Venetoclax to enhance apoptotic death of BCL2 inhibitor-resistant NB4 cells through MCL1 inhibition in vitro and in vivo. A The protein levels of DDIT3 were increased in NB4 cells after the treatment of tunicamycin (100 ng/ml, 24 h) and sorafenib (3 μM, 24 h). B-C The protein level of MCL1 could be up-regulated by venetoclax, while down-regulated by tunicamycin (B) and sorafenib (C). The MCL1 levels of NB4 cells were further decreased by treatment with tunicamycin + venetocalx and sorafenib + venetoclax. D Combined treatment with tunicamycin and venetoclax resulted in increased apoptosis/cell death of NB4 cells compared to each drug alone. E The combination indexes (CIs) were calculated for each concentration combination of tunicamycin and venetoclax, and the CIs were all less than 1, indicating the synergistic effect. F Combined treatment with sorafenib and venetoclax resulted in increased apoptosis/cell death of NB4 cells compared to each drug alone. G The CIs were also calculated for each concentration combination of sorafenib and venetoclax, and the values were all less than 1. H NB4 cells treated with combination of tunicamycin (100 ng/ml, 24 h) and venetoclax (5 μM, 48 h) had a significantly increased proportion of cells in the G0/G1 phase and a significantly decreased proportion of cells in the G2/M phase compared to cells treated with a single drug or DMSO. I NB4 cells treated with combination of sorafenib (3 μM, 24 h) and venetoclax (5 μM, 24 h) had a significantly increased proportion of cells in the G0/G1 phase and a significantly decreased proportion of cells in the S phase compared to cells treated with a single drug or DMSO. J Treatment schedule and experimental set-up. K Quantification of bioluminescence showed the lowest tumor load in mice after treatment with venetocalx (100 mg/kg/d) + sorafenib (40 mg/kg/d) on day 7. L During the process of treatment, the group with co-administration of venetoclax and sorafenib continuously showed the lowest leukemia load by quantification of bioluminescence, and was the only group that showed a significantly decreased AML burden compared to vehicle group on day 14. M Kaplan-Meyer survival curve revealed that co-administration of venetoclax and sorafenib substantially prolonged animal survival compared to vehicle group (statistical analysis by Log-rank test, P = 0.0063). Data represent Mean ± SD (n = 3); Each animal group included 5 mice; *P < 0.05; **P < 0.01, ***P < 0.001, ****P < 0.0001