Fig. 5: Role of the intrinsic apoptotic pathway in VEN + CTL treatment.

A OCI-AML2 cells were placed in co-culture with WT1-CTLs (Hem14 donor, E:T 1) ± VEN (100 nM). Prior to co-culture, CTLs were labeled with cell trace violet (CTV). At the indicated time points, CTV-negative (AML cells) were separated from CTV+ cells by FACS (5 min sorting per sample) and CTV-negative cells were immediately placed in the indicated Caspase-Glo buffer for 45 min prior to assessing luciferase activity. Equivalent numbers of CTV-negative cells were sorted for each condition at each timepoint. RLU = relative light unit. B Genes for BAX and/or BAK1 were knocked out from OCI-AML2 cells using CRISPR gRNA/Cas9 ribonucleoprotein (RNP) nucleofection. Knockout efficacy was evaluated by western blot for Bax (top) and Bak (bottom) protein, with α-tubulin used as a loading control. SCR = negative control KO (irrelevant scramble sgRNA). Molecular weight markers (15, 25, 50 kDa) are shown. Full western blots available in supplementary file. C OCI-AML2 cells based on knockout status (negative control vs BAX/BAK1 double KO) were treated with VEN (100 nM) ± WT1-CTLs (E:T 0.5) concurrently and AML cell viability was assessed at 24 h using annexin V/PI staining. D THP-1 cells were placed in co-culture with WT1-CTLs (Hem14 donor, E:T2) for 4 h. During co-culture, cells were treated with or without inhibitors against caspase 2 and caspase 9 (each at 100 μM). Conditions that did not have caspase inhibitors were treated with an equivalent volume of DMSO as was used for the caspase inhibitor conditions. Results are shown as mean ± standard deviation of n = 4 (A, D) or n = 3 samples (C) and are representative of 3 (A) or 2 (C, D) independent experiments. Asterisks in (C) are comparisons of co-culture with control OCI-AML2 cells vs knockout OCI-AML2 cells. **p < 0.01 using unpaired t-test. Schematic of the experimental setup is shown for (C).