Venetoclax

ABT-737: The mice were treated with either 100 mg/kg/d ABT-737 solution or an equal volume of carrier solution administrated via intraperitoneal injections (i.p.) for 21 consecutive days. ABT-737 was received from Abbott (now AbbVie Inc). After the treatment the mice were followed up for 21 days. Navitoclax, venetoclax, metformin: The mice were treated with vehicle (5% EtOH, 20% Phosal PG in MQ), 100 mg/kg/d Navitoclax or venetoclax, 300 or 600 g/kg/d metformin or with drug combinations delivered via intragastric (i.g.) route for 21 days. The cohorts were followed up until 60 days after transplantation. Navitoclax and venetoclax were from AbbVie Inc. Drugs were sonicated daily for better solubility. In the experiments including anti-PD-1, mice received i.p. injections of either 200 µg control IgG (bxcell, #BE0089) or 200 µg anti-PD-1 (bxcell, #BE0146) every third day, in total four times, together with the 1-week daily adjuvant i.g. treatments of either vehicle, venetoclax, metformin or the combination of venetoclax+metformin. Paclitaxel was administrated every third day, i.p., 10 mg/kg in cremphor EL-ethanol-saline. Plasma ALAT levels were measured in the Biochemical Analysis Core for Experimental Research, University of Helsinki.

BH3 profiling was carried out as previously described, using protocols adapted from Ryan et al. [57 (link), 58 (link)]. Briefly, 10 million MOLM-14 cells in 20 mL of culture media were treated for 24 h. with 2.5 nM CR-1-31-B or vehicle control. After treatment, cells were stained using aqua live/dead (Invitrogen, #L34966). Following exposure to digitonin, cells were incubated with varying concentrations of BH3 peptides and inhibitors involved in apoptotic activation for 90 min. Moreover, cells were incubated with inhibitors targeting BCL2 (Venetoclax, AbbVie), MCL1 (S63845, SelleckChem) and BCL-XL (A-1331852, SelleckChem) or multiple BCL2 proteins (ABT-737, SelleckChem). After incubation, cells were fixed and stained overnight at 4 °C for cytochrome C (BD Biosciences, #558709). Retention of cytochrome C was measured using a Fortessa Flow Cytometer (BD Biosciences) where DMSO is a control for cytochrome C retention and alamethicin (ALA) is a control for cytochrome C release. BH3 results (cytochrome C release) are expressed as a percentage of the inverse of staining cytochrome C retention. Acquired data was normalized to DMSO in each experiment. Results from untreated conditions were subtracted from treated ones to calculate a change in response (“apoptotic priming”) upon 24 h exposure to CR-1-31-B. Four independent experiments were performed.

Naproxen was supplied by Cayman Chemical (Ann Arbor, MI, USA), and venetoclax was obtained from AbbVie Inc. (Chicago, IL, USA). Vinylpyrrolidone-vinyl acetate copolymer (PVPVA64, Kollidon^® VA 64) was purchased from BASF SE (Ludwigshafen, Germany). Methanol, water (LS-MS Grade), trifluoroacetic acid, and acetonitrile were purchased from Merck KGaA (Darmstadt, Germany). For the dissolution experiments, purified water without buffer was used. Unbuffered water was chosen because the modeling calculations in Section 2 were performed using water without any buffer system.

CD8⁺ T cells, CD4⁺FOXP3⁻ T cells (CD4⁺ T cells), and CD4⁺FOXP3⁺GFP⁺ T cells (Tregs) were cultured as previously described [11 (link)] and expanded using CD3/CD28 Dynabeads (Life Technologies, Carlsbad, CA) at a 1:1 bead:cell ratio and 500 U/mL of recombinant IL-2 (Prometheus Therapeutics & Diagnostics, San Diego, CA) (CD8⁺ and CD4⁺ T cells) or with a 3.5:1 bead:cell ratio and 2,000 U/mL IL- 2 (Tregs). Cells were treated with DMSO (vehicle) or 500 nM venetoclax (AbbVie, North Chicago, IL) daily for five days after reaching logarithmic growth and >95% viable (day 3). BH3 profiling was performed as previously described [11 (link)]. % Depolarization was calculated using the following formula: % Depolarization = (1−(sample-FCCP)/(DMSO-FCCP)) × 100.