JAK1/2 inhibitor restores activity of a BCL2 antagonist against AML cells in stroma-based conditions. (A-B) Combinatorial treatment of AML cells with ruxolitinib and venetoclax shows synergistic activity between the inhibitors in the presence of CM, whereas the synergy is less pronounced in MCM. AML cells were cultured either in 25% HS-5 CM or MCM for 72 hours in the presence of various concentrations of ruxolitinib, venetoclax, or a combination of the 2 agents, and cell viability was measured using the CTG assay. For the combination matrices, the interaction landscapes are shown in 2 dimensional. δ, the difference in percentage inhibition compared with the expected additive compound effect calculated by the ZIP model. (C) Ruxolitinib and venetoclax combination reduces colony-forming ability of primary AML cells in CM. AML cells from 2 patients were left untreated (0.1% dimethyl sulfoxide [DMSO] = control) or treated with ruxolitinib (300 nM), venetoclax (100 nM), or their combination in MCM or 25% CM medium for 72 hours and plated in methylcellulose progenitor assay. Total CFC output was recorded after 14 days. (D) CD34⁺ expression after culture of primary AML cells for 48 hours with or without drug treatment (300 nM ruxolitinib, 100 nM venetoclax, or their combination) with RPMI, 25% HS-5 CM, direct contact with AML-derived BM MSCs, or separated from stroma with a 0.4-µm pore membrane. Error bars represent standard deviation from 2 independent experiments.