High-throughput drug screening approach identifies gilteritinib as a synergistic combination partner for venetoclax. (A) Experimental setup of the drug screening approach. A total of 31 high-risk AML patient samples were treated with venetoclax (0, 1, and 20 nM) with 64 different drugs in 5 different concentrations for 48 hours. Maximum concentrations used in the drug screen were 50% inhibitory concentrations found in literature; all other concentrations were deduced from division steps by 5. Viability was determined as a readout using CellTiter-Glo, and synergism scores (Bliss and zero interaction potency) were calculated using the synergyfinder R package (version 2.4.13).²¹ (B) Waterfall plot of mean Bliss scores of all drugs combined with venetoclax (calculated as the mean over all Bliss scores reached with each drug in 5 concentrations combined with venetoclax in 2 concentrations). Bliss synergy score was calculated as described by Bliss et al.¹⁹ Colors indicate targets of the respective drugs. Waterfall plot is shown for all primary AML samples (n = 31). (C) RI reached by gilteritinib monotherapy (left) and maximum Bliss synergy scores for venetoclax-gilteritinib reached in all tested concentrations (right) in FLT3 ITD mutated (n = 10) or FLT3 wild-type (WT; n = 17) samples. RI scores were computed according to the area under the curve of the viability curves. The RI scores indicate the proportion of the maximum possible inhibition of each drug independent of a single concentration. Mean RI and mean maximum Bliss scores, respectively, of individual patient samples are shown. Colors indicate the FLT3 mutational status. Statistical significance was assessed using a 2-tailed Student unpaired t test. (D) Heat map depicting Bliss scores for venetoclax combined with different FLT3 inhibitors in FLT3 WT and FLT3-mutated patient samples. (E) Waterfall plot of mean Bliss scores of all drugs combined with venetoclax (calculated as the mean over all Bliss scores reached with each drug in 5 concentrations combined with venetoclax in 2 concentrations). Bliss synergy score was calculated as described by Bliss et al.¹⁹ Colors indicate targets of the respective drugs. Waterfall plot is shown for the subgroup of patients with TP53 mutations obtained at first diagnosis (n = 6). (F) Bliss synergy scores of venetoclax in combination with gilteritinib, azacitidine, cytarabine, and daunorubicin, respectively, in a patient sample with TP53 mutation and FLT3 WT. Colors indicate synergism calculated as described by Bliss et al.¹⁹ Synergy scores of ≥0 are regarded as synergistic. ∗P ≤ .05. ITD, internal tandem duplication.