Inhibition of group I PAKs causes cytotoxicity in various ALL and shows synergism with FLT3 inhibition in FLT3-driven ALL. (A) Apoptosis of pro-B SEM cells under PAK-directed tetracycline-induced RNAi. Mean fold-change plus or minus SD of apoptosis is shown in YFP⁺ shPAK1- or shPAK2-expressing single-cell clones vs mCherry and YFP double-positive shPAK1 and shPAK2–coexpressing cells compared with shSCR-YFP (scrambled RNA) control (also see supplemental Figure 8G-I). shRNA sequences are listed according to indicated shRNA number in supplemental Methods. Mann-Whitney U test; *P ≤ .05; **P < .01. (B) Proliferation of pro-B SEM cells under treatment with titrated concentrations of midostaurin (PKC412) and simultaneous PAK1 or PAK2-directed RNAi compared with shSCR control shown by MTT assay. (C) Apoptosis of FLT3^WT-dependent XT-ALL114 under treatment with titrated concentrations of ATP-competitive group I PAK inhibitor FRAX486 or midostaurin (PKC412) and combination of both at equimolar concentrations. Early apoptosis measured by annexin V FACS analysis (also see supplemental Figure 9I). (B-C) Two-way ANOVA test; *P < .05; **P < .01; ****P < .0001). (D-E) Analysis of PAK1 and PAK2 activities in FLT3^D835H and PDGFRB^WT coexpressing XT-ALL22089 under ligand stimulation (D, FLT3L; E, PDGF-BB) and treatment with FRAX486 or PKC412 or a combination of FRAX486 and PKC412 using radioactive IVK with H4 as kinase substrate after PAK1/2 IP. (F-G) MTT assays depicting pharmacosensitivities of genetically distinct BCP-ALL including XT-ALL and ALL lines toward FRAX486 (F) and midostaurin (PKC412) (G). FLT3^ITD-carrying monocytic MV4-11 and endothelial HUVEC cell lines included as controls. Mean plus or minus SD of technical triplicates is given. Biological replicates yielded similar results. (H) Synergism between FRAX486 and midostaurin (PKC412) or gilteritinib in different FLT3-dependent leukemia models. Color bar represents degree of synergism vs antagonism. Assay was based on IC₅₀ measurements of leukemia cells exposed to single drug. Synergy plots were generated using the Combenefit tool.¹⁸