PP2A activation further improves the effectiveness of venetoclax plus azacitidine therapy in AML. (A) Multidrug synergy graphs show the average ZIP synergy score for double and triple combinations between FTY720, venetoclax, and azacitidine. Analyses were performed with the SynergyFinder Plus webtool. Synergy scores >10 (gray boxes) were considered synergistic. (B) Colonization potential of HL-60 cells in zebrafish xenograft models upon treatment with control, venetoclax plus azacitidine, or the triple therapy. Experiments were done in triplicate; number of embryos used is indicated as “n.” Data were analyzed by χ2 tests; ∗∗P < .01, ∗∗∗P < .001. (C) Representative images of HL-60 invading cells throughout the tail of zebrafish xenograft embryos at 72 hpi upon treatment with control, venetoclax plus azacitidine, or the triple therapy. AML cells were tracked as red-emitting fluorescent cells. Arrows show migrating HL-60 cells. Scale bar: 0.25 mm. (D) Western blot analyses of Ser70p-BCL2, BCL2, MCL1, Thr202/Tyr204p-ERK, and ERK protein expression levels in HL-60 cells after 24 hours of treatment with control vehicle, venetoclax plus azacitidine, or the triple combination. Only upon treatment with the triple combination were the levels of all 3 proteins p-BCL2, MCL1, and p-ERK significantly decreased. (E) Multidrug synergy graphs show the average ZIP score for double and triple combinations between FTY720, venetoclax, and azacitidine in 2 AML primary samples. Analyses were performed with the SynergyFinder Plus webtool. Synergy scores >10 (gray boxes) were considered synergistic. (F) Western blot analyses of Ser70p-BCL2, BCL2, MCL1, Thr202/Tyr204p-ERK, and ERK protein expression levels in AML-2 cells after 36 hours of treatment with venetoclax plus azacitidine or the triple combination. Only upon triple treatment was the expression levels of MCL1, Ser70p-BCL2, and Thr202/Tyr204p-ERK significantly decreased. (G) NSG mice were injected intravenously with AML-2 primary cells. Vehicle or double or triple-combination treatments were administrated by oral gavage starting 5 days postinjection. (H) Survival curve of an aggressive AML PDX model in NSG mice treated with vehicle, venetoclax plus azacitidine, or FTY720, venetoclax, and azacitidine combination (n = 7 mice per group). Triple-combination treatment induced a statistically significant increase in OS in treated mice compared with control animals (median OS, 57 vs 39 days; P = .007), or animals treated with venetoclax plus azacitidine (median OS, 47 vs 57 days; P = .01). Data were analyzed by the log-rank (Mantel-Cox) test. ZIP, zero interaction potency.
