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. Author manuscript; available in PMC: 2025 Dec 24.
Published in final edited form as: Blood. 2025 Feb 13;145(7):748–764. doi: 10.1182/blood.2022019306

Figure 4. Sequential pharmacological BET-followed by p300-inhibition synergistically/optimally suppresses AML proliferation.

Figure 4

A. Schema of the approach adopted to inhibit the proposed feedback rescue loop to p300 after BETi. In total, 6 cells lines were treated, each in 3 biological replicates with the two inhibitors at each 4 dosages and DMSO (20 combinations) in 3 temporal sequences: mode i) BETi sequentially followed by p300i, mode ii) both inhibitors concomitantly and mode iii) p300i sequentially followed by BETi.

B.-D. Three-dimensional diffusion plots indicating maximal synergy scores (right panels) of combined treatment with BETi and p300i in the indicated orders and cell lines. Treatment efficiency was measured with CellTiterGlo® at day 5 after begin of the first treatment. For the sequential treatment modes, the second compound was added 48hr after initial treatment commencement. Shown are averages and (for bar plots) SD from 3 biological/experimental replicates. ZIP scores > +10 were considered synergistic, while ZIP scores < -10 were antagonistic.

E. Plots of synergy scores of combined treatment with BETi and p300i in the indicated orders and cell lines. Treatment efficiency was measured with CellTiterGlo® at day 5 after the beginning of the first treatment. For the sequential treatment modes, the second compound was added 48hr after treatment began. Shown are averages and SD from 3 biological/experimental replicates.

F. Analysis of colony formation of Aml1-Eto9a-transformed murine AML cells after 2 rounds of plating and treatment with either DMSO in both plates, BETi followed by p300i or vice versa. Shown are mean percentages normalized to DMSO and SD from 3 biological replicates. In each plating, treatment was performed for 7 days in methylcellulose.

G. Analysis of colony formation of Npm1c/Flt3-ITD murine AML cells after 2 rounds of plating and treatment with either DMSO in both plates, BETi followed by p300i or vice versa. Shown are mean percentages normalized to DMSO and SD from 3 biological replicates. In each plating, treatment was performed for 7 days in methylcellulose.

H. Tertiary Aml1-Eto9a-transplanted nod-scid gamma (NSG) mice were allocated to 4 treatment groups (untreated (n=5), BETi-treated (n=4), p300i-treated (n=4) and sequentially combined treated (n=4)). Shown is the Kaplan-Meier plot of survival with log-rank p values (Mantel-Cox and trend). One mouse in the sequential combined treatment group was censored due to death to AML-unrelated reasons at d23.