Sequential pharmacological BET inhibition followed by p300 inhibition synergistically/optimally suppresses AML proliferation. (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 2 inhibitors at each 4 doses and DMSO (20 combinations) in 3 temporal sequences: mode 1, BETi sequentially followed by p300i; mode 2, both inhibitors concomitantly; and mode 3, p300i sequentially followed by BETi. (B-D) Three-dimensional diffusion plots indicating maximal synergy scores (right) 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 48 hours after the initial treatment commencement. Shown are averages and (for bar plots) SD from 3 biological/experimental replicates. ZIP scores >10 were considered synergistic, whereas ZIP scores less than –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 48 hours 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) Nod-scid gamma (NSG) mice that underwent tertiary transplant with Aml1-Eto9a 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 on day 23.
Figure 4.

Sequential pharmacological BET inhibition followed by p300 inhibition synergistically/optimally suppresses AML proliferation. (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 2 inhibitors at each 4 doses and DMSO (20 combinations) in 3 temporal sequences: mode 1, BETi sequentially followed by p300i; mode 2, both inhibitors concomitantly; and mode 3, p300i sequentially followed by BETi. (B-D) Three-dimensional diffusion plots indicating maximal synergy scores (right) 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 48 hours after the initial treatment commencement. Shown are averages and (for bar plots) SD from 3 biological/experimental replicates. ZIP scores >10 were considered synergistic, whereas ZIP scores less than –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 48 hours 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) Nod-scid gamma (NSG) mice that underwent tertiary transplant with Aml1-Eto9a 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 on day 23.

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