MEK blockade and Bcl-2 inhibition have synergistic effects in AML cell lines and primary AML samples and overcome Bcl-2 overexpression-mediated resistance.

(A) OCI-AML3 (▿, dotted line), HL-60 (▪, solid line), and KG1 (○, dashed line) cells were cultured in the presence of escalating doses of HA14-1 (2.5-20 μM), PD184352 (0.016-10 μM), or combinations of the 2 agents at a 10:1 ratio (2.5/0.25, 5/0.5, 10/1, 15/1.5). After 48 hours, apoptosis was measured by sub-G₁ DNA content. CI plots were then generated using the Calcusyn software (symbols represent actual data points for the combination). CI values of less than 1.0 indicate synergism. (B) HL-60 was cultured in the presence of vehicle control (■), 16 μM Bcl-2 NS (▨), 16 μM Bcl-2 AS (▥), 2 μM PD184352 (), Bcl-2 NS+PD184352 (), and Bcl-2 AS+PD184352 (▪). After 96 hours, cells were analyzed for DNA content. Results are presented as the percentage of cells with a sub-G₁ DNA content and represent the average ± SD of 3 independent experiments. The inset shows the CI plot for the combination of escalating doses of Bcl-2 AS and PD184352 at a 8:1 ratio, obtained as described in panel A; the dotted line indicates CI = 1. (C) HL-60 cells stably transfected with either empty vector control (HL-60/neo) or Bcl-2 expression vector (HL-60/Bcl-2) were cultured in the presence of HA14-1 (20 μM) alone or in combination with PD184352 (1.2 μM). After 48 hours, cells were harvested and stained for annexin V binding (x-axis) while simultaneously assessing membrane integrity by propidium iodide staining (y-axis). Numbers indicate the percentage of cells in the corresponding quadrant. Results from one experiment representative of 2 performed are shown. (D) Bone marrow cells obtained at diagnosis (all but 1 patient progressing from myelodysplastic syndrome (MDS)) from 5 AML (M0, complex karyotype; M2, diploid; M4, inv(16); M4, diploid; previous MDS, monosomy 7) patients with more than 50% (median 61%, range 51%-79%) blasts were subjected to AML blast colony assay15 in the presence of the indicated doses of HA14-1, alone (○) or in combination with a fixed dose of PD184352 (1.25 μM, ●). Results shown represent the average ± SD of the results obtained in each individual patient and are expressed as a percentage of the colony-forming unit blast in the untreated control. The mean ± SD numbers of colonies in the untreated control for each individual patient were 608 ± 29, 627 ± 22, 584 ± 27, 392 ± 32, and 643 ± 18, respectively (*P = .015 and **P < .008 by Studentt test).