MEK inhibitors and retinoids synergistically inhibit the growth and induce apoptosis in AML cell lines with constitutive MAPK activation. OCI-AML3 cells were pretreated with the MEK inhibitor CI-1040 (0.5 μM) for 30 minutes and subsequently exposed to either ATRA (1 μM) or 9-cis RA (0.1 μM) for 96 hours. (A) Viability was then assessed by measuring the intracellular ATP content using the Vialight assay. Results are expressed as percentage of viable cells relative to vehicle control–treated cells and represent the average ± SD of 6 independent experiments (for the comparison between CI-1040 and combination treatments, P ≤ .02; for the comparison between ATRA or 9-cis RA and combination treatments, P ≤ .05). (B) Apoptosis was evaluated by flow cytometric analysis of FITC-conjugated annexin V binding, while simultaneously assessing membrane integrity by PI exclusion. Results are expressed as the net apoptosis induction (percentage of apoptosis in treated cells minus percentage of apoptosis in control cells) and represent the average ± SD of 9 independent experiments (for the comparison between CI-1040 and combination treatments, P < .001; for the comparison between ATRA or 9-cis RA and combination treatments, P < .001). (C) Primary data from one representative experiment. Annexin V–positive cells are highlighted in the box, and their percentage is shown in the figure. (D) AML cell lines with (OCI-AML2, NB4, HL-60) or without (U937, KG1) constitutive MAPK activation were pretreated with the MEK inhibitor CI-1040 (0.5 μM) for 30 minutes and subsequently exposed to either ATRA (1 μM) or 9-cis RA (0.1 μM) for 96 hours. Apoptosis was then evaluated as described for panel B. Results are expressed as the net apoptosis induction and represent the average ± SD of at least 3 independent experiments for each cell line. Synergism analysis for (E) viability reduction and (F) apoptosis induction was carried out using a fixed dose of CI-1040 (0.5 μM) and escalating doses of ATRA (0.1-1 μM, ○) and 9-cis RA (0.1-1 μM, •);CI values were then derived using the Chou-Talalay method⁴⁴  and are plotted against retinoid concentrations. The gray area indicates additive effects (CI = 0.9-1.2), while synergism and antagonism fall below and above the area, respectively.