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. 2001 Sep;108(6):851-9.
doi: 10.1172/JCI12807.

Therapeutic targeting of the MEK/MAPK signal transduction module in acute myeloid leukemia

M Milella  1 , S M Kornblau, Z Estrov, B Z Carter, H Lapillonne, D Harris, M Konopleva, S Zhao, E Estey, M Andreeff
Affiliations

Therapeutic targeting of the MEK/MAPK signal transduction module in acute myeloid leukemia

M Milella et al. J Clin Invest. 2001 Sep.

Abstract

The mitogen-activated protein kinase (MAPK) pathway regulates growth and survival of many cell types, and its constitutive activation has been implicated in the pathogenesis of a variety of malignancies. In this study we demonstrate that small-molecule MEK inhibitors (PD98059 and PD184352) profoundly impair cell growth and survival of acute myeloid leukemia (AML) cell lines and primary samples with constitutive MAPK activation. These agents abrogate the clonogenicity of leukemic cells but have minimal effects on normal hematopoietic progenitors. MEK blockade also results in sensitization to spontaneous and drug-induced apoptosis. At a molecular level, these effects correlate with modulation of the expression of cyclin-dependent kinase inhibitors (p27(Kip1) and p21(Waf1/CIP1)) and antiapoptotic proteins of the inhibitor of apoptosis proteins (IAP) and Bcl-2 families. Interruption of constitutive MEK/MAPK signaling therefore represents a promising therapeutic strategy in AML.

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Figures

Figure 1
Figure 1
MEK blockade inhibits the growth of leukemic, but not normal, hematopoietic cells. (a) AML cell lines were assessed for MAPK phosphorylation by Western blot. (b) OCI-AML3 cells were exposed to DMSO (C) or PD98059 at the indicated doses for 6 hours. MAPK phosphorylation was then assessed by Western blot. (c) OCI-AML3 cells were treated as in b. MAPK enzymatic activity was measured by the ability to phosphorylate the specific substrate Elk1 in an in vitro kinase assay. (d) Mononuclear cells from patients 5 (filled circles), 9 (open squares), 10 (filled triangles), and 11 (open circles) were grown in colony assays in the presence of DMSO or PD98059 at the indicated doses. Results show AML blast colonies in the PD98059-treated group, expressed as a percentage of the colonies in the DMSO-treated group, and represent the average of quadruplicate cultures (SEM was constantly less than 10%). (e) MACS-sorted CD34+ cells from healthy donors were cultured in the presence of DMSO or 20 μM PD98059. Viability was then assessed by trypan blue dye exclusion. Results show viable cells in the PD98059-treated group, expressed as a percentage of the viable cells in the DMSO-treated group (four bars at left; each bar represents a single donor). Bone marrow cells from healthy donors were grown in colony assays in the presence of DMSO or 50 μM PD98059. Results show CFU-GM and burst-forming units–erythroid (BRU-E) in the PD98059-treated group, expressed as a percentage of the colonies in the DMSO-treated group (four bars at right; two donors).
Figure 2
Figure 2
MEK inhibition causes cell cycle arrest and modulates p27Kip1 and p21Waf1/CIP1 expression. (a) OCI-AML3 (open circles), HL-60 (filled circles), and freshly isolated leukemic blasts from patient 3 (filled squares) were cultured for 48 hours in the presence of DMSO or PD98059 at the indicated concentrations, and then stained for DNA content. Results are expressed as percentage of S-phase cells in the DMSO-treated group. Cell line results are representative of one of three independent experiments. (b) OCI-AML3, HL-60, and U937 cells were cultured for 24 hours in the presence of DMSO or PD98059 (20 μM), and subjected to Western blot analysis with mAb’s specific for the indicated cell cycle–regulating proteins. Results are representative of one of three independent experiments. (c) OCI-AML3 cells were cultured for the indicated times in the presence of DMSO or PD98059 (20 μM), and subjected to quantitative real-time PCR analysis of p21Waf1/CIP1 (filled circles) and p27Kip1 (open circles) RNA transcripts. Results are expressed as percentage of specific RNA transcripts in the DMSO-treated group and are representative of one of two independent experiments. (d) OCI-AML3 cells were cultured for the indicated times in the presence of DMSO or PD98059 (20 μM) and assessed for DNA content. Results are expressed as percentage of S-phase cells in the DMSO-treated group. Two hours after the addition of PD98059, cells were lysed, and the phosphorylation status of p27 Kip1 was analyzed by Western blot using T187-phosphospecific Ab’s (p-p27Kip1).
Figure 3
Figure 3
MEK inhibition sensitizes to spontaneous and drug-induced apoptosis. (a) OCI-AML3 cells were cultured in the presence of DMSO or PD98059 (20 μM) and stained for annexin V binding at the indicated times. Matched DMSO control at 96 hours is shown (C). Results are representative of one of three independent experiments. (b) Primary AML blasts were cultured in the presence of DMSO or PD98059 at the indicated doses for 96 hours. Apoptosis was then measured as percentage of cells with hypodiploid DNA content. Results are expressed as the net apoptosis induction (percentage of apoptosis in treated cells minus percentage of apoptosis in DMSO-treated cells) and represent the mean ± SD of the results obtained in three different patient samples (patients 3, 14, and 16). (c) AML cell lines were cultured for 96 hours in the presence of DMSO (white bars), 20 μM PD98059 (hatched bars), 1 μM ATRA (gray bars), or a combination of PD98059 and ATRA (black bars), and stained for annexin V binding. Results are representative of one of three independent experiments. (d) HL-60 cells were exposed to 10 μM Ara-C, washed, and cultured in the presence of DMSO (gray bars) or 20 μM PD98059 (black bars). White bars and hatched bars represent DMSO- and PD98059-treated HL-60 cells, respectively, in the absence of prior exposure to Ara-C. At the indicated times, cells were stained for annexin V binding. *DMSO-treated cultures were discontinued after 144 hours due to overgrowth. Results are representative of one of three independent experiments.
Figure 4
Figure 4
MEK inhibition downregulates antiapoptotic proteins of the IAP and Bcl-2 families. (a) OCI-AML3, HL-60, and U937 cells were cultured under standard conditions for 48 hours in the presence of DMSO or PD98059 (20 μM), and then subjected to Western blot analysis with Ab’s specific for the indicated antiapoptotic proteins of IAP (survivin, XIAP) and Bcl-2 (Mcl-1, Bcl-XL, Bcl-2) families. Results are representative of one of three independent experiments. (b) The MAPK phosphorylation status of AML cell lines was assessed by Western blot analysis. The ratio of phosphorylated to total p42MAPK (Phospho/total p42MAPK) was then plotted against the percent reduction in survivin expression observed in the corresponding cell lines after PD98059 treatment, and regression analysis was performed (R2 = 0.9104, P = 0.012). Results are representative of one of three independent experiments.
Figure 5
Figure 5
Effect of PD184352 on AML cell lines and primary samples. (a) OCI-AML3 cells were exposed to DMSO (C) or PD184352 for 6 hours and analyzed for MAPK phosphorylation. (b) OCI-AML3 cells were cultured in the presence of DMSO or PD184352. After 96 hours, cells were assessed for viability (open circles) and DNA content (filled circles). Results show viable cells in the PD98059-treated group, expressed as percentage of the viable cells in the DMSO-treated group, and percentage of cells with a hypodiploid DNA content, respectively, and are representative of one of three independent experiments. (c) OCI-AML3 (open circles), HL-60 (filled circles), and leukemic blasts from patient 19 (open squares) were cultured in the presence of DMSO or PD184352 for 48 hours and stained for DNA content. Results are expressed as percentage of S-phase cells in the DMSO-treated group. Cell line results are representative of one of three independent experiments. (d) The MAPK phosphorylation status of AML cell lines was assessed by Western blot. The ratio of phosphorylated to total p42MAPK was then plotted against the IC50 of PD184352 for the corresponding cell lines, and regression analysis was performed (R2 = 0.8933, P = 0.015). (e) CD34+ cells from healthy donors (n = 3) and blasts from AML patients (n = 7) were analyzed for phosphorylated MAPK by Western blot. Patient samples showing a greater than 25% decrease (from patients 14, 19, 21, and 24) and a less than 25% decrease (from patients 17, 20, and 22) in cell viability after in vitro exposure to MEK inhibitors are labeled as sensitive and resistant, respectively.
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