The MEK inhibitor, PD98059, reduces survival but does not block acute myeloid leukemia blast maturation in vitro

The appearance of blasts in acute myeloid leukemia (AML) reflects a shift f rom cellular processes inducing maturation and cell death to those favourin g survival and accumulation. We have monitored changes in the growth factor signalling molecule MAPKinase, in the cytoprotective protein Bcl-2 and in the cell death protein Bar, during maturation of proliferating and non-prol iferating AML blasts in vitro. Eighteen AML samples were cultured for 7 d i n serum-free medium with or without a supplement of recombinant cytokines c omprising c-kit ligand, IL3 and GMCSF. Maturation of AML blasts, as assesse d by morphology on Romanowsky-stained slides of 7/18 samples and by changes in surface CD markers on all 18 leukemias, occurred in both the absence an d presence of cytokines. Cell numbers decreased to a mean of 71% after 7 d of cytokine-free culture, but increased to 210% in cytokine-supplemented cu ltures. The proportion of CD15-positive cells, assessed by flow cytometry, increased over 7 d in 17/18 samples, from a mean of 22% to 68% in cytokine- free cultures and to 72% in cytokine-supplemented cultures (p = < 0.0001 fo r both). By immunofluorescence/ flow cytometry, there was no significant ch ange: in Bcl-2 over 7 d of culture, while Bar increased, particularly in cy tokine-free cultures (2.2-fold), which led to a significant decrease in the Bcl-2/Bax ratio. Immunoblotting demonstrated that ERK was briefly phosphor ylated after seeding AML blasts into culture. PD98059, an inhibitor of MAPK inase kinase (MEK) which activates MAPKinase, inhibited this transient ERK phosphorylation but was unable to block maturation as measured by acquisiti on of CD15 in samples from 12 patients with low starting numbers of CD 15-p ositive cells. PD98059, however, reduced cell numbers in 7-d liquid culture and, in cytokine-supplemented cultures, this was associated with a 1.3-fol d increase in Bcl-2 (p =0.012) and a 1.4-fold increase in Bar (p=0.02). Ove rall, these data demonstrate that most leukemic populations can partially d ifferentiate in vitro without the need for cytokines or inducers. The MAPKi nase pathway is not required for this maturation, but it does maintain cell viability in the absence or presence of cytokines. A rise in Bcl-2 may not protect AML blasts in the face of elevated Bar.