The MEK inhibitor, PD98059, reduces survival but does not block acute myeloid leukemia blast maturation in vitro Baines P, Fisher J, Truran L, Davies E, Hallett M, Hoy T, Burnett AK. The MEK inhibitor, PD98059, reduces survival but does not block acute myeloid leukemia blast maturation in vitro. Eur J Haematol 2000: 64: 211±218. # Munksgaard 2000. Abstract: The appearance of blasts in acute myeloid leukemia (AML) re¯ects a shift from cellular processes inducing maturation and cell death to those favouring 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 Bax, during maturation of proliferating and non-proliferating AML blasts in vitro. Eighteen AML samples were cultured for 7 d in serum-free medium with or without a supplement of recombinant cytokines comprising c-kit ligand, IL3 and GMCSF. Maturation of AML blasts, as assessed 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 and 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 cultures. The proportion of CD15-positive cells, assessed by ¯ow 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 for both). By immuno¯uorescence/ ¯ow cytometry, there was no signi®cant change in Bcl-2 over 7 d of culture, while Bax increased, particularly in cytokine-free cultures (2.2- fold), which led to a signi®cant decrease in the Bcl-2/Bax ratio. Immunoblotting demonstrated that ERK was brie¯y phosphorylated after seeding AML blasts into culture. PD98059, an inhibitor of MAPKinase kinase (MEK) which activates MAPKinase, inhibited this transient ERK phosphorylation but was unable to block maturation as measured by acquisition of CD15 in samples from 12 patients with low starting numbers of CD15-positive cells. PD98059, however, reduced cell numbers in 7-d liquid culture and, in cytokine-supplemented cultures, this was associated with a 1.3-fold increase in Bcl-2 (p=0.012) and a 1.4- fold increase in Bax (p=0.02). Overall, these data demonstrate that most leukemic populations can partially differentiate in vitro without the need for cytokines or inducers. The MAPKinase 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 Bax. Paul Baines, Janet Fisher, Louise Truran, Eryl Davies, Maurice Hallett, Terry Hoy, Alan K. Burnett Haematology Department and Molecular Signalling Group, University Hospital of Wales and University of Wales College of Medicine, Cardiff, UK Key words: acute myeloid leukemia; differentiation; PD98059; MEK; MAPKinase; Bcl-2; Bax Correspondence: Dr Paul Baines, Department of Haematology, University Hospital of Wales, Cardiff CF4 4XW, UK Tel: +1222 743486 Fax: +1222 744655 e-mail: BainesP@CF.Ac.UK Accepted for publication 10 November 1999 One way of reducing blast numbers in acute myeloid leukemia (AML) is to overcome the maturation block in these cells (1±3). Mature cells eventually apoptose, perhaps because levels of the anti-apoptotic Bcl-2 protein fall (4±6). Maturation can also be accompanied by a decline in the activity of drug ef¯ux pumps, which would reduce the resistance of leukemic cells to cytotoxic drugs (7). Differentiation therapy, using all-trans retinoic acid (ATRA), has been particularly successful in Eur J Haematol 2000: 64: 211±218 Printed in UK. All rights reserved Copyright # Munksgaard 2000 EUROPEAN JOURNAL OF HAEMATOLOGY ISSN 0902-4441 211