[CANCER RESEARCH 62, 188 –199, January 1, 2002] Pharmacologic Mitogen-activated Protein/Extracellular Signal-regulated Kinase Kinase/Mitogen-activated Protein Kinase Inhibitors Interact Synergistically with STI571 to Induce Apoptosis in Bcr/Abl-expressing Human Leukemia Cells 1 Chunrong Yu, Geoffrey Krystal, Lyuba Varticovksi, Robert McKinstry, Mohamed Rahmani, Paul Dent, and Steven Grant 2 Division of Hematology/Oncology [C. Y., G. K., M. R., S. G.], and the Department of Radiation Oncology [P. D., R. M.], Medical College of Virginia, Richmond, Virginia 23298, and the Department of Medicine, Tufts University, Boston, Massachusetts 02135 [L. V.] ABSTRACT Interactions between the kinase inhibitor STI571 and pharmacological antagonists of the mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK)/mitogen-activated protein kinase (MAPK) cascade have been examined in human myeloid leukemia cells (K562 and LAMA 84) that express the Bcr-Abl kinase. Exposure of K562 cells to concentra- tions of STI571 that minimally induced apoptosis (e.g., 200 nM) resulted in early suppression (i.e., at 6 h) of p42/44 MAPK phosphorylation fol- lowed at later intervals (i.e., >24 h) by a marked increase in p42/44 MAPK phosphorylation/activation. Coadministration of a nontoxic con- centration of the MEK1/2 inhibitor PD184352 (5 M) prevented STI571- mediated activation of p42/44 MAPK. Cells exposed to STI571 in combi- nation with PD184352 for 48 h demonstrated a very dramatic increase in mitochondrial dysfunction (e.g., loss of m and cytosolic cytochrome c release) associated with procaspase-3 activation, poly(ADP-ribose) polym- erase cleavage, and the appearance of the characteristic morphological features of apoptosis. Similar results were obtained using other pharma- cological MEK1/2 inhibitors (e.g., PD 98059 and U0126) as well as another leukemic cell line that expresses Bcr-Abl (e.g., LAMA 84). However, synergistic induction of apoptosis by STI571 and PD184352 was not observed in human myeloid leukemia cells that do not express the Bcr-Abl kinase (e.g., HL-60 and U937) nor in normal human peripheral blood mononuclear cells. Synergistic potentiation of STI571-mediated lethality by PD184352 was associated with multiple perturbations in signaling and apoptotic regulatory pathways, including caspase-dependent down-regu- lation of Bcr-Abl and Bcl-2; caspase-independent down-regulation of Bcl-x L and Mcl-1; activation of JNK, p38 MAPK, and p34 cdc2 ; and diminished phosphorylation of Stat5 and CREB. Significantly, coexposure to PD184352 strikingly increased the lethality of a pharmacologically achievable concentration of STI571 (i.e., 1–2 M) in resistant K562 cells expressing marked increases in Bcr-Abl protein levels. Together, these findings raise the possibility that treatment of Bcr-Abl-expressing cells with STI571 elicits a cytoprotective MAPK activation response and that interruption of the latter pathway (e.g., by pharmacological MEK1/2 inhibitors) is associated with a highly synergistic induction of mitochon- drial damage and apoptosis. They also indicate that in the case of Bcr- Abl-positive cells, simultaneous interruption of two signal transduction pathways may represent an effective antileukemic strategy. INTRODUCTION The Bcr/Abl kinase is an oncogenic fusion protein that arises as a consequence of the joining of varying NH 2 -terminal sequences of the Bcr 3 gene on chromosome 22 with COOH-terminal sequences (exons 2–11) of the abl gene on chromosome 9 (1). This fusion protein occurs in 95% of CML and 10 –15% of acute lymphoblastic leukemia pa- tients (2). As a result of alterations in both inter- and intramolecular interactions, the protein tyrosine kinase domain of the Bcr/Abl fusion protein becomes constitutively activated, an event that is required for malignant transformation (3). There is compelling evidence implicat- ing constitutive activation of the Bcr/Abl oncogene in the pathogen- esis of these disorders, including the observations that expression of the p210 Bcr/Abl fusion protein in murine hematopoietic cells results in a disease resembling CML in host mice (4 – 6). Among other actions, the Bcr/Abl kinase protects hematopoietic progenitor cells from spontaneous apoptosis as well as that induced by various nox- ious stimuli, including chemotherapeutic drugs (7). Although the precise mechanism(s) by which Bcr/Abl exerts its antiapoptotic prop- erties is (are) unknown, several candidate downstream mediators have been proposed, including increased expression of the antiapoptotic proteins Bcl-x L (8) and induction of nuclear factor B (9). Given the well-defined role of the Bcr/Abl kinase in CML and related disorders, it represents a very attractive molecular target for pharmacological intervention. Recently, considerable attention has focused on CGP57148B, currently referred to as STI571, an inhibitor of the Bcr/Abl, Kit, and platelet-derived growth factor receptor ki- nases (10). In in vitro studies, STI571 has been shown to inhibit the growth of Bcr/Abl-positive leukemic cells at micromolar concentra- tions (11). Interestingly, exposure of such cells to STI571 promotes leukemic cell apoptosis (12), suggesting that Bcr/Abl not only confers on these cells a growth advantage but is at the same time required for their survival. Importantly, clinical trials have now demonstrated that STI571, when administered at doses of 300 mg/day, achieves clin- ical remissions in the large majority of patients with CML (e.g., 96%; Ref. 13). In addition, preclinical studies have demonstrated that the combination of STI571 with established chemotherapeutic drugs (e.g., ara-C) results in enhanced toxicity in Bcr/Abl-positive leukemias (14, 15). These findings raise the possibility that combining STI571 with such agents might lead to enhanced activity in CML and/or circum- vention of drug resistance. In this context, Vigneri and Wang (16) reported recently that coadministration of STI571 with leptomycin, an inhibitor of the nuclear export sequence receptor, resulted in increased killing of cells expressing Bcr/Abl. However, in this study optimal killing occurred in cells exposed to 10 M STI571, which is above Received 6/25/01; accepted 11/1/01. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by Grants CA63753 and CA 83705 from the NIH, by a Merit Award from the Veteran’s Administration, and Grant 6630-01 from the Leukemia and Lymphoma Society of America. 2 To whom requests for reprints should be addressed, at Division of Hematology/ Oncology, Medical College of Virginia, MCV Station Box 230, Richmond VA, 23298. Phone: (804) 828-5211; Fax: (804) 828-8079; E-mail: stgrant@hsc.vcu.edu. The abbreviations used are: BCR, breakpoint cluster region; MEK, mitogen-activated protein/extracellular signal-regulated kinase kinase; ERK, extracellular signal-regulated kinase; MAP, mitogen-activated protein; MAPK, mitogen-activated protein kinase; PARP, poly(ADP-ribose) polymerase; Abl, Ableson murine leukemia; CML, chronic myelogenous leukemia; JNK, c-Jun-NH 2 -terminal kinase; TUNEL, terminal deoxynucle- otidyl transferase (Tdt)-mediated nick end labeling; DiOC6, 3,3-dihexyloxacarbocyanine iodide; PBS-T, PBS-Tween; EMSA, electrophoretic mobility shift assay; MMP, mito- chondrial membrane potential. 188 Research. on February 18, 2016. © 2002 American Association for Cancer cancerres.aacrjournals.org Downloaded from