Leukemia (2002) 16, 486–507 2002 Nature Publishing Group All rights reserved 0887-6924/02 $25.00 www.nature.com/leu REVIEW The Raf/MEK/ERK signal transduction cascade as a target for chemotherapeutic intervention in leukemia JT Lee Jr1 and JA McCubrey1,2 1Department of Microbiology and Immunology, Brody School of Medicine at East Carolina University, Greenville, NC, USA; and 2Leo Jenkins Cancer Center, Brody School of Medicine at East Carolina University, Greenville, NC, USA The Raf/MEK/ERK (MAPK) signal transduction cascade is a constituents of these pathways are also ideal candidates for vital mediator of a number of cellular fates including growth, novel anti-neoplastic chemotherapies. This review will focus proliferation and survival, among others. The focus of this review centers on the MAPK signal transduction pathway, its on the MAP kinase cascades, the repertoire of proteins which mechanisms of activation, downstream mediators of signaling, comprise these pathways, and possible routes of chemothera- and the transcription factors that ultimately alter gene peutic intervention for leukemic disorders within these expression. Furthermore, negative regulators of this cascade, signaling cascades. SPOTLIGHT including phosphatases, are discussed with an emphasis placed upon chemotherapeutic intervention at various points along the pathway. In addition, mounting evidence suggests that the PI3K/Akt pathway may play a role in the effects elicited 2 Overviewof the MAP kinases via MAPK signaling; as such, potential interactions and their possible cellular ramifications are discussed. The serine/threonine MAP kinases are activated in response Leukemia (2002) 16, 486–507. DOI: 10.1038/sj/leu/2402460 to upstream receptor tyrosine kinases and/or cytokine recep- Keywords: Ras; Raf; MEK; ERK; phosphatase; transcription factor; tors that associate with heterotrimeric G proteins so as to trig- inhibitors; IL-3; signal transduction; leukemia; oncogenes; PI3K; Akt ger downstream pathways. The most extensively studied MAP kinase pathway is the Raf-MEK-ERK cascade. Just over a dec- ade ago, the extracellular signal-regulated kinases (ERKs) were 1 Introduction identified as a novel protein kinase family that exhibited pro- liferative effects when activated.2–5 Subsequent studies soon Hematopoiesis occurs through a complex series of intricate, identified the upstream activators of ERK, the MEK (MAP/ERK highly regulated intracellular steps, each of which ultimately kinase) family of kinases; the MEKs are dual-specificity kinases contribute to the proper development of the body’s myeloid, that can phosphorylate both serine/threonine and tyrosine lymphoid and erythroid lineages. Unfortunately though, these residues on the MAP kinases.6–8 A kinase responsible for acti- processes sometimes become dysregulated due to over- vation of MEK is Raf and directly upstream of Raf is the expression of certain oncogenes, aberrant behavior of onco- GTPase, Ras.9,10 The Ras oncoprotein has been noted to be proteins, and expression of chromosomal abnormalities, mutated in approximately 20% of all human malignancies.11 among others. Thus, the elaborate process of hematopoiesis Together, these proteins relay various stimuli to the cell can be changed from one that is necessary for life to another nucleus, each of which ultimately contribute to cellular that is able to endanger it. proliferation.12 Our understanding of the cellular changes that occur in Another member of the MAP kinase family of proteins is the order for leukemogenesis to arise is essential for the develop- p38 kinase. The p38 group of MAPKs has been found to be ment of successful therapies to treat these types of disorders. involved in inflammation, cell growth, cell differentiation, cell A prime example of how comprehending a cell’s demise can cycle progression, and cell death.13 p38 exists in four different lead to the development of an effective drug is the Bcr-Abl isoforms: ␣, ␤, ␥ and ␦.14–17 Although each of these isoforms tyrosine kinase inhibitor STI571 (trademark name, Gleevec); is moderately different in sequence(s) and structure(s), their STI571 functions by competitively inhibiting the binding site activation loops each contain a TGY sequence motif. The p38 of ATP on the kinase thus preventing downstream activation MAP kinases are selectively activated by two MEK isoforms, and amplification of proliferative signals. In its first phase of either MEK3 or MEK6. MEK6 appears to phosphorylate each clinical trials, STI571 proved to have hematologic responses of the p38 isoforms with little to no discrimination between in 53 out of 54 patients with chronic myelogenous leukemia each of them. However, MEK3 is only able to activate the (CML) within a 4-week period.1 Furthermore, extensive testing p38␣ and p38␤ isoforms.18 is now underway to explore these promising results. The third MAPK of interest is the c-JUN NH2-terminal pro- As alluded to above, the exploitation of knowledge gained tein kinase (JNK) which is also commonly referred to as the from studying cell signaling pathways can be extremely stress-activated protein kinase (SAPK). The JNK kinases are attractive when considering chemotherapeutic options for tre- similar to the p38 family of kinases in that although many of ating a variety of neoplasia. The mitogen-activated protein the stimuli that activate these pathways are pro-apoptotic, the kinases (MAPKs) are key regulators of embryogenesis, cell dif- biological outcome of the aforementioned stimuli appears to ferentiation, cell proliferation, and even apoptosis; thus, the be largely dependent on cell type. There are three JNK family members (JNK1, JNK2, JNK3) which are activated by two dis- tinct MAPK kinases: MKK4/SEK1 and MKK7.19–21 Each of the Correspondence: JA McCubrey, Department of Microbiology and Immunology, Brody School of Medicine at East Carolina University, upstream mediators of JNK phosphorylation are activated in Greenville, NC 27858 USA; Fax: 252 816 3104 response to a variable number of stimuli including inflamma- Received 21 December 2001; accepted 16 January 2002 tory cytokines (ie TNF and IL-1␤), UV light, reactive oxygen Therapeutic intervention in leukemia via Raf/MEK/ERK pathway JT Lee and JA McCubrey 487 species (ROS), heat, osmotic shock and withdrawal of growth increased tyrosine kinase activity on the intracellular portion factors.22 A simplistic overview of the MAP kinase cascades of the receptor. Much of the tyrosine kinase activity is due to is presented in Figure 1. autophosphorylation that occurs within the two units of the receptor, where one acts as a kinase and the other as a sub- strate.26 These phosphorylated residues then become docking 3 The Ras-Raf-MEK-ERK signal transduction cascade sites for intracellular proteins that contain Src Homology-2 (SH-2) domains. Through this process, an intracellular con- Target: ligand–receptor interaction glomerate of proteins can initiate immediate signaling, in a variety of cascades, from the binding of one ligand to its As previously mentioned, the Raf-MEK-ERK signal transduc- receptor.27–29 tion cascade is primarily activated in response to various Dysregulated expression of either the ligand or the receptor extracellular growth factors which are able to initiate intra- can contribute to the development of neoplastic growth. Thus, cellular signaling. This mitogenic signal most often occurs at targeting either the ligand or the receptor with chemotherapy the level of a ligand–receptor interaction, followed by down- is an ideal candidate for regulating uncontrolled signaling stream signaling, which ultimately causes altered regulation from within these complexes. This tactic would most likely be of the genes responsible for oncogenesis. For this reason, com- employed so as to neutralize the receptor with a synthesized prehending the complex, interwoven signaling that occurs ligand (or vice versa) by overexpression of such a molecule. from receptor engagement to the onset of gene transcription This section contains a discussion of several growth factors, can help us unlock the mysteries of leukemogenesis and allow their receptors, and their roles in oncogenesis. Furthermore, for the development of chemotherapeutic drugs. possible sites for chemotherapeutic intervention will also be Cytokine and growth factor receptors are composed of at addressed. least two subunits which, when alike, form homodimers (ie granulocyte colony-stimulating factor (G-CSF) and EPO receptor). These receptor subunits may also be different (ie IL- Insulin-like growth factor-1 (IGF-1) receptor 3, GM-CSF, IL-5 receptors), which are collectively known as heterodimers.23,24 Some receptors appear to be non-discrimi- IGF-1-R has been associated with a variety of types of carci- nate with respect to their dimerization preference, in which nomas including breast cancer,30 medullocarcinomas,31 case they may form either hetero- or homodimers (ie epider- Ewing’s sarcoma,32 pancreatic tumors,33 and a variety of leu- mal growth factor receptor (EGF-R) family members).25 kemias,34 among others. Most recently, researchers have been Finally, some receptors exist as trimer entities, such as the investigating the correlation between IGF-1-R/IGF-1-binding tumor necrosis factor receptor (TNFR). When a growth factor, protein (IGF-1-BP) expression and survival in child patients such as EGF, binds to its respective receptor, it induces both with acute lymphoblastic leukemia (ALL).35,36 The mitogenic the aforementioned dimerization of the receptor and signal