The Raf/MEK/ERK Signal Transduction Cascade As a Target for Chemotherapeutic Intervention in Leukemia JT Lee Jr1 and JA Mccubrey1,2

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 decade ago, the extracellular signal-regulated kinases (ERKs) were 1 Introduction identified as a novel protein kinase family that exhibited proliferative 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 distinct 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 substrate.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 of the IGF-1 receptor/protein interaction is most often transduced via the PI3K pathway,37 but sometimes signaling is relayed via either the MAPK pathway or by mitochondrial translocation of Raf-1.38 Furthermore, it has been reported that two of these three pathways must be active for the mitogenic survival signal to proceed.39 Because signaling through this receptor is able to lead to tumor development, it is an ideal candidate for chemotherapeutic intervention. A schematic representation of potential signal transduction pathways activated by IGF-1 is described in Figure 2. The IGF-1-R gene encodes for a polypeptide gene product that is subsequently proteolytically cleaved into ␣- and ␤- chains that ultimately comprise the receptor. Theoretically speaking, protease inhibitors should therefore be able to inhibit formation of the cleaved subunits of the IGF-1 receptor. The IGF-1 receptor, specifically the ␤-subunit, has been SPOTLIGHT found to be in an altered state in human leukemic cells where it exists as a larger (105 kDa), fetal version of the protein, thereby causing malignancy.40 Accordingly, it has been a target of high priority for neutralization in leukemic disorders. As such, it has been shown, that when evaluating the potential role of IGF-1 as a growth-promoting factor in malignant lym- phocyte development, that ALL cell lines will halt proliferation in response to the addition of anti-IGF-1R antibodies.41 These results had been predicted from previous research which stated that both insulin and IGF-1 were able to stimulate growth of cultured, serum-starved human leukemia cells via their respective receptors.42 Also, IGF-1R overexpression has been shown to be sufficient to abrogate the cytokine depen- dence of FDC-P1 hematopoietic precursor cells, an essential Figure 1 Overview of the MAPK cascades. The MAPK signal trans- 43 duction pathways can be induced by either cytokines or stress. Acti- step in leukemogenesis. Most recently, research has vation of the different cascades can have diverse effects on cell revealed that in patients with CML, treatment with interferon- growth, inflammatory responses, and/or apoptosis. ␣ is able to increase expression of IGF-1R on lymphoid cells.

Leukemia Therapeutic intervention in leukemia via Raf/MEK/ERK pathway JT Lee and JA McCubrey 488 SPOTLIGHT

Figure 3 Mechanisms of regulation of PDGF receptor (PDGF-R) Figure 2 Mechanism and diversity of anti-apoptotic signaling elic- activity. (a) Normal activation of the PDGF receptor by PDGF; (b–d) ited via IGF-1/IGF-1 receptor interactions. (a) Inactive IGF-1 receptor; Different mechanisms to regulate PDGF-R activity by targeting: (b) (b) Induction of the PI3K cascade via an active IGF-1 receptor; (c) ligand binding; (c) receptor dimerization; and (d) receptor tyrosine Induction of the Ras/Raf/MEK/ERK cascade via an active IGF-1 kinase activity. receptor.

ation.51,52 Furthermore, the development of chronic myelo- Thus, overexpression of the IGF-1R may enable the immune monocytic leukemia and progression to AML is directly system to mount a more efﬁcient attack on the malignant cell related to the constitutive activation of PDGF ␤ receptors as clone by enhancing immunocompetent cell proliferation a result of an aberrant chromosomal translocation.53,54 The and action.44 aforementioned activation of the PDGF receptor is a direct Perhaps the most intriguing aspect of the mitogenic sig- result of its translocation with the ets-like transcription factor, naling that occurs from the IGF-1 receptor is the level of cross- TEL.53,55 This association fuses the amino terminus of TEL, talk that is initiated through the PI3K pathway. As previously which contains the helix–loop–helix domain, to the trans- mentioned, the vast majority of signaling through IGF-1R membrane and cytoplasmic domains of the PDGF ␤ receptor occurs at the level of PI3K. PI3K is then able to activate causing constitutive PDGF-R activity. A similar oncogenic Akt/PKB in an indirect manner so as to elicit anti-apoptotic chromosomal translocation with TEL has been noted with the effects.45,46 It is at this point that synergistic signaling has been Janus associated kinase (JAK), whereby cytokine-independent reported. Recent evidence shows that when both Akt and Raf cellular proliferation resulted.56 Moreover, it has also been are active, the frequency of isolation of cytokine-independent discovered that the PDGF-B (ligand) subunit is associated with hematopoietic cells increases over 100-fold.47 A more in- the pathogenesis of marrow ﬁbrosis associated with CML.57 depth discussion about PI3K/MAPK interactions is discussed Lastly, the PDGF-A growth factor has been isolated from two later in this review. pre-B ALL cell lines, implying a possible autocrine component to the transformation process.58 Taken together, these data suggest that PDGF and its cognate receptor are ideal targets Platelet-derived growth factor (PDGF) receptor for drug therapies. Researchers have developed PDGF antagonists at each level PDGF family members are dimeric molecules with a con- of PDGF-induced activation (ie ligand binding, receptor served sequence of roughly 100 amino acid residues contain- dimerization, and activation of the receptor kinase). An over- ing a motif of eight cysteine residues. There are three known view of these antagonists is depicted in Figure 3. Neutralizing existing isoforms, those being homo- and heterodimers of the antibodies against both the PDGF protein isomers and the A and B polypeptide chains.48 More recently, though, PDGF receptor proteins have been raised and tested in various additional PDGF isoforms have been discovered, known as animal systems.59–64 Nucleic acid ligands, known as aptamers, PDGF-C and D, respectively.49,50 can also be used to neutralize PDGF signaling. Aptamers are PDGF, like IGF-1, has been shown to activate both the Raf- generated by a process that selects for molecules with the MEK-ERK and PI3K pathways to induce cellular transform- highest binding capabilities over several rounds of binding to

Leukemia Therapeutic intervention in leukemia via Raf/MEK/ERK pathway JT Lee and JA McCubrey 489 the protein of choice. After numerous selective rounds, a high- of the IL-3R ␣-subunit allows for hematopoietic cellular pro- affinity molecule specific for the target is eventually isolated. liferation in suboptimal concentrations of the cytokine, IL-3.88 This process is commercially known as systematic evolution This observation has recently been supported by researchers of ligands by exponential enrichment (SELEX) and is currently who have found that the IL-3R ␣-subunit is a unique marker under extensive investigation. Recently, a group from Gilead for detection of acute myelogenous leukemia (AML) cells, as Sciences isolated an aptamer specific for PDGF that has an it is overexpressed in primitive leukemia cells.89 Conversely, ␤ astonishingly low equilibrium dissociation constant of less constitutive expression of the c chain of IL-3R or GM-CSFR than 3 × 10−10.65 mutants imparts acute, chronic myeloproliferative disorder(s) Scientists have also found that PDGF-A-mediated DNA syn- in murine hematopoietic cells.90–92 It is also interesting to note ␤ thesis can be halted by overexpressing a segment of the extra- that a truncated isoform of the c chain of the GM-CSF, IL-3, cellular portion of the PDGF receptor.66 These proteins have and IL-5 receptors has been found overexpressed in patients been shown to inhibit PDGF mainly by preventing autophos- with AML and ALL; this isoform appears to block signal trans- phorylation as described earlier. Furthermore, these com- duction in an unexplained manner resulting in malignant 93 pounds are able to block PDGF activity with IC50 values rang- transformation. ing from 100 nM up to 1 ␮M.67–69 The development of monoclonal antibodies specific for dis- As denoted previously, receptor dimerization is a critical tinct domains of the aforementioned receptors has shown step in transducing extracellular stimuli to the cell. This stage promise over the past few years. A monoclonal antibody has of mitogenic signal transduction also provides a potential for been developed that is specific for a region of the IL-3R.94 chemotherapeutic intervention. To illustrate this point, the use Shortly thereafter, monoclonal antibodies were generated to of monoclonal antibodies against specified domains of the target the IL-3R, but these also bound the GM-CSF and IL- PDGF receptor have been shown to significantly reduce and 5 receptors due to their high degree of homology on their ␤ 95 sometimes abolish PDGF-mediated signaling at IC50 values of c chains. under 1 nM.64 Furthermore, it was observed that antibodies The vast majority of the research done to antagonize the IL- directed at specific regions of the PDGF-R could inhibit 3 and GM-CSF receptors over the past few years has been in autophosphorylation without interfering with actual PDGF the area of toxic fusion proteins. DT388-GM-CSF, a fusion binding.64,70 protein derived from a truncated diphtheria toxin and GM- Ultimately, it is the kinase activity of the PDGF receptor CSF, has been shown to cause death in AML cells expressing that transduces the extracellular signal to activate intracellular the GM-CSF receptor.96 This effect was not evident in non- oncogenes. As such, several compounds have been developed transformed hematopoietic progenitor cells, thus providing a that specifically target the PDGF-R kinase. These tyrosine kin- rational chemotherapy for the treatment of AML. These experi- ase inhibitors are normally low molecular weight proteins that ments have been extended to an array of leukemic disorders, function to compete with ATP-binding at the kinase active including juvenile and chronic myelomonocytic leukemias, site. Over the past decade, a number of compounds have each of which is susceptible to DT388-GM-CSF.97,98 Nearly been developed including AG1296,71,72 CGP53716,73 STI- identical studies have been performed using the same trunc- 571 (also known as Gleevec, CGP57148),74,75 SU101,76 ated diphtheria toxin attached to IL-3. These studies have also Ki689677 and RPR101511A,78 each of which binds non-dis- yielded positive results with respect to purging leukemic cells criminately to the kinase(s) of the different PDGF receptor iso- from bone marrow grafts.99,100 forms. Each of the aforementioned inhibitors has been applied Recently, modified and chimeric cytokines have been to animal models with some success; but, by far, STI-571 has developed to aid in the repopulation of hematopoietic cell been pegged as the next miracle drug in leukemic ther- compartments following myelosuppressive chemotherapy. apies.79,80 Lastly, other similar compounds have been synthe- Daniplestim is an engineered IL-3 receptor agonist created by sized and used successfully in in vitro models, but have not truncations and amino acid substitutions to the native IL-3 yet been tested in animal models.81,82 protein. It can bind to the IL-3 receptor with significantly higher affinity than native IL-3.101,102 Myelopoietin-1 belongs to a family of engineered chimeric molecules with dual agon- IL-3 and GM-CSF receptors istic capabilities for the IL-3 and G-CSF receptors.101,102 When fused together to produce a chimeric cytokine, these proteins IL-3 and GM-CSF possess unique receptors, in that they lack have an enhanced ability to suppress apoptosis in hematopo- SPOTLIGHT intrinsic tyrosine kinase activity as compared to the PDGF-R ietic cells.101,102 Furthermore, chimeras between G-CSF and ␤ and IGF-R. To compensate for this shortcoming, the c chain FLT-3 ligand have also been developed and show potential of the receptor is able to recruit specific kinases in order to for preventing apoptosis in certain cells, as well as dendritic transduce extracellularly derived signals. One of the prototypi- cells.103,104 These types of studies should provide much ␤ cal kinases that serves this function is the JAK2 kinase. The c needed insight into post-chemotherapeutic care in leukemia chain is also able to attract the adaptor molecule, Shc, which patients. allows for the association of growth factor receptor-bound (Grb) protein and mammalian son of sevenless (SOS), each of which enables subsequent activation of the Ras oncoprotein. Target: Ras It is thought that an 85-kDa protein, identified as the p85 subunit of PI3K (described in more detail later) is also able to bind The 21-kDa Ras gene product is a monomeric membrane- ␤ to the c chain, thus enabling activation of the PI3K signal localized G-protein that initiates MAPK signal transduction. It transduction pathway.83 These biochemical events each lead is approximated that Ras is mutated in nearly 20% of all can- to DNA synthesis and prevention of apoptosis. cers.11 Within hematopoietic cancers, it has been seen that Hematopoietic cells can become transformed in response Ras is mutated in approximately 20% to 60% of AMLs.105–108 to mutation(s) occurring within the IL-3 gene which impart Because Ras is widely mutated in a variety of human malig- constitutive IL-3 expression.84–87 Furthermore, overexpression nancies and is a molecular switch in a network of signaling

Leukemia Therapeutic intervention in leukemia via Raf/MEK/ERK pathway JT Lee and JA McCubrey 490 pathways, it has become an important target for the develop- mediates proliferative signaling (Figure 4). Therefore, the ment of anticancer drugs. development of FT inhibitors is a viable strategy used to Ras is a membrane-bound guanosine triphosphate inhibit the oncogenic effects of Ras. At the time of publication, (GTP)/guanosine diphosphate (GDP)-binding (G) protein that there are currently four FTIs in clinical trials worldwide: acts to relay signals from the lipid-rich cellular membrane to R115777, SCH66336, L-778123 and BMS-214662.115 the nucleus. These signals lead to protein synthesis, regulation R115777 has undergone phase I clinical trials and was found of cell survival, cellular proliferation, and differentiation. After to significantly reduce Ras-mediated signaling.116,117 Similar binding of a ligand to its cognate receptor, dimerization of results were observed for the compound SCH66336.118 that receptor occurs, as described previously. Dimerization There are some data that argue that the effects of FTIs are causes the intracellular tyrosine kinase(s) to become phos- not only due to the inhibition of Ras farnesylation, but also phorylated, which then becomes bound by the SH2 domains due to the inhibition of farnesylation of other proteins essential 109 of the adaptor proteins, such as Grb2. Not only does Grb2 for cellular proliferation. RhoB is a 21-kDa G-protein that possess an SH2 domain, but it also has an SH3 domain that helps regulate receptor signaling and, in addition, is a target binds to proline-rich motifs of other peptides (ie son of seven- of FT inhibitors. Du and Prendergast119 showed that in the less (SOS)). SOS is a guanine dissociation stimulator of Ras presence of FTIs, RhoB became geranylgeranylated (RhoB- which binds to Ras after receptor activation. Ras’ native con- GG) in high quantities. At these concentrations, it was found formation is then compromised and GDP is dissociated from that RhoB-GG became toxic to malignant cells, but not nor- Ras, allowing for the subsequent association of GTP. In the mal cells. A second hypothesis dealing with the mechanism GTP-bound form, Ras is in its active state and able to recruit SPOTLIGHT of anti-tumor activity elicited by FTIs has also recently been Raf to the cell membrane, thus enabling activation of developed. Jiang et al120 have demonstrated that FT inhibitors Raf.110–113 However, there exist several GTPase-activating are able to inhibit PI3K-mediated signaling and induce proteins (GAPs) that are able to stimulate the GTPase activity apoptosis in cancer cells that overexpress the AKT2 isoform. of Ras, which convert it back to its original inactive, GDP- bound state.114 Supporting this hypothesis are data that indicate that FTIs are able to block a downstream kinase of PI3K, specifically There have been three main approaches taken to inhibit S6K 121 Ras-mediated signaling: (1) prevention of membrane localiz- p70 . ation of Ras; (2) inhibition of Ras protein expression via anti- Research has also been done to examine the effects of tar- sense nucleotides or RNAs; and (3) inhibition of kinases geting specific RNA sequences to inhibit translation of the Ras downstream of Ras (to be covered at each individual kinase RNA message into a functional protein. This antisense level). Each of these approaches has been successful, to vary- approach uses oligonucleotides, which are complementary to ing degrees, in blocking the proliferative effects of Ras mRNA transcripts of the Ras oncogene. These oligonucleo- activation. tides hybridize to the complementary mRNAs and inhibit their In order for Ras to become active, it must undergo post- subsequent translation into protein(s). Malignant phenotypes translational modifications that add farnesyl or geranylgeranyl have been observed to be suppressed in response to a retrovi- isoprenoid moieties to its carboxy-terminus. These modifi- ral vector-induced transduction of K-Ras antisense RNA of cations are generated by three different enzymes, namely far- human cancer cells.122,123 A second, similar approach has nesyltransferase (FT), geranyltransferase type I (GGTI), and also been used to inhibit abnormal Ras expression. This GGTII. By blocking the enzymatic action of these enzymes, method employs the use of plasmid vectors encoding anti- Ras is unable to localize to the cellular membrane where it sense RNAs that are able to inhibit tumorigenic growth.124

Figure 4 Activation of the Ras/Raf/MEK/ERK pathway by IL-3 and inhibition by farnesyltransferase (FT) inhibitors. (a) Activation of Ras/Raf/MEK/ERK by binding of IL-3 to its cognate receptor and subsequent signaling through coupling to Ras. (b) Uncoupling of the IL-3- mediated response by FT inhibitors which prevent the farnesylation of Ras and resultant targeting of Ras to the lipid-rich cell membrane.

Leukemia Therapeutic intervention in leukemia via Raf/MEK/ERK pathway JT Lee and JA McCubrey 491 Target: Raf Alternative strategies exist to inhibit Raf-mediated signaling. RNA aptamers have been developed that bind to the Ras-bind- Raf-1 was the first identified downstream effector kinase of ing domain of the Raf-1 isoform. These RNA molecules bind Ras11–113 and is currently the most characterized substrate of with fairly high dissociation constants (300 nM) and may be Ras. However, two other Raf isoforms are known to exist, able to inhibit Ras-Raf signaling in mammalian cells.154 those being A-Raf and B-Raf. This family of kinases has been Another approach has been to synthesize proteins that are suggested to have both overlapping and unique regulatory able to antagonize the binding between Ras and Raf.155 An functions.125–127 All three of the Raf isomers have been found additional method devised to inhibit Raf signaling is the devel- to be expressed in hematopoietic cells of either lymphoid or opment of antisense oligodeoxynucleotides that are specific myeloid lineages.23,128,129 The level of Raf expression in trans- for each isomer of Raf.156 Further research into these types of formed hematopoietic cells has been noted to be elevated.130–133 compounds has yielded marginal results, though.157 However, it is not known whether the heightened level of Raf It is of critical importance to mention that Ras also signals expression actually caused the cells to become transformed through parallel pathways in addition to the Raf-MEK-ERK because the cell lines of interest were established tumors. pathway. As such, chemotherapeutic intervention at any point Conversely, it is known that several mechanisms exist to elicit along the pathway may affect the signaling capacities of these dysregulated Raf expression. They include point mutations, alternative pathways. However, discussing the cellular rami- deletions, gene rearrangements and gene amplifications.134–138 fications of these types of interactions is outside the realm of Thus, the collective evidence suggests that Raf is a viable this review. anticancer target for chemotherapeutic intervention. Raf activation occurs on a dual level. In the first step, active GTP-bound Ras recruits Raf to the plasma membrane, which Target: alternative Raf kinases allows subsequent interaction(s) with additional activators that are not well-described.139–141 Though this step is necessary for Raf is activated by more than just Ras; the kinase suppressor activation of Raf, it is not sufficient to cause Raf activation. of Ras (KSR), PKC and the Src and JAK families of kinases Therefore, a secondary stage of activation occurs when Raf is have all been implicated in the Ras-independent activation phosphorylated on tyrosine and/or serine/threonine residue(s). of Raf.158–163 Each of these kinases activates Raf in a unique, This is normally accomplished on tyrosine residues by a mem- deliberate manner. Targeting their inactivation, collectively or ber of the Src family of kinases and on serine/threonine resi- individually, would result in decreased levels of Raf activity dues by protein kinase C (PKC) isoforms.142–144 It is worth- and the subsequent abrogation of unharnessed proliferation. while to note that the S43, S259 and S621 residues found on mutant, non-active Raf are still able to be phosphorylated, suggesting that these residues are not distinct autophosphorylation sites.145 Lastly, dimerization of Raf is also known to JAK kinases induce Raf activity. Therefore, drugs which are able to prevent potential dimerization(s) may be effective anti-signaling The Janus family of kinases (JAKs) is a receptor-associated compounds.146 group of kinases that become phosphorylated in response to Recently, it was reported that Ras is able to induce the het- ligand-receptor binding. After dimerization of the receptor erodimerization of the Raf-1 and B-Raf isoforms.139 The role occurs, the JAKs become phosphorylated on tyrosine residues, that Raf heterodimers play in signal transduction is largely most likely a result of cross-phosphorylation. After they are unknown. As such, elucidation of the role that each of the Raf activated, the JAKs then are able to phosphorylate other tyro- oncogenes plays in tumor development is imperative for the sine residues on the receptor. The phosphorylated residues development of drugs that can effectively treat leukemic dis- then attract a variety of signaling molecules that each contain orders. This is an increasingly crucial issue because the differ- an SH2 domain which enables them to dock to the phos- ent Raf oncogenes have been shown to possess diverse sig- phorylation site. The signal transducers and activators of tran- naling properties in various cell types.147 For example, A-Raf scription (STATs) are a family of transcription factors that fall may promote cellular proliferation, whereas a different iso- into the aforementioned group of SH2-containing signaling form such as B-Raf induces apoptosis and arrests cell cycle molecules.164 When a STAT molecule binds to a phosphoryl- progression.23,148 Rationale mandates, then, that inhibitors to ated residue, it then becomes phosphorylated by a JAK due SPOTLIGHT each kinase must be developed in order to properly regulate to their close proximity. Phosphorylation allows the STATs to these kinases, as they may have opposing roles in either dimerize and enter the nucleus to act as transcription factors preventing or stimulating cellular proliferation. for genes that lead to altered phenotypes.165,166 Geldanamycin has been the mainstay with respect to Raf WHI-P131 is a compound that has been observed to target inhibition over the previous decade. In effect, though, geldan- JAK3 activity.167 JAK3 has been noted to have a role in lymph- amycin does not inhibit Raf activity, rather it destabilizes a oid development and function and thus is a viable candidate protein–protein interaction between Raf and heat shock pro- for chemotherapeutic intervention. WHI-P131 was able to tein (hsp) 90.149 By binding to hsp90, geldanamycin precludes induce apoptosis in the NALM-6 and LC1;19 human leukemia binding between Raf and hsp90 which ultimately prevents cell lines, suggesting that this compound may be an attractive proper Raf signaling functions.150 However, since many pro- candidate for future pharmaceutical endeavors.168 teins interact with hsp90, geldanamycin is not a specific Raf A tryphostin derivative that acts as a kinase inhibitor, AG- inhibitor.151 More recently, a second type of compound has 490, has also been used to inhibit JAK activity. It was first been shown to destabilize the Raf protein: KF25706 and observed in 1996 that this novel compound had apoptotic- KF58333 are oxime derivatives of radicol that have been inducing properties in ALL cells.169 Since then, AG-490 has shown to exhibit anti-tumor activities by acting in a manner been widely noted to act by inhibiting STAT3 activation and similar to geldanamycin.152,153 The chemical structures of inducing apoptosis in leukemic cells.170,171 As such, this com- these and other kinase inhibitors are presented in Figure 5. pound may be ideal for pharmacologically induced inhibition

Leukemia Therapeutic intervention in leukemia via Raf/MEK/ERK pathway JT Lee and JA McCubrey 492 SPOTLIGHT

Figure 5 Chemical structures of various signal transduction inhibitor molecules outlined in this review. (a) PDGF-R and BCR-Abl inhibitors; (b) Raf inhibitors; (c) JAK inhibitors; (d) PKC inhibitors; (e) MEK inhibitors.

of the JAK-STAT pathway and consequent inhibition of the folding of the MAPK proteins of the Raf-MEK-ERK pathway. It Raf-MEK-ERK cascade. has been noted that the kinase domain (CA5) of KSR is able to mediate direct interactions between both MEK1/2 and KSR.183 A similar type of binding interaction is seen with the Protein kinase C (PKC) ERK1/2 kinases; KSR binds to these proteins via a serine-threonine rich domain (CA4) located adjacent to the kinase PKC has been shown to be a potent activator of Raf-1.172 As domain.184 Interestingly, the interaction with ERK is depen- a result, blocking PKC has become another approach to halt dent on Ras activation, whereas the binding to MEK seems to Raf-MEK-ERK signaling in leukemic disorders. Staurosporine be constitutive.185,186 Raf-1/KSR interactions appear to be both is a non-specific protein kinase inhibitor that has been shown Ras-dependent and located at the cytoplasmic membrane.187 to halt cell cycle progression at concentrations ranging from These data, taken together, suggest a unique signaling mech- 1 to 100 nM.173 Staurosporine analogues exist, namely UCN- anism where KSR is able to promote the assembly on a multi- 101, which have a higher affinity for PKC isoforms.174,175 Fur- protein complex that brings MEK into close proximity with thermore, UCN-101 elicits both cell cycle inhibitory and anti- both its upstream activator, Raf, and its downstream substrate, proliferative effects in several tumor cell lines.176,177 Another ERK. Although much of the research done on KSR is relatively staurosporine derivative, CGP 41251, has been developed novel, one might hypothesize that inhibition of one or more and has also shown promise as a potential anti-tumor of these interactions may have anti-neoplastic ramifications. agent.178–180

Target: mitogen-activated protein kinase kinase-1/2 Kinase suppressor of Ras (KSR) (MEK-1/2)

The kinase suppressor of Ras (KSR, also known as CAPK) has Targeting the molecule directly downstream of Raf is another recently been identiﬁed to be a possible scaffolding protein possibility for the rational design of anticancer drugs. The for the entire Raf-MEK-ERK transduction cascade.181 It was MEKs belong to a multi-gene family that includes MEK-1 to originally reported that KSR was a positive effector of Ras sig- MEK-5. Of these isoforms, MEKs-3, 4 and 5 are relatively less naling that appeared to act either upstream of Raf or in a paral- characterized than either MEK-1 or MEK-2. The dearth of lel pathway, but the exact nature of these interactions was not research associated with the former proteins may be directly known.182 However, more recent studies have indicated that correlated to the lack of detectable kinase activity observed KSR may play an integral role in membrane-associated scaf- with the initial research that has been completed.188,189 Con-

Leukemia Therapeutic intervention in leukemia via Raf/MEK/ERK pathway JT Lee and JA McCubrey 493 versely, much research has been done on the MEK-1 and directly blame constitutive ERK activation for the onset of AML MEK-2 isoforms, some of which has shown that they exhibit and/or CML.224,225 Others have reported conflicting results, similar homologies to each other including a proline-rich suggesting that CML cell growth and differentiation are inde- region, which appears to be essential for signal transduction pendent of ERK activation.226 Another group has recently capabilities.190 Furthermore, MEK-1 and MEK-2 (hereafter shown that Ras-mediated ERK activation is absent in primary MEK), are phosphorylated on distinct serine residues, S218 AMLs, indicating that ERK is not an essential mediator of the and S222 by the upstream Raf molecules.191,192 Phosphoryl- development of leukemia(s).227 Crosstalk between parallel ation of MEK on both of these residues has been reported to pathways and/or the use of a variety of cell lines may help to increase its activity by more than 7000-fold.193 Moreover, explain the apparent contradiction in these reports. mammalian cells have been observed to be transformed as a One approach taken to inhibit ERK phosphorylation is result of constitutively activated MEK.194 Increased MEK through the generation of a competitive inhibitor. A 15-amino activity has also been seen in a variety of leukemias including acid peptide was synthesized resembling the ␣-C helix of ERK- AML and CML.195,196 Additionally, aberrant MEK-1 activity 1 and then kinetic analyses were performed to determine the has also been shown to have anti-apoptotic effects in hemato- efficacy of ERK inhibition mediated by this molecule. The poietic cells.197–200 In all, these data suggest that targeting authors found that this peptide was able to inhibit ERK from MEK with specific inhibitors may be sufficient to abrogate binding to MEK via a competitive inhibition mechanism.228 aberrant MAPK cell signaling. More recently, it was reported that ERK activation is inhibited PD098059 is a commercially available MEK inhibitor that by parallel activation of the stress-activated p38MAPK. Upon functions by binding to, and thus preventing activation of, the phosphorylation, p38MAPK binds to ERK, sequestering it away dephosphorylated form of MEK-1.201,202 Studies have shown from MEK-mediated activation, effectively inhibiting the Raf- that PD098059 is not only able to impair cellular proliferation, MEK-ERK signaling pathway at the level of the ERK protein.229 but is also able to impact other cellular events including angiogenesis, differentiation, and apoptosis.203–207 A second MEK inhibitor, U0126, was identified using a cell- Target: 90 kDa ribosomal S6 kinase (p90RSK) based screen seeking inhibitors of phorbol ester-stimulated AP-1 transactivation.208 This inhibitor, like PD098059, does The p90RSK kinase is a serine-threonine kinase that contains not compete with ATP suggesting that its mechanism of action two functional kinase domains: an amino terminal kinase that is not by binding the ATP site of the enzyme. The basis for phosphorylates substrates of RSK and a carboxy terminal kin- the interactions that occur between these molecules is ase that enables the activation of RSK by ERK.230 Three iso- unknown due to the fact that there is a lack of any known forms of p90RSK are known to exist including RSK-1, RSK-2 crystal structure for either MEK-1 or MEK-2.193 and RSK-3, each of which exhibit approximately 75–80% Recently, it was reported that MEK inhibition via the use of sequence homology to one another.231–233 p90RSK is fully acti- a novel inhibitor, PD184352, is able to suppress growth of vated after phosphorylation on three distinct residues which tumors in mice.209 Accordingly, this compound is presently requires three concerted steps. First, ERK phosphorylates undergoing phase I clinical trials in cancer patients.210 Inter- p90RSK on S369, which triggers phosphorylation of S386 by estingly, it has also been recently seen that the PKC inhibitor, the carboxy terminal kinase. Lastly, the amino terminal kinase UCN-01, when combined with PD184352 (or alternate MEK is phosphorylated on S227 by 3-phosphoinositide-dependent inhibitors, PD098059 and U0126) is able to cause mitochon- protein kinase-1 (PDK-1), which enhances RSK activity 100- drial dysfunction and subsequent induction of apoptosis.211 fold.234 It was later found that the S386 residue is located in Therefore, evidence suggests that targeting the MEK compo- a hydrophobic motif that acts as a docking site and is essential nent of the Raf-MEK-ERK signal transduction cascade may be for PDK-1-mediated phosphorylation of S227.235 This is an a viable option for chemotherapeutic intervention in apparent linker that bridges both the PI3K and Raf-MEK-ERK leukemic disorders. pathways, as PDK-1 also serves to phosphorylate and activate the downstream mediator of PI3K, Akt, on T308. This is an area of increasing interest and thus, this type of crosstalk Target: extracellular signal-regulated kinase (ERK) research merits further, more detailed investigations. The substrates of p90RSK include an array of transcription ERK-1 and ERK-2 are 43/44 and 41/42 kDa kinases that are factors including cAMP-response element-binding protein SPOTLIGHT directly downstream of the MEKs. Phosphorylation of the ERKs (CREB), estrogen receptor-␣ (ER-␣), I␬-B/NF-␬B, and c-Fos.236–240 mainly occurs as a result of growth factor/cytokine stimu- The roles that each of these transcription factors (and others) lation, but has also been shown to be activated by hydrogen play in the onset of leukemogenesis and cell cycle progression peroxide, UV light, and ionizing radiation.212–216 ERK-1 and will be briefly discussed in the following section. ERK-2 phosphorylation occurs on residues T183 and Y185 for maximal activity.6,7,188 When activated, ERK-2 is able to dimerize with other ERK-2 proteins (regardless of their acti- Target: nuclear transcription factors vation state) and induce nuclear translocation.217,218 Once inside the nucleus, ERK-2 is thought to phosphorylate and sub- Aberrant gene expression and the consequential development sequently activate a variety of nuclear targets including topiso- of neoplasia can be targeted at the transcriptional level by merase II-␣, a cell cycle progression mediator.219,220 The pri- examining the mechanisms by which these dysregulated mary cytoplasmic target of the ERK kinases is p90RSK, also genes become activated. Transcription factors (TFs) are the known as the ribosomal protein S6 kinase, but a wide array nuclear proteins that directly influence specific gene of other targets are known to exist.221–223 expression by stabilizing the interactions between the RNA Despite the abundance of information that directly corre- polymerase and the DNA template. In the unphosphorylated lates Ras-Raf-MEK activation with leukemogenesis, ERK acti- state, most TFs neither promote nor antagonize transcription vation is not as readily implicated in the process. Some reports of their respective genes. However, upon phosphorylation,

Leukemia Therapeutic intervention in leukemia via Raf/MEK/ERK pathway JT Lee and JA McCubrey 494 some TFs translocate from the cytosol to the nucleus so as to and ERK are able to phosphorylate c-Fos on a defined region activate transcription of their respective genes (Table 1). of its C-terminus, thereby enhancing the proliferation of Others, originating in the nucleus, merely localize to their fibroblastic cell lines.285,286 Also, activation of both the JNK respective sequence(s) on the DNA template in order to and MAPK pathways are sufficient to raise the protein levels enhance transcriptional efficiency. Research to antagonize and/or activity of c-Jun and c-Fos.287,288 Lastly, both p38MAPK these types of interactions between TFs and DNA is underway and JNK activation are necessary to elicit transformation and preliminary findings are revealing that targeting induced by c-Jun.289 TFs may be a sensible approach for the development of anti-neoplastic drugs. Nuclear factor-␬B

Cyclic AMP (cAMP)-response element binding protein NF-␬B is the generic name for a broad array of heterodimeric (CREB) transcription factors that are key modulators of a number of genes involved in cellular proliferation and apoptosis.290 NF- Activation of p90RSK can lead to phosphorylation of the CREB ␬B is found in the cytosol complexed with the inhibitory pro- transcription factor on S133.241,269,270 CREB, when phos- tein, I␬-B. A number of stimuli are able to induce phosphoryl- phorylated on this residue, is bound by the CREB-binding pro- ation of I-␬B, which allows for the dissociation of the com- tein (CBP) that enhances stabilization of the post-trans- plex, I-␬B degradation by the proteasome, and subsequent lationally altered CREB protein.271,272 Furthermore, this NF-␬B activation. Upon activation, NF-␬B is able to translo- SPOTLIGHT phosphorylation event is correlated with activation of immedi- cate to the nucleus to enhance transcription of a variety of ate–early genes that can cause tumorgenesis, such as c-fos.243 genes. Over 150 potential target genes have been identified for NF-␬B, a large proportion of which are cytokines required for immune responses.291 Activator protein-1 family of transcription factors (AP- The MAPKs can activate NF-␬B through several different 1) mechanisms. MEKK-1, a kinase of the JNK pathway, has been reported to phosphorylate I␬-B both in vitro and in vivo.292 The AP-1 family of transcription factors include c-Jun, c-Fos Raf-1 and p90RSK have also been shown to be I␬-B kinases, and activating transcription factor-2 (ATF-2).273 These compo- thereby inducing NF-␬B activation.293–296 Furthermore, the nents are able to dimerize with each other via leucine zipper constitutively active RelA/p65 subunit of the NF-␬B protein domains in order to activate transcription of specific genes.274 has been isolated from adenocarcinomas. This activated tran- c-Jun is phosphorylated on serines 63 and 73 by JNK and is scriptional activator was found to be phosphorylated by phosphorylated on C-terminal inhibitory sites by ERK1/2.275– MAPKs of the Raf/MEK/ERK pathway.297 279 Phosphorylation of the N-terminal amino acids (S63/S73) leads to enhanced c-Jun activity, which is coordinated with Ha-Ras-mediated oncogenic transformation.280 c-Myc The AP-1 transcription factors are defined targets of the MAPKs. For example, Jun:Fos and Jun:ATF dimers have been The c-Myc oncogene product is a transcription factor that is implicated in Ras-mediated oncogenesis.281 Moreover, both able to regulate gene expression in both normal and neoplas- c-Jun and Fos are necessary for cellular transformation tic cells.298–300 c-Myc is known to heterodimerize with two induced by Ras.282–284 Evidence exists suggesting that p90RSK proteins: Mad and Max.301 These proteins, when complexed,

Table 1 Common transcription factors of the RAf/MEK/ERK pathway

Name Consensus sequence Target gene Gene product

CREB241 TGACGTCA242 c-fos243 Transcription factor egr-1244 Transcription factor bcl-2245 Anti-apoptotic factor c-Myc246 CACGTG247 C/EBP␣248 Transcription factor Cyclin D1249 Cell cycle regulator p53250 Tumor suppressor eIF-4E251 Translation initiation factor ODC251 Cell cycle regulator Ets253 C/AGGAA/T253 u/PA254 Angiogenic factor MMP1255 Angiogenic factor MET256 Oncogene PTHrP257 Tumor regulator AP-1258 TGACTCA259 HB-EGF260 Growth factor SSeCKS261 Cell cycle regulator SPARC262 Oncogenic factor

263 264 265 NF-␬B GGGA/GNT/ATTCC BCL-XL Anti-apoptotic factor GM-CSF266 Growth factor c-Myc267 Transcription factor Cyclin D1268 Cell cycle regulator

Leukemia Therapeutic intervention in leukemia via Raf/MEK/ERK pathway JT Lee and JA McCubrey 495 have diverse cellular effects. Myc:Max heterodimers activate activation is dependent on the delicate balance between the transcription, while Mad:Max complexes repress gene activating signal(s) and inactivation mechanism(s). As pre- expression from the same promoter.302,303 Recently, another viously mentioned, ERK is fully activated by phosphorylation c-Myc binding protein was discovered that possessed tumor- on both a tyrosine and a threonine residue. Removing a phos- suppressor characteristics. This peptide, MM-1, complexes phate through the action of phosphatases from either of these with c-Myc’s transactivation domain to suppress its regulat- residues results in decreased ERK activity. The predominant ory capacities.304,305 problem with phosphatase research to date has been the Activated ERK dimers are c-Myc kinases that traverse the determination of their cellular localization. Consequently, nuclear membrane to phosphorylate c-Myc on S62, thus sti- there is little evidence that unequivocably implicates any mulating activity of the transactivation domain.306,307 In con- particular phosphatase with any definite kinase. However, trast, another group reported that although MAPK phosphoryl- research into the actions and modalities of these negative ation of c-Myc occurs in vitro, the actual in vivo kinase(s) of regulators is underway and thus, is presented hereafter. c-Myc are still unknown.308 Upstream of ERK, Ras is a There is an array of dual-specificity phosphatases known as mediator of c-Myc activation as it increases c-Myc transactiv- MAP kinase phosphatases (MKPs) that serve to dephosphorylation over five-fold by interacting with the N-terminal region, ate ERK.320,321 At the time of publication, there are seven perhaps by stabilizing the activated protein.309,310 In addition, known MKPs (MKPs1–7). MKP-1 is the original family mem- a Raf-1 mutant which blocked Ras signaling caused by down- ber and was the first ERK phosphatase described.322 MKP-2 regulation of c-Myc expression directly implicates the MAPK (also known as TYP-1) was later isolated and also found to be cascade in c-Myc regulation.311 Interestingly, the stress-acti- an effective regulator of ERK activity.323,324 MKP-3 and MKP- vated pathway, headed by the JNK kinase(s), has also been 4 were each soon described as ERK phosphatases.325–327 The implicated in c-Myc activation, but in these cases, c-Myc acti- isolation of MKP-5 was the first phosphatase to be isolated vation can lead to apoptosis.312 Together these data suggest that was not effective in inhibiting ERK activity. Rather, MKP- that a degree of interplay can occur, perhaps mediated by dis- 5 was observed to inactivate the stress-activated kinases, JNK tinct extracellular stimuli, that ultimately leads to either cell and p38MAPK.328,329 Most recently, MKP-7 has been isolated death or cellular proliferation by c-Myc. and found to be similar in function to that of MKP-5.330 Lastly, MKP-6 is unique in that it functions as a novel negative-feedback regulator of CD28 costimulatory signaling that controls Ets family of transcription factors the activation of MAP kinases in peripheral blood T cells.331 As previously alluded to, each of these phosphatases have dis- The Ets family of transcription factors has been implicated as tinct subcellular locales which imply the varying functions of one of the many downstream targets of the MAPK family these relatively homologous proteins. The utilization (ie (p38MAPK, JNK and ERK), as well as the PI3K pathway.313 This overexpression) of one or several of the previously mentioned group of TFs is characteristically defined by a conserved phosphatases in a pharmaco-preventative nature may help winged helix–turn–helix DNA binding motif.314 Current litera- inhibit MAPK signaling and thus, halt uncontrolled cellular ture suggests that the Ets TFs are responsible for targeting over proliferation. 200 different genes.315 The targeted gene is a function of the Of the above MKPs, two have been extensively reported to protein heterodimers that occur as a result of activation. Two be effective in the dephosphorylation and subsequent inacti- major groups of these TFs are currently defined: the Ets group vation of ERK molecules: MKP-1 and MKP-3. Activation of the which includes Ets1, Ets2 and Pointed, and also the TCF Raf/MEK/ERK cascade has been reported to be of instrumental (ternary complex factors) family which include Elk1, Sap1a, importance in the induction of MKP-1 expression to dephos- Sap1b, Fli1 and Net.316 phorylate ERK, which denotes the presence of an inhibitory The Ets subgroup has only one phosphorylation site located feedback loop.332 Further studies have endorsed this theory near the N-terminal domain, which is primarily activated by by showing that overexpression of MKP-1 is sufficient to the Ras/Raf/MEK/ERK family.317 The TCFs, though, are unique increase both Raf and MEK kinase activities.333 MKP-3 is in that they have multiple serines and threonines within a another phosphatase that has been shown to dephosphorylate transactivation domain that can be phosphorylated for acti- ERK. MKP-3 is activated merely by binding to ERK and func- vation.318 This phosphorylation event is sufficient to allow for tions primarily in the cytoplasm, whereas MKP-1 expression

efﬁcient binding to Ras-responsive elements (RREs) and/or SPOTLIGHT is reserved for nuclear activities.334,335 serum response elements (SSEs) that exist in the promoters of Protein phosphatases-1 and 2a (PP1 and PP2a) are a duo of multiple immediate–early genes, including c-fos, egr-1 and dephosphorylating agents with highly conserved amino-acid junB.319 RREs require the binding of both an AP-1 dimer sequences that remove phosphate groups from a wide array (previously discussed) and an Ets component. Therefore, gene of phosphoproteins.336,337 It has been seen that these two expression controlled by these promoter sequences is phosphatases have an afﬁnity for interaction with each of the immeasurably complex, considering that over 25 Ets members components of the MAPK pathway (Raf, MEK and ERK).335–340 exist and that both the AP1 and Ets regulation are controlled The role that these phosphatases play in regulating each of coordinately by Ras.316 The multifaceted and interwoven nat- these proteins may be quite similar. One report showed that ure of the signaling pathways combined with the number of PP2a inactivation and MAPK activation are strongly corre- unique TF heterodimers that can occur help explain why these lated, indicating that PP2a plays a negative modulatory role transcriptional activators and their mode(s) of action are not in the regulation of MAP kinase activity.341 Other reports, well understood. dealing with the Raf kinase, state that PP1 and PP2a dephosphorylate phospho-Raf, when it is bound to the 14-3-3 pro- Negative regulation of MAPKs: phosphatases teins, in order to allow for subsequent Ras-mediated activation of Raf.342,343 In each case, PP1 and PP2a activity permit the The premise of this review has been based on phosphoregul- initiation of additional MAPK signaling. In addition, okadaic ation of signaling cascades. The duration and degree of MAPK acid has been noted to be a potent inhibitor of both PP1 and

Leukemia Therapeutic intervention in leukemia via Raf/MEK/ERK pathway JT Lee and JA McCubrey 496 PP2a phosphatase activities, albeit through a currently unde- PDKs and Akt are not brought into close proximity to each scribed mechanism.336,344 The cellular phosphatases that act other. Thus, activation of Akt is minimal, if at all. The theory upon p90RSK have not been identified as of yet. that presence of a functional PTEN phosphatase is essential for the prevention of tumorigenesis is endorsed by data that showed that PTEN−/− mice either die during embryogenesis or Crosstalk: MAPK interactions with PI3K/Akt spontaneously develop a variety of tumors later in life.363,364 Two pharmacological inhibitors of PI3K have been used As alluded to previously, the PI3K/Akt pathway is one that extensively over the past decade. Wortmannin, originally often transduces signals that are similar in nature to that of known as an inhibitor of respiratory burst activities, has been the Raf/MEK/ERK pathway. It has been shown that a certain shown to be a potent inhibitor of PI3K-mediated sig- degree of crosstalk exists between these pathways, which naling.365,366 LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1- either enhance or sometimes negate oncogenic extracellular benzopyran-4-one] is another inhibitor of PI3K that has been ␮ 367 signals. By understanding the interactions that occur between used with high degrees of efficacy at low IC50s (1.4 M). the components of these pathways, we will be better advised The two aforementioned inhibitors have been useful in block- in the development of treatment strategies that are able to ing PI3K signaling, but have no capacity to directly inhibit Akt more efficiently target leukemic cells within the body. activity. More recently though, CalBioChem (San Diego, CA, Phosphatidylinositol 3-kinase (PI3K) is located in the cyto- USA) has developed a highly potent, quasi-specific inhibitor plasm in resting cells. It exists as a dimer of two subunits: a of Akt known as IL-6-hydroxymethyl-chiro-inositol-2(R)-2-O- regulatory p85 subunit combined with a catalytic p110 methyl-3-O-octadecylcarbonate. This compound has been SPOTLIGHT 345,346 component. Upon activation of a receptor tyrosine kin- seen to selectively inhibit Akt activity with an IC50 of approxi- ase and subsequent autophosphorylation, the SH-2 domain(s) mately 5.0 ␮M, while only faintly affecting PI3K activity.368 of the p85 regulatory subunit bind to the receptor’s phos- Further studies using this inhibitor should provide valuable phorylated Y residues.83 PI3K then generates a series of phos- insight into Akt-mediated signaling, especially that inde- phorylated lipids at the cytoplasmic membrane that act to pendent of PI3K. recruit the phosphatidylinositol-dependent kinases (PDKs) and Crosstalk between the PI3K and Raf/MEK/ERK pathways has their downstream substrate Akt/PKB to the membrane.347,348 been reported on multiple levels, with some research stating PDK1 serves to phosphorylate Akt on T308, whereas PDK2 that PI3K activity is essential for induction of Raf/MEK/ERK phosphorylates S473 of Akt.349–352 Activated Akt is then able activity.369–371 Additional studies suggest that the PI3K path- to phosphorylate a variety of downstream substrates each of way enhances and/or synergizes with Raf/MEK/ERK signaling which enable the cell to proliferate including the pro-apop- to provide a more robust signal through the lower components totic molecule BAD, caspase-9 (human cell lines), the for- of the MAPK cascade (ie ERK).47,371–373 However, there is con- khead family transcription factors, and I-K, a kinase that regu- flicting evidence that states that Akt is able to phosphorylate lates the NF-B transcription factor.352–357 An overview of the Raf, thereby efficiently abrogating Raf activity on downstream potential interactions within this pathway are depicted in substrates.374–376 Furthermore, some data suggest that this Figure 6. effect may be dependent upon the differentiation state of the Abrogation of oncogenic signals from the PI3K pathway cell, which may account for the apparent discrepancies in the occurs via the tumor suppressor gene product, PTEN.358 PTEN current literature.377 Regardless of cell type, differentiation is a phosphatase that acts by removing a phosphate group state, or assay conditions, it is quite apparent that there is a

from the 3 position of the inositol ring of the PIP3,4,5 phosphol- varying degree of crosstalk that occurs between these path- ipids located at the cellular membrane.359–362 In doing so, the ways. Examining the level, nature and circumstances by which these interactions occur will be instrumental in the development of new agents designed to battle leukemia.

Conclusions

The Raf/MEK/ERK signal transduction cascade is responsible for many of the phenotypes observed in a wide array of oncogenic disorders, including leukemia. Drug intervention and/or the use of pharmacological inhibitors will be practical at each level of this cascade, from the cell receptor at the plasma membrane to the transcription factors at the nucleus. This review would be remiss if it did not address the importance of acknowledging the complexities of the signal transduction and apoptotic pathways discussed (a more detailed, but not all-encompassing schematic is shown in Figure 7). All conclusions drawn are not necessarily true for all cell types and the degree of crosstalk that occurs between parallel pathways remains largely unknown.378,379 Furthermore, blocking the activity of a protein far upstream of others (ie Ras) may have other injurious effects that were not intended, as inhibiting its activity may also suppress other vital functions. Conversely, by inhibiting the activity of a downstream compo- RSK Figure 6 Roles of the PI3K/Akt pathway in growth and the preven- nent of the MAPK pathway (ie p90 ), one may overlook tion of apoptosis. alternative mechanisms by which the cell is able to bypass

Leukemia Therapeutic intervention in leukemia via Raf/MEK/ERK pathway JT Lee and JA McCubrey 497

Figure 7 Sites of interaction of various inhibitors of cytokine and stress-mediated signal transduction. that signaling component. For that reason, a multi-tiered GD. ERKs: a family of protein-serine/threonine kinases that are approach targeting the inhibition of uncontrolled cell activated and tyrosine phosphorylated in response to insulin and signaling may be merited. NGF. Cell 1991; 65: 663–675. 4 Rossomando AJ, Payne DM, Weber MJ, Sturgill TW. Evidence that The hallmark of every neoplasia is uncontrolled cellular pp42, a major tyrosine kinase target protein, is a mitogen-activated proliferation. This aberrant behavior relies on a series of over- serine/threonine protein kinase. Proc Natl Acad Sci USA 1989; 86: expressed oncogenes and on the accumulation of tumor-sup- 6940–6943. pressor gene mutations. The combinatorial effects of these 5 Payne DM, Rossomando AJ, Martino P, Erickson AK, Her JH, Shab- genotypes are detrimental not only to the cell, but also to the anowitz J, Hunt DF, Weber MJ, Sturgill TW. Identification of the entire body. If the cellular processes by which these anomalies regulatory phosphorylation sites in pp42/mitogen-activated pro- occur are better understood, then scientists can begin to tein kinase (MAP kinase). EMBO J 1991; 10: 885–892. 6 Seger R, Seger D, Lozeman FJ, Ahn NG, Graves LM, Campbell exploit that knowledge so as to develop more effective, JS, Ericsson L, Harrylock M, Jensen AM, Krebs EG. Human T-cell non-invasive chemotherapeutic treatments. mitogen-activated protein kinase kinases are related to yeast signal Transduction kinases. J Biol Chem 1992; 267: 25628–25631. 7 Crews C, Alesandrini A, Erikson R. The primary structure of MEK, a Acknowledgements protein kinase that phosphorylates the ERK gene product. Science 1992; 258: 478–480. SPOTLIGHT This work was supported in part by RO1CA51025 from the 8 Kosako H, Gotoh Y, Matsuda S, Ishikawa M, Nishida E. Xenopus MAP kinase activator is a serine/threonine/tyrosine kinase acti- National Cancer Institute and 2000-ARG-0003 from the North vated by threonine phosphorylation. EMBO J 1992; 11: 2903– Carolina Biotechnology Center. We wish to thank Ms Cather- 2908. ine Spruill for the excellent artwork. 9 Kyriakis JM, App H, Zhang XF, Banerjee P, Brautigan DL, Rapp UR, Avruch J. Raf-1 activates MAP kinase-kinase. Nature 1992; 358: 417–421. References 10 Dent P, Haser W, Haystead TAJ, Vincent LA, Roberts TM, Sturgill TW. Activation of mitogen-activated protein kinase kinase by v- 1 Druker BJ, Talpaz M, Resta DJ, Peng B, Buchdunger E, Ford JM, Raf in NIH 3T3 cells and in vitro. Science 1992; 257: 1404–1407. Lydon NB, Kantarjian H, Capdeville R, Ohno-Jones S, Sawyers CL. 11 Khleif SN, Abrams SI, Hamilton JM, Bergmann-Leitner E, Chen A, Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine Bastian A, Bernstein S, Chung Y, Allegra CH, Schlom J. A phase kinase in chronic myeloid leukemia. N Engl J Med 2001; 344: I vaccine trial with peptides reflecting ras oncogene mutations of 1031–1037. solid tumors. J Immunother 1999; 22: 155–165. 2 Boulton TG, Yancopoulos GD, Gregory JS, Slaughter C, Moomaw 12 McCubrey JA, May WS, Duronio V, Mufson A. Serine/Threonine C, Hsu J, Cobb MH. An insulin-stimulated protein kinase similar phosphorylation in cytokine signal transduction. Leukemia 2000; to yeast kinases involved in cell cycle control. Science 1990; 249: 14: 9–21. 64–67. 13 New L, Han J. The p38 MAP kinase pathway and its biological 3 Boulton TG, Nye SH, Robbins DJ, Ip NY, Radziejewska E, Morgen- function. Trends Cardiovasc Med 1998; 8: 220–228. besser SD, DePinho RA, Ranayotatos N, Cobb MH, Yancopoulos 14 Lee JC, Laydon JT, McDonnell PC, Gallagher TF, Kumary S, Green

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