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Oncogene (2007) 26, 3291–3310 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc REVIEW Targeting the Raf-MEK-ERK mitogen-activated protein kinase cascade for the treatment of cancer PJ Roberts1 and CJ Der2 1 Division of Pharmacotherapy and Experimental Therapeutics, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA and 2Department of Pharmacology, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA Mitogen-activated protein kinase (MAPK) cascades are key signaling pathways involved in the regulation of normal cell proliferation, survival and differentiation. Aberrant regulation of MAPK cascades contribute to cancer and other human diseases. In particular, the extracellular signal-regulated kinase (ERK) MAPK pathway has been the subject of intense research scrutiny leading to the development of pharmacologic inhibitors for the treatment of cancer. ERK is a downstream component of an evolutionarily conserved signaling module that is activated by the Raf serine/threonine kinases. Raf activates the MAPK/ERK kinase (MEK)1/2 dual-specificity protein kinases, which then activate ERK1/2. The mutational activation of Raf in human cancers supports the important role of this pathway in human oncogenesis. Additionally, the Raf-MEK-ERK pathway is a key downstream effector of the Ras small GTPase, the most frequently mutated oncogene in human cancers. Finally, Ras is a key downstream effector of the epidermal growth factor receptor (EGFR), which is mutationally activated and/or overexpressed in a wide variety of human cancers. ERK activation also promotes upregulated expression of EGFR ligands, promoting an autocrine growth loop critical for tumor growth. Thus, the EGFR-Ras-RafMEK-ERK signaling network has been the subject of intense research and pharmaceutical scrutiny to identify novel target-based approaches for cancer treatment. In this review, we summarize the current status of the different approaches and targets that are under evaluation and development for the therapeutic intervention of this key signaling pathway in human disease. Oncogene (2007) 26, 3291–3310. doi:10.1038/sj.onc.1210422 Keywords: mitogen-activated protein kinases; ERK; Ras; epidermal growth factor receptor; small molecule inhibitors; monoclonal antibodies of death in the United States in people under the age of 85 (Jemal et al., 2005). Although we have made great strides in unraveling the mysteries of cancer genetics and biology, the important task now facing researchers and clinicians is to translate these discoveries into novel therapeutics that will improve patient outcomes. As many of the genetic alterations found in cancers involve genes whose products are regulators of signal transduction (Hanahan and Weinberg, 2000), much of the focus in the development of novel therapeutics has involved inhibitors of signal transduction molecules, in particular protein kinases (Arslan et al., 2006; Davies et al., 2006; Sebolt-Leopold and English, 2006). The human kinome is comprised of over 518 protein kinases (http:// kinase.com), with disease associations reported for over 150 kinases (http://www.cellsignal.com/reference/kinase_disease.asp). Presently, the Food and Drug Administration (FDA) has approved 10 protein kinase inhibitors and over 100 kinase-targeted agents are currently undergoing clinical evaluation. Target-based therapies are widely considered to be the future of cancer treatment and much attention has been focused on developing inhibitors of the Raf–mitogenactivated protein kinase (MAPK)/extracellular signalregulated kinase (ERK) kinase MEK–ERK–MAPK signaling pathway and its upstream activators (SeboltLeopold and Herrera, 2004). Evidence that ERK MAPK signaling promotes cell proliferation, cell survival and metastasis, along with the overwhelming frequency in which this pathway is aberrantly activated in cancer, in particular by upstream activation by the epidermal growth factor receptor (EGFR) and the Ras small guanosine triphosphatases (GTPases) (Figure 1), support current efforts to identify approaches to inhibit this pathway. In this review, we will summarize the current status of the approaches and development of pharmacologic inhibitors to block EGFR–Ras–Raf–MEK–ERK MAPK signaling for the treatment of cancer. Introduction In 2005, the American Cancer Society reported that cancer has surpassed heart disease as the leading cause Correspondence: CJ Der, Department of Pharmacology, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, CB 7295, Chapel Hill, NC 27599-7295, USA. E-mail: cjder@med.unc.edu The Raf–MEK–ERK MAPK cascade Mammalian cells possess four well characterized and widely studied MAPKs (Figure 2). These cascades are comprised of three protein kinases that act as a signaling relay controlled, in part, by protein phosphorylation: a MAPK kinase kinase (MAPKKK), a MAPK kinase Targeting the Raf-MEK-ERK MAPK cascade PJ Roberts and CJ Der 3292 EGFR overexpression: • Colorectal cancer (27-77%) • Pancreatic cancer (30-50%) • Lung cancer (40-80%) • Non-small cell lung cancer (14-91%) TGFα Sos EGFR* Ras* Grb2 Raf* EGFR mutation: • NSCLC (10%) • Glioblastoma (20%) MEK Ras mutation: • Pancreatic cancer (90%) • Papillary thyroid cancer (60%) • Colon cancer (50%) • Non-small cell lung cancer (30%) B-Raf mutation: • Melanoma (70%) • Papillary thyroid cancer (50%) • Colon cancer (10%) ERK *Mutated in human cancers Figure 1 Oncogene activation of the ERK MAPK cascade. Mutationally activated B-Raf, Ras and mutationally activated (by missense mutations in the cytoplasmic kinase domain in NSCLC or by extracellular domain truncations (e.g., VIII) in glioblastomas) and/or overexpressed EGFR causes persistent activation of the ERK MAPK cascade in human cancers. Activated ERKs translocate to the nucleus, where they phosphorylate and regulate various transcription factors leading to changes in gene expression. In particular, ERK-mediated transcription can result in the upregulation of EGFR ligands, such as TGFa, thus creating an autocrine feedback loop that is critical for Ras-mediated transformation and Raf-mediated gene expression changes. (MAPKK) and a MAPK (Johnson and Lapadat, 2002). The terminal serine/threonine kinases (MAPKs) are the ERK1/2, the c-Jun amino-terminal kinases (JNK12/3; also called SAPKs), p38 kinases (p38a/b/g/d) and ERK5. Generally, the ERK pathway is activated by growth factor-stimulated cell surface receptors, whereas the JNK, p38 and ERK5 pathways are activated by stress and growth factors. MAPK cascades function downstream of cell surface receptors and other cytoplasmic signaling proteins whose functions are deregulated in cancer and other human pathologic disorders. In light of the recent success in the clinical development of small molecule inhibitors of protein kinases, components of MAPK cascades have been the subject of intense research and drug discovery efforts. Of these, the p44 ERK1 and p42 ERK2 MAPKs have attracted intense research interest because of their critical involvement in the regulation of cell proliferation and survival. In particular, the mutational activation and/or overexpression of upstream signaling components that activate the ERK MAPKs (Figure 1), together with the substantial body of experimental observations demonstrating the necessity of this pathway in oncogene function, has ‘validated’ this pathway for drug discovery (Benson et al., 2006). This has stimulated intensive efforts by the research community and pharmaceutical industry to develop inhibitors of ERK signaling for cancer treatment. The validated involvement of the related JNK and p38 MAPK signaling cascades in cancer is less clearly established, and consequently, the status of inhibitor Oncogene development for these MAPK cascades will only be discussed briefly. Quite an extensive array of potent and specific inhibitors of p38 are being evaluated in phase I and II clinical trials (Dominguez et al., 2005; Hynes and Leftheri, 2005; O’Neill, 2006) (Table 1). They have been developed primarily for the treatment of chronic inflammatory diseases (e.g., rheumatoid arthritis, Crohn’s disease), although some trials are also evaluating possible applications in cancer. One consequence of p38 inhibitors is blockage of p38-induced transcriptional expression of genes that encode proinflammatory cytokines. In contrast to p38 inhibitors, only a handful of JNK inhibitors have been developed, and are being considered for the treatment of cancer, as well as inflammatory, vascular, neurodegenerative and metabolic disorders (Manning and Davis, 2003). Unlike the considerable volume of preclinical studies that utilized highly specific inhibitors of MEK1/2 (PD98059, U0126 and CI-1040/PD184352) or p38 (SB203580 and SB 202190) (Davies et al., 2000), the preclinical observations made with the SP600125 JNK inhibitor are likely to be less informative regarding the biological roles of JNK, owing to the considerable off-target activity of this compound (Bain et al., 2003). One JNK inhibitor, CC-401, is currently in phase II evaluation in acute myelogenous leukemia, and has also been considered for the treatment of respiratory diseases (Table 1). The MAPKKK component of the ERK cascade is comprised of the Raf serine/threonine kinases (c-Raf-1, Targeting the Raf-MEK-ERK MAPK cascade PJ Roberts and CJ Der 3293 Stimulus Growth Factors Stress, Cytokines Growth Factors Stress, Cytokines Growth Factors Stress, Growth Factors MAPKKK A-Raf, B-Raf, c-Raf-1, Mos, Tpl-2 MLK3, TAK1, MEKK4, ASK1 MEKK1/4, DLK, MLK1-4, LZK, TAK1, ASK1, ZAK MEKK2/3 MAPKK MEK1/2 MKK3/6 MKK4/7 MEK5 MAPK ERK1/2 p38α/β/γ/δ JNK1,2,3 ERK5 Transcription Factors, Etc. Elk-1, Ets-2 RSK, MNK, MSK, cPLA2 CHOP, ATF2, MNK, MSK, MEF2, Elk-1 Jun, ATF2, RNPK, p53, NFAT 4, Shc MEF2 Figure 2 Mammalian MAPK cacades. There are four major mammalian MAPKKK–MAPKK–MAPK protein kinase cascades. Whereas the ERK pathway is commonly activated by growth factors, the JNK, p38 and ERK5 pathways are activated by environmental stress, including osmotic shock, ionizing radiation. Many of the substrates for MAPKs are nuclear transcription factors. Interactions and substrates were compiled from information from STKE (http://stke.sciencemag.org/index.dtl). Table 1 Germline mutation of the Ras-Raf-MEK-ERK cascade in human disorders Syndrome Protein Mutations References Noonan syndrome Costello syndrome K-Rasa H-Rasa P34R, T58I, V14 I, V152G, D153V G12A, G12C G12E, G12S, G12V, G13C, G13D, K117R, A146T Cardio–facio–cutaneous syndrome K-Rasa G60R, D153V Carta et al. (2006); Schubbert et al. (2006) Aoki et al. (2005); Estep et al. (2006); Gripp et al. (2006); Kerr et al. (2006); van Steensel et al. (2006); Carta et al. (2006) Niihori et al. (2006); Schubbert et al. (2006) B-Rafb A246P, Q257R, S467A, F468S, G469E, L485F, K499E, E501G, E501K, N518D, F595L, G596V F35S, Y130C F57C MEK1c MEK2c Niihori et al. (2006); Rodriguez-Viciana et al. (2006) Rodriguez-Viciana et al. (2006) Rodriguez-Viciana et al. (2006) Abbreviations: MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase. aThe most common RAS mutations found in human cancers are seen at residues G12, then Q61, and infrequently at G13; bOf the over 40 missense mutations, the V600E mutation in the kinase domain accounts for B90% of human cancers; cNo MEK1/2 mutations have been identified in human cancers. A-Raf and B-Raf) (Wellbrock et al., 2004; Schreck and Rapp, 2006). Raf kinases phosphorylate and activate the MEK1 and MEK2 dual-specificity protein kinases. Although Raf has been reported to phosphorylate other proteins, to date, the only validated physiologically relevant substrates remain the two closely related MEK1 and MEK2 proteins. MEK1/2 (MAPKK) then phosphorylate and activate the ERK1 and ERK2 MAPKs. Activated ERKs phosphorylate and regulate the activities of an ever growing roster of substrates that are estimated to comprise over 160 proteins (Yoon and Seger, 2006). The majority of ERK substrates are nuclear proteins, but others are found in the cytoplasm and other organelles. Activated ERKs can translocate to the nucleus, where they phosphorylate and regulate various transcription factors, such as Ets family transcription factors (e.g., Elk-1), ultimately leading to changes in gene expression (Zuber et al., 2000; Schulze et al., 2004). There is substantial evidence validating the importance of Raf and MEK in cancer progression and in promoting cancer growth (Shields et al., 2000). The importance of this pathway in oncogenesis was first suggested by the initial identification of Raf as potent retrovirus oncogenes (Schreck and Rapp, 2006). Subsequently, laboratory generated constitutively activated Oncogene Targeting the Raf-MEK-ERK MAPK cascade PJ Roberts and CJ Der 3294 mutants of Raf and MEK were shown to potently transform rodent fibroblasts and other cell types. Furthermore, studies using genetic or pharmacologic approaches have shown that MEK and ERK are required for the transforming activities of Ras and other oncogenes. More recently, mutationally activated B-Raf has been identified in a variety of human cancers (Davies et al., 2002) and the finding that mutationally activated Ras and Raf occur in a non-overlapping occurrence in melanomas, colorectal carcinomas, papillary thyroid carcinomas, serous ovarian carcinomas and lung cancers suggests that Ras function is facilitated primarily by activation of Raf (Rajagopalan et al., 2002; Mercer and Pritchard, 2003; Singer et al., 2003; Vos et al., 2003; Sieben et al., 2004). Interfering RNA suppression of mutant B-Raf demonstrated the importance of continued B-Raf activity for the transformed and tumorigenic growth of melanomas (Hingorani et al., 2003; Sharma et al., 2005; Hoeflich et al., 2006; Sumimoto et al., 2006). Recently, germline de novo mutational activation of H-Ras and K-Ras, B-Raf, as well as MEK1 and MEK2, has been found in patients that comprise a group of Table 2 related developmental disorders (Costello, cardio–facio– cutaneous and Noonan’s syndromes) (Table 2), and suggests that aberrant ERK activation will contribute to other human disorders as well as cancer (Aoki et al., 2005; Niihori et al., 2006; Rodriguez-Viciana et al., 2006; Schubbert et al., 2006). These syndromes are associated with similar consequences that include facial dysmorphia, cardiomyopathy, as well as increased incidence of cancer (Duesbery and Vande Woude, 2006). Interestingly, the mutational spectrum seen in these syndromes differ from those seen in cancer, and generally lead to weakly activated proteins. This reflects the likelihood that the more potent activating mutations found in cancer cannot be tolerated during development. Taken together, these observations provided strong support for the therapeutic value of blocking ERK signaling in cancer and developmental disorders. Although the Raf–MEK–ERK cascade is typically drawn as a simple linear, unidirectional cascade of protein kinases, the more appropriate depiction of this cascade is that it is a key core element of a complex signaling network, with many other interactions Small molecule MAPK inhibitorsa Agent Company Target Status RO4402257 RO3201195 PH-797804 AZD-6703 GW681323 (SB-681323) Hoffmann-LaRoche Hoffmann-LaRoche Pfizer AstraZeneca GlaxoSmithKline p38 p38 p38 p38 p38 Phase Phase Phase Phase Phase VX-745 VX-702 Vertex Pharmaceuticals Vertex Pharmaceuticals/ Kissei Scios/Johnson & Johnson p38 p38 Phase IIb Phase II p38a Phase II p38a p38 p38 Phase I Phase IIb Phase I RWJ-67657 BIRB-796 KC706 Scios/Johnson & Johnson Takeda Pharmaceuticals Pharmacopeia/ Bristol-Myers Squibb Johnson & Johnson Boehringer Ingelheim Ke´mia p38 p38a p38 Phase Ib Phase IIb/IIIb Phase II ARRY-797 AMG-548 CC-401 SP600125 CEP-1347 Sorafenib (Nexavar) Array BioPharma Amgen Celgene Celgene Cephalon Bayer/Onyx p38 p38a JNK JNK1, JNK2, JNK3 MLK-1, MLK-2, MLK-3 B-Raf, Raf-1c Phase I Phase Ib Phase II Preclinical Phase II/IIIb Approved Phase II–III Raf265 (CHIR-265) PLX4032 PD0325901 AZD6244 (ARRY-142886) ARRY-438162 Novartis Roche/Plexxikon Pfizer AstraZeneca/Array BioPharma Array BioPharma A-Raf, B-Raf, c-Raf-1d Mutant B-Raf(V600E) MEK,MEK2 MEK,MEK2 Phase Phase Phase Phase MEK, MEK2 Phase I Scio-469 Scio-323 TAK-715 PS540446 Indication/target population II I II I II I I II II Rheumatoid arthritis Rheumatoid arthritis Rheumatoid arthritis Rheumatoid arthritis Patients with coronary heart disease undergoing percutaneous coronary interventions; neuropathic pain following nerve trauma Rheumatoid arthritis Rheumatoid arthritis, acute coronary syndromes Rheumatoid arthritis, multiple myeloma, myelodysplastic syndromes Rheumatoid arthritis Rheumatoid arthritis Rheumatoid arthritis Inflammatory disease Inflammatory and autoimmune diseases Rheumatoid arthritis, metabolic disorders, cardiovascular disease Inflammatory disease, cancer Inflammatory disease AML, inflammatory disease Parkinson’s disease, asthma Parkinson’s disease, Alzheimer’s disease Advanced RCC CRC, NSCLC, pancreatic cancer, melanoma, ovarian cancer, AML, as well as other solid and hematological malignancies Locally advanced or metastatic melanoma Cancer Breast cancer, CRC, NSCLC, melanoma NSCLC, melanoma, pancreatic cancer; inflammation Rheumatoid arthritis Abbreviations: AML, acute myelogenous leukemia; CRC, colorectal cancer; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; NSCLC, non-small-cell lung cancer; RCC, renal cell cancer, aCompiled from studies cited in reviews that we have referenced in the text, from company websites and from www.clinicaltrials.gov; AML bDiscontinued, cAlso inhibitory activity for VEGFR-2, PDGFR-b, Flt-3, c-Kit and FGFR-1, dAlso inhibitory activity for VEGFR-2. Oncogene Targeting the Raf-MEK-ERK MAPK cascade PJ Roberts and CJ Der 3295 (Figure 3) (Kolch, 2005). This complexity is best represented at the level of Raf, where multiple signals converge to regulate Raf activation. As described below, a major activator of Raf kinases are the Ras small GTPases. However, Raf activation is complex, with Ras facilitating the plasma membrane association of normally cytosolic Raf, where additional signaling activities, including phosphorylation (e.g., p21-activated protein kinases (PAK) serine/threonine and Src family tyrosine kinases) and dephosphorylation (protein phosphatase 2A) events are required to fully activate the kinase function. Raf function is also regulated by interaction with other proteins, including 14-3-3 proteins and heat shock protein 90 (Hsp90). Hence, inhibitors of Raf and MEK are not likely to cause similar consequences and show equivalent clinical responses. These regulatory events also suggest other approaches for antagonizing Raf–MEK–ERK signaling. Another layer of regulation of the Raf–MEK–ERK cascade involves scaffolding proteins that help to regulate the activity, specificity and spatial regulation of MEK/ERK signaling in mammalian cells. These include kinase suppressor of Ras 1 (KSR1), MEK partner-1 (MP1), b-arrestins, IQ motif-containing GTPase activating protein 1 (IQGAP1) and others (Kolch, 2005). Of these, KSR has been one of the best studied, and KSR function has been shown to be critical for Ras activation of Raf. Mouse embryonic fibroblasts (MEF) deficient in KSR1 function showed impaired Golgi Late Endosomes MEK Ras Src PAK Sef MEK1 ERK IMP Raf RKIP MP1 Hsp90 ERK1 KSR MEK Mos Actin ERK MEK MKP1 Plasma Membrane IQGAP1 ERK Nuclear – Elk-1, Fos, Myc, STAT3 Membrane – Syk, CD120a, calnexin Cytoskeletal – paxillin, neurofilaments Cytosolic - RSK Figure 3 The Raf–MEK–ERK cascade is a core signaling component of a complex signaling network. The ERK MAPK cascade is regulated by a complex array of functionally diverse proteins. In particular, Raf function is regulated by both positive and negative signal inputs. In particular, scaffold proteins (e.g., KSR, Sef, MP1 and IQGAP1) bind to distinct subsets of components of this cascade to create unique ERK MAPK signaling complexes at specific and distinct subcellular locations that result in preferential utilization of only some of the many possible ERK substrates. This arrangement channels the unique input signals that activate the pathway into the specific output signaling in distinct spatio-temporal patterns. ERKs then phosphorylate multiple targets, including nuclear, cytosolic, membrane-associated and cytoskeletal substrates. sensitivity to Ras transformation (Kortum and Lewis, 2004). Furthermore, antisense suppression of KSR1 expression was found to impair the growth of Ras mutation-positive human tumor cells, supporting KSR as a possible therapeutic target for blocking Ras (Xing et al., 2003). Ras family small GTPases are key upstream activators of Raf Ras proteins (H-, K- and N-Ras) function as a GDP/ GTP-regulated switch. GDP/GTP cycling is regulated by guanine nucleotide exchange factors (RasGEFs; e.g., Sos) that promote formation of active Ras-GTP, whereas GTPase-activating proteins (GAPs; e.g., NF1 neurofibromin) stimulate GTP hydrolysis and formation of inactive Ras-GDP (Mitin et al., 2005). In normal quiescent cells, Ras is GDP-bound and inactive. Extracellular stimuli (e.g., EGF) cause transient formation of the active, GTP-bound form of Ras. Activated Ras-GTP binds to a spectrum of downstream effector targets, of which the Raf kinases are the best characterized. Ras is mutationally activated in 30% of all cancers, with pancreas (90%), colon (50%), thyroid (50%), lung (30%) and melanoma (25%) having the highest prevalence (Malumbres and Barbacid, 2003). The mutant Ras genes in human cancers encode mutated proteins that harbor single amino-acid substitutions primarily at residues G12 or Q61. Mutant Ras proteins are GAPinsensitive, rendering the proteins constitutively GTPbound and activated, leading to stimulus-independent, persistent activation of downstream effectors, in particular, the Raf–MEK–ERK cascade (Figure 1). Although there is considerable experimental evidence that the Raf–MEK–ERK cascade is a critical mediator of Ras-induced oncogenesis, recent studies have also clearly demonstrated that Ras utilizes additional effectors to promote tumorigenesis (Repasky et al., 2004). Currently, at least four other effector classes have demonstrated roles in Ras transformation: the p110 catalytic subunits (p110a, b, g and d) of class I phosphatidylinositol 3-kinases (PI3K), the Tiam1 Rac small GTPase-specific GEF, the Ral small GTPasespecific GEFs (RalGDS, Rgl, Rgl2 and Rgl3) and phospholipase C epsilon. The evidence for their involvement and roles in Ras-mediated oncogenesis has been summarized in recent reviews (Repasky et al., 2004; Shaw and Cantley, 2006). The existence of nonRaf mechanisms of Ras-mediated oncogenesis prompts the question of whether blocking the Raf–MEK–ERK pathway alone be sufficient to effectively block oncogenic Ras function, or will concurrent inhibition of multiple effector pathways be required? Alternatively, as this protein kinase pathway is central to many signaling networks beyond Ras, will blocking the Raf–MEK–ERK pathway be too deleterious and result in significant normal cell toxicity? Instead, will blocking a subset of downstream functions of the ERK pathway be a more effective approach? Oncogene Targeting the Raf-MEK-ERK MAPK cascade PJ Roberts and CJ Der 3296 The epidermal growth factor receptor: upstream activation of Ras and the ERK cascade and a component of a Ras-mediated autocrine growth loop In addition to activating mutations of the Ras oncogene, the ERK MAPK signaling pathway can also be activated by perturbation of components upstream of Ras. For example, aberrant overexpression or mutational activation of receptor tyrosine kinases (RTKs; e.g., EGFR and HER2) can cause hyperactivation of Ras leading to upregulated MAPK signaling (Lynch et al., 2004; Stephens et al., 2004; Hynes and Lane, 2005). In particular, the EGFR is overexpressed or mutationally activated in many human cancers (Grandis and Sok, 2004). The regularity with which this signaling cascade is activated suggests that it is critical in oncogenesis and makes it an appealing pathway for drug development. Another linkage between Ras and the EGFR receptor is mediated by the upregulation of expression of EGFR ligands by Ras signaling (Figure 1). One important gene target of Ras activation involves transcriptional activation of the gene for transforming growth factor alpha (TGFa), a ligand for the EGFR. Increased TGFa gene expression and secretion of TGFa in turn causes persistent stimulation of the EGFR. The upregulated expression and secretion of TGFa and other EGFR ligands (e.g., heparin-binding EGF, amphiregulin) has been observed in a wide variety of Ras- or Raf-transformed cell types (McCarthy et al., 1995; Gangarosa et al., 1997; Schulze et al., 2001). The importance of this autocrine signaling loop for Ras transformation has been demonstrated by the ability of inhibitors of EGFR to block oncogenic Ras transformation. Additionally, the majority of Raf-induced changes in gene expression were found to be dependent on EGFR function (Schulze et al., 2004). Hence, the EGFR can function both upstream, as well as downstream, of Ras and the ERK MAPK cascade. Inhibitors of the Raf–MEK–ERK cascade Currently, inhibitors of the kinase function of Raf and MEK represent the most studied and advanced approaches for blocking ERK signaling, with several inhibitors under evaluation in clinical trials and additional inhibitors in preclinical analyses (Table 1). To date, no inhibitors of ERK1 or ERK2 have been described. Raf inhibitors The three Raf kinases exhibit the same substrate specificity, with MEK1 and MEK2 the only known substrates. However, these highly related isoforms do exhibit differences in regulation and biological function (Wellbrock et al., 2004; Schreck and Rapp, 2006). Furthermore, functions independent of MEK activation have been described, although these activities remain poorly characterized. Several structurally distinct classes of compounds have been developed as potential Raf Oncogene kinase inhibitors (Smith et al., 2006)). In addition to small molecule inhibitors of Raf kinase, other anti-Raf efforts include the development of antisense inhibitors of Raf expression, particularly ISIS-5132, a 20base phosphorothioate DNA oligonucleotide that inhibits c-Raf-1 protein expression. ISIS-5132 showed antitumor activity in preclinical xenograph models and early clinical trials (Monia et al., 1996; Cripps et al., 2002; Tolcher et al., 2002; Oza et al., 2003). However, no patient response or antitumor efficacy was seen in phase II clinical trials (Cripps et al., 2002; Tolcher et al., 2002; Oza et al., 2003) and it has been withdrawn from further clinical evaluation. A related approach, using liposomeencapsulated antisense c-raf-1 oligonucleotide (Gokhale et al., 2002), showed antitumor activity in xenograft analyses (Mewani et al., 2004). Liposome entrapment serves to protect the oligonucleotide from degradation and to improve delivery. LErafAON has completed initial phase I clinical trials where it was evaluated as monotherapy and in combination with radiation or chemotherapy (Rudin et al., 2004; Dritschilo et al., 2006). Further clinical development of LErafAON is ongoing. Small molecule inhibitors of Ras–Raf interaction (MCP1 and derivatives) have been described and shown to have antitumor activity in cell culture studies (KatoStankiewicz et al., 2002). MCP1 was identified as an inhibitor of Ras interaction with Raf in a yeast twohybrid-based screen. The exact mechanism by which MCP1 functions is currently unclear and this information will be important for further clinical development of this novel class of Raf inhibitors. Currently, preclinical evaluation of MCP continues in human tumor xenograft mouse models in combination with other chemotherapeutic agents (Skobeleva et al., 2007). Hsp90 functions as a chaperone that is required for the stability and function of Raf and other oncogene proteins, including HER2 and the Met RTK, as well as steroid hormone receptors that include the androgen and estrogen receptors (Neckers, 2006). The antitumor activity of the antibiotic benzoquinone ansamycin geldanamycin is due to binding to and promoting Hsp90 degradation, and hence, indirectly inhibiting the function of Raf and other client proteins by promoting their proteosomal degradation. Interestingly, a recent study found that Hsp90 is required for wild-type c-Raf-1 and A-Raf, but not B-Raf, stability (Grbovic et al., 2006). However, mutant B-Raf showed dependency on Hsp90 for function. Treatment of melanoma cells with the geldanamycin analog 17-allylamino-17-demethoxygeldanamycin (17-AAG) caused degradation of mutant B-Raf and inhibition of ERK, and showed antitumor activity. Another study also found preferential sensitivity of mutant B-Raf to 17-AAG treatment (da Rocha Dias et al., 2005). 17-AAG is currently in phase II clinical trials (Sharp and Workman, 2006). Although 17-AAG has shown promising activities in preclinical and clinical trials, the further clinical development may be limited by problems with solubility, stability and hepatotoxicity. Hsp90 inhibitors are relatively unique antitumor agents in that they simultaneously inhibit Targeting the Raf-MEK-ERK MAPK cascade PJ Roberts and CJ Der 3297 multiple, functionally diverse, signaling components that promote oncogenesis. Hence, the precise biomarker for defining and monitoring antitumor response may be complicated. To date, the most successful anti-Raf inhibitor has been sorafenib (tosylate salt of BAY 43-9006; Nexavar), an orally available compound that received FDA approval in 2005 for the use in advanced renal cell carcinoma (RCC). Sorafenib is a bi-aryl urea compound (Smith et al., 2006) that was originally developed as an inhibitor of Raf-1 (Lyons et al., 2001). Subsequent analysis revealed that sorafenib was a potent inhibitor of both wild-type and mutant (e.g., V600E, the most frequent mutation found in human cancers) B-Raf kinases in vitro. Crystallographic analyses of sorafenib complexed with the kinase domain of B-Raf showed that the inhibitor bound to the ATP-binding pocket and prevented kinase activation, thus preventing substrate binding and phosphorylation (Wan et al., 2004). Shortly after clinical analyses begin, it was revealed that sorafenib also showed very potent activity for other protein kinases in vitro and in vivo, in particular, for the proangiogenic RTKs such as VEGFR-2, VEGFR-3, PDGFR-b, Flt-3, c-Kit and FGFR-1 (Wilhelm et al., 2004). Although sorafenib demonstrated multikinase inhibition, it was also found to maintain some specificity, as it did not inhibit other protein kinases such as MEK1, ERK1, EGFR, HER2 and others. In cell culture and mouse models representing a wide range of tumor cell types, sorafenib exhibited broad antitumor activity and was associated with reduced MEK and ERK activation, supporting the possibility that its antitumor activity involves, in part, inhibition of Raf (Wilhelm et al., 2004). However, despite its association with reduced ERK activation, sorafenib antitumor activity must also be a consequence of its ability to inhibit angiogenesis-related kinases as well as other non-Raf kinases. This possibility is supported by the potent antitumor activity that was independent of Ras or B-Raf mutation status that was seen with sorafenib in xenograft studies. Phase I clinical trials established sorafenib as a safe and well-tolerated oral agent with skin rash and diarrhea as the most common adverse effects (Awada et al., 2005) (Moore et al., 2005a). Results from phase I clinical trials suggested clinical activity in several patients with RCC, resulting in subsequent clinical trials focused on RCC. Ultimately, this effort led to a large phase III clinical trial that enrolled more than 900 patients with advanced RCC, who previously failed prior systemic therapy. The primary endpoint of this study was improved survival. This trial met its surrogate endpoint of significantly longer progression-free survival in the sorafenib arm compared to the placebo arm of the study. In addition, sorafenib treatment doubled the disease progression-free survival from 12 to 24 months when compared to the placebo-control arm (Escudier et al., 2005; Nexavar website has updated survival data). In addition to RCC, several single agent and combination clinical studies are ongoing in hepatocelluar carcinoma, non-small-cell lung cancer (NSCLC), prostate cancer, breast cancer, ovarian cancer, pancreatic cancer, melanoma and hematological malignancies (Hahn and Stadler, 2006; Rini, 2006). The results of clinical trial analyses of sorafenib have not provided sufficient information to conclude that inhibition of Raf provides a clinical value. As B-Raf mutations are not seen in RCC, and as sorafenib is not a potent Raf inhibitor, the antitumor activity seen may be attributed to the antiangiogenic, rather than anti-Raf, activity of sorafenib. Although preclinical cell culture and mouse model analyses showed that continued expression of mutant B-Raf is critical for melanoma growth and tumorigenicity, phase II clinical trial analyses with sorafenib observed little or no antitumor activity when evaluated as monotherapy for advanced melanomas (Eisen et al., 2006). Some clinical efficacy had been seen for melanomas when sorafenib was used in combination, with clinical trials ongoing for advanced melanomas with sorafenib combination with bevacizumab or carboplatin and paclitaxel. However, a recently completed phase III trial administering sorafenib or placebo tablets in combination with carboplatin and paclitaxel in patients with advanced melanoma found no difference in the primary end point of improving progression-free survival. RAF265 (formerly CHIR-265) is another orally bioavailable Raf inhibitor currently being investigated in phase I clinical trials in locally advanced or metastatic melanoma (http://www.clinicaltrials.gov/ct/show/ NCT00304525). RAF265 inhibits all three Raf isoforms as well as mutated B-Raf. Like sorafenib, RAF265 may also have antiangiogenic activity through inhibition of VEGFR2. PLX4032 is a potent and selective inhibitor of mutant B-Raf that is currently in phase I clinical evaluation. Other Raf inhibitors are also in preclinical evaluation and should be entering clinical evaluation in the near future. The clinical success of sorafenib and another approved multikinase inhibitor (sunitinib) has prompted a debate regarding the advantages and disadvantages of highly specific versus broad specificity inhibitors (Sebolt-Leopold and English, 2006). As cancer is a multistep process, requiring multiple alterations, it is expected that effective cancer treatment requires concurrent activities that target different defects (Hanahan and Weinberg, 2000). The greater success of combination chemotherapy is consistent with this premise. The ability to optimize the pharmacokinetics and pharmacodynamic properties of a single agent with multiple activities is also a great advantage for the successful clinical development of a drug. In the development of sorafenib, the intention was the identification of a Raf inhibitor, with the activity against other protein kinases fortuitous and unplanned. Hence, this has complicated a full understanding of the mechanism of action of sorafenib and the importance of its anti-Raf activities for its clinical efficacy. Therefore, whether blocking Raf will be a clinically effective approach will require future clinical evaluation of more specific and potent Raf kinase inhibitors. The clinical success of highly selective protein kinase inhibitors, in particular monoclonal Oncogene Targeting the Raf-MEK-ERK MAPK cascade PJ Roberts and CJ Der 3298 antibody (mAb)-based drugs (e.g., trastuzumab, bevacizumab), demonstrates that there is clinical value for both highly selective and multitargeted inhibitors. MEK inhibitors MEK1 and MEK2 are closely related dual-specificity kinases, capable of phosphorylating both serine/threonine and tyrosine residues of their substrates ERK1 and ERK2. They are the only known catalytic substrates of Raf kinases. The fact that ERK is the only known substrate of MEK, when coupled with the observation that ERK is commonly activated in both tumor cell lines and patient tumors, has fueled strong interest in developing pharmacological inhibitors of MEK as a means to block ERK activation (Hoshino et al., 1999). In contrast to sorafenib, small molecule inhibitors of MEK1/2 are highly specific protein kinase inhibitors. Although the first two MEK inhibitors, PD98059 and U0126, were highly specific (Davies et al., 2000) they lacked the pharmaceutical properties needed to be successful clinical candidates. Nonetheless, these compounds have been invaluable academic research tools for dissecting the MEK–ERK pathway and have provided enormous insight into the importance of ERK MAPK signaling in cancer (Cox and Der, 2002b; Sebolt-Leopold and Herrera, 2004). The first MEK inhibitor to enter clinical trials was CI-1040 (PD184352), an orally active, highly potent and selective inhibitor of MEK1 and MEK2 (SeboltLeopold et al., 1999). Preclinical evaluation found that CI-1040 inhibited the growth of human colon cancer cells and human melanoma cells in athymic nude mice (Sebolt-Leopold et al., 1999; Collisson et al., 2003). Subsequent phase I and II clinical trials reported the most common toxicities were mild skin rash, diarrhea and fatigue (Lorusso et al., 2005) (Rinehart et al., 2004). During the phase I trial, a partial response was seen in one patient with pancreatic cancer and 25% of patients with a variety of tumors had stable disease for greater than 3 months (Lorusso et al., 2005). Tumor tissues from treated patients showed significant reduction in activated phosphorylated ERK, indicating that the target was inhibited. These encouraging results prompted a phase II study in patients with advanced NSCLC, breast cancer, colorectal cancer (CRC) and pancreatic cancer. Unfortunately, the results of this trial were negative and CI-1040 was determined to have poor pharmacokinetic properties (Rinehart et al., 2004). However, when considered together with the significant body of positive preclinical data as well as early indications from the phase I trial, it is still believed that MEK is a valid therapeutic target for the treatment of cancer. Thus, two second generation MEK1/2-specific inhibitors (PD325901 and ADZ6244) believed to have superior pharmacological and biopharmaceutical properties have been developed and are currently in clinical trials (Table 1). In contrast to the majority of protein kinase inhibitors, MEK inhibitors are non-ATP competitive inhibitors, which may account for their highly selective Oncogene properties. Structural studies with an analog of CI-1040 in complex with MEK1 or MEK2 showed inhibitor binding did not perturb ATP binding, and instead, bound to a unique inhibitor binding pocket adjacent to the ATP-binding site (Ohren et al., 2004). Inhibitor binding locked MEK in a catalytically inactive conformation. This recognition of MEK sequences that are not shared with other protein kinases, and their association with an inactivate conformation, account for MEK inhibitor target selectively. PD0325901 is a derivative of CI-1040 where several slight modifications to the chemical structure have resulted in more than a 50-fold increase in potency against MEK1/2, improved bioavailability, and longer duration of target suppression compared to CI-1040 (Sebolt-Leopold and Herrera, 2004). Antitumor activity for PD0325901 was demonstrated for a variety of tumor xenografts and this inhibitor is now being evaluated in phase I/II clinical trials with a focus on tumors expected to have activated ERK MAPK signaling (Thompson and Lyons, 2005; Solit et al., 2006). AZD6244 (ARRY-142886) is an orally bioavailable benzimidazole derivative known to potently inhibit MEK1/2 in vitro and in cell-based assays (Lyssikatos et al., 2004; Yeh et al., 2004). Like other MEK inhibitors, AZD6244 is ATP non-competitive. Preclinical evaluation of AZD6244 showed antitumor activity in several human xenograft models including colon, pancreas, breast, NSCLC and melanoma (Lee et al., 2004). Additionally, AZD6244 antitumor activity was found to correlate with suppression of ERK activation, which further validates that its mechanism of action is MEK-dependent. Results from preclinical analysis have been extremely promising and thus AZD6244 has moved into clinical development. Recently, initial results of a first in human dose-ranging study to assess the pharmacokinetics, pharmacodynamics and toxicities of AZD6244 in patients with advanced solid tumors concluded that AZD6244 is well tolerated, and the most common treatment-related adverse events were rash, diarrhea, nausea, fatigue, peripheral edema and vomiting. The best clinical response seen in the 57 patients was stable disease (19 patients at the end of cycle two; nine of whom achieved stable disease for 5 months or greater). The pharmacokinetic and pharmacodynamic analyses showed good systemic exposure, which correlated with high levels of ERK inhibition in peripheral blood mononuclear cells (PBMCs) (http://www.arraybiopharma. com). AZD6244 is now being evaluated in multiple phase II trials in a variety of solid tumors. Solit et al., (2006) recently reported that B-Raf mutant tumors are exquisitely sensitive to MEK inhibition (Solit et al., 2006). In this study, the authors used genetic and pharmacological (CI-1040 and PD0325901) approaches to evaluate MEK dependence in a variety of tumor types and found that human tumor cell lines possessing mutant B-Raf were much more sensitive to MEK inhibition than cells with wild-type B-Raf or mutant K-Ras. In their xenograft models, they found B-Raf mutation-positive tumor xenografts had completely abrogated tumor growth whereas Targeting the Raf-MEK-ERK MAPK cascade PJ Roberts and CJ Der 3299 xenographs of Ras mutation-positive tumor cells were only partially inhibited, perhaps reflecting the fact that Ras also utilizes non-Raf effector pathways to promote oncogenesis. Whether Ras or B-Raf mutation status will accurately identify the patients who will respond to MEK inhibitor treatment, and whether suppression of ERK activity is an accurate measure of drug inhibition of MEK, are issues that remain to be determined. In addition to small molecule inhibitors of MEK kinase activation, bacterial toxins have been identified that inhibit MEK function by unique biochemical mechanisms. Anthrax lethal factor (LF) is a protease, a component of Bacillus anthracis exotoxin, the Grampositive bacterium responsible for the disease anthrax. LF, together with a second exotoxin component (protective antigen), comprise lethal toxin (LeTx). LeTx inactivates multiple MAPKKs, including MEK1 and MEK2 by proteolytic cleavage and inactivation of kinase function (Bodart et al., 2002). LeTx was shown to block the transformed and tumorigenic growth of Ras-transformed rodent fibroblasts (Duesbery et al., 2001). LeTx also showed preferential inhibition of growth of melanoma cell lines that harbored mutated B-Raf and elevated ERK activity (Abi-Habib et al., 2005). Yersinia outer protein J (YopJ) is another bacterial toxin that inhibits MEK function. The bacterial pathogen Yersinia pestis, the causative agent for the plague (Black Death), uses a type III secretion system to inject YopJ and other virulence factors into target cells. YopJ was shown to bind directly to MEK1, MEK2 and other MAPKKs and block their phosphorylation and activation (Orth et al., 1999). YopJ functions as an acetyltransferase, using acetyl-coenzyme A (CoA) to modify the critical serine and threonine residues in the activation loop of MEKs and thereby blocking MAPKKK phosphorylation and activation (Mukherjee et al., 2006). YopJ also causes acetylation of a threonine residue in the activation loop of inhibitor of nuclear Factor kappa B (NF-kB) (IkB) kinase b, preventing its phosphorylation and inactivation of IkB, thus preventing nuclear translocation and activation of the NF-kB transcription factor. As inhibition of NF-kB also has an antitumor consequences (Karin, 2006), YopJ can inhibit concurrently at least two important pathways that promote oncogenesis. The unique biochemical activities of these bacterial proteins may identify novel approaches for blocking MEK–ERK signaling. Inhibitors of Ras As the most commonly mutated oncogene in cancer, Ras has been thought of as the ‘Holy Grail’ of cancer drug development (Cox and Der, 2002b). As the key biochemical defect of mutated Ras proteins is the GAPinsensitivity and persistent GTP binding, early efforts attempted to develop GAP-based approaches to reactivate the intrinsic GTP hydrolysis activity or to antagonize GTP binding, but with no success reported in these efforts. Instead, over the years two main strategies have been vigorously pursued to identify anti-Ras inhibitors. As described above, the first approach has been the development of inhibitors of Ras downstream effector signaling, with efforts focused on the ERK MAPK pathway. The second approach has focused in blocking the post-translational modifications that promote Ras membrane association. For proper signaling, Ras proteins must be localized to the inner surface of the plasma membrane where they interact with upstream activators (receptor tyrosine kinases) and downstream effectors. Ras proteins are synthesized as inactive, cytosolic proteins that quickly undergo a series of post-translational modifications, which targets Ras to the plasma membrane (Figure 4) (Basso et al., 2006). This series of modifications is initiated by enzymes that recognize the Ras carboxylterminal CAAX tetrapeptide (C ¼ cysteine, A ¼ aliphatic amino acid, and X ¼ terminal amino acid) motif. The first step is catalysed by the cytosolic farnesyltransferase (FTase) enzyme and results in the covalent addition of a farnesyl isoprenoid lipid to the cysteine residue of the CAAX sequence. Next, the AAX peptide is cleaved by the Ras converting enzyme 1 (Rce1) endoprotease. Finally, isoprenylcysteine-O-carboxyl methyltransferase (ICMT) covalently attaches a methyl group to the farnesylated cysteine residue (Sebti and Der, 2003). Both Rce1 and ICMT are associated with the endoplasmic reticulum. Each modification is important for increasing Ras affinity for membranes and inhibition of the initial CAAX-signaled processing step (farnesylation) has been shown to inhibit all subsequent steps. With this knowledge, drug discovery efforts have focused on the development of FTase inhibitors (FTIs) in hopes that they would render Ras completely cytosolic and inactive. Although FTIs have shown some success in treating hematologic cancers, their use as Ras inhibitors has been complicated by the fact that K-Ras and N-Ras (the Ras isoforms commonly mutated in cancer) undergo alternative prenylation by a related enzyme, geranylgeranyltransferase I (GGTaseI), when FTase activity is inhibited by FTIs (Figure 4) (Sebti and Der, 2003; Rowinsky, 2006). Hence, in contrast to popular perception, FTIs are not effective inhibitors of Ras function. Nevertheless, although FTIs are no longer considered Ras inhibitors (except for H-Ras), they have continued to progress through preclinical and clinical development, and preclinical studies are focused on identifying the true targets that contribute to the antitumor activities of FTIs. To date, many potent and specific FTIs have been developed, with SCH-66336 (lonafarnib; Sarasar) and R115777 (tipifarnib; Zarnestra) the most advanced and are currently in phase II/III clinical trials for hematologic and other cancers (Lancet et al., 2007). Finally, inhibitors of GGTaseI have also been considered, to block the activities of K-Ras and N-Ras when treated with FTIs. Initial evaluation of the importance of each of the three post-translational modifications of Ras concluded that inhibition of AAX proteolysis or carboxylmethylation Oncogene Targeting the Raf-MEK-ERK MAPK cascade PJ Roberts and CJ Der 3300 Normal Ras Processing PM Alternative K/N-Ras Processing PM Ras -C-OMe ICMT ER -C-OMe Ras -C Ras -CAAX ICMT Ras -C Ras -CAAX ER Rce1 Rce1 FTI FTase Cytosol Ras Ras FTase -CAAX Farnesyl isoprenoid Cytosol GGTaseI Ras -CAAX Geranylgeranyl isoprenoid Figure 4 Post-translational processing of Ras. Normal Ras processing – Under normal physiological conditions Ras undergoes three CAAX-motif signaled post-translational modifications catalysed by the cytosolic farnesyltransferase (FTase), which catalyses covalent addition of the C15 farnesyl isoprenoid. This modification is followed by reactions catalysed by the endoplasmic reticulum (ER)-associated Ras converting enzyme 1 (Rce1) endoprotease and isoprenylcysteine-O-carboxyl methyltransferase (ICMT). The CAAX modifications, together with a second membrane targeting signal upstream of the CAAX motif (palmitoylated cyteines, for HRas, N-Ras and K-Ras4A; lysine-rich sequence, for K-Ras4B). Alternative K/N-Ras processing – When FTase activity is blocked by farnesyltransferase inhibitors (FTIs), N-Ras and K-Ras (the Ras isoforms most commonly mutated in cancer) will undergo alternative prenylation by the related geranylgeranyltransferase I (GGTase) enzyme, which adds the more hydrophobic C20 geranylgeranyl isoprenoid to the cysteine residue of the CAAX motif. As alternatively prenylated Ras retains normal membrane association and function, FTIs fail to block the function of these Ras isoforms. H-Ras does not undergo alternative prenylation, and consequently, its function is effectively blocked by FTI treatment. was not sufficient to fully block Ras transformation and thus attention was focused on developing inhibitors of FTase (Cox and Der, 2002a). However, with the realization that FTIs are not effective anti-Ras drugs, attention has shifted back to evaluating Rce1 and ICMT as targets for inhibiting Ras membrane association and localization (Clarke and Tamanoi, 2004; Winter-Vann and Casey, 2005). Studies by Young, Casey and coworkers found that conditional deletion of either Rce1 or ICMT in fibroblasts derived from Rce1- or ICMTdeficient MEFs impaired their sensitivity to K-Rasmediated transformation. Although knockout of Rce1 or ICMT function was critical for mouse development, lack of Rce1 or ICMT expression was not essential for the growth of adult tissue or for MEFs in cell culture (Kim et al., 1999; Bergo et al., 2000). When mutated K-Ras(12V) was expressed ectopically in fibroblasts isolated from Rce1- or ICMT-deficient mouse embryos, K-Ras membrane association was partially impaired, Ras activation of ERK was reduced, and Ras-mediated growth transformation was impaired (Bergo et al., 2001, 2002, 2004). Further evidence for the importance of ICMT in Ras transformation came from studies by Casey and co-workers, who found that the anti-neoplastic drug Oncogene methotrexate, an inhibitor of DNA synthesis, may also act, in part, by inhibition of ICMT to block Ras transformation (Winter-Vann et al., 2003). Furthermore, this group recently reported the identification of a selective small-molecule inhibitor of ICMT, 2-(5-(3-methylphenyl)-1-octyl-1H-indol-3-yl)acetamide (cysmethynil) (Winter-Vann and Casey, 2005). Early preclinical evaluation indicated that cysmethynil treatment inhibited cell growth in an ICMT-dependent fashion, resulted in cytosolic mislocalization of Ras proteins in MDCK cells and blocked EGFR-mediated signaling. In addition, cysmethynil treatment was shown to inhibit anchorage-independent growth of DKOB8 cells (Winter-Vann and Casey, 2005). Although an obvious concern with Rce1 and Icmt inhibitors is the fact that the number of substrates for these enzymes is extensive, with several hundred CAAXterminating proteins that are known or putative substrates for FTase or GGTaseI (Reid et al., 2004), the fact that a number of these CAAX-terminating proteins also display functions in oncogenesis (e.g., Rheb, Ral, RhoC and Rac1b) also suggest that their multi-targeted actions may also be advantageous for cancer treatment. However, it is possible that ICMTmediated methylation may be critical for the function of only farnesylated proteins, thus greatly reducing the number of targets for ICMT inhibitors (Michaelson et al., 2005). Finally, ICMT-mediated methylation is also needed for the function of a subset of Rab family small GTPases, regulators of vesicular trafficking (Leung et al., 2006), thus expanding the candidate targets for ICMT inhibitors beyond CAAX-terminating proteins. Some Rab proteins have also been implicated in oncogenesis (Cheng et al., 2005). Inhibitors of EGFR signaling The EGFR family consists of four transmembrane receptors: EGFR (HER1/erbB-1), HER2 (erbB-2/neu), HER3 (erbB-3) and HER4 (erbB-4) (Mendelsohn and Baselga, 2006; Scaltriti and Baselga, 2006). Multiple ligands bind to and activate EGFR, HER3 and HER4. Ligands for EGFR include EGF, TGFa, HB-EGF, betacellulin and epiregulin. Ligand binding results in the formation of receptor homodomers and heterdimers leading to subsequent receptor activation and ultimately the activation of downstream signal transduction pathways (Yarden, 2001; Wiley, 2003). The best characterized cytoplasmic signaling pathway activated by the EGFR is the ERK MAPK pathway, and ERK activation has been utilized as a biomarker for EGFR inhibitor action. Additional EGFR-activated pathways include the PI3K and AKT serine/threonine kinase, signal transducer and activator of transcription (STAT), as well as protein kinase C and phospholipase D pathways (Citri et al., 2003). EGFR activation of these pathways results in enhanced proliferation, angiogenesis, invasion and metastasis, as well as inhibition of apoptosis (Jimeno and Hidalgo 2006). Aberrant Targeting the Raf-MEK-ERK MAPK cascade PJ Roberts and CJ Der 3301 activation of the EGFR signaling commonly occurs in cancer (NSCLC, CRC, breast cancer, gastric cancer and others), and multiple mechanisms describing its activation have been reported including EGFR overexpression, EGFR gene amplification, acquisition of activating mutations and overexpression of EGFR ligands (Baselga and Arteaga, 2005). Two strategies to inhibit EGFR signaling have been successfully developed and include mAbs directed against the extracellular domain of EGFR (Table 3) and small molecule tyrosine kinase inhibitors (TKIs) of the intracellular tyrosine kinase domain (Table 4). By blocking ligand–receptor interactions, mAbs inhibit receptor homo- and heterodimerization resulting in receptor internalization and inhibition of EGFR signaling pathways. Additionally, the clinical activity of some EGFR mAbs may also be attributed to their ability to stimulate an immune response. Alternatively, EGFR receptor TKIs bind to the intracellular tyrosine kinase domain preventing ATP binding and receptor activation, thus blocking EGFR activation of ERK and other signaling pathways. Mab and small molecule inhibitors of EGFR possess shared and distinct features that distinguish their mechanistic and clinical activities (Table 5). Hence, it is believed that their use in combination may act synergistically and improve antiEGFR therapy. As with other signal-transduction inhibitors, EGFR inhibitors lack the severe myelosuppressive toxicities seen with conventional cytotoxic drugs. Toxicities are Table 3 most evident in tissues that are dependent on EGFR function, in particular the skin (Lacouture, 2006). This includes a papulopustular rash that affects the face and upper trunk, abnormalities in hair growth, and dry and itchy skin. There is incomplete evidence that the strength of skin rash may be a good indication of drug activity and possibly favorable patient response and survival (Perez-Soler and Saltz, 2005). Several EGFR mAbs have been developed that recognize the extracellular domain of the EGFR (Figure 5). However, as they recognize distinct sequences and vary in binding affinities and composition (Figure 5), they also vary in their biological activities. The first FDA-approved EGFR mAb was cetuximab, a chimeric monoclonal IgG1 antibody initially approved for use in combination with irinotecan for the treatment of EGFR-detectable metastatic CRC refractory to irinotecan, as well as monotherapy for the treatment of EGFRdetectable metastatic CRC in patients intolerant to irinotecan (Cunningham et al., 2004). More recently, cetuximab has received additional approval for use in combination with radiation to treat inoperable squamous cell cancer of the head and neck (Bonner et al., 2006). Finally, although EGFR expression was an initial basis for patient selection, patient response has not correlated with the degree of overexpression. Thus, a reliable biomarker for defining patient response remains elusive. Although cetuximab has proven to be an effective treatment in the aforementioned indications and continues to be investigated for use in other tumor types, Monoclonal antibody EGFR-directed therapeutic agentsa Compound Generic (Trade) name Characteristics Company Status Tumors IMC-C225 Cetuximab (Erbitux) Chimeric IgG1 Imclone/Bristol-Myers Squibb/Merck KGaA Approved CRC, SCCHN ABX-EGF Panitumumab (Vectibix) Fully human IgG2 Abgenix/Amgen Phase II–III Pancreatic cancer, NSCLC, breast cancer, and other solid tumors Approved CRC Phase I–III EMD-72000 h-R3 Matuzumab Nimotuzumab (TheraCIM) mAb 2F8 Zalutumumab (HuMax-EGFr) rhuMAb 2C4 Pertuzumab (Omnitarg) ch806 MDX-214 Phase III NSCLC, rectal cancer, bladder cancer, breast cancer and ovarian cancer Various solid tumors including NSCLC, cervical cancer and gastric cancer Various solid tumors with a focus in NSCLC SCCHN, NSCLC Phase II Breast cancer, ovarian cancer Phase I Advanced solid tumors Phase II Advanced solid tumors Humanized IgG1 EMD Pharmaceuticals/ Phase II Merck KGaA Humanized IgG1 YM Biosciences/ CIMAB S.A. Medarex/Genmab Fully human IgG1 Humanized mAb inhibits HER2 dimerization with other HER family members Chimeric anti-EGFR vIIIb IgG2 Genentech/HoffmannLa Roche Ludwig Institute/ Life Science Pharmaceuticals EGF fusion with Fab fragment of Medarex a fully human anti-CD89 (IgA FcR) mAbc Phase III Abbreviations: CRC, colorectal cancer; SCCHN, squamous cell carcinoma of the head and neck; NSCLC, non-small-cell lung cancer. aCompiled from studies cited in reviews that we have referenced in the text, from company websites, and from www.clinicaltrials.gov, bGenerated against the deletion mutant of EGFR lacking exons 2–7 of the extracellular domain, cActivation of cytotoxic neutrophils via CD89 stimulation. Oncogene Targeting the Raf-MEK-ERK MAPK cascade PJ Roberts and CJ Der 3302 Table 4 Small molecule tyrosine kinase inhibitor EGFR-directed therapeutic agentsa Compound Generic (Trade) name Characteristics Company Status Tumors ZD1839 Gefitinib (Iressa) EGFR-specific; reversible quinazoline AstraZeneca Approvedb NSCLC OSI-774 Erlotinib (Tarceva) GW572016/GW2016 Lapatinib (Tykerb) EKB-569 PKI-166/PKI116 ARRY-334543 BMS-599626 CI-1033 HKI-272 AEE788 Canertinib ZD6474 Vandetanib (Zactima) XL647 Phase II–III Breast cancer, hepatocellular carcinoma, esophageal cancer, SCCHN, brain and CNS tumors, and other solid tumors EGFR-specific, reversible quinazoline OSI Pharmaceu- Approved NSCLC, pancreatic cancer ticals/Genentech/ Roche Phase II–III CRC, brain and CNS tumors, bladder cancer, RCC, SSCHN, prostate cancer and other solid tumors. EGFR, HER2; reversible quinazoline GlaxoSmithKPhase III Breast cancer, RCC, head and line neck cancer EGFR, HER2; irreversible quinazoline Wyeth Phase III Breast cancer, NSCLC EGFR, HER2 pyrrolotriazine Novartis Phase I Prostate, RCC EGFR, HER2 reversible Array Phase I Advanced tumors pyridopyrimidine Biopharma Pan-HER,reversible pyrrolotriazine Bristol-Myers Phase I Advanced solid tumors Squibb Pan-HER, irreversible quinazoline Pfizer Phase I/II NSCLC, breast cancer Pan-HER, irreversible quinazoline Wyeth Phase II breast cancer, NSCLC EGFR, HER2 and VEGFR2; Novartis Phase I Advanced solid tumors reversible pyrrolotriazine EGFR, VEGFR2, RET; reversible AstraZeneca Phase III NSCLC, medullary thyroid quinazoline cancer EGFR, HER2, VEGFR2, EphB4 Exelixis Phase II NSCLC Abbreviations: CRC, colorectal cancer; CNS, central nervous system; NSCLC, non-small-cell lung cancer; SCCHN, squamous cell carcinoma of the head and neck; RCC, renal cell cancer. aCompiled studies cited in reviews that we have referenced in the text, from company websites and from www.clinicaltrials.gov; bCurrently approved for patients with past treatment; not available to new patients. Table 5 Properties of monoclonal antibody and small molecule tyrosine kinase inhibitors of EGFR Characteristics Monoclonal antibodies Tyrosine kinase inhibitors mAb versus TKI Mechanism of action Blockade of EGFR ligand binding, receptor internalization, Inhibition of receptor dimerization, stimulation of antibody-dependent immune response (IgG1 isotypes only) Multiple mechanisms of mAb may lead to enhanced antitumor activity and more robust EGFR inhibition, whereas TKIs can inhibit ligand-independent constitutively activated EGFR. Route of administration Frequency of administration Size Specificity Intravenous Binding to or adjacent to ATPbinding pocket to inhibit kinase activity and phosphorylation of downstream substrates, preventing activation of the ERK MAPK and other signaling pathways. Oral Weekly to every 3 weeks Daily Large and bulky Highly specific, no off-target activities Small molecule Likely to have off-target activity, ranging from limited to very significant. Yes Effectiveness against constitutively active EGFR No Common toxicities Rash, fatigue, infusion reactions (for some) Rash, fatigue and diarrhea Oral administration of TKIs decreases time in the clinic mAbs have longer elimination halflife, allowing for less frequent dosing TKIs have better tumor penetration. mAb specificity may reduce toxicity, while TKI off-target activity may enhance anti-tumor activity TKIs are active on mutated, constitutively active EGFRs that no longer rely on ligand activation; mAbs will not be effective in these tumors. Toxicity profiles are similar with the EGFR-specific mAb and TKIs; however, the pan-HER and TKIs with additional antiangeogenesis activities may exhibit greater toxicities. Abbreviations: EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase. one of its major drawbacks is the associated risk of anaphylactic reactions during cetuximab infusion (Cunningham et al., 2004; Bonner et al., 2006). This Oncogene has led to the development of two humanized EGFR (matuzumab and nimotuzumab) and two fully human (panitumumab and zalutumumab) mAbs. Targeting the Raf-MEK-ERK MAPK cascade PJ Roberts and CJ Der 3303 Mouse (-o mab) Chimeric (-xi mab) Humanize (-zu mab) Human (-u mab) 100% Mouse 225 ~33% Mouse Cetuximab ~10% Mouse Matuzumab Nimotuzumab 100% Human Panitumumab Zalutumumab Figure 5 EGFR monoclonal antibodies. Nomenclature of mAbs has been devised for assigning generic or nonproprietary names. All monoclonal antibodies end with the suffix -mab, whereas different infixes are used to differentiate the agents depending on the structure and function of the therapeutic agent. The infix immediately preceding the -mab suffix denotes the animal origin of the antibody (o, mouse; xi, chimera; zu, humanized; u, human). The first mAbs were produced in mice and are recognized as foreign by the human immune system (e.g., 225), which can lead to increased clearance and allergic/anaphylactic reactions. Therefore, sequences that comprise the constant regions of the antibody, which are not involved in epitope recognition, may be replaced with human sequences, to create mAbs that are chimeric mouse–human proteins (e.g., IMC-C225 was derived from 225) or humanized mAbs. Recently, the use of transgenic mice with human immunoglobulin heavy and light chain coding sequences (e.g., XenoMouse) has facilitated the generation of fully human mAbs. The infix preceding the source of the antibodies refers to the disease condition the agent is used to treat. Most of these consist of a consonant, vowel and another consonant. To simplify the naming scheme, the final consonant is commonly dropped if the following infix begins with a consonant (such as -zu- or -xi-). For mAbs used for the treatment of cancer ‘-tu(m)-’ is used as the infix to designate tumor (* designates EGFR mAbs that are FDA approved). Panitumumab (Vectibix, Amgen, Inc.) was approved in 2006 for the treatment of patients with EGFR expressing, metastatic CRC with disease progression on or following fluoropyrimidine-, oxaliplatin- and irinotecan-containing chemotherapy regimens. Panitumumab is a fully humanized mAb that is similar to cetuximab except that panitumumab is an IgG2 antibody that does not elicit antibody-dependent cell-mediated cytotoxicity (ADCC) (Yang et al., 1999). In the pivotal phase III trial that led to its approval, panitumumab treatment significantly improved progression-free survival and resulted in an 8% overall response rate (Gibson et al., 2006). The most common toxicities seen include rash, fatigue, hypomagnesemia, abdominal pain and diarrhea. As expected with a fully humanized mAb, no grade 3/4 infusion-related reactions were seen in this trial. Matuzumab is a humanized IgG1 anti-EGFR mAb. As a humanized mAb matuzumab, like panitumumab, is expected to have reduced infusion-related anaphylactic reactions. Furthermore, as an IgG1 antibody matuzumab-induced potent ADCC against tumor cells in vivo, which distinguishes matuzumab from the recently approved panitumumab (Bier et al., 1998). Although results from several phase II trials are expected within the next year, initial phase I trials have shown matuzumab is well tolerated, with rash and diarrhea the most common toxicities. In these trials, evaluation of both tumor tissue and skin biopsies determined that matuzumab inhibited phosphorylation of EGFR, ERK and AKT (Tabernero et al., 2003; Salazar et al., 2004; Vanhoefer et al., 2004; Doi et al., 2005). In phase I trials, activity has been evaluated in colorectal, cervical and esophageal cancers and in squamous cell cancer of the head and neck. Current therapeutic targets in phase II trials include cervical and gastric cancers, and NSCLC. Pertuzumab is a novel mAb with a unique mechanism of action designed to inhibit the heterodimerization of HER2 with EGFR and other HERs. Similar to other EGFR mAb, the most common toxicities include rash and diarrhea (Agus et al., 2005). To date several phase II trials evaluating pertuzumab have been completed. Initial results evaluating pertuzumab as monotherapy in metastatic breast cancer and hormone-refractory prostate cancer were not promising as there was limited evidence of activity in breast cancer patients and no prostate-specific antigen responses in the prostate cancer (Cortes et al., 2005; de Bono et al., 2005). However, more promising results in refractory or recurrent ovarian cancer have been seen in which clinical activity was seen in 15% of patients (Gordon et al., 2005). Although single-agent use of pertuzumab has shown limited clinical activity, our experience with targetedtherapies along with preclinical xenograft data suggest that pertuzumab may have greater clinical benefit when used in combination with other therapies (Friess et al., 2005). Thus, current phase II trials of pertuzumab are focused on combination therapy, including a study of pertuzumab in combination with erlotinib in patients with locally advanced or metastatic NSCLC. Two EGFR small molecule kinase inhibitors have received approval for use in NSCLC (Table 4). The first was gefitinib, which received accelerated approval as third-line monotherapy in NSCLC based on its 12% response rate and 43% rate of tumor control. However, gefitinib was subsequently evaluated in a large phase III trial comparing gefitinib to placebo in NSCLC patients who had failed previous treatment regimens, and results Oncogene Targeting the Raf-MEK-ERK MAPK cascade PJ Roberts and CJ Der 3304 from this trial showed no improvement in survival compared to placebo resulting in a FDA-mandated labeling change. Currently, gefitinib is only approved for the treatment of cancer patients who have previously received and benefited from treatment. Ongoing clinical trials are evaluating the use of gefitinib for the treatment of other cancers. As apposed to gefitinib, erlotinib has demonstrated improved survival in a randomized phase III placebocontrolled trial. Although the overall response rate was only 9% in the erlotinib arm, erlotinib prolonged overall survival (6.7 vs 4.7 months) and increased 1-year survival (31 vs 22%). These results led to FDA approval in 2004. More recently, erlotinib has also gained FDA approval for use in combination with gemcitabine for the first-line treatment of patients with locally advanced, unresectable or metastatic pancreatic cancer (Moore et al., 2005b). As is seen with some recent mAb-based EGFR strategies (e.g., zatumumab), newer generation TKIs are now being developed to inhibit multiple RTKs (Table 4). In particular, laptinib (Tykerb) is a dual EGFR and HER2 tyrosine kinase inhibitor that has been extensively evaluated in multiple solid tumors including breast, NSCLC, CRC, head and neck cancer, hepatocellular carcinoma and billary carcinoma (Nelson and Dolder, 2006). Initial evaluation of this compound revealed diarrhea and skin rash as the most common toxicities (Nelson and Dolder, 2006). Although the clinical activity of this agent has been evaluated in multiple tumor types, the main focus has been in the treatment of breast cancer a disease known to rely on both HER-2 and EGFR signaling. Numerous phase I and II clinical trials have been reported, including several looking at the use of lapatinib in refractory metastatic breast cancer and in first line treatment of advanced breast cancer (Moy and Goss, 2006). An international, multicenter, randomized, open label phase III trial in patients with documented HER2 overexpressing refractory advanced or metastatic breast cancer treated with lapatinib in combination with capecitabine versus capecitabine alone was stopped after the interim analysis. Of the 321 evaluable patients, 161 were treated in the combination arm and 160 in the monotherapy arm. Median time to progression in the combination arm was 8.5 months, compared with 4.5 months in the capecitabine alone arm (Tykerb at ASCO 2006, http://www.gsk.com/investors/presentations_webcasts.html). Although these data need to mature so that an overall survival advantage can be determined, this compound is likely to become part of the standard of care in breast cancer and possibly other tumor types as well. The results of this phase III trial are so promising that it has been made available through an Expanded Access Protocol of lapatinib combined with capecitabine in metastatic breast cancer in a non-randomized, open label, uncontrolled trial. In addition to lapatinib, EKB-569 and PKI-166 are two additional dual EGFR/HER2 inhibitors in clinical trials (Table 4). Furthermore, canertinib (CI-1033), BMS-599626 and HKI-272 are pan-HER inhibitors, Oncogene whereas several EGFR inhibitors also inhibit RTKs involved in tumor angiogenesis. Although targeting multiple protein kinases may enhance antitumor activity, it may also result in greater normal cell toxicity. Biomarkers: verifying target inhibition and predicting patient response The important issues that MAPK drug discovery faces are those that have accompanied the new era of targetbased anticancer drug discovery. Historically, oncology drug development has focused on determining the maximum tolerated dose (MTD), safety profile and efficacy of a compound whereas mechanistic studies and efforts to preselect responders were almost never undertaken. In fact, the mechanisms of action that accounts for the antitumor activity of many of our commonly used chemotherapeutic agents used today are still poorly understood. However, with an ever-growing understanding of cancer biology, new targeted-based or mechanism-based therapies are making there way into the clinic. These compounds differ greatly from the majority of the conventional cytotoxic antineoplastic drugs in that they are rationally designed to mechanistically inhibit a particular protein target that is hypothesized to be critical for cancer growth and progression. Although these novel agents offer great promise for significant improvements in cancer chemotherapy, they have also brought researchers and clinicians several new challenges and issues for drug discovery. First, the identification of useful and predictive biomarkers has become an important endeavor. The development of methodology to accurately verify that a targeted therapy is actually blocking the function of its target is crucial for effective clinical evaluation. If the compound efficiently inhibits the target but has no clinical response, then it allows researchers to conclude that the target is not important for that particular cancer. For example, will a reduction in ERK activation be a reliable marker for monitoring the effectiveness of inhibitors of Raf or MEK to block target function? Will ERK inhibition correlate with patient response? A related issue involves the tissue source for monitoring drug action. The most direct and relevant measure of target inhibition is to use pre- and posttreatment tumor samples to measure the biomarker of interest. The obvious limitation of this approach is that tumor tissue is not readily accessible in many types of cancer and putting patients through multiple biopsies is not reasonable. Therefore, other methods to measure target inhibition have been devised and include the use of surrogate tissues such as PBMCs, skin and buccal mucosa (Parulekar and Eisenhauer, 2004). However, while feasible, whether drug inhibition in the surrogate tissue accurately reflects drug activity in the tumor remains an important unresolved issue. A second crucial issue is the identification of genetic or biochemical markers that define the patient population that will respond to inhibitor treatment. Despite Targeting the Raf-MEK-ERK MAPK cascade PJ Roberts and CJ Der 3305 their target-based development, patient response to signal transduction inhibitors remains disappointingly poor and modest. For example, EGFR is overexpressed in 50–80% of NSCLCs yet initial clinical trials evaluating EGFR TKIs found only a 10% response rate. Furthermore, early correlative studies were unable to establish a positive correlation between phospho-EGFR immunohistochemistry and response, thus complicating our ability to predict which patients will respond. Eventually, retrospective analysis revealed the presence of EGFR mutations in the majority of responders thus identifying a molecular marker with a high-predictive value of response (Lynch et al., 2004). However, more recent analyses have found that EGFR mutation status may not be as strongly associated with patient response as initially believed 17045403 (Jimeno and Hidalgo, 2006). Additionally, repeated studies have shown that patients with K-Ras mutations are resistant to EGFR inhibition in both single-agent and combined therapy regimens (Eberhard et al., 2005; Pao et al., 2005). To further complicate patient selection, recent correlative analysis suggests that in addition to EGFR/K-Ras mutational analysis, EGFR gene amplification and protein expression may also serve as important molecular markers (Cappuzzo et al., 2005). Specifically, EGFR mutations may be predictive of response, whereas EGFR gene amplification and protein expression may be better markers of survival. An important direction in this area of research is the determination of gene array profiles that may be more effective at predicting patient response to a particular drug. Finally, as the successful application of Raf or MEK inhibitors will almost certainly require their use in combination with other drugs, what other signaling inhibitors or conventional cytotoxic drugs should be used? As the potential combinations are daunting, effective preclinical analyses are needed to better focus clinical trial design. Will our current preclinical cell culture or mouse models allow reliable determination of the best combination approaches? Although still the standard of the pharmaceutical industry, the value of human tumor xenograft mouse models in predicting drug activity in the patient remains a hotly debated issue (Sausville and Burger, 2006). Although the new generation of genetically engineered mouse models are optimistically believed to provide more accurate systems for anticancer agent evaluation, this remains to be validated (Sharpless and Depinho, 2006). With limited patient populations for phase I/II clinical trials, and limited research and development budgets, this remains an important limitation in anticancer drug discovery. Conclusions and future directions The considerable genetic and experimental observations provide strong validation that inhibitors of the ERK MAPK cascade will provide effective antineoplastic agents for the treatment of a wild range of human cancers, including those where our current regimen of drugs are disappointingly ineffective. Many inhibitors of EGFR, Ras, Raf and MEK have been developed that target different components of ERK signaling, with a handful of agents already approved and added to our expanding repertoire of anticancer agents. However, many questions and issues remain, and whether inhibitors of ERK signaling will provide drugs that significantly advance cancer treatment is far from certain. As these efforts continue, in parallel, research efforts have also revealed a considerably greater complexity to the once simple linear Raf–MEK–ERK signaling cascade. These complexities suggest that targeting this pathway will not be as straightforward as once imagined. It has become evident that cancer cell resistance will limit the effectiveness of target-based signal-transduction inhibitors as it has with conventional cytotoxic anticancer drugs. Therefore, developing mechanistically distinct inhibitors of the same targets will be essential. Additionally, as the clinical analyses of these inhibitors progress, in parallel, we have also developed a greater appreciation of the complex biology and genetics of the cancer cell, and a better understanding of how clinical trial design can be improved. Hence, our learning curve in the development of target-based anticancer drugs is steep and we are in the very early part of that curve. Despite the continued twists and turns and bumps in the path of drug discovery, optimism remains cautiously high that we are still heading in the right direction. Finally, as reflected in current efforts, cell surface available molecules and protein kinases continue to be the most favored targets for anticancer drug discovery. Perhaps an important emphasis for future MAPK drug discovery should be to consider targets that are not classically considered attractive and ‘druggable’ targets (Overington et al., 2006), yet are clearly important modulators of MAPK function. Acknowledgements We thank Misha Rand for assistance in figure and manuscript preparation, and Robert Campbell for helpful discussions. We apologize to all colleagues whose work could not be cited due to space limitations. Research in the authors’ laboratory was supported by grants from the National Institutes of Health (to CJD). References Abi-Habib RJ, Urieto JO, Liu S, Leppla SH, Duesbery NS, Frankel AE. (2005). 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