Development of Rationally Designed, Target-Based Agents for the Treatment of Advanced Colorectal Cancer

Development of Rationally Designed, Target-Based Agents for the Treatment of Advanced Colorectal Cancer

Translational Medicine Development of Rationally Designed, Target-Based Agents for the Treatment of Advanced Colorectal Cancer Alain C. Mita, Monica...

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Translational

Medicine

Development of Rationally Designed, Target-Based Agents for the Treatment of Advanced Colorectal Cancer Alain C. Mita, Monica M. Mita, Eric K. Rowinsky Abstract Although there have been several recent additions to the conventional armamentarium used to treat patients with advanced colorectal cancer, principally as a result of the development of selective and nonselective pharmacologic agents and antibodies, the general outcome of patients with advanced disease is still poor. However, a greater understanding of cancer biology, as well as major advances in biotechnology, is beginning to identify and characterize molecular aberrations that are common in patients with colorectal cancer.These advances have resulted in the development of a wide range of rationally designed, target-based anticancer therapeutic agents, which, by virtue of their selectivity, would be expected to produce less nonspecific toxicity and therefore higher therapeutic indices compared with nonspecific cytotoxic agents. This review will discuss several novel targets and therapeutic agents, particularly those designed to interrupt aberrant signal transduction and apoptotic processes. It will also emphasize the complexity of these systems and the need to incorporate novel clinical development paradigms based on a thorough scientific understanding of these targets. Clinical Colorectal Cancer, Vol. 4, No. 2, 107-123, 2004 Key words: Apoptosis, ErbB, Mitogen-activated protein kinase, Ras, Signal transduction inhibitors, TRAIL receptors

Introduction Colorectal cancer (CRC) is the second leading cause of cancer death in developed countries.1 It is clear that the outcome of patients with CRC has improved in recent years, largely because of the optimization of surgical techniques, use of multimodal approaches in some clinical settings, and incorporation of nonselective cytotoxic chemotherapeutic agents such as the fluoropyrimidines in the adjuvant setting. Except for rare highly selective surgical and/or combined surgical/chemotherapeutic approaches, however, the probability of achieving long-term survival is low in patients with metastatic disease. Until recently, 5-fluorouracil (5FU), used as a single agent or in combination with leucovorin (LV), was the only reasonable systemic therapeutic option for patients with advanced disease. During the past few years, however, several nonspecific cytotoxic agents, including irinotecan, oxaliplatin, capecitabine, raltitrexed, and uracil/tegafur, have demonstrated consistent, albeit low-level, antitumor activity and increInstitute for Drug Development, Cancer Therapy and Research Center, University of Texas Health Science Center at San Antonio Submitted: Dec 24, 2003; Revised: Apr 7, 2004; Accepted: Apr 9, 2004 Address for correspondence: Alain C. Mita, MD, Institute for Drug Development, Cancer Therapy and Research Center, 7979 Wurzbach Rd, 4th Floor Zeller Building, San Antonio, TX 78229 Fax: 210-692-7502; e-mail: [email protected]

mental benefit.2-6 Combination regimens consisting of these drugs have often resulted in higher response rates and longer progression-free and overall survival times.7-9 Despite the encouraging results, however, the overall survival of patients with advanced disease remains poor, and there is a critical need to develop more effective therapies for patients with CRC. Recently, a large number of molecular and genetic aberrations that occur during the transition from normal colonic epithelium to a colorectal adenocarcinoma have been characterized, and several of these aberrations represent potential targets for therapy. Included in these targets are those involving strategic facets of growth signal transduction, malignant angiogenesis, survival, metastasis, and cell cycle regulation. The discovery of new subcellular targets and the development of target-based agents have opened an era of new opportunities along with extraordinary developmental challenges. Because these agents may target inherent abnormalities of cancer cells, they may produce less toxicity at effective doses compared with more traditional nonselective cytotoxic agents, which would make them more suitable for chronic, uninterrupted administration. However, based on preclinical and early clinical studies, the most common antitumor effect noted after interruption of these processes is a decreased rate of tumor growth, which may not be readily detected or quantified with traditional methodologies used to evaluate clinical benefit in early nonrandomized studies.10 Therefore, regulatory and clinical practice endpoints such as in-

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Targeted Therapy Agents for Colorectal Cancer Figure 1

Schematic Representation of Critical Signal Transduction Pathways Targeted with Therapeutic Strategies Including Small Molecules, Antibodies, and Antisense Oligonucleotides Antiligand Therapeutics Receptor Tyrosine Kinase Inhibitors

PTEN

PI-3 SH2 P Kinase

PLC-g

PI3K Akt

P

P SH2

SH3

P

p70s6k

mTOR Inhibitors

SH3

Grb2

TK

PI3K Inhibitors ERK Akt Inhibitors 4E-BP1 Inhibitors

mTOR

Activation of Antiapoptotic Proteins

SH2

Inhibitors of Ras Farnesylation

Antireceptor Antibodies

Transcription Factors

Ras

SOS GTP Raf

Raf kinase Inhibitors

Erk

Ras GDP

Ras Inhibitors

MAPK MAPK Inhibitors Mitosis, Gene Expression, Anabolism, Growth, Angiogenesis

Abbreviations: 4E-BP1 = 4E-binding protein-1; ERK = extracellular signal–regulated kinase; GDP = guanosine diphosphate; GTP = guanosine triphosphate; MAPK = mitogenactivated protein kinase; mTOR = mammalian target of rapamycin; PI-3 = phosphatidylinositol 3-kinase; PLC = phospholipase C; SOS = son of sevenless

creased time to progression, reduced disease-related symptoms, and improved quality of life, which are generally considered secondary endpoints in the development of nonselective cytotoxic agents, may evolve into primary endpoints for newer classes of agents if they are associated with substantially less toxicity, similar to endpoints used for the development of agents in other therapeutic areas. Selecting an optimal dose for targeted therapies is especially challenging, as objective antitumor activity is generally near its maximum above a “threshold” level, whereas toxicity generally increases with increasing dose. Therefore, the development of biologic assays or biomarkers that reflect target activity or surrogates that reflect relevant clinical endpoints would facilitate optimal dose selection for clinical trials. Finally, prospective identification of patients whose tumors have functional target aberrations could increase the chances of selecting the patients who are most likely to benefit from these highly selective therapies.

Proliferative Signal Transduction Elements as Therapeutic Targets The broad term “signal transduction” refers to the means by which regulatory molecules that govern the fundamental processes of cell growth, differentiation, and survival (eg, extracellular hormones, growth factors, cytokines, specialized proteins) communicate and induce responses within cells, resulting in the tight coordination of proliferative and other essential processes in various tissues. Cell signaling is extremely complex, with a wide array of components interacting through cascades of chemical signals arranged in overlapping networks.11,12 These networks, consisting of parallel tracks and intricate interconnections, enhance the robustness and diversity of signaling and permit fine tuning, amplification, and diminution of output, which may not be accomplished as efficiently by simpler linear cascades. However, the in-

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herent redundancy and complexity of networks also confer protection against cytotoxins, thereby decreasing the likelihood that any therapeutic manipulation against a single element will be highly successful unless the element is a significant contributor to the tumor’s proliferative advantage. Targeting aberrant and/or overactive proliferative cell signaling elements is perhaps the most important ongoing developmental therapeutic endeavor against cancer, as aberrations in signal transduction processes have been consistently demonstrated to enhance proliferation, invasiveness, metastasis, and angiogenesis, and to confer shortened survival and poor response to nonspecific cytotoxic modalities.11-13 In addition, the development of therapeutic agents against such processes is projected to yield broadly generalizable results because most malignancies, including gastrointestinal cancers, have at least one aberrant signaling element that confers a proliferative or survival advantage.11-13 However, the most common aberrations are those involving loss of protein function, which must be approached differently. Nevertheless, less common gain-of-function aberrations result in unchecked, autonomous, or constitutive activity of elements that normally regulate cell signaling.11,12,14,15 In contrast to the situations represented by chronic myelogenous leukemia and gastrointestinal stromal cell tumors, in which single aberrations such as a bcr-abl translocation or c-kit mutation are the principal drivers of tumor proliferation and successful targeting results in profound cytoreductive effects, most malignancies possess multiple aberrations, several of which confer a proliferative advantage.14,16,17 However, targeting any single specific “driver” in a tumor that has multiple relevant aberrations may result in therapeutic efficacy, the magnitude of which relates to the importance of the driver itself and its contribution to the tumor’s proliferative and/or survival advantage. Nevertheless, even if the overall efficacy achieved by targeting only one of many drivers may be somewhat limited, the innate importance of many types of signaling elements, several of which are shown in Figure 1, as well as the selectivity of their cognate therapeutic agents, may impart minimal toxicity and high therapeutic indices, rendering the agent attractive for clinical use.

Targeting the ErbB Receptor Family Most current efforts at developing therapeutic agents against signal transduction processes involve membrane receptors or elements that comprise downstream signaling cascades. With regard to signal transduction receptors, developmental efforts are predominantly being directed against receptor tyrosine kinases (RTKs) and G-protein–coupled receptors (GPCRs), which have secondary relay systems that permit signal amplification, diversification, and crosstalk.11,12,15 The complexity of signal transduction networks and the challenges related to the development of therapeutic agents against these intricate systems are exemplified by the complex structural and functional aspects of the ErbB receptor family and related downstream processes, as well as the multifactorial determinants of each specific signal. The overexpression and/or constitutive activation of ErbB receptors favor cell proliferation, invasiveness, angiogenesis, and resistance to chemotherapy and radiation therapy.11,12,15 Members of the ErbB receptor family include ErbB1 (also called EGFR or HER1), ErbB2

Alain C. Mita et al (HER2/neu), ErbB3 (HER3), and ErbB4 (HER4),18 which are commonly overexpressed, overactive, or aberrant in colorectal and other epidermoid gastrointestinal malignancies. The most relevant ErbB with regard to therapeutic targeting is ErbB1. The ErbB1 gene encodes a 170-kd membrane-spanning glycoprotein expressed on the cell surface of many normal tissues. Aberrant ErbB1 activation can result from gene amplification, increased transcription, excess ligand, or receptor mutations that cause dysregulated signaling in the absence of ligand. ErbB1 expression or overexpression has been documented in 25%-77% of human colon cancers,19,20 and the percentage of EGFR-expressing cells has been inversely related to prognosis.20 In addition to the extracellular domain of the ErbB that binds to growth factors and serves as a target for therapeutic antibodies, the ErbB receptor includes a transmembrane domain and intracellular portions that consist of a RTK domain and a domain that regulates RTK activity (Figure 2).11,12,15,21 Ligand binding induces conformational changes in the receptor, which in turn activates the RTK, thereby facilitating dimerization with other ErbB receptors.11,12,15,21-24 After dimerization, conformational changes result in phosphorylation or activation of specific tyrosine residues. Activated ErbB receptors in turn phosphorylate or activate specific downstream signaling elements, thereby transducing mitogenic and other types of signals in the cell. The specificity, potency, and, in essence, the diversity of intracellular signals are determined in part by the effectors of ErbB, as well as by the identity of the ligand, dimer, and specific structural determinants of the receptors. However, the principal determinant is the vast array of phosphotyrosine-binding (PTB) proteins that associate with the C-terminal domain of each ErbB receptor.11,12,15,21-24 The critical sequences of the C-terminal domains, which contain tyrosine residues that undergo phosphorylation, represent docking sites for various proteins involved in signal transduction. Docking sites are provided for proteins that recognize specific phosphotyrosine residues in the context of their surrounding amino acids. Each ErbB receptor displays a distinct pattern of C-terminal autophosphorylation sites. At least for ErbB2, which does not have a direct activating ligand, PTB sites are essential for the transforming properties of the receptor. It is now evident that there is a great deal of overlap in the signaling pathways activated by the 4 ErbB receptors. For example, the mitogen-activated protein kinase (MAPK) signaling pathway is an invariable target of all ErbB family members. There are also many examples of preferential modulation of specific pathways, such as the presence of multiple binding sites for the regulatory subunit of phosphatidylinositol-3 kinase (PI3K) on ErbB3 and ErbB4, rendering these receptors the most efficient activators of the PI3K “cell survival” pathway.21-24 Signals arising from the simultaneous activation of linear cascades, including the MAPK, stress-activated protein kinase cascade (SAPK), protein kinase C, and PI3K pathways, are integrated in the nucleus into distinct transcriptional programs, the culmination of which is the net cellular response.

Therapeutics Targeting ErbB1 and Other ErbB Family Members Several therapeutic approaches aimed at inhibiting ErbB function are being pursued. To date, most efforts have been

Figure 2

Structure of the ErbB Family Receptors and Their Cognate Ligands

EGF TGF-a Amphiregulin Betacellulin Heparin-Binding EGF Epiregulin

No Known Ligands

Heparin Binding EGF Neuregulins 1-4 Heregulins Betacellulin Epiregulin

Heregulins Neuregulins 1-4 Epiregulin

CR1

LigandBinding Domain

CR2 Extracellular Ligand-Binding Domain

Extracellular

Extracellular Tyrosine Kinase Domain

Intracellular

ErbB-1 HER1 EGFR

ErbB-2 HER2/neu

ErbB-3 HER3

ErbB-4 HER4

The receptor consists of 3 domains: a ligand-binding extacellular domain containing 2 cysteine-rich regions (CR1 and CR2), a transmembrane domain, and an intracellular domain containing a tyrosine kinase region. Abbreviations: EGF = epithelial growth factor; EGFR = epithelial growth factor receptor; TGF = transforming growth factor

directed toward ErbB1 (EGFR) and ErbB2, but therapeutic agents targeting all members of the ErbB receptor family are also being evaluated.

Anti-EGFR Antibodies Based on the clinical success of monoclonal antibodies (MoAbs) targeting ErbB2, other MoAbs directed against the EGFR are being evaluated. These antibodies block ligand binding to the EGFR and inhibit EGF-stimulated RTK activity.25-29 The murine antibodies used in the early studies have been progressively replaced by chimeric or partially humanized MoAbs that retain only a small portion of the murine protein sequences responsible for antigen binding, with the remainder of the molecule composed of the human immunoglobulin. Among the most promising of this new generation of MoAbs are the chimeric cetuximab and matuzumab (EMD-72000), the totally humanized panitumumab (ABX-EGF), and the bispecific MDX-214. Cetuximab received regulatory approval in the United States for previously treated advanced CRC in February 2004. The aforementioned antibodies against EGFR have been shown to inhibit the growth of EGFR-expressing human tumor xenografts of colorectal, pancreatic, prostate, renal, and breast origin, and they have also been demonstrated to induce profound apoptosis in some models.26,2830 There is also experimental and circumstantial clinical evidence that MoAbs to EGFR enhance the cytotoxic effects of many types of nonselective chemotherapeutic agents and ionizing radiation.27,31-34 Because DNA alteration caused by many cytotoxic

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Targeted Therapy Agents for Colorectal Cancer agents and radiation results in cell cycle arrest in G1, in which cells repair damage, followed by apoptosis if DNA repair does not occur, growth factor restriction combined with DNA-damaging agents may preferentially enhance apoptosis in tumor cells.35 The inhibition of the EGFR may also abrogate the downstream signals driven by the MAPK and the PI3K cell survival pathway, thereby sensitizing cells to radiation and cytotoxic drugs. Therefore, several anti-EGFR MoAbs are currently under evaluation in combination with various chemotherapy regimens.

Cetuximab Cetuximab is a chimeric MoAb with a binding affinity 10 times greater than that of the natural EGFR ligand. Although cetuximab induces receptor dimerization, EGF-induced activation, autophosphorylation, and receptor internalization are blocked.18 Cetuximab was reported to be active as a single agent in patients with CRC refractory to treatment with irinotecan.36 All tumors expressed EGFR as assessed by immunohistochemistry. In an initial single-agent study, cetuximab was administered as a 400-mg/m2 intravenous (I.V.) loading dose followed by 250 mg/m2 I.V. weekly. Six of 57 patients (11%) had a partial response (PR), and 13 patients had stable disease (SD) as their best response. The principal side effects included an acneiform skin rash in 86% of patients, which was severe (grade 3) in 16%, and asthenia in 53% of patients, which was severe in 7%. The combination of cetuximab and irinotecan demonstrated impressive antitumor activity in patients with EGFR-expressing metastatic CRC whose tumors had failed to respond to previous treatment with irinotecan.37 The preliminary report indicated that 21 of 121 patients (17%) had a PR and 37 patients (31%) had SD as their best response. In addition, the combination of cetuximab and irinotecan did not appear to result in a higher rate of toxicity than irinotecan as a single agent. Similar results were reported in a European randomized phase II trial in which patients with metastatic EGFR-expressing CRC that progressed during treatment with irinotecan were randomized to treatment with cetuximab alone or cetuximab/irinotecan.38 The overall response rate and median time to progression were 17.9% and 126 days, respectively, in patients treated with the cetuximab/irinotecan regimen and 9.9% and 45 days, respectively, in patients treated with cetuximab alone. These impressive preliminary results have led to a variety of feasibility and efficacy studies of cetuximab-based combination regimens in untreated CRC. In a phase II trial of cetuximab plus irinotecan 125 mg/m2 I.V., 5-FU 500 mg/m2 I.V., and LV 20 mg/m2 I.V., with all agents administered weekly, 11 of 25 previously untreated patients (44%) with metastatic disease experienced PRs, and 5 additional patients had minor responses.39 The most common side effects were diarrhea (severe in 33% of patients) and neutropenia (severe in 33% of patients). Because the doses of irinotecan and/or 5-FU had to be reduced in 89% of patients, slightly lower doses (irinotecan 100 mg/m2, 5-FU 400 mg/m2, and LV 20 mg/m2 each week) were recommended for subsequent evaluations. In another study of cetuximab combined with irinotecan 80 mg/m2 per week I.V., 5-FU 1500-2000 mg/m2 I.V. over 24 hours, and LV 500 mg/m2, 5 of the first 13 patients (38%) experienced PR.40 In addition, toxicity was quite acceptable, with

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only 1 patient experiencing grade 3 diarrhea and 2 patients developing grade 3-4 skin toxicity. Finally, a European trial assessing the feasibility of cetuximab in combination with the FOLFIRI regimen (biweekly irinotecan, infusional 5-FU, and LV), 8 of the first 18 patients (44%) experienced PRs, and toxicity was also reported to be acceptable.41 Based on these encouraging results, several randomized phase III trials evaluating the benefit of adding cetuximab to conventional oxaliplatin- or irinotecan-based multiagent regimens have begun. Although antitumor activity of cetuximab and of other EGFRtargeting agents in CRC and other tumor types does not appear to relate to the magnitude of EGFR expression as determined immunohistochemically,36,37,42,43 the preponderance of preliminary clinical data from studies involving patients with CRC and other tumor types indicate that drug-induced cutaneous toxicity may be related to clinical benefit.44,45 However, it will be important to confirm these results in prospective evaluations in which the doses of cetuximab and other EGFR-targeted therapeutic agents are titrated to various grades of cutaneous toxicity and clinical benefit is assessed in patients who have experienced rash of various grades.

Matuzumab Matuzumab, a humanized MoAb directed at the EGFR, is undergoing early clinical development. An initial phase I pharmacokinetic and pharmacodynamic study explored a weekly schedule of matuzumab administered as a 1-hour I.V. infusion at fixed dose in patients with EGFR-expressing tumors.46 The maximum tolerated dose was determined to be 1600 mg per week, with further dose escalation precluded by severe headache and fever. Thirteen of the 22 evaluable patients (59%) developed an acneiform rash of mild to moderate severity. As a result of the relatively long half-life of the antibody, the feasibility of administering matuzumab on less frequent dosing schedules (eg, every 2 or 3 weeks) at a fixed dose of 1200 mg is being evaluated.47 To date, severe toxicities have not occurred. In contrast, antitumor activity has been noted with matuzumab on all 3 schedules. In the phase I study, 2 PRs and 1 minor response were reported in the 15 patients with CRC enrolled. Correlative biologic studies demonstrated that treatment with matuzumab completely inhibited phosphorylated (activated) ErbB1 as well as several critical signaling elements of the MAPK pathway in skin biopsy specimens tested during treatment on all 3 schedules. The mean trough matuzumab concentrations on the every-3-week schedule was 10 times higher than required to maximally inhibit cancer growth in vitro, which seems to support this convenient treatment schedule for further studies.

Panitumumab Panitumumab is a fully human immunoglobulin (Ig) G2 MoAb with high affinity for the EGFR (5 × 10–10 mol/L). The principal toxicity observed in phase I/II trials, a mild to moderate acneiform skin rash, is similar to that observed with cetuximab and matuzumab.48,49 In contrast to cetuximab treatment, which requires premedication with corticosteroids because of hypersensitivity reactions related to the murine components of the MoAb, hypersensitivity reactions have not been reported

Alain C. Mita et al with panitumumab and premedication is not required. Major antitumor activity has been reported in patients with advanced CRC and other types of advanced and drug-refractory malignancies in phase I/II studies.48 The preliminary results of a multicenter phase II study of panitumumab 2.5 mg/kg I.V. weekly in patients with progressive CRC after treatment with fluoropyrimidines and irinotecan with or without oxaliplatin have been reported.49 Major antitumor responses and disease stabilization were noted in 13% and 39% of patients, respectively. The feasibility of administering panitumumab on a less frequent schedule (eg, every 2 weeks) and in combination with common first-line regimens used to treat patients with CRC is being studied.

MDX-214 In contrast to the aforementioned MoAbs against EGFR, MDX-214 is a bifunctional antibody that consists of human EGF fused to a Fab´ antibody fragment that is specific for the human receptor for the Fc portion of human IgA (FcαR1, CD89). CD89 is expressed on the surface of myeloid cells, and its cross-linking has been shown to trigger immune processes including phagocytosis, superoxide generation, and antibodydependent cellular cytotoxicity. Therefore, it has been proposed that MDX-214 could stimulate an immune-mediated antitumor effect that could potentially compound the intrinsic tumor growth–inhibitory effects induced by the EGFR blockade. In the presence of CD89-expressing immune effector cells, MDX214 has been demonstrated to induce CD89-mediated cytotoxicity of EGFR-overexpressing cell lines in a dose-dependent manner.50 The principal toxicities of MDX-214 in animal studies were mild to moderate emesis, decreased activity, and hypotension. Early clinical evaluations are in progress.

Small-Molecule EGFR Tyrosine Kinase Inhibitors Another approach to blocking EGFR and downstream signaling involves the use of small-molecule EGFR–tyrosine kinase (TK) inhibitors. Inhibition of EGFR TK prevents receptor autophosphorylation and activation of downstream pathways, particularly MAPK and PI3K. This approach may inhibit signaling induced by EGF and TGF-α, as well as signaling that is independent of growth factors such as deletions/mutations resulting in constitutively active EGFR.51,52 Several small-molecule inhibitors of EGFR TK have demonstrated significant antitumor activity in preclinical studies, blocking the proliferation of EGFR-expressing cancer cells in vitro and the growth of well-established EGFR-expressing human tumor xenografts. Most of these small molecules are quinazolines that competitively and reversibly bind to the adenosine triphosphate–binding site in the TK domain of the ErbB receptors, inhibiting the TK activity of ErbB1 (eg, gefitinib, erlotinib) or multiple ErbB receptor subfamilies (eg, lapatinib [GW572016] inhibits RTK of ErbB1 and ErbB2).15,53-56 Other small molecules form irreversible covalent linkages with cysteine residues in the receptor TK domains of several types of ErbB receptors. For example, canertinib (CI-1033) is an irreversible inhibitor of all 4 ErbB family members, whereas EKB-569 irreversibly inhibits the TK

activity of ErbB1 and ErbB2.15,53-56 The relative therapeutic merits of antibodies versus small-molecule inhibitors of a single ErbB TK versus multiple RTKs, and those of reversible receptor binding versus irreversible receptor binding, are not known, but antibodies do result in rapid receptor internalization and degradation, and antitumor activity has not been observed with the small molecules in CRC to the extent that it has been observed with the MoAbs to ErbB1. Similar to the ErbB-targeting antibodies, an acneiform rash is the principal toxicity of most of these pharmacologic agents. However, unlike the MoAbs to EGFR, diarrhea is also a predominant dose-related toxicity of the small-molecule EGFR-TK inhibitors. Interestingly, disease-directed evaluations of the small-molecule TK inhibitors erlotinib and gefitinib as single agents in patients with advanced CRC have been disappointing to date. Two phase II trials of single-agent gefitinb in heavily pretreated CRC have failed to demonstrate antitumor activity57 (Chris Takimoto, MD, personal communication, September 2003). In these studies, patients were treated with gefitinib on an uninterrupted oral daily schedule at doses of 750 mg or 500 mg. Although 4 patients demonstrated minor tumor regression on radiographic scanning, neither PRs nor evidence of prolonged SD were noted in either study, which enrolled a combined total of 45 patients. Ongoing biologic studies will hopefully provide clues to explain these results, particularly relative to the apparent activity of the MoAbs. Of particular note, however, are the intriguing preliminary results reported in patients with advanced CRC treated with the combination of gefitinib 500 mg per day combined with oxaliplatin, 5-FU, and LV (ie, the FOLFOX4 regimen) in metastatic CRC.58 In this pilot study of this regimen, 75% of patients with advanced CRC who had not yet received chemotherapy in the metastatic setting had objective responses, whereas 23% of previously treated patients had responses. Severe diarrhea, nausea, and neutropenia were experienced by 34%, 31%, and 28% of patients, respectively. Similar to the experience with gefitinib, objective responses were not observed in 25 patients with previously treated metastatic CRC in a phase II study of erlotinib 150 mg per day; 8 patients (32%) experienced SD as their best response.59 However, the preliminary results of a feasibility study of erlotinib administered at daily doses as high as 150 mg combined with the FOLFOX4 regimen in patients with metastatic CRC are encouraging.60 Although most patients received previous treatment for metastatic disease, 5 of 15 patients (33%) experienced PRs and 6 patients had SD as their best response. In addition, the toxicity noted with the regimen was considered acceptable, with skin rash and diarrhea noted as principal toxicities, and there were no major pharmacokinetic interactions. In another feasibility study of an erlotinib combination with capecitabine, 2 of 8 patients (25%) with metastatic CRC who had previously been treated with 5-FU experienced PRs.61 Phase I studies of erlotinib in combination with a variety of other chemotherapy regimens that are relevant in this setting are ongoing. Although the preliminary results of studies of small-molecule EGFR-TK inhibitors as single agents in patients with metastatic CRC have not been encouraging, caution should be exercised in in-

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Targeted Therapy Agents for Colorectal Cancer terpreting the results of preliminary studies because of limitations in their size and design that limit their statistical power. Because the requirements for antitumor activity are much more stringent for targeted therapeutic agents compared with nonspecific cytotoxic agents, the statistical power of traditional 2-stage phase II studies, particularly those that involve negligible screening for the target and/or lack a rational enrichment strategy, is likely insufficient to detect a relevant degree of antitumor activity. Most of the aforementioned nonrandomized clinical evaluations with EGFR inhibitors were not designed to robustly assess the degree to which these agents may affect clinically relevant endpoints that may reflect tumor growth inhibition (ie, time to tumor progression and survival). Additionally, it is plausible that the real benefit of some targeted therapeutic agents will be evident only when they are combined with cytotoxic agents or other targeted therapeutic agents. In addition, it will be important to discern which patients have the greatest likelihood of responding to these therapies based on the biologic features of their tumors and the optimal means to develop combination regimens with these agents.

Targeting the MAPK Pathway Following ligand-receptor binding, receptor activation, and activation of membrane-bound members of the Ras family of small GPCRs, proliferative signals are relayed downstream to intracellular signaling elements along many pathways, including the growth-stimulatory MAPK pathway. Ras is a potent, efficient, and prominent activator of the Raf family of kinases, which in turn trigger MAPK kinase (MEK)/extracellular signal–regulated kinase (ERK1/ERK2).62-64 Likewise, Ras can directly relay survival signals via activation of the PI3K cell survival or antiapoptotic pathway.63,64 Ras activation through the MAPK pathway modulates the activity of nuclear transcription factors such as Fos, Jun, and AP-1, which in turn regulate the transcription of genes required for proliferation.65-68 MAPK is a convergence point for a broad array of signals from membrane receptors, and the network of phosphorylation-mediated signals emanating from MAPKs is equally expansive. The 3tiered MAPK kinase module, the Raf1-MEK-ERK module, is employed ubiquitously in the transduction of cell type–specific growth and differentiation signals from RTKs and GPCRs.65-68 Signaling through MAPK mediates inflammatory and stress responses to stimuli such as cytokines, FasL, and tumor necrosis factor (TNF), whereas the SAPK pathway transduces these signals and is involved with growth, differentiation, and cellular stress induced by oxidation and DNA damage. The MAPK pathway does not function as an isolated linear cascade, but is instead integrated into other cellular signaling networks that impact MAPK signaling. The importance of the MAPK signaling pathway in almost all aspects of cellular growth and anabolism is highlighted by the profound pathologic consequences incurred by minimal perturbations in MAPK signaling. Therefore, therapeutic efforts are currently being directed at several components of the MAPK pathway.

Targeting Ras Ras proteins are an extended family of G proteins, which are involved in protein synthesis and signal transduction69 Three ras

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proto-oncogenes have been identified: H-ras, K-ras, and N-ras, which encode four 21-kd proteins called p21ras or Ras (H-Ras, N-Ras, K-Ras4A, and K-Ras4B).70,71 Ras is synthesized as an inactive cytosolic peptide and undergoes a series of post-translational modifications at the C-terminus, which increase its hydrophobicity and facilitate association with the cell membrane.69,72 The first and most critical step of these modifications is farnesylation, catalyzed by the farnesyltransferase (FTase). Ras functions as a chemical switch, cycling between inactive guanosine diphosphate–bound and active guanosine triphosphate (GTP)–bound states. Upon activation, Ras transmits proliferative signals from cell surface receptors (including the ErbB family and hormone and cytokine receptors) to the cytoplasm and nucleus, initiating a cascade of protein kinases that ultimately regulate nuclear, cytoskeletal, and cytoplasmic processes. In its GTP-bound state, Ras activates several downstream effector pathways, particularly the MAPK pathway, through the Raf-1 serine-threonine kinase. There are other multiple branch points in the Ras pathway, such as the small GTP-binding proteins Rac and Rho, PI3K, and downstream elements in the MAPK pathway.73,74 Mutations of ras occur in approximately 30% of all human cancers, including approximately 50% of CRCs.75 Most mutationally activated forms of ras in tumors result in disrupted guanine nucleotide regulation and constitutive activation of Ras.76 Therefore, novel therapies targeting ras, including FTase inhibitors (FTI) and ras antisense oligonucleotides (ASONs), emerged as promising novel approaches in the treatment of advanced CRC.

Farnesyltransferase Inhibitors Strategies that are capable of blocking FTase and preventing farnesylation were initially pursued as a means of inhibiting the maturation of Ras into a biologically active molecule, thus turning off signal transduction. Most of the efforts targeting FTase have sought to selectively develop inhibitors that are > 1000 times more potent at inhibiting FTase than geranylgeranyl transferase (GGTase) I or II. Various FTIs including tipifarnib (R115777; Zarnestra®), lonafarnib (SCH66336), and BMS214662 are undergoing broad clinical evaluations. Nevertheless, the results of disease-directed phase II/III studies in CRC have been disappointing. The results from a randomized, double-blind, placebo-controlled trial of tipifarnib 300 mg twice daily for 21 days every 4 weeks in patients with advanced, refractory CRC have been presented.77 The study was designed to detect a 50% improvement in overall survival and accrued a total of 368 patients who had received ≥ 2 previous chemotherapy regimens for metastatic disease. The study, in which patients were randomized in a 2:1 fashion to treatment or placebo, failed to demonstrate an effect of tipifarnib treatment on survival. However, the study design may have overestimated the impact of the drug on survival in this unselected, heterogeneous, and very heavily pretreated population, with some patients having received as many as 8 previous treatment regimens. Therefore, it is still possible that tipifarnib and other FTase inhibitors may confer clinical benefit to highly selected or enriched patient populations whose tumors have the optimal determinants of drug sensitivity. However, the results of a Southwest Oncology Group phase II trial of tipifarnib administered on an identical schedule as first-line treatment in patients with

Alain C. Mita et al metastatic CRC were also disappointing.78 The endpoints of the trial included response rate, time to treatment failure, survival, and toxicity. Of 51 evaluable patients, 1 confirmed and 2 unconfirmed PRs were noted, and the median time to treatment failure was 1.7 months. A possible explanation for the disappointing clinical results with FTase inhibitors in patients with CRC is the predominance of K-ras mutations in this malignancy. Similar disappointing results have been observed in a phase III study of tipifarnib and gemcitabine versus gemcitabine alone in previously untreated patients with advanced pancreatic cancer, in which K-ras mutations also predominate.79 Although tumors with K-ras mutations have been demonstrated to be relatively sensitive to FTase inhibitors in preclinical studies, their sensitivity is substantially less than that of tumors with H-ras mutations.80 The most likely explanation for these observations is the fact that K-Ras can be alternatively prenylated by other prenyl transferases, particularly GGTases.81

Antisense Oligonucleotides of Ras Although most efforts aimed at blocking the activation of mutant Ras have focused on inhibiting FTase, ASONs that block Ras function have also been designed and evaluated. Antisense oligonucleotides hybridize to the complementary sequence present on the target messenger RNA (mRNA), followed by RNase Hmediated degradation of mRNA. A principal advantage of this approach is the specificity of the ASON to block the expression of mutated forms of Ras. Unfortunately, the therapeutic use of these compounds was initially limited, as earlier ASONs were rapidly degraded by intracellular nucleases. Recently, chemical modifications of the oligonucleotide backbone by replacement of one oxygen atom of each phosphorus moiety by sulphur (phosphorothioates), a methyl group (methylphosphonates), or amines (phosphoramidates) have overcome this hurdle. However, toxicities caused by the modality itself rather than the specific antisense target, including severe immune reactions, have limited the clinical development of ASONs.82 Despite these limitations, some phosphorothioate oligonucleotides have undergone clinical evaluations. For example, the 20-mer oligonucleotide ISIS 2503, which binds to the translation-initiation region of human H-ras mRNA, has entered early clinical trials, including evaluations of its role in the first-line treatment for patients with advanced CRC.83 ISIS 2503 was well tolerated when administered at 6 mg/kg per day by 14-day continuous I.V. infusion repeated every 3 weeks. No antitumor responses were reported, but 2 of 12 patients experienced SD as their best response. Because most CRCs harbor K-ras and not H-ras, alternative ASONs against K-Ras would likely be more attractive and are currently under evaluation.84

Targeting Raf Because the Raf family of signaling elements is immediately downstream of Ras, and the first committed step in the MAPK pathway, and raf mutations are associated with proliferative and transforming properties, Raf has become an important target for therapeutic development. The Raf family is composed of 3 related serine/threonine protein kinases, Raf-1, A-Raf, and B-Raf, which act in part as downstream effectors of Ras signaling. Raf-1, the protein product of the c-raf proto-oncogene, is

ubiquitous; whereas B-Raf is found mainly in neural tissue, and A-Raf is most abundant in urogenital organs, including the ovary, kidney, testes, and prostate. B-Raf and A-Raf, like Raf-1, are Ras effectors, but the specificity of their activity is not well understood. Raf mutations have also been recently described. B-raf mutations that confer elevated kinase activity and transforming properties occur in 66% of malignant melanomas and at a lower frequency in other types of human cancers.85 In addition, Raf may play a broader role in tumorigenesis, as it can be activated independently of Ras by protein kinase C-α and promotes the expression of the multidrug resistance gene.86-88 Activated Ras interacts directly with the aminoterminal regulatory domain of the Raf kinase, resulting in a cascade of reactions such as the direct activation of MEK. The serine/threonine kinase, Raf-1, the best-characterized downstream effector of Ras, is activated in a number of steps, including phosphorylation, recruitment to the plasma membrane, and binding to activated Ras.65-68 Additional steps, including interactions with other proteins, are required, although these steps are less well defined. After activation, Raf-1 in turn activates MEK through phosphorylation of 2 serine moieties.86-88 Because Raf kinase is the first committed step in the MAPK pathway, it is an attractive target for therapeutic development, and its successful inhibition may block signals from a diverse array of growth stimuli. In addition, there is a large body of experimental data demonstrating that the inhibition of Raf kinase can reverse the phenotype of Ras-transformed cells and block tumor growth. Moreover, decreased tumorigenicity has been demonstrated in cell lines in which the activation of MEK is disrupted as a result of a variety of mutations. Several approaches to targeting Raf, including the use of ASONs and small molecules, are being evaluated.

Antisense Oligonucleotides to Raf ISIS 5132 (CGP 69846A) is a 20-base phosphorothioate ASON that was designed to hybridize to the 3' untranslated sequences of the c-raf-1 gene.89-90 The binding of ISIS 5132 to Raf-1 mRNA promotes RNAase H–mediated mRNA degradation and reduces Raf-1 protein synthesis in a nucleotide sequence–specific and concentration-dependent fashion. ISIS-5132 has been shown to inhibit the expression of c-Raf mRNA, as well as the proliferation of ovarian, lung, colon, cervical, prostate, and colon carcinomas in preclinical studies.86,89,90 Acute toxicities include fever, fatigue, transient asymptomatic prolongation of activated partial thromboplastin time, and alternate complement activation, and have been ascribed to the phosphorothioate backbone. Disease-directed phase II evaluations of ISIS 5132 as a single agent and a component of multiple-agent regimens are being performed.86,88 The antitumor activities of ISIS 5132 and ISIS 3521, an ASON to protein kinase C-α, in patients with recurrent or refractory metastatic CRC were evaluated in a randomized phase II trial.91 Patients were treated with ISIS 5132 or ISIS 3521 at 2 mg/kg per day as a 21-day continuous I.V. infusion every 28 days. No major responses were reported among 32 evaluable patients, and 5 of 15 patients and 4 of 17 patients treated with ISIS 5132 and ISIS 3521, respectively, had SD as their best response. The investigators concluded that these ASONs possessed negligible utility as single agents on this schedule in patients with recurrent or refractory CRC.

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Targeted Therapy Agents for Colorectal Cancer Small-Molecule Raf Kinase Inhibitors Another approach to targeting Raf involves the use of smallmolecule inhibitors of Raf kinase. Sorafenib (BAY 43-9006), a small-molecule inhibitor belonging to a class of compounds defined as bis-aryl ureas that was designed to specifically target Raf kinase, inhibits the adenosine triphosphate–binding site of Raf kinase and is active in cancers that overexpress growth factor receptor and bear K-ras.86,92,93 Prominent antitumor activity with sorafenib in human colon, pancreatic, lung, and ovarian carcinomas in vitro has been reported.86,92,93 Sorafenib has demonstrated potent growth inhibitory activity against HCT 116 colon (mutant Kras), MIA PaCa-2 pancreatic (mutant K-ras), H460 non–smallcell lung (mutant K-ras), and SKOV-3 ovarian (wild-type ras) carcinomas. Sorafenib and irinotecan also demonstrated therapeutic synergism against a human DLD-1 colon xenograft, which suggests a rationale for the clinical evaluations of sorafenib-based combination regimens.94 In early clinical trials, stomatitis, vomiting, diarrhea, skin rash (erythema, folliculitis, dry skin, desquamation), and hand-foot syndrome have been reported; whereas moderate lymphopenia and anemia have been the most common hematologic toxicities.86 Significant inhibition of ERK phosphorylation in peripheral blood mononuclear cells was demonstrated at sorafenib doses > 200 mg twice daily on an uninterrupted oral schedule. The recommended dose for disease-directed evaluations is 400 mg twice daily. In phase I evaluations, antitumor activity was reported in patients with a wide range of tumor types, including colorectal, hepatocelluar, and renal carcinomas, and broad diseasedirected evaluations are ongoing.86 An innovative multicenter, placebo-controlled randomized discontinuation phase II study for patients with a wide range of solid malignancies, but with a principal focus on CRC, has recently been completed (Figure 3). In this study, all patients were treated with sorafenib 400 mg twice daily in the first 12-week lead-in stage. In essence, the rates of tumor regression and other traditional phase II endpoints could be assessed in this study stage. The trial design also permitted the tailoring and dynamic adjustment of the size of each disease-specific patient cohort to a prespecified level of statistical power. At the end of the 12-week period, patients who had developed progressive disease (> 25% tumor growth) were taken off study, which enriched the remaining patient population with subjects who were most likely to benefit from continuing treatment. Patients in the remaining population were then randomized to treatment with the study drug or placebo to ascertain whether the study drug is inherently capable of delaying tumor growth as measured by the ratio of time to tumor progression between the treatment and placebo arms. At the time of development of progressive disease, subjects who were randomized to placebo were able to cross over to study drug treatment. The randomized discontinuation design has been proposed as a means of assessing whether further resource-intensive phase III evaluations of agents with predominant growth-inhibitory effects are warranted, particularly when optimal enrichment strategies are not clear. The “natural” enrichment of the randomized population increases the efficiency of the trial, with as few as 20% of the standard number of randomized patients. This results in the overall number of patients with this design being lower than that required for a classic randomized trial. Thus, this

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design has the potential of markedly shortening phase II development. However, if there is a clear difference in disease progression between patients who continue to receive treatment and patients randomized off treatment, a general conclusion can still be made regarding the activity of the agent. Because the major development question is whether a target-based agent has activity, this design allows a rapid route to that answer. Patient accrual for the randomized discontinuation trial of sorafenib has been completed. A preliminary report detailed the results in the first 290 patients, including CRC (n = 138), renal cell carcinoma (n = 57), melanoma (n = 37), pancreatic carcinoma, and other tumor types.95 The principal toxicities, consisting of skin rash, hand-foot syndrome, anorexia, diarrhea, and fatigue, were similar to those noted in previous trials. Interestingly, somewhat robust activity was noted in the first 18 evaluable patients with renal cell carcinoma, with 8 patients (44%) experiencing disease regression of ≥ 25% and 7 patients (39%) experiencing SD as their best response. Antitumor activity was also noted in patients with CRC, melanoma, thyroid and pancreatic carcinomas, and sarcoma, but the rate of objective antitumor activity in CRC appears somewhat low. Because sorafenib is known to inhibit vascular endothelial growth factor receptor TK and other TKs, in addition to Raf kinase, it is possible that its prominent activity in renal cell carcinoma is a result of its kinase promiscuity.

Targeting MEK MEK is a dual-specificity kinase in that it activates ERK by phosphorylating tyrosine and threonine residues. Two related genes code for MEK1 and MEK2.65-68 Both MEK proteins play critical roles in the Ras signaling pathways. However, MEK1 and MEK2 differ in ERK binding affinities and possibly in their abilities to activate ERK. In the mitogen-activated Ras/Raf/MEK/ERK cascade, Raf usually activates the dual-specific serine/threonine and tyrosine kinases MEK1 and MEK2, which then activate ERK1 and ERK2. MEK has not been identified as an oncogene product in human malignancies; however, it is a critical point of convergence that integrates input from a variety of protein kinases through Ras. In addition, Ras is very restricted in its substrate specificity, with the MAPKs being the sole known substrates of importance. Therefore, MEK is a target of great interest for the development of oncologic therapeutic agents. CI-1040, an orally administered selective small-molecule inhibitor of MEK, significantly inhibited the growth of a variety of human cancer cell lines and xenografts, particularly those derived from human colon carcinoma.96,97 Antitumor activity, which has been related to levels of MAPK expression, has been achieved without notable toxicity. In a phase I trial of oral CI-1040 administered daily for 21 days every 4 weeks, inhibition of phosphorylation of ERK, which is downstream of MEK, has been documented. Diarrhea, rash, and fatigue appear to be the principal toxicities. A patient with advanced pancreatic carcinoma was reported to have had a PR, and 30% of patients had SD lasting > 12 weeks.98 No major antitumor activity has been observed in a limited number of phase II trials in previously treated patients with colorectal, breast, pancreatic, and non–small-cell lung cancers. Because CI1040 has suboptimal pharmaceutical properties, a more potent backup compound, PD0325901, has entered early clinical evalu-

Alain C. Mita et al ations. In addition, several other specific MEK inhibitors such as ARRY-142886 (AZD6244) are undergoing clinical development.

Figure 3

Targeting ERK In mammalian cells, there are 2 closely related genes that code for ERK1 and ERK2. After activation, ERKs enter the nucleus of cells, where they become phosphorylated and in turn activate transcription factors, which leads to the expression of genes involved in growth and differentiation.65-68 Although no direct inhibitors of ERK are currently in clinical development, the kinase is actively being pursued as a strategic target for therapeutic development.

Signaling Through the PI3K Cell Survival Pathway Cellular survival is an active process that is monitored continuously and regulated by signals that promote survival or programmed cell death (apoptosis). These signals relay information about the availability of growth and survival factors, supply of nutrients and oxygen, cellular stress, and genomic integrity, and activate death receptors. Amplified, overexpressed, and aberrant signaling elements in the PI3K/protein kinase B (ie, Akt) pathway result in unchecked or aberrant proliferative, anabolic, and survival signals in many types of malignancies.99-101 Phosphatidylinositol 3-kinase phosphorylates phosphoinosiditides, which generate 3-phosphorylated phospholipids that act as membrane tethers for proteins with pleckstrin homology regions, such as the serine/threonine protein kinase known as Akt and phosphoinositide-dependent kinase–1 (PDK1). Akt is the focal point for survival signals from growth and survival factor receptors.99102 After activation, Akt prevents apoptosis through the inhibition of proapoptotic factors including BAD, caspase 9, glycogen synthase kinase–3, and the Forkhead family of transcription factors (FKHRL1, FKHR, AFX), or by activating antiapoptotic factors such as Bcl-2 and nuclear factor–κB (NFκB).103-110 The PI3Ks, Akt, and PDK1 play important roles in regulating many cellular processes including proliferation, carbohydrate metabolism, and motility. Evidence that these kinases are important components of the molecular mechanisms of many diseases, including cancer, diabetes, and chronic inflammation, is emerging.101,102 The initial discovery of Akt as a retroviral oncogene reinforces its role in oncogenesis. Several lines of evidence suggest that increased signaling through Akt confers a clear survival advantage to tumor cells in the face of inappropriate activation of oncogenes, hypoxia, DNA damage, exposure to cytokines, and Fas ligand. The survival function of Akt is also suggested by the frequent and causal role of activating mutations of several components of the Akt pathway in the development of many types of cancer.97,98,111 The tumor-suppressor gene PTEN, which encodes the tumor suppressor phosphatase PTEN, is a negative regulator of Akt activation. The PTEN protein dephosphorylates the PI3K effector product PIP3, thereby inactivating it. PTEN inactivation in tumor cells causes increased intracellular levels of PIP3 and general enhancement of PI3K signaling. Deletions or mutations of PTEN in many cancers, particularly endometrial, colon, ovarian, prostate, and small-cell lung and breast carcinomas, and melanoma, meningioma, glioblastoma, and anaplastic astrocytoma, allow ge-

Randomized Discontinuation Study of the c-Raf Kinase Inhibitor Sorafenib in Advanced Colorectal Cancer Regression ³ 25% (Continue BAY 43-9006)

Specific Refractory Cancers (CRC) Sorafenib

Initial Evaluation (12 weeks) Stopping rule based on risk of PD Disease Progression > 25% (Off Study)

Regression or Growth < 25%

R A N D O M I Z E

Continue until PD

Continue Sorafenib Placebo Unblind for PD (May restart Sorafenib)

Abbreviation: CRC = colorectal cancer; PD = progressive disease

nomically compromised cells to survive and accumulate further DNA damage, which in turn leads to neoplastic transformation.99102,112 In contrast, ectopic expression of wild-type PTEN reduces Akt activity and restores sensitivity to apoptotic stimuli. Additionally, persistent signaling through the PI3K/Akt pathway by stimulation of the insulin-like growth factor receptor appears to be a mechanism of resistance to inhibitors of ErbB1 and ErbB2.113,114 Therefore, this pathway is an attractive target for therapeutic development in colorectal and many other types of cancers, as such agents might inhibit proliferation and reverse the repression of apoptosis and the resistance to cytotoxic therapy in cancer cells. The potential importance of the PI3K survival pathway as a target in the treatment of CRC has been supported by recent preclinical data linking the pathway to carcinogenesis of CRC. First, the gene coding for the p85α regulatory subunit of PI3K has been identified as an oncogene in CRC.115 Somatic mutations in this gene have been associated with constitutive activation of PI3K and tumorigenesis. In addition, Akt proto-oncogene activation has been identified as an early event in sporadic carcinogenesis of CRC.116,117 Because there are 2 sites containing an (A)6 repeat in the PTEN coding sequence, and because mononucleotide repeat sequences constitute frequent targets for mutations in tumors with microsatellite instability (MI), mutations of the PTEN gene have been demonstrated in approximately 18% of MI-positive CRCs, including sporadic CRC and/or CRC related to the hereditary nonpolyposis colon cancer syndrome, and may play a role in tumorigenesis.118,119 In addition, decreased or undetectable cytoplasmatic PTEN has been found in as many as 35% of MI-positive tumors and in some of the MI-negative tumors, suggesting that mechanisms other than structural PTEN alteration occur in CRC.119 Although many efforts are being directed at developing specific inhibitors of PI3K, PDK1, and Akt, these efforts have largely resulted in nonspecific kinase inhibitors. However, therapeutic agents directed against signaling elements downstream of PI3K and Akt, such as molecular target of rapamycin (mTOR) and the antiapoptotic protein Bcl-2, which will be discussed in the next section, appear more fruitful at this juncture.13

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Targeted Therapy Agents for Colorectal Cancer Targeting the mTOR-Dependent and Rapamycin-Sensitive Pathways The mTOR—also called FRAP, RAFT1, and RAPT1—is a member of a recently identified family of protein kinases termed phosphoinositide-3 kinase–related kinases (PIKKs), which preferentially link proliferative stimuli to cell cycle progression and nutrient use.13,120,121 Molecular target of rapamycin plays a critical role in the transduction of proliferative signals mediated through the PI3K/Akt signal transduction pathway. Its main function is to regulate the initiation of protein translation and ribosomal biosynthesis by altering the phosphorylation states of the translational regulator eukaryotic initiation factor 4E-binding protein and a 70-kd S6 kinase known as p70s6k (Figure 4). Inhibition of mTOR function abolishes the proliferative and nutrient-use signals mediated through the PI3K/Akt signaling pathway and results in profound arrest of cycling cells in the G1 phase of the cell cycle, tumor growth inhibition, and apoptosis.13,120,121

Targeting mTOR Because mTOR appears to be a critical proliferative signaling element, it has become a principal target for therapeutic agent developmental efforts. The macrolide fungicide rapamycin, which was originally isolated from the soil organism Streptomyces hygroscopicus, is a specific inhibitor of mTOR that exerts potent antimicrobial, immunosuppressant, and antineoplastic actions.13,120 Because of its profound immunosuppressive actions, rapamycin was initially developed and received regulatory approval for prevention of allograft rejection after organ transplantation. However, impressive antiproliferative activity occurs after treatment of a diverse range of experimental tumors. The antiproliferative effects of rapamycin appear to result from its ability to bind to the intracellular immunophilin FKBP-12, and the complex then binds to and inhibits the activity of mTOR.13,120,121 However, the poor solubility and chemical stability of rapamycin have precluded its administration on a variety of administration schedules, and several rapamycin analogues that are more amenable to parenteral administration, such as temsirolimus (CCI-779), RAD001, and AP23573, are under development.13,122 The toxicities of temsirolimus, which is the furthest along in clinical development, are similar to those induced by rapamycin (eg, mild cytopenias, fatigue, and elevated levels of serum triglyceride and liver enzyme); and regression of several types of advanced cancers (eg, breast, renal, and lung carcinomas and soft tissue sarcoma) have been reported.123,124 Clinical evaluations in patients with a wide variety of solid malignancies are ongoing. As for many targeted therapies, the in vitro activity of mTOR inhibitors in different cell lines of a given tumor type is quite variable and dependent on the importance and the integrity of the PI3K/Akt/mTOR axis in the tumor proliferation and/or survival. For example, mTOR inhibitors have demonstrated notable antitumor activity against CaCo2 colorectal cancer in vitro (50% inhibitory concentration [IC50] of 4 nmol/L); whereas other colorectal cancers, such as CX-1, COLO 205, and SW480, have demonstrated profound resistance (IC50 values of 4.4-5.9 μmol/L).125 Interestingly, the low level of 4E-BP-1, which is an important downstream effector of mTOR, appears to account for the resistance to

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rapamycin in HCT8 colorectal cancer.126 In addition, rapamycin sensitivity has been directly related to various upstream aberrations that activate mTOR, including ErbB overexpression/overactivation; PTEN deletions, mutations, or loss; or constitutively activated Akt and PI3K signaling elements.122,127,128 Several lines of preclinical evidence have also suggested that innate and acquired resistance to inhibitors of ErbB1 and ErbB2 in many types of malignancies is associated with increased signaling through the PI3K pathway and possibly mTOR.129-131 Similarly, experimental data have also indicated that the sensitivity to many types of ErbB inhibitors, such as gefitinib, cetuximab, and the dual ErbB1/ErbB2 TK inhibitor lapatinib, requires intact growth factor receptor–stimulated Akt activity.130,132 Moreover, drug sensitivity can be restored in resistant cells in vitro by genetic and pharmacologic means such as by introducing the PTEN gene into PTEN-deficient cells or using inhibitors of PI3K/Akt.130,131 These studies suggest that rapamycin analogues and more direct inhibitors of the PI3K pathway may be used with various inhibitors of ErbB and other signaling elements to prevent the development of drug resistance and/or augment sensitivity.

Regulators of Apoptosis as Anticancer Targets Cellular aberrations that enhance cell survival, in addition to those that result an increased rate of cell proliferation, are commonly found in most human malignancies.14 Therefore, it can be reasoned that enhancing or restoring apoptosis to improve the effectiveness of chemotherapy, irradiation, and hormone therapy may be an effective overall strategy and approach to therapeutic development against cancers, particularly against malignancies with aberrant survival mechanisms resulting from aberrations in triggering apoptosis under appropriate conditions. There is a hierarchical organization of the pathways of cellular apoptosis. The final common biochemical pathway that executes this process requires the orderly activation of a family of tightly regulated intracellular cysteine proteases called caspases. These proteases exist as inactive zymogen forms, which, after activation upstream, induce enzymatic cleavage of many proteins involved in apoptosis. This process leads to an orderly cascade of proteolysis downstream, including autoactivation of other caspases. Among these, caspases 3, 6, 7, 8, and 9 have well-described function in cell death pathways. The downstream caspases mediate cellular destruction of various “housekeeping” functions through cleavage of protein kinases and other signal transduction proteins, cytoskeletal proteins, chromatin modifying proteins, DNA repair proteins, and inhibitory subunits of endonucleases. The activation of upstream caspases is mediated by at least 2 known pathways termed the intrinsic and extrinsic pathways of apoptosis (Figure 5). Although these pathways appear to be linked, each pathway uses a separate upstream caspase family member to regulate the activation of caspase-3 and other downstream caspases. Rationally derived therapeutic agents directed at the regulation of Bcl-2 family members, which are the principal components of the intrinsic pathway of apoptosis, are in late clinical trials in patients with CRC and other malignancies, whereas strategies aimed at targeting the extrinsic pathway of apoptosis via the TNF receptor (TNFR) family are in earlier developmental stages.

Alain C. Mita et al Targeting the Intrinsic Pathway of Apoptosis The intrinsic pathway is a mitochondrial membrane–dependent pathway mediated by the Bcl-2 family of proteins.133 The bcl-2 gene was originally identified as the chromosomal breakpoint of the translocation of a portion of chromosome 18 to 14 in follicular B cell non-Hodgkin’s lymphoma. Bcl-2 belongs to a superfamily of apoptosis regulatory gene products, which may be death antagonists (Bcl-2, Bcl-XL, Bcl-2, Bfl-1, and Mcl-1) or death agonists (Bax, Bak, Bcl-Xs, Bad, Bid, Bik, Bim, and Hrk).134 Although somewhat oversimplified, the balance of death antagonists and agonists, and the interactions of these proteins, influence the cellular response to apoptotic and survival signals. This death/life balance is mediated, at least in part, by the selective and competitive dimerization of antagonists and agonists.135 Many of the members of this regulatory family, such as the prototypic death agonist Bax, act at the level of the outer mitochondrial membrane.133 The disruption of the mitochondrial membrane is one of the first universal manifestations of the apoptotic process, resulting in the release of cytochrome c and other protease activators (including caspases 2, 3, and 9) and apoptosis-inducing factors (eg, apoptosis protease–activating factor–1 [Apaf-1]). Cytochrome c, along with deoxyadenosine triphosphate, binds to and changes the conformation of the Apaf-1 complex, which results in the activation of caspase-9 and irreversible commitment to apoptosis. As the dynamic equilibrium between proapoptotic and antiapoptotic proteins ultimately determines the susceptibility of the cell to apoptotic death, many novel therapeutic agents that perturb this equilibrium may enhance the effectiveness of chemotherapy, irradiation, and hormone therapy.

Figure 4

Rapamycin-Sensitive Signal Transduction Pathway

Growth Factors, Nutrients

P

P

P

P

P-12

P PI-3 SH2 RA Kinase SH2

PTEN

FKB

G1

4E-BP1 Translation of critical proteins (eg, G1 checkpoint proteins)

PI3K Akt

mTOR

P Bad

Bad

p70s6k

Transcriptional Regulation

S Ribosome Biogenesis

P Apoptosis

Survival

Targeting Bcl-2

Rapamycin and its analogues bind to the immunophilin FKBP-12. The RAP-FKBP-12 complex blocks the kinase activity of mTOR, which, in turn, inhibits 4E-BP1, p70s6k, and other translational regulators. The inhibition of 4E-BP1 and p70s6k decreases ribosomal biosynthesis and the translation of specific proteins essential for cellcycle progression from G1 to S phase. Abbreviations: 4E-BP1 = 4E binding protein–1; FKBP-12 = FK506-binding protein–12; mTOR = molecular target of rapamycin; P-I3 = phosphatidylinositol-3 kinase; RAP = rapamycin

Targeting the Bcl-2 antiapoptotic protein, which is overexpressed and dysfunctional in CRC and other common human malignancies and plays a pivotal role in regulating cell death following apoptotic stimuli from anticancer therapeutics, is an attractive approach for therapeutic intervention.134,136-140 In addition, Bcl-2 overexpression confers a multidrug-resistant phenotype, and bcl-2 transfection results in tumor cells that are resistant to radiation and many cytotoxic agents, such as irinotecan and platinum derivates, which are commonly used to treat patients with advanced CRC.141,142 Bcl-2 overexpression, which has been demonstrated in 30%-94% of human CRC specimens, appears to be an early event in colorectal tumorigenesis and tumor proliferation, even though its prognostic significance is controversial.143-145 Other factors, such as decreased Bax expression and/or Bax dysfunction caused by inactivating mutations of the bax gene, have also been demonstrated and may be important in colon carcinogenesis.146 Moreover, CRCs with the microsatellite mutator phenotype (MMP) are often associated with somatic frameshift mutations in the bax gene, resulting in reduced Bax protein expression and/or dysfunction, which provide a survival advantage and are selected for during the progression of MMP-positive tumors.147-149 Oblimersen sodium is an 18-mer phosphorotheioate ASON directed at the first 6 codons of the human bcl-2 open reading frame. Extensive preclinical investigations indicate that oblimersen sodium treatment leads to sequence-specific and dose-dependent

degradation of Bcl-2 mRNA with subsequent inhibition of Bcl-2 protein expression in vitro and in vivo.150,151 In addition, several lines of evidence indicate that oblimersen sodium treatment enhances the effectiveness of other therapeutic modalities, including cytotoxic therapeutic agents such as irinotecan, taxanes, and platinating and alkylating agents.152,153 The feasibility of administering oblimersen sodium alone or in combination with relevant therapeutic regimens in patients with advanced CRC and other malignancies is being evaluated.154-160 The principal toxicities of oblimersen sodium administered as a protracted I.V. infusion or subcutaneously include hyperglycemia, transient hepatic aminotransferase elevations, fever, fatigue, thrombocytopenia, leukopenia, and local inflammation at the injection site. The early observations of clinical activity with oblimersen sodium administered as a single agent against lymphomas and leukemias may be a result of a pivotal role of Bcl-2 in the etiology of these disorders and provide proof of principle that Bcl-2 inhibition may restore apoptosis in tumors in which Bcl-2 regulation is aberrant. In CRC and other malignancies, however, Bcl-2 expression may not be fundamental to cell survival, except in the presence of external apoptotic stimuli such as chemotherapy and irradiation. In several clinical trials, oblimersen sodium was administered as a 7-day continuous I.V. infusion before chemotherapy, based on the hypothesis and early preclinical evidence

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Targeted Therapy Agents for Colorectal Cancer Figure 5

TNF Family of Death Receptors

Schematic Representation of Intrinsic and Extrinsic Pathways of Apoptosis DcR1 DR4

DR5

OPG DcR2 Extrinsic Pathway

Mitochondria

FLIP

Apoptotic Stimuli

FADD caspase-8 BID caspase-3

caspase-6

cytochrome c Apaf-1

Bcl-2 Bcl-XL

caspase-9 caspase-7

Cascade of Effector Caspases Apoptosis

Intrinsic Pathway Apoptosis Inhibitory Proteins

The intrinsic pathway is mediated by Bcl-2 family members at the mitochondria that releases cytochrome c and activates the proapoptotic protein Apaf-1 and the cascade of caspases. The extrinsic pathway is mediated by the TNF family of receptors at the cellular membrane. Abbreviations: Apaf-1 = apoptotic protease activating factor–1; DcR = decoy receptor; DR = death receptor; FADD = Fas-associated death domain; FLIP = FADD-like inhibitory protein; TNF = tumor necrosis factor

that several types of chemotherapeutic agents phosphorylate Bcl2, thereby decreasing the availability of Bcl-2 and possibly augmenting the overall effects of oblimersen sodium.154-160 The preliminary results of these studies indicate that oblimersen sodium doses as high as 5 mg/kg per day as a 7-day continuous I.V. infusion, which result in oblimersen sodium plasma concentrations in excess of those required for Bcl-2 modulation in tumor xenograft models, could be administered safely with chemotherapy. The combination of oblimersen sodium administered on this schedule with irinotecan 350 mg/m2 I.V. on day 8 every 3 weeks was demonstrated to be well tolerated.157 Mild fever and fatigue were the principal side effects attributable to oblimersen sodium, and there was no notable enhancement of irinotecan toxicity. Of the 20 patients treated in this study, 1 patient (5%) experienced a PR and 10 other patients, including those with tumors that were clearly refractory to irinotecan, had SD lasting 3-10 months. In addition, consistent decrements in Bcl-2 expression were noted in peripheral blood mononuclear cells and in serial tumor biopsies of patients undergoing treatment with oblimersen sodium in this study.157,158 A National Cancer Institute–sponsored phase I/II study of the combination of oblimersen sodium with the FOLFOX4 regimen in oxaliplatin-naive patients with metastatic CRC is being conducted. Oblimersen sodium is being administered as a 5-day continuous I.V. infusion to provide optimal downregulation of Bcl-2 before treatment with FOLFOX4. The combination of oblimersen sodium at doses as high as 7 mg/kg per day with standard doses of FOLFOX4 appears well tolerated. One of 11 evaluable patients had a complete response. Pharmacokinetic and biologic correlative studies on tumor samples are also being performed. Although none are in clinical development, peptide and smallmolecule inhibitors of Bcl-2-Bax heterodimer formation have

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been described.161,162 One small molecule inhibitor, HA14-1, which was discovered with a computer screening strategy based on the predicted structure of the Bcl-2 protein surface, binds to a surface pocket of the Bcl-2 protein.162 Such work may ultimately lead to a series of small-molecule therapeutic agents that replace Bcl-2 antisense strategies. Several agents targeting other components of the intrinsic pathway of apoptosis are in preclinical development. Among these are ASONs directed at Bcl-XL and other antiapoptotic regulatory proteins such as clusterin, survivin, and testosterone-repressed prostatic message–2. Other therapies are directed at enhancing the expression of the proapoptotic protein Bax, and include inhibition of the proteasome and/or ubiquitin pathway and bax gene transfection.163-166

Targeting the Extrinsic Pathway of Apoptosis The extrinsic pathway of apoptosis refers to the activation of caspase-8 as a result of apoptosis-inducing ligands that bind to the TNFR family. The TNFR protein complex includes the receptors TNF R1, Fas (Apo1), DR3 (Apo2), DR4 (TNF-related apoptosisinducing ligand [TRAIL] R1), DR5 (TRAILR2), and DR6, bundled with an intracytoplasmic death domain protein and critical adaptor proteins.167 These adaptor proteins, particularly TRADD or FADD (TNF-α–associated or Fas-associated death domain protein, respectively), mediate intracytoplasmic signals from the receptor using death domain proteins to interact with the receptor and death effector domains to interact with procaspase-8.168-170 These proteins engage proteases and cleave the N-terminal of caspase-8, thereby activating the caspase cascade. There are several homeostatic mechanisms for the regulation of cell death in the extrinsic pathway. Decoy receptors for Fas ligands and TRAIL (TRAIL R3/DcR1 and TRAIL R-4/DcR2) compete for ligand and modulate apoptotic signals. Moreover, several intracellular proteins interact with death domain proteins to inhibit the signal transduction of apoptosis and include the protein silencer of death domains and FAP1, which may represent a mechanism of resistance to Fas-inducing apoptosis.171 In addition, members of the death effect domain family (eg, FLIP) compete with procaspase-8 for the binding with FADD and inhibit apoptosis.167 Dysregulation of these mediators may lead to malignant transformation. Mutations or deletions in the Fas gene have been found in cells from patients with many types of malignancies. It is also likely that the intrinsic and extrinsic apoptotic pathways are linked at many critical juncture points. For example, abrogation of TRAIL-mediated apoptosis occurs in some cancer cell lines secondary to overexpression of Bcl-2 family proteins; whereas TNFα–mediated expression of NFκB activates several bcl-2 family genes that have antiapoptotic functions.172,173 Comparative studies of the expression pattern of these receptors in normal and malignant colon epithelium have been performed.174 In normal colon mucosa, TRAIL and TRAIL-R2 are expressed in the surface epithelium, whereas TRAIL-R1 and TRAIL-R4 are found mainly along the crypt axis. Whereas, in adenomas, this expression pattern is mostly retained, in carcinomas, the expression of TRAIL and TRAIL receptors is much more variable. Of all these receptors, only TRAIL-R1 expression was significantly associated with dis-

Alain C. Mita et al ease-free survival in patients with stage II/III CRC who have undergone resection (P = 0.003). These findings suggest a potential use of TRAIL as anticancer agent in CRC alone or, more likely, in combination with chemotherapy.

Targeting TRAIL Receptors Tumor necrosis factor, which has the potential to induce apoptosis in tumor cells and mediates inflammatory processes, is the prototypic ligand for the TNFR family.175 Tumor necrosis factor–α and Fas ligands are not candidates for drug therapy because of their nonspecific activation of multiple TNFRs and their causality of septic shock and fulminant hepatic failure in animals.176 In contrast, Apo2 ligand (Apo2L; also called TRAIL), a member of the tumor TNF gene superfamily discovered on the basis of its sequence homology to Fas/Apo1 ligand (Fas/Apo1L) and TNF, is a candidate for further clinical development.177,178 Native Apo2L is expressed as a type II transmembrane protein, which is cleaved proteolytically to form a soluble homotrimer. TRAIL binds to the death receptors DR4 and DR5, the decoy receptors DcR 1 and 2, and OPG. After ligand binding to DR4 or DR5, the cascade of caspases is activated, ultimately resulting in cell death. Interestingly, Apo2L induces apoptosis in a variety of human cancer cell lines but not in normal cells.176,179 In addition, many human tissues express Apo2L mRNA, suggesting that normal cells may have physiological mechanisms (such as expression of decoy receptors) that can protect many normal cell types from induction of apoptosis specifically by Apo2L.176,179 In fact, further studies of recombinant soluble human TRAIL suggest that it is a prime candidate for clinical development based on its potential to induce apoptosis in a broad spectrum of human cancer cell lines, including COLO205, HCT15, and HCT116 colon cancers.171,179,180 In the National Cancer Institute cell line screening panel, the majority of the tested tumors demonstrated a relevant degree of sensitivity to recombinant Apo2L including colon, lung, central nervous system, ovarian, renal, prostate, and breast cancers, as well as leukemia and melanoma.176 Sensitivity to TRAIL was independent of p53 status. In line with this observation, TRAIL has been consistently shown to enhance the cytotoxic effects of a wide range of chemotherapeutic agents in vitro, including irinotecan and 5-FU.181,182 Moreover, impressive antitumor activity without toxicity has been observed in human tumor xenograft models, including effects that compare favorably with those of single-agent 5-FU in a human HCT16 colon carcinoma xenograft.183 In addition, favorable interactions between TRAIL and 5-FU and irinotecan have also been noted in vivo.184 The selectivity of TRAIL in mediating apoptosis in tumor cells and not in normal cells has not been elucidated, but the overexpression of death receptors and/or a relative absence of decoy receptors in tumor cells have been proposed as explanations. However, preclinical studies demonstrating apoptosis in human hepatic cells in vitro raised concerns about clinical evaluations. Recent evidence indicates that different versions of recombinant soluble human TRAIL may exhibit a different propensity for hepatocyte toxicity. Recombinant soluble human TRAIL is undergoing late-stage preclinical and toxicologic evaluations before entry into the clinic.185

Monoclonal antibodies with agonist-like properties at the DR4 and DR5 sites represent alternate strategies for the induction of apoptosis via the extrinsic pathway. After antibody–antigen complex formation, caspase activation and apoptosis induction occur, resulting in tumor regression in several xenograft models.186,187 The affinity for DR4 binding appears to be less important for agonist activity than the specific binding site on the receptor.186 This implies that the specific agonist site exists within the receptor and suggests that widely divergent results may be obtained with different antibodies directed to the same target DR4.186 TRM-1 and TRM-2 are humanized MoAbs that undergo highaffinity binding to DR4 and DR5, respectively.188 TRM-1 is active against cancer cells expressing DR4, with IC50 values in the nanomolar range. The MoAb also induces apoptosis in CRC in vitro and in vivo.188 However, it appears that the most profound activity of both MoAbs may involve their extraordinary potential to enhance the cytotoxicity of chemotherapeutic agents, particularly topoisomerase I–targeting agents including irinotecan.188 Phase I studies of TRM-1 and TRM-2 are in progress. It may also be reasonable to consider treating CRC and other malignancies with hybrid antibodies directed at both death receptors or with cocktails of monoclonal antibodies against DR4 and DR5 based on the death receptor profile of individual malignancies.

Conclusion During the past few years, a plethora of rationally designed, target-based therapeutic options has become available for patients with CRC and other malignancies as a result of a greater understanding of cancer biology and advances in biotechnology. However, the optimal integration of these novel agents into available treatment regimens is an inordinate challenge. There must be a greater emphasis placed on understanding the precise mechanisms of action of these rationally designed targeted therapies, as well as on identifying the molecular aberrations that are principally driving the proliferation of CRC. It is likely that a greater emphasis of efforts in this direction will result in an enhanced selection of patients who are likely to benefit from these therapies, which are expected to be much more restrictive than the use of nonspecific cytotoxic agents. Moreover, because of the redundancy of the signaling pathways and extensive crosstalk, it is unlikely that blocking any single pathway will lead to robust and durable antitumor activity. Therefore, the development of optimal combination regimens based on a solid preclinical foundation must be pursued. Finally, radical changes in clinical evaluation paradigms must be considered to fully exploit the potential of these novel therapies.

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