71 Molecular Targeted Therapy
Table 71-1 lists the types of molecular targeted therapies discussed in this chapter. New small-molecule inhibitors, monoclonal antibodies, and viral therapies evolve rapidly and continuously, so the chapter is not intended to delineate a complete list of all agents under development. Instead, we will try to provide an overview of the different strategies available, paying particular attention to the better-known agents likely to affect the clinical approach to cancer.
Table 71-1 Categories and Examples of Molecular Targeted Therapies
| Agent | ||
|---|---|---|
| ErbB family inhibitors | Gefitinib | Erlotinib |
| Cetuximab | ||
| Trastuzumab | ||
| Lapatinib | ||
| PDGF receptor inhibitors | Imatinib | |
| VEGF receptor inhibitors | Sorafenib | Sunitinib |
| Bevacizumab | ||
| Farnesyltransferase inhibitors (FTIs) | Tipifarnib | |
| Lonafarnib | ||
| mTOR inhibitors | Sirolimus | |
| Temsirolimus | ||
| Everolimus | ||
| Histone deacetylase inhibitors | SAHA | |
| Nonreplicating viral therapy | Gene replacement | p53 |
| Suicide gene therapy | HSV-tk | |
| Radiation-inducible viral therapy | Egr-TNFα | |
| Replicating viral therapy | Oncolytic adenoviral therapy | Onyx-015 |
| CV707 | ||
| CV787 | ||
| Oncolytic herpesvirus therapy | G207 | |
| G92a |
EGF, Epidermal growth factor; HSV-tk, herpes simplex virus tyrosine kinase; PDGF, platelet-derived growth factor receptor; SAHA, suberoylanilide hydroxamic acid; TNF, tumor-necrosis factor; VEGF, vascular endothelial growth factor.
Molecular Targets
Inhibition of Tyrosine Kinase Receptors
Receptor tyrosine kinases (RTKs) play pleiotropic roles in maintaining homeostasis of individual cells, specific tissues, and entire organisms. The function of RTKs must be tightly regulated, since they mediate fundamental cellular functions including proliferation, survival, adhesion, and differentiation. RTK molecules share a ligand-binding extracellular portion, a transmembrane section, and an intracellular portion that contains the tyrosine kinase catalytic domain.1–3 The family of RTKs includes the epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR), fibroblast growth factor receptor (FGFR), stem-cell factor receptor (SCFR), and nerve growth factor receptor (NGFR). Inactivating mutations in various domains of the protein, including cytoplasmic juxtamembrane and extracellular and kinase domains, confer ligand-independent phosphorylation and kinase activation.4 Activation of RTKs directly contributes to the initiation, progression, and prognosis of several human malignancies.5,6
Monoclonal antibodies (mAbs) can prevent binding of ligands to their receptors, impede the subsequent phosphorylation loop, and thus block signaling through growth factor receptors.7,8 Technical difficulties such as detrimental antibodies patients develop against rodent antibodies have been addressed by advances in antibody construction.9
Small-molecule inhibitors compete with adenosine triphosphate (ATP) for binding sites in the receptor, blocking signaling through RTKs.2 In comparison to mAbs, small-molecular inhibitors against RTKs tend to have less specificity and multiple target receptors. They also have significantly shorter half-lives than mAbs.
Epidermal Growth Factor Receptor Inhibitors
The ErbB family includes four different receptors: ErbB-1 (also known as EGFR), ErbB-2 (also known as HER2/neu), ErbB-3 (also known as HER3), and ErbB-4 (also known as HER4).10–12 EGFR is located on chromosome 7p12, and its activation occurs via several mechanisms including amplification, overexpression, and expression of a truncated constitutively active form. EGFR plays a key role in the pathogenesis of many human malignancies: non–small-cell lung, breast, head and neck, gastric, colorectal, esophageal, prostate, bladder, renal, pancreatic, ovarian, and brain malignancies. EGFR is the most commonly amplified oncogene in glioblastoma (GM), with amplification seen in 40% of tumors.13–15 One-third of GMs in which EGFR is amplified contain a mutant form, most commonly the EGFRvIII mutant, in which deletion in the extracellular domain results in constitutive tyrosine kinase activity.16,17
EGFR offers a promising and exciting target for therapeutic intervention. Several small-molecule inhibitors of EGFR signaling have entered clinical trials for a variety of neoplasms. Gefitinib and erlotinib are two such oral drugs that inhibit EGFR signaling by targeting the ATP-binding site of the receptor.18,19 In a phase III trial of refractory non–small cell large carcinoma (NSCLC) patients, gefitinib did not improve survival in the overall study population, but a subgroup analysis revealed a significant survival benefit in patients of Asian origin or in those who had never smoked.20 Surprisingly, a phase III trial of erlotinib (BR.21) showed an increase in median survival of 2 months in a population of previously treated NSCLC patients.21 Although a survival benefit was found in all subgroups, it was most robust in women, patients with adenocarcinoma, and patients who had never smoked. It is hypothesized that the differences in responses between erlotinib and gefitinib may be the result of the doses used in the trials. In pancreatic cancer patients, a phase III trial of erlotinib in combination with gemcitabine showed impressive improvement in response and overall survival, resulting in approval of this combination as first-line therapy for these patients.22
The efficacies of erlotinib and gefitinib have also been investigated in glioma patients. In unselected GMs, 10% to 20% of patients respond to EGFR inhibitors, and while this proportion is modest, some patients experience dramatic responses.23 Studies have sought to understand the molecular underpinnings that render certain tumors responsive to EGFR inhibitors.24,25 Two complementary studies found that overexpression of wild-type or mutant EGFR promotes sensitivity to EGFR inhibitors, and activation of the phosphoinositide 3-kinase (PI3K)-mediated signaling pathway leads to resistance to such small-molecular inhibitors.26–28
Inhibition of EGFR signaling has been accomplished not only with small molecules but also with mAbs directed against the extracellular domain of EGFR. Human/murine chimeric antibodies offer the advantage of reduced immunogenicity while preserving potency. Cetuximab is such an antibody. Its administration in animal models delays growth of xenograft tumors overexpressing ErbB-1.29 Like the small-molecular inhibitors, the clinical efficacy of Cetuximab has also been investigated in a variety of neoplasms. Phase II trials have shown limited activity for cetuximab in the treatment of NSCLC.30 However, more definitive conclusions must await publication of the results of a recently completed phase III trial (NCT00148798). The benefits of cetuximab for colorectal cancer patients have been thoroughly explored. In phase II trials in metastatic colorectal cancer, response rates to cetuximab alone were between 9% and 12% and in combination with irinotecan rose to 20%.31 A phase III trial of cetuximab plus irinotecan compared to irinotecan alone as second-line therapy showed significant improvements in response rates, progression-free survival, and quality of life.32 More impressively, a phase III trial involving patients who had failed all available therapies showed that cetuximab, compared to best supportive care, improved progression-free survival, overall survival, and quality of life.33 Cetuximab has also proved promising in the treatment of squamous cell carcinoma of the head and neck. When used in combination with radiotherapy, the addition of cetuximab resulted in a significant prolongation of progression-free survival, duration of locoregional control, and overall survival when compared to RT alone.34 Furthermore, the addition of cetuximab to a fluorouracil or platinum-based chemotherapy in recurrent or metastatic disease improved progression-free and overall survival.35
Platelet-Derived Growth Factor Receptor Inhibitors
Whereas some novel signaling inhibitors exhibit specificity toward a given receptor, other drugs can inhibit a number of RTKs. Imatinib is a small-molecule drug that inhibits the tyrosine kinases Abl, PDGFR, and Kit.36 Clinical trials have documented efficacy of imatinib in the treatment of chronic myeloid leukemia, a malignancy driven by constitutive activation of Abl resulting from a chromosomal translocation called Bcr-Abl. Imatinib induces durable responses in the vast majority of these patients, although resistance can develop.37 On the basis of impressive phase II trial results wherein 54% of patients had a partial response to it, imatinib was also approved for the treatment of gastrointestinal stromal tumor (GIST).38
Clinical trials are incorporating treatment with imatinib in the approach to several adult and pediatric malignancies. Studies of gliomas are capitalizing on the inhibitory function of imatinib against PDGFR. The relevance of imatinib for glioma therapy rests in molecular aberrations involving PDGF and PDGFR. PDGFR-α and PDGFR-β are two distinct receptors that bind PDGF ligand, which in turn consists of various dimers of PDGFA and PDGFB chains. Overexpression of the PDGF ligand and receptor are seen in all grades of gliomas, suggesting activation of an autocrine loop that drives glioma proliferation.39,40 In the minority of cases, amplification of the PDGFR-α gene underlies overexpression, but in the majority, the mechanism of overexpression remains unknown.39,41,42 These molecular aberrations in the PDGFR signaling pathway have provided the rationale for treating gliomas of all grades with PDGFR inhibitors. To date, two phase II trials of imatinib in patients with recurrent high-grade gliomas have shown promising results.43,44 Imatinib has also shown some efficacy when combined with docetaxel in treating ovarian neoplasms.45
Vascular Endothelial Growth Factor Receptor Inhibitors
Initiation and maintenance of malignancies rely not only on cell proliferation and survival but also on concomitant development of a blood supply to support the enlarging tumor. Therefore, much effort has focused on developing molecules that block proangiogenic factors and inhibit angiogenesis. The best-described proangiogenic factors are VEGFs that transmit their signals through Flt-1 (also known as VEGF-R1) and Flk-1/KDR (also known as VEGF-R2) receptors.46 Whereas VEGF ligands are secreted by tumor and stromal cells, VEGF receptors are expressed mostly by endothelial cells.
Strategies to block signaling through VEGFR follow the familiar dual paths of small-molecule inhibitors and antibodies directed against receptors or ligands. This theme applies to all RTKs targeted to date. Sorafenib is a small-molecule inhibitor that targets the Faf/MEK/Erk, VEGFR, and PDGFR pathways.47 A second pharmacologic molecule, sunitinib, exhibits wider specificity toward potentially proangiogenic RTKs, including VEGFR2, FLT-3, PDGF, and c-KIT receptors.48 In addition to small molecule inhibitors, antibodies directed against VEGF or its receptors have entered clinical trials.51 Bevacizumab, directed against the receptor ligand VEGF, is the most studied of these recombinant mAbs.
Despite widespread excitement over the positive preclinical results associated with antiangiogenic therapies, early trials proved surprisingly disappointing.52 However, more recent clinical evidence gives reason for optimism. Based upon the encouraging results of a phase III trial of bevacizumab in combination with other chemotherapeutics, the U.S. Food and Drug Administration (FDA) approved its use for treatment of metastatic colorectal cancer. In these patients, bevacizumab increased median survival, progression-free survival, objective response, and duration of response when compared to irinotecan, fluorouracil, and leucovorin treatment alone.53 Bevacizumab has also been approved for treatment of NSCLC in combination with paclitaxel, having demonstrated an ability to increase median and progression-free survival in these patients.54 It is currently undergoing evaluation for possible use in treating kidney, breast, prostate, brain, and ovarian cancers. Besides the mAb therapies, the small-molecule inhibitors sorafenib and sunitinib have also gained approval for the treatment of advanced renal cell carcinoma and GIST.
Inhibition of Downstream Effectors
Farnesyltransferase Inhibitors
Intermediate molecules transmit downstream signals that emanate from engagement of growth factor receptors. The Ras proteins are such intermediaries that help propagate downstream signaling cascades. Ras is a GTPase that cycles between its active guanosine 5-triphosphate (GTP)-bound state and its inactive guanosine 5-biphosphate (GDP)-bound state. Ras regulates many physiologic cellular functions. Biological functions of Ras, including proliferation, survival, cytoskeletal organization, differentiation, and membrane trafficking, rely on its association with the inner surface of the plasma membrane.55–57 Ras proteins are synthesized as cytosolic precursors and converted to membrane-bound forms through posttranslational modifications that begin with the addition of a 15-carbon farnesyl moiety (an isoprene lipid) to a specific motif at the carboxyl-terminus of Ras proteins. Farnesyltransferase catalyzes the transfer of a farnesyl group from farnesyl diphosphate to a cysteine residue within the CAAX box (A is an aliphatic amino acid and X is methionine or serine) of Ras.58
By blocking farnesylation of Ras and inhibiting its function, farnesyltransferase inhibitors (FTIs) can interrupt the effects of tyrosine kinase receptors that signal through Ras. Thus, FTIs may demonstrate antineoplastic activity not only against tumors that contain oncogenic Ras mutations but also against malignancies driven by aberrant signaling through receptor tyrosine kinases. For example, although gliomas rarely possess mutated oncogenic forms of Ras, common genetic aberrations such as EGFR overexpression may be susceptible to therapeutic targeting by FTIs. The precise mode of FTI action remains unclear, since the mutational status of Ras does not consistently correlate with response of cells to FTI treatment. An enlarging body of evidence suggests that FTI activity is mediated in part through inhibition of farnesylation of other Ras family members such as RhoB.59–61 As a result, it remains difficult to use biochemical characteristics of individual tumors to predict which malignancies will prove most susceptible to FTI therapy. These ambiguities notwithstanding, FTI treatment has resulted in objective tumor responses in early clinical trials of various human malignancies.
Tipifarnib and lonafarnib are the two small-molecule FTIs that have been most extensively investigated. Although tipifarnib failed to improve survival and demonstrated minimal activity as a monotherapy in treating metastatic colorectal cancer and NSCLC, it has shown impressive single-agent activity in a variety of hematologic malignancies.62–64 Moreover, tipifarnib, in combination with imatinib, has shown impressive activity in chronic myelogenous leukemia patients with Abl kinase mutations.65
As a monotherapy, lonafarnib has also disappointed, failing to show tumoricidal activity in metastatic colorectal, urothelial, or squamous-cell head and neck cancers.66–68 However, as with tipifarnib, lonafarnib has shown impressive results in several hematologic malignancies.69
Mammalian Target of Rapamycin (mTOR) Inhibitors
The mammalian target of rapamycin (mTOR) is a 289-kD serine/threonine kinase that is critical in regulating cell growth and metabolism during conditions of nutrient abundance. Activation of mTOR itself is mediated by a variety of growth factors and nutrients. Upon activation, mTOR complexes with raptor and mSLT8 to form mTORC1. The mTORC1 complex then, by phosphorylating a variety of downstream effectors, upregulates protein biosynthesis, ribosome biogenesis, and the transcription of genes crucial to cell growth. Because of its intimate involvement in cell growth, and thus cell division, it is not surprising to find mTOR frequently upregulated in certain tumors.70
Because of its importance in tumorigenesis, there has been a recent rash of interest in inhibitors of mTOR. A phase II trial of temsirolimus in advanced refractory renal cell carcinoma demonstrated antitumor activity and encouraging survival, specifically a dose-dependent increase in overall survival.71 In GM, although temsirolimus was well tolerated, it showed only moderate antitumor activity.72,73 In advanced previously treated breast cancer, a phase II trial found a 9.2% objective response rate to treatment with temsirolimus monotherapy. Increasingly, the focus has turned to the benefit of mTOR inhibitors in combination with tyrosine kinase inhibitors, with the two drugs acting synergistically to arrest tumor growth. There are a variety of phase II trials underway to evaluate this novel combination.
Epigenetic Therapies
Although associated with significant toxicities at higher doses, lower doses of the DNA demethylating agents, 5-aza cytidine and 5-aza 2-deoxycytidine, by reactivating tumor-suppressor genes and inducing differentiation, have shown promise in treatment of hematologic malignancies. Recently the FDA approved these two agents for treating myelodysplastic syndrome.74
Histone deacetylase inhibitors (HDACI) represent another promising therapeutic strategy. The acetylation of certain lysine residues on histones results in increased expression of associated genes. In some tumors, the enzyme histone deacetylase is responsible for abrogation of tumor-suppressor expression.75 Suberoylanilide hydroxamic acid (SAHA) is an HDACI that recently has been approved for the treatment of cutaneous T-cell lymphoma. Other HDACIs are under investigation for treatment of a variety of hematologic and solid malignancies.76 Histone methylation is another modification that can, depending upon the residue involved, result in transcriptional repression. Again, certain compounds that result in removal of these methylation marks have shown promise in cancer cell lines.77
Combining Molecularly Targeted Therapies with Conventional Antineoplastic Treatment
Novel inhibitors of signaling cascades hold great promise in treating human malignancies. Inhibitors of cell signaling, whether antibodies or small-molecule agents, are most likely to affect the treatment of human cancers when combined with standard forms of antineoplastic therapy such as chemotherapy and radiation. Clinical impact will be maximized for combinations of signaling inhibitors and standard cytotoxic agents that result in synergistic effects. Many studies of cell lines in vitro and xenograft tumor models in vivo indicate that treatment with signaling inhibitors augments tumor response to radiation and chemotherapy.78 Effects on cell proliferation, survival, migration, invasion, angiogenesis, and DNA repair constitute molecular mechanisms that may underlie radiosensitization associated with signaling inhibitors. The precise molecular mechanisms, however, remain elusive.
Consistent results in multiple published studies demonstrate that blocking EGFR signaling sensitizes tumor cells to ionizing radiation. In several tumor types, overexpression of EGFR correlates with resistance to radiation in vitro and in vivo.79–82 Furthermore, EGFR overexpression correlates with radiographically measured radiation response in tumors such as GMs in vivo.83 In a model of human squamous-cell carcinoma cells grown in mice, administration of cetuximab together with radiation resulted in complete regression of established xenograft tumors.84,85 Impressive efficacy of concurrent cetuximab and radiation has also been documented in intracranial tumors of human glioma cells grown as xenografts in athymic mice. Small-molecule inhibitors of EGFR similarly sensitize human malignancies to radiation in cell lines in vitro and in animal models of human malignancies in vivo. Gefitinib sensitizes several human cell lines to radiation, as does an additional pharmacologic tyrosine kinase inhibitor, CI-1033, which blocks activities of all four types of ErbB receptors.11 These studies have established the rationale for current clinical trials in which EGFR inhibitors are administered concurrently with radiation in the treatment of adult as well as pediatric malignancies.
Yet more complex combinations of cytotoxic agents and targeted therapies are entering clinical practice. In an open-label, prospective single-arm phase II study, investigators combined erlotinib with radiation therapy and temozolomide. For the 65 patients in this study, median survival was 86 weeks compared to 61 weeks in the two combined historical control studies, with a hazard ratio for survival of 1.72 (CI 1.16-2.54) adjusted for age, extent of resection, and KPS (P < .01). An abstract reporting early results of this study suggested provocative associations between response to multiagent therapy and molecular characteristics in newly diagnosed GMs.86 Among those patients with MGMT (O6-methylguanine-DNA-methyltransferase) promoter methylation, providing a permissive background for response to temozolomide, expression of PTEN was associated with longer survival (P = .06). Although preliminary, these results highlight potential interactions between MGMT methylation and PTEN status within the context of promising clinical results of multimodality therapy. Clearly, acquisition of molecular data is of critical importance in studies that employ targeted inhibitors, particularly as the benefit of multiagent treatment emerges.
Agents that inhibit EGFR signaling offer the dual benefit of potentially inhibiting tumor growth as well as sensitizing resistant tumors to radiation. Similarly promising results indicate that treatment with FTIs sensitizes human cancer cell lines to irradiation. FTIs reverse the radiation resistance of cell lines containing mutant Ras without affecting the radiosensitivity of cells expressing wild-type Ras.78 A critical unanswered question is whether FTIs will also preferentially radiosensitize cells with aberrant signaling cascades that rely on Ras as an intermediary. In a recently reported study, the Pediatric Brain Tumor Consortium conducted a phase II study to assess the efficacy of tipifarnib, an FTI, which was administered concurrently with radiation therapy in children with newly diagnosed intrinsic diffuse brainstem gliomas (BSG). In this setting, tipifarnib exhibited significant toxicities involving primarily neurologic, hematologic, and metabolic systems. Unfortunately, when combined with radiation, tipifarnib offered no benefit in progression-free survival or overall survival compared to historical controls treated with radiation alone.
Antiangiogenic therapy has also shown great promise in enhancing the effects of radiation therapy. Studies in which VEGF mediates resistance to radiation predict that blocking VEGF signaling will augment the cytotoxic effects of radiation. Furthermore, antiangiogenic drugs synergize with radiation to block tumor growth in vivo.87 The in vivo mechanism by which antiangiogenic agents like bevacizumab radiosensitized tumors was at first difficult to discern; after all, antiangiogenic agents, insofar as they intensify hypoxia, would be expected to promote radioresistance.88 However, recent work has unearthed another effect of antiangiogenic therapy on tumor vessels. It has been shown that angiogenesis inhibitors act transiently to normalize tumor vasculature, improving oxygen delivery and thereby enhancing radiation sensitivity.89,90 The preclinical promise of combined treatment with antiangiogenic agents and radiation is currently undergoing intense investigation.
One of the newest strategies for radiosensitization involves the use of HDACIs. Multiple different classes of HDACIs have been shown to sensitize a variety of tumor cell lines to radiation, including the benzamide, MS-275, the short-chain fatty acid derivative, valproic acid, as well as SAHA, CBHA, and the cyclic peptide, depsipeptide.91–93 Clinical trials testing the efficacy of HDACIs are currently ongoing, with one of the more anticipated trials looking at HDACI in combination with temozolomide and fractionated radiotherapy in the treatment of glioblastoma.
Nonreplicating Viral Therapy
Gene Replacement
Initial interest in gene replacement therapy has focused on the tumor-suppressor gene p53. This gene is an attractive option for gene replacement therapy because p53 is mutated in a majority of human tumors and plays a central role in growth arrest, apoptosis, and subsequent response to radiation or chemotherapy or both. Using a replication-defective adenovirus carrying wild-type p53, scientists have shown that this therapy can sensitize many tumor types to radiation therapy.94,95 Phase I clinical trials have demonstrated feasibility and safety of delivering adenoviral vectors expressing p53 through repeated endobronchial injections for non–small-cell lung cancer.96,97 Current trials are investigating the combination of this approach with radiation therapy. Unfortunately, this gene therapy approach only affects a small number of tumor cells near the injection site of the virus. There is no effect on disease at distant sites and therefore no effect on the ultimate outcome of the patient.
p53 Tumor-Suppressor Protein
The p53 tumor-suppressor protein stands at the crossroads of cell death, growth, and differentiation and therefore sustains homeostasis of individual cells, specific tissues, and entire organisms. It regulates apoptosis, proliferation, differentiation, angiogenesis, and cell-matrix interactions.98–100 Each individual tissue and cell lineage incorporates a distinct balance among the various biological processes mediated by p53.101 For example, p53-dependent apoptosis dominates the behavior of hematologic cells, so irradiation of hematologic malignancies such as leukemia and lymphoma produces rapid apoptosis. A clinical corollary of this laboratory observation is that irradiation of hematologic malignancies produces rapid and durable responses. Similarly, in many pediatric tissues, apoptosis plays a key role during organogenesis, and pediatric solid tumors such as Wilms’ tumor and neuroblastoma exhibit significant apoptosis and excellent cure rates when treated with radiation. In contradistinction, in many adult solid tumors, apoptosis plays a minor role and the balance of p53-mediated functions tilts toward proliferation and differentiation. Thus, documentation of p53-mediated apoptosis in vivo following irradiation of adult solid tumors such as GMs and sarcomas has remained elusive.
Despite these complex considerations, the presence of p53 mutations in more than half of human cancers has provided enthusiasm for harnessing p53 to overcome tumor resistance to conventional therapies such as radiation.102 Expression of the protein in cells lacking wild-type p53 enhances cellular sensitivity to ionizing radiation.
In the quest to restore wild-type p53 function and overcome resistance to radiation, two main approaches have been used: gene therapy and pharmacologic molecules that confer wild-type p53 function on mutant forms of p53.103 Genetic introduction of wild-type p53 with viral or nonviral vectors may restore physiologic roles of p53, and such functions, particularly p53-mediated apoptosis, may radiosensitize human tumors. However, many impediments to gene therapy have arisen, including inefficient delivery and detrimental immune responses.
Suicide Gene Therapy
Another therapeutic approach is to use viral vectors to introduce an enzyme that converts an inactive prodrug to a toxic antimetabolite, aiming to increase intratumoral concentration and efficacy of the drug. One such strategy uses adenovirus to introduce the herpes simplex virus tyrosine kinase (HSV-tk) gene into tumor cells followed by administration of acyclovir or ganciclovir. HSV-tk phosphorylates the prodrug, acyclovir or ganciclovir, to an active metabolite. The active drug can diffuse to neighboring uninfected cells through cellular gap junctions, causing toxicity in cells that do not contain the HSV-tk gene. This bystander effect increases tumor kill beyond that observed due to direct infection of tumor cells. A second example of this approach is the use of adenoviral vectors to introduce the bacterial gene cytosine deaminase (CD) followed by 5-fluorocytosine (5FC) administration. CD converts 5FC to the toxic metabolite 5-fluorouracil (5FU). Active 5FU may diffuse throughout the tumor, thereby causing direct cytotoxicity or radiosensitization or both of tumor cells in vitro.104–106 Clinical trials are currently underway with and without radiation therapy in several human tumors.107–112
Radiation-Inducible Viral Therapy
A large number of genes are involved in the response to ionizing radiation: p53, p21, ATM, c-jun, egr-1, c-fos, GADD45, and NF-κB. The induction of genes in response to radiation has led to the engineering of viral vectors to deliver gene therapy within the radiation field, hoping to improve local tumor control while minimizing systemic toxicity. By investigating the genes induced by radiation, radiation response elements (promoters and enhancers) have been identified. These radiation response elements can be inserted upstream from a gene of interest and introduced into a viral vector, creating a radiation-inducible viral therapy. Using this principle, the egr-1 promoter was linked to tumor necrosis factor alpha (TNFα). TNFα is directly cytotoxic to some tumors, activates the immune response, and can increase sensitivity to radiation,113,114 but it is limited in its clinical application because of systemic toxicity. A replication-deficient adenovirus carrying the egr-TNFα construct in combination with radiotherapy has shown dramatic effects on animal models.115,116 Phase I clinical trials with this virus in combination with radiation are currently underway at multiple institutions, with preliminary results demonstrating gene expression in human tumors.117,118
Replicating Viral Therapy
Oncolytic Adenoviral Therapy
Replicating adenoviruses are being investigated as a new method of delivering targeted therapy. Adenoviruses are nonenveloped, linear, double-stranded DNA viruses with a genome size of 38 kb. Adenoviruses can infect dividing and nondividing epithelial cells. Once inside the cell, the virus uses the cell’s normal proteins to produce and package more virus particles. The virus then lyses and kills the cell, exposing neighboring cells to new virus particles. When adenovirus infects normal cells, p53 levels are increased and the cell undergoes either apoptosis or growth arrest, attempting to prevent viral replication. Wild-type adenovirus evades apoptosis and growth arrest by expressing the E1B 55-kD gene, which encodes a protein that binds to and inactivates p53 and thus allows viral replication to proceed.119 One strategy of generating a tumor-specific adenovirus is to mutate the E1B 55-kD gene. ONYX-015 is an adenovirus that lacks the E1B gene; therefore, viral replication is prevented in cells with normal p53 for the previously mentioned reasons.120 Many tumor cell types lack functional p53, which accounts for the tumor-specific replication of the ONYX-015 virus. Loss of p14(arf), a tumor-suppressor gene whose product functionally stabilizes p53, also allows for replication of ONYX-015.121 Put together, the ONYX-015 virus is able to replicate and kill tumor cells that have mutation in the p53 pathway as a whole.
ONYX-015 is now under study for the treatment of p53-deficient malignancies. Recurrent squamous-cell carcinomas of the head and neck region have been treated with the combination of ONYX-015 and chemotherapy.122,123 Head and neck cancers often have mutation in the p53 gene. This fact, in combination with easy access for direct injection of the virus, has made head and neck tumors an ideal place to start investigating the effect of ONYX-015. The phase II trial of ONYX-015 and chemotherapy demonstrated limited toxicity and significant antitumor activity.122 A phase III randomized trial of chemotherapy with and without ONYX-015 showed that the virotherapy group had a significantly greater response rate compared to control for patients with recurrent squamous-cell carcinoma of the head and neck region.125 This virus has also been investigated in primary liver and ovarian tumors, unresectable pancreatic tumors, and malignant gliomas.126–129
A similar approach in developing a tumor-specific adenovirus involves deletion of the E1A gene from the adenoviral genome. E1A binds to and inhibits the action of the tumor-suppressor gene, pRB.130 This serves to release pRB from the transcription factor, E2F, and facilitate entry into the G1 phase of the cell cycle. E2F mediates the transcription of several cellular enzymes necessary for DNA synthesis. The ultimate effect of E1A expression is to create a cellular environment favorable for the synthesis of multiple copies of the viral genome. An adenovirus containing a deletion in the E1A gene will therefore replicate in and lyse cells containing mutations in pRB. The E1A-deleted virus can also replicate in cells mutant in p16, indicating that a deficiency in the pRB pathway as a whole is sufficient to allow for viral replication.131
A second approach to achieve tumor-selective adenoviral replication is the use of tumor- or tissue-specific promoters to drive the expression of an adenoviral gene that is critical for efficient replication, such as E1A. One example of this strategy is CV706, an engineered virus specific for prostate cancer. By placing the prostate-specific antigen (PSA) promoter-enhancer region upstream of the E1A gene, scientists have developed an adenovirus that will replicate only within PSA-producing cells.132 This virus has been used in a phase I trial of intraprostatic injection of patients with locally recurrent prostate cancer following definitive radiation therapy.133 A more potent oncolytic virus, CV787, has been generated using the prostate-specific rat probasin promoter driving E1A expression and the human PSA enhancer/promoter driving the E1B gene. Currently, CV787 is being investigated in patients with either organ-confined disease or hormone-refractory metastatic prostate cancer. In addition, laboratory experiments have shown synergy between radiation therapy and this prostate-specific adenovirus.134 In future studies, this virus may be used in conjunction with more traditional therapies such as surgery or radiation therapy.
Oncolytic Herpesvirus Therapy
Herpes simplex virus 1 (HSV-1) is an enveloped double-stranded DNA virus with a genome size of 152 kb. As with the oncolytic adenoviruses, deletion or inactivation of essential viral genes may lead to tumor specificity. A deletion in the HSV tyrosine kinase (tk) gene prevents replication of the virus in normal cells. Malignant glioma cells are actively dividing and therefore have high endogenous levels of tk, allowing replication of the virus and destruction of the tumor cells. This tk-mutant HSV has not been used in clinical trials because of neurotoxicity and resistance to antiviral therapies (acyclovir and ganciclovir). Investigators have looked instead at developing HSV-1 mutants that maintain sensitivity to acyclovir and ganciclovir and exhibit less neurovirulence by deletion of the γ134.5 gene. The protein product of γ134.5 blocks the inhibition of host protein synthesis in infected cells by interacting with cellular phosphatase 1α to dephosphorylate eIF2α. This leads to production of more progeny virion from each infected cell. Replication of the γ134.5-null mutant is attenuated in normal cells such as adult neurons, thereby minimizing the risk of viral encephalitis. However, γ134.5-null mutants replicate in actively dividing tumor cells. Without disruption of the HSV-tk gene, these mutants remain sensitive to acyclovir and ganciclovir.135,136
G207 is an HSV-1 mutant in γ134.5 with an additional inactivation of the ICP6 gene that encodes for a subunit of the viral ribonucleotide reductase. The presence of two mutations makes spontaneous reversion to a wild-type virus nearly impossible, therefore providing an added level of safety. Mammalian ribonucleotide reductase is elevated in tumor cells relative to normal cells, so HSV-1 mutants defective in ribonucleotide reductase replicate preferentially in tumor cells. G207 HSV is being used in clinical trials for recurrent glioma, with no toxicity seen.137 The γ134.5-null mutant HSV is also being combined with transgene expression of interleukin 4 (IL-4) and IL-12 to combine the oncolytic property of the virus with enhancement of the host’s endogenous T-cell immunity.
Another approach to achieve preferential viral replication in tumor cells is to use tumor- or tissue-specific promoters to drive expression of a critical viral gene. G92a is one such vector in which the ICP4 gene is under transcriptional control of the albumin promoter/enhancer.138 Replication of G92a is several logs more efficient in albumin-expressing hepatoma cells. So far, this virus has only been used in animal models.139
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