Molecular/Targeted Therapy of Cancer

Efficient Gene Delivery: The Major Hurdle to Clinical Benefits

In simple terms, gene therapy is the introduction of nucleic acid into a cell to ameliorate a disease process. For this to be effective, the gene has to be delivered to sufficient numbers of target cells in the body, and this requires a vehicle or vector for delivery. In addition, the gene has to be expressed at a sufficient level and for a length of time appropriate for the disease.²

Early studies on how viruses were able to cause tumors by delivery of their own DNA to foreign cells made them ideal candidates for vectors for gene therapy (i.e., the vehicles by which we could transfer therapeutic genes to patients). However, it was not until the 1980s when work on the retroviral life cycle started to revolutionize the development of gene therapy. In these early studies, it was demonstrated that retroviruses could transfer DNA to cells and this DNA could be stably integrated into the host cell’s genome. Since these early experiments, a number of viruses have been manipulated to act as vehicles for gene delivery. In addition, because of concerns over safety of viral vectors for gene delivery, a number of workers have also explored the possibility of using naked DNA (a therapeutic gene delivered in a bacterial plasmid or lipid/DNA complexes) with some success. The vehicles used for gene delivery and their associated problems are shown in Table 14-1.

The ideal vector would efficiently deliver the gene of interest (transgene) specifically to the cancer cell. It would be easy and cheap to manufacture and would be nonimmunogenic. Obviously the ideal vector does not exist, but there have been great advances in both viral and nonviral delivery systems.²

Targeted Gene Delivery

One of the major barriers to gene therapy becoming accepted in clinical practice is the ability to give vectors systemically and to ensure that therapeutic transgenes are not expressed in normal cells. Numerous strategies have been attempted to provide levels of targeting to spare normal tissue. In a later section, we describe the use of conditionally replicating viruses, which is one method of targeting. Surface modification of viruses (transductional targeting) is also being explored. An example of this is the use of modified fibers on the surface of adenoviruses that only allow the virus to enter cells with specific receptors (Figure 14-2).

A further targeting strategy is transcriptional targeting once the vector has entered the cell.^19-26 Although every gene is represented in every cell of the body, expression of any one gene requires specific transcription factors that may be unique to a particular cell or tissue type. Certain genes have been identified that are expressed in cancer cells but are not expressed in normal cells (e.g., telomerase) or are only expressed in a specific tissue type (e.g. prostate-specific antigen [PSA]). By using the promoter sequences for these genes to drive transgene expression, targeted expression in cancer cells only (e.g., using the promoter for telomerase) or to a specific tissue type (e.g., to the prostate using the promoter for PSA) can be achieved (see Figure 14-2).

Gene Therapy Strategies for Cancer

Despite advances in surgical techniques and the use of radiotherapy and chemotherapy, cancer still remains a disease of high mortality in both human and veterinary medicine, warranting the investigation of alternative treatments. Gene therapy has the potential to play a major role in the development of new cancer therapeutic agents, and there are four broad approaches that can be applied, as follows.

Safety Considerations in Gene Therapy

One of the major considerations in gene therapy revolves around issues of safety, in particular the safety of the vectors used for gene delivery. In 1999, gene therapy suffered a major setback with the death of a patient as a direct result of adenovirus gene therapy. Problems associated with vector delivery include inappropriate inflammatory responses caused by vector delivery (e.g., adenoviruses), the generation of replication-competent viruses (although this is unlikely with new generation vectors), and insertional mutagenesis caused by integrating viruses (e.g., retroviruses).

Until recently, many gene therapy trials had utilized retroviral vectors for gene delivery. There are many advantages to using retroviruses as outlined in Table 14-1. However, retroviruses are also associated with serious diseases of domestic animals and the use of these in gene therapy poses a risk of insertional mutagenesis or the production of replication-competent viruses during the manufacturing process. Realistically, insertional mutagenesis leading to a malignant transformation is an unlikely event because cancer is a multistep process. In fact, there may be a greater risk of malignant transformation from external beam radiation than from the use of retroviruses to treat cancer. The production of replication-competent retroviruses during the production process would also be unlikely because of the rigorous testing that is required prior to clinical application. Many of these issues are resolving with the development of new generation vectors.¹ As an example, in the use of retroviruses and to prevent insertional mutagenesis in normal tissues, one group recently described the use of zinc finger nucleases (engineered DNA-editing enzymes) that allows the insertion of DNA to a site of choice within the genome.⁴⁹ This adds a further level of safety in a high-risk procedure.

In recent years, there has been a shift from using retroviruses in gene therapy to using viruses such as adenovirus and the human adeno-associated viruses (AAV). Adenoviruses may also pose some risks, including inappropriate inflammatory responses leading to serious clinical complications. However, a great deal of work is currently underway to improve viral vectors by removing most of their genetic material to produce “ghost vectors.” These would be potentially less toxic and offer a brighter future for virally mediated gene transfer.

One might imagine that the delivery of naked DNA may offer a safer alternative. However, all of the potential safety issues using this technology are still not fully answered. These include potential risks of autoimmunity and also the actual fate of the DNA when it has been delivered to the patient; in the case of the former, however, there would appear to be no evidence of autoimmunity being a problem in preclinical models.

One of the most exciting developments in cancer gene therapy is the use of conditionally replicating viruses. However, the safety of these vector systems needs special consideration as many of them in their native form could pose a risk to both human and animal health.

New Horizons

Gene therapy promises a completely new approach to the treatment of cancer and represents the newest area of pharmacology. It has suffered over the past 10 years as clinical trials in human medicine have not delivered what they had originally promised. However, one has to put this into context in that many of these studies were conducted in patients with high-grade or end-stage disease and many studies were conducted prematurely without refining the delivery technologies. Clearly, there are a number of technical issues such as safety issues surrounding the delivery and efficiency of the vectors that need to be resolved before gene therapy becomes established clinical practice. Despite gene therapy being very much in its infancy, the field is advancing at a rapid rate. A number of clinical trials have begun in companion animals, and products are in development for clinical application. However, although these treatments would appear to be powerful in preclinical models, it is likely that their greatest benefit will be in the management of patients with minimal disease states. Thus gene therapy will probably have its greatest advantage not as a stand-alone treatment but as an adjunct to more conventional therapies such as surgery, radiation, or chemotherapy.

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24. Song, JS. Adenovirus-mediated suicide SCLC gene therapy using the increased activity of the hTERT promoter by the MMRE and SV40 enhancer. Biosci Biotechnol Biochem. 2005;69(1):56–62.

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26. Fullerton, NE, Boyd, M, Mairs, RJ, et al. Combining a targeted radiotherapy and gene therapy approach for adenocarcinoma of prostate. Prostate Cancer Prostatic Dis. 2004;7(4):355–363.

27. Edelman, J, Edelman, J, Nemunaitis, J. Adenoviral p53 gene therapy in squamous cell cancer of the head and neck region. Curr Opin Mol Ther. 2003;5(6):611–617.

28. Bortolanza, S, Hernandez-Alcoceba, R, Kramer, G, et al. Evaluation of the tumor specificity of a conditionally replicative adenovirus controlled by a modified human core telomerase promoter. Mol Ther. 2004;9:S375.

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35. Yamazaki, M, Straus, FH, Messina, M, et al. Adenovirus-mediated tumor-specific combined gene therapy using Herpes simplex virus thymidine/ganciclovir system and murine interleukin-12 induces effective antitumor activity against medullary thyroid carcinoma. Cancer Gene Ther. 2004;11(1):8–15.

36. Nagayama, Y, Nakao, K, Mizuguchi, H, et al. Enhanced antitumor effect of combined replicative adenovirus and nonreplicative adenovirus expressing interleukin-12 in an immunocompetent mouse model. Gene Ther. 2003;10(16):1400–1403.

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38. Goto, H, Osaki, T, Nishino, K, et al. Construction and analysis of new vector systems with improved interleukin-18 secretion in a xenogeneic human tumor model. J Immunother. 2002;25:S35–S41.

39. Quintin-Colonna, F, Devauchelle, P, Fradelizi, D, et al. Gene therapy of spontaneous canine melanoma and feline fibrosarcoma by intratumoral administration of histoincompatible cells expressing human interleukin-2. Gene Ther. 1996;3(12):1104–1112.

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43. Chiocca, EA, Abbed, KM, Tatter, S, et al. A phase I open-label, dose-escalation, multi-institutional trial of injection with an E1B-attenuated adenovirus, ONYX-015, into the peritumoral region of recurrent malignant gliomas, in the adjuvant setting. Mol Ther. 2004;10(5):958–966.

44. Post, DE, Fulci, G, Chiocca, EA, et al. Replicative oncolytic herpes simplex viruses in combination cancer therapies. Curr Gene Ther. 2004;4(1):41–51.

45. Shah, AC, Benos, D, Gillespie, GY, et al. Oncolytic viruses: clinical applications as vectors for the treatment of malignant gliomas. J Neuroncol. 2003;65(3):203–226.

46. Dirven, CMF, van Beusechem, VW, Lamfers, MLM, et al. Oncolytic adenoviruses for treatment of brain tumours. Exp Opin Biol Ther. 2002;2(8):943–952.

47. Hemminki, A, Kanerva, A, Kremer, EJ, et al. A canine conditionally replicating adenovirus for evaluating oncolytic virotherapy in a syngeneic animal model. Mol Ther. 2003;7(2):163–173.

48. Zhan, JH, Gao, Y, Wang, WS, et al. Tumor-specific intravenous gene delivery using oncolytic adenoviruses. Cancer Gene Therapy. 2005;12(1):19–25.

49. Lombardo, A, Genovese, P, Beausejour, CM, et al. Gene editing in human stem cells using zinc finger nucleases and integrase-defective lentiviral vector delivery. Nat Biotechnol. 2007;25:1298–1306.

Protein Kinases and Normal Cells

Protein kinases play critical roles in normal cell signal transduction, acting to tightly regulate critical cellular processes such as growth and differentiation. These proteins work through phosphorylation; that is, they bind adenosine triphosphate (ATP) and use it to add phosphate groups to key residues on themselves (a process called autophosphorylation) and on other molecules, thereby stimulating a downstream signal inside the cell. This process typically occurs in response to external signals generated by growth factors (GFs) or other stimuli that initiate the cascade. Protein kinases are classified as tyrosine kinases (TKs) if they phosphorylate proteins on tyrosine residues or serine/threonine kinases if they phosphorylate proteins on serine and threonine residues. In some cases the kinases perform both functions (i.e., dual function kinases). These kinases can be expressed on the cell surface, in the cytoplasm, and in the nucleus. The human genome encodes approximately 518 kinases, of which 90 are classified as TKs.¹

TKs on the cell surface that are activated through binding of GFs are called receptor TKs (RTKs). Of the 90 identified TKs, 58 are known to be RTKs. Each RTK contains an extracellular domain that binds the GF, a transmembrane domain, and a cytoplasmic kinase domain that positively and negatively regulates phosphorylation of the RTK (Figure 14-6).^2-4 Most RTKs are monomers on the cell surface and are dimerized through the act of GF binding; this changes the three-dimensional structure of the receptor, permitting ATP to bind and autophosphorylation to occur, resulting in generation of a downstream signal through subsequent binding of adaptor proteins and nonreceptor kinases.² Dysregulation of RTKs resulting in pathway activation/uncontrolled signaling is known to contribute to several human cancers, and work is ongoing to characterize such abnormalities in canine and feline cancers. Examples of RTKs known to play prominent roles in specific cancers include KIT, MET, epidermal growth factor receptor (EGFR), and anaplastic lymphoma kinase (ALK), which can be activated by overexpression, mutation, and chromosomal translocation.^5-9

RKT signaling is critical for regulating typical cell functions and is also an important regulator of angiogenesis, a process known to be essential for continued tumor cell growth. The RTKs involved in angiogenesis include vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), fibroblast growth factor receptor (FGFR), and Tie-1 and Tie-2 (receptors for angiopoietin).^10-13 VEGFRs are expressed on vascular endothelium, and VEGFR signaling drives endothelial migration and proliferation.¹⁰ PDGFR is expressed on stroma and pericytes that are critical for the maintenance of newly formed blood vessels. It also supports angiogenesis by inducing VEGF transcription and secretion.^12,13 FGFR is expressed on vascular endothelium and works with VEGFR to promote increased expression of VEGF.¹² Tie-1 and Tie-2 are expressed on blood vessels in tumors and are important in the recruitment of pericytes and smooth muscle cells to the newly forming vascular channels.¹⁴

Kinases in the cytoplasm act as a bridge, conducting signals generated by RTKs to the nucleus through a series of intermediates that become phosphorylated.¹⁵ The cytoplasmic kinases may be directly on the inside of the cell membrane or free in the cytoplasm. With respect to tumor cell biology, two particular cytoplasmic pathways are often dysregulated in a number of cancers. The first includes members of the RAS-RAF-MEK-ERK/p38/JNK families (Figure 14-7).^16,17 Most of these are serine/threonine kinases, and their activation leads to ERK phosphorylation, translocation into the nucleus, and subsequent alteration of transcription factors and nuclear kinase activity important for controlling the cell cycle. Examples of dysregulation in human cancers include RAS mutations in lung cancer, colon cancer, and several hematologic malignancies and BRAF mutations in cutaneous melanomas and papillary thyroid carcinomas.^18-20

The second cytoplasmic pathway includes phosphatidyl inositol-3 kinase (PI3K) and its associated downstream signal transducers AKT, nuclear factor κB (NFκB), and mTOR, among others (Figure 14-8).^21,22 PI3K is activated by RTKs and in turn activates AKT, which alters several additional proteins involved in the regulation of cell survival, cycling, and growth.²³ AKT phosphorylates targets that promote apoptosis (BAD, procaspase-9, and Forkhead transcription factors) and activates NFκB, a transcription factor that has antiapoptotic activity.^21-23 AKT also phosphorylates other proteins such as mTOR, p21, p27, and glycogen synthase kinase 3 (GSK3). This leads to redistribution of these proteins either in or out of the nucleus, ultimately inhibiting apoptosis while promoting cell cycling.^21-23 Abnormalities of PI3K resulting in pathway activation are commonly found in human cancers, including mutations (breast and colorectal cancers and glioblastoma) and gene amplification (gastric, lung, ovarian cancers).²⁴ This pathway may also become dysregulated through loss of activity of PTEN, a phosphatase that normally acts to dephosphorylate AKT and terminate signaling.^21,25,26 PTEN mutations and/or decreased PTEN expression are found in several human cancers (e.g., glioblastoma and prostate cancer)^24,25 and have been documented in canine cancers (OSA, melanoma).^27-29

RTK-induced signaling ultimately influences cellular events by affecting transcription and the proteins that control cell cycling. The cyclins and their kinase partners (cyclin-dependent kinases [CDKs]) act to regulate the progression of cells through various phases of the cell cycle (Figure 14-9).^30-32 The cyclins comprise several families. Cyclins D and E control restriction point passage by activating their respective CDKs (CDK4 and CDK6 for cyclin D and CDK2 for cyclin E). Coordinated function of cyclins D and E is required for cells to progress from G₁ into S phase (see Figure 14-9). In many cases, RTK-generated signals induce expression of cyclin D, which complexes with CDK4 and CDK6, resulting in phosphorylation of the tumor suppressor Rb, partially repressing its function.^31,32 Functional cyclin D/CDK complexes induce transcription of cyclin E, and active cyclin E/CDK complexes further reduce Rb activity through phosphorylation. This in turn initiates the process of DNA replication important for cell cycling. Dysregulation of the cyclins and CDKs is common in human cancers; for example, overexpression of cyclins D and E is often present in breast, pancreas, and head and neck carcinomas.³²

Protein Kinases and Cancer Cells

Dysfunction of protein kinases is now recognized to be a common event in tumors. Although this has been best characterized in humans, recent data indicate that dog and cat cancers experience similar dysregulation (Table 14-2). Kinases may be dysregulated through a variety of mechanisms, including mutation, overexpression, fusion proteins, or autocrine loops. In the case of mutations, these may result in phosphorylation of the kinase in the absence of an appropriate signal. Such mutations can consist of a single amino acid change through a point mutation, deletion of amino acids, or insertion of amino acids, usually in the form of an internal tandem duplication (ITD). For example, a point mutation occurs in the BRAF gene (V600E, exon 15) in approximately 60% of human cutaneous melanomas.^18,33,34 This amino acid change causes a conformation change in BRAF that mimics its activated form, thereby inducing constitutive downstream ERK signaling and abnormal promotion of cell growth and survival.^35,36 RAS is another kinase that is dysregulated through point mutation in several hematopoietic neoplasms (multiple myeloma, juvenile chronic myelogenous leukemia [CML], acute myelogenous leukemia [AML], and chronic myelomonocytic leukemia [CMML]) and in lung cancer, colon cancer, and many others.^17,37,38

Another example of a mutation involves KIT, an RTK that normally is expressed on hematopoietic stem cells, on melanocytes, in the central nervous system, and on mast cells.³⁹ In approximately 25% to 30% of canine grade 2 and grade 3 mast cell tumors (MCTs), mutations consisting of ITDs are found in the juxtamembrane domain of KIT, resulting in constitutive activation in the absence of ligand binding. These mutations are associated with a higher risk of local recurrence and metastasis.^40-42 KIT mutations consisting of deletions in the juxtamembrane domain are also found in approximately 50% of human patients with gastrointestinal stromal tumors (GISTs) and are also found in GISTs in dogs.^43-45 There are now several well-characterized mutations involving RTKs in human cancers, including FLT3 ITDs in AML,^46-49 EGFR point mutations in lung carcinomas,^50,51 and PI3K mutations in several types of carcinomas.²⁴

Overexpression of kinases usually involves the RTKs and may result in enhanced response of the cancer cells to normal levels of growth factor; or, if the levels are high enough, the kinase may become activated through spontaneous dimerization in the absence of signal/growth factor. In humans, the RTK human epidermal growth factor receptor 2 (HER2; also known as ErbB2, a member of the EGFR family) is overexpressed in both breast and ovarian carcinomas and often correlates with a more aggressive phenotype.^4,52,53 EGFR is also overexpressed in human lung, bladder, cervical, ovarian, renal, and pancreatic cancers, and some tumors have as many as 60 copies of the gene per cell.^7,54,55 As with HER2, such overexpression is linked to a worse outcome in affected patients.⁷

Fusion proteins are generated when a portion of the kinase becomes attached to another gene through chromosomal rearrangement and the normal mechanisms that control protein function are disrupted. One of the best characterized fusion proteins is BCR-ABL, which is found in 90% of patients with CML.^56-59 ABL is a cytoplasmic tyrosine kinase that, when fused to BCR, results in dysregulation of ABL, inappropriate activity of the protein, and resultant malignant transformation. Other examples include TEL-PDGFRβ in CMML, FIP1-PDGFRα in hypereosinophilic syndrome with mastocytosis, and (EML4)-ALK in non–small-cell lung cancer (NSCLC).⁶⁰

Autocrine loops of activation primarily occur when the tumor cell expresses both the RTK and the growth factor; in most cases, one or the other usually is also overexpressed, resulting in constitutive activation of the RTK. Examples include coexpression of transforming growth factor α (TGFα) and EGFR in glioblastoma and squamous cell carcinoma, insulin-like growth factor (IGF) and its ligand, IGF-1R, in breast and colorectal cancer, and VEGF and VEGFR in melanoma.^4,61-63 In canine cancers, possible autocrine loops have been documented in OSA (co-expression of MET and its ligand, HGF) and hemangiosarcoma (HSA; co-expression of KIT and its ligand, SCF).^64-66

Inhibition of Kinases

Given the detailed molecular characterization of signal transducer dysregulation in cancer cells, significant efforts have been directed at developing strategies to inhibit those transducers that participate in tumorigenesis through direct effects on the cancer cell or through modulation of tumor microenvironment (stroma and neovasculature). The two most successful approaches to date have been monoclonal antibodies (MAbs) and small molecule inhibitors.

Several antibodies have been developed to target the extracellular domain of RTKs known to be important in a variety of tumors. These antibodies may prevent the growth factor from binding, may promote internalization of the RTK and subsequent degradation, or may induce an immune response against the cancer cell. One of the most successful examples is a humanized MAb called trastuzumab (Herceptin). This antibody targets HER2, which as previously discussed is overexpressed in approximately 30% of breast cancers, as well as other cancers, including prostate cancer, ovarian cancer, and NSCLC.⁶⁷ In initial clinical trials, trastuzumab treatment of HER2-positive breast cancer resulted in a response rate of approximately 25% in patients with metastatic disease.⁶⁸ The response rate approached 50% when trastuzumab was combined with chemotherapy.⁶⁹ When used in the adjuvant setting, multiple studies have demonstrated that trastuzumab markedly improves survival rates of women with HER2-positive disease; trastuzumab is now part of the routine standard of care for this disease.^70,71 Other examples of MAbs that have demonstrated significant activity in human cancers include rituximab (Rituxan) that targets CD20 expressed in a number of B-cell lymphomas^72,73 and cetuximab (Erbitux) that targets ERBB1/HER1 EGFR known to be overexpressed in several carcinomas.^7,67,74

Small molecule inhibitors work primarily by blocking the ATP-binding site of kinases, essentially acting as competitive inhibitors; a smaller number of these inhibitors work by preventing necessary protein-protein interactions (allosteric inhibition).⁷⁵ In the absence of ATP, the kinase is unable to phosphorylate itself or downstream signaling elements, thereby interrupting a survival/growth signal essential to the tumor cell, ultimately resulting in cell death. As the molecular characterization of tumors has improved, the development and application of small molecule inhibitors have rapidly expanded in human oncology and their use is markedly altering how cancers are managed. Such inhibitors often are easy to synthesize in large quantities, frequently orally bioavailable, and can readily enter cells to bind the intended target.

The first small molecule inhibitor to be approved for human use was imatinib (Gleevec), an orally administered drug that binds the ATP pocket of ABL, as well as the RTKs KIT and PDGFRα.⁷⁶ As previously discussed, BCR-ABL fusion proteins are present in 90% of human patients with CML, making ABL a good target for therapeutic intervention. The application of imatinib to CML has been transformative, with significant biologic activity demonstrated in several clinical trials, resulting in the approval of imatinib for up-front care of affected individuals.^77-82 In the chronic phase of CML, imatinib induces a remission rate of close to 95%, and most patients remain in remission for longer than 1 year. Unfortunately, the remission rate is much lower for patients in blast crisis (20% to 50%), often lasting less than 10 months. Resistance to imatinib has been well characterized and is primarily due to the development of mutations in ABL that preclude drug binding, although gene amplification has also been documented.^83,84 Imatinib also has clinical activity against human GIST in which 60% to 80% of the tumors have point mutations or deletions in the juxtamembrane domain of KIT, resulting in constitutive activation.^85,86 Response rates of 50% to 70% have been reported with imatinib, far better than the 5% response rate seen with standard chemotherapy.^87,88 A small number of GISTs have activating mutations in PDGFRα instead of KIT mutations; these patients also respond to imatinib.⁸⁹

There are now several small molecule inhibitors approved for the treatment of human cancers that possess specific mutations in kinases known to drive tumor growth and survival. A subset of patients with NSCLC have tumors with activating mutations in EGFR that respond to erlotinib (Tarceva) or gefitinib (Iressa), small molecule inhibitors of EGFR.⁹⁰ Response rates in patients with EGFR mutations can be as high as 80% compared to less than 10% to 20% for those without, demonstrating that efficacy of targeted therapies often depends on the presence of a known activated signaling element. A small number of patients with NSCLC also exhibit activation of the RTK ALK through its fusion to EML4.⁹¹ A small molecule inhibitor of ALK, crizotinib (Xalkori), has demonstrated significant activity against lung cancer patients whose tumors express the EML4-ALK translocation. In a recent phase II study, an objective response rate of 56% was achieved in this subset of patients, with another 31% of patients experiencing stable disease.^91,92 As expected with a targeted therapeutic that disrupts key cell-signaling events, responses were rapid with most occurring by 8 weeks of treatment.

Vemurafenib (Zelboraf) is a small molecule inhibitor of BRAF that has shown significant activity against cutaneous malignant melanomas that possess activating mutations in BRAF. Most patients treated with vemurafenib experienced tumor shrinkage, with close to 50% meeting the criteria for objective response.⁹³ This compares to an objective response rate of only 5% in patients treated with dacarbazine. Inhibition of mTOR has become of interest in several cancers given the activation of the PI3K pathway and the critical role of mTOR in mediating its effects. Rapamycin, a drug used for many years as an immunosuppressive agent, is the prototypical mTOR inhibitor.^94,95 Temsirolimus and everolimus, two rapamycin analogs, have already been approved for use in patients with metastatic renal carcinoma and other mTOR inhibitors are currently under investigation for their potential utility in treating soft tissue sarcomas and bone sarcomas.^94,95

Flavopiridol, a partly synthetic flavonoid derived from an indigenous plant (rohitukine) found in India, sits in the ATP-binding pocket of CDK2, acting as a competitive inhibitor.^89,90 This compound has been shown to inhibit most of the CDKs evaluated, although some less potently than others.^91,92 Although flavopiridol initially failed to demonstrate significant efficacy when used as a single agent,⁸⁹ recent pharmacokinetic data demonstrated activity in some patients, particularly those with chronic lymphocytic leukemia (CLL), when an alternative dosing schedule that enhanced the area under the curve was employed.⁹⁶

The inhibitors discussed previously tend to affect a restricted set of kinases, although other drugs exhibit much more broadly targeted inhibition. Sunitinib (Sutent) is a small molecule inhibitor of several RTKs, including VEGFR1, VEGFR2, PDGFRα/β, KIT, FLT3, receptor of colony-stimulating factor 1 (CSFR1), and rearranged during transfection receptor (RET).⁹⁷ The multitargeted nature of this inhibitor may be responsible for its observed activity in several types of cancer, including GIST, renal cell carcinoma, thyroid carcinoma, and insulinoma, among others.⁹⁷ Although such agents often have significant clinical activity, they are typically associated with a broader range of toxicities that may limit their use.

Kinase Inhibitors in Veterinary Medicine

There are now two small molecule inhibitors approved for use in dogs in veterinary medicine. Toceranib (Palladia) is a multitargeted inhibitor closely related to sunitinib that exhibits a similar target profile, including VEGFR, PDGFR, KIT, FLT3, and CSF1R. Toceranib has demonstrated activity against MCT, as well as sarcomas and carcinomas. In the original phase I study, 28% of dogs experienced objective responses to treatment, with an additional 26% experiencing stable disease for an overall biologic activity of 54%.⁹⁸ A pivotal study of toceranib was subsequently conducted in dogs with recurrent or metastatic intermediate or high grade MCT resulting in an objective response rate of 42.8% (21 complete responses, 42 partial responses), with an additional 16 dogs experiencing stable disease for an overall biologic activity of 60%.⁹⁹ Dogs whose MCT harbored activating mutations in KIT were roughly twice as likely to respond to toceranib than those without mutation (69% versus 37%). Following approval of toceranib in 2009, it has been used to treat a number of different solid tumors.¹⁰⁰ Preliminary observations of biologic activity were reported in dogs with anal sac adenocarcinoma, thyroid carcinoma, head and neck carcinoma, nasal carcinoma, and OSA. Several studies are ongoing to more clearly define the role of toceranib in the treatment of canine and feline cancer.

Masitinib (Kinavet) is a small molecule inhibitor of KIT, PDGFRα/β, and Lyn. In dogs with MCTs, masitinib significantly improved time to progression compared to placebo, and outcome was improved in dogs with MCT harboring KIT mutations.¹⁰¹ Subsequent follow-up of patients treated with long-term masitinib identified an increased number of patients with long-term disease control compared to those treated with placebo (40% versus 15% alive at 2 years).¹⁰² Finally, small studies have evaluated the efficacy of imatinib for the treatment of canine and feline MCT.^103-105 Imatinib was well tolerated, and objective antitumor responses were observed in dogs with both mutant and wild-type KIT. Responses have also been observed in cats with MCT.^106,107

Conclusion

With the advent of molecular techniques, the characterization of signal transduction pathways that are dysfunctional in cancer cells has become commonplace. Advances in computer modeling and small molecule engineering have led to the rapid development of inhibitors capable of blocking specific pathways critical for cancer cell survival. The success of inhibitors such as imatinib and crizotinib indicate that the application of this therapeutic strategy can markedly improve clinical outcome. Perhaps the greatest challenges will be determining how these novel therapeutics can be effectively combined with standard treatment regimens such as chemotherapy and radiation therapy to provide optimal anticancer efficacy without enhancing toxicity and identifying strategies to use these therapeutics in ways that are less likely to result in drug resistance.

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Tumor Angiogenesis

Angiogenesis has emerged as a validated therapeutic target in oncology over the last several years, particularly with the widespread approval of drugs that target the proangiogenic vascular endothelial growth factor (VEGF) signaling pathway.^1-5 Drugs such as bevacizumab (Avastin), the humanized anti-VEGF monoclonal antibody, and small molecule RTK inhibitors (RTKIs) such as sunitinib (Sutent) and sorafenib (Nexavar) in human oncology and toceranib (Palladia) in veterinary medicine are all considered to be efficacious, at least in part, because of an antiangiogenic mechanism of action.^6,7 The guiding principle of tumor angiogenesis states that in order for a solid tumor to grow beyond a few millimeters in size, it must recruit its own blood supply to provide adequate nutrients and oxygen to the dividing cell mass, as well as the removal of waste products.^8,9 Inducing angiogenesis is considered a hallmark of cancer progression,¹⁰ yet angiogenesis is a prominent aspect of normal physiology and transient pathology (e.g., in wound healing) and is tightly regulated. A large number of proangiogenic growth factors and signaling pathways promote the process of blood vessel growth.⁸ Similarly, endogenous inhibitors of angiogenesis are temporally expressed to suppress vessel expansion and maintain angiogenic balance.¹¹ The net effect depends on this relative balance, which is tipped toward promoting angiogenesis during tumor growth. Therapeutic interventions aim to tip the scales back toward inhibition of tumor angiogenesis.¹²

Angiogenesis occurs through the sprouting of new vessels from existing vasculature, whereas the term vasculogenesis is generally used to describe the de novo formation of new blood vessels from bone marrow–derived progenitor cell populations that respond to locally produced proangiogenic signals.^13,14 In addition, although controversial, circulating endothelial progenitor cells (CEPs) from the bone marrow may contribute to tumor angiogenesis by travelling to tumor sites and incorporating into existing vessel walls.^15-18 Finally, mature endothelial cells originating from the growing vasculature itself may be shed into the bloodstream, and are referred to as circulating endothelial cells (CECs).¹⁹ These CECs may represent a byproduct of local angiogenesis rather than an indicator of bone marrow involvement. At the present time, there is still much to learn regarding the morphology, surface marker expression, and relative contribution circulating cell populations make to tumor blood vessel growth and maintenance.^20,21

Despite the principles of angiogenesis finding extensive application in the field of solid tumor growth, blood vessels are also a normal component of the bone marrow microenvironment, and angiogenesis is a prominent feature of malignant bone marrow disorders. Many of the same growth factor and inhibitory pathway components described in solid tumor angiogenesis contribute to the biology of myelodysplasia, leukemias, myeloma, and lymphoma, among other “liquid” tumors.²² As a result, targeting angiogenesis is being investigated for the treatment of blood and bone marrow neoplasia and, indeed, one such agent with demonstrated antiangiogenic effects, thalidomide, has been approved for treatment of human multiple myeloma.²³

In addition to blood vessels, lymphatics are an important component of the tumor microenvironment and a prominent feature of tissue homeostasis, immunosurveillance, and a gateway to metastatic spread.²⁴ The regulation of lymphatic vessel growth is physiologically similar in principle to that of blood vessels, although the contribution of cells from the bone marrow to the formation and maintenance of lymphatic channels may be equally controversial.²⁵ Similar to angiogenesis, targeting lymphangiogenesis is an attractive prospect in clinical oncology that is receiving an increased research focus.²⁶

Antiangiogenic Therapy

There are multiple theories to explain the potential antitumor effects that result from antiangiogenic therapies.³ The first and most intuitive is vascular collapse resulting in impaired oxygen delivery to the tumor, leading to nutrient starvation, hypoxia, and death of cancer cells that cannot survive in this oxygen-deprived environment. Conversely, a second mechanism actually involves more efficient delivery of oxygen, nutrients, and indeed other drugs (such as chemotherapeutics) to tumor cells by a process termed vessel normalization.^27,28 Normalization occurs when smaller, tortuous, inefficient, and leaky tumor vessels are selectively destroyed, resulting in improved blood flow through the tumor as a whole.²⁹ Vascular normalization has been demonstrated clinically using functional magnetic resonance imaging (fMRI) methods in patients undergoing treatment with VEGF pathway inhibitors.^30,31 The most common clinical approach to targeting tumor angiogenesis has been either inhibition of overexpressed proangiogenic stimuli or supplementation of factors that inhibit angiogenesis. Targeting vasculogenesis through neutralization of CEPs is also a potential treatment strategy.³²

Direct Targeting of Tumor Endothelial Cell Surface Markers

Tumor endothelial cells differ from normal endothelia in their genetic and protein composition.⁵⁰ These differences may represent an opportunity for a favorable therapeutic index when drugs are designed to bind differentially expressed targets on tumor-specific endothelium. If not naturally destructive to these cells, drugs can be designed to carry a payload to induce local cytotoxicity, resulting in an antiangiogenic effect. A phage vector delivering tumor necrosis factor (RGD-A-TNF) to αV integrins on tumor endothelium is one example of such a strategy that has undergone evaluation in dogs.⁵¹ Through serial biopsy, this dose escalation trial was able to demonstrate selective targeting of tumor endothelium via αV integrin-targeted expression of TNF, and treatment of a cohort of tumor-bearing dogs with the maximum tolerated dose (MTD) resulting in partial remission in 2/14 and stable disease in 6/14 dogs.

Other Antiangiogenic Agents

A vast array of drugs not necessarily designed as anticancer therapeutics may derive at least a portion of their activity through inhibition of angiogenesis. Two examples investigated in a veterinary setting include inhibitors of matrix metalloproteinase (MMP) and cyclooxygenase (COX). The MMPs are a family of enzymes that degrade the extracellular matrix and basement membrane, thereby mediating tumor invasion, angiogenesis, and metastasis.⁵² Unfortunately, clinical evaluation of compounds that inhibit these enzymes has to date been largely unrewarding,^53,54 including results from a large randomized trial of 303 dogs with OSA, in which treatment with the MMP inhibitor Bay12-9566 or placebo after doxorubicin chemotherapy did not improve overall survival.⁵⁵ Inhibition of the proinflammatory COX enzymes has been commonly studied in numerous tumor types in veterinary oncology.^56-58 The effects of COX inhibition on reducing angiogenesis specifically have been evaluated with piroxicam treatment of canine transitional cell carcinoma (TCC). In a study of 18 dogs, piroxicam treatment was associated with reduced urinary concentrations of the proangiogenic growth factor basic fibroblast growth factor (bFGF) and induction of apoptosis.⁵⁹ In a subsequent study evaluating these parameters in 12 dogs treated with piroxicam in combination with cisplatin, reductions in urinary bFGF and VEGF were associated with response to the combination regimen.⁶⁰

Metronomic Chemotherapy

Conventional cytotoxic chemotherapy agents that have been in clinical use for many decades also achieve at least a portion of their therapeutic efficacy by inhibition of tumor angiogenesis.^61,62 The basis for this notion likely originated with the observation that tumor endothelial cell populations are much more proliferative than the quiescent endothelium found elsewhere in the body. These rapidly dividing endothelial cells would be targets of chemotherapy in the same way that other proliferative cells such as intestinal epithelia and bone marrow precursors are damaged. The key to maximizing the antiangiogenic potential of chemotherapeutic drugs was considered to be through reduction or elimination of the break period between doses. It is during this break period that repair and repopulation of endothelial cells were likely to occur, as they do for other cell populations.⁶¹ However, conventional chemotherapeutic protocols are largely based on the principle of MTD, which originated through models of optimized tumor cell kill that require a break period to allow recovery of normal cell populations.^63,64 Despite the success of MTD chemotherapy protocols for the treatment and outright cure of certain cancers, critics of such models point out that the preclinical experiments were performed in exponential growth phase cell culture models, and the exposure time of the cells to chemotherapy was not an included variable.⁶⁵ More continuous exposure to chemotherapy, with reduction or elimination of the break period between each dose, has come to be known as “metronomic” delivery.⁶⁶ Not surprisingly, dose reduction is required to provide a more continuous treatment schedule. Generally speaking, metronomic chemotherapy protocols are well tolerated. The low toxicity profile, combined with ease of administration when oral drugs are used, and often lower cost make metronomic chemotherapy protocols appealing in veterinary oncology. However, mechanistic and clinical evaluation in veterinary patients is still at an early stage.

Clinical Trial Evaluation

Recent small retrospective and phase II clinical trials have been reported in human and veterinary oncology with a variety of chemotherapy drugs and combinations.^80-86 Most clinical trial evaluations to date have paired conventional chemotherapy drugs with noncytotoxic drugs that target angiogenesis directly or indirectly. Preclinical results demonstrated over a decade ago that such combinations resulted in superior outcomes, likely due to more robust neutralization of the prosurvival functions of VEGF in the tumor endothelium.^87,88 As a result, most clinical trials have evaluated combination approaches without necessarily any clear prior documentation of single-agent clinical activity for each drug. Bevacizumab has been a popular choice to pair with human metronomic chemotherapy schedules and is FDA-approved for multiple indications.^83,89-92 Other, more readily available “targeted” drugs, shown to have at least indirect antiangiogenic effects, are also commonly utilized. Examples include COX inhibitors,^81,93-97 tetracyclines, thalidomide,^23,98 and others.⁹⁹ Generally speaking, in contrast to MTD chemotherapy protocols, targeted drugs are often given for longer periods of time at their optimal biologic dose (OBD). As a result, targeted drug combinations with metronomic chemotherapy may be an effective treatment strategy, as each follows a similar dose and scheduling principle. In addition, it is possible that there may be a role for targeted antiangiogenic treatment or metronomic chemotherapy in combination with MTD chemotherapy schedules to neutralize rebound angiogenic responses that occur during the break period. This approach has been demonstrated successfully in preclinical studies.^72,100 Ultimately, rigorous clinical trials are necessary to identify and optimize successful drug combinations and schedules of antiangiogenic drugs and chemotherapy.

Biomarkers for Antiangiogenic and Metronomic Therapy

The identification and application of biomarkers is becoming increasingly important as the field of oncology moves further into clinical application of targeted therapeutics.¹¹³ The aims of validated biomarkers are to (1) predict patient populations that will or will not respond to treatment, (2) monitor response to therapy on a cellular or molecular level, or (3) determine the proper therapeutic dose for targeted agents that often possess optimal biologic activity at doses well below the traditionally defined MTD. The use of biomarkers for antiangiogenic agents is valid in all three categories.

Tumor tissue expression and/or blood-based circulating growth factor levels have been the most popular approaches to predicting response to angiogenesis inhibitors, with VEGF being the most studied molecule.^114-116 For example, in the study of metronomic cyclophosphamide and celecoxib by Marchetti and colleagues, low baseline plasma VEGF was predictive of response.¹⁰⁶ In many cases it may be through comparison of pretreatment to posttreatment levels that insight will be gained from this surrogate marker, as was demonstrated with urinary bFGF and VEGF with piroxicam treatment of canine TCC.^59,60 The most important biomarker in angiogenesis may be one that defines tumor response because inhibition of angiogenesis does not necessarily correspond to a reduction in tumor volume. This static response sits in sharp contrast to MTD chemotherapy approaches that result in measureable tumor shrinkage. Systems for clinical evaluation of tumor response to therapy are often formally based on World Health Organization (WHO) criteria or response evaluation criteria in solid tumors (RECIST), which equate tumor shrinkage with positive outcome. Therefore there may be a need for both recognition of sustained stable disease as a desirable clinical trial endpoint and biomarkers to evaluate angiogenic function during treatment. Imaging modalities that can provide information about vascular function are a potentially valuable tool for monitoring tumor angiogenesis and the effects of antiangiogenic therapy.¹¹⁴ Dynamic contrast-enhanced MRI (DCE-MRI) has been studied for its ability to quantitatively assess vascular parameters.¹¹⁷ A study by MacLeod and colleagues demonstrated blood volume and permeability measurements in canine intracranial masses using this modality.¹¹⁸

The area of dose optimization also represents an unmet need for biomarkers in antiangiogenic and metronomic chemotherapy. Tregs are providing insight in this regard. Recent results from Burton and coworkers demonstrated a dose-dependent reduction in Tregs with metronomic cyclophosphamide treatment, observed at 15 mg/m² but not 12.5 mg/m² doses, leading the investigators to conclude that 15 mg/m² may be a more appropriate dose for future clinical trials.⁷⁹ In addition to Treg levels, temporal evaluation of other markers has been studied. Assessment of CECs and/or CEPs has been applied for biomarker analysis of dosing antiangiogenic and metronomic therapy.^75,97,119 For example, in the study by Rusk and colleagues, decreasing CEC levels in dogs treated with the thrombospondin-1 mimetic peptide ABT-526 may have indicated adequate exposure to the antiangiogenic drug, which in this study was utilized at a single dose.⁴⁵

Side Effects (Adverse Events)

Despite the therapeutic index achieved by treating activated versus dormant vasculature, there is still potential risk that drug effects on vessels have unwanted consequences. For bevacizumab, potential side effects of a vascular etiology include hypertension, edema, hemorrhage, thromboembolism, proteinuria, intestinal perforation, and impaired wound healing.¹²⁰ Through further clinical evaluation, we will learn the relative risks for different antiangiogenic drugs applied to a number of tumor settings. Resistance to antiangiogenic therapy has also become evident from early clinical data. Given the genetically stable nature of the blood vessel/endothelial cell target compared to the mutationally prone cancer cell population, antiangiogenic treatment was originally postulated to be less likely to demonstrate drug resistance.⁷¹ Proposed mechanisms of resistance to antiangiogenic therapies involve both tumor and host-mediated pathways that can be intrinsic or induced by treatment.¹²¹ One alarming consequence of antiangiogenic treatment that has emerged from recent preclinical studies is the potential that certain drugs may alter the host microenvironment, leading to metastatic conditioning.^122,123 This phenomenon of increased invasion and metastasis as a result of antiangiogenic drug treatment, despite successful suppression of primary tumor growth, has been demonstrated in preclinical models with certain antiangiogenic RTKIs. The drugs studied include sunitinib, an RTKI with a similar target spectrum to toceranib.¹²³ Further validation and investigation into potential explanations for these provocative results are ongoing.

Finally, it is worth noting that similar to most other drugs, antiangiogenic agents are not innocuous and may have a unique side-effect profile that is independent of their effects on blood vessels or the tumor microenvironment. Side effects for toceranib include dose-dependent gastrointestinal upset, including bleeding, myelosuppression, azotemia, anemia, lethargy, or lameness.^124-126 Masitinib’s side effects include gastrointestinal upset, regenerative or nonregenerative anemia, and protein-losing nephropathy.^35,124 Cyclophosphamide has become a popular drug for metronomic scheduling but is associated with the potential for sterile hemorrhagic cystitis.¹²⁷ It is currently unknown whether cystitis is more frequent in metronomic protocols; however, close urinary monitoring is strongly recommended for these patients. Similarly, chlorambucil has been clinically evaluated for metronomic use¹⁰⁴ and can be used as an alternative to cyclophosphamide, although it is still not known whether drug efficacy will be similar for different drugs applied across multiple tumor types.

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111. Havlicek, M, Straw, RS, Langova, V, et al. Intra-operative cisplatin for the treatment of canine extremity soft tissue sarcomas. Vet Comp Oncol. 2009;7(2):122–129.

112. Selting, KA, Wang, X, Gustafson, DL, et al. Evaluation of satraplatin in dogs with spontaneously occurring malignant tumors. J Vet Intern Med. 2011;25:909–915.

113. Park, JW, Kerbel, RS, Kelloff, GJ, et al. Rationale for biomarkers and surrogate end points in mechanism-driven oncology drug development. Clin Cancer Res. 2004;10:3885–3896.

114. Drevs, J, Schneider, V. The use of vascular biomarkers and imaging studies in the early clinical development of anti-tumour agents targeting angiogenesis. J Intern Med. 2006;260:517–529.

115. Sandri, MT, Johansson, HA, Zorzino, L, et al. Serum EGFR and serum HER-2/neu are useful predictive and prognostic markers in metastatic breast cancer patients treated with metronomic chemotherapy. Cancer. 2007;110:509–517.

116. Lindauer, A, Di, GP, Kanefendt, F, et al. Pharmacokinetic/Pharmacodynamic modeling of biomarker response to sunitinib in healthy volunteers. Clin Pharmacol Ther. 2010;87:601–608.

117. Morgan, B, Thomas, AL, Drevs, J, et al. Dynamic contrast-enhanced magnetic resonance imaging as a biomarker for the pharmacological response of PTK787/ZK 222584, an inhibitor of the vascular endothelial growth factor receptor tyrosine kinases, in patients with advanced colorectal cancer and liver metastases: results from two phase I studies. J Clin Oncol. 2003;21:3955–3964.

118. MacLeod, AG, Dickinson, PJ, LeCouteur, RA, et al. Quantitative assessment of blood volume and permeability in cerebral mass lesions using dynamic contrast-enhanced computed tomography in the dog. Acad Radiol. 2009;16:1187–1195.

119. Bertolini, F, Mancuso, P, Shaked, Y, et al. Molecular and cellular biomarkers for angiogenesis in clinical oncology. Drug Discov Today. 2007;12:806–812.

120. Shih, T, Lindley, C. Bevacizumab: an angiogenesis inhibitor for the treatment of solid malignancies. Clin Ther. 2006;28:1779–1802.

121. Ebos, JM, Lee, CR, Kerbel, RS. Tumor and host-mediated pathways of resistance and disease progression in response to antiangiogenic therapy. Clin Cancer Res. 2009;15:5020–5025.

122. Paez-Ribes, M, Allen, E, Hudock, J, et al. Antiangiogenic therapy elicits malignant progression of tumors to increased local invasion and distant metastasis. Cancer Cell. 2009;15:220–231.

123. Ebos, JM, Lee, CR, Cruz-Munoz, W, et al. Accelerated metastasis after short-term treatment with a potent inhibitor of tumor angiogenesis. Cancer Cell. 2009;15:232–239.

124. London, CA. Tyrosine kinase inhibitors in veterinary medicine. Top Companion Anim Med. 2009;24:106–112.

125. London, CA, Hannah, AL, Zadovoskaya, R, et al. Phase I dose-escalating study of SU11654, a small molecule receptor tyrosine kinase inhibitor, in dogs with spontaneous malignancies. Clin Cancer Res. 2003;9:2755–2768.

126. London, CA, Malpas, PB, Wood-Follis, SL, et al. Multi-center, placebo-controlled, double-blind, randomized study of oral toceranib phosphate (SU11654), a receptor tyrosine kinase inhibitor, for the treatment of dogs with recurrent (either local or distant) mast cell tumor following surgical excision. Clin Cancer Res. 2009;15:3856–3865.

127. Charney, SC, Bergman, PJ, Hohenhaus, AE, et al. Risk factors for sterile hemorrhagic cystitis in dogs with lymphoma receiving cyclophosphamide with or without concurrent administration of furosemide: 216 cases (1990-1996). J Am Vet Med Assoc. 2003;222:1388–1393.

DNA Methylation

In addition to the information encoded within the genome sequence, it has been shown that epigenetic changes are of great importance in the modification and maintenance of gene expression. These changes take place through a number of mechanisms, including polymerase enzyme modulation, chromatin condensation, and DNA methylation. Mammalian DNA is methylated at cytosines within cytosine-guanine (CpG) dinucleotide sequences. During tissue differentiation, the methylation pattern is one governor of tissue-specific gene expression and thus phenotype.^1-3

Two different methylation-related phenomena have been identified in cancer. First, tumor cell DNA has been shown in both dogs and other mammals to be globally hypomethylated,^4,5 specifically in pericentromeric satellite sequences. This may lead to decreased genome stability and an increase in the incidence of oncogenic chromosome defects. Indeed, the purposeful induction of genomic hypomethylation by reduction in germline DNA methyltransferase-1 (DNMT1) levels in genetically engineered mice is associated with a high incidence of T-cell lymphomas displaying trisomy 15.⁶ Second, cancer cells also acquire sequence-specific promoter hypermethylation and transcriptional repression in normally unmethylated regions, several of which have been shown to be associated with known tumor suppressor genes, including Rb, p16, p73, and the von Hippel-Lindau protein (VHL),^1,2,7-9 or other important tumor-associated genes such as E-cadherin and estrogen and retinoic acid receptors.¹⁰

The methylation of DNA is controlled by four known DNMTs, of which DNMT1 may be the most important in cancer.^1-3 A variety of agents can inhibit DNMT function. The two best studied are 5-azacytidine (Vidaza) and 5-aza-deoxycitidine (decitabine, Dacogen), which are nucleoside analogs that incorporate into DNA and inhibit DNMT activity but allow replication to proceed. A large number of single-agent clinical trials with these agents have been reported, and significant activity has been demonstrated in hematopoietic neoplasia, leading to the Food and Drug Administration (FDA) approval of 5-azacytidine and decitabine for the treatment of myelodysplastic syndrome.^11,12 Encouraging response rates to the nucleoside analog decitabine have also been seen in human patients with imatinib-refractory CML.^13,14 Results in advanced solid tumors have been generally disappointing^15,16; however, studies in combination with standard antineoplastic therapy and other targeted agents (e.g., histone deacetylase inhibitors) are ongoing.¹⁷ One drawback of these drugs is the requirement of DNA replication for activity. There are various other drugs that have DNMT inhibitory activity in nonreplicating cells, including green tea polyphenols¹ and marine products of the psammaplin class,¹⁸ a synthetic derivative of which concurrently inhibits histone deacetylase (see later) and is in human clinical trials.¹⁹ Interestingly, the commonly used cardiac medications procainamide and hydralazine also possess demethylating activity,^10,20 and clinical trials have demonstrated alterations in promoter methylation and reactivation of silenced genes following administration of well-tolerated doses of hydralazine to human cervical cancer patients.²¹ Hydralazine-valproic acid combinations have demonstrated activity in myelodysplastic syndrome in early human trials.²² Procainamide and hydralazine have a long track record of use in veterinary medicine and as such could serve as interesting and readily available drugs for the initial evaluation of methylation inhibition in veterinary cancer patients.

Two potential problems exist regarding the wide clinical implementation of DNMT inhibitors for cancer treatment. As discussed previously, induction of long-term genome-wide hypomethylation could decrease chromosome stability leading to potentially tumorigenic chromosome rearrangements.³ Demethylation could also trigger the reactivation of genes promoting a more aggressive or metastatic phenotype.³ In support of this theory, treatment of nonmetastatic breast cancer cells with 5-azacytidine was shown to upregulate expression of urokinase-like plasminogen activator, an enzyme important in tumor invasion and metastasis, leading to enhanced metastatic potential.²³

Histone Deacetylase

Another critical determinant of gene expression is the condensation of chromatin in the form of heterochromatin, which results in transcriptional silencing and enhanced genome stability. This is accomplished by a number of pathways, one of which is the acetylation and deacetylation of histones, controlled by histone acetyltransferases and histone deacetylases (HDACs).²⁴ The HDACs specifically maintain chromatin in a condensed form, and can associate with specific transcription factors resulting in transcription repression. The acetylation of histones may be key in regulating the expression of genes associated with cellular proliferation, differentiation, and survival, both in development and carcinogenesis.^25,26 Studies suggest that histone acetylation reduces electrostatic charge interactions between histones, leading to chromatin decondensation, and more recent work has suggested that HDAC enzymes’ effects may also be indirect through modulation of other proteins important in chromatin condensation, including DNMT1.²⁷ Induction of HDAC expression, leading to transcriptional repression, is a common feature in human cancers such as colon cancer²⁸ and negatively regulates the expression of several tumor suppressor genes, including p53 and VHL.²⁹ Differential expression of certain HDAC isoforms has been associated with outcome in a variety of human tumors, with HDAC2 and 6 studied most completely.³⁰

Pharmacologic inhibition of HDAC can affect multiple facets of the malignant phenotype. HDAC inhibition inhibits colon carcinogenesis in the adenomatosis polyposis coli (APC) mouse model.²⁸ Angiogenesis can be inhibited through upregulation of VHL and subsequent inhibition of hypoxia-inducible factor-1α (HIF-1α) function and VEGF production^29,31,32; decreased expression of other proangiogenic factors such as bFGF, angiopoietin-2, and Tie2^31,32; inhibition of endothelial nitric oxide (NO) synthase and endothelial cell proliferation and tube formation^33,34; and inhibition of the commitment of endothelial progenitor cells to the endothelial lineage.³⁵ Inhibition of HDAC can enhance apoptosis in tumor and endothelial cells^32,36-38 and directly inhibit tumor cell proliferation.^37,39-41 Consistent with its role as a transcription repressor, inhibition of HDAC has been shown to induce differentiation in thyroid and prostate cancers, neuroblastoma, and the leukemias.^42-46 HDAC inhibition dramatically enhances the in vitro and in vivo efficacy of multiple standard cytotoxic therapies, as well as novel antibodies and small molecules.^{27,32,38,47-54}

Two HDAC inhibitors, vorinostat (suberoylanilide hydroxamic acid [SAHA], Zolinza) and romidepsin (FK228, Istodax), have recently been approved by the FDA for the treatment of cutaneous T-cell lymphoma (CTCL),^55-57 and a large number of additional agents are in intermediate to late-stage clinical trials. A number of studies are being conducted evaluating HDAC inhibitors for various other hematologic and solid tumors, and early studies combining these drugs with other targeted and cytotoxic therapies have been reported.^58-61

A recent in vitro study demonstrated potent induction of histone acetylation, growth inhibition, and induction of apoptosis in a variety of canine tumor cell lines treated with either vorinostat or the novel HDAC inhibitor OSU-HDAC42 (AR42).⁶² The commercially available anticonvulsant drug valproic acid (VPA) can function as an HDAC inhibitor and is capable of inhibiting tumor cell invasion, P-glycoprotein expression, proliferation, and angiogenesis and enhancing chemosensitivity.^24,27,43 VPA was recently shown to enhance the antiproliferative and proapoptotic effects of doxorubicin in canine OSA cell lines in vitro and to synergize with doxorubicin in a canine OSA xenograft.⁵³

There is a single case report describing administration of vorinostat to a dog with microscopic HSA.⁶³ Although apparently well tolerated, the dose was empirically chosen, there was no pharmacokinetic analysis or demonstration of target inhibition, and the drug’s impact on the course of the disease could not be unequivocally determined. A recent study demonstrated that VPA could be administered prior to a standard dose of doxorubicin in tumor-bearing dogs, at dosages sufficient to enhance histone acetylation in tumor and peripheral blood mononuclear cells, but was not associated with toxicity or apparent potentiation of doxorubicin’s adverse effects.⁵⁴

The Proteasome

The abundance of cellular proteins is tightly controlled at the levels of both production and destruction. Protein production can be modified at the transcriptional and posttranscriptional level, and therapies based on these approaches are relatively abundant. Until recently, relatively little attention has been paid to the manipulation of protein degradation as a therapeutic modality. The ubiquitin-proteasome pathway (UPP) is responsible for the degradation of the majority of intracellular proteins and for the regulation of many proteins with key roles in cancer.

The 26S proteasome is a large multiprotein complex containing ubiquitin recognition domains, which bind ubiquitinated proteins tagged for degradation, and proteolytic domains with trypsin-like, chymotrypsin-like, and caspase-like activity, which degrade proteins into short polypeptide sequences.⁶⁴ It is responsible for the degradation of a variety of proteins responsible for cell-cycle regulation, angiogenesis, apoptosis, and chemotherapy and radiation sensitivity (Table 14-4).^64-66

Tumor cells are generally more sensitive to the effects of proteasome inhibition than are normal cells. Various studies have demonstrated a threefold to fortyfold increase in susceptibility to proteasome inhibitor–associated apoptosis when comparing tumor cells to corresponding normal tissues.^66-70 The mechanisms for this differential sensitivity are unclear, but proliferating cells generally appear more sensitive than do quiescent cells.^64,66 Additionally, dysregulation of UPP function appears to occur in many cancer cells, thus potentially rendering them more sensitive to inhibition.^66,71 Recent studies implicate profound upregulation of the proapoptotic factor Noxa as being a key mediator of proteasome inhibitor–induced tumor cell apoptosis.^72,73

Although a number of chemicals appear capable of proteasome inhibition in vitro, only the boronic acid derivatives appear suitable for clinical use and only one drug, bortezomib (Velcade, PS-341), has received FDA approval for the treatment of multiple myeloma.^74-77 Several additional proteasome inhibitors are in clinical development. Meaningful antitumor activity has been observed in patients with other hematopoietic neoplasms,^78-80 but less activity has been seen in solid tumors to date.^81-84 Although toxicology studies with bortezomib have been performed in dogs,⁸⁵ there is no information available to date regarding the safety or biologic effect of proteasome inhibitors in veterinary patients or tumor cells.

Heat Shock Protein 90

Given the complex nature of cancer and the multiple pathways that can be subjugated to contribute to the malignant phenotype, an optimal cancer drug might target a variety of oncogenic pathways simultaneously. One molecular target that has the potential to interrupt a wide variety of pathways important in cancer is heat shock protein 90 (HSP90), a molecular chaperone responsible for the conformational maturation of many proteins involved in diverse oncogenic activities such as cell adhesion/migration/invasion, signal transduction, cell cycle progression, angiogenesis, and survival (Table 14-5).^86-111 HSP90 and other chaperones are responsible for ensuring the correct folding and prevention of aggregation of their client proteins.¹¹² Misfolding and aggregation of proteins lead to ubiquitination and proteasomal destruction, resulting in proteins with diminished function and greatly shortened half-lives.¹¹³ Although several classes of compound are capable of inhibiting HSP90 chaperone function,^97,98,103 the best studied are ansamycin antibiotics of the geldanamycin class.

Many HSP90 inhibitors appear to demonstrate significant preferential activity against malignant versus normal somatic cells. The HSP90 derived from most tumor cells has a binding affinity for 17-allylaminogeldanamycin (17-AAG) approximately 100-fold higher than HSP90 derived from normal cells. This may occur as a result of the overaccumulation of mutated, misfolded, and overexpressed signaling proteins in tumor cells, leading to increased HSP90 chaperone activity and a greater proportion of the molecule in the bound, active, and 17-AAG–sensitive state.¹¹⁴

Tumor cells display considerable variation in sensitivities to HSP90 inhibition. Although the mechanisms underlying this differential sensitivity are incompletely characterized, some important characteristics include reliance on certain kinase cascades, expression of apoptotic and cell-cycle regulators, and p-glycoprotein expression.⁹⁰

Many RTKs targeted by the geldanamycins may have an important role in canine and feline tumors. For example, they are capable of inhibiting the function of mutant and wild-type KIT,⁹¹ important in canine mast cell neoplasia¹¹⁵; Met,⁹² expressed in multiple canine tumor types^116,117; PDGFR,¹¹⁸ expressed in FVAS and OSA^119,120; and IGF-1R,⁹¹ which is expressed and functional in canine OSA and melanoma.^121-123 The geldanamycins are likewise able to attenuate the function of the HIF-1α protein, a key transcription factor responsible for sensing and responding to hypoxia and activating the angiogenic switch.^97,99,100 They are additionally able to deplete key antiapoptotic proteins such as mutant p53 and survivin,^104-107 contributing to enhanced in vitro sensitivity to standard cytotoxic therapies such as radiation and chemotherapy when used in combination.^{93,94,101,124-126}

Under certain circumstances, HSP90 inhibitors may have undesirable effects from the standpoint of cancer therapy. For example, 17-AAG has been shown to protect colon carcinoma cells from cisplatin-mediated toxicity,⁹⁵ whereas it has additive or synergistic activity when combined with cisplatin against human neuroblastoma and OSA cells.¹²⁴ Additionally, 17-AAG inhibited primary tumor formation, although it potentiated bone-specific mammary carcinoma metastasis by enhancing osteoclastogenesis in one murine model.¹²⁷

The impressive preclinical data generated with compounds such as HSP90 inhibitors have led to phase I human clinical trials of multiple agents,^128-132 including some early combinatorial studies.^130-132 Evidence of biologic effect in the form of upregulation of HSP70 chaperone expression in peripheral blood cells has been observed. There is in vitro evidence of antitumor activity of HSP90 inhibitors in canine OSA and mast cell tumor cell lines^133,134; however, no clinical evaluation of these agents has been reported to date.

Poly Adenosine Diphosphate Ribose Polymerase and Poly Adenosine Diphosphate Ribose Glycohydrolase

Poly adenosine diphosphate (ADP)-ribose polymerase (PARP) is a “nick-sensor” that signals the presence of DNA damage and facilitates DNA repair.¹³⁵ The first PARP enzyme was discovered by Chambon et al and is now recognized as a superfamily of 18 members,¹³⁶ although only PARP-1 and PARP-2 are known to act in DNA damage.¹³⁷ The PARP family is also involved in the regulation of several transcription factors such as NFκB in modulating the expression of chemokines, adhesion molecules, inflammatory cytokines, and mediators.¹³⁵ Poly ADP-ribose glycohydrolase (PARG) is the main enzyme in catabolizing poly ADP-ribose to ADP-ribose. To date, only one single PARG gene has been detected in mammals, encoding for three complementary DNAs (cDNA), which generate three isoforms.¹³⁵

PARP has multiple intracellular functions, including signaling DNA damage, and recognizing and binding to DNA strand breaks generated by DNA-damaging agents (cytotoxic drugs and ionizing radiation).¹³⁸ Activation of PARP is one of the earliest DNA damage responses. PARP is also a modulator of DNA base excision repair (BER), which constitutes a major mechanism for genomic stability. There is increasing evidence demonstrating that both PARP and PARG repair DNA.¹³⁶ When PARP binds to DNA strand breaks, it activates an enzyme causing shuttling of PARP and, subsequently, opening of the chromatin. PARG enters the nucleus, it moves to the PARP substrate, and DNA strand breaks are repaired. Due to excessive PARG, poly ADP-ribose decreases and thus chromatin reverts back to its original structure.

PARP inhibition has been suggested as an important approach in sensitizing cancer cells to conventional cancer therapy, leading to early clinical trials with PARP inhibitors.¹³⁸ PARP inhibitors have been shown to be lethal in BRCA-deficient cells due to persistence of DNA lesions that would normally be repaired in a BRCA-dependent fashion,¹³⁹ suggesting that PARP inhibitors might be an effective monotherapy in these cancers. However, one might expect that their major benefit would be to enhance conventional cytotoxic drug treatment or radiation therapy, and studies have demonstrated that PARP inhibition potentiates the cytotoxicity of anticancer drugs and ionizing radiation through inhibition of DNA repair in cancer cells. PARG inhibition could also be one of the pathways selected for cancer management due to its effects on increased sensitivity to both radiation and chemotherapy.

Although PARG inhibitors have lagged behind PARP inhibitors, a number of molecules have been developed that target these pathways, with varying degrees of specificity. In experimental mouse models, these have shown promise in breast, colon, lung, and brain tumors and in melanoma, either as monotherapy or combined with conventional drugs or radiation.^140-144 Only PARP inhibitors have been used in early human clinical trials (phase I). It is too early to pass judgment on these early trials, and the next 5 years will demonstrate whether PARP inhibitors will have a place in the drug arsenal for cancer treatment.

Carbonic Anhydrase

Hypoxia in cancer tissues is emerging as a key negative prognostic marker in cancer survival. Carbonic anhydrase functions to interconvert carbon dioxide and bicarbonate to maintain acid-base balance in tissues and may play important roles in hypoxic states. In humans, there are 13 active isoenzymes of carbonic anhydrase (CA), and the transmembrane isoform CA IX has been linked with carcinogenesis.¹⁴⁵ High levels of CA IX expression have been demonstrated in human epithelial tumors such as carcinomas of the cervix, uterus, kidney, lung, esophagus, breast, and colon. Normally, this isoenzyme has restricted expression to the epithelia of the gastrointestinal tract, which is in contrast to studies in cancer where ectopic expression has been demonstrated in hypoxic tumors, participating in tumor cell environment acidosis and contributing to malignant progression and poor treatment outcome.¹⁴⁵ Modulation of extracellular tumor pH via inhibition of CA IX activity has been suggested as a promising approach to novel anticancer therapies. Much attention has recently been paid to the CA IX inhibitors’ drug design, and efforts have been made to obtain isozyme IX inhibitors, with putative applications as antitumor drugs or diagnostic agents. A large number of selective CA IX inhibitors have been developed in the past 5 years in the sulfonamide, sulfamide, and sulfamate series.¹⁴⁶ Some of these compounds can constitute interesting leads for further development. Furthermore, new classes of prodrug have emerged in the design of new anticancer compounds, which can be activated under hypoxia. As yet, there is little clinical information regarding the efficacy of any of these compounds, but one may anticipate that these drugs could be beneficial in large hypoxic tumors that have failed conventional treatments or as possible sensitizers to radiation damage.^145,147

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