Chapter 6.2

Kinase dysfunction and kinase inhibitors

Cheryl A. London

Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH 43214, USA

Correspondence: Cheryl A. London, Department of Veterinary Biosciences, The Ohio State University, 454 VMAB, 1925 Coffey Road, Columbus, OH 43210, USA. E-mail: cheryl.london@cvm.osu.edu

With recent advances in molecular biology, abnormalities in cancer cells that contribute to dysregulation of cell survival and proliferation are being characterized with greater precision. Through this process, key abnormalities in cancer cells involving proteins that regulate signal transduction, migration, mitosis and other critical processes have been identified. Such abnormalities often involve a class of proteins called kinases that act to phosphorylate other proteins in the cell, resulting in activation of these proteins in the absence of appropriate stimulation/regulation. Given their role in tumour biology, substantial effort has been directed at blocking the function of these proteins. Several approaches have been used, including monoclonal antibodies and small molecule inhibitors. While antibodies are primarily directed at cell surface proteins, small molecule inhibitors, also known as kinase inhibitors, target proteins throughout the cell.

A variety of kinase inhibitors have been approved for the treatment of human cancers. In some instances, these inhibitors have exhibited significant clinical efficacy, and it is likely that their biological activity will be further enhanced as combination regimens with standard treatment modalities are explored.

The use of kinase inhibitors in dogs and cats is relatively recent, although two inhibitors, toceranib (Palladia; Pfizer Animal Health, Madison, NJ, USA) and masitinib (Kinavet; Catalent Pharma Solutions, Somerset, NJ, USA) have been approved by the Federal Drug Administration (USA) for use in dogs. This article reviews the biology of protein kinase dysfunction in human and animal cancers, and the application of specific kinase inhibitors to veterinary cancer patients.

Protein kinases and normal cell biology

Protein kinases are critical regulators of normal cell signalling, because they control several key processes, such as cell survival, growth, differentiation and migration. The kinases act through phosphorylation of other proteins. They accomplish this by binding adenosine triphosphate (ATP) and using this to add phosphate groups to key amino acids on themselves (also known as autophosphorylation) and on other proteins, thereby promoting the transmission of cellular signals. This process usually occurs in response to stimuli generated by growth factors (GFs) or other substances outside of the cell that intiate the process. Protein kinases are termed tyrosine kinases (TKs) if they phosphorylate proteins on the amino acid tyrosine or serine/threonine kinases if they phosphorylate proteins on the amino acids serine and threonine. These kinases can be expressed on the cell surface, in the cytoplasm and in the nucleus.

Receptor tyrosine kinases (RTKs) are those TKs expressed on the cell surface that are stimluated by binding of GFs. Receptor tyrosine kinases are structured to contain an extracellular ligand binding domain, a transmembrane domain that anchors the RTK in the membrane, and a cytoplasmic kinase domain that positively and negatively regulates phosphorylation through inhibition of spontaneous dimerization.^1–3 Binding of a GF to the RTK induces dimerization of the receptor, thereby inducing a conformational change that allows ATP binding, autophosphorylation, and the initiation of a downstream signal through subsequent binding of adaptor proteins and additional TKs.¹ It is now understood that dysregulation of RTKs through mutation, overexpression or chromosomal translocation results in pathway activation and subsequent uncontrolled signalling. While the molecular aspects of these events have been extensively researched in human cancers, characterization of similar abnormalities in canine and feline cancers is just beginning. Examples of RTKs known to be dysregulated in human and canine cancers include KIT, MET, anaplastic lymphoma kinase (ALK) and epidermal growth factor receptor (EGFR). ^4–8

Receptor tyrosine kinase signalling is also an important driver of the process of new blood vessel growth, known as angiogenesis, which is essential for expansion of tumours beyond 1-2 mm in size. 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).^9–12 Vascular endothelial growth factor receptors are expressed on new blood vessel cells, and signalling promotes migration and proliferation;⁹ PDGFR is expressed on pericytes, promoting their maturation;^11,12 and FGFR is expressed on endothelial cells, enhancing expression of VEGF.¹¹ Both Tie-1 and Tie-2 are expressed on blood vessels in tumours and are important in the recruitment of pericytes and smooth muscle cells to the newly forming vascular channels.¹³

Kinases present in the cytoplasm act to transmit signals generated by RTKs to the nucleus through a series of intermediates that also become phosphorylated.¹⁴ In cancer cells, two specific cytoplasmic pathways are known to be dysregulated with signficant frequency. The first includes members of the RAS-RAF-MEK-ERK/p38/JNK families,^15,16 most of which are serine/threonine kinases. Their activation results in ERK phosphorylation, translocation into the nucleus, and subsequent alteration of transcription factor and nuclear kinase activity important for controlling the cell cycle. Specific members known to be mutated in human tumours include RAS (lung cancer, colon cancer and several haematologic malignancies) and BRAF (melanomas, thyroid carcinomas and colon cancer).^16–18

The second cytoplasmic pathway frequently altered in cancer includes phosphatidyl inositol-3 kinase (PI3K) and its downstream signal transducers AKT, nuclear factor κB and mTOR, among others.^19,20 Phosphatidyl inositol-3 kinase is activated by RTK stimulation and in turn phosphorylates AKT, which alters several additional proteins involved in the regulation of cell survival, cycling and growth.²¹ AKT phosphorylates targets that promote apoptosis (BAD, procaspase-9 and Forkhead transcription factors) and activates nuclear factor κB, a transcription factor that regulates multiple cellular processes.^19–21 AKT also phosphorylates other proteins such as mTOR, p21, p27 and GSK3, resulting in redistribution of these proteins either in or out of the nucleus, ultimately inhibiting apoptosis while stimulating cell cycling.^19–21 Abnormalities of PI3K, including mutations and gene amplification, are found in many human cancers, including breast, colorectal, lung and ovarian carcinomas.²² Another manner in which this pathway can become activated is through loss of activity of PTEN, a phosphatase that normally acts to regulate AKT and terminate signalling.^19,23,24 PTEN mutations and/or decreased PTEN expression occur in many human cancers (e.g. glioblastoma and prostate cancer)^22,23 and have been documented in canine cancers as well (osteosarcoma and melanoma).^25–27

The act of signal transduction serves to influence cellular events by affecting gene transcription and multiple proteins that regulate cell cycling. The cyclins and their kinase partners [cyclin-dependent kinases (CDKs)] control progression of cells through various phases of the cell cycle.^28–30 Cyclins D and E and their CDK partners (CDK2, 4 and 6) are the primary regulators of entry into the cell cycle, as co-ordinated function of these is necessary for cells to progress from G1 into S phase. Signals generated by RTKs often induce expression and activation of cyclin D/CDK4,6 complexes that act to phosphorylate the tumour suppressor Rb, partly repressing its activity.^29,30 This is further enhanced by cyclin E/CDK complexes, thereby initiating the process of DNA replication necessary for cell division. Dysregulation of the cylins and CDKs occurs frequently in human cancers, with overexpression of cyclins D and E found in breast, pancreas, and head and neck carcinomas.³⁰

Kinases and cancer cells

Dysfunction of protein kinases occurs frequently in human cancers, and recent data indicate that dog and cat cancers may also experience a similar level of dysfunction. Mutation, overexpression, the generation of fusion proteins and the presence of autocrine loops of activation are all mechanisms by which this may take place. Mutations often alter the structure of a kinase such that phosphorylation occurs in the absence of an appropriate stimulus. For example, a point mutation occurs in the BRAF gene in approximately 60% of human cutaneous melanomas^18,31,32 that induces a conformational change in the protein, resulting in constitutive activation, downstream ERK signalling, and promotion of cell growth and survival.^33,34 RAS is another kinase that is dysregulated through point mutation in several haematopoietic neoplasms, lung cancer, colon cancer and many others.^16,31,32

KIT, an RTK normally expressed on haematopoietic stem cells, melanocytes, interstitial cells of Cajal, in the central nervous system and on mast cells, has been shown to be dysregulated in several cancers.³⁵ In approximately 25-30% of canine grade 2 and 3 mast cell tumours, mutations consisting of internal tandem duplications are found in the juxtamembrane domain of KIT, resulting in ligand-independent activation. These mutations are associated with a higher risk of local recurrence and metastasis.^36–38 Deletions in the juxtamembrane domain of KIT are found in approximately 50-80% of human patients with gastrointestinal stromal tumours and have been identified in canine gastrointestinal stromal tumours as well.^39–4 Other cancers in which KIT mutations have been identified include melanoma and acute myelogenous leukaemia. Other examples include FLT3 internal tandem duplications in acute myelogenous leukaemia,^42–45 EGFR point mutations in lung carcinomas,^46,47 and PI3Ka mutations in several types of carcinomas.²²

Overexpression of kinases often involves the RTKs and either promotes an enhanced response to normal levels of growth factor or, more commonly, induces spontaneous receptor dimerization in the absence of ligand binding. Perhaps the most well-described example is overexpression of the RTK HER2/Neu (a member of the EGFR family) in breast and ovarian carcinomas.^3,48,49 EGFR is also overexpressed in human lung, bladder, cervical, ovarian, renal and pancreatic cancers, and some tumours have as many as 60 copies of the gene per cell.⁶ Fusion proteins are generated when a portion of the kinase becomes attached to another gene through chromosomal rearrangement, thereby disrupting the mechanisms that typically control protein function. One of the best-characterized fusion proteins is BCR-ABL, which is found in 90% of patients with chronic myelogenous leukaemia (CML).^50,51 This fusion induces constitutive activation of the cytoplasmic kinase ABL, contributing to malignant transformation. Other examples include TEL-PDGFRα in leukaemia and EML4-ALK in non-small-cell lung cancer.⁵² Lastly, autocrine loops of activation primarily occur when tumour cells express both the RTK and the growth factor resulting in constitutive receptor activation. Examples include coexpression of transforming growth factor β 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 and glioblastoma.^3,53-55 In canine cancers, possible autocrine loops have been documented in osteosarcoma (OSA, MET and HGF) and haemangiosarcoma (HSA, KIT and SCF).^56–58

Kinase inhibitors

With the understanding that certain molecular events can act as drivers of uncontrolled cancer cell growth and survival, substantial effort has been directed at blocking the specific proteins that initiate this process either directly, at the level of the tumour cell, or indirectly, at the level of the tumour microenvironment. The two approaches most commonly used are monoclonal antibodies and small molecule inhibitors.

Technology now exists to engineer antibodies to recognize specific epitopes on a variety of proteins. This method has been successfully used to generate antibodies that can bind to the extracellular domains of RTKs or circulating growth factors, thereby inhibiting function of these proteins. One of the most successful examples is a humanized monoclonal antibody called trastuzumab (Herceptin; Genentech, South San Francisco, CA, USA) that targets HER2/Neu, which is overexpressed in approximately 30% of breast cancers, as well as other epithelial cancers.⁵⁹ In women with metastatic HER2-positive breast cancer, trastuzumab resulted in a response rate of approximately 25%⁶⁰ that improved to approximately 50% when trastuzumab was combined with chemotherapy.⁶¹ When used in the adjuvant setting, multiple studies have demonstrated that trastuzumab markedly improves survival of women with HER2-positive breast cancer and, consequently, it is now part of the routine standard of care.^62,63 Other examples of monoclonal antibodies approved for use in human cancers include rituxumab (Rituxan; Genentech), which targets CD20 expressed in B-cell malignancies^64,65 and cetuximab (Erbitux; Bristol-Meyers Squibb, Princeton, NJ, USA), which targets ErbB1/EGFR overexpressed in several human carcinomas.^6,59,66

The second major method for blocking the function of a protein is through the use of small molecule inhibitors. These work either by blocking the ATP binding site of kinases, essentially acting as competitive inhibitors, or by blocking protein-protein interactions, known as allosteric inhibition.⁶⁷ Those small molecule compounds that block ATP binding to the kinase prevent autophosphorylation, as well as downstream phosphorylation, thereby interrupting the survival/growth signal essential to the tumour cell, ultimately resulting in cell death. In contrast to the monoclonal antibodies, the small molecule inhibitors are often easy to synthesize in large quantities, are usually 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; Novartis Oncology US, East Hanover, NJ, USA), an orally administered drug that binds the ATP pocket of ABL, as well as the RTKs KIT and PDGFRα.⁶⁸ As previously mentioned, BCR-ABL fusion proteins are present in over 90% of human patients with CML, making this a good target for therapy. The use of imatinib for treatment of patients with CML has been transformative, with significant biological activity resulting in the approval of this drug as standard of care for affected individuals.^69–74 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. The activity of imatinib is much lower in patients with blast crisis (20-50%), often lasting <10 months. Resistance to therapy is primarily due to the development of mutations in ABL that preclude drug binding, although gene amplification has also been documented.^75,76 Through its ability to inhibit KIT, imatinib also has substantial single-agent activity against human gastrointestinal stromal tumours, in which up to 80% of the tumours have activating mutations in KIT.^77,78 Response rates of 50-70% have been reported to imatinib, far better than the 5% response rate seen with standard chemotherapy.^79,80

There are now several small molecule inhibitors approved for use in the treatment of a variety of human cancers, and many more currently undergoing clinical investigation. A subset of people with non-small-cell lung cancer (NSCLC) have tumours with activating mutations in EGFR

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