Affiliation of author: Developmental Therapeutics Program,Division of Cancer Treatment and Diagnosis, National Cancer Institute, Bethesda, MD.
Correspondence to: Edward A. Sausville, M.D., Ph.D., National Institutes of Health, EPN Bldg., Rm. 843, Bethesda, MD 20892-7458.
Protein kinases catalyze the transfer of the -phosphate of
adenosine triphosphate (ATP) to protein acceptors. Over the past 40
years, we have learned that protein phosphorylation is a central
regulatory strategy to alter cellular function. Proteins can be
phosphorylated on serine, threonine, tyrosine, and rarely histidine
residues. However, tyrosine kinases have come to be understood as
critical regulators of cell proliferation, invasion, metastasis, and
cell survival. Tyrosine kinases exist as two major classes. In receptor
tyrosine kinases, including platelet-derived growth factor receptor,
epidermal growth factor receptor, and its homologue the c-erbB2
oncogene product, the kinase activity is actually part of the receptor,
which has one extracellular domain to bind molecules promoting
proliferation at the cell surface, a second domain that traverses the
cell membrane, and an intracellular catalytic domain that acts to cause
tyrosine phosphorylation. Nonreceptor tyrosine kinases exist in the
cytoplasm, but they can be recruited to distinct subcellular locations
after receipt of various cellular signals. Tyrosine kinases
phosphorylate proteins that change cell function, either by directly
activating or inhibiting "downstream" kinases, or by creating
tyrosine phosphates that serve as "scaffold" sites for the
assembly of regulatory molecules (1).
The importance of tyrosine kinases to cancer growth is underscored by the fact that many of these molecules were first detected as transforming oncogene products in tumor-causing viruses, carrying mutated forms of kinases present in the normal animal genome. The paradigm setting, archetypal chicken Rous sarcoma virus genome, encodes a tyrosine kinase (v-src) whose activity is essential for tumor formation and which is a mutated form of the normal c-src gene present in all animal genomes (2). However, one of the first tyrosine kinases to be clearly associated with a human neoplasm was the c-abl proto-oncogene product, first detected in humans by its similarity to the v-abl oncogene, which in turn was originally defined as the transforming oncogene of the Abelson murine leukemia virus (3). Classical cytogenetic studies had established the near-ubiquitous presence in patients afflicted with chronic myeloid leukemia (CML) of the Philadelphia chromosome (4), which was subsequently characterized to be the result of a translocation between chromosome 9 and chromosome 22 (5). The molecular basis for CML was established by the demonstration that the c-abl proto-oncogene was fused as a result of the translocation from chromosome 9 to sequences deriving from chromosome 22 (the breakpoint cluster region) to yield a fused gene encoding a novel p210bcr-abl chimeric fusion protein (6), with a very active protein tyrosine kinase activity (7). The neoplasm-promoting function of p210bcr-abl requires tyrosine kinase activity. Introduction of p210bcr-abl into mouse cells causes a disease similar to human CML to occur in the animals, concordant with the idea that the disease process derives from the action of the fusion protein (8). From a clinical perspective, demonstration of p210bcr-abl is the sine qua non for diagnosis of the usually encountered type of CML, and an analogous protein, p185bcr-abl, occurs in 30%-50% of adult acute lymphoblastic leukemia (9).
The importance of tyrosine kinase signaling as a potential target for novel cancer treatments is promised by the near-ubiquitous deregulation of tyrosine kinase signaling in human cancers (10). It is further supported by recent evidence of a clinically useful effect from antibodies directed against the c-erbB2 cell surface receptor tyrosine kinase (11). Unfortunately, intracellular, nonreceptor tyrosine kinases such as p210bcr-abl are not approachable by such easily generated reagents but require campaigns to define "small molecules" that can enter cells to affect their function. Drugs directed against protein tyrosine kinases might affect their catalytic utilization of ATP or protein substrate or might affect the capacity of the kinases to associate with cellular components important for their function. CML is at present curable by allogeneic marrow transplantation (12). That therapy is available to at most 30% of all patients. While useful benefit may derive from "standard" treatments with interferon (13) or hydroxyurea (9) or from investigational strategies to treat patients with high-dose chemotherapy and autologous stem cell rescue (14), clearly, better treatments are necessary.
In this issue of the Journal, le Coutre et al. (15) describe preclinical experiments that offer great promise on the part of the 2-phenylaminopyrimidine derivative CGP57148B as a p210bcr-abl -directed tyrosine kinase antagonist for treatment of CML. Their results represent a milestone, a guide for developing protein kinase antagonists for cancer treatment, and convey excitement for the promise of these molecules. These experiments are of importance for a number of theoretical and practical reasons. First, although the general chemotype of CGP57148 was detected empirically in a biochemical screen for novel structures active against protein kinases, the structure of CGP57148B was refined after consideration of the molecular structure of the ATP-binding site of protein kinases (16,17). This drug thus represents what hopefully will be common in the new generation of rationally designed and selected drugs directed against the molecular basis of a particular cancer. Second, although selective for p210bcr-abl, CGP57148B is not absolutely specific because it is an ATP-binding site antagonist potentially able to interact with other cellular ATP-binding sites. (It actually does have some activity as a platelet-derived growth factor receptor antagonist.) The experiments presented by le Coutre et al. therefore, illustrate clearly that an ATP site-directed antagonist can have a meaningful therapeutic index in a living creature and that absolute selectivity for the target is not essential for a useful outcome. Third, the biologic effect of single doses, uniquely defined by le Coutre et al. as a reduction in tyrosine phosphorylation of p210bcr-abl in tumors growing in animals, was transient and suggested the necessity for multiple, daily dosing of animals.
Persistence in developing this regimen paid off with remarkable success (perhaps cures) in animals with "early stage," low-volume CML tumor grafts as well as very gratifying responses in animals with bulky disease. The latter result is particularly noteworthy in that it emphasizes that p210bcr-abl not only mediates proliferation, but also may govern susceptibility to cell death. This drug therefore, shows great promise in patients with CML. While the drug may be usefully employed with expectation of response in patients with clinically bulky disease, the experiments reported here also suggest a perhaps equally important application may be in patients who have prior reduction of their leukemia burden by chemotherapeutic approaches but then receive CGP57148B in an effort to promote long-term suppression and perhaps to eradicate the leukemic clone, after either allogeneic or autologous stem cell support.
Caution must be urged. Mouse models have been notoriously unreliable in predicting the efficacy of human cancer treatments. The concentrations of drug achieved in the mouse to elicit these favorable results are not described and might not be achievable in humans. One must be very careful never to imply that tumor "eradication" in a mouse necessarily signifies "cure" in humans. In particular, eradication of the p210bcr-abl-bearing leukemia cells must be accompanied by re-population of bone marrow by normal cells not bearing the fusion protein, an event not addressed by the mouse model. Moreover, CML does not grow as a subcutaneous lump, as studied here. However, the mouse model does represent the "substance of things hoped for" when CGP57148B enters human clinical trials. The experiments of le Coutre et al. (15) are meticulously conducted, use statistically meaningful treatment cohort sizes, are properly controlled, use a well-defined drug product, and optimize the drug's use with reference to a pathogenically relevant target.
These experiments, therefore, promise a new paradigm for cancer drug development, where early phase I clinical trials will not be governed by toxicity as an end point but rather by an effect of the test drug on a molecular mediator of the drug's effect and hopeful utility. Patients with CML were among the first to teach us the importance of oncogenes in human disease. With CGP57148B, it is hoped that these patients will reap the first fruits of therapeutic efforts rationally designed and directed against these targets.
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