Walking the Bridge Between Idea and Cureâ€”Seventeenth Bruce F. Cain Memorial Award Lecture1

[CANCER RESEARCH 58. 4211-4216, October 1. I998| Special Lecture

Translational Research: Walking the Bridge between Idea and Cureâ€”Seventeenth Bruce F. Cain Memorial Award Lecture1

Bruce A. Chabner,2 Anthony L. Boral, and Pratik Multani

Division of Hematology/Oncology, Massachusetts General Hospital and Han'ard Medical School. Boulon, Massachusetts 02114

Abstract New Biology, New Targets

Advances in the understanding of normal and malignant cell biology Drug discovery for the treatment of cancer has undergone dramatic are allowing the development of biologically targeted drugs directed at changes in recent years, and a new cadre of agents designed to interact specific differences between host and tumor. The array of potential new with the tumor cell at unique sites is approaching the clinic. In the past targets is vast, but drugs currently in development are targeted at cell- 50 years, since the initial discovery of the antitumor activity of cycle regulators, growth factors and their receptors, signal transduction alkylating agents and antimetabolites, anticancer drugs have been intermediates, angiogenesis, and the mechanisms that mediate apoptosis designed with DNA synthesis as their target. For classical cytotoxic and DNA repair. Recent results raise the possibility that novel biologically drugs, the end points for their clinical evaluation were straightfor targeted agents, perhaps in combination with traditional cytotoxic agents, ward: tumor regression on the one hand and toxicity to host tissues, may finally cure cancer. However, the development of a biologically predominantly bone marrow and gastrointestinal mucosa, on the targeted drug raises unique challenges in the design of clinical trials to other. The newer agents presently under development have targets demonstrate its efficacy, and despite the promising preclinical data that removed from the direct synthesis of DNA; they affect the signals that exist for most of the agents in development, the clinical trial remains the promote or regulate DNA synthesis, including growth factors and critical, final step across the bridge from basic research to clinical appli their receptors, signal transduction pathways, as well as pathways that cation. In this review, we discuss some of the challenges in the clinical determine susceptibility to apoptosis and DNA repair. Each of these development of biologically targeted agents and the implications for clin ical trial design. pathways may be affected by activating mutations that predispose to cancer in selected tumor types and, thus, offer potential as a target for Introduction inhibition (Fig. 1). In addition, there are important steps in tumor progression, including angiogenesis and metastasis, that form the With dramatic advances in the understanding of normal and ma basis for alternative anticancer strategies. A compelling rationale, as lignant cell biology over the past decade, the possibility has grown well as strong preclinical evidence, supports the focus of attention on that new drugs, directed at specific differences between host and these pathways as the best hope for the cure of cancer. However, the tumor, may finally cure cancer. The spectrum of new targets is vast, transition from concept to drug is a tortuous path involving the including the newly discovered controls for cell proliferation, the refinement of leads to enhance antitumor effects, incorporate desirable pharmacologie properties (good oral absorption, long half-life in vivo, pathways for transmission of positive and negative signals from cell ability to cross the blood-brain barrier, and lack of susceptibility to surface to nucleus, the stimulation of new vessel growth, and the complex processes that determine apoptosis and DNA repair. Each of multidrug resistance), and decrease toxicity to host organs. The final step of the journey, across the bridge from basic research to clinical these may be disrupted by mutations in selected human tumors and application, is the clinical trial (Fig. 2). Many promising candidates presents opportunities for new drug design. It is the purpose of this fail in this final, but most important, step. study to explore the challenge of the evaluation of these new thera pies. The Clinical Trial Basic science and clinical medicine have long existed within sep arate worlds that only occasionally meet in an uneasy partnership. There are a number of elements in an appropriately designed Basic research is necessary to achieve an understanding of the normal development plan for a new anticancer drug. These include the defi and pathological state, but cannot, by itself, put new knowledge to nition of a safe dose and schedule, in which the parameters: (a) satisfy useful applications. The bridge to application is created by the devel end points of toxicity, drug exposure, or molecular action (Phase I); (b) have a choice of an appropriate patient population for testing of opment of diagnostic tests or new drugs that allow an intervention in efficacy and establishing antitumor effects (Phase II); and (c) have a patients. The clinical trial then becomes the medium through which comparison with standard therapies in a marketing-directed trial the value of the new intervention is measured, but the trial only fulfills (Phase III). For each phase of clinical testing, the new molecular its purpose if it provides a valid test of the application. The bridge entities may require a rethinking of common strategies for drug between these two worlds is difficult but necessary to build, in order evaluation. to assure that our clinical trials have meaningful and specific end The end points for Phase I trials may be quite different from those points and are designed to prove the effectiveness of such targeted of cytotoxic compounds. In Phase I, clinical cancer researchers have therapies. sought to determine the maximum tolerated dose for a schedule shown to be effective in preclinical models of human cancer. However, for noncytotoxic drugs targeted at specific molecular events, one can Received 7/21/98; accepted 8/3/98. imagine alternatives. The end point might be pharmacokinetic: an area 1 Presented by Dr. Bruce A. Chabner at the 89th Annual Meeting of the American under the curve of drug concentration as a function of time and a Association for Cancer Research. March 31. 1998. New Orleans, LA. 2 To whom requests for reprints should be addressed, at the Massachusetts General measure of cumulative exposure, associated with response in a pre Hospital, Cox 640. 100 Blossom Street. Boston, MA 02114. clinical system. Alternatively, the aim may be to maintain plasma 4211

established in animals, then drug concentrations in themselves could become an end point for the Phase I trial. Limiting drug concentration to the "biologically effective range" has a strong rationale, as the specificity of effect is likely to be lost as drug concentrations vastly exceed the binding constant for the intended target. For example, this consideration becomes particularly important in dealing with inhibi tors designed to block the ATP binding pocket of a specific protein kinase. While exquisite specificity of binding is achieved at or near the Ki, broad cross reaction with other kinases occurs at high con centrations of drug (2). The same cross-reactivity with alternative targets may occur with tight-binding enzyme inhibitors, as for exam

GrowthFactor ple with methotrexate (3), and can lead to unanticipated therapeutic or Receptor toxic effects, such as hepatic or neurologic toxicity. In planning a Phase I clinical trial, where the primary objective is to establish a safe dose, therapeutic benefit is also sought, but often not seen (4). In contrast to the usual Phase I trial of a cytotoxic drug, for which most cancer patients would be a candidate, it is important to choose a more restricted patient population for trials of biologically targeted drugs. These agents are often directed at a molecular lesion found in very specific types of cancer (bcr-abl translocations in chronic myelocytic leukemia, k-rai mutations in pancreatic or colon cancer, cyclin D overexpression in mantle cell lymphomas, and other human tumors). In some cases, the molecular change is found in only a subset of patients with a specific histopathological diagnosis. In order to maximize the chance of response and to interpret response as Fig. 1. Sites of action of several molecularly targeted drugs. Some of the intermediates between a membrane-bound growth factor receptor and effects on cellular proliferation a function of the presence of the target in the clinical setting, it is are illustrated. Several molecularly targeted drugs are depicted in ellipses, and the enzyme critical to have information on the presence and/or level of expression or process that each inhibits is indicated. FPT inhibitor, farnesyl protein transferase of these targets in the patients recruited to such trials. Such informa inhibitor: MAP kinase cascade, mitogen-activaled protein kinase cascade. tion can be obtained from testing the initial biopsy material, provided that the test is available, the tissue is adequate, and the test can be concentrations of drug above a trough concentration shown preclini- performed on tissue blocks or preserved specimens. For most molec cally to be adequate to block a specific pathway. Alternatively, the ular testing, the presence of a tissue bank with fresh frozen material is trial might have a molecular/biochemical end point: to maintain in critical to identifying the appropriate patient population in a prelim vivo inhibition of an enzymatic reaction or signaling pathway for a inary fashion. given duration of time. The latter is more difficult to confirm than the An example of the unique pathology and the expression of molec former, in that drug concentrations in plasma are easily measured over ular targets relevant to therapy is illustrated in Fig. 3. This tumor, a time, whereas the activity of an enzyme or expression of a message hemangioendothelioma, expresses markers characteristic of human cannot be repeatedly sampled in tumor over time. endothelial cells (CD-34, Factor 8; Ref. 5). A number of new thera pies, including Folkman's angiostatin-endostatin combination (6), an The strategy of setting dose according to pharmacokinetic end points has a strong rationale, although it has been rarely used in ti-vascular endothelial growth factor antibody (7), anti-endothelial cell antibodies (8), Ruoslahti's doxorubicin-peptide conjugates (9) and, anticancer therapy in the past. More commonly, pharmacokinetics has been used as a surrogate end point to allow more rapid dose escalation potentially at least, vascular endothelial growth factor receptor antag to the maximum tolerated dose (1). However, if the relationship of onists (10) target endothelial cells. If these trials establish the value of drug concentration in plasma to intracellular molecular action can be molecularly targeted therapy, major cancer centers will need to es-

â€¢¿Predmicalefficacy â€¢¿Refineleadactivity Phaselili trial PhaseIIItrial incelllinesand â€¢¿Tumorspecificity â€¢¿Optimizedose â€¢¿Proveefficacy xenografts â€¢¿AccesstoCMS â€¢¿Delineatetoxcity â€¢¿Survival/QOL â€¢¿Preclinicalpharmacology â€¢¿MDRresistance â€¢¿Demonstratemolecular â€¢¿Toxicology activity â€¢¿Formulation â€¢¿Evaluatefortumor response

Fig. 2. The bridge from basic research to clinical application. Some of the key steps in the development of a molecularly targeted drug are illustrated. 4212

Fig. 3. Hemangioendothelioma expressing factor VIII- related antigen. Photomicrograph of a hemangioendothelioma. visualized with peroxidase, after immunohistochemical detection of factor Vlll-related antigen. Multiple tumor cells stain darkly, indicative of factor Vlll-relaled antigen expres sion. â€¢¿-St

' - . tablish laboratories to perform routine molecular pathology on clinical cultured cells, their ability to cause regression of established tumors in specimens in conjunction with clinical trials, and for routine patient mice is variable. Many of the new agents entering trials in man have care. shown the ability to inhibit growth, but a lesser ability to eradicate Beyond the identification of an appropriate patient population, the established tumors. Will signal transduction inhibitors that block the initial Phase I and Phase II trials must address the "proof of principle," pathways from membrane growth factor receptors to the nucleus that is the ability of the drug to interact with and activate or block its induce cell death or simply hold tumors static? The answer to this target. To get that information, novel strategies for the Phase I or question will determine the ultimate strategy for establishing efficacy. Phase II study may be required. One strategy would be to introduce If an agent is cytostatic, then it may be difficult to determine major the new drug in a "window of opportunity" after initial biopsy effects in a nonrandomized Phase II setting. End points such as time diagnosis of lung, colon or breast cancer, but just before definitive to progression or survival are difficult to assess in the absence of a surgical removal of the tumor. This strategy would allow the inves control group. A large Phase III trial may be required even in the tigator to compare pre- and posttreatment activity of the molecular absence of positive Phase II data, with the ultimate end point being target and determine the impact of the drug on the target in vivo, and, time to progression or survival. To give the therapy the best chance thus, could prove the principle upon which the therapy is based. Such possible, it may be necessary to conduct the study in patients with critical proof of principle is often lacking in the initial evaluation minimal disease or with no evident disease, but often such patients are strategy of new cancer drugs. The usual dose escalation to the limits aggressively treated with chemotherapy. In that instance, the trial of tolerance produces a toxicity end point, but offers no direct proof design might require a combination of standard chemotherapy with or that the drug's target is inhibited. without the molecular agent. Such a strategy has established the value Since tumor sampling after drug administration will not be possible of the anti-HER-2-Neu monoclonal antibody in combination with in many patients, noninvasive alternatives for monitoring drug action chemotherapy for metastatic breast cancer (17, 18). will be necessary. Assessment of drug effects in normal tissues, such as circulating mononuclear cells in the peripheral blood or in bone Antisense Oligonucleotides, Molecularly Targeted Drugs marrow, can provide evidence for biological effects, but the relevance of this information to drug effects on tumor tissue are at best tenuous, The problems and pitfalls of taking a molecule from the tissue given the variable contribution of other factors such as drug transport, culture dish to man are clearly reflected in the development of level of expression of the target, and metabolic transformation in antisense Oligonucleotides. Antisense deoxyribonucleotides comple tumor versus normal tissues. A more direct assessment of drug effect mentary to specific RNA sequences bind to and initiate the degrada on tumor may depend on imaging, such as magnetic resonance im tion of message and, thus, prevent the expression of a single, specific aging (11) or positron emission tomography (12-14). For positron gene. Thus, they possess the specificity desired for anticancer therapy emission tomography studies, it is possible to determine tumor uptake and, at least in theory, could be targeted to block the expression of a of drugs labeled with unstable isotopes, or to follow changes in tumor mutant, cancer-causing gene or a gene downstream in an essential metabolism (glucose uptake) using fluorine-18 labeled 2-deoxyglu- signaling pathway. In practical terms, however, sequential scanning of cose or blood flow using oxygen-15 labeled H2O. Magnetic resonance the message is necessary to identify the most vulnerable 20-base imaging may be useful to evaluate vessel size, vascular permeability, sequence, against which the antisense molecule is made. To further and vascular density. complicate their development, these molecules have a number of An overall strategy for developing molecularly targeted drugs must intrinsically undesirable properties as drugs. Oligonucleotides have a ultimately establish antitumor effects in Phase II and Phase III trials. high molecular weight, which deters cellular uptake and oral absorp Although many of the newer drugs such as the antisense oligonucleo- tion. Furthermore, they are difficult to synthesize in large quantity, tides, farnesyl transferase inhibitors (15), and flavopiridol (an inhib unstable in biological fluids, and because they are highly charged they itor of cyclin-dependent kinases; Ref. 16) may induce apoptosis in are slowly taken up by normal and tumor cells. Advances in oligo- 4213

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1998 American Association for Cancer Research. TRANSLATIONAL RESEARCH: BRIDGE BETWEEN IDEA AND CURE nucleotide chemistry have led to the efficient synthesis of the more additional studies of both normal and malignant tissues must be stable phosphorothioate oligonucleotides, which resist exonuclease undertaken in Phase II. which will explore both single agent and degradation and penetrate cells with greater ease (19). combination therapy regimens. A number of antisense ODNs3 are now in clinical trial against cancer, AIDS, other viral infections, and as anti-inflammatory drugs. The Potential of Combination Therapies Those under study in cancer Phase I and II trials block various steps in signal transduction, including Raf-1 (20), Ras, and protein kinase There is strong experimental evidence to support the combination C-a (21), and interact with tumor-specific mutations such as the of anti-signaling agent and chemotherapy. The theoretical basis for bcr-ahl translocation in chronic myelocytic leukemia (22) and the these combinations is the hypothesis, for which there is considerable bcl-2 translocation in small cell lymphomas (23). Because these supporting data, that cell death and cell proliferation are opposing molecules have a high molecular weight, undergo hydrolysis in the limbs of a common signaling system; signals that promote prolifera stomach's acid environment, and when injected i.V., have a short tion simultaneously protect cells against apoptosis induced by DNA halt-life in plasma, they are usually administered by the inconvenient damage, whereas a block in the same proliferative pathways would schedule and route of continuous i.v. infusion. The anticancer ODNs lower the threshold for apoptosis (30, 31). As mentioned previously, directed against Raf-1 and PKC are primarily cytostatic, delaying signaling inhibitors such as flavopiridol and farnesylation blockers tumor growth, and, therefore, must be given for multiple consecutive can induce apoptosis in tumor cells (15). Since many of the newer days (24, 25). In addition, the ODNs, perhaps as a consequence of anticancer drugs are designed to block the signals that promote their si/e, charge, and avid uptake in the reticulo-endothelial system, neoplastic proliferation, if this theory is correct, they would be ex activate, complement, and cause thrombocytopenia. a property that pected to lower the threshold for apoptosis induced by cytotoxic limits dose escalation (26). agents, and such is the case for several combinations studied thus far. The molecules directed against PKC and against Raf-1 have Thus, antibodies that block the HER-2-Neu receptor, an analogue of the EGF-a receptor, promote cell killing by cisplatin (17), paclitaxel reached the Phase II stage of development. A brief history of the Raf antisense trials sponsored by Isis Pharmaceuticals illustrates many of (18), and doxorubicin (18) in mice and in vitro, and enhance the the problems in targeted molecular trials. As shown in Fig. 1, both response rate to paclitaxel and doxorubicin chemotherapy in patients Raf-1 and PKC participate in signaling pathways believed to be with metastatic breast cancer. In a recently completed trial in patients essential in the pathogenesis of some forms of cancer. Raf-1 is the with metastatic breast cancer, the same HER-2-Neu antibody nearly doubled the response rate (62% versus 36%) to paclitaxel or cyclo- immediate downstream partner of Ras. a frequently mutated oncogene phosphamide/doxorubicin (32). Anti-EGF receptor antibody enhances in solid tumors, and is itself overexpressed in lung, breast, and other cancers. In addition, the Ras-Raf-mitogen-activated protein kinase the response to chemotherapy in mice (33), and the combination is pathway transmits signals from a number of growth factor receptors of undergoing trials in man. the tyrosine kinase class, including EOF, insulin-like growth factor-1, There are additional examples of the interaction of signaling inhib platelet-derived growth factor, and fibroblast growth factor (27, 28). itors and cytotoxics. Ras farnesylation inhibitors and paclitaxel dem Potentially, overactivity of any upstream component of the Ras sig onstrate synergy in cell culture (15). In cell culture experiments with naling pathway could be blocked by Raf-1 antisense. human cervical carcinoma cell lines, antisense to raf reverses pacli The Raf-1 antisense drug currently in clinical trials, ISIS 5132, was taxel resistance (34). Other small molecules, that function as inhibi tors of platelet-derived growth factor receptor (35), EOF receptor selected for clinical development based on its ability to inhibit the growth of experimental tumors in vitro (IC50 = 50 nM for sensitive (36), and the fusion protein Bcr-Abl (37), are in early stages of tumors) and in xenografts in mice. Furthermore, it caused a disap development, and have the same potential for synergy with cytotoxic pearance of the Raf-1 message, an effect absolutely dependent on the drugs. For all these compounds, combination trial of traditional cyto complementarity of its antisense sequence. However, tumors regrew toxics and the molecularly targeted signal inhibitors are likely to when drug treatment ceased (24, 29). In the initial human trials, become pathways to early drug approval. continuous i.v. infusion for 21 days was tolerated at drug concentra Other molecularly targeted drugs offer the potential of enhancing tions in plasma of >5(K) nM and, at this concentration c-raf RNA response to cytotoxic drugs through their effects on cell cycle pro expression in peripheral blood mononuclear cells, was diminished gression. When cells are exposed to agents that damage DNA, check points at the G,-S or G-.-M transitions monitor the integrity of DNA during the infusion (20, 26). There are no corresponding studies of effects on Raf-1 activity or expression in tumor cells. Dose-limiting and promote either DNA repair or apoptosis (38). The relationship toxicity was not observed in humans, but dose escalation was stopped between cell cycle transition and sensitivity to chemotherapy is quite as peak plasma concentrations of ODN approached those that caused complex, and seems to differ for the various classes of anticancer complement activation in monkeys (26). During the trial, the inves drugs now in the clinic. Intact p53, which monitors the transition from tigators observed suggestive evidence of drug activity, including G| to synthetic phase and promotes apoptosis of cells with damaged stable disease in a colon cancer patient and two renal cancer patients DNA, confers sensitivity to radiation therapy, alkylating agents, and for more than 7 months, and a marked drop in CA-125 from 1490-47 antimetabolites (39). but not to paclitaxel or the camptothecins. In the units/ml in an ovarian cancer patient. However, there were no partial clinic, the loss of p53 function has been correlated with tumor insen- or complete clinical responses. sitivity to drugs in vitro (40) and a failure to respond to clinical In these trials, a number of important goals were achieved, includ treatment (41). ing the pharmacokinetic end point of exceeding the in vitro IC5(, with To date, no small molecules have been developed that up-regulate prolonged infusion, with possible evidence for antitumor effects. or restore p53 function in mutant cells, although adenovirus vectors Toxicity to normal tissues was tolerable, although the evidence for containing the wild type p53 gene are able to induce apoptosis in effective blockade of the targeted pathway in tumors was lacking. experimental tumors (42) and are being tested alone and in conjunc Given the importance of establishing its effect on Raf-1 expression, tion with irradiation or chemotherapy in man. Recent work by Chu and Allegra (43) suggests that 5-fluorouracil, by binding to thymidy- 1 The abbreviations used are: ODN. oligodeoxyribonucleotide: PKC. protein kinase C; late synthase, may block thymidylate synthase inhibition of p53 EOF, epidermal growth factor: MDR. multidrug resistance: MRP. MDR protein. translation and. thus, might up-regulate p53 in cultured cells. This 4214

action might explain the clinical synergy of 5-fluorouracil with cis- known to be affected by MDR, and a few have been evaluated for platin, alkylating agents, and irradiation, but has the obvious draw susceptibility to MDR (66). Those that target a specific protein in a back that it requires an intact p53 gene. cancer cell are likely to be affected by overexpression, amplification, Although checkpoint function has the important property of pro or mutation of the target protein. Additional problems, such as the moting apoptosis in damaged cells, it also allows time for repair of redundancy of signaling pathways and the multiplicity of proliferative DNA injury. Loss of checkpoint function might sensitize cells to signals for a tumor cell, could negate the effects of a signal transduc- agents (such as the camptothecins and taxanes) that require ongoing tion inhibitor on its target. DNA synthesis to express their lethality (44). Camptothecins promote Perhaps angiogenesis inhibitors, which suppress tumor-induced pro DNA strand breaks, and become lethal to cells when the cells enter liferation of normal endothelial cells, will not be susceptible to drug DNA synthesis. UNC-01, an inhibitor of protein kinase C that abol resistance to the same degree as are approaches that target the malignant ishes the residual G,-S checkpoint in p53 mutant cells, promotes S cell. However, although the normal endothelial cell should be genetically phase entry and sensitizes cells to camptothecin (45). stable, the possibility exists that the tumor may use several of the The logical conclusion of these experiments is that the new molec- alternative signaling pathways that promote angiogenesis. ularly targeted inhibitors of proliferation signals and cell cycle con Conclusion trols must be tested with conventional chemotherapy. Indeed, such combinations may represent a vehicle for early drug approval, in that The biotechnology revolution has created immense opportunities the evaluation of drug combinations with end points such as clinical for advancing the effort to prevent, diagnose, and treat cancer. These response or tumor regression might significantly short circuit the more opportunities require that we rethink our strategies for proving effi complex and time-consuming development plan of the molecularly cacy through clinical trials, so that we obtain full value from the targeted drug by itself. precious molecules that are arriving at the clinical doorstep. Attention to trial design and patient selection, confirmation of mechanisms of Drug Resistance action in man, and imaginative interplay with existing cytotoxic therapies will surely lead to improvements in the management of What implications do molecularly targeted drugs have for the more cancer and ultimately its cure. 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