Annals of Oncology 3: 527-531, 1992. © 1992 Kluwer Academic Publishers. Primed in the Netherlands.
Commentary Signal transduction inhibitors as novel anticancer drugs: Where are we? P. Workman Cancer Research Campaign Beatson Laboratories, CRC Department of Medical Oncology, University of Glasgow, Scotland
Introduction
A landmark in the recognition of signal transduction pathways as fertile ground for new drug development was provided by the AACR/BACR/EORTC meeting on 'The Cell Membrane and Cell Signals as Targets in Cancer Chemotherapy' which was held in Cambridge, UK, in September 1989 [1-3]. Some 2'/2 years later the progress in this area was selectively reviewed in a session of the 7th NCI-EORTC Symposium on New Drugs in Cancer Therapy, Amsterdam, The Netherlands, March 17-20, 1992 [4]. It seems appropriate therefore to ask the question: Where are we? But before that let us briefly consider another question: Where were we? There were several reasons which prompted the sea change towards cancer cell signalling as a viable new approach. There was frustration at that time over the lack of a new breakthrough drug and a growing feeling that conventional 'anti-DNA/cell replication' poisons may have run their course [5|. Linked to that was a mounting concern about the quality of our tumour models and the limitations of the screening approach to drug development [6]. This led to a accelerating enthusiasm for the mechanism-based, biology-driven approach which was beginning to dominate other areas of medicinal chemistry [7, 8]. At the same time enormous progress was taking place in our understanding of the molecular basis of malignant transformation. In particular it became clear that cancer is a series of diseases caused by an accumulation of genetic changes which involve both the mutation or increased expression of growth-promoting cellular oncogenes and the mutation and loss of growth-inhibiting tumour suppressor genes |9|. Of particular excitement to the hungry drug hunter was the realization that the protein products of the various cancer genes were essentially all involved in some way or other with signal transduction [10]. Whether these be growth factor receptors, transducing proteins, signalling enzymes or transcription factors, all have clear potential as drug targets. Moreover, not only has the recombinant DNA revolution led to quantum
leaps in our molecular comprehension of cancer, it has also provided us with unlimited supplies of purified reagents and genetically engineered cell lines [8]. Sitedirected mutagenesis has allowed us to demonstrate for example that disabling the tyrosine kinase activity of the epidermal growth factor receptor blocks not only the phosphorylation of downstream signalling proteins such as phospholipase Cy, but also its growth-promoting ability in the presence of ligand. Thus the drug hunter can rest assured that if he can identify appropriate tyrosine kinase inhibitors these will almost certainly exhibit growth-inhibitory activity. At the outset there were concerns about normal tissue toxicity of anti-signalling drugs. These were answered, initially in a somewhat vague way, by reference to the differential involvement of multiple isoforms of signalling proteins (e.g. protein kinase C) and to the success with other anti-signalling drugs, including most notably of course the antiestrogen tamoxifen. The move into the signalling area was clearly unstoppable given the molecular Zeitgeist prevailing in the late 1980s, and the correctness of this approach was in my view confirmed by the presentations in the Signal Transduction session in Amsterdam [4]. Interestingly, however, it was clear too from the meeting |4] that also rewarded were those who argued the case for retaining a modified screening approach which placed a greater emphasis on solid tumours [11] or human tumour lines [12] rather than rodent leukaemias. There can be no doubt about the highly promising clinical activity of the DNA-interactive anthrapyrazoles, of the camptothecins as inhibitors of topoisomerase 1, or of the antitubulin drugs taxol, taxotere and rhizoxin. The clinical results reported on these new agents at the meeting were certainly impressive. Nevertheless, these agents are hitting conventional targets and one wonders about rediscovering the wheel! There was therefore a considerable degree of interest in the Signal Transduction session - no doubt from the committed, the sceptical and the don't-knows. There are so many potential targets in the cell signalling area [13] that not all could be covered. However, those
528
chosen illustrate the potential of the approach, the progress made and the obstacles still to be overcome. The session was chaired by J. A. Hickman (Manchester University) and M. Moolenar (Netherlands Cancer Institute, Amsterdam). Cell cycle control genes as drug targets In the opening presentation, J. Roberts (Fred Hutchinson Cancer Research Center, Seattle) reviewed our current knowledge of the genes and proteins involved in the control of the replication cycle in yeast and mammalian cells. He argued that whereas in normal cells proliferation is restricted at entry into S-phase, this was not the case in malignant cells. Thus cell cycle regulation is perturbed as a result of oncogenic transformation. Consequently, a detailed analysis of the molecular components involved in cell cycle control may identify novel targets for drug action. The Gl/S checkpoint appears to be rate-limited by the activation of p34 CDC2 kinase or the related p33 CDK 2 kinases at the end of Gl phase. We now know that this activation requires the association of the kinases with positively acting regulatory subunits called cyclins, including cyclins A and E. Specific CDC or CDK kinases complex with specific cyclins at particular phases of the cycle. Thus the activity of the cyclin E/CDK 2 complex peaks in the late Gl and falls during S and G2, whereas the cyclin A/CDC 2 complex activity increases alongside DNA replication and then peaks in late G2. Of great interest is the fact that the latter complex binds to the replication initiation complex and phosphorylates specific proteins involved in this process. Although there is a lot more to be learned about the complex molecular controls on the cell cycle, inhibitors of the players identified so far are already emerging (see below). Very interestingly, the retinoblastoma tumour suppressor gene product and the p53 protein may both receive afferent signals via phosphorylation by CDC kinases [14).
free medium was 0.4 uM for the 3-chloro derivative and ten-fold lower for the 3-azido analogue. Interestingly these agents are incorporated into phosphoinositides in the cell and allow regular calcium ion signalling to occur. Thus growth inhibition was hypothesized to be due to the phosphoinositides acting on phosphatidyl inositol 3'-kinase (PI-3'-kinase). Evidence suggests that this enzyme is important for transformation, although the precise role of the phosphatidylinositol-3-phosphate products of the enzyme is not known. Unfortunately physiological concentrations of myo-inositol antagonize the effects of the analogues and in order to circumvent this the group is now developing 3-substituted phosphatidylinositols. With one such agent, D-3deoxy-3-fluoro-phosphatidylinostol, relatively weak inhibition of cell growth was seen (IC50 ~ 100 uM) and although this was not reversed by myo-inositol the differential activity against oncogene transformed cells was lost. Given the potential importance of the PI-3'kinase this seems an important target to pursue. Powis reported other new data which showed that the PI-3' kinase is also inhibited by ether lipids such as ET-I8-OCH3, SRI 62-834 and the related agent hexadecyclphosphocholine, with IC50s in the range 17-43 \iM. These tend to be a little higher than the cytotoxic concentrations. Ether lipids also inhibit both phosphoinositol-phospholipase C (PI-PLC) and protein kinase C. Both the fS and y forms of PLC are inhibited by concentrations below 1 \iM. PLCp is activated by G-protein-linked receptors and PLCy by association with receptor tyrosine kinases and thus both types of signalling pathways would be blocked by ether lipids. These drugs, which are already in clinical trial, are in fact the most potent known inhibitors of PLC. Powis concluded with a plea for signal transduction inhibitors to be evaluated in relevant tumour models, and also pointed out that such agents might require administration in combination with other drugs, in order to inhibit multiple signalling targets most effectively (see below).
Growth factor and oncogene signalling
Ether lipids as pleiomorphic signalling inhibitors
G. Powis (Mayo Clinic, Rochester and more recently the Arizona Cancer Center, Tucson) reviewed progress in this somewhat more 'established' area of drug hunting. Agents of particular interest he felt included inhibitors of growth factor-receptor binding, modulators of protein kinase C, inhibitors or protein tyrosine kinases, antagonists of calcium ion signalling and compounds which interfere with myo-inositol signals. Powis chose to concentrate on his recent results with two classes of drugs in the latter category. Analogues of wyo-inositol have been made containing substitutions such as fluorine at the 3-position. These were shown to act as selective inhibitors of oncogene-transformed cell lines. For example, in v-sis transformed NIH 3T3 cells the IC50 in /riyo-inositol
P. Workman (Cancer Research Campaign Beatson Laboratories, University of Glasgow) continued the ether lipid theme by underlining the fact that ether lipids are important leads for drug hunting, but act on multiple signalling targets. They are also of interest because such ether lipids as ET-18-OCH3 (eldofosine), BM 41.440 (ilmofosine) and the related hexadecyclphosphocholine (miltefosine) have all entered clinical studies. Another member of the series SRI 62-834 will enter phase I trials shortly with the Cancer Research Campaign Phase I/II Clinical Trials Committee and a further group is under preclinical evaluation by the EORTC New Drug Development Coordinating Committee and Office. A number of responses have been seen although it remains unclear how we should admin-
529
ister these agents for optimal activity. Activity is seen with topical administration in advanced breast cancer for example. There may also be a role in bone marrow purging. Clinical results were presented in a number of posters. Although ether lipids do not interfere directly with DNA they cause a cell cycle arrest at the G2M interphase. In addition to the effects on PLC and protein kinase C mentioned above they also cause direct membrane damage and changes in intracellular calcium levels. They alter phospholipid metabolism and antagonize growth factor-induced signal transduction. The role of endocytotoxic uptake is also a factor in the sensitivity of tumour cells to ether lipids. Antitumour ether lipids are related structurally to the inflammatory mediator platelet activating factor (PAF). However, the antitumour analogues are very much potent in terms of antitumour cytotoxicity and recent evidence shows that PAF receptors are not involved in their mechanism of action. The antitumour ether lipids can induce a growth factor-like increase in intracellular calcium. Again this is not involved in tumour cell killing and it was speculated that it may even be a mitogenic signal, as with lysophosphatidic acid. It appears to be much more significant that ether lipids antagonise the calcium rise induced by mitogenic growth factors, presumably via inhibition of PLC. The combined effects on PLC and other targets may be important (see below). Antitumour ether lipids retain activity in cell lines made resistant to drugs like anthracyclines and cisplatin. Moreover, they may also act as resistance modulators, and this appears to involve the inhibition of signal transduction.
Bryostatin and protein kinase C
P. M. Blumberg (National Cancer Institute, Bethesda) emphasised the importance of this key enzyme in cell signalling and therefore as a target for drug development. Protein kinase C (PKC) is the major receptor of the tumour-promoting phorbol esters, is implicated in drug resistance and is modulated by a wide variety of growth factors and oncogenes. Because of its ubiquitous involvement in multiple growth and differentiation pathways, concern has been expressed about the potentially limited selectivity of PKC drugs. However it is becoming clear that not all PKC modulators have the same effects, possibly implicating specific effects on different PKC isoforms. At least seven genes have been identified. Blumberg concentrated on the PKC modulator bryostatin 1. This is an extremely potent partial agonist of PKC and is currently in clinical trial (see below). Bryostatin induces only some of typical responses of phorbol esters, and indeed it antagonises those phorbol responses which it cannot itself elicit. Certain phorbol derivatives also behave like bryostatin. Thus the partial
agonist prostratin (12-deoxyphorbol 13-acetate) blocks the hyperplasia induced in mouse skin by conventional phorbol esters and also inhibits the associated inflammatory response. Bryostatins cause a faster down-regulation of PKC, exhibit a higher affinity for the enzyme and have a slower off-rate from the enzyme than regular phorbol esters. Many questions remain unanswered about PKC approaches to cancer drug development. Will it be feasible to inhibit particular isoforms of the enzyme? Can we simplify the complex structure of natural PKC modulators and yet still retain activity? How tumour selective will they be? While we await answers to these questions, important results are being obtained from the clinical trials which have now started with the ether lipids and bryostatin 1. A. Harris (Imperial Cancer Research Fund, University of Oxford) reported the latest results from the Cancer Research Campaign clinical trial run jointly between Oxford and Manchester University. The high potency of bryostatin is such that pharmacokinetic studies have not been possible. However, bryostatin 1 elicits a wide range of biological activities including haemopoietic stimulation and immunoenhancing activity so various biological responses have been monitored using modern techniques as a pharmacodynamic guide to dose escalation. These sophisticated studies include: 1) release of 5HT by platelets after ionophore stimulation; 2) cytokine production, particularly TNF and IL2, by individual peripheral lymphoid cells; and 3) changes in peripheral T lymphocytes analysed by flow cytometry and cytokine production by polymerase chain reaction. Platelet cells were also monitored carefully because of the platelet aggregating effects of bryostatin observed in vitro. Results were presented on patients treated at 5, 10, 20, 35 and 50 (ig/rn2- A rapid drop in platelets was seen at 2 hours, but no bleeding problems were observed. Increased TNFa was detected in the serum indicating that bryostatin 1 does have effects on the cytokines. Flu-like symptoms with headache are seen and the dose-limiting toxicity will be myalgia which can be prolonged. The mechanisms involved are uncertain. It is also not clear to what extent protein kinase C modulation is responsible. This study illustrates the biological sophistication which is required in modern phase I studies of signal transduction modulators and at the same time highlight limitations such as bioanalytical problems with highly potent natural products.
Tyrosine kinase inhibitors
E. A. Sausville (National Cancer Institute, Bethesda) reviewed his group's progress with the development of inhibitors of protein tyrosine kinase. The justification for this approach is that the products of numerous viral oncogenes and human proto-oncogenes exhibit protein tyrosine kinase activity. Sausville classified the tyrosine
530
kinase inhibitors as those which compete with ATP and GTP such as flavones and isoflavones, those which compete with the protein substrate typified by the tyrphostins, and those with mixed or unclear mechanisms like the benzoquinoid ansamycins and staurosporins. To date most interest has focused on the tyrphostins because of their potential for greater specificity than the nucleotide triphosphate competitive inhibitors, which would be expected to inhibit a wide range of kinases. In fact, significant specificity against individual kinases can be seen even for the latter agents. Moreover, although tyrphostins with considerable specificity for say the epidermal growth factor versus the insulin receptor kinase can be seen, the number of kinases in which individual agents are evaluated is generally rather limited. Nevertheless, these agents are quite rightly generating very considerable interest, though none has yet entered clinical trial. Sausville described his recent experience with the novel flavone L86-8275 which potently inhibits the proliferation of breast and lung cancer cell lines. G2 and possibly G1S cell cycle arrest is seen. An extremely interesting observation was that whereas a generalised inhibition of protein tyrosine phosphorylation was not observed, phosphorylation of a protein related to p34 CDC 2 kinase was blocked at concentrations around the IC50. This raises the possibility that cell cycle-related protein tyrosine kinases may be achievable targets for inhibition in addition to the receptor and scc/abl type non-receptor kinases. Turning to the tyrophostins, two of these (AG 17 and AG 592) exhibited IC50 values below 10 uM although there was no clear pattern of selectivity for breast cancer lines exhibiting different kinase profiles. Staurosporins were active at less than 1 \iM in breast cancer cell lines, and an inhibition of the phosphorylation of a 30 kd protein is seen. Sausville urged caution in concluding that tyrosine kinase inhibitors were necessarily operating through this mode of action alone. He also introduced a theme which was subsequently taken up by Workman and in the discussion. This was the desirability or otherwise for developing a 'clean' — that is highly molecular target-specific - anti-signalling agents. This has intellectual appeal and offers the opportunity that molecular specificity may translate into rumour selectivity. On the other hand, the multiplicity and redundancy of mitogenic signalling pathways in cancer cells might suggest that drugs working on two or more critical targets could be of great therapeutic value. In support of this view, recent results with broad-spectrum antagonists of neuropeptide mitogen receptors in small cell lung cancer suggests an advantage over specific antagonists for the bombesin/gastrin releasing peptide (poster by Langdon et al., Imperial Cancer Research Fund, Edinburgh University). Suramin also has multiple effects (poster by Myers et al., National Cancer Institute, Bethesda and by others) including inhibition of the IGF-1 receptor and anti-angiogenic activity via block-
ade of the bFGF receptor. Similarly, the ether lipids may owe their activity to their simultaneous inhibition of say PLC and PKC. Thus kinase inhibitors which block multiple tyrosine kinases, or even inhibit serine/ threonine kinases such as raf-1, should not necessarily be discounted. 'Dirty' drugs acting promiscuously on several targets should probably be developed alongside counterparts which act as more monogamous inhibitors. The relative value of the two approaches can then be compared. Of course, 'clean' anti-signalling drugs could always be used in appropriately selected combinations. About half of the posters in the Signal Transduction session dealt with ether lipids, including both basic science and clinical aspects. Also of interest M. Toi (Imperial Cancer Research Fund, University of Oxford) reported that the phosphinositol kinase inhibitors 2, 3-dihydroxybenzaldehyde and psi-tectorigenin were able to provide potent inhibition of microvascular endothelial growth and thus have the potential for novel anti-angiogenic therapy. Some new 3'5'-cyclic nucleotide diesterase inhibitors were presented by M. Drees (University of Kauserslautern). In other sessions, J. Hickman (University of Manchester) reported on the resistance to antimetabolite induced cell death involving the bcl-2 gene as a potential new signalling target and V. Brunton (Cancer Research Campaign, Beatson Laboratories, University of Glasgow) demonstrated growth factor antagonism by the PAF antagonist SDZ 62-434 which is in phase I clinical trial with the Cancer Research Campaign. Several posters also covered differentiation induction, in which signalling clearly plays a key role. Concluding remarks
Although considerable progress is being made in the area of anti-signalling drugs a number of questions remain to be answered. This will require careful molecular pharmacology and experimental chemotherapy studies. Drug resistance mechanisms remain to be elucidated. While several of the well known signalling targets were well covered at the congress, other areas such as ras inhibiting drugs and src homology domains as targets were not represented. As a proportion of the whole meeting, the anti-signalling abstracts represented a relatively small proportion, particularly if the ether lipid presentations were excluded. 'Conventional' agents such as anti-tubulin drugs and topoisomerase inhibitors were more predominant. However, there is no doubt that interest in the signalling area is growing, and the pharmaceutical companies clearly have leads which are not yet sufficiently mature. On the other hand, there is undoubtedly a major contribution to be made by the research institutes and universities. Moreover, collaboration between these two spheres will be increasingly productive. The developing interest in signal transduction targets will accelerate as signalling proteins like the epidermal growth factor receptor, erbB-2, p53 and the
531 nm 23 metastasis suppressor gene product turn out to have prognostic significance, emphasising their likely importance in the malignant transformation. It seems a reasonable prediction that the next NCI-EORTC meeting will see a boom in the number of drugs entering clinical trial as anti-signalling agents for cancer.
8. 9. 10. 11.
References 1. Workman P. The cell membrane and cell signals: New targets for novel anticancer drugs. Ann Oncol 1990; 1: 100-11. 2. Powis G, Hickman J, Workman P et al. The cell membrane and cell signals as targets in cancer chemotherapy. Cancer Res 1990; 50: 2203-11. 3. Proceedings: The cell membrane and cell signals as targets in cancer chemotherapy. Cancer Chemother Pharmac 1989; 24 (Suppl 2). 4. Proceedings: 7th NCI-EORTC symposium on new drugs in cancer therapy. Ann Oncol 1992; 3 (Suppl 1). 5. Tritton TR, Hickman JA. How to kill cancer cells: Membranes and cell signalling as targets in cancer chemotherapy. Cancer Cells 1990; 2: 95-105. 6. Johnson RK. Screening methods in antineoplastic drugs discovery. J Natl Cancer Inst 1990; 82: 1082-3. 7. Johnson RK, Hertzberg RP. Mechanism-based discovery of
Book review Vascular tumors and malformations of the ocular fundus. J. J. de Laey, M. Hanssens (eds). Kluwer Academic Publishers, Dordrecht/Boston/London, 1990. 256 pp, ill., $ 121.00, £67.00, Dfl. 195.00 Edited by two ophthalmologists from the Department of Ophthalmology in Ghent, Belgium, this book is a comprehensive summary of available information about vascular tumors and malformations. It contains chapters on a type of ocular pathology, vascular tumors of the fundus oculi, with which ophthalmologists working outside specialized centers are very rarely confronted. Despite its relative brevity - a little over 200 pages the book is subdivided into various chapters describing several tumorous disorders of vascular origin. Special emphasis is placed on various tests and instruments used for diagnosis. The lists of differential diagnoses are detailed, and an appropriate amount of space is devoted to therapeutic approaches for each pathological entity. There are also detailed descriptions of the histopathological features, some of them with illustrations. In fact, the numerous well-chosen black-and-white illustrations - usually angiograms - are critical for comprehending the text.
12. 13. 14.
anticancer agents. Ann Reports in Medicinal Chemistry 1989; 25: 129-40. Venuti M. The impact of biotechnology on drug discovery. Ann Reports in Medicinal Chemistry 1989; 25: 289-98. Fearon ER, Vogelstein B. A genetic model for colorectal tumorigenesis.Cell 1990;61: 759-67. Cantley LC, Auger KR, Carpenter C et al. Oncogenes and signal transduction. Cell 1991; 64: 281 -302. Grindey GB. Current status of cancer drug development: Failure or limited success? Cancer Cells 1990; 2: 163-71. Boyd MR. Status of the NCI preclinical antitumour drug discovery screen. Principles and Practices of Oncology Updates 1989; 3: No. 10. Powis G. Signalling targets for anticancer drug development. Trends in Pharmac Sci 1991; 12: 188-94. Weinberg RA. Tumour suppressor genes. Science 1991; 254: 1138-45.
Received 24 April 1992; accepted 24 April 1992. Correspondence to: Paul Workman, Ph.D. Cancer Research Campaign Beatson Laboratories CRC Department of Medical Oncology University of Glasgow Garscube Estate, Switchback Road Bearsden, Glasgow G61 1BD Scotland, U.K.
Annals of Oncology 3: 531, 1992.
The book is an excellent reference for this rare type of pathology and, in the rich bibliographies which conclude each chapter, provides sources of specific information. Of special interest for ophthalmologists is the account of systemic aspects of ocular diseases, awareness of which makes possible a complete evaluation as opposed to an eye-focused one; it can also provide invaluable insights into the type of interaction to be sought with colleagues in mutual efforts to provide optimal care to patients. A few of the topics will be of interest to the medical oncologist: von Hippel-Lindau's disease, capillary haemangioma of the optic disc, cavernous haemangioma of the retina and of the optic disc, von Recklinghausen's disease, tuberous sclerosis, and aneurysms which may stimulate malignant tumors due to their anatomical aspect. The book should be available in the libraries of referral institutions where tumorous vascular disorders of the eye are seen more often than once a decade, and which routinely take an interdisciplinary approach to patient treatment. Francesca Stucchi-Goldhirsch Lugano
Commentary Signal transduction inhibitors as novel anticancer drugs: Where are we? P. Workman Cancer Research Campaign Beatson Laboratories, CRC Department of Medical Oncology, University of Glasgow, Scotland
Introduction
A landmark in the recognition of signal transduction pathways as fertile ground for new drug development was provided by the AACR/BACR/EORTC meeting on 'The Cell Membrane and Cell Signals as Targets in Cancer Chemotherapy' which was held in Cambridge, UK, in September 1989 [1-3]. Some 2'/2 years later the progress in this area was selectively reviewed in a session of the 7th NCI-EORTC Symposium on New Drugs in Cancer Therapy, Amsterdam, The Netherlands, March 17-20, 1992 [4]. It seems appropriate therefore to ask the question: Where are we? But before that let us briefly consider another question: Where were we? There were several reasons which prompted the sea change towards cancer cell signalling as a viable new approach. There was frustration at that time over the lack of a new breakthrough drug and a growing feeling that conventional 'anti-DNA/cell replication' poisons may have run their course [5|. Linked to that was a mounting concern about the quality of our tumour models and the limitations of the screening approach to drug development [6]. This led to a accelerating enthusiasm for the mechanism-based, biology-driven approach which was beginning to dominate other areas of medicinal chemistry [7, 8]. At the same time enormous progress was taking place in our understanding of the molecular basis of malignant transformation. In particular it became clear that cancer is a series of diseases caused by an accumulation of genetic changes which involve both the mutation or increased expression of growth-promoting cellular oncogenes and the mutation and loss of growth-inhibiting tumour suppressor genes |9|. Of particular excitement to the hungry drug hunter was the realization that the protein products of the various cancer genes were essentially all involved in some way or other with signal transduction [10]. Whether these be growth factor receptors, transducing proteins, signalling enzymes or transcription factors, all have clear potential as drug targets. Moreover, not only has the recombinant DNA revolution led to quantum
leaps in our molecular comprehension of cancer, it has also provided us with unlimited supplies of purified reagents and genetically engineered cell lines [8]. Sitedirected mutagenesis has allowed us to demonstrate for example that disabling the tyrosine kinase activity of the epidermal growth factor receptor blocks not only the phosphorylation of downstream signalling proteins such as phospholipase Cy, but also its growth-promoting ability in the presence of ligand. Thus the drug hunter can rest assured that if he can identify appropriate tyrosine kinase inhibitors these will almost certainly exhibit growth-inhibitory activity. At the outset there were concerns about normal tissue toxicity of anti-signalling drugs. These were answered, initially in a somewhat vague way, by reference to the differential involvement of multiple isoforms of signalling proteins (e.g. protein kinase C) and to the success with other anti-signalling drugs, including most notably of course the antiestrogen tamoxifen. The move into the signalling area was clearly unstoppable given the molecular Zeitgeist prevailing in the late 1980s, and the correctness of this approach was in my view confirmed by the presentations in the Signal Transduction session in Amsterdam [4]. Interestingly, however, it was clear too from the meeting |4] that also rewarded were those who argued the case for retaining a modified screening approach which placed a greater emphasis on solid tumours [11] or human tumour lines [12] rather than rodent leukaemias. There can be no doubt about the highly promising clinical activity of the DNA-interactive anthrapyrazoles, of the camptothecins as inhibitors of topoisomerase 1, or of the antitubulin drugs taxol, taxotere and rhizoxin. The clinical results reported on these new agents at the meeting were certainly impressive. Nevertheless, these agents are hitting conventional targets and one wonders about rediscovering the wheel! There was therefore a considerable degree of interest in the Signal Transduction session - no doubt from the committed, the sceptical and the don't-knows. There are so many potential targets in the cell signalling area [13] that not all could be covered. However, those
528
chosen illustrate the potential of the approach, the progress made and the obstacles still to be overcome. The session was chaired by J. A. Hickman (Manchester University) and M. Moolenar (Netherlands Cancer Institute, Amsterdam). Cell cycle control genes as drug targets In the opening presentation, J. Roberts (Fred Hutchinson Cancer Research Center, Seattle) reviewed our current knowledge of the genes and proteins involved in the control of the replication cycle in yeast and mammalian cells. He argued that whereas in normal cells proliferation is restricted at entry into S-phase, this was not the case in malignant cells. Thus cell cycle regulation is perturbed as a result of oncogenic transformation. Consequently, a detailed analysis of the molecular components involved in cell cycle control may identify novel targets for drug action. The Gl/S checkpoint appears to be rate-limited by the activation of p34 CDC2 kinase or the related p33 CDK 2 kinases at the end of Gl phase. We now know that this activation requires the association of the kinases with positively acting regulatory subunits called cyclins, including cyclins A and E. Specific CDC or CDK kinases complex with specific cyclins at particular phases of the cycle. Thus the activity of the cyclin E/CDK 2 complex peaks in the late Gl and falls during S and G2, whereas the cyclin A/CDC 2 complex activity increases alongside DNA replication and then peaks in late G2. Of great interest is the fact that the latter complex binds to the replication initiation complex and phosphorylates specific proteins involved in this process. Although there is a lot more to be learned about the complex molecular controls on the cell cycle, inhibitors of the players identified so far are already emerging (see below). Very interestingly, the retinoblastoma tumour suppressor gene product and the p53 protein may both receive afferent signals via phosphorylation by CDC kinases [14).
free medium was 0.4 uM for the 3-chloro derivative and ten-fold lower for the 3-azido analogue. Interestingly these agents are incorporated into phosphoinositides in the cell and allow regular calcium ion signalling to occur. Thus growth inhibition was hypothesized to be due to the phosphoinositides acting on phosphatidyl inositol 3'-kinase (PI-3'-kinase). Evidence suggests that this enzyme is important for transformation, although the precise role of the phosphatidylinositol-3-phosphate products of the enzyme is not known. Unfortunately physiological concentrations of myo-inositol antagonize the effects of the analogues and in order to circumvent this the group is now developing 3-substituted phosphatidylinositols. With one such agent, D-3deoxy-3-fluoro-phosphatidylinostol, relatively weak inhibition of cell growth was seen (IC50 ~ 100 uM) and although this was not reversed by myo-inositol the differential activity against oncogene transformed cells was lost. Given the potential importance of the PI-3'kinase this seems an important target to pursue. Powis reported other new data which showed that the PI-3' kinase is also inhibited by ether lipids such as ET-I8-OCH3, SRI 62-834 and the related agent hexadecyclphosphocholine, with IC50s in the range 17-43 \iM. These tend to be a little higher than the cytotoxic concentrations. Ether lipids also inhibit both phosphoinositol-phospholipase C (PI-PLC) and protein kinase C. Both the fS and y forms of PLC are inhibited by concentrations below 1 \iM. PLCp is activated by G-protein-linked receptors and PLCy by association with receptor tyrosine kinases and thus both types of signalling pathways would be blocked by ether lipids. These drugs, which are already in clinical trial, are in fact the most potent known inhibitors of PLC. Powis concluded with a plea for signal transduction inhibitors to be evaluated in relevant tumour models, and also pointed out that such agents might require administration in combination with other drugs, in order to inhibit multiple signalling targets most effectively (see below).
Growth factor and oncogene signalling
Ether lipids as pleiomorphic signalling inhibitors
G. Powis (Mayo Clinic, Rochester and more recently the Arizona Cancer Center, Tucson) reviewed progress in this somewhat more 'established' area of drug hunting. Agents of particular interest he felt included inhibitors of growth factor-receptor binding, modulators of protein kinase C, inhibitors or protein tyrosine kinases, antagonists of calcium ion signalling and compounds which interfere with myo-inositol signals. Powis chose to concentrate on his recent results with two classes of drugs in the latter category. Analogues of wyo-inositol have been made containing substitutions such as fluorine at the 3-position. These were shown to act as selective inhibitors of oncogene-transformed cell lines. For example, in v-sis transformed NIH 3T3 cells the IC50 in /riyo-inositol
P. Workman (Cancer Research Campaign Beatson Laboratories, University of Glasgow) continued the ether lipid theme by underlining the fact that ether lipids are important leads for drug hunting, but act on multiple signalling targets. They are also of interest because such ether lipids as ET-18-OCH3 (eldofosine), BM 41.440 (ilmofosine) and the related hexadecyclphosphocholine (miltefosine) have all entered clinical studies. Another member of the series SRI 62-834 will enter phase I trials shortly with the Cancer Research Campaign Phase I/II Clinical Trials Committee and a further group is under preclinical evaluation by the EORTC New Drug Development Coordinating Committee and Office. A number of responses have been seen although it remains unclear how we should admin-
529
ister these agents for optimal activity. Activity is seen with topical administration in advanced breast cancer for example. There may also be a role in bone marrow purging. Clinical results were presented in a number of posters. Although ether lipids do not interfere directly with DNA they cause a cell cycle arrest at the G2M interphase. In addition to the effects on PLC and protein kinase C mentioned above they also cause direct membrane damage and changes in intracellular calcium levels. They alter phospholipid metabolism and antagonize growth factor-induced signal transduction. The role of endocytotoxic uptake is also a factor in the sensitivity of tumour cells to ether lipids. Antitumour ether lipids are related structurally to the inflammatory mediator platelet activating factor (PAF). However, the antitumour analogues are very much potent in terms of antitumour cytotoxicity and recent evidence shows that PAF receptors are not involved in their mechanism of action. The antitumour ether lipids can induce a growth factor-like increase in intracellular calcium. Again this is not involved in tumour cell killing and it was speculated that it may even be a mitogenic signal, as with lysophosphatidic acid. It appears to be much more significant that ether lipids antagonise the calcium rise induced by mitogenic growth factors, presumably via inhibition of PLC. The combined effects on PLC and other targets may be important (see below). Antitumour ether lipids retain activity in cell lines made resistant to drugs like anthracyclines and cisplatin. Moreover, they may also act as resistance modulators, and this appears to involve the inhibition of signal transduction.
Bryostatin and protein kinase C
P. M. Blumberg (National Cancer Institute, Bethesda) emphasised the importance of this key enzyme in cell signalling and therefore as a target for drug development. Protein kinase C (PKC) is the major receptor of the tumour-promoting phorbol esters, is implicated in drug resistance and is modulated by a wide variety of growth factors and oncogenes. Because of its ubiquitous involvement in multiple growth and differentiation pathways, concern has been expressed about the potentially limited selectivity of PKC drugs. However it is becoming clear that not all PKC modulators have the same effects, possibly implicating specific effects on different PKC isoforms. At least seven genes have been identified. Blumberg concentrated on the PKC modulator bryostatin 1. This is an extremely potent partial agonist of PKC and is currently in clinical trial (see below). Bryostatin induces only some of typical responses of phorbol esters, and indeed it antagonises those phorbol responses which it cannot itself elicit. Certain phorbol derivatives also behave like bryostatin. Thus the partial
agonist prostratin (12-deoxyphorbol 13-acetate) blocks the hyperplasia induced in mouse skin by conventional phorbol esters and also inhibits the associated inflammatory response. Bryostatins cause a faster down-regulation of PKC, exhibit a higher affinity for the enzyme and have a slower off-rate from the enzyme than regular phorbol esters. Many questions remain unanswered about PKC approaches to cancer drug development. Will it be feasible to inhibit particular isoforms of the enzyme? Can we simplify the complex structure of natural PKC modulators and yet still retain activity? How tumour selective will they be? While we await answers to these questions, important results are being obtained from the clinical trials which have now started with the ether lipids and bryostatin 1. A. Harris (Imperial Cancer Research Fund, University of Oxford) reported the latest results from the Cancer Research Campaign clinical trial run jointly between Oxford and Manchester University. The high potency of bryostatin is such that pharmacokinetic studies have not been possible. However, bryostatin 1 elicits a wide range of biological activities including haemopoietic stimulation and immunoenhancing activity so various biological responses have been monitored using modern techniques as a pharmacodynamic guide to dose escalation. These sophisticated studies include: 1) release of 5HT by platelets after ionophore stimulation; 2) cytokine production, particularly TNF and IL2, by individual peripheral lymphoid cells; and 3) changes in peripheral T lymphocytes analysed by flow cytometry and cytokine production by polymerase chain reaction. Platelet cells were also monitored carefully because of the platelet aggregating effects of bryostatin observed in vitro. Results were presented on patients treated at 5, 10, 20, 35 and 50 (ig/rn2- A rapid drop in platelets was seen at 2 hours, but no bleeding problems were observed. Increased TNFa was detected in the serum indicating that bryostatin 1 does have effects on the cytokines. Flu-like symptoms with headache are seen and the dose-limiting toxicity will be myalgia which can be prolonged. The mechanisms involved are uncertain. It is also not clear to what extent protein kinase C modulation is responsible. This study illustrates the biological sophistication which is required in modern phase I studies of signal transduction modulators and at the same time highlight limitations such as bioanalytical problems with highly potent natural products.
Tyrosine kinase inhibitors
E. A. Sausville (National Cancer Institute, Bethesda) reviewed his group's progress with the development of inhibitors of protein tyrosine kinase. The justification for this approach is that the products of numerous viral oncogenes and human proto-oncogenes exhibit protein tyrosine kinase activity. Sausville classified the tyrosine
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kinase inhibitors as those which compete with ATP and GTP such as flavones and isoflavones, those which compete with the protein substrate typified by the tyrphostins, and those with mixed or unclear mechanisms like the benzoquinoid ansamycins and staurosporins. To date most interest has focused on the tyrphostins because of their potential for greater specificity than the nucleotide triphosphate competitive inhibitors, which would be expected to inhibit a wide range of kinases. In fact, significant specificity against individual kinases can be seen even for the latter agents. Moreover, although tyrphostins with considerable specificity for say the epidermal growth factor versus the insulin receptor kinase can be seen, the number of kinases in which individual agents are evaluated is generally rather limited. Nevertheless, these agents are quite rightly generating very considerable interest, though none has yet entered clinical trial. Sausville described his recent experience with the novel flavone L86-8275 which potently inhibits the proliferation of breast and lung cancer cell lines. G2 and possibly G1S cell cycle arrest is seen. An extremely interesting observation was that whereas a generalised inhibition of protein tyrosine phosphorylation was not observed, phosphorylation of a protein related to p34 CDC 2 kinase was blocked at concentrations around the IC50. This raises the possibility that cell cycle-related protein tyrosine kinases may be achievable targets for inhibition in addition to the receptor and scc/abl type non-receptor kinases. Turning to the tyrophostins, two of these (AG 17 and AG 592) exhibited IC50 values below 10 uM although there was no clear pattern of selectivity for breast cancer lines exhibiting different kinase profiles. Staurosporins were active at less than 1 \iM in breast cancer cell lines, and an inhibition of the phosphorylation of a 30 kd protein is seen. Sausville urged caution in concluding that tyrosine kinase inhibitors were necessarily operating through this mode of action alone. He also introduced a theme which was subsequently taken up by Workman and in the discussion. This was the desirability or otherwise for developing a 'clean' — that is highly molecular target-specific - anti-signalling agents. This has intellectual appeal and offers the opportunity that molecular specificity may translate into rumour selectivity. On the other hand, the multiplicity and redundancy of mitogenic signalling pathways in cancer cells might suggest that drugs working on two or more critical targets could be of great therapeutic value. In support of this view, recent results with broad-spectrum antagonists of neuropeptide mitogen receptors in small cell lung cancer suggests an advantage over specific antagonists for the bombesin/gastrin releasing peptide (poster by Langdon et al., Imperial Cancer Research Fund, Edinburgh University). Suramin also has multiple effects (poster by Myers et al., National Cancer Institute, Bethesda and by others) including inhibition of the IGF-1 receptor and anti-angiogenic activity via block-
ade of the bFGF receptor. Similarly, the ether lipids may owe their activity to their simultaneous inhibition of say PLC and PKC. Thus kinase inhibitors which block multiple tyrosine kinases, or even inhibit serine/ threonine kinases such as raf-1, should not necessarily be discounted. 'Dirty' drugs acting promiscuously on several targets should probably be developed alongside counterparts which act as more monogamous inhibitors. The relative value of the two approaches can then be compared. Of course, 'clean' anti-signalling drugs could always be used in appropriately selected combinations. About half of the posters in the Signal Transduction session dealt with ether lipids, including both basic science and clinical aspects. Also of interest M. Toi (Imperial Cancer Research Fund, University of Oxford) reported that the phosphinositol kinase inhibitors 2, 3-dihydroxybenzaldehyde and psi-tectorigenin were able to provide potent inhibition of microvascular endothelial growth and thus have the potential for novel anti-angiogenic therapy. Some new 3'5'-cyclic nucleotide diesterase inhibitors were presented by M. Drees (University of Kauserslautern). In other sessions, J. Hickman (University of Manchester) reported on the resistance to antimetabolite induced cell death involving the bcl-2 gene as a potential new signalling target and V. Brunton (Cancer Research Campaign, Beatson Laboratories, University of Glasgow) demonstrated growth factor antagonism by the PAF antagonist SDZ 62-434 which is in phase I clinical trial with the Cancer Research Campaign. Several posters also covered differentiation induction, in which signalling clearly plays a key role. Concluding remarks
Although considerable progress is being made in the area of anti-signalling drugs a number of questions remain to be answered. This will require careful molecular pharmacology and experimental chemotherapy studies. Drug resistance mechanisms remain to be elucidated. While several of the well known signalling targets were well covered at the congress, other areas such as ras inhibiting drugs and src homology domains as targets were not represented. As a proportion of the whole meeting, the anti-signalling abstracts represented a relatively small proportion, particularly if the ether lipid presentations were excluded. 'Conventional' agents such as anti-tubulin drugs and topoisomerase inhibitors were more predominant. However, there is no doubt that interest in the signalling area is growing, and the pharmaceutical companies clearly have leads which are not yet sufficiently mature. On the other hand, there is undoubtedly a major contribution to be made by the research institutes and universities. Moreover, collaboration between these two spheres will be increasingly productive. The developing interest in signal transduction targets will accelerate as signalling proteins like the epidermal growth factor receptor, erbB-2, p53 and the
531 nm 23 metastasis suppressor gene product turn out to have prognostic significance, emphasising their likely importance in the malignant transformation. It seems a reasonable prediction that the next NCI-EORTC meeting will see a boom in the number of drugs entering clinical trial as anti-signalling agents for cancer.
8. 9. 10. 11.
References 1. Workman P. The cell membrane and cell signals: New targets for novel anticancer drugs. Ann Oncol 1990; 1: 100-11. 2. Powis G, Hickman J, Workman P et al. The cell membrane and cell signals as targets in cancer chemotherapy. Cancer Res 1990; 50: 2203-11. 3. Proceedings: The cell membrane and cell signals as targets in cancer chemotherapy. Cancer Chemother Pharmac 1989; 24 (Suppl 2). 4. Proceedings: 7th NCI-EORTC symposium on new drugs in cancer therapy. Ann Oncol 1992; 3 (Suppl 1). 5. Tritton TR, Hickman JA. How to kill cancer cells: Membranes and cell signalling as targets in cancer chemotherapy. Cancer Cells 1990; 2: 95-105. 6. Johnson RK. Screening methods in antineoplastic drugs discovery. J Natl Cancer Inst 1990; 82: 1082-3. 7. Johnson RK, Hertzberg RP. Mechanism-based discovery of
Book review Vascular tumors and malformations of the ocular fundus. J. J. de Laey, M. Hanssens (eds). Kluwer Academic Publishers, Dordrecht/Boston/London, 1990. 256 pp, ill., $ 121.00, £67.00, Dfl. 195.00 Edited by two ophthalmologists from the Department of Ophthalmology in Ghent, Belgium, this book is a comprehensive summary of available information about vascular tumors and malformations. It contains chapters on a type of ocular pathology, vascular tumors of the fundus oculi, with which ophthalmologists working outside specialized centers are very rarely confronted. Despite its relative brevity - a little over 200 pages the book is subdivided into various chapters describing several tumorous disorders of vascular origin. Special emphasis is placed on various tests and instruments used for diagnosis. The lists of differential diagnoses are detailed, and an appropriate amount of space is devoted to therapeutic approaches for each pathological entity. There are also detailed descriptions of the histopathological features, some of them with illustrations. In fact, the numerous well-chosen black-and-white illustrations - usually angiograms - are critical for comprehending the text.
12. 13. 14.
anticancer agents. Ann Reports in Medicinal Chemistry 1989; 25: 129-40. Venuti M. The impact of biotechnology on drug discovery. Ann Reports in Medicinal Chemistry 1989; 25: 289-98. Fearon ER, Vogelstein B. A genetic model for colorectal tumorigenesis.Cell 1990;61: 759-67. Cantley LC, Auger KR, Carpenter C et al. Oncogenes and signal transduction. Cell 1991; 64: 281 -302. Grindey GB. Current status of cancer drug development: Failure or limited success? Cancer Cells 1990; 2: 163-71. Boyd MR. Status of the NCI preclinical antitumour drug discovery screen. Principles and Practices of Oncology Updates 1989; 3: No. 10. Powis G. Signalling targets for anticancer drug development. Trends in Pharmac Sci 1991; 12: 188-94. Weinberg RA. Tumour suppressor genes. Science 1991; 254: 1138-45.
Received 24 April 1992; accepted 24 April 1992. Correspondence to: Paul Workman, Ph.D. Cancer Research Campaign Beatson Laboratories CRC Department of Medical Oncology University of Glasgow Garscube Estate, Switchback Road Bearsden, Glasgow G61 1BD Scotland, U.K.
Annals of Oncology 3: 531, 1992.
The book is an excellent reference for this rare type of pathology and, in the rich bibliographies which conclude each chapter, provides sources of specific information. Of special interest for ophthalmologists is the account of systemic aspects of ocular diseases, awareness of which makes possible a complete evaluation as opposed to an eye-focused one; it can also provide invaluable insights into the type of interaction to be sought with colleagues in mutual efforts to provide optimal care to patients. A few of the topics will be of interest to the medical oncologist: von Hippel-Lindau's disease, capillary haemangioma of the optic disc, cavernous haemangioma of the retina and of the optic disc, von Recklinghausen's disease, tuberous sclerosis, and aneurysms which may stimulate malignant tumors due to their anatomical aspect. The book should be available in the libraries of referral institutions where tumorous vascular disorders of the eye are seen more often than once a decade, and which routinely take an interdisciplinary approach to patient treatment. Francesca Stucchi-Goldhirsch Lugano
