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Targeting the cell cycle
Vol. 1, No. 4 2004
Drug Discovery Today: Therapeutic Strategies Editors-in-Chief Raymond Baker – formerly University of Southampton, UK and Merck Sharp & Dohme, UK Eliot Ohlstein – GlaxoSmithKline, USA DRUG DISCOVERY
TODAY THERAPEUTIC
STRATEGIES
Cancer
Targeting the cell cycle Peter M. Fischer1,*, David M. Glover2, David P. Lane1 1 2
Cyclacel, James Lindsay Place, Dundee, UK DD1 5JJ Cyclacel, Polgen Division, Babraham Bioincubator, Babraham Hall, Cambridge, UK CB2 4AT
Many existing cancer chemotherapies interfere with DNA replication and formation of the mitotic spindle, that is, the processes central to the cell cycle. Alternative strategies aimed at targeting cell-cycle components that are specifically deregulated in tumour cells are now being explored. Several groups of protein kinases associated with aberrant tumour cell-cycle checkpoints are used as targets for the discovery of small-molecule inhibitor leads. The first of these compounds are now being evaluated in the clinic.
Section Editors: Lance Liotta – National Institutes of Health, Bethesda, MD, USA Neil Gibson – OSI Pharmaceuticals, NY, USA The emergence of molecular targeted therapies (MTTs) provides an excellent opportunity to pharmacologically modulate the aberrant cellcycle processes that are dysregulated in many human tumours. It is fortunate that several proteins that are found in this dysfunctional process are enzymes and are, thus, suitable targets for drug discovery. Here, Peter Fisher and colleagues describe several potential new targets, explain their role in cell-cycle progression and review the current status of known inhibitors. Thus, it seems that the development of MTTs that selectively target the cancer rather than all cell types might soon become a reality.
Introduction The continued integrity of every organism relies on the ability of its component cells to hand down faithfully their genetic material during cell division. The main tasks of the cell cycle are, therefore, to enable and ensure error-free DNA replication, chromosome segregation and cytokinesis. In response to extracellular proliferative signals, a cell enters the cycle from the resting state (G0) and becomes committed to division once it passes the restriction point (R) late in the first gap phase (G1), the point at which it prepares for DNA replication (Fig. 1). Beyond this point, the cell-cycle programme becomes autonomous and its fidelity is interrogated at various stages. DNA-damage checkpoints operate throughout the cycle, especially before (G1–S transition), during and after the DNA-synthesis phase (S), where the cell enters the second gap phase (G2) and prepares for mitosis (M). During mitosis, CENTROSOME (see Glossary) separation, chromosome condensation and formation of the MITOTIC SPINDLE (see Glossary) are instrumental prerequisites in separating the sister chromatids and in dispatching them to the nascent daughter *Corresponding author: (P.M. Fischer) [email protected] 1740-6773/$ ß 2004 Elsevier Ltd. All rights reserved.
DOI: 10.1016/j.ddstr.2004.11.014
cells. These processes are also subject to checkpoints before the cell-cycle programme can be resumed. Depending on the circumstances, activation of a checkpoint enables extra time for repairing the detected lesion or prevents progression to cell division altogether, either by imposing a cycle arrest or by induction of APOPTOSIS (see Glossary).
Conventional cell-cycle-related strategies in oncology Cancer cells arise as a result of somatic mutations, and many of the affected genes are directly or indirectly involved in cell proliferation. In fact, oncogenic processes exert their strongest effects by targeting regulators of the G1 phase. Whereas normal cells rely on mitogenic stimulation to enter the cycle, cancer cells enter and stay in cycle permanently, leading to the unchecked growth of tumours. Furthermore, DNA and spindle checkpoints are also frequently overridden in transformed cells, resulting in additional genetic instability and proliferative advantages of cancer cells compared with normally dividing cells. This situation suggests that reinstating cell-cycle control via pharmacological targeting of deregulated components of the checkpoint pathways should be a www.drugdiscoverytoday.com
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Glossary Anuploidy: chromosome imbalance in daughter cells arising from faulty mitosis. Apoptosis: physiological manifestation of the activated cell death programme, that is, destruction and elimination of cellular components mediated by proteases known as caspases. Centrosome: microtubule organizing centre that promotes recruitment of mitotic proteins and establishment of bipolar spindles during cell division. Kinetochore: structure on either side of the centromeres (condensed regions of the chromosomes where chromatids are held together) for microtubule attachment. Mitotic spindle: a network of microtubules (hollow cylinders made up of a,b-tubulin heterodimers) formed during prophase, which provides the basic structure for chromosome alignment and pulling apart of the sister chromatids before cytokinesis.
viable strategy in anticancer therapy. Many of the traditional chemotherapeutic agents in current clinical use can also be regarded as cell-cycle agents. However, DNA-damaging agents, antimetabolites, topoisomerase inhibitors, as well as agents that disrupt the microtubules of the mitotic spindle are poorly selective for transformed versus normal cells because they interfere directly with cell-cycle processes that are essential to all proliferating cells. The main limitations of existing anticancer drugs arise from adaptive modulation of the levels of both drug and drug targets in tumour cells, as
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well as defects in apoptosis and heightened DNA repair, leading to drug resistance and narrow therapeutic margins. Here, we take a ‘whistle-stop tour’ around the cell cycle and introduce the salient strategies and experimental drugs that have recently been adopted in an effort to target directly some of the deregulated cell-cycle control components rather than the mechanics of cellular proliferation.
Targeting G1/S Upon receipt of mitogenic signals, cells accumulate D-type cyclins (GenBank accession numbers X59798 and M90813). These, in turn, free the cyclin-dependent kinases (CDKs) CDK4 (GenBank accession number M14505) and CDK6 (GenBank accession number X66365) from their inhibitors, mainly INK4 tumour-suppressor proteins (especially p16INK4a; GenBank accession number L27211). The chief function of the cyclin D-CDK complexes is to activate DNA-binding proteins of the E2F family (most importantly E2F1; GenBank accession number M96577) by phosphorylation of the retinoblastoma-associated gene product (pRb; GenBank accession number L41870) and related pocket proteins, which leads to dissociation of inactive pRb–E2F complexes. E2F proteins are the key transcription factors that drive proliferating cells into S phase. Because tumour cells must be able to proliferate in the absence of mitogenic
Figure 1. The mammalian cell cycle. The key processes, that is, DNA replication, chromosome segregation and cell division are indicated diagrammatically, and the phases of the cycle are labelled. Key cell-cycle components that currently serve as targets in drug discovery and development are shown in red. Abbreviations: ARK, aurora kinase; CDK, cyclin-dependent kinase; CHK, checkpoint kinase; PLK, polo-like kinase. The cell-cycle phases are as follows: G0, quiescent phase; G1, first gap phase; S, DNA-synthesis phase; G2, second gap phase; M, mitosis.
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signals, the cyclin D-CDK–INK4–pRb–E2F cascade is altered in >80% of human neoplasias by mutations either within the genes encoding these proteins or in their upstream regulators [1]. Later in G1, pRb is maintained in a hyper-phosphorylated state by the action of the complexes between E-type cyclins (GenBank accession numbers M73812 and AF106690), themselves products of E2F-responsive genes, and CDK2 (GenBank accession number X61622). However, many growth inhibitory signals – such as that from the cell stress-induced p53 (GenBank accession number X02469) or p21 (GenBank accession number L25610) checkpoint – also converge on pRb, which thus determines whether a cell should divide. CDK2 also opposes the inhibitory action of another tumour suppressor, p27 (GenBank accession number U10906), which is phosphorylated and subsequently degraded. A-type cyclins (GenBank accession numbers U66838 and X51688) are expressed next and they replace E-type cyclins as the partners to CDK2; these CDK2–cyclin A complexes continue to keep E2F active by pRb phosphorylation and, thus, permits S-phase entry. Later in S phase, however, the same CDK2–cyclin A complexes phosphorylate E2F, which brings activation of E2F targets to a halt and sets the stage for the next cell-cycle transition. Unscheduled E2F activity in S phase constitutes a potent apoptotic trigger and E2F can thereby act as a tumour suppressor or an oncogene, depending on the context. Because transcription from E2F-responsive promoters is already enhanced in tumour cells, they are closer to the brink of E2F-mediated apoptosis than normal cells and are therefore selectively sensitive to CDK2–cyclin A inhibition [2]. The mainstay of cell-cycle-targeted therapeutic strategies, until recently, has been pharmacological reinstatement of G1/S checks in tumour cells. Early approaches were based on gene therapy with CDK-directed tumour-suppressor proteins (p16, p53/p21 and p27) and oncolytic viruses targeting E2F via p53 or pRb [3]. More recently, these indirect approaches have been supplanted by small-molecule agents, especially ATP-antagonist CDK inhibitors. Several of these agents are currently being evaluated in the clinic (Table 1). At this stage, it is too early to assess objectively the clinical antitumour activity of CDK inhibitors in monotherapy or as combinations with existing chemotherapy, and it also remains unclear if the proposed mechanistic selectivity of CDK inhibitors discussed here will translate into therapeutic margins. Nevertheless, preclinical studies and early clinical results are promising [4]. Another clinical agent that targets G1/S is E7070; its molecular target is not known but CDK activity appears to be inhibited indirectly (Table 1). The effects on tumour cells by the clinical CDK inhibitor agents, as well as the majority of the many preclinical CDKinhibitory pharmacophores reported to date, are owing to inhibition not only of CDK2, but also of CDK1 (GenBank
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accession number X05360; see later), and to a lesser extent of CDK4 or CDK6. As expected, such compounds are capable of inducing apoptosis in G1/S-defective transformed cells more or less selectively. It has transpired recently, however, that CDK1 and/or CDK2 inhibitors also frequently affect CDKs involved not in regulation of the cell cycle, but in transcription. This is the case, at least, for complexes of CDK7 (GenBank accession number X79193) with cyclin H (GenBank accession number U11791) and complexes of CDK9 (GenBank accession number L25676) with cyclin T (GenBank accession numbers AF045161 and AF048732). This activity probably contributes to the pro-apoptotic effects, especially in haematological malignancies, the survival of which depends heavily on expression of short-lived antiapoptotic proteins. The inhibition of additional CDKs by such compounds probably also explains why their pharmacological action leads to cell death even in tumours in which CDK2 activity appears to be redundant [5]. At present, the only truly selective CDK inhibitors known are those that target CDK4 and CDK6. Because cells have not irreversibly committed to division in G1 before R has been traversed, it has been believed that selective CDK4 and CDK6 inhibition should lead to reversible cell cycle arrest, that is, tumour stasis at best. This notion has recently been challenged by the demonstration that a biochemically and mechanistically highly CDK4and CDK6-selective inhibitor can cause marked tumour regression in certain xenograft models [6].
G2 checkpoint abrogators Following DNA replication, cells enter G2, the point at which damaged DNA is detected by two groups of sensor proteins. The main purpose of the G2 checkpoint is to allow time for the cell to repair such damage before mitotic entry. The sensor complexes communicate with the DNA-repair machinery to initiate this process. At the same time, damage caused by replication stress and UV light is signalled via ataxia telangiectasia mutated kinase (ATM; GenBank accession number U33841)-related and Rad3-related kinase (ATR; GenBank accession number AF325699) to the checkpoint kinase1 (CHK1; GenBank accession number AF016582), whereas DNA lesions caused by ionising radiation are relayed via ATM to CHK2 (GenBank accession number AJ131197). The CHK kinases then phosphorylate and deactivate CDC25C (GenBank accession number M34065), a phosphatase whose action on the CDK1–cyclin B complex (GenBank accession numbers M25753, AF002822 and AJ416458), the master switch for G2–M transition, is crucial for progression beyond G2. As a consequence of G1 checkpoint defects, tumour cells appear to depend on the G2 checkpoint more than normally proliferating cells. Abrogation of this checkpoint should, therefore, deny tumour cells the opportunity to repair damaged DNA and should sensitise them towards DNAdamaging chemotherapy. This, indeed, appears to be the www.drugdiscoverytoday.com
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Strategy
Pros
Cons
Latest developments
Who is working on this strategy
References
CDK inhibition
Holds promise as non-genotoxic and non-myelosuppressive therapy modality; first signs from clinical studies show that apoptosis is induced in cancer cells; many compounds identified and selectivity design improving
Most desirable CDK selectivity profile remains unclear (13 human CDKs with varying functions identified so far)
CYC202 (seliciclib) and flavopiridol (alvocidib, HMR1275) in phase II monotherapy and combination clinical studies; BMS 387032, AZD-5438 and ON 01910.Na in phase I studies; many more at preclinical and discovery stages
Groups with compounds currently in clinical evaluation:
[22,25–30]
CHK inhibition
Potential for improved applications Few selective of existing chemotherapies compounds known
KSP inhibition
Expect similar efficacy as established microtubule inhibitors but no neurotoxicity
PLK inhibition
ARK inhibition
Cell-cycle agents with unknown molecular target
UCN-01 in various phase II combinations with chemotherapy
Cyclacel (http://www.cyclacel.com/) Aventis and NCI (http://aventisoncology.com/pipeline.htm)
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Table 1. Overview of new cell-cycle-targeted experimental cancer drugs
Bristol–Myers Squibb (http://www.bms.com/landing/data/index.html) Onconova (http://www.onconova.com/) AstraZeneca (http://www.astrazeneca.com/node/home.aspx) Kyowa Hakko Kogyo (http://www.kyowa.co.jp/eng/)
[9,10]
Possibly myelosuppressive SB-715992 in phase I/II owing to lack of selectivity for tumour cells
Cytokinetics and GlaxoSmithKline (http://www.cytokinetics.com/, http://science.gsk.com/)
[31]
Suppression of PLK expression in proof-of-principle studies provides good target validation
Very few inhibitors known
HMN-214 in phase I
Nippon Shinyaku (http://www.nippon-shinyaku.co.jp/english/)
[32,33]
Strong association of ARK function with cancer; several selective compounds identified
Effect on proliferating normal cells unclear
VX-680; R763, and AZD1152 in preclinical development
Vertex and Merck (http://www.vrtx.com/, http://www.merck.com/)
[34–37]
Rigel (http://www.rigel.com/rigel/oncology) AstraZeneca (http://www.astrazeneca.com/node/researchoncology.aspx) G1/S inhibitor indisulam (E7070) in phase I/II; M-phase inhibitor Ro 31-7453 in phase I
Eisai (http://www.eisai.com/) F. Hoffmann-La Roche (http://www.roche.com/home.htm)
[38–40]
Abbreviations: ARK: aurora kinase; CDK: cyclin-dependent kinase; CHK: checkpoint kinase; KSP: kinesin-like spindle protein; PLK: polo-like kinase.
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case because membrane-permeable CDC25C substrate peptides have been observed to sensitise p53-defective cancer cells to DNA-damaging agents without obvious cytotoxic effects on normal cells [7]. All four G2 checkpoint kinases, that is, ATM, ATR, CHK1 and CHK2, represent potential drug targets, but CHK1 is especially promising because its depletion in somatic cells is non-toxic and demonstrably increases the sensitivity of tumour cells to DNA-damaging agents [8]. Various kinase inhibitors with G2-checkpoint abrogating properties are known, including, the methylxanthine derivatives caffeine and pentoxyfilline (ATM and ATR inhibitors), indolocarbazoles such as UCN-01 and SB-218078, and debromohymenialdisine (CHK inhibitors), as well as inhibitors with other G2 targets and compounds with unknown mode of action [9]. Unfortunately, these agents do not appear to be specific, and only UCN-01 is currently being developed clinically (Table 1). This compound is structurally related to the promiscuous kinase inhibitor staurosporine; apart from CHKs, it also potently inhibits for example, CDKs and protein kinase C. Multiple phase-I and -II trials are currently under way, both as monotherapy, as well as combination studies with platins, topotecan and gemcitabine [10]. Despite the sound rationale for chemosensitisation, the current paucity of experimental agents with G2-checkpoint abrogative activity and the uncertain antiproliferative mechanism of UCN-01 suggest that the clinical potential of G2 drug targets is unlikely to become clear in the near future.
Mitosis and its drug targets Mitosis involves profound reorganization of the cell architecture to permit chromosome segregation and cytokinesis. The orchestration of these events requires extensive regulation because errors leading to ANUPLOIDY (see Glossary) can have catastrophic consequences for an organism. The following is a brief mitotic diary: during prophase, the replicated chromosomes are condensed and the two centrosomes of the cell move to opposite poles. Following nuclear envelope breakdown, the mitotic spindle begins to grow. In pro-metaphase, the microtubules of the spindle start to interact with the chromosomes, culminating in their congression at the centre of the spindle, at a site known as the metaphase plate, with each KINETOCHORE (see Glossary) of the replicated chromosome pointing towards one side of the spindle. Anaphase commences with the initial splitting of sister chromatids at their centromeres. These daughter chromosomes then begin to separate from one another, each moving away from the metaphase plate and towards one of the two spindle pole regions. Telophase completes mitosis before cytokinesis; the chromosomes have now moved close to the spindle pole regions, and the midzone of the central spindle begins to form to orchestrate cytokinesis. A central actin ring forms and
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constricts and a new cell membrane is then incorporated to form a boundary between the newly separating daughter cells (Fig. 1). Once the mitosis-promoting factor (MPF) – the active CDK1–cyclin B complex – is formed a cell enters mitosis. MPF formation is tightly regulated and involves, among many other components, yet another CDK, namely the CDK-activating kinase (CAK, a complex that includes CDK7). For mitotic progression, MPF activity must be sustained from prophase to metaphase, and advance to anaphase requires MPF inactivation by the multi-subunit anaphase-promoting complex (APC). The regulation of activity and spatiotemporal distribution of the CDK1–cyclin B complex is very important for normal cells, not only in preventing CDK1 activation upon triggering of the DNAstructure checkpoints, but also in mitosis at the spindleassembly checkpoint. Its inactivation, once the checkpoint is satisfied, occurs together with the destruction of securin (GenBank accession number AJ223953), an inhibitor of the protease that cleaves chromosome-cohesion proteins, enabling even partitioning of the genetic materials into two daughter nuclei. Direct inhibition of the mitotic spindle with such agents as paclitaxel, docetaxel and vinblastine has been successful therapeutically, although the exact reasons for the observed selectivity between cancerous and normal cells remain elusive. This is probably due, at least in part, to the fact that these drugs exploit defects in the spindle checkpoint of many cancer cells. However, toxicity, drug resistance, complex galenic formulations and poor bioavailability limit the clinical use of spindle poisons in cancer therapy. For these reasons, improved microtubule inhibitors continue to be developed [11] and alternative antimitotic strategies are now emerging. The most advanced of these are as discussed here.
Kinesins Kinesins constitute a fairly diverse family of microtubule motor proteins that are required for bipolar spindle formation. The fact that reversible and selective inhibition of kinesin-like spindle protein (KSP; GenBank accession number X85137), which leads to mitotic arrest and apoptosis, could be achieved with a small molecule was first demonstrated with monastrol [12], which allosterically blocks the ATPase activity of KSP [13]. As KSP is selectively overexpressed in malignant cells and functions exclusively in mitosis, KSP inhibitors might be useful as cancer drugs. Furthermore, KSP is not expressed in differentiated neurons, therefore, the neurotoxicity associated with some existing therapies based on microtubule inhibition should be avoided. This appears to be the case of a KSP inhibitor, SB715992, which is currently being evaluated in early clinical trials (Table 1). www.drugdiscoverytoday.com
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PLKs The four non-redundant polo-like kinases (PLKs) 1–4 (GenBank accession numbers U01038, AF059617, AJ293866 and Y13115) have recently emerged as important cell-cycle regulators. In fact, their leading role in directing mitosis mirrors that of the CDKs in the earlier cell-cycle phases. At the G2–M transition, PLKs appear to oppose CHK activity by activating CDC25C, as well as relieving CDK1 inhibition by blocking the two kinases WEE1 (GenBank accession number U10564) and MYT1 (GenBank accession number U56816), thereby activating CDK1–cyclin B. It remains somewhat unclear, however, whether PLKs are triggers for M-phase entry or if their role is more important in cell-cycle resumption from a state of G2 arrest [14]. Adaptation of cancer cells to proliferation in the presence of damaged DNA might, therefore, depend on PLK activity and this, in turn, would explain why PLKs – especially PLK1 – are frequently overexpressed in transformed cells. PLKs have many additional functions in mitosis, including regulation of centrosome maturation, spindle assembly, APC activities and cytokinesis. It has already been demonstrated that depletion of PLKs in tumour cells induces apoptosis, although the development of pharmacological PLK inhibitors is still at an early stage [15]. Nevertheless, HMN-214 – the antiproliferative and pro-apoptotic effects of which are probably partly owing to indirect PLK inhibition [16] – is currently in early clinical examination (Table 1).
Aurora kinases Another group of mitotic kinases are the Aurora kinases, of which at least Aurora A kinase (also known as aurora-2 and ARK1; GenBank accession number D84212) and Aurora B kinase (also known as aurora-1 and ARK2; GenBank accession number AF008552) have important roles in regulating mitotic progression, especially at the level of centrosome maturation and chromosome segregation. The gene for Aurora A kinase has been shown to be an oncogene and certain mutants are associated with tumour susceptibility [17]. Various genetic studies demonstrate that both Aurora A and B kinases are essential for cell proliferation and the former is highly expressed in a variety of cancer types, including colorectal and pancreatic tumours [18]. For these reasons, specific Aurora kinase inhibitors have been sought as new antimitotic agents [19]. Following the identification of hesperadin as one of the first small-molecule Aurora kinase inhibitors [20], convincing preclinical proof of principle has recently been demonstrated with another molecule, VX-680 (Table 1).
Conclusions The majority of the new mechanism-based cell-cycle-targeting agents are ATP-antagonist serine/threonine kinase inhibitors. Recent success with similar inhibitors of signal422
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Related articles Nurse, P. (2000) A long twentieth century of the cell cycle and beyond. Cell 100, 71–78 Dancey, J. and Sausville, E.A. (2003) Issues and progress with protein kinase inhibitors for cancer treatment. Nat. Rev. Drug Discov. 2, 296– 313 Blagosklonny, M.V. (2004) Prospective strategies to enforce selectively cell death in cancer cells. Oncogene 23, 2967–2975 Dai, W. and Cogswell, J.P. (2003) Polo-like kinases and the microtubule organization center: targets for cancer therapies. Progr. Cell Cycle Res. 5, 327–334 Warner, S.L. et al. (2003) Targeting Aurora-2 kinase in cancer. Mol. Cancer Ther. 2, 589–595
transduction tyrosine kinases [21] suggests that kinase inhibition might also be a viable strategy in cell-cycle modulation. Taken together with the strong association with cancer of many of the cell-cycle kinases discussed above, this permits an optimistic outlook. In principle, agents that target the regulatory components of the cell cycle, which are only activated once the cycle has been entered, should be able to distinguish between quiescent and proliferating cells and, for this reason, should be devoid of some of the systemic toxicities of conventional chemotherapy. Furthermore, the argument that transformed cells become ‘addicted’ to certain cell-cycle checkpoint facilitators to be able to sustain proliferation despite genetic instability and anuploidy means that inhibition of such dysregulated functions should result in selective antiproliferative effects in tumour cells, whereas sparing non-transformed cells that are normally highly proliferative, such as certain stem cells. However, whether or not these expectations – confirmed in many cases in preclinical cell-biology studies – will provide improved therapeutic margins, compared with current treatment modalities, remains to be seen. The most advanced new cell-cycle agents are the CDK inhibitors. Whether or not these will become useful new cancer drugs should emerge soon because two have now reached phase II clinical trials as monotherapy and in combination with existing chemotherapy. Many more ATP-antagonist CDK inhibitor molecules are currently at the late discovery and preclinical development stage [22]. Outstanding questions regarding overall kinase and optimal CDK isoform selectivity should be able to be addressed with these as the structural understanding and design of kinase selectivity continue to progress [15]. The comparative successes and limitations of therapies based on interference with the mitotic spindle have also now spurred the search for alternative targets in mitosis. Again, a host of kinases that are involved in the regulation of the intricate mitotic events are at the forefront [23]. Many more strategies, more or less directly associated with cell-cycle regulation but too numerous to discuss here, also hold promise [24].
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Outstanding issues Can the promise of cyclin-dependent kinase inhibitors in cell-biology and preclinical efficacy model studies be translated into clinical success? How can cell-cycle agents best be used to synergise with established chemotherapy, new signal-transduction modulators, antiangiogenic agents, and so on? What are appropriate pharmacodynamic end-points for effective clinical development of cell-cycle agents? Will mitotic kinase inhibitors be able to build on the success of existing mitotic agents?
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Drug Discovery Today: Therapeutic Strategies Editors-in-Chief Raymond Baker – formerly University of Southampton, UK and Merck Sharp & Dohme, UK Eliot Ohlstein – GlaxoSmithKline, USA DRUG DISCOVERY
TODAY THERAPEUTIC
STRATEGIES
Cancer
Targeting the cell cycle Peter M. Fischer1,*, David M. Glover2, David P. Lane1 1 2
Cyclacel, James Lindsay Place, Dundee, UK DD1 5JJ Cyclacel, Polgen Division, Babraham Bioincubator, Babraham Hall, Cambridge, UK CB2 4AT
Many existing cancer chemotherapies interfere with DNA replication and formation of the mitotic spindle, that is, the processes central to the cell cycle. Alternative strategies aimed at targeting cell-cycle components that are specifically deregulated in tumour cells are now being explored. Several groups of protein kinases associated with aberrant tumour cell-cycle checkpoints are used as targets for the discovery of small-molecule inhibitor leads. The first of these compounds are now being evaluated in the clinic.
Section Editors: Lance Liotta – National Institutes of Health, Bethesda, MD, USA Neil Gibson – OSI Pharmaceuticals, NY, USA The emergence of molecular targeted therapies (MTTs) provides an excellent opportunity to pharmacologically modulate the aberrant cellcycle processes that are dysregulated in many human tumours. It is fortunate that several proteins that are found in this dysfunctional process are enzymes and are, thus, suitable targets for drug discovery. Here, Peter Fisher and colleagues describe several potential new targets, explain their role in cell-cycle progression and review the current status of known inhibitors. Thus, it seems that the development of MTTs that selectively target the cancer rather than all cell types might soon become a reality.
Introduction The continued integrity of every organism relies on the ability of its component cells to hand down faithfully their genetic material during cell division. The main tasks of the cell cycle are, therefore, to enable and ensure error-free DNA replication, chromosome segregation and cytokinesis. In response to extracellular proliferative signals, a cell enters the cycle from the resting state (G0) and becomes committed to division once it passes the restriction point (R) late in the first gap phase (G1), the point at which it prepares for DNA replication (Fig. 1). Beyond this point, the cell-cycle programme becomes autonomous and its fidelity is interrogated at various stages. DNA-damage checkpoints operate throughout the cycle, especially before (G1–S transition), during and after the DNA-synthesis phase (S), where the cell enters the second gap phase (G2) and prepares for mitosis (M). During mitosis, CENTROSOME (see Glossary) separation, chromosome condensation and formation of the MITOTIC SPINDLE (see Glossary) are instrumental prerequisites in separating the sister chromatids and in dispatching them to the nascent daughter *Corresponding author: (P.M. Fischer) [email protected] 1740-6773/$ ß 2004 Elsevier Ltd. All rights reserved.
DOI: 10.1016/j.ddstr.2004.11.014
cells. These processes are also subject to checkpoints before the cell-cycle programme can be resumed. Depending on the circumstances, activation of a checkpoint enables extra time for repairing the detected lesion or prevents progression to cell division altogether, either by imposing a cycle arrest or by induction of APOPTOSIS (see Glossary).
Conventional cell-cycle-related strategies in oncology Cancer cells arise as a result of somatic mutations, and many of the affected genes are directly or indirectly involved in cell proliferation. In fact, oncogenic processes exert their strongest effects by targeting regulators of the G1 phase. Whereas normal cells rely on mitogenic stimulation to enter the cycle, cancer cells enter and stay in cycle permanently, leading to the unchecked growth of tumours. Furthermore, DNA and spindle checkpoints are also frequently overridden in transformed cells, resulting in additional genetic instability and proliferative advantages of cancer cells compared with normally dividing cells. This situation suggests that reinstating cell-cycle control via pharmacological targeting of deregulated components of the checkpoint pathways should be a www.drugdiscoverytoday.com
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Glossary Anuploidy: chromosome imbalance in daughter cells arising from faulty mitosis. Apoptosis: physiological manifestation of the activated cell death programme, that is, destruction and elimination of cellular components mediated by proteases known as caspases. Centrosome: microtubule organizing centre that promotes recruitment of mitotic proteins and establishment of bipolar spindles during cell division. Kinetochore: structure on either side of the centromeres (condensed regions of the chromosomes where chromatids are held together) for microtubule attachment. Mitotic spindle: a network of microtubules (hollow cylinders made up of a,b-tubulin heterodimers) formed during prophase, which provides the basic structure for chromosome alignment and pulling apart of the sister chromatids before cytokinesis.
viable strategy in anticancer therapy. Many of the traditional chemotherapeutic agents in current clinical use can also be regarded as cell-cycle agents. However, DNA-damaging agents, antimetabolites, topoisomerase inhibitors, as well as agents that disrupt the microtubules of the mitotic spindle are poorly selective for transformed versus normal cells because they interfere directly with cell-cycle processes that are essential to all proliferating cells. The main limitations of existing anticancer drugs arise from adaptive modulation of the levels of both drug and drug targets in tumour cells, as
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well as defects in apoptosis and heightened DNA repair, leading to drug resistance and narrow therapeutic margins. Here, we take a ‘whistle-stop tour’ around the cell cycle and introduce the salient strategies and experimental drugs that have recently been adopted in an effort to target directly some of the deregulated cell-cycle control components rather than the mechanics of cellular proliferation.
Targeting G1/S Upon receipt of mitogenic signals, cells accumulate D-type cyclins (GenBank accession numbers X59798 and M90813). These, in turn, free the cyclin-dependent kinases (CDKs) CDK4 (GenBank accession number M14505) and CDK6 (GenBank accession number X66365) from their inhibitors, mainly INK4 tumour-suppressor proteins (especially p16INK4a; GenBank accession number L27211). The chief function of the cyclin D-CDK complexes is to activate DNA-binding proteins of the E2F family (most importantly E2F1; GenBank accession number M96577) by phosphorylation of the retinoblastoma-associated gene product (pRb; GenBank accession number L41870) and related pocket proteins, which leads to dissociation of inactive pRb–E2F complexes. E2F proteins are the key transcription factors that drive proliferating cells into S phase. Because tumour cells must be able to proliferate in the absence of mitogenic
Figure 1. The mammalian cell cycle. The key processes, that is, DNA replication, chromosome segregation and cell division are indicated diagrammatically, and the phases of the cycle are labelled. Key cell-cycle components that currently serve as targets in drug discovery and development are shown in red. Abbreviations: ARK, aurora kinase; CDK, cyclin-dependent kinase; CHK, checkpoint kinase; PLK, polo-like kinase. The cell-cycle phases are as follows: G0, quiescent phase; G1, first gap phase; S, DNA-synthesis phase; G2, second gap phase; M, mitosis.
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signals, the cyclin D-CDK–INK4–pRb–E2F cascade is altered in >80% of human neoplasias by mutations either within the genes encoding these proteins or in their upstream regulators [1]. Later in G1, pRb is maintained in a hyper-phosphorylated state by the action of the complexes between E-type cyclins (GenBank accession numbers M73812 and AF106690), themselves products of E2F-responsive genes, and CDK2 (GenBank accession number X61622). However, many growth inhibitory signals – such as that from the cell stress-induced p53 (GenBank accession number X02469) or p21 (GenBank accession number L25610) checkpoint – also converge on pRb, which thus determines whether a cell should divide. CDK2 also opposes the inhibitory action of another tumour suppressor, p27 (GenBank accession number U10906), which is phosphorylated and subsequently degraded. A-type cyclins (GenBank accession numbers U66838 and X51688) are expressed next and they replace E-type cyclins as the partners to CDK2; these CDK2–cyclin A complexes continue to keep E2F active by pRb phosphorylation and, thus, permits S-phase entry. Later in S phase, however, the same CDK2–cyclin A complexes phosphorylate E2F, which brings activation of E2F targets to a halt and sets the stage for the next cell-cycle transition. Unscheduled E2F activity in S phase constitutes a potent apoptotic trigger and E2F can thereby act as a tumour suppressor or an oncogene, depending on the context. Because transcription from E2F-responsive promoters is already enhanced in tumour cells, they are closer to the brink of E2F-mediated apoptosis than normal cells and are therefore selectively sensitive to CDK2–cyclin A inhibition [2]. The mainstay of cell-cycle-targeted therapeutic strategies, until recently, has been pharmacological reinstatement of G1/S checks in tumour cells. Early approaches were based on gene therapy with CDK-directed tumour-suppressor proteins (p16, p53/p21 and p27) and oncolytic viruses targeting E2F via p53 or pRb [3]. More recently, these indirect approaches have been supplanted by small-molecule agents, especially ATP-antagonist CDK inhibitors. Several of these agents are currently being evaluated in the clinic (Table 1). At this stage, it is too early to assess objectively the clinical antitumour activity of CDK inhibitors in monotherapy or as combinations with existing chemotherapy, and it also remains unclear if the proposed mechanistic selectivity of CDK inhibitors discussed here will translate into therapeutic margins. Nevertheless, preclinical studies and early clinical results are promising [4]. Another clinical agent that targets G1/S is E7070; its molecular target is not known but CDK activity appears to be inhibited indirectly (Table 1). The effects on tumour cells by the clinical CDK inhibitor agents, as well as the majority of the many preclinical CDKinhibitory pharmacophores reported to date, are owing to inhibition not only of CDK2, but also of CDK1 (GenBank
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accession number X05360; see later), and to a lesser extent of CDK4 or CDK6. As expected, such compounds are capable of inducing apoptosis in G1/S-defective transformed cells more or less selectively. It has transpired recently, however, that CDK1 and/or CDK2 inhibitors also frequently affect CDKs involved not in regulation of the cell cycle, but in transcription. This is the case, at least, for complexes of CDK7 (GenBank accession number X79193) with cyclin H (GenBank accession number U11791) and complexes of CDK9 (GenBank accession number L25676) with cyclin T (GenBank accession numbers AF045161 and AF048732). This activity probably contributes to the pro-apoptotic effects, especially in haematological malignancies, the survival of which depends heavily on expression of short-lived antiapoptotic proteins. The inhibition of additional CDKs by such compounds probably also explains why their pharmacological action leads to cell death even in tumours in which CDK2 activity appears to be redundant [5]. At present, the only truly selective CDK inhibitors known are those that target CDK4 and CDK6. Because cells have not irreversibly committed to division in G1 before R has been traversed, it has been believed that selective CDK4 and CDK6 inhibition should lead to reversible cell cycle arrest, that is, tumour stasis at best. This notion has recently been challenged by the demonstration that a biochemically and mechanistically highly CDK4and CDK6-selective inhibitor can cause marked tumour regression in certain xenograft models [6].
G2 checkpoint abrogators Following DNA replication, cells enter G2, the point at which damaged DNA is detected by two groups of sensor proteins. The main purpose of the G2 checkpoint is to allow time for the cell to repair such damage before mitotic entry. The sensor complexes communicate with the DNA-repair machinery to initiate this process. At the same time, damage caused by replication stress and UV light is signalled via ataxia telangiectasia mutated kinase (ATM; GenBank accession number U33841)-related and Rad3-related kinase (ATR; GenBank accession number AF325699) to the checkpoint kinase1 (CHK1; GenBank accession number AF016582), whereas DNA lesions caused by ionising radiation are relayed via ATM to CHK2 (GenBank accession number AJ131197). The CHK kinases then phosphorylate and deactivate CDC25C (GenBank accession number M34065), a phosphatase whose action on the CDK1–cyclin B complex (GenBank accession numbers M25753, AF002822 and AJ416458), the master switch for G2–M transition, is crucial for progression beyond G2. As a consequence of G1 checkpoint defects, tumour cells appear to depend on the G2 checkpoint more than normally proliferating cells. Abrogation of this checkpoint should, therefore, deny tumour cells the opportunity to repair damaged DNA and should sensitise them towards DNAdamaging chemotherapy. This, indeed, appears to be the www.drugdiscoverytoday.com
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Strategy
Pros
Cons
Latest developments
Who is working on this strategy
References
CDK inhibition
Holds promise as non-genotoxic and non-myelosuppressive therapy modality; first signs from clinical studies show that apoptosis is induced in cancer cells; many compounds identified and selectivity design improving
Most desirable CDK selectivity profile remains unclear (13 human CDKs with varying functions identified so far)
CYC202 (seliciclib) and flavopiridol (alvocidib, HMR1275) in phase II monotherapy and combination clinical studies; BMS 387032, AZD-5438 and ON 01910.Na in phase I studies; many more at preclinical and discovery stages
Groups with compounds currently in clinical evaluation:
[22,25–30]
CHK inhibition
Potential for improved applications Few selective of existing chemotherapies compounds known
KSP inhibition
Expect similar efficacy as established microtubule inhibitors but no neurotoxicity
PLK inhibition
ARK inhibition
Cell-cycle agents with unknown molecular target
UCN-01 in various phase II combinations with chemotherapy
Cyclacel (http://www.cyclacel.com/) Aventis and NCI (http://aventisoncology.com/pipeline.htm)
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Table 1. Overview of new cell-cycle-targeted experimental cancer drugs
Bristol–Myers Squibb (http://www.bms.com/landing/data/index.html) Onconova (http://www.onconova.com/) AstraZeneca (http://www.astrazeneca.com/node/home.aspx) Kyowa Hakko Kogyo (http://www.kyowa.co.jp/eng/)
[9,10]
Possibly myelosuppressive SB-715992 in phase I/II owing to lack of selectivity for tumour cells
Cytokinetics and GlaxoSmithKline (http://www.cytokinetics.com/, http://science.gsk.com/)
[31]
Suppression of PLK expression in proof-of-principle studies provides good target validation
Very few inhibitors known
HMN-214 in phase I
Nippon Shinyaku (http://www.nippon-shinyaku.co.jp/english/)
[32,33]
Strong association of ARK function with cancer; several selective compounds identified
Effect on proliferating normal cells unclear
VX-680; R763, and AZD1152 in preclinical development
Vertex and Merck (http://www.vrtx.com/, http://www.merck.com/)
[34–37]
Rigel (http://www.rigel.com/rigel/oncology) AstraZeneca (http://www.astrazeneca.com/node/researchoncology.aspx) G1/S inhibitor indisulam (E7070) in phase I/II; M-phase inhibitor Ro 31-7453 in phase I
Eisai (http://www.eisai.com/) F. Hoffmann-La Roche (http://www.roche.com/home.htm)
[38–40]
Abbreviations: ARK: aurora kinase; CDK: cyclin-dependent kinase; CHK: checkpoint kinase; KSP: kinesin-like spindle protein; PLK: polo-like kinase.
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case because membrane-permeable CDC25C substrate peptides have been observed to sensitise p53-defective cancer cells to DNA-damaging agents without obvious cytotoxic effects on normal cells [7]. All four G2 checkpoint kinases, that is, ATM, ATR, CHK1 and CHK2, represent potential drug targets, but CHK1 is especially promising because its depletion in somatic cells is non-toxic and demonstrably increases the sensitivity of tumour cells to DNA-damaging agents [8]. Various kinase inhibitors with G2-checkpoint abrogating properties are known, including, the methylxanthine derivatives caffeine and pentoxyfilline (ATM and ATR inhibitors), indolocarbazoles such as UCN-01 and SB-218078, and debromohymenialdisine (CHK inhibitors), as well as inhibitors with other G2 targets and compounds with unknown mode of action [9]. Unfortunately, these agents do not appear to be specific, and only UCN-01 is currently being developed clinically (Table 1). This compound is structurally related to the promiscuous kinase inhibitor staurosporine; apart from CHKs, it also potently inhibits for example, CDKs and protein kinase C. Multiple phase-I and -II trials are currently under way, both as monotherapy, as well as combination studies with platins, topotecan and gemcitabine [10]. Despite the sound rationale for chemosensitisation, the current paucity of experimental agents with G2-checkpoint abrogative activity and the uncertain antiproliferative mechanism of UCN-01 suggest that the clinical potential of G2 drug targets is unlikely to become clear in the near future.
Mitosis and its drug targets Mitosis involves profound reorganization of the cell architecture to permit chromosome segregation and cytokinesis. The orchestration of these events requires extensive regulation because errors leading to ANUPLOIDY (see Glossary) can have catastrophic consequences for an organism. The following is a brief mitotic diary: during prophase, the replicated chromosomes are condensed and the two centrosomes of the cell move to opposite poles. Following nuclear envelope breakdown, the mitotic spindle begins to grow. In pro-metaphase, the microtubules of the spindle start to interact with the chromosomes, culminating in their congression at the centre of the spindle, at a site known as the metaphase plate, with each KINETOCHORE (see Glossary) of the replicated chromosome pointing towards one side of the spindle. Anaphase commences with the initial splitting of sister chromatids at their centromeres. These daughter chromosomes then begin to separate from one another, each moving away from the metaphase plate and towards one of the two spindle pole regions. Telophase completes mitosis before cytokinesis; the chromosomes have now moved close to the spindle pole regions, and the midzone of the central spindle begins to form to orchestrate cytokinesis. A central actin ring forms and
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constricts and a new cell membrane is then incorporated to form a boundary between the newly separating daughter cells (Fig. 1). Once the mitosis-promoting factor (MPF) – the active CDK1–cyclin B complex – is formed a cell enters mitosis. MPF formation is tightly regulated and involves, among many other components, yet another CDK, namely the CDK-activating kinase (CAK, a complex that includes CDK7). For mitotic progression, MPF activity must be sustained from prophase to metaphase, and advance to anaphase requires MPF inactivation by the multi-subunit anaphase-promoting complex (APC). The regulation of activity and spatiotemporal distribution of the CDK1–cyclin B complex is very important for normal cells, not only in preventing CDK1 activation upon triggering of the DNAstructure checkpoints, but also in mitosis at the spindleassembly checkpoint. Its inactivation, once the checkpoint is satisfied, occurs together with the destruction of securin (GenBank accession number AJ223953), an inhibitor of the protease that cleaves chromosome-cohesion proteins, enabling even partitioning of the genetic materials into two daughter nuclei. Direct inhibition of the mitotic spindle with such agents as paclitaxel, docetaxel and vinblastine has been successful therapeutically, although the exact reasons for the observed selectivity between cancerous and normal cells remain elusive. This is probably due, at least in part, to the fact that these drugs exploit defects in the spindle checkpoint of many cancer cells. However, toxicity, drug resistance, complex galenic formulations and poor bioavailability limit the clinical use of spindle poisons in cancer therapy. For these reasons, improved microtubule inhibitors continue to be developed [11] and alternative antimitotic strategies are now emerging. The most advanced of these are as discussed here.
Kinesins Kinesins constitute a fairly diverse family of microtubule motor proteins that are required for bipolar spindle formation. The fact that reversible and selective inhibition of kinesin-like spindle protein (KSP; GenBank accession number X85137), which leads to mitotic arrest and apoptosis, could be achieved with a small molecule was first demonstrated with monastrol [12], which allosterically blocks the ATPase activity of KSP [13]. As KSP is selectively overexpressed in malignant cells and functions exclusively in mitosis, KSP inhibitors might be useful as cancer drugs. Furthermore, KSP is not expressed in differentiated neurons, therefore, the neurotoxicity associated with some existing therapies based on microtubule inhibition should be avoided. This appears to be the case of a KSP inhibitor, SB715992, which is currently being evaluated in early clinical trials (Table 1). www.drugdiscoverytoday.com
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PLKs The four non-redundant polo-like kinases (PLKs) 1–4 (GenBank accession numbers U01038, AF059617, AJ293866 and Y13115) have recently emerged as important cell-cycle regulators. In fact, their leading role in directing mitosis mirrors that of the CDKs in the earlier cell-cycle phases. At the G2–M transition, PLKs appear to oppose CHK activity by activating CDC25C, as well as relieving CDK1 inhibition by blocking the two kinases WEE1 (GenBank accession number U10564) and MYT1 (GenBank accession number U56816), thereby activating CDK1–cyclin B. It remains somewhat unclear, however, whether PLKs are triggers for M-phase entry or if their role is more important in cell-cycle resumption from a state of G2 arrest [14]. Adaptation of cancer cells to proliferation in the presence of damaged DNA might, therefore, depend on PLK activity and this, in turn, would explain why PLKs – especially PLK1 – are frequently overexpressed in transformed cells. PLKs have many additional functions in mitosis, including regulation of centrosome maturation, spindle assembly, APC activities and cytokinesis. It has already been demonstrated that depletion of PLKs in tumour cells induces apoptosis, although the development of pharmacological PLK inhibitors is still at an early stage [15]. Nevertheless, HMN-214 – the antiproliferative and pro-apoptotic effects of which are probably partly owing to indirect PLK inhibition [16] – is currently in early clinical examination (Table 1).
Aurora kinases Another group of mitotic kinases are the Aurora kinases, of which at least Aurora A kinase (also known as aurora-2 and ARK1; GenBank accession number D84212) and Aurora B kinase (also known as aurora-1 and ARK2; GenBank accession number AF008552) have important roles in regulating mitotic progression, especially at the level of centrosome maturation and chromosome segregation. The gene for Aurora A kinase has been shown to be an oncogene and certain mutants are associated with tumour susceptibility [17]. Various genetic studies demonstrate that both Aurora A and B kinases are essential for cell proliferation and the former is highly expressed in a variety of cancer types, including colorectal and pancreatic tumours [18]. For these reasons, specific Aurora kinase inhibitors have been sought as new antimitotic agents [19]. Following the identification of hesperadin as one of the first small-molecule Aurora kinase inhibitors [20], convincing preclinical proof of principle has recently been demonstrated with another molecule, VX-680 (Table 1).
Conclusions The majority of the new mechanism-based cell-cycle-targeting agents are ATP-antagonist serine/threonine kinase inhibitors. Recent success with similar inhibitors of signal422
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Related articles Nurse, P. (2000) A long twentieth century of the cell cycle and beyond. Cell 100, 71–78 Dancey, J. and Sausville, E.A. (2003) Issues and progress with protein kinase inhibitors for cancer treatment. Nat. Rev. Drug Discov. 2, 296– 313 Blagosklonny, M.V. (2004) Prospective strategies to enforce selectively cell death in cancer cells. Oncogene 23, 2967–2975 Dai, W. and Cogswell, J.P. (2003) Polo-like kinases and the microtubule organization center: targets for cancer therapies. Progr. Cell Cycle Res. 5, 327–334 Warner, S.L. et al. (2003) Targeting Aurora-2 kinase in cancer. Mol. Cancer Ther. 2, 589–595
transduction tyrosine kinases [21] suggests that kinase inhibition might also be a viable strategy in cell-cycle modulation. Taken together with the strong association with cancer of many of the cell-cycle kinases discussed above, this permits an optimistic outlook. In principle, agents that target the regulatory components of the cell cycle, which are only activated once the cycle has been entered, should be able to distinguish between quiescent and proliferating cells and, for this reason, should be devoid of some of the systemic toxicities of conventional chemotherapy. Furthermore, the argument that transformed cells become ‘addicted’ to certain cell-cycle checkpoint facilitators to be able to sustain proliferation despite genetic instability and anuploidy means that inhibition of such dysregulated functions should result in selective antiproliferative effects in tumour cells, whereas sparing non-transformed cells that are normally highly proliferative, such as certain stem cells. However, whether or not these expectations – confirmed in many cases in preclinical cell-biology studies – will provide improved therapeutic margins, compared with current treatment modalities, remains to be seen. The most advanced new cell-cycle agents are the CDK inhibitors. Whether or not these will become useful new cancer drugs should emerge soon because two have now reached phase II clinical trials as monotherapy and in combination with existing chemotherapy. Many more ATP-antagonist CDK inhibitor molecules are currently at the late discovery and preclinical development stage [22]. Outstanding questions regarding overall kinase and optimal CDK isoform selectivity should be able to be addressed with these as the structural understanding and design of kinase selectivity continue to progress [15]. The comparative successes and limitations of therapies based on interference with the mitotic spindle have also now spurred the search for alternative targets in mitosis. Again, a host of kinases that are involved in the regulation of the intricate mitotic events are at the forefront [23]. Many more strategies, more or less directly associated with cell-cycle regulation but too numerous to discuss here, also hold promise [24].
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Outstanding issues Can the promise of cyclin-dependent kinase inhibitors in cell-biology and preclinical efficacy model studies be translated into clinical success? How can cell-cycle agents best be used to synergise with established chemotherapy, new signal-transduction modulators, antiangiogenic agents, and so on? What are appropriate pharmacodynamic end-points for effective clinical development of cell-cycle agents? Will mitotic kinase inhibitors be able to build on the success of existing mitotic agents?
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