Complexities in the development of cyclin-dependent kinase inhibitor drugs

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Complexities in the development of cyclin-dependent kinase inhibitor drugs Edward A. Sausville Abnormalities in the normal regulation of the cell cycle are a hallmark of neoplasia. Drugs directed against the cyclindependent kinases (CDKs), which govern the normal orderly progression through the cell cycle, have been proposed to address the pathogenic defect in tumors. Recently, CDK family members that do not regulate the cell cycle directly but instead influence transcription (CDK7, CDK8, and CDK9) and neuronal and secretory cell function (CDK5) have been described. Continued synthetic chemistry efforts have defined important new selective inhibitors of CDKs, and strategies directed at newly described CDK-related targets, such as transcription control, can now be envisaged. CDKs remain important and novel targets whose potential needs to be more fully explored, albeit in light of the newly emerging complexities of their cellular physiology.

Edward A. Sausville Developmental Therapeutics Program, National Cancer Institute, Executive Plaza North Room 8018, 6130 Executive Boulevard, Rockville, MD 20852, USA. e-mail: [email protected]

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CDKs, the cell cycle and cancer Deregulation of the mechanisms controlling cell-cycle progression is a hallmark of neoplasia [1]. The enzymes governing normal cellular events are shown schematically in Fig. 1a. Cyclins are proteins originally defined by their periodic occurrence correlating with transition from one cell-cycle phase to another. They associate with and activate cyclin-dependent kinases (CDKs), which, as monomeric kinase subunits, are without activity. The orderly progression through the G1 phase (which is driven by cyclins D/CDK4 or D/CDK6 in response to growth factor stimulation) and to the S phase (which is driven by cyclin E/CDK2), leads to phosphorylation of the retinoblastoma susceptibility protein (Rb). This in turn enables transition to the S phase and activation of genes responsible for completing DNA synthesis through unopposed action of the E2F family of transcription factors [2]. Progression through the S phase is marked by the appearance of cyclin A/CDK2 and of inactive cyclin A/CDK1 and cyclin B/CDK1 complexes. Transition from the G2 to M phase is marked by the activation of CDC25 phosphatase (originally defined by a yeast cell division control mutant) to remove inhibitory phosphorylations from and activation of cyclin B/CDK1, with breakdown of the nuclear envelope and chromosome condensation. Cell-cycle ‘checkpoints’ assess the completion of respective stages before progressing to the next phase [3]. Negative regulators of CDK activity include the endogenous inhibitors of CDK catalytic activity, p15, p16, p21, p27, and p57 [4]. Other regulators include activating kinases of CDKs (including the CDK-activating kinase (CAK), which is itself homologous to other CDKs and designated CDK7/cyclinH) and,

indirectly, the chk1 and chk2 modulators of CDC25 phosphatase function. Clinical tumor specimens demonstrate deletion of negative regulatory molecules such as p16, amplification or overexpression of positive regulators, including cyclins D and E, and mutations or deletions of the Rb protein itself [5]. At least one of these alterations occurs in essentially all cancers.This knowledge has provided a collective rationale for regarding CDKs and their associated regulatory molecules as targets for drug discovery efforts. The most simplistic view of how success might be achieved for this type of approach focuses on a molecule that could inhibit CDK4 or CDK6/cyclin D, enabling restoration of normal cell-cycle control to neoplastic cells. Alternatively, model systems have suggested that inhibition of CDK2/cyclin E might promote apoptosis by deregulation of the transcription factor E2F [6].This review outlines recently emerging issues in CDK cellular physiology, with reference to drugs designed to address these targets, in an effort to highlight issues that might aid future development. CDKs: really specific for cell-cycle control? A recent series of developments has highlighted a basis for uncertainty regarding the suitability of CDKs as drug targets directly and specifically related to cell-cycle progression. First, although the term ‘cyclin’ originally referred to a periodically occurring and transiently acting activating subunit, homology cloning has revealed CDKs with activating subunits analogous to the classically defined cyclins that do not, however, vary in expression during the cell cycle. These ‘non-cycling cyclins/CDKs’ are frequently associated with non-cell-cycle-related functions.

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(a)

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For example, the CAK activity associated with CDK7/ cyclin H was originally thought to regulate CDK activity by maintaining the orderly appearance of an activating phosphorylation at a site in the ‘activation loop’ of the kinase corresponding to Thr 160 in CDK1. Analogous sites are known in CDK2, CDK4, and CDK6. However, recent studies have identified CDK7/cyclin H as having a non-cell-cycle role because it also possesses kinase activity for the carboxy-terminal domain (CTD) of RNA polymerase II (RNA pol II). CAK is present in the TFIIH complex, a general transcription factor also containing ERCC3/XPB, ERCC2/XPD, p62, p52, p 44, MAT1, and p34 (summarized in [7]). Thus, many structural motifs responsible for activating cell-cycle-related CDK 1, CDK2, CDK4, and CDK6 implicit in the active site of CDK7/ cyclin H are also relevant to regulating RNA transcription. Further complicating the diversity of roles played by the CDK family, was the subsequent observation that a molecule that binds to the HIV tat gene product also facilitates phosphorylation of the RNA pol II CTD by activating a kinase – CDK9 – that has obvious similarity to the CDKs [8–13]. The tat-binding molecule – cyclin T1 – is the activating cyclin partner of CDK9, and another example of a non-cycling cyclin that does not vary in expression through the cell cycle. Recent studies have confirmed that CDK9/cyclin T1 actually corresponds to the previously described PTEFb transcription factor, and plays a key role in promoting the high efficiency transcription of HIV1 from the integrated pro-virus long terminal repeat (LTR). CDK9/cyclin T1 achieves this as a result of its RNA http://www.trends.com

Review

Figure 1. Roles of cyclin-dependent kinases

Inefficient elongation

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(a) Cyclin-dependent kinases (CDKs) catalytic subunits and cyclins responsible for progression through the indicated cell-cycle phase. Arrowheads indicate activation, and flat bars indicate inhibition by the specific endogenous negative regulator. (b) Transcription-related CDKs promote elongation of nascent transcripts by phosphorylating the carboxy-terminal domain (CTD) of RNA polymerase II. (c) CDK5/p25 facilitates secretion of insulin by phosphorylating an as-yet incompletely defined set of substrates, possibly including munc 18, synapsin-1, and syntaxin, all of which are responsible for secretory vesicle alignment with, for example, the cytoskeleton and membrane (described in detail in [17]).

pol II, CTD-directed kinase activity, which results in an alteration of the state of RNA pol II to produce very efficient elongation of the nascent HIV RNAs (Fig. 1b). A general role for PTEFb (CDK9/cyclin T1) in regulating transcription is suggested by the recent discovery that a very prevalent small nuclear RNA – 7SK – interacts directly with and inhibits CDK9/cyclin T1 kinase activity [14,15], and thus RNA transcription. Indeed, up to 50% of all CDK9/cyclin T (PTEFb) complexes could be constantly bound to 7SK RNA. Degradation of 7SK RNA in response to cellular stresses is proposed to enable activation of stress-response genes. Another recent insight concerns CDK5, which has long been known as a non-cell-cycle-related CDK ‘cousin’. Its cognate activating partner, p35, was not even thought of as a cyclin and had a unique activation strategy – cleavage resulting in a p25 subunit that, as a CDK5/p25 activity, is highly expressed in neural tissue. Indeed, the identification of CDK5/p25 as a prominent tau (microtubule-associated protein) kinase suggested that inhibitors of CDK5/p25 might be useful as therapeutics for Alzheimer’s disease, where phosphorylated tau protein appears to be a prominent component of neurofibrillary tangles underlying the pathogenesis of that disease (discussed in [16]). Recent studies have shown, however, that in addition to a role in the central nervous system, CDK5/p25 also appears to have a prominent role in promoting insulin secretion in the pancreatic β-cell (Fig. 1c) [17]. Thus, modulation of CDK function could have unintended consequences for maintenance of glucose homeostasis.

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Non-specific protein kinase inhibitors Staurosporine (UCN-01)

Selective but ‘pan’ CDK antagonist (first generation) Flavopiridol (CDK1,2,4,6,9)

‘Semi-selective’ second generation antagonists CDK1,2,5 Olomoucine Roscovitine Purvalanol Paullones Butyrolactone CDK1 Thio/oxoflavopiridols

CDK4 Aminothiazoles Benzocarbazoles Pyrimidines CDK2 Oxindoles TRENDS in Molecular Medicine

Figure 2. Generations of cyclin-dependent kinase inhibitor compounds The ‘pan’ cyclin-dependent kinase (CDK) inhibitor flavopiridol potently inhibits all known CDKs to an approximately equal degree. It possibly has a greater potency for CDK9 owing to the capacity to form an ultra-tight binding complex. This has inspired design efforts to derive a second generation of agents with a spectrum of selectivity for the indicated kinase.

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Current anti-CDK drugs: how truly cell-cycle directed? Figure 2 is a schematic depiction of the current known classes of CDK inhibitor drugs. Staurosporine and related indolocarbazole drugs certainly can affect CDKs but are not selective in comparison to their effects on other kinases. Flavopiridol was the first relatively selective inhibitor of CDKs to be elucidated [5], with Ki or IC50s in the 40–400 nM range for all CDKs tested. Comparable inhibitory potency against glycogen synthase kinase (GSK)-3β has recently been found [18], but other kinases, such as protein kinase C, epidermal growth factor receptor, and cAMP-dependant kinase are susceptible to inhibition only with drug concentrations in the midto-high µM concentration range. A detailed account of the discovery, biochemical and cellular pharmacology, and initial clinical studies with flavopiridol has been given elsewhere [5,19]. Although early evaluations of flavopiridol documented its ability to inhibit labeled uridine incorporation into RNA over a period of several hours, as well as its capacity to decrease cyclin D1 and its mRNA [20,21], the basis for these phenomena were not clear. The association of CDKs with the transcriptional regulation described previously prompted studies that indeed revealed that flavopiridol is a potent inhibitor of CDK9/ cyclin T1 (or PTEFb) activity [22], resulting in a potent ability to inhibit HIV transcription. Subsequent studies have gone on to show the capacity of flavopiridol to block RNA pol-II-related transcription in non-HIV-infected cells [23], and therefore its potential to affect cell-cycle progression by this rather indirect mechanism, as well as its potential to affect the catalytic activity of the bona fide

cell-cycle-related CDKs. Indeed, certain features of the interaction of flavopiridol with CDK9/cyclinT1 are unique, in that, in contrast to the kinetic features of the actions of the drug with CDK1, CDK2, and CDK4, flavopiridol interacts with CDK9/cyclin T1 with the properties of a ‘tightbinding’ inhibitor. These features are distinct from findings with other CDKs, where there is simple competitive inhibition of ATP [23]. It is apparent that a most informed understanding of flavopiridol and other CDK-directed drug activities would consider their actions against transcription-related CDKs as well as activities directed against the cell-cycle CDKs. Apoptosis mechanisms from CDK inhibitors Indeed, these findings have prompted the suggestion that all of the growth-regulatory features of flavopiridol action might be related to the alteration of RNA polymerase function [23]. Although from a simple affinity and stoichiometric point of view this possibility has to be considered quite seriously, several ambiguities remain. First, drug concentrations that clearly do not appear to inhibit α-amanitin-sensitive RNA synthesis, for example in HeLa cells [23], are known to cause cell-cycle arrest in other cell types [21]. Second, even HIV-related RNA synthesis appears to be inordinately sensitive to flavopiridol in comparison to total RNA synthesis. It is therefore not clear that CDK9/cyclin T is always uniformly susceptible to flavopiridol in all its physiological states. Nonetheless, this should be regarded as a very open question at this point, and one that might require elaboration for each cell type. An argument in favor of the importance of the RNA-synthesis-related mechanism for the prominent proapoptotic effects of flavopiridol in lymphoid cells, was the recent discovery in activated large-cell lymphoma cells that flavopiridol prominently and selectively affects the expression of certain cell-cycle and survival-related genes such as mcl1 and c-myc. Indeed, microarray analysis shows that, at certain concentrations, the actions of flavopiridol closely resemble those of actinomycin D or DRB – both recognized inhibitors of RNA polymerase action [24]. This analysis therefore suggests a CDK-related basis for the poorly understood capacity of flavopiridol to evoke an apoptotic response in certain cell types, for example, certain leukemia/lymphoma [25] and squamous carcinoma [26] cells. According to this point of view, rapid downregulation of the expression of cell survival genes would enable susceptible cells to become predisposed to apoptosis, as shown previously in hematopoeitc cell types with evidence of downregulated bcl2 [27] and mcl1 [28], although these responses were not observed in all cell types [25]. It should be noted that the RNA-directed effects of flavopiridol do not imply therapeutic equivalence to RNA http://www.trends.com

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CDK1 CDK2 CDK5 GSK3β

10 | EKIGEGTYGVVYKG EKIGEGTYGVVYKA EKIGEGTYGTVFKA KVIGNGSFGVVYQA

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synthesis inhibitors such as actinomycin D; the latter has a very distinctive human toxicology spectrum and is poorly reversible after short exposures in model systems or in clinical use. Efforts for greater CDK selectivity The ‘pan’-CDK nature of the inhibitory capacity of flavopiridol has prompted a continuing search for molecules that are perhaps related more specifically to inhibition, ideally of single CDKs. These second-generation CDK inhibitors can be grouped into families related to their respective patterns of CDK inhibition (Fig. 2). For example, a large series of purine-related structures (including olomoucine, roscovitine, purvanolols, the recently described benzyl-purine and pyrimidine derivatives, and non-purine structures such as the paullones and indigo-derived compounds [16,29,18]), have relative selectivity for CDK1, CDK2, and CDK5. Relative (approximately 10-fold) selectivity for CDK1 has been displayed by oxoflavopiridols and thioflavopiridols [30], and for CDK2 by oxindoles [31]. Isolated CDK4 or CDK6 selectivity has been less frequently apparent but, recently, CDK4selective antagonists have been described, including aminothiazoles, benzocarbozoles, and pyrimidine-based compounds ([32,33] and reviewed in 34]). A problem that emerges immediately, however, is that if the goal of these synthetic efforts has been to promote the evolution of structures with relative selectivity for the cell-cycle-related CDKs, little or no information is available concerning the capacity of these newer molecules to inhibit the RNA-polymerase-regulating CDK7, CDK8, and CDK9. Indeed, studying the ATP-binding domains of CDKs and GSK-3β (Fig. 3), similar – if not identical – regions of homology are prominent. Thus, at least with respect to ATP-site-directed agents, it will be difficult to evolve selective inhibitors of CDK1 and CDK2 versus CDK9. As might be expected, therefore, the CDK1-, CDK2-, CDK5-directed molecule roscovitine has recently been demonstrated to inhibit HIV replication, implying at least some capacity to inhibit CDK9/cyclin T [35]. http://www.trends.com

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Review

Figure 3. Primary sequence of CDKs Sections of each cyclindependent kinase (CDK) related to an idealized ATP-binding domain are highlighted. Plain text indicates identical residues; bold text denotes varying residues; and underlined text denotes potentially similar functionalities to consensus residues. Specific sources for sequences: CDK1 [48], CDK2 [49], CDK5 [50], GSK3β [51], CDK4 [52], CDK6 [53], CDK9 [54].

CDK inhibitors: tools or therapeutics? The recent progress in linking CDKs with control of transcription poses the question of whether the compounds so far derived are the best molecules for clinical use if the therapeutic goal is specifically to ‘re-regulate’ the enzymatic machinery responsible for altered cell-cycle control in neoplasia. On the one hand, although there is no question that the molecules produced to date are fascinating for the dissection of cellular pharmacology, and now perhaps transcriptional control, the potential for global effects on transcription and exocytosis by CDK-related mechanisms could be perceived as detracting from their eventual value as therapeutics. On the other hand, the potential capacity of CDK 9/cyclin T to regulate gene expression, apoptosis, and retroviral transcription might lead to valuable new strategies to better exploit drug regulation of CDK activity. Indeed, there is great interest in defining CDK9/cyclin T-related inhibitors as novel approaches to HIV therapy [23]. Although the parenteral mode of administration of flavopiridol, along with its inflammatory, systemic, and gastrointestinal toxicities would argue against the suitability of flavopiridol in this regard, an optimally designed CDK9-directed molecule might be devoid of these effects. A distinct strategy for the use of CDK inhibitors relates to early observations that, as antiproliferative agents used in combination with cytotoxic agents, they were most effective when given after conventional cytotoxic agents (see, for example, [36]). When the CDK inhibitor preceded the cytotoxic agent there was actually a decrease in toxicity, ascribed to CDK-inhibitor-related cellular quiescence. Recently, a clinical application for this phenomenon has been proposed where a CDK2 selective inhibitor significantly ameliorated chemotherapy-induced alopecia after topical application [37]. Analogous approaches to protect cycling hematopoetic cells can be envisaged, using an appropriately specific CDK4-directed therapeutic in responsive tumors that have a defective Rb gene product [e.g. small-cell lung cancer (SCLC)]. Balanced against the possibility of persuing the latter approach is the frequent

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Box 1. Therapeutic opportunities and directions of future research • CDK regulation is altered in every tumor, either directly or indirectly. • Re-regulation of CDK activity could lead to clinically useful cytostasis or induction of apoptosis. • Some newer CDKs have housekeeping as well as cell-cycle-related roles: CDK7, CDK8, and CDK9 regulate transcription; CDK5 influences neuronal function and insulin secretion from pancreatic β cells. • A crucial feature for further CDK inhibitor drugs is to have a clear definition of the ‘anti-housekeeping’ properties of the drugs, as well as their effects on the cell-cycleregulatory proteins. • Selectivity for avoiding effects on housekeeping CDK functions might be difficult with ATP-site-directed drugs. • CDK7, CDK8, and CDK9 might represent novel approaches to regulating gene expression of rapidly turning-over genes, leading to clinically useful induction of apoptosis. In addition, CDK9 has emerged as a very novel target for anti-retroviral strategies.

clinical evidence against the use of dose intensification in many solid tumors, including SCLC. Have the best strategies for CDK modulation been defined? The concerns mentioned above must raise the question of whether selective modulation of cell-cycle-related CDKs (without effects on transcription or secretion) might not be achievable by strategies that are directed at the ATP-binding site of these kinases. Although distinctive strategies to derive ATP and substrate-selective molecules [38] have been proposed, this is a relatively underdeveloped approach. Alternatively, modulation of CDK function by non-ATP-sitedirected molecules must be considered.The large number of regulatory features built into CDK regulation (activating cyclins, endogenous inhibitors, degradation by proteosomedirected ubiquitination, and activating and de-activating phosphorylations, which are reviewed extensively elsewhere [39,40]) do suggest additional alternatives that might be pursued. Although the actual interaction between an activating cyclin and a CDK has proved quite difficult to prevent (because of the molecular topography of the interaction), the clear specificity for certain cyclins to certain types of cancer (e.g. the recently defined relation of cyclin D1 – as opposed to other D cyclins – to certain breast tumors [41]), highlights the need for continued definition of the pathways governing cyclin D expression. Pathways influenced by βcatenin, rapamycin, and geldanamycin [42–44] have been defined, but which is the most important in a given tissue, and how these might be more finely manipulated by small molecules, remains to be elucidated. Evidence has recently been presented for a non-cyclin-associated CDK – CDK10 – which can interact with the Ets2 transcription factor [45]. The physiological role of CDK10 is still being defined and the identification of inhibitors of the non-cyclin- or CDKbinding partners of CDKs and cyclins, respectively, would also be a direction of considerable interest to pursue.

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An equally interesting emerging opportunity relates to the recognition that several novel differentiating agents, as well as certain proteasome inhibitors, have the ability to induce expression of the endogenous CDK inhibitor p21 [46,47]. Their potential for resulting in functionally useful cell-cycle arrest is still to be explored in the clinical setting. Thus, regulation of p21 or related endogenous CDK inhibitors would emerge as a key fulcrum with which to lever CDK-directed strategies. This brings us to the question of whether the intended outcome of the action of the drug is protracted cytostasis with continued drug exposure, or whether induction of cytotoxicity via an apoptotic response (and leading to actual shrinkage of tumor masses) will be the criterion of success. Although these outcomes require potentially different clinical strategies, they are also likely to require different molecular features to be built-in to the drug in question. A transiently acting pro-apoptosis stimulus might be possible with some of the drugs in hand (e.g. a flavopiridol-like, CDK9-related inhibition of the elaboration of cell survival gene products). An alternative strategy could use a CDKdirected static agent, which ideally would have metabolic features leading to long-term persistence in the tumor milieu of interest during continuous dosing. Molecular features of drugs that enable selective distribution or uptake of the drugs into tumors could become increasingly possible with the incorporation of gene-expressionderived understanding of drug transport, and activation and detoxification pathways present in tumor cells. Although these pathways might not be responsible for tumor pathogenesis, they are crucial for enabling the success of small-molecule treatment strategies. Concluding remarks Recent research has revealed that the CDK family of cellcycle regulators is structurally similar to (and in some cases shares function with) CDKs regulating RNA synthesis and exocrine secretion. Currently available CDKinhibitor drugs have not been evaluated thoroughly in relation to these newly discovered targets. Despite the many remaining questions, it is also clear that a wide variety of potential CDK-related strategies for deriving useful anti-neoplastic agents remain ripe for exploitation (Box 1). Success in capitalizing on these emerging opportunities must be based on the evolving understanding of the biological roles of these targets and, ideally, will build assessments of molecular success in achieving endpoints related to these targets into early clinical trials. Acknowledgements I would like to thank Cheryl Seibert, Sarah Brannen, and Shannon O’Barr-Decker for help with the manuscript and http://www.trends.com

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figures, and to Rick Gussio and Connor McGrath for the CDK alignments.

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