Mitotic drug targets and the development of novel anti-mitotic anticancer drugs

Drug Resistance Updates 10 (2007) 162–181

Mitotic drug targets and the development of novel anti-mitotic anticancer drugs Mathias Schmidt a , Holger Bastians b,∗ a

Altana Pharma AG, Therapeutic Area Oncology, Byk-Gulden Strasse 2, Konstanz, Germany Philipps University Marburg, Institute for Molecular Biology and Tumor Research (IMT), Emil-Mannkopff-Strasse 2, 35032 Marburg, Germany

b

Received 6 June 2007; accepted 18 June 2007

Abstract Drugs that interfere with the normal progression of mitosis belong to the most successful chemotherapeutic compounds currently used for anti-cancer treatment. Classically, these drugs are represented by microtubule binding drugs that inhibit the function of the mitotic spindle in order to halt the cell cycle in mitosis and to induce apoptosis in tumor cells. However, these compounds act not only on proliferating tumor cells, but exhibit significant side effects on non-proliferating cells including neurons that are highly dependent on intracellular transport processes mediated by microtubules. Therefore, there is a particular interest in developing novel anti-mitotic drugs that target non-microtubule structures. In fact, recently several novel drugs that target mitotic kinesins or the Aurora and polo-like kinases have been developed and are currently tested in clinical trials. In addition, approaches of cell cycle checkpoint abrogation during mitosis and at the G2/M transition inducing mitosis-associated tumor cell death are promising new strategies for anti-cancer therapy. It is expected that this “next generation” of anti-mitotic drugs will be as successful as the classical anti-microtubule drugs, while avoiding some of the adverse side effects. © 2007 Elsevier Ltd. All rights reserved. Keywords: Mitosis; Chemotherapy; Spindle checkpoint; Aurora kinase; Polo-like kinase; KSP; Eg5; Microtubules; Taxanes; Vinca alkaloids; UCN-01; G2 checkpoint; Anticancer drug resistance

1. Introduction Most chemotherapeutic anti-cancer drugs used in the clinic today include agents that target the cell cycle in order to inhibit the hyperproliferation state of tumor cells and – subsequently – to induce apoptosis, which is the desired outcome of chemotherapy (Lee and Schmitt, 2003). Based on their mode of action these chemotherapeutic drugs can be subdivided into distinct groups: (i) drugs that interfere with DNA synthesis, (ii) drugs that introduce DNA damage and (iii) drugs that inhibit the function of the mitotic spindle. The latter have been proven to be exceptionally successful in the clinic and are classically represented by microtubule binding drugs frequently referred to as spindle ∗

Corresponding author. E-mail address: [email protected] (H. Bastians).

1368-7646/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.drup.2007.06.003

poisons (Jordan and Wilson, 2004; Mollinedo and Gajate, 2003; Zhou and Giannakakou, 2005; Pasquier et al., 2006). These drugs, which include taxanes and various Vinca alkaloids, bind to and inhibit the function of microtubules of the mitotic spindle apparatus, which leads to a stop of the cell cycle in mitosis and subsequently to the induction of tumor cell death. However, since microtubules fulfill important functions in resting and differentiated cells by mediating, e.g. intracellular transport processes, anti-microtubule drugs exhibit a plethora of unwanted side effects including severe peripheral neuropathies. Therefore, novel drug targets that spare microtubules, but inhibit the progression of mitosis are highly desired and already exploited for the development of novel anti-mitotic drugs. Thus, current drug development programs focus not only on improved novel anti-microtubule drugs, but also on novel mitotic drug targets that include mitotic kinesins and mitotic kinases (Jackson et

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al., 2007; Jiang et al., 2006). In addition, the use of drugs that abrogate the cell cycle arrest imposed by DNA damaging agents leading to an unscheduled entry into mitosis in the presence of DNA damage is a promising strategy to induce mitosis-associated cell death in tumor cells (Kawabe, 2004). This review summarizes the evaluation of novel antimitotic drug targets and the latest development of anti-mitotic drugs.

2. The mitotic spindle: a classical target for anti-cancer drugs 2.1. Microtubules Microtubules, together with actin and intermediate filaments are the major components of the cytoskeleton of eukaryotic cells. In interphase and differentiated cells, microtubules form fibers that serve as tracks for the intracellular transport of organelles and vesicles. When cells enter mitosis, this interphase network is dissolved and reorganized into a mitotic spindle that is required for the congression of chromosomes and the subsequent segregation of sister chromatids. Microtubules are long, stiff and cylindrical tubes with a diameter of about 25 nm consisting of ␣- and ␤-tubulin heterodimers that polymerize into 13 protofilaments, which form the wall of the microtubule. Microtubules are highly dynamic structures that constantly grow and shrink, a process termed dynamic instability (Cassimeris, 1993; Desai and Mitchison, 1997). This dynamic polymerization behavior is driven by the hydrolysis of GTP, which is bound at tubulin subunits. The two ends of microtubules are distinct: one end, the plus end, is much more dynamic and can show a net growth while the other, the minus end, is anchored in the centrosome or microtubule organizing center (MTOC), and is less dynamic and can show net shrinkage. Thus, at a given time, microtubules can show no change in their polymer mass, yet exhibit very high dynamics. The second important dynamic behavior of microtubules is termed treadmilling, which is a net growth at the plus end and a balanced net shortening at the minus end. Consequently, treadmilling results in a flow of tubulin subunits from the plus to the minus end of microtubules (Waterman-Storer and Salmon, 1997). Both, dynamic instability and treadmilling are important features for the function of microtubules, especially during mitosis. Many different proteins can bind to microtubules. Some of them are structural proteins, termed microtubule-associated proteins (MAPs) that regulate the stability and the dynamic behavior of microtubules. The other large group of microtubule-associated proteins is represented by motor proteins, which can be grouped into kinesins and dyneins. Some of these proteins move along microtubules mediating intracellular cargo transport, others have specific functions in mitosis in centrosome positioning, chromosome congression and segregation (Cassimeris and Spittle, 2001).

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2.2. Mitosis When cells enter mitosis, the microtubules become 20–100-fold more dynamic with a 7-fold enhancement in the microtubule nucleation rate at the centrosomes and a half-life of polymerized tubulin of 10–30 s (Hayden et al., 1990; Piehl et al., 2004; Saxton et al., 1984). This enhanced dynamics is especially required in the early phases of mitosis after breakdown of the nuclear envelope (prophase and prometaphase), when microtubules must grow for long distances and then almost completely shorten in order to search for kinetochores to be attached to the spindle microtubules (Hayden et al., 1990). This stochastic search-and-capture process leads to the attachment of one kinetochore of a given chromosome to microtubules and the chromosome is then pulled to the pole from which the microtubule is emanating. Proper alignment of the chromosomes requires that the second kinetochore of the same chromosome is subsequently captured by microtubules from the opposite pole enabling chromosomes to congress at the so-called metaphase plate, which exhibits fully aligned chromosomes whose kinetochores are set under tension. Only when all chromosomes are properly aligned (metaphase) the cohesion between the sister chromatids is dissolved and the chromatids are pulled towards the opposite poles driven by the net shortening of kinetochore-attached microtubules (anaphase). Several microtubule and kinetochore associated microtubule motor proteins contribute to the congression and the poleward movement of chromosomes (Maiato et al., 2004). The segregated chromosomes decondense in telophase, the last phase of mitosis, and two new nuclei are formed. Mitosis is followed by cytokinesis during which the cytoplasm is divided by the contractile action of actin-myosin ring structures. A schematic view of the normal mitotic progression is presented in Fig. 1. 2.3. The spindle assembly checkpoint It is essential that a mitotic cell has to ensure that chromosomes are first properly aligned during metaphase before anaphase is initiated in order to ensure the equal distribution of chromosomes onto the newly established daughter cells. Failure of this principle allows chromosome segregation in the presence of unaligned chromosomes and thus, results in chromosomal instability associated with aneuploidy. To prevent chromosome missegregation a signal transduction pathway known as “the spindle assembly checkpoint” inhibits the onset of anaphase until all kinetochores are properly attached to spindle microtubules and set under tension during metaphase. The spindle checkpoint signaling involves the function of several highly conserved proteins and includes Mad1, Mad2, Mad3/BubR1, Bub1, Bub3 and Mps1 and several other less well-characterized proteins (for review see Bharadwaj and Yu, 2004). Activation of the spindle checkpoint soon after nuclear envelope breakdown is associated with the recruitment of spindle checkpoint proteins to those kinetochores that lack either microtubule

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Fig. 1. Stages of mitosis and phenotypes of chemotherapeutic drug treatment. The proteins that are promising targets for cancer therapy are indicated and include polo-like kinase 1 (Plk1), kinesin-spindle protein (KSP/Eg5), centromeric protein E (CENP-E), Aurora-A kinase (Aur-A), Aurora-B kinase (Aur-B) and the mitotic spindle assembly checkpoint pathway that controls the metaphase to anaphase transition. Inhibitors of the mitotic spindle (spindle poisons) are frequently used for anti-cancer treatment and inhibitors for KSP/Eg5, Plk1 and the Aurora kinases are currently in clinical development. The mitotic phenotypes caused by these inhibitors are schematically shown. Interference with the mitotic spindle results in a prometaphase-like state, while inhibition of KSP/Eg5, Plk1 and Aurora-A result in a mitotic arrest phenotype with predominant monopolar spindles. In contrast, inhibition of Aurora-B prevents cytokinesis and induces polyploidization. Upon prolonged treatment with spindle poisons and kinesin inhibitors, cells exhibit a mitotic slippage, which is required for the subsequent induction of apoptosis in the postmitotic G1 phase. So far, it is not clear whether cells treated with Aurora A or Plk1 inhibitors undergo apoptosis during the mitotic arrest or whether a mitotic slippage is required. In addition, the induction of DNA damage during mitosis by abrogation of the G2 cell cycle arrest by inhibitors of the Chk1 kinase (e.g. UCN-01) leads to a spindle checkpoint mediated mitotic arrest followed by the induction of a mitotic form of apoptosis.

attachment or kinetochore tension and results in an inhibition of the anaphase-promoting-complex/cyclosome (APC/C), an E3 ubiquitin-ligase that marks key mitotic proteins for degradation through the 26S proteasome (Yu, 2002). The key substrates of the APC/C are securin and cyclin B, whose degradation is required for the onset of anaphase and the exit from mitosis, respectively. The degradation of securin is required at the metaphase to anaphase transition to liberate the active form of separase, a protease cleaving a subunit of the cohesion complex that holds the sister chromatids together (Peters, 2006; Pines, 2006). Thus, an activated spindle checkpoint prevents the onset of anaphase through inhibition of protein proteolysis and the maintenance of the chromatid cohesion.

Although little is known about the molecular mechanism of spindle checkpoint activation, the recruitment of the checkpoint proteins to kinetochores as well as the activities of the kinases Bub1, BubR1 and Mps1 are needed to activate the terminal effector protein, Mad2, that directly binds to and inhibits the ubiquitin ligase activity of the APC/C (Fang et al., 1998; Yu, 2002). However, it is still unclear how the lack of microtubule attachment or the lack of kinetochore tension is translated into an active checkpoint signal. The kinetochore-based kinesin protein CENP-E might have a function in sensing the attachment of microtubules to kinetochores and might be involved in initiating the checkpoint signal by activating the BubR1 kinase (Weaver et al., 2003; Yao et al., 2000). In addition, the so-called chromosomal pas-

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senger complex comprised of the Aurora-B kinase, INCENP, Borealin and survivin might have a role in activating the spindle checkpoint selectively upon a lack of kinetochore tension (Carvalho et al., 2003; Ditchfield et al., 2003; Kallio et al., 2002; Lens et al., 2003). Failure of the spindle checkpoint results in premature separation of sister chromatids even in the presence of misaligned chromosomes, which directly gives rise to chromosomal instability (CIN), the perpetual gain or loss of chromosomes or large parts thereof. This is associated with aneuploidy, which is a major hallmark of human cancer. In fact, in many tumor cells the spindle checkpoint function is weakened and the checkpoint signal is not sustained (Weaver and Cleveland, 2006). Thus, an impaired spindle checkpoint might directly contribute to the generation of chromosomal instability and tumorigenesis in human cancer. 2.4. Microtubule binding anti-cancer drugs The rationale for the use of anti-cancer drugs that inhibit the function of microtubules is to inhibit normal mitotic progression by interfering with the normal function of the mitotic spindle. In fact, many chemical compounds targeting microtubules, mostly derived from natural sources, exert their primary mode of action on proliferating tumor cells by a blockade of mitosis, which subsequently leads to the induction of cell death (for review see Jordan and Wilson, 2004; Zhou and Giannakakou, 2005). Traditionally, microtubule-binding drugs are classified into two groups: agents that stabilize microtubules including various taxanes and epothilones and agents that destabilize microtubules including various Vinca alkaloids and colchicine. However, it is now established that all these drugs at low, clinically relevant concentrations in the nanomolar range suppress the dynamics of microtubules rather than changing the net polymer mass of microtubules (Jordan et al., 1996; Jordan and Wilson, 2004; Yvon et al., 1999). Thus, it is more appropriate to refer to these agents as drugs that suppress the dynamic instability of microtubules. However, they are grouped based on their binding sites on microtubules rather than on the mode of action. The well-characterized binding sites include the taxane site on ␤-tubulin within the lumen of the microtubules, the Vinca domain close to the GTP binding site on ␤-tubulin and the colchicine binding site at the interface between the ␣- and ␤-tubulin dimers (Downing, 2000). 2.4.1. Taxanes Drugs that bind to the taxane site include different analogues of paclitaxel (Taxol® ) and docetaxel (Taxotere® ) and several others (for extensive summaries on taxanes and related compounds see Geney et al., 2005; Larkin and Kaye, 2006). Paclitaxel was originally isolated from the bark of the pacific yew tree Taxus brevifolia, while Docetaxel is a semi-synthetic analogue synthesized from a precursor isolated from the European yew tree Taxus baccata. Meanwhile, both taxanes can be produced semi-synthetically from precur-

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sor molecules, which warrants their supply for their frequent and successful clinical use for the treatment of breast, ovarian, prostate and non-small cell lung cancer. Both compounds bind to the taxane site and exhibit microtubule-stabilizing activity at relatively high concentrations. However, at rather low concentrations, which are most relevant for their use in patients, they suppress efficiently the dynamic instability of microtubules resulting in a failure of chromosome alignment causing mitotic arrest that subsequently leads to apoptosis. 2.4.2. Epothilones and discodermolide The epothilones, originally isolated from the myxobacterium Sorangium cellulosum, bind to the taxane site of microtubules and show microtubule stabilizing activity (Larkin and Kaye, 2006). Compounds belonging to this chemical family include patupilone, ixabepilone, BMS-310705, ZK-EPO and KOS-862 and are currently investigated in clinical phase I to phase II trials. Discodermolide, isolated from the marine sponge Discodermia dissoluta, is another example of a natural product that stabilizes microtubules. Both, epothilones and discodermolide, are much more potent than taxanes and show a strong antitumor activity in vitro and in vivo. 2.4.3. Vinca alkaloids Various Vinca alkaloids originally isolated form the periwinkle plant Vinca rosea as well as several other naturally occurring compounds like dolastatins (from the sea hare Dolabella auricularia), halichondrins (from the marine sponge Halichondria okadai) and spongistatins (from the matine sponge Spirastrella spinispirulifera) bind to the Vinca binding site near the plus ends of microtubules and exhibit microtubule depolymerizing activity at relatively high concentrations. Vinblastine and vincristine are the founding members of the Vinca alkaloids that were already introduced into the clinic in the late 1950s (Johnson et al., 1960). Subsequently, several semi-synthetic analogues notably vinorelbine, vindesine and vinflunine have been introduced into the clinic for the treatment of leukemias, lymphomas and solid tumors. As for the taxanes, drugs that bind to the Vinca domain provoke a suppression of microtubule dynamics at low, clinically relevant concentrations. Nocodazole is another drug that binds to ␤-tubulin. Although very efficient in destabilization of microtubules nocodazole failed to become an efficient anti-tumor drug probably due to its high toxicity. 2.4.4. Colchicine Similar to Vinca alkaloids colchicine can destabilize microtubules at high concentrations by binding to the colchicine site of microtubules. Despite the fact that colchicine was one of first microtubule-bindings drug identified (isolated from the meadow saffron plant Colchicum autumnale) a clinical development of this drug for the treatment of cancer failed. Colchicine is, however, approved for the treatment of gouty diseases. Interestingly, serious signs

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of peripheral neuropathy do not occur in patients treated with colchicine. Therefore, it may be of therapeutic benefit in prevention of liver cancer in high-risk patients (Arrieta et al., 2006). Other drugs that bind to the colchicine site like combretastatins (isolated form the African willow tree Combretum caffrum) are currently undergoing clinical trials for the treatment of cancer (Cirla and Mann, 2003). 2.5. Principal side effects of anti-microtubule drugs The primary target of microtubule binding drugs is the mitotic spindle. However, since interphase, resting and differentiated cells also require dynamic microtubules for the maintenance of cytoskeletal functions and intracellular transport processes (in particular in neuronal cells where axonal transport of neurotransmitter vesicles is guided by microtubules) there are adverse effects caused by microtubule drugs (Trudeau, 1996; Zhou and Giannakakou, 2005). Peripheral neuropathy might be explained by a disruption of microtubule mediated axonal flow and includes numbness, jaw pain, vocal cord dysfunction, constipation and abdominal cramps. Suppression of the mitotic microtubule function also inhibits the proliferation of non-transformed cells including hematopoetic precursor cells, which might explain the severe myelosuppression and neutropenia observed in patients during therapy. Thus, targeting microtubules during chemotherapy is not selective for tumor cells, but affects also non-proliferating cells as well. In addition, hypersensitivities to solvents (cremophor EL for taxanes and Tween-80 for docetaxel) might contribute to the side effects observed upon treatment with anti-microtubule drugs. 2.6. Mechanisms of cell death induced by microtubule-binding drugs Although anti-microtubule drugs are used in the clinic for many years the mechanisms of how these drugs induce tumor cell death are not well understood. This lack of knowledge makes it very difficult to explain many of the resistance phenotypes observed in patients. In this line, it is not clear why taxanes are efficient for example in breast, ovarian and lung carcinomas, but not, e.g. in colon carcinomas. Thus, it is most important to understand how taxanes and other anti-mitotic drugs induce apoptosis in order to predict the efficacy of these drugs for individual patients. It is well established that the treatment of cancer cells with anti-microtubule drugs results predominantly in an accumulation of mitotic cells and it is assumed that this mitotic arrest is tightly associated with cell death (Jordan et al., 1996; Milross et al., 1996). At clinically relevant concentrations, taxanes, epothilones and Vinca alkaloids suppress the dynamics of the mitotic spindle and thereby inhibit kinetochore capture and chromosome alignment. The presence of partially aligned chromosomes that lack microtubule attachment or kinetochore tension chronically activates the mitotic spindle checkpoint leading to the mitotic arrest in a

prometaphase-like state (Fig. 1). In fact, the mitotic arrest observed upon treatment with anti-microtubule drugs is dependent on the spindle checkpoint, but is not permanent. Instead, upon prolonged treatment, cells exit from mitosis in the presence of misaligned chromosomes, a process known as mitotic slippage leading to multinucleated cells with a 4N DNA content. It is not clear how cells can escape from the mitotic arrest in the presence of an activated spindle checkpoint. A slow, but continuous degradation of cyclin B in the presence of an active checkpoint might contribute to the exit from mitosis, but other mechanisms are also possible (Brito and Rieder, 2006). Once these tetraploid cells have exited from mitosis aberrantly, an activation of p53 and subsequent induction of its target gene p21 is observed indicating that failure of mitosis associated with tetraploidy can trigger a p53-dependent checkpoint response in G1, which can act as a second fail-safe mechanism to prevent further polyploidization (Lanni and Jacks, 1998; Margolis et al., 2003; Vogel et al., 2004). Interestingly, it has been shown that apoptosis induced by nocodazole, taxol or KSP/Eg5 inhibitors (see later) requires the activation of the spindle checkpoint as well as the subsequent slippage from the mitotic arrest (Kasai et al., 2002; Kienitz et al., 2005; Masuda et al., 2003; Tao et al., 2005). However, it is not clear whether the subsequent activation of the G1 checkpoint has a role in the initiation of apoptosis. Notably, it has been reported that p53-deficient tumor cells show a higher sensitivity towards anti-microtubule drugs (Stewart et al., 1999; Wahl et al., 1996), but contrasting results using isogenic cell lines have also been described (Kienitz et al., 2005). Unfortunately, the functional cross-talk between spindle checkpoint activation and the initiation of apoptosis is not well understood, but possibly a subset of spindle checkpoint genes have specific pro-apoptotic functions depending on the nature of spindle damage (Kienitz et al., 2005). Interestingly, components of the chromosomal passenger complex that include the Aurora-B kinase, INCENP, Borealin and survivin are required for spindle checkpoint function and mitotic arrest upon treatment with paclitaxel (Carvalho et al., 2003; Ditchfield et al., 2003; Hauf et al., 2003; Lens et al., 2003; Vogel et al., 2007). Moreover, survivin has been shown to act as an anti-apoptotic protein during mitosis (Altieri, 2003) and its stability is maintained by a mitosis-specific phosphorylation on Thr-34 by the CDK1-cyclin B kinase (O’Connor et al., 2000). Consistently, small molecule inhibitors of CDK1 act highly synergistically with taxol by destabilizing survivin during mitosis (O’Connor et al., 2000, 2002). Thus, while some components of the spindle checkpoint might act as pro-apoptotic regulators, others might be part of a survival pathway during the drug induced mitotic arrest. In this context it is interesting to note that mitotically arrested cells with an activated spindle checkpoint do not initiate apoptosis until they slip out of mitosis (Jordan et al., 1996; Tao et al., 2005). The mitotic arrest is associated with a hyperphosphorylation of the anti-apoptotic protein bcl-2, which might be associated with an enhanced anti-

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apoptotic activity (Deng et al., 2004), although the opposite has also been reported (Scatena et al., 1998). Bcl-2 counteracts the pro-apoptotic function of bax by preventing its conformational activation (Willis et al., 2003). Indeed, overexpression of bcl-2 in frequently observed in human cancer (e.g. Higashiyama et al., 1995) and antisense-mediated downregulation of bcl-2 sensitizes cells to paclitaxel treatment (Kim et al., 2004). Remarkably, bcl-2 is also hyperphosphorylated and the survivin containing chromosomal passenger complex is active and localized at kinetochores during an unperturbed mitosis (Ling et al., 1998). Therefore, it seems possible that these components might constitute an active survival pathway that is needed to suppress the initiation of a default apoptosis pathway during a normal mitosis. This would also explain why anti-mitotic drugs are such efficient apoptosis-inducing agents. Intriguingly, it has been suggested that the inhibition of active transcription during the mitotic arrest might be responsible for the depletion of anti-apoptotic proteins lading to the initiation of apoptosis upon a prolonged treatment with anti-microtubule drugs (Blagosklonny, 2007). Another important player in this regard is the bcl-2 family member bim. Bim is associated with microtubules during an unperturbed mitosis, while it dissociates from microtubules and binds to and inhibits the anti-apoptotic function of bcl-2 after paclitaxel treatment (Puthalakath et al., 1999). So far, there is no consistent view on how bcl-2 family proteins are regulated during mitosis and upon spindle damage. Several stress-induced kinases including JNK and p38 become activated upon mitotic damage, but the roles of these kinases are not clear (for review see Mollinedo and Gajate, 2003).

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also to a hyperactive mitotic survival checkpoint rendering tumor cells resistant to paclitaxel treatment (Tirro et al., 2006; Zaffaroni et al., 2002). Another cause of resistance towards anti-microtubule drugs might be a modulation of the microtubule composition and a change in microtubule dynamics. Resistant tumor cells were shown to express mutant forms of ␣- and ␤-tubulin, in which the drug binding sites are mutated (Giannakakou et al., 2000; Kavallaris et al., 2001a). Alternatively, resistant tumor cells were shown to overexpress a certain isoform of ␤-tubulin (␤III-tubulin), which results in significant higher microtubule dynamics (Goncalves et al., 2001). The same effect is produced by mutations in ␣-tubulin or by overexpression of microtubule-destabilizing proteins (e.g. op18/stathmin) or by loss of microtubule-stabilizing proteins (e.g. MAP4). In fact, a deregulated expression of microtubule-associated proteins is detected in cancer cells (Balachandran et al., 2003; Martello et al., 2003; Zhang et al., 1998). Although changes in the composition and dynamics of microtubules can clearly contribute to resistance towards taxanes and other anti-microtubule drugs in vitro, it is not clear whether these mechanisms indeed account for resistance in patients (Kavallaris et al., 2001b). Importantly, taxanes and Vinca alkaloids are very good substrates for the P-glycoprotein drug efflux pump, the product of the multidrug-resistance (MDR1) gene, which directly contributes to a low cellular concentration of the drug (Galletti et al., 2007). However, epothilones escape from MDR mediated efflux and are therefore active even in many taxol resistant tumor cell lines. Thus, several other microtubule-binding drugs that are not substrates for the Pglycoprotein are now under investigation (Lee and Swain, 2005).

2.7. Possible mechanisms of resistance From the mechanisms of apoptosis as described above, several routes of resistance towards spindle-damaging drugs are conceivable. It has been shown in various cell systems that cells with an impaired mitotic spindle checkpoint escape from apoptosis upon treatment with paclitaxel and other antimitotic drugs that activate the spindle checkpoint. Although inactivating mutations in the known spindle checkpoint genes appear to be rather rare (Hernando et al., 2001; Sato et al., 2000) deregulated expression of spindle checkpoint genes such as MAD1 or MAD2 might weaken the spindle checkpoint function in human cancer (Hernando et al., 2004; Kienitz et al., 2005; Michel et al., 2001; Wang et al., 2002). In addition, overexpression of Aurora-A, which acts as an oncogene, has been demonstrated to result in an abrogation of the spindle checkpoint leading to resistance towards taxol (Anand et al., 2003). Since colon carcinomas exhibit a very high incidence of chromosomal instability, which might be associated with spindle checkpoint malfunction, a weakened checkpoint might explain the poor efficacy of paclitaxel or related drugs in this entity. Moreover, survivin is frequently overexpressed in cancer cells (Wheatley and McNeish, 2005) and this might contribute not only to spindle checkpoint malfunction, but

3. Mitotic kinesin: KSP/Eg5 3.1. The role of KSP/Eg5 Given the fact that anti-microtubule drugs significantly interfere with the function of microtubules in resting and differentiated cells, which can lead to e.g. peripheral neuropathies, there is an urgent need to identify novel drug targets that interfere with the normal progression of mitosis without modulating the function of microtubules. Kinesin proteins represent promising candidates. Kinesins are a family of proteins that bind to and move along microtubules via their ATP-dependent motor domain. In interphase, kinesin family members are responsible for the transport of cargo and, during mitosis, several kinesins are essential for the proper chromosome alignment, segregation and centrosome separation (Miki et al., 2005). To date, there are 14 mitosis specific kinesins known that contribute to the proper execution of mitosis. Some of them regulate the congression and segregation of chromosomes, others mediate the positioning of centrosomes. One of the mitosis-specific kinesins is KSP (kinesin spindle protein) also known as kinesin-5 or Eg5.

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KSP/Eg5 is required for the generation of a bipolar spindle and for proper segregation of sister chromatids (Blangy et al., 1995). Ablation of KSP/Eg5 prevents the separation of the two mitotic centrosomes resulting in the formation of a monopolar spindle. Although a monopolar spindle allows the attachment of chromosomes, a bipolar attachment and thus, the generation of kinetochore tension is prevented (Kapoor et al., 2000). This explains why a functional inhibition of KSP/Eg5 activates the mitotic spindle checkpoint leading to a cell cycle arrest in mitosis. Importantly, it is now evident that mitotic kinesins are well druggable targets, by both, competitive and allosteric inhibitors (Bergnes et al., 2005; DeBonis et al., 2004; Maliga et al., 2002). 3.2. Anti-cancer drugs targeting KSP/Eg5 A chemical genetics screen has led to the identification of monastrol as the first inhibitor of the mitotic kinesin KSP/Eg5. The target of monastrol has been identified through its intriguing phenotype, namely arresting target cells in mitosis with monastrol spindles (Mayer et al., 1999), which is compatible with KSP/Eg5’s function for centrosome separation (Fig. 1). Although monastrol has been the prototype of KSP/Eg5 inhibitors, its relatively low cellular activity combined with other non-drug like properties has hampered further development. Meanwhile, the field of KSP/Eg5 inhibitor discovery and development has exploded and therefore, we focus here on KSP/Eg5 kinesin inhibitors that are already in clinical development (Table 1). Cytokinetics has been the leader in the development of KSP/Eg5 kinesin inhibitors. In 2001, Cytokinetics and GlaxoSmithKline agreed to jointly develop kinesin inhibitors and ispinesib (SB-715992) has been the first candidate to enter clinical trials. Since then, Ispinesib underwent several phase II trials and it is probably the relatively long half-life (29–54 h) that led to the re-initiation of phase I trials with different dose-escalation schedules. Most of the phase II trials have been designed as an 18 mg/m2 every 3 weeks schedule. Partial responses were observed in three breast cancer patients and the dose limiting toxicity was determined to be neutropenia. A follow-up derivative (SB-743921) with a five-fold higher activity has been nominated and is currently undergoing phase I/II trials in patients with non-Hodgkin’s lymphoma (NHL) as a 1-h intravenous infusion on days 1 and 15 of a 28-day schedule. SB-743921 currently also undergoes early clinical trials in patients with solid tumors. Mk-0731 (Merck) is another potent KSP/Eg5 inhibitor currently undergoing phase I clinical trials in patients with advanced cancers. In vitro, MK-0731 causes mitotic arrest in various cancer cell lines in the range between 3 and 5 nM and results in mitotic accumulation of the target cells. ARRY-520 (Array Biopharma) is a KSP/Eg5 inhibitor with approximately five times higher biochemical and cellular potency than ispinesib and advantageous physicochemical properties. The compound is active in various tumor xenograft models, and HT29 xenografts of treated animals

display an increased mitotic index with monoastral spindles (Woessner et al., 2007). ARRY-520 is currently undergoing phase I clinical trials in patients with advanced solid tumors and leukemias. The mechanism of apoptosis induction in response to KSP/Eg5 inhibition appears to be very similar to taxol. KSP/Eg5 inhibitors activate the mitotic spindle checkpoint and cells arrest in mitosis with monoastral spindles. Upon prolonged treatment cells escape from the mitotic arrest and initiate apoptosis. Remarkably, both, the spindle checkpoint activation and the subsequent slippage from the mitotic arrest are required for the efficient activation of the proapoptotic bax and the induction of apoptosis (Tao et al., 2005). Moreover, de novo protein synthesis is not required for the induction of apoptosis (Tao et al., 2007) and KSP/Eg5 inhibitors are effective even in taxol resistant cancer cells that express the product of the MDR1 gene regardless of the p53 status of the cells (Marcus et al., 2005; Tao et al., 2007). Given the mitosis-specific function of KPS/Eg5, inhibitors act specifically on proliferating cells. Therefore, the dose-limiting toxicities for KSP/Eg5 inhibitors are mainly neutropenia (Stein et al., 2006), but also diarrhea, alopecia, nail changes, nausea/vomiting, mucositis, abdominal pain, anorexia, or phlebitis have been reported. 3.3. Other mitotic kinesins In addition to KSP/Eg5, several other mitotic kinesin motor proteins contribute to the proper alignment and segregation of chromosomes (Moore and Wordeman, 2004). These include MKLP1, Kif4, Kid, MCAK and CENP-E, among others. All these kinesin proteins might be effective as drug targets since their inhibition is associated with defects in mitotic progression. CENP-E (centromeric protein E) is of particular interest in this regard. CENP-E is a 312 kDa protein localized at the kinetochore and harboring a N-terminal motor domain, which is required for its microtubule motor activity. CENP-E is essential for the normal progression of mitosis by contributing to normal chromosome congression (Wood et al., 1997; Yao et al., 2000). In addition, it stabilizes microtubulekinetochore attachments (Putkey et al., 2002) and it might have a function as an attachment sensor of the mitotic spindle checkpoint by directly regulating the activity of the spindle checkpoint kinase BubR1 (Abrieu et al., 2000; Weaver et al., 2003). Importantly, no function has been so far assigned to CENP-E outside of mitosis. Interference with its function by the use of siRNA or in mouse knockout models leads to severe misalignment of chromosomes, which is associated with a mitotic delay (Putkey et al., 2002; Tanudji et al., 2004). Thus, similar to spindle disrupting drugs, a lack of CENP-E function leads to severe mitotic defects suggesting that inhibition of CENP-E is an attractive strategy for cancer therapy. In fact, Cytokinetics and GlaxoSmithKline recently disclosed nonclinical data on the CENP-E inhibitor GSK923295A, which induces a severe anti-mitotic phenotype associated with a

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169

Table 1 Selected inhibitors of KSP/Eg5 in clinical development Structure

n.a.

Name (originator)

Properties

Reference

Ispinesib, SB-715992 (Cytokinetics, GlaxoSmithKline) returned from C. to GSK in 2006

IC50 = 0.6 nM, phase II trial in breast, ovarian, NSCLC and other indications

www.Cytokinetics.com

SB-743921 (Cytokinetics, GlaxoSmithKline)

Derivative of ispinesib, IC50 = 0.1 nM, highly effective in Colo205 xenografts, less effective on HT29 and MX-1 xenografts, phase I/II trials

www.Cytokinetics.com

MK-0731 (Merck)

IC50 = 2.2 nM, 20,000-fold selectivity over other kinesins, inhibits proliferation with IC50 = 3–5 nM, phase I trials

Stein et al. (2006)

ARRY-520 (Array Biopharma)

Inhibition of Eg5 and mitotic arrest in low nM range, highly effective on HT29, A2780, K-562 and HL-60 xenografts, phase I in advanced solid tumors

www.arraybiopharm.com

strong anti-tumor activity in vitro and in vivo (Sutton et al., 2007). Another less-well characterized mitotic kinesin-like ATPase is QN1/KIAA1009 (Leon et al., 2006). It is localized at centrosomes and essential for faithful mitotic progression since siRNA-mediated depletion of QN1/KIAA1009 results in abnormal mitoses with chromosome segregation defects and abnormal centrosome separation, ultimately resulting in apoptosis. It has to be awaited if QN1/KIAA1009 represents a promising candidate for drug development.

4. Polo-like kinases 4.1. The role of polo-like kinases The family of polo-like kinases comprises four members: Plk1, Plk2 (Snk), Plk3 (Fnk or Prk), and Plk4 (Sak; for review see Barr et al., 2004). Members of this family are characterized by a C-terminal region containing two polo boxes, each being 60–70 amino acids in length. Despite a limited amino acid sequence identity (12%), both polo box domains form an

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intramolecular dimer with identical folds of a six-stranded ␤sheet and an ␣-helix (Cheng et al., 2003). Interestingly, Sak, the fourth member of the polo-like kinase family, contains only one polo box. Crystal structure analyses of the polo box motif have shown that the Sak polo box forms a homodimer in vitro and in vivo and localizes to centrosomes and the cleavage furrow during cytokinesis (Leung et al., 2002). The most thoroughly studied polo-like kinase family member is Plk1 with numerous publications highlighting the essential and non-redundant roles of Plk1 during mitosis for centrosome maturation, spindle assembly, chromosome segregation, activation of the APC/C, cytokinesis and the activation of the spindle checkpoint as well as for cdk1 activation at the G2/M transition (reviewed in Strebhardt and Ullrich, 2006; van Vugt and Medema, 2005). The attractiveness of Plk1 as a target for directed tumor therapy is endorsed by two observations: first, Plk1 overexpression has been observed in a variety of cancers of different pathological origin including breast, ovary, colon, pancreas, lung, endometrium, brain, skin, head and neck, esophagus, gastric tract, and prostate (Takai et al., 2005; Weichert et al., 2005a,b,c). Second, targeted interference with Plk1 function on its own in cancer cells by antisense molecules, RNA interference technologies or small molecule inhibitors induces congruent molecular alterations namely arrest in mitosis and subsequent onset of cell death (Guan et al., 2005; Liu and Erikson, 2003; Schmidt et al., 2006; Spankuch-Schmitt et al., 2002; Sumara et al., 2004; van Vugt et al., 2004). Therefore, it is expected that targeted inhibition of Plk1 may be of therapeutic benefit for cancer patients. In this regard, Plk1 inhibitors fulfill the same premise of mitotic targeting as microtubule interfering agents with the potential of being active in taxane-resistant tumors, being applicable in indications in which spindle poisons have not shown efficacy at all (e.g. colon cancer), circumventing peripheral neuropathy due to lack of tubulin interference, as well as sparing solvent-associated adverse effects (hypersensitivity reactions) as observed with formulations containing Cremophor (e.g. Taxol® ) or Tween 80 (e.g. Taxotere® ). The recent disclosure of the Plk1 crystal structure (Kothe et al., 2007) may further encourage the discovery of selective Plk1 small molecule inhibitors. 4.2. Anti-cancer drugs targeting polo-like kinases Several small molecules with Plk1 inhibitory activities have been described. These include compounds such as Scytonemin, Wortmannin, LY294002, or certain CDK inhibitors with Plk1 inhibitory activity and also some recent patent literature disclosures (McInnes et al., 2005). One of the first potent Plk1 inhibitors reported in the literature was ON01910Na (Temple University; Gumireddy et al., 2005). However, we (unpublished observations) and others (Steegmaier et al., 2007) have been unable to reproduce the results using ON01910Na and several lines of experimental evidence strongly suggest that this molecule is an inhibitor of tubulin

polymerization rather than a Plk1 inhibitor. Similar caution should also to be taken regarding the mode of action of HMN214 (University of Arizona; Garland et al., 2006), to which Plk1-inhibiting properties have been ascribed. In contrast, several compounds represent indeed validated Plk1 inhibitors (Table 2) and the most advanced compound of those is BI2536 (Boehringer Ingelheim). BI2536 inhibits Plk1 in vitro with an IC50 value below 1 nM and the cellular phenotypes reflect those upon Plk1 knockdown by RNAi, namely mitotic arrest with predominantly monopolar spindles (Fig. 1; Lenart et al., 2007; Sumara et al., 2004). In vitro, BI2536 inhibits the growth of multiple tumor cell lines in an IC50 range between approximately 2 and 30 nM (Steegmaier et al., 2005, 2007). Particularly, a HCT116 (human colon carcinoma) xenograft model was shown to be highly sensitive to BI2536 and complete tumor regression has been reported on a schedule of twice weekly administration on two consecutive days for 5 weeks. Based on the published crystal structure of Plk1 (containing the T210V mutation; Kothe et al., 2007), BI2536 docks into the catalytic domain of Plk1. The close proximity of the pteridinone core to Val114 and Cys67 may account for the selectivity of BI2536. The original crystal structure has been obtained in complex with the non-hydrolyzable ATP analog adenylylimidodiphosphate and with PHA-680626, a pyrrolo-pyrazole inhibitor of both, Aurora and Plk1 (Fancelli et al., 2005). Results of phase I trials have been reported (Mross et al., 2005) with neutropenia as dose-limiting toxicity and BI2536 is currently in phase II clinical trials for various tumor indications. Another recently disclosed inhibitor of Plk1 is GSK 461364A (GlaxoSmithKline; Madden et al., 2007). This benzimidazolyl-thiophene has been chosen as Plk1 clinical candidate molecule and emanated through chemical optimization from a benzimidazolyl-thiophene precursor molecule called compound 1 (Lansing et al., 2007). GSK461364A inhibits Plk1 in the low nanomolar range in an ATP-competitive manner. This compound arrests tumor cells in mitosis in a dose-dependent fashion. Application of higher concentrations results in a G2 arrest rather than mitotic accumulation in U2OS cells (Laquerre et al., 2007). Dose-dependent in vivo activity has been observed on various established human tumor xenografts with Colo205 being most sensitive with a partial regression at the highest tolerated dose (Sutton et al., 2007). In contrast to BI2536 and GSK461364A, cellular phenotypes obtained with an optimized benzthiazole N-oxide, cyclapolin 1 (Cyclacel), have not been congruent with RNAi phenotypes (McInnes et al., 2005). The lead structure for this compound was initially identified using a structurebased approach on a Plk1 kinase homology model derived from cdk2. Virtual screening identified the benzthiazole N-oxide core structure, which was then chemically optimized. Cyclapolin inhibits Plk1 with an IC50 of 20 nM. However, cellular effects were observed at concentrations >10 ␮M. Surprisingly, Hela or Drosophila S2 cells displayed only a slight increase in mitotic cells and impaired micro-

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171

Table 2 Selected inhibitors of Plk1 in preclinical or clinical development Structure

Name (originator)

Properties

Reference

BI2536 (Boehringer Ingelheim)

IC50 = 0.8 nM, ATP competitive, cellular phenotype compatible with Plk1 RNAi data, phase II clinical trials

Steegmaier et al. (2007)

GSK461364A (GlaxoSmithKline)

Ki = 2.2nM, ATP competitive, highly selective for Plk1, some cross-inhibition of Nek2, selected as clinical candidate

Madden et al. (2007)

Cyclapolin 1 (Cyclacel)

IC50 = 20 nM, only little mitotic arrest observed

McInnes et al. (2005)

DAP-81 (Rockefeller University)

IC50 = 0.9 ␮M, mitotic arrest observed

Peters et al. (2006)

tubule nucleating activity at the centrosome upon cyclapolin treatment. DAP-81 (Rockefeller University) was identified in a cellbased screen for mitotic phenotypes using a small library of diaminopyrimidines (Peters et al., 2006). Although this compound inhibits Plk1 at relatively high concentrations (IC50 ∼ 1 ␮M), the cellular phenotypes observed are congruent with RNAi phenotypes including strongly impaired spindle bipolarity. Data about cell selectivity, induction of apoptosis, or cytotoxicity are not provided yet. Further Plk1 inhibitors highlighted in the patent literature or project data bases (Pharmaprojects, Prous) include imidazole derivatives from Banyu (WO2006/025567; WO2006/049339), aminopyrimidines from Amgen (WO2006/066172), lactam derivatives from Millenium (WO2006/041773), thiazolidinones from Schering AG (WO2006114334), substances from Cyclacel (developed

either in house or with Bayer as originator) and from SuperGen (WO2006124996). In summary, despite an extensive lag period in the development of Plk1 inhibitors, significant progress has been made in this field and clinical phase II data are now awaited for BI2536 and GSK461364A. However, caution has to be taken in interpreting the wealth of data on Plk1 inhibitors in the literature, since inhibition of Plk1 in a biochemical assay plus induction of a mitotic phenotype does not preclude other targets apart from Plk1. Therefore validated Plk1 specific cellular read-outs would lend significantly more credibility to the postulated mode of action of Plk1 inhibitors (Schmidt et al., 2006). So far, very little is known about the mechanisms of apoptosis induced by Plk1 inhibitor compounds. Since inhibition of Plk1 prevents the formation of a bipolar spindle, the mitotic spindle checkpoint is responsible for the mitotic arrest phe-

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notype observed (Sumara et al., 2004; van Vugt et al., 2004). Therefore, it seems possible that similar mechanisms account for the induction of apoptosis after drug induced spindle damage and Plk1 inhibition. It is interesting to note that downregulation of Plk1 elevates the drug sensitivity of cancer cells towards taxol (Spankuch et al., 2006). The molecular basis for this observation, however, is not clear.

5. Aurora kinases 5.1. The role of Aurora kinases The Aurora kinases have attracted much attention during the last couple of years, both, in academia and in the pharmaceutical industry. They fulfill important roles during mitosis to ensure proper centrosome function, chromosome alignment and segregation (for reviews see Crane et al., 2004; Marumoto et al., 2005; Vader et al., 2006). Moreover, Aurora kinases are frequently overexpressed in human cancer and Aurora A has been shown to be amplified in several tumors and can act as an oncogene (Bischoff et al., 1998). Thus, Aurora kinases represent attractive targets for anti-cancer therapy (Keen and Taylor, 2004). While yeasts and invertebrates have only one or two forms of Aurora kinases, mammalian cells comprise three family members, namely Aurora A, B, and C, which arose most likely through gene duplication as they show high sequence homology in their kinase domains (Brown et al., 2004). Caution has to be taken with respect to the sometimes confusing alternative names for Aurora A (Aurora 2, STK6, ARK1, AIK), Aurora B (Aurora 1, STK12, AIM-1, ARK2), and Aurora C (Aurora 3, STK13, AIK3, AIE-2). Multiple essential mitotic roles have been assigned to Aurora A. At the G2/M transition, Aurora A complexes with Ajuba and appears to play an essential role in the progression from G2 into mitosis (Hirota et al., 2003). During mitosis, Aurora A binds to the regulatory protein TPX2 and is localized to centrosomes and the spindle poles (Kufer et al., 2002). There, it is involved in the regulation of centrosomal proteins such as TACC3, which are required for microtubule nucleation and normal spindle assembly (Kinoshita et al., 2005). Ablation or pharmacological inhibition of Aurora A leads to defects in centrosome maturation associated with severe spindle defects and to the formation of monopolar spindles suggesting a role in the maintenance of spindle bipolarity (Girdler et al., 2006; Liu and Ruderman, 2006; Manfredi et al., 2007). Moreover, overexpression of Aurora A has been shown to override the spindle checkpoint after taxol treatment (Anand et al., 2003). More recently, a role in the promotion of nuclear envelope breakdown has been assigned to Aurora A (Portier et al., 2007) and inactivation of Aurora A by proteasomal degradation accompanies the exit from mitosis (Littlepage and Ruderman, 2002). Importantly, while Aurora A is frequently overexpressed in human cancer, its ablation strongly inhibits tumor cell growth in vitro and tumorigenicity

in vivo. Moreover, inhibition of Aurora A greatly sensitizes cells towards taxol treatment (Hata et al., 2005). Aurora B is part of the chromosomal passenger protein complex, which comprises INCENP, borealin and survivin (comprehensive overviews are given in Vader et al., 2006; Vagnarelli and Earnshaw, 2004; Wheatley and McNeish, 2005). Aurora B is found at multiple localizations depending on the different stages of mitosis. In the early phases of mitosis, it localizes to chromosome arms and the inner centromere region, in anaphase within the spindle midzone and in telophase at the midbody. Important roles have been assigned to Aurora B in chromatin protein modification with histone H3 and CENP-A being important physiological substrates of Aurora B (Hsu et al., 2000; Zeitlin et al., 2001). At centromeres, inhibition of the microtubule-destabilizing activity of op18/stathmin by Aurora B mediated phosphorylation might be required for proper spindle assembly (Gadea and Ruderman, 2006). Moreover, Aurora B is required for resolving synthetic microtubule-kinetochore attachments, thereby correcting monooriented attachments and ensuring a proper bipolar chromosome alignment (Ditchfield et al., 2003; Hauf et al., 2003). This function might be mediated in part by regulating the activity of the mitotic kinesin MCAK, which can destabilize microtubules at the kinetochore (Lan et al., 2004). In addition, Aurora B is required for spindle checkpoint activation in response to agents that interfere with the generation of tension across sister kinetochores (Ditchfield et al., 2003; Hauf et al., 2003; Vogel et al., 2007). In late telophase, Aurora B relocalizes to the contractile ring and to the midbody and is essential for cytokinesis. Ablation of Aurora B activity, either by siRNA or by pharmacological inhibitors strongly interferes with chromosome alignment and blocks cell division, but not cell cycle progression, which results in polyploidy (Ditchfield et al., 2003; Hauf et al., 2003; Terada et al., 1998). Aurora C, which is less well-studied than Aurora A or B, appears to have somewhat redundant functions to Aurora B as it might also be part of the chromosomal passenger protein complex and can partially complement the loss of function of Aurora B (Sasai et al., 2004). 5.2. Anti-cancer drugs targeting Aurora kinases Since the kinase domains of the Aurora kinases are highly homologous it is difficult to obtain inhibitors that are able to discriminate between Aurora family members over several orders of magnitude. Nevertheless, many Aurora inhibitors are currently in preclinical and clinical development (a selection is summarized in Table 3). The first Aurora kinase inhibitors described (Hesperadin, Boehringer Ingelheim and ZM447439, AstraZeneca) were shown to be potent panAurora inhibitors in vitro. Interestingly, although not very selective in vitro, the phenotypes observed after treatment of tissue culture cells were largely consistent with those observed upon loss of Aurora B, namely misalignment of chromosomes, inhibition of spindle checkpoint activation after treatment with taxol (but to a lower extent after treat-

Table 3 Selected inhibitors of aurora kinases in clinical development Structure

Properties

Reference

VX-680 (vertex)

IC50 = 0.7 nM (Aur-A), 18 nM (Aur-B), 4.6 nM (Aur-C), regression of leukemia xenograft in vivo, phase II trials in advanced colorectal cancer and NSCLC

Harrington et al. (2004)

PHA-739358 (Pfizer, Nerviano)

Pan-aurora inhibitor, phase I trials in hematological malignancies, phase II in Glivec-resistant CML

www.clinicaltrials.gov

AZD1152 (AstraZeneca)

IC50 = 687 nM (Aur-A), 3.7 nM (Aur-B), selective for Aur-B ATP competitive, phase I trials

www.clinicaltrials.gov

MLN-8054 (Millenium)

IC50 = 4 nM (Aur-A), 172 nM (Aur-B), selective for Aur-A in vivo, orally active, phase II trials

Manfredi et al. (2007)

M. Schmidt, H. Bastians / Drug Resistance Updates 10 (2007) 162–181

Name (originator)

173

www.clinicaltrials.gov

www.cyclacel.com, Hauf et al. (2003) www.clinicaltrials.gov PF-03814735 (Pfizer)

AT-9283 (Astex)

n.a.

n.a.

Pan-aurora inhibitor, phase I trials announced Orally available, phase I with solid tumors Pan-aurora inhibitor, phase I/II CYC116 (Cyclacel)

Pan-aurora inhibitor, phase I in CML, AML and MDS R763 (Rigel, licenced to Merck Serono)

n.a.

Properties Name (originator) Structure

Table 3 (Continued)

www.rigel.com

M. Schmidt, H. Bastians / Drug Resistance Updates 10 (2007) 162–181

Reference

174

ment with agents that prevent microtubule attachment), loss of phosphorylation of histone H3 and severe polyploidization due to inhibition of cytokinesis (Ditchfield et al., 2003; Girdler et al., 2006; Hauf et al., 2003). Very similar results were obtained by using VX-680 (Vertex), which is currently used in clinical phase I and II trials (Harrington et al., 2004). Several other pan-Aurora kinase inhibitors are now investigated in clinical trials (Table 3). It appears that all these inhibitors, although inhibiting both, Aurora A and B, act mostly through inhibition of Aurora B. Most recently, an inhibitor with a particular selectivity towards Aurora A (MLN-8054, Millenium) was presented. MLN-8054 inhibits Aurora A with an IC50 of 4 nM, while inhibiting Aurora B at 172 nM (Manfredi et al., 2007). Consistently, at low concentrations, MLN-8054 does not inhibit the phosphorylation of histone H3 on serine-10 indicating that Aurora B activity is not modulated in vivo. However, it blocks the autophosphorylation of Aurora A on threonine288, which was suggested to represent an in vivo marker for Aurora A activity. However, other kinases might also phosphorylate this residue on Aurora A indicating that this is rather an unreliable marker (Ohashi et al., 2006). In addition, treatment of cells with MLN-8054 induces monopolar spindles and a moderate accumulation of mitotic cells (Manfredi et al., 2007), which is consistent with some reports using siRNAs targeting Aurora A in human cells (Girdler et al., 2006), while not with others (Kunitoku et al., 2003). At higher concentrations, MLN-8054 induces polyploidy indicating that Aurora B might also be targeted in vivo. In human tumor xenografts, MLN-8054 was shown to be effective and clinical studies are now underway (Manfredi et al., 2007). AZD1152 (AstraZeneca) is an Aurora inhibitor that may be selective for Aurora B. As expected, this inhibitor causes a failure in cytokinesis and the occurrence of cells with >4N DNA content. The list of Aurora inhibitors in preclinical development is long: preclinical activities are among others reported by Sunesis (SNS-314), Montigen (MP-529), Avalon, GPC (RGB-311400), EntreMed/Miikana, Chroma Therapeutics (CHR3520), Ambit (AB-038), Banyu, Roche, SanofiAventis, Janssen, Johnson and Johnson (JNJ-7706621), GSK, SuperGen, Imclone, Boehringer Ingelheim (Hesperadin), Takeda, Rigel, Cyclacel, Amgen, Mitsubishi Pharma, Amphora, Pfizer, Astex, Astra Zeneca, Nerviano Medical Sciences and 4SC/Proqinase. Several crystal structures of Aurora A (in complex with ADP, adenosine, TPX2 or the small molecule inhibitors PHA680632 or PHA-739358) and Aurora B (in complex with INCENP or Hesperadin/INCENP) have been reported (see overview in Matthews et al., 2006), possibly opening up the avenue for the development of novel, highly selective inhibitors for the Aurora kinases. Currently, it is not clear how apoptosis is induced upon treatment with Aurora kinase inhibitors. It appears that most Aurora inhibitors act through the induction of polyploidy and it has been shown that loss of p53 facilitates endoredu-

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plication by abrogation of the postmitotic G1 checkpoint (Gizatullin et al., 2006). This could indicate that inhibition of Aurora kinases might target p53 deficient tumor cells preferentially. However, it is presently unclear how polyploidization could initiate apoptosis. On the other hand, some concern arose as to whether this would increase the risk of therapy-related secondary cancers since induction of polyploidy would also occur in proliferating non-transformed cells, which might predispose them to oncogenic transformation. Although the cellular targets cannot be clearly discriminated and the mechanisms of the induction of apoptosis are not clear, many Aurora inhibitors are currently in phase I or II clinical trials for the treatment of solid or hematological malignancies and the results are eagerly awaited (Table 3).

6. The spindle checkpoint-signaling pathway as a novel therapeutic target The mitotic spindle checkpoint-signaling pathway might also represent an attractive target for anti-cancer treatment. The rationale for this therapeutic concept is based on the following observations: (i) While a partial downregulation of spindle checkpoint gene expression in human tumor cell lines leads to aneuploidy and drug resistance, a severe repression of MAD2 or BUBR1 results in massive missegregation of chromosomes during mitosis, which is associated with apoptosis (Kienitz et al., 2005; Kops et al., 2004; Michel et al., 2004; Michel et al., 2001). (ii) Heterozygous deletion of MAD2, BUB3 or BUBR1 in mice generates chromosomal instability, but homozygous deletion produces early embryonic lethality (Babu et al., 2003; Dai et al., 2004; Dobles et al., 2000; Kalitsis et al., 2000; Michel et al., 2001). (iii) Partial depletion of the kinase activity of BubR1 is sufficient to impair the spindle checkpoint to a level no longer compatible with survival (Kops et al., 2004). These observations suggest that a certain degree of spindle checkpoint activity is required for cell survival and therefore, a targeted inactivation of the spindle checkpoint signaling pathway mediated by pharmacological inhibitors could be sufficient to induce apoptosis in proliferating tumor cells. Moreover, it is expected that abrogation of spindle checkpoint activity and the subsequent induction of apoptosis is independent of checkpoint activity in tumor cells and should therefore also be efficient in tumor cells with a weakened spindle checkpoint. Since protein kinases are well-validated targets for drug development, the different kinases known to function in the spindle checkpoint, namely Bub1, BubR1 and Mps1 are among the most favored drug targets. Recently, inhibitors for Mps1 in yeast and Mps1/TTK in mammalian cells have been described, but the induction of apoptosis upon Mps1 inhibition was not investigated (Dorer et al., 2005;

175

Schmidt et al., 2005). The identification and characterization of novel spindle checkpoint inhibitors is eagerly awaited. In addition to targeting the spindle checkpoint directly, upstream regulatory pathways of the spindle checkpoint are of great interest for drug development. Interestingly, the spindle checkpoint protein and kinesin CENP-E and the kinetochore component CENP-F are regulated by farnesylation (Ashar et al., 2000; Hussein and Taylor, 2002). In fact, inhibition of a mitotic farnesylation by the use of farnesyltransferase inhibitors induces mitotic defects (Ashar et al., 2000) and possibly concomitantly a malfunction of the spindle checkpoint, which is expected to be deleterious to tumor cells. Interestingly, it has been shown that farnesyltransferase inhibitor act highly synergistic with taxanes and epothilones (Moasser et al., 1998). Several potent farnesyltransferase inhibitors have been identified and are undergoing already clinical trials (reviewed in Pan and Yeung, 2005). However, the mode of action of farnesyltransferase inhibitors and their effects on the mitotic checkpoint has not been investigated yet.

7. Mitotic catastrophe The ultimate goal of chemotherapeutic treatment of cancer is the induction of apoptosis (Mashima and Tsuruo, 2005). In addition, a second form of tumor cell death, termed mitotic catastrophe, which originates from an abnormal mitosis, is often referred to as a form of cell death unrelated to apoptosis (Okada and Mak, 2004). Most recently, however, it has been shown that mitotic catastrophe might represent a mitotic form of apoptosis that can be induced by chemotherapeutic treatment (Castedo et al., 2004; Vogel et al., 2007). One important form of drug regimen that leads to mitotic catastrophe is the induction of DNA damage during mitosis, a condition, which is the result of an abrogation of the DNA damage checkpoint in G2 (Kawabe, 2004). 7.1. G2-DNA damage checkpoint pathways Anti-cancer therapies that employ irradiation or chemotherapeutic treatment with platinum drugs or topoisomerase inhibitors (e.g. etoposide, topotecan, doxorubicin) induce severe DNA damage, thereby activating DNA damage checkpoints resulting in halt of the cell cycle allowing DNA repair to occur (for review see Kastan and Bartek, 2004). The first step of the DNA damage response is the recruitment of sensor complexes to the sites of DNA damage where the ATM/ATR kinases are activated. These kinases can phosphorylate and activate either the transcription factor p53 directly or they activate the Chk2 kinase, which in turn can phosphorylate and activate p53. The transcriptional activation of p53 results in an induction of its target gene p21, which encodes a CDK inhibitor protein that binds to and inhibits CDK-cyclin complexes in G1. Since CDK activity is required for progression into S phase, activation

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of this checkpoint results in a cell cycle arrest in late G1 and thus, this checkpoint is termed G1 checkpoint. In addition, a second DNA damage checkpoint exists, which acts in G2: the ATM and ATR kinases can also phosphorylate and activate the Chk1 kinase, which phosphorylates and inactivates the dual specificity phosphatase Cdc25C leading to its cytoplasmic sequestration. Cdc25C is the phosphatase responsible for removing two inhibitory phosphates from CDK1, which is required for activation of CDK1 and subsequent entry into mitosis. Thus, DNA damage induced inhibition of Cdc25C is mediated by Chk1 and prevents the entry into mitosis constituting the G2 DNA damage checkpoint (Kastan and Bartek, 2004). Since most human tumor cells have lost the function of the G1 checkpoint (e.g. by loss of p53 or by other alterations), treatment of these tumor cells with DNA damaging agents results in a cell cycle arrest exclusively in G2. This circumstance allows the selective targeting of tumor cells by abrogating the remaining G2 arrest by the use of pharmacological inhibitors of the G2 checkpoint, thereby forcing cells into mitosis in the presence of DNA damage leading to the induction of a mitotic catastrophe associated with cell death (Kawabe, 2004). 7.2. UCN-01: a G2-checkpoint abrogator and inducer of mitotic catastrophe The Chk1 kinase is essential for the G2 arrest upon chemotherapy induced DNA damage and can be efficiently inhibited by the indolocarbazole compound UCN-01 (7hydroxystaurosporine) that acts as an ATP competitive inhibitor (Eastman, 2004; Wang et al., 1996). Sequential treatment of p53 deficient tumor cells with DNA damaging drugs and UCN-01 results in an efficient abrogation of the G2 arrest and entry into mitosis. Similar to the treatment with anti-mitotic drugs, cells activate the spindle checkpoint once they enter mitosis with damaged chromosomes, most likely due to the inability of a proper chromosome congression (Vogel et al., 2007). Subsequently, mitotically arrested cells activate apoptotic pathways that involve the release of pro-apoptotic proteins from mitochondria and the subsequent activation of caspases. Thus, mitotic catastrophe upon G2 checkpoint abrogation represents a form of apoptosis triggered in mitosis. Notably, as seen for the induction of apoptosis after treatment with anti-microtubule drugs, mitotic apoptosis is also dependent on a functional spindle checkpoint (Vogel et al., 2007). Interestingly, mitotic apoptosis appears to be counteracted by mitotic survival pathways that involve the chromosomal passenger complex, which itself is part of the mitotic spindle checkpoint. Consistently, inhibition of components of the chromosomal passenger complex including survivin and the Aurora-B kinase (e.g. by small molecule inhibitors) greatly enhances the efficacy of the UCN-01 mediated therapy (Vogel et al., 2007). UCN-01 has completed several phase I clinical trials in the U.S. and in Japan as a stand-alone therapy and phase II trials are cur-

rently underway to investigate the efficacy of UCN-01 in lymphomas (National Cancer Institute). In addition, due to the promising preclinical results that support the concept of G2 checkpoint abrogation, several phase I and II clinical trials for leukemia, lung cancer and advanced solid tumors are now underway to explore the efficacy of UCN-01 in combination with various DNA damaging agents including platinum compounds and topoisomerase inhibitors (National Cancer Institute, USA). So far, as a G2 checkpoint abrogator, UCN-01 is the most advanced drug, but several other Chk1 inhibitors are currently tested in preclinical or clinical investigations. Clinical candidates include XL-844 from Exelixis, AZD7762 from AstraZeneca and PF-477736 from Pfizer.

8. Novel mitotic targets Genome wide screens for molecules essential for cell cycle regulation and progression have yielded a large number of potential targets whose selective inhibition might result in phenotypes similar to the ones observed for Plk1, Eg5 or Aurora kinases (Bettencourt-Dias et al., 2004; Mukherji et al., 2006). A novel target in this context may be Haspin, a kinase that appears to be essential for sister chromatid cohesion (Dai and Higgins, 2005). Ablation of Haspin results in spindle checkpoint activation and mitotic arrest. Another highly interesting target appears to be the p58 isoform of cdk11, which is localized at mitotic centrosomes. Targeted depletion of cdk11 results in the occurrence of monopolar spindles with shortened microtubules, a phenotype that could be rescued by the p58 isoform, but not the p110 isoform (Petretti et al., 2006). The list of potentially druggable proteins for mitotic targeting is far from being complete. Since mitosis is such a tightly regulated process and cancer cells have only very limited mechanisms to evade targeted pharmacological interference during mitosis it is expected that the quest for further essential mitotic targets will be rewarding and it will be interesting to see which targets will then be validated by the use of specific inhibitors displaying the expected pharmacological and therapeutic phenotype.

9. Concluding remarks The identification of druggable proteins whose function is indispensable for faithful mitotic progression has been of outstanding interest in academia and in the pharmaceutical industry. Driven by the particular clinical and commercial success of the taxanes the goal is to develop novel therapeutics that fulfill the same premise, namely irreversibly arresting cancer cells in mitosis and the subsequent onset of apoptosis. The need is defined by the fact to (i) circumvent peripheral neuropathy as side effect of targeting tubulin polymerization,

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(ii) prevent adverse events due to hypersensitivity to solvents used for taxanes (cremophor EL or Tween-80), (iii) overcome the inherited resistance of tumors extensively treated with taxanes or (iv) to address indications in which taxanes have shown not to be efficient (e.g. in colon cancer). It now appears that targets such as Plk1 or Eg5 might potentially fulfill these requirements. Interestingly, inhibitors of Aurora kinases, although acting during mitosis, display a clearly distinguishable phenotype from Plk1 or KSP/Eg5 inhibitors and possibly also a distinct mode of apoptosis induction by causing severe polyploidization of cells. Another type of therapeutic window may be attributable to Plk1 inhibitors, since differential roles for Plk1 have been described for normal cells compared to transformed cells (Guan et al., 2005; Liu et al., 2006). Thus, although these novel inhibitors are promising tools for cancer therapy that might be superior to the classical anti-mitotic drugs, there is much more work ahead to understand the mechanisms of cell death induced by these drugs. Only a complete definition of the pathways required to induce a mitotic arrest and subsequent apoptosis will allow a further directed development of novel and highly efficient anti-mitotic drugs.

Acknowledgements M.S. acknowledges Swen Hoelder and Markus Boehm for critical discussion on the Aurora and kinesin inhibitors, respectively. H.B. thanks the members of the Bastians lab and Heike Krebber for comments on the manuscript. We apologize to all colleagues whose work could not be cited in this review due to space limitations.

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