Abstract: The loss of ability in controlling cell cycle leads to aberrant cell growth and is a hallmark of cancer cells. Cell cycle regulation and progression mainly rely on protein phosphorylation events, therefore cell cycle kinases have long been viewed as potential targets for anticancer strategies. Consistently, cell cycle kinases are often dysregulated in different types of human cancer. Despite years of research and attempts directed at inhibiting cell cycle kinases, none of these approaches has been successfully translated to the clinic to halt tumorigenesis. Here, we review several currently pursued strategies and highlight both current challenges and some recent findings, which might help to develop new, better conceived therapeutic approaches based on cell-cycle kinase inhibition.

Aberrant cell growth, due to the loss of ability to control cell cycle, is the main feature of cancer cells. The first insights into the mechanisms underlying cell cycle control date back to the late 60s, with the seminal work of Lee Hartwell, Paul Nurse, and Tim Hunt (Boye and Grallert, 2001), and it soon became evident that proteins involved in cell cycle regulation could be potential targets for anticancer strategies. As progression through the cycle largely relies on protein phosphorylation events, and cell cycle kinases are often deregulated in cancer, many have pursued strategies aimed at inhibiting cell cycle kinases to halt tumorigenesis. So far, unfortunately, none of these approaches has successfully worked its way into the clinical practice. However, recent findings seem to indicate that this is a road worth further traveling.

Protein Kinases Regulate Mammalian Cell Cycle

Progression through cell cycle depends on a series of precisely coordinated sequential events that, following mitogenic cues, lead to cell division. Key players in this process are the cyclin-dependent kinases (CDKs), the activity of which is regulated in a timely manner by their association with specific proteins, which are named cyclins because they are synthesized and destroyed at specific times during cell cycle. Mitogenic signals converge on and activate the G1 phase CDK-cyclin complexes, which initiate the phosphorylation of their targets, thereby inducing the transcriptional activation of genes required for the DNA synthesis phase (S) and of cyclins that will activate the CDKs required for the later stages. Once the DNA is replicated, mitotic CDK-cyclin complexes trigger mitosis initiation by inducing proteins involved in chromosome condensation and mitotic spindle assembly. Crucial to this phase are also other serine-threonine kinases, such as aurora A and polo-like kinase 1 (PLK1), which contribute to centrosome maturation and spindle assembly (Archambault and Glover, 2009; Barr and Gergely, 2007). Subsequently, activation of the anaphase-promoting complex (APC) promotes the degradation of mitotic cyclins and of structural proteins required for chromosome segregation, halting cell cycle and allowing cytokinesis to proceed (van Leuken et al., 2008).

Different checkpoints regulate progression through the cycle preventing cell duplication when something goes wrong, for instance when the DNA is damaged or has not been synthesized completely, or if chromosomes do not segregate properly. Several protein kinases are crucial to the function of cell cycle checkpoints. ATM and ATR, for example, are two kinases that, following DNA damage, phosphorylate a myriad of substrates including the checkpoint kinases CHK1 and CHK2 in order to arrest cell cycle and allow DNA repair or, depending on the extent of the damage, induce apoptosis to avoid transmission of a mutated genotype to the next generation (Cimprich and Cortez, 2008; Lavin, 2008). The same scope of due diligence acts on the mitotic checkpoint, or spindle assembly checkpoint (SAC), which is also regulated by protein kinases such as BUB1, BUB1B, and aurora B (Musacchio and Salmon, 2007).

Protein Kinases Are Often Dysregulated in Cancer

Cell cycle kinases dysregulation is a common feature of cancer cells, resulting in uncontrolled proliferation and loss of checkpoint integrity that favors genomic instability.

CDKs are found overexpressed in several types of tumors, including breast cancer, sarcoma, and lymphoma. CDK overexpression can be caused by gene amplification, chromosome translocation, or point mutations that impair the kinase interaction with CDK inhibitors (as the case for CDK6 in some melanoma patients). CDK activity can also be dysregulated by overexpression of the cyclin partner or inactivation of CDK inhibitors, both events being quite common in tumors (Lapenna and Giordano, 2009; Malumbres and Barbacid, 2009).

Inactivation of ATM and ATR has also been detected in several cancers and is usually caused by truncation or missense mutations. Individuals lacking these kinases are more prone to cancer development owing to the increased genomic instability resulting from a defective DNA damage checkpoint. Similarly, CHK1 and CHK2 are found to be mutated in some cancer types, which might account for the enhanced genomic instability in these tumors (Lapenna and Giordano, 2009).

Also the kinases involved in mitotic entry and spindle progression are found to be mutated in cancer by gene amplification (aurora A) or point mutations (PLK1, BUB1, BUB1B), with the event being likely to contribute to the chromosomal instability underlying these tumours (Lapenna and Giordano, 2009).

However, abrogation of the cell cycle checkpoints, through the pharmacological inhibition of checkpoint kinases, in combination with DNA damaging drugs or ionizing radiations is an attractive antitumor strategy. This, in fact, should force cancer cells into mitosis with heavy damaged DNA, inducing mitotic catastrophe and associated cell death.

Targeting Protein Kinases in Cancer

So far, several approaches have been attempted to arrest cancer cell proliferation by inhibiting cell cycle kinases. In particular, the search for drugs aimed at blocking CDKs has been intensively pursued in the past two decades, although none of these drugs proved to be effective in clinical trials. The first generation of CDK inhibitors, such as flavopiridol, olomoucine, and roscovitin, showed low activity and/or toxicity in clinical trials (Shapiro, 2006). Several hypotheses have been made to explain this failure. It could be that these drugs have other key targets or that off-target effects prevent them to reach therapeutic concentrations (Malumbres et al., 2008). Also, it is possible that the dosing schedule and the pharmacokinetics of the drugs had not been optimized. Being pan-CDK inhibitors, these molecules target different CDKs and therefore may act at multiple steps during cell cycle. This should be taken into account when deciding the drug administration schedule. For instance, in a Phase I clinical trial a 4-hour infusion of flavopiridol, preceded by a 30-minute bolus dose, achieved encouraging results in patients with leukemia (Byrd et al., 2007). Moreover, CDK inhibitors could improve the antitumor efficacy of other cytotoxic agents, such as cisplatin and doxorubicin, as suggested by combination studies (Malumbres et al., 2008). For instance, the aminothiazole SNS-032, a second generation CDK inhibitor, sensitized radioresistant lung cancer cells to ionizing radiation, in a ‘cell-cycle’ independent manner (Kodym et al., 2009), suggesting that CDK inhibitors could act also through other mechanisms. Another current challenge to the use of CDK inhibitors is that, by targeting multiple CDKs, they often inhibit also transcription-related CDKs, such as CDK9 (Shapiro, 2006). Although it could be appealing to globally inhibit cellular transcription in cancer cells, this could have toxic effects in normal cells.

Recent landmark studies in mouse models have provided genetic evidence that, although CDK1 is indeed required for cell cycle, the interphase CDKs are only essential for specialized cells. These findings, which have been elegantly reviewed elsewhere (Hochegger et al., 2008; Malumbres and Barbacid, 2009; Malumbres et al., 2008), have important implications in the development of antitumor strategies based on CDK inhibition. They suggest that it is crucial to define what the specific CDKs are that are required to drive proliferation of normal and cancer cells in order to eliminate undesired toxicities in normal cells. Also, it emerged that different tumor types could have different sensitivity to CDK inhibitors depending on the pattern of oncogenic mutations. Such mutations would determine what specific CDKs the tumor cell relies on. Our increasing understanding of these issues, along with the development of a new generation of more potent and specific agents (some of which are already in clinical trials), will hopefully allow us to move a step forward towards better conceived therapeutic strategies.

More recently, efforts have been made to inhibit kinases involved in the DNA damage checkpoints. Two small molecules acting as ATM inhibitors sensitized cells to DNA damaging agents (such as topoisomerase inhibitors and ionizing radiation) and seem to be promising agents. Several drugs have been developed to inhibit CHK1 and CHK2 and some of these entered clinical trials, either as single agents or in combination with other drugs, for the treatment of different types of tumors. However, in contrast with CHK1 inhibitors, which abrogate DNA damage checkpoints and increase cell killing, CHK2 inhibitors had no effect on cytotoxicity following exposure to DNA damaging drugs. Mouse models indicate that loss of CHK2 function could instead protect from the effects of genotoxic agents. Further studies should be performed to evaluate the possibility of using checkpoint kinase inhibitors in specific tumors.

Cells are particularly sensitive to apoptosis during mitosis, therefore many cancer patients are treated with antimitotic agents, such as taxanes and vinca alkaloids, as a front-line therapy. These drugs, by affecting microtubule dynamics, interfere with the formation of the mitotic spindle and induce a prolonged mitosis, which often results in cell death (Jordan and Wilson, 2004; Jackson et al., 2007). However, as the case with other chemotherapy agents, they are quite toxic for normal cells, and as a result many studies in the past few years have searched for new mitotic targets that could improve specificity towards cancer cells. Most work so far focused on aurora kinases (Keen and Taylor, 2004) and PLKs (Strebhardt and Ullrich, 2006) resulting in the development of compounds that inhibit these proteins. Many of these are currently undergoing clinical trials for the treatment of patients with different types of tumors.

Aurora inhibition has a different effect depending on which family member is targeted. Aurora A inhibition is characterized by a SAC-induced mitotic arrest, which can eventually result in apoptosis. Aurora B inhibition, instead, abrogates the SAC; anaphase proceeds despite misaligned chromosomes and cell division does not occur, resulting in cells with enlarged polyploidy nuclei that eventually become senescent or undergo apoptosis. The mechanism of action of aurora B inhibitors differs from that of traditional antimitotic agents because cells continue to cycle instead of arresting in mitosis. So, aurora B inhibitors could prove successful in combination with agents that require exposure during other phases of the cell cycle (Jackson et al., 2007).

Preclinical studies, however, showed that aurora kinase inhibitors are able to suppress tumor growth regardless of their specificity, suggesting that both aurora A and B hold the potential for being effective anticancer target. One possible hurdle is that, most aurora inhibitors being ATP-competitive, cancer cells may develop resistance, as occurs with other kinase inhibitors. Indeed, a recent study identified aurora B mutants in the ATP-binding site in cancer cells which were challenged with competitive inhibitors of different chemical classes (Girdler et al., 2008). Further characterization of such mutants, along with the development of non-ATP competitive aurora inhibitors, should help to confront the eventual resistance.

The best characterized polo-like kinase is PLK1, which is overexpressed in many tumors and is often associated with a poor prognosis. PLK1 represents an attractive anticancer target because, similarly to other antimitotic agents, it causes cell cycle arrest but does not disrupt microtubule dynamics. Therefore PLK1 inhibition should be effective in cancer cells without causing heavy toxicity in normal cells. Both ATP-competitive and non-ATP-competitive PLK1 inhibitors have been developed and are currently investigated in clinical trials. The presence of a characteristic motif in PLKs, the polo box, which is required for the kinase activity, offers the possibility for a more specific approach for targeting PLK1. The first non-peptidic inhibitor targeting the polo box, poloxin, proved successful for the suppression of a cancer cell line in vitro (Reindl et al., 2008).

Other strategies aimed at silencing the mitotic checkpoint to selectively kill tumors by increased genomic instability are currently pursued through the specific targeting of other SAC kinases. Our understanding of cell cycle regulation continues to increase, leading to the identification of new promising therapeutic targets. Based on the previous experience, it seems crucial to investigate the different responses of tumor versus normal cells in order to identify a possible Achilles heel in cancer and a way to tackle it. Also, it will be necessary to determine what types of tumors are most likely to respond to specific kinase inhibitors to identify the subsets of patients who will probably benefit from these treatments. It will be also important to determine the rationale for new combination studies that might synergize in improving patient outcome and reducing toxicities. The future holds great promise with many new, more potent and more selective targeted agents that are currently under investigation or in the development pipeline.

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