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The Oncologist, Vol. 7, Suppl 3, 12-19, August 2002
© 2002 AlphaMed Press

The Cell Cycle as a Target for Cancer Therapy: Basic and Clinical Findings with the Small Molecule Inhibitors Flavopiridol and UCN-01

Adrian M. Senderowicz

Molecular Therapeutics Unit, Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland, USA

Correspondence: Adrian M. Senderowicz, M.D., National Institutes of Health, Oral and Pharyngeal Cancer Branch, NIDCR Building 30; Room 211, 30 Convent Drive, Bethesda, Maryland 20892-4340, USA. Telephone: 301-594-5270; Fax: 301-402-0823; e-mail: asenderowicz{at}dir.nidcr.nih.gov


   LEARNING OBJECTIVES
Top
 Learning Objectives
Abstract
 Introduction
 Flavopiridol
 Conclusions
 References
 
After completing this course, the reader will be able to:

  1. Describe the basics of cell-cycle regulation.
  2. Understand how Rb pathway derangement causes cancer.
  3. Relate the clinical findings with flavopiridol and UCN-01 to their mechanisms of action.

Access and take the CME test online and receive one hour of AMA PRA category 1 credit at CME.TheOncologist.com


   ABSTRACT
Top
 Learning Objectives
 Abstract
Introduction
 Flavopiridol
 Conclusions
 References
 
Many tumor types are associated with genetic changes in the retinoblastoma pathway, leading to hyperactivation of cyclin-dependent kinases and incorrect progression through the cell cycle. Small-molecule cyclin-dependent kinase inhibitors are being developed as therapeutic agents. Of these, flavopiridol and UCN-01 are being explored in cancer patients in phase I and phase II clinical trials, both as single agents and in combination with conventional chemotherapeutic agents. The present article discusses the mechanisms of action of flavopiridol and UCN-01 as well as the outcome of clinical trials with these novel agents.

Key Words. Cell cycle • Cyclin-dependent kinases • Flavopiridol • UCN-01 • Phase I trials • Clinical trials


   INTRODUCTION
Top
 Learning Objectives
 Abstract
 Introduction
Flavopiridol
 Conclusions
 References
 
During progression through the phases of the cell cycle (G1, S [DNA synthesis], G2, M [mitosis]), DNA is duplicated and the chromosome sets are distributed evenly over the two daughter cells (Fig. 1AGo). To ensure accuracy of the cell-cycle progression, cells need to go through several pauses or "checkpoints." At the checkpoint in late G1, the cell either exits to G0 and becomes quiescent or commits to the cell cycle [1]. The G2 checkpoint allows the cell to repair DNA damage before entering mitosis. Cell-cycle progression is regulated by cyclin-dependent kinases (cdks), a family of serine/threonine kinases, which during G1 progressively phosphorylate the retinoblastoma (Rb) protein [2–4]. Upon phosphorylation, Rb is inactivated and releases transcription factors of the E2F family [reviewed in 5], which subsequently induce transcription of genes needed for S-phase entry (Fig. 1AGo) [6]. Cdks are positively regulated by cyclins with which they form holoenzymes (Fig. 1Go) [7, 8]. In addition, cdks are positively regulated by cdk7, which, in a complex with cyclin H, phosphorylates cdks at threonine 160/161 [9]. Negative regulation of cdks is performed by two families of cdk inhibitors (Fig. 1Go). The INK4a family of cdk inhibitors [10] includes p16INK4a, p15INK4b, p18INK4c, and p19INK4d and specifically inhibits cdk4 and cdk6, which form complexes with D-type cyclins. In contrast, the Cip/Kip family [10, 11] consists of p21Cip1, p27Kip1, and p57Kip2 and inhibits most cdks. Cdk1 (cdc2), the cdk responsible for the G2/M transition, forms complexes with cyclin B and regulation of this cdk involves reversible phosphorylation at tyrosine 15 and threonine 14 [12].





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  		Figure 1. Progression through the stages of the cell cycle is regulated by cyclin-dependent kinases (cdks). Cdks are positively regulated by cyclins, levels of which fluctuate throughout the cell cycle, and negatively regulated by the endogenous cdk inhibitors of the INK4a and Cip/Kip families. Arrows indicate sites of action of flavopiridol and UCN-01. (A) Depiction of cell cycle control and phases of the cell cycle where flavopiridol or UCN-01 act. (B) Mechanism by which flavopiridol blocks cell cycle progression. (C) Abrogation of G2 checkpoint by UCN-01. Following DNA damage, the G2 checkpoint is activated, which allows the cell to remain in G2 until all DNA damage is repaired and, thus, to enter M phase with "intact" DNA. However, UCN-01 treatment of DNA-damaged cells abrogates the G2 checkpoint (IC50 ~50 nM), which allows the cells to progress into M prior to completion of DNA repair, leading to apoptosis. The UCN-01 G2 checkpoint abrogation was found to involve Cdc25C, the cdk1 (Cdc2)-activating phosphatase, which is modulated by chk1, a protein kinase directly affected by UCN-01.

 
In human cancer, the Rb pathway (cyclin D1/cdk4/ p16INK4a/Rb) frequently is nonfunctional [7, 13, 14]. Although few human tumors contain a mutation of the Rb gene itself, the majority of human malignancies have derangement in Rb function due to "hyperactivation" of cdks due to an increase in the catalytic subunit (cdk) or the cofactors (cyclins), or loss of inhibitors (endogenous cdk inhibitors) [15, 16]. Thus, cdks are suitable targets for cancer therapy. Many approaches are being designed to interfere with cdk activation [15, 16]. The most effective inhibition of cdk activity appears to come from rationally designed small molecule inhibitors. Of these inhibitors, flavopiridol and UCN-01 are the most advanced in clinical development. This article discusses the mechanisms of action of flavopiridol and UCN-01 and the outcome of initial clinical trials with these compounds.


   FLAVOPIRIDOL
Top
 Learning Objectives
 Abstract
 Introduction
 Flavopiridol
Conclusions
 References
 
Flavopiridol (L86-8275 or HMR 1275) is a semisynthetic derivative of rohitukine, an alkaloid isolated from a plant indigenous to India.

Mechanisms of Action
Initially, flavopiridol was found to inhibit the epidermal growth factor receptor and protein kinase A (inhibitory concentration 50% [IC50] = 21 and 122 µM, respectively) [15, 17]. Flavopiridol was later shown to inhibit cell proliferation, at more physiologically relevant concentrations (IC50 = 66 nM) when the drug was tested in the National Cancer Institute Developmental Therapeutics Program panel of 60 human tumor cell lines [15, 18]. The various mechanisms of action of flavopiridol are described below.

Cdk Inhibition
Studies using purified cdks showed that flavopiridol inhibits cdk1, cdk2, cdk4, and cdk6 (all IC50 ~41 nM) as well as cdk7 (IC50 = 300 nM) by competing with ATP [17, 19–23]. Analysis of the crystal structure of deschloro-flavopiridol bound to cdk2 showed that this flavopiridol congener, which has a phenyl ring instead of flavopiridol’s chlorophenyl, binds to the ATP-binding pocket of cdk2 [24]. Because cdks have a conserved structure, flavopiridol is expected to inhibit all cdks by docking in the ATP-binding site (Fig. 1BGo). In addition to binding to the ATP site of cdks, flavopiridol prevents the activation of most cdks due to inhibition of the cdk-activating kinase "CAK," also known as cdk7, leading to the loss in phosphorylation at threonine 160/161, phosphorylation necessary for activation of most cdks, including cdk1, cdk2, cdk4, and cdk6 (Fig. 1BGo) [19, 23]. Flavopiridol also has been shown to inhibit cdk5, which is expressed in many cells but is only active in neuronal cells. Thus, flavopiridol may have therapeutic potential for Alzheimer’s disease, which is associated with increased cdk5 activation [25]. Furthermore, flavopiridol inhibits cdk9, which together with T-type cyclins forms a complex known as positive transcription elongation factor b (P-TEFb), a kinase required for elongation control of RNA polymerase II [26, 27]. This binding does not appear to be competitive with ATP, suggesting that flavopiridol binds P-TEFb very tightly [27]. Binding of flavopiridol to P-TEFb leads to inhibition of RNA polymerase II transcription [27, 28].

Depletion of Cyclin D1
Exposure of MCF-7 breast cancer cells to flavopiridol resulted in a decrease in cyclin D1 protein within 3 hours, followed by a decrease in levels of cyclin D3 but not of cyclin D2 or cyclin E (IC50 = 100-1000 nM) [29]. This depletion occurs at the mRNA level. Using luciferase reporter assays, we have shown that the depletion of cyclin D1 was preceded by a decline in cyclin D1 promoter activity leading to loss in cyclin D1 mRNA [29]. Another study from our laboratory, using the nonmalignant breast epithelial cell line MCF10A, showed a G1/S cell-cycle arrest 12 hours after administration of flavopiridol, which was accompanied by a loss in cdk6 activity as measured by reduced Rb phosphorylation [22]. Again, the loss in cdk6 activity was preceded by decline in cyclin D1 [22]. Cyclin D1 transcriptional repression is likely to be related to the inhibition of P-TEFb by flavopiridol (see "Inhibition of Transcription," below).

Inhibition of Transcription
In addition to the effects of flavopiridol on cyclin D1 transcription, flavopiridol also modulates transcription in yeast, with clear changes in the families of genes involved in regulation of cell-cycle progression, phosphate and cellular energy metabolism, and guanosine 5'-triphospate (GTP)- and ATP-binding proteins [30]. These findings confirm that flavopiridol modulates transcription in several eukaryotic systems. To determine the exact mechanism by which flavopiridol modulates transcription, we studied the putative effects of flavopiridol on the activity of P-TEFb, a complex of cdk9 and T-type cyclins [26, 31]. P-TEFb phosphorylates the carboxyl-terminal domain of the large subunit of RNA polymerase II, thereby facilitating transcription elongation [31]. Two recent reports have demonstrated that inhibition of P-TEFb by flavopiridol at a concentration of <100 nM, which is easily achieved in human clinical trials [32], results in blockage of RNA polymerase II transcription [27, 28]. Affected genes may include those involved in the regulation of apoptosis and cell cycle [28].

P-TEFb is required for activation of transcription of the HIV-1 genome by the viral transactivator Tat [31]. Flavopiridol binding of P-TEFb was found to inhibit HIV replication at low concentrations (IC50 = 8 nM) [26], whereas flavopiridol concentrations of up to 100 nM did not inhibit cellular transcription [27]. Thus, flavopiridol may have promising potential for AIDS therapy.

Angiogenesis Inhibition
Studies from our laboratory using human monocytes have demonstrated that flavopiridol prevents the vascular endothelial growth factor (VEGF) upregulation induced by hypoxia (IC50 = 50-100 nM) [33]. Flavopiridol modulates VEGF through a decrease in VEGF mRNA stability [33]. Studies from other laboratories also have shown an antiangiogenic effect of flavopiridol in various preclinical models: flavopiridol induced apoptosis in human umbilical vein endothelial cells and decreased blood vessel formation in the mouse Matrigel model of angiogenesis [34, 35]. It is not clear yet whether the antiangiogenic effect of flavopiridol is related to its cdk inhibitory effect.

Apoptosis Induction
Studies in our laboratory using head and neck squamous cell carcinoma (HNSCC) cell lines have shown that flavopiridol induces apoptosis as evidenced by the increase in sub-G1 DNA content (IC50 = 100-1,000 nM) [36]. Flavopiridol even induced apoptosis in HN30, a HNSCC cell line that is resistant to apoptosis induction by DNA-damaging agents such as bleomycin and {gamma}-irradiation. Flavopiridol treatment (i.p., daily for 5 days) induced apoptosis in the HNSCC xenograft HN12 as detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin end labeling (or TUNEL), with significant reduction in tumor size. Furthermore, flavopiridol resulted in depleted cyclin D1 levels in the HN12 tumor xenograft, whereas levels of cyclin D3 and cyclin E remained constant [36]. The mechanism(s) for apoptosis induction by flavopiridol [16, 37] are still under investigation. It is unclear whether the cdk-inhibiting activity of flavopiridol is required for induction of apoptosis. A recent report showed that flavopiridol inhibits transcription of genes that encode apoptosis regulators [28]. Further studies into the mechanism of apoptosis by this agent are warranted.

Induction of Differentiation
Flavopiridol was shown to induce mucinous differentiation in lung carcinoma cells accompanied by loss in cdk2 activity [38]. Again, it is unclear whether the induction of differentiation is related to the cdk-inhibitory properties of flavopiridol.

Clinical Studies
The first clinical trial using flavopiridol was conducted at the National Cancer Institute (NCI) [32]. The promising results of this trial prompted the initiation of many clinical trials testing flavopiridol with different schedules, as well as in combination with standard chemotherapeutic agents.

Seventy-Two-Hour Continuous Infusion Studies
In the first trial of flavopiridol at NCI, 76 patients received a 72-hour continuous infusion of flavopiridol every 2 weeks. This initial schedule was chosen based on the "cytostatic effects" observed in preclinical models with prostate and other solid tumor models [16, 17]. Thus, using this schedule, tumor regressions in patients were not likely. However, significant stability was the expected outcome with this schedule. The maximum tolerated dose (MTD) was 50 mg/m2/day, with a dose-limiting toxicity of secretory diarrhea [32]. To understand this side effect, mechanistic in vitro studies were conducted that found that flavopiridol modifies chloride secretion by intestinal epithelial cells [39]. Using antidiarrheal prophylaxis, the MTD could be increased to 78 mg/m2/day, which resulted in a dose-limiting toxicity of symptomatic hypotension and proinflammatory syndrome [32]. This toxicity was reversible and its etiology is under study. A patient with refractory renal cancer exhibited a partial response (tumor shrinkage >50%), while minor responses (tumor shrinkage <50%) were observed in one patient with non-Hodgkin’s lymphoma, one patient with colon cancer, and one patient with renal cancer. Cdk-inhibitory plasma concentrations of flavopiridol (300-500 nM) were achieved [32].

The 72-hour continuous infusion of flavopiridol 50 mg/m2/day every 2 weeks has been used in other phase I/II clinical trials [40–44]. In addition to diarrhea and fatigue, a few patients exhibited arterial and venous thromboses following flavopiridol treatment [41–44]. A complete response was observed in a patient with refractory metastatic gastric cancer [40]. In contrast, in a phase II trial conducted in 14 patients with metastatic gastric cancer, one minor response was observed while histology and radiography showed tumor necrosis in several patients [44]. There were two partial responses among 35 patients with metastatic renal cancer [41]. In this trial, pharmacokinetic analysis demonstrated that the systemic glucuronidation of flavopiridol is inversely associated with the risk of developing diarrhea [45]. In another trial, 20 patients with metastatic non-small cell lung cancer (NSCLC) were treated with 72 hour infusional flavopiridol (50 mg/m2/day) [42]. Although no objective responses were observed, six patients exhibited significant disease stabilization (>=3 months) with an overall survival of ~7 months [42]. Preliminary results from a phase II trial in patients with metastatic colorectal cancer showed no objective responses among 10 evaluable patients [43]. Flavopiridol plasma concentrations in these trials [40, 42, 44], except for two in which no drug plasma concentrations were published [41, 43], were in the range of those reported for the NCI trial [32].

One-Hour Infusion Studies
In order to obtain a higher therapeutic index of flavopiridol, we administered flavopiridol to leukemia/ lymphoma and HNSCC xenografts as a bolus for 5 consecutive days. A strong induction of apoptosis and antitumor effects was observed in these models [36, 46]. Therefore, we initiated another phase I trial in which patients received flavopiridol as a 1-hour infusion for 5 consecutive days every 3 weeks [47, 48]. The MTD was 37.5 mg/m2/day and dose-limiting toxicities were neutropenia, fatigue, and diarrhea. The five daily 1-hour infusions every 3 weeks resulted in a flavopiridol concentration of ~1.5 µM. To reach higher drug plasma concentrations, flavopiridol was administered as 1-hour infusions at a higher dose per day (50 mg/m2/day) for 3 consecutive days every 3 weeks. This treatment resulted in a flavopiridol plasma concentration of ~4 µM and associated toxicities of neutropenia, vomiting, diarrhea, and pro-inflammatory syndrome. Although no objective responses were observed, this treatment stabilized disease in 3 (one mantle-cell lymphoma, one NSCLC, one melanoma) of 12 patients [47, 48].

Flavopiridol at the 1-hour infusion schedule is being explored in ongoing phase I and phase II clinical trials in hematologic malignancies, melanoma, renal cell cancer, endometrial carcinoma, and HNSCC (source: http://www.cancer.gov/search/clinical_trials/).

Combination Therapy
A phase I trial infusional flavopiridol in combination with paclitaxel was well tolerated [49]. Combination therapy trials using flavopiridol and various standard chemotherapeutic agents have been initiated (for more information, see http://www.cancer.gov/search/clinical_trials/). These trials are based on in vitro preclinical models in which most synergism was observed when the cytotoxic agent was applied before flavopiridol [50, 51].

UCN-01
UCN-01 (7-hydroxystaurosporine, NSC638850) is a derivative of staurosporine, a nonspecific protein kinase C (PKC) inhibitor. Although UCN-01 inhibits certain PKC isozymes (see below), its strong antiproliferative effects involving cdk inhibition, "inappropriate" activation of cdks, and induction of apoptosis are thought to be unrelated to PKC inhibition [reviewed in 24, 34, 52].

Mechanisms of Action

PKC-Inhibition
UCN-01 inhibits Ca2+-dependent PKC isozymes (IC50 ~30 nM) and, to a lesser extent, the Ca2+-independent PKC isozymes (IC50 ~ 600 nM). UCN-01 does not inhibit the atypical PKC [53–55]. Several studies indicate that PKC-inhibition by UCN-01 is unrelated to its effects on the cell cycle and apoptosis [24, 34, 52].

Cdk Inhibition/"Inappropriate" Cdk Activation
UCN-01 can either inhibit or activate cdks, which may result in cell-cycle arrest or apoptosis [reviewed in 24, 34, 52]. UCN-01 has been reported to induce G1 cell-cycle arrest at low nM concentrations (IC50 = 100-300 nM) [56, 57]. Although the mechanism is still under investigation, the cell-cycle arrest is associated with induction of endogenous cdk inhibitor p21waf1 and p27kip1. However, at higher (IC50 = 300-600 nM) concentrations, UCN-01 has been shown to inhibit cdk1 (Cdc2) and cdk2 in in vitro H1 kinase assays [58].

Following DNA damage, the G2 checkpoint is activated, which allows the cell to remain in G2 until all DNA damage is repaired, allowing cells to enter M phase with "intact" DNA (Fig. 1CGo). However, UCN-01 treatment of DNA-damaged cells abrogates the G2 checkpoint (IC50 ~50 nM), which allows the cells to progress into M prior to completion of DNA repair, leading to apoptosis (Fig. 1CGo). The UCN-01 G2 checkpoint abrogation was found to involve Cdc25C, the cdk1 (Cdc2)-activating phosphatase [59]. Recent studies have shown that UCN-01 inhibits phosphorylation of Cdc25C by the kinase chk1 (Fig. 1CGo) [60–62]. The ability of UCN-01 to induce apoptosis in response to DNA damage is being explored in combination trials with standard chemotherapeutic agents.

UCN-01 has also been shown to abrogate the Sphase checkpoint, which is activated upon DNA damage [63, 64]. The target of UCN-01 is likely to be chk1, which recently was shown to be involved in the S-phase checkpoint [65].

Induction of Apoptosis
Our laboratory and others have demonstrated apoptosis induction by UCN-01, particularly in a panel of HNSCC cell lines. Furthermore, UCN-01 has potent antitumor effects on HN12 tumor xenograft after treatment for 5 days (A. Senderowicz, submitted for publication). This antitumor effect was associated with depletion of cyclin D3 and an increase in p27Kip1 and p21waf1. Although the mechanism of UCN-01-induced apoptosis (IC50 = 100-1,000 nM) is still unknown [34, 52], several reports demonstrate that, in some in vitro models, UCN-01 can downregulate some antiapoptotic proteins, similar to flavopiridol [28].

Clinical Studies
The first clinical trial with UCN-01 was recently completed at NCI [66]. The initial schedule was a 72-hour continuous infusion every 2 weeks. However, in the first nine patients who received infusional UCN-01, the half-life appeared to be 100-fold longer than that observed in preclinical models. Furthermore, the initial concentrations achieved in plasma were ~4 to 7 µM [66]. This again differed greatly from the findings in animal models, in which drug plasma concentrations of >=1 µM were universally lethal [67]. It appeared that UCN-01 in humans strongly binds to plasma {alpha}1-acidic glycoprotein [68–70]. Therefore, treatment schedules were successfully changed to 36-hour continuous infusion in patients receiving >=12 mg/m2/day every 4 weeks [66]. Dose-limiting toxicities were nausea/vomiting, symptomatic hyperglycemia, and pulmonary toxicity. The mean half-life was approximately 588 hours and the total drug plasma concentration ranged from 30 to 40 µM. The concentration of "free" salivary UCN-01 concentrations at MTD was ~100 nM, which has been shown to modulate cell-cycle processes in vitro. Similar results were obtained in plasma samples after ultracentrifugation [66]. A partial response was observed in a patient with melanoma. A complete response was observed in a patient with refractory alk-positive anaplastic large-cell lymphoma [66]. This patient’s treatment was discontinued >=1 year ago and he is still disease-free after 4 years of therapy.

Plasma samples from patients who received UCN-01 were shown to induce a 40% to 70% abrogation in an ex vivo G2 checkpoint assay, reflecting free plasma UCN-01 [66]. Furthermore, target modulation by UCN-01 was demonstrated by the level of phosphorylation of the PKC substrate adducin. Adducin phosphorylation in bone marrow and tumor samples taken during UCN-01 treatment was significantly reduced compared with pretreatment samples [66].

Currently, phase I clinical trials are exploring novel schedules of UCN-01 (1- to 3-hour infusion every 4 weeks) [71, 72]. Furthermore, phase I clinical trials using a combination of cytotoxic agents (cisplatin, 5-fluorouracil, fludarabine) with UCN-01 are ongoing (source: http://www.cancer.gov/search/clinical_trials/).


   CONCLUSIONS
Top
 Learning Objectives
 Abstract
 Introduction
 Flavopiridol
 Conclusions
References
 
Most human malignancies display aberrations in the Rb pathway due to cdk hyperactivation. Thus, cdks may be an excellent therapeutic target. The cdk inhibitors flavopiridol and UCN-01 are the first small molecules that target cdks. Their therapeutic value in cancer patients currently is being evaluated in phase I/II clinical trials. In addition, several novel compounds that target molecular pathways involved in cancer progression are under development. Extensive testing of effective novel compounds, including flavopiridol and UCN-01, in phase II clinical trials is needed to assess appropriate treatment schedules as well as the possibility of their use in combination with conventional cytotoxic agents.


   ACKNOWLEDGMENT
Top
 Learning Objectives
 Abstract
 Introduction
 Flavopiridol
 Conclusions
 References
 
The author acknowledges Anita Frijhoff (University of Texas, M.D. Anderson Cancer Center) as technical writer for editing the manuscript and recompiling literature for this article. He also recognizes Margaret Hayes for transcription.

Disclaimer: The views expressed in this manuscript do not necessarily represent the views of the National Institutes of Health and/or the Department of Health and Human Services.


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 Abstract
 Introduction
 Flavopiridol
 Conclusions
 References
 

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Received May 23, 2002; accepted for publication July 2, 2002.




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