Cell Cycle Modulators for the Treatment of Lung Malignancies

c omprehensive review Cell Cycle Modulators for the Treatment of Lung Malignancies Adrian M. Senderowicz Abstract It has become clear in the past decade that most human malignancies, including lung neoplasms, have aberrations in cell cycle control. The tumor suppressor gene retinoblastoma is an important player in the G1/S transition and its function is abnormal in most human neoplasms. Retinoblastoma function is lost as a result of phosphorylation by the cyclin-dependent kinases (CDKs). Thus, modulation of CDKs may have an important use for the therapy and prevention of human neoplasms. Direct CDK modulators are small molecules that target specifically the adenosine triphosphate binding site of CDKs. In contrast, indirect CDK modulators affect CDK function by modulation of upstream pathways required for CDK activation. The first example of a direct smallmolecule CDK modulator tested in the clinic, flavopiridol, is a pan-CDK inhibitor that not only promotes cell cycle arrest but also halts transcriptional elongation, promotes apoptosis, induces differentiation, and has antiangiogenic properties. The second example of direct small-molecule CDK modulators tested in clinical trials is UCN-01 (7-hydroxystaurosporine). UCN-01 has interesting preclinical features: it inhibits Ca2+-dependent protein kinase C, promotes apoptosis, arrests cell cycle progression at G1/S, and abrogates checkpoints upon DNA damage. In summary, novel small-molecule CDK modulators are being tested in the clinic with interesting results. Although these small molecules are directed toward a very prevalent cause of carcinogenesis, their role in the clinical armamentarium is still uncertain. Clinical Lung Cancer, Vol. 5, No. 3, 158-168, 2003

Key words: Angiogenesis, Apoptosis, Cyclin-dependent kinases, Differentiation, Flavopiridol, 7-Hydroxystaurosporine, Retinoblastoma

Introduction

INK (p16INK4a, p15INK4b, p18INK4c, p19INK4d) and CIP/KIP families (p21CIP1, p27KIP1, and p57KIP2).1,2 Retinoblastoma proteins are pocket proteins that sequester E2F transcription factors, preventing them from activating critical genes in cell proliferation.3,4 After Rb phosphorylation by CDK4 and/or CDK6 complexes during G1-phase and by CDK2 at G1/S interphase, E2F proteins are released and promote the transcription of genes essential for the transition to S-phase of the cell cycle.5,6 CDK4,6/D-type cyclins therefore execute their critical functions during mid to late G1-phase, as cells cross a G1 restriction point and become independent of mitogens for completion of the division cycle. These features suggest that the fundamental role of these complexes is to integrate extracellular signals with the cell cycle machinery.1,2 Other important points of regulation have been described in G2 and mitosis. In these phases, the specific expression of certain regulators is essential to control the correct sequence of events that leads to cell division. Basically, cyclins B1 and B2 and their partner cdc2 (ie, CDK1), together with other kinases and phosphatases (eg, wee1, cdc25), regulate the final phases of the cell cycle (Figure 1).

Brief Overview of Cell Cycle Regulation Regulation of the cell cycle and cell proliferation has been extensively studied in the past few years, and a consensus paradigm of the regulation of the cell cycle has been developed.1,2 According to this paradigm, the “master switch” of the cell cycle is the retinoblastoma (Rb) family of proteins. Proliferation occurs by the phosphorylation of these proteins by the cyclin-dependent kinases (CDKs; Figure 1).1 At least 9 CDKs are activated by D-type cyclins (D1, D2, and D3) and cyclin E and inhibited by 2 families of CDK inhibitors, the

Molecular Therapeutics Unit, Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD Submitted: May 29, 2003; Revised: Sep 23, 2003; Accepted: Oct 1, 2003 Address for correspondence: Adrian M. Senderowicz, MD, Molecular Therapeutics Unit, Oral and Pharyngeal Cancer Branch, National Institute of Craniofacial and Dental Research, National Institutes of Health, 30 Convent Dr, Bldg 30, Rm 212, Bethesda, MD 20892-4330 Fax: 301-402-0823; e-mail: [email protected]

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In the past decade, several proteins that participate in the tight control of cell division have been found to be mutated, deleted, amplified, or overexpressed in human tumors. In the first part of this review, the principal points of deregulation found in human tumors will be summarized, with particular emphasis on lung cancer.

Figure 1

Regulation of G1-, S-, G2-, and M-Phases of Cell Cycle Growth Factors (ras, myc, fos, jun) Cyclin B CDK 1

Cell Cycle Alterations in Neoplasia In the past few years, it has become clear that cyclins, CDK complexes, and other cell cycle regulators are mechanistically involved in the development of human tumors.7-11 This is consistent with a large body of literature showing the importance of inactivation of the Rb pathway in tumor development.9,12,13 The inactivation of Rb can be produced by direct mutation of the Rb protein, but this is a relatively rare event, occurring only in Rbs, osteosarcomas, and a minority of breast and some other tumors.1,9,14 More frequent alterations of this pathway occur by functional inactivation of Rb by hyperphosphorylation. This is normally the result of increased CDK activities caused by overexpression of cyclins. For example, several laboratories have reported that some tumors show loss of Rb or, alternatively, overexpression of cyclin D1.15-17 Similarly, in other tumors, loss of p16INK4a and Rb are mutually exclusive.18-20 This observation led to the hypothesis that inactivation of the cyclin D/CDK/p16/Rb protein pathway can promote tumor development and that either (1) loss of the suppressor activity of Rb or p16INK4a or (2) overexpression of cyclin D1 can override this checkpoint.1,9 Several reports have implicated D-type cyclins in the neoplastic development, although limited information is available on the participation of its partner, CDK4, in these events. The involvement of CDK4 in the neoplastic process was suggested by the fact that CDK4 amplification and/or overexpression was detected in human glioblastomas, but overexpression and/or amplification of D-type cyclins was not detected in these tumors.21,22 In addition, CDK4 mutations were identified in patients with familial melanoma,23 and recently, amplification and overexpression of CDK4 were also detected in sporadic breast carcinomas,24 ovarian carcinomas,25 and sarcomas.26 In light of all these findings, proteins that govern cell cycle control are reasonable targets for cancer therapy.13,27

Specific Cell Cycle Aberrations in Lung Cancer The Rb tumor-suppressor gene is located on chromosome 13q14. Cytogenetic abnormalities of chromosome 13 and loss of heterozygosity at the Rb locus have been reported in a variety of human cancers, including non–small-cell lung cancer (NSCLC), and the frequency of Rb abnormalities detected by immunohistochemistry in NSCLC has reached 30%.28,29 Furthermore, functional Rb protein is absent in 90% of cases of small-cell lung cancer (SCLC) and in as many as 30% of NSCLC primary lesions and cell lines.28 Thus, the G1/S checkpoint transition is lost in the majority of patients with lung cancer. As mentioned earlier, the molecular events that lead to deregulated Rb function include sustained activity of CDKs as a result of loss of CDK inhibitors such as p16INK4a,

p16INK4a family

M Rb

p21CIP1 family P

CDK 4/6

P

G2 Rb~ P E2F

Cyclin A

Cyclin D

CDK 1

G1 Cyclin E CDK 2

S Cyclin A CDK 2

p21CIP1 family

Abbreviations: CDK = cyclin-dependent kinase; Rb = retinoblastoma

and persistent upregulation of several cyclins such as cyclin D1, cyclin A, and cyclin E. Therefore, sustained hyperphosphorylation and inactivation of Rb also contribute to the transformation of normal bronchial epithelium to autonomously growing cancer cells. Additional mechanisms of Rb inactivation include Rb point mutations and chromosomal deletions.30,31 Although Rb plays an important role in lung tumorigenesis, it appears that Rb status does not represent a significant prognostic factor in adenocarcinoma of the lung. Indeed, among 90 patients, 56.7% were shown to have reduced expression of Rb as measured by immunohistochemistry; however, there were no statistical differences among expression of Rb and clinical survival.32 Similar results were observed in a larger study in 207 surgically resected primary NSCLCs.33 Members of the INK4 protein family such as p16, p15, p18, and p19 inhibit CDK4- and CDK6-mediated phosphorylation of Rb;34,35 inactivation of p16INK4a in tumors expressing wild-type Rb is required for malignant cells to enter the Sphase or escape senescence.36 The occurrence of p16INK4a lesions is second only to p53 abnormalities in human cancer and is a frequent event in premalignant lesions of the upper digestive tract.37 The CDKN2A gene is inactivated by a 2-hit mechanism that can involve CpG island methylation, 9p21 loss of heterozygosity, mutation, or homozygous deletion.14,38 The CDKN2A locus on chromosome 9p21 encodes 2 proteins translated by alternative splicing of messenger RNA (mRNA): p16INK4a, which inhibits phosphorylation of Rb through cyclin D1/CDK4 and p14ARF, a protein that binds MDM2, thereby stabilizing wild-type p53.39 Analysis of p16INK4a, p14ARF, and p53 from 38 primary NSCLC specimens showed that p16INK4a was inactivated in 58% of tumors by homozy-

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Cyclin-Dependent Kinase Inhibitors in Lung Cancer Figure 2 Modes of Action for Cell Cycle Modulators

A P

CDK7

T160 PO4-

CDK

CKI

ATP Y15

Cyclin

T14

wee1/myt1

P

PO4-

cdc25

B Perifosine

Manipulation of Cell Cycle Machinery for Therapeutic Purposes

UCN-01 T160

CKI CKI CKI CDK

Flavopiridol Alsterpaullone

ATP

Cyclin

Y15 T14

Loss in CDK function may occur as a result of loss in mass of catalytic subunit and/or cofactors, increased endogenous inhibitors, increased wee1/myt1, or loss in CDK7 or cdc25 activity (A). However, the most successful way to modulate CDK is by competing with ATP binding in CDK and upregulating endogenous CDK inhibitors (B). Abbreviations: ATP = adenosine triphosphate; CDK = cyclin-dependent kinase, CKI = cyclin-dependent kinase inhibitor

gous deletions, promoter hypermethylation, and point mutation in exon 2.40 Fourteen tumors had simultaneous p16INK4a and p14ARF inactivation, most frequently because of homozygous deletions extending into the INK4a/ARF locus. A confirmatory study by Spanakis et al demonstrated decreased p16INK4a in 46% of NSCLCs examined.41 Gorgoulis et al investigated the alterations of G1-phase proteins using immunohistochemical and molecular methods in a series of 68 NSCLCs and performed correlation with clinicopathologic findings and prognosis.42 Abnormal expression in p16INK4a and Rb was observed in 33 (49%) and 27 (40%) cases, respectively. The main mechanisms responsible for loss in p16INK4a were deletions and transcriptional silencing by methylation. In addition, similar deregulation may occur in other tumor-suppressor genes that reside at the 9p21-22 region, CDKN2/MTS1/p16INK4a, p14ARF, and MTS2/p15INK4b. The lack of correlation with clinical tumor-stage and prevalence aberrations in several G1 proteins suggests that multiple hits of this network may represent an early event in lung carcinogenesis. In another interesting study, Shapiro et al compared the expression of G1 checkpoint proteins in NSCLC, which is predominantly Rb-positive, and in SCLC, which is Rb-negative.43 Most NSCLC and SCLC resection specimens and cell lines overexpress cyclin D1 (indicating that cyclin D1 overexpression and Rb inactivation can coexist in SCLC). However, 9 of 9 Rbpositive NSCLC cell lines have absent or low levels of p16, whereas an Rb-negative NSCLC line and 5 of 5 SCLC cell lines have high levels of p16. In primary resection specimens, p16

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was undetectable in 18 of 27 NSCLC samples and abundant in 4 of 5 SCLC samples. Thus, a reciprocal relationship between Rb inactivation and p16 expression is common in several malignancies (including NSCLC), and a clear distinction is apparent in the expression of p16 in SCLC and NSCLC. In summary, most lung carcinomas have aberrations in the G1/S transition. Although decreased expression in Rb occurs in a significant number of cases, the majority of lung tumors have loss in Rb function caused by aberrations in proteins that regulate the activity of CDKs, leading to inactivation and phosphorylation of Rb. This information suggests that smallmolecule CDK modulators may be useful for the prevention or treatment of lung cancer.

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Several strategies could be considered to modulate CDK activity (Figure 2). These strategies are divided into direct effects on the catalytic CDK subunit and indirect modulation of regulatory pathways that govern CDK activity.44,45 As depicted in Figure 2A, the small molecular CDK inhibitors are compounds that directly target the catalytic CDK subunit. Most of these compounds modulate CDK activity by interacting specifically with the adenosine triphosphate (ATP) binding site of CDKs.44-48 Examples of this class include flavopiridol and alsterpaullone. The second class of CDK inhibitors are compounds that inhibit CDK activity by targeting the regulatory upstream pathways that modulate CDK activity by altering the expression and synthesis of the CDK/cyclin subunits or the CDK inhibitory proteins; by modulating the phosphorylation of CDKs; by targeting CDK-activating kinase, cdc25, and wee1/myt1; or by manipulating the proteolytic machinery that regulates the catabolism of CDK/cyclin complexes or their regulators (Figure 2B).44,45,49 Examples of this class of compounds include perifosine and UCN-01.

Modulators of Cyclin-Dependent Kinase Activity As mentioned previously, CDK can be modulated by direct effects on the catalytic subunit and/or by disruption of upstream regulatory pathways. Several examples and mechanisms are described in Table 145,47,50-66 and elsewhere.13,27,44,49,67-70

Flavopiridol Mechanism of Antiproliferative Effects. Flavopiridol (L868275; HMR 1275) is a semisynthetic flavonoid derived from rohitukine, an indigenous plant of India. Initial studies with flavopiridol demonstrated modest in vitro inhibitory activity with respect to epidermal growth factor receptor (EGFR) and protein kinase A (PKA; inhibitory concentrations [IC50s] of 21 and 122 μmol/L, respectively).71 However, when this compound was tested in the NCI 60 cell line anticancer drug screen panel, it demonstrated very potent growth inhibition (IC50, 66 nmol/L), a concentration approximately 1000 times lower than the concentration required to inhibit PKA and EGFR.71 Initial studies with this flavonoid revealed clear evidence of G1/S or G2/M arrest caused by loss in CDK1 and

Adrian M. Senderowicz CDK2.72-74 Studies using purified CDKs showed that the inhibition observed is reversible and competitively blocked by ATP, with a Ki of 41 nmol/L.72-76 Furthermore, the crystal structure of the complex of deschloroflavopiridol and CDK2 showed that flavopiridol binds to the ATP binding pocket, with the benzopyrene occupying the same region as the purine ring of ATP,77 confirming the results of earlier biochemical studies with flavopiridol.73 Flavopiridol inhibits all CDKs thus far examined (IC50, approximately 100 nmol/L), but the inhibition potential of flavopiridol on CDK7 (CDK-activating kinase) was found to be much lower than that against other CDKs (IC50, approximately 300 nmol/L).73,75,76 In addition to directly inhibiting CDKs, flavopiridol promotes a decrease in the level of cyclin D1, an oncogene that is overexpressed in many human neoplasms. Of note, neoplasms that overexpress cyclin D1 have a poor prognosis.78-80 Depletion of cyclin D1 appears to lead to the loss of CDK activity.81 Cyclin D1 decrease is caused by depletion of cyclin D1 mRNA and was associated with a specific decrease in cyclin D1 promoter as measured by a luciferase reporter assay. The transcriptional repression of cyclin D1 observed after treatment with flavopiridol is consistent with the effects of flavopiridol on yeast cells (as described earlier) and underscores the conserved effect of flavopiridol on eukaryotic cyclin transcription.50 In summary, flavopiridol can induce cell cycle arrest by at least these 3 mechanisms: (1) direct inhibition of CDK activities by binding to the ATP binding site; (2) prevention of the phosphorylation of CDKs at threonine-160/161 by inhibition of CDK7/cyclin H;74,75 and (3) decrease in the amount of cyclin D1, an important cofactor for CDK4 and CDK6 activation (G1/S arrest only). Another effect of flavopiridol on transcription is attenuation of the induction of vascular endothelial growth factor (VEGF) mRNA in monocytes after hypoxia (as described later). This effect is a result of alterations in the stability of VEGF mRNA.82 We recently demonstrated that flavopiridol potently inhibits P-TEFb (also known as CDK9/cyclin T), with a Ki of 3 nmol/L, leading to inhibition of transcription by RNA polymerase II by blocking the transition into productive elongation. Interestingly, in contrast with all CDKs tested so far, flavopiridol was not competitive with ATP in this reaction. P-TEFb is a required cellular cofactor for the human immunodeficiency virus (HIV)–1 transactivator Tat. Consistent with its ability to inhibit P-TEFb, flavopiridol blocked Tat transactivation of the viral promoter in vitro. Furthermore, flavopiridol blocked HIV1 replication in single-round and viral spread assays with an IC50 of < 10 nmol/L.83 These actions of the drug led to the testing of flavopiridol in clinical trials in patients with HIV-related malignancies.84 Apoptosis. An important biochemical effect involved in the antiproliferative effect of flavopiridol is the induction of apoptotic cell death. Hematopoietic cell lines are often quite sensitive to flavopiridol-induced apoptotic cell death,85-88 but the mechanism(s) by which flavopiridol induces apoptosis have not yet been elucidated. Flavopiridol does not modulate topoi-

Table 1

Direct Cyclin-Dependent Kinase Modulators

Kinase Modulated

CDK1/CDK2/CDK5

Examples Purvalanol and Compound 5250,63 Roscovitine55,56 Olomoucine55,57,59 Butyrolactone I58 CVT-31360

Nonspecific CDK

Flavopiridol45,65 UCN-0145,51-54 Staurosporine53,65 Oxindole I61

Unknown

Paullones47,55,64 Toyocamycin62 Myricetin66

Abbreviation: CDK = cyclin-dependent kinase

somerase I/II activity.87 In certain hematopoietic cell lines, neither Bcl-2/BAX nor p53 appeared to be affected;87,89 whereas, in other systems, Bcl-2 may be inhibited.88 Preliminary evidence demonstrated that flavopiridol-induced apoptosis in leukemia cells is associated with early activation of the mitogen-activated protein kinase family of proteins (MEK, p38, and JNK).90 This activation may lead to activation of caspases.90 As seen in this and other models, caspase inhibitors prevent flavopiridol-induced apoptosis.86,90 It is unclear whether the putative flavopiridol-induced inhibition of CDK activity is required for induction of apoptosis. Clear evidence of cell cycle arrest along with apoptosis was observed in a panel of squamous head and neck cancer cell lines, including a cell line (HN30) that is refractory to several DNA-damaging agents, such as γ-irradiation and bleomycin.91 Again, the apoptotic effect was independent of p53 status and was associated with the depletion of cyclin D1. These findings have been corroborated in other preclinical models.89,92-94 Efforts to understand flavopiridol-induced apoptosis are under intense investigation. Angiogenesis. Flavopiridol targets not only tumor cells but also angiogenesis pathways. Brusselbach et al incubated primary human umbilical vein endothelial cells with flavopiridol and observed apoptotic cell death even in cells that were not cycling, leading to the notion that flavopiridol may have antiangiogenic properties as a result of endothelial cytotoxicity.95 In other model systems, Kerr et al tested flavopiridol in an in vivo Matrigel™ model of angiogenesis and found that flavopiridol decreased blood vessel formation, which is a surrogate marker for antiangiogenic effect of this compound.96 Furthermore, as mentioned earlier, Melillo et al demonstrated that, at low nanomolar concentrations, flavopiridol prevented the induction of VEGF by hypoxic conditions in human monocytes.82 This effect was caused by a decreased stability of VEGF mRNA, which paralleled the decrease in VEGF protein. Thus, the antitumor activity of flavopiridol observed may be caused in part by antiangiogenic effects. Whether various antiangiogenic actions of flavopiridol result from its interaction with a CDK target or other targets requires further study.

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Cyclin-Dependent Kinase Inhibitors in Lung Cancer Tumor Cell Differentiation. The antitumor effect observed with flavopiridol can also be explained by activation of differentiation pathways. It has become clear that cells become differentiated when exit of the cell cycle (G0) and loss of CDK2 activity occurs. Based on this information, Lee and colleagues tested flavopiridol and roscovitine, both known CDK2 inhibitors, to determine whether they induce a differentiated phenotype.97 For this purpose, NCI-H358 lung carcinoma cell lines were exposed to CDK2 antisense construct, flavopiridol, or roscovitine. Clear evidence of mucinous differentiation along with loss in CDK2 activity was observed in this lung carcinoma model. Thus, it is plausible that the antitumor effect of flavopiridol in lung carcinoma models may be a result of induction of differentiation, among other causes. Synergistic Effect with Chemotherapy. Several investigators have attempted to determine whether flavopiridol has synergistic effects with standard chemotherapeutic agents. For example, synergistic effects in A549 lung carcinoma cells were demonstrated when treatment with flavopiridol followed treatment with paclitaxel, cytarabine, topotecan, doxorubicin, or etoposide.98,99 In contrast, a synergistic effect was observed with 5-fluorouracil (5FU) only when cells were treated with flavopiridol for 24 hours before addition of 5-FU. Furthermore, synergistic effects with cisplatin were not schedule dependent.99 However, Chien et al failed to demonstrate a synergistic effect between flavopiridol and cisplatin and/or γ-irradiation in bladder carcinoma models.92 One important issue to mention is that most of these studies were performed in in vitro models. Thus, confirmatory in vivo studies in animal models are needed. Animal Studies. Experiments using colorectal (Colo205) and prostate (LnCaP/DU-145) carcinoma xenograft models in which flavopiridol was administered frequently over a protracted period demonstrated that flavopiridol is cytostatic.71,100 These demonstrations led to human clinical trials of flavopiridol administered as a 72-hour continuous infusion every 2 weeks101. Subsequent studies in human leukemia/lymphoma xenografts demonstrated that flavopiridol administered intravenously as a bolus rendered animals tumor free, whereas flavopiridol administered as an infusion only delayed tumor growth.85 Moreover, in a head and neck cancer xenograft (HN12), flavopiridol administered as an intraperitoneal bolus daily at 5 mg/kg for 5 days demonstrated substantial growth delay.91 Again, apoptotic cell death and cyclin D1 depletion were observed in tissues from xenografts treated with flavopiridol.85 Based on these results, a phase I trial of 1hour daily infusional flavopiridol every 3 weeks was conducted at the National Cancer Institute (NCI).102 Phase I Trial of 72-Hour Continuous Infusion of Flavopiridol. Two phase I clinical trials of flavopiridol administered as a 72-hour continuous infusion every 2 weeks have been completed.101,103 In the NCI phase I trial of infusional flavopiridol (N = 76), the dose-limiting toxicity (DLT) was diarrhea, with a maximum tolerated dose (MTD) of 50 mg/m2/day for 3 days. In the presence of antidiar-

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rheal prophylaxis (a combination of cholestyramine and loperamide), patients tolerated higher doses and a second MTD of 78 mg/m2/day for 3 days was defined. The DLTs observed at the higher dose level were a substantial proinflammatory syndrome (fever, fatigue, local tumor pain, and modulation of acute phase reactants) and reversible hypotension.101 Minor responses were observed in patients with non-Hodgkin's lymphoma (NHL), colon cancer, and kidney cancer for > 6 months. Moreover, 1 patient with refractory renal cancer experienced a partial response for > 8 months. Of 14 patients who received flavopiridol for > 6 months, 5 patients received flavopiridol for > 1 year and 1 patient received flavopiridol for > 2 years. Plasma concentrations of 300-500 nmol/L flavopiridol, which inhibit CDK activity in vitro, were safely achieved during this trial. In a complementary phase I trial exploring the same schedule (72-hour continuous infusion every 2 weeks), Thomas et al found that the DLT was diarrhea, corroborating the NCI experience.103 Moreover, plasma concentrations of 300-500 nmol/L flavopiridol were also observed. Interestingly, there was one patient in this trial with refractory metastatic gastric cancer that progressed after a treatment regimen containing 5-FU. When treated with flavopiridol, this patient experienced a sustained complete response without any evidence of disease for > 2 years after treatment was completed. Phase I Trial of 1-Hour Flavopiridol Infusion for 5 Days. The first phase I trial of a daily 1-hour infusion of flavopiridol for 5 consecutive days every 3 weeks was recently completed.101,102 This schedule was based on antitumor results observed in leukemia/lymphoma and head and neck cancer xenografts treated with flavopiridol.85,91 A total of 55 patients were treated in this trial. The recommended phase II dose is 37.5 mg/m2 per day for 5 consecutive days. Dose-limiting toxicities observed at 52.5 mg/m2/day are nausea/vomiting, neutropenia, fatigue, and diarrhea.101,102 Other (non–dose-limiting) side effects are local tumor pain and anorexia. To reach higher flavopiridol concentrations, the protocol was amended to administer flavopiridol for 3 days and then for 1 day only every 3 weeks. With these protocol modifications, we were able to achieve concentrations necessary to induce apoptosis in xenograft models (approximately 4 μmol/L).85,91,102,104 Of note, the half-life observed in this trial was much shorter (approximately 3 hours) than that observed in the infusional trial (approximately 10 hours). Thus, the high micromolar concentrations achieved in the 1-hour infusional trial could be maintained for short periods of time.102,104 Several phase 2 trials in patients with refractory head and neck cancer, chronic lymphocytic leukemia (CLL), or mantle-cell lymphoma (MCL) are currently being tested using this schedule (described in later sections). A phase I trial testing the combination of paclitaxel and 24-hour infusional flavopiridol demonstrated good tolerability with a pulmonary DLT.105 Phase II Trials of 72-Hour Continuous Infusion of Flavopiridol. Current research includes phase II trials of flavopiridol given as a 72-hour continuous infusion in CLL, NSCLC, NHL, and colon, prostate, gastric, head and neck, and kidney cancers;

Adrian M. Senderowicz and phase I trials of flavopiridol administered on novel schedules and in combination with standard chemotherapeutic agents.84,106-109 In a recently published phase II trial of flavopiridol in metastatic renal cancer, 2 objective responses were observed (response rate, 6%; 95% CI, 1%-20%). Most patients developed grade 1/2 diarrhea and asthenia.108 In this trial, patients who demonstrated glucuronide flavopiridol metabolites in plasma as measured by high-performance liquid chromatography had less pronounced diarrhea than patients who demonstrated no glucuronide flavopiridol metabolites.110 Thus, it may be possible that patients with higher metabolic rates may tolerate higher doses of flavopiridol. Phase II trials of shorter infusions of flavopiridol (ie, 1 hour) are being conducted in MCL, CLL, and head and neck squamous cell carcinoma. Of interest, several patients with refractory CLL or MCL have demonstrated clear evidence of responses (partial responses) in these trials (J.R. Suarez, MD, oral communication, May 2002). Preclinical Studies of Flavopiridol in Lung Carcinoma Models. A panel of NSCLC cell lines expressing wild-type Rb protein and lacking p16INK4A were exposed to flavopiridol for 72 hours.89 Flavopiridol demonstrated cytotoxicity in all 7 cell lines, with evidence of apoptosis. As mentioned before, the induction of apoptosis was p53-independent, a clinical situation that occurs in ≥ 50% of solid tumors. Although cells in all phases are sensitive to flavopiridol, cells in S-phase are preferentially sensitive to this agent. In addition, cells are sensitized to flavopiridol following recruitment to S-phase, whether this is accomplished by synchronization or by treatment with noncytotoxic concentrations of chemotherapy agents that impose an S-phase delay.89 Combinations of gemcitabine or cisplatin, followed by flavopiridol at concentrations that correlate with CDK inhibition, produce sequence-dependent cytotoxic synergy. Thus, administration of flavopiridol after chemotherapy agents that cause S-phase accumulation may be an efficacious antitumor strategy. Another interesting feature observed with flavopiridol is the induction of differentiation in lung carcinoma models. Lee and colleagues tested whether flavopiridol and roscovitine, both known CDK2 inhibitors or antisense CDK2 constructs, block cell cycle progression in NCI-H358 lung carcinoma cell lines.97 Clear evidence of mucinous differentiation, along with loss in CDK2 activity, was observed in this lung carcinoma model. Clinical Studies of Flavopiridol in NSCLC. Based on the interesting results obtained in preclinical lung carcinoma models, a phase II trial in patients with metastatic lung carcinoma was performed.111 A total of 20 patients were treated with a 72-hour continuous infusion of flavopiridol every 14 days at a dosage of 50 mg/m2/day. The most common toxicities included grade ≤ 2 diarrhea, asthenia, and venous thromboses. Median overall survival for the 20 patients who received treatment was 7.5 months. Of note, the median survival of 7.5 months achieved with flavopiridol is similar to the range of 7.4-8.2 months reported recently in a randomized trial of 4 chemotherapy regimens containing platinum analogues in combination with tax-

anes or gemcitabine.112 Moreover, a similar overall survival was obtained with gefitinib, a recently approved EGFR inhibitor for the treatment of advanced lung cancer.113 Based on these encouraging results, a phase III trial comparing standard combination chemotherapy versus combination chemotherapy plus flavopiridol is currently under investigation.

UCN-01 Mechanism of Antiproliferative Activity. Staurosporine is a potent nonspecific protein and tyrosine kinase inhibitor with a very low therapeutic index in animals.114 Thus, efforts to find staurosporine analogues have identified compounds specific for protein kinases. One staurosporine analogue, UCN-01 (7hydroxystaurosporine), has potent activity against several protein kinase C isoenzymes, particularly the Ca2+-dependent protein kinase C, with an IC50 of approximately 30 nmol/L.52,115,116 In addition to its effects on protein kinase C, UCN-01 has antiproliferative activity in several human tumor cell lines.51,53,54,117,118 In contrast, another highly selective potent protein kinase C inhibitor, GF-109203X, has minimal antiproliferative activity, despite a similar capacity to inhibit protein kinase C in vitro.117 These results suggest that the antiproliferative activity of UCN-01 cannot be explained solely by inhibition of protein kinase C. Although UCN-01 moderately inhibited the activity of immunoprecipitated CDK1 (cdc2) and CDK2 (IC50, 300-600 nmol/L), exposure of UCN-01 to intact cells leads to “inappropriate activation” of the same kinases.117 This phenomenon correlates with the G2 abrogation checkpoint observed with this agent. Experimental evidence suggests that DNA damage leads to cell cycle arrest to allow DNA repair. In cells in which the G1-phase checkpoint is not active because of p53 inactivation, irradiated cells accumulate in G2-phase as a result of activation of the G2 checkpoint (inhibition of cdc2). In contrast, Wang et al exposed CA46 cell lines to radiation followed by UCN-01, promoting the inappropriate activation of cdc2/cyclin B and early mitosis with the onset of apoptotic cell death.119 These effects could be partially explained by the inactivation of wee1, the kinase that negatively regulates the G2/M-phase transition.120 Moreover, UCN-01 can have a direct effect on chk1, a protein kinase that regulates the G2 checkpoint.121-123 Thus, although UCN-01 at high concentrations can directly inhibit CDKs in vitro, UCN-01 can modulate cellular upstream regulators at much lower concentrations, leading to inappropriate CDC2 activation. Studies from other groups suggest that not only is UCN-01 able to abrogate the G2 checkpoint induced by DNA-damaging agents, but also, in some circumstances, it is able to abrogate the DNA damage–induced S-phase checkpoint.124,125 Cell Arrests in G1 Phase. Another interesting property of UCN-01 is its ability to arrest cells in G1-phase of the cell cycle.51,54,97,118,126-130 When human epidermoid carcinoma A431 cells (mutated p53) or HN12 head and neck carcinoma cell lines are incubated with UCN-01, these cells were arrested in G1-phase with Rb hypophosphorylation and p21waf1/p27kip1

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Cyclin-Dependent Kinase Inhibitors in Lung Cancer accumulation.118,131 Chen et al suggested that Rb function, but not p53 function, is essential for UCN-01–mediated G1 arrest.129 However, Shimizu et al demonstrated that lung carcinoma cell lines with absent, mutant, or wild-type Rb exposed to UCN-01 displayed G1 arrest and antiproliferative effects irrespective of Rb function.128 Thus, the exact role of Rb or p53 in the G1 arrest induced by UCN-01 is still unknown. Further studies of the putative target(s) for UCN-01 in the G1-phase arrest of cells are warranted. Cytotoxicity in Tumor Cells with Mutated p53. Another interesting pharmacologic feature of UCN-01 is the observed increased cytotoxicity in cells that harbor mutated p53.119 In CA46 and HT-29 tumor cell lines carrying mutated p53 genes, potent cytotoxicity results following exposure to UCN-01. To further extend these observations, the MCF-7 cell line with no endogenous p53 because of the ectopic expression of E6, a human papilloma virus (HPV) type-16 protein, showed enhanced cytotoxicity when treated with a DNA-damaging agent, such as cisplatin, and UCN-01, compared with the isogenic wild-type MCF-7 cell line. Thus, a common feature observed in > 50% of human neoplasias that is associated with poor outcome and refractoriness to standard chemotherapies may render tumor cells more sensitive to UCN-01.132,133 Inhibition of PI3 Kinase/Akt Survival Pathway. A very exciting recent development is that of the effects of UCN-01 on the PI3 kinase/Akt survival pathway.134,135 UCN-01 displays a very potent inhibition in vitro of the phosphoinositide-dependent kinase 1 serine/threonine kinase, leading to dephosphorylation and inactivation of Akt.135 Although this is an exciting novel feature of UCN-01, it is of utmost importance to demonstrate whether the antitumor effects of UCN-01 are mediated by this action. Moreover, demonstration that these effects also occur in in vivo settings is crucial. Synergy with Chemotherapy. As previously mentioned, synergistic effects of UCN-01 have been observed with many chemotherapeutic agents, including mitomycin-C, 5-FU, carmustine, and camptothecin, among others.124,136-143 Therefore, it is possible that combining UCN-01 with these or other agents could improve the agents’ therapeutic indexes. Clinical trials exploring these possibilities are currently being developed. Animal Studies. UCN-01 administered by an intravenous or intraperitoneal route displayed antitumor activity in xenograft model systems with breast carcinoma (MCF-7 cells), renal carcinoma (A498 cells), and leukemia (MOLT-4 and HL-60 cells; A.S., unpublished data, 2003). The antitumor effect was greater when UCN-01 was given over a longer period of time. This requirement for a longer period of treatment was also observed in in vitro models, with the greatest antitumor activity observed when UCN-01 was present for 72 hours.51 Thus, a clinical trial using a 72-hour continuous infusion every 2 weeks was conducted.

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Clinical Trials of UCN-01. The first phase I trial of UCN-01 was recently completed.101,144 UCN-01 was initially administered as a 72-hour continuous infusion every 2 weeks based on data from in vitro and xenograft preclinical models. However, it became apparent in the first few patients that the drug had an unexpectedly long half-life (approximately 30 days). This half-life was 100 times longer than the half-life observed in preclinical models, most likely because of the avid binding of UCN-01 to α1-acid glycoprotein.145,146 Thus, the protocol was modified to administer UCN-01 every 4 weeks (1 halflife), and on subsequent courses, the duration of infusion was decreased by half (total of 36 hours). Thus, it was possible to reach similar peak plasma concentrations in subsequent courses with no evidence of drug accumulation. There was no evidence of myelotoxicity or gastrointestinal toxicity, which were prominent side effects observed in animal models, despite very high plasma concentrations achieved (35-50 μmol/L).101,144-146 Dose-limiting toxicities were nausea/vomiting (amenable to standard antiemetic treatment), symptomatic hyperglycemia associated with an insulin-resistant state (increase in insulin and c-peptide levels while receiving UCN-01), and pulmonary toxicity characterized by substantial hypoxemia without obvious radiologic changes. The recommended phase II dosage of UCN-01 on a 72-hour continuous infusion schedule was 42.5 mg/m 2/day. 144 One patient with refractory metastatic melanoma showed a partial response that lasted 8 months. Another patient with refractory anaplastic large-cell lymphoma that had failed multiple chemotherapeutic regimens including high-dose chemotherapy had no evidence of disease 4 years after the initiation of UCN-01 therapy. Moreover, several patients with leiomyosarcoma, NHL, or lung cancer demonstrated stable disease for ≥ 6 months.144,147 In order to estimate free UCN-01 concentrations in body fluids, several efforts were considered. Plasma ultracentrifugation and salivary determination of UCN-01 revealed similar results. At the recommended phase II dosage (37.5 mg/m2/day over 72 hours), concentrations of free salivary UCN-01 that may cause G2 checkpoint abrogation (approximately 100 nmol/L) can be achieved. As mentioned earlier, UCN-01 is a potent protein kinase C inhibitor. In order to determine the putative signaling effects of UCN-01 in tissues, bone marrow aspirates and tumor cells were obtained from patients before and during the first cycle of UCN-01 administration. Western blot studies were performed in those samples against phosphorylated adducin, a cytoskeletal membrane protein, a specific substrate phosphorylated by protein kinase C.148 Clear loss in phosphoadducin content in the posttreatment samples was observed in all tumor and bone marrow samples tested, and the conclusion was drawn that UCN-01 could modulate protein kinase C activity in tissues from patients in this trial.144,147 Several groups are conducting shorter-duration (3 hours) infusional trials of UCN-01. Interestingly, the toxicity profiles of shorter infusions are similar to the toxicities observed in the 72hour infusion trials.149,150 However, with shorter infusions, more pronounced hypotension was observed. Determination of free UCN-01 in these trials is of utmost importance because

Adrian M. Senderowicz higher free concentrations for shorter periods may be more or less beneficial than the free concentrations observed in the 72hour infusion trial. Based on the unique pharmacologic features and anecdotal clinical evidence of synergistic effects in 1 patient with refractory disease,151 several combination trials with standard chemotherapeutic agents recently commenced. A phase I/II trial of gemcitabine followed by 72-hour infusional UCN-01 in CLL was started at the NCI. Other studies of UCN-01 in combination with cisplatin and 5-FU, among other agents, also commenced recently. Studies of UCN-01 in Preclinical Lung Carcinoma Models. The use of UCN-01 in lung cancer models was investigated by several groups, particularly in combination studies with DNAdamaging agents.127,128,130,152-159 Shimizu et al studied the cell cycle effects of UCN-01 in a large panel of NSCLC cell lines.128 Clear loss in Rb phosphorylation was associated with G1/S arrest in the majority of cells exposed to UCN-01. In another study, Usuda et al investigated the expression of cell cycle proteins in sensitive and resistant SB3 NSCLC clones.130 They observed that SB3-resistant clones have significantly lower levels of baseline and induced p21waf1/cip1, a known endogenous CDK inhibitor. They concluded that the lack of p21waf1/cip1 may result from loss of IRF-1, a protein that may be associated with modulation of p21waf1/cip1.130 Another group using A549 cells demonstrated that A549-resistant clones were not cross-resistant to doxorubicin, paclitaxel, staurosporine, and UCN-02, but they displayed significant resistance to cisplatin and mitomycin-C.160 The expression of cyclin A, cyclin B1, Rb, and CDK2 proteins were downregulated in the resistant clones. Moreover, the antiapoptotic protein bcl-2 was apparently upregulated in A549/UCN cells; however, bcl-xL, another antiapoptotic protein, was downregulated and there were no changes in bak and bax.160 Thus, multiple cell cycle and apoptotic proteins may be associated with UCN01 resistance in lung cancer models. Mack et al studied several NSCLC cell lines with different genotypes (ie, Rb and p53 mutant, null, or wild type) using the combination of cisplatin and UCN-01.158 Clear synergistic effects (apoptosis and G2 checkpoint abrogation) were observed when NSCLC cells were treated with cisplatin followed by UCN-01, particularly in cells with mutant p53, a situation that occurs in the majority of cases of NSCLC.158 Similar synergism was observed in another study using UCN-01 and 5-fluorodeoxyuridine in A431 human epidermoid cancer cells.159 Finally, Xiao et al combined UCN-01 with γ-irradiation in several wild-type and mutant p53 lung carcinoma models.157 They observed, as expected, that cells with loss in p53 resulting from transfection in the HPV E6 protein were more resistant to radiation. However, the same radiation-resistant cells were more sensitive to the combination of UCN-01 and radiation.157 Taken together, trials using a combination of DNA-damaging agents followed by UCN-01 (particularly in p53 mutant lung cancer) are very reasonable to consider. Clinical trials in several tumor types including lung cancer are being planned using cisplatin or γ-irradiation in combination with UCN-01.

Conclusion Based on the frequent aberration in cell cycle regulatory pathways in human cancer by CDK hyperactivation, novel ATPcompetitive CDK inhibitors are being developed. The first 2 tested in clinical trials, flavopiridol and UCN-01, showed promising results with evidence of antitumor activity and plasma concentrations sufficient to inhibit CDK-related functions. The best administration schedule, combination with standard chemotherapeutic agents, tumor types to be targeted, and demonstration of CDK modulation from tumor samples from patients in these trials are important issues that need to be answered in order to advance these agents to the clinic.

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