A phase I/II study of KW-2478, an Hsp90 inhibitor, in combination with bortezomib in patients with relapsed/refractory multiple myeloma.

BACKGROUND: KW-2478 is a novel non-ansamycin Hsp90 inhibitor with modest single-agent activity in relapsed/refractory myeloma but which shows synergistic antimyeloma activity with bortezomib (BTZ) in preclinical studies. This study determined the safety, preliminary clinical activity, and pharmacokinetics of KW-2478, an Hsp90 inhibitor, in combination with BTZ in patients with relapsed/refractory multiple myeloma (MM). METHODS: Phase I dose escalation determined the recommended phase II dose (RP2D) of KW-2478 plus BTZ, which was then used during phase II. RESULTS: The maximum tolerated dose was not reached during phase I and the RP2D was KW-2478 175 mg m-2 plus BTZ 1.3 mg m-2 on days 1, 4, 8, and 11 every 3 weeks. In the efficacy evaluable phase I/II population treated at the RP2D (n=79), the objective response rate was 39.2% (95% confidence interval: 28.4-50.9%), clinical benefit rate 51.9% (40.4-63.3%), median progression-free survival 6.7 (5.9-not reached (NR)) months, and median duration of response 5.5 (4.9-NR) months. In the phase I/II safety population (n=95), the most frequently observed treatment-related grade 3/4 adverse events were diarrhoea, fatigue, and neutropenia (each in 7.4% of patients), and nausea and thrombocytopenia (each in 5.3%). CONCLUSIONS: KW-2478 plus BTZ was well tolerated with no apparent overlapping toxicity in patients with relapsed/refractory MM. The antimyeloma activity of KW-2478 in combination with BTZ as scheduled in this trial appeared relatively modest; however, the good tolerability of the combination would support further exploration of alternate dosing schedules and combinations.

Phase I study of KW-2478, a novel Hsp90 inhibitor, in patients with B-cell malignancies.

BACKGROUND: KW-2478 is a novel, non-ansamycin, non-purine heat-shock protein 90 (Hsp90) inhibitor. METHODS: In this phase I, multicentre study, KW-2478 was administered intravenously over 1 h at doses ranging from 14 to 176 mg m(-2) once daily on days 1-5 of a 14-day cycle in a standard 3+3 design in 27 patients (22 with multiple myeloma and 5 with non-Hodgkin lymphoma). Patients enrolled had relapsed/refractory disease previously treated with ⩾2 regimens. RESULTS: There were no dose-limiting toxicities, thus the maximum-tolerated dose was not reached. KW-2478 was well tolerated and did not manifest significant retinal or ocular toxicity. The most common treatment-related adverse events were diarrhoea (33.3%), fatigue (29.6%), headache (25.9%), hypertension (22.2%), nausea (14.8%), vomiting (7.4%), and dizziness (7.4%). Plasma concentrations peaked at the end of infusion and decayed in a biphasic manner with a terminal half-life of ∼6 h. Target inhibition was inferred from the increase in Hsp70 levels in peripheral blood mononuclear cells at doses ⩾71 mg m(-2). Twenty-four of 25 (96%) evaluable patients showed stable disease, with five being free of disease progression for ⩾6 months. CONCLUSIONS: Preliminary clinical response data were encouraging and warrant further investigation of KW-2478 in combination regimens for relapsed/refractory B-cell malignancies.

A randomized, double-blind, placebo-controlled, phase III trial of erlotinib with or without a c-Met inhibitor tivantinib (ARQ 197) in Asian patients with previously treated stage IIIB/IV nonsquamous nonsmall-cell lung cancer harboring wild-type epidermal growth factor receptor (ATTENTION study).

BACKGROUND: A previous randomized phase II study demonstrated that the addition of a c-Met inhibitor tivantinib to an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor erlotinib might prolong progression-free survival (PFS) in patients with previously treated, nonsquamous nonsmall-cell lung cancer (NSCLC). On a subset analysis, the survival benefit was greater in patients with wild-type EGFR (WT-EGFR) than in those with activating EGFR mutations. Herein, this phase III study compared overall survival (OS) between Asian nonsquamous NSCLC patients with WT-EGFR who received erlotinib plus tivantinib (tivantinib group) or erlotinib plus placebo (placebo group). METHODS: A total of 460 NSCLC patients were planned to be randomized to the tivantinib or placebo group. Primary end point was OS. Secondary end points were PFS, tumor response, and safety. Tissue was collected for biomarker analysis, including c-Met and HGF expression. RESULTS: Enrollment was stopped when 307 patients were randomized, following the Safety Review Committee's recommendation based on an imbalance in the interstitial lung disease (ILD) incidence between the groups. ILD developed in 14 patients (3 deaths) and 6 patients (0 deaths) in the tivantinib and the placebo groups, respectively. In the enrolled patients, median OS was 12.7 and 11.1 months in the tivantinib and the placebo groups, respectively [hazard ratio (HR) = 0.891, P = 0.427]. Median PFS was 2.9 and 2.0 months in the tivantinib and the placebo groups, respectively (HR = 0.719, P = 0.019). The commonly observed grade ≥ 3 adverse events in the tivantinib group were neutropenia (24.3%), leukopenia (18.4%), febrile neutropenia (13.8%), and anemia (13.2%). CONCLUSIONS: This study was prematurely terminated due to the increased ILD incidence in the tivantinib group. Although this study lacked statistical power because of the premature termination and did not demonstrate an improvement in OS, our results suggest that tivantinib plus erlotinib might improve PFS than erlotinib alone in nonsquamous NSCLC patients with WT-EGFR. TRIAL REGISTRATION NUMBER: NCT01377376.

CYP2C19 genotype-based phase I studies of a c-Met inhibitor tivantinib in combination with erlotinib, in advanced/metastatic non-small cell lung cancer.

BACKGROUND: A previous clinical study in non-small cell lung cancer (NSCLC) patients in Western countries suggested the potential for combination of a first-in-class non-ATP-competitive c-Met inhibitor tivantinib with an epidermal growth factor receptor-tyrosine kinase inhibitor erlotinib. Polymorphisms of CYP2C19, the key metabolic enzyme for tivantinib, should be addressed to translate the previous Western study to Asian population, because higher incidence of poor metabolisers (PMs) is reported in Asian population. METHODS: Japanese patients with advanced/metastatic NSCLC received tivantinib in combination with erlotinib to evaluate safety and pharmacokinetics. Doses of tivantinib were escalated separately for extensive metabolisers (EMs) and PMs. RESULTS: Tivantinib, when combined with erlotinib, was well tolerated up to 360 mg BID for EMs and 240 mg BID for PMs, respectively. Among 25 patients (16 EMs and 9 PMs), the adverse events (AEs) related to tivantinib and/or erlotinib (>20%, any grade) were rash, diarrhoea, dry skin and nausea. Grade ≥3 AEs were leukopenia, anaemia and neutropenia. No dose-limiting toxicity was observed. Pharmacokinetics profile of tivantinib was not clearly different between the combination and monotherapy. Three partial response and three long-term stable disease (≥24 weeks) were reported. CONCLUSION: Two doses of tivantinib in combination with erlotinib were recommended based on CYP2C19 genotype: 360 mg BID for EMs and 240 mg BID for PMs.

The effect of CYP2C19 polymorphism on the safety, tolerability, and pharmacokinetics of tivantinib (ARQ 197): results from a phase I trial in advanced solid tumors.

BACKGROUND: Tivantinib (formerly ARQ 197) is a selective inhibitor of c-Met mainly metabolized by CYP2C19. CYP2C19 is known for genetic polymorphisms, and ~20% of Asians are poor metabolizers (PMs), while others are extensive metabolizers (EMs). In this study, we examined the safety, pharmacokinetics (PK), and preliminary efficacy of tivantinib as a single agent to determine recommended phase II doses (RPIIDs). PATIENTS AND METHODS: Forty-seven patients (EMs, 33; PMs, 14) with solid tumors were orally treated with tivantinib, from 70 to 360 mg bid in a 3 + 3 dose-escalation scheme. EMs and PMs were separately enrolled at the doses >120 mg bid. RESULTS: Tivantinib was well tolerated up to 360 mg bid for EMs and 240 mg bid for PMs. Neutropenia, leukopenia, anemia, fatigue, and anorexia were the frequent adverse events related to tivantinib and were commonly observed in both EMs and PMs. PMs had 1.9-fold higher AUC(0-12) compared with EMs at 240 mg bid. Regardless of CYP2C19 phenotype, Gr.4 neutropenia occurred in patients with relatively high exposure to tivantinib. A confirmed partial response was achieved in two non-small-cell lung cancer (NSCLC) patients. CONCLUSION: Two different settings of RPIIDs, 360 mg bid for EMs and 240 mg bid for PMs, were determined.

Anti-tumor activity against multiple myeloma by combination of KW-2478, an Hsp90 inhibitor, with bortezomib.

Heat shock protein 90 (Hsp90) is a promising target for anti-tumor therapy. We previously reported the anti-tumor activity of a novel Hsp90 inhibitor, KW-2478, in multiple myeloma (MM) as a single agent. In this study, we examined the combinational effect of KW-2478 and bortezomib, a proteasome inhibitor, in vitro and in vivo. In vitro, KW-2478 enhanced bortezomib-induced cell growth inhibition, both in MM cell lines and primary patient MM cells. The combination of KW-2478 and bortezomib also induced caspase activation in MM cell lines. Interestingly, the combination synergistically enhanced the expression of Hsp70B, a homolog of Hsp70, in human MM cells and peripheral blood mononuclear cells, indicating Hsp70B could be a surrogate biomarker for the combination of Hsp90 and proteasome inhibitors. In vivo, the combination of KW-2478 with bortezomib showed synergistic anti-tumor activity without significant body weight loss in a subcutaneously inoculated human myeloma model. Furthermore, the combination also showed synergistic reduction of tumor burden in bone marrow in an orthotopic myeloma model. Our results strongly suggest that combination of KW-2478 with bortezomib could exhibit enhanced anti-tumor activity against human myeloma.

A radicicol derivative, KF58333, inhibits expression of hypoxia-inducible factor-1alpha and vascular endothelial growth factor, angiogenesis and growth of human breast cancer xenografts.

A novel oxime derivative of radicicol, KF58333, binds to the heat shock protein 90 (Hsp90) and destabilizes its associated signaling molecules. These effects play a critical role in the growth inhibition of tumor cells. To further investigate the effects of this agent, it was administered to two human breast cancer cell lines, KPL-1 and KPL-4, both in vitro and in vivo. KF58333 dose-dependently inhibited the growth and vascular endothelial growth factor (VEGF) secretion, concomitantly with a decrease in VEGF mRNA expression, in each cell line. This agent also suppressed the increase of VEGF secretion and expression induced by hypoxia (1% O(2)). Intravenous injections of this agent into nude mice bearing either KPL-1 or KPL-4 xenografts significantly inhibited the tumor growth associated with a decrease in the Ki67 labeling index and microvascular area and an increase in apoptosis and the necrotic area. These findings indicate that the antitumor activity of this radicicol derivative may be partly mediated by decreasing VEGF secretion from tumor cells and inhibiting tumor angiogenesis. To explore the action mechanisms of the anti-angiogenic effect, the expression level of hypoxia-inducible factor (HIF)-1alpha was investigated. KF58333 provided a significant decrease in the HIF-1alpha protein expression under both normoxic and hypoxic conditions. In contrast, the mRNA expression of HIF-1alpha was not decreased by this agent. It is suggested that the post-transcriptional down-regulation of HIF-1alpha expression by this agent may result in a decrease of VEGF expression and tumor angiogenesis.

Destabilization of steroid receptors by heat shock protein 90-binding drugs: a ligand-independent approach to hormonal therapy of breast cancer.

Steroid hormone receptors have become an important target in the management of breast cancers. Despite a good initial response rate, however, most tumors become refractory to current hormonal therapies within a year of starting treatment. To address this problem, we evaluated the effects of agents that bind the molecular chaperone heat shock protein 90 (Hsp90) on estrogen receptor function in breast cancer. Unstimulated estrogen and progesterone receptors exist as multimolecular complexes consisting of the hormone-binding protein itself and several essential molecular chaperones including Hsp90. We found that interaction of the Hsp90-binding drugs geldanamycin and radicicol with the chaperone destabilizes these hormone receptors in a ligand-independent manner, leading to profound and prolonged depletion of their levels in breast cancer cells cultured in vitro. Consistent with these findings, in vivo administration of the geldanamycin derivative 17-allylaminogeldanamycin (17AAG; NSC330507) to estrogen-supplemented, tumor-bearing SCID mice resulted in marked depletion of progesterone receptor levels in both uterus and tumor. Drug administration also delayed the growth of established, hormone-responsive MCF-7 and T47D human tumor xenografts for up to 3 weeks after the initiation of therapy. We conclude that in light of their novel mechanism of anti-hormone action, consideration should be given to examining the activity of 17AAG and other Hsp90-binding agents in patients with refractory breast cancer in future clinical trials, either alone or in combination with conventional hormone antagonists.

UCN-01 (7-hydoxystaurosporine) inhibits in vivo growth of human cancer cells through selective perturbation of G1 phase checkpoint machinery.

Mechanisms underlying tumor sensitivity to the antitumor agent UCN-01 (7-hydroxystaurosporine) were examined in the nude mouse model using three human tumor xenografts, two pancreatic cancers (PAN-3-JCK and CRL 1420) and a breast cancer (MX-1). UCN-01 antitumor activity was evaluated in terms of relative tumor weights in treated and untreated mice bearing the tumor xenografts. The activity of cyclin-dependent kinase 2 (CDK2), levels of p21 and p27 proteins, pRb status and cell cycle were evaluated. Induction of p21 and apoptosis were also assessed immunohistochemically in CRL 1420. UCN-01 was administered intraperitoneally at a dose of either 5 or 10 mg / kg daily for 5 days followed by a further 5 injections after an interval of 2 days. UCN-01 significantly suppressed the growth of both pancreatic cancers, but was ineffective against MX-1. p21 protein expression was markedly induced in the UCN-01-sensitive pancreatic carcinoma xenografts at both doses, but p21 induction was only evident in the UCN-01-resistant MX-1 at 10 mg / kg. MX-1 exhibited CDK2 activity that was 6-fold higher than that of pancreatic cancer strains, which may explain the resistance of MX-1 to UCN-01 despite the induction of p21 at the dose of 10 mg / kg. The UCN-01-sensitive tumors exhibited G1 arrest and increased levels of apoptosis, changes not observed in resistant MX-1. In conclusion, it appears that a determining factor of in vivo UCN-01 sensitivity involves the balance of CDK2 kinase activity and p21 protein induction, resulting in augmented pRb phosphorylation, G1 cell cycle arrest and apoptosis.

Stereospecific antitumor activity of radicicol oxime derivatives.

PURPOSE: Radicicol is a novel hsp90 antagonist, distinct from the chemically unrelated benzoquinone ansamycin compounds, geldanamycin and herbimycin. Both geldanamycin and radicicol bind in the aminoterminal nucleotide-binding pocket of hsp90, destabilizing the hsp90 client proteins, many of which are essential for tumor cell growth. We describe here antitumor activity of a novel oxime derivative of radicicol, KF58333. We also investigated the mechanism of antitumor activity of KF58333 in comparison with its oxime isomer KF58332. METHODS: Antiproliferative activities were determined in a panel of breast cancer cell lines in vitro. We also examined inhibition of hsp90 function and apoptosis induction in erbB2-overexpressing human breast carcinoma KPL-4 cells in vitro. Direct binding activity to hsp90 was assessed by hsp90-binding assays using geldanamycin or radicicol beads. In animal studies, we investigated plasma concentrations of these compounds after i.v. injection in BALB/c mice and antitumor activity against KPL-4 cells transplanted into nude mice. Inhibition of hsp90 function and induction of apoptosis in vivo were investigated using tumor specimens from drug-treated animals. RESULTS: KF58333 showed potent antiproliferative activity against all breast cancer cell lines tested in vitro, and was more potent than its stereoisomer KF58332. These results are consistent with the ability of KF58333 to deplete hsp90 client proteins and the induction of apoptosis in KPL-4 cells in vitro. Interestingly, KF58333, but not KF58332, showed significant in vivo antitumor activity accompanied by induction of apoptosis in KPL-4 human breast cancer xenografts. Although the plasma concentrations of these compounds were equivalent, KF58333, but not KF58332, depleted hsp90 client proteins such as erbB2, raf-1 and Akt in the tumor specimen recovered from nude mice. CONCLUSIONS: These results suggest that inhibition of hsp90 function, which causes depletion of hsp90 client proteins in tumor, contributes to the antitumor activity of KF58333, and that the stereochemistry of the oxime moiety is important for the biological activity of radicicol oxime derivatives.

Novel oxime derivatives of radicicol induce erythroid differentiation associated with preferential G(1) phase accumulation against chronic myelogenous leukemia cells through destabilization of Bcr-Abl with Hsp90 complex.

Chronic myelogenous leukemia (CML) is a clonal disorder of a pluripotent hematopoietic stem cells characterized by a chimeric bcr-abl gene giving rise to a p210(Bcr-Abl) protein with dysregulated tyrosine kinase activity. Radicicol, a macrocyclic antifungal antibiotic, binds to the N-terminal of heat shock protein 90 (Hsp90) and destabilizes Hsp90-associated proteins such as Raf-1. This study investigated the effect of radicicol, novel oxime derivatives of radicicol (KF25706 and KF58333), and herbimycin A (HA), a benzoquinoid ansamycin antibiotic, on the growth and differentiation of human K562 CML cells. Although KF25706 and KF58333 induced the expression of glycophorin A in K562 cells, radicicol and HA caused erythroid differentiation transiently. Cell cycle analysis showed that G(1) phase accumulation was observed in K562 cells treated with KF58333. KF58333 treatment depleted p210(Bcr-Abl), Raf-1, and cellular tyrosine phosphorylated proteins in K562 cells, whereas radicicol and HA showed transient depletion of these proteins. KF58333 also down-regulated the level of cell cycle-dependent kinases 4 and 6 and up-regulated cell cycle-dependent kinase inhibitor p27(Kip1) protein without an effect on the level of Erk and Hsp90 proteins. Immunoprecipitation analysis showed that p210(Bcr-Abl) formed multiple complexes with Hsp90, some containing p23 and others Hsp70; KF58333 treatment dissociated p210(Bcr-Abl) from Hsp90/p23 chaperone complexes. Furthermore, KF58333 induced apoptosis in K562 cells and administration of KF58333 prolonged the survival time of SCID mice inoculated with K562 cells. These results suggest that KF58333 may have therapeutic potential for the treatment of CML that involves abnormal cellular proliferation induced by p210(Bcr-Abl).

Retinoblastoma protein-initiated cellular growth arrest overcomes the ability of cotransfected wild-type p53 to induce apoptosis.

The retinoblastoma gene, RB, participates in the regulation of the G1/S-phase transition and in p53-mediated apoptosis. We have previously reported that stably transfected RB functions as a growth and tumour suppressor in HTB9 human bladder carcinoma cells, which carry a mutation of the p53 gene at codon 280 and lack RB expression. To elucidate the potential role of RB in the regulation of p53-mediated apoptosis, we transfected a wt p53 expression plasmid under the control of the human cytomegalovirus promoter into parental and RB-transfected HTB9 cells. The p53(+)/RB(-)cells were susceptible to apoptosis under various experimental conditions: 1) incubation in serum-free culture for 72 h, 2) short-term (6 h) or long-term (48 h) exposure to etoposide, and 3) culturing in soft agar. In contrast, p53(+)/RB(+)cells were significantly resistant to apoptosis under similar conditions and exhibited efficient growth arrest, as measured by laser scanning cytometry. Tumorigenicity in nude mice of parental HTB9 cells was lost by exogenous expression of wt p53. Likewise, none of mice injected subcutaneously with either p53(-)/RB(+)or p53(+)/RB(+)cells developed tumours, indicating that RB allows suppression of tumorigenesis, regardless of p53 status. These results suggest that the growth-inhibitory function of RB may overcome the ability of wt p53 to induce apoptosis.

Induction of a heat shock factor 1-dependent stress response alters the cytotoxic activity of hsp90-binding agents.

In addition to its classic role in the cellular stress response, heat shock protein 90 (Hsp90) plays a critical but less well appreciated role in regulating signal transduction pathways that control cell growth and survival under basal, nonstress conditions. Over the past 5 years, the antitumor antibiotics geldanamycin and radicicol have become recognized as selective Hsp90-binding agents (HBA) with a novel ability to alter the activity of many of the receptors, kinases, and transcription factors involved in these cancer-associated pathways. As a consequence of their interaction with Hsp90, however, these agents also induce a marked cellular heat shock response. To study the mechanism of this response and assess its relevance to the anticancer action of the HBA, we verified that the compounds could activate a reporter construct containing consensus binding sites for heat shock factor 1 (HSF1), the major transcriptional regulator of the vertebrate heat shock response. We then used transformed fibroblasts derived from HSF1 knock-out mice to show that unlike conventional chemotherapeutics, HBA increased the synthesis and cellular levels of heat shock proteins in an HSF1-dependent manner. Compared with transformed fibroblasts derived from wild-type mice, HSF1 knock-out cells were significantly more sensitive to the cytotoxic effects of HBA but not to doxorubicin or cisplatin. Consistent with these in vitro data, we found that systemic administration of an HBA led to marked increases in the level of Hsp72 in both normal mouse tissues and human tumor xenografts. We conclude that HBA are useful probes for studying molecular mechanisms regulating the heat shock response both in cells and in whole animals. Moreover, induction of the heat shock response by HBA will be an important consideration in the clinical application of these drugs, both in terms of modulating their cytotoxic activity as well as monitoring their biological activity in individual patients.

Therapeutic potential of chimeric anti-(ganglioside GD3) antibody KM871: antitumor activity in xenograft model of melanoma and effector function analysis.

KM871 is a chimeric antibody recognizing ganglioside GD3, which is one of the major gangliosides expressed on the cell surface of human tumors of neuroectodermal origin. This study demonstrates the antitumor activity of KM871 against human melanoma xenografts in nude mice, and analyzes the effector function operating in mice. In a well-established tumor model, KM871 showed antitumor activity against H-15 and SK-MEL-28 human melanoma but not against H-187 and G361 human melanoma when administered intravenously 5 days/week for 2 weeks. The G361 tumor became sensitive when KM871 was first administered on the day of tumor inoculation. In this assay, it was observed that almost all the mice were tumor-free, but a few mice developed tumors. Therefore, we examined the amount and expression pattern of GD3 antigen on G361 tumors escaping from KM871 treatment, but no change was observed. Next we examined the optimal administration schedule for KM871 in mice, using H-15 melanoma. KM871 showed antitumor activity when administered intravenously either 5 days/week for 2 weeks or three biweekly doses. However, the effect of the former schedule was stronger than three biweekly doses. To compare the effector function in humans and mice, we studied the complement-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity and antibody-dependent macrophage-mediated cytotoxicity of KM871 using complement or effector cells prepared from humans and mice. It was found that the antibody-dependent cell-mediated cytotoxicity exerted by polymorphonuclear cells and antibody-dependent macrophage-mediated cytotoxicity were the only antitumor mechanism of KM871 in mice. However their action was very weak compared with that in humans, and complement-mediated cytotoxicity, which was strong in humans, was not observed in mice. Therefore, the antitumor activity of KM871 against human melanomas evaluated by the nude mouse model might be underestimated. These results indicate that KM871 shows good antitumor activity against GD3-positive human melanoma and the antitumor activity expected in humans might be superior to that of the nude mouse model.

UCN-01 (7-hydroxystaurosporine) and other indolocarbazole compounds: a new generation of anti-cancer agents for the new century?

UCN-01 (7-hydroxystaurosporine) is a protein kinase inhibitor which is under development as an anti-cancer agent in the USA and Japan. Although UCN-01 was originally isolated from the culture broth of Streptomyces sp. as a protein kinase C-selective inhibitor, its ultimate target as an anti-cancer agent remains elusive. As a single agent, UCN-01 exhibits two key biochemical effects, namely accumulation of cells in the G1 phase of the cell cycle and induction of apoptosis. Both these effects may be important for its anti-cancer activity. As a modulator, UCN-01 enhances the cytotoxicity of other anti-cancer drugs such as DNA-damaging agents and anti-metabolite drugs through putative abrogation of G2 and/or S phase accumulation induced by these anti-cancer agents. Currently, in addition to UCN-01, four other indolocarbazole anti-cancer drugs-two protein kinase inhibitors, CGP 41251, CEP-751, and two DNA-damaging agents, NB-506 and a Rebeccamycin analog-are undergoing clinical investigations in the USA, Europe or Japan. In this review, we would like to address the differences and similarities of these indolocarbazole compounds as anti-cancer agents with regard to their mechanism(s) of action, the effects on cell cycle progression, induction of apoptosis and modulation of drug sensitivity.

Chemoprotective effects of KF41399, a derivative of carbazole compounds, on nimustine-induced thrombocytopenia.

We examined the chemoprotective effects of KF41399, a novel derivative of carbazole compounds, on severe thrombocytopenia induced by nimustine (ACNU, 45 mg/kg administered for 2 consecutive days intravenously) in mice. Administration schedule studies revealed that pretreatment of mice with KF41399 was necessary to improve thrombocytopenia. Oral administration of KF41399 ameliorated thrombocytopenia induced by ACNU and accelerated the rate of platelet recovery in a dose-dependent fashion. In addition, KF41399 pretreatment improved the decrease in body weight and spleen weight and in the colony-forming activity of bone marrow mononuclear cells (MNC). Oral administration of KF41399 to normal mice induced G(0)/G(1)-phase accumulation of MNC as well as hematopoietic progenitor cells (lineage negative cells [Lin(-)]) and reduced the colony-forming activity of MNC. In Lin(-) cells derived from KF41399-treated mice, up-regulation of Bcl-2 and down-regulation of cyclin E and cyclin A proteins were observed. In the same cells, a decrease in the phosphorylated form of Rb protein and an increase in the p130 protein were observed without changes in the protein level of cell cycle-dependent kinase 2 (Cdk2), Cdk4, and Cdk6. More important, KF41399 did not affect the antitumor activity of ACNU against mouse Sarcoma180 and human lung cancer LC-6. However, 25-mg/kg KF41399 treatment reduced the antitumor activity of ACNU against human lung cancer Lu-65, and 5 mg/kg KF41399 caused a slight reduction of the antitumor activity of ACNU without inducing thrombocytopenia. These results suggest that KF41399 might be useful as a chemoprotective agent to improve chemotherapy-induced thrombocytopenia and types of other toxicity. (Blood. 2000;95:3771-3780)

UCN-01 selectively enhances mitomycin C cytotoxicity in p53 defective cells which is mediated through S and/or G(2) checkpoint abrogation.

We have previously reported that UCN-01 (7-hydroxystaurosporine), a protein kinase inhibitor that is under clinical trials as an anti-cancer agent in the USA and Japan, enhanced the anti-tumor activity of mitomycin C (MMC) in vitro and in vivo. Subsequent studies from other laboratories revealed that UCN-01 could selectively enhance cytotoxicity of DNA damaging agents in p53 defective cells and that this was mediated by abrogation of S and /or G(2) arrest by UCN-01. In this study, we report that UCN-01 selectively enhances the cytotoxicity of MMC in human p53 mutant cell lines. In contrast, UCN-01 showed little, if any, effect on MMC cytotoxicity in human p53 wild-type cell lines. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-nick end-labeling (TUNEL) assay revealed that the combination of MMC with UCN-01 increased DNA breaks consistent with apoptosis in p53 defective A431 epidermoid carcinoma cells. In p53 wild-type MCF-7 breast carcinoma cells, the cyclin-dependent kinase inhibitor protein p21/WAF1 was markedly induced after the treatment with MMC alone, although this response was significantly delayed from the time of MMC treatment. Detailed cell-cycle studies revealed that UCN-01 abrogated S and G(2) phase accumulation induced by MMC in p53 defective cells and to a lesser extent in p53 wild-type cell lines. The abrogation of arrest in p53 wild-type cells was observed prior to significant induction of the p53 response. Since MMC was less effective against p53 defective cell lines than against p53 wild-type cell lines and UCN-01 selectively enhanced MMC cytotoxicity in p53 defective cell lines, UCN-01 may provide a new modality of MMC-based cancer chemotherapy, particularly in p53 defective cancer patients.

Decrease in susceptibility toward induction of apoptosis and alteration in G1 checkpoint function as determinants of resistance of human lung cancer cells against the antisignaling drug UCN-01 (7-Hydroxystaurosporine).

7-Hydroxystaurosporine (UCN-01) is a protein kinase inhibitor that is under development as an anticancer agent in the United States and Japan. Long-term exposure of human A549 non-small cell lung cancer cells to UCN-01 furnished cells (A549/UCN) with acquired resistance against UCN-01. In this study, the sensitivity of these cells toward the growth-arresting properties of certain conventional cytotoxic agents was explored. Cells were not cross-resistant against adriamycin, Taxol, staurosporine, and UCN-02, but they displayed 14- and 4.4-fold resistance against cisplatin and mitomycin C, respectively. Previous studies on the mechanism(s) of action of UCN-01 suggest that induction of apoptosis and G1 phase accumulation are important for its anticancer activity; therefore, we compared induction of apoptosis and cell cycle distribution caused by UCN-01 in wild-type A549 and A549/UCN cells using flow cytometry. UCN-01 (0.4 microM) induced apoptosis (62% terminal deoxynucleotidyl transferase-mediated nick end labeling-positive cells) in A549 cells, but not in A549/UCN cells. The percentages of cells that accumulated in G1 when exposed to UCN-01 (0.4 microM) were 22% in A549 cells and 67% in A549/UCN cells. These results suggest that acquired resistance of cancer cells against UCN-01 is characterized by attenuation of apoptosis induction associated with reinforcement of the G1 checkpoint and that apoptosis regulation is drastically altered in A549/UCN cells as compared with A549 cells. Cyclin-dependent kinase (CDK) inhibitor proteins p21 and p27 in A549/UCN cells were up-regulated, which was accompanied by overexpression of G1 cyclins D1 and E, but UCN-01 hardly affected levels of these proteins. In contrast, cyclin A, cyclin B1, retinoblastoma, and CDK2 proteins were apparently down-regulated, without changes in CDK4/6. UCN-01 hardly affected the expression level of cyclin B1 and induced dephosphorylation of retinoblastoma in both cell types. UCN-01 induced down-regulation of cyclin A level and CDK2 activity accompanied with its dephosphorylation in A549/UCN cells, but not in A549 cells. The antiapoptotic protein bcl-2 was apparently up-regulated in A549/UCN cells, however, bcl-xL, another antiapoptotic protein, was down-regulated, without changes in bak and bax. Taken together, these results are consistent with the notion that induction of apoptosis and block of cell cycle in G1 are important determinants of the sensitivity of cancer cells to UCN-01 and suggest that inhibition of CDK2 activity accompanied by its dephosphorylation and decrease of expression level of cyclin A might play an important role in the G1 phase accumulation induced by UCN-01.