A phase II study of cell cycle inhibitor UCN-01 in patients with metastatic melanoma: a California Cancer Consortium trial

Background Genetic abnormalities in cell cycle control are common in malignant melanoma. UCN-01 (7-hydroxystaurosporine) is an investigational agent that exhibits antitumor activity by perturbing the cancer cell cycle. A patient with advanced melanoma experienced a partial response in a phase I trial of single agent UCN-01. We sought to determine the activity of UCN-01 against refractory metastatic melanoma in a phase II study. Patients and methods Patients with advanced melanoma received UCN-01 at 90 mg/m2 over 3 h on cycle 1, reduced to 45 mg/m2 over 3 h for subsequent cycles, every 21 days. Primary endpoint was tumor response. Secondary endpoints included progression-free survival (PFS) and overall survival (OS). A two-stage (17 + 16), single arm phase II design was employed. A true response rate of ≥20% (i.e., at least one responder in the first stage, or at least four responders overall) was to be considered promising for further development of UCN-01 in this setting. Results Seventeen patients were accrued in the first stage. One patient was inevaluable for response. Four (24%) patients had stable disease, and 12 (71%) had disease progression. As there were no responders in the first stage, the study was closed to further accrual. Median PFS was 1.3 months (95% CI, 1.2–3.0) while median OS was 7.3 months (95% CI, 3.4–18.4). One-year and two year OS rates were 41% and 12%, respectively. A median of two cycles were delivered (range, 1–18). Grade 3 treatment-related toxicities include hyperglycemia (N = 2), fatigue (N = 1), and diarrhea (N = 1). One patient experienced grade 4 creatinine elevation and grade 4 anemia possibly due to UCN-01. No dose modification was required as these patients had disease progression. Conclusion Although well tolerated, UCN-01 as a single agent did not have sufficient clinical activity to warrant further study in refractory melanoma

Cyclic dermal BMP signalling regulates stem cell activation during hair regeneration

In the age of stem cell engineering it is critical to understand how stem cell activity is regulated during regeneration. Hairs are mini-organs that undergo cyclic regeneration throughout adult life1, and are an important model for organ regeneration. Hair stem cells located in the follicle bulge2 are regulated by the surrounding microenvironment, or niche3. The activation of such stem cells is cyclic, involving periodic -catenin activity4, 5, 6, 7. In the adult mouse, regeneration occurs in waves in a follicle population, implying coordination among adjacent follicles and the extrafollicular environment. Here we show that unexpected periodic expression of bone morphogenetic protein 2 (Bmp2) and Bmp4 in the dermis regulates this process. This BMP cycle is out of phase with the WNT/-catenin cycle, thus dividing the conventional telogen into new functional phases: one refractory and the other competent for hair regeneration, characterized by high and low BMP signalling, respectively. Overexpression of noggin, a BMP antagonist, in mouse skin resulted in a markedly shortened refractory phase and faster propagation of the regenerative wave. Transplantation of skin from this mutant onto a wild-type host showed that follicles in donor and host can affect their cycling behaviours mutually, with the outcome depending on the equilibrium of BMP activity in the dermis. Administration of BMP4 protein caused the competent region to become refractory. These results show that BMPs may be the long-sought 'chalone' inhibitors of hair growth postulated by classical experiments. Taken together, results presented in this study provide an example of hierarchical regulation of local organ stem cell homeostasis by the inter-organ macroenvironment. The expression of Bmp2 in subcutaneous adipocytes indicates physiological integration between these two thermo-regulatory organs. Our findings have practical importance for studies using mouse skin as a model for carcinogenesis, intra-cutaneous drug delivery and stem cell engineering studies, because they highlight the acute need to differentiate supportive versus inhibitory regions in the host skin

A phase I trial of the selective oral cyclin-dependent kinase inhibitor seliciclib (CYC202; R-Roscovitine), administered twice daily for 7 days every 21 days

Seliciclib (CYC202; R-roscovitine) is the first selective, orally bioavailable inhibitor of cyclin-dependent kinases 1, 2, 7 and 9 to enter clinical trial. Preclinical studies showed antitumour activity in a broad range of human tumour xenografts. A phase I trial was performed with a 7-day b.i.d. p.o. schedule. Twenty-one patients (median age 62 years, range: 39–73 years) were treated with doses of 100, 200 and 800 b.i.d. Dose-limiting toxicities were seen at 800 mg b.i.d.; grade 3 fatigue, grade 3 skin rash, grade 3 hyponatraemia and grade 4 hypokalaemia. Other toxicities included reversible raised creatinine (grade 2), reversible grade 3 abnormal liver function and grade 2 emesis. An 800 mg portion was investigated further in 12 patients, three of whom had MAG3 renograms. One patient with a rapid increase in creatinine on day 3 had a reversible fall in renal perfusion, with full recovery by day 14, and no changes suggestive of renal tubular damage. Further dose escalation was precluded by hypokalaemia. Seliciclib reached peak plasma concentrations between 1 and 4 h and elimination half-life was 2–5 h. Inhibition of retinoblastoma protein phosphorylation was not demonstrated in peripheral blood mononuclear cells. No objective tumour responses were noted, but disease stabilisation was recorded in eight patients; this lasted for a total of six courses (18 weeks) in a patient with ovarian cancer

Clinical anticancer drug development: targeting the cyclin-dependent kinases

Cell division involves a cyclical biochemical process composed of several step-wise reactions that have to occur once per cell cycle. Dysregulation of cell division is a hallmark of all cancers. Genetic and epigenetic mechanisms frequently result in deranged expression and/or activity of cell-cycle proteins including the cyclins, cyclin-dependent kinases (Cdks), Cdk inhibitors and checkpoint control proteins. The critical nature of these proteins in cell cycling raises hope that targeting them may result in selective cytotoxicity and valuable anticancer activity

Human Solid Tumor Xenografts in Immunodeficient Mice Are Vulnerable to Lymphomagenesis Associated with Epstein-Barr Virus

Xenografting primary human solid tumor tissue into immunodeficient mice is a widely used tool in studies of human cancer biology; however, care must be taken to prove that the tumors obtained recapitulate parent tissue. We xenografted primary human hepatocellular carcinoma (HCC) tumor fragments or bulk tumor cell suspensions into immunodeficient mice. We unexpectedly observed that 11 of 21 xenografts generated from 16 independent patient samples resembled lymphoid neoplasms rather than HCC. Immunohistochemistry and flow cytometry analyses revealed that the lymphoid neoplasms were comprised of cells expressing human CD45 and CD19/20, consistent with human B lymphocytes. In situ hybridization was strongly positive for Epstein-Barr virus (EBV) encoded RNA. Genomic analysis revealed unique monoclonal or oligoclonal immunoglobulin heavy chain gene rearrangements in each B-cell neoplasm. These data demonstrate that the lymphoid neoplasms were EBV-associated human B-cell lymphomas. Analogous to EBV-associated lymphoproliferative disorders in immunocompromised humans, the human lymphomas in these HCC xenografts likely developed from reactivation of latent EBV in intratumoral passenger B lymphocytes following their xenotransplantation into immunodeficient recipient mice. Given the high prevalence of latent EBV infection in humans and the universal presence of B lymphocytes in solid tumors, this potentially confounding process represents an important pitfall of human solid tumor xenografting. This phenomenon can be recognized and avoided by routine phenotyping of primary tumors and xenografts with human leukocyte markers, and provides a compelling biological rationale for exclusion of these cells from human solid tumor xenotransplantation assays

Inhibition of constitutive and cxc-chemokine-induced NF-κB activity potentiates ansamycin-based HSP90-inhibitor cytotoxicity in castrate-resistant prostate cancer cells

Background: We determined how CXC-chemokine signalling and necrosis factor-B (NF-B) activity affected heat-shock protein 90 (Hsp90) inhibitor (geldanamycin (GA) and 17-allylamino-demethoxygeldanamycin (17-AAG)) cytotoxicity in castrate-resistant prostate cancer (CRPC).Methods:Geldanamycin and 17-AAG toxicity, together with the CXCR2 antagonist AZ10397767 or NF-B inhibitor BAY11-7082, was assessed by 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay in two CRPC lines, DU145 and PC3. Flow cytometry quantified apoptotic or necrosis profiles. Necrosis factor-B activity was determined by luciferase readouts or indirectly by quantitative PCR and ELISA-based determination of CXCL8 expression.Results:Geldanamycin and 17-AAG reduced PC3 and DU145 cell viability, although PC3 cells were less sensitive. Addition of AZ10397767 increased GA (e.g., PC3 IC 20: from 1.670.4 to 0.180.2 nM) and 17-AAG (PC3 IC 20: 43.77.8 to 0.641.8 nM) potency in PC3 but not DU145 cells. Similarly, BAY11-7082 increased the potency of 17-AAG in PC3 but not in DU145 cells, correlating with the elevated constitutive NF-B activity in PC3 cells. AZ10397767 increased 17-AAG-induced apoptosis and necrosis and decreased NF-B activity/CXCL8 expression in 17-AAG-treated PC3 cells.Conclusion:Ansamycin cytotoxicity is enhanced by inhibiting NF-B activity and/or CXC-chemokine signalling in CRPC cells. Detecting and/or inhibiting NF-B activity may aid the selection and treatment response of CRPC patients to Hsp90 inhibitors.</p

Histone Deacetylase Inhibitors Downregulate Checkpoint Kinase 1 Expression to Induce Cell Death in Non-Small Cell Lung Cancer Cells

Background: Histone deacetylase inhibitors (HDACis) are promising anticancer drugs; however, the molecular mechanisms leading to HDACi-induced cell death have not been well understood and no clear mechanism of resistance has been elucidated to explain limited efficacy of HDACis in clinical trials. Methods and Findings: Here, we show that protein levels of checkpoint kinase 1 (Chk1), which has a major role in G2 cell cycle checkpoint regulation, was markedly reduced at the protein and transcriptional levels in lung cancer cells treated with pan-and selective HDACis LBH589, scriptaid, valproic acid, apicidin, and MS-275. In HDACi treated cells Chk1 function was impaired as determined by decreased inhibitory phosphorylation of cdc25c and its downstream target cdc2 and increased expression of cdc25A and phosphorylated histone H3, a marker of mitotic entry. In time course experiments, Chk1 downregulation occurred after HDACi treatment, preceding apoptosis. Ectopic expression of Chk1 overcame HDACiinduced cell death, and pretreating cells with the cdc2 inhibitor purvalanol A blocked entry into mitosis and prevented cell death by HDACis. Finally, pharmacological inhibition of Chk1 showed strong synergistic effect with LBH589 in lung cancer cells. Conclusions: These results define a pathway through which Chk1 inhibition can mediate HDACi-induced mitotic entry and cell death and suggest that Chk1 could be an early pharmacodynamic marker to assess HDACi efficacy in clinical samples

Effective Inhibition of Xenografts of Hepatocellular Carcinoma (HepG2) by Rapamycin and Bevacizumab in an Intrahepatic Model

10.1007/s11307-009-0213-4Molecular Imaging and Biology115334-342CPIM

Cellular Growth Kinetics Distinguish a Cyclophilin Inhibitor from an HSP90 Inhibitor as a Selective Inhibitor of Hepatitis C Virus

During antiviral drug discovery, it is critical to distinguish molecules that selectively interrupt viral replication from those that reduce virus replication by adversely affecting host cell viability. In this report we investigate the selectivity of inhibitors of the host chaperone proteins cyclophilin A (CypA) and heat-shock protein 90 (HSP90) which have each been reported to inhibit replication of hepatitis C virus (HCV). By comparing the toxicity of the HSP90 inhibitor, 17-(Allylamino)-17-demethoxygeldanamycin (17-AAG) to two known cytostatic compounds, colchicine and gemcitabine, we provide evidence that 17-AAG exerts its antiviral effects indirectly through slowing cell growth. In contrast, a cyclophilin inhibitor, cyclosporin A (CsA), exhibited selective antiviral activity without slowing cell proliferation. Furthermore, we observed that 17-AAG had little antiviral effect in a non-dividing cell-culture model of HCV replication, while CsA reduced HCV titer by more than two orders of magnitude in the same model. The assays we describe here are useful for discriminating selective antivirals from compounds that indirectly affect virus replication by reducing host cell viability or slowing cell growth

Aurora kinases are expressed in medullary thyroid carcinoma (MTC) and their inhibition suppresses in vitro growth and tumorigenicity of the MTC derived cell line TT

International audienceBACKGROUND: The Aurora kinase family members, Aurora-A, -B and -C, are involved in the regulation of mitosis, and alterations in their expression are associated with cell malignant transformation. To date no information on the expression of these proteins in medullary thyroid carcinoma (MTC) are available. We here investigated the expression of the Aurora kinases in human MTC tissues and their potential use as therapeutic targets. METHODS: The expression of the Aurora kinases in 26 MTC tissues at different TNM stages was analyzed at the mRNA level by quantitative RT-PCR. We then evaluated the effects of the Aurora kinase inhibitor MK-0457 on the MTC derived TT cell line proliferation, apoptosis, soft agar colony formation, cell cycle and ploidy. RESULTS: The results showed the absence of correlation between tumor tissue levels of any Aurora kinase and tumor stage indicating the lack of prognostic value for these proteins. Treatment with MK-0457 inhibited TT cell proliferation in a time- and dose-dependent manner with IC50 = 49.8 ± 6.6 nM, as well as Aurora kinases phosphorylation of substrates relevant to the mitotic progression. Time-lapse experiments demonstrated that MK-0457-treated cells entered mitosis but were unable to complete it. Cytofluorimetric analysis confirmed that MK-0457 induced accumulation of cells with ≥ 4N DNA content without inducing apoptosis. Finally, MK-0457 prevented the capability of the TT cells to form colonies in soft agar. CONCLUSIONS: We demonstrate that Aurora kinases inhibition hampered growth and tumorigenicity of TT cells, suggesting its potential therapeutic value for MTC treatment