Purpose: The silencing of gene expression through DNA methylation contributes to defects in antigen presentation and apoptosis in melanoma and renal cell cancer. To determine how a hypomethylating agent would modulate the toxicity and antitumor activity of immunotherapy, we initiated a phase I trial of 5-aza-2 ¶-deoxycytidine (decitabine) plus high-dose interleukin 2 (IL-2). Experimental Design: Patients received s.c. decitabine daily  5 days on weeks 1 and 2 of a 12-week cycle. High-dose IL-2, consisting of two cycles of IL-2 600,000 IU/kg i.v. q8 hours  14 doses separated by a 2-week break, was administered starting on week 3. Decitabine was escalated from 0.1 to 0.25 mg/kg. The hypomethylating activity of decitabine was assessed during cycle 1 by measuring hemoglobin F levels and changes in DNA methylation in peripheral blood mononuclear cells. Results: Twenty-one patients with melanoma or renal cell cancer were enrolled. Decitabine did not alter the tolerability of IL-2 but caused grade 4 neutropenia in most patients. Grade 4 neutropenia lasting more than 7 days was the only dose-limiting toxicity, with a trend toward a higher incidence with increasing decitabine doses. Infection occurred in only one patient despite the high incidence of neutropenia, and granulocyte colony-stimulating factor use in several patients expedited neutrophil recovery. Decitabine augmented hemoglobin F levels and altered DNA methylation and gene expression in peripheral blood mononuclear cells in a doseindependent manner that overlapped with the administration of IL-2. Objective responses occurred in 31% of melanoma patients. Conclusions: Decitabine can be safely administered with high-dose IL-2 and may enhance the activity of IL-2 in melanoma.

IFN-; plays a role in the response to melanoma indirectly through its effect on the immune system and directly through its antiproliferative and proapoptotic effects on melanoma cells. To understand the molecular basis for the direct antimelanoma effect of IFN-;, we studied IFN-induced changes in gene expression and signaling among three human melanoma cell lines (DM6, DM93, and 501mel). These were resistant to the antimelanoma effect of IFN-A, and only DM6 cells exhibited growth inhibition and apoptosis with IFN-;. Through DNA microarray analysis, we found that the antimelanoma effect of IFN-; in DM6 was associated with the down-regulation of multiple genes involved in G-protein signaling and phospholipase C activation (including Rap2B and calpain 3) as well as the down-regulation of genes involved in melanocyte/melanoma survival (MITF and SLUG), apoptosis inhibition (Bcl2A1 and galectin-3), and cell cycling (CDK2). The antimelanoma effect of IFN-; was also associated with the up-regulation of the proapoptotic dependence receptor UNC5H2 and the Wnt inhibitor Dkk-1. Whereas both IFNs were able to activate Stat1 in all cell lines, the delayed activation of the extracellular signal-regulated kinase, p38, and c-Jun NH 2 -terminal kinase mitogen-activated protein kinases occurred only in DM6 with IFN-;, and the effect of IFN-; on cell growth and survival as well as gene expression in DM6 was dependent on the coordinate activation of MEK1 and p38. These findings provide new insights into the signaling events and gene expression changes associated with growth inhibition and apoptosis in melanoma and may thereby assist in identifying new targets for the treatment of melanoma.

Purpose: Metastatic uveal melanoma is resistant to conventional chemotherapy and immunotherapy. In this study, we investigated the responsiveness of uveal melanoma cell lines to IFNs and the hypomethylating agent decitabine. Experimental Design: The uveal melanoma cell lines 92-1, UW-1, OCM-1, and MKT-BR were exposed to varying concentrations of IFN-a, IFN-g, and decitabine, alone and in combination. The effects of decitabine on gene expression were examined using DNA microarray analysis. Results: We found that IFN-g and decitabine induced cell death in uveal melanoma. Whereas a high concentration of IFN-g (1,000 units/mL) was required to induce cell death, we observed a dose-related increase in cell death when decitabine was used at a range of 0.1 to 10 Amol/L. Strikingly, 1 Amol/L decitabine synergized with 10 to 1,000 units/mL IFN-g to induce massive cell death. In contrast, decitabine had no effect on three cutaneous melanoma cell lines and exhibited no synergy with either IFN. In uveal melanoma, decitabine up-regulated the expression of genes involved in growth control and apoptosis and down-regulated genes that have been implicated in the malignant phenotype of cutaneous melanoma. The gene up-regulated to the greatest degree by decitabine and whose expression showed a dose-effect across the three concentrations of decitabine was S100A2, a putative tumor suppressor.The genes modulated by decitabine in uveal melanoma were largely unaffected in cutaneous melanoma. Conclusions: These findings form a basis for testing the decitabine/IFN-g combination in metastatic uveal melanoma and for exploring the role of S100A2 in the susceptibility of uveal melanoma to IFN-mediated cell death.Metastatic uveal melanoma is an aggressive malignancy with a median survival of <6 months (1). Patients with locally advanced primary lesions also have a poor prognosis because up to 50% will subsequently develop disseminated metastases within 10 years (2). Although hepatic metastases can respond partially to intra-arterial chemotherapy or chemoembolization, metastatic uveal melanoma is largely resistant to systemic chemotherapy (3). Immunomodulatory drugs used to treat advanced cutaneous melanoma, including interleukin 2 (IL-2) and IFN-a, have also had very limited activity in uveal melanoma (4).The mechanism underlying the antitumor effect of IFN-a or IL-2 in select patients with advanced cutaneous melanoma remains poorly defined. The association of good outcome with the development of autoimmune breakthrough events (5,6) suggests that overcoming tolerance to self-antigens plays a role in cytokine-mediated antitumor immunity. However, IFN-a and IFN-g can also exert direct antiproliferative and/or proapoptotic effects on cutaneous melanoma cell lines (7, 8), illustrating how certain cytokines can have antitumor effects that are independent of the induction of antigen-specific cellular or humoral immunity.The epigenetic suppression of gene expression through DNA methylation has been linked to the relative resistance of a cuta...

Spindle assembly and elongation involve poleward and away‐from‐the‐pole forces produced by microtubule dynamics and spindle‐associated motors. Here, we show that a bidirectional Drosophila Kinesin‐14 motor that moves either to the microtubule plus or minus end in vitro unexpectedly causes only minor spindle defects in vivo. However, spindles of mutant embryos are longer than wild type, consistent with increased plus‐end motor activity. Strikingly, suppressing spindle dynamics by depriving embryos of oxygen causes the bidirectional motor to show increased accumulation at distal or plus ends of astral microtubules relative to wild type, an effect not observed for a mutant motor defective in motility. Increased motor accumulation at microtubule plus ends may be due to increased slow plus‐end movement of the bidirectional motor under hypoxia, caused by perturbation of microtubule dynamics or inactivation of the only other known Drosophila minus‐end spindle motor, cytoplasmic dynein. Negative‐stain electron microscopy images are consistent with highly cooperative motor binding to microtubules, and gliding assays show dependence on motor density for motility. Mutant effects of the bidirectional motor on spindle function may be suppressed under normal conditions by motor: motor interactions and minus‐end movement induced by spindle dynamics. These forces may also bias wild‐type motor movement toward microtubule minus ends in live cells. Our findings link motor : motor interactions to function in vivo by showing that motor density, together with cellular dynamics, may influence motor function in live cells.