Scientific Report 2003-2004 - Cleveland Clinic Lerner Research ...

Development of Clinical Anti-TumorAgents Based on Antisense Knockout ofRID Genes, Interferon Role in CytotoxicityAbetter understanding of how interferons(IFNs) induce apoptosis may allow theirimproved clinical utilization as antitumoragents. Though they have traditionally beenconsidered cytostatic agents, IFNs inducecytotoxicity in several tumor cell lines in cultureand in vivo. The mechanism by which this occursrequires the function of IFN-induced geneproducts. To identifyfunctionally relevant deathassociatedgene products,our laboratory has employedan antisense technicalknockout strategy. In thisapproach, specific deathinducinggenes, termedRegulators of InterferoninducedDeath (RIDs), areinactivated by antisensegene products, thusproviding a growthadvantage to transfectedcells in the presence ofIFNs. Because an IFNstimulatedpromoter drivesthe expression of theantisense inserts, a functionalJAK-STAT pathwayis required. Therefore,antisense RNAs directedagainst the genes encodingIFN receptor chains, or JAKs, or STATs cannotaccount for cell survival in the presence of IFNs.Consistent with the possibility that multiple geneproducts participate in IFN-induced cell death,several different cDNAs have been isolated usingthis approach, many of them novel. One RIDgene is identical to inositol hexakisphosphatekinase 2, (IP6K2) a kinase that creates pyrophosphatelinkages on inositol phosphates.Overexpression of IP6K2 sensitizes cells to avariety of cytotoxic stimuli. Dr. Bei Morrisonperforms functional analysis of two RID genes toelucidate their enzymatic activity. In collaborationwith our clinical colleagues, we are performing aPhase II trial of IFN-beta in ovarian carcinoma.RID gene expression will be evaluated in bloodand tumor samples from these patients.To potentiate their clinical efficacy, IFNsare increasingly being used in combinationtherapy. In collaboration with Drs. ErnestBorden and Dhan Kalvakolanu, we have shownthat combining IFNs with antiestrogens (e.g.,tamoxifen) or retinoids (e.g., all-trans retinoicacid) results in enhanced antitumor effects, bothin cell culture and in athymic nude mice xenograftmodels. Part of the increased antitumor activityis due to direct anti-cellular effects mediated byDaniel J. Lindner, M.D., Ph.D.the drug combination, and a portion of theantitumor effect is secondary to enhanced hosteffects. One of the most important anti-tumoreffects mediated by IFNs is the inhibition oftumor-induced angiogenesis. On a molar basis,IFNs are the most powerful anti-angiogenicagents currently known. Current angio-genesisstudies are probing the antiangio-genic effects ofsecond generation IFNs such asPEG-IFN, a polyethylene glycolmodification of the IFNmolecule that results in aprolonged half life and hencemay provide enhancedbiological activity. We are alsotesting IFNs in combinationwith Angiozyme, an antisensemolecule directed againstVEGFR1/flt-1, a criticalreceptor that mediates bloodvessel growth.Our laboratory is alsoactive in the area of newcancer drug development. Dr.Joseph Bauer has synthesized anovel chemotherapeuticcompound, nitrosylcobalamin(NO-Cbl), that consists ofnitric oxide bound to vitaminB12. Inactive until taken up bycells, once inside lysosomes, thedrug releases nitric oxide and induces apoptosis.Importantly, NO-Cbl causes very little toxicity tonormal tissues. We believe this “Trojan Horse”approach may target tumors through their highrequirement for vitamin B12.THE LINDNERLABORATORYPRINCIPAL INVESTIGATORDaniel J. Lindner, M.D., Ph.D.RESEARCH ASSOCIATESJoseph A. Bauer, Ph.D.Bei Hu Morrison, M.D.COLLABORATORSAlexandru Almasan, Ph.D.Ernest C. Borden, M.D.Dhananjaya Kalvakolanu, Ph.D. 1Bellur Seetharam, Ph.D. 2Ganes C. Sen, Ph.D.Deborah J. Vestal, Ph.D.1Dept. of Microbiology, Univ. ofMaryland, Baltimore, MD2Dept. of Biochemistry, Med.College of Wisconsin,Milwaukee, WICLINICAL COLLABORATORSJerome Belinson, M.D.Maurie Markman, M.D.Kenneth Webster, M.D.Lindner, D.J. (2002) Interferons as antiangiogenic agents. Curr. Oncol. Rep. 4:510-514.Yi, T., Pathak, M.K., Lindner, D.J., Ketterer, M.E., Farver, C., and E.C. Borden(2002) Anticancer activity of sodium stibogluconate in synergy with IFNs. J. Immunol.169:5978-5985.Pathak, M.K., Dhawan, D., Lindner, D.J., Borden, E.C., Farver, C., and T. Yi(2002) Pentamidine is an inhibitor of PRL phosphatases with anticancer activity.Mol. Cancer Ther. 1:1255-1264.Bauer, J.A., Morrison, B.H., Grane, R.W., Jacobs, B.S., Borden, E.C., and D.J.Lindner (2003) IFN-alpha2b and thalidomide synergistically inhibit tumor-inducedangiogenesis. J. Interferon Cytokine Res. 23:3-10.Chawla-Sarkar, M., Lindner, D.J., Liu, Y.F., Williams, B.R., Sen, G.C., Silverman,R.H., and E.C. Borden (2003) Apoptosis and interferons: Role of interferon-stimulatedgenes as mediators of apoptosis. Apoptosis 8:237-249.173