Multiple myeloma (MM) is a B cell malignancy characterized by the accumulation in the bone marrow of secretory plasma cells with a low proliferation index and extended survival. Despite improvements in treatment, most patients (approximately 11,000/year) die from their disease. Interleukin-6 (IL-6) is the key growth and survival factor for MM cells. IL-6 activates the Jak/STAT and Ras/MAPK signal transduction pathways which lead to the induction of genes responsible for cell cycle progression and resistance to apoptosis. STAT3 has been shown to play central role in these events. The goal of this proposal is to develop a high-throughput drug screen for inhibitors of STAT3 signaling in MM cells. A cell-based assay will be developed to test for drugs that inhibit the expression of indicator genes from STAT3-specific promoters. The STAT3- specific promoters include a construct encoding 7 repeats of STAT3 binding sites (acute phase response elements; APRE) upstream of a minimal TK promoter, and 5'-regulatory sequences from the C-reactive protein (CRP) gene that contains an APRE element in the context of a natural gene. STAT3-specific promoters will drive expression of the destabilized, enhanced-cyan fluorescent protein (d2ECFP) which has a short half-life and is suited for gene expression studies of this type. A second plasmid with the SV40 promoter driving expression of destabilized, enhanced-yellow fluorescent protein (d2EYFP) will be co-transfected with the STAT3 reporter construct; and will serve as a control for nonspecific toxic effects of drugs. STAT3-specific and control vectors will be introduced into MM cells and clones expressing d2ECFP in a STAT3-dependent manner will be used in the drug screen. A transient assay system utilizing the same expression vectors will be developed in parallel. A 96-well plate assay will be developed in which natural products or synthetic drugs will be screened for those that inhibit expression from the STAT3- specific promoter without inhibition of the control vector. The sites of action of novel drugs within the IL-6/gpl30/STAT3 pathway will be determined.
