Abstract A201: Development of new assay systems for screening inhibitory molecules of c-Myb.

Abstract

Abstract c-myb is a proto-oncogene encoding a transcription factor which is highly expressed in hematopoietic progenitor cells. It is primarily involved in regulation of the expression of genes important for differentiation, cell proliferation and lineage determination. The c-Myb protein is an interesting therapeutic target because emerging evidence suggests that deregulated c-Myb expression is involved in several types of leukemia and development of human tumors such as breast and colon cancer, adenoid cystic carcinoma and subtypes of glioblastoma. So far therapeutic low molecular weight inhibitors for c-Myb have not been reported. Based on the fact that the ability of c-Myb to act as a transcription factor is highly dependent upon its interaction with the coactivator CBP/p300, we have developed novel in vivo and in vitro assays which allow screening of chemical inhibitors of Myb. The in vivo assay consists of the N- and C-terminal halves of firefly luciferase which are expressed as fusion proteins with either the KIX domain of p300 or the p300 binding region of c-Myb. KIX and c-Myb interaction leads to complementation of the luciferase molecule rendering it active, which can be monitored by a standard luciferase assay. The in vitro system employs a bacterial auto-display system. The KIX domain of p300 is expressed in E coli in such a way that it is displayed on the surface of the bacterial cells. Myb is expressed as a soluble GFP fusion protein which is added to the bacterial cells. The interaction between both binding partners renders the bacterial cells fluorescent which can be quantified by flow-cytometry. These assays should be useful to screen large libraries of compounds as they both allow high throughput techniques. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A201. Citation Format: Sagar R. Uttarkar, Michael Goblirsch, Joachim Jose, Karl-Heinz Klempnauer. Development of new assay systems for screening inhibitory molecules of c-Myb. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A201.