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Limited options provided by the current standard of care for the patients suffering from myeloproliferative diseases (MPDs) prompted an extensive search for the underlying molecular mechanisms of these disorders. Recent discovery of a single activating mutation (V617F) in JAK2 kinase gene associated with the development of the polycythemia vera (PV), essential thrombocythemia (ET) and chronic idiopathic myelofibrosis (CIMF) opened up a possibility to develop highly targeted therapies against these debilitating ailments. To that end, we engineered cytokine-independent Ba/F3 cell line expressing the V617F mutant of JAK2 to screen a focused small molecule library for potential inhibitors of V617F JAK2-dependent proliferation. We confirmed the ability of hit compounds to inhibit proliferation of JAK2-dependent tumor cell lines using UKE-1 cells carrying the V617F JAK2 mutation. A FACS-based phosphoSTAT5 assay was then used to demonstrate that the hits directly targeted mutant JAK2. To avoid compounds with nonspecific antiproliferative activity, the hits were tested in JAK2-independent MOLT4 and TF1a cell lines. To probe the specificity of the compounds against other members of the JAK kinase family, we generated Ba/F3 cell lines expressing TEL-JAK1, TEL-JAK2 and TEL-JAK3 fusion proteins and used them in proliferation assays. Compound hits with the desirable properties were further evaluated for their ability to inhibit JAK2, JAK3 and other kinases in the context of T cell, B cell, or mast cell activation using a variety of cell-based assays. We identified a number of compounds that potently inhibit growth of the two V617F mutant cell lines with EC50s varying from 100 to 500 nM, but do not affect proliferation of control cell lines MOLT4 and TF-1a to the same degree. These compounds induce strong and highly specific suppression of the JAK-STAT pathway with IC50s of 50 to 200 nM, according to FACS analysis of STAT5 phosphorylation. The results of the hit profiling will be discussed.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA