Abstract A242: Identification of novel and specific Pim‐1 kinase inhibitors with potent anticancer activity in hematological and solid tumor malignancies

The Pim serine‐threonine kinase family is composed of three members which play an important role in intracellular signaling and contribute to pathways involved in cell survival, proliferation, stress response and cellular motility. Out the three family members, Pim‐1 has been studied most extensively and was shown to be a crucial downstream effector of several oncogenes like Jak2 and FLT3 kinases. Pim‐1 overexpression has been also reported in a variety of cancers such as diffuse B cell lymphoma, chronic lymphocytic leukemia, Flt3‐mediated acute myelogenous leukemia and solid tumors such as prostate and pancreatic cancers. For this reason, Pim‐1 kinase emerged as a novel and interesting target of significant potential for therapeutic intervention. In the current study we have performed lead optimization of a new small molecule Pim‐1 kinase inhibitor. Initial compound exerted moderate potency in Pim‐1 kinase inhibition accompanied by high selectivity for the Pim kinase family. In parallel to biochemical characterization, the original compound had been extensively tested in vitro and in vivo for anticancer activity. In order to improve potency and selectivity of the original compound a series of new derivatives was synthesized and screened for Pim‐1 inhibition. Among the newly synthesized compounds several exerted increased potency in Pim‐1 inhibition with IC50 values as low as 4,8 nM. Further biochemical profiling of best inhibitors on a 440 KINOMEscanTM Max kinase panel revealed superior selectivity in binding and inhibition of the Pim‐1 kinase with over 20‐fold off‐target selectivity. Anticancer effect of new derivatives was investigated in several cancer cell lines of hematological and solid tumor origin where the compounds was shown to induce of apoptosis. As Pim‐1 kinase was shown to regulate cell cycle progression by phosphorylation of several key proteins such as p27 and cdc25A in the G1/S checkpoint and cdc25C and c‐TAK‐1 in the G2/M checkpoint, we investigated cell cycle effects after compounds administration. We could observe two phenotypes in cell that did not undergo apoptosis, namely a time and dose dependent arrest of the cells either in the G0/G1 or G2/M phase, depending on the cell line. To confirm that the observed cytotoxic and cell cycle effects were due to inhibition of the Pim‐1 kinase by the new derivatives, a biomarker analysis was performed. Pim‐1 kinase was shown to phosphorylate p27KIP1 at threonine 198 both in vitro and in the cells. As soon as 1h after treatment with the compounds, a dramatic inhibition of phospho‐p27KIP1 was observed on K562 cells proving the expected mechanism of action of Pim‐1 kinase inhibiting compounds in the cells. Results of the Selvita9s Pim‐1 inhibitor lead optimization efforts are shown and discussed, supporting further development of this class of inhibitors in oncology indications. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A242.