Abstract 2530: Antimyeloma activity of a small molecule multi-targeted kinase inhibitor, AT9283, via potent aurora kinase and STAT3 inhibition
Authors: Santo Loredana , Hideshima Teru , Cirstea Diana , Nelson Erik A. , Bandi Madhavi L. , Gorgun Gullu , Vallet Sonia , Pozzi Samantha , Patel Kishan , Ikeda Hiroshi , Perrone Giulia , Hu Yiguo , Chauhan Dharminder , Squires Matthew , Munshi Nikhil C. , Anderson Kenneth C. , Raje Noopur 
Published: February 2011

Abstract
Aurora Kinases are a family of mitotic regulators. Aurora Kinase A (AURKA) plays a crucial role in centrosome separation and spindle assembly and is required for mitosis and bipolar mitotic spindle formation. Aurora Kinase B (AURKB), a member of the chromosomal passenger complex, is required for chromosome segregation, spindle assembly checkpoint and cytokinesis. Both AURKA and AURKB are significantly overexpressed in MM cells, which has prompted the investigation of aurora kinase inhibitors as a therapeutic strategy in MM. Here, we investigated the preclinical activity of a small molecule multitargeted inhibitor, AT9283, with potent in vitro kinase activity against AURKA and AURKB kinases (3 nM), JAK2 and 3 (at 1.2 and 1.1 nM) and Abl T315I (at 4 nM). Growth inhibitory effects of AT9283 on MM cell lines and patient derived cells was observed with IC50 values of 0.25µM −0.5 µM at 48 hours using a [3H]thymidine incorporation assay. Cell cycle analysis following AT9283 treatment resulted in increased G2/M phase and polyploidy consistent with failed cytokinesis (associated with AURKB inhibition) confirmed by immunofluorescence assay. This was followed by induction of apoptosis assessed by Annexin V+PI+ staining peaking at 48 − 72 hours with associated −8-9 cleavage. Decreased levels of phosphorylated histone H3 at serine-10, a direct downstream substrate of AURKB, confirmed the role of AURKB inhibition by AT9283. Importantly, besides aurora kinase inhibition, we observed that AT9283 also inhibited STAT3 tyrosine phosphorylation in MM cells within 30 minutes of treatment. The effect of AT9283 on STAT3 inhibition was further investigated by using U3A cells stably expressing a luciferase reporter gene under the control of a STAT-dependent promoter. AT9283 inhibited STAT3-dependent luciferase activity with an EC50 of approximately 0.125 μM. Since MM cell lines with constitutive STAT3 tyrosine phosphorylation were more sensitive to AT9283, we investigated whether AT9283-induced effects on the JAK/STAT pathway correlated with Aurora inhibition. Genetic depletion by RNA interference showed that STAT3 knockdown in U266 cells did not affect the expression levels of AURKA and AURKB. In contrast, in cells with knocked-down AURK A and B, we observed a downregulation in the expression level of STAT3, due to either an off-target effect or the possibility that STAT3 is downstream of Aurora Kinases. Ongoing studies are aimed at understanding whether AT9283-induced effects on the JAK/STAT pathway enhance the efficacy of aurora kinase inhibition in the context of MM. Finally, in vivo data using a xenograft mouse model of human MM show that mice treated with AT9283 demonstrated slower tumor growth compared to the control group without adverse effects. In conclusion, these results show significant anti-MM activity of AT9283, and provide the rationale for its clinical evaluation in MM.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2530.