Preclinical activity of ENMD-981693, an inhibitor of Aurora A kinase and multiple receptor tyrosine kinases, against multiple myeloma (MM) cells

Abstract

8609 Background: ENMD-981693 is a novel, orally-active molecule that has been shown to have significant activity against Aurora A kinase as well as multiple receptor tyrosine kinases (RTKs). We investigated the activity of ENMD-981693 against MM cells. Methods: Human MM cells (IM-9, ARH-77, RPMI-8226, U266, MM.1S, MM.1R, H929) were treated with ENMD-981693 for 6–72h. Cytotoxicity was assessed using MTT assay and annexin V/PI staining, and intracellular signaling by Western blotting. Results: ENMD- 981693 showed significant cytotoxicity against MM cells with mean LC50 of 7.15±2.97 μM and 3.84±0.86 μM at 24 and 48 hours, respectively. Cytotoxicity was associated with cleavage of caspases 3, 8, 9 and PARP, and loss of mitochondrial membrane potential as early as 6 hours. Inhibition of caspase 9, but not of caspase 8, protected against cell death at 6 and 24 hours indicating that apoptosis was predominantly mediated via a mitochondrial pathway. ENMD-981693 also downregulated survivin and X-linked inhibitor of apoptosis at 6 hours, although Bcl-2, Bcl-xL, and Bax remained unchanged. At the earliest time point evaluated (6h), inhibition of VEGFR-1 and 2, c-kit, and FGFR-1 and 3 was also observed following treatment. Consistent with activity against RTKs, inhibition of the PI3K/AKT pathway was observed at 6 hours with downregulation of downstream targets including phosphorylated (p)-AKT, p-p70s6k, p-4EBP1, p-BAD, p-Foxo1a, p- GSK-3β. Although downregulation of JAK-2 was also observed, there was minimal effect on p-Stat-3(Tyr 705) and cyclin D1. With longer treatment (24–48h), ENMD-981693 also inhibited Aurora A kinase, with downregulation of p-Aurora A, G2 cell cycle arrest, and reduction in cyclins A and B. Conclusions: ENMD-981693 shows significant activity against MM cells. Cell death appears to follow a biphasic response, with apoptosis mediated early by a caspase 9-dependent mitochondrial pathway and RTK inhibition with consequent effects predominantly on the PI3K/AKT pathway. Later, G2 cell cycle arrest occurs due to Aurora kinase A inhibition. These results provide pre-clinical rationale for clinical investigation of ENMD-981693 in MM. No significant financial relationships to disclose.