Poster Board III-769
Aurora kinases, a family of mitotic regulators, whose expression has been recently linked to genetic instability and cellular proliferation in several cancers including multiple myeloma (MM), are being studied as novel mitotic therapeutic targets. Aurora A plays a crucial role in centrosome separation and spindle assembly and is required for mitosis and bipolar mitotic spindle formation. Aurora B, a member of the chromosomal passenger complex, is required for chromosome segregation, spindle assembly checkpoint and cytokinesis. Both aurora kinase A and B are significantly overexpressed in MM cells prompting the investigation of aurora kinase inhibitors as a therapeutic strategy in MM. Here, we investigated the preclinical activity of a small molecule multi-targeted inhibitor, AT9283, with potent in vitro kinase activity against aurora A and B kinases (3 nM), JAK2 and 3 (at 1.2 and 1.1 nM, respectively) 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 aurora kinase B 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 caspase-8/-9 cleavage. The cellular inhibition of aurora kinase activity by AT9283 was confirmed by evaluating the phosphorylation of histone H3 at serine-10, a direct downstream substrate of aurora B kinase. Pretreatment of MM.1S cells with nocodazole, known to induce maximal phosphorylation of histone H3 by causing an M-phase block, resulted in decreased levels of phosphorylated histone H3 after AT9283 treatment suggesting the role of aurora B kinase inhibition by AT9283. Importantly, in addition to aurora kinase inhibition, we observed that AT9283 also inhibited signal transducer and activator of transcription (STAT3) tyrosine phosphorylation in MM cells within 30 minutes of treatment. Janus Kinase (JAK)2/STAT3 pathway is one of the major signaling cascades activated by gp130 family member cytokines that promotes MM cell survival. The effect of AT9283 on pSTAT3 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 suggesting that STAT3 was functionally inhibited by AT9283. Since MM cell lines with the constitutive STAT3 tyrosine phosphorylation were more sensitive to AT9283, ongoing studies are aimed at understanding whether AT9283-induced effects on the JAK/STAT pathway enhances the efficacy of the 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. Our results show pleiotropic effects of AT9283 in MM and warrant further study to determine its suitability for clinical evaluation in MM.
Squires:Astex Therapeutics, Ldt: Employment. Yule:Astex Therapeutics Ldt: Employment. Anderson:Novartis, Millennium, Celgene: Consultancy, Honoraria, Research Funding. Raje:Celgene, Norvartis, Astrazeneca: Research Funding.
Asterisk with author names denotes non-ASH members.