Background: XL228 is a small molecule protein kinase inhibitor currently in Phase 1 clinical development targeting Ph+ leukemias, multiple myeloma, and solid tumors. Cellular and in vivo data have demonstrated activity of XL228 against IGF1R, ABL (including the T315I variant) and SRC family kinases. XL228 was tested in a panel of biochemical protein kinase assays, and additional putative targets were identified, including Aurora family kinases, FGFR1‐3, and ALK. As these potential targets are known to enhance cell proliferation and survival in certain malignancies, the validation of their inhibition by XL228 through in vitro studies and in clinical pharmacodynamic samples was performed.

Results: A panel of kinase inhibitors including XL228 was profiled against a series of cancer cell lines with known alterations in major signaling pathways. Approximately 30% of the lines demonstrated XL228 IC50 values of <100nM in viability assays, including many lines with characterized ALK or FGFR mutations or amplifications. Cell lines showing the highest level of response to XL228 were often sensitive to other Aurora, ALK or FGFR inhibitors as well. Sub‐20nM activity was demonstrated against FGFR2 in a cellular kinase ELISA assay, and confirmed by immunoblot analysis. Potency against ALK in the cell kinase assay was lower (approximately 200 nM). The Aurora inhibitory activity of XL228 was examined through the use of immunoblots, immunofluorescence, and substrate phosphorylation assays. XL228 eliminated the phosphorylation of Aurora A and B at concentrations above 10 nM. Short‐term treatment of HeLa cells led to disruption of mitotic spindle formation, with the majority of mitotic cells exhibiting a unipolar spindle and disorganized chromosomes. After exposure to XL228 for 24 hours, there was a significant increase in cells with >4N DNA content, suggesting a failure of cytokinesis and consequent endoreduplication. Activity of XL228 against Aurora kinases was further verified by dose‐dependent inhibition of histone H3 phosphorylation, where it showed approximately 5‐fold higher potency than the control compound VX‐680. To confirm the relevance of the in vitro observations to the clinical activity of XL228, tumor biopsy specimens obtained from the solid tumor Phase 1 clinical study were evaluated for changes in phosphorylation of pathway markers by immunofluorescence. Inhibition of Aurora, FGFR, IGF1R, and SRC family kinases was observed, as measured by decreases in kinase substrate phosphorylation in these specimens.

Conclusions: XL228 shows a broad pattern of protein kinase inhibition, including the tyrosine kinases IGF1R, SRC, ABL, FGFR1‐3, and ALK and the serine/threonine kinases Aurora A and Aurora B. The clinical impact of this target profile is currently being assessed in two Phase 1 studies.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C192.