Abstract
Accumulating evidence suggests important roles for the receptor tyrosine kinase Axl in cancer progression, invasion, metastasis, drug resistance, and patient mortality, highlighting Axl as an attractive target for therapeutic development. We have generated and characterized a potent and selective small-molecule inhibitor, R428, that blocks the catalytic and procancerous activities of Axl. R428 inhibits Axl with low nanomolar activity and blocked Axl-dependent events, including Akt phosphorylation, breast cancer cell invasion, and proinflammatory cytokine production. Pharmacologic investigations revealed favorable exposure after oral administration such that R428-treated tumors displayed a dose-dependent reduction in expression of the cytokine granulocyte macrophage colony-stimulating factor and the epithelial-mesenchymal transition transcriptional regulator Snail. In support of an earlier study, R428 inhibited angiogenesis in corneal micropocket and tumor models. R428 administration reduced metastatic burden and extended survival in MDA-MB-231 intracardiac and 4T1 orthotopic (median survival, >80 days compared with 52 days; P < 0.05) mouse models of breast cancer metastasis. Additionally, R428 synergized with cisplatin to enhance suppression of liver micrometastasis. Our results show that Axl signaling regulates breast cancer metastasis at multiple levels in tumor cells and tumor stromal cells and that selective Axl blockade confers therapeutic value in prolonging survival of animals bearing metastatic tumors.
Highlights
Axl is a member of the TAM (Tyro3, Axl, Mer) receptor tyrosine kinase (RTK) family and was originally identified as a transforming gene expressed in cells from patients with chronic myelogenous leukemia [1] or chronic myeloproliferative disorder [2]
R428 activity was evaluated in a broad panel of 133 tyrosine and serine/threonine kinases using in vitro enzymatic assays (Table 1)
Axl inhibition has been observed in some multitargeted tyrosine kinase inhibitors (Supplementary Table S2; refs. 12, 24, 25), R428 is, to the best of our knowledge, unique in its nanomolar on-target activity and restricted selectivity profile
Summary
Axl is a member of the TAM (Tyro, Axl, Mer) receptor tyrosine kinase (RTK) family and was originally identified as a transforming gene expressed in cells from patients with chronic myelogenous leukemia [1] or chronic myeloproliferative disorder [2]. Axl activation occurs by binding of its cognate protein ligand, growth arrest specific 6 (Gas6), homotypic dimerization through its extracellular domain or cross-talk via the interleukin (IL)-15 receptor [3, 4] or HER2 [5]. Axl signaling stimulates cellular responses, including activation of phosphoinositide 3-kinase–Akt, extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase cascades, the NF-κB pathway, and signal transducer and activator of transcription (STAT) signaling [3]. Including invasion, migration, survival signaling, angiogenesis, resistance to chemotherapeutic and targeted drugs, cell transformation, and proliferation, represent undesirable traits associated with cancer [4]. We previously uncovered the association between Axl and regulation of cell migration using a functional genetic screen designed to identify regulators of haptotaxis in primary human endothelial cells [6]. Axldependent changes in invasive cell behavior are observed on manipulation of Axl signaling in gliomas in vivo [7]
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