Significant cross-talk between tumor cells and the surrounding stromal tissue are essential for tumor vascularization, survival, immunotolerance, invasion, and metastasis. The angiopoietin (ANG)/TIE2 kinase signaling pathway is a pivotal cross talk axis in the tumor microenvironment. It has been demonstrated that TIE2-expressing macrophages (TEMs) mediate invasion and metastasis in the PyMT syngeneic breast cancer model, and that TIE2 expression correlates with poor overall survival and high risk of metastasis in breast cancer patients. In this study, rebastinib was evaluated as a TIE2 inhibitor in in vitro and in vivo models, and was shown to have profound effects on the structure and function of perivascular TEMs.


TIE2 kinase assays used a standard PK/LDH coupled spectrophotometric continuous assay. CHO cells were transiently transfected to express human TIE2 for cellular studies. CHOs, HUVECs and EA.hy926 cells were used to evaluate rebastinib for inhibition of ANG1-stimulated TIE2 phosphorylation. In vitro intravasation studies were performed in a transwell transendothelial migration chamber wherein TIE2HI macrophages interact with breast tumor cells to cause transendothelial migration of tumor cells across a sealed HUVEC endothelial monolayer. In vivo evaluations of rebastinib were performed using intravital high-resolution two-photon microscopy in the murine PyMT breast cancer model to evaluate effects on tumor vascular permeability and tumor cell intravasation.


Rebastinib is a potent inhibitor of TIE2 kinase (IC50 = 0.63 nM). Rebastinib slowly dissociated from TIE2 (koff = 0.0012 minutes−1; T1/2 = 10 hr). In HUVECs or EA.hy926 cells, rebastinib inhibited ANG1-stimulated TIE2 kinase activity (IC50s of 0.018 and 0.091 nM, respectively). In TIE2 CHO cells, rebastinib inhibited TIE2 phosphorylation (IC50 2.0 nM), and demonstrated a prolonged off-rate (> 24 hr) against TIE2 after inhibitor washout. Rebastinib exhibited an IC50 < 5 nM for inhibiting macrophage-inducedbreast tumor cell intravasation in the in vitro transwell transendothelial migration assay.

Rebastinib was evaluated in vivo in the PyMT syngeneic breast cancer model. Rebastinib dosed at 10 mg/kg orally twice weekly impaired tumoral perivascular TEMs, resulting in a significant reduction in vascular permeability and in tumor cell intravasation as quantified by CTCs.

Conclusion Rebastinib is a potent inhibitor of TIE2 kinase and exhibits durable cellular inhibition in endothelial cells and in TIE2 macrophages. Oral dosing of rebastinib resulted in a significant reduction in TIE2-macrophage mediated tumor vascular permeability and in the intravasation of tumor cells into the circulation. Rebastinib is currently in Phase 1 clinical evaluation in solid tumors.

Citation Format: Allison Harney, Jeanine Pignatelli, Edison Leung, Maja Oktay, Yarong Wang, Bryan D. Smith, Daniel L. Flynn, John S. Condeelis. Rebastinib potently inhibits function of perivascular TIE2 expressing macrophages in vitro and in vivo. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 397. doi:10.1158/1538-7445.AM2015-397