Extracellular Granzyme K Modulates Angiogenesis by Regulating Soluble VEGFR1 Release From Endothelial Cells

Angiogenesis is crucial for normal development and homeostasis, but also plays a role in many diseases including cardiovascular diseases, autoimmune diseases, and cancer. Granzymes are serine proteases stored in the granules of cytotoxic cells, and have predominantly been studied for their pro-apoptotic role upon delivery in target cells. A growing body of evidence is emerging that granzymes also display extracellular functions, which largely remain unknown. In the present study, we show that extracellular granzyme K (GrK) inhibits angiogenesis and triggers endothelial cells to release soluble VEGFR1 (sVEGFR1), a decoy receptor that inhibits angiogenesis by sequestering VEGF-A. GrK does not cleave off membrane-bound VEGFR1 from the cell surface, does not release potential sVEGFR1 storage pools from endothelial cells, and does not trigger sVEGFR1 release via protease activating receptor-1 (PAR-1) activation. GrK induces de novo sVEGFR1 mRNA and protein expression and subsequent release of sVEGFR1 from endothelial cells. GrK protein is detectable in human colorectal tumor tissue and its levels positively correlate with sVEGFR1 protein levels and negatively correlate with T4 intratumoral angiogenesis and tumor size. In conclusion, extracellular GrK can inhibit angiogenesis via secretion of sVEGFR1 from endothelial cells, thereby sequestering VEGF-A and impairing VEGFR signaling. Our observation that GrK positively correlates with sVEGFR1 and negatively correlates with angiogenesis in colorectal cancer, suggest that the GrK-sVEGFR1-angiogenesis axis may be a valid target for development of novel anti-angiogenic therapies in cancer.

Effect of TKI258 on plasma biomarkers and pharmcokinetics in patients with advanced melanoma

9020 Background: TKI258 (dovitinib lactate), is a multi-tyrosine kinase inhibitor of VEGF receptors-1,2,3, FGF receptors-1, 2, 3, PDGFR-β, and c-KIT. A phase I study was conducted to determine the maximum tolerated dose (MTD), the biological activity and the preliminary efficacy of TKI258 in patients with advanced melanoma. A panel of plasma biomarkers of angiogenesis and soluble receptors were evaluated to determine the pharmacodynamic effect of TKI258. Methods: Patients were treated orally with 200, 300, 400 or 500 mg/day on a once daily continuous dose schedule. The MTD was defined at 400 mg/day. Plasma samples from 43 patients were collected. Plasma concentration of TKI258 was measured by LC/MS/MS. Plasma VEGF, placental growth factor (PLGF), basic FGF (bFGF), and soluble VEGFR1 and VEGFR2 (sVEGFR1 and 2), and c-Kit were measured by multiplex assays using the Meso-Scale Discovery platform. Plasma FGF23 was evaluated by ELISA as a pharmcodynamic marker of FGFR1 inhibition. Results: Following 400 mg or 500 mg continuous daily dosing, the mean plasma exposure (AUC24hr) was approximately 3000 ng/mL*h and 4100 ng/mL*h, respectively. No accumulation in TKI258 plasma exposure was observed at doses of 400mg or below, while accumulation up to 2.5-fold was observed on day 15 following the 500 mg daily dose. At the end of the first treatment cycle, mean plasma VEGF, PLGF and FGF23 levels increased over baseline by 100%, 198% and 68%, respectively, while mean plasma sVEGFR2 levels decreased by 15% in patients treated with 400 and 500 mg/day TKI258. Further analysis of correlations with pharmacokinetic and clinical parameters is ongoing. Conclusions: TKI258 therapy is associated with increases of plasma VEGF and PLGF as well as decreases of sVEGFR2 suggesting VEGFR inhibition. Induction of plasma FGF23 suggest that FGFR may be inhibited at doses of 400 mg/day and above. This panel of circulating proteins may have utility as pharmacodynamic biomarkers of TKI258 activity in patients with advanced melanoma. [Table: see text]