CONFIDENTIAL 1 Small molecule-mediated inhibition of Factor VIIa catalytic activity blocks fVIIa:TF cellular signaling and chemokine secretion in human tumor cell lines Leslie J. Holsinger , William D. Shrader, Catherine Magill, Meire C.D. Bremer, Jennifer Kattler, James W. Janc, Peter R. Young, Wendy B. Young. Celera Genomics, 180 Kimball Way, South San Francisco, CA 94080 SUMMARY • FVIIa addition to TF+ tumor cell lines stimulates AKT/ ERK pathway activation and pro-angiogenic chemokine secretion, and this activation is specific to the FVIIa protease. • FVIIa catalytic activity is required for the activation of intracellular signaling, and small molecule inhibitors are able to block these signaling events. • FVIIa was readily detected in primary human pancreatic tumors. • These data indicate that FVIIa inhibitors may have utility as anti-tumor agents in TF+ malignancies. ABSTRACT INTRODUCTION RESULTS: 7. FVII/FVIIa detected in primary pancreatic carcinoma (Staining done with Lifespan Biosciences) 1. CRA-027483 is a potent, competitive, and reversible selective small molecule inhibitor of FVIIa In addition to its role in coagulation, Factor VIIa (fVIIa) binding to TF has been demonstrated to activate a variety of intracellular signaling pathways including MAPK, AKT, and Src. This activity has been shown to require TF binding as well as fVIIa protease activity. TF overexpression in tumor epithelial cells and tumor vasculature has been linked to prognosis. However a precise role for fVIIa:TF signaling in tumor cell growth and survival has not yet been established. We have demonstrated that fVIIa is able to activate MAPK, AKT, and early- response gene induction in the BxPC3 pancreatic human tumor cell line at physiologic (0.1-10nM) concentrations. TF expression in the tumor cell line is required for this intracellular signaling. Preincubation of fVIIa with potent, fVIIa-selective, reversible small molecule inhibitors of fVIIa protease activity inhibited the activation of MAPK and early-response gene induction in a dose-dependent manner. These data demonstrate that fVIIa catalytic activity is required for its effect on intracellular signaling, and indicate that small molecule inhibitors of fVIIa:TF activity on tumor epithelial cells may have direct anti-tumor efficacy. fVIIa also induced the secretion of chemokines IL-8, GROα, and VEGF in the human breast tumor cell line MDA-MB-231. fVIIai was unable to induce chemokine secretion. fVIIa-specific small molecule inhibitors of fVIIa protease activity inhibited this secretion in the presence or absence of serum components. IL-8 is an autocrine growth factor in tumors that stimulates chemotaxis and invasion, and also exhibits effects on endothelial cell survival and proliferation and may directly effect the formation and maintenance of tumor vasculature. IL-8 secretion was not induced by fXa or a number of other serine proteases including urokinase, thrombin, and tryptase indicating that this activity is specific to the fVIIa protease. In summary, fVIIa mediated secretion of pro- angiogenic chemokines as well as signaling events which regulate tumor cell growth and survival. These fVIIa-specific effects were blocked with small molecule inhibitors of fVIIa protease activity. Thus, small molecule inhibitors of fVIIa may be expected to have utility as anti-tumor agents in the treatment of malignancies. Malignant Cells 40X Malignant Cell at Pushing Margin of Invasion 60X Malignant Cells and Surrounding Fibrocollagenous Matrix 40X • FVIIa detected in 12/13 pancreatic carcinomas with IHC staining • Staining detected primarily in malignant cells with normal surrounding fibrous tissue negative • Staining often detected at the pushing margins of tumor invasion Enzyme (human) Potency: (µM) Ki fVIIa/TF 0.0017 fIXa 1.05 fXa 5.4 fXIIa 140 fIIa >150 p-kallikrein 0.195 trypsin 11.0 2xPT(µM) human 1.4 mouse 29 Cell Surface VII proteolytic activation VIIa CRA TF VIIa CRA TF X Xa binding site is blocked & further coagulation propagation is inhibited CRA-027483 Mode of Binding: Schematic diagram of the mode of interaction of CRA-027483 with FVIIa. CRA-027483 inhibitor interacts with the FVIIa/TF complex, inhibits catalytic activity, and competes for the FX binding site. CRA-027483 is a competitive, reversible active site inhibitor with fast on and fast off kinetics. 2. FVIIa activates ERK and c-fos/egr-1 gene induction in the TF positive BxPC3 pancreatic tumor cell line • Celera has developed a series of potent small molecule inhibitors of FVIIa with anticoagulant activity . • Efficacy has been demonstrated in a baboon thrombosis model, and no alerts found in preliminary tox evaluations. • CRA-027483 selected as a development candidate as an anticoagulant. • We investigated the function of FVIIa:TF in human tumor cell lines to to better understand its proposed role in tumorigenesis and to provide suppport for the use of FVIIa inhibitors in oncology. Smooth Muscle Cells Activated Endothelial Cells Tumor epithelial cells Proliferation, migration Vascular remodeling and angiogenesis VEGF, IL8, uPAR, growth factors, cytokines Tumor growth and survival signals Proliferation migration Vascular remodeling Tumor growth metastasis PAR-2 TF fVIIa 0 5 10 15 20 40 60 90 180 360 min 100nM FVIIa Blot: pERK Blot: egr-1 Blot: c-fos Blot: ERK nM FVIIa 15’ serum 15’ 0 .001 .01 .1 1 10 100 Blot: pAKT (pSer473) Blot: AKT Blot: pERK 0 500 1000 1500 2000 2500 3000 3500 4000 4500 - fVIIa +CRA X1 +CRA X2 +CRA X3 +CRA X4 +CRA X5 +CRA X6 - + + + + + + + - - + + + + + + 1µM cmpd FVIIa FVIIa inhibitors block IL-8 secretion in MDA-MB-231 Dose response, and comparison to MEK inhibitor 0 200 400 600 800 1000 1200 1400 1600 1800 2000 IL-8 (pg/ml) MEK inhibitor * - + + + + + + 100nM FVIIa + nM CRA X2 - - - 1000 10 1 .1 IL-8 (pg/ml) *MEK inhibitor: U0126 10 µM IL-8 pg/ml 0 100 200 300 400 500 600 700 800 Control FVIIa 100nm FVIIa 10nm FVII 100nm FVII 10nm urokinase 100nm urokinase 10nm thrombin 100nm thrombin 10nm tyrptase 100nm tryptase 10nm 0 100 200 300 400 500 600 700 Control FVIIa 100nm FVIIa 10nm trypsin 100nm trypsin 10nm FXa 100nm FXa 10nm 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000 pg/ml 0 1 10 100 nM fVIIa IL-8 (CXCL8) 0 100 200 300 400 500 600 700 800 900 1000 0 1 10 100 nM fVIIa GROα (CXCL1) 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000 0 10 100 nM fVIIa VEGF Blot: pΑKT Blot: AKT BxPC3 (TF positive ) MiaPaCa2 (TF negative ) Blot: pAKT Blot: AKT nM FVIIa 15’ 0 .001 01 .1 1 10 100 serum 15’ 3. FVIIa actives ERK and AKT in a dose-dependent and TF- dependent manner Pancreatic tumor cell line BxPC3 (positive for TF expression) was serum-starved for 24h, and purified FVIIa added in serum-free media at the indicated concentrations and times listed. Cell lysates were analyzed by immunoblotting for ERK and AKT phosphorylation, and c-fos and egr-1 gene induction. Stimulation with 15% serum was used as a control. 5. FVIIa activates pro-angiogenic chemokine secretion in the MDA-MB-231 breast tumor cell line. 0 15% serum 10nM FVIIa 100nM FVIIa CRA X2 0 50000 100000 150000 200000 250000 0 10nM FVIIa 100nM FVIIa 15% serum µM CRA X2 0 1 10 Blot: c-fos 0 1 10 0 1 10 BxPC3 cells were serum-starved for 24h. FVIIa (or serum) at the indicated concentrations were preincubated with FVIIa inhibitor for 30 minutes, and added to cells. 60 minutes after addition cell lysates were analyzed for c-fos induction. 4. A small molecule FVIIa inhibitor blocks FVIIa- dependent c-fos induction MDA-MB-231 cells were stimulated with purified protein preparations of the indicated proteases in serum free or serum-containing media. After 24hr supernatants were harvested and analyzed by ELISA for IL-8, GROa, and VEGF levels. MDA-MB-231 cells were stimulated 100nM FVIIa that had been preincubated with vehicle control or the indicated concentrations of FVIIa inhibitiorsin serum-free media for 30 minutes. MEK inhibitor was added as indicated at the time of FVIIa addition. 24h after stimulation with FVIIa supernatants analyzed for IL-8 secretion. 6. FVIIa inhibitors block protease-dependent IL-8 secretion in MDA-MB-231 cells. View publication stats View publication stats