VEGF Receptor Family Research Articles

Identification of an osteopontin-derived peptide that binds neuropilin-1 and activates vascular repair responses and angiogenesis

The osteopontin-derived peptide FOL-005 stimulates hair growth. Using ligand-receptor glyco-capture technology we identified neuropilin-1 (NRP-1), a known co-receptor for vascular endothelial growth factor (VEGF) receptors, as the most probable receptor for FOL-005 and the more stable analogue FOL-026. X-ray diffraction and microscale thermophoresis analysis revealed that FOL-026 shares binding site with VEGF in the NRP-1 b1-subdomain. Stimulation of human umbilical vein endothelial cells with FOL-026 resulted in phosphorylation of VEGFR-2, ERK1/2 and AKT, increased cell growth and migration, stimulation of endothelial tube formation and inhibition of apoptosis in vitro. FOL-026 also promoted angiogenesis in vivo as assessed by subcutaneous Matrigel plug and hind limb ischemia models. NRP-1 knock-down or treatment of NRP-1 antagonist EG00229 blocked the stimulatory effects of FOL-026 on endothelial cells. Exposure of human coronary artery smooth muscle cells to FOL-026 stimulated cell growth, migration, inhibited apoptosis, and induced VEGF gene expression and VEGFR-2/AKT phosphorylation by an NRP-1-dependent mechanism. RNA sequencing showed that FOL-026 activated pathways involved in tissue repair. These findings identify NRP-1 as the receptor for FOL-026 and show that its biological effects mimic that of growth factors binding to the VEGF receptor family. They also suggest that FOL-026 may have therapeutical potential in conditions that require vascular repair and/or enhanced angiogenesis.

An intuitionistic approach for the predictability of anti-angiogenic inhibitors in cancer diagnosis

Malignant cancer angiogenesis has historically attracted enormous scientific attention. Although angiogenesis is requisite for a child’s development and conducive to tissue homeostasis, it is deleterious when cancer lurks. Today, anti-angiogenic biomolecular receptor tyrosine kinase inhibitors (RTKIs) to target angiogenesis have been prolific in treating various carcinomas. Angiogenesis is a pivotal component in malignant transformation, oncogenesis, and metastasis that can be activated by a multiplicity of factors (e.g., VEGF (Vascular endothelial growth factor), (FGF) Fibroblast growth factor, (PDGF) Platelet-derived growth factor and others). The advent of RTKIs, which primarily target members of the VEGFR (VEGF Receptor) family of angiogenic receptors has greatly ameliorated the outlook for some cancer forms, including hepatocellular carcinoma, malignant tumors, and gastrointestinal carcinoma. Cancer therapeutics have evolved steadily with active metabolites and strong multi-targeted RTK inhibitors such as E7080, CHIR-258, SU 5402, etc. This research intends to determine the efficacious anti-angiogenesis inhibitors and rank them by using the Preference Ranking Organization Method for Enrichment Evaluation (PROMETHEE- II) decision-making algorithm. The PROMETHEE-II approach assesses the influence of growth factors (GFs) in relation to the anti-angiogenesis inhibitors. Due to their capacity to cope with the frequently present vagueness while ranking alternatives, fuzzy models constitute the most suitable tools for producing results for analyzing qualitative information. This research’s quantitative methodology focuses on ranking the inhibitors according to their significance concerning criteria. The evaluation findings indicate the most efficacious and idle alternative for inhibiting angiogenesis in cancer.

Antitumor immune effects of preoperative sitravatinib and nivolumab in oral cavity cancer: SNOW window-of-opportunity study

BackgroundSitravatinib, a tyrosine kinase inhibitor that targets TYRO3, AXL, MERTK and the VEGF receptor family, is predicted to increase the M1 to M2-polarized tumor-associated macrophages ratio in the tumor microenvironment...

VEGFR-3 signaling is regulated by a G-protein activator, activator of G-protein signaling 8, in lymphatic endothelial cells

Vascular endothelial growth factor C (VEGFC) and its cognate receptor VEGFR-3 play a key role in lymphangiogenesis. We previously reported that an ischemia-inducible Gβγ signal regulator, activator of G-protein signaling 8 (AGS8), regulated the subcellular distribution of vascular endothelial growth factor receptor-2 (VEGFR-2) and influenced VEGFA-induced signaling in vascular endothelial cells. Here, we report that AGS8 regulates VEGFR-3, which is another subtype of the VEGF receptor family, and mediates VEGFC signaling in human dermal lymphatic endothelial cells (HDLECs). VEGFC stimulated the proliferation of HDLECs and tube formation by HDLECs, which were inhibited by knocking down AGS8 by small interfering RNA (siRNA). AGS8 siRNA inhibited VEGFC-mediated phosphorylation of VEGFR-3 and its downstream molecules, including ERK1/2 and AKT. Analysis of fluorescence-activated cell sorting and immunofluorescence staining demonstrated that AGS8 knockdown was associated with a reduction of VEGFR-3 at the cell surface. Endocytosis inhibitors did not rescue the decrease of cell-surface VEGFR-3, suggesting that AGS8 regulated the trafficking of VEGFR-3 to the plasma membrane. An immunoprecipitation assay indicated that VEGFR-3 formed a complex including AGS8 and Gβγ in cells. These data suggest the novel regulation of VEGFC-VEGFR-3 by AGS8 in HDLECs and a potential role for AGS8 in lymphangiogenesis.

Clinical pharmacodynamic/exposure characterisation of the multikinase inhibitor ilorasertib (ABT-348) in a phase 1 dose-escalation trial

BackgroundIlorasertib (ABT-348) inhibits Aurora and VEGF receptor (VEGFR) kinases. Patients with advanced solid tumours participated in a phase 1 dose-escalation trial to profile the safety, tolerability, and pharmacokinetics of ilorasertib.MethodsIlorasertib monotherapy was administered at 10–180 mg orally once daily (Arm I, n = 23), 40–340 mg orally twice daily (Arm II, n = 28), or 8–32 mg intravenously once daily (Arm III, n = 7), on days 1, 8, and 15 of each 28-day cycle.ResultsDose-limiting toxicities were predominantly related to VEGFR inhibition. The most frequent treatment-emergent adverse events ( > 30%) were: fatigue (48%), anorexia (34%), and hypertension (34%). Pharmacodynamic markers suggested that ilorasertib engaged VEGFR2 and Aurora B kinase, with the VEGFR2 effects reached at lower doses and exposures than Aurora inhibition effects. In Arm II, one basal cell carcinoma patient (40 mg twice daily (BID)) and one patient with adenocarcinoma of unknown primary site (230 mg BID) had partial responses.ConclusionsIn patients with advanced solid tumours, ilorasertib treatment resulted in evidence of engagement of the intended targets and antitumour activity, but with maximum inhibition of VEGFR family kinases occurring at lower exposures than typically required for inhibition of Aurora B in tissue.Clinical Trial Registration: NCT01110486

Targeting VEGFR and FGFR in head and neck squamous cell carcinoma in vitro.

Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous disease characterized by a tumor microenvironment (TME) that overexpresses vascular endothelial growth factor receptor (VEGFR) and fibroblast growth factor receptor (FGFR), which can lead to neovascularization, tumor growth and metastasis. Therapeutic strategies inhibiting these signaling pathways might lead to innovative HNSCC treatments. Five HNSCC cell lines were characterized based on VEGFR1-3 and FGFR1-4 expression by sqRT-PCR and treated with three different tyrosine kinase inhibitors (TKIs) (nintedanib, dovitinib and pazopanib), all of which are effective against VEGFR and FGFR family members. Crystal violet assays were performed to analyze the effect of the treatments on cell growth (viability). Additionally, VEGFR1-3 and FGFR1-4 expression data were retrieved from The Cancer Genome Atlas (TCGA), and statistical analyses were performed to investigate the receptor expression level in the different cell lines and the efficacy of the single-agent treatments. A correlation analysis was performed to quantify the degree of relationship between receptor expression and drug efficacy. With the exception of VEGFR2, the targeted receptors were expressed at different levels in all of the cell lines. The cell lines exhibited concentration-dependent responses with cell line-specific differences toward two of the three TKIs (nintedanib and dovitinib). Notably, all of the cell lines were resistant to pazopanib. TKIs have potential as therapeutic agents for HNSCC. Cell line-specific differences were observed in our invitro experiments. The observed pazopanib resistance could be explained by receptor expression. Further investigation is required to determine TKI efficacy in HNSCC.

Lipoxins exert antiangiogenic and anti-inflammatory effects on Kaposi's sarcoma cells

Lipoxin A4 (LXA4) is an endogenously produced host molecule with anti-inflammatory resolution effects. Previous studies demonstrated it to be involved in anti-vascular endothelial growth factor (VEGF)-mediated angiogenesis and in a possible anticancer role via interaction with its receptor, lipoxin A 4 receptor (ALXR). Here, we examined the effects of LXA4 and its epimer 15-epi-LXA4 in inhibiting proinflammatory and angiogenic functions in a human Kaposi's sarcoma tumor-derived cell line (KS-IMM). KS-IMM cells expressed increased levels of inflammatory cyclooxygenase 2 (COX-2) and 5-lipoxygenase (5-LO) pathway enzymes when compared with human microvascular dermal endothelial cells (HMVEC-d). KS-IMM cells secreted high levels of prostaglandin E2 (PGE2) and chemotactic leukotriene B4 (LTB4). Treatment with LXA4 or 15-epi-LXA4 effectively reduced the levels of COX-2, 5-LO proteins, and secretion of PGE2 and LTB4 in KS-IMM cells. LXA4 or 15-epi-LXA4 treatment also decreased secretion of proinflammatory interleukin 6 (IL-6) and IL-8 cytokines but induced the secretion of anti-inflammatory IL-10. LXA4 treatment reduced the phosphorylation of VEGF receptor (VEGFR) and ephrin family receptor tyrosine kinases. LXA4 treatment effectively induced dephosphorylation of multiple cellular kinases such as Focal Adhesion Kinase, Protein kinase B, nuclear factor kappa-light-chain-enhancer of activated B cells, and Extracellular signal-regulated kinases (ERK)1/2, and reduced angiogenic factor VEGF-C secretion in KS cells. LX treatment drastically induced the Src-homology 2 domain-containing phosphatase tyrosine (Y542) phosphatase and reduced VEGFR-2 phosphorylation at sites Y1059, Y1175, and Y1212. Treatment of KS-IMM cells with LXA4 resulted in selective localization of VEGFR-2 in nonlipid raft (non-LR) and ALXR to LR fractions. These results demonstrated that LXA4 or 15-epi-LXA4 induce anti-inflammatory and antiangiogenic effects in KS cells and suggest that treatment with LXs is an attractive novel strategy against KS.

Vascular endothelial growth factor receptor 3 controls neural stem cell activation in mice and humans.

SUMMARYNeural stem cells (NSCs) continuously produce new neurons within the adult mammalian hippocampus. NSCs are typically quiescent but activated to self-renew or differentiate into neural progenitor cells. The molecular mechanisms of NSC activation remain poorly understood. Here, we show that adult hippocampal NSCs express vascular endothelial growth factor receptor (VEGFR) 3 and its ligand VEGF-C, which activates quiescent NSCs to enter the cell cycle and generate progenitor cells. Hippocampal NSC activation and neurogenesis are impaired by conditional deletion of Vegfr3 in NSCs. Functionally, this is associated with compromised NSC activation in response to VEGF-C and physical activity. In NSCs derived from human embryonic stem cells (hESCs), VEGF-C/VEGFR3 mediates intracellular activation of AKT and ERK pathways that control cell fate and proliferation. These findings identify VEGF-C/VEGFR3 signaling as a specific regulator of NSC activation and neurogenesis in mammals.

Evidence for New Homotypic and Heterotypic Interactions between Transmembrane Helices of Proteins Involved in Receptor Tyrosine Kinase and Neuropilin Signaling

Signaling in eukaryotic cells frequently relies on dynamic interactions of single-pass membrane receptors involving their transmembrane (TM) domains. To search for new such interactions, we have developed a bacterial two-hybrid system to screen for both homotypic and heterotypic interactions between TM helices. We have explored the dimerization of TM domains from 16 proteins involved in both receptor tyrosine kinase and neuropilin signaling. This study has revealed several new interactions. We found that the TM domain of Mucin-4, a putative intramembrane ligand for erbB2, dimerizes not only with erbB2 but also with all four members of the erbB family. In the Neuropilin/Plexin family of receptors, we showed that the TM domains of Neuropilins 1 and 2 dimerize with themselves and also with Plexin-A1, Plexin-B1, and L1CAM, but we were unable to observe interactions with several other TM domains notably those of members of the VEGF receptor family. The potentially important Neuropilin 1/Plexin-A1 interaction was confirmed using a surface plasmon resonance assay. This work shows that TM domain interactions can be highly specific. Exploring further the propensities of TM helix–helix association in cell membrane should have important practical implications related to our understanding of the structure–function of bitopic proteins' assembly and subsequent function, especially in the regulation of signal transduction.

The inhibition of tyrosine kinase receptor signalling in leiomyosarcoma cells using the small molecule kinase inhibitor PTK787/ZK222584(Vatalanib®).

Leiomyosarcomas remain challenging tumors to manage and novel therapy strategies besides radiation and conventional chemotherapy are needed. Targeting angiogenesis by inhibition of vascular endothelial growth factor (VEGF) receptor tyrosine kinases (RTKs) of the tumor vasculature with small molecules is a promising new therapy. It has been shown recently that these receptors are not only expressed on tumor endothelium but also on tumor cells themselves. Thus, we investigated the expression of members of the VEGF receptor (VEGFR) family and corresponding growth factors in leiomyosarcoma tissue specimens and in the leiomyosarcoma cell lines SK-LMS-1 and SK-UT-1. We evaluated the influence of the VEGFR inhibitor PTK787/ZK222584 (PTK787) on cell growth, migration, apoptosis and phosphorylation of intracellular signalling molecules. In human leiomyosarcoma tissue specimens VEGFR-1/-2 and platelet-derived growth factor receptor (PDGFR-β) were strongly expressed. Both leiomyosarcoma cell lines expressed VEGFR-1/-3 and PDGFR-β but VEGFR-2 protein expression was positive only in SK-UT-1. SK-LMS-1 and SK-UT-1 cells secreted high and low amounts of VEGF-A, respectively, whereas PDGF-BB secretion was similar in both cell lines. Application of PTK787 led to partial inhibition of PDGF-BB-activated AKT/p90RSK and ERK1/2 signalling pathways. In contrast, protein phosphorylation was not affected by PTK787 in VEGF-A-treated cells. PTK787 turned out to inhibit cell migration even though no effects were observed upon stimulation with VEGF-A or PDGF-BB. In line, cell growth in leiomyosarcoma cell lines remained unchanged upon PTK787 treatment alone and with subsequent VEGF-A- or PDGF-BB-stimulation. However, VEGF-A, but not PDGF-BB-treated cells showed increased cell death upon PTK787 treatment. VEGFR family members are expressed in leiomyosarcomas in vivo and in vitro. Upon receptor stimulation, PTK787 is able to inhibit subsequent phosphorylation events and influences cell survival but not metabolic activity and migration. Thus, the inhibitor is possibly an additional option in the treatment of leiomyosarcomas.

Systematic evaluation of vascular endothelial growth factor receptor expression in primary ovarian cancer and its clinical relevance

Objectives: The identification of novel molecular biomarkers, predicting outcome of ovarian cancer, is highly desirable. Considering that angiogenesis is a critical factor for ascites development and peritoneal dissemination in ovarian cancer and given that the vascular endothelial growth factor (VEGF) receptor signaling axis is a major driver of angiogenesis, we sought to analyze expression and compartmental distribution of VEGF-receptor family in ovarian cancer and to assess its clinical relevance with regard to established clinicopathological parameters, tumor cell dissemination to the bone marrow (BM) and the patient's survival.

Preparation and antitumor effect of a toxin-linked conjugate targeting vascular endothelial growth factor receptor and urokinase plasminogen activator.

The aberrant signaling activation of vascular endothelial growth factor receptor (VEGFR) and urokinase plasminogen activator (uPA) is a common characteristic of many tumors, including lung cancer. Accordingly, VEGFR and uPA have emerged as attractive targets for tumor. KDR (Flk-1/VEGFR-2), a member of the VEGFR family, has been recognized as an important target for antiangiogenesis in tumor. In this study, a recombinant immunotoxin was produced to specifically target KDR-expressing tumor vascular endothelial cells and uPA-expressing tumor cells and mediate antitumor angiogenesis and antitumor effect. Based on its potent inhibitory effect on protein synthesis, Luffin-beta (Lβ) ribosome-inactivating protein was selected as part of a recombinant fusion protein, a single-chain variable fragment against KDR (KDRscFv)-uPA cleavage site (uPAcs)-Lβ-KDEL (named as KPLK). The KDRscFv-uPAcs-Lβ-KDEL (KPLK) contained a single-chain variable fragment (scFv) against KDR, uPAcs, Lβ, and the retention signal for endoplasmic reticulum proteins KDEL (Lys-Asp-Glu-Leu). The KPLK-expressing vector was expressed in Escherichia coli, and the KPLK protein was isolated with nickel affinity chromatography and gel filtration chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis test demonstrated KPLK was effectively expressed. Result of invitro cell viability assay on non-small cell lung cancer (NSCLC) H460 cell line (uPA-positive cell) revealed that KPLK significantly inhibited cell proliferation, induced apoptosis, and accumulated cells in S and G2/M phases, but the normal cell line (human submandibular gland cell) was unaffected. These effects were enhanced when uPA was added to digest KPLK to release Lβ. For invivo assay of KPLK, subcutaneous xenograft tumor model of nude mice were established with H460 cells. Growth of solid tumors was significantly inhibited in animals treated with KPLK up to 21 days, tumor weights were decreased, and the expression of angiogenesis marker CD31 was downregulated; meanwhile, the apoptosis-related protein casspase-3 was upregulated. These results suggested that the recombinant KPLK may have therapeutic applications on tumors, especially uPA-overexpressing ones.

Linifanib (ABT-869), enhances cytotoxicity with poly (ADP-ribose) polymerase inhibitor, veliparib (ABT-888), in head and neck carcinoma cells

PARP inhibitors (PARPi) may provide an opportunity to gain selective killing of tumor cells which have deficiencies in cellular DNA repair systems. We previously demonstrated linifanib (ABT-869), a multi-receptor tyrosine kinase inhibitor of VEGF and PDGF receptor families, radiosensitized Head and Neck Squamous Cell Carcinoma cells (HNSCC) via inhibiting STAT3 activation. Given that STAT3 can modulate DNA damage response (DDR) pathway, in this study, we evaluate the effects of linifanib to enhance cytotoxicity with the PARPi, veliparib (ABT-888), in HNSCC. UMSCC-22A and UMSCC-22B cells were treated with linifanib (ABT-869) and veliparib (ABT-888). Cell viability, cytotoxicity, apoptosis induction, DNA single strand break (SSB) and double strand break (DSB) damages were examined by MTT assay, colony formation assay, flow cytometry and comet assay. In addition, the expression of DNA homologous recombination repair protein Rad51, γH2AX, a double strand break marker and cleaved PARP, an apoptotic cell death marker, were assessed using western immunoblotting. Combination treatment resulted in more cell growth inhibition, induction of apoptosis, DNA damages and double strand breaks, lower expression of Rad51, increase γH2AX expression and PARP cleavage. These data suggest the possibility of combining targeted therapeutic such as linifanib with veliparib to augment the inhibition of cell growth and apoptosis via synthetic lethality in HNSCC cells. Thus, it may provide a novel therapeutic strategy and improve efficacy and outcome in HNSCC.

Explorative investigation of vascular endothelial growth factor receptor expression in primary ovarian cancer and its clinical relevance

ObjectivesThe identification of novel molecular biomarkers, predicting outcome of ovarian cancer, is highly desirable. Considering that angiogenesis is a critical factor for ascites development and peritoneal dissemination in ovarian cancer and given that the vascular endothelial growth factor (VEGF) receptor signaling axis is a major driver of angiogenesis, we sought to analyze expression and compartmental distribution of VEGF-receptor family in ovarian cancer and to assess its clinical relevance with regard to established clinicopathological parameters, tumor cell dissemination to the bone marrow (BM) and the patient's survival. MethodsA total of 73 patients with primary ovarian cancer were enrolled into this study. Primary tumor tissue was analyzed for the expression of VEGF-R1, VEGF-R2 and VEGF-R3 by immunohistochemistry. The presence of disseminated tumor cells (DTC) in the BM was analyzed by immunocytochemistry using the pancytokeratin antibody A45B/B3 and subsequent automatic detection based on staining and cytomorphology. ResultsIn primary ovarian cancer tissue, VEGF-receptor expression, detected with an overall frequency of 44%, was mostly located in the vascular wall and across the stroma; positivity rates for VEGF-R1, VEGF-R2 and VEGF-R3 were 34%, 18% and 26%, respectively. Total VEGF-receptor expression correlated with residual tumor after primary debulking surgery and the presence of DTC at primary diagnosis (p=0.035, p=0.023, respectively). Interestingly, VEGF-R1 positivity significantly correlated with decreased progression-free survival (p=0.026). ConclusionsThis is the first report, suggesting total VEGF-receptor status as a molecular biomarker for monitoring tumor cell spread to the BM and, particularly, revealing prognostic significance of VEGF-R1.

The progression free survival-plateau with vascular endothelial growth factor receptor inhibitors – Is there more to come?

The field of renal cell carcinoma (RCC) treatment has changed dramatically during recent years. Sunitinib, sorafenib and pazopanib were the first generation of vascular endothelial growth factor receptor (VEGFR) tyrosine kinase inhibitors (TKIs) to have significant clinical activity in metastatic clear cell RCC. These TKI share inhibition of VEGFR family members, but differ in their ability to block other cellular kinases. Axitinib and tivozanib are 2nd generation TKIs, which show high specificity for VEGFR inhibition and exert a favourable toxicity profile. Both agents have succeeded in pivotal clinical trials, which were the first studies to compare two distinct TKIs. Progression free survival (PFS) shows an advantage for the 2nd generation TKIs, but also curbs enthusiasm for limitless PFS expectations with a PFS plateau of 13–14months in 1st line treatment. More recently, novel targets have gained attention in RCC, such as the mesenchymal epithelial transition factor also known as the MET receptor and the fibroblast growth factor receptor (FGFR). These receptors are included into the inhibitory profiles of third generation TKIs such as cabozantinib or dovitinib, which showed promising activity in early clinical trials. Randomised controlled trials explore the role of these agents, and whether the expansion of targets inhibited may lead to more effective treatments in RCC.

Abstract 4544: Novel TDA technology and high content imaging as complementary assay platforms to identify VEGFR-2 inhibitors.

Abstract Vascular endothelial growth factor (VEGF) is potent mitogen for endothelial cells that signals through VEGF Receptor-type2 (VEGFR-2)/Flk-1/KDR. Binding of VEGF to VEGFR-2, a membrane bound receptor tyrosine kinase of the VEGF-receptor family, leads to receptor dimerization, protein kinase activation, trans-autophosphorylation, and initiation of signaling pathways stimulating angiogenesis. In adults, angiogenesis normally occurs during wound healing to restore blood flow post injury and during menstruation and pregnancy. Angiogenesis is also involved in several pathological disease states such as rheumatoid arthritis, psoriasis, diabetic retinopathy and cancer. While physiological angiogenesis involves interplay between pro- and anti-angiogenic factors, the ability to inhibit pathological angiogenesis has therapeutic potential. Inhibitors of VEGFR-2 provide targeted treatments for tumor growth as well as abnormal neovascularization related diseases. The caveat of traditional biochemical VEGFR-2 assays is that only the catalytic domain of the receptor is used to identify hits. One strategy to identify true novel inhibitors against membrane associated proteins is to screen these targets in a more physiological environment, where lipid-protein and protein-protein interactions are included in the assay. We achieved this goal by docking the entire cytoplasmic domain of the receptor on a liposome. Template Directed Assembly (TDA) is a screening technology designed to accomplish this objective. TDA technology utilizes recombinant VEGFR-2 cytoplasmic domain tagged with polyhistidine at the juxtamembrane position and docked to the liposome via the nickel residues. The lipids in TDA are able to diffuse within the surface of the liposome and, therefore, proteins can diffuse to form oligomeric complexes such as homo- or hetero-dimers formation. To compliment the TDA biochemical screen, we are implementing an image based high content screen (HCS) utilizing human umbilical vein endothelial cells (HUVECs) that express native VEGFR-2. The HCS screen is designed to identify potent and specific inhibitors of VEGFR-2 as assessed by a reduction in the proliferation marker Ki67. We are currently (1) validating the TDA assay with known VEGFR-2 inhibitors (2) screening a focus small molecule library to identify novel hits (3) testing the primary hits against a kinase selectivity panel and (4) designing and developing a VEGFR2 cell-based HCS assay to confirm the primary hits. The hit to lead program will be guided by using both physiologically relevant biochemical-based and cell-based assays. The combination of our novel TDA screening platform and the HCS assay technology permits us to identify unique VEGFR-2 inhibitors. Citation Format: Kelvin Lam, Edward Esposito, Deanna Navaroli, Ryan Clinton, Kerrin Beovich, Norman Garceau. Novel TDA technology and high content imaging as complementary assay platforms to identify VEGFR-2 inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4544. doi:10.1158/1538-7445.AM2013-4544

Post-axitinib systemic therapy in metastatic renal cell carcinoma (mRCC).

4609 Background: Axitinib is a potent and selective inhibitor of the VEGF receptor family with efficacy as a second-line therapy in mRCC. Evaluation of further systemic therapy following axitinib has not been previously reported. Methods: The medical records ofmRCCpatients (pts) treated at The Cleveland Clinic or MD Anderson Cancer Center who received axitinib were retrospectively reviewed. Patient characteristics including best response and duration of treatment on axitinib and each subsequent line of therapy were recorded. Results: Eighty-one patients were initially identified; 29 patients received at least one approved targeted agent following axitinib and are included in this analysis. The remainder of pts were excluded due to ongoing axitinib (n=16), lack of subsequent therapy (n=13), lost to follow up (n=11), received experimental agents post-axitinib (n=7) or were inevaluable (n=2). Three pts were excluded due to ≤ 21 days on axitinib. Pt characteristics at start of axitinib included 76% male, median age 59 (range, 44-78); 97% clear cell; 93% prior nephrectomy; 64% previously-treated. Pts were favorable (39%) or intermediate (60%) risk based on Heng criteria. Overall response rate to axitinib was 52% and median duration of treatment of 11.2 months (range, 1.1-90). Thirty eight percent of patients had new brain, bone, and/or liver metastases at disease progression on axitinib. For the first subsequent therapy post axitinib (n=25), pts were treated with tyrosine kinase inhibitors (72%) or mTOR inhibitors (28%). Overall 8% of pts achieved partial responses (one pt each to sunitinib and pazopanib) and 52% had a best response of stable disease. Estimated median duration of therapy was 4.4 months (range, 0.2-27.5+). Fifteen pts received a second post-axitinib targeted therapy with mTOR inhibitors (60%) or TKIs (40%). One patient (7%) achieved a partial response to pazopanib and 8 patients (53%) had a best response of SD. Estimated median treatment duration was 4.8 months (range, 0.7-19.1+). Conclusions: There are objective responses and stable disease to post-axitinib targeted therapies, consistent with previous series and trials in refractory RCC patients.

Abstract 3970: Model-driven investigation of VEGF binding to endothelial cells and implications of the spatial organization of the cell membrane

Abstract VEGF signaling plays an important role in the development and maintenance of blood vessels and it has received much attention due to its role in tumor development and other pathologies. Recent developments in therapeutics have highlighted the complex and often contradictory features of cell signaling mediated by the VEGF ligand and receptor family. The aim of this study is to constrain the effective parameters of a minimal mathematical model [1] of the first steps in VEGF signaling, namely, the molecular transformations that occur from the presentation of VEGF ligand in the extracellular space to the formation of activated ligand-receptor complexes on the cell membrane. This study addresses unique properties of VEGF due to its bivalent nature. Bivalence complicates signal initiation, which requires the formation of a complex of two receptors cross-linked by a ligand. Unlike the initial capture of ligand from the surrounding volume, the second binding step occurs between proteins bound to the cell membrane, and is therefore modulated by the lateral mobility of the receptors, which in turn reflects the limitations to free movement due to various features of the cell membrane. The kinetics of cross-linking are reflected in (i) the degree of reversibility of VEGF binding to the cell, and (ii) the phenomenon of high dose inhibition. Thus, the empirically derived kinetics that defines the well mixed model [1] help integrate binding data with high resolution imaging and single particle tracking studies of the cell membrane, two classes of experimental information that are hard to compare directly. The binding of VEGF to human umbilical vascular endothelial cells (HUVEC) has been well studied in the literature, and was the basis of the original parameterization of the model [1]. However, those experiments did not pursue the distinction between the two different binding steps, and the kinetics of cross-linking was not well determined. To address this, we use a synthetic compound, single-chain VEGF [2], conjugated to a fluorescent tag (scVEGF-Cy5.5). This ligand has two functional binding sites and its binding affinity and physiological effect on cells expressing VEGFR-2 closely reproduce those of VEGF165. We used flow cytometry to quantify the binding of scVEGF-Cy5.5 to HUVEC in vitro. We obtained dose response curves and time courses for total binding in solution, for incubation times from 30 - 120 minutes and ligand concentrations from 0.1 - 200 nM. The data show possible signs of high dose inhibition in the 100 nM range, and allow us to estimate a range for the characteristic time of cross-linking. Subsequent experiments will directly probe this time range, based on use of this versatile probe for imaging studies of receptor localization and mobility in the cell membrane.

Abstract 2337: Regorafenib (BAY 73-4506): Anti-metastatic activity in a mouse model of colorectal cancer

Abstract Regorafenib is an oral multi-kinase inhibitor which has recently been reported to extend overall survival in a Phase III trial in patients with metastatic colorectal tumors who have failed standard therapies (CORRECT trial) when given with best supportive care (BSC) vs placebo plus BSC. It is also examined in a Phase III trial for the treatment of patients with metastatic and/or unresectable gastrointestinal stromal tumors who have failed imatinib and sunitinib therapy (GRID trial). Regorafenib deactivates tumors across three dimensions (angiogenesis, oncogenesis, and stromatogenesis) by inhibition of angiogenic receptor tyrosine kinases (RTK) (e.g., VEGFR1-3, TIE2), oncogenic (e.g., KIT, RET) and stromal RTKs (e.g., PDGFR, FGFR). In preclinical studies it was previously shown that regorafenib inhibits a broad spectrum of different tumor types including colorectal cancer (CRC) models. Regorafenib was efficacious as a single agent and in combination with irinotecan in CRC models and demonstrated anti-metastatic activities in preclinical models of breast cancer. VEGFR-3, a member of the VEGF receptor family, is thought to play an important role in tumor lymphangiogenesis and metastasis. Regorafenib potently inhibited (4-8 nM) VEGF-C stimulated VEGFR-3 receptor autophosphorylation in human dermal lymphatic endothelial cells (LECs) and inhibited VEGF-C stimulated LEC cell migration in a scratch assay at concentrations below 100 nM. In vivo regorafenib's antimetastatic potential was examined in a syngeneic CRC liver metastasis model. In this model murine MC38 CRC cells were injected into the mouse spleen with following ablation of the organ. Significant survival of mice treated orally at a daily dose of 10 mg/kg regorafenib was observed compared to vehicle-treated control mice. Liver weights, which were used as read-out for the degree of metastases formation, was reduced in mice treated with regorafenib for thirteen days vs vehicle treated animals. This was accompanied by significantly reduced number of metastases observed outside the liver. Finally, the direct effects of regorafenib in CRC cell lines was evaluated in vitro in a panel of 25 different human CRC cell lines where it inhibited the proliferation of 19 cell lines with IC50 values between 1 and 10µM and no correlation with mutated KRAS or BRAF was observed. Effects of regorafenib on intracellular MAPK signalling in CRC cell lines was analyzed by western blotting. Regorafenib, which also inhibits Raf kinases, reduced pERK by 50% at concentrations of about 500-2000 nM in HT29, HCT116 and Colo-205 CRC cell lines. In summary, these results suggest that regorafenib may exert its anti-tumor activity in colorectal cancer by inhibition of multiple kinases across its three dimensions within the tumor environment. Additional analyses in particular of clinical tumor samples are necessary to more clearly understand regorafenib's molecular mechanisms of action. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2337. doi:1538-7445.AM2012-2337

Role of Angiogenesis Inhibitors in Colorectal Cancer: Sensitive and Insensitive Tumors

Angiogenesis is a key factor in the carcinogenesis process. In oncological practice, angiogenesis inhibition, mainly through the blockade of the VEGF family and its receptors, has been robustly demonstrated to produce clinical benefits and, in specific disease subsets such as colorectal cancer, to extend the overall survival of treated patients. VEGF is a multifunctional growth factor that mediates its functions through cognate receptors on endothelial cells and it has been discovered for its capability to induce macromolecule hyperpermeability in veins and venules. Several approaches have been taken to target angiogenesis in cancer: drugs that target one or more soluble ligands of the VEGF family, drugs that selectively inhibit one or more receptors of the VEGF receptor family, and drugs that inhibit VEGF receptor(s) among other, non VEGF-related targets. At present, two compounds have shown significant clinical activity, bevacizumab, Avastin® and aflibercept, Zaltrap®, and only one of these (bevacizumab) has so far been registered for use in clinical practice. In the present review, we explore and summarize the main features of the angiogenetic process, concerning in particular a common and potentially lethal disease as colorectal cancer. We overview the molecular pathways that characterize angiogenesis, focusing on VEGF family, the current applications and limitations of its blockade in oncology, and the hypothetical future perspectives of anti-angiogenic therapy.