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Figure S1: Impedance barrier function assay: The effect of S1P and Ex82 was analyzed using a transendothelial electrical impedance assay. S1P treatment (10 nM) of an endothelial monolayer strongly increases electrical impedance (B) vs control (A) whereas Ex82 has the opposite effect and significantly decreases electrical impedance (D). These results are consistent with the known barrier function of S1P1. In addition, pretreatment with Ex82 blocked the S1P dependent increase in electrical impedance (C).

Inhibition of VEGFR signaling is an effective treatment for renal cell carcinoma, but resistance continues to be a major problem. Recently, the sphingosine phosphate (S1P) signaling pathway has been implicated in tumor growth, angiogenesis, and resistance to antiangiogenic therapy. S1P is a bioactive lipid that serves an essential role in developmental and pathologic angiogenesis via activation of the S1P receptor 1 (S1P1). S1P1 signaling counteracts VEGF signaling and is required for vascular stabilization. We used in vivo and in vitro angiogenesis models including a postnatal retinal angiogenesis model and a renal cell carcinoma murine tumor model to test whether simultaneous inhibition of S1P1 and VEGF leads to improved angiogenic inhibition. Here, we show that inhibition of S1P signaling reduces the endothelial cell barrier and leads to excessive angiogenic sprouting. Simultaneous inhibition of S1P and VEGF signaling further disrupts the tumor vascular beds, decreases tumor volume, and increases tumor cell death compared with monotherapies. These studies suggest that inhibition of angiogenesis at two stages of the multistep process may maximize the effects of antiangiogenic therapy. Together, these data suggest that combination of S1P1 and VEGFR-targeted therapy may be a useful therapeutic strategy for the treatment of renal cell carcinoma and other tumor types.

Figure S2: Co-targeting S1P1 and VEGFR2 pathways induced reduction in permeability of retinal vessels. Ter 119 staining (panels A) visualize red blood cells and show that the combination of DC101 and the S1P1 antagonist in the retina led to reduction of the areas of hemorrhage within the vascular plexus and restricted only to the angiogenic front indicating that the remaining vessels in the remodeled area are less permeable.

These figures contain extensions of data that would not fit in the primary manuscript but are important for a complete understanding of the work. These include analysis of other data sets, correlations between genomic profiling our stromal subtyping and information on bioinformatics and animal models.

Treatment of metastatic gastric cancer typically involves chemotherapy and monoclonal antibodies targeting HER2 (ERBB2) and VEGFR2 (KDR). However, reliable methods to identify patients who would benefit most from a combination of treatment modalities targeting the tumor stroma, including new immunotherapy approaches, are still lacking. Therefore, we integrated a mouse model of stromal activation and gastric cancer genomic information to identify gene expression signatures that may inform treatment strategies. We generated a mouse model in which VEGF-A is expressed via adenovirus, enabling a stromal response marked by immune infiltration and angiogenesis at the injection site, and identified distinct stromal gene expression signatures. With these data, we designed multiplexed IHC assays that were applied to human primary gastric tumors and classified each tumor to a dominant stromal phenotype representative of the vascular and immune diversity found in gastric cancer. We also refined the stromal gene signatures and explored their relation to the dominant patient phenotypes identified by recent large-scale studies of gastric cancer genomics (The Cancer Genome Atlas and Asian Cancer Research Group), revealing four distinct stromal phenotypes. Collectively, these findings suggest that a genomics-based systems approach focused on the tumor stroma can be used to discover putative predictive biomarkers of treatment response, especially to antiangiogenesis agents and immunotherapy, thus offering an opportunity to improve patient stratification. Cancer Res; 76(9); 2573–86. ©2016 AACR.

These figures contain extensions of data that would not fit in the primary manuscript but are important for a complete understanding of the work. These include analysis of other data sets, correlations between genomic profiling our stromal subtyping and information on bioinformatics and animal models.

This file contains details too lengthy to put in the primary manuscript file as well as methods for supplemental data

Supplemental methodology, specifically the in vitro cord formation assay and the enzyme-linked immunosorbent assay.

These are the figure legends for the supplemental figures and tables.

Purpose: Checkpoint kinase 1 (CHK1) is a key regulator of the DNA damage response and a mediator of replication stress through modulation of replication fork licensing and activation of S and G2–M cell-cycle checkpoints. We evaluated prexasertib (LY2606368), a small-molecule CHK1 inhibitor currently in clinical testing, in multiple preclinical models of pediatric cancer. Following an initial assessment of prexasertib activity, this study focused on the preclinical models of neuroblastoma.Experimental Design: We evaluated the antiproliferative activity of prexasertib in a panel of cancer cell lines; neuroblastoma cell lines were among the most sensitive. Subsequent Western blot and immunofluorescence analyses measured DNA damage and DNA repair protein activation. Prexasertib was investigated in several cell line–derived xenograft mouse models of neuroblastoma.Results: Within 24 hours, single-agent prexasertib promoted γH2AX–positive double-strand DNA breaks and phosphorylation of DNA damage sensors ATM and DNA–PKcs, leading to neuroblastoma cell death. Knockdown of CHK1 and/or CHK2 by siRNA verified that the double-strand DNA breaks and cell death elicited by prexasertib were due to specific CHK1 inhibition. Neuroblastoma xenografts rapidly regressed following prexasertib administration, independent of starting tumor volume. Decreased Ki67 and increased immunostaining of endothelial and pericyte markers were observed in xenografts after only 6 days of exposure to prexasertib, potentially indicating a swift reduction in tumor volume and/or a direct effect on tumor vasculature.Conclusions: Overall, these data demonstrate that prexasertib is a specific inhibitor of CHK1 in neuroblastoma and leads to DNA damage and cell death in preclinical models of this devastating pediatric malignancy. Clin Cancer Res; 23(15); 4354–63. ©2017 AACR.

The mammalian target of rapamycin (mTOR) pathway is implicated widely in cancer pathophysiology. Dual inhibition of the mTOR kinase complexes mTORC1 and mTORC2 decreases tumor xenograft growth in vivo and VEGF secretion in vitro, but the relationship between these two effects are unclear. In this study, we examined the effects of mTORC1/2 dual inhibition on VEGF production, tumor angiogenesis, vascular regression, and vascular regrowth, and we compared the effects of dual inhibition to mTORC1 inhibition alone. ATP-competitive inhibitors OSI-027 and OXA-01 targeted both mTORC1 and mTORC2 signaling in vitro and in vivo, unlike rapamycin that only inhibited mTORC1 signaling. OXA-01 reduced VEGF production in tumors in a manner associated with decreased vessel sprouting but little vascular regression. In contrast, rapamycin exerted less effect on tumoral production of VEGF. Treatment with the selective VEGFR inhibitor OSI-930 reduced vessel sprouting and caused substantial vascular regression in tumors. However, following discontinuation of OSI-930 administration tumor regrowth could be slowed by OXA-01 treatment. Combining dual inhibitors of mTORC1 and mTORC2 with a VEGFR2 inhibitor decreased tumor growth more than either inhibitor alone. Together, these results indicate that dual inhibition of mTORC1/2 exerts antiangiogenic and antitumoral effects that are even more efficacious when combined with a VEGFR antagonist. Cancer Res; 71(5); 1573–83. ©2011 AACR.

Supplementary Figure 2 from Reduced VEGF Production, Angiogenesis, and Vascular Regrowth Contribute to the Antitumor Properties of Dual mTORC1/mTORC2 Inhibitors

Supplementary Figure 1 from Reduced VEGF Production, Angiogenesis, and Vascular Regrowth Contribute to the Antitumor Properties of Dual mTORC1/mTORC2 Inhibitors

Supplementary Figure Legends 1-2, Methods from Reduced VEGF Production, Angiogenesis, and Vascular Regrowth Contribute to the Antitumor Properties of Dual mTORC1/mTORC2 Inhibitors

Merestinib is an oral multi-kinase inhibitor targeting a limited number of oncokinases including MET, AXL, RON and MKNK1/2. Here, we report that merestinib inhibits neurotrophic receptor tyrosine kinases NTRK1/2/3 which are oncogenic drivers in tumors bearing NTRK fusion resulting from chromosomal rearrangements. Merestinib is shown to be a type II NTRK1 kinase inhibitor as determined by x-ray crystallography. In KM-12 cells harboring TPM3-NTRK1 fusion, merestinib exhibits potent p-NTRK1 inhibition in vitro by western blot and elicits an anti-proliferative response in two- and three-dimensional growth. Merestinib treatment demonstrated profound tumor growth inhibition in in vivo cancer models harboring either a TPM3-NTRK1 or an ETV6-NTRK3 gene fusion. To recapitulate resistance observed from type I NTRK kinase inhibitors entrectinib and larotrectinib, we generated NIH-3T3 cells exogenously expressing TPM3-NTRK1 wild-type, or acquired mutations G595R and G667C in vitro and in vivo. Merestinib blocks tumor growth of both wild-type and mutant G667C TPM3-NTRK1 expressing NIH-3T3 cell-derived tumors. These preclinical data support the clinical evaluation of merestinib, a type II NTRK kinase inhibitor (NCT02920996), both in treatment naïve patients and in patients progressed on type I NTRK kinase inhibitors with acquired secondary G667C mutation in NTRK fusion bearing tumors.

Vascular endothelial growth factor receptor 2 (VEGFR2) is an attractive therapeutic target in solid malignancies due to its central role in tumor angiogenesis. Ramucirumab (Cyramza®, LY3009806) is a human monoclonal antibody specific for VEGFR2 approved for several adult indications and currently in a phase 1 clinical trial for pediatric patients with solid tumors (NCT02564198). Here, we evaluated ramucirumab in vitro and the anti-murine VEGFR2 antibody DC101 in vivo with or without chemotherapy across a range of pediatric cancer models. Ramucirumab abrogated in vitro endothelial cord formation driven by cancer cell lines representing multiple pediatric histologies; this response was independent of the origin of the tumor cell-line. Several pediatric cancer mouse models responded to single agent DC101-mediated VEGFR2 inhibition with tumor growth delay. Preclinical stable disease and partial xenograft regressions were observed in mouse models of Ewing's sarcoma, synovial sarcoma, neuroblastoma, and desmoplastic small round cell tumor treated with DC101 and cytotoxic chemotherapy. In contrast, DC101 treatment in osteosarcoma models had limited efficacy alone or in combination with chemotherapeutics. Our data indicate differential efficacy of targeting the VEGFR2 pathway in pediatric models and support the continued evaluation of VEGFR2 inhibition in combination with cytotoxic chemotherapy in multiple pediatric indications.

Interaction of numerous signaling pathways in endothelial and mesangial cells results in exquisite control of the process of physiological angiogenesis, with a central role played by vascular endothelial growth factor receptor 2 (VEGFR-2) and its cognate ligands. However, deregulated angiogenesis participates in numerous pathological processes. Excessive activation of VEGFR-2 has been found to mediate tissue-damaging vascular changes as well as the induction of blood vessel expansion to support the growth of solid tumors. Consequently, therapeutic intervention aimed at inhibiting the VEGFR-2 pathway has become a mainstay of treatment in cancer and retinal diseases. In this review, we introduce the concepts of physiological and pathological angiogenesis, the crucial role played by the VEGFR-2 pathway in these processes, and the various inhibitors of its activity that have entered the clinical practice. We primarily focus on the development of ramucirumab, the antagonist monoclonal antibody (mAb) that inhibits VEGFR-2 and has recently been approved for use in patients with gastric, colorectal, and lung cancers. We examine in-depth the pre-clinical studies using DC101, the mAb to mouse VEGFR-2, which provided a conceptual foundation for the role of VEGFR-2 in physiological and pathological angiogenesis. Finally, we discuss further clinical development of ramucirumab and the future of targeting the VEGF pathway for the treatment of cancer.

Treatment of metastatic gastric cancer typically involves chemotherapy and monoclonal antibodies targeting HER2 (ERBB2) and VEGFR2 (KDR). However, reliable methods to identify patients who would benefit most from a combination of treatment modalities targeting the tumor stroma, including new immunotherapy approaches, are still lacking. Therefore, we integrated a mouse model of stromal activation and gastric cancer genomic information to identify gene expression signatures that may inform treatment strategies. We generated a mouse model in which VEGF-A is expressed via adenovirus, enabling a stromal response marked by immune infiltration and angiogenesis at the injection site, and identified distinct stromal gene expression signatures. With these data, we designed multiplexed IHC assays that were applied to human primary gastric tumors and classified each tumor to a dominant stromal phenotype representative of the vascular and immune diversity found in gastric cancer. We also refined the stromal gene signatures and explored their relation to the dominant patient phenotypes identified by recent large-scale studies of gastric cancer genomics (The Cancer Genome Atlas and Asian Cancer Research Group), revealing four distinct stromal phenotypes. Collectively, these findings suggest that a genomics-based systems approach focused on the tumor stroma can be used to discover putative predictive biomarkers of treatment response, especially to antiangiogenesis agents and immunotherapy, thus offering an opportunity to improve patient stratification. Cancer Res; 76(9); 2573–86. ©2016 AACR.

Antiangiogenesis–based cancer therapies, specifically those targeting the VEGF-A/VEGFR2 pathway, have been approved for subsets of solid tumors. However, these therapies result in an increase in hematologic adverse events. We surmised that both the bone marrow vasculature and VEGF receptor–positive hematopoietic cells could be impacted by VEGF pathway–targeted therapies. We used a mouse model of spontaneous breast cancer to decipher the mechanism by which VEGF pathway inhibition alters hematopoiesis. Tumor-bearing animals, while exhibiting increased angiogenesis at the primary tumor site, showed signs of shrinkage in the sinusoidal bone marrow vasculature accompanied by an increase in the hematopoietic stem cell–containing Lin-cKit+Sca1+ (LKS) progenitor population. Therapeutic intervention by targeting VEGF-A, VEGFR2, and VEGFR3 inhibited tumor growth, consistent with observed alterations in the primary tumor vascular bed. These treatments also displayed systemic effects, including reversal of the tumor-induced shrinkage of sinusoidal vessels and altered population balance of hematopoietic stem cells in the bone marrow, manifested by the restoration of sinusoidal vessel morphology and hematopoietic homeostasis. These data indicate that tumor cells exert an aberrant systemic effect on the bone marrow microenvironment and VEGF-A/VEGFR targeting restores bone marrow function. Cancer Res; 76(3); 517–24. ©2015 AACR.

Inhibition of VEGFR signaling is an effective treatment for renal cell carcinoma, but resistance continues to be a major problem. Recently, the sphingosine phosphate (S1P) signaling pathway has been implicated in tumor growth, angiogenesis, and resistance to antiangiogenic therapy. S1P is a bioactive lipid that serves an essential role in developmental and pathologic angiogenesis via activation of the S1P receptor 1 (S1P1). S1P1 signaling counteracts VEGF signaling and is required for vascular stabilization. We used in vivo and in vitro angiogenesis models including a postnatal retinal angiogenesis model and a renal cell carcinoma murine tumor model to test whether simultaneous inhibition of S1P1 and VEGF leads to improved angiogenic inhibition. Here, we show that inhibition of S1P signaling reduces the endothelial cell barrier and leads to excessive angiogenic sprouting. Simultaneous inhibition of S1P and VEGF signaling further disrupts the tumor vascular beds, decreases tumor volume, and increases tumor cell death compared with monotherapies. These studies suggest that inhibition of angiogenesis at two stages of the multistep process may maximize the effects of antiangiogenic therapy. Together, these data suggest that combination of S1P1 and VEGFR-targeted therapy may be a useful therapeutic strategy for the treatment of renal cell carcinoma and other tumor types.

Blood vessels are crucial components for normal tissue development and homeostasis, so it is not surprising that endothelial dysfunction and dysregulation results in a variety of different pathophysiological conditions. The large number of vascular-related disorders and the emergence of angiogenesis as a major hallmark of cancer has led to significant interest in the development of drugs that target the vasculature. While several in vivo models exist to study developmental and pathological states of blood vessels, few in vitro assays have been developed that capture the significant complexity of the vascular microenvironment. Here, we describe a high content endothelial colony forming cells (ECFC)/adipose-derived stem cell (ADSC) coculture assay that captures many elements of in vivo vascular biology and is ideal for in vitro screening of compounds for pro- or anti-angiogenic activities.

Targeting multiple hallmarks of cancer with drug combinations may provide unique opportunities for cancer therapeutics; however, phenotypic quantification is necessary to understand in vivo mechanisms of action of each drug alone or in combination. Immunohistochemistry (IHC) can quantify phenotypic changes, but traditional methods are not amenable for high-throughput drug discovery. In this article, we describe a high-content method to quantify changes in tumor angiogenesis, vascular normalization, hypoxia, tumor cell proliferation, and apoptosis using IHC. This method to quantify tumor model phenotypes can be useful for cancer drug discovery by increasing the understanding of: (i) tumor models used in efficacy studies, (ii) changes occurring during the growth of the tumor, and (iii) novel mechanisms of actions of cancer therapeutics.

Purpose: Checkpoint kinase 1 (CHK1) is a key regulator of the DNA damage response and a mediator of replication stress through modulation of replication fork licensing and activation of S and G2–M cell-cycle checkpoints. We evaluated prexasertib (LY2606368), a small-molecule CHK1 inhibitor currently in clinical testing, in multiple preclinical models of pediatric cancer. Following an initial assessment of prexasertib activity, this study focused on the preclinical models of neuroblastoma. Experimental Design: We evaluated the antiproliferative activity of prexasertib in a panel of cancer cell lines; neuroblastoma cell lines were among the most sensitive. Subsequent Western blot and immunofluorescence analyses measured DNA damage and DNA repair protein activation. Prexasertib was investigated in several cell line–derived xenograft mouse models of neuroblastoma. Results: Within 24 hours, single-agent prexasertib promoted γH2AX–positive double-strand DNA breaks and phosphorylation of DNA damage sensors ATM and DNA–PKcs, leading to neuroblastoma cell death. Knockdown of CHK1 and/or CHK2 by siRNA verified that the double-strand DNA breaks and cell death elicited by prexasertib were due to specific CHK1 inhibition. Neuroblastoma xenografts rapidly regressed following prexasertib administration, independent of starting tumor volume. Decreased Ki67 and increased immunostaining of endothelial and pericyte markers were observed in xenografts after only 6 days of exposure to prexasertib, potentially indicating a swift reduction in tumor volume and/or a direct effect on tumor vasculature. Conclusions: Overall, these data demonstrate that prexasertib is a specific inhibitor of CHK1 in neuroblastoma and leads to DNA damage and cell death in preclinical models of this devastating pediatric malignancy. Clin Cancer Res; 23(15); 4354–63. ©2017 AACR.

Background Antiangiogenic agents have established efficacy in the treatment of metastatic colorectal cancer. We investigated whether bevacizumab could improve disease-free survival in the adjuvant setting after resection of the primary tumour. Methods For the open-label, randomised, controlled QUASAR 2 trial, which was done at 170 hospitals in seven countries, we recruited patients aged 18 years or older with WHO performance status scores of 0 or 1 who had undergone potentially curative surgery for histologically proven stage III or high-risk stage II colorectal cancer. Patients were randomly assigned (1:1) to receive eight 3-week cycles of oral capecitabine alone (1250 mg/m2 twice daily for 14 days followed by a break for 7 days) or the same regimen of oral capecitabine plus 16 cycles of 7·5 mg/kg bevacizumab by intravenous infusion over 90 min on day 1 of each cycle. Randomisation was done by a computer-generated schedule with use of minimisation with a random element stratified by age, disease stage, tumour site, and country. The study was open label and no-one was masked to treatment assignment. The primary endpoint was 3-year disease-free survival, assessed in the intention-to-treat population. Toxic effects were assessed in patients who received at least one dose of randomised treatment. This trial is registered with the ISRCTN registry, number ISRCTN45133151. Findings Between April 25, 2005, and Oct 12, 2010, 1952 eligible patients were enrolled, of whom 1941 had assessable data (968 in the capecitabine alone group and 973 in the capecitabine and bevacizumab group). Median follow-up was 4·92 years (IQR 4·00–5·16). Disease-free survival at 3 years did not differ between the groups (75·4%, 95% CI 72·5–78·0 in the capecitabine and bevacizumab group vs 78·4%, 75·7–80·9 in the capecitabine alone group; hazard ratio 1·06, 95% CI 0·89–1·25, p=0·54). The most common grade 3–4 adverse events were hand–foot syndrome (201 [21%] of 963 in the capecitabine alone group vs 257 [27%] of 959 in the capecitabine and bevacizumab group) and diarrhoea (102 [11%] vs 104 [11%]), and, with the addition of bevacizumab, expected increases were recorded in all-grade hypertension (320 [33%] vs 75 [8%]), proteinuria (197 [21%] vs 49 [5%]), and wound healing problems (30 [3%] vs 17 [2%]). 571 serious adverse events were reported (221 with capecitabine alone and 350 with capecitabine and bevacizumab). Most of these were gastrointestinal (n=245) or cardiovascular (n=169). 23 deaths within 6 months of randomisation were classified as being related to treatment, eight in the capecitabine alone group and 15 in the capecitabine and bevacizumab group. Interpretation The addition of bevacizumab to capecitabine in the adjuvant setting for colorectal cancer yielded no benefit in the treatment of an unselected population and should not be used.

Lung cancer is a leading cause of cancer death worldwide. ERK1/2, a key downstream effector of RAS mutations, is involved in the signaling network which drives cell proliferation, survival, metastasis and cancer resistance to drug treatment. KRAS mutation driven MAPK activation is present in ~ 30% of lung cancer patients. Non-small cell lung cancer (NSCLC) patients with KRAS mutation have poor prognosis and represents an unmet medical need. LY3214996, an ERK1/2 inhibitor which is in a phase I clinical trial, has anti-tumor activities in RAS mutant tumor cells in vitro and in vivo (http://cancerres.aacrjournals.org/content/77/13_Supplement/4973). Ramucirumab, a fully-human monoclonal antibody to human VEGFR-2 was approved as an anti-angiogenic treatment for several cancer indications including second-line NSCLC. Combination strategies in cancer including targeting both tumor cells and the surrounding stroma including endothelial cells have been shown to be effective in various tumor subtypes. In this study, the combination effect of LY3214996 with VEGFR-2 inhibitor DC101 (a monoclonal antibody specific for murine VEGFR-2 and a surrogate for ramucirumab) were evaluated in KRAS mutant NSCLC models, including NCI-H2122 (G-12C), A549 (G-12S) and NCI-H441 (G-12V). LY3214996 treatment alone resulted in 41%, 91% and 101% tumor growth inhibition in H2122, A549 and H441 xenograft tumors, respectively. DC101 treatment alone resulted in 64%, 75% and 102% tumor growth inhibition in H2122, A549 and H441, respectively. The combination of LY3214996 with DC101 led to better tumor growth inhibition 83%, 115% (i.e. 15% regression) and 146% (i.e. 46% regression) for H2122, A549 and H441, respectively when compared with single agent treatment. The molecular mechanism was further investigated in H2122 tumor xenograft tumors in terms of MAPK signaling, MAPK gene signatures, tumor vascularization, cell proliferation and apoptosis. LY3214996 together with DC101 led to greater reduction in tumor blood vessels density compared to DC101 alone. Similarly, the analysis of multiple cell cycle markers (including pRb, pH3 and Ki67) indicated that the combination treatment resulted in greater inhibited of cell proliferation compared to single agent. Moreover, the combination effect of LY3214996 with ramucirumab was also investigated via tumor cell driven cord formation assays in vitro; and the data indicated that the combination enhanced the inhibition of cord formation when compared to single agent. Overall, combined inhibition of ERK1/2 and VEGFR-2 enhanced both anti-angiogenesis and antitumor effects. Taken together, these data support further clinical development of the combination of an ERK1/2 inhibitor, LY3214996 with ramucirumab in the treatment of KRAS mutant NSCLC. Citation Format: Wenjuan Wu, Shripad V. Bhagwat, Bonita D. Jones, Michelle L. Swearingen, Beverly L. Falcon, William T. McMillen, Sajan Joseph, Sean Buchanan, Sheng-Bin Peng, Christoph Reinhard, Ramon V. Tiu. Combination of an ERK1/2 inhibitor (LY3214996) with VEGFR-2 inhibitor enhances anti-tumor activity in KRAS mutant non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-185.

497 Background: Treatment approaches combining anti-angiogenic therapy with chemotherapy have shown promising clinical results in metastatic bladder cancer (BC). In order to interrogate the potential clinico-pathologic and molecular basis of these findings, we evaluated VEGFR2, vascular density and molecular subtyping in a series of BC patients. Methods: A custom-TMA with primary BC tissues from 117 patients (mean age 71 yrs; range 38-99 yrs; M:F = 83:34) treated at a single institution, was stained and scored (0-3) for CD34 (vascular density) and for VEGFR2 on tumor vessels and cells. CK5/6 and GATA3 IHC was scored by a pathologist to identify main molecular classes of BC. The association between clinico-pathologic variables, VEGFR2, CD34 and molecular subtypes was analyzed by Fisher’s exact test and ANOVA. Univariate and multivariate Cox proportional models were used for survival analysis. Results: Of 112 analyzable BC tissues, 41% were muscle-invasive (MIBC) vs. 56% non-muscle invasive (NMIBC) and 3% undetermined. Compared to NMIBC patients those with MIBC had shorter overall survival (p < 0.001). The main molecular subtypes included basal (11%), mixed baso-luminal (28%) and luminal (61%). Compared to luminal and baso-luminal subtypes, the majority of basal BCs were muscle invasive (p = 0.036). VEGFR2 expression was higher in tumor vessels but variable in tumor cells. 74% of BCs showed high-medium levels of VEGFR2 (scores 3-2) in tumor vessels and 88% had high-medium levels of tumor vascular density. Within basal, luminal and baso-luminal subtypes, 58%, 78% and 71% had high-medium levels of vascular VEGFR2 (p = 0.52), while 83%, 91% and 82% had high-medium levels of tumor vascular density (p = 0.08). Survival analyses showed increasing patient age, higher stage and lower VEGFR2 levels as independent predictors of shorter survival. Conclusions: Given the observed complexity of BC regarding VEGFR2 expression and vascular density across molecular subtypes, further investigation is warranted to understand how the frequent expression of VEGFR2 on tumor vasculature and variable expression in tumor cells relates to the efficacy of anti-angiogenic agents across these subtypes in bladder cancer.

The combination of tumor targeted therapeutics with PD-L1 checkpoint blockade is being explored as a method to increase the clinical benefits of immunotherapy, and expand response to additional cancer types. Merestinib (Mer) is a kinase inhibitor targeting several oncokinases1 (including MET, MST1R, AXL, MERTK, and MKNK1/2) that can potentially modulate immune function, angiogenesis, as well as target the tumor 1-5. To determine the combinatorial potential with immunotherapy, the effects of Mer were evaluated in vitro on human T cells, PBMCs and murine tumor lines CT26 colon carcinoma (harbors KRASmt G12D expresses low Met/no p-Met/high Axl/p-Axl) and B16F10 melanoma (expressing high Met/pMet/peIF4E). Additionally, the anti-tumor effect of Mer was tested in vivo on established CT26 and B16F10 tumors compared to MET specific TKIs (savolitinib, PF4217903) alone or in combination with PD-L1 antibody (Ab) blockade. In vitro, Mer showed no significant effects on either T cells or PBMCs, but was able to inhibit downstream signaling in both CT26 and B16F10 showing activity on murine tumor cell lines. In vivo, daily Mer monotherapy (6, 12 or 24 mg/kg) showed significant anti-tumor effect at all doses in both CT26 and B16F10, that was not seen with either savolitinib or PF4217903. Concurrent combination of Mer (12 mg/kg) and anti-PD-L1 Ab (0.5 mg qw) in CT26 was found to have anti-tumor activity that was synergistic as compared to each single agent alone. While the effect of Mer monotherapy was lost when treatment ended, tumors continued to regress in the combination group even upon cessation of therapy. The combination was well tolerated and resulted in 90% complete responders compared to 30% with anti-PD-L1 Ab alone, 35 days after completing dosing. To test the ability to generate immunologic memory, complete responders were re-challenged with CT26 cells on the contralateral side. All mice in the combination group resisted re-challenge, showing that Mer/PD-L1 Ab combination was triggering immunologic memory. Although there was no significant change in intra-tumor immune cell populations between groups, combination therapy showed an enhanced and unique intra-tumor immune activation/inflammation gene expression signature compared to PD-L1 Ab monotherapy. The enhanced immune activation of the combination therapy, leading to synergistic anti-tumor efficacy, demonstrates that merestinib has the potential to augment immunotherapy while targeting the tumor directly. This preclinical data provides the rationale for the clinical investigation of merestinib in combination with checkpoint therapies targeting the PD-L1/PD1 axis (NCT02791334). 1 - Yan et al. Invest New Drugs 2013;31:833-44 2 - Balan et al. J Biol Chem 2015;290:8110-20 3 - Eyob et al. Cancer Discov 2013;3:751-60 4 - Lemke G. CSH Persp Biol 2013;5:a009076 5 - Piccirillo et al. Nat Immunol 2014;15:503-11 Citation Format: Sau-Chi Betty Yan, Victoria L. Peek, Jennifer R. Stephens, Um L. Um, Amaladas Nelusha, Colleen A. Burns, Kelly M. Credille, Thompson N. Doman, Scott W. Eastman, Beverly L. Falcon, Gerald E. Hall, Philip W. Iversen, Bruce W. Konicek, Jason R. Manro, Any T. Pappas, Julie A. Stewart, Michael B. Topper, Swee-Seong Wong, Michael Kalos, Ruslan D. Novosiadly, Richard A. Walgren, David Schaer. Combination of an oncokinase inhibitor merestinib with anti-PD-L1 results in enhanced immune mediated antitumor activity in CT26 murine tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5590. doi:10.1158/1538-7445.AM2017-5590

Merestinib (LY2801653) is an orally bioavailable small molecule inhibitor of several oncokinases, including MET, AXL, DDR1/2, MERTK, ROS1, Tie2 (aka TEK), and MKNK1/2. Merestinib has been extensively characterized in a wide range of preclinical tumor xenograft models and shown to potently inhibit MET driven and non-MET driven tumor growth. In addition to its direct antitumor activity, merestinib inhibits angiogenesis and induces a tumor vessel normalization phenotype in xenograft tumors1. While MET signaling is important for angiogenesis, the effect of merestinib on angiogenesis is likely not exclusively driven by MET inhibition. In co-culture angiogenesis assays, merestinib inhibited VEGF-dependent and VEGF-independent endothelial cell cord formation2,3 and sprouting4 with potencies in the low nM range (3-30 nM). In contrast, the MET-specific kinase inhibitor, PF04217903, only weakly inhibited cord formation and endothelial sprouting. In an established in vivo matrigel co-implant vasculogenesis model where VEGFR2 or MET selective inhibition had minimal effect, merestinib decreased vascular density by 69%. In addition, while MET antibody emibetuzumab (human anti-MET antibody) plus ramucirumab (human anti-VEGFR2 antibody) decreased vascular density by 64%, merestinib plus ramucirumab decreased it by 92%. In a mouse adenovirus-driven VEGF-A ear angiogenesis model5, treatment with DC101, a mouse anti-VEGFR2 antibody, or merestinib inhibited angiogenesis; however the combination of DC101 and merestinib appeared to inhibit it even more. Finally, in the MKN45 gastric tumor xenograft model, merestinib (T/C = 4.8%) and DC101 (T/C = 15.3%) each significantly inhibited tumor growth alone and the combination resulted in 27.6% tumor regression and was significantly better than either single agent alone. Together, these studies indicate that merestinib has greater effects on angiogenesis than selective MET inhibition and its actions are not dependent on VEGFR2. In addition, while in vitro studies show reductions in VEGFR2 phosphorylation with high concentration of merestinib, treatment with merestinib did not inhibit VEGF dependent phosphorylation of VEGFR2 in mouse lung tissue at clinically relevant exposures. These data suggest that the anti-angiogenic activity of merestinib includes activities of other kinases targeted by merestinib. These data provide rationale and support for the clinical evaluation of combination of merestinib with ramucirumab (NCT02745769). 1-Yan et. al. Invest New Drugs. 2013;31:833-844, 2- Falcon et. al. J Hematol Oncol. 2013;6:31, 3- Falcon et. al. PLoS ONE. 2014;9:e106901, 4- Nakatsu et. al. Methods Enzymol. 2008;433:65-82, 5- Nagy et. al. Methods Enzymol. 2008;444:43-64. Citation Format: Diane M. Bodenmiller, Julie A. Stewart, Glenn F. Evans, Victoria L. Peek, Jennifer R. Stephens, Xi Lin, Seema Iyer, Beverly L. Falcon, Sudhakar Chintharlapalli, Sau-Chi Betty Yan, Anthony S. Fischl. Characterization of the anti-angiogenic properties of merestinib (LY2801653), an oncokinase inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1823. doi:10.1158/1538-7445.AM2017-1823

ERK1/2, a key downstream effector of RAS mutations, is involved in the signaling network which drives cell proliferation, survival, metastasis and cancer resistance to drug treatment (including MEK and BRAF inhibitors). Lung cancer is a leading cause of cancer death worldwide. KRAS mutation present in up to 30% of NSCLC patients is associated with a poor prognosis and represents an unmet medical need. In KRAS mutant NSCLC, enhanced ERK activation cooperates with dysregulation of the cell cycle checkpoint (e.g., cyclin D, CDK4 and CDK6 complex), and contributes to tumor progression; thus, the simultaneous inhibition of ERK and the CDK4/6 pathway is hypothesized to augment tumor growth inhibition. LY3214996, a novel and highly selective small molecule inhibitor of ERK1 and ERK2, is currently in phase I clinical trial and has been shown to inhibit cell proliferation in RAS or BRAF mutant tumor cells in vitro and xenograft tumor growth in vivo. Abemaciclib, a CDK4 and CDK6-selective inhibitor is currently in phase III studies for ER positive breast cancer and KRAS mutant NSCLC. In this study we explore the potential efficacy of combined inhibition of ERK1/2 and CDK4 and CDK6 in KRAS mutant NSCLC. The combination of LY3214996 and abemaciclib synergistically inhibited cell proliferation in 85% of KRAS mutant cells in an unbiased NSCLC panel. Combination treatment with LY3214996 and abemaciclib significantly decreased levels of phospho- p90RSK, phospho-Rb, phospho-S6 and Ki67; and synergistically inhibited cell proliferation and survival in KRAS mutant NSCLC cell lines including NCI-H2122 (G-12C), A549 (G-12S) and NCI-H441 (G-12V). Subsequent in vivo studies showed that the combination treatment with LY3214996 and abemaciclib was well tolerated and led to more robust tumor growth inhibition or regression in all KRAS mutant NSCLC xenograft models (H2122, A549 and H441) compared with either single agent treatment (p≤0.002). Furthermore, in xenograft tumors the combination of LY3214996 and abemaciclib resulted in more significant reduction of phospho-p90RSK, phospho-Rb, phospho-S6 and Ki67 in H2122 tumors compared with either single agent. Overall, the combined inhibition of ERK1/2 and CDK4 and CDK6 was tolerated and enhanced antitumor efficacy in several KRAS mutant NSCLC preclinical models. These data support the feasibility of combining ERK inhibitor LY3214996 with CDK4 and CDK6 inhibitor abemaciclib as a promising strategy for the treatment of KRAS mutant NSCLC patients, and provides the rationale for the combination study in the on-going phase I LY3214996 clinic trial (NCT02857270). Citation Format: Wenjuan Wu, Shripad V. Bhagwat, Constance King, Susan Pratt, Xueqian Gong, Julie Stewart, Bonita Jones, Robert Flack, Richard Beckman, Beverly Falcon, Jason Manro, William T. McMillen, Ramon V. Tiu, Sheng-Bin Peng, Christoph Reinhard, Sajan Joseph, Sean Buchanan. Combination of a novel ERK1/2 inhibitor (LY3214996) with CDK4 and CDK6 inhibitor (abemaciclib) enhances antitumor efficacy in KRAS mutant non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 317. doi:10.1158/1538-7445.AM2017-317

Lung cancer is the leading cause of cancer death worldwide. MAPK activation via KRAS mutation is present in up to 30% of lung cancer patients. NSCLC patients with KRAS mutation is associated with poor prognosis and represents an unmet medical need. LY3009120, a pan-RAF and RAF dimer inhibitor which is in phase I clinical trial, was previously demonstrated to have anti-tumor activities in BRAF or RAS mutant tumor cells in vitro and in vivo. Ramucirumab, a fully-human antagonist monoclonal antibody to human VEGFR-2 was recently approved as an anti-angiogenic treatment for several cancer indications including second-line NSCLC. Combination strategies in cancer including targeting both tumor cells and the surrounding stroma cells have been shown to be effective in various disease subtypes. In this study, the combination effect of LSN3074753 (a surrogate and an analogue of LY3009120) with VEGFR-2 inhibitor DC101 (a monoclonal antibody specific for murine VEGFR-2 and a surrogate for ramucirumab) were evaluated in KRAS mutant NSCLC models, including NCI-H2122 (G-12C), A549 (G-12S) and NCI-H441 (G-12V). LSN3074753 treatment alone resulted in 66.9% and 82.4% tumor growth inhibition in H2122 and A549 xenograft tumors, respectively; and 41.4% tumor regression in H441 xenograft tumors. DC101 treatment alone resulted in 64.5%, 75% and 102.2% tumor growth inhibition in H2122, A549 and H441, respectively. The combination of LSN3074753 and DC101 led to more significant tumor growth inhibition (87.2% of tumor growth inhibition for H2122, p Citation Format: Wenjuan Wu, Julie Stewart, Constance King, Bonita Jones, Robert Flack, Susan Pratt, Randi Berryman, Michelle Swearingen, Diane Bodenmiller, Xi Lin, Mark Uhlik, Beverly Falcon, Anthony Fischl, Jason Manro, Ramon Tiu, Sudhakar Chintharlapalli, Bronislaw Pytowski, Shripad V. Bhagwat, Sean Buchanan, Sheng-Bin Peng. Combined inhibition of pan-RAF and VEGFR-2 mediates antitumor activity in KRAS mutant non-small cell lung cancer (NSCLC) through enhanced inhibition of tumor angiogenesis and growth. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 930.

CHK1 is a serine/threonine protein kinase essential for S-phase and G2/M cell cycle checkpoint regulation following DNA damage. Targeted inhibition of CHK1 in several tumor types increases DNA damage and replication stress, culminating in cell death through mitotic catastrophe. Recent studies have identified CHK1 as a therapeutic target in several pediatric tumor types. We evaluated the antitumor efficacy of LY2606368 mesylate monohydrate (“LY”), a checkpoint kinase 1 (CHK1)/CHK2 inhibitor currently in early phase clinical trials for adult solid cancers, in a panel of pediatric tumor cell lines and mouse models of embryonal tumors and pediatric sarcoma. In vitro effects of LY were assessed via Cell Titer Glo, immunoblotting, and cell cycle analysis by flow cytometry. For in vivo studies, mice bearing cell-derived (CDX) or patient-derived xenografts (PDX) of several pediatric tumor types were treated with four weekly cycles of 10 mg/kg LY BID for 3 consecutive days, followed by a 4 day dosing holiday. Tumor volume and body weight were measured 2x weekly. Xenograft tumor health following LY, chemotherapy, or combination treatment was evaluated by fluorescent immunohistochemistry (IHC) for a panel of markers for cell proliferation (Ki67), apoptosis (TUNEL), and angiogenesis (CD31, smooth muscle actin [SMA], MECA32). Single digit nanomolar sensitivity to LY was observed in the majority of pediatric cancer cell lines evaluated in vitro. A more detailed analysis of LY-treated neuroblastoma and pediatric sarcoma cell lines showed increased DNA damage, CHK1 phosphorylation, and MAPK pathway activation. Significant single agent LY activity was observed in mouse models of neuroblastoma and pediatric sarcoma, but not in models of hepatoblastoma or retinoblastoma. Acquired resistance to LY was observed in the ST162 and SJCRH30 models of alveolar rhabdomyosarcoma. Interestingly, more stroma was observed following LY single agent treatment as measured by CD31, SMA, and MECA32 IHC staining; co-treatment with chemotherapy reduced the amount of SMA expressing-cells. Overall, our data demonstrate that LY is highly effective as a single agent in murine in vivo models of human neuroblastoma and several pediatric sarcoma subtypes. Current studies include further evaluation of the LY mechanism of action; investigation into the mechanism of intrinsic and acquired resistance; and identification of possible biomarkers for LY sensitivity. Citation Format: Caitlin D. May, Richard Beckmann, Wayne Blosser, Michele Dowless, Alle VanWye, Teresa Burke, Gerard Oakley, Jennifer Stephens, Julie Stewart, Beverly Falcon, Louis Stancato. Targeting checkpoint kinase 1 (CHK1) with the small molecule inhibitor LY2606368 mesylate monohydrate in models of high-risk pediatric cancer yields significant antitumor effects. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2458.

Although anti-angiogenic therapy has emerged as a leading modality in treating human cancer, further improvements in the duration and frequency of clinical response of various human cancers remain important clinical needs. Maturity of the tumor vasculature has been identified as one of the major determinants of response of cancers to anti-angiogenic treatments. We have developed and optimized the application of a quantitative, high throughput, hyper-plexed, fluorescence imaging technology to refine our understanding of the complexity of vascular phenotypes in various human malignancies. The technology was used to quantify tumor blood vessels and expression of thirteen proteins of known roles in blood vessel biology in single sections from archival primary tumor tissues from 64 prostate cancer patients. CD31 was used to segment vascular objects in each image. CD31 and CD34 endothelial cell staining, SMA pericyte staining, and collagen IV basement membrane staining were used to classify detected vessels using K-means cluster analysis. Segmented vessels were clustered into 2-20 cluster sets, and the reproducibility of vessel classification of each cluster set was determined using the consensus clustering algorithm. A six cluster set that reflected biologically relevant tumor vascular subsets with high consensus clustering concordance was selected for further analysis. Clusters consistent with different stages of vessel development were obtained, including clusters with CD34 high/SMA low and CD34 low/SMA high profiles, reflecting immature and mature vascular phenotypes. Additional clusters representing phenotypes consistent with transitional vascular developmental stages were also identified. Nine additional proteins involved in angiogenesis were also quantified in each vessel and expression profiles for each cluster were determined. The enrichment of blood vessel clusters was then analyzed for each patient. This revealed differential patterns of vascular maturity phenotypes in the prostate cancer tissues analyzed. We have demonstrated that high-throughput, quantitative characterization of vascular maturity phenotypes is feasible in human cancer tissue specimens. Such immunofluorescence hyper-plex profiling of vascular-related proteins has the potential to illuminate the complex biology of tumor angiogenesis and to enable novel approaches for patient tailoring in clinical trials of anti-angiogenic therapeutics. Citation Format: Chris Sevinsky, Alberto Santamaria-Pang, Jingyu Zhang, Christina Lowes, Dipen Sangurdekar, Beverly Falcon, Qing Li, Bronek Pytowski, Laura Benjamin, Jeremy Graff, Fiona Ginty, Aejaz Nasir, Mark T. Uhlik. Quantification of biologically relevant vascular phenotypes in human prostate cancer: automated image analysis using hyperplexed immunofluorescence. [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 1709. doi:10.1158/1538-7445.AM2015-1709

Therapeutic targeting of the VEGF pathway has not matched the efficacy suggested by preclinical tumor models. To date, the limitations of this form of therapy stem either from inherent or acquired resistance of the tumor vasculature to the reduction of VEGF signaling. Previous studies have described morphological phenotypes of different blood vessel types in human and mouse tumors. Recapitulation of these vascular phenotypes with a surrogate Ad-VEGFA164 model has shown differential sensitivity of these vascular subclasses to inhibitors of the VEGF pathway. The goal of the current study was to identify molecular markers for the various subtypes of pathological vessels and to assess whether similar molecular signatures are found in blood vessels in mouse tumor models and human disease. Using the Ad-VEGF-A164 flank model, microarray analysis was performed to identify molecular markers at each time-point, representing distinct stages of vessel development and maturation. We identified unique gene signatures at the various time-points and confirmed differential expression of the genes by qRT-PCR and/or immunohistochemistry. We found that vascular markers such as CD31, Ang2, and Tie1 were pan-endothelial markers at all time-points. However, other markers such as CD34, MECA-32, vWF, SMA, Tie2, CD105, and AQP1 were expressed on subclasses of endothelial cells. Expression of the molecular markers in tumors models revealed different subclasses of tumor vessels in patient-derived xenografts which were sensitive or resistant to anti-VEGF-A or anti-VEGFR-2 treatment. These vascular subtypes were also found in tissues from human breast and gastric cancers. Together these results indicate that multiple molecular markers can be used to identify unique pathological subclasses of tumor vessels. Some of these vascular subtypes may be insensitive to inhibitors targeting the VEGF pathways and their signatures may be useful biomarkers to predict vascular sensitivity to anti-angiogenic therapy. Citation Format: Beverly L. Falcon, Marguerita O'Mahony, Julie Stewart, Jiangang Liu, Janice A. Nagy, Qi Xue, Yong Pan, Cynthia Jeffries, Emma Hatten, Tim R. Holzer, Harold F. Dvorak, Aejaz Nasir, Bronek Pytowski, Laura Benjamin, Mark T. Uhlik. Identification of molecular markers of pathological vascular subtypes with differential sensitivity to therapies targeting the VEGF pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4810. doi:10.1158/1538-7445.AM2014-4810

Vascular Endothelial Growth Factor A (VEGF-A) is the predominant growth factor expressed by tumor cells to drive angiogenesis and solid tumor growth. Antiangiogenesis therapies have been clinically demonstrated to be effective, however, there are still not effective means to preselect those patients most likely to derive optimal benefit. To identify blood vessel markers that may differentiate patients responsive to VEGF pathway blockade, we used a well-established in vivo model to generate pathological angiogenesis following the delivery of adenovirus engineered to express VEGF-A164 (Ad-VEGF) to a localized subcutaneous site within the flank of a nude mouse. This model provides a temporally controlled induction of angiogenesis and subsequent remodeling and maturation of the vasculature. This enabled us to study discrete subclasses of vessel which have been previously shown to have differential sensitivities to anti-angiogenic therapy. Here we report gene expression profiling on a time course of the Ad-VEGF flank model which was used to characterize molecular and genetic changes associated with these distinct vessel subtypes. Using a nearest centroid-based classification algorithm we identified unique endothelial cell specific gene signatures representing ‘early’, ‘middle' and ‘late' stages of vessel development and maturation. To evaluate the utility of these gene signatures in predicting sensitivity to anti-VEGF therapy, we profiled a publically available gene array dataset generated from a mouse xenograft model following anti-VEGF treatment with our vessel subtype gene signatures. A majority of the early and middle vessel genes were significantly reduced in bevacizumab-treated animals, demonstrating they respond to VEGF pathway inhibition. To further evaluate the potential value of these gene signatures as predictive biomarkers we profiled a dataset of 248 gastric tumors using hierarchical clustering. Our analysis shows the middle vessel signature was highly expressed in a mesenchymal molecular subtype of gastric cancer. Interestingly, the majority of the mesenchymal gastric tumors were ‘diffuse' subtype in Lauren's classification. Together, our data demonstrate the potential usefulness of our vessel subtype gene signatures in identifying tumor subtypes that may benefit from this anti-angiogenic therapy. Citation Format: Jiangang Liu, Beverly L. Falcon, Janice A. Nagy, Shou-Ching S. Jaminet, Dan Li, Thompson N. Doman, Sudhakar Chintharlapalli, Bronek Pytowski, Mark T. Uhlik, Harold Dvorak, Laura Benjamin, Anthony S. Fischl. Bioinformatics analysis of an Ad-VEGF flank angiogenesis model identifies vessel subtype gene signatures: Implications for anti-VEGF therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2359. doi:10.1158/1538-7445.AM2014-2359

The Vascular Endothelial Growth Factor (VEGF) pathway plays an important role in the genesis, growth and progression of human cancer, including colorectal carcinomas (CRC). The key mediators of VEGF signaling are VEGFR1, VEGFR2, and VEGFR3, part of a family of related receptor tyrosine kinases. The relative expression, activity, or interplay among these receptors may determine the response of CRC patients to anti-angiogenic therapies. Using high-affinity, specific monoclonal primary anti-VEGFR1, 2 3 antibodies, we have developed robust imunohistochemical assays in our laboratory to quantify VEGFR1, 2 and 3 in archival human tissues. Using a well-annotated CRC tissue microarray (TMA), we carried out comprehensive comparative evaluation of immunohistochemical (IHC) expression of the three VEGFRs in archival primary CRC tissues (n=84). VEGFR1 immunoreactivity was reported as H-score (range 0-300); VEGFR2 positive vessels were counted and normalized to the number of CD34-positive vessels and reported as vascular positivity index (VEGFR2-VPI) and VEGFR3-positive vessels were counted in each core by the same solid tumor immunopathologist, who was blinded to the clinico-pathologic details. Based on immunoreactivity for VEGFRs, each case was scored as negative, low, medium or high. Thresholds were selected based on the overall range of expression of each receptor in the CRC tissues examined: 0, 1-50, 51-100, >100 (VEGFR1 H-scores); 0, 1-25, 26-49, 50-100 (VEGFR2-VPI); 0, 1-5, 6-10, >10 (VEGFR3+ vascular count). Based on VEGFR (1,2,3) expression, a set of eight VEGFR staining profiles were noted: Triple VEGFR positive (n=9, 11%), VEGFR1 predominant (17, 20%), VEGFR2 predominant (7, 8%), VEGFR3 predominant (1, 1%), VEGFR1/2 predominant (42, 50%), VEGFR1/3 predominant (2, 2%), VEGFR2/3 predominant (3, 4%), and triple VEGFR negative (3, 4%). These new data provide original insights on the distribution, subcellular localization and heterogeneity of expression of VEGFRs in human CRC stromal vessels and tumor cells. The proposed human CRC sub-classification, based on the observed differential VEGFR 1, 2, 3 expression profiles, has identified various subsets of human CRCs. Clinical trials incorporating IHC profiling would be required to test whether these subsets show differential responsiveness to targeted agents in the VEGF/VEGFR2 family. Citation Format: Timothy R. Holzer, Leslie A. O'Neill, Drew M. Nedderman, Angie D. Fulford, Beverly L. Falcon, Mark T. Uhlik, Laura E. Benjamin, Andrew E. Schade, Aejaz Nasir. Heterogeneity of vascular endothelial growth factor receptors 1, 2, and 3 in primary human colorectal adenocarcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3007. doi:10.1158/1538-7445.AM2014-3007

Vascular endothelial growth factor (VEGF) plays a dominant role in angiogenesis. While inhibitors of the VEGF pathway are approved for the treatment of a number of tumor types, the effectiveness is limited and evasive resistance is common. One mechanism of evasive resistance to inhibition of the VEGF pathway is upregulation of other pro-angiogenic factors such as fibroblast growth factor (FGF) and epidermal growth factor (EGF). Numerous in vitro assays examine angiogenesis, but many of these assays are performed in media or matrix with multiple growth factors or are driven by VEGF. In order to study angiogenesis driven by other growth factors, we developed a basal medium to use on a co-culture cord formation system of adipose derived stem cells (ADSCs) and endothelial colony forming cells (ECFCs). We found that cord formation driven by different angiogenic factors led to unique phenotypes that could be differentiated and combination studies indicate dominant phenotypes elicited by some growth factors. VEGF-driven cords were highly covered by smooth muscle actin, and bFGF-driven cords had thicker nodes, while EGF-driven cords were highly branched. Multiparametric analysis indicated that when combined EGF has a dominant phenotype. In addition, because this assay system is run in minimal medium, potential proangiogenic molecules can be screened. Using this assay we identified an inhibitor that promoted cord formation, which was translated into in vivo tumor models. Together this study illustrates the unique roles of multiple anti-angiogenic agents, which may lead to improvements in therapeutic angiogenesis efforts and better rational for anti-angiogenic therapy.