Laura E. Benjamin

Angiopoietin-2: An Attractive Target for Improved Antiangiogenic Tumor Therapy

Anti-VEGF pathway therapies primarily target immature blood vessels in tumors. However, emerging approaches to combine with targeted therapies impacting the later stages of remodeling and vessel maturation are expected to improve clinical efficacy by expanding the target vessel population. The angiopoietin/Tie ligand/receptor system is a prototypic regulator of vessel remodeling and maturation. Angiopoietin-2 (Ang2) appears to be a particularly attractive therapeutic target. In fact, the experimental proof-of-concept showing improved efficacy when VEGF and Ang2-targeting therapies are combined has been solidly established in preclinical models, and several Ang2-targeting drugs are in clinical trials. However, rational development of these second-generation combination therapies is hampered by a limited understanding of the biological complexity that is generated from agonistic and antagonistic Ang/Tie signaling. This review discusses recent mechanistic advances in angiopoietin signaling, particularly in light of the recent study published on REGN910 and summarizes the status quo of Ang2-targeting therapies. In light of the clarified partial agonist function of Ang2, we propose that clarity on the expression profile of the angiopoietin ligands and Tie1 and Tie2 receptors in subsets of cancer vessels and cancer cells will provide clearer hypotheses for more focused rational clinical trials to exploit this seminal pathway and improve current antiangiogenic therapies.

Akt1 and Akt3 exert opposing roles in the regulation of vascular tumor growth

Vascular tumors are endothelial cell neoplasms whose mechanisms of tumorigenesis are poorly understood. Moreover, current therapies, particularly those for malignant lesions, have little beneficial effect on clinical outcomes. In this study, we show that endothelial activation of the Akt1 kinase is sufficient to drive de novo tumor formation. Mechanistic investigations uncovered opposing functions for different Akt isoforms in this regulation, where Akt1 promotes and Akt3 inhibits vascular tumor growth. Akt3 exerted negative effects on tumor endothelial cell growth and migration by inhibiting activation of the translation regulatory kinase S6-Kinase (S6K) through modulation of Rictor expression. S6K in turn acted through a negative feedback loop to restrain Akt3 expression. Conversely, S6K signaling was increased in vascular tumor cells where Akt3 was silenced, and the growth of these tumor cells was inhibited by a novel S6K inhibitor. Overall, our findings offer a preclinical proof of concept for the therapeutic utility of treating vascular tumors, such as angiosarcomas, with S6K inhibitors.

Tumor Cell Expression of Vascular Endothelial Growth Factor Receptor 2 Is an Adverse Prognostic Factor in Patients with Squamous Cell Carcinoma of the Lung

A robust immunohistochemical (IHC) assay for VEGFR2 was developed to investigate its utility for patient tailoring in clinical trials. The sensitivity, specificity, and selectivity of the IHC assay were established by siRNA knockdown, immunoblotting, mass spectrometry, and pre-absorption experiments. Characterization of the assay included screening a panel of multiple human cancer tissues and an independent cohort of non-small cell lung carcinoma (NSCLC, nu200a=u200a118) characterized by TTF-1, p63, CK5/6, and CK7 IHC. VEGFR2 immunoreactivity was interpreted qualitatively (VEGFR2 positive/negative) in blood vessels and by semi-quantitative evaluation using H-scores in tumor cells (0–300). Associations were determined among combinations of VEGFR2 expression in blood vessels and tumor cells, and clinico-pathologic characteristics (age, sex, race, histologic subtype, disease stage) and overall survival using Kaplan-Meier analyses and appropriate statistical models. VEGFR2 expression both in blood vessels and in tumor cells in carcinomas of the lung, cervix, larynx, breast, and others was demonstrated. In the validation cohort, 99/118 (83.9%) NSCLC tissues expressed VEGFR2 in the blood vessels and 46/118 (39.0%) showed high tumor cell positivity (H-score ≥10). Vascular and tumor cell expression were inversely correlated (pu200a=u200a0.0175). High tumor cell expression of VEGFR2 was associated with a 3.7-fold reduction in median overall survival in lung squamous-cell carcinoma (SCC, nu200a=u200a25, pu200a=u200a0.0134). The inverse correlation between vascular and tumor cell expression of VEGFR2 and the adverse prognosis associated with high VEGFR2 expression in immunohistochemically characterized pulmonary SCC are new findings with potential therapeutic implications. The robustness of this novel IHC assay will support further evaluation of its utility for patient tailoring in clinical trials of antiangiogenic agents.

RhoB differentially controls Akt function in tumor cells and stromal endothelial cells during breast tumorigenesis

Tumors are composed of cancer cells but also a larger number of diverse stromal cells in the tumor microenvironment. Stromal cells provide essential supports to tumor pathophysiology but the distinct characteristics of their signaling networks are not usually considered in developing drugs to target tumors. This oversight potentially confounds proof-of-concept studies and increases drug development risks. Here, we show in established murine and human models of breast cancer how differential regulation of Akt by the small GTPase RhoB in cancer cells or stromal endothelial cells determines their dormancy versus outgrowth when angiogenesis becomes critical. In cancer cells in vitro or in vivo, RhoB functions as a tumor suppressor that restricts EGF receptor (EGFR) cell surface occupancy as well as Akt signaling. However, after activation of the angiogenic switch, RhoB functions as a tumor promoter by sustaining endothelial Akt signaling, growth, and survival of stromal endothelial cells that mediate tumor neoangiogenesis. Altogether, the positive impact of RhoB on angiogenesis and progression supercedes its negative impact in cancer cells themselves. Our findings elucidate the dominant positive role of RhoB in cancer. More generally, they illustrate how differential gene function effects on signaling pathways in the tumor stromal component can complicate the challenge of developing therapeutics to target cancer pathophysiology.

VEGF-A/VEGFR Inhibition Restores Hematopoietic Homeostasis in the Bone Marrow and Attenuates Tumor Growth

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.

Stromal-Based Signatures for the Classification of Gastric 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.

Abstract 3259: LY3127804, a novel anti-Angiopoietin-2 antibody in combination with an anti-VEGFR2 antibody potently inhibits angiogenesis, tumor growth and metastasis

Angiopoeitin-2 (Ang2) is released from endothelial cells only in response to stimulus (e.g. wound healing, tumor growth) and facilitates blood vessel sprouting and inhibits pericyte-endothelial cell interaction via Tie2 signaling. Combination of an anti-Ang2 antibody and aflibercept, a VEGF trap, has been shown to inhibit tumor growth and decrease tumor vascularity in mouse xenograft tumor models (Daly et al., Cancer Res (2013) 73(1):108). Multiple investigational anti-Ang2 antibody therapies are currently in clinical trials. LY3127804 is a humanized and engineered IgG4 isotype antibody that selectively binds to Ang2 with high affinity and neutralizes Ang2 induced phospho-Tie2. LY3127804 inhibits sprouting angiogenesis and increases pericyte coverage in a mouse developmental retinal angiogenesis model and in mice bearing PC3 xenograft tumors. Combination of LY3127804 and DC101, a potent anti-VEGFR2 antibody, exhibits enhanced efficacy when compared to monotherapy in multiple patient derived xenograft models including NSCLC and ovarian cancers. Anti-Ang2 antibody monotherapy alone resulted in marginal reduction of tumor growth and improved overall survival, while DC101monotherapy had greater reduction in tumor volume with no survival benefit in MDA-MB-231 breast orthotopic model. Combination of anti-Ang2 antibody with anti-VEGFR2 antibody shows reduction in tumor volume and improved overall survival. This robust pre-clinical evidence supports testing the combination of anti-Ang2 and anti-VEGFR2 antibodies in the clinic. LY3127804 is currently in Phase 1 clinical trials (NCT02597036) Citation Format: Sudhakar R. Chintharlapalli, Johnny E. Croy, Donmienne Leung, Damien Gerald, Jirong Lu, Philip W. Iversen, Linda N. Lee, Lysiane Huber, Jonathan Tetreault, Rowena Almonte-Baldonado, Jianghuai Xu, Bharathi Ramamurthy, Jennifer A. Pereira, Chi-Kin Chow, Axel-Rainer Hanauske, Volker Wacheck, Laura Benjamin, Ling Liu. LY3127804, a novel anti-Angiopoietin-2 antibody in combination with an anti-VEGFR2 antibody potently inhibits angiogenesis, tumor growth and metastasis. [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 3259.

Abstract IA21: Vascular phenotyping: Using genomics to inform antiangiogenesis

Anti-angiogenesis therapy has proven to be an important tool for oncologists particularly in metastatic disease. To date much of the success has been built on targeting the VEGF/VEGFR pathways and FDA approvals have been granted in many disease settings including CRC, RCC, NSCLC, and others. In 2014 Cervical Cancer and Gastric cancer were new cancers added to this list. Still, it9s fair to say that we have a limited understanding of how to identify the patients who will receive optimal benefit from the available therapeutic agents, and how inhibitors of non-VEGF pathways will compare or combine with currently approved therapies. This presentation will discuss new findings that use molecular analysis to understand heterogeneity in cancer vascular beds and how they relate to preclinical efficacy for VEGF pathway inhibitors. Linking these findings to clinical outcomes in the future may provide a bridge between the wealth of data being generated in cancer genomics and patients most likely to derive benefit from anti-angiogenesis. Citation Format: Laura E. Benjamin. Vascular phenotyping: Using genomics to inform antiangiogenesis. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Angiogenesis and Vascular Normalization: Bench to Bedside to Biomarkers; Mar 5-8, 2015; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl):Abstract nr IA21.

Abstract 4157: Heterogeneity of vascular endothelial growth factor receptors 1, 2, and 3 in human non-small cell lung carcinomas

The Vascular Endothelial Growth Factor (VEGF) pathway plays a prominent role in the growth and progression of human cancers, including non-small cell lung carcinoma (NSCLC). The key mediators of VEGF signaling are a family of related receptor tyrosine kinases that include VEGFR1, VEGFR2, and VEGFR3. The relative expression levels, activity and cross-talk among these receptors may contribute to clinical response of NSCLC patients to anti-angiogenic therapies. Using a well-annotated tissue microarray (TMA) and robust immunohistochemical (IHC) assays developed, standardized and implemented in our laboratory, we comparatively evaluated expression of the three VEGFRs in archival primary NSCLC tissues (n = 97). VEGFR1 and VEGFR2 were localized both in tumor vessels and cells, while VEGFR3 was only localized in tumor vessels. VEGFR1 immunoreactivity was reported as negative/low, medium, or high, based on intensity and proportion of stained tumor cell by an experienced solid tumor immunopathologist (AN), who was blinded to clinico-pathologic details. VEGFR2 and VEGFR3 positive vessels were counted by manual assessment of each core by the same immunopathologist. For systematic comparative analysis of VEGFR data, IHC expression thresholds were selected based on the 25% and 75% quartiles around the median of the range of counts for VEGFR2 and VEGFR3: 0-2, 3-10 and >10 (VEGFR2+ vascular count) respectively; and 0-1, 2-9 and >9 (VEGFR3+ vascular count) respectively. Based on VEGFR (1,2 and3) expression levels defined above, a set of eight VEGFR staining profiles were identified: Triple VEGFR positive (n = 11, 11.3%), VEGFR1 predominant (22, 22.7%), VEGFR2 predominant (9, 9.3%), VEGFR3 predominant (3, 3.1%), VEGFR1/2 predominant (13, 13.4%), VEGFR1/3 predominant (2, 2.1%), VEGFR2/3 predominant (9, 9.3%), and triple VEGFR negative (28, 28.9%). These new data provide original insights on the tissue distribution, subcellular localization and heterogeneity of expression of VEGFRs in human NSCLC cells and stromal vessels. The proposed human NSCLC sub-classification, based on the observed differential VEGFR 1, 2, 3 expression profiles, has identified various subsets of human NSCLC, especially triple VEGFR +, triple VEGFR -, VEGFR1 predominant and VEGFR 1 / 2 predominant. These profiles are distinct from the VEGF receptor profiles that we reported in a prior study on colorectal carcinomas (Holzer, Nasir et al., AACR 2014), of which VEGFR1/2 predominant subset was 50% and triple VEGFR-negative subset was only 4%. This work suggests distinct patterns of heterogeneity of VEGF receptor profiles in human NSCLC and CRCs. These data also support further evaluation of whether the reported VEGFR profiles correlate with differential sensitivity to therapeutic VEGF/VEGFR pathway inhibition. Citation Format: Timothy R. Holzer, Angie D. Fulford, Leslie O9Neill Reising, Drew M. Nedderman, Laura E. Benjamin, Andrew E. Schade, Aejaz Nasir. Heterogeneity of vascular endothelial growth factor receptors 1, 2, and 3 in human non-small cell lung carcinomas. [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 4157. doi:10.1158/1538-7445.AM2015-4157

Abstract 4161: Differential expression of VEGFR2 Protein in HER2 positive primary human breast cancer: Potential relevance to newer anti-angiogenic therapies

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PAnnPreviously, we demonstrated immunohistochemical expression of VEGFR2 protein and vascular phenotypes in human breast carcinomas (Holzer, Nasir et al AACR 2014). Here, we carried out a large-scale immunopathologic analysis of tumor vascular density and differential expression of VEGFR2 protein in various subsets of primary human breast carcinomas from 186 females (Mean age: 59 years; range 33-88 years). Sections from a primary breast cancer tissue microarray (Yale University), were stained for a sensitive tumor vascular marker (CD34) and for VEGFR2 protein using a novel VEGFR2 IHC assay developed and standardized in our laboratory (Holzer, Nasir et al PLOS One 2013). Discrete VEGFR2+ and CD34+ tumor vessels were counted in each analyzable TMA core by an experienced breast pathologist, excluding any suboptimal/inadequate cores or any areas of tumor necrosis. Histologic subtypes of primary breast carcinomas included invasive ductal, lobular, mixed ductal-lobular and colloid (N = 139, 22, 18, 7) respectively. Based on hormone receptor (HR) and Her2 status, cases were grouped into HR+, Her2+ and triple-negative subsets. Pathologic assessments of vascular density (CD34 counts) and imunohistochemical expression of VEGFR2 protein were made only in the viable invasive carcinoma tissue. The observed CD34+ and VEGFR2+ tumor vascular counts in individual cases were heterogeneous, ranging from high vascular density and high VEGFR2 expression to minimal vascular density and/or minimal or no VEGFR2 expression. Among all histologies, mean CD34+ and VEGFR2+ tumor vascular counts were 11 and 3.4 per tumor TMA core respectively. Eighty-nine of 186 (48%) cases had >10 CD34+ tumor vessels, while 97/186 (52%) had fewer CD34+ vessels in each TMA core. Of 169 analyzable cores in the VEGFR2 stained TMA, 90 (53%) showed 1 to 5 VEGFR2+ tumor vessels/TMA core, while 42/169 (25%) cores had no VEGFR2+ tumor vessels. Thirteen of 169 (8%) cases also showed tumor cell (cytoplasmic and/or membrane) expression of VEGFR2 protein. Triple-negative breast cancers (TNBCs) appeared to be less vascular (Mean VD = 9.8, range 0-34) than other breast cancer subtypes. Overall, VEGFR2+ tumor vessel counts were significantly higher in Her2+ as compared to HR+ (p = 0.03) and TNBC (p = 0.01) tissues. Compared to Her2- cases, Her2+ breast cancers had higher VEGFR2+ tumor vessel counts (p = 0.006). These data provide insights about tumor vascular density and vascular VEGFR2 expression in primary human breast cancer subtypes, especially Her2 positive breast cancer, and offer a pathobiologic hypothesis for the patterns of clinical response of breast cancer patients to newer anti-angiogenic therapies.nnCitation Format: Aejaz Nasir, Timothy R. Holzer, Michael Man, Laura E. Benjamin, Allen S. Melemed, Andrerw E. Schade. Differential expression of VEGFR2 Protein in HER2 positive primary human breast cancer: Potential relevance to newer anti-angiogenic therapies. [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 4161. doi:10.1158/1538-7445.AM2015-4161