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Hepatocyte growth factor (HGF) signaling through its receptor Met has been implicated in hepatocellular carcinoma tumorigenesis and progression. Met interaction with integrins is shown to modulate the downstream signaling to Akt and ERK (extracellular-regulated kinase). In this study, we developed a mechanistically detailed systems biology model of HGF/Met signaling pathway that incorporated specific interactions with integrins to investigate the efficacy of integrin-binding peptide, AXT050, as monotherapy and in combination with other therapeutics targeting this pathway. Here we report that the modeled dynamics of the response to AXT050 revealed that receptor trafficking is sufficient to explain the effect of Met–integrin interactions on HGF signaling. Furthermore, the model predicted patient-specific synergy and antagonism of efficacy and potency for combination of AXT050 with sorafenib, cabozantinib, and rilotumumab. Overall, the model provides a valuable framework for studying the efficacy of drugs targeting receptor tyrosine kinase interaction with integrins, and identification of synergistic drug combinations for the patients., Oncology: Scientists predict efficacy of cancer therapies Mathematical modeling can be used to predict the efficacy of drug therapies in liver cancer for individual patients. A team lead by Mohammad Jafarnejad at Johns Hopkins University developed a mathematical model of cellular signaling in liver cells that can predict drug interactions affecting the growth pathways in liver cells found to be responsible for tumor formation in liver cancer. The model was calibrated based on available data and was able to predict the efficacy of common therapies and predicted a synergy for a combination of therapies. Overall, the mathematical modeling allowed us to study the differences in efficacy between different drugs and the efficacy of combining drugs. With the expected increase in the availability of patient-specific data, the methods developed here can be used to benefit individual patients by determining which therapies are most likely to be effective.

Background Retinal vascular diseases such as neovascular age-related macular degeneration, diabetic retinopathy and/or diabetic macular edema, and retinal vein occlusion with macular edema—share several key pathophysiologic aspects including neovascularization, vascular permeability, and inflammation. The role of vascular endothelial growth factor (VEGF) in these processes, and the therapeutic benefits of VEGF inhibition, have been well characterized. Anti-VEGF therapy is highly effective for many patients but is not uniformly effective in all patients and imposes a significant treatment burden. More recently, the role of the Tie2 signaling pathway in the pathophysiology of retinal vascular diseases has been investigated, and the Tie2 pathway represents a novel therapeutic target for these conditions. Areas covered The index review describes the Tie2 pathway and its complementary role to the VEGF pathway in the angiogenesis cascade and will summarize studies of molecules in development to therapeutically modulate the Tie2 pathway in retinal vascular diseases. Conclusions Activation of the Tie2 pathway leads to downstream signaling that promotes vascular health and stability and decreases vascular permeability and inflammation. AXT107 is a collagen IV–derived synthetic peptide with a dual mechanism of action that involves suppression of VEGF signaling and activation of the Tie2 pathway; these actions are accomplished by AXT107 binding to and disrupting different integrin, leading to blockade of the VEGF receptor and rearrangement of cellular Tie2 rendering it susceptible to Ang2 agonism. Other Tie2 agonist compounds are also in development, including faricimab and razuprotafib. Tie2 activation only modestly impacts angiogenesis on its own but significantly potentiates VEGF suppression. Co-regulation of the VEGF and Tie2 signaling pathways has the potential to improve functional and structural outcomes in eyes with retinal vascular diseases.

Triple-negative breast cancer (TNBC) is a highly metastatic and aggressive disease with limited treatment options. Recently, the combination of the immune checkpoint inhibitor (ICI) atezolizumab (anti-PD-L1) with nab-paclitaxel was approved following a clinical trial that showed response rates in at least 43% of patients. While this approval marks a major advance in the treatment of TNBC it may be possible to improve the efficacy of ICI therapies through further modulation of the suppressive tumor immune microenvironment (TIME). Several factors may limit immune response in TNBC including aberrant growth factor signaling, such as VEGFR2 and cMet signaling, inefficient vascularization, poor delivery of drugs and immune cells, and the skewing of immune cell populations toward immunosuppressive phenotypes. Here we investigate the immune-modulating properties of AXT201, a novel 20 amino-acid integrin-binding peptide in two syngeneic mouse TNBC models: 4T1-BALB/c and NT4-FVB. AXT201 treatment improved survival in the NT4 model by 20% and inhibited the growth of 4T1 tumors by 47% over 22 days post-inoculation. Subsequent immunohistochemical analyses of 4T1 tumors also showed a 53% reduction in vascular density and a 184% increase in pericyte coverage following peptide treatment. Flow cytometry analyses demonstrated evidence of a more favorable anti-tumor immune microenvironment following treatment with AXT201, including significant decreases in the populations of T regulatory cells, monocytic myeloid-derived suppressor cells, and PD-L1 expressing cells and increased expression of T cell functional markers. Together, these findings demonstrate immune-activating properties of AXT201 that could be developed in combination with other immunomodulatory agents in the treatment of TNBC.

// Mustafa A. Barbhuiya 1, * , Adam C. Mirando 2, * , Brian W. Simons 3 , Ghali Lemtiri-Chlieh 1 , Jordan J. Green 2 , Aleksander S. Popel 2 , Niranjan B. Pandey 2 and Phuoc T. Tran 1, 3 1 Department of Radiation Oncology and Molecular and Radiation Sciences, Sidney Kimmel Comprehensive Cancer Centre, Johns Hopkins School of Medicine, Baltimore, MD, USA 2 Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD, USA 3 Department of Medical Oncology, Sidney Kimmel Comprehensive Cancer Centre and Department of Urology, The Brady Urological Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA * These authors have contributed equally to this work Correspondence to: Phuoc T. Tran, email: tranp@jhmi.edu , tranpt21@sbcglobal.net Niranjan B. Pandey, email: npandey4@jhmi.edu , npandey@asclepix.com Aleksander S. Popel, email: apopel@jhu.edu Keywords: hepatocellular carcinoma; angiogenesis; c-Met; HGF; IGF1R Received: April 07, 2017 Accepted: August 26, 2017 Published: September 21, 2017 ABSTRACT Hepatocellular carcinoma (HCC) is a major cause of cancer-related death worldwide. Due to inadequate screening methods and the common coexistence of limited functional liver reserves, curative treatment options are limited. Liver transplantation is the only curative treatment modality for early HCC. There are multidisciplinary treatment options like ablative treatments, radiation and systemic therapy available for more advanced patients or those that are inoperable. Treatment resistance and progression is inevitable for these HCC patients. Newer therapeutics need to be explored for better management of HCC. HCC is a hypervascular tumor and many pro-angiogenic proteins are found significantly overexpressed in HCC. Here we explored the therapeutic potential of the anti-angiogenic, anti-lymphangiogenic, and directly anti-tumorigenic biomimetic collagen IV-derived peptide developed by our group. Human HCC cell lines HuH7, Hep3b and HepG2 showed significant disruption of cell adhesion and migration upon treatment with the peptide. Consistent with previously described multimodal inhibitory properties, the peptide was found to inhibit both c-Met and IGF1R signaling in HepG2 cells and blocked HepG2 conditioned media stimulation of microvascular endothelial cell (MEC) tube formation. Furthermore, the peptide treatment of mouse HepG2 tumor xenografts significantly inhibited growth relative to untreated controls. The peptide was also found to improve the survival of autochthonous Myc-induced HCC in a transgenic mouse model. Mechanistically, we found that the peptide treatment reduced microvascular density in the autochthonous liver tumors with increased apoptosis. This study shows the promising therapeutic potential of our biomimetic peptide in the treatment of HCC.

Persistent inflammation is a complication associated with many ocular diseases. Changes in ocular vessels can amplify disease responses and contribute to vision loss by influencing the delivery of leukocytes to the eye, vascular leakage, and perfusion. Here, we report the anti-inflammatory activity for AXT107, a non-RGD, 20-mer &alpha, v&beta, 3 and &alpha, 5&beta, 1 integrin-binding peptide that blocks vascular endothelial growth factor (VEGF)-signaling and activates tyrosine kinase with immunoglobulin and EGF-like domains 2 (Tie2) using the normally inhibitory ligand angiopoietin 2 (Ang2). Tumor necrosis factor &alpha, (TNF&alpha, ), a central inflammation mediator, induces Ang2 release from endothelial cells to enhance its stimulation of inflammation and vascular leakage. AXT107 resolves TNF&alpha, induced vascular inflammation in endothelial cells by converting the endogenously released Ang2 into an agonist of Tie2 signaling, thereby disrupting both the synergism between TNF&alpha, and Ang2 while also preventing inhibitor of nuclear factor-&kappa, B &alpha, (I&kappa, B&alpha, ) degradation directly through Tie2 signaling. This recovery of I&kappa, prevents nuclear factor kappa-light-chain-enhancer of activated B cells (NF-&kappa, B) nuclear localization, thereby blocking NF-&kappa, B-induced inflammatory responses, including the production of VCAM-1 and ICAM-1, leukostasis, and vascular leakage in cell and mouse models. AXT107 also decreased the levels of pro-inflammatory TNF receptor 1 (TNFR1) without affecting levels of the more protective TNFR2. These data suggest that AXT107 may provide multiple benefits in the treatment of retinal/choroidal and other vascular diseases by suppressing inflammation and promoting vascular stabilization.

The angiopoietin (Ang)/Tie2 signaling pathway is essential for maintaining vascular homeostasis, and its dysregulation is associated with several diseases. Interactions between Tie2 and α5 β1 integrin have emerged as part of this control; however, the mechanism is incompletely understood. AXT107, a collagen IV–derived peptide, has strong antipermeability activity and has enabled the elucidation of this previously undetermined mechanism. Previously, AXT107 was shown to inhibit VEGFR2 and other growth factor signaling via receptor tyrosine kinase association with specific integrins. AXT107 disrupts α5 β1 and stimulates the relocation of Tie2 and α5 to cell junctions. In the presence of Ang2 and AXT107, junctional Tie2 is activated, downstream survival signals are upregulated, F-actin is rearranged to strengthen junctions, and, as a result, endothelial junctional permeability is reduced. These data suggest that α5 β1 sequesters Tie2 in nonjunctional locations in endothelial cell membranes and that AXT107-induced disruption of α5 β1 promotes clustering of Tie2 at junctions and converts Ang2 into a strong agonist, similar to responses observed when Ang1 levels greatly exceed those of Ang2. The potentiation of Tie2 activation by Ang2 even extended to mouse models in which AXT107 induced Tie2 phosphorylation in a model of hypoxia and inhibited vascular leakage in an Ang2-overexpression transgenic model and an LPS-induced inflammation model. Because Ang2 levels are very high in ischemic diseases, such as diabetic macular edema, neovascular age-related macular degeneration, uveitis, and cancer, targeting α5 β1 with AXT107 provides a potentially more effective approach to treat these diseases. Fil: Mirando, Adam C.. University Johns Hopkins; Estados Unidos Fil: Shen, Jikui. University Johns Hopkins; Estados Unidos Fil: Silva, Raquel Lima E.. University Johns Hopkins; Estados Unidos Fil: Chu, Zenny. University Johns Hopkins; Estados Unidos Fil: Sass, Nicholas C.. University Johns Hopkins; Estados Unidos Fil: Lorenc, Valeria Erika. University Johns Hopkins; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Green, Jordan J.. University Johns Hopkins; Estados Unidos. AsclepiX Therapeutics; Estados Unidos Fil: Campochiaro, Peter A.. University Johns Hopkins; Estados Unidos Fil: Popel, Aleksander S.. University Johns Hopkins; Estados Unidos Fil: Pandey, Niranjan B.. University Johns Hopkins; Estados Unidos. AsclepiX Therapeutics; Estados Unidos

Macrophages respond to signals in the microenvironment by changing their functional phenotypes, a process known as polarization. Depending on the context, they acquire different patterns of transcriptional activation, cytokine expression and cellular metabolism which collectively constitute a continuous spectrum of phenotypes, of which the two extremes are denoted as classical (M1) and alternative (M2) activation. To quantitatively decode the underlying principles governing macrophage phenotypic polarization and thereby harness its therapeutic potential in human diseases, a systems-level approach is needed given the multitude of signaling pathways and intracellular regulation involved. Here we develop the first mechanism-based, multi-pathway computational model that describes the integrated signal transduction and macrophage programming under M1 (IFN-γ), M2 (IL-4) and cell stress (hypoxia) stimulation. Our model was calibrated extensively against experimental data, and we mechanistically elucidated several signature feedbacks behind the M1-M2 antagonism and investigated the dynamical shaping of macrophage phenotypes within the M1-M2 spectrum. Model sensitivity analysis also revealed key molecular nodes and interactions as targets with potential therapeutic values for the pathophysiology of peripheral arterial disease and cancer. Through simulations that dynamically capture the signal integration and phenotypic marker expression in the differential macrophage polarization responses, our model provides an important computational basis toward a more quantitative and network-centric understanding of the complex physiology and versatile functions of macrophages in human diseases., Author summary As essential regulators of the immune system, macrophages can be polarized to acquire distinct phenotypes in response to a wide range of signals in the tissue microenvironment, such as bacterial products, endogenous cytokines, cell damage and stress. Decades of research has shown that a number of signaling pathways can regulate this process and determine the functional phenotypes of macrophages in physiology as well as various disease scenarios, and recent studies suggest that macrophage polarization is indeed a dynamic process and that the canonical dichotomous notion with only classical (M1) and alternative (M2) activation states is oversimplifying the continuous spectrum of polarized macrophage phenotypes observed in health and disease. To investigate the mechanistic and therapeutic aspects associated with differentially polarized macrophages, we formulated and calibrated a multi-pathway computational model based on literature knowledge and quantitative experimental datasets to systematically describe the integrative regulation of macrophage transcriptional programs and phenotype markers under different stimuli combinations. Our systems-level model is a key building block of a potential “virtual macrophage” simulation platform that can enable researchers to efficiently generate mechanistic hypotheses and assess macrophage-based therapeutic strategies for human diseases.

Vascular endothelial growth factor (VEGF)–neutralizing proteins provide benefit in several retinal and choroidal vascular diseases, but some patients still experience suboptimal outcomes, and the need for frequent intraocular injections is a barrier to good outcomes. A mimetic peptide derived from collagen IV, AXT107, suppressed subretinal neovascularization (NV) in two mouse models predictive of effects in neovascular age-related macular degeneration (NVAMD) and inhibited retinal NV in a model predictive of effects in ischemic retinopathies. A combination of AXT107 and the current treatment aflibercept suppressed subretinal NV better than either agent alone. Furthermore, AXT107 caused regression of choroidal NV. AXT107 reduced the VEGF-induced vascular leakage that underlies macular edema in ischemic retinopathies and NVAMD. In rabbit eyes, which are closer to the size of human eyes, intraocular injection of AXT107 significantly reduced VEGF-induced vascular leakage by 86% at 1 month and 70% at 2 months; aflibercept significantly reduced leakage by 69% at 1 month and did not reduce leakage at 2 months, demonstrating the longer effectiveness of AXT107. AXT107 reduced ligand-induced phosphorylation of multiple receptors: VEGFR2, c-Met, and PDGFRβ. Optimal signaling through these receptors requires complex formation with β3 integrin, which was reduced by AXT107 binding to αvβ3. AXT107 also reduced total VEGFR2 levels by increasing internalization, ubiquitination, and degradation. This biomimetic peptide is a sustained, multitargeted therapy that may provide advantages over intraocular injections of specific VEGF-neutralizing proteins.

The polyketide natural product borrelidin displays antibacterial, antifungal, antimalarial, anticancer, insecticidal and herbicidal activities through the selective inhibition of threonyl-tRNA synthetase (ThrRS). How borrelidin simultaneously attenuates bacterial growth and suppresses a variety of infections in plants and animals is not known. Here we show, using X-ray crystal structures and functional analyses, that a single molecule of borrelidin simultaneously occupies four distinct subsites within the catalytic domain of bacterial and human ThrRSs. These include the three substrate-binding sites for amino acid, ATP and tRNA associated with aminoacylation, and a fourth ‘orthogonal’ subsite created as a consequence of binding. Thus, borrelidin competes with all three aminoacylation substrates, providing a potent and redundant mechanism to inhibit ThrRS during protein synthesis. These results highlight a surprising natural design to achieve the quadrivalent inhibition of translation through a highly conserved family of enzymes., Borrelidin is an antibiotic with antimicrobial, antifungal, antimalarial and immunosuppressive activity that targets threonyl-tRNA synthetase. Here the authors show that borrelidin functions by preventing binding of all three ThrRS substrates and inducing a distinct, non-productive, conformation of the enzyme.

In addition to their canonical roles in translation the aminoacyl-tRNA synthetases (ARSs) have developed secondary functions over the course of evolution. Many of these activities are associated with cellular survival and nutritional stress responses essential for homeostatic processes in higher eukaryotes. In particular, six ARSs and one associated factor have documented functions in angiogenesis. However, despite their connection to this process, the ARSs are mechanistically distinct and exhibit a range of positive or negative effects on aspects of endothelial cell migration, proliferation, and survival. This variability is achieved through the appearance of appended domains and interplay with inflammatory pathways not found in prokaryotic systems. Complete knowledge of the non-canonical functions of ARSs is necessary to understand the mechanisms underlying the physiological regulation of angiogenesis.

Aminoacyl-tRNA synthetases classically regulate protein synthesis but some also engage in alternative signaling functions related to immune responses and angiogenesis. Threonyl-tRNA synthetase (TARS) is an autoantigen in the autoimmune disorder myositis, and borrelidin, a potent inhibitor of TARS, inhibits angiogenesis. We explored a mechanistic link between these findings by testing whether TARS directly affects angiogenesis through inflammatory mediators. When human vascular endothelial cells were exposed to tumor necrosis factor-α (TNF-α) or vascular endothelial growth factor (VEGF), TARS was secreted into the cell media. Furthermore, exogenous TARS stimulated endothelial cell migration and angiogenesis in both in vitro and in vivo assays. The borrelidin derivative BC194 reduced the angiogenic effect of both VEGF and TARS, but not a borrelidin-resistant TARS mutant. Our findings reveal a previously undiscovered function for TARS as an angiogenic, pro-migratory extracellular signaling molecule. TARS thus provides a potential target for detecting or interdicting disease-related inflammatory or angiogenic responses.