VEGF Receptor Research Articles

Inhibition of PCSK9 Improves the Development of Pulmonary Arterial Hypertension Via Down-Regulating Notch3 Expression.

Pulmonary arterial hypertension (PAH) is a fatal disease characterized by continuous constriction and occlusion of small pulmonary arteries, leading to the development of right ventricular failure and death. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a kind of serine protease enzyme that increases low-density lipoprotein cholesterol (LDLC) levels through degrading low-density lipoprotein cholesterol receptors (LDLr). However, whether inhibition of PCSK9 can alleviate PAH has not been reported. We reported that PCSK9 expression was up-regulated in lung tissues of PAH patients. In addition, we used PCSK9 monoclonal antibody subcutaneously to inhibit PCSK9 expression in mice exposed to chronic hypoxia (10%) in combination with SU5416, a VEGF receptor inhibitor. Hypoxia plus SU5416-induced PAH was attenuated in PCSK9 monoclonal antibody-treated mice compared with wild-type mice. PCSK9 inhibited pulmonary vascular remodeling in mice. Moreover, PCSK9 knockdown significantly altered the proliferation and migration of hypoxia-induced PASMCs. We also found that PCSK9 monoclonal antibody inhibited Notch3 expression in vivo and in vitro. Our results suggest that the PCSK9-Notch3 signaling pathway is critical for the proliferation and migration of PASMCs and provides a potential drug target for the treatment of PAH.

Aflibercept Suppression of angiopoietin-2 in a Rabbit Retinal Vascular Hyperpermeability Model.

Anti-vascular endothelial growth factor (anti-VEGF) therapies, which attenuate the capacity of VEGF to bind to VEGF receptors, are standard-of-care options for various retinal disorders that are characterized by pathologic retinal angiogenesis and vascular permeability. Multiple receptors and ligands have also been reported as being involved in these pathways, including angiopoietin-1 (ANG1) and angiopoietin-2 (ANG2). Electrochemiluminescence immunoassays were used to detect human VEGF (hVEGF), as well as rabbit ANG2 and basic fibroblast growth factor protein levels in vitreous samples derived from a study evaluating the efficacy of the anti-VEGF agents ranibizumab, aflibercept, and brolucizumab in an hVEGF165-induced rabbit retinal vascular hyperpermeability model. hVEGF was completely suppressed in rabbit vitreous after anti-VEGF treatment for 28 days. ANG2 protein in vitreous and ANGPT2 mRNA in retina tissue were similarly suppressed, although the anti-VEGF agents do not directly bind to ANG2. Aflibercept demonstrated the greatest inhibitory effect in ANG2 levels in vitreous, which correlated with strong, durable suppression of intraocular hVEGF levels. This study explored the effects of anti-VEGF therapies beyond direct binding of VEGF by evaluating protein levels and the expression of target genes involved in angiogenesis and associated molecular mechanisms in the rabbit retina and choroid. In vivo data suggest that anti-VEGF agents currently used for the treatment of retinal diseases could provide beneficial effects beyond direct binding of VEGF, including suppression of ANG2 protein and ANGPT2 mRNA.

Insights into Overlappings of Fibrosis and Cancer: Exploring the Tumor-related Cardinal Genes in Idiopathic Pulmonary Fibrosis.

The pathogenesis of idiopathic pulmonary fibrosis (IPF) is quite similar to that of cancer pathogenesis, and several pathways appear to be involved in both disorders. The mammalian target of the rapamycin (mTOR) pathway harbors several established oncogenes and tumor suppressors. The same signaling molecules and growth factors, such as vascular endothelial growth factor (VEGF), contributing to cancer development and progression play a part in fibroblast proliferation, myofibroblast differentiation, and the production of extracellular matrix in IPF development as well. The expression of candidate genes acting upstream and downstream of mTORC1, as well as Vegf and low-density lipoprotein receptor related protein 1(Lrp1), was assessed using specific primers and quantitative polymerase chain reaction (qPCR) within the lung tissues of bleomycin (BLM)-induced IPF mouse models. Lung fibrosis was evaluated by histological examinations and hydroxyproline colorimetric assay. BLM-exposed mice developed lung injuries characterized by inflammatory manifestations and fibrotic features, along with higher levels of collagen and hydroxyproline. Gene expression analyses indicated a significant elevation of regulatory associated protein of mTOR (Raptor), Ras homolog enriched in brain (Rheb), S6 kinase 1, and Eukaryotic translation initiation factor 4E-binding protein 1 (4Ebp1), as well as a significant reduction of Vegfa, Tuberous sclerosis complex (Tsc2), and Lrp1; no changes were observed in the Tsc1 mRNA level. Our findings support the elevation of S6K1 and 4EBP1 in response to the TSC/RHEB/mTORC1 axis, which profoundly encourages the development and establishment of IPF and cancer. In addition, this study suggests a possible preventive role for VEGF-A and LRP1 in the development of IPF.

Analysis of differentially expressed genes in bile acid-treated liver cancer cells

Liver cancer is one of the tumors of digestive system. Bile acids are derivatives of choline acids and play a regulatory role in tumors. However, the gene expression profiles in liver cancer cells after bile acid treatment remain unclear. Human hepatoma cell line SMMC7721 was herein cultured. mRNA expression profile was detected by mRNA suppression subtractive hybridization. SMMC7721 cells were divided into 3 groups: control group, DLC1 (deleted in hepatocellular carcinoma 1) group and DLC1 siRNA group. The expression of DLC1, cell proliferation, cell apoptosis and cell invasion were detected by Transwell chamber method. The expressions of VEGF, MMP-2 and DLC1 were detected by Western blot. After bile acid treatment, DLC1, B-cell receptor-associated protein 31 (BCAP31), extension factor 1-α1 (eEF1a1), cell division cycle 20 (CDC20), WD repeat containing protein 6 (WDR6), extension factor Tu and mitochondria (TUFM) were the most significantly increased genes. DLC1 gene was selected with most significant changes. Overexpression of DLC1 significantly decreased expression of VEGF, MMP-2, and ephrin type-A receptor 2 (EphA2) (P <0.05). Transfection of DLC1 siRNA significantly down-regulated DLC1, promoting cell proliferation, decreasing Caspase3 activity, and increasing cell invasion, expression of VEGF, MMP-2 and EphA2 (P <0.05). Bile acid can cause differential gene expressions in liver cancer cells with DLC1 changes being most significant. DLC1 can influence the invasion and of proliferation hepatoma cells by regulating the expression of VEGF, MMP-2 and EphA2.

Investigation into the role of Apj and Vegf receptor signaling in the regulation of coronary angiogenesis

Coronary artery disease (CAD) is one of the leading causes of death worldwide; however, a therapeutic remedy has not been developed. Coronary angiogenesis, a process by which new coronary vessels are formed in the embryos, is primarily a function of endothelial cells which form the inner lining of the vessel wall. Therefore, understanding coronary endothelial cells' cellular and molecular biology can teach us how to effectively stimulate the repair and regeneration of damaged coronary vessels. Investigations in mice have identified the Apelin receptor (alias: APJ) and Vascular endothelial growth factor receptor 2 (VEGFR2) signaling as regulators of coronary angiogenesis. However, it is unclear whether APJ and VEGFR2 signaling interact with one another in the regulation of coronary angiogenesis. To investigate this, we performed in vitro cell culture experiments using isolated coronary endothelial cells and in vivo experiments with APJ KO mice. We hypothesize that ELABELA/APJ signaling downregulates VEGFR2 expression in coronary endothelial cells. Our expression analysis using immunostaining and qPCR show that APJ loss-of-function in coronary endothelial cells results in increased VEGFR2 expression. Collectively, our results show that ELABELA/APJ suppresses VEGFR2 signaling during coronary angiogenesis. This study is supported by the NIH R15 (1R15HL159660-01) research grant to BS. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

BIODEGRADABLE MAGNESIUM COMBINED WITH DISTRACTION OSTEOGENESIS SYNERGISTICALLY STIMULATES BONE TISSUE REGENERATION

Critical size bone defects are frequently caused by accidental trauma, oncologic surgery, and infection. Distraction osteogenesis (DO) is a useful technique to promote the repair of critical size bone defects. However, DO is usually a lengthy treatment, therefore accompanied with increased risks of complications such as infections and delayed union.Herein, we developed an innovative intramedullary biodegradable magnesium (Mg) nail to accelerate bone regeneration in critical size bone defect repair during DO.We observed that Mg nail induced almost 4-fold increase of new bone formation and over 5-fold of new vessel formation at 2 weeks after distraction. Mg nail upregulated the expression of calcitonin gene-related peptide (CGRP) in the new bone as compared with the DO alone group. We further revealed that blockade of the sensory nerve by overdose capsaicin blunted Mg nail enhanced critical size bone defect repair during the DO process. Moreover, inhibitors/antagonist of CGRP receptor, FAK, and VEGF receptor blocked the Mg nail stimulated vessel and bone formation.In summary, we revealed, for the first time, a CGRP-FAK-VEGF signaling axis linking sensory nerve and endothelial cells, which may be the main mechanism underlying Mg-enhanced critical size bone defect repair when combined with DO, suggesting a great potential of Mg implants in reducing DO treatment time for clinical applications.

Blockade of VEGFR3 signaling leads to functional impairment of dural lymphatic vessels without affecting autoimmune neuroinflammation.

The recent discovery of lymphatic vessels (LVs) in the dura mater, the outermost layer of meninges around the central nervous system (CNS), has opened a possibility for the development of alternative therapeutics for CNS disorders. The vascular endothelial growth factor C (VEGF-C)/VEGF receptor 3 (VEGFR3) signaling pathway is essential for the development and maintenance of dural LVs. However, its significance in mediating dural lymphatic function in CNS autoimmunity is unclear. We show that inhibition of the VEGF-C/VEGFR3 signaling pathway using a monoclonal VEGFR3-blocking antibody, a soluble VEGF-C/D trap, or deletion of the Vegfr3 gene in adult lymphatic endothelium causes notable regression and functional impairment of dural LVs but has no effect on the development of CNS autoimmunity in mice. During autoimmune neuroinflammation, the dura mater was only minimally affected, and neuroinflammation-induced helper T (TH) cell recruitment, activation, and polarization were significantly less pronounced in the dura mater than in the CNS. In support of this notion, during autoimmune neuroinflammation, blood vascular endothelial cells in the cranial and spinal dura expressed lower levels of cell adhesion molecules and chemokines, and antigen-presenting cells (i.e., macrophages and dendritic cells) had lower expression of chemokines, MHC class II-associated molecules, and costimulatory molecules than their counterparts in the brain and spinal cord, respectively. The significantly weaker TH cell responses in the dura mater may explain why dural LVs do not contribute directly to CNS autoimmunity.

Abstract LB208: A potent macrophage switching drug D-4559 reduces tumor GROWTH in a hepatocellular carcinoma mouse model

Abstract Tumor-associated macrophages (TAMs) play a role in cancer progression and are associated with Sorafenib resistance in hepatocellular carcinoma (HCC)1. D-4559 is a new potent macrophage switching nanomedicine technology that selectively inhibits VEGF receptor tyrosine kinases (VEGFR1, 2, 3) in TAMs leading to a functional reprogramming of TAMs toward a pro-inflammatory activated phenotype. Here, we evaluate the effect of D-4559 on the M1 and M2 polarization of TAMs and its anti-tumor efficacy in a murine HCC tumor model. In vivo efficacy of D-4559 was examined in the subcutaneous Hepa 1-6 liver tumor model in C57BL/6 mice. Animals (n=15/group) were treated with D-4559 (i.p., 200 mg/kg daily) and free drug Sorafenib (p.o., 40 mg/kg daily) as a positive control for 4 weeks. The treatment started when the mean tumor size reached approximately 100 mm3, then the animals were randomly allocated into study groups. The day of randomization and treatment was denoted as day 0. Tumor sizes were measured using a caliper for 27 days, M1/M2 macrophage polarization was examined by flow cytometry at day 16, and cytokine biomarkers were evaluated with MSD Cytokine Multiplex Assay at day 16 and 27.D-4559 significantly reduced Hepa 1-6 tumor growth (**p<0.01 vs. vehicle). D-4559 and Sorafenib efficacy were similar with 40% of mice with a tumor volume less than 500 mm3 after 27 days of treatment. At day 16, D-4559 significantly increased M1/M2 ratio by inducing M1 macrophage infiltration and reprogramming of TAMs into M1 macrophages compared to vehicle treatment groups (* p<0.05). In contrast, Sorafenib did not switch TAM polarization from M2 to M1 phenotype. Moreover, D-4559 significantly increased TNF-a and IL-8 cytokines in the tumor microenvironment at days 16 and 27, indicative of a M1 signature. Increased levels of TNF-a and IL-8 have also been correlated with better survival in HCC patients2,3. D-4559 significantly increased M1 infiltration, shifted TAM polarization from M2 to M1 phenotype and favored pro-inflammatory cytokines suggesting that D-4559 creates an immunopermissive tumor microenvironment leading to a reduction in tumor growth. This preclinical study supports the development of the potent macrophage switching drug D-4559 as a safe and effective agent that can be used systemically for the treatment of HCC or in combination therapies to improve anti-tumor immune response.

Investigation of the Role of Carcinoembryonic Antigen-Related Cell Adhesion Molecule-1 in Diabetic Retinopathy

ABSTRACT Purpose Diabetic retinopathy (DR) has become a major cause of blindness with increased prevalence of diabetic mellitus. Carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1) plays a part in pathological neovascularization. This study aimed to investigate the role of CEACAM1 in the progression of DR. Methods Aqueous and vitreous samples were collected from proliferative or non-proliferative DR and the control group. Multiplex fluorescent bead-based immunoassays were used to detect the levels of Cytokines. Expression of CEACAM1, VEGF, VEGF receptor 2 (VEGFR2) and hypoxia-induced factor-1α (HIF-1α) were detected in human retinal microvascular endothelial cells (HRECs). Results CEACAM1 and VEGF levels were significantly upregulated in PDR group and positively correlated with PDR progression. Expression CEACAM1 and VEGFR2 were increased in HRECs under hypoxic conditions. The HIF-1α/VEGFA/VEGFR2 pathway was blocked by CEACAM1 siRNA in vitro. Conclusions CEACAM1 might play a role in the pathology of PDR. CEACAM1 might be a therapeutic target for retinal neovasculariztion.

Abstract 5071: Olverembatinib (HQP1351) enhances antitumor effects of immunotherapy in renal cell carcinoma (RCC)

Abstract In solid tumors, resistance to checkpoint inhibitors (CPIs) is frequently observed, partially due to upregulation of vascular endothelial growth factor A (VEGFA) and programmed death-ligand 1 (PD-L1). This culminates in an immunosuppressive tumor microenvironment and immune escape. Inhibitors against VEGF and the VEGF receptor (VEGFR) foster tumor vessel normalization and immunostimulatory reprogramming, in turn promoting treatment effects of immunotherapies. In recent years, TKIs, including axitinib, lenvatinib, and cabozantinib, plus immunotherapy have been approved to treat advanced RCC. Currently under clinical development for relapsed or refractory chronic myeloid leukemia and gastrointestinal tumor, olverembatinib (HQP1351) is a new-generation multikinase inhibitor with targets including VEGFR, fibroblast growth factor receptor (FGFR), SRC, BCR-ABL1, c-KIT, and platelet-derived growth factor receptor. The aim of this study was to assess whether olverembatinib combined with immunotherapy can promote inhibitory effects on RCC. In cell-free kinase assays, olverembatinib inhibited VEGFR1, -2, and -3 with IC50 values of 4.2, 6.1, and 4.1 nM, respectively. Compared to lenvatinib, olverembatinib had more potent antiproliferative effects on human umbilical vein endothelial cells. Olverembatinib also had antiproliferative activity in murine RCC lines RANCA and RAG, with IC50 values of 141 and 53 nM, respectively. When olverembatinib was coadministered with an anti-PD-1 antibody in a RANCA-derived syngeneic model, both agents exerted synergistic effects, with tumor growth inhibition rates reaching 60.6%. Mechanistically, olverembatinib influenced cancer cell proliferation directly by inhibiting phosphorylation of FGFR and downstream proteins. Increased cleavage of caspase-3 and poly (ADP-ribose) polymerase 1 were observed, suggesting induction of apoptosis. Olverembatinib also influenced proliferation of vascular endothelial cells by inhibiting phosphorylation of VEGFR, SRC, and downstream proteins Akt and extracellular signal-regulated kinases. Olverembatinib also reduced expression of PD-L1 in RCC cells. In tumor-infiltrating lymphocyte assays, olverembatinib increased numbers of cytotoxic T cells (CTL, CD8+) and natural-killer cells (NK, CD3−/CD49B+) in RANCA tumor tissues. Combined with an anti-PD-1 antibody, olverembatinib increased CTLs, NK cells, dendritic cells (DCs, MHC-II+/CD11C+), and M1 macrophages (F4/80+/CD11B+/CD86+) in RANCA tumor tissues, indicating an immunoregulatory effect of olverembatinib. Taken together, these data suggest that combining olverembatinib with a CPI confers synergistic antitumor effects in an RCC cancer mouse model by targeting tumor growth, angiogenesis, and immune regulation. This novel combination may provide an alternative approach to enhance treatment effects with CPIs in renal cancers. Citation Format: Guangfeng Wang, Eric Liang, Ping Min, Huidan Yu, Bingxing Wu, Dajun Yang, Yifan Zhai. Olverembatinib (HQP1351) enhances antitumor effects of immunotherapy in renal cell carcinoma (RCC). [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5071.

Abstract 5054: The study of synergy mechanism between surufatinib and vinorelbine in the treatment of non-small cell lung cancer

Abstract Background: The incidence and mortality rate of lung cancer ranks first among malignant tumors. Non-small cell lung cancer (NSCLC) is the most common pathological type of lung cancer. Combined use of targeted therapy can enhance the efficacy of chemotherapy drugs while reduce the related side effects in clinical practice. Surufatinib is a potent, small-molecule tyrosine kinase inhibitor (TKI), selectively targeting VEGF receptors (VEGFR) 1, 2, and 3, Fibroblast growth factor receptor 1 (FGFR1), and CSF-1R, which simultaneously prevents tumor angiogenesis and tumor immune evasion. Our clinical study (NCT04922658) has showed the promising effect and safety of surufatinib plus vinorelbine in NSCLC patients, but the potential mechanism is still to be revealed. Herein, tumor-bearing models and cancer cell culture platform would be established for further studying, which may shed light on the synergy mechanism between surufatinib and vinorelbine, and guide the clinical treatment ultimately. Methods: Subcutaneous tumorigenesis in nude mice were successfully established, and then treated with related experimental drugs. The tumor size was observed and CD31 expression of tumor tissue was measured using immunohistochemical analysis. In vitro study, the A549 and PC9 cell multiplication capacities were measured by CCK8 assay and plate cloning assay. The abilities of cancer cell migration and apoptosis were detected using cell scratch assay and flow cytometry kit, respectively. Results: Monotherapy with surufatinib or vinorelbine showed a moderate tumor suppression, whereas the combination therapy of surufatinib and vinorelbine showed the remarkable inhibition of tumor growth in vivo. Surufatinib and the combination therapy obviously decreased the expression of CD31 in tumor issue which might cause the anti-angiogenesis. Monotherapy with surufatinib or vinorelbine significantly inhibited the proliferation of lung cancer cells (A549 cells and PC9 cells) in a concentration-dependent manner, and surufatinib could memorably promote the anti-proliferation activity of vinorelbine. As a result, combination therapy of surufatinib and vinorelbine distinctly inhibited lung cancer cell proliferation when compared with monotherapy with vinorelbine. Consistently, the synergistic effects of surufatinib plus vinorelbine were also observed in cell migration and apoptosis analysis. Conclusions: Synergistic effect of surufatinib and vinorelbine resulted in a conspicuous tumor regression, which is consistent with the clinical observation. In a depth data analysis, surufatinib enhanced the antitumor activity of vinorelbine probably by inhibiting tumor cell proliferation and migration, suppressing tumor angiogenesis and promoting tumor apoptosis in NSCLC patients. Citation Format: Xiaoran Wu, Yanfang Zheng, Lingyu Qin, Ying Li, Zhongjian Yu, Xiongjie Zhu, Rui Cai, Feiyu Niu, Yan Yuan, Peng Jiang, Shaoshi Wen, Xing Lv, Jiancai Zhou. The study of synergy mechanism between surufatinib and vinorelbine in the treatment of non-small cell lung cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5054.

Abstract 4898: Imipridones and EZH2 inhibitors induce similar changes in cytokines and regulated genes in GBM and DMG while vorinostat potentiates anti-tumor efficacy despite variability in cytokine profiles

Abstract TIC10/ONC201 and ONC206 are small molecule imipridones with anti-cancer activity. ONC201, EZH2i and HDACi can modulate tumor microenvironment (TME) and antitumor immunity. We studied the impact of these drugs or their combinations on the TME or tumor immunity by investigating cytokine profiles. We treated U251 GBM and HCT116 CRC cells with vorinostat and found upregulation of CXCL11, CXCL14, INF-γ and TRAIL, and downregulation of prolactin, CXCL9, VEGF and CCL2 in U251. CXCL11 promotes anti-tumor immunity by increasing activated CD8+ T cells in tumors but was associated with metastasis of GBM. CXCL13 induces tertiary lymphoid structures and enhances infiltration of CD8+ T cells in tumors. INF-γ enhances antigen presentation and promotes activation of NK cells. Vorinostat upregulated the secretion of pro-immune cytokines and TRAIL which induces tumor apoptosis. Downregulated cytokines in U251 were prolactin, CXCL9 involved in tumor growth and metastasis, VEGF, and CCL2 involved in immunosuppression in GBM. Cytokines upregulated in HCT116 included IL-8 contributing to CRC progression, and CXCL13, and CXCL11 which correlates with anti-tumor immunity in colon cancer. Downregulated cytokines in HCT116 were soluble TRAIL R2 functioning as a decoy receptor for TRAIL, VEGF, and prolactin. We treated DMG, GBM and HCC cells with imipridones, EHZ2i tazemetostat or HDACi panobinostat alone or combination of imipridones plus tazemetostat or panobinostat or the triple combination of imipridone plus tazemetostat and panobinostat. We observed that cytokines impairing immunity were downregulated and cytokines promoting immunity were upregulated by individual drugs or combinations. Hep3B HCC cells showed the most robust changes among the mentioned tumors with respect to the changes of cytokine profile following the treatments. Imipridone or EZH2i treated cells showed similar cytokine profile changes in tumor cells. Imipridones downregulated EZH1 and EZH2 proteins in tumor cells. We RNA-seq to investigate gene expression profiles following treatment with ONC201 or tazemetostat. In GBM and DMG, ONC201 and tazemetostat shared similar top regulated genes. GO enrichment analysis showed overlap of top regulated pathways between ONC201 and EZH2i treated cells. Shared regulated pathways in U251 included cell cycle arrest, cell adhesion, nervous system development, cell proliferation, negative regulation of proliferation, PERK-mediated unfolded protein response, extracellular matrix organization, regulation of transcription from RNA polymerase II promoter, and cellular response to hypoxia pathways. We conclude that imipridones ONC201, ONC206, and ONC212 which reduce EZH1 and EZH2 proteins share similar cytokine alterations, gene expression targets and actions with EZH2 inhibitors. Citation Format: Yiqun Zhang, Kelsey Huntington, Wafik S. El-Deiry. Imipridones and EZH2 inhibitors induce similar changes in cytokines and regulated genes in GBM and DMG while vorinostat potentiates anti-tumor efficacy despite variability in cytokine profiles. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4898.

Abstract 6315: Human granzyme B fusions targeting VEGFR on tumor cells and activated vasculature

Abstract VEGF121 is a naturally-occurring splice variant that binds to VEGF receptors R-1 and R-2, which are over-expressed on the endothelium of tumor vasculature but not normal vasculature, as well as several tumor cells. While anti-VEGF therapy is a key therapeutic in several cancer treatment regimens, none of the current therapeutics (bevacizumab, VEGF-trap, tyrosine kinase inhibitors) are directly cytotoxic against tumor cells. The serine protease Granzyme B (GrB) is a key effector in immune-mediated cell killing by inducing apoptosis through both caspase-dependent and -independent mechanisms. We previously reported the development and characterization of a fusion protein composed of GrB and VEGF121 (GrB/VEGF121). Here, we report on GrB-Fc-VEGF121, a fusion protein that incorporates a human IgG heavy chain Fc fragment for dimerization which increases the molecular weight and improves in vivo circulation and targeting potential. GrB-Fc-VEGF121 was expressed in HEK-293E cells under serum-free conditions and purified from the conditioned medium with a final yield of approximately 40 mg/L. The enzymatic activity of GrB in GrB-Fc-VEGF121 was comparable to that of commercially available human GrB. In vitro studies of GrB-Fc-VEGF121 showed cytotoxicity in the nanomolar range against tumor and endothelial cell lines expressing high levels of VEGFR-1 or VEGFR-2. Receptor-negative cells demonstrated IC50 levels in the high micromolar range. Immunofluorescence and confocal microscopy demonstrated that GrB-Fc-VEGF121 readily internalized into VEGFR-2+ tumor cells and endothelial cells within 2 hours of treatment while untargeted GrB did not internalize, suggesting internalization was receptor-mediated. Treatment of VEGFR-2+ endothelial and tumor cells at the IC50 dose resulted in >50% cell death via apoptosis and/or necrosis within 48 h. Ex vivo serum stability of GrB-Fc-VEGF121 indicated a gradual protein loss of GrB-Fc-VEGF121, with an overall stability of about 50% over 96 hours. No toxicity was observed in BALB/c mice treated with a total 475 mg/kg given IP every other day over 5 doses, indicating that the maximum tolerated dose of GrB-Fc-VEGF121 exceeds this dose level. In vivo efficacy studies in an OVCAR8 tumor xenograft model with GrB-Fc-VEGF121 (100 mg/kg over 5 doses) resulted in significant growth inhibition of established tumors compared to vehicle controls. We observed a significant decrease in the number of CD31+ blood vessels and Ki-67+ proliferating tumor cells in treated tumors compared to controls. Thus, GrB-Fc-VEGF121 has significant anti-tumor effects in vivo with virtually no toxicity against normal tissues at doses 4-5 times higher than therapeutic levels. This tumor- and vascular-targeting agent appears to have significant potential as a new class of targeted therapeutic agents with a unique mechanism of action. Research conducted, in part, by the Clayton Foundation for Research. Citation Format: Khalid A. Mohamedali, Lawrence H. Cheung, Ana Alvarez-Cienfuegos, Walter N. Hittelman, Michael G. Rosenblum. Human granzyme B fusions targeting VEGFR on tumor cells and activated vasculature [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6315.

Abstract 571: Angiopoietin-like 4 as a potential therapeutic target for clear cell renal carcinoma

Abstract Renal cell carcinoma (RCC) is the 7th most common type of cancer, with over 400,000 new cases each year and 170,000 deaths worldwide. RCCs are grouped into several histological subtypes, with clear cell renal cell carcinoma (ccRCC) being the most common and most aggressive type. The first line of therapy for ccRCC is tyrosine kinase inhibitors (TKIs) which target multiple kinases including the VEGF receptor. The treatment can be used alone or in combination with immune checkpoint inhibitors. However, most patients develop resistance to the TKIs within six months of treatment. Therefore, our aim is to develop novel single or combinatorial therapies and discover ways to overcome the resistance to TKIs. By analyzing TCGA and published datasets from GEO, we found that angiopoietin-like 4 (ANGPTL4) is significantly elevated in both the WT and TKI-resistant ccRCC tumors. ANGPTL4 has been shown to participate in angiogenesis in several other tumor models, and blocking ANGPTL4 with an antibody in vitro, reduces the angiogenic potential of 786-O and RCC4 human ccRCC cell lines, suggesting a pro-angiogenic effect of ANGPTL4 in ccRCC. Knockout of ANGPTL4 in Caki-1 xenograft tumor model significantly reduced tumor angiogenesis and increased vessel normalization, as evidenced by increased expression of VEGFR2 and adhesion molecules on endothelial cells. The increased normalization of blood vessels is an indication of the therapeutic potential of combing anti-ANGPTL4 therapy with immune checkpoint inhibitors. Furthermore, both knockout of ANGPTL4 in 786-O cells and treatment of 786-O tumor-bearing mice with an ANGPTL4 blocking antibody significantly reduced tumor growth. To illustrate the mechanism of ANGPTL4-induced angiogenesis, we performed live cell receptor capturing to find cANGPTL4 binding partners on the cell membrane of human endothelial cells. After the mass spectrometry analysis, several novel receptors were found, and the top candidate is endothelial cell surface chemotaxis receptor (ECSCR), which is highly expressed on endothelial and adipocytes. Previous studies have shown that ECSCR can potentiate angiogenic signaling, which is similar to our data showing that ANGPTL4 can synergistically increase FGF2-induced angiogenesis. Thus, ANGPTL4 may promote angiogenesis in ccRCC by potentiating other angiogenic signaling via interaction with ECSCR. To summarize, we demonstrated the pro-angiogenic potential of ANGPTL4 in ccRCC and its critical role in tumor development. Knockout or antibody-mediated inhibition of ANGPTL4 reduced tumor angiogenesis and growth and increased vessel normalization. This data supports the therapeutic potential of ANGPTL4 targeted treatment in ccRCC. Citation Format: Zeng Jin, Umasankar De, Lei Wang, Jeremy Kleberg, Weizhou Zhang, Ryan Kolb. Angiopoietin-like 4 as a potential therapeutic target for clear cell renal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 571.

Impact of a TAK-1 inhibitor as a single or as an add-on therapy to riociguat on the metabolic reprograming and pulmonary hypertension in the SUGEN5416/hypoxia rat model.

Background: Despite increasing evidence suggesting that pulmonary arterial hypertension (PAH) is a complex disease involving vasoconstriction, thrombosis, inflammation, metabolic dysregulation and vascular proliferation, all the drugs approved for PAH mainly act as vasodilating agents. Since excessive TGF-β signaling is believed to be a critical factor in pulmonary vascular remodeling, we hypothesized that blocking TGFβ-activated kinase 1 (TAK-1), alone or in combination with a vasodilator therapy (i.e., riociguat) could achieve a greater therapeutic benefit. Methods: PAH was induced in male Wistar rats by a single injection of the VEGF receptor antagonist SU5416 (20mg/kg) followed by exposure to hypoxia (10%O2) for 21 days. Two weeks after SU5416 administration, vehicle, riociguat (3mg/kg/day), the TAK-1 inhibitor 5Z-7-oxozeaenol (OXO, 3mg/kg/day), or both drugs combined were administered for 7days. Metabolic profiling of right ventricle (RV), lung tissues and PA smooth muscle cells (PASMCs) extracts were performed by magnetic resonance spectroscopy, and the differences between groups analyzed by multivariate statistical methods. Results: In vitro, riociguat induced potent vasodilator effects in isolated pulmonary arteries (PA) with negligible antiproliferative effects and metabolic changes in PASMCs. In contrast, 5Z-7-oxozeaenol effectively inhibited the proliferation of PASMCs characterized by a broad metabolic reprogramming but had no acute vasodilator effects. In vivo, treatment with riociguat partially reduced the increase in pulmonary arterial pressure (PAP), RV hypertrophy (RVH), and pulmonary vascular remodeling, attenuated the dysregulation of inosine, glucose, creatine and phosphocholine (PC) in RV and fully abolished the increase in lung IL-1β expression. By contrast, 5Z-7-oxozeaenol significantly reduced pulmonary vascular remodeling and attenuated the metabolic shifts of glucose and PC in RV but had no effects on PAP or RVH. Importantly, combined therapy had an additive effect on pulmonary vascular remodeling and induced a significant metabolic effect over taurine, amino acids, glycolysis, and TCA cycle metabolism via glycine-serine-threonine metabolism. However, it did not improve the effects induced by riociguat alone on pulmonary pressure or RV remodeling. None of the treatments attenuated pulmonary endothelial dysfunction and hyperresponsiveness to serotonin in isolated PA. Conclusion: Our results suggest that inhibition of TAK-1 induces antiproliferative effects and its addition to short-term vasodilator therapy enhances the beneficial effects on pulmonary vascular remodeling and RV metabolic reprogramming in experimental PAH.

Non-Small Cell Lung Cancer Treatment with Molecularly Targeted Therapy and Concurrent Radiotherapy—A Review

Lung cancer is the leading cause of death worldwide for both men and women. Surgery can be offered as a radical treatment at stages I and II and selected cases of stage III (III A). Whereas at more advanced stages, combined modalities of treatment are applied: radiochemotherapy (IIIB) and molecularly targeted treatment (small molecule tyrosine kinase inhibitors, VEGF receptor inhibitors, monoclonal antibodies, and immunological treatment with monoclonal antibodies). Combination treatment, composed of radiotherapy and molecular therapy, is increasingly employed in locally advanced and metastatic lung cancer management. Recent studies have indicated a synergistic effect of such treatment and modification of immune response. The combination of immunotherapy and radiotherapy may result in the enhancement of the abscopal effect. Anti-angiogenic therapy, in combination with RT, is associated with high toxicity and should be not recommended. In this paper, the authors discuss the role of molecular treatment and the possibility of its concurrent use with radiotherapy in non-small cell lung cancer (NSCLC).

Inhibition of VEGF receptors induces pituitary apoplexy: An experimental study in mice.

Anti-vascular endothelial growth factor (VEGF) therapy has been developed for the treatment of a variety of cancers. Although this therapy may be a promising alternative treatment for refractory pituitary adenomas and pituitary carcinomas, the effects of anti-VEGF agents on the pituitary gland are not yet well understood. Here, we found that mice administered with OSI-930, an inhibitor of receptor tyrosine kinases including VEGF receptor 1 and 2, frequently exhibited hemorrhage in the pituitary gland. This is the first report that anti-VEGF therapy can cause pituitary apoplexy. C57BL/6 mice were daily injected intraperitoneally with 100 mg/kg body weight of OSI-930 for one to six days. Pituitary glands were immunohistochemically examined. Four of six mice treated for three days and all of five mice treated for six days exhibited hemorrhage in the pituitary gland. In all cases, the hemorrhage occurred just around Rathke's cleft. In OSI-930-administered mice, the vascular coverage and branching were reduced in the anterior lobe, and capillary networks were also decreased in the intermediate lobe in a treatment-day dependent manner. Few blood vessels around Rathke's cleft of the intermediate lobe express VE-cadherin and are covered with platelet-derived growth factor receptor-β (PDGFR-β)-positive cells, which suggests that capillaries around Rathke's cleft of the intermediate lobe were VE-cadherin-negative and not covered with pericytes. The reduction of capillary plexus around Rathke's cleft was observed at the site where hemorrhage occurred, suggesting a causal relationship with the pathogenesis of pituitary hemorrhage. Our study demonstrates that anti-VEGF agents have a risk of pituitary apoplexy. Pituitary apoplexy should be kept in mind as an adverse effect of anti-VEGF therapy.

Analysis of Efficacy-To-Safety Ratio of Angiogenesis-Inhibitors Based Therapies in Ovarian Cancer: A Systematic Review and Meta-Analysis

(1) Background: Among new anti-angiogenesis agents being developed and ever-changing guidelines indications, the question of the benefits/safety ratio remains unclear. (2) Methods: We performed a systematic review combined with a meta-analysis of 23 randomized controlled trials (12,081 patients), evaluating overall survival (OS), progression free survival (PFS) and toxicity (grade ≥ 3 toxic effects, type, and number of all adverse effects. (3) Results: The analysis showed improvement of pooled-PFS (HR, 0.71; 95% CI, 0.64-0.78; I2 = 77%; p < 0.00001) in first-line (HR, 0.85; 95% CI, 0.78-0.93; p = 0.0003) or recurrent cancer (HR, 0.62; 95% CI, 0.56-0.70; p < 0.00001) and regardless of the type of anti-angiogenesis drug used (Vascular endothelial growth factor (VEGF) inhibitors, VEGF-receptors (VEGF-R) inhibitors or angiopoietin inhibitors). Improved OS was also observed (HR, 0.95; 95% CI, 0.90-0.99; p = 0.03). OS benefits were only observed in recurrent neoplasms, both platinum-sensitive and platinum-resistant neoplasms. Grade ≥ 3 adverse effects were increased across all trials. Anti-angiogenetic therapy increased the risk of hypertension, infection, thromboembolic/hemorrhagic events, and gastro-intestinal perforations but not the risk of wound-related issues, anemia or posterior leukoencephalopathy syndrome. (4) Conclusions: Although angiogenesis inhibitors improve PFS, there are little-to-no OS benefits. Given the high risk of severe adverse reactions, a careful selection of patients is required for obtaining the best results possible.

Smooth Muscle Mineralocorticoid Receptor Promotes Hypertension After Preeclampsia.

Preeclampsia is a syndrome of high blood pressure (BP) with end organ damage in late pregnancy that is associated with high circulating soluble VEGF receptor (sFlt1 [soluble Fms-like tyrosine kinase 1]). Women exposed to preeclampsia have a substantially increased risk of hypertension after pregnancy, but the mechanism remains unknown, leaving a missed interventional opportunity. After preeclampsia, women have enhanced sensitivity to hypertensive stress. Since smooth muscle cell mineralocorticoid receptors (SMC-MR) are activated by hypertensive stimuli, we hypothesized that high sFlt1 exposure in pregnancy induces a postpartum state of enhanced SMC-MR responsiveness. Postpartum BP response to high salt intake was studied in women with prior preeclampsia. MR transcriptional activity was assessed in vitro in sFlt1-treated SMC by reporter assays and PCR. Preeclampsia was modeled by transient sFlt1 expression in pregnant mice. Two months post-partum, mice were exposed to high salt and then to AngII (angiotensin II) and BP and vasoconstriction were measured. Women exposed to preeclampsia had significantly enhanced salt sensitivity of BP verses those with a normotensive pregnancy. sFlt1 overexpression during pregnancy in mice induced elevated BP and glomerular endotheliosis, which resolved post-partum. The sFlt1 exposed post-partum mice had significantly increased BP response to 4% salt diet and to AngII infusion. In vitro, SMC-MR transcriptional activity in response to aldosterone or AngII was significantly increased after transient exposure to sFlt1 as was aldosterone-induced expression of AngII type 1 receptor. Post-partum, SMC-MR-KO mice were protected from the enhanced response to hypertensive stimuli after preeclampsia. Mechanistically, preeclampsia mice exposed to postpartum hypertensive stimuli develop enhanced aortic stiffness, microvascular myogenic tone, AngII constriction, and AngII type 1 receptor expression, all of which were prevented in SMC-MR-KO littermates. These data support that sFlt1-induced vascular injury during preeclampsia produces a persistent state of enhanced sensitivity of SMC-MR to activation. This contributes to postpartum hypertension in response to common stresses and supports testing of MR antagonism to mitigate the increased cardiovascular risk in women after PE.

LITESPARK-024: A randomized phase 1/2 study of belzutifan with or without palbociclib in patients with advanced renal cell carcinoma.

TPS747 Background: The combination of immunotherapy with antiangiogenic agents is a well-established first-line treatment option for patients (pts) with advanced renal cell carcinoma (RCC), but many pts develop resistance, and effective second- or subsequent-line options are needed. The von Hippel-Lindau ( VHL) gene is inactivated in approximately 90% of RCC cases, which results in the constitutive activation of hypoxia-inducible factor 2α (HIF-2α) signaling. HIF-2α is involved in angiogenesis, tumor growth, proliferation, and metastasis, and is a key oncogenic driver in RCC. The HIF-2α inhibitor belzutifan has demonstrated promising antitumor activity with manageable safety in pts with heavily pretreated RCC. The cyclin-dependent kinase (CDK) pathway is altered in several cancer types, including RCC, and is associated with poor clinical outcomes. The CDK 4/6 inhibitor palbociclib inhibited cell growth in RCC cell lines, and the antiproliferative effects of CDK 4/6 inhibition were synergistic with HIF-2α inhibition in HIF-2α–dependent VHL -/- clear cell RCC cell lines. We hypothesized palbociclib could potentially enhance the efficacy of belzutifan as combination therapy for previously treated pts with advanced RCC. Methods: LITESPARK-024 (NCT05468697) is an open-label, multicenter, phase 1/2 randomized study of belzutifan + palbociclib versus belzutifan monotherapy in pts with advanced RCC. Pts must have histologically confirmed unresectable stage IV RCC with a clear cell component, received at least 2 prior systemic regimens (both an anti–PD-1/PD-L1 monoclonal antibody and a VEGF receptor–targeted TKI, in sequence or in combination), have measurable disease per RECIST v1.1 by BICR, have KPS score of ≥70%, and have radiographic disease progression on or after the most recent regimen per investigator. Part 1 will evaluate the safety of belzutifan + palbociclib and determine the recommended phase 2 dose (RP2D) for the combination using a modified toxicity probability interval design. Part 2 will evaluate the safety and efficacy of belzutifan + palbociclib versus belzutifan alone. In part 1, ≤30 pts will be enrolled into 3 dose groups and receive belzutifan 120 mg once daily + palbociclib (75, 100, or 125 mg) daily for 21 consecutive days followed by 7 days off. In part 2, approximately 150 pts will be randomly assigned 2:1 to receive belzutifan 120 mg once daily + palbociclib RP2D (21 consecutive days/7 days off) or belzutifan 120 mg once daily. Pts will be stratified by IMDC risk (0 vs 1-2 vs 3-6) and sarcomatoid histology (yes vs no) at randomization in part 2. The primary end point for part 1 is to assess dose-limiting toxicities and adverse events to determine the RP2D of belzutifan + palbociclib. The primary end point for part 2 is ORR per RECIST v1.1 by investigator assessment. Secondary end points for part 2 are clinical benefit rate, DOR, PFS, OS, and safety and tolerability. Clinical trial information: NCT05468697 .