Kinase Tie2 Research Articles

Abstract We098: Target-Specific Novel Therapeutic Approach for Prevention of Arteriovenous Malformations in Hereditary Hemorrhagic Telangiectasia

Background: Hereditary hemorrhagic telangiectasia (HHT) is a rare genetic vascular disease with no cure. HHT is characterized by abnormalities in blood vessels that occur in mucocutaneous tissues (telangiectasias) and deep tissues (arteriovenous malformations, AVMs). The tangled, enlarged, fragile, and aberrant vasculature in HHT patients is prone to chronic bleeding or hemorrhage and may cause serious complications, especially in the brain. Problem Statement: While diagnosed AVMs can be surgically treated, there are no FDA-approved drugs to prevent or manage them. Developing targeted therapeutics to manage AVMs has been incredibly challenging as the mechanism of AVM formation is not clearly understood. Rationale: Dysregulation of pro-angiogenic signaling pathways is considered to play a major role in AVM development. For instance, VEGFA-VEGFR2, ANG2–TIE2 and MAP Kinase 1/2 signaling have been implicated individually in HHT pathogenesis. While MAPK 1/2 signaling is difficult to target because of its universal presence, its upstream activators VEGFA and ANG2 are lucrative targets for their relative specificity primarily affecting vasculature (Figure 1). Innovation: We use a novel approach to target VEGFA and ANG2 simultaneously using IgG CrossMab bispecific inhibitor, allowing superior efficacy by inhibiting dual specific targets to prevent genetic vasculature abnormalities. Approach: We use vascular latex casting and molecular quantification techniques to assess the benefits of inhibiting abnormal pro-angiogenic signaling in HHT mice models. Our preliminary data shows improved vasculature development with small doses of MAPK 1/2 inhibitor, but early mortality due to its damaging side effects. Preliminarily, the combined effect of the bispecific inhibitor in smaller doses improves the vasculature defects in the mutant brain more robustly than the individual inhibitors alone, while not negatively affecting the wild-type model (Figure 2). We further expect this dual inhibition to normalize the transcriptomic signature of mutants to mimic the wild-type setting. Societal Impact: The expected beneficial effects of the bispecific inhibitor will provide a pre-clinical assessment of the cerebrovascular AVM management strategies for 1.4 million HHT patients. It will further the mechanistic understanding of AVM development and prevention in HHT, the 2 nd most common bleeding disorder.

Identification of naturally occurring flavonoids as anticancer agents: In silico studies

It is recognized that flavonoids have a variety of functions in nature, including anti-inflammatory, antibacterial, antioxidant, anticancer, anti-HIV and neuroprotective properties. In the present study, molecular docking experiment was performed on 15 naturally occurring flavonoids as anticancer agents. The analysis of results was performed on the basis of docking score, glide energy and interaction with amino acids. Each flavonoid showed a specific interaction with target protein. Naringin showed interactions with amino acids GLN131, ASP145, PHE80, VAL83, GLU8, ILE10, THR89 (docking score −12.48) involved with the protein PDB ID: 1gij (inhibitor of cyclin-dependent kinase 4). Naringin further showed interactions with THR887, THR886, LYS833, ASP836 amino acids (docking score −15.078) involved with PDB ID: 5jhb (inhibitor of PI3K and tubulin). Orientin showed interactions with amino acids ASN150, ASP163, LYS43, VAL101 (docking score −13.89) with PDB ID: 2euf as inhibitor of cyclin-dependent kinase 6. Quercetin showed interactions with amino acids PHE1047, LEU840, CYS919, GLU917 (docking score −11.7) with PDB ID: 4agd as inhibitor of VEGF receptor tyrosine kinase inhibitor. Epigallocatechin gallate showed interactions with amino acids LYS868, GLU885, THR916, ASP1046 using protein PDB ID: 3ewh (docking score −10.004) as inhibitor of Tie-2 kinase. The given flavonoid drugs interaction with cancer targets shows their possible potential against various forms of cancer. Epigallocatechin gallate when docked with 3ewh showed best docking score with important interaction required for anticancer activity. In molecular dynamic study, Epigallocatechin gallate in complex with VEGFR2 kinase protein (PDB ID: 3ewh) with RMSD indicates the protein remained stable through the simulation of 100ns with fluctuation in the range of 1.5–3.5 Å. The provided docking results may be useful for the development of novel drug candidate for anticancer targets.

Activity of a TIE2 inhibitor (rebastinib) in a patient with a life-threatening cervicofacial venous malformation.

Targeted therapy has become the first therapeutic option in many patients with vascular anomalies. A male 28-year-old patient presented a severe cervicofacial venous malformation involving half-lower face, anterior neck, and oral cavity with progression despite multiple previous treatments, with a somatic variant in TEK (endothelial-specific protein receptor tyrosine kinase) (c.2740C>T; p.Leu914Phe). The patient had facial deformity, daily episodes of pain and inflammation needing massive amount of medication, and difficulty in speech and swallowing, so rebastinib (a TIE2 kinase inhibitor) was approved for compassionate use. After 6months of treatment, venous malformation had decreased in size and lightened, as well as improved quality-of-life scores.

Abstract 3457: Rebastinib, a TIE2 antagonist improves chemotherapy response in homologous recombination proficient epithelial ovarian cancer murine models

Abstract Objectives: The Angiopoietin (ANGPT)/TIE2 kinase axis plays a critical role in cancer angiogenesis. Rebastinib, an investigational TIE2 inhibitor inhibits Tie2 receptor in endothelial cells and macrophages. M2-like Tie2+ macrophages, being pro-tumorigenic, are an attractive target for cancer therapy. Current clinical trials are testing rebastinib with chemotherapy in platinum-resistant ovarian cancer. However, the mechanism is not fully understood. The objective of this study was to determine the effects of rebastinib alone and combined with chemotherapy in preclinical models of ovarian cancer. Methods: RNA-Seq was carried out to determine rebastinib-induced gene expression in ID8 cells and PMJ2R murine peritoneal macrophages. To determine the pharmacodynamics of immune cells in ascites, syngeneic ID8 mice were pre-treated with 10 mg/kg/day rebastinib/control for a week, followed by rebastinib with or without 20 mg/kg carboplatin + 12 mg/kg paclitaxel (chemo) for two weeks. Mice were sacrificed 24h after the last treatment to harvest ascites for flow cytometry. Cohorts of mice from syngeneic ID8 murine model and from a patient derived xenograft (PDX) model of ovarian cancer were used for survival analysis. Results: RNA-Seq showed that in ID8 cells, 1528 genes were upregulated and 3115 genes were downregulated by rebastinib. In macrophages, 2302 genes were upregulated and 2970 genes were downregulated. Several ANGPT-like genes (ANGPTL2, ANGPTL4, ANGPTL6) involved in tumorigenesis, angiogenesis, proliferation were downregulated 2X-10X (p<0.001) in ID8 cells and macrophages. ANGPTL1, an anti-angiogenic and anti-apoptotic gene, was increased 10X in ID8 cells (p<0.001) but not altered in macrophages. ANGPT2, a context-dependent agonist/antagonist of TIE2 pathway, was increased 1.42X in ID8 (P<0.05) cells and 4.6X in macrophages (p<0.001). Flow studies showed that rebastinib significantly increased CD45+ macrophage (p<0.03 vs. chemo; p<0.02 vs. rebastinib+chemo), maintained Th leukocytes (p<0.03, vs. chemo) and significantly increased cytotoxic T cells (p<0.02 vs. control; p<0.0004, p<0.0001 vs. chemo and rebastinib+chemo, respectively). Rebastinib had no significant effect on regulatory T cells, Tie2+ macrophages or Tie2+ M2 macrophages. Rebastinib combined with chemotherapy improved median survival significantly in murine models of ovarian cancer - ID8 (p<0.01) and PDX (p<0.0001). Conclusions: Rebastinib exerts differential effects on tumor cells and macrophages that could contribute to its mechanism of action. Rebastinib alters immune cells and increases cytotoxic T cells in ascites. Rebastinib combined with chemotherapy extends survival in PDX and syngeneic ID8 murine models of ovarian cancer. Further investigation using rational combinations of rebastinib and chemotherapy are underway. Citation Format: Vijayalaxmi G. Gupta, Katherine F. Roby, Harsh B. Pathak, Andrew K. Godwin, Sumedha Gunewardena, Dineo Khabele. Rebastinib, a TIE2 antagonist improves chemotherapy response in homologous recombination proficient epithelial ovarian cancer murine models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3457.

728P A phase Ib/II study of rebastinib and paclitaxel in advanced/metastatic platinum-resistant ovarian cancer

Rebastinib is a switch-control inhibitor of TIE2 kinase. TIE2 is expressed in endothelial cells and certain macrophages with pro-angiogenic, pro-metastatic, and immunosuppressive properties associated with chemotherapy resistance. This is a 2-part, open-label, phase Ib/II study with orally administered rebastinib in combination with weekly paclitaxel. In part 1, we observed antitumor activity across multiple tumor types, including 3 partial responses (PRs) in platinum-resistant ovarian cancer (PROC).

Ionizing radiation induces endothelial transdifferentiation of glioblastoma stem-like cells through the Tie2 signaling pathway

Glioblastomas (GBM) are brain tumors with a poor prognosis despite treatment that combines surgical resection and radio-chemotherapy. These tumors are characterized by abundant vascularization and significant cellular heterogeneity including GBM stem-like cells (GSC) which contribute to tumor aggressiveness, resistance, and recurrence. Recent data has demonstrated that GSC are directly involved in the formation of new vessels via their transdifferentiation into Tumor Derived Endothelial Cells (TDEC). We postulate that cellular stress such as ionizing radiation (IR) could enhance the transdifferentiation of GSC into TDEC. GSC neurospheres isolated from 3 different patients were irradiated or not and were then transdifferentiated into TDEC. In fact, TDEC obtained from irradiated GSC (TDEC IR + ) migrate more towards VEGF, form more pseudotubes in MatrigelTM in vitro and develop more functional blood vessels in MatrigelTM plugs implanted in Nude mice than TDEC obtained from non-irradiated GSC. Transcriptomic analysis allows us to highlight an overexpression of Tie2 in TDEC IR+. All IR-induced effects on TDEC were abolished by using a Tie2 kinase inhibitor, which confirms the role of the Tie2 signaling pathway in this process. Finally, by analyzing Tie2 expression in patient GBMs by immunohistochemistry, we demonstrated that the number of Tie2+ vessels increases in recurrent GBM compared with matched untreated tumors. In conclusion, we demonstrate that IR potentiates proangiogenic features of TDEC through the Tie2 signaling pathway, which indicates a new pathway of treatment-induced tumor adaptation. New therapeutic strategies that associate standard treatment and a Tie2 signaling pathway inhibitor should be considered for future trials.

Relation Between Tissue Iron Content and Polarization of Macrophages in Diabetic Ulcer and the Transitional Zone of Diabetic Ulcers with Major Amputation.

Most diabetic lower-limb amputations probably result from combinations of contributing causes rather than from unitary causes. Iron-induced damage might modulate the development of chronic diabetes complications. In this study, the relationship between tissue iron levels and polarization of macrophages in induction of angiogenesis was investigated in diabetic ulcer samples and the transitional zone of diabetic ulcers. Patients with diabetic ulcers who underwent amputation were included. The transitional zone of diabetic ulcers, from the same diabetic patients, was used as a control group. After tissue preparation, Perls Prussian blue staining and immunohistochemistry for CD11c, CD163, and CD68 markers were done. Vascular endothelial growth factor (VEGF), hypoxia-inducible factor (HIF), Tie2, and protein kinase B (also known as AKT) transcription of genes were measured by real-time polymerase chain reaction. For statistical analysis, we used independent samples t-test or its nonparametric equivalents, Mann-Whitney U test was used for quantitative variables, and chi-square (or Fisher's exact test) for qualitative variables. According to the results, the ratio of M2 to M1 macrophages was decreased in ulcers tissue compared to the transitional zone of diabetic ulcers. The expression of angiogenesis-related genes was increased due to hypoxia induction such as HIF and VEGF in ulcer tissue (P < .0001), but the expression of vascular stability-related genes such as Tie2 was decreased (P < .0001).In amputated diabetic ulcers, the polarization of macrophages is toward the classic type, but no connection was found in terms of tissue iron and help in the polarization of macrophages.

AUTOSTERE: Systematic Search for Scaffold Replacement Opportunities within Structural Databases.

Medicinal chemists often bias toward working with scaffolds with which previously they have had direct experience and successes. In this way, it is often the case that scaffolds which have proven tractable within a research group are "reused" across multiple and sometimes unrelated drug targets. With this concept in mind, we designed a new computer algorithm AUTOSTERE which could systematically assess the opportunities to replace any part of any molecule within an entire database of known ligand structures with a target scaffold and automatically evaluate the potential designs in the context of the original ligand's protein environment. As such, it performs scaffold replacement on an unprecedented scale and suggests new target opportunities for preferred chemistries rather than the conventional reverse situation. The results of this approach for one scaffold, a substituted triazolinone, applied to a set of 10 426 ligand conformations extracted from the PDB are described. This led to the identification of ∼600 novel ligands incorporating the triazolinone scaffolds in complex with their predicted drug targets. From these, design examples are provided for HSP-90, cathepsin K, and TIE-2 kinase. A further study involved the searching for possible drug targets for unusual pyridopyrimidine cores. This process resulted in the identification of potential novel HIV reverse transcriptase inhibitors which were synthesized and shown to exhibit similar in vitro potencies to marketed compounds. Overall, the methodology described provides a powerful new approach to identify new target opportunities for scaffolds of provenance.

839P A phase Ib/II study of rebastinib and paclitaxel in advanced or metastatic platinum-resistant ovarian cancer

Rebastinib is a switch control inhibitor of TIE2 kinase. TIE2 is expressed in endothelial cells and in some macrophages with pro-angiogenic, pro-metastatic and immunosuppressive properties associated with chemotherapy resistance. This is a 2-part open-label, phase Ib/II study with orally administered rebastinib in combination with weekly paclitaxel 80 mg/m2. In part 1, we observed antitumor activity across multiple tumor types (5 PRs in 24 pts at 50 mg BID and 3 PRs in 19 pts at 100 mg BID), including 3 PRs in platinum-resistant ovarian cancer (PROC). Part 2 has 5 cohorts: TNBC, inflammatory breast cancer, PROC, endometrial cancer and carcinosarcoma. Here, we present preliminary results from the PROC cohort. Part 2 is a Simon 2-stage design enrolling 18 pts into the first stage, and if ≥5 responses, an additional 15 pts in the second stage. Pts were treated with rebastinib in combination with paclitaxel 80 mg/m2 intravenous weekly (D1, D8, D15 of repeated 28-day cycles) and evaluated for safety (CTCAE v5.0) and efficacy (RECIST v1.1). As of March 20, 2020, 20 pts were enrolled; the median age was 58 years. All received ≥ 1 prior regimen with paclitaxel/carboplatin; the median number of prior therapies was 5 (2, 7). Ten pts were initially treated with 100 mg BID rebastinib (reduced to 50 mg BID due to muscular weakness [4 pts in this cohort]) and 10 pts with 50 mg BID with a treatment median duration of 16 weeks (0.7, 31.9). In 17 evaluable pts, there were 5 PRs and 9 SDs (at 8 weeks) for an ORR of 29% and CBR of 82%. In addition, 8/13 (62%) pts had a CA-125 response. TEAEs (≥n=5) were mostly ≤ grade 2, including fatigue (n=8), nausea (n=8), diarrhea, dry mouth, peripheral sensory neuropathy, vomiting (n=6 each); abdominal pain, alopecia, constipation, peripheral edema and stomatitis (n=5 each). Serious AEs possibly related to rebastinib included grade 2, reversible muscular weakness (at 100 mg BID with no additional occurrences at rebastinib 50 mg BID), fatigue and constipation (n=1 each). The preliminary activity and safety of rebastinib at 50 mg BID in combination with weekly paclitaxel for heavily pretreated pts with PROC, all of whom received prior carboplatin/paclitaxel, is encouraging. Simon stage 2 enrollment is ongoing.

Angiopoietin-1/Tie-2 signal after focal traumatic brain injury is potentiated by BQ788, an ETB receptor antagonist, in the mouse cerebrum: Involvement in recovery of blood-brain barrier function.

Angiopoietin-1, an angiogenic factor, stabilizes brain microvessels through Tie-2 receptor tyrosine kinase. In traumatic brain injury, blood-brain barrier (BBB) disruption is an aggravating factor that induces brain edema and neuroinflammation. We previously showed that BQ788, an endothelin ETB receptor antagonist, promoted recovery of BBB function after lateral fluid percussion injury (FPI) in mice. To clarify the mechanisms underlying BBB recovery mediated by BQ788, we examined the involvements of the angiopoietin-1/Tie-2 signal. When angiopoietin-1 production and Tie-2 phosphorylation were assayed by quantitative reverse transcription polymerase chain reaction and western blotting, increased angiopoietin-1 production and Tie-2 phosphorylation were observed in 7-10days after FPI in the mouse cerebrum, whereas no significant effects were obtained at 5days. When BQ788 (15nmol/day, i.c.v.) were administered in 2-5days after FPI, increased angiopoietin-1 production and Tie-2 phosphorylation were observed. Immunohistochemical observations showed that brain microvessels and astrocytes contained angiopoietin-1 after FPI, and brain microvessels also contained phosphorylated Tie-2. Treatment with endothelin-1 (100nM) decreased angiopoietin-1 production in cultured astrocytes and the effect was inhibited by BQ788 (1μM). Five days after FPI, increased extravasation of Evans blue dye accompanied by reduction in claudin-5, occludin, and zonula occludens-1 proteins were observed in mouse cerebrum while these effects of FPI were reduced by BQ788 and exogenous angiopoietin-1 (1μg/day, i.c.v.). The effects of BQ788 were inhibited by co-administration of a Tie-2 kinase inhibitor (40nmol/day, i.c.v.). These results suggest that BQ788 administration after traumatic brain injury promotes recovery of BBB function through activation of the angiopoietin-1/Tie-2 signal.

C16 Peptide Promotes Vascular Growth and Reduces Inflammation in a Neuromyelitis Optica Model.

The goal of this study was to elucidate the mechanism of action of C16, a laminin-1 peptide that competes with αvβ3 for integrin binding, in treating neuromyelitis optica (NMO). A NMO rat model was established and specific inhibitors were used to investigate the effect of Tie2 kinase, integrin, and PI3K/Akt signaling pathways on C16 function in NMO using histological, immunohistochemical, immunofluorescence, Western blot, and ELISA assays. A total of 150 rats were divided into five groups: a control untreated group (n = 18) and four test groups (n = 33 per group) including vehicle-treated control, C16, Tie2 kinase inhibitor + C16, and PI3K/Akt inhibitor LY294002 + C16. We found that inhibiting Tie2 kinase resulted in partial loss of C16 peptide-mediated effects, while suppressing PI3K/Akt signaling reduced C16 peptide-mediated effects. In addition, activation of the αvβ3 integrin axis and Tie2 kinase promoted PI3K/Akt signaling. Our study showed that the Tie2-PI3K/Akt, Tie2 integrin, and integrin-PI3K/Akt signaling pathways regulate C16 peptide function in vascular growth and stabilization as well as inflammation in NMO.

Ionizing radiation induces endothelial transdifferentiation of glioblastoma stem-like cells through the Tie2 signaling pathway

Glioblastomas (GBM) are brain tumors with a poor prognosis despite treatment that combines surgical resection and radio-chemotherapy. These tumors are characterized by abundant vascularization and significant cellular heterogeneity including GBM stem-like cells (GSC) which contribute to tumor aggressiveness, resistance, and recurrence. Recent data has demonstrated that GSC are directly involved in the formation of new vessels via their transdifferentiation into Tumor Derived Endothelial Cells (TDEC). We postulate that cellular stress such as ionizing radiation (IR) could enhance the transdifferentiation of GSC into TDEC. GSC neurospheres isolated from 3 different patients were irradiated or not and were then transdifferentiated into TDEC. In fact, TDEC obtained from irradiated GSC (TDEC IR+) migrate more towards VEGF, form more pseudotubes in MatrigelTM in vitro and develop more functional blood vessels in MatrigelTM plugs implanted in Nude mice than TDEC obtained from non-irradiated GSC. Transcriptomic analysis allows us to highlight an overexpression of Tie2 in TDEC IR+. All IR-induced effects on TDEC were abolished by using a Tie2 kinase inhibitor, which confirms the role of the Tie2 signaling pathway in this process. Finally, by analyzing Tie2 expression in patient GBMs by immunohistochemistry, we demonstrated that the number of Tie2+ vessels increases in recurrent GBM compared with matched untreated tumors. In conclusion, we demonstrate that IR potentiates proangiogenic features of TDEC through the Tie2 signaling pathway, which indicates a new pathway of treatment-induced tumor adaptation. New therapeutic strategies that associate standard treatment and a Tie2 signaling pathway inhibitor should be considered for future trials.

Probing 2H-Indazoles as Templates for SGK1, Tie2, and SRC Kinase Inhibitors.

The broader and systematic application of a novel scaffold is often hampered by the unavailability of a short and reliable synthetic access. We investigated a new strategy for the design and synthesis of an array of N2-substituted aza-2H-indazole derivatives as potential kinase inhibitors. Guided by a rational ligand alignment approach to qualify the so-far underrepresented aza-2H-indazole scaffold, indazoles were connected at the N2 position with a phenyl spacer and an arylsulfonamide or amide linkage. Initial profiling against a panel of 30 kinases confirmed the in silico predicted selectivity bias. A synthesized focused library of 52 different aza-2H-indazole derivatives showed good initial selective inhibition against SGK1, Tie2, and SRC kinases, with the best representatives having IC50 values in the range of 500 nm. In a comparative computational study, these data were analyzed and rationalized in light of docking studies.

Activated protein C induces suppression and regression of choroidal neovascularization– A murine model

Activated protein C (APC) exerts diverse cell signaling pathways which results in multiple distinct cytoprotective actions. These include anti-apoptotic and anti-inflammatory activities and stabilization of endothelial and epithelial barriers. We studied the ability of APC to inhibit the leakage and the growth of newly formed as well as pre-existing choroidal neovascularization (CNV) and examined the ability of APC to stabilize the Retinal Pigmented Epithelium (RPE). We explored the contribution of Tie2 receptor to the protective effects of APC.CNV was induced by laser photocoagulation in C57BL/6J mice. APC was injected intravitreally immediately or 7 days after CNV induction. Neovascularization was evaluated on RPE-choroidal flatmounts using FITC-dextran perfusion and CD31 immunofluorescence. CNV leakage was measured by fluorescein angiography (FA). The ability of APC to stabilize the RPE barrier was evaluated in-vitro by dextran permeability and zonula occludens 1 (ZO1) immunostaining. Tie2 blocking was induced in-vivo by intraperitoneal injection of Tie2 kinase inhibitor and in-vitro by incubation with anti Tie2 antibodies.APC treatment dramatically inhibited the generation of newly formed CNV leakage sites and reversed leakage in 85% of the pre-existing CNV leaking sites. In RPE cell culture, APC induced translocation of ZO1 to the cell membrane, accompanied by reduction in permeability of the monolayer. Inhibition of Tie2 significantly decreased APC protective activities in both the mouse model and the RPE cell culture.Our results show that APC treatment significantly inhibits the leakage and growth of newly formed, as well as pre-existing CNV, and its protective activities are partially mediated via the Tie2 receptor.The data suggest that APC should be further investigated as a possible effective treatment for CNV.

Pro-Angiogenic Actions of CMC-Derived Extracellular Vesicles Rely on Selective Packaging of Angiopoietin 1 and 2, but Not FGF-2 and VEGF.

While the fundamental mechanism by which cardiac cell therapy mitigates ventricular dysfunction in the post ischemic heart remains poorly defined, donor cell paracrine signaling is presumed to be a chief contributor to the afforded benefits. Of the many bioactive molecules secreted by transplanted cells, extracellular vesicles (EVs) and their proteinaceous, nucleic acid, and lipid rich contents, comprise a heterogeneous assortment of prospective cardiotrophic factors-whose involvement in the activation of endogenous cardiac repair mechanism(s), including reducing fibrosis and promoting angiogenesis, have yet to be fully explained. In the current study we aimed to interrogate potential mechanisms by which cardiac mesenchymal stromal cell (CMC)-derived EVs contribute to the CMC pro-angiogenic paracrine signaling capacity in vitro. Vesicular transmission and biological activity of human CMC-derived EVs was evaluated in in vitro assays for human umbilical vein endothelial cell (HUVEC) function, including EV uptake, cell survival, migration, tube formation, and intracellular pathway activation. HUVECs incubated with EVs exhibited augmented cell migration, tube formation, and survival under peroxide exposure; findings which paralleled enhanced activation of the archetypal pro-survival/pro-angiogenic pathways, STAT3 and PI3K-AKT. Cytokine array analyses revealed preferential enrichment of a subset of prototypical angiogenic factors, Ang-1 and Ang-2, in CMC EVs. Interestingly, pharmacologic inhibition of Tie2 in HUVECs, the cognate receptors of angiopoietins, efficiently attenuated CMC-EV-induced HUVEC migration. Further, in additional assays a Tie2 kinase inhibitor exhibited specificity to inhibit Ang-1-, but not Ang-2-, induced HUVEC migration. Overall, these findings suggest that the pro-angiogenic activities of CMC EVs are principally mediated by Ang-1-Tie2 signaling.

Kaolin-SO3H nanoparticles: A new efficient and reusable catalyst for synthesis of 2-substituted benzimidazoles at room temperature

Kaolinite clay found its application in medicine, in toothpaste, in cosmetic and as a food additive. Recently, a specially formulated spray is used in fruit and vegetable production to repel the insects and prevent sunburn. Kaolin-SO3H nanoparticles were prepared via reaction of kaolin and chlorosulfonic acid and characterized by FT-IR, XRD, FESEM, TEM, XRF, EDS, BET and TGA. 2-Substituted benzimidazoles have been used as selective neuro-peptides YY, receptor antagonists, antitumor, antivirus, antimicrobial, antioxidant, antiparasitic, antihelmintics, antiproliferative, anti-HIV, anticonvulsant, anti-inflammatory, antihypertensive, antineoplastic, analgesicand antitrichinellosis, topoisomerase IV inhibitors, potent inhibitors of TiE-2 and VEGFER-2 tyrosine kinase receptor, and 5-HT3 antagonists. 2-Substituted benzimidazoles are prepared via condensation of o-phenylenediamines and aldehydes. In this article, we have used Kaolin-SO3H nanoparticles for the synthesis of 2-substituted benzimidazoles under mild reaction conditions. The structure of products were identified by FT-IR, 1H-NMR and 13C-NMR. This method has the advantages of high yields, short reaction times and easy work-up.

Abstract P6-18-22: Phase Ib study of rebastinib plus antitubulin therapy with paclitaxel (P) or eribulin (E) in patients with HER2-negative metastatic breast cancer (MBC)

Abstract BACKGROUND: TMEM (Tumor Microenvironment of Metastasis) are the portal for tumor cell intravasation into the circulation and subsequent metastasis (Harney et al Cancer Discov 2015). The potent Tie2 kinase inhibitor rebastinib inhibits intravasation at TMEM sites, reduces circulating tumor cell (CTC) burden, increases angiopoietin (Ang) 1/2 levels, prevents distant metastases, and improves survival in breast cancer animal models when added to either P or E (Harney et al MCT 2017), and circumvent chemotherapy-induced pro-metastatic changes in the tumor microenvironment mediated by TMEM (Karagiannis et al STM 2017). We sought to determine the safety of rebastinib combined with antitubulin therapy (P or E) in patients with HER2- MBC. We also hypothesized that addition of rebastinib would reduce CTC burden and increase Ang levels by blocking Ang-mediated stimulation of VEGF release from TMEM-associated macrophages. METHODS: We aimed to determine the safety and recommended phase 2 dose (RP2D) of rebastinib (2 dose levels: 50 mg or 100 mg PO BID) in combination with P (80 mg/m2 x 12 weeks) or E (1.4 mg/m2 on day 1 &amp; 8 q 21 days) using a standard 3+3 design (1 cycle = 21 days). Secondary objectives included evaluating the effect of the P/E + rebastinib combination on CTCs (TelomeScan) and Ang levels. Dose limiting toxicity (DLT) was defined as grade 3-4 febrile neutropenia, thrombocytopenia, and non-hematologic toxicity during the first 6 weeks of therapy. Eligibility included HER2- MBC, ECOG PS 0-1, CDK4/6 inhibitor progression if ER+. Patients with ≤ 2 prior non-taxane chemotherapy regimens received P+ rebastinib, whereas those with ≥ 2 chemo regimens (including a taxane) received E+ rebastinib. RESULTS: Of 11 treated patients, 6 received rebastinib + P and 5 received rebastinib + E (2 non-evaluable due to rapid disease progression and non-compliance). Among 11 patients who received 60 treatment cycles, only 1 patient (treated with eribulin) had grade 3 events (anemia and neuropathy after week 6) potentially related to treatment. When combined with P, the RP2D of rebastinib was 100 mg PO BID, with DLT occurring in 0/6 patients. When combined with E, 0/3 evaluable patients had a DLT at 50 mg BID of rebastinib (accrual ongoing for 100mg BID). Best response included partial response/stable disease in 4(2PR/2SD) of 6 treated with P+ rebastinib, and 1(1PR) of 5 treated with E+ rebastinib. CTCs decreased during therapy (median decrease 99.7 %) and 4/8 patients converted from CTC+ to CTC-. Ang1 levels increased during therapy in 8 patients (0.2-7.0 fold), while Ang2 levels were also increased in 8 patients (0.2-1.4 fold). CONCLUSIONS: When combined with P x 12 weeks, the RP2D of rebastinib is 100 mg PO BID. When combined with E, the RP2D of rebastinib is at least 50mg PO BID; however, the 100 mg PO BID dose level is still accruing patients. The P/E + rebastinib combinations are associated with antitumor activity and exhibit pharmacodynamic evidence indicating blockade of Tie2 (increased Ang) and TMEM function (reduced CTCs) We plan to further evaluate the P+ rebastinib combination as neoadjuvant therapy in the I-SPY program, and continue further evaluation of P/E + rebastinib combinations in MBC. Citation Format: Anampa JD, Xue X, Oh S-y, Kornblum N, Sadan S, Oktay MH, Condeelis J, Sparano JA. Phase Ib study of rebastinib plus antitubulin therapy with paclitaxel (P) or eribulin (E) in patients with HER2-negative metastatic breast cancer (MBC) [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-18-22.

A Tie2 kinase mutation causing venous malformations increases phosphorylation rates and enhances cooperativity

The endothelial receptor tyrosine kinase Tie2 plays an important role in vascular formation and maintenance. Mutations in Tie2 lead to vascular malformations, which are painful vascular lesions that cause disfigurement, bleeding, and thrombosis. R849W Tie2 is the most common mutation implicated in an inherited form of vascular malformations and has been shown to be activating, though little is known about the kinetic features of catalysis. Here we undertake a steady-state kinetic analysis of heterologously expressed and purified wild type (WT) and R849W Tie2. While the catalytic efficiencies of the two forms are not significantly different, the observed maximal rate of phosphorylation, kcat,obs, is > 3-fold higher for R849W Tie2 compared to WT. Notably, steady-state catalysis by R849W Tie2 has more striking sigmoidal features compared to WT, suggesting enhanced positive cooperativity. We propose that activating catalytic features are one important consequence of the R849W mutation, though likely other factors such as increased protein binding affinity also contribute to the phenotypes observed in patients.

Activated Protein C Induces Stabilization of the Outer Retinal Barrier and Inhibits Laser Induced Choroidal Neovascularization in a Murine Model

Introduction: Age-related macular degeneration (AMD) is considered one of the main causes of severe vision loss in older adults. Wet AMD is characterized by choroidal neovascular membrane developed under or into the sensory retina, breaking the outer blood-retina barrier (oBRB) and causing leakage and bleeding. Protein C (PC) is a plasma serine protease zymogen whose active form, activated protein C (APC), exerts diverse homeostatic cell signaling pathways apart from its anticoagulant activity. APC activities are mediated mainly through endothelial PC receptor (EPCR) and protease activated receptor (PAR)-1. APC was recently shown to act as a novel ligand for Tie2, a tyrosine kinase receptor playing an important role in angiogenesis. Thus, APC may modulate pathological angiogenesis in relevant ocular conditions, such as choroidal neovascularization (CNV), via its interaction with Tie2. To the best of our knowledge, there are no studies addressing the potential use of APC as oBRB stabilizer and CNV inhibitor.Aim: To assess the protective effect of APC as a retinal barrier stabilizer and its ability to reduce CNV growth, leakage and penetration from the choroid into the sensory retina. Moreover, to study the role of Tie2 receptor upon APC mode of action in the retina.Methods: CNV development was induced in C57BL/6J mice by laser applications. Immediately following CNV induction APC (0.1-10 µg/µl) was injected intravitrealy. Seven to 14 days later, the retina was separated from the underlying tissues and the 2 segments were flattened: sclera-choroid-retinal pigment epithelium (RPE) segment and the photoreceptors-sensory retina segment. Blood vessels were stained using CD31 (an endothelial marker) or FITC-dextran and analyzed by using a confocal microscopy. Fluorescein angiography (FA) was performed to detect leakage from CNV. The ability of APC to stabilize the oBRB and prevent leakage was studied using ARPE-19 tissue culture (simulating the oBRB). FITC-Dextran leakage through the cultured cell monolayers was measured and the tight junction protein, Zonula occludens-1 (ZO1) cellular distribution was followed using immunostaining. Tie2 Kinase inhibitor and anti-Tie2 antibodies were used for Tie2 inhibition in the murine and cell culture models, respectively.Results: Quantitative analysis of the CNV area and volume revealed that 7-14 days after laser applications, APC treatment resulted in a statistically significant reduction in CNV growth, area and volume as compared to saline. Moreover, APC inhibited the penetration of CNV from the choroid throughout the RPE into the sensory retina in a dose-dependent manner. Figure 1 summarize the quantitative assessment of APC suppression of blood vessel penetration from the choroid (Fig.1A) to the sensory retina segment (Fig.1B). FA analysis showed that APC significantly suppressed the leakage of fluorescein from the laser lesion and preserved the retinal architecture. Incubation of RPE cells with APC resulted in dose dependent translocation of ZO1 to the cell membrane region, accompanied by reduction in permeability throughout the RPE monolayer. ZO1 translocation formed a typical ZO1 honeycomb pattern that is characteristic for the stabilization of tight junctions (Fig.1C). Inhibition of Tie2, in the laser induced CNV mice model and in RPE cell culture, significantly decreased APC protective activities in both models.Conclusions: Several Chorio-retinal pathologies are accompanied by damage to the oBRB and pathological growth of CNV. Our present data provide evidence that APC can protect the choroid and the retina from CNV growth, can suppress leakage from the newly formed choroidal blood vessels and may act as oBRB stabilizer. The protective function of APC in the eye provides direction for new strategies to treat chorio-retinal diseases affected by abnormal oBRB function and CNV growth. Our preliminary results warrant further investigation to extend the data regarding the potential use of APC as a novel anti-CNV treatment and oBRB protector. DisclosuresNo relevant conflicts of interest to declare.

Phosphorylation of serine/arginine-rich splicing factor 1 at tyrosine 19 promotes cell proliferation in pediatric acute lymphoblastic leukemia.

Serine/arginine‐rich splicing factor 1 (SRSF1) has been linked to various human cancers including pediatric acute lymphoblastic leukemia (ALL). Our previous study has shown that SRSF1 potentially contributes to leukemogenesis; however, its underlying mechanism remains unclear. In this study, leukemic cells were isolated from pediatric ALL bone marrow samples, followed by immunoprecipitation assays and mass spectrometry analysis specific to SRSF1. Subcellular localization of the SRSF1 protein and its mutants were analyzed by immunofluorescence staining. Cell growth, colony formation, cell apoptosis, and the cell cycle were investigated using stable leukemic cell lines generated with lentivirus‐mediated overexpressed WT or mutant plasmids. Cytotoxicity of the Tie2 kinase inhibitor was also evaluated. Our results showed the phosphorylation of SRSF1 at tyrosine 19 (Tyr‐19) was identified in newly diagnosed ALL samples, but not in complete remission or normal control samples. Compared to the SRSF1 WT cells, the missense mutants of the Tyr‐19 phosphorylation affected the subcellular localization of SRSF1. In addition, the Tyr‐19 phosphorylation of SRSF1 also led to increased cell proliferation and enhanced colony‐forming properties by promoting the cell cycle. Remarkably, we further identified the kinase Tie2 as a potential therapeutic target in leukemia cells. In conclusion, we identify for the first time that the phosphorylation state of SRSF1 is linked to different phases in pediatric ALL. The Tyr‐19 phosphorylation of SRSF1 disrupts its subcellular localization and promotes proliferation in leukemia cells by driving cell‐cycle progression. Inhibitors targeting Tie2 kinase that could catalyze Tyr‐19 phosphorylation of SRSF1 offer a promising therapeutic target for treatment of pediatric ALL.