Targeted therapy, immunotherapy, and small molecules and peptidomimetics as emerging immunoregulatory agents for melanoma.

Primary cutaneous melanoma is the most lethal of all skin neoplasms and its incidence is increasing. Clinical management of advanced melanoma in the last decade has been revolutionised by the availability of immunotherapies and targeted therapies, used alone and in combination. This article summarizes advances in the treatment of late-stage melanoma including use of protein kinase inhibitors, antibody-based immune checkpoint inhibitors, adoptive immunotherapy, vaccines and more recently, small molecules and peptidomimetics as emerging immunoregulatory agents.

The MEK-ERK-Egr-1 axis and its regulation in cardiovascular disease.

Cardiovascular disease (CVD) is the primary cause of morbidity and mortality in the Western world. Multiple molecular and cellular processes underpinning the pathogenesis of CVD are regulated by the zinc finger transcription factor and product of an immediate-early gene, early growth response-1 (Egr-1). Egr-1 regulates multiple pro-inflammatory processes that underpin the manifestation of CVD. The activity of Egr-1 itself is influenced by a range of post-translational modifications including sumoylation, ubiquitination and acetylation. Egr-1 also undergoes phosphorylation by protein kinases, such as extracellular-signal regulated kinase (ERK) which is itself phosphorylated by MEK. This article reviews recent progress on the MEK-ERK-Egr-1 cascade, notably regulation in conjunction with factors and agents such as TET2, TRIB2, MIAT, SphK1, cAMP, teneligliptin, cholinergic drugs, red wine and flavonoids, wogonin, febuxostat, docosahexaenoic acid and AT1R blockade. Such insights should provide new opportunity for therapeutic intervention in CVD.

Flubendazole inhibits PD-1 and suppresses melanoma growth in immunocompetent mice.

BACKGROUND: Immune checkpoint inhibitor therapy has revolutionized the clinical management of a diverse range of cancer types, including advanced cutaneous melanoma. While immunotherapy targeting the PD-1/PD-L1 system has become standard of care, overall response rates remain unsatisfactory for most patients and there are no approved small molecule inhibitors of the PD-1/PD-L1 system. Flubendazole (FLU) is an anthelmintic that has been used to treat worm infections in humans and animals for decades. METHODS: Here we tested the anti-cancer activity of systemically delivered FLU with suppression of PD-1 in immunocompetent mice. RESULTS: In C57BL/6J mice bearing subcutaneous B16F10 melanoma, FLU reduced both tumor growth and PD-1 protein levels without affecting levels of PD-L1. FLU's suppression of PD-1 was accompanied by increased CD3+ T cell infiltration. Western blotting with extracts from human Jurkat T cells showed that FLU inhibited PD-1 protein expression, findings confirmed by flow cytometry. To gain mechanistic insights on FLU's ability to suppress PD-1 protein levels, we performed bulk RNA sequencing on extracts of Jurkat T cells exposed to the benzimidazole for 4 h. From a pool of 14,475 genes there were 1218 differentially-expressed genes; 687 with increased expression and 531 with decreased expression. Among the genes induced by FLU was the AP-1 family member, JUN and surprisingly, pdcd1. KEGG pathway analysis showed FLU up-regulated genes over-represented in multiple pathways (p < 0.01), the top hit being amoebiasis. FLU also affected the expression of genes in cancer-associated pathways, both through down-regulation and up-regulation. Gene set enrichment analysis revealed a large number of immunological signature gene sets correlated with FLU treatment, including gene sets associated with T cell differentiation, proliferation and function. The AP-1 inhibitor T5224 rescued PD-1 protein expression from inhibition by FLU. CONCLUSION: This study is the first to show that FLU can inhibit melanoma growth with PD-1 suppression in immunocompetent mice.

Immune Checkpoint Inhibitors, Small-Molecule Immunotherapies and the Emerging Role of Neutrophil Extracellular Traps in Therapeutic Strategies for Head and Neck Cancer.

Immune checkpoint inhibitor (ICI) therapy has revolutionized the treatment of many cancer types, including head and neck cancers (HNC). When checkpoint and partner proteins bind, these send an "off" signal to T cells, which prevents the immune system from destroying tumor cells. However, in HNC, and indeed many other cancers, more people do not respond and/or suffer from toxic effects than those who do respond. Hence, newer, more effective approaches are needed. The challenge to durable therapy lies in a deeper understanding of the complex interactions between immune cells, tumor cells and the tumor microenvironment. This will help develop therapies that promote lasting tumorlysis by overcoming T-cell exhaustion. Here we explore the strengths and limitations of current ICI therapy in head and neck squamous cell carcinoma (HNSCC). We also review emerging small-molecule immunotherapies and the growing promise of neutrophil extracellular traps in controlling tumor progression and metastasis.

Gene Therapeutic Strategies for Peripheral Artery Disease and New Opportunities Provided by Adeno-Associated Virus Vectors.

Peripheral artery disease (PAD) is a vascular disorder caused by occlusive atherosclerosis, which commonly impairs blood flow to the lower extremities. The prevalence of PAD is increasing globally with >200 million people affected. PAD remains a growing global health problem as the population continues to age and diabetes incidence grows. Many patients with PAD, most notably those with critical limb ischemia, fail attempts at surgical and percutaneous intervention to improve blood flow and are at risk of amputation. Gene therapy provides an opportunity to change the clinical course of PAD in these patients via strategies that increase vascular supply through angiogenesis and arteriogenesis improving muscle perfusion and function in ischemic legs. This article discusses gene therapy approaches in the context of PAD, both intermittent claudication and critical limb ischemia, and the promise of adeno-associated virus-based strategies delivering not just VEGFs (vascular endothelial growth factors) but a range of other mediators as potential new therapeutics. We also highlight challenges and failures in the clinical translation of gene therapy for PAD and how at least some of these obstacles may be overcome using adeno-associated virus.

Early Growth Response-1: Friend or Foe in the Heart?

Cardiovascular disease is a major cause of mortality and morbidity worldwide. Early growth response-1 (Egr-1) plays a critical regulatory role in a range of experimental models of cardiovascular diseases. Egr-1 is an immediate-early gene and is upregulated by various stimuli including shear stress, oxygen deprivation, oxidative stress and nutrient deprivation. However, recent research suggests a new, underexplored cardioprotective side of Egr-1. The main purpose of this review is to explore and summarise the dual nature of Egr-1 in cardiovascular pathobiology.

Emerging therapeutic strategies for unmet need in neovascular age-related macular degeneration.

Neovascular age-related macular degeneration (nAMD) is a major cause of visual impairment and blindness. Anti-vascular endothelial growth factor (VEGF) agents, such as ranibizumab, bevacizumab, aflibercept, brolucizumab and faricimab have revolutionized the clinical management of nAMD. However, there remains an unmet clinical need for new and improved therapies for nAMD, since many patients do not respond optimally, may lose response over time or exhibit sub-optimal durability, impacting on real world effectiveness. Evidence is emerging that targeting VEGF-A alone, as most agents have done until recently, may be insufficient and agents that target multiple pathways (e.g., aflibercept, faricimab and others in development) may be more efficacious. This article reviews issues and limitations that have arisen from the use of existing anti-VEGF agents, and argues that the future may lie in multi-targeted therapies including alternative agents and modalities that target both the VEGF ligand/receptor system as well as other pathways.

Personalised Medicine and the Potential Role of Electrospinning for Targeted Immunotherapeutics in Head and Neck Cancer.

Advanced head and neck cancer (HNC) is functionally and aesthetically destructive, and despite significant advances in therapy, overall survival is poor, financial toxicity is high, and treatment commonly exacerbates tissue damage. Although response and durability concerns remain, antibody-based immunotherapies have heralded a paradigm shift in systemic treatment. To overcome limitations associated with antibody-based immunotherapies, exploration into de novo and repurposed small molecule immunotherapies is expanding at a rapid rate. Small molecule immunotherapies also have the capacity for chelation to biodegradable, bioadherent, electrospun scaffolds. This article focuses on the novel concept of targeted, sustained release immunotherapies and their potential to improve outcomes in poorly accessible and risk for positive margin HNC cases.

Transcriptional regulation of vascular smooth muscle cell proliferation, differentiation and senescence: Novel targets for therapy.

Vascular smooth muscle cells (SMC) possess a unique cytoplasticity, regulated by transcriptional, translational and phenotypic transformation in response to a diverse range of extrinsic and intrinsic pathogenic factors. The mature, differentiated SMC phenotype is physiologically typified transcriptionally by expression of genes encoding "contractile" proteins, such as SMα-actin (ACTA2), SM-MHC (myosin-11) and SM22α (transgelin). When exposed to various pathological conditions (e.g., pro-atherogenic risk factors, hypertension), SMC undergo phenotypic modulation, a bioprocess enabling SMC to de-differentiate in immature stages or trans-differentiate into other cell phenotypes. As recent studies suggest, the process of SMC phenotypic transformation involves five distinct states characterized by different patterns of cell growth, differentiation, migration, matrix protein expression and declined contractility. These changes are mediated via the action of several transcriptional regulators, including myocardin and serum response factor. Conversely, other factors, including Kruppel-like factor 4 and nuclear factor-κB, can inhibit SMC differentiation and growth arrest, while factors such as yin yang-1, can promote SMC differentiation whilst inhibiting proliferation. This article reviews recent advances in our understanding of regulatory mechanisms governing SMC phenotypic modulation. We propose the concept that transcription factors mediating this switching are important biomarkers and potential pharmacological targets for therapeutic intervention in cardiovascular disease.

RNA sequencing identifies genes reliant upon Ser26 in the zinc finger transcription factor, early growth response-1.

Early growth response-1 (Egr-1) is an inducible master regulatory transcription factor that orchestrates gene expression in vascular endothelial cells. We recently determined that Ser26 in Egr-1 undergoes phosphorylation and plays a critical functional role in a range of pro-angiogenic processes. To better understand the effect of Ser26 on Egr-1-dependent gene expression, in this study, we performed RNA-seq and bioinformatics analysis with human microvascular endothelial cells bearing a germline mutation (M) in Ser26 to Ala (M26 cells) exposed to the mitogen and chemoattractant fibroblast growth factor-2 (FGF2) as compared with wildtype (WT) cells. In WT cells, FGF2 increased the expression of numerous growth factors and hormones, cytokines, signaling molecules and transcriptional regulators. Comparison of FGF2-inducible WT and M26 cells enabled identification of differentially expressed genes, including genes reliant or not reliant upon Ser26. For example, Ser26 in Egr-1 was required for FGF2 inducible LIF expression but not for FGF2 inducible IL11. Ser26 was also required for FGF2 inducible NKX2-8 and RIPK2 expression but not for FGF2 inducible CREB5 or ALPK2 expression. Conversely, FGF2 inhibited genes such as TIE1, GPR146 and EPHB3, and Ser26 was required for FGF2's effect on TIE1 and GPR146 but not for EPHB3. Enrichment analysis also identified a range of gene ontologies upregulated and downregulated by FGF2. These findings demonstrate the importance of Ser26 in Egr-1 in programs of endothelial gene expression modulated by FGF2.

Early Growth Response-1, an Integrative Sensor in Cardiovascular and Inflammatory Disease.

Early growth response-1 (Egr-1) is a master regulator and transcriptional sensor in vascular dysfunction and disease. This article reviews recent developments in our understanding of the regulatory roles this zinc finger protein and product of an immediate-early gene plays in a range of cardiovascular and inflammatory disorders. Egr-1 can amplify pathologic signals from the extracellular environment by serving as a molecular conduit in the inducible expression of proliferative, migratory and proinflammatory genes driving disease progression. Strategies targeting Egr-1 may provide therapeutic benefit in cardiovascular and inflammatory disorders.

Serine 26 in Early Growth Response-1 Is Critical for Endothelial Proliferation, Migration, and Network Formation.

Background Vascular endothelial cell proliferation, migration, and network formation are key proangiogenic processes involving the prototypic immediate early gene product, Egr-1 (early growth response-1). Egr-1 undergoes phosphorylation at a conserved Ser26 but its function is completely unknown in endothelial cells or any other cell type. Methods and Results A CRISPR/Cas9 strategy was used to introduce a homozygous Ser26>Ala mutation into endogenous Egr-1 in human microvascular endothelial cells. In the course of generating mutant cells, we produced cells with homozygous deletion in Egr-1 caused by frameshift and premature termination. We found that Ser26 mutation in Egr-1, or Egr-1 deletion, perturbed endothelial cell proliferation in models of cell counting or real-time growth using the xCELLigence System. We found that Ser26 mutation or Egr-1 deletion ameliorated endothelial cell migration toward VEGF-A165 (vascular endothelial growth factor-A) in a dual-chamber model. On solubilized basement membrane preparations, Ser26 mutation or Egr-1 deletion prevented endothelial network (or tubule) formation, an in vitro model of angiogenesis. Flow cytometry further revealed that Ser26 mutation or Egr-1 deletion elevated early and late apoptosis. Finally, we demonstrated that Ser26 mutation or Egr-1 deletion increased VE-cadherin (vascular endothelial cadherin) expression, a regulator of endothelial adhesion and signaling, permeability, and angiogenesis. Conclusions These findings not only indicate that Egr-1 is essential for endothelial cell proliferation, migration, and network formation, but also show that point mutation in Ser26 is sufficient to impair each of these processes and trigger apoptosis as effectively as the absence of Egr-1. This highlights the importance of Ser26 in Egr-1 for a range of proangiogenic processes.

Emerging insights on functions of the anthelmintic flubendazole as a repurposed anticancer agent.

While flubendazole has been used as a macrofilaricide in humans and animals for some 40 years, work in vitro and in preclinical models over the last decade has suggested its potential use as an anticancer agent. This article reviews recent studies in a range of tumor types indicating novel functions for flubendazole in its control of processes associated with tumor growth, spread and renewal including ferroptosis, autophagy, cancer stem-like cell killing and suppression of intratumoral myeloid-derived suppressor cell accumulation and programmed cell death protein 1. Flubendazole's potential use in clinical oncology will require further understanding of its mechanistic roles, range of inhibition of cancer types, capacity for adjunctive therapy and possible reformulation for enhanced solubility, bioavailability and potency.

BT2 Suppresses Human Monocytic-Endothelial Cell Adhesion, Bone Erosion and Inflammation.

INTRODUCTION: Inflammation and bone erosion are processes key to the pathogenesis of rheumatoid arthritis, a systemic autoimmune disease causing progressive disability and pain, impacting around 1.3 million people in the United States alone. However, many patients do not respond sufficiently to existing therapies or benefit is not sustained and alternate therapeutic approaches are lacking. We recently identified the dibenzoxazepinone BT2, which inhibits ERK phosphorylation, from a high-throughput chemical screen and identified its ability to inhibit angiogenesis and vascular leakiness. METHODS: Here we evaluated BT2 for potential anti-inflammatory activity in in vitro models of human monocytic-endothelial cell adhesion, monocytic cell extravasation and collagen antibody-induced arthritis in mice. RESULTS: BT2 inhibits human monocytic cell adhesion to IL-1ß-treated human endothelial cells and inhibits monocytic transendothelial migration toward MCP-1. In mice rendered arthritic, single systemic administration of BT2 prevented footpad swelling, bone destruction and TRAP+ cells in the joints. BT2 suppressed inducible circulating levels of IL-1ß, IL-2 and IL-6 to normal levels without affecting levels of IL-4 or IL-10 among other cytokines. BT2 also inhibited the expression of pro-inflammatory adhesion molecules ICAM-1 and VCAM-1 in arthritic joints. There was no evidence of toxicity following intraperitoneal, gavage or intraarticular administration of BT2. CONCLUSION: BT2 is a novel small molecule inhibitor of joint inflammation, bone erosion, pro-inflammatory cytokine and adhesion molecule expression. This suggests the potential clinical utility of BT2 as a new anti-inflammatory agent.

Truncated YY1 interacts with BASP1 through a 339KLK341 motif in YY1 and suppresses vascular smooth muscle cell growth and intimal hyperplasia after vascular injury.

AIMS: In-stent restenosis and late stent thrombosis are complications associated with the use of metallic and drug-coated stents. Strategies that inhibit vascular smooth muscle cell (SMC) proliferation without affecting endothelial cell (EC) growth would be helpful in reducing complications arising from percutaneous interventions. SMC hyperplasia is also a pathologic feature of graft stenosis and fistula failure. Our group previously showed that forced expression of the injury-inducible zinc finger (ZNF) transcription factor, yin yang-1 (YY1), comprising 414 residues inhibits neointima formation in carotid arteries of rabbits and rats. YY1 inhibits SMC proliferation without affecting EC growth in vitro. Identifying a shorter version of YY1 retaining cell-selective inhibition would make it more amenable for potential use as a gene therapeutic agent. METHODS AND RESULTS: We dissected YY1 into a range of shorter fragments (YY1A-D, YY1Δ) and found that the first two ZNFs in YY1 (construct YY1B, spanning 52 residues) repressed SMC proliferation. Receptor binding domain analysis predicts a three-residue (339KLK341) interaction domain. Mutation of 339KLK341 to 339AAA341 in YY1B (called YY1Bm) abrogated YY1B's ability to inhibit SMC but not EC proliferation and migration. Incubation of recombinant GST-YY1B and GST-YY1Bm with SMC lysates followed by precipitation with glutathione-agarose beads and mass spectrometric analysis identified a novel interaction between YY1B and BASP1. Overexpression of BASP1, like YY1, inhibited SMC but not EC proliferation and migration. BASP1 siRNA partially rescued SMC from growth inhibition by YY1B. In the rat carotid balloon injury model, adenoviral overexpression of YY1B, like full-length YY1, reduced neointima formation, whereas YY1Bm had no such effect. CD31+ immunostaining suggested YY1B could increase re-endothelialization in a 339KLK341-dependent manner. CONCLUSION: These studies identify a truncated form of YY1 (YY1B) that can interact with BASP1 and inhibit SMC proliferation, migration, and intimal hyperplasia after balloon injury of rat carotid arteries as effectively as full length YY1. We demonstrate the therapeutic potential of YY1B in vascular proliferative disease.

Neutralizing the pathological effects of extracellular histones with small polyanions.

Extracellular histones in neutrophil extracellular traps (NETs) or in chromatin from injured tissues are highly pathological, particularly when liberated by DNases. We report the development of small polyanions (SPAs) (~0.9-1.4 kDa) that interact electrostatically with histones, neutralizing their pathological effects. In vitro, SPAs inhibited the cytotoxic, platelet-activating and erythrocyte-damaging effects of histones, mechanistic studies revealing that SPAs block disruption of lipid-bilayers by histones. In vivo, SPAs significantly inhibited sepsis, deep-vein thrombosis, and cardiac and tissue-flap models of ischemia-reperfusion injury (IRI), but appeared to differ in their capacity to neutralize NET-bound versus free histones. Analysis of sera from sepsis and cardiac IRI patients supported these differential findings. Further investigations revealed this effect was likely due to the ability of certain SPAs to displace histones from NETs, thus destabilising the structure. Finally, based on our work, a non-toxic SPA that inhibits both NET-bound and free histone mediated pathologies was identified for clinical development.

Thermostable small-molecule inhibitor of angiogenesis and vascular permeability that suppresses a pERK-FosB/ΔFosB-VCAM-1 axis.

Vascular permeability and angiogenesis underpin neovascular age-related macular degeneration and diabetic retinopathy. While anti-VEGF therapies are widely used clinically, many patients do not respond optimally, or at all, and small-molecule therapies are lacking. Here, we identified a dibenzoxazepinone BT2 that inhibits endothelial cell proliferation, migration, wound repair in vitro, network formation, and angiogenesis in mice bearing Matrigel plugs. BT2 interacts with MEK1 and inhibits ERK phosphorylation and the expression of FosB/ΔFosB, VCAM-1, and many genes involved in proliferation, migration, angiogenesis, and inflammation. BT2 reduced retinal vascular leakage following rat choroidal laser trauma and rabbit intravitreal VEGF-A165 administration. BT2 suppressed retinal CD31, pERK, VCAM-1, and VEGF-A165 expression. BT2 reduced retinal leakage in rats at least as effectively as aflibercept, a first-line therapy for nAMD/DR. BT2 withstands boiling or autoclaving and several months' storage at 22°C. BT2 is a new small-molecule inhibitor of vascular permeability and angiogenesis.

Recruitment and maturation of the coronary collateral circulation: Current understanding and perspectives in arteriogenesis.

The coronary collateral circulation is a rich anastomotic network of primitive vessels which have the ability to augment in size and function through the process of arteriogenesis. In this review, we evaluate the current understandings of the molecular and cellular mechanisms by which this process occurs, specifically focussing on elevated fluid shear stress (FSS), inflammation, the redox state and gene expression along with the integrative, parallel and simultaneous process by which this occurs. The initiating step of arteriogenesis occurs following occlusion of an epicardial coronary artery, with an increase in FSS detected by mechanoreceptors within the endothelium. This must occur within a 'redox window' where an equilibrium of oxidative and reductive factors are present. These factors initially result in an inflammatory milieu, mediated by neutrophils as well as lymphocytes, with resultant activation of a number of downstream molecular pathways resulting in increased expression of proteins involved in monocyte attraction and adherence; namely vascular cell adhesion molecule 1 (VCAM-1), monocyte chemoattractant protein 1 (MCP-1) and transforming growth factor beta (TGF-ß). Once monocytes and other inflammatory cells adhere to the endothelium they enter the extracellular matrix and differentiate into macrophages in an effort to create a favourable environment for vessel growth and development. Activated macrophages secrete inflammatory cytokines such as tumour necrosis factor-α (TNF-α), growth factors such as fibroblast growth factor-2 (FGF-2) and matrix metalloproteinases. Finally, vascular smooth muscle cells proliferate and switch to a contractile phenotype, resulting in an increased diameter and functionality of the collateral vessel, thereby allowing improved perfusion of the distal myocardium subtended by the occluded vessel. This simultaneously reduces FSS within the collateral vessel, inhibiting further vessel growth.