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.

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.

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.

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.

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.

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.

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.

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.

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.

Pharmaceutical patents: reconciling the human right to health with the incentive to invent.

In developed countries that protect core aspects of the fundamental human right to the highest attainable standard of health, how does that right intersect with intellectual property rights? Here, the human rights implication of providing access to all cancer drugs recommended by experts in a developed country is considered in the context of conflict between the incentive to invent and the rights of others to access medicines. Effective incentives to innovate in developed countries can lead to global improvements in access to medicine if the intellectual property system is calibrated to permit this. This depends partly on the usefulness of compulsory licensing and alternative mechanisms facilitating global access to drugs. This review considers tensions between fundamental rights to access essential medicines and rights of the inventor and investors, including the pharmaceutical industry.

Remote Ischemic Preconditioning induces Cardioprotective Autophagy and Signals through the IL-6-Dependent JAK-STAT Pathway.

: Autophagy is a cellular process by which mammalian cells degrade and assist in recycling damaged organelles and proteins. This study aimed to ascertain the role of autophagy in remote ischemic preconditioning (RIPC)-induced cardioprotection. Sprague Dawley rats were subjected to RIPC at the hindlimb followed by a 30-min transient blockade of the left coronary artery to simulate ischemia reperfusion (I/R) injury. Hindlimb muscle and the heart were excised 24 h post reperfusion. RIPC prior to I/R upregulated autophagy in the rat heart at 24 h post reperfusion. In vitro, autophagy inhibition or stimulation prior to RIPC, respectively, either ameliorated or stimulated the cardioprotective effect, measured as improved cell viability to mimic the preconditioning effect. Recombinant interleukin-6 (IL-6) treatment prior to I/R increased in vitro autophagy in a dose-dependent manner, activating the Janus kinase/signal transducers and activators of transcription (JAK-STAT) pathway without affecting the other kinase pathways, such as p38 mitogen-activated protein kinases (MAPK), and glycogen synthase kinase 3 Beta (GSK-3ß) pathways. Prior to I/R, in vitro inhibition of the JAK-STAT pathway reduced autophagy upregulation despite recombinant IL-6 pre-treatment. Autophagy is an essential component of RIPC-induced cardioprotection that may upregulate autophagy through an IL-6/JAK-STAT-dependent mechanism, thus identifying a potentially new therapeutic option for the treatment of ischemic heart disease.

Repurposing Drugs for Skin Cancer.

Drug repurposing is the process of developing existing or abandoned drugs for a different disease. Repurposing can circumvent higher costs and times associated with conventional drug discovery strategies because toxicity and pharmacokinetics profiles are typically already established. This brief review focuses on efforts to repurpose drugs for skin cancer and includes reuse of antihypertensives, anthelmintics and antifungals among a range of other medicines. Repurposing not only ushers promising known drugs for new indications, the process of repurposing can uncover new mechanistic insights in the pathogenesis of disease and uncover new opportunities for pharmaceutical intervention.

The anthelmintic flubendazole blocks human melanoma growth and metastasis and suppresses programmed cell death protein-1 and myeloid-derived suppressor cell accumulation.

The incidence of melanoma is increasing faster than any other cancer. In recent years, treatment of melanoma and a range of other deadly cancers has involved immunotherapy with programmed cell death protein-1 (PD-1)/PD-1 ligand (PD-L1) checkpoint blockade which has improved survival. However, many patients do not respond or have partial response, survival benefit is in the order of months and all available PD-1/PD-L1 strategies are antibodies requiring intravenous infusion. There are no clinically approved small molecule pharmacologic inhibitors of the PD-1/PD-L1 system. The benzimidazole derivative flubendazole is a widely used anthelmintic available over the counter in Europe. Here we demonstrate the ability of flubendazole to inhibit human melanoma growth and spread in mice. Flubendazole's ability to block tumor growth and spread was comparable to paclitaxel. Anti-tumor effects were observed when flubendazole was delivered systemically not locally. Flubendazole inhibited CD31/PECAM-1 staining indicating suppression of tumor angiogenesis. Most surprisingly, flubendazole inhibited PD-1 levels within the tumors, but not PD-L1. Western blotting and flow cytometry revealed that flubendazole inhibits PD-1 expression in cultured melanoma cells. Flubendazole also reduced myeloid-derived suppressor cell (MDSC) levels in tumor tissue. Further we found that flubendazole inhibited active (phospho-Tyr705) signal transducer and activator of transcription (STAT3), an upstream regulator of PD-1 expression. These findings uncover that flubendazole is a novel small molecule inhibitor of not only melanoma growth and spread but also of PD-1 and MDSC.

Deoxyribozymes as Catalytic Nanotherapeutic Agents.

RNA-cleaving deoxyribozymes (DNAzymes) are synthetic single-stranded DNA-based catalytic molecules that can be engineered to bind to and cleave target mRNA at predetermined sites. These have been used as therapeutic agents in a range of preclinical cancer models and have entered clinical trials in Europe, China, and Australia. This review surveys regulatory insights into mechanisms of disease brought about by use of catalytic DNA in vitro and in vivo, including recent uses as nanosensors, nanoflowers, and nanosponges, and the emerging role of adaptive immunity underlying DNAzyme inhibition of cancer growth. DNAzymes represent a promising new class of nucleic acid-based therapeutics in cancer. This article discusses mechanistic and therapeutic insights brought about by DNAzyme use as nanotools and reagents in a range of basic science, experimental therapeutic and clinical applications. Current limitations and future perspectives are also discussed.

Promoter Usage and Dynamics in Vascular Smooth Muscle Cells Exposed to Fibroblast Growth Factor-2 or Interleukin-1ß.

Smooth muscle cells (SMC) in blood vessels are normally growth quiescent and transcriptionally inactive. Our objective was to understand promoter usage and dynamics in SMC acutely exposed to a prototypic growth factor or pro-inflammatory cytokine. Using cap analysis gene expression (FANTOM5 project) we report differences in promoter dynamics for immediate-early genes (IEG) and other genes when SMC are exposed to fibroblast growth factor-2 or interleukin-1ß. Of the 1871 promoters responding to FGF2 or IL-1ß considerably more responded to FGF2 (68.4%) than IL-1ß (18.5%) and 13.2% responded to both. Expression clustering reveals sets of genes induced, repressed or unchanged. Among IEG responding rapidly to FGF2 or IL-1ß were FOS, FOSB and EGR-1, which mediates human SMC migration. Motif activity response analysis (MARA) indicates most transcription factor binding motifs in response to FGF2 were associated with a sharp induction at 1 h, whereas in response to IL-1ß, most motifs were associated with a biphasic change peaking generally later. MARA revealed motifs for FOS_FOS{B,L1}_JUN{B,D} and EGR-1..3 in the cluster peaking 1 h after FGF2 exposure whereas these motifs were in clusters peaking 1 h or later in response to IL-1ß. Our findings interrogating CAGE data demonstrate important differences in promoter usage and dynamics in SMC exposed to FGF2 or IL-1ß.

Targeted therapies in the management of locally advanced and metastatic pancreatic cancer: a systematic review.

Pancreatic cancer has a dismal prognosis particularly in patients presenting with unresectable tumors. We performed a bibliometric analysis of clinical trials for pancreatic cancer conducted between 2014-2016 focusing on patients that presented with unresectable (locally advanced or metastatic) tumors. We discuss a range of studies that employed FOLFIRINOX, the gemcitabine + nab-paclitaxel combination and studies that used molecularly-targeted therapy. Major areas of focus have been dual targeting of EGFR and VEGFR, immunotherapy or a multimodal approach - combining chemotherapy with radiotherapy. We also point out the need for molecular selection for low prevalence subtypes. Key insights sourced from these pivotal trials should improve clinical outcomes for this devastating cancer.

Melanoma protective antitumor immunity activated by catalytic DNA.

Melanoma incidence is increasing worldwide, and although drugs such as BRAF/MEK small-molecule inhibitors and immune checkpoint antibodies improve patient outcomes, most patients ultimately fail these therapies and alternative treatment strategies are urgently needed. DNAzymes have recently undergone clinical trials with signs of efficacy and no serious adverse events attributable to the DNAzyme. Here we investigated c-Jun expression in human primary and metastatic melanoma. We also explored the role of T cell immunity in DNAzyme inhibition of primary melanoma growth and the prevention of growth in non-treated tumors after the cessation of treatment in a mouse model. c-Jun was expressed in 80% of melanoma cells in human primary melanomas (n = 17) and in 83% of metastatic melanoma cells (n = 38). In contrast, c-Jun was expressed in only 11% of melanocytes in benign nevi (n = 24). Dz13, a DNAzyme targeting c-Jun/AP-1, suppressed both Dz13-injected and untreated B16F10 melanoma growth in the same mice, an abscopal effect relieved in each case by administration of anti-CD4/anti-CD8 antibodies. Dz13 increased levels of cleaved caspase-3 within the tumors. New, untreated melanomas grew poorly in mice previously treated with Dz13. Administration of anti-CD4/anti-CD8 antibodies ablated this inhibitory effect and the tumors grew rapidly. Dz13 inhibited c-Jun expression, reduced intratumoral vascularity (vascular lumina area defined by CD31 staining), and increased CD4+ cells within the tumors. This study provides the first demonstration of an abscopal effect of a DNAzyme on tumor growth and shows that Dz13 treatment prevents growth of subsequent new tumors in the same animal. Dz13 may be useful clinically as a therapeutic antitumor agent by preventing tumor relapse through adaptive immunity.