Lysosomal Biogenesis and Implications for Hydroxychloroquine Disposition.

Lysosomes act as a cellular drug sink for weakly basic, lipophilic (lysosomotropic) xenobiotics, with many instances of lysosomal trapping associated with multiple drug resistance. Lysosomotropic agents have also been shown to activate master lysosomal biogenesis transcription factor EB (TFEB) and ultimately lysosomal biogenesis. We investigated the role of lysosomal biogenesis in the disposition of hydroxychloroquine (HCQ), a hallmark lysosomotropic agent, and observed that modulating the lysosomal volume of human breast cancer cell lines can account for differences in disposition of HCQ. Through use of an in vitro pharmacokinetic (PK) model, we characterized total cellular uptake of HCQ within the duration of static equilibrium (1 hour), as well as extended exposure to HCQ that is subject to dynamic equilibrium (>1 hour), wherein HCQ increases the size of the lysosomal compartment through swelling and TFEB-induced lysosomal biogenesis. In addition, we observe that pretreatment of cell lines with TFEB-activating agent Torin1 contributed to an increase of whole-cell HCQ concentrations by 1.4- to 1.6-fold, which were also characterized by the in vitro PK model. This investigation into the role of lysosomal volume dynamics in lysosomotropic drug disposition, including the ability of HCQ to modify its own disposition, advances our understanding of how chemically similar agents may distribute on the cellular level and examines a key area of lysosomal-mediated multiple drug resistance and drug-drug interaction. SIGNIFICANCE STATEMENT: Hydroxychloroquine is able to modulate its own cellular pharmacokinetic uptake by increasing the cellular lysosomal volume fraction through activation of lysosomal biogenesis master transcription factor EB and through lysosomal swelling. This concept can be applied to many other lysosomotropic drugs that activate transcription factor EB, such as doxorubicin and other tyrosine kinase inhibitor drugs, as these drugs may actively increase their own sequestration within the lysosome to further exacerbate multiple drug resistance and lead to potential acquired resistance.

The Angiotensin Receptor Blocker Losartan Suppresses Growth of Pulmonary Metastases via AT1R-Independent Inhibition of CCR2 Signaling and Monocyte Recruitment.

Inflammatory monocytes have been shown to play key roles in cancer metastasis through promotion of tumor cell extravasation, growth, and angiogenesis. Monocyte recruitment to metastases is mediated primarily via the CCL2-CCR2 chemotactic axis. Thus, disruption of this axis represents an attractive therapeutic target for the treatment of metastatic disease. Losartan, a type I angiotensin II receptor (AT1R) antagonist, has been previously shown to have immunomodulatory actions involving monocyte and macrophage activity. However, the exact mechanisms accounting for these effects have not been fully elucidated. Therefore, we investigated the effects of losartan and its primary metabolite on CCL2-mediated monocyte recruitment and CCR2 receptor function using mouse tumor models and in vitro human monocyte cultures. We show, in this study, that losartan and its metabolite potently inhibit monocyte recruitment through the noncompetitive inhibition of CCL2-induced ERK1/2 activation, independent of AT1R activity. Studies in experimental metastasis models demonstrated that losartan treatment significantly reduced the metastatic burden in mice, an effect associated with a significant decrease in CD11b+/Ly6C+-recruited monocytes in the lungs. Collectively, these results indicate that losartan can exert antimetastatic activity by inhibiting CCR2 signaling and suppressing monocyte recruitment and therefore suggest that losartan (and potentially other AT1R blocker drugs) could be repurposed for use in cancer immunotherapy.

Role of Periostin Expression in Canine Osteosarcoma Biology and Clinical Outcome.

Periostin is a matricellular protein important in regulating bone, tooth, and cardiac development. In pathologic conditions, periostin drives allergic and fibrotic inflammatory diseases and is also overexpressed in certain cancers. Periostin signaling in tumors has been shown to promote angiogenesis, metastasis, and cancer stem cell survival in rodent models, and its overexpression is associated with poor prognosis in human glioblastoma. However, the role of periostin in regulating tumorigenesis of canine cancers has not been evaluated. Given its role in bone development, we sought to evaluate mRNA and protein expression of periostin in canine osteosarcoma (OS) and assess its association with patient outcome. We validated an anti-human periostin antibody cross-reactive to canine periostin via western blot and immunohistochemistry and evaluated periostin expression in microarray data from 49 primary canine OS tumors and 8 normal bone samples. Periostin mRNA was upregulated greater than 40-fold in canine OS tumors compared to normal bone and was significantly correlated with periostin protein expression based on quantitative image analysis. However, neither periostin mRNA nor protein expression were associated with time to metastasis in this cohort. Gene Set Enrichment Analysis demonstrated significant enhancement of pro-tumorigenic pathways including canonical WNT signaling, epithelial-mesenchymal transition, and angiogenesis in periostin-high tumors, while periostin-low tumors demonstrated evidence of heightened antitumor immune responses. Overall, these data identify a novel antibody that can be used as a tool for evaluation of periostin expression in dogs and suggest that investigation of Wnt pathway-targeted drugs in periostin overexpressing canine OS may be a potential therapeutic target.

Evaluation of immunological parameters in pit bull terrier-type dogs with juvenile onset generalized demodicosis and age-matched healthy pit bull terrier-type dogs.

BACKGROUND: Juvenile onset generalized demodicosis (JOGD) is thought to occur due to immunological abnormalities and is over-represented in pit bull terrier-type dogs. ANIMALS: Twelve pit bull terrier-type dogs with JOGD and 12 age-matched healthy pit bull terrier-type dogs. OBJECTIVE: To investigate immunological differences between age-matched healthy and JOGD pit bull terrier-type dogs by flow cytometry, multiplex, molecular and serological assays. METHODS AND MATERIALS: Flow cytometry quantified B cells expressing MHCII or surface-bound IgG, CD4+ T cells expressing MHCII, CD8 T cells expressing MHCII or CD11a, neutrophil and monocyte markers. Surface expression was quantified by calculating the geometric mean fluorescence index. The Wilcoxon rank sum test was used to compare median results for IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-13, IL-18, FOXP3, monocyte chemoattractant protein-1, GM-CSF, KC, IgE, IgA, IgG, IgM, C-reactive protein, lymphocyte, neutrophil and monocyte in the groups. IFN-gamma, IP-10, IL-15, IL-31 and TNF-alpha also were measured; however, insufficient dogs (<5) had values that were in range of the assay to allow for statistical evaluation. Significance was defined as P < 0.05. RESULTS: Serum concentrations of IL-2, IL-18 and MCP-1 were significantly higher (P = 0.01, P = 0.01, P = 0.04) in the JOGD group. Also, IgA median value was significantly higher (P = 0.002) in pit bull terrier-type dogs with JOGD. Flow cytometry revealed that T-cell, neutrophil and monocyte markers were not different between groups. CONCLUSIONS: Results suggest an appropriate compensatory immune response by pit bull terrier-type dogs in the JOGD group and do not support the explanation of global immune deficiency in these dogs.

Immunohistochemical and transcriptomic characterization of T and myeloid cell infiltrates in canine malignant melanoma.

Immune checkpoint inhibitor therapy can provide significant clinical benefit in patients with certain cancer types including melanoma; however, objective responses are only observed for a subset of patients. Mucosal melanoma is a rare melanoma subtype associated with a poor prognosis and, compared with cutaneous melanoma, is significantly less responsive to immune checkpoint inhibitors. Spontaneous canine tumours have emerged as valuable models to inform human cancer studies. In contrast to human melanoma, most canine melanomas are mucosal-an incidence that may be leveraged to better understand the subtype in humans. However, a more comprehensive understanding of the immune landscape of the canine disease is required. Here, we quantify tumour infiltrative T and myeloid cells in canine mucosal (n = 13) and cutaneous (n = 5) melanomas using immunohistochemical analysis of CD3 and MAC387 expression, respectively. Gene expression analysis using the Canine IO NanoString panel was also performed to identify genes and pathways associated with immune cell infiltration. T and myeloid cell densities were variable with geometric means of 158.7 cells/mm2 and 166.7 cells/mm2, respectively. Elevated T cell infiltration was associated with increased expression of cytolytic genes as well as genes encoding the coinhibitory checkpoint molecules PD-1, CTLA-4, TIM-3 and TIGIT; whereas increased myeloid cell infiltration was associated with elevated expression of protumourigenic cytokines. These data provide a basic characterization of the tumour microenvironment of canine malignant melanoma and suggest that, like human melanoma, inherent variability in anti-tumour T cell responses exists and that a subset of canine melanomas may respond better to immunomodulation.

Downregulation of cannabinoid receptor 1 from neuropeptide Y interneurons in the basal ganglia of patients with Huntington's disease and mouse models.

Cannabinoid receptor 1 (CB(1) receptor) controls several neuronal functions, including neurotransmitter release, synaptic plasticity, gene expression and neuronal viability. Downregulation of CB(1) expression in the basal ganglia of patients with Huntington's disease (HD) and animal models represents one of the earliest molecular events induced by mutant huntingtin (mHtt). This early disruption of neuronal CB(1) signaling is thought to contribute to HD symptoms and neurodegeneration. Here we determined whether CB(1) downregulation measured in patients with HD and mouse models was ubiquitous or restricted to specific striatal neuronal subpopulations. Using unbiased semi-quantitative immunohistochemistry, we confirmed previous studies showing that CB(1) expression is downregulated in medium spiny neurons of the indirect pathway, and found that CB(1) is also downregulated in neuropeptide Y (NPY)/neuronal nitric oxide synthase (nNOS)-expressing interneurons while remaining unchanged in parvalbumin- and calretinin-expressing interneurons. CB(1) downregulation in striatal NPY/nNOS-expressing interneurons occurs in R6/2 mice, Hdh(Q150/Q150) mice and the caudate nucleus of patients with HD. In R6/2 mice, CB(1) downregulation in NPY/nNOS-expressing interneurons correlates with diffuse expression of mHtt in the soma. This downregulation also occludes the ability of cannabinoid agonists to activate the pro-survival signaling molecule cAMP response element-binding protein in NPY/nNOS-expressing interneurons. Loss of CB(1) signaling in NPY/nNOS-expressing interneurons could contribute to the impairment of basal ganglia functions linked to HD.

Losartan Blocks Osteosarcoma-Elicited Monocyte Recruitment, and Combined With the Kinase Inhibitor Toceranib, Exerts Significant Clinical Benefit in Canine Metastatic Osteosarcoma.

PURPOSE: There is increasing recognition that progress in immuno-oncology could be accelerated by evaluating immune-based therapies in dogs with spontaneous cancers. Osteosarcoma (OS) is one tumor for which limited clinical benefit has been observed with the use of immune checkpoint inhibitors. We previously reported the angiotensin receptor blocker losartan suppressed metastasis in preclinical mouse models through blockade of CCL2-CCR2 monocyte recruitment. Here we leverage dogs with spontaneous OS to determine losartan's safety and pharmacokinetics associated with monocyte pharmacodynamic endpoints, and assess its antitumor activity, in combination with the kinase inhibitor toceranib. PATIENTS AND METHODS: CCL2 expression, monocyte infiltration, and monocyte recruitment by human and canine OS tumors and cell lines were assessed by gene expression, ELISA, and transwell migration assays. Safety and efficacy of losartan-toceranib therapy were evaluated in 28 dogs with lung metastatic OS. Losartan PK and monocyte PD responses were assessed in three dose cohorts of dogs by chemotaxis, plasma CCL2, and multiplex cytokine assays, and RNA-seq of losartan-treated human peripheral blood mononuclear cells. RESULTS: Human and canine OS cells secrete CCL2 and elicit monocyte migration, which is inhibited by losartan. Losartan PK/PD studies in dogs revealed that a 10-fold-higher dose than typical antihypertensive dosing was required for blockade of monocyte migration. Treatment with high-dose losartan and toceranib was well-tolerated and induced a clinical benefit rate of 50% in dogs with lung metastases. CONCLUSIONS: Losartan inhibits the CCL2-CCR2 axis, and in combination with toceranib, exerts significant biological activity in dogs with metastatic osteosarcoma, supporting evaluation of this drug combination in patients with pediatric osteosarcoma. See related commentary by Weiss et al., p. 571.

Antibacterial activity of human mesenchymal stem cells mediated directly by constitutively secreted factors and indirectly by activation of innate immune effector cells.

Mesenchymal stem cells (MSC) have been shown to improve wound healing and suppress inflammatory immune responses. Newer research also indicates that MSC exhibit antimicrobial activity, although the mechanisms underlying this activity have not been fully elucidated. Therefore, we conducted in vitro and in vivo studies to examine the ability of resting and activated MSC to kill bacteria, including multidrug resistant strains. We investigated direct bacterial killing mechanisms and the interaction of MSC with host innate immune responses to infection. In addition, the activity of MSC against chronic bacterial infections was investigated in a mouse biofilm infection model. We found that MSC exhibited high levels of spontaneous direct bactericidal activity in vitro. Moreover, soluble factors secreted by MSC inhibited Staphylococcus aureus biofilm formation in vitro and disrupted the growth of established biofilms. Secreted factors from MSC also elicited synergistic killing of drug-resistant bacteria when combined with several major classes of antibiotics. Other studies demonstrated interactions of activated MSC with host innate immune responses, including triggering of neutrophil extracellular trap formation and increased phagocytosis of bacteria. Finally, activated MSC administered systemically to mice with established S. aureus biofilm infections significantly reduced bacterial numbers at the wound site and improved wound healing when combined with antibiotic therapy. These results indicate that MSC generate multiple direct and indirect, immunologically mediated antimicrobial activities that combine to help eliminate chronic bacterial infections when the cells are administered therapeutically.

Activation of upper respiratory tract mucosal innate immune responses in cats by liposomal toll-like receptor ligand complexes delivered topically.

BACKGROUND: Nonspecific induction of local innate immune responses by mucosally administered immunotherapy is a new approach to protection from upper respiratory tract infections. Therefore, a new liposome-toll-like receptor complex (LTC) immune stimulant was developed and investigated for its ability to activate innate immune responses in cats, both in vitro and in vivo, as part of an initial evaluation of LTC for use as an immunotherapeutic agent in cats. OBJECTIVES: We hypothesized that LTC could activate innate immune responses in cats after topical application to nasal and oropharyngeal mucosal surfaces. ANIMALS: Mucosal immune responses to topical administration of LTC were assessed in 7 healthy, purpose-bred cats, and in vitro responses were assessed using blood samples from healthy cats. METHODS: Cytokine and cellular immune responses to LTC were evaluated in blood samples, nasal lavage specimens, and pharyngeal swabs from cats, using reverse transcriptase polymerase chain reaction assays, ELISA assays, and flow cytometry. RESULTS: Liposome-TLR complexes rapidly activated leukocytes in vitro, including upregulation of costimulatory molecule expression and cytokine production. Topical administration of LTC in healthy cats triggered rapid recruitment of monocytes to the nasal and oropharyngeal mucosa. CONCLUSIONS AND CLINICAL IMPORTANCE: Liposome-TLR complexes were found to effectively activate innate immune responses in cats after mucosal administration. These findings suggest that LTC have potential for use as a new mucosally administered immunotherapy for nonspecific protection from viral and bacterial respiratory tract infections.

Humoral immune responses against gut bacteria in dogs with inflammatory bowel disease.

Inflammatory bowel disease (IBD) in dogs is associated with clinical signs of intestinal dysfunction, as well as abnormal lymphocytic and myeloid cell infiltrates in the small and/or large intestine. Thus, in many respects IBD in dogs resembles IBD in humans. However, the factors that trigger intestinal inflammation in dogs with IBD are not well understood and have been variously attributed to immune responses against dietary antigens or intestinal antigens. Previous studies in humans with IBD have documented increased production of IgG and IgA antibodies specific to intestinal bacteria, and this abnormal immune response has been linked to disease pathogenesis. Therefore, we investigated the humoral immune response against gut bacteria in dogs with IBD, using flow cytometry to quantitate IgG and IgA binding. Studies were also done to investigate the source of these antibodies (locally produced versus systemic production) and whether greater antibody binding to bacteria is associated with increased inflammatory responses. We found that dogs with IBD had significantly higher percentages and overall amounts of IgG bound to their intestinal bacteria compared to healthy dogs. Similarly, significantly higher percentages of bacteria were IgA+ bacteria were also found in dogs with IBD. Serum antibody recognition of gut bacteria was not different between healthy dogs and dogs with IBD, suggesting that anti-bacterial antibodies were primarily produced locally in the gut rather than systemically. Importantly, bacteria in the Actinobacteria phylum and in particular the genus Collinsella had significantly greater levels of antibody binding in dogs with IBD. Based on these findings, we concluded that antibody binding to commensal gut bacteria was significantly increased in dogs with IBD, that particular phyla were preferential targets for gut antibodies, and that anti-bacterial antibody responses may play an important role in regulating gut inflammation.

Mesenchymal Stem Cells (MSC) Derived from Induced Pluripotent Stem Cells (iPSC) Equivalent to Adipose-Derived MSC in Promoting Intestinal Healing and Microbiome Normalization in Mouse Inflammatory Bowel Disease Model.

Cellular therapy with allogeneic or autologous mesenchymal stem cells (MSC) has emerged as a promising new therapeutic strategy for managing inflammatory bowel disease (IBD). However, MSC therapy ideally requires a convenient and relatively homogenous cell source (typically bone marrow or adipose tissues) and the ability to generate cells with stable phenotype and function. An alternative means of generating allogeneic MSC is to derive them from induced pluripotent stem cells (iPSC), which could in theory provide an indefinite supply of MSC with well-defined phenotype and function. Therefore, we compared the effectiveness of iPSC-derived MSC (iMSC) and adipose-derived MSC (adMSC) in a mouse model of IBD (dextran sodium sulfate-induced colitis), and investigated mechanisms of intestinal protection. We found that iMSC were equivalent to adMSC in terms of significantly improving clinical abnormalities in treated mice and reducing lesion scores and inflammation in the gut. Administration of iMSC also stimulated significant intestinal epithelial cell proliferation, increased in the numbers of Lgr5+ intestinal stem cells, and increased intestinal angiogenesis. In addition, the microbiome alterations present in mice with colitis were partially restored to resemble those of healthy mice following treatment with iMSC or adMSC. Thus, iMSC administration improved overall intestinal health and healing with equivalent potency to treatment with adMSC. This therefore is the first report of the effectiveness of iMSC in the treatment of IBD, along with a description of unique mechanisms of action with respect to intestinal healing and microbiome restoration. Stem Cells Translational Medicine 2018;7:456-467.

Mechanisms of Immune Suppression Utilized by Canine Adipose and Bone Marrow-Derived Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) from rodents and humans have been shown to suppress T cells by distinct primary pathways, with nitric oxide (NO)-dependent pathways dominating in rodents and indoleamine 2,3-deoxygenase (IDO)-dependent pathways dominating in humans. However, the immune suppressive pathways utilized by canine MSC have not been thoroughly studied, nor have bone marrow-derived MSC (BM-MSC) and adipose-derived MSC (Ad-MSC) been directly compared for their immune modulatory potency or pathway utilization. Therefore, canine BM-MSC and Ad-MSC were generated in vitro and their potency in suppressing T cell proliferation and cytokine production was compared, and differential gene expression. Mechanisms of T cells suppression were also investigated for both MSC types. We found that BM-MSC and Ad-MSC were roughly equivalent in terms of their ability to suppress T cell activation. However, the two MSC types used both shared and distinct biochemical pathways to suppress T cell activation. Ad-MSC utilized TGF-ß signaling pathways and adenosine signaling to suppress T cell activation, whereas BM-MSC used cyclooxygenase, TGF-ß and adenosine signaling pathways to suppress T cell activation. These results indicate that canine MSC are distinct from human and rodent MSC terms of their immune suppressive pathways, relying primarily on cyclooxygenase and TGF-ß pathways for T cell suppression, rather than on NO or IDO-mediated pathways.

Activated Mesenchymal Stem Cells Interact with Antibiotics and Host Innate Immune Responses to Control Chronic Bacterial Infections.

Chronic bacterial infections associated with biofilm formation are often difficult to resolve without extended courses of antibiotic therapy. Mesenchymal stem cells (MSC) exert antibacterial activity in vitro and in acute bacterial infection models, but their activity in chronic infection with biofilm models has not been previously investigated. Therefore, we studied the effects of MSC administration in mouse and dog models of chronic infections associated with biofilms. Mice with chronic Staphylococcus aureus implant infections were treated by i.v. administration of activated or non-activated MSC, with or without antibiotic therapy. The most effective treatment protocol was identified as activated MSC co-administered with antibiotic therapy. Activated MSC were found to accumulate in the wound margins several days after i.v. administration. Macrophages in infected tissues assumed an M2 phenotype, compared to untreated infections which contained predominately M1 macrophages. Bacterial killing by MSC was found to be mediated in part by secretion of cathelicidin and was significantly increased by antibiotics. Studies in pet dogs with spontaneous chronic multi drug-resistant wound infections demonstrated clearance of bacteria and wound healing following repeated i.v. administration of activated allogeneic canine MSC. Thus, systemic therapy with activated MSC may be an effective new, non-antimicrobial approach to treatment of chronic, drug-resistant infections.

Comparison of proliferative and immunomodulatory potential of adipose-derived mesenchymal stem cells from young and geriatric cats.

Objectives The objective of this study was to compare the ability of adipose-derived mesenchymal stem cells (aMSCs) generated from young vs geriatric cats to proliferate in culture, suppress lymphocyte proliferation and undergo senescence. Methods Adipose tissues from five young (<5 years) and six geriatric (>10 years) cats were harvested and cryopreserved for subsequent aMSC isolation and culture. aMSC proliferation in culture was compared via determination of time until passage two and by 3-(4,5-demethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The immunomodulatory capacity of aMSCs was assessed using lymphocyte proliferation assays, and senescence was evaluated using senescence-associated B-galactosidase (SABG) expression. All assays were performed on aMSCs between passage two and passage three. Results aMSCs from geriatric cats took significantly longer ( P = 0.008) to reach passage two (median 11 days, range 9-22 days) compared with aMSCs from young healthy cats (median 7 days, range 6-8 days). No significant difference was detected between young and geriatric cats in terms of their ability to suppress lymphocyte proliferation. SABG expression was not significantly different between young and geriatric aMSCs. Conclusions and relevance Compared with young feline aMSCs, geriatric aMSCs are significantly impaired in their ability to rapidly proliferate to passage two following initial culture, presenting a concern for autologous therapy. Nonetheless, once the cells are expanded, young and geriatric cat aMSCs appear to be equivalent in terms of their ability to functionally suppress T-cell activation and proliferation.

Suppression of Canine Dendritic Cell Activation/Maturation and Inflammatory Cytokine Release by Mesenchymal Stem Cells Occurs Through Multiple Distinct Biochemical Pathways.

Mesenchymal stem cells (MSC) represent a readily accessible source of cells with potent immune modulatory activity. MSC can suppress ongoing inflammatory responses by suppressing T cell function, while fewer studies have examined the impact of MSC on dendritic cell (DC) function. The dog spontaneous disease model represents an important animal model with which to evaluate the safety and effectiveness of cellular therapy with MSC. This study evaluated the effects of canine MSC on the activation and maturation of canine monocyte-derived DC, as well as mechanisms underlying these effects. Adipose-derived canine MSC were cocultured with canine DC, and the MSC effects on DC maturation and activation were assessed by flow cytometry, cytokine ELISA, and confocal microscopy. We found that canine MSC significantly suppressed lipopolysaccharide (LPS)-stimulated upregulation of DC activation markers such as major histocompatibility class II (MHCII), CD86, and CD40. Furthermore, pretreatment of MSC with interferon gamma (IFNγ) augmented this suppressive activity. IFNγ-activated MSC also significantly reduced LPS-elicited DC secretion of tumor necrosis factor alpha without reducing secretion of interleukin-10. The suppressive effect of IFNγ-treated MSC on LPS-induced DC activation was mediated by soluble factors secreted by both MSC and DC. Pathways of DC functional suppression included programmed death ligand-1 expression and secretion of nitrous oxide, prostaglandin E2, and adenosine by activated MSC. Coculture of DC with IFNγ-treated MSC maintained DC in an immature state and prolonged DC antigen uptake during LPS maturation stimulus. Taken together, canine MSC are capable of potently suppressing DC function in a potentially inflammatory microenvironment through several separate immunological pathways and confirm the potential for immune therapy with MSC in canine immune-mediated disease models.

Cancer immunotherapy in veterinary medicine: Current options and new developments.

Excitement in the field of tumor immunotherapy is being driven by several remarkable breakthroughs in recent years. This review will cover recent advances in cancer immunotherapy, including the use of T cell checkpoint inhibitors, engineered T cells, cancer vaccines, and anti-B cell and T cell antibodies. Inhibition of T cell checkpoint molecules such as PD-1 and CTLA-4 using monoclonal antibodies has achieved notable success against advanced tumors in humans, including melanoma, renal cell carcinoma, and non-small cell lung cancer. Therapy with engineered T cells has also demonstrated remarkable tumor control and regression in human trials. Autologous cancer vaccines have recently demonstrated impressive prolongation of disease-free intervals and survival times in dogs with lymphoma. In addition, caninized monoclonal antibodies targeting CD20 and CD52 just recently received either full (CD20) or conditional (CD52) licensing by the United States Department of Agriculture for clinical use in the treatment of canine B-cell and T-cell lymphomas, respectively. Thus, immunotherapy for cancer is rapidly moving to the forefront of cancer treatment options in veterinary medicine as well as human medicine.

ST-11: A New Brain-Penetrant Microtubule-Destabilizing Agent with Therapeutic Potential for Glioblastoma Multiforme.

Glioblastoma multiforme is a devastating and intractable type of cancer. Current antineoplastic drugs do not improve the median survival of patients diagnosed with glioblastoma multiforme beyond 14 to 15 months, in part because the blood-brain barrier is generally impermeable to many therapeutic agents. Drugs that target microtubules (MT) have shown remarkable efficacy in a variety of cancers, yet their use as glioblastoma multiforme treatments has also been hindered by the scarcity of brain-penetrant MT-targeting compounds. We have discovered a new alkylindole compound, ST-11, that acts directly on MTs and rapidly attenuates their rate of assembly. Accordingly, ST-11 arrests glioblastoma multiforme cells in prometaphase and triggers apoptosis. In vivo analyses reveal that unlike current antitubulin agents, ST-11 readily crosses the blood-brain barrier. Further investigation in a syngeneic orthotopic mouse model of glioblastoma multiforme shows that ST-11 activates caspase-3 in tumors to reduce tumor volume without overt toxicity. Thus, ST-11 represents the first member of a new class of brain-penetrant antitubulin therapeutic agents. Mol Cancer Ther; 15(9); 2018-29. ©2016 AACR.

The serine hydrolase ABHD6 controls the accumulation and efficacy of 2-AG at cannabinoid receptors.

The endocannabinoid 2-arachidonoylglycerol (2-AG) regulates neurotransmission and neuroinflammation by activating CB1 cannabinoid receptors on neurons and CB2 cannabinoid receptors on microglia. Enzymes that hydrolyze 2-AG, such as monoacylglycerol lipase, regulate the accumulation and efficacy of 2-AG at cannabinoid receptors. We found that the recently described serine hydrolase alpha-beta-hydrolase domain 6 (ABHD6) also controls the accumulation and efficacy of 2-AG at cannabinoid receptors. In cells from the BV-2 microglia cell line, ABHD6 knockdown reduced hydrolysis of 2-AG and increased the efficacy with which 2-AG can stimulate CB2-mediated cell migration. ABHD6 was expressed by neurons in primary culture and its inhibition led to activity-dependent accumulation of 2-AG. In adult mouse cortex, ABHD6 was located postsynaptically and its selective inhibition allowed the induction of CB1-dependent long-term depression by otherwise subthreshold stimulation. Our results indicate that ABHD6 is a rate-limiting step of 2-AG signaling and is therefore a bona fide member of the endocannabinoid signaling system.