Targeting Tumor Angiogenesis with the Selective VEGFR-3 Inhibitor EVT801 in Combination with Cancer Immunotherapy.

The receptor tyrosine kinase VEGFR-3 plays a crucial role in cancer-induced angiogenesis and lymphangiogenesis, promoting tumor development and metastasis. Here, we report the novel VEGFR-3 inhibitor EVT801 that presents a more selective and less toxic profile than two major inhibitors of VEGFRs (i.e., sorafenib and pazopanib). As monotherapy, EVT801 showed a potent antitumor effect in VEGFR-3-positive tumors, and in tumors with VEGFR-3-positive microenvironments. EVT801 suppressed VEGF-C-induced human endothelial cell proliferation in vitro and tumor (lymph)angiogenesis in different tumor mouse models. In addition to reduced tumor growth, EVT801 decreased tumor hypoxia, favored sustained tumor blood vessel homogenization (i.e., leaving fewer and overall larger vessels), and reduced important immunosuppressive cytokines (CCL4, CCL5) and myeloid-derived suppressor cells (MDSC) in circulation. Furthermore, in carcinoma mouse models, the combination of EVT801 with immune checkpoint therapy (ICT) yielded superior outcomes to either single treatment. Moreover, tumor growth inhibition was inversely correlated with levels of CCL4, CCL5, and MDSCs after treatment with EVT801, either alone or combined with ICT. Taken together, EVT801 represents a promising anti(lymph)angiogenic drug for improving ICT response rates in patients with VEGFR-3 positive tumors. Significance: The VEGFR-3 inhibitor EVT801 demonstrates superior selectivity and toxicity profile than other VEGFR-3 tyrosine kinase inhibitors. EVT801 showed potent antitumor effects in VEGFR-3-positive tumors, and tumors with VEGFR-3-positive microenvironments through blood vessel homogenization, and reduction of tumor hypoxia and limited immunosuppression. EVT801 increases immune checkpoint inhibitors' antitumor effects.

EVT-701 is a novel selective and safe mitochondrial complex 1 inhibitor with potent anti-tumor activity in models of solid cancers.

Targeting the first protein complex of the mitochondrial electron transport chain (MC1) in cancer has become an attractive therapeutic approach in the recent years, given the metabolic vulnerabilities of cancer cells. The anticancer effect exerted by the pleiotropic drug metformin and the associated reduction in hypoxia-inducible factor 1α (HIF-1α) levels putatively mediated by MC1 inhibition led to the development of HIF-1α inhibitors, such as BAY87-2243, with a more specific MC1 targeting. However, the development of BAY87-2243 was stopped early in phase 1 due to dose-independent emesis and thus there is still no clinical proof of concept for the approach. Given the importance of mitochondrial metabolism during cancer progression, there is still a strong therapeutic need to develop specific and safe MC1 inhibitors. We recently reported the synthesis of compounds with a novel chemotype and potent action on HIF-1α degradation and MC1 inhibition. We describe here the selectivity, safety profile and anti-cancer activity in solid tumors of lead compound EVT-701. In addition, using murine models of lung cancer and of Non-Hodgkin's B cell lymphoma we demonstrated that EVT-701 reduced tumor growth and lymph node invasion when used as a single agent therapy. LKB1 deficiency in lung cancer was identified as a potential indicator of accrued sensitivity to EVT-701, allowing stratification and selection of patients in clinical trials. Altogether these results support further evaluation of EVT-701 alone or in combination in preclinical models and eventually in patients.

Disrupting Mitochondrial Electron Transfer Chain Complex I Decreases Immune Checkpoints in Murine and Human Acute Myeloid Leukemic Cells.

Oxidative metabolism is crucial for leukemic stem cell (LSC) function and drug resistance in acute myeloid leukemia (AML). Mitochondrial metabolism also affects the immune system and therefore the anti-tumor response. The modulation of oxidative phosphorylation (OxPHOS) has emerged as a promising approach to improve the therapy outcome for AML patients. However, the effect of mitochondrial inhibitors on the immune compartment in the context of AML is yet to be explored. Immune checkpoints such as ectonucleotidase CD39 and programmed dead ligand 1 (PD-L1) have been reported to be expressed in AML and linked to chemo-resistance and a poor prognosis. In the present study, we first demonstrated that a novel selective electron transfer chain complex (ETC) I inhibitor, EVT-701, decreased the OxPHOS metabolism of murine and human cytarabine (AraC)-resistant leukemic cell lines. Furthermore, we showed that while AraC induced an immune response regulation by increasing CD39 expression and by reinforcing the interferon-γ/PD-L1 axis, EVT-701 reduced CD39 and PD-L1 expression in vitro in a panel of both murine and human AML cell lines, especially upon AraC treatment. Altogether, this work uncovers a non-canonical function of ETCI in controlling CD39 and PD-L1 immune checkpoints, thereby improving the anti-tumor response in AML.

Rimonabant, a selective cannabinoid CB1 receptor antagonist, inhibits atherosclerosis in LDL receptor-deficient mice.

OBJECTIVE: The objective of this study was to determine whether the potent selective cannabinoid receptor-1 antagonist rimonabant has antiatherosclerotic properties. METHODS AND RESULTS: Rimonabant (50 mg/kg/d in the diet) significantly reduced food intake (from 3.35+/-.04 to 2.80+/-0.03 g/d), weight gain (from 14.6+/-0.7 g to -0.6+/-0.3 g), serum total cholesterol (from 8.39+/-0.54 to 5.32+/-0.18 g/L), and atherosclerotic lesion development in the aorta (from 1.7+/-0.22 to 0.21+/-0.037 mm(2)) and aortic sinus (from 101,000+/-7800 to 27,000+/-2900 microm(2)) of LDLR(-/-) mice fed a Western-type diet for 3 months. Rimonabant also reduced plasma levels of the proinflammatory cytokines MCP-1 and IL12 by 85% (P<0.05) and 76% (P<0.05), respectively. Pair-fed animals had reduced weight gain (6.2+/-0.6 g gain), but developed atherosclerotic lesions which were as large as those of untreated animals, showing that the antiatherosclerotic effect of rimonabant is not related to reduced food intake. Interestingly, rimonabant at a lower dose (30 mg/kg/d in the diet) reduced atherosclerosis development in the aortic sinus (from 121,000+/-20,000 to 62,000+/-11,000 microm(2), 49% reduction, P<0.05), without affecting serum total cholesterol (7.8+/-0.7 g/L versus 8.1+/-1.3 g/L in the control group). Rimonabant decreased lipopolysaccharide (LPS)- and IL1beta-induced proinflammatory gene expression in mouse peritoneal macrophages in vitro as well as thioglycollate-induced recruitment of macrophages in vivo (10 mg/kg, p.o. bolus). CONCLUSIONS: These results show that rimonabant has antiatherosclerotic effects in LDLR(-/-) mice. These effects are partly unrelated to serum cholesterol modulation and could be related to an antiinflammatory effect.

Lipolysis is altered in MHC class I zinc-alpha(2)-glycoprotein deficient mice.

Non-conventional major histocompatibility complex class I molecules are involved in a variety of physiological functions, most at the periphery of the immune system per se. Zinc-alpha(2)-glycoprotein (ZAG), the sole soluble member of this superfamily has been implicated in cachexia, a poorly understood yet life-threatening, severe wasting syndrome. To further ascertain the role of ZAG in lipid metabolism and perhaps the immune system, we inactivated both ZAG alleles by gene targeting in mice. Subjecting these ZAG deficient animals to standard or lipid rich food regimens led to increased body weight in comparison to identically treated wild-type mice. This phenotype appeared to correlate with a significant decrease in adipocytic lipolysis that could not be rescued by several pharmacological agents including beta(3)-adrenoreceptor agonists. Furthermore, in contrast to previously reported data, ZAG was found to be ubiquitously and constitutively expressed, with an especially high level in the mouse liver. No overt immunological phenotype was identified in these animals.

The imidazoline I2-site ligands BU 224 and 2-BFI inhibit MAO-A and MAO-B activities, hydrogen peroxide production, and lipolysis in rodent and human adipocytes.

Numerous imidazolinic agents exhibit antihyperglycaemic properties and have been described to promote insulin secretion, however their effects on adipose tissue development have been poorly investigated. Since white adipose tissue (WAT) plays an important role in glucose homeostasis and expresses imidazoline (I(2)) binding sites abundantly, this work aimed at studying extrapancreatic actions of two I(2)-site ligands, BU 224 and 2-BFI in adipocytes. Interaction with monoamine oxidase (MAO) was investigated by measuring the ability to modulate [(14)C]tyramine oxidation and hydrogen peroxide production. Direct influence on glucose uptake or on lipolytic activity was tested on mouse, rat, rabbit and human adipocytes. BU 224 and 2-BFI behaved as reversible inhibitors of both MAO-A and -B, as demonstrated by total inhibition of tyramine oxidation in human adipocytes and platelets or in liver from rats previously treated with selective MAO-inhibitors. Moreover, they weakly inhibited semicarbazide-sensitive amine oxidase. Like classical MAO-inhibitors, they were unable to produce hydrogen peroxide and to activate glucose uptake but prevented tyramine to do so in rodent or human adipocytes. BU 224 and 2-BFI also differed from MAO-inhibitors since they inhibited lipolysis at millimolar concentrations via a still undefined pathway independent of alpha(2)-adrenoceptor stimulation, beta-adrenergic antagonism and MAO activation. However, chronic treatment of obese Zucker rats with 2-BFI did not modify the maximal lipolytic capacity or the mild insulin resistance status of their adipocytes. Taken together, our observations demonstrate on WAT novel effects of BU 224 and 2-BFI different from their already reported actions on brain or endocrine pancreas.

Short- and long-term insulin-like effects of monoamine oxidases and semicarbazide-sensitive amine oxidase substrates in cultured adipocytes.

Semicarbazide-sensitive amine oxidase (SSAO) is known to increase during in vitro adipogenesis and to be one of the most highly expressed membrane proteins of white adipocytes. Although less well documented, mitochondrial monoamine oxidases (MAOs) are also present in adipocytes and share with SSAO the capacity to generate hydrogen peroxide. This work therefore aimed to compare several biologic effects of MAO and SSAO substrates in 3T3-F442A adipocytes. In differentiated cells, tyramine oxidation was predominantly MAO dependent, whereas benzylamine oxidation was SSAO dependent. Both amines partially mimicked insulin actions, including stimulation of Akt phosphorylation and glucose uptake. In addition, tyramine and benzylamine impaired tumor necrosis factor alpha-dependent nitric oxide formation in a pargyline- and semicarbazide-sensitive manner, respectively. Various biogenic amines were tested in competition for tyramine or benzylamine oxidation and classified as MAO-preferring (methoxytyramine, tryptamine) or SSAO-preferring substrates (methylamine, octopamine). Short-term incubation with 1 mmol/L of all amines except histamine stimulated glucose uptake up to 20% to 50% of maximal insulin activation. One-week treatment with either MAO or SSAO substrates alone allowed postconfluent cells to differentiate into adipocytes, reproducing 60% of insulin-promoted lipid accumulation. All amines also exerted a slight improvement in the adipogenic action of insulin. Therefore, like SSAO, substrate activation of MAO can interact with adipocyte metabolism by mimicking diverse effects of insulin in addition to preventing tumor necrosis factor alpha-dependent responses.

Glucose handling in streptozotocin-induced diabetic rats is improved by tyramine but not by the amine oxidase inhibitor semicarbazide.

A soluble form of semicarbazide-sensitive amine oxidase (SSAO) circulating in plasma is known to increase in type 1 and 2 diabetes. This cuproenzyme generates hydrogen peroxide, ammonia, and aldehydes when oxidizing circulating biogenic or exogenous amines. Based on the angiotoxicity of these products, inhibition of SSAO has been proposed to prevent vascular complications of diabetes. However, substrates of SSAO and monoamine oxidase (MAO) have been recently evidenced to activate glucose utilisation in insulin-sensitive tissues and to exhibit antihyperglycemic actions. To determine whether amine oxidase blockade or activation could be beneficial for diabetes, we aimed at comparing the influence of prolonged treatments with semicarbazide (SSAO-inhibitor), pargyline (MAO-inhibitor), or tyramine (amine oxidase substrate) on amine oxidase activities and glycemic control in streptozotocin-induced diabetic rats. The increase in plasma SSAO was confirmed in diabetic rats, while MAO and SSAO were decreased in subcutaneous adipose tissue when compared with normoglycemic controls. Among the diabetic rats, only those receiving tyramine exhibited slightly decreased hyperglycemia and improved glucose tolerance. Adipocytes from untreated or treated diabetic rats shared similar sensitivity to insulin. However glucose uptake activation and lipolysis inhibition in response to amine oxidase substrates combined with vanadate were impaired in rats treated with amine oxidase inhibitors. Thus, amine oxidase inhibition does not improve metabolic control while prolonged administration of tyramine slightly improves glucose disposal. It is therefore concluded that amine oxidase activation by increased substrate supply elicits insulin-like actions that may be more beneficial in diabetes than SSAO inhibition formerly proposed to prevent vascular complications.

Methylamine but not mafenide mimics insulin-like activity of the semicarbazide-sensitive amine oxidase-substrate benzylamine on glucose tolerance and on human adipocyte metabolism.

It has been reported that benzylamine reduces blood glucose in rabbits, stimulates hexose uptake, and inhibits lipolysis in mouse, rabbit, and human adipocytes. In the presence of vanadate, benzylamine is also able to improve glucose disposal in normoglycaemic and diabetic rats. Such insulin-mimicking properties are the consequence of hydrogen peroxide production during benzylamine oxidation by semicarbazide-sensitive amine oxidase (SSAO). The aim of the study was to determine whether other SSAO-substrates could share such potential antidiabetic properties. Thus, mafenide, a synthetic antimicrobial sulfonamide structurally related to benzylamine, and which has been recently reported to interact with SSAO, was tested in the above mentioned models, in parallel with methylamine, a proposed endogenous SSAO-substrate. All tested amines stimulated glucose uptake and inhibited lipolysis in rat and mouse fat cells. Methylamine and benzylamine, but not mafenide, reduced the hyperglycaemic response during a glucose tolerance test in rabbits while the three amines tested were devoid of insulin-releasing activity under both in vivo and in vitro conditions. In human adipocytes, mafenide did not stimulate glucose transport since it was not a high-affinity substrate for SSAO and generated less hydrogen peroxide than benzylamine or methylamine. Therefore, mafenide could not be considered as an antidiabetic drug despite being oxidized and exhibiting insulin-mimicking effects in rat and mouse adipocytes. By contrast, the endogenous substrate methylamine improved glucose utilization in all in vitro and in vivo models, leading to consider novel SSAO substrates as drugs with potential anti-hyperglycaemic properties.

Alteration of amine oxidase activity in the adipose tissue of obese subjects.

OBJECTIVE: To explore the activity of monoamine oxidases (MAOs) and semicarbazide-sensitive amine oxidases (SSAOs) in adipose tissue and blood of lean and moderately obese subjects and to study whether there is a link between these hydrogen peroxide-generating enzymes and blood markers of oxidative stress. RESEARCH METHODS AND PROCEDURES: Nine obese male subjects (BMI 32.6 +/- 0.4 kg/m(2)) and nine controls (BMI 23.4 +/- 0.5) of 24- to 40-year-old subjects were included in the study. MAO and SSAO activities were measured on microbiopsies of abdominal subcutaneous adipose tissue by quantifying (14)C-tyramine and (14)C-benzylamine oxidation. Levels of soluble SSAO, lipid peroxidation products, and antioxidant agents were measured in plasma, whereas cytoprotective enzymes were determined in blood lysates. RESULTS: The high MAO activity found in adipose tissue was diminished by one-half in obese subjects (maximum initial velocity of 1.2 vs. 2.3 nmol tyramine oxidized/mg protein/min). There was no change in SSAO activity, either under its adipose tissue-bound or plasma-soluble form. Plasma levels of lipid peroxidation products and antioxidant vitamins remained unmodified, as well as erythrocyte antioxidant enzymes, whereas circulating triglycerides, insulin, and leptin were increased. DISCUSSION: Although they already exhibited several signs of endocrino-metabolic disorders, the obese men did not exhibit the increase in blood markers of oxidative stress or the decrease in antioxidant defenses reported to occur in very obese or diabetic subjects. The reduced MAO and the unchanged SSAO activities found in obesity suggest that these hydrogen peroxide-generating enzymes expressed in adipocytes are probably not involved in the onset of the oxidative stress found in severe obesity and/or in its complications.

Benzylamine exhibits insulin-like effects on glucose disposal, glucose transport, and fat cell lipolysis in rabbits and diabetic mice.

Benzylamine, a substrate of semicarbazide-sensitive amine oxidase (SSAO), stimulates glucose transport in rat adipocytes and improves glucose disposal in diabetic rats only in the presence of vanadate. These effects have been described to result from a synergism between the hydrogen peroxide formed during amine oxidation and vanadate, via the generation of pervanadate, a powerful insulin mimicker. However, it has also been reported that benzylamine alone can stimulate glucose uptake and inhibit lipolysis in human fat cells. In this work, we therefore investigated whether benzylamine on its own was able to induce both in vivo and in vitro insulin-like responses in animal models other than rat. In rabbits, the i.v. infusion of 7 micromol/kg benzylamine before a glucose tolerance test resulted in a net reduction of the hyperglycemic response without a change in insulin secretion. Benzylamine also improved glucose tolerance and reduced lipid mobilization in hyperglycemic/obese mice. In vitro, 0.1 mM benzylamine stimulated glucose transport and inhibited lipolysis in mouse and rabbit adipocytes. These effects were blocked by previous treatments with semicarbazide, a SSAO inhibitor. Levels of benzylamine oxidation were more elevated in mouse than in rabbit adipose tissues, whereas the reverse was observed for skeletal muscles. Finally, benzylamine was unable to stimulate insulin secretion by isolated pancreatic islets from both species and SSAO activity was hardly detectable in pancreas. Together, our results bring evidence that benzylamine on its own can improve glucose tolerance in rabbit and mouse, likely by stimulating glucose uptake via amine oxidase activation in insulin-sensitive tissues.

Inhibition of rat fat cell lipolysis by monoamine oxidase and semicarbazide-sensitive amine oxidase substrates.

It has been demonstrated that amine oxidase substrates stimulate glucose transport in cardiomyocytes and adipocytes, promote adipogenesis in pre-adipose cell lines and lower blood glucose in diabetic rats. These insulin-like effects are dependent on amine oxidation by semicarbazide-sensitive amine oxidase or by monoamine oxidase. The present study aimed to investigate whether amine oxidase substrates also exhibit another insulin-like property, the inhibition of lipolysis. We therefore tested the influence of tyramine and benzylamine on lipolytic activity in rat adipocytes. These amines did not modify basal lipolysis but dose-dependently counteracted the stimulation induced by lipolytic agents. The response to 10 nM isoprenaline was totally inhibited by tyramine 1 mM. The blockade produced by inhibition of amine oxidase activity or by 1 mM glutathione suggested that the generation of oxidative species, which occurs during amine oxidation, was involved in tyramine antilipolytic effect. Among the products resulting from amine oxidation, only hydrogen peroxide was antilipolytic in a manner that was potentiated by vanadate, as for tyramine or benzylamine. Antilipolytic responses to tyramine and to insulin were sensitive to wortmannin. These data suggest that inhibition of lipolysis is a novel insulin-like effect of amine oxidase substrates which is mediated by hydrogen peroxide generated during amine oxidation.

Tyramine stimulates glucose uptake in insulin-sensitive tissues in vitro and in vivo via its oxidation by amine oxidases.

Tyramine and benzylamine have been described as stimulators of glucose transport in adipocytes. This effect is dependent on amine oxidation by monoamine oxidase (MAO) or semicarbazide-sensitive amine oxidase (SSAO) and on the subsequent hydrogen peroxide formation as already demonstrated by blockade with oxidase inhibitors or antioxidants and potentiation with vanadate. In this work, we extended these observations to skeletal muscle and cardiac myocytes using in vitro and in vivo approaches. Tissue distribution studies showed that substantial extrahepatic peripheral MAO activities exist in kidney and gut, but also in insulin-sensitive tissues: heart, adipose tissue, and skeletal muscles. SSAO activity is also widely distributed and present at a lower level than MAO, except in fat depots where both oxidases were equally involved in tyramine oxidation. When tested in vitro at millimolar doses, tyramine caused a large stimulation of glucose transport in rat adipocytes and in skeletal and cardiac muscles. In vivo administration of tyramine (4 mg/kg i.p.) lowered the hyperglycemic responses to a glucose challenge in control and in streptozotocin-treated rats. This positive effect on glucose disposal was obtained without vanadate and was abolished by SSAO and MAO inhibitors. Tyramine increased hexose uptake in vivo in insulin-sensitive tissues, whereas it induced only transient effects on plasma insulin or cardiovascular parameters. In conclusion, activation of the amine oxidases present in insulin-sensitive tissues induces insulin-like effects, readily detectable in vitro, and increasing peripheral glucose utilization in vivo.