Immunometabolic Adaptation of CD19-Targeted CAR T Cells in the Central Nervous System Microenvironment of Patients Promotes Memory Development.

Metabolic reprogramming is a hallmark of T-cell activation, and metabolic fitness is fundamental for T-cell-mediated antitumor immunity. Insights into the metabolic plasticity of chimeric antigen receptor (CAR) T cells in patients could help identify approaches to improve their efficacy in treating cancer. Here, we investigated the spatiotemporal immunometabolic adaptation of CD19-targeted CAR T cells using clinical samples from CAR T-cell-treated patients. Context-dependent immunometabolic adaptation of CAR T cells demonstrated the link between their metabolism, activation, differentiation, function, and local microenvironment. Specifically, compared with the peripheral blood, low lipid availability, high IL15, and low TGFß in the central nervous system microenvironment promoted immunometabolic adaptation of CAR T cells, including upregulation of a lipolytic signature and memory properties. Pharmacologic inhibition of lipolysis in cerebrospinal fluid led to decreased CAR T-cell survival. Furthermore, manufacturing CAR T cells in cerebrospinal fluid enhanced their metabolic fitness and antileukemic activity. Overall, this study elucidates spatiotemporal immunometabolic rewiring of CAR T cells in patients and demonstrates that these adaptations can be exploited to maximize the therapeutic efficacy of CAR T cells. SIGNIFICANCE: The spatiotemporal immunometabolic landscape of CD19-targeted CAR T cells from patients reveals metabolic adaptations in specific microenvironments that can be exploited to maximize the therapeutic efficacy of CAR T cells.

Bioenergetic modulation with the mitochondria uncouplers SR4 and niclosamide prevents proliferation and growth of treatment-naïve and vemurafenib-resistant melanomas.

BRAF mutations are detected in >50% of all melanomas. These mutations impair the LKB1-AMPK signaling, an important metabolic pathway associated with cell growth, proliferation and survival. Melanoma patients with BRAF mutations are usually treated with BRAF inhibitors such as vemurafenib, but responses are short-lived as drug resistant tumors metabolically switch to mitochondrial oxidative phosphorylation (OXPHOS) to escape metabolic stress-induced BRAF inhibition. Additionally, a large subset of melanoma utilizes OXPHOS in their metabolism, which can confer de novo resistance to BRAF inhibitors. Therefore, uncoupling of OXPHOS to perturb energy homeostasis and to indirectly stimulate AMPK could be a novel treatment for melanoma and to overcome intrinsic and acquired resistance to BRAF inhibitors. Here, we investigated the effects of SR4 and niclosamide, two small molecule mitochondria uncouplers, on the growth and proliferation of treatment-naïve and vemurafenib-resistant melanomas in vitro and in vivo. SR4 and niclosamide inhibited melanoma proliferation irrespective of BRAF/NRAS status. Melanomas with greater OXPHOS phenotype (higher OCR/ECAR), with LKB1 mutation, or with acquired resistance to vemurafenib displayed greater sensitivity to both uncouplers. More importantly, SR4 and niclosamide inhibited tumor growth in both treatment-naïve and vemurafenib-resistant xenograft mice models. Mechanistic studies indicate both uncouplers induced energetic stress, modulated the AMPK-mTOR pathway, and promoted apoptosis without affecting MEK-ERK MAPK signaling. These results suggest that uncouplers such as SR4 and niclosamide may be useful as first line treatment against melanoma regardless of BRAF/NRAS status, and as an adjuvant therapy for patients failing MAPK inhibitors.

SR4 Uncouples Mitochondrial Oxidative Phosphorylation, Modulates AMP-dependent Kinase (AMPK)-Mammalian Target of Rapamycin (mTOR) Signaling, and Inhibits Proliferation of HepG2 Hepatocarcinoma Cells.

Mitochondrial oxidative phosphorylation produces most of the energy in aerobic cells by coupling respiration to the production of ATP. Mitochondrial uncouplers, which reduce the proton gradient across the mitochondrial inner membrane, create a futile cycle of nutrient oxidation without generating ATP. Regulation of mitochondrial dysfunction and associated cellular bioenergetics has been recently identified as a promising target for anticancer therapy. Here, we show that SR4 is a novel mitochondrial uncoupler that causes dose-dependent increase in mitochondrial respiration and dissipation of mitochondrial membrane potential in HepG2 hepatocarcinoma cells. These effects were reversed by the recoupling agent 6-ketocholestanol but not cyclosporin A and were nonexistent in mitochondrial DNA-depleted HepG2 cells. In isolated mouse liver mitochondria, SR4 similarly increased oxygen consumption independent of adenine nucleotide translocase and uncoupling proteins, decreased mitochondrial membrane potential, and promoted swelling of valinomycin-treated mitochondria in potassium acetate medium. Mitochondrial uncoupling in HepG2 cells by SR4 results in the reduction of cellular ATP production, increased ROS production, activation of the energy-sensing enzyme AMPK, and inhibition of acetyl-CoA carboxylase and mammalian target of rapamycin signaling pathways, leading to cell cycle arrest and apoptosis. Global analysis of SR4-associated differential gene expression confirms these observations, including significant induction of apoptotic genes and down-regulation of cell cycle, mitochondrial, and oxidative phosphorylation pathway transcripts at 24 h post-treatment. Collectively, our studies demonstrate that the previously reported indirect activation of AMPK and in vitro anticancer properties of SR4 as well as its beneficial effects in both animal xenograft and obese mice models could be a direct consequence of its mitochondrial uncoupling activity.

2'-Hydroxyflavanone: A promising molecule for kidney cancer prevention.

Kidney cancer, also known as renal cell carcinoma (RCC), is one of the top 10 diagnosed cancers in the USA, and the incidence is rising. Despite major improvements in drug therapy strategies, RCC remains a deadly malignancy if not found and removed in its early stages. RCC is so highly drug-resistant that no effective life-prolonging regimen of cytotoxic chemotherapy has been demonstrated for RCC, despite several decades of effort. It is also highly radiation-resistant, thus circumventing therapies to prevent local recurrence or to control metastatic disease. In the last few years, extensive research has been conducted to elucidate the functional significance of the plant-derived compounds, and their derivatives, as anticancer agents. This review is focussed on a chemo-dietary prevention strategy against RCC using a citrus-derived compound called 2'-hydroxyflavanone. RCC is frequently caused by VHL gene mutations, which contribute to 75% of all RCCs. These mutations are positively linked to cigarette smoking, and exposure to the tobacco carcinogen, N-nitrosodimethylamine and benzopyrene, can disrupt VHL. According to in vitro and preclinical mouse studies, 2'-hydroxyflavanone can both protect the VHL locus and prevent the progression of VHL-mutant cancer. Human clinical trials examining the effect of supplementation of 2'-hydroxyflavanone, either alone or in combination with chemotherapeutic drugs, on RCC prevention have not been conducted, although there is considerable potential for 2'-hydroxyflavanone and its derivatives to be developed as RCC chemoprevention agents. Therefore, the discovery of plant-derived cancer therapies, such as 2'-hydroxyflavanone, offers a new strategy for combating this highly resistant cancer.

LR-90 prevents methylglyoxal-induced oxidative stress and apoptosis in human endothelial cells.

Methylglyoxal (MGO) is a highly reactive dicarbonyl compound known to induce cellular injury and cytoxicity, including apoptosis in vascular cells. Vascular endothelial cell apoptosis has been implicated in the pathophysiology and progression of atherosclerosis. We investigated whether the advanced glycation end-product inhibitor LR-90 could prevent MGO-induced apoptosis in human umbilical vascular endothelial cells (HUVECs). HUVECs were pre-treated with LR-90 and then stimulated with MGO. Cell morphology, cytotoxicity and apoptosis were evaluated by light microscopy, MTT assay, and Annexin V-FITC and propidium iodide double staining, respectively. Levels of Bax, Bcl-2, cytochrome c, mitogen-activated protein kinases (MAPKs) and caspase activities were assessed by Western blotting. Reactive oxygen species (ROS) generation and mitochondrial membrane potential (MMP) were measured with fluorescent probes. LR-90 dose-dependently prevented MGO-associated HUVEC cytotoxicity and apoptotic biochemical changes such as loss of MMP, increased Bax/Bcl-2 protein ratio, mitochondrial cytochrome c release and activation of caspase-3 and 9. Additionally, LR-90 blocked intracellular ROS formation and MAPK (p44/p42, p38, JNK) activation, though the latter seem to be not directly involved in MGO-induced HUVEC apoptosis. LR-90 prevents MGO-induced HUVEC apoptosis by inhibiting ROS and associated mitochondrial-dependent apoptotic signaling cascades, suggesting that LR-90 possess cytoprotective ability which could be beneficial in prevention of diabetic related-atherosclerosis.

Inter- and intraspecific identification of the screwworm, Cochliomyia hominivorax, using random amplified polymorphic DNA-polymerase chain reaction.

The screwworm, Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae), is one of the most devastating arthropod pests of livestock in the Western Hemisphere. Early instars are very difficult to distinguish morphologically from several closely related blow fly species. Random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) markers were developed for identifying C. hominivorax from other wound inhabiting species. Forty decameric primers were screened; nine showed clear reproducible RAPD profiles suitable for distinguishing all life stages of C. hominivorax from 7 other species, including C. macellaria (Fabricius). The results from RAPD-PCR with field-collected samples of unknown first instars agreed with morphological identification that the samples were not C. hominivorax. Three different primers showed DNA polymorphisms (intraspecific) for samples originating from Mexico, Costa Rica, Panama, Jamaica, and Brazil. Therefore, RAPD-PCR may be useful for determining the geographic origin of C. hominivorax samples. Comparing products from these primers, used with known and unknown screwworm samples from an outbreak in Mexico, clearly showed that the outbreak did not originate from the mass rearing facility. Accurate identification of suspected C. hominivorax samples is possible using RAPD-PCR. Further development to identify the geographic origin of samples would benefit the ongoing surveillance programs against C. hominivorax and the decision process during suspected outbreaks of this important pest.

Small­molecule COH-SR4 inhibits adipocyte differentiation via AMPK activation.

Obesity is a chronic metabolic disorder caused by an imbalance between energy intake and expenditure. It is one of the principal causative factors involved in the development of metabolic syndrome and cancer. Inhibition of adipocyte differentiation has often been a target of anti-obesity strategies since obesity is caused not only by hypertrophy but also by adipocyte hyperplasia. In this study, we investigated the effects of COH-SR4, a novel compound with anticancer properties, on the adipogenesis in 3T3-L1 cells. Treatment with COH-SR4 significantly inhibited adipocyte differentiation in a dose-dependent manner. This inhibitory effect mainly occurred at the early phase of differentiation through inhibition of mitotic clonal expansion and cell cycle arrest at the G1/S phase transition. In differentiating adipocytes, COH-SR4 significantly reduced intracellular lipid accumulation and downregulated the expression of key adipogenesis-related transcription factors and lipogenic proteins. COH-SR4 exhibited no cytotoxic effects in 3T3-L1 cells, but indirectly activated AMP-activated protein kinase (AMPK). AMPK activation by COH-SR4 also resulted in the phosphorylation of raptor and tuberous sclerosis protein 2 (TSC2), two proteins involved in the mammalian target of rapamycin (mTOR) signaling pathways. Additionally, COH-SR4 decreased the phosphorylation of p70 kDa ribosomal protein S6 kinase (S6K) and initiation factor 4E (eIF4E) binding protein 1 (4EB­P1), two downstream effectors of mTOR that regulate protein synthesis. Interestingly, knockdown of AMPKα1/α2 prevented the ability of COH-SR4 to inhibit cell cycle arrest and overall adipogenesis and lipid accumulation in the differentiating 3T3-L1 cells. Taken together, these results suggest that COH-SR4 inhibits 3T3-L1 adipogenesis via AMPK activation. COH-SR4 may be a promising compound for the treatment of obesity and related metabolic disorders.

Novel dichlorophenyl urea compounds inhibit proliferation of human leukemia HL-60 cells by inducing cell cycle arrest, differentiation and apoptosis.

Two novel dichlorophenyl urea compounds, SR4 and SR9, were synthesized in our laboratory and evaluated for anti-cancer activities. Specifically, we investigated the antiproliferative properties of these new compounds on promyelocytic HL-60 leukemia cells by analyzing their effects on cell differentiation, cell cycle progression and apoptosis. SR4 and SR9 were both cytotoxic to HL-60 cells in a dose-and time-dependent manner, with IC(50) of 1.2 µM and 2.2 µM, respectively, after 72 h treatment. Both compounds strongly suppressed growth of HL-60 cells by promoting cell cycle arrest at the G0/G1 transition, with concomitant decrease in protein levels of cyclins D1 and E2 and cyclin-dependent kinases (CDK 2 and CDK 4), and increased protein expression of CDK inhibitors p21(WAF1/Cip1) and p27(Kip1). In addition, either compounds induce cell differentiation as detected by increased NBT staining and expression of CD11b and CD14. Treatment with SR compounds also promoted mitochondrial-dependent apoptosis as confirmed by Annexin V-FITC double staining, DNA fragmentation, increased expression of caspase 3, 7 and 9, cytochrome c release, PARP degradation, and collapse in mitochondrial membrane potential (ΔΨ(MT)). Collectively, these results provide evidence that SR4 and SR9 have the potential for the treatment of human leukemia and merit further investigation as therapeutic agents against other types of cancer.

Advanced glycation end products of DNA: quantification of N2-(1-Carboxyethyl)-2'-deoxyguanosine in biological samples by liquid chromatography electrospray ionization tandem mass spectrometry.

Methylglyoxal (MG) and related alpha-oxoaldehydes react with proteins, lipids, and DNA to give rise to covalent adducts known as advanced glycation end products (AGEs). Elevated levels of AGEs have been implicated in the pathological complications of diabetes, uremia, Alzheimer's disease, and possibly cancer. There is therefore widespread interest in developing sensitive methods for the in vivo measurement of AGEs as prognostic biomarkers and for treatment monitoring. The two diastereomeric MG-DNA adducts of N(2)-(1-carboxyethyl)-2'-deoxyguanosine (CEdG) are the primary glycation products formed in DNA; however, accurate assessment of their distribution in vivo has not been possible since there is no readily available quantitative method for CEdG determination in biological samples. To address these issues, we have developed a sensitive and quantitative liquid chromatography electrospray ionization tandem mass spectrometry assay using the stable isotope dilution method with an (15)N(5)-CEdG standard. Methods for CEdG determination in urine or tissue extracted DNA are described. Changes in urinary CEdG in diabetic rats in response to oral administration of the AGE inhibitor LR-90 are used to demonstrate the potential utility of the method for treatment monitoring. Both stereoisomeric CEdG adducts were detected in a human breast tumor and normal adjacent tissue at levels of 3-12 adducts/10(7) dG, suggesting that this lesion may be widely distributed in vivo. Strategies for dealing with artifactual adduct formation due to oxoaldehyde generation during DNA isolation and enzymatic workup procedures are described.

Proinflammatory effects of advanced lipoxidation end products in monocytes.

OBJECTIVE: The reactions of carbohydrate- or lipid-derived intermediates with proteins lead to the formation of Maillard reaction products, which subsequently leads to the formation of advanced glycation/lipoxidation end products (AGE/ALEs). Levels of AGE/ALEs are increased in diseases like diabetes. Unlike AGEs, very little is known about ALE effects in vitro. We hypothesized that ALEs can have proinflammatory effects in monocytes. RESEARCH DESIGN AND METHODS: In a profiling approach, conditioned media from THP-1 cells either cultured in normal glucose (5.5 mmol/l) or treated with MDA-Lys or MDA alone were hybridized to arrays containing antibodies to 120 known human cytokines/chemokines. Pathway analyses with bioinformatics software were used to identify signalling networks. RESULTS: Synthetic ALE (malondialdehyde-lysine [MDA-Lys]) (50 micromol/l) could induce oxidant stress and also activate the transcriptional factor nuclear factor-kappaB (NF-kappaB) in THP-1 monocytes. MDA-Lys also significantly increased the expression of key candidate proinflammatory genes, interferon-gamma-inducible protein-10, beta1- and beta2-integrins, cyclooxygenase-2 (COX-2), monocyte chemoattractant protein-1 (MCP-1), interleukin-6 and -8, and inducible nitric-oxide synthase, which are also associated with monocyte dysfunction. Several key target proinflammatory proteins were significantly induced by MDA-Lys relative to normal glucose or MDA alone, including MCP-1; tumor necrosis factor ligand superfamily member-14; chemokine CC motif ligand-11 (CCL11); growth-related oncogene-alpha, -beta, and -gamma; and chemokine CXC motif ligand-13. Bioinformatics analyses identified a network of chemokine signaling among MDA-Lys-regulated genes. MDA-Lys also increased monocyte binding to vascular smooth muscle and endothelial cells. Furthermore, plasma from diabetic rats showed significantly higher levels of MDA-Lys and CCL11. CONCLUSIONS: These new results suggest that ALEs can promote monocyte activation and vascular complications via induction of inflammatory pathways and networks.

Anti-inflammatory effects of the advanced glycation end product inhibitor LR-90 in human monocytes.

Ligation of advanced glycation end products (AGEs) with their receptor (RAGE) plays an important role in the development of various diabetes complications, including atherosclerosis. Monocyte activation, adhesion, and migration are key events in the pathogenesis of atherosclerosis. Previous studies showed that AGEs and S100b, a specific RAGE ligand, could augment monocyte inflammatory responses via RAGE. In this study, we examined whether LR-90, a compound belonging to a new class of AGE inhibitor, could inhibit inflammatory responses in human monocytes. Human THP-1 cells were pretreated with LR-90 and then stimulated with S100b. LR-90 significantly inhibited S100b-induced expression of RAGE and other proinflammatory genes including monocyte chemoattractant protein-1, interferon-gamma-inducible protein-10, and cyclooxygenase-2 in a dose-dependent manner. These inhibitory effects may be exerted via inhibition of nuclear factor-kappaB (NF-kappaB) activation, as LR-90 suppressed both S100b-and tumor necrosis factor-alpha-induced IkappaB-alpha degradation as well as NF-kappaB promoter transcriptional activity. LR-90 also prevented oxidative stress in activated monocytes, as demonstrated by its inhibitory effects on S100b-induced expression of NADPH oxidase and intracellular superoxide production. In addition, LR-90 blocked S100b-induced monocyte adhesion to human umbilical vein endothelial cell. These new data show that, in addition to its AGE inhibitory effects, LR-90 has novel anti-inflammatory properties and might therefore have additional protective effects against diabetic vascular complications.

Novel inhibitors of advanced glycation endproducts.

A number of natural or synthetic compounds as AGE inhibitors have been proposed, discovered or currently being advanced by others and us. We have identified two new classes of aromatic compounds; aryl- (and heterocyclic) ureido and aryl (and heterocyclic) carboxamido phenoxyisobutyric acids, and benzoic acid derivatives and related compounds, as potential inhibitors of glycation and AGE formation. Some of these novel compounds also showed "AGE-breaking" activities in vitro. Current evidence is that chelation of transition metals and/or trapping or indirect inhibition of formation of reactive carbonyl compounds are involved in the mechanisms of action of these novel AGE inhibitors and breakers. Here, we review the inhibitors of glycation and AGE-breakers published to date and present the results of our in vitro and in vivo investigations on a number of these novel AGE inhibitors. These AGE-inhibitors and AGE-breakers may find therapeutic use in the treatment of diseases that AGE formation and accumulation may be responsible for their pathogenesis such as diabetes, Alzheimer's, rheumatoid arthritis, and atherosclerosis.