International Union of Basic and Clinical Pharmacology CXIII: Nuclear Receptor Superfamily-Update 2023.

The NR superfamily comprises 48 transcription factors in humans that control a plethora of gene network programs involved in a wide range of physiologic processes. This review will summarize and discuss recent progress in NR biology and drug development derived from integrating various approaches, including biophysical techniques, structural studies, and translational investigation. We also highlight how defective NR signaling results in various diseases and disorders and how NRs can be targeted for therapeutic intervention via modulation via binding to synthetic lipophilic ligands. Furthermore, we also review recent studies that improved our understanding of NR structure and signaling. SIGNIFICANCE STATEMENT: Nuclear receptors (NRs) are ligand-regulated transcription factors that are critical regulators of myriad physiological processes. NRs serve as receptors for an array of drugs, and in this review, we provide an update on recent research into the roles of these drug targets.

Retinal Degeneration: Short-Term Options and Long-Term Vision for Future Therapy.

The leading cause blindness is the loss of retinal ganglion cells which connect the retina to the brain. Degenerative retinal diseases include retinal dystrophy, macular degeneration and diabetic retinopathy, which are currently incurable as the mammalian retina has no intrinsic regenerative capacity. By utilizing insight gained from retinal regeneration in simpler species we define an approach that may unlock regenerative programs in the mammalian retina that potentially facilitate the clinical restoration of retinal function.

REV-ERB and ROR: therapeutic targets for treating myopathies.

Muscle is primarily known for its mechanical roles in locomotion, maintenance of posture, and regulation of cardiac and respiratory function. There are numerous medical conditions that adversely affect muscle, myopathies that disrupt muscle development, regeneration and protein turnover to detrimental effect. Skeletal muscle is also a vital secretory organ that regulates thermogenesis, inflammatory signaling and directs context specific global metabolic changes in energy substrate preference on a daily basis. Myopathies differ in the causative factors that drive them but share common features including severe reduction in quality of life and significantly increased mortality all due irrefutably to the loss of muscle mass. Thus far clinically viable approaches for preserving muscle proteins and stimulating new muscle growth without unwanted side effects or limited efficacy has been elusive. Over the last few decades, evidence has emerged through in vitro and in vivo studies that suggest the nuclear receptors REV-ERB and ROR might modulate pathways involved in myogenesis and mitochondrial biogenesis. Hinting that REV-ERB and ROR might be targeted to treat myopathies. However there is still a need for substantial investigation into the roles of these nuclear receptors in in vivo rodent models of degenerative muscle diseases and acute injury. Although exciting, REV-ERB and ROR have somewhat confounding roles in muscle physiology and therefore more studies utilizing in vivo models of skeletal muscle myopathies are needed. In this review we highlight the molecular forces driving some of the major degenerative muscular diseases and showcase two promising molecular targets that may have the potential to treat myopathies: ROR and REV-ERB.

Combined regenerative rehabilitation improves recovery following volumetric muscle loss injury in a rat model.

Volumetric muscle loss (VML) injury causes irreversible deficits in muscle mass and function, often resulting in permanent disability. The current standard of care is physical therapy, but it is limited in mitigating functional deficits. We have previously optimized a rehabilitation technique using electrically stimulated eccentric contraction training (EST) that improved muscle mass, strength, and size in VML-injured rats. A biosponge scaffold composed of extracellular matrix proteins has previously enhanced muscle function postVML. This study aimed to determine whether combining a regenerative therapy (i.e., biosponge) with a novel rehabilitation technique (i.e., EST) could enhance recovery in a rat model of VML. A VML defect was created by removing ~20% of muscle mass from the tibialis anterior muscle in adult male Lewis rats. Experimental groups included VML-injured rats treated with biosponge with EST or biosponge alone (n = 6/group). EST was implemented 2 weeks postinjury at 150 Hz and was continued for 4 weeks. A linear increase in eccentric torque over 4 weeks showed the adaptability of the VML-injured muscle to EST. Combining biosponge with EST improved peak isometric torque by ~52% compared with biosponge treatment alone at 6 weeks postinjury. Application of EST increased MyoD gene expression and the percentage of large (>2000 µm2) type 2B myofibers but reduced fibrotic tissue deposition in VML-injured muscles. Together, these changes may provide the basis for improved torque production. This study demonstrates the potential for combined regenerative and rehabilitative therapy to improve muscle recovery following VML.

Suppression of cytokine-mediated complement factor gene expression through selective activation of the Ah receptor with 3',4'-dimethoxy-α-naphthoflavone.

We have characterized previously a class of aryl hydrocarbon receptor (AHR) ligand termed selective AHR modulators (SAhRMs). SAhRMs exhibit anti-inflammatory properties, including suppression of cytokine-mediated acute phase genes (e.g., Saa1), through dissociation of non-dioxin-response element (DRE) AHR activity from DRE-dependent xenobiotic gene expression. The partial AHR agonist α-naphthoflavone (αNF) mediates the suppressive, non-DRE dependent effects on SAA1 expression and partial DRE-mediated CYP1A1 induction. These observations suggest that αNF may be structurally modified to a derivative exhibiting only SAhRM activity. A screen of αNF derivatives identifies 3',4'-dimethoxy-αNF (DiMNF) as a candidate SAhRM. Competitive ligand binding validates DiMNF as an AHR ligand, and DRE-dependent reporter assays with quantitative mRNA analysis of AHR target genes reveal minimal agonist activity associated with AHR binding. Consistent with loss of agonist activity, DiMNF fails to promote AHR binding to DRE probes as determined through electromobility shift assay. Importantly, mRNA analysis indicates that DiMNF retains the suppressive capacity of αNF regarding cytokine-mediated SAA1 expression in Huh7 cells. Interestingly, predictive docking modeling suggests that DiMNF adopts a unique orientation within the AHR ligand binding pocket relative to αNF and may facilitate the rational design of additional SAhRMs. Microarray studies with a non-DRE binding but otherwise functional AHR mutant identified complement factor C3 as a potential SAhRM target. We confirmed this observation in Huh7 cells using 10 µM DiMNF, which significantly repressed C3 mRNA and protein. These data expand the classes of AHR ligands exerting DRE-independent anti-inflammatory SAhRM activity, suggesting SAhRMs may have application in the amelioration of inflammatory disorders.

A two-hit model of alcoholic liver disease that exhibits rapid, severe fibrosis.

Alcoholic liver disease (ALD) is responsible for an average of 50.4% and 44.2%of liver disease deaths among males and females respectively. Driven by alcohol misuse, ALD is often reversible by cessation of consumption. However, abstinence programs can have limited success at curtailing abuse, and the loss of life. ALD, therefore, remains a significant clinical challenge. There is a need for effective treatments that prevent or reverse alcohol-induced liver damage to complement or supplant behavioral interventions. Metabolic syndrome, which is disproportionally prevalent in ALD patients, accelerates the progression of ALD and increases liver disease mortality. Current rodent models of ALD unfortunately do not account for the contribution of the western diet to ALD pathology. To address this, we have developed a rodent model of ALD that integrates the impact of the western diet and alcohol; the WASH-diet model. We show here that the WASH diet, either chronically or in small time-restricted bouts, accelerated ALD pathology with severe steatohepatitis, elevated inflammation and increased fibrosis compared to mice receiving chronic alcohol alone. We also validated our WASH-diet model as an in vivo system for testing the efficacy of experimental ALD treatments. The efficacy of the inverse-agonist SR9238, previously shown to inhibit both non-alcohol and alcohol-induced steatohepatitis progression, was conserved in our WASH-diet model. These findings suggested that the WASH-diet may be useful for in vivo pre-clinical assessment of novel therapies.

CRTC1/MAML2 directs a PGC-1α-IGF-1 circuit that confers vulnerability to PPARγ inhibition.

Mucoepidermoid carcinoma (MEC) is a life-threatening salivary gland cancer that is driven primarily by a transcriptional coactivator fusion composed of cyclic AMP-regulated transcriptional coactivator 1 (CRTC1) and mastermind-like 2 (MAML2). The mechanisms by which the chimeric CRTC1/MAML2 (C1/M2) oncoprotein rewires gene expression programs that promote tumorigenesis remain poorly understood. Here, we show that C1/M2 induces transcriptional activation of the non-canonical peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) splice variant PGC-1α4, which regulates peroxisome proliferator-activated receptor gamma (PPARγ)-mediated insulin-like growth factor 1 (IGF-1) expression. This mitogenic transcriptional circuitry is consistent across cell lines and primary tumors. C1/M2-positive tumors exhibit IGF-1 pathway activation, and small-molecule drug screens reveal that tumor cells harboring the fusion gene are selectively sensitive to IGF-1 receptor (IGF-1R) inhibition. Furthermore, this dependence on autocrine regulation of IGF-1 transcription renders MEC cells susceptible to PPARγ inhibition with inverse agonists. These results yield insights into the aberrant coregulatory functions of C1/M2 and identify a specific vulnerability that can be exploited for precision therapy.

The uremic toxin 3-indoxyl sulfate is a potent endogenous agonist for the human aryl hydrocarbon receptor.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor involved in the regulation of multiple cellular pathways, such as xenobiotic metabolism and Th17 cell differentiation. Identification of key physiologically relevant ligands that regulate AHR function remains to be accomplished. Screening of indole metabolites has identified indoxyl 3-sulfate (I3S) as a potent endogenous ligand that selectively activates the human AHR at nanomolar concentrations in primary human hepatocytes, regulating transcription of multiple genes, including CYP1A1, CYP1A2, CYP1B1, UGT1A1, UGT1A6, IL6, and SAA1. Furthermore, I3S exhibits an approximately 500-fold greater potency in terms of transcriptional activation of the human AHR relative to the mouse AHR in cell lines. Structure-function studies reveal that the sulfate group is an important determinant for efficient AHR activation. This is the first phase II enzymatic product identified that can significantly activate the AHR, and ligand competition binding assays indicate that I3S is a direct AHR ligand. I3S failed to activate either CAR or PXR. The physiological importance of I3S lies in the fact that it is a key uremic toxin that accumulates to high micromolar concentrations in kidney dialysis patients, but its mechanism of action is unknown. I3S represents the first identified relatively high potency endogenous AHR ligand that plays a key role in human disease progression. These studies provide evidence that the production of I3S can lead to AHR activation and altered drug metabolism. Our results also suggest that prolonged activation of the AHR by I3S may contribute to toxicity observed in kidney dialysis patients and thus represent a possible therapeutic target.

Evidence for ligand-mediated selective modulation of aryl hydrocarbon receptor activity.

The concept of selective receptor modulators has been established for the nuclear steroid hormone receptors. Such selective modulators have been used therapeutically with great success in the treatment of cancer. However, this concept has not been examined with regard to the aryl hydrocarbon receptor (AHR) because of the latent toxicity commonly associated with AHR activation. AHR-mediated toxicity is primarily derived from AHR binding to its dioxin response element (DRE) and driving expression of CYP1 family members, which have the capacity to metabolize procarcinogens to genotoxic carcinogens. Recent evidence using a non-DRE binding AHR mutant has established the DRE-independent suppression of inflammatory markers by the AHR. We wished to determine whether such DRE-independent repression with wild-type AHR could be dissociated from canonical DRE-dependent transactivation in a ligand-dependent manner and, in doing so, prove the concept of a selective AHR modulator (SAhRM). Here, we identify the selective estrogen receptor (ER) modulator Way-169916 as a dually selective modulator, binding both ER and AHR. Inflammatory gene expression associated with the cytokine-inducible acute-phase response (e.g., SAA1 and CRP) are diminished by Way-169916 in an AHR-dependent manner. Furthermore, activation of AHR by Way-169916 fails to stimulate canonical DRE-driven AHR-mediated CYP1A1 expression, thus eliminating the potential for AHR-mediated genotoxic stress. Such anti-inflammatory activity in the absence of DRE-mediated expression fulfills the major criteria of an SAhRM, which suggests that selective modulation of AHR is possible and renders the AHR a therapeutically viable drug target for the amelioration of inflammatory disease.

Antagonism of aryl hydrocarbon receptor signaling by 6,2',4'-trimethoxyflavone.

The aryl hydrocarbon receptor (AHR) is regarded as an important homeostatic transcriptional regulator within physiological and pathophysiological processes, including xenobiotic metabolism, endocrine function, immunity, and cancer. Agonist activation of the AHR is considered deleterious based on toxicological evidence obtained with environmental pollutants, which mediate toxic effects through AHR. However, a multitude of plant-derived constituents, e.g., polyphenols that exhibit beneficial properties, have also been described as ligands for the AHR. It is conceivable that some of the positive aspects of such compounds can be attributed to suppression of AHR activity through antagonism. Therefore, we conducted a dioxin response element reporter-based screen to assess the AHR activity associated with a range of flavonoid compounds. Our screen identified two flavonoids (5-methoxyflavone and 7,4'-dimethoxyisoflavone) with previously unidentified AHR agonist potential. In addition, we have identified and characterized 6,2',4'-trimethoxyflavone (TMF) as an AHR ligand that possesses the characteristics of an antagonist having the capacity to compete with agonists, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin and benzo[a]pyrene, thus effectively inhibiting AHR-mediated transactivation of a heterologous reporter and endogenous targets, e.g., CYP1A1, independent of cell lineage or species. Furthermore, TMF displays superior action by virtue of having no partial agonist activity, in contrast to other documented antagonists, e.g., alpha-napthoflavone, which are partial weak agonists. TMF also exhibits no species or promoter dependence with regard to AHR antagonism. TMF therefore represents an improved tool allowing for more precise dissection of AHR function in the absence of any conflicting agonist activity.

Rev-erbα heterozygosity produces a dose-dependent phenotypic advantage in mice.

Numerous mutational studies have demonstrated that circadian clock proteins regulate behavior and metabolism. Nr1d1(Rev-erbα) is a key regulator of circadian gene expression and a pleiotropic regulator of skeletal muscle homeostasis and lipid metabolism. Loss of Rev-erbα expression induces muscular atrophy, high adiposity, and metabolic syndrome in mice. Here we show that, unlike knockout mice, Nr1d1 heterozygous mice are not susceptible to muscular atrophy and in fact paradoxically possess larger myofiber diameters and improved neuromuscular function, compared to wildtype mice. Heterozygous mice lacked dyslipidemia, a characteristic of Nr1d1 knockout mice and displayed increased whole-body fatty-acid oxidation during periods of inactivity (light cycle). Heterozygous mice also exhibited higher rates of glucose uptake when fasted, and had elevated basal rates of gluconeogenesis compared to wildtype and knockout littermates. Rev-erbα ablation suppressed glycolysis and fatty acid-oxidation in white-adipose tissue (WAT), whereas partial Rev-erbα loss, curiously stimulated these processes. Our investigations revealed that Rev-erbα dose-dependently regulates glucose metabolism and fatty acid oxidation in WAT and muscle.

Ligand selectivity and gene regulation by the human aryl hydrocarbon receptor in transgenic mice.

The aryl hydrocarbon receptor (AHR) is a ligand-inducible transcription factor that displays interspecies differences with the human and mouse AHR C-terminal region sequences sharing only 58% amino acid sequence identity. Compared with the mouse AHR (mAHR), the human AHR (hAHR) displays approximately 10-fold lower relative affinity for prototypical AHR ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxin, which has been attributed to the amino acid residue valine 381 (alanine 375 in the mAHR) in the ligand binding domain of the hAHR. We investigated whether the 10-fold difference in ligand-binding affinity between the mAHR and hAHR would be observed with a diverse range of AHR ligands. To test this hypothesis, ligand binding assays were performed using the photo-affinity ligand 2-azido-3-[(125)I]iodo-7,8-dibromodibenzo-p-dioxin and liver cytosol isolated from hepatocyte-specific transgenic hAHR mice and C57BL/6J mice. It is noteworthy that competitive ligand-binding assays revealed that, compared with the mAHR, the hAHR has a higher relative affinity for certain compounds, including indirubin [(2Z)-2,3-biindole-2,3 (1'H,1'H)-dione and quercetin (2-(3,4dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-one]. Electrophoretic mobility shift assays revealed that indirubin was more efficient at transforming the hAHR compared with the mAHR. Indirubin was also a more potent inducer of Cyp1a1 expression in transgenic hAHR mouse hepatocytes compared with C57BL/6J mouse hepatocytes. These observations suggest that indirubin is a potent hAHR ligand that is able to selectively bind to and activate the hAHR. These discoveries imply that there may be a significant degree of structural divergence between mAHR and hAHR ligands and highlights the importance of the hAHR transgenic mouse as a model to study the hAHR in vivo.

Ah receptor represses acute-phase response gene expression without binding to its cognate response element.

Repression of the nuclear factor-kappaB (NF-kappaB) pathway has been extensively researched because of its pivotal role in inflammation. We investigated the potential of the aryl hydrocarbon receptor (AHR) to suppress NF-kappaB regulated-gene expression, especially acute-phase genes, such as serum amyloid A (Saa). Using AHR mutants, it was determined that nuclear translocation and heterodimerization with AHR-nuclear translocator are essential, but DNA binding is not involved in AHR-mediated Saa repression. A number of AHR ligands were capable of repressing Saa3 expression. AHR activation leads to a decrease in RELA and C/EBP/beta recruitment to and histone acetylation at Saa3 gene promoter. A battery of acute-phase genes (eg C-reactive protein and haptoglobin) induced by cytokine exposure was repressed by AHR activation in mouse hepatocytes. Dietary exposure to an AHR ligand represses cytokine-induced acute-phase response in the liver. Use of a human liver-derived cell line revealed similar repression of Saa mRNA levels and secreted protein. Repression of AHR expression also enhanced Saa induction in response to cytokines, suggesting that AHR is capable of constitutively repressing Saa gene expression. These results establish a role for AHR in inflammatory signaling within the liver, presenting a new therapeutic opportunity, and signify AHR's ability to function in a DNA-independent manner.

LXR-inverse agonism stimulates immune-mediated tumor destruction by enhancing CD8 T-cell activity in triple negative breast cancer.

Triple-negative breast cancer (TNBC) is a highly aggressive subtype that is untreatable with hormonal or HER2-targeted therapies and is also typically unresponsive to checkpoint-blockade immunotherapy. Within the tumor microenvironment dysregulated immune cell metabolism has emerged as a key mechanism of tumor immune-evasion. We have discovered that the Liver-X-Receptors (LXRα and LXRß), nuclear receptors known to regulate lipid metabolism and tumor-immune interaction, are highly activated in TNBC tumor associated myeloid cells. We therefore theorized that inhibiting LXR would induce immune-mediated TNBC-tumor clearance. Here we show that pharmacological inhibition of LXR activity induces tumor destruction primarily through stimulation of CD8+ T-cell cytotoxic activity and mitochondrial metabolism. Our results imply that LXR inverse agonists may be a promising new class of TNBC immunotherapies.

Inhibition of Hepatotoxicity by a LXR Inverse Agonist in a Model of Alcoholic Liver Disease.

Alcohol abuse is a major cause of liver disease and mortality worldwide and is a significant public health issue. Patients with alcoholic liver disease (ALD) have severe hepatic lipid accumulation, inflammation, and fibrosis. Therapies for ALD are very limited and even abstinence from alcohol consumption does not necessarily protect patients from progression of the disease. We sought to evaluate the efficacy of a liver X receptor (LXR) inverse agonist, SR9238, in an animal model of ALD. SR9238 suppresses hepatic lipogenesis, a pathological hallmark of ALD, and we hypothesized that targeting suppression of hepatic metabolic pathways that are activated in ALD may be an effective treatment for the disease. A chronic ethanol diet with or without a final ethanol binge treatment was used to induce ALD in mice. Mice were administered the liver specific LXR inverse agonist SR9238 for 4 weeks after the mice had been maintained on the ethanol diet for 14 days. Mice developed all the hallmarks of advanced ALD demonstrating significant pathophysiology and hepatotoxicity. SR9238 significantly attenuated liver injury and hepatic steatosis and fibrosis was nearly eliminated in SR9238 treated mice. SR9238 treatment reversed the damage associated with chronic ethanol use returning the liver to near normal morphology. These results indicate that inhibiting LXR activity using the inverse agonist has a hepatoprotective effect in rodent models of ALD; thus, this pharmacological approach may be efficacious for treatment of ALD in humans.

Pharmacological inhibition of REV-ERB stimulates differentiation, inhibits turnover and reduces fibrosis in dystrophic muscle.

Duchenne muscular dystrophy (DMD) is a debilitating X-linked disorder that is fatal. DMD patients lack the expression of the structural protein dystrophin caused by mutations within the DMD gene. The absence of functional dystrophin protein results in excessive damage from normal muscle use due to the compromised structural integrity of the dystrophin associated glycoprotein complex. As a result, DMD patients exhibit ongoing cycles of muscle destruction and regeneration that promote inflammation, fibrosis, mitochondrial dysfunction, satellite cell (SC) exhaustion and loss of skeletal and cardiac muscle function. The nuclear receptor REV-ERB suppresses myoblast differentiation and recently we have demonstrated that the REV-ERB antagonist, SR8278, stimulates muscle regeneration after acute injury. Therefore, we decided to explore whether the REV-ERB antagonist SR8278 could slow the progression of muscular dystrophy. In mdx mice SR8278 increased lean mass and muscle function, and decreased muscle fibrosis and muscle protein degradation. Interestingly, we also found that SR8278 increased the SC pool through stimulation of Notch and Wnt signaling. These results suggest that REV-ERB is a potent target for the treatment of DMD.

Rev-Erb co-regulates muscle regeneration via tethered interaction with the NF-Y cistrome.

OBJECTIVE: The loss of skeletal muscle mass and strength are a central feature of traumatic injury and degenerative myopathies. Unfortunately, pharmacological interventions typically fail to stem the long-term decline in quality of life. Reduced Rev-Erb-mediated gene suppression in cultured C2C12 myoblasts has been shown to stimulate myoblast differentiation. Yet the mechanisms that allow Rev-Erb to pleiotropically inhibit muscle differentiation are not well understood. In this study, we sought to elucidate the role of Rev-Erb in the regulation of muscle differentiation and regeneration in vivo. METHODS: Using Rev-Erbα/ß shRNAs, pharmacological ligands, and Rev-Erbα null and heterozygous mice, we probed the mechanism of Rev-Erbα/ß regulation of muscle differentiation and muscle regeneration. RESULTS: ChIP seq analysis of Rev-Erb in differentiating myoblasts showed that Rev-Erbα did not transcriptionally regulate muscle differentiation through cognate Rev-Erb/ROR-response elements but through possible interaction with the cell fate regulator NF-Y at CCAAT-motifs. Muscle differentiation is stimulated by Rev-Erb release from CCAAT-motifs at promoter and enhancer elements of a number of myogenesis proteins. Partial loss of Rev-Erb expression in mice heterozygous for Rev-Erbα accelerated muscle repair in vivo whereas Rev-Erb knockout mice showed deficiencies in regenerative repair compared to wild type mice. These phenotypic differences between heterozygous and knockout mice were not apparently dependent on MRF induction in response to injury. Similarly, pharmacological disruption of Rev-Erb suppressive activity in injured muscle accelerated regenerative repair in response to acute injury. CONCLUSIONS: Disrupting Rev-Erb activity in injured muscle accelerates regenerative muscle repair/differentiation through transcriptional de-repression of myogenic programs. Rev-Erb, therefore, may be a potent therapeutic target for a myriad of muscular disorders.

Fluorophore-NanoLuc BRET Reporters Enable Sensitive In Vivo Optical Imaging and Flow Cytometry for Monitoring Tumorigenesis.

Fluorescent proteins are widely used to study molecular and cellular events, yet this traditionally relies on delivery of excitation light, which can trigger autofluorescence, photoxicity, and photobleaching, impairing their use in vivo. Accordingly, chemiluminescent light sources such as those generated by luciferases have emerged, as they do not require excitation light. However, current luciferase reporters lack the brightness needed to visualize events in deep tissues. We report the creation of chimeric eGFP-NanoLuc (GpNLuc) and LSSmOrange-NanoLuc (OgNLuc) fusion reporter proteins coined LumiFluors, which combine the benefits of eGFP or LSSmOrange fluorescent proteins with the bright, glow-type bioluminescent light generated by an enhanced small luciferase subunit (NanoLuc) of the deep-sea shrimp Oplophorus gracilirostris. The intramolecular bioluminescence resonance energy transfer that occurs between NanoLuc and the fused fluorophore generates the brightest bioluminescent signal known to date, including improved intensity, sensitivity, and durable spectral properties, thereby dramatically reducing image acquisition times and permitting highly sensitive in vivo imaging. Notably, the self-illuminating and bifunctional nature of these LumiFluor reporters enables greatly improved spatiotemporal monitoring of very small numbers of tumor cells via in vivo optical imaging and also allows the isolation and analyses of single cells by flow cytometry. Thus, LumiFluor reporters are inexpensive, robust, noninvasive tools that allow for markedly improved in vivo optical imaging of tumorigenic processes.

The LXR inverse agonist SR9238 suppresses fibrosis in a model of non-alcoholic steatohepatitis.

OBJECTIVE: Non-alcoholic steatohepatitis (NASH) is characterized by hepatic steatosis, inflammation and fibrosis. There are currently no targeted therapies for NASH. We developed a liver-specific LXR inverse agonist, SR9238, which effectively reduces hepatic lipogenesis in models of obesity and hepatic steatosis. We hypothesized that suppression of lipogenesis, which is pathologically elevated in NASH may suppress progression of hepatic steatosis to NASH. METHODS: NASH was induced in B6 V-lep (ob)/J (ob/ob) mice using a custom complete rodent diet (HTF) containing high amounts of trans-fat, fructose, and cholesterol. Once NASH was induced, mice were treated with SR9238 for one month by i.p. injection. Plasma lipid levels and liver health were analyzed by clinical chemistry. QPCR, western blot, and immunohistochemistry were used to assess disease severity. RESULTS: Ob/ob mice are obese and diabetic thus they are commonly used as models for the study of metabolic diseases. These mice quickly developed the NASH phenotype when provided the HTF diet. The mice develop hepatic steatosis, severe hepatic inflammation and fibrosis on the HTF diet. Treatment with SR9238 significantly reduced the severity of hepatic steatosis and most importantly reduced hepatic inflammation and ameliorated hepatic fibrosis. CONCLUSIONS: Here, we demonstrate that an LXR inverse agonist, SR9238, is effective in reduction of hepatic steatosis, inflammation and fibrosis in an animal model of NASH. These results have important implications for the development of therapeutics for treatment NASH in humans.

Broad Anti-tumor Activity of a Small Molecule that Selectively Targets the Warburg Effect and Lipogenesis.

Malignant cells exhibit aerobic glycolysis (the Warburg effect) and become dependent on de novo lipogenesis, which sustains rapid proliferation and resistance to cellular stress. The nuclear receptor liver-X-receptor (LXR) directly regulates expression of key glycolytic and lipogenic genes. To disrupt these oncogenic metabolism pathways, we designed an LXR inverse agonist SR9243 that induces LXR-corepressor interaction. In cancer cells, SR9243 significantly inhibited the Warburg effect and lipogenesis by reducing glycolytic and lipogenic gene expression. SR9243 induced apoptosis in tumors without inducing weight loss, hepatotoxicity, or inflammation. Our results suggest that LXR inverse agonists may be an effective cancer treatment approach.