Bacterial Outer Membrane Vesicles Loaded with Perhexiline Suppress Tumor Development by Regulating Tumor-Associated Macrophages Repolarization in a Synergistic Way.

Tumor-associated macrophages (TAMs) promote tumor development and metastasis and are categorized into M1-like macrophages, suppressing tumor cells, and M2-like macrophages. M2-like macrophages, occupying a major role in TAMs, can be repolarized into anti-tumoral phenotypes. In this study, outer membrane vesicles (OMVs) secreted by Escherichia coli Nissle 1917 carry perhexiline (OMV@Perhx) to explore the influence of OMVs and perhexiline on TAM repolarization. OMV@Perhx was internalized by macrophages and regulated the phenotype of TAMs from M2-like to M1-like efficiently to increase the level of tumor suppressor accordingly. Re-polarized macrophages promoted apoptosis and inhibited the mobility of tumor, cells including invasion and migration. The results indicate that OMVs improve the efficacy of perhexiline and also represent a promising natural immunomodulator. Combining OMVs with perhexiline treatments shows powerfully synergistic anti-tumor effects through co-culturing with re-polarized macrophages. This work is promising to exploit the extensive applications of OMVs and chemical drugs, therefore developing a meaningful drug carrier and immunomodulator as well as expanding the purposes of traditional chemical drugs.

Proteomic characterisation of perhexiline treatment on THP-1 M1 macrophage differentiation.

Background: Dysregulated inflammation is important in the pathogenesis of many diseases including cancer, allergy, and autoimmunity. Macrophage activation and polarisation are commonly involved in the initiation, maintenance and resolution of inflammation. Perhexiline (PHX), an antianginal drug, has been suggested to modulate macrophage function, but the molecular effects of PHX on macrophages are unknown. In this study we investigated the effect of PHX treatment on macrophage activation and polarization and reveal the underlying proteomic changes induced. Methods: We used an established protocol to differentiate human THP-1 monocytes into M1 or M2 macrophages involving three distinct, sequential stages (priming, rest, and differentiation). We examined the effect of PHX treatment at each stage on the polarization into either M1 or M2 macrophages using flow cytometry, quantitative polymerase chain reaction (qPCR) and enzyme linked immunosorbent assay (ELISA). Quantitative changes in the proteome were investigated using data independent acquisition mass spectrometry (DIA MS). Results: PHX treatment promoted M1 macrophage polarization, including increased STAT1 and CCL2 expression and IL-1ß secretion. This effect occurred when PHX was added at the differentiation stage of the M1 cultures. Proteomic profiling of PHX treated M1 cultures identified changes in metabolic (fatty acid metabolism, cholesterol homeostasis and oxidative phosphorylation) and immune signalling (Receptor Tyrosine Kinase, Rho GTPase and interferon) pathways. Conclusion: This is the first study to report on the action of PHX on THP-1 macrophage polarization and the associated changes in the proteome of these cells.

Electrophysiological and ECG Effects of Perhexiline, a Mixed Cardiac Ion Channel Inhibitor, Evaluated in Nonclinical Assays and in Healthy Subjects.

Perhexiline has been used to treat hypertrophic cardiomyopathy. In addition to its effect on carnitine-palmitoyltransferase-1, it has mixed ion channel effects through inhibition of several cardiac ion currents. Effects on cardiac ion channels expressed in mammalian cells were assayed using a manual patch-clamp technique, action potential duration (APD) was measured in ventricular trabeculae of human donor hearts, and electrocardiogram effects were evaluated in healthy subjects in a thorough QT (TQT) study. Perhexiline blocked several cardiac ion currents at concentrations within the therapeutic range (150-600 ng/mL) with IC50 for hCav1.2 ∼ hERG < late hNav1.5. A significant APD shortening was observed in perhexiline-treated cardiomyocytes. The TQT study was conducted with a pilot part in 9 subjects to evaluate a dosing schedule that would achieve therapeutic and supratherapeutic perhexiline plasma concentrations on days 4 and 6, respectively. Guided by the results from the pilot, 104 subjects were enrolled in a parallel-designed part with a nested crossover comparison for the positive control. Perhexiline caused QTc prolongation, with the largest effect on ΔΔQTcF, 14.7 milliseconds at therapeutic concentrations and 25.6 milliseconds at supratherapeutic concentrations and a positive and statistically significant slope of the concentration-ΔΔQTcF relationship (0.018 milliseconds per ng/mL; 90%CI, 0.0119-0.0237 milliseconds per ng/mL). In contrast, the JTpeak interval was shortened with a negative concentration-JTpeak relationship, a pattern consistent with multichannel block. Further studies are needed to evaluate whether this results in a low proarrhythmic risk.

Identification of hit compounds with anti-schistosomal activity on in vitro generated juvenile worms in cell-free medium.

BACKGROUND: Anthelminthic treatment options against schistosomiasis are limited. The current treatment relies almost exclusively on a single drug, praziquantel (PZQ). As a consequence, the development of resistance to PZQ and limited activity of PZQ against earlier development stages are respectively a risk and a limitation to achieving the goals of the new WHO roadmap towards elimination. For the discovery of new chemical starting points, the in vitro drug screening on Schistosoma mansoni (S. mansoni) against newly transformed schistosomula (NTS) is still the most predominant approach. The use of only NTS in the initial screening limits sensitivity to potential new compounds which are predominantly active in later developmental stages. Using our recently described highly standardized, straightforward and reliable culture method that generates high rates of juvenile worms, we aimed to repurpose a subset of the National Center for Advancing Translational Sciences (NCATS) Pharmaceutical Collection (340 compounds) to identify new hits with an in vitro worm culture assay. METHODOLOGY/PRINCIPAL FINDINGS: Cercariae were mechanically transformed into skin-stage (SkS) schistosomula and continuously cultured for 3-6 weeks to the liver stage (LiS). A commercial source of serum was identified, and decrease of NTS/well along with optimal drug testing conditions was established to test compounds on early and late LiS worms. The library was screened in 96-well format assays using praziquantel (PZQ) as a positive control. Primary screening allowed a 5.9% hit rate and generated two confirmed hits on adult worms; a prophylactic antianginal agent and an antihistaminic drug. CONCLUSION: With this standardized and reliable in vitro assay, important S. mansoni developmental stages up to LiS worms can be generated and cultured over an extended period. When exposed to a subset of the NCATS Pharmaceutical Collection, 3 compounds yielded a defined anti-schistosomal phenotype on juvenile worms. Translation of activity on perfused adult S. mansoni worms was achieved only for perhexiline (a prophylactic antianginal agent) and astemizole (an antihistaminic drug).

A mechanism of perhexiline's cytotoxicity in hepatic cells involves endoplasmic reticulum stress and p38 signaling pathway.

Perhexiline is a coronary vasodilator for angina treatment that was first developed in the 1960s. Perhexiline enjoyed worldwide success before reports of severe side effects, such as hepatotoxicity and neurotoxicity, caused its withdrawal from most of the markets. The underlying mechanism of the cytotoxicity of perhexiline, however, is not yet well understood. Here we demonstrated that perhexiline induced cellular damage in primary human hepatocytes, HepaRG cells and HepG2 cells. Analysis of gene and protein expression levels of endoplasmic reticulum (ER) stress markers showed that perhexiline caused ER stress in primary human hepatocytes and HepG2 cells. The splicing of XBP1 mRNA, a hallmark of ER stress, was observed upon perhexiline treatment. Using Gluc-Fluc-HepG2 cell line, we demonstrated that protein secretion was impaired upon perhexiline treatment, suggesting functional deficits in ER. Inhibition of ER stress using ER inhibitor 4-PBA or salubrinal attenuated the cytotoxicity of perhexiline. Directly knocking down ATF4 using siRNA also partially rescued HepG2 cells upon perhexiline exposure. In addition, inhibition of ER stress using either inhibitors or siRNA transfection attenuated perhexiline-induced increase in caspase 3/7 activity, indicating that ER stress contributed to perhexiline-induced apoptosis. Moreover, perhexiline treatment resulted in activation of p38 and JNK signaling pathways, two branches of MAPK cascade. Pre-treating HepG2 cells with p38 inhibitor SB239063 attenuated perhexiline-induced apoptosis and cell death. The inhibitor also prevented the activation of CHOP and ATF4. Overall, our study demonstrated that ER stress is one important mechanism underlying the hepatotoxicity of perhexiline, and p38 signaling pathway contributes to this process. Our finding shed light on the role of both ER stress and p38 signaling pathway in drug-induced liver injury.

Perhexiline Demonstrates FYN-mediated Antitumor Activity in Glioblastoma.

Glioblastoma is the most common primary malignant brain tumor in adults. Despite aggressive treatment, outcomes remain poor with few long-term survivors. Therefore, considerable effort is being made to identify novel therapies for this malignancy. Targeting tumor metabolism represents a promising therapeutic strategy and activation of fatty acid oxidation (FAO) has been identified as a central metabolic node contributing toward gliomagenesis. Perhexiline is a compound with a long clinical track record in angina treatment and commonly described as an FAO inhibitor. We therefore sought to determine whether this compound might be repurposed to serve as a novel therapy in glioblastoma. Perhexiline demonstrated potent in vitro cytotoxicity, induction of redox stress and apoptosis in a panel of glioblastoma cell lines. However, the antitumor activity of perhexiline was distinct when compared with the established FAO inhibitor etomoxir. By evaluating mitochondrial respiration and lipid dynamics in glioblastoma cells following treatment with perhexiline, we confirmed this compound did not inhibit FAO in our models. Using in silico approaches, we identified FYN as a probable target of perhexiline and validated the role of this protein in perhexiline sensitivity. We extended studies to patient samples, validating the potential of FYN to serve as therapeutic target in glioma. When evaluated in vivo, perhexiline demonstrated the capacity to cross the blood-brain barrier and antitumor activity in both flank and orthotopic glioblastoma models. Collectively, we identified potent FYN-dependent antitumor activity of perhexiline in glioblastoma, thereby, representing a promising agent to be repurposed for the treatment of this devastating malignancy.

Inhibition of fatty acid catabolism augments the efficacy of oxaliplatin-based chemotherapy in gastrointestinal cancers.

Gastrointestinal cancer causes countless deaths every year due to therapeutic resistance. However, whether metabolic alterations contribute to chemoresistance is not well understood. In this study, we report that fatty acid (FA) catabolism was activated in gastrointestinal cancer cells treated with oxaliplatin, which exhibited higher expression of the rate-limiting enzymes carnitine palmitoyltransferase 1B (CPT1B) and CPT2. The clinical analysis also showed that high expression of these enzymes was associated with poor oxaliplatin-based chemotherapy outcomes in patients. Furthermore, genetic or pharmacological inhibition of CPT2 with perhexiline disturbed NADPH and redox homeostasis and increased reactive oxygen species (ROS) generation and cell apoptosis in gastrointestinal cancer cells following oxaliplatin treatment. Specifically, the combination of oxaliplatin and perhexiline significantly suppressed the progression of gastrointestinal cancer in cell-based xenograft and patient-derived xenograft (PDX) models. Mechanistically, CPT2 was transcriptionally upregulated by nuclear factor of activated T cells 3 (NFATc3), which translocated to the nucleus in response to oxaliplatin treatment. In summary, our study suggests that the inhibition of CPT-mediated FA catabolism combined with conventional chemotherapy is a promising therapeutic strategy for patients with gastrointestinal cancers.

A Tumor Agnostic Therapeutic Strategy for Hexokinase 1-Null/Hexokinase 2-Positive Cancers.

Since Warburg's observation that most cancers exhibit elevated glycolysis, decades of research have attempted to reduce tumor glucose utilization as a therapeutic approach. Hexokinase (HK) activity is the first glycolytic enzymatic step; despite many attempts to inhibit HK activity, none has reached clinical application. Identification of HK isoforms, and recognition that most tissues express only HK1 while most tumors express HK1 and HK2, stimulated reducing HK2 activity as a therapeutic option. However, studies using HK2 shRNA and isogenic HK1+HK2- and HK1+HK2+ tumor cell pairs demonstrated that tumors expressing only HK1, while exhibiting reduced glucose consumption, progressed in vivo as well as tumors expressing both HK1 and HK2. However, HK1-HK2+ tumor subpopulations exist among many cancers. shRNA HK2 suppression in HK1-HK2+ liver cancer cells reduced xenograft tumor progression, in contrast to HK1+HK2+ cells. HK2 inhibition, and partial inhibition of both oxidative phosphorylation and fatty acid oxidation using HK2 shRNA and small-molecule drugs, prevented human liver HK1-HK2+ cancer xenograft progression. Using human multiple myeloma xenografts and mouse allogeneic models to identify potential clinical translational agents, triple therapies that include antisense HK2 oligonucleotides, metformin, and perhexiline prevent progression. These results suggest an agnostic approach for HK1-HK2+ cancers, regardless of tissue origin.

Enantioselectivity in the tissue distribution of perhexiline contributes to different effects on hepatic histology and peripheral neural function in rats.

Perhexiline, a chiral drug, is a potent antiischemic agent whose clinical utility is limited by hepatic and neural toxicities. It inhibits mitochondrial carnitine palmitoyltransferase-1, however, excessive inhibition predisposes toward tissue steatosis. This pilot study investigated the distribution of the two enantiomers and their toxicological potential. Dark Agouti rats (n = 4 per group) were administered vehicle or 200 mg/kg daily of racemic, (+)- or (-)-perhexiline maleate orally for 8 weeks. Plasma biochemical liver function tests and Von Frey assessments of peripheral neural function were performed. Hepatic and neuronal histology, including lipid and glycogen content, was assessed using electron microscopy. Concentrations of the perhexiline enantiomers and metabolites were quantified in plasma, liver and heart. Plasma perhexiline concentrations following administration of racemate, (+)- or (-)-enantiomer were within the mid-upper clinical therapeutic range. There was extensive uptake of both enantiomers into liver and heart, with 2.5- to 4.5-fold greater net uptake of (+)- compared to (-)-perhexiline (P < .05) when administered as pure enantiomers, but not when administered as racemate. There was no biochemical or gross histological evidence of hepatotoxicity. However, livers of animals administered (+)-perhexiline had higher lipid (P < .01) and lower glycogen (P < .05) content, compared to those administered (-)-perhexiline. Animals administered racemic perhexiline had reduced peripheral neural function (P < .05) compared to controls or animals administered (-)-perhexiline. For the same plasma concentrations, differences in tissue distribution may contribute to disparities in the effects of (+)- and (-)-perhexiline on hepatic histology and neural function.

Carnitine palmitoyltransferase gene upregulation by linoleic acid induces CD4+ T cell apoptosis promoting HCC development.

Hepatocellular carcinoma (HCC) is a common cause of cancer-related death worldwide. As obesity and diabetes become more prevalent, the contribution of non-alcoholic fatty liver disease (NAFLD) to HCC is rising. Recently, we reported intrahepatic CD4+ T cells are critical for anti-tumor surveillance in NAFLD. Lipid accumulation in the liver is the hallmark of NAFLD, which may perturb T cell function. We sought to investigate how the lipid-rich liver environment influences CD4+ T cells by focusing on carnitine palmitoyltransferase (CPT) family members, which control the mitochondrial ß-oxidation of fatty acids and act as key molecules in lipid catabolism. Linoleic acid (C18:2) co-localized within the mitochondria along with a corresponding increase in CPT gene upregulation. This CPT upregulation can be recapitulated by feeding mice with a high-C18:2 diet or the NAFLD promoting methionine-choline-deficient (MCD) diet. Using an agonist and antagonist, the induction of CPT genes was found to be mediated by peroxisome proliferator-activated receptor alpha (PPAR-α). CPT gene upregulation increased mitochondrial reactive oxygen species (ROS) and led to cell apoptosis. In vivo, using liver-specific inducible MYC transgenic mice fed MCD diet, blocking CPT with the pharmacological inhibitor perhexiline decreased apoptosis of intrahepatic CD4+ T cells and inhibited HCC tumor formation. These results provide useful information for potentially targeting the CPT family to rescue intrahepatic CD4+ T cells and to aid immunotherapy for NAFLD-promoted HCC.

Lipid degradation promotes prostate cancer cell survival.

Prostate cancer is the most common male cancer and androgen receptor (AR) is the major driver of the disease. Here we show that Enoyl-CoA delta isomerase 2 (ECI2) is a novel AR-target that promotes prostate cancer cell survival. Increased ECI2 expression predicts mortality in prostate cancer patients (p = 0.0086). ECI2 encodes for an enzyme involved in lipid metabolism, and we use multiple metabolite profiling platforms and RNA-seq to show that inhibition of ECI2 expression leads to decreased glucose utilization, accumulation of fatty acids and down-regulation of cell cycle related genes. In normal cells, decrease in fatty acid degradation is compensated by increased consumption of glucose, and here we demonstrate that prostate cancer cells are not able to respond to decreased fatty acid degradation. Instead, prostate cancer cells activate incomplete autophagy, which is followed by activation of the cell death response. Finally, we identified a clinically approved compound, perhexiline, which inhibits fatty acid degradation, and replicates the major findings for ECI2 knockdown. This work shows that prostate cancer cells require lipid degradation for survival and identifies a small molecule inhibitor with therapeutic potential.

Development of Fluorinated Analogues of Perhexiline with Improved Pharmacokinetic Properties and Retained Efficacy.

We designed and synthesized perhexiline analogues that have the same therapeutic profile as the parent cardiovascular drug but lacking its metabolic liability associated with CYP2D6 metabolism. Cycloalkyl perhexiline analogues 6a-j were found to be unsuitable for further development, as they retained a pharmacokinetic profile very similar to that shown by the parent compound. Multistep synthesis of perhexiline analogues incorporating fluorine atoms onto the cyclohexyl ring(s) provided a range of different fluoroperhexiline analogues. Of these, analogues 50 (4,4-gem-difluoro) and 62 (4,4,4',4'-tetrafluoro) were highly stable and showed greatly reduced susceptibility to CYP2D6-mediated metabolism. In vitro efficacy studies demonstrated that a number of derivatives retained acceptable potency against CPT-1. Having the best balance of properties, 50 was selected for further evaluation. Like perhexiline, it was shown to be selectively concentrated in the myocardium and, using the Langendorff model, to be effective in improving both cardiac contractility and relaxation when challenged with high fat buffer.

Discovery and Characterization of Novel Anti-schistosomal Properties of the Anti-anginal Drug, Perhexiline and Its Impact on Schistosoma mansoni Male and Female Reproductive Systems.

BACKGROUND: Schistosomiasis, one of the world's greatest human neglected tropical diseases, is caused by parasitic trematodes of the genus Schistosoma. A unique feature of schistosome biology is that the induction of sexual maturation as well as the maintenance of the differentiation status of female reproductive organs and egg production, necessary for both disease transmission and pathogenesis, are strictly dependent on the male. The treatment and most control initiatives of schistosomiasis rely today on the long-term application of a single drug, praziquantel (PZQ), mostly by campaigns of mass drug administration. PZQ, while very active on adult parasites, has much lower activity against juvenile worms. Monotherapy also favors the selection of drug resistance and, therefore, new drugs are urgently needed. METHODS AND FINDINGS: Following the screening of a small compound library with an ATP-based luminescent assay on Schistosoma mansoni schistosomula, we here report the identification and characterization of novel antischistosomal properties of the anti-anginal drug perhexiline maleate (PHX). By phenotypic worm survival assays and confocal microscopy studies we show that PHX, in vitro, has a marked lethal effect on all S. mansoni parasite life stages (newly transformed schistosomula, juvenile and adult worms) of the definitive host. We further demonstrate that sub-lethal doses of PHX significantly impair egg production and lipid depletion within the vitellarium of adult female worms. Moreover, we highlighted tegumental damage in adult male worms and remarkable reproductive system alterations in both female and male adult parasites. The in vivo study in S. mansoni-patent mice showed a notable variability of worm burdens in the individual experiments, with an overall minimal schistosomicidal effect upon PHX treatment. The short PHX half-life in mice, together with its very high rodent plasma proteins binding could be the cause of the modest efficacy of PHX in the schistosomiasis murine model. CONCLUSIONS/SIGNIFICANCE: Overall, our data indicate that PHX could represent a promising starting point for novel schistosomicidal drug discovery programmes.

Pleiotropic mechanisms of action of perhexiline in heart failure.

INTRODUCTION: The re-purposing of the anti-anginal drug perhexiline (PHX) has resulted in symptomatic improvements in heart failure (HF) patients. The inhibition of carnitine palmitoyltransferase-1 (CPT-1) has been proposed as the primary mechanism underlying the therapeutic benefit of PHX. This hypothesis is contentious. AREAS COVERED: We reviewed the primary literature and patent landscape of PHX from its initial development in the 1960s through to its emergence as a drug beneficial for HF. We focused on its physico-chemistry, molecular targets, tissue accumulation and clinical dosing. EXPERT OPINION: Dogma that the beneficial effects of PHX are due primarily to potent myocardial CPT-1 inhibition is not supported by the literature and all available evidence point to it being extremely unlikely that the major effects of PHX occur via this mechanism. In vivo PHX is much more likely to be an inhibitor of surface membrane ion channels and also to have effects on other components of cellular metabolism and reactive oxygen species (ROS) generation across the cardiovascular system. However, the possibility that minor effects of PHX on CPT-1 underpin disproportionately large effects on myocardial function cannot be entirely excluded, especially given the massive accumulation of the drug in heart tissue.

Elimination of chronic lymphocytic leukemia cells in stromal microenvironment by targeting CPT with an antiangina drug perhexiline.

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the western countries and is currently incurable due, in part, to difficulty in eliminating the leukemia cells protected by stromal microenvironment. Based on previous observations that CLL cells exhibit mitochondrial dysfunction and altered lipid metabolism and that carnitine palmitoyltransferases (CPT) have a major role in transporting fatty acid into mitochondria to support cancer cell metabolism, we tested several clinically relevant inhibitors of lipid metabolism for their ability to eliminate primary CLL cells. We discovered that perhexiline, an antiangina agent that inhibits CPT, was highly effective in killing CLL cells in stromal microenvironment at clinically achievable concentrations. These effective concentrations caused low toxicity to normal lymphocytes and normal stromal cells. Mechanistic study revealed that CLL cells expressed high levels of CPT1 and CPT2. Suppression of fatty acid transport into mitochondria by inhibiting CPT using perhexiline resulted in a depletion of cardiolipin, a key component of mitochondrial membranes, and compromised mitochondrial integrity, leading to rapid depolarization and massive CLL cell death. The therapeutic activity of perhexiline was further demonstrated in vivo using a CLL transgenic mouse model. Perhexiline significantly prolonged the overall animal survival by only four drug injections. Our study suggests that targeting CPT using an antiangina drug is able to effectively eliminate leukemia cells in vivo, and is a novel therapeutic strategy for potential clinical treatment of CLL.

Drugs that Affect Cardiac Metabolism: Focus on Perhexiline.

Approaches to the pharmacotherapy of angina pectoris have previously centred on the concept that a transient imbalance between myocardial oxygen "demand" and supply within the myocardium can best be addressed by reducing demand (for example, with ß-adrenoceptor antagonist) or by increasing availability of blood (via coronary vasomotor reactivity adjustment or coronary revascularization). However, this principle is potentially challenged by the emergence of cases of angina unsuitable for such therapies (for example because of concomitant severe systolic heart failure) and by the recognition that impaired myocardial energetics may precipitate angina in the absence of fixed or variable coronary obstruction (for example in hypertrophic cardiomyopathy). The past 20 years have seen the re-emergence of a class of anti-anginal agents which act primarily by improving efficiency of myocardial oxygen utilization, and thus can correct impaired energetics, simultaneously treating angina and heart failure symptoms. We review the principles underlying the safe use of such agents, beginning with the prototype drug perhexiline maleate, which despite complex pharmacokinetics and potential hepato- or neuro-toxicity has emerged as an attractive management option in many "complicated" cases of angina pectoris.

Perhexiline maleate enhances antitumor efficacy of cisplatin in neuroblastoma by inducing over-expression of NDM29 ncRNA.

High Risk Neuroblastoma (HR-NB) is a pediatric cancer characterized by high malignancy and remarkable cell heterogeneity within the tumour nodules. In a recent study, we demonstrated that in vitro and in vivo over-expression of the non-coding RNA NDM29 (neuroblastoma differentiation marker 29) induces NB cell differentiation, dramatically reducing their malignancy. Among gene expression changes, differentiated phenotype induced by NDM29 is characterized by decrease of the expression of ABC transporters responsible for anticancer drug resistance. Thus, the pharmacological induction of NDM29, in principle, might represent a possible novel strategy to increase cytotoxic drug responses. In this work, we identify a small molecule able to induce the expression of NDM29 in NB cells, conferring to malignant cells increased susceptibility to cisplatin cytotoxic effects. We demonstrate that the pharmacological induction of NDM29 expression in vivo enhances the antitumoral effects of chemotherapy specifically on tumour initiating/cancer stem cells sub-population, usually refractory to therapies and responsible for tumour relapse. In summary, we suggest a novel therapeutical approach possibly useful to treat very aggressive NB cases with poor prognosis. This novel pharmacological strategy aims to promote differentiation of "stem-like" cells to render them more susceptible to the killing action of cytotoxic anticancer drugs.

Perhexiline activates KLF14 and reduces atherosclerosis by modulating ApoA-I production.

Recent genome-wide association studies have revealed that variations near the gene locus encoding the transcription factor Krüppel-like factor 14 (KLF14) are strongly associated with HDL cholesterol (HDL-C) levels, metabolic syndrome, and coronary heart disease. However, the precise mechanisms by which KLF14 regulates lipid metabolism and affects atherosclerosis remain largely unexplored. Here, we report that KLF14 is dysregulated in the liver of 2 dyslipidemia mouse models. We evaluated the effects of both KLF14 overexpression and genetic inactivation and determined that KLF14 regulates plasma HDL-C levels and cholesterol efflux capacity by modulating hepatic ApoA-I production. Hepatic-specific Klf14 deletion in mice resulted in decreased circulating HDL-C levels. In an attempt to pharmacologically target KLF14 as an experimental therapeutic approach, we identified perhexiline, an approved therapeutic small molecule presently in clinical use to treat angina and heart failure, as a KLF14 activator. Indeed, in WT mice, treatment with perhexiline increased HDL-C levels and cholesterol efflux capacity via KLF14-mediated upregulation of ApoA-I expression. Moreover, perhexiline administration reduced atherosclerotic lesion development in apolipoprotein E-deficient mice. Together, these results provide comprehensive insight into the KLF14-dependent regulation of HDL-C and subsequent atherosclerosis and indicate that interventions that target the KLF14 pathway should be further explored for the treatment of atherosclerosis.

Perhexiline promotes HER3 ablation through receptor internalization and inhibits tumor growth.

INTRODUCTION: Human epidermal growth factor receptor HER3 has been implicated in promoting the aggressiveness and metastatic potential of breast cancer. Upregulation of HER3 has been found to be a major mechanism underlying drug resistance to EGFR and HER2 tyrosine kinase inhibitors and to endocrine therapy in the treatment of breast cancer. Thus, agents that reduce HER3 expression at the plasma membrane may synergize with current therapies and offer a novel therapeutic strategy to improve treatment. METHODS: We devised an image-based screening platform using membrane localized HER3-YFP to identify small molecules that promote HER3 internalization and degradation. In vitro and in vivo tumor models were used to characterize the signaling effects of perhexiline, an anti-anginal drug, identified by the screening platform. RESULTS: We found perhexiline, an anti-anginal drug, selectively internalized HER3, decreased HER3 expression, and subsequently inhibited signaling downstream of HER3. Consistent with these results, perhexiline inhibited breast cancer cell proliferation in vitro and tumor growth in vivo. CONCLUSIONS: This is the first demonstration that HER3 can be targeted with small molecules by eliminating it from the cell membrane. The novel approach used here led to the discovery that perhexiline ablates HER3 expression, and offers an opportunity to identify HER3 ablation modulators as innovative therapeutics to improve survival in breast cancer patients.

Hydroxycarboxylic acid receptors are essential for breast cancer cells to control their lipid/fatty acid metabolism.

Cancer cells exhibit characteristic changes in their metabolism with efforts being made to address them therapeutically. However, targeting metabolic enzymes as such is a major challenge due to their essentiality for normal proliferating cells. The most successful pharmaceutical targets are G protein-coupled receptors (GPCRs), with more than 40% of all currently available drugs acting through them.We show that, a family of metabolite-sensing GPCRs, the Hydroxycarboxylic acid receptor family (HCAs), is crucial for breast cancer cells to control their metabolism and proliferation.We found HCA1 and HCA3 mRNA expression were significantly increased in breast cancer patient samples and detectable in primary human breast cancer patient cells. Furthermore, siRNA mediated knock-down of HCA3 induced considerable breast cancer cell death as did knock-down of HCA1, although to a lesser extent. Liquid Chromatography Mass Spectrometry based analyses of breast cancer cell medium revealed a role for HCA3 in controlling intracellular lipid/fatty acid metabolism. The presence of etomoxir or perhexiline, both inhibitors of fatty acid ß-oxidation rescues breast cancer cells with knocked-down HCA3 from cell death.Our data encourages the development of drugs acting on cancer-specific metabolite-sensing GPCRs as novel anti-proliferative agents for cancer therapy.