Glutamine Metabolism Mediates Sensitivity to Respiratory Complex II Inhibition in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is a hematologic malignancy metabolically dependent on oxidative phosphorylation and mitochondrial electron transport chain (ETC) activity. AML cells are distinct from their normal hematopoietic counterparts by this metabolic reprogramming, which presents targets for new selective therapies. Here, metabolic changes in AML cells after ETC impairment are investigated. Genetic knockdown of the ETC complex II (CII) chaperone protein SDHAF1 (succinate dehydrogenase assembly factor 1) suppressed CII activity and delayed AML cell growth in vitro and in vivo. As a result, a novel small molecule that directly binds to the ubiquinone binding site of CII and inhibits its activity was identified. Pharmacologic inhibition of CII induced selective death of AML cells while sparing normal hematopoietic progenitors. Through stable isotope tracing, results show that genetic or pharmacologic inhibition of CII truncates the tricarboxylic acid cycle (TCA) and leads to anaplerotic glutamine metabolism to reestablish the truncated cycle. The inhibition of CII showed divergent fates, as AML cells lacked the metabolic plasticity to adequately utilize glutamine metabolism, resulting in preferential depletion of key TCA metabolites and death; normal cells were unaffected. These findings provide insight into the metabolic mechanisms that underlie AML's selective inhibition of CII. IMPLICATIONS: This work highlights the effects of direct CII inhibition in mediating selective AML cell death and provides insights into glutamine anaplerosis as a metabolic adaptation that can be therapeutically targeted.

Novel Mango Ginger Bioactive (2,4,6-Trihydroxy-3,5-diprenyldihydrochalcone) Inhibits Mitochondrial Metabolism in Combination with Avocatin B.

Acute myeloid leukemia (AML) is an aggressive blood cancer with limited effective chemotherapy options and negative patient outcomes. Food-derived molecules such as avocatin B (Avo B), a fatty-acid oxidation (FAO) inhibitor, are promising novel therapeutics. The roots of the Curcuma amada plants have been historically used in traditional medicine, but isolated bioactive compounds have seldom been studied. Here, we report that 2,4,6-trihydroxy-3,5-diprenyldihydrochalcone (M1), a bioactive from C. Amada, possesses novel anticancer activity. This in vitro study investigated the antileukemia properties of M1 and its effects on mitochondrial metabolism. In combination with Avo B, M1 synergistically reduced AML cell line viability and patient-derived clonogenic growth with no effect on normal peripheral blood stem cells. Mechanistically, M1 alone inhibited mitochondria complex I, while the M1/Avo B combination inhibited FAO by 60%, a process essential to the synergy. These results identified a novel food-derived bioactive and its potential as a novel chemotherapeutic for AML.

Avocado-derived avocadyne is a potent inhibitor of fatty acid oxidation.

Avocatin-B (Avo-B), an avocado-derived 1:1 mixture of the polyhydroxylated alcohols avocadyne (AYNE) and avocadene, eliminated leukemia cells by suppressing fatty acid oxidation (FAO) in vivo and in vitro while sparing healthy blood cells. In this study, we identified AYNE as the most potent FAO inhibitor within the Avo-B mixture capable of inducing cell death in leukemia cells lines (IC50 : 3.10 ± 0.14 µM in TEX cells; 11.53 ± 3.32 µM in OCI-AML2) and patient-derived acute myeloid leukemia cells. When added individually, the two Avo-B constituents demonstrated antagonism (Combination Index values >1), highlighting the need for future studies to assess AYNE alone. Together, this work highlighted AYNE as the most potent FAO inhibitor within the Avo-B mixture. PRACTICAL APPLICATIONS: This work identifies which of the two molecules in avocatin B (Avo-B), an avocado-derived mixture of two molecules with demonstrated human safety, utility against leukemia, insulin resistance and diabetes, is most useful. Therefore, it provides the basis for future clinical studies that will focus on testing and developing the most active Avo-B constituent.

Structure-activity relationship of avocadyne.

Avocado consumption is associated with numerous health benefits. Avocadyne is a terminally unsaturated, 17-carbon long acetogenin found almost exclusively in avocados with noted anti-leukemia and anti-viral properties. In this study, specific structural features such as the terminal triple bond, odd number of carbons, and stereochemistry are shown to be critical to its ability to suppress mitochondrial fatty acid oxidation and impart selective activity in vitro and in vivo. Together, this is the first study to conduct a structure-activity analysis on avocadyne and outline the chemical moieties critical to fatty acid oxidation suppression.

Very long chain fatty acid metabolism is required in acute myeloid leukemia.

Acute myeloid leukemia (AML) cells have an atypical metabolic phenotype characterized by increased mitochondrial mass, as well as a greater reliance on oxidative phosphorylation and fatty acid oxidation (FAO) for survival. To exploit this altered metabolism, we assessed publicly available databases to identify FAO enzyme overexpression. Very long chain acyl-CoA dehydrogenase (VLCAD; ACADVL) was found to be overexpressed and critical to leukemia cell mitochondrial metabolism. Genetic attenuation or pharmacological inhibition of VLCAD hindered mitochondrial respiration and FAO contribution to the tricarboxylic acid cycle, resulting in decreased viability, proliferation, clonogenic growth, and AML cell engraftment. Suppression of FAO at VLCAD triggered an increase in pyruvate dehydrogenase activity that was insufficient to increase glycolysis but resulted in adenosine triphosphate depletion and AML cell death, with no effect on normal hematopoietic cells. Together, these results demonstrate the importance of VLCAD in AML cell biology and highlight a novel metabolic vulnerability for this devastating disease.

Estrogen Receptors Alpha and Beta in Acute Myeloid Leukemia.

Estrogen receptor (ER) signaling has been widely studied in a variety of solid tumors, where the differential expression of ERα and ERß subtypes can impact prognosis. ER signaling has only recently emerged as a target of interest in acute myeloid leukemia (AML), an aggressive hematological malignancy with sub-optimal therapeutic options and poor clinical outcomes. In a variety of tumors, ERα activation has proliferative effects, while ERß targeting results in cell senescence or death. Aberrant ER expression and hypermethylation have been characterized in AML, making ER targeting in this disease of great interest. This review describes the expression patterns of ERα and ERß in AML and discusses the differing signaling pathways associated with each of these receptors. Furthermore, we assess how these signaling pathways can be targeted by various selective estrogen receptor modulators to induce AML cell death. We also provide insight into ER targeting in AML and discuss pending questions that require further study.

Avocado-derived polyols for use as novel co-surfactants in low energy self-emulsifying microemulsions.

Avocado (Persea americana Mill.; Lauraceae) seed-derived polyhydroxylated fatty alcohols (PFAs) or polyols (i.e., avocadene and avocadyne) are metabolic modulators that selectively induce apoptosis of leukemia stem cells and reverse pathologies associated with diet-induced obesity. Delivery systems containing avocado polyols have not been described. Herein, natural surface active properties of these polyols are characterized and incorporated into self-emulsifying drug delivery systems (SEDDS) that rely on molecular self-assembly to form fine, transparent, oil-in-water (O/W) microemulsions as small as 20 nanometers in diameter. Mechanistically, a 1:1 molar ratio of avocadene and avocadyne (i.e., avocatin B or AVO was shown to be a eutectic mixture which can be employed as a novel, bioactive, co-surfactant that significantly reduces droplet size of medium-chain triglyceride O/W emulsions stabilized with polysorbate 80. In vitro cytotoxicity of avocado polyol-SEDDS in acute myeloid leukemia cell lines indicated significant increases in potency and bioactivity compared to conventional cell culture delivery systems. A pilot pharmacokinetic evaluation of AVO SEDDS in C57BL/6J mice revealed appreciable accumulation in whole blood and biodistribution in key target tissues. Lastly, incorporation of AVO in SEDDS significantly improved encapsulation of the poorly water-soluble drugs naproxen and curcumin.

Avocatin B Protects Against Lipotoxicity and Improves Insulin Sensitivity in Diet-Induced Obesity.

SCOPE: The effects of an avocado-derived fatty acid oxidation (FAO) inhibitor, avocatin B (AvoB), on glucose and lipid metabolism in models of diet-induced obesity (DIO) and in vitro models of lipotoxicity are evaluated. The safety of its oral consumption in humans is also determined. METHODS AND RESULTS: Mice are given high-fat diets (HFD) for 8 weeks. Thereafter, AvoB or vehicle is administered orally twice weekly for 5 weeks. AvoB inhibits FAO which led to improved glucose tolerance, glucose utilization, and insulin sensitivity. AvoB's effects on metabolism under lipotoxic conditions are evaluated in vitro in pancreatic ß-islet cells and C2C12 myotubes. AvoB inhibits FAO and increases glucose oxidation, resulting in lowering of mitochondrial reactive oxygen species that improves insulin responsiveness in C2C12 myotubes and insulin secretion in INS-1 (832/13) cells, respectively. A randomized, double-blind, placebo-controlled clinical trial in healthy human participants is conducted to assess the safety of AvoB consumption (50 mg or 200 mg per day for 60 days). AvoB is well-tolerated and not associated with any dose-limiting toxicity. CONCLUSION: Therapeutic agents that are safe and effectively inhibit FAO and improve DIO-associated pathologies are currently not available. AvoB's mechanism of action and favorable safety profile highlight its nutritional and clinical importance.

Knockdown of BNIP3L or SQSTM1 alters cellular response to mitochondria target drugs.

Macroautophagy/autophagy, a pathway by which cellular components are sequestered and degraded in response to homeostatic and cell stress-related signals, is required to preserve hematopoietic stem and progenitor cell function. Loss of chromosomal regions carrying autophagy genes and decreased autophagy gene expression are characteristic of acute myeloid leukemia (AML) cells. Deficiency of autophagy proteins is also linked to an altered AML metabolic profile; altered metabolism has recently emerged as a potential druggable target in AML. Here, we sought to understand the mitochondria-specific changes that occur in leukemia cells after knockdown of BNIP3L/Nix or SQSTM1/p62, which are two autophagy genes involved in mitochondrial clearance and are downregulated in primary AML cells. Mitochondrial function, as measured by changes in endogenous levels of reactive oxygen species (ROS) and mitochondrial membrane potential, was altered in leukemia cells deficient in these autophagy genes. Further, these AML cells were increasingly sensitive to mitochondria-targeting drugs while displaying little change in sensitivity to DNA-targeting agents. These findings suggest that BNIP3L or SQSTM1 may be useful prognostic markers to identify AML patients suitable for mitochondria-targeted therapies. Abbreviations: AML: acute myeloid leukemia; DHE: dihydroethidium; mtDNA: mitochondrial DNA; NAO: 10-N-nonyl acridine orange; PD: population doubling; R123: rhodamine 123; ROS: reactive oxygen species; TRC: transduced scramble controls.

Inhibition of FAO in AML co-cultured with BM adipocytes: mechanisms of survival and chemosensitization to cytarabine.

Adipocytes are the prevalent stromal cell type in adult bone marrow (BM), and leukemia cells continuously adapt to deficiency of nutrients acquiring chemoresistant profiles in the BM microenvironment. We have previously shown that fatty acid metabolism is a key energy pathway for survival of acute myeloid leukemia (AML) cells in the adipocyte-abundant BM microenvironment. The novel fatty acid ß-oxidation (FAO) inhibitor avocatin B, an odd-numbered carbon lipid derived from the avocado fruit, induced apoptosis and growth inhibition in mono-cultured AML cells. In AML cells co-cultured with BM adipocytes, FAO inhibition with avocatin B caused adaptive stimulation of free fatty acid (FFA) uptake through upregulation of FABP4 mRNA, enhanced glucose uptake and switch to glycolysis. These changes reflect the compensatory response to a shortage of FFA supply to the mitochondria, and facilitate the protection of AML cells from avocatin B-induced apoptosis in the presence of BM adipocytes. However, the combination treatment of avocatin B and conventional anti-AML therapeutic agent cytarabine (AraC) increased reactive oxygen species and demonstrated highly synergistic effects on AML cells under BM adipocyte co-culture condition. These findings highlight the potential for combination regimens of AraC and FAO inhibitors that target bone marrow-resident chemoresistant AML cells.

Drosophila melanogaster as a function-based high-throughput screening model for antinephrolithiasis agents in kidney stone patients.

Kidney stone disease involves the aggregation of stone-forming salts consequent to solute supersaturation in urine. The development of novel therapeutic agents for this predominantly metabolic and biochemical disorder have been hampered by the lack of a practical pre-clinical model amenable to drug screening. Here, Drosophila melanogaster, an emerging model for kidney stone disease research, was adapted as a high-throughput functional drug screening platform independent of the multifactorial nature of mammalian nephrolithiasis. Through functional screening, the therapeutic potential of a novel compound commonly known as arbutin that specifically binds to oxalate, a key component of kidney calculi, was identified. Through isothermal titration calorimetry, high-performance liquid chromatography and atomic force microscopy, arbutin was determined to interact with calcium and oxalate in both free and bound states, disrupting crystal lattice structure, growth and crystallization. When used to treat patient urine samples, arbutin significantly abrogated calculus formation in vivo and outperformed potassium citrate in low pH urine conditions, owing to its oxalate-centric mode of action. The discovery of this novel antilithogenic compound via D. melanogaster, independent of a mammalian model, brings greater recognition to this platform, for which metabolic features are primary outcomes, underscoring the power of D. melanogaster as a high-throughput drug screening platform in similar disorders. This is the first description of the use of D. melanogaster as the model system for a high-throughput chemical library screen. This article has an associated First Person interview with the first authors of the paper.

Analytical Method To Detect and Quantify Avocatin B in Hass Avocado Seed and Pulp Matter.

Avocatin B, an avocado-derived compound mixture, was demonstrated recently to possess potent anticancer activity by selectively targeting and eliminating leukemia stem cells. Avocatin B is a mixture of avocadene and avocadyne, two 17-carbon polyhydroxylated fatty alcohols (PFAs), first discovered in avocado seeds; their quantities in avocado pulp are unknown. Analytical methods to detect avocado seed PFAs have utilized NMR spectroscopy and GC-MS; both of these lack quantitative capacity and accuracy. Herein, we report a sensitive LC-MS method for the quantitation of avocadene and avocadyne in avocado seed and pulp. The method has a reliable and linear response range of 0.1-50 µM (0.03-17.2 ng/µL) for both avocadene and avocadyne ( r2 > 0.990) with a lower limit of quantitation (LLOQ) of 0.1 µM. The intra- and interassay accuracy and precision of the quality control (QC) samples at LLOQ showed ≤18.2% percentage error and ≤14.4% coefficient of variation (CV). The intra- and interassay accuracy and precision for QC samples at low and high concentrations were well below 10% error and CV. This method was successfully applied to quantify avocadene and avocadyne in total lipid extracts of Hass avocado pulp and seed matter.

Diosmetin Induces Apoptosis of Acute Myeloid Leukemia Cells.

Acute myeloid leukemia is an aggressive disease with limited and nonselective therapeutic options. This study explored the bioactivity and cell death inducing mechanism of diosmetin, a novel compound identified in a nutraceutical screen to impart selective anti-AML activity. Diosmetin, a citrus flavone, induced apoptosis characterized by increases in caspases 8 and 3/7 and the death inducing cytokine TNFα. In fact, through protein and mRNA expression analysis, activity was shown to be dependent on expression of estrogen receptor (ER) ß. Treatment with diosmetin also delayed tumor growth in AML mouse xenografts. In summary, these studies highlight diosmetin as a novel therapeutic that induces apoptosis through estrogen receptor ß.

Estrogen Receptor ß Is a Novel Target in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is a devastating disease characterized by poor patient outcome and suboptimal chemotherapeutics. Here, a high-throughput screen identified diosmetin, a citrus flavonoid, with anti-AML activity. Diosmetin imparted selective toxicity against leukemia and leukemia stem cells in vitro and in vivo with no effect on normal hematopoietic stem cells. Mechanistically, we demonstrated that diosmetin targets estrogen receptor (ER) ß. ERß expression conferred cell sensitivity, as patient-derived AML cells with high levels of ERß were sensitive, whereas cells with low ERß were insensitive to diosmetin. Knockdown of ERß confirmed resistance, whereas overexpression enhanced sensitivity to diosmetin, which was demonstrated to be mediated by reactive oxygen species signaling. In summary, these studies highlight targeting of ERß with diosmetin as a potential novel therapeutic strategy for the treatment of AML. Mol Cancer Ther; 16(11); 2618-26. ©2017 AACR.

A potential bioactive hard-stock fat replacer comprised of a molecular gel.

Short-chain ceramides, such as N-acetoyl-d-erythro-sphingosine (C2), have a remarkable ability to structure edible oils, such as canola oil, into self-standing organogels without any added saturated or trans fats. These short-chain ceramides are ubiquitously found in foods ranging from eggs to soybeans. As the ceramide fatty acid chain length increases, there is an increase in the melting temperature of the organogel and a decrease in the elastic modulus. Gelation ability is lost at 2 wt% when the fatty acid chain length increases to six carbons; however, organogels form at 5 wt% up to 18 carbons. Short-chain ceramides, C2, decrease cell viability of colon, prostate, ovarian, and leukemia cell lines, while ceramides with long-chain fatty acids, C18, do not affect the viability of these cancer cell lines. This suggests that a bioactive spreadable fat, with no trans or added saturated fat, with the potential to alter the viability of cancer cell growth, is possible.

Synthesis and evaluation of alendronate-modified gelatin biopolymer as a novel osteotropic nanocarrier for gene therapy.

AIM: To synthesize an osteotropic alendronate functionalized gelatin (ALN-gelatin) biopolymer for nanoparticle preparation and targeted delivery of DNA to osteoblasts for gene therapy applications. MATERIALS & METHODS: Alendronate coupling to gelatin was confirmed using Fourier transform IR, (31)PNMR, x-ray diffraction (XRD) and differential scanning calorimetry. ALN-gelatin biopolymers prepared at various alendronate/gelatin ratios were utilized to prepare nanoparticles and were optimized in combination with DNA and gemini surfactant for transfecting both HEK-293 and MG-63 cell lines. RESULTS: Gelatin functionalization was confirmed using the above methods. Uniform nanoparticles were obtained from a nanoprecipitation technique. ALN-gelatin/gemini/DNA complexes exhibited higher transfection efficiency in MG-63 osteosarcoma cell line compared with the positive control. CONCLUSION: ALN-gelatin is a promising biopolymer for bone targeting of either small molecules or gene therapy applications.