Publications by authors named "Aimee L Edinger"

50 Publications

Drug-like sphingolipid SH-BC-893 opposes ceramide-induced mitochondrial fission and corrects diet-induced obesity.

EMBO Mol Med 2021 Aug 7;13(8):e13086. Epub 2021 Jul 7.

Department of Developmental and Cell Biology, University of California Irvine, Irvine, CA, USA.

Ceramide-induced mitochondrial fission drives high-fat diet (HFD)-induced obesity. However, molecules targeting mitochondrial dynamics have shown limited benefits in murine obesity models. Here, we reveal that these compounds are either unable to block ceramide-induced mitochondrial fission or require extended incubation periods to be effective. In contrast, targeting endolysosomal trafficking events important for mitochondrial fission rapidly and robustly prevented ceramide-induced disruptions in mitochondrial form and function. By simultaneously inhibiting ARF6- and PIKfyve-dependent trafficking events, the synthetic sphingolipid SH-BC-893 blocked palmitate- and ceramide-induced mitochondrial fission, preserved mitochondrial function, and prevented ER stress in vitro. Similar benefits were observed in the tissues of HFD-fed mice. Within 4 h of oral administration, SH-BC-893 normalized mitochondrial morphology in the livers and brains of HFD-fed mice, improved mitochondrial function in white adipose tissue, and corrected aberrant plasma leptin and adiponectin levels. As an interventional agent, SH-BC-893 restored normal body weight, glucose disposal, and hepatic lipid levels in mice consuming a HFD. In sum, the sphingolipid analog SH-BC-893 robustly and acutely blocks ceramide-induced mitochondrial dysfunction, correcting diet-induced obesity and its metabolic sequelae.
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http://dx.doi.org/10.15252/emmm.202013086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8350895PMC
August 2021

Synthetic Sphingolipids with 1,2-Pyridazine Appendages Improve Antiproliferative Activity in Human Cancer Cell Lines.

ACS Med Chem Lett 2020 May 12;11(5):686-690. Epub 2020 Feb 12.

Department of Chemistry, Université de Montréal, P.O. Box 6128, Station Centre-Ville, Montréal, Quebec H3C 3J7, Canada.

A synthetic sphingolipid related to a ring-constrained hydroxymethyl pyrrolidine analog of FTY720 that was known to starve cancer cells to death was chemically modified to include a series of alkoxy-tethered 3,6-substituted 1,2-pyridazines. These derivatives exhibited excellent antiproliferative activity against eight human cancer cell lines from four different cancer types. A 2.5- to 9-fold reduction in IC in these cell lines was observed relative to the lead compound, which lacked the appended heterocycle.
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http://dx.doi.org/10.1021/acsmedchemlett.9b00553DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7236038PMC
May 2020

Macropinocytosis confers resistance to therapies targeting cancer anabolism.

Nat Commun 2020 02 28;11(1):1121. Epub 2020 Feb 28.

Department of Developmental and Cell Biology, University of California Irvine, Irvine, CA, 92697, USA.

Macropinocytic cancer cells scavenge amino acids from extracellular proteins. Here, we show that consuming necrotic cell debris via macropinocytosis (necrocytosis) offers additional anabolic benefits. A click chemistry-based flux assay reveals that necrocytosis provides not only amino acids, but sugars, fatty acids and nucleotides for biosynthesis, conferring resistance to therapies targeting anabolic pathways. Indeed, necrotic cell debris allow macropinocytic breast and prostate cancer cells to proliferate, despite fatty acid synthase inhibition. Standard therapies such as gemcitabine, 5-fluorouracil (5-FU), doxorubicin and gamma-irradiation directly or indirectly target nucleotide biosynthesis, creating stress that is relieved by scavenged nucleotides. Strikingly, necrotic debris also render macropinocytic, but not non-macropinocytic, pancreas and breast cancer cells resistant to these treatments. Selective, genetic inhibition of macropinocytosis confirms that necrocytosis both supports tumor growth and limits the effectiveness of 5-FU in vivo. Therefore, this study establishes necrocytosis as a mechanism for drug resistance.
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http://dx.doi.org/10.1038/s41467-020-14928-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7048872PMC
February 2020

Biocompatible Chemotherapy for Leukemia by Acid-Cleavable, PEGylated FTY720.

Bioconjug Chem 2020 03 27;31(3):673-684. Epub 2020 Jan 27.

Zymo Research Corporation, Irvine, California 92604, United States.

Targeting the inability of cancerous cells to adapt to metabolic stress is a promising alternative to conventional cancer chemotherapy. FTY720 (Gilenya), an FDA-approved drug for the treatment of multiple sclerosis, has recently been shown to inhibit cancer progression through the down-regulation of essential nutrient transport proteins, selectively starving cancer cells to death. However, the clinical use of FTY720 for cancer therapy is prohibited because of its capability of inducing immunosuppression (lymphopenia) and bradycardia when phosphorylated upon administration. A prodrug to specifically prevent phosphorylation during circulation, hence avoiding bradycardia and lymphopenia, was synthesized by capping its hydroxyl groups with polyethylene glycol (PEG) via an acid-cleavable ketal linkage. Improved aqueous solubility was also accomplished by PEGylation. The prodrug reduces to fully potent FTY720 upon cellular uptake and induces metabolic stress in cancer cells. Enhanced release of FTY720 at a mildly acidic endosomal pH and the ability to substantially down-regulate cell-surface nutrient transporter proteins in leukemia cells only by an acid-cleaved drug were confirmed. Importantly, the prodrug demonstrated nearly identical efficacy to FTY720 in an animal model of BCR-Abl-driven leukemia without inducing bradycardia or lymphopenia in vivo, highlighting its potential clinical value. The prodrug formulation of FTY720 demonstrates the utility of precisely engineering a drug to avoid undesirable effects by tackling specific molecular mechanisms as well as a financially favorable alternative to new drug development. A multitude of existing cancer therapeutics may be explored for prodrug formulation to avoid specific side effects and preserve or enhance therapeutic efficacy.
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http://dx.doi.org/10.1021/acs.bioconjchem.9b00822DOI Listing
March 2020

Design, synthesis and anticancer activity of constrained sphingolipid-phenoxazine/phenothiazine hybrid constructs targeting protein phosphatase 2A.

Bioorg Med Chem Lett 2019 09 19;29(18):2681-2685. Epub 2019 Jul 19.

Department of Developmental and Cell Biology, University of California, Irvine, 2128 Natural Sciences 1, CA 92697-2300, USA. Electronic address:

Inspired by the cytotoxicity of perphenazine toward cancer cells and its ability to activate the serine/threonine protein phosphatase 2A (PP2A), we prepared series of ether-carbon linked analogs of a constrained synthetic sphingolipid analog 3, known for its cytotoxicity, nutrient transporter down-regulation and vacuolation properties, incorporating the tricyclic neuroleptics phenoxazine and phenothiazine to represent hybrid structures with possible synergistic cytotoxic activity. While the original activity of the lead compound 3 was diminished by fusion with the phenoxazine or phenothiazine tethered moieties, the corresponding 3-pyridyltetryl ether analog 10 showed cytotoxicity and nutrient transporter down-regulation similar to the lead compound 3, although it separated these PP2A-dependent phenotypes from that of vacuolation.
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http://dx.doi.org/10.1016/j.bmcl.2019.07.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7784717PMC
September 2019

Dynamic Phosphoproteomics Uncovers Signaling Pathways Modulated by Anti-oncogenic Sphingolipid Analogs.

Mol Cell Proteomics 2019 Mar 22;18(3):408-422. Epub 2020 Sep 22.

Institute for Research in Immunology and Cancer, Université de Montréal, C.P. 6128, Succursale centre-ville, Montréal, Québec, H3C 3J7, Canada;; Department of Chemistry, Université de Montréal, Quebec, H3C 3J7, Canada;; Department of Biochemistry, Université de Montréal, C.P. 6128, Succursale centre-ville, Montréal, Québec, H3C 3J7, Canada. Electronic address:

The anti-neoplastic sphingolipid analog SH-BC-893 starves cancer cells to death by down-regulating cell surface nutrient transporters and blocking lysosomal trafficking events. These effects are mediated by the activation of protein phosphatase 2A (PP2A). To identify putative PP2A substrates, we used quantitative phosphoproteomics to profile the temporal changes in protein phosphorylation in FL5.12 cells following incubation with SH-BC-893 or the specific PP2A inhibitor LB-100. These analyses enabled the profiling of more than 15,000 phosphorylation sites, of which 958 sites on 644 proteins were dynamically regulated. We identified 114 putative PP2A substrates including several nutrient transporter proteins, GTPase regulators (e.g. Agap2, Git1), and proteins associated with actin cytoskeletal remodeling (e.g. Vim, Pxn). To identify SH-BC-893-induced cell signaling events that disrupt lysosomal trafficking, we compared phosphorylation profiles in cells treated with SH-BC-893 or C2-ceramide, a non-vacuolating sphingolipid that does not impair lysosomal fusion. These analyses combined with functional assays uncovered the differential regulation of Akt and Gsk3b by SH-BC-893 (vacuolating) and C2-ceramide (non-vacuolating). Dynamic phosphoproteomics of cells treated with compounds affecting PP2A activity thus enabled the correlation of cell signaling with phenotypes to rationalize their mode of action.
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http://dx.doi.org/10.1074/mcp.RA118.001053DOI Listing
March 2019

Dynamic Phosphoproteomics Uncovers Signaling Pathways Modulated by Anti-oncogenic Sphingolipid Analogs.

Mol Cell Proteomics 2019 03 27;18(3):408-422. Epub 2018 Nov 27.

From the ‡Institute for Research in Immunology and Cancer, Université de Montréal, C.P. 6128, Succursale centre-ville, Montréal, Québec, H3C 3J7, Canada;

The anti-neoplastic sphingolipid analog SH-BC-893 starves cancer cells to death by down-regulating cell surface nutrient transporters and blocking lysosomal trafficking events. These effects are mediated by the activation of protein phosphatase 2A (PP2A). To identify putative PP2A substrates, we used quantitative phosphoproteomics to profile the temporal changes in protein phosphorylation in FL5.12 cells following incubation with SH-BC-893 or the specific PP2A inhibitor LB-100. These analyses enabled the profiling of more than 15,000 phosphorylation sites, of which 958 sites on 644 proteins were dynamically regulated. We identified 114 putative PP2A substrates including several nutrient transporter proteins, GTPase regulators ( Agap2, Git1), and proteins associated with actin cytoskeletal remodeling ( Vim, Pxn). To identify SH-BC-893-induced cell signaling events that disrupt lysosomal trafficking, we compared phosphorylation profiles in cells treated with SH-BC-893 or C2-ceramide, a non-vacuolating sphingolipid that does not impair lysosomal fusion. These analyses combined with functional assays uncovered the differential regulation of Akt and Gsk3b by SH-BC-893 (vacuolating) and C2-ceramide (non-vacuolating). Dynamic phosphoproteomics of cells treated with compounds affecting PP2A activity thus enabled the correlation of cell signaling with phenotypes to rationalize their mode of action.
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http://dx.doi.org/10.1074/mcp.RA118.001053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6398214PMC
March 2019

In search of constrained FTY720 and phytosphingosine analogs as dual acting anticancer agents targeting metabolic and epigenetic pathways.

Eur J Med Chem 2018 Nov 21;159:217-242. Epub 2018 Sep 21.

Department of Developmental and Cell Biology, University of California, Irvine, 2128 Natural Sciences 1, CA, 92697-2300, USA. Electronic address:

A series of compounds containing pyrrolidine and pyrrolizidine cores with appended hydrophobic substituents were prepared as constrained analogs of FTY720 and phytosphingosine. The effect of these compounds on the viability of cancer cells, on downregulation of the nutrient transport systems, and on their ability to cause vacuolation was studied. An attempt to inhibit HDACs with some phosphate esters of our analogs was thwarted by our failure to reproduce the reported inhibitory action of FTY720-phosphate.
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http://dx.doi.org/10.1016/j.ejmech.2018.09.043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6217821PMC
November 2018

Starving PTEN-deficient prostate cancer cells thrive under nutrient stress by scavenging corpses for their supper.

Mol Cell Oncol 2018 17;5(4):e1472060. Epub 2018 Aug 17.

Department of Developmental and Cell Biology, University of California, Irvine, CA, USA.

Our recent work demonstrates that inactivating mutations in phosphatase and tensin homolog () are sufficient to drive macropinocytosis in the context of AMP-activated protein kinase (AMPK) activation. Given that blocking macropinocytosis limits PTEN-deficient prostate tumor growth, AMPK or phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) inhibitors could have therapeutic value in castration-resistant prostate cancer patients, particularly when used in combination with standard of care therapies. ATG5: autophagy related 5; NHE: Na(+)/H(+) exchanger; PAK1: p21-activated kinase 1; PI3K: phosphatidylinositol-4,5-bisphosphate 3-kinase; PIP: phosphatidylinositol (3,4,5)-trisphosphate; PIP: phosphatidylinositol (4,5)-bisphosphate; RAC1: Rac family small GTPase 1.
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http://dx.doi.org/10.1080/23723556.2018.1472060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6149707PMC
August 2018

Nutrient scavenging in cancer.

Nat Rev Cancer 2018 10;18(10):619-633

Department of Developmental and Cell Biology, University of California Irvine, Irvine, CA, USA.

While cancer cell proliferation depends on access to extracellular nutrients, inadequate tumour perfusion means that glucose, amino acids and lipids are often in short supply. To overcome this obstacle to growth, cancer cells utilize multiple scavenging strategies, obtaining macromolecules from the microenvironment and breaking them down in the lysosome to produce substrates for ATP generation and anabolism. Recent studies have revealed four scavenging pathways that support cancer cell proliferation in low-nutrient environments: scavenging of extracellular matrix proteins via integrins, receptor-mediated albumin uptake and catabolism, macropinocytic consumption of multiple components of the tumour microenvironment and the engulfment and degradation of entire live cells via entosis. New evidence suggests that blocking these pathways alone or in combination could provide substantial benefits to patients with incurable solid tumours. Both US Food and Drug Administration (FDA)-approved drugs and several agents in preclinical or clinical development shut down individual or multiple scavenging pathways. These therapies may increase the extent and durability of tumour growth inhibition and/or prevent the development of resistance when used in combination with existing treatments. This Review summarizes the evidence suggesting that scavenging pathways drive tumour growth, highlights recent advances that define the oncogenic signal transduction pathways that regulate scavenging and considers the benefits and detriments of therapeutic strategies targeting scavenging that are currently under development.
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http://dx.doi.org/10.1038/s41568-018-0048-xDOI Listing
October 2018

Sphingolipids inhibit endosomal recycling of nutrient transporters by inactivating ARF6.

J Cell Sci 2018 06 25;131(12). Epub 2018 Jun 25.

Department of Developmental and Cell Biology, University of California Irvine, Irvine, CA 92697, USA

Endogenous sphingolipids (ceramide) and related synthetic molecules (FTY720, SH-BC-893) reduce nutrient access by decreasing cell surface expression of a subset of nutrient transporter proteins. Here, we report that these sphingolipids disrupt endocytic recycling by inactivating the small GTPase ARF6. Consistent with reported roles for ARF6 in maintaining the tubular recycling endosome, MICAL-L1-positive tubules were lost from sphingolipid-treated cells. We propose that ARF6 inactivation may occur downstream of PP2A activation since: (1) sphingolipids that fail to activate PP2A did not reduce ARF6-GTP levels; (2) a structurally unrelated PP2A activator disrupted tubular recycling endosome morphology and transporter localization; and (3) overexpression of a phosphomimetic mutant of the ARF6 GEF GRP1 prevented nutrient transporter loss. ARF6 inhibition alone was not toxic; however, the ARF6 inhibitors SecinH3 and NAV2729 dramatically enhanced the killing of cancer cells by SH-BC-893 without increasing toxicity to peripheral blood mononuclear cells, suggesting that ARF6 inactivation contributes to the anti-neoplastic actions of sphingolipids. Taken together, these studies provide mechanistic insight into how ceramide and sphingolipid-like molecules limit nutrient access and suppress tumor cell growth and survival.
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http://dx.doi.org/10.1242/jcs.213314DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6031383PMC
June 2018

PTEN Deficiency and AMPK Activation Promote Nutrient Scavenging and Anabolism in Prostate Cancer Cells.

Cancer Discov 2018 07 23;8(7):866-883. Epub 2018 Mar 23.

Department of Developmental and Cell Biology, University of California, Irvine, California.

We report that PTEN-deficient prostate cancer cells use macropinocytosis to survive and proliferate under nutrient stress. PTEN loss increased macropinocytosis only in the context of AMPK activation, revealing a general requirement for AMPK in macropinocytosis and a novel mechanism by which AMPK promotes survival under stress. In prostate cancer cells, albumin uptake did not require macropinocytosis, but necrotic cell debris proved a specific macropinocytic cargo. Isotopic labeling confirmed that macropinocytosed necrotic cell proteins fueled new protein synthesis in prostate cancer cells. Supplementation with necrotic debris, but not albumin, also maintained lipid stores, suggesting that macropinocytosis can supply nutrients other than amino acids. Nontransformed prostatic epithelial cells were not macropinocytic, but patient-derived prostate cancer organoids and xenografts and autochthonous prostate tumors all exhibited constitutive macropinocytosis, and blocking macropinocytosis limited prostate tumor growth. Macropinocytosis of extracellular material by prostate cancer cells is a previously unappreciated tumor-microenvironment interaction that could be targeted therapeutically. As PTEN-deficient prostate cancer cells proliferate in low-nutrient environments by scavenging necrotic debris and extracellular protein via macropinocytosis, blocking macropinocytosis by inhibiting AMPK, RAC1, or PI3K may have therapeutic value, particularly in necrotic tumors and in combination with therapies that cause nutrient stress. .
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http://dx.doi.org/10.1158/2159-8290.CD-17-1215DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6030497PMC
July 2018

Branched chain amino acid metabolism and cancer: the importance of keeping things in context.

Transl Cancer Res 2017 May;6(Suppl 3):S578-S584

Department of Developmental and Cell Biology, University of California Irvine, Irvine, CA 92697-2300, USA.

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http://dx.doi.org/10.21037/tcr.2017.05.05DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6319930PMC
May 2017

Rab7-a novel redox target that modulates inflammatory pain processing.

Pain 2017 07;158(7):1354-1365

Institute of Pharmacology, College of Pharmacy, Goethe University, Frankfurt am Main, Germany.

Chronic pain is accompanied by production of reactive oxygen species (ROS) in various cells that are important for nociceptive processing. Recent data indicate that ROS can trigger specific redox-dependent signaling processes, but the molecular targets of ROS signaling in the nociceptive system remain largely elusive. Here, we performed a proteome screen for pain-dependent redox regulation using an OxICAT approach, thereby identifying the small GTPase Rab7 as a redox-modified target during inflammatory pain in mice. Prevention of Rab7 oxidation by replacement of the redox-sensing thiols modulates its GTPase activity. Immunofluorescence studies revealed Rab7 expression to be enriched in central terminals of sensory neurons. Knockout mice lacking Rab7 in sensory neurons showed normal responses to noxious thermal and mechanical stimuli; however, their pain behavior during inflammatory pain and in response to ROS donors was reduced. The data suggest that redox-dependent changes in Rab7 activity modulate inflammatory pain sensitivity.
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http://dx.doi.org/10.1097/j.pain.0000000000000920DOI Listing
July 2017

Oligodendroglial deletion of ESCRT-I component TSG101 causes spongiform encephalopathy.

Biol Cell 2016 Nov 23;108(11):324-337. Epub 2016 Aug 23.

McLaughlin Research Institute, Great Falls, MT, 59405, USA.

Background Information: Vacuolation of the central nervous system (CNS) is observed in patients with transmissible spongiform encephalopathy, HIV-related encephalopathy and some inherited diseases, but the underlying cellular mechanisms remain poorly understood. Mice lacking the mahogunin ring finger-1 (MGRN1) E3 ubiquitin ligase develop progressive, widespread spongiform degeneration of the CNS. MGRN1 ubiquitinates and regulates tumour susceptibility gene 101 (TSG101), a central component of the endosomal trafficking machinery. As loss of MGRN1 is predicted to cause partial TSG101 loss-of-function, we hypothesised that CNS vacuolation in Mgrn1 null mice may be caused by the accumulation of multi-cisternal endosome-like 'class E' vacuolar protein sorting (vps) compartments similar to those observed in Tsg101-depleted cells in culture.

Results: To test this hypothesis, Tsg101 was deleted from mature oligodendroglia in vivo. This resulted in severe spongiform encephalopathy, histopathologically similar to that observed in Mgrn1 null mutant mice but with a more rapid onset. Vacuoles in the brains of Tsg101-deleted and Mgrn1 mutant mice labelled with endosomal markers, consistent with an endosomal origin. Vacuoles in the brains of mice inoculated with Rocky Mountain Laboratory (RML) prions did not label with these markers, indicating a different origin, consistent with previously published studies that indicate RML prions have a primary effect on neurons and cause vacuolation in an MGRN1-independent manner. Oligodendroglial deletion of Rab7, which mediates late endosome-to-lysosome trafficking and autophagosome-lysosome fusion, did not cause spongiform change.

Conclusions: Our data suggest that the formation of multi-cisternal 'class E' vps endosomal structures in oligodendroglia leads to vacuolation.

Significance: This work provides the first evidence that disrupting multi-vesicular body formation in oligodendroglia can cause white matter vacuolation and demyelination. HIV is known to hijack the endosomal sorting machinery, suggesting that HIV infection of the CNS may also act through this pathway to cause encephalopathy.
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http://dx.doi.org/10.1111/boc.201600014DOI Listing
November 2016

Vps34 regulates Rab7 and late endocytic trafficking through recruitment of the GTPase-activating protein Armus.

J Cell Sci 2016 12 28;129(23):4424-4435. Epub 2016 Oct 28.

Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook NY11794, USA

The class III phosphoinositide 3-kinase (PI3K) Vps34 (also known as PIK3C3 in mammals) produces phosphatidylinositol 3-phosphate [PI(3)P] on both early and late endosome membranes to control membrane dynamics. We used Vps34-deficient cells to delineate whether Vps34 has additional roles in endocytic trafficking. In Vps34 mouse embryonic fibroblasts (MEFs), transferrin recycling and EEA1 membrane localization were unaffected despite elevated Rab5-GTP levels. Strikingly, a large increase in Rab7-GTP levels, an accumulation of enlarged late endosomes, and decreased EGFR degradation were observed in Vps34-deficient cells. The hyperactivation of Rab7 in Vps34-deficient cells stemmed from the failure to recruit the Rab7 GTPase-activating protein (GAP) Armus (also known as TBC1D2), which binds to PI(3)P, to late endosomes. Protein-lipid overlay and liposome-binding assays reveal that the putative pleckstrin homology (PH) domain in Armus can directly bind to PI(3)P. Elevated Rab7-GTP led to the failure of intraluminal vesicle (ILV) formation and lysosomal maturation. Rab7 silencing and Armus overexpression alleviated the vacuolization seen in Vps34-deficient cells. Taken together, these results demonstrate that Vps34 has a previously unknown role in regulating Rab7 activity and late endosomal trafficking.
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http://dx.doi.org/10.1242/jcs.192260DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5201010PMC
December 2016

Targeting cancer metabolism by simultaneously disrupting parallel nutrient access pathways.

J Clin Invest 2016 11 26;126(11):4088-4102. Epub 2016 Sep 26.

Oncogenic mutations drive anabolic metabolism, creating a dependency on nutrient influx through transporters, receptors, and macropinocytosis. While sphingolipids suppress tumor growth by downregulating nutrient transporters, macropinocytosis and autophagy still provide cancer cells with fuel. Therapeutics that simultaneously disrupt these parallel nutrient access pathways have potential as powerful starvation agents. Here, we describe a water-soluble, orally bioavailable synthetic sphingolipid, SH-BC-893, that triggers nutrient transporter internalization and also blocks lysosome-dependent nutrient generation pathways. SH-BC-893 activated protein phosphatase 2A (PP2A), leading to mislocalization of the lipid kinase PIKfyve. The concomitant mislocalization of the PIKfyve product PI(3,5)P2 triggered cytosolic vacuolation and blocked lysosomal fusion reactions essential for LDL, autophagosome, and macropinosome degradation. By simultaneously limiting access to both extracellular and intracellular nutrients, SH-BC-893 selectively killed cells expressing an activated form of the anabolic oncogene Ras in vitro and in vivo. However, slower-growing, autochthonous PTEN-deficient prostate tumors that did not exhibit a classic Warburg phenotype were equally sensitive. Remarkably, normal proliferative tissues were unaffected by doses of SH-BC-893 that profoundly inhibited tumor growth. These studies demonstrate that simultaneously blocking parallel nutrient access pathways with sphingolipid-based drugs is broadly effective and cancer selective, suggesting a potential strategy for overcoming the resistance conferred by tumor heterogeneity.
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http://dx.doi.org/10.1172/JCI87148DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5096903PMC
November 2016

Effects of stereochemistry, saturation, and hydrocarbon chain length on the ability of synthetic constrained azacyclic sphingolipids to trigger nutrient transporter down-regulation, vacuolation, and cell death.

Bioorg Med Chem 2016 09 18;24(18):4390-4397. Epub 2016 Jul 18.

Department of Chemistry, Université de Montréal, PO Box 6128, Station Centre-Ville, Montréal, QC H3C 3J7, Canada. Electronic address:

Constrained analogs containing a 2-hydroxymethylpyrrolidine core of the natural sphingolipids sphingosine, sphinganine, N,N-dimethylsphingosine and N-acetyl variants of sphingosine and sphinganine (C2-ceramide and dihydro-C2-ceramide) were synthesized and evaluated for their ability to down-regulate nutrient transporter proteins and trigger cytoplasmic vacuolation in mammalian cells. In cancer cells, the disruptions in intracellular trafficking produced by these sphingolipids lead to cancer cell death by starvation. Structure activity studies were conducted by varying the length of the hydrocarbon chain, the degree of unsaturation and the presence or absence of an aryl moiety on the appended chains, and stereochemistry at two stereogenic centers. In general, cytotoxicity was positively correlated with nutrient transporter down-regulation and vacuolation. This study was intended to identify structural and functional features in lead compounds that best contribute to potency, and to develop chemical biology tools that could be used to isolate the different protein targets responsible for nutrient transporter loss and cytoplasmic vacuolation. A molecule that produces maximal vacuolation and transporter loss is expected to have the maximal anti-cancer activity and would be a lead compound.
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http://dx.doi.org/10.1016/j.bmc.2016.07.038DOI Listing
September 2016

Age-related myelin degradation burdens the clearance function of microglia during aging.

Nat Neurosci 2016 08 13;19(8):995-8. Epub 2016 Jun 13.

Max Planck Institute of Experimental Medicine, Göttingen, Germany.

Myelin is synthesized as a multilamellar membrane, but the mechanisms of membrane turnover are unknown. We found that myelin pieces were gradually released from aging myelin sheaths and were subsequently cleared by microglia. Myelin fragmentation increased with age and led to the formation of insoluble, lipofuscin-like lysosomal inclusions in microglia. Thus, age-related myelin fragmentation is substantial, leading to lysosomal storage and contributing to microglial senescence and immune dysfunction in aging.
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http://dx.doi.org/10.1038/nn.4325DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7116794PMC
August 2016

Attacking the supply wagons to starve cancer cells to death.

FEBS Lett 2016 Apr 22;590(7):885-907. Epub 2016 Mar 22.

Department of Developmental and Cell Biology, University of California Irvine, CA, USA.

The constitutive anabolism of cancer cells not only supports proliferation but also addicts tumor cells to a steady influx of exogenous nutrients. Limiting access to metabolic substrates could be an effective and selective means to block cancer growth. In this review, we define the pathways by which cancer cells acquire the raw materials for anabolism, highlight the actionable proteins in each pathway, and discuss the status of therapeutic interventions that disrupt nutrient acquisition. Critical open questions to be answered before apical metabolic inhibitors can be successfully and safely deployed in the clinic are also outlined. In summary, recent studies provide strong support that substrate limitation is a powerful therapeutic strategy to effectively, and safely, starve cancer cells to death.
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http://dx.doi.org/10.1002/1873-3468.12121DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4833639PMC
April 2016

Azacyclic FTY720 Analogues That Limit Nutrient Transporter Expression but Lack S1P Receptor Activity and Negative Chronotropic Effects Offer a Novel and Effective Strategy to Kill Cancer Cells in Vivo.

ACS Chem Biol 2016 Feb 14;11(2):409-14. Epub 2015 Dec 14.

Department of Chemistry, Université de Montréal , P.O. Box 6128, Station Centre-Ville, Montréal, Quebec H3C 3J7, Canada.

FTY720 sequesters lymphocytes in secondary lymphoid organs through effects on sphingosine-1-phosphate (S1P) receptors. However, at higher doses than are required for immunosuppression, FTY720 also functions as an anticancer agent in multiple animal models. Our published work indicates that the anticancer effects of FTY720 do not depend on actions at S1P receptors but instead stem from FTY720s ability to restrict access to extracellular nutrients by down-regulating nutrient transporter proteins. This result was significant because S1P receptor activation is responsible for FTY720s dose-limiting toxicity, bradycardia, that prevents its use in cancer patients. Here, we describe diastereomeric and enantiomeric 3- and 4-C-aryl 2-hydroxymethyl pyrrolidines that are more active than the previously known analogues. Of importance is that these compounds fail to activate S1P1 or S1P3 receptors in vivo but retain inhibitory effects on nutrient transporter proteins and anticancer activity in solid tumor xenograft models. Our studies reaffirm that the anticancer activity of FTY720 does not depend upon S1P receptor activation and uphold the promise of using S1P receptor-inactive azacyclic FTY720 analogues in human cancer patients.
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http://dx.doi.org/10.1021/acschembio.5b00761DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4862402PMC
February 2016

Targeting cancer metabolism at the plasma membrane by limiting amino acid access through SLC6A14.

Biochem J 2015 Sep;470(3):e17-9

Department of Developmental and Cell Biology, University of California Irvine, Irvine, CA 92697-2300, U.S.A.

Rapidly proliferating cancer cells increase flux through anabolic pathways to build the mass necessary to support cell division. Imported amino acids and glucose lie at the apex of the anabolic pyramid. Consistent with this, elevated expression of nutrient transporter proteins is characteristic of aggressive and highly malignant cancers. Because tumour cells are more dependent than their normal neighbours on accelerated nutrient import, these up-regulated transporters could be excellent targets for selective anti-cancer therapies. A study by Babu et al. in a recent issue of the Biochemical Journal definitively shows that SLC6A14 (where SLC is solute carrier) is one such cancer-specific amino acid transporter. Although mice completely lacking SLC6A14 are viable and exhibit normal mammary gland development, these animals are highly resistant to mammary tumour initiation and progression driven by potent oncogenes. Because SLC6A14 is essential for tumour growth yet dispensable for normal development and tissue maintenance, small molecules that block amino acid import through this transporter could be effective and selective anti-cancer agents, particularly as components of rational drug combinations.
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http://dx.doi.org/10.1042/BJ20150721DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4613721PMC
September 2015

Nitric Oxide Synthase as a Target for Methicillin-Resistant Staphylococcus aureus.

Chem Biol 2015 Jun;22(6):785-92

Departments of Molecular Biology and Biochemistry, Pharmaceutical Sciences, and Chemistry, University of California, Irvine, CA 92697-3900, USA. Electronic address:

Bacterial infections associated with methicillin-resistant Staphylococcus aureus (MRSA) are a major economic burden to hospitals, and confer high rates of morbidity and mortality among those infected. Exploitation of novel therapeutic targets is thus necessary to combat this dangerous pathogen. Here, we report on the identification and characterization, including crystal structures, of two nitric oxide synthase (NOS) inhibitors that function as antimicrobials against MRSA. These data provide the first evidence that bacterial NOS (bNOS) inhibitors can work synergistically with oxidative stress to enhance MRSA killing. Crystal structures show that each inhibitor contacts an active site Ile residue in bNOS that is Val in the mammalian NOS isoforms. Mutagenesis studies show that the additional nonpolar contacts provided by the Ile in bNOS contribute to tighter binding toward the bacterial enzyme.
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http://dx.doi.org/10.1016/j.chembiol.2015.05.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4475277PMC
June 2015

B cell Rab7 mediates induction of activation-induced cytidine deaminase expression and class-switching in T-dependent and T-independent antibody responses.

J Immunol 2015 Apr 4;194(7):3065-78. Epub 2015 Mar 4.

Department of Microbiology and Immunology, School of Medicine, University of Texas Health Science Center, San Antonio, TX 78229;

Class switch DNA recombination (CSR) is central to the maturation of the Ab response because it diversifies Ab effector functions. Like somatic hypermutation, CSR requires activation-induced cytidine deaminase (AID), whose expression is restricted to B cells, as induced by CD40 engagement or dual TLR-BCR engagement (primary CSR-inducing stimuli). By constructing conditional knockout Igh(+/C)γ(1-cre)Rab7(fl/fl) mice, we identified a B cell-intrinsic role for Rab7, a small GTPase involved in intracellular membrane functions, in mediating AID induction and CSR. Igh(+/C)γ(1-cre)Rab7(fl/fl) mice displayed normal B and T cell development and were deficient in Rab7 only in B cells undergoing Igh(C)γ(1-cre) Iγ1-Sγ1-Cγ1-cre transcription, as induced--like Igh germline Iγ1-Sγ1-Cγ1 and Iε-Sε-Cε transcription--by IL-4 in conjunction with a primary CSR-inducing stimulus. These mice could not mount T-independent or T-dependent class-switched IgG1 or IgE responses while maintaining normal IgM levels. Igh(+/C)γ(1-cre)Rab7(fl/fl) B cells showed, in vivo and in vitro, normal proliferation and survival, normal Blimp-1 expression and plasma cell differentiation, as well as intact activation of the noncanonical NF-κB, p38 kinase, and ERK1/2 kinase pathways. They, however, were defective in AID expression and CSR in vivo and in vitro, as induced by CD40 engagement or dual TLR1/2-, TLR4-, TLR7-, or TLR9-BCR engagement. In Igh(+/C)γ(1-cre)Rab7(fl/fl) B cells, CSR was rescued by enforced AID expression. These findings, together with our demonstration that Rab7-mediated canonical NF-κB activation, as critical to AID induction, outline a novel role of Rab7 in signaling pathways that lead to AID expression and CSR, likely by promoting assembly of signaling complexes along intracellular membranes.
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http://dx.doi.org/10.4049/jimmunol.1401896DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4643723PMC
April 2015

Design, Synthesis, and Anti-leukemic Activity of Stereochemically Defined Constrained Analogs of FTY720 (Gilenya).

ACS Med Chem Lett 2013 Oct;4(10)

Department of Chemistry, Université de Montréal, PO Box 6128, Station Centre-Ville, Montréal, Que., H3C 3J7, Canada.

FTY720 functions as an immunosuppressant due to its effect on sphingosine-1-phosphate receptors. At doses well above those needed for immunosuppression, FTY720 also has anti-neoplastic actions. Our published work suggests that at least some of FTY720's anti-cancer activity is independent of its effects on S1P receptors and due instead to its ability to induce nutrient transporter down-regulation. Compounds that trigger nutrient transporter loss but lack FTY720's S1P receptor-related, dose-limiting toxicity have the potential to be effective and selective anti-tumor agents. In this study, a series of enantiomerically pure and stereochemically diverse O-substituted benzyl ethers of pyrrolidines was generated and tested for the ability to kill human leukemia cells. The stereochemistry of the hydroxymethyl was found to be a key determinant of compound activity. Moreover, phosphorylation of this group was not required for anti-leukemic activity.
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http://dx.doi.org/10.1021/ml4002425DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3834958PMC
October 2013

Reciprocal effects of rab7 deletion in activated and neglected T cells.

Autophagy 2013 Jul 15;9(7):1009-23. Epub 2013 Apr 15.

Department of Developmental and Cell Biology; University of California, Irvine; Irvine, CA USA.

Mouse models lacking proteins essential for autophagosome formation have demonstrated that autophagy plays a critical role in T cell development and activation. To better understand the function of autophagy in quiescent and activated lymphocytes, we have generated a mouse deficient in rab7 selectively in T cells and compared the effects of blocking autophagy at an early (atg5(-/-)) or late (rab7(-/-)) stage on T cell biology. rab7(-/-) murine embryonic fibroblasts (MEFs) and T cells generated from these mice exhibit a profound block in autophagosome degradation and are as sensitive as atg5(-/-) cells to extracellular nutrient limitation. Rab7(flox/flox)CD4-Cre(+) mice lacking the RAB7 protein in both CD4 and CD8 T cells had reduced numbers of peripheral T cells, but this defect was not as severe as in Atg5(flox/flox)CD4-Cre(+) mice despite efficient rab7 deletion and inhibition of autophagic flux. This difference may stem from the reduced ROS generation and enhanced survival of rab7(-/-) T cells compared with wild-type and atg5(-/-) T cells in the absence of cytokine stimulation. rab7(-/-) and atg5(-/-) T cells exhibited similar defects in proliferation both following antibody-mediated T cell receptor (TCR) cross-linking and using a more physiologic activation protocol, allogeneic stimulation. Interestingly, autophagy was not required to provide building blocks for the upregulation of nutrient transporter proteins immediately following activation. Together, these studies suggest that autophagosome degradation is required for the survival of activated T cells, but that loss of rab7 is better tolerated in naïve T cells than the loss of atg5.
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http://dx.doi.org/10.4161/auto.24468DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3722312PMC
July 2013

Nutrient transporters: the Achilles' heel of anabolism.

Trends Endocrinol Metab 2013 Apr 8;24(4):200-8. Epub 2013 Feb 8.

Department of Developmental and Cell Biology, University of California, Irvine, CA 92697, USA.

Highly proliferative cells, including cancer cells, require a constant supply of molecular building blocks to support their growth. To acquire substrates such as glucose and amino acids from the extracellular space, dividing cells rely on transporter proteins in the plasma membrane. Numerous studies link transcriptional and post-translational control of nutrient transporter expression with proliferation, highlighting the importance of nutrient transporters in both physiologic and pathologic growth. Here we review recent work that spotlights the crucial role of nutrient transporters in cell growth and proliferation, discuss post-translational mechanisms for coordinating expression of different transporters, and consider the therapeutic potential of targeting these proteins in cancer and other diseases characterized by inappropriate cell division.
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http://dx.doi.org/10.1016/j.tem.2013.01.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3617053PMC
April 2013

Guidelines for the use and interpretation of assays for monitoring autophagy.

Autophagy 2012 Apr;8(4):445-544

Life Sciences Institute, University of Michigan, Ann Arbor, MI, USA.

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3404883PMC
http://dx.doi.org/10.4161/auto.19496DOI Listing
April 2012

Flow injection tandem mass spectrometric measurement of ceramides of multiple chain lengths in biological samples.

J Chromatogr B Analyt Technol Biomed Life Sci 2012 Feb 18;883-884:136-40. Epub 2011 Nov 18.

Department of Pathology and Laboratory Medicine, Children's Hospital of Philadelphia, Philadelphia, PA 19104, United States.

A method is presented for the measurement of ceramide species in biological fluids using flow injection tandem mass spectrometry. Ceramides are important signaling compounds in a number of cell:cell interactions including apoptosis and neurodegeneration. Because of the large number of potential fatty acid constituent moieties on ceramide molecules, a method which accurately distinguishes different chain-length species was required. The present method does not require HPLC separation and is designed to be applicable to high throughput analysis required for clinical studies. We provide a reference range for all measurable ceramide species in normal human plasma and an example of the utility of the assay in providing biomarkers in an in vitro apoptotic cell death study using murine hematopoietic cells treated with daunorubicin.
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http://dx.doi.org/10.1016/j.jchromb.2011.11.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3491661PMC
February 2012
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