A high dose KRP203 induces cytoplasmic vacuoles associated with altered phosphoinositide segregation and endosome expansion.

In animal cells, vacuoles are absent, but can be induced by diseases and drugs. While phosphoinositides are critical for membrane trafficking, their role in the formation of these vacuoles remains unclear. The immunosuppressive KRP203/Mocravimod, which antagonizes sphingosine-1-phosphate receptors, has been identified as having novel multimodal activity against phosphoinositide kinases. However, the impact of this novel KRP203 activity is unknown. Here, we show that KRP203 disrupts the spatial organization of phosphoinositides and induces extensive vacuolization in tumor cells and immortalized fibroblasts. The KRP203-induced vacuoles are primarily from endosomes, and augmented by inhibition of PIKFYVE and VPS34. Conversely, overexpression of PTEN decreased KRP203-induced vacuole formation. Furthermore, V-ATPase inhibition completely blunted KRP203-induced vacuolization, pointing to a critical requirement of the endosomal maturation process. Importantly, nearly a half of KRP203-induced vacuoles are significantly decorated with PI4P, a phosphoinositide typically enriched at the plasma membrane and Golgi. These results suggest a model that noncanonical spatial reorganization of phosphoinositides by KRP203 alters the endosomal maturation process, leading to vacuolization. Taken together, this study reveals a previously unrecognized bioactivity of KRP203 as a vacuole-inducing agent and its unique mechanism of phosphoinositide modulation, providing a new insight of phosphoinositide regulation into vacuolization-associated diseases and their molecular pathologies.

Diversifying faculty recruitment in biomedical sciences.

A wealth of data has consistently demonstrated that a diverse faculty maximizes productivity and innovation in the research enterprise and increases the persistence and success of groups that are underrepresented in STEM. While the diversity of students in graduate programs has steadily increased, faculty diversity, particularly in the biomedical sciences, continues to remain relatively flat. Several issues contribute to this mismatch between the pipeline and the professoriate including biases in search and hiring practices, lack of equity and equal opportunities for individuals from underrepresented backgrounds, and unwelcoming campus climates that lead to marginalization and isolation in academic life. A comprehensive approach that addresses these challenges is necessary for institutions of higher education to achieve their faculty diversity goals and create a climate where individuals from all groups feel welcomed and succeed. This article focuses on the first step in this approach-diversifying faculty recruitment through adopting search practices that generate an applicant pool that matches national availability, ensures equity in evaluation and hiring practices, and promotes inclusion and belonging in the hiring experience. These strategies have been recently used at the University of California, Irvine's School of Biological Sciences and while the long-term impact remains unknown, short-term outcomes in recruitment and hiring have demonstrated significant improvement over previous years.

Rab7a activation promotes degradation of select tight junction proteins at the blood-brain barrier after ischemic stroke.

The stability of tight junctions (TJs) between endothelial cells (ECs) is essential to maintain blood-brain barrier (BBB) function in the healthy brain. Following ischemic stroke, TJ strand dismantlement due to protein degradation leads to BBB dysfunction, yet the mechanisms driving this process are poorly understood. Here, we show that endothelial-specific ablation of Rab7a, a small GTPase that regulates endolysosomal protein degradation, reduces stroke-induced TJ strand disassembly resulting in decreased paracellular BBB permeability and improved neuronal outcomes. Two pro-inflammatory cytokines, TNFα and IL1ß, but not glucose and oxygen deprivation, induce Rab7a activation via Ccz1 in brain ECs in vitro, leading to increased TJ protein degradation and impaired paracellular barrier function. Silencing Rab7a in brain ECs in vitro reduces cytokine-driven endothelial barrier dysfunction by suppressing degradation of a key BBB TJ protein, Claudin-5. Thus, Rab7a activation by inflammatory cytokines promotes degradation of select TJ proteins leading to BBB dysfunction after ischemic stroke.

Disruption of the Rab7-Dependent Final Common Pathway of Endosomal and Autophagic Processing Results in a Severe Podocytopathy.

SIGNIFICANCE STATEMENT: Endocytosis, recycling, and degradation of proteins are essential functions of mammalian cells, especially for terminally differentiated cells with limited regeneration rates and complex morphology, such as podocytes. To improve our understanding on how disturbances of these trafficking pathways are linked to podocyte depletion and slit diaphragm (SD) injury, the authors explored the role of the small GTPase Rab7, which is linked to endosomal, lysosomal, and autophagic pathways, using as model systems mice and Drosophila with podocyte-specific or nephrocyte-specific loss of Rab7, and a human podocyte cell line depleted for Rab7. Their findings point to maturation and fusion events during endolysosomal and autophagic maturation as key processes for podocyte homeostasis and function and identify altered lysosomal pH values as a putative novel mechanism for podocytopathies. BACKGROUND: Endocytosis, recycling, and degradation of proteins are essential functions of mammalian cells, especially for terminally differentiated cells with limited regeneration rates, such as podocytes. How disturbances within these trafficking pathways may act as factors in proteinuric glomerular diseases is poorly understood. METHODS: To explore how disturbances in trafficking pathways may act as factors in proteinuric glomerular diseases, we focused on Rab7, a highly conserved GTPase that controls the homeostasis of late endolysosomal and autophagic processes. We generated mouse and Drosophila in vivo models lacking Rab7 exclusively in podocytes or nephrocytes, and performed histologic and ultrastructural analyses. To further investigate Rab7 function on lysosomal and autophagic structures, we used immortalized human cell lines depleted for Rab7. RESULTS: Depletion of Rab7 in mice, Drosophila , and immortalized human cell lines resulted in an accumulation of diverse vesicular structures resembling multivesicular bodies, autophagosomes, and autoendolysosomes. Mice lacking Rab7 developed a severe and lethal renal phenotype with early-onset proteinuria and global or focal segmental glomerulosclerosis, accompanied by an altered distribution of slit diaphragm proteins. Remarkably, structures resembling multivesicular bodies began forming within 2 weeks after birth, prior to the glomerular injuries. In Drosophila nephrocytes, Rab7 knockdown resulted in the accumulation of vesicles and reduced slit diaphragms. In vitro , Rab7 knockout led to similar enlarged vesicles and altered lysosomal pH values, accompanied by an accumulation of lysosomal marker proteins. CONCLUSIONS: Disruption within the final common pathway of endocytic and autophagic processes may be a novel and insufficiently understood mechanism regulating podocyte health and disease.

Simultaneous inhibition of endocytic recycling and lysosomal fusion sensitizes cells and tissues to oligonucleotide therapeutics.

Inefficient endosomal escape remains the primary barrier to the broad application of oligonucleotide therapeutics. Liver uptake after systemic administration is sufficiently robust that a therapeutic effect can be achieved but targeting extrahepatic tissues remains challenging. Prior attempts to improve oligonucleotide activity using small molecules that increase the leakiness of endosomes have failed due to unacceptable toxicity. Here, we show that the well-tolerated and orally bioavailable synthetic sphingolipid analog, SH-BC-893, increases the activity of antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs) up to 200-fold in vitro without permeabilizing endosomes. SH-BC-893 treatment trapped endocytosed oligonucleotides within extra-lysosomal compartments thought to be more permeable due to frequent membrane fission and fusion events. Simultaneous disruption of ARF6-dependent endocytic recycling and PIKfyve-dependent lysosomal fusion was necessary and sufficient for SH-BC-893 to increase non-lysosomal oligonucleotide levels and enhance their activity. In mice, oral administration of SH-BC-893 increased ASO potency in the liver by 15-fold without toxicity. More importantly, SH-BC-893 enabled target RNA knockdown in the CNS and lungs of mice treated subcutaneously with cholesterol-functionalized duplexed oligonucleotides or unmodified ASOs, respectively. Together, these results establish the feasibility of using a small molecule that disrupts endolysosomal trafficking to improve the activity of oligonucleotides in extrahepatic tissues.

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

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.

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

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 IC50 in these cell lines was observed relative to the lead compound, which lacked the appended heterocycle.

Macropinocytosis confers resistance to therapies targeting cancer anabolism.

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.

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

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.

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

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.

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

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.

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

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.

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

Our recent work demonstrates that inactivating mutations in phosphatase and tensin homolog (PTEN) 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. Abbreviations: ATG5: autophagy related 5; NHE: Na(+)/H(+) exchanger; PAK1: p21-activated kinase 1; PI3K: phosphatidylinositol-4,5-bisphosphate 3-kinase; PIP3: phosphatidylinositol (3,4,5)-trisphosphate; PIP2: phosphatidylinositol (4,5)-bisphosphate; RAC1: Rac family small GTPase 1.

Nutrient scavenging in cancer.

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.

Sphingolipids inhibit endosomal recycling of nutrient transporters by inactivating ARF6.

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.

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

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.Significance: 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. Cancer Discov; 8(7); 866-83. ©2018 AACR.See related commentary by Commisso and Debnath, p. 800This article is highlighted in the In This Issue feature, p. 781.

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

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.

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

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.

Targeting cancer metabolism by simultaneously disrupting parallel nutrient access pathways.

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.