Lysosomal Inhibitor Research Articles

Acute exercise dynamically modulates the hepatic mitochondrial proteome.

Exercise powerfully increases energy metabolism and substrate flux in tissues, a process reliant on dramatic changes in mitochondrial energetics. Liver mitochondria play a multi-factorial role during exercise to fuel hepatic glucose output. We previously showed acute exercise activates hepatic mitophagy, a pathway to recycle low-functioning/damaged mitochondria, however little is known how individual bouts of exercise alters the hepatic mitochondrial proteome. Here we leveraged proteomics to examine changes in isolated hepatic mitochondria both immediately after and 2 hours post an acute, 1 hour bout of treadmill exercise in female mice. Further, we utilized leupeptin, a lysosomal inhibitor, to capture and measure exercise-induced changes in mitochondrial proteins that would have been unmeasured due to their targeting for lysosomal degradation. Proteomic analysis of enriched hepatic mitochondria identified 3241 total proteins. Functional enrichment analysis revealed robust enrichment for proteins critical to the mitochondria including metabolic pathways, tricarboxylic acid cycle, and electron transport system. Compared to the sedentary condition, exercise elevated processes regulating lipid localization, Il-5 signaling, and protein phosphorylation in isolated mitochondria. t-SNE analysis identified 4 unique expressional clusters driven by time-dependent changes in protein expression. Isolation of proteins significantly altered with exercise from each cluster revealed influences of leupeptin and exercise both independently and cooperatively modulating mitochondrial protein expressional profiles. Overall, we provide evidence that acute exercise rapidly modulates changes in the proteins/pathways of isolated hepatic mitochondria that include fatty acid metabolism/storage, post-translational protein modification, inflammation, and oxidative stress. In conclusion, the hepatic mitochondrial proteome undergoes extensive remodeling with a bout of exercise.

GIF-2209, an Oxindole Derivative, Accelerates Melanogenesis and Melanosome Secretion via the Modification of Lysosomes in B16F10 Mouse Melanoma Cells.

Melanogenesis and melanosome secretion are regulated by several mechanisms. In this study, we found that the oxindole derivative GIF-2209 accelerated melanogenesis associated with the discrimination in the expression and intracellular distributions of two melanogenic enzymes, tyrosinase (TYR) and tyrosinase-related protein-1 (TYRP-1). GIF-2209 upregulated the expression of TYR via a microphthalmia transcription factor (MITF)-independent mechanism, leading to high expression of protein. In contrast, GIF-2209 did not alter the mRNA levels of TYRP-1 and suppressed its protein levels. GIF-2209 induced the dissociation of TYR from TYRP-1 but did not alter the association between TYR and CD63, a melanosome and lysosome marker. The protein levels of CD63 were also upregulated by GIF-2209. GIF-2209 induced lysosome expansion and redistribution in all areas of the cytosol, accompanied by autophagy acceleration (upregulation of LC3BII protein levels and downregulation of p62 protein levels). In addition, GIF-2209 stimulated the secretion of melanosomes containing high levels of TYR, TYRP-1, and CD63 proteins. The GIF-2209 mediated melanosome secretion was sensitive to the lysosome inhibitor chloroquine. These results suggest that GIF-2209 may activate lysosomal functions with TYR gene expression, while it accelerates melanosome secretion, which finally leads to the depletion of intracellular melanogenic enzyme, especially TYRP-1 protein.

Emerging Lysosomal Functions for Photoreceptor Cell Homeostasis and Survival.

Lysosomes are membrane-bound cell organelles that respond to nutrient changes and are implicated in cell homeostasis and clearance mechanisms, allowing effective adaptation to specific cellular needs. The relevance of the lysosome has been elucidated in a number of different contexts. Of these, the retina represents an interesting scenario to appreciate the various functions of this organelle in both physiological and pathological conditions. Growing evidence suggests a role for lysosome-related mechanisms in retinal degeneration. Abnormal lysosomal activation or inhibition has dramatic consequences on photoreceptor cell homeostasis and impacts extensive cellular function, which in turn affects vision. Based on these findings, a series of therapeutic methods targeting lysosomal processes could offer treatment for blindness conditions. Here, we review the recent findings on membrane trafficking, subcellular organization, mechanisms by which lysosome/autophagy pathway impairment affects photoreceptor cell homeostasis and the recent advances on developing efficient lysosomal-based therapies for retinal disorders.

Misrouting of glucagon and stathmin-2 towards lysosomal system of α-cells in glucagon hypersecretion of diabetes

ABSTRACT Glucagon hypersecretion from the pancreatic α-cell is a characteristic sign of diabetes, which exacerbates fasting hyperglycemia. Thus, targeting glucagon secretion from α-cells may be a promising approach for combating hyperglucagonemia. We have recently identified stathmin-2 as an α-cell protein that regulates glucagon secretion by directing glucagon toward the endolysosomal system in αTC1-6 cells. We hypothesized that disruption of Stmn2-mediated trafficking of glucagon to the endolysosomes in diabetes contributes to hyperglucagonemia. In isolated islets from male mice treated with streptozotocin (STZ), glucagon secretion and cellular content were augmented, but cellular Stmn2 levels were reduced (p < .01), as measured by both ELISA and immunofluorescence intensity. Using confocal immunofluorescence microscopy, the colocalization of glucagon and Stmn2 in Lamp2A+ lysosomes was dramatically reduced (p < .001) in islets from diabetic mice, and the colocalization of Stmn2, but not glucagon, with the late endosome marker, Rab7, significantly (p < .01) increased. Further studies were conducted in αTC1-6 cells cultured in media containing high glucose (16.7 mM) for 2 weeks to mimic glucagon hypersecretion of diabetes. Surprisingly, treatment of αTC1-6 cells with the lysosomal inhibitor bafilomycin A1 reduced K+-induced glucagon secretion, suggesting that high glucose may induce glucagon secretion from another lysosomal compartment. Both glucagon and Stmn2 co-localized with Lamp1, which marks secretory lysosomes, in cells cultured in high glucose. We propose that, in addition to enhanced trafficking and secretion through the regulated secretory pathway, the hyperglucagonemia of diabetes may also be due to re-routing of glucagon from the degradative Lamp2A+ lysosome toward the secretory Lamp1+ lysosome.

Chronobiological activity of cysteinyl leukotriene receptor 1 during basal and induced autophagy in the ARPE-19 retinal pigment epithelial cell line.

Autophagy is an important cellular mechanism for maintaining cellular homeostasis, and its impairment correlates highly with age and age-related diseases. Retinal pigment epithelial (RPE) cells of the eye represent a crucial model for studying autophagy, as RPE functions and integrity are highly dependent on an efficient autophagic process. Cysteinyl leukotriene receptor 1 (CysLTR1) acts in immunoregulation and cellular stress responses and is a potential regulator of basal and adaptive autophagy. As basal autophagy is a dynamic process, the aim of this study was to define the role of CysLTR1 in autophagy regulation in a chronobiologic context using the ARPE-19 human RPE cell line.Effects of CysLTR1 inhibition on basal autophagic activity were analyzed at inactive/low and high lysosomal degradation activity with the antagonists zafirlukast (ZTK) and montelukast (MTK) at a dosage of 100 nM for 3 hours. Abundances of the autophagy markers LC3-II and SQSTM1 and LC3B particles were analyzed in the absence and presence of lysosomal inhibitors using western blot analysis and immunofluorescence microscopy.CysLTR1 antagonization revealed a biphasic effect of CysLTR1 on autophagosome formation and lysosomal degradation that depended on the autophagic activity of cells at treatment initiation. ZTK and MTK affected lysosomal degradation, but only ZTK regulated autophagosome formation. In addition, dexamethasone treatment and serum shock induced autophagy, which was repressed by CysLTR1 antagonization. As a newly identified autophagy modulator, CysLTR1 appears to be a key player in the chronobiological regulation of basal autophagy and adaptive autophagy in RPE cells.

Concurrent Inhibition of IGF1R and ERK Increases Pancreatic Cancer Sensitivity to Autophagy Inhibitors.

Compensatory upregulation of IGF1R and ERK-MAPK signaling limits the efficacy of autophagy inhibitors chloroquine and hydroxychloroquine, and their concurrent inhibition synergistically increases autophagy dependence and chloroquine sensitivity in pancreatic ductal adenocarcinoma.

Hyperphosphatemia-induced degradation of transcription factor EB exacerbates vascular calcification

AimsChronic kidney disease (CKD) and subsequent hyperphosphatemia causes vascular calcification (VC), a strong predictor of mortality. Dysregulation of the autophagy-lysosomal pathway in vascular smooth muscle cells (VSMCs) mediates hyperphosphatemia-dependent VC. However, the process through which lysosomes become dysfunctional remains unknown. Transcription factor EB (TFEB) is a master regulator of lysosome biogenesis. The present study examined the hypothesis that TFEB dysfunction causes VC progression. Methods and resultsInorganic phosphate (Pi) dose-dependently promoted VC in mouse aorta ex vivo, in rat VSMCs in vitro, and in human aortic smooth muscle cells in vitro, all accompanied by a decrease in TFEB protein. Lysosomal inhibitors or TFEB knockdown using small interfering RNA exacerbated Pi-induced VC in VSMCs. Conversely, TFEB downregulation was not observed in the hypercalcemia-sensitive VC model induced by excessive vitamin D dosages. Feeding rats an adenine-containing diet caused CKD and hyperphosphatemia. VC occurred in the adenine-fed rat aorta and regressed after adenine cessation. In this CKD model, aortic TFEB expression decreased at VC onset but recovered to average levels during recovery from VC after adenine cessation. The calcified area of the CKD rat aorta exhibited lysosomal damage and enhanced TFEB ubiquitination. Hyperphosphatemia in vitro increased insoluble TFEB and decreased soluble TFEB in VSMCs, both of which were abrogated by the proteasome inhibitor, MG-132. ConclusionHyperphosphatemia caused VC via TFEB downregulation in VSMCs. Under hyperphosphatemia, TFEB was insolubilized and degraded via the ubiquitin–proteasome system. Our results suggest a new mechanism for the pathogenesis of VC under CKD and hyperphosphatemia.

A unique NLRC4 receptor from echinoderms mediates Vibrio phagocytosis via rearrangement of the cytoskeleton and polymerization of F-actin.

Many members of the nucleotide-binding and oligomerization domain (NACHT)- and leucine-rich-repeat-containing protein (NLR) family play crucial roles in pathogen recognition and innate immune response regulation. In our previous work, a unique and Vibrio splendidus-inducible NLRC4 receptor comprising Ig and NACHT domains was identified from the sea cucumber Apostichopus japonicus, and this receptor lacked the CARD and LRR domains that are typical of common cytoplasmic NLRs. To better understand the functional role of AjNLRC4, we confirmed that AjNLRC4 was a bona fide membrane PRR with two transmembrane structures. AjNLRC4 was able to directly bind microbes and polysaccharides via its extracellular Ig domain and agglutinate a variety of microbes in a Ca2+-dependent manner. Knockdown of AjNLRC4 by RNA interference and blockade of AjNLRC4 by antibodies in coelomocytes both could significantly inhibit the phagocytic activity and elimination of V. splendidus. Conversely, overexpression of AjNLRC4 enhanced the phagocytic activity of V. splendidus, and this effect could be specifically blocked by treatment with the actin-mediated endocytosis inhibitor cytochalasin D but not other endocytosis inhibitors. Moreover, AjNLRC4-mediated phagocytic activity was dependent on the interaction between the intracellular domain of AjNLRC4 and the β-actin protein and further regulated the Arp2/3 complex to mediate the rearrangement of the cytoskeleton and the polymerization of F-actin. V. splendidus was found to be colocalized with lysosomes in coelomocytes, and the bacterial quantities were increased after injection of chloroquine, a lysosome inhibitor. Collectively, these results suggested that AjNLRC4 served as a novel membrane PRR in mediating coelomocyte phagocytosis and further clearing intracellular Vibrio through the AjNLRC4-β-actin-Arp2/3 complex-lysosome pathway.

CDK9 inhibition blocks the initiation of PINK1-PRKN-mediated mitophagy by regulating the SIRT1-FOXO3-BNIP3 axis and enhances the therapeutic effects involving mitochondrial dysfunction in hepatocellular carcinoma

ABSTRACT Mitophagy is a type of selective macroautophagy/autophagy that degrades dysfunctional or excessive mitochondria. Regulation of this process is critical for maintaining cellular homeostasis and has been closely implicated in acquired drug resistance. However, the regulatory mechanisms and influences of mitophagy in cancer are still unclear. Here, we reported that inhibition of CDK9 blocked PINK1-PRKN-mediated mitophagy in HCC (hepatocellular carcinoma) by interrupting mitophagy initiation. We demonstrated that CDK9 inhibitors promoted dephosphorylation of SIRT1 and promoted FOXO3 protein degradation, which was regulated by its acetylation, leading to the transcriptional repression of FOXO3-driven BNIP3 and impairing the BNIP3-mediated stability of the PINK1 protein. Lysosomal degradation inhibitors could not rescue mitophagy flux blocked by CDK9 inhibitors. Thus, CDK9 inhibitors inactivated the SIRT1-FOXO3-BNIP3 axis and PINK1-PRKN pathway to subsequently block mitophagy initiation. Moreover, CDK9 inhibitors facilitated mitochondrial dysfunction. The dual effects of CDK9 inhibitors resulted in the destruction of mitochondrial homeostasis and cell death in HCC. Importantly, a novel CDK9 inhibitor, oroxylin A (OA), from Scutellaria baicalensis was investigated, and it showed strong therapeutic potential against HCC and a striking capacity to overcome drug resistance by downregulating PINK1-PRKN-mediated mitophagy. Additionally, because of the moderate and controlled inhibition of CDK9, OA not led to extreme repression of general transcription and appeared to overcome the inconsistent anti-HCC efficacy and high normal tissue toxicity that was associated with existing CDK9 inhibitors. All of the findings reveal that mitophagy disruption is a promising strategy for HCC treatment and OA is a potential candidate for the development of mitophagy inhibitors. Abbreviations: BNIP3: BCL2 interacting protein 3; CCCP: carbonyl cyanide p-trichloromethoxy-phenylhydrazone; CDK9: cyclin dependent kinase 9; CHX: cycloheximide; CQ, chloroquine; DFP: deferiprone; DOX: doxorubicin; EBSS: Earle’s balanced salt solution; E64d: aloxistatin; FOXO3: forkhead box O3; HCC: hepatocellular carcinoma; HepG2/ADR: adriamycin-resistant HepG2 cells; MMP: mitochondrial membrane potential; mito-Keima: mitochondria-targeted and pH-sensitive fluorescent protein; MitoSOX: mitochondrial reactive oxygen species; OA: oroxylin A; PB: phosphate buffer; PDX: patient-derived tumor xenograft; PINK1: PTEN induced kinase 1; POLR2A: RNA polymerase II subunit A; p-POLR2A-S2: Ser2 phosphorylation of RNA polymerase II subunit A; PRKN: parkin RBR E3 ubiquitin protein ligase; SIRT1: sirtuin 1.

Docetaxel-triggered SIDT2/NOX4/JNK/HuR signaling axis is associated with TNF-α-mediated apoptosis of cancer cells

Previous studies have confirmed that docetaxel (DTX) treatment increases TNF-α production in cancer cells, but its mechanism of action remains unclear. Therefore, this study aimed to determine the signaling axis by which DTX induced the expression of TNF-α in U937 leukemia and MCF-7 breast carcinoma cells. DTX treatment promoted Ca2+-controlled autophagy and SIDT2 expression, resulting in lysosomal degradation of miR-25 in U937 cells. Downregulation of miR-25 increased NOX4 mRNA stability and protein expression. NOX4-stimulated ROS generation led to JNK-mediated phosphorylation of cytosolic HuR at Ser221, thereby increasing TNF-α protein expression by stabilizing TNF-α mRNA. Consequently, DTX induced TNF-α-dependent death in U937 cells. Depletion of HuR using siRNA or abolishment of JNK activation reduced TNF-α expression and eliminated DTX-mediated cytotoxicity. Knockdown of SIDT2 or pretreatment with chloroquine (a lysosome inhibitor) reduced DTX-induced NOX4 and TNF-α expression and mitigated JNK-mediated HuR phosphorylation. Altogether, our data indicate that DTX triggers HuR-mediated TNF-α mRNA stabilization through the Ca2+/SIDT2/NOX4/ROS/JNK axis, thereby inducing TNF-α-dependent apoptosis in U937 cells. In addition, DTX induces apoptosis in MCF-7 cells through SIDT2/NOX4/JNK/HuR axis-mediated TNF-α expression.

Quantitative super-resolution microscopy reveals promoting mitochondrial interconnectivity protects against AKI.

The root of many kidney diseases in humans can be traced to alterations or damage to subcellular organelles. Mitochondrial fragmentation, endoplasmic reticulum (ER) stress, and lysosomal inhibition, among others, ultimately contribute to kidney injury and are the target of therapeutics in development. Although recent technological advancements allow for the understanding of disease states at the cellular level, investigating changes in subcellular organelles from kidney tissue remains challenging. Using structured illumination microscopy, we imaged mitochondria and other organelles from paraffin sections of mouse tissue and human kidney biopsy specimens. The resulting images were 3D rendered to quantify mitochondrial size, content, and morphology. Results were compared with those from transmission electron microscopy and segmentation. Super-resolution imaging reveals kidney tubular epithelial cell mitochondria in rodent and human kidney tissue form large, interconnected networks under basal conditions, which are fragmented with injury. This approach can be expanded to other organelles and cellular structures including autophagosomes, ER, brush border, and cell morphology. We find that, during unilateral ischemia, mitochondrial fragmentation occurs in most tubule cells, and they remain fragmented for >96 hours. Promoting mitochondrial fusion with the fusion promotor M1 preserves mitochondrial morphology and interconnectivity and protects against cisplatin-induced kidney injury. We provide, for the first time, a nonbiased, semiautomated approach for quantification of the 3D morphology of mitochondria in kidney tissue. Maintaining mitochondrial interconnectivity and morphology protects against kidney injury. Super-resolution imaging has the potential to both drive discovery of novel pathobiologic mechanisms in kidney tissue and broaden the diagnoses that can be made on human biopsy specimens.

Abstract P175: Investigating inositol polyphosphate-4-phosphatase, type II (INPP4B) signaling and role in acute myeloid leukemia

Abstract Acute myeloid leukemia (AML) is an aggressive clonal stem cell disorder characterized by invasion of bone marrow, impaired hematopoiesis and accumulation of functionally immature myeloblasts. Our previous studies demonstrated that the expression level of the lipid phosphatase, INPP4B, is associated with poor outcomes in AML. To gain an understanding of the role of Inpp4b in AML progression, we performed RNA sequencing on Inpp4b+/+ and Inpp4b−/− MLL-AF9 leukemias. Analysis of RNA sequencing data revealed that a disproportionately large number of lysosomal gene transcripts are decreased in Inpp4b−/− leukemias; these include cathepsins and lysosomal specific proteases responsible for proteolysis. This discovery provided evidence supporting a potential role for INPP4B in the lysosomal pathway. To validate these findings, immunofluorescence staining of the lysosomal protein LAMP1 was performed, which revealed a greater number of lysosomes in U2OS cells overexpressing INPP4B, and a decrease in lysosome number upon siRNA knockdown of INPP4B. Subsequently the DQ-BSA staining was used to assess INPP4B’s effect on lysosomal function. In both U2OS and the leukemia cells, we observed that INPP4B expression is positively associated with the proteolytic activity of lysosomes. Currently we are testing the role of INPP4B expression on sensitivity to lysosomal inhibitors; Lys05, Chloroquine, and Mefloquine. Overall, we aim to uncover how INPP4B expression in AML controls lysosomal mechanisms and biology and to test putative therapeutic strategies to exploit this pathway. Citation Format: Keyue Chen, Gizem E. Genc, John F. Woolley, Daniel K.C. Lee, Roberto J. Botelho, Leonardo Salmena. Investigating inositol polyphosphate-4-phosphatase, type II (INPP4B) signaling and role in acute myeloid leukemia [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P175.

Neuregulin 4 Downregulation Induces Insulin Resistance in 3T3-L1 Adipocytes through Inflammation and Autophagic Degradation of GLUT4 Vesicles

The adipokine Neuregulin 4 (Nrg4) protects against obesity-induced insulin resistance. Here, we analyze how the downregulation of Nrg4 influences insulin action and the underlying mechanisms in adipocytes. Validated shRNA lentiviral vectors were used to generate scramble (Scr) and Nrg4 knockdown (KD) 3T3-L1 adipocytes. Adipogenesis was unaffected in Nrg4 KD adipocytes, but there was a complete impairment of the insulin-induced 2-deoxyglucose uptake, which was likely the result of reduced insulin receptor and Glut4 protein. Downregulation of Nrg4 enhanced the expression of proinflammatory cytokines. Anti-inflammatory agents recovered the insulin receptor, but not Glut4, content. Proteins enriched in Glut4 storage vesicles such as the insulin-responsive aminopeptidase (IRAP) and Syntaxin-6 as well as TBC1D4, a protein involved in the intracellular retention of Glut4 vesicles, also decreased by Nrg4 KD. Insulin failed to reduce autophagy in Nrg4 KD adipocytes, observed by a minor effect on mTOR phosphorylation, at the time that proteins involved in autophagy such as LC3-II, Rab11, and Clathrin were markedly upregulated. The lysosomal activity inhibitor bafilomycin A1 restored Glut4, IRAP, Syntaxin-6, and TBC1D4 content to those found in control adipocytes. Our study reveals that Nrg4 preserves the insulin responsiveness by preventing inflammation and, in turn, benefits the insulin regulation of autophagy.

Regulatory mechanism of interferon regulatory factor 1 by α-synuclein in mouse Parkinson's disease model

To investigate the molecular mechanism by which α-synuclein (α-Syn) regulates interferon regulatory factor 1 (IRF-1) expression. SH-SY5Y cells overexpressing α-Syn and transgenic mouse model carrying human α-Syn gene with A53T mutation (3 and 6 months old) were examined for IRF-1 mRNA and protein expressions using real-time PCR and Western blotting, respectively. The subcellular localization of IRF-1 was determined with immunofluorescence staining and cytoplasmic/nuclear protein isolation. The optimal concentrations of the proteasome inhibitor MG132 (0.01-2.0 μmol/L) and lysosomal inhibitor chloroquine (5-200 μmol/L) for treatment of SH-SY5Y cells for 24 h were determined by examining the cell viability. SH-SY5Y cells were treated with 0.2 μmol/L MG132 and 30 μmol/L chloroquine for 24 h (the maximum dose that did not cause cell damage), and the changes of IRF-1 protein expressions was analyzed. The effects of α-Syn on MDM2 protein expression and IRF-1 ubiquitylation were analyzed using Western blotting and ubiquitylation assay. α-Syn overexpression did not affect the mRNA level of IRF-1 but significantly increased its protein level (P < 0.01). In α-Synoverexpressing SH-SY5Y cells, IRF-1 translocation was observed from the cytoplasm to the nucleus (P < 0.001). Treatment of the cells with 0.2 μmol/L MG132 significantly aggravated α-Syn-induced increase of IRF-1 protein expression (P < 0.01) while 30 μmol/L chloroquine produced no significant changes in IRF-1 level. α-Syn overexpression caused an obvious decrease of MDM2 protein level and further inhibited the ubiquitylation of IRF-1 (P < 0.01). α-Syn blocks MDM2-mediated ubiquitylation of IRF-1 through ubiquitin proteasome pathway, thereby enhancing IRF-1 protein expression.

Abstract PO-031: Lysosome inhibition overcomes resistance to CDK4/6 inhibition in PDA

Abstract The current FDA-approved chemotherapies for patients with Pancreatic Ductal Adenocarcinoma (PDA) demonstrate substantial toxicities with only a modest survival benefit highlighting the urgent need to better understand the molecular mechanisms driving PDA and how they interact in order to develop more precise and less toxic pathway-targeted therapies. PDA often harbors oncogenic mutations in KRAS and CDKN2A that drive a dysregulated cell cycle through activation of CDK4/6 (Cyclin-Dependent Kinases 4 and 6), which mono-phosphorylate RB1 to govern progression through G0◊G1◊S phases. Currently, there are three FDA-approved CDK4/6 inhibitors, palbo-, abema-, and ribociclib (X-ciclibs), that are predicted to have anti-cancer activity in PDA cells. However, the benefits of X-ciclib treatment in PDA patients are largely underwhelming. We found that inherent resistance to X-ciclib treatment in vitro is due to compensatory activation of protective autophagy, a critical lysosomal degradation process that generates a source of nutrients during periods of cellular stress. We observed that X-ciclib treatment induces autophagic flux in PDA cell lines. This pro-cancer process can be targeted with the lysosomal inhibitors, chloroquine and hydroxychloroquine. The addition of chloroquine sensitizes PDA cells to X-ciclib treatment, resulting in diminished proliferative ability. Additionally, a late-stage PDA patient was treated with the combination of an X-ciclib and hydroxychloroquine and displayed a dramatic decrease in the bloodborne PDA biomarker, Cancer Antigen 19-9. These data may suggest a novel combination treatment strategy for late-stage PDA patients, but the molecular mechanisms underlying are yet to be studied in PDA. Future work will explore the role RB1 plays in CDK4/6 regulated autophagy by investigating how phosphorylation at distinct RB1 sites modulates autophagy-related gene expression and the extent to which mTORC1 deactivation is necessary for CDK4/6 regulated autophagy. Citation Format: Dilru Silva, Conan Kinsey, Martin McMahon. Lysosome inhibition overcomes resistance to CDK4/6 inhibition in PDA [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-031.

How Antimalarials and Antineoplastic Drugs can Interact in Combination Therapies: A Perspective on the Role of PPT1 Enzyme.

Antimalarial drugs from different classes have demonstrated anticancer effects in different types of cancer cells, but their complete mode of action in cancer remains unknown. Recently, several studies reported the important role of palmitoyl-protein thioesterase 1 (PPT1), a lysosomal enzyme, as the molecular target of chloroquine and its derivates in cancer. It was also found that PPT1 is overexpressed in different types of cancer, such as breast, colon, etc. Our group has found a synergistic interaction between antimalarial drugs, such as mefloquine, artesunate and chloroquine and antineoplastic drugs in breast cancer cells, but the mechanism of action was not determined. Here, we describe the importance of autophagy and lysosomal inhibitors in tumorigenesis and hypothesize that other antimalarial agents besides chloroquine could also interact with PPT1 and inhibit the mechanistic target of rapamycin (mTOR) signalling, an important pathway in cancer progression. We believe that PPT1 inhibition results in changes in the lysosomal metabolism that result in less accumulation of antineoplastic drugs in lysosomes, which increases the bioavailability of the antineoplastic agents. Taken together, these mechanisms help to explain the synergism of antimalarial and antineoplastic agents in cancer cells.

G protein pathway suppressor 2 suppresses gastric cancer by destabilizing epidermal growth factor receptor.

G protein pathway suppressor 2 (GPS2) is expressed in most human tissues, including the stomach. However, the biological functions of GPS2 in cancer, as well as the underlying molecular mechanisms, remain poorly understood. Here, we report that GPS2 expression was aberrantly downregulated in gastric cancer (GC) tissues compared with control tissues. Clinicopathologic analysis showed that low GPS2 expression was significantly correlated with pathological grade, lymph node stage, and invasive depth. Kaplan‐Meier analysis indicated that patients with low GPS2 expression showed poorer overall survival rates than those with high GPS2 expression. Moreover, GPS2 overexpression decreased GC cell proliferation, colony formation, tumorigenesis, and invasion. Overexpression of GPS2 reduced the protein expression of epidermal growth factor receptor (EGFR) and inhibited its downstream signaling in GC cells. Interestingly, GPS2 decreased EGFR protein expression, which was reversed by a lysosome inhibitor. Furthermore, GPS2 reduced EGFR protein stability by enhancing the binding of EGFR and an E3 ligase, c‐Cbl, which promoted the ubiquitination of EGFR, ultimately leading to its degradation through the lysosomal pathway. Further analysis indicated that GPS2 activated autophagy and promoted the autophagic flux by destabilizing EGFR. Taken together, these results suggest that low GPS2 expression is associated with GC progression and provide insights into the applicability of the GPS2‐EGFR axis as a potential therapeutic target in GC.

Autophagy-dependent glutaminolysis drives superior IL21 production in HIV-1-specific CD4 T cells

ABSTRACT The maintenance of a strong IL21 production in memory CD4 T cells, especially in HIV-1-specific cells, represents a major correlate of natural immune protection against the virus. However, the molecular mechanisms underlying IL21 production during HIV-1 infection, which is only elevated among the naturally protected elite controllers (EC), are still unknown. We recently found out that lipophagy is a critical immune mediator that control an antiviral metabolic state following CD8A T cell receptor engagement, playing an important role in the natural control of HIV-1 infection. This led us to investigate whether the beneficial role of a strong macroautophagy/autophagy, could also be used to ensure effective IL21 production as well. Herein, we confirm that after both polyclonal and HIV-1-specific activation, memory CD4 T cells (Mem) from EC display enhanced activity of the autophagy-mediated proteolysis compared to ART. Our results indicate that the enhanced autophagy activity in EC was controlled by the energy-sensing PRKAA1 (protein kinase AMP-activated catalytic subunit alpha 1). We further confirmed the critical role of the autophagy-mediated proteolysis in the strong IL21 production in EC by using BECN1 gene silencing as well as protease, PRKAA1, and lysosomal inhibitors. Finally, we established that high autophagy-mediated proteolysis in EC fuels their cellular rates of mitochondrial respiration due to glutaminolysis. Our data confirm the critical role of autophagy in dictating the metabolic input, which is required not only to ensure protective cytotoxic CD8A T cell responses, but also to provide strong IL21 production among antiviral CD4 T cells. Abbreviations: AKG: alpha-ketoglutarate; ART: patients under antiretroviral therapy; ATG7: autophagy related 7; BaF: bafilomycin A1; BECN1: beclin 1; Chloro.: chloroquine; EC: elite controllers; EIF4EBP1: eukaryotic translation initiation factor 4E binding protein 1; FOXO3: forkhead box O3; GLS: glutaminase; GLUD1: glutamate dehydrogenase 1; HIVneg: HIV-1-uninfected control donors; IFNG/IFN-γ: interferon gamma; IL21: interleukin 21; MTOR: mechanistic target of rapamycin kinase; PBMC: peripheral blood mononuclear cells; PRKAA1: protein kinase AMP-activated catalytic subunit alpha 1; SQSTM1: sequestosome 1; TCA: tricarboxylic acid cycle; ULK1: unc-51 like autophagy activating kinase

Human cytomegalovirus UL42 protein inhibits the degradation of glycoprotein B through inhibition of Nedd4 family ubiquitin E3 ligases.

Human cytomegalovirus (HCMV) is a globally ubiquitous pathogen and causes congenital infection as well as opportunistic infection in immunocompromised patients. The HCMV UL42 gene encodes a membrane protein that regulates the function of Nedd4 family ubiquitin E3 ligases through its PPxY motif. As HCMV envelope glycoprotein B (gB) also has a PPxY motif at its C-terminal cytoplasmic domain, we examined whether there was any relationship between UL42 protein and gB. Among the Nedd4 family proteins, Nedd4, Nedd4L, and Itch induced the degradation of gB in transiently expressing cells. The degradation of gB by Nedd4 was inhibited by proteasome inhibitor MG132, lysosome inhibitor chloroquine, and the co-expression of UL42 proteins. Among those Nedd4 family proteins, Itch was relocalized by the co-expression of gB to the perinuclear region of the cytoplasm. A co-immunoprecipitation assay demonstrated an interaction between gB and Itch through its PPxY motif. The 150 kDa gB precursor was aberrantly ubiquitinated, and the total amount of gB was quickly decreased in the absence of UL42. Our results indicate that UL42 prevents the degradation of gB by the inhibition of Nedd4 family proteins.

AMPK S-sulfuration contributes to H2S donors-induced AMPK phosphorylation and autophagy activation in dopaminergic cells

Hydrogen sulfide (H2S) serves as a neuromodulator and regulator of neuroinflammation. It is reported to be therapeutic for Parkinson's disease (PD) animal and cellular models. However, whether it affects α-synuclein accumulation in dopaminergic cells, the key pathological feature in PD, is poorly understood. In this study we reported that exogenous H2S donors NaHS and GYY4137 (GYY) enhanced the autophagy activity, as indicated by the increases of autophagy marker LC3-II expression and LC3 dots formation even during lysosome inhibition in dopaminergic cell lines and HEK293 cells. The enhancement of H2S donors on autophagic flux was mediated by adenosine 5′-monophosphate-activated protein kinase (AMPK)-dependent mammalian target of rapamycin (mTOR) inhibition, as H2S donors activated AMPK but reduced the mTOR activity and H2S donors-induced LC3-II increase was diminished by mTOR activator. Moreover, point mutation of Cys302 into alanine (C302A) in AMPKα2 subunit abolished the AMPK activation and mTOR inhibition, as well as autophagic flux increase elicited by NaHS. Interestingly, NaHS triggered AMPK S-sulfuration, which was not observed in AMPK C302A-transfected cells. Further, NaHS was able to attenuate α-synuclein accumulation in a cellular model induced by dopamine oxidized metabolite 3, 4-dihydroxyphenylacetaldehyde (DOPAL), and this effect was interfered by autophagy inhibitor wortmannin and also eliminated in AMPK Cys302A-transfected cells. In sum, the findings identified a role of Cys302 S-sulfuration in AMPK activation induced by exogenous H2S and demonstrated that H2S donors could enhance the autophagic flux via AMPK-mTOR signaling and thus reduce α-synuclein accumulation in vitro.