Cellular stresses trigger autophagy to remove damaged macromolecules and organelles. Lysosomes 'host' multiple stress-sensing mechanisms that trigger the coordinated biogenesis of autophagosomes and ...lysosomes. For example, transcription factor (TF)EB, which regulates autophagy and lysosome biogenesis, is activated following the inhibition of mTOR, a lysosome-localized nutrient sensor. Here we show that reactive oxygen species (ROS) activate TFEB via a lysosomal Ca(2+)-dependent mechanism independent of mTOR. Exogenous oxidants or increasing mitochondrial ROS levels directly and specifically activate lysosomal TRPML1 channels, inducing lysosomal Ca(2+) release. This activation triggers calcineurin-dependent TFEB-nuclear translocation, autophagy induction and lysosome biogenesis. When TRPML1 is genetically inactivated or pharmacologically inhibited, clearance of damaged mitochondria and removal of excess ROS are blocked. Furthermore, TRPML1's ROS sensitivity is specifically required for lysosome adaptation to mitochondrial damage. Hence, TRPML1 is a ROS sensor localized on the lysosomal membrane that orchestrates an autophagy-dependent negative-feedback programme to mitigate oxidative stress in the cell.
Targeting exosome biogenesis and release may have potential clinical implications for cancer therapy. Herein, we have optimized a quantitative high throughput screen (qHTS) assay to identify ...compounds that modulate exosome biogenesis and/or release by aggressive prostate cancer (PCa) CD63-GFP-expressing C4-2B cells. A total of 4,580 compounds were screened from the LOPAC library (a collection of 1,280 pharmacologically active compounds) and the NPC library (NCGC collection of 3,300 compounds approved for clinical use). Twenty-two compounds were found to be either potent activators or inhibitors of intracellular GFP signal in the CD63-GFP-expressing C4-2B cells. The activity of lead compounds in modulating the secretion of exosomes was validated by a tunable resistive pulse sensing (TRPS) system (qNano-IZON) and flow cytometry. The mechanism of action of the lead compounds in modulating exosome biogenesis and/or secretion were delineated by immunoblot analysis of protein markers of the endosomal sorting complex required for transport (ESCRT)-dependent and ESCRT-independent pathways. The lead compounds tipifarnib, neticonazole, climbazole, ketoconazole, and triademenol were validated as potent inhibitors and sitafloxacin, forskolin, SB218795, fenoterol, nitrefazole and pentetrazol as activators of exosome biogenesis and/or secretion in PC cells. Our findings implicate the potential utility of drug-repurposing as novel adjunct therapeutic strategies in advanced cancer.
Significance Lysosomes are the cell’s degradation center. To adapt to different environmental conditions, the cell has evolved a set of delicate mechanisms to rapidly change lysosome function, which ...is referred to as lysosomal adaptation. Notably, lysosomal adaptation is required for cell survival under low nutrient conditions. In this study, we identified TRPML1, a lysosomal Ca ²⁺-permeant ion channel, as an essential player required for lysosomal adaptation. The activity of TRPML1 is potently (up to 10-fold) and rapidly increased upon nutrient starvation. Furthermore, pharmacological inhibition or genetic deletion of TRPML1 completely abolished the effects of starvation on boosting the degradation capability of lysosomes.
Upon nutrient starvation, autophagy digests unwanted cellular components to generate catabolites that are required for housekeeping biosynthesis processes. A complete execution of autophagy demands an enhancement in lysosome function and biogenesis to match the increase in autophagosome formation. Here, we report that mucolipin-1 (also known as TRPML1 or ML1), a Ca ²⁺ channel in the lysosome that regulates many aspects of lysosomal trafficking, plays a central role in this quality-control process. By using Ca ²⁺ imaging and whole-lysosome patch clamping, lysosomal Ca ²⁺ release and ML1 currents were detected within hours of nutrient starvation and were potently up-regulated. In contrast, lysosomal Na ⁺-selective currents were not up-regulated. Inhibition of mammalian target of rapamycin (mTOR) or activation of transcription factor EB (TFEB) mimicked a starvation effect in fed cells. The starvation effect also included an increase in lysosomal proteostasis and enhanced clearance of lysosomal storage, including cholesterol accumulation in Niemann–Pick disease type C (NPC) cells. However, this effect was not observed when ML1 was pharmacologically inhibited or genetically deleted. Furthermore, overexpression of ML1 mimicked the starvation effect. Hence, lysosomal adaptation to environmental cues such as nutrient levels requires mTOR/TFEB-dependent, lysosome-to-nucleus regulation of lysosomal ML1 channels and Ca ²⁺ signaling.
Loss of function mutations in Kelch-like ECH Associated Protein 1 (KEAP1), or gain-of-function mutations in nuclear factor erythroid 2-related factor 2 (NRF2), are common in non-small cell lung ...cancer (NSCLC) and associated with therapeutic resistance. To discover novel NRF2 inhibitors for targeted therapy, we conducted a quantitative high-throughput screen using a diverse set of ∼400 000 small molecules (Molecular Libraries Small Molecule Repository Library, MLSMR) at the National Center for Advancing Translational Sciences. We identified ML385 as a probe molecule that binds to NRF2 and inhibits its downstream target gene expression. Specifically, ML385 binds to Neh1, the Cap ‘N’ Collar Basic Leucine Zipper (CNC-bZIP) domain of NRF2, and interferes with the binding of the V-Maf Avian Musculoaponeurotic Fibrosarcoma Oncogene Homologue G (MAFG)-NRF2 protein complex to regulatory DNA binding sequences. In clonogenic assays, when used in combination with platinum-based drugs, doxorubicin or taxol, ML385 substantially enhances cytotoxicity in NSCLC cells, as compared to single agents. ML385 shows specificity and selectivity for NSCLC cells with KEAP1 mutation, leading to gain of NRF2 function. In preclinical models of NSCLC with gain of NRF2 function, ML385 in combination with carboplatin showed significant antitumor activity. We demonstrate the discovery and validation of ML385 as a novel and specific NRF2 inhibitor and conclude that targeting NRF2 may represent a promising strategy for the treatment of advanced NSCLC.
Alveolar rhabdomyosarcoma is a life-threatening myogenic cancer of children and adolescent young adults, driven primarily by the chimeric transcription factor PAX3-FOXO1. The mechanisms by which ...PAX3-FOXO1 dysregulates chromatin are unknown. We find PAX3-FOXO1 reprograms the
-regulatory landscape by inducing
super enhancers. PAX3-FOXO1 uses super enhancers to set up autoregulatory loops in collaboration with the master transcription factors MYOG, MYOD, and MYCN. This myogenic super enhancer circuitry is consistent across cell lines and primary tumors. Cells harboring the fusion gene are selectively sensitive to small-molecule inhibition of protein targets induced by, or bound to, PAX3-FOXO1-occupied super enhancers. Furthermore, PAX3-FOXO1 recruits and requires the BET bromodomain protein BRD4 to function at super enhancers, resulting in a complete dependence on BRD4 and a significant susceptibility to BRD inhibition. These results yield insights into the epigenetic functions of PAX3-FOXO1 and reveal a specific vulnerability that can be exploited for precision therapy.
PAX3-FOXO1 drives pediatric fusion-positive rhabdomyosarcoma, and its chromatin-level functions are critical to understanding its oncogenic activity. We find that PAX3-FOXO1 establishes a myoblastic super enhancer landscape and creates a profound subtype-unique dependence on BET bromodomains, the inhibition of which ablates PAX3-FOXO1 function, providing a mechanistic rationale for exploring BET inhibitors for patients bearing PAX-fusion rhabdomyosarcoma.
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Knowledge of oncogenic mutations can inspire therapeutic strategies that are synthetically lethal, affecting cancer cells while sparing normal cells. Lenalidomide is an active agent in the activated ...B cell-like (ABC) subtype of diffuse large B cell lymphoma (DLBCL), but its mechanism of action is unknown. Lenalidomide kills ABC DLBCL cells by augmenting interferon β (IFNβ) production, owing to the oncogenic MYD88 mutations in these lymphomas. In a cereblon-dependent fashion, lenalidomide downregulates IRF4 and SPIB, transcription factors that together prevent IFNβ production by repressing IRF7 and amplify prosurvival NF-κB signaling by transactivating CARD11. Blockade of B cell receptor signaling using the BTK inhibitor ibrutinib also downregulates IRF4 and consequently synergizes with lenalidomide in killing ABC DLBCLs, suggesting attractive therapeutic strategies.
► Lenalidomide kills ABC DLBCLs by decreasing expression of IRF4 and SPIB ► IRF4 and SPIB maintain ABC DLBCL viability ► IRF4 and SPIB block toxic IFNβ production while enhancing prosurvival NF-κB activity ► Lenalidomide and BCR pathway drugs synergize to inhibit IRF4 and kill ABC DLBCLs
The identification of cell-penetrating peptides (CPPs) as vectors for the intracellular delivery of conjugated molecules such as peptides, proteins, and oligonucleotides has emerged as a significant ...tool to modulate biological activities inside cells. The mechanism of CPP uptake by the cells is still unclear, and appears to be both endocytotic and non-endocytotic, depending on the CPP and cell type. Moreover, it is also unknown whether cargo sequences have an effect on the uptake and cellular distribution properties of CPP sequences. Here, we combine results from quantitative fluorescence microscopy and binding to lipid membrane models to determine the effect of cargo peptide molecules on the cellular uptake and distribution of the arginine-rich CPPs, R
7, and R
7W, in live cells. Image analysis algorithms that quantify fluorescence were used to measure the relative amount of peptide taken up by the cell, as well as the extent to which the uptake was endocytotic in nature. The results presented here indicate that fusion of arginine-rich CPPs to peptide sequences reduces the efficiency of uptake, and dramatically changes the cellular distribution of the CPP from a diffuse pattern to one in which the peptides are mostly retained in endosomal compartments.
The tumour microenvironment contributes to cancer metastasis and drug resistance. However, most high throughput screening (HTS) assays for drug discovery use cancer cells grown in monolayers. Here we ...show that a multilayered culture containing primary human fibroblasts, mesothelial cells and extracellular matrix can be adapted into a reliable 384- and 1,536-multi-well HTS assay that reproduces the human ovarian cancer (OvCa) metastatic microenvironment. We validate the identified inhibitors in secondary in vitro and in vivo biological assays using three OvCa cell lines: HeyA8, SKOV3ip1 and Tyk-nu. The active compounds directly inhibit at least two of the three OvCa functions: adhesion, invasion and growth. In vivo, these compounds prevent OvCa adhesion, invasion and metastasis, and improve survival in mouse models. Collectively, these data indicate that a complex three-dimensional culture of the tumour microenvironment can be adapted for quantitative HTS and may improve the disease relevance of assays used for drug screening.
Human immunodeficiency virus (HIV)-1 depends on the host cell machinery to support its replication. To discover cellular factors associated with HIV-1 replication, we conducted a genome-scale siRNA ...screen, revealing more than 311 host factors, including 267 that were not previously linked to HIV. Surprisingly, there was little overlap between these genes and the HIV dependency factors described recently. However, an analysis of the genes identified in both screens revealed overlaps in several of the associated pathways or protein complexes, including the SP1/mediator complex and the NF-kappaB signaling pathway. cDNAs for a subset of the identified genes were used to rescue HIV replication following knockdown of the cellular mRNA providing strong evidence that the following six genes are previously uncharacterized host factors for HIV: AKT1, PRKAA1, CD97, NEIL3, BMP2K, and SERPINB6. This study highlights both the power and shortcomings of large scale loss-of-function screens in discovering host-pathogen interactions.
Phagocytosis of large extracellular particles such as apoptotic bodies requires delivery of the intracellular endosomal and lysosomal membranes to form plasmalemmal pseudopods. Here, we identified ...mucolipin TRP channel 1 (TRPML1) as the key lysosomal Ca2+ channel regulating focal exocytosis and phagosome biogenesis. Both particle ingestion and lysosomal exocytosis are inhibited by synthetic TRPML1 blockers and are defective in macrophages isolated from TRPML1 knockout mice. Furthermore, TRPML1 overexpression and TRPML1 agonists facilitate both lysosomal exocytosis and particle uptake. Using time-lapse confocal imaging and direct patch clamping of phagosomal membranes, we found that particle binding induces lysosomal PI(3,5)P2 elevation to trigger TRPML1-mediated lysosomal Ca2+ release specifically at the site of uptake, rapidly delivering TRPML1-resident lysosomal membranes to nascent phagosomes via lysosomal exocytosis. Thus phagocytic ingestion of large particles activates a phosphoinositide- and Ca2+-dependent exocytosis pathway to provide membranes necessary for pseudopod extension, leading to clearance of senescent and apoptotic cells in vivo.
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•Phagocytic uptake of apoptotic bodies is defective in TRPML1 knockout macrophages•Large particle binding induces lysosomal PI(3,5)P2 elevation in macrophages•Particle binding activates TRPML1, lysosomal Ca2+ release, and focal exocytosis•Focal exocytosis provides intracellular membranes for phagosome biogenesis
Phagocytosis of large extracellular particles such as apoptotic bodies requires delivery of the intracellular endosomal and lysosomal membranes to form plasmalemmal pseudopods. In this study, Samie et al. identified TRPML1 as the key lysosomal Ca2+ channel regulating focal lysosomal exocytosis, providing the membranes necessary for pseudopod extension and phagosome biogenesis.
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