Clotrimazole-Mediated Autophagy to Protect Against Cisplatin-Induced Ototoxicity via the AMPK/mTOR/TFEB Pathway in Mice.

Mammalian target of rapamycin (mTOR) complex 1 (mTORC1) is an important, highly conserved, regulator of cell growth. Ancient among the signals that regulate mTORC1 are nutrients. Amino acids direct mTORC1 to the surface of the late endosome/lysosome, where mTORC1 becomes receptive to other inputs. However, the interplay between endosomes and mTORC1 is poorly understood. Here, we report the discovery of a network that links mTORC1 to a critical component of the late endosome/lysosome, the V-ATPase. In an unbiased screen, we found that mTORC1 regulated the expression of, among other lysosomal genes, the V-ATPases. mTORC1 regulates V-ATPase expression both in cells and in mice. V-ATPase regulation by mTORC1 involves a transcription factor translocated in renal cancer, TFEB. TFEB is required for the expression of a large subset of mTORC1 responsive genes. mTORC1 coordinately regulates TFEB phosphorylation and nuclear localization and in a manner dependent on both TFEB and V-ATPases, mTORC1 promotes endocytosis. These data uncover a regulatory network linking an oncogenic transcription factor that is a master regulator of lysosomal biogenesis, TFEB, to mTORC1 and endocytosis.

Cell growth is accompanied by the synthesis of macromolecules and biogenesis of organelles. The protein kinase mTOR (mechanistic or mammalian target of rapamycin) controls these processes by sensing availability of growth signals. The targeting of macromolecules and trafficking of cargo‐containing vesicles into appropriate cellular compartments are also important processes that are highly controlled during growth versus stress conditions. In this issue of The EMBO Journal , Pena‐Llopis et al demonstrate that mTOR complex 1 (mTORC1) could regulate endocytosis by controlling the expression of endosomal proteins such as the vacuolar (V)‐ATPases. mTORC1 performs this novel function by modulating the phosphorylation and activity of the transcription factor EB (TFEB), which is required for expression of genes involved in autophagosome and lysosome biogenesis. This study, along with a related study in Science by Settembre et al , reveals how growth signals mediated by mTOR and other protein kinases such as mitogen‐activated protein kinase (MAPK) can converge on TFEB to direct endosome biogenesis and trafficking.

Postischemic angiogenesis is critical to limit the ischemic tissue damage and improve the blood flow recovery. The regulation and the underlying molecular mechanisms of postischemic angiogenesis are not fully unraveled. TFEB (transcription factor EB) is emerging as a master gene for autophagy and lysosome biogenesis. However, the role of TFEB in vascular disease is less understood. We aimed to determine the role of endothelial TFEB in postischemic angiogenesis and its underlying molecular mechanism. In primary human endothelial cells (ECs), serum starvation induced TFEB nuclear translocation. VEGF (vascular endothelial growth factor) increased TFEB expression level and nuclear translocation. Utilizing genetically engineered EC-specific TFEB transgenic and KO (knockout) mice, we investigated the role of TFEB in postischemic angiogenesis in the mouse hindlimb ischemia model. We observed improved blood perfusion and increased capillary density in the EC-specific TFEB transgenic mice compared with the wild-type littermates. Furthermore, blood flow recovery was attenuated in EC-TFEB KO mice compared with control mice. In aortic ring cultures, the TFEB transgene significantly increased vessel sprouting, whereas TFEB deficiency impaired the vessel sprouting. In vitro, adenovirus-mediated TFEB overexpression promoted EC tube formation, migration, and survival, whereas siRNA-mediated TFEB knockdown had the opposite effect. Mechanistically, TFEB activated AMPK (AMP-activated protein kinase)-α signaling and upregulated autophagy. Through inactivation of AMPKα or inhibition of autophagy, we demonstrated that the AMPKα and autophagy are necessary for TFEB to regulate angiogenesis in ECs. Finally, the positive effect of TFEB on AMPKα activation and EC tube formation was mediated by TFEB-dependent transcriptional upregulation of MCOLN1 (mucolipin-1). In summary, our data demonstrate that TFEB is a positive regulator of angiogenesis through activation of AMPKα and autophagy, suggesting that TFEB constitutes a novel molecular target for ischemic vascular disease.

Rationale: Postischemic angiogenesis is critical to limit the ischemic tissue damage and improve the blood flow recovery. The regulation and the underlying molecular mechanisms of angiogenesis are not fully unraveled. Transcription factor-EB (TFEB) is emerging as a master gene for autophagy and lysosome biogenesis. However, the role of TFEB in the vascular disease is less understood. Objective: We aim to determine the role of endothelial TFEB in postischemic angiogenesis and underlying molecular mechanism. Methods and Results: In a murine hindlimb ischemic model, we demonstrated that TFEB was upregulated in the ischemic skeletal muscle tissue. Utilizing genetically-engineered endothelial cell (EC) specific TFEB transgenic mice, we investigated the function of TFEB in postischemic angiogenesis. We observed improved blood perfusion and increased capillary density in the EC-specific TFEB transgenic mice compared with the wild-type littermates (n = 8-9 for each group, p < 0.01). Furthermore, we found that blood flow recovery was attenuated in EC-selective TFEB deficient mice compared with control mice (n =8-9 for each group, p< 0.01). In aortic ring cultures, we found that TFEB transgene significantly increased the vessel sprouting. Adenovirus-mediated TFEB overexpression promoted EC tube formation whereas small interfering RNA (siRNA)-mediated TFEB knockdown suppressed tube formation in ECs. Mechanistically, TFEB activated calcium/calmodulin-dependent protein kinase kinase-β and AMP-activated protein kinase (AMPK)-α signaling pathway. Through pharmacological inactivation and siRNA-mediated knockdown of AMPKα, we demonstrated that AMPKα is necessary for TFEB to regulate tube formation in ECs. Conclusions: In summary, our data demonstrate that TFEB is a positive regulator of angiogenesis through activation of AMPKα signaling, suggesting that TFEB constitutes a novel molecular target for ischemic vascular disease.

The anti-tumor agent, Dp44mT, promotes nuclear translocation of TFEB via inhibition of the AMPK-mTORC1 axis

An Evolutionarily Conserved PLC-PKD-TFEB Pathway for Host Defense.

Comment on: Peña-Llopis S, et al. EMBO J 2011; 30:3242-58.

AMP-Activated protein kinase (AMPK) acts as a central metabolic sensor at the interface of metabolic and signaling networks. Activated AMPK promotes multiple catabolic processes to generate ATP, such as glucose uptake, glycolysis, fatty acid uptake and oxidation, and mitochondrial biogenesis. In addition, AMPK activation suppresses the cell cycle, and anabolic processes such as the mammalian Target of Rapamycin Complex 1 (mTORC1)-dependent protein synthesis and fatty acid biosynthesis via inactivating phosphorylation of cytosolic acetyl-CoA carboxylase 1 (ACC1) and mitochondrial ACC2. The activation of AMPK under conditions of energetic stress is modulated by an allosteric mechanism where the depletion of ATP promotes AMP binding at the γ subunit of AMPK with a subsequent conformational change in the catalytic α subunit. In the context of cancer AMPK has been reported to promote tumor survival and progression under hypoxia and the maintenance of cancer stem cells (CSCs). A major challenge to interrogating the therapeutic potential of targeting AMPK in cancer is the lack of potent and selective small molecule inhibitors. Compound C has been widely used as an AMPK inhibitor, but it lacks potency and has a poor selectivity profile. The multi-kinase inhibitor, sunitinib, has demonstrated potent nanomolar inhibition of AMPK activity, but has broad-spectrum activity across the kinome. Although sunitinib targets multiple kinases, its nanomolar potency for AMPK inhibition and large scope for chemical substitution on the core oxindole ring make it an attractive lead for AMPK inhibitor development. We have used a computational model of sunitinib docked into the ATP-binding site of the α subunit of AMPK to design and synthesize several series of oxindoles to examine structural modifications to improve AMPK inhibition and selectivity. These candidate inhibitors were evaluated against the activity of the α1 and α2 isoforms of AMPK using the TR-FRET assay and cell engagement was determined by ELISA, measuring p-ACC(Ser79), in the chronic myeloid leukemia K562 cell line. Our assays identified two novel, potent oxindole-based AMPK inhibitors that hadsignificantly reduced binding affinity when compared with sunitinib against members of the receptor tyrosine kinase (RTK) family, including VEGFR1, VEGFR2 and CSF1R, as well as BTK, a member of the B cell receptor (BCR) signaling pathway. Interestingly, cellular AMPK inhibition did not impact cell viability or result in cytotoxicity in K562 cells and may only sensitize these cells to chemotherapy. AMPK inhibition may be a more effective strategy to eliminate cancer cells in hypoxic microenvironments or against certain cell types, such as CSCs, that may be more reliant on AMPK for survival. Our current studies are designed to examine our AMPK inhibitors as single agents and in combination therapy in cancer cells grown under hypoxia and in CSC populations in order to eliminate these drug-resistant cancer cell populations with an aim to prevent cancer recurrence. In summary, we have developed two novel, potent AMPK inhibitors that were designed to interact with the DFG motif in the ATP-binding site of the α subunit of AMPK that are undergoing evaluation in advanced models of leukemia. Citation Format: Christopher J Matheson, Kimberly A Casalvieri, Donald S Backos, Craig T Jordan, Philip Reigan. Substituted oxindoles as AMP-activated protein kinase (AMPK) inhibitors and their evaluation in models of leukemia [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C078. doi:10.1158/1535-7163.TARG-19-C078

TFEB (transcription factor EB) is a critical regulator of lysosomal biogenesis, macroautophagy/autophagy and energy homeostasis through controlling expression of genes belonging to the coordinated lysosomal expression and regulation network. AMP-activated protein kinase (AMPK) has been reported to phosphorylate TFEB at three conserved C-terminal serine residues (S466, S467, S469) and these phosphorylation events were reported to be essential for transcriptional activation of TFEB. In sharp contrast to this proposition, we demonstrate that AMPK activation leads to the dephosphorylation of the C-terminal sites. We show that a synthetic peptide encompassing the C-terminal serine residues of TFEB is a poor substrate of AMPK in vitro. Treatment of cells with an AMPK activator (MK-8722), glucose deprivation or MTOR inhibitor (torin1) robustly dephosphorylated TFEB not only at the MTORC1-targeted N-terminal serine sites, but also at the C-terminal sites. Loss of function of AMPK abrogated MK-8722- but not torin1-induced dephosphorylation and induction of the TFEB target genes. Abbreviations: AMPK: 5’-adenosine monophosphate-activated protein kinase; ACAC/ACC: acetyl-CoA carboxylase; AICAR: 5-aminoimidazole-4-carbox-amide ribonucleotide; CLEAR: coordinated lysosomal expression and regulation; DKO: double knockout; DMEM: Dulbecco’s modified Eagle’s medium; DMSO: dimethyl sulfoxide; DQ-BSA: self-quenched BODIPY® dye conjugates of bovine serum albumin; KI: knock-in; KO: knockout; MEFs: mouse embryonic fibroblasts; MTORC1: mechanistic target of rapamycin kinase complex 1; RRAGC: Ras related GTP binding C; RPTOR: regulatory associated protein of MTOR complex 1; RPS6KA/RSK: ribosomal protein S6 kinase A; RPS6KB1/S6K1: ribosomal protein S6 kinase B1; RT-qPCR: reverse transcription quantitative polymerase chain reaction; TFE3: transcription factor binding to IGHM enhancer 3; TFEB: transcription factor EB; ULK1: unc-51 like autophagy activating kinase 1; WT: wild-type

Nonalcoholic steatohepatitis (NASH) is a combination of hepatic steatosis, inflammation, and fibrosis, and it often follows simple hepatic steatosis in nonalcoholic fatty liver disease (NAFLD). However, no pharmacological treatment is currently available for NASH. Given the important role of TFEB (transcription factor EB) in regulating the macroautophagy/autophagy-lysosomal pathway, TFEB is potentially a novel therapeutic target for treatment of NASH, which function can be regulated by AMP-activated protein kinase (AMPK) and MTOR (mechanistic target of rapamycin kinase) complex 1 (MTORC1). Buddleoside (Bud), a natural flavonoid compound, has recently emerged as a promising drug candidate for liver diseases. Here, we shown that Bud treatment alleviated hepatic steatosis, insulin resistance, inflammation, and fibrosis in mice fed a high-fat and high-cholesterol (HFHC) diet. Notably, Bud activated AMPK, inhibited MTORC1, and enhanced TFEB transcriptional activity as well as autophagic flux in vivo and in vitro. Inhibition of AMPK or knockout of hepatic Tfeb abrogated the alleviation effects of Bud on hepatic steatosis, insulin resistance, inflammation, and fibrosis. Mechanistic investigation revealed that Bud bound to the PRKAB1 subunit via Val81, Arg83, and Ser108 residues and activated AMPK, thereby eliciting phosphorylation of RPTOR (regulatory associated protein of MTOR complex 1) and inhibiting the kinase MTORC1, which activated the TFEB-mediated autophagy-lysosomal pathway and further ameliorated HFHC-induced NASH in mice. Altogether, our results indicate that Bud ameliorates NASH by activating hepatic the AMPK-TFEB axis, suggesting that Bud is a potential therapeutic strategy for NASH. Abbreviations: ACAC, acetyl-CoA carboxylase; ADaM, allosteric drug and metabolite; AICAR, 5-aminoimidazole-4-carboxamide1-β-D-ribofuranoside; AKT, AKT serine/threonine kinase; ALP, autophagy-lysosomal pathway; AMPK, AMP-activated protein kinase; Bud, buddleoside; CAMKK2, calcium/calmodulin dependent protein kinase kinase 2; CC, compound C; CETSA, cellular thermal shift assay; Cmax, maximum concentration; CQ, chloroquine; DARTS, drug affinity responsive target stability assay; EIF4EBP1, eukaryotic translation factor 4E binding protein 1; GOT1, glutamic-oxaloacetic transaminase 1; GPT, glutamic–pyruvic transaminase; GSK3B, glycogen synthase kinase 3 beta; GTT, glucose-tolerance test; HFD, high fat diet; HFHC, high-fat and high-cholesterol; HOMA-IR, homeostasis model assessment of insulin resistance; IKBKB, inhibitor of nuclear factor kappa B kinase subunit beta; INSR, insulin receptor; ITT, insulin-tolerance test; LDH, lactate dehydrogenase; STK11, serine/threonine kinase 11; MAP1LC3/LC3, microtubule associated protein 1 light chain 3; MTORC1, MTOR complex 1; NAFLD, non-alcoholic fatty liver disease; NASH, non-alcoholic steatohepatitis; ND, normal diet; NFKB, nuclear factor kappa B; PA, palmitic acid; PSR, picrosirius red; RRAG, Ras related GTP binding; RPTOR, regulatory associated protein of MTOR complex 1; RPS6, ribosomal protein S6; RPS6KB, ribosomal protein S6 kinase B; SMAD2, SMAD family member 2; SMAD3, SMAD family member 3; SQSTM1, sequestosome 1; TFEB, transcription factor EB; tfeb-HKO, hepatocyte-specific tfeb knockout; TSC2, TSC complex subunit 2.

Cells respond to mitochondrial poisons with rapid activation of the adenosine monophosphate-activated protein kinase (AMPK), causing acute metabolic changes through phosphorylation and prolonged adaptation of metabolism through transcriptional effects. Transcription factor EB (TFEB) is a major effector of AMPK that increases expression of lysosome genes in response to energetic stress, but how AMPK activates TFEB remains unresolved. We demonstrate that AMPK directly phosphorylates five conserved serine residues in folliculin-interacting protein 1 (FNIP1), suppressing the function of the folliculin (FLCN)-FNIP1 complex. FNIP1 phosphorylation is required for AMPK to induce nuclear translocation of TFEB and TFEB-dependent increases of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) and estrogen-related receptor alpha (ERRα) messenger RNAs. Thus, mitochondrial damage triggers AMPK-FNIP1-dependent nuclear translocation of TFEB, inducing sequential waves of lysosomal and mitochondrial biogenesis.

Mitogen-activated protein kinase (MAPK) pathways are involved in the regulation of cellular responses, including cell proliferation, differentiation, cell growth, and apoptosis. Because these responses are tightly related to cellular energy level, AMP-activated protein kinase (AMPK), which plays an essential role in energy homeostasis, has emerged as another key regulator. In the present study, we demonstrate a novel signal network between AMPK and MAPK in HCT116 human colon carcinoma. Glucose deprivation activated AMPK and three MAPK subfamilies, extracellular signal-regulated kinase (ERK), c-Jun NH(2)-terminal kinase (JNK), and p38 MAPK. Under these conditions, inhibition of endogenous AMPK by expressing a dominant-negative form significantly potentiated ERK activation, indicating that glucose deprivation-induced AMPK is specifically antagonizing ERK activity in HCT116 cells. Moreover, we provide novel evidence that AMPK activity is critical for p53-dependent expression of dual-specificity phosphatase (DUSP) 1 & 2, which are negative regulators of ERK. Notably, ERK exhibits pro-apoptotic effects in HCT116 cells under glucose deprivation. Collectively, our data suggest that AMPK protects HCT116 cancer cells from glucose deprivation, in part, via inducing DUSPs, which suppresses pro-apoptotic ERK, further implying that a signal network between AMPK and ERK is a critical regulatory point in coupling the energy status of the cell to the regulation of cell survival.

The transcription factor EB (TFEB) was identified as a master regulator of autophagy and lysosomal biogenesis. We recently demonstrated that TFEB is aberrantly regulated in pancreatic cancer (PDAC) cells. Interference with TFEB impairs PDAC cell growth supporting an important role for TFEB in maintaining PDAC cell phenotype. Given the limited impact of chemotherapeutic drugs in the context of PDAC, we aimed at testing whether the aberrantly regulated TFEB in PDAC cells could confer resistance to DNA damage agents, commonly used in PDAC therapeutic regimens. Methods: Experiments were performed in PDAC cells (MIA PaCa2, PANC1). The DNA damage agents gemcitabine, 5-FU, cisplatin and doxorubicin were used. Autophagic flux was measured upon 4-hours treatment with bafilomycin A1. Results: Treatment with DNA damage agents 1) increased the autophagic flux in PDAC cells. This autophagic signal was associated with 2) the dephosphorylation and nuclear accumulation of TFEB. The mTORC1 pathway is well-known to regulate TFEB phosphorylation and nuclear localization of TFEB. 3) However, the impact of the DNA damage agents on TFEB appeared independent of the mTORC1 pathway since no modulation in the phosphorylation of the mTORC1 target S6K1 was observed. To delineate whether TFEB influenced the response to DNA damage agents, we generated stable populations of PDAC cells expressing a non-targeting or shRNA targeting TFEB. Interfering with TFEB function 4) prevented the autophagic response upon treatment with DNA damage agents. This correlated with 5) increased accumulation of DNA damage and 6) increased sensitivity to the DNA damage agents. Conclusion: Our results suggest that DNA damage agents can induce a pro-survival autophagic response involving TFEB. Interfering with TFEB function limits the autophagic response and leads to increased sensitivity to DNA damage agents. Altogether, our results suggest that the aberrantly expressed TFEB in PDAC cells could confer resistance to DNA damage agents by, among others, promoting autophagy and limiting DNA damage accumulation. Citation Format: Benoit Marchand, Marie-Josée Boucher. DNA damage agents promote TFEB function and induce pro-survival autophagy signals [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2619.

Rationale: Abdominal aortic aneurysm (AAA) is a vascular disease with a very high mortality rate in the case of rupture. Vascular smooth muscle cells (VSMCs) are crucial to maintaining vascular integrity and function. Transcription Factor EB (TFEB) is a master regulator of autophagy and lysosomal biogenesis in a variety of cell types via inducing the transcription of a coordinated lysosomal regulatory gene network. TFEB shows anti-inflammatory and anti-atherosclerotic effects in vascular endothelial cells and macrophages. However, the role of TFEB in vascular disease remains to be further explored. Objectives: To investigate the role of VSMC TFEB in AAA. Methods and Results: We found that TFEB was down-regulated in human aortic aneurysmal lesion compared with the non-lesion area by QRT-PCR and immunostaining. In human aortic smooth muscle cells (HASMCs), TFEB mRNA and protein abundance were decreased upon treatment with pro-inflammatory factors while adenovirus-mediated TFEB overexpression potently inhibits inflammation, apoptosis (caspase 3 cleavage and Annexin V staining) and matrix metalloproteinase activity (zymography). A consistent phenotype was observed in the TFEB knockdown HASMCs. Mechanistically, TFEB activates the PI3K-Akt pathway, and PI3K inhibitors (wortmannin and LY294002) abolished the anti-apoptotic effect of TFEB in HASMCs. Utilizing VSMC-specific TFEB deficiency mice (floxed-TFEB/myh11-ERT2 cre+), we determined the effect of TFEB on AAA formation in vivo. In the mouse aneurysm model induced by the combination of angiotensin II and 3-aminopropionitrile infusion, TFEB VSMC-deletion significantly increases aneurysm formation, rupture, and mortality (n =13-14 for each group, p< 0.01). Conclusions: Our data reveal a critical protective role of TFEB in VSMC homeostasis, suggesting TFEB to be a potential target to treat aortic aneurysm.

A stress response p38 MAP kinase inhibitor SB202190 promoted TFEB/TFE3-dependent autophagy and lysosomal biogenesis independent of p38