Kinase ULK1 Research Articles

RCE‑4, a potential anti‑cervical cancer drug isolated from Reineckia carnea, induces autophagy via the dual blockade of PI3K and ERK pathways in cervical cancer CaSki cells.

The steroidal saponin RCE‑4 (1β, 3β, 5β, 25S)‑spirostan‑1, 3‑diol 1‑[α‑L‑rhamnopyranosyl‑(1→2)‑β‑D‑xylopyranoside], isolated from Reineckia carnea, exerts significant anti‑cervical cancer activity by inducing apoptosis. The potential effect of RCE‑4 on proliferation inhibition and autophagy induction has rarely been studied. Therefore, the focus of the present study was to investigate the effects of RCE‑4 on proliferation, and to elucidate the detailed mechanisms involved in autophagy induction in cervical cancer cells. CaSki cells were treated with RCE‑4 or/and autophagy inhibitors, and the effect of RCE‑4 on cellular proliferation was assessed by MTT assay. The pro‑autophagic properties of RCE‑4 were subsequently confirmed using monomeric red fluorescent protein‑green fluorescent protein‑microtubule‑associated proteins1A/1B light chain3B (LC3) adenoviruses and CYTO‑ID autophagy assays, and by assessing the accumulation of lipid‑modifiedLC3 (LC3II). The mechanisms of RCE‑4‑induced autophagy were investigated by western blot analysis. The results demonstrated that inhibiting autophagy significantly promoted RCE‑4‑induced cell death, indicating that autophagy served a protective role following RCE‑4 treatment. In addition, RCE‑4‑induced autophagy was reflected by increased expression levels of the serine/threonine‑protein kinase ULK1, phosphorylated (p)‑ULK1, p‑Beclin‑1 and LC3II, the formation of autophagosomes and autolysosomes, and sequestosome1 (p62) degradation. Subsequent analysis indicated that RCE‑4 activated the AMP‑activated protein kinase (AMPK) pathway by upregulating AMPK and p‑AMPK, and also inhibited the PI3K and extracellular signal‑regulated kinase (ERK) signaling pathways by downregulating p‑PI3K, p‑Akt, p‑mTOR, Ras, c‑Raf, p‑c‑Raf, dual specificity mitogen‑activated protein kinase kinase (MEK)1/2, p‑MEK1/2 and p‑Erk1/2. Additionally, with increased treatment times RCE‑4 may impair lysosomal cathepsin activity and inhibit autophagy flux by suppressing the expression of AMPK, p‑AMPK, ULK1, p‑ULK1 and p‑Beclin‑1, and upregulating that of p62. These results indicated that the dual RCE‑4‑induced inhibition of the PI3K and ERK pathways may result in a more significant anti‑tumor effect and prevent chemoresistance, compared with the inhibition of either single pathway; furthermore, dual blockade of PI3K and ERK, and the AMPK pathway may be involved in the regulation of autophagy caused by RCE‑4. Taken together, RCE‑4 induced autophagy to protect cancer cells against apoptosis, but AMPK‑mediated autophagy was inhibited in the later stages of RCE‑4 treatment. In addition, autophagy inhibition improved the therapeutic effect of RCE‑4. These data highlight RCE‑4 as a potential candidate for cervical cancer treatment.

P.80Imbalances in protein homeostasis caused by mutant desmin

Mutations of the human desmin gene cause myopathies and cardiomyopathies. We investigated immortalized R349P desmin knock-in myoblasts and myotubes in conjunction with the corresponding desminopathy mice as models for desminopathies to analyse major protein quality control processes in response to the presence of R349P mutant desmin. Desmin immunoblots and immunostains of differentiated myotubes revealed that these desminopathy cell models closely mirror central aspects of the desmin pathology observed in our desminopathy mice. Proteasomal activity was increased in muscle tissue lysates derived from hetero- and homozygous R349P desmin knock-in mice. Notably, treatment of the desminopathy myoblasts with the proteasome inhibitor MG132 did not alter the levels of both wild-type and R349P mutant desmin protein. Immunoblotting of tissue and myotube lysates demonstrated markedly increased levels of the autophagy-related markers serin/threonin protein kinase Ulk-1 and sequestosome 1 (p62/SQSTM1) as well as of p62/SQSTM1 and LC3-II. Furthermore, we detected increased protein levels of filamin-C and BAG-3. Finally, we found increased protein levels of αB-crystallin, Hsp27, and Hsp90 in skeletal muscle homogenates from homozygous mice, while only αB-crystallin and Hsp27, but not Hsp90, were increased in both desminopathy myotube genotypes. Both αB-crystallin and Hsp27 as well as Hsp90 displayed translocation patterns from Z-discs as well as Z-I junctions, respectively, to the level of sarcomeric I-bands. Our findings demonstrate that the presence of R349P mutant desmin causes a general imbalance in skeletal muscle protein homeostasis via aberrant activity of all major protein quality control systems. The augmented activity of these systems and the subcellular shift of essential heat shock proteins may deleteriously contribute to the previously observed increased turnover of desmin itself and desmin-binding partners, which triggers progressive dysfunction of the extrasarcomeric cytoskeleton and the myofibrillar apparatus in the course of the development of desminopathies.

ALS-FTLD-linked mutations of SQSTM1/p62 disrupt selective autophagy and NFE2L2/NRF2 anti-oxidative stress pathway

ABSTRACTMacroautophagy (autophagy) is a key catabolic pathway for the maintenance of proteostasis through constant digestion of selective cargoes. The selectivity of autophagy is mediated by autophagy receptors that recognize and recruit cargoes to autophagosomes. SQSTM1/p62 is a prototype autophagy receptor, which is commonly found in protein aggregates associated with major neurodegenerative diseases. While accumulation of SQSTM1 implicates a disturbance of selective autophagy pathway, the pathogenic mechanism that contributes to impaired autophagy degradation remains poorly characterized. Herein we show that amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD)-linked mutations of TBK1 and SQSTM1 disrupt selective autophagy and cause neurotoxicity. Our data demonstrates that proteotoxic stress activates serine/threonine kinase TBK1, which coordinates with autophagy kinase ULK1 to promote concerted phosphorylation of autophagy receptor SQSTM1 at the UBA domain and activation of selective autophagy. In contrast, ALS-FTLD-linked mutations of TBK1 or SQSTM1 reduce SQSTM1 phosphorylation and compromise ubiquitinated cargo binding and clearance. Moreover, disease mutation SQSTM1G427R abolishes phosphorylation of Ser351 and impairs KEAP1-SQSTM1 interaction, thus diminishing NFE2L2/Nrf2-targeted gene expression and increasing TARDBP/TDP-43 associated stress granule formation under oxidative stress. Furthermore, expression of SQSTM1G427R in neurons impairs dendrite morphology and KEAP1-NFE2L2 signaling. Therefore, our results reveal a mechanism whereby pathogenic SQSTM1 mutants inhibit selective autophagy and disrupt NFE2L2 anti-oxidative stress response underlying the neurotoxicity in ALS-FTLD.Abbreviations: ALS: amyotrophic lateral sclerosis; FTLD: frontotemporal lobar degeneration; G3BP1: GTPase-activating protein (SH3 domain) binding protein 1; GSTM1: glutathione S-transferase, mu 1; HMOX/HO-1: Heme oxygenase 1; IP: immunoprecipitation; KEAP1: kelch-like ECH associated protein 1; KI: kinase inactive; KIR: KEAP1 interaction region; KO: knockout; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MBP: maltose binding protein; NBR1: NBR1, autophagy cargo receptor; NFE2L2/Nrf2: nuclear factor, erythroid derived 2, like 2; NQO1: NAD(P)H quinone dehydrogenase 1; SQSTM1/p62: sequestosome 1; SOD1: superoxide dismutase 1, soluble; S.S.: serum starvation; TARDBP/TDP-43: TAR DNA binding protein; TBK1: TANK binding kinase 1; UBA: ubiquitin association; ULK1: unc-51 like autophagy activating kinase 1; WT: wild type.

ULK1-mediated phosphorylation of ATG16L1 promotes xenophagy, but destabilizes the ATG16L1 Crohn's mutant.

Autophagy is a highly regulated catabolic pathway that is potently induced by stressors including starvation and infection. An essential component of the autophagy pathway is an ATG16L1-containing E3-like enzyme, which is responsible for lipidating LC3B and driving autophagosome formation. ATG16L1 polymorphisms have been linked to the development of Crohn's disease (CD), and phosphorylation of CD-associated ATG16L1 T300A (caATG16L1) has been hypothesized to contribute to cleavage and autophagy dysfunction. Here we show that ULK1 kinase directly phosphorylates ATG16L1 in response to infection and starvation. Phosphorylated ATG16L1 localizes to the site of internalized bacteria and stable cell lines harbouring a phospho-dead mutant of ATG16L1 have impaired xenophagy, indicating a role for ATG16L1 phosphorylation in the promotion of anti-bacterial autophagy. In contrast to wild-type ATG16L1, ULK1-mediated phosphorylation of caATG16L1 drives its destabilization in response to stress. In summary, our results show that ATG16L1 is a novel target of ULK1 kinase and that ULK1 signalling to ATG16L1 is a double-edged sword, enhancing the function of the wild-type ATG16L1, but promoting degradation of caATG16L1.

ER-Targeted Beclin 1 Supports Autophagosome Biogenesis in the Absence of ULK1 and ULK2 Kinases.

Autophagy transports cytoplasmic material and organelles to lysosomes for degradation and recycling. Beclin 1 forms a complex with several other autophagy proteins and functions in the initiation phase of autophagy, but the exact role of Beclin 1 subcellular localization in autophagy initiation is still unclear. In order to elucidate the role of Beclin 1 localization in autophagosome biogenesis, we generated constructs that target Beclin 1 to the endoplasmic reticulum (ER) or mitochondria. Our results confirmed the proper organelle-specific targeting of the engineered Beclin 1 constructs, and the proper formation of autophagy-regulatory Beclin 1 complexes. The ULK kinases are required for autophagy initiation upstream of Beclin 1, and autophagosome biogenesis is severely impaired in ULK1/ULK2 double knockout cells. We tested whether Beclin 1 targeting facilitated its ability to rescue autophagosome formation in ULK1/ULK2 double knockout cells. ER-targeted Beclin 1 was most effective in the rescue experiments, while mitochondria-targeted and non-targeted Beclin 1 also showed an ability to rescue, but with lower activity. However, none of the constructs was able to increase autophagic flux in the knockout cells. We also showed that wild type Beclin 1 was enriched on the ER during autophagy induction, and that ULK1/ULK2 facilitated the ER-enrichment of Beclin 1 under basal conditions. The results suggest that one of the functions of ULK kinases may be to enhance Beclin 1 recruitment to the ER to drive autophagosome formation.

Loss of BRUCE reduces cellular energy level and induces autophagy by driving activation of the AMPK-ULK1 autophagic initiating axis.

Autophagy is an intracellular catabolic system. It delivers cellular components to lysosomes for degradation and supplies nutrients that promote cell survival under stress conditions. Although much is known regarding starvation-induced autophagy, the regulation of autophagy by cellular energy level is less clear. BRUCE is an ubiquitin conjugase and ligase with multi-functionality. It has been reported that depletion of BRUCE inhibits starvation-induced autophagy by blockage of the fusion step. Herein we report a new function for BRUCE in the dual regulation of autophagy and cellular energy. Depletion of BRUCE alone (without starvation) in human osteosarcoma U2OS cells elevated autophagic activity as indicted by the increased LC3B-II protein and its autophagic puncta as well as further increase of both by chloroquine treatment. Such elevation results from enhanced induction of autophagy since the numbers of both autophagosomes and autolysosomes were increased, and recruitment of ATG16L onto the initiating membrane structure phagophores was increased. This concept is further supported by elevated lysosomal enzyme activities. In contrast to starvation-induced autophagy, BRUCE depletion did not block fusion of autophagosomes with lysosomes as indicated by increased lysosomal cleavage of the GFP-LC3 fusion protein. Mechanistically, BRUCE depletion lowered the cellular energy level as indicated by both a higher ratio of AMP/ATP and the subsequent activation of the cellular energy sensor AMPK (pThr-172). The lower energy status co-occurred with AMPK-specific phosphorylation and activation of the autophagy initiating kinase ULK1 (pSer-555). Interestingly, the higher autophagic activity by BRUCE depletion is coupled with enhanced cisplatin resistance in human ovarian cancer PEO4 cells. Taken together, BRUCE depletion promotes induction of autophagy by lowering cellular energy and activating the AMPK-ULK1-autophagy axis, which could contribute to ovarian cancer chemo-resistance. This study establishes a BRUCE-AMPK-ULK1 axis in the regulation of energy metabolism and autophagy, as well as provides insights into cancer chemo-resistance.

Autophagy-induced senescence is regulated by p38\u03b1 signaling

Apoptosis and senescence are two mutually exclusive cell fate programs that can be activated by stress. The factors that instruct cells to enter into senescence or apoptosis are not fully understood, but both programs can be regulated by the stress kinase p38α. Using an inducible system that specifically activates this pathway, we show that sustained p38α activation suffices to trigger massive autophagosome formation and to enhance the basal autophagic flux. This requires the concurrent effect of increased mitochondrial reactive oxygen species production and the phosphorylation of the ULK1 kinase on Ser-555 by p38α. Moreover, we demonstrate that macroautophagy induction by p38α signaling determines that cancer cells preferentially enter senescence instead of undergoing apoptosis. In agreement with these results, we present evidence that the induction of autophagy by p38α protects cancer cells from chemotherapy-induced apoptosis by promoting senescence. Our results identify a new mechanism of p38α-regulated basal autophagy that controls the fate of cancer cells in response to stress.

MDH1 and MPP7 Regulate Autophagy in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is driven by metabolic changes in pancreatic cells caused by oncogenic mutations and dysregulation of p53. PDAC cell lines and PDAC-derived xenografts grow as a result of altered metabolic pathways, changes in stroma, and autophagy. Selective targeting and inhibition of one of these may open avenues for the development of new therapeutic strategies. In this study, we performed a genome-wide siRNA screen in a PDAC cell line using endogenous autophagy as a readout and identified several regulators of autophagy that were required for autophagy-dependent PDAC cell survival. Validation of two promising candidates, MPP7 (MAGUK p55 subfamily member 7, a scaffolding protein involved in cell-cell contacts) and MDH1 (cytosolic Malate dehydrogenase 1), revealed their role in early stages of autophagy during autophagosome formation. MPP7 was involved in the activation of YAP1 (a transcriptional coactivator in the Hippo pathway), which in turn promoted autophagy, whereas MDH1 was required for maintenance of the levels of the essential autophagy initiator serine-threonine kinase ULK1, and increased in the activity upon induction of autophagy. Our results provide a possible explanation for how autophagy is regulated by MPP7 and MDH1, which adds to our understanding of autophagy regulation in PDAC. SIGNIFICANCE: This study identifies and characterizes MPP7 and MDH1 as novel regulators of autophagy, which is thought to be responsible for pancreatic cancer cell survival.

RAB2 regulates the formation of autophagosome and autolysosome in mammalian cells

ABSTRACT Multiple sources contribute membrane and protein machineries to construct functional macroautophagic/autophagic structures. However, the underlying molecular mechanisms remain elusive. Here, we show that RAB2 connects the Golgi network to autophagy pathway by delivering membrane and by sequentially engaging distinct autophagy machineries. In unstressed cells, RAB2 resides primarily in the Golgi apparatus, as evidenced by its interaction and colocalization with GOLGA2/GM130. Importantly, autophagy stimuli dissociate RAB2 from GOLGA2 to interact with ULK1 complex, which facilitates the recruitment of ULK1 complex to form phagophores. Intriguingly, RAB2 appears to modulate ULK1 kinase activity to propagate signals for autophagosome formation. Subsequently, RAB2 switches to interact with autophagosomal RUBCNL/PACER and STX17 to further specify the recruitment of HOPS complex for autolysosome formation. Together, our study reveals a multivalent pathway in bulk autophagy regulation, and provides mechanistic insights into how the Golgi apparatus contributes to the formation of different autophagic structures. Abbreviations ACTB: actin beta; ATG9: autophagy related 9A; ATG14: autophagy related 14; ATG16L1: autophagy related 16 like 1; BCAP31: B cell receptor associated protein 31; BECN1: beclin 1; Ctrl: control; CQ: chloroquine; CTSD: cathepsin D; DMSO: dimethyl sulfoxide; EBSS: Earle’s balanced salt solution; EEA1: early endosome antigen 1; GDI: guanine nucleotide dissociation inhibitor; GFP: green fluorescent protein; GOLGA2: golgin A2; HOPS: homotypic fusion and protein sorting complex; IP: immunoprecipitation; KD: knockdown; KO: knockout; LAMP1: lysosomal associated membrane protein 1; LC3: microtubule-associated protein 1 light chain 3; OE: overexpression; PtdIns3K: class III phosphatidylinositol 3-kinase; SQSTM1/p62: sequestosome 1; RAB2: RAB2A, member RAS oncogene family; RAB7: RAB7A, member RAS oncogene family; RAB11: RAB11A, member RAS oncogene family; RUBCNL/PACER: rubicon like autophagy enhancer; STX17: syntaxin 17; TBC1D14: TBC1 domain family member 14; TFRC: transferrin receptor; TGOLN2: trans-golgi network protein 2; TUBB: tubulin beta class I; ULK1: unc-51 like autophagy activating kinase 1; VPS41: VPS41, HOPS complex subunit; WB: western blot; WT: wild type; YPT1: GTP-binding protein YPT1.

Conservation of structure, function and inhibitor binding in UNC-51-like kinase 1 and 2 (ULK1/2).

Autophagy is essential for cellular homeostasis and when deregulated this survival mechanism has been associated with disease development. Inhibition of autophagy initiation by inhibiting the kinase ULK1 (Unc-51-like autophagy activating kinase 1) has been proposed as a potential cancer therapy. While inhibitors and crystal structures of ULK1 have been reported, little is known about the other closely related kinase ULK2 (Unc-51-like autophagy activating kinase 2). Here, we present the crystal structure of ULK2 in complex with ATP competitive inhibitors. Surprisingly, the ULK2 structure revealed a dimeric assembly reminiscent of dimeric arrangements of auto-activating kinases suggesting a role for this association in ULK activation. Screening of a kinase focused library of pre-clinical and clinical compounds revealed several potent ULK1/2 inhibitors and good correlation of inhibitor-binding behavior with both ULK kinases. Aurora A was identified as a major off-target of currently used ULK1 inhibitors. Autophagic flux assays demonstrated that this off-target activity by strongly inducing autophagy in different cellular systems conferred an additional layer of complexity in the interpretation of cellular data. The data presented here provide structural models and chemical starting points for the development of ULK1/2 dual inhibitors with improved selectivity for future exploitation of autophagy inhibition.

Spatiotemporal Control of ULK1 Activation by NDP52 and TBK1 during Selective Autophagy

SummarySelective autophagy recycles damaged organelles and clears intracellular pathogens to prevent their aberrant accumulation. How ULK1 kinase is targeted and activated during selective autophagic events remains to be elucidated. In this study, we used chemically inducible dimerization (CID) assays in tandem with CRISPR KO lines to systematically analyze the molecular basis of selective autophagosome biogenesis. We demonstrate that ectopic placement of NDP52 on mitochondria or peroxisomes is sufficient to initiate selective autophagy by focally localizing and activating the ULK1 complex. The capability of NDP52 to induce mitophagy is dependent on its interaction with the FIP200/ULK1 complex, which is facilitated by TBK1. Ectopically tethering ULK1 to cargo bypasses the requirement for autophagy receptors and TBK1. Focal activation of ULK1 occurs independently of AMPK and mTOR. Our findings provide a parsimonious model of selective autophagy, which highlights the coordination of ULK1 complex localization by autophagy receptors and TBK1 as principal drivers of targeted autophagosome biogenesis.

Discovery of human autophagy initiation kinase ULK1 inhibitors by multi-directional in silico screening strategies

Autophagy is a self-catabolic mechanism employed by cancer cells to acquire nutrients and energy in times of stress conditions, thereby leading to its progression and survival. Thus, autophagy inhibition has emerged as a new paradigm in the area of cancer treatment. Here, we leverage multi-dimensional screening campaigns aim to identify potent inhibitors against an early and an essential autophagic kinase, ULK1 from DrugBank database. In particular, receptor-based hypothesis, pharmacophore hypothesis, e-pharmacophore hypothesis and shape similarity-based screening algorithm were employed. Of note, the results of the different algorithm were then integrated to eliminate the false positive prediction. Moreover, the inhibitory activities and PK/PD parameters of the leads were tested by Glide and Qikprop algorithm. This resulted in a set of four hits namely; DB12686, DB08341, DB07936, and DB07163. Finally, molecular dynamics simulation was performed using the GROMACS package, to validate the binding kinetics of the hit compound. The compound activity in vitro was assessed by PASS algorithm, highlights the anti-cancer activities of the hits. The structural insights reveal existence of functional moieties such as piperidine carboxamide, benzenesulfonamide, benzamide, and isoindolone in the resultant hits which plays a major role in the anti-cancer activity. Overall, we strongly believe that these ULK1 antagonists could be novel and potent drug candidates for future cancer therapeutics.

Autophagy induction via STING trafficking is a primordial function of the cGAS pathway.

Cyclic GMP-AMP (cGAMP) synthase (cGAS) detects infections or tissue damage by binding to microbial or self-DNA in the cytoplasm1. Upon binding DNA, cGAS produces cGAMP that binds to and activates the adaptor protein STING, which then activates the kinases IKK and TBK1 to induce interferons and other cytokines2–6. Here, we report that STING also activates autophagy through a mechanism independent of TBK1 activation and interferon induction. Upon binding cGAMP, STING translocates to the ER-Golgi intermediate compartment (ERGIC) and the Golgi in a process dependent on the COP-II complex and ARF GTPases. STING-containing ERGIC serves as a membrane source for LC3 lipidation, a key step in autophagosome biogenesis. cGAMP induced LC3 lipidation through a pathway dependent on WIPI2 and ATG5 but independent of the ULK and VPS34/BECLIN kinase complexes. Furthermore, we show that cGAMP-induced autophagy is important for the clearance of DNA and viruses in the cytosol. Interestingly, STING from the sea anemone Nematostella vectensis induces autophagy but not interferons in response to stimulation by cGAMP, suggesting that induction of autophagy is a primordial function of the cGAS-STING pathway.

The ALS-FTD-linked gene product, C9orf72, regulates neuronal morphogenesis via autophagy

ABSTRACTMutations in C9orf72 leading to hexanucleotide expansions are the most common genetic causes for amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). A phenotype resembling ALS and FTD is seen in transgenic mice overexpressing the hexanucleotide expansions, but is absent in C9orf72-deficient mice. Thus, the exact function of C9orf72 in neurons and how loss of C9orf72 may contribute to neuronal dysfunction remains to be clearly defined. Here, we showed that primary hippocampal neurons cultured from c9orf72 knockout mice have reduced dendritic arborization and spine density. Quantitative proteomic analysis identified C9orf72 as a component of the macroautophagy/autophagy initiation complex composed of ULK1-RB1CC1-ATG13-ATG101. The association was mediated through the direct interaction with ATG13 via the isoform-specific carboxyl-terminal DENN and dDENN domain of C9orf72. Furthermore, c9orf72 knockout neurons showed reduced LC3-II puncta accompanied by reduced ULK1 levels, suggesting that loss of C9orf72 impairs basal autophagy. Conversely, wild-type neurons treated with a ULK1 kinase inhibitor showed a dose-dependent reduction of dendritic arborization and spine density. Furthermore, expression of the long isoform of human C9orf72 that interacts with the ULK1 complex, but not the short isoform, rescues autophagy and the dendritic arborization phenotypes of c9orf72 knockout neurons. Taken together, our data suggests that C9orf72 has a cell-autonomous role in neuronal and dendritic morphogenesis through promotion of ULK1-mediated autophagy.

Differential effects of ginkgol C17:1 on cisplatin-induced cytotoxicity: Protecting human normal L02 hepatocytes versus sensitizing human hepatoma HepG2 cells.

Liver cancer is a major healthcare problem and one of the leading causes of cancer-associated mortality in the world. To date, chemotherapy remains a common method for treating cancer and cisplatin is one of the most widely used chemotherapeutics. However, owing to drug resistance and side effects, it is imperative to identify a novel approach to improve the anticancer effect of cisplatin. Auxiliary chemotherapy drugs with minor toxicity to normal cells may represent a novel strategy for cancer therapy. Previous studies have indicated that ginkgol C17:1 exhibits anticancer effects in liver cancer cells in vitro and in vivo. The antitumor activity of ginkgol C17:1 has been reported in combination with cisplatin in human liver cancer cells. Owing to the route of systemic administration, liver cancer cells and normal hepatocytes were exposed to chemotherapeutics and auxiliary chemotherapy drugs. However, the effects of ginkgol C17:1 in normal hepatocytes remain unclear. In the present study, the biological effects of ginkgol C17:1 alone and as co-treatment with cisplatin were compared in human hepatoma cells and normal hepatocytes. Consistently, the results confirmed that in human hepatoma HepG2 cells, ginkgol C17:1 or cisplatin alone induced autophagy and apoptosis. The co-treatment increased cisplatin-induced apoptosis and inhibited cisplatin-induced autophagy. In comparison, the treatments in human normal L02 hepatocytes indicated that ginkgol C17:1 alone induced autophagy, whereas cisplatin alone induced apoptosis. The co-treatment inhibited cisplatin-induced apoptosis, but enhanced autophagy in L02 cells. Further investigation revealed that the AMP-activated protein kinase/serine/threonine protein kinase ULK1 and phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin signaling pathways were involved in the underlying regulatory mechanisms. Taken together, the results of the present study provide the first evidence that ginkgol C17:1 protects normal hepatocytes against cisplatin-induced cytotoxicity while potentiating the anticancer effect of cisplatin chemotherapy. The differential effects on normal and cancer cells suggest that ginkgol C17:1 is a promising candidate for auxiliary chemotherapy.

Syncytia generated by hemagglutinin-neuraminidase and fusion proteins of virulent Newcastle disease virus induce complete autophagy by activating AMPK-mTORC1-ULK1 signaling>

Autophagy triggered by glycoprotein-mediated membrane fusion has been reported for several paramyxoviruses. However, the function of HN and F glycoproteins of NDV and their role in autophagy induction have not been studied. Here, we found that co-transfection of HN and F of virulent NDV rapidly induced syncytium formation and triggered a steady state autophagy flux in adenocarcinomic human alveolar basal epithelial (A549) cells and chicken embryo fibroblast (DF-1) cells. Furthermore, we clearly identified that F and HN synergistically induced autophagosome fusion with lysosomes for subsequent degradation. The seven cleavage site mutations of F significantly decreased the autophagy induction, compared with those of wildtype virulent F. RNAi and pharmacological experiments suggested that autophagy benefitted membrane fusion and syncytium formation induced by F and HN of NDV. Activated F1 co-operated with HN to stimulate AMPK kinase and downstream ULK1 activation to suppress mTORC1 signaling. Our data described the synergistic role of HN and F in the induction of completed autophagic flux through the activation of AMPK- mTORC1- ULK1 pathway.

Targeting Autophagy Kinase ULK1 Can Reverse Bcl2 Inhibitor (ABT-199) Induced Autophagy to Overcome Acquired Resistance in Acute Myeloid Leukemia

IntroductionAnti-apoptotic Bcl2 family members mediate resistance to therapies in acute myeloid leukemia (AML)1. The small molecule Bcl2 inhibitor ABT-199 (venetoclax) promotes mitochondria driven intrinsic apoptosis, and in combination with hypomethylating agents or chemotherapy, has been highly promising in the clinic as treatment of AML2-4. The response rate to ABT-199 is very impressive, but acquired resistance is a major problem. Compensatory upregulation of Mcl1 is an important mechanism of such acquired resistance to mitochondrial apoptosis5. Autophagy is vital for mitochondrial health, mediates resistance to apoptosis and is induced by Bcl2 inhibition6. We performed mechanistic studies to address our hypothesis that disabling autophagy by targeting the apical autophagy kinase ULK1 can reverse resistance to ABT-199.MethodsULK1 was genetically modified in OCIAML3 (human AML cell line), by shRNA knockdown (KD) or CRISPR-Cas9 knockout (KO). In addition, AML cell lines (including ABT-199 resistant) and patient samples were treated with ABT-199 and ULK1 inhibitor SBI-02069657. Combination index (CI) for drug synergy was calculated based on Chou-Talalay method8. Drug-treated or genetically manipulated cells were profiled by reverse phase protein array (RPPA), mass cytometry (CyTOF) and gene expression profiling (GEP). Autophagy was detected by LC3 quantification by western blot (WB) and flow cytometry, and monodansylcadaverine assay. Mitochondrial functions were analyzed by Seahorse Cell Mito Stress test, and MTG, TMRE and ROS assays (flow cytometry). For in vivo studies ULK1 KO and corresponding control cells were injected in NSG mice and monitored by bioluminescent imaging (BLI) and quantification of human CD45 cells.ResultsABT-199 induced autophagy in OCIAML3 (increase by 175±27%, p=0.01 - LC3 flow; 4X increase in LC3 II/I ratio - WB). Apoptosis induction by ABT-199 was enhanced by ULK1 KD (36±1.9% over control, p<0.01) or KO (77±1.3%, p<0.01). ULK1 inhibitor SBI-0206965 demonstrated high synergy with ABT-199 at inducing apoptosis in OCIAML3 (CI = 0.51, p<0.001) and MOLM13 (CI = 0.24, p<0.001). The combination was also effective in eliminating bulk and CD34+ stem/progenitor cells in primary AML samples (CI - Bulk: 0.69; CD34: 0.74; p<0.05) (Fig 1).Mcl1 was significantly downregulated by ULK1 inhibitor alone and in combination with ABT-199. ULK1 inhibition lowered Mcl1 transcription, as measured by qRT-PCR: 43±0.03% with SBI-0206965 and 63±0.3% in KO cells (both p<0.01). SBI-0206965 inhibited Mcl1 transcription regulators Stat3 and Erk1/2 and enhanced DNA damage in combination with ABT-199 (WB) (Fig 2).Since ABT-199 modulates mitochondrial function, we examined the effect of inhibiting ULK1 in this context. By Seahorse assay, the combination decreased basal OCR and ATP production by 62 and 58% respectively, p<0.01. This was accompanied by an increase in membrane depolarization (TMRE change - OCIAML3: 62%, MOLM13: 82%; p<0.01) and mitochondrial ROS generation (62% increase, p=0.01) compared to control cells (Fig 3). Interestingly, ULK1 inhibition increased mitochondrial mass (30%, p=0.012) by MTG assay, which may be due to impaired mitophagy. Thus, the combination impairs mitochondrial metabolism and function, which results in the observed increase in ROS that may account for the observed DNA damage and apoptosis. CD44/44v is critical for mitigating ROS through reduced glutathione (GSH)9, and ULK1 inhibition lowered CD44/44v transcription (qRT-PCR) and intracellular GSH in AML cells.Corroborating our earlier data, the ABT-199 resistant cells (OCIAML2R & MOLM13R) show enhanced autophagy as compared to parental cells (OCIAML2: 83%, MOLM13: 35% increase; p=0.001 & 0.009). SBI-0206965 reversed ABT-199 induced autophagy and restored ABT-199 sensitivity in these cells (Fig 4).In a pilot in vivo experiment control and ULK1 KO cells were injected in NSG mice and leukemia engraftment was markedly delayed in the ULK1 KO group (Fig 5). The therapeutic combination study is ongoing.ConclusionResults indicate concomitant targeting of autophagy by ULK1 inhibition and Bcl2 inhibition by ABT-199 can overcome acquired resistance to ABT-199. Hence, with the emergence of Bcl2 inhibitors in frontline therapy for AML and efforts at developing ULK1 inhibitors, this study informs the development of novel apoptosis/autophagy targeting approaches to improve AML therapy. [Display omitted] DisclosuresKonopleva:Stemline Therapeutics: Research Funding; abbvie: Research Funding; Immunogen: Research Funding; cellectis: Research Funding. Andreeff:Daiichi-Sankyo: Consultancy, Patents & Royalties: MDM2 inhibitor activity patent, Research Funding; United Therapeutics: Patents & Royalties: GD2 inhibition in breast cancer ; Aptose: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Research Funding; Jazz Pharma: Consultancy; SentiBio: Equity Ownership; Oncolyze: Equity Ownership; Celgene: Consultancy; Reata: Equity Ownership; Oncoceutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Eutropics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Research Funding.

Activation of ULK1 Kinase Mediates Clearance of Free Alpha-Globin in Human Beta-Thalassemic Erythroblasts

▪β-Thalassemia is a common, frequently debilitating, inherited anemia caused by HBB gene mutations that reduce or eliminate the expression of the β-globin subunit of adult hemoglobin (HbA, α2β2). Consequently, excess free α-globin forms toxic precipitates in red blood cells (RBCs) and their precursors, leading to ineffective erythropoiesis and hemolytic anemia. Previously, we showed that free α-globin is eliminated by protein quality-control pathways, including the ubiquitin-proteasome system and autophagy (Khandros et al., Blood 2012;119:5265). In β-thalassemic mice, disruption of the Unc-51-like autophagy activating kinase gene (Ulk1) increased α-globin precipitates and worsened the pathologies of β-thalassemia. Treatment of β-thalassemic mice with rapamycin to inhibit mTOR (an ULK1 inhibitor) reduced α-globin precipitates, lessened ineffective erythropoiesis, and increased the lifespan of circulating RBCs in an Ulk1-dependent fashion.To investigate the therapeutic potential of rapamycin in human β-thalassemia, we treated erythroid precursors generated by in vitro differentiation of patient-derived CD34+ hematopoietic stem and progenitor cells. Reverse-phase high-performance liquid chromatography (HPLC) analysis of hemoglobinized erythroblasts generated from transfusion-dependent (TD, n = 5) or non-transfusion-dependent (NTD, n = 5) β-thalassemia patients revealed α-chain excesses (α-chain/β-like [β + γ + δ] chain) of approximately 40% and 15%, respectively (compared to 7 normal donors; P < 0.001). Rapamycin (10µM or 20µM) or the proteasome inhibitor MG132 (2.5µM) was added to day 13 cultures, which contained mid- to late-stage erythroblasts, and α-globin accumulation was determined by HPLC 2 days later. As expected, proteasome inhibition by MG132 raised free α-globin levels in thalassemic erythroblasts (P < 0.01) and induced cell death (P < 0.01). In contrast, rapamycin reduced free α-globin in a dose-dependent manner by 40% and 85% in TD (P < 0.0001) and NTD β-thalassemia (P < 0.001), respectively, but had no effect on erythroblasts derived from normal CD34+ cells (figure). We also observed decreases in the accumulation of autophagic markers, such as SQSTM1/p62 protein, by Western blotting. We observed no negative effects of rapamycin on the survival of patient-derived erythroblasts. Also of note, under our experimental conditions, rapamycin treatment of erythroblasts did not induce fetal hemoglobin production, as has been previously reported, thereby excluding this potential mechanism for reducing globin chain imbalances. Overall, rapamycin treatment significantly reduced the accumulation of free α-globin in TD β-thalassemia and almost fully corrected the imbalance in NTD β-thalassemia cells.Our findings identify a new drug-regulatable pathway for ameliorating β-thalassemia. Rapamycin is approved and well studied, and it has a generally manageable toxicity profile. Moreover, there are additional pharmacologic approaches to activating ULK via mTOR inhibition or other pathways. These approaches may lead to effective drug therapies for β-thalassemia, particularly NTD or intermittently TD forms of the disease. [Display omitted] DisclosuresCappellini:Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Vifor: Membership on an entity's Board of Directors or advisory committees; Sanofi/Genzyme: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria.

IFN-γ-inducible antiviral responses require ULK1-mediated activation of MLK3 and ERK5.

It is well established that activation of the transcription factor signal transducer and activator of transcription 1 (STAT1) is required for the interferon-γ (IFN-γ)-mediated antiviral response. Here, we found that IFN-γ receptor stimulation also activated Unc-51-like kinase 1 (ULK1), an initiator of Beclin-1-mediated autophagy. Furthermore, the interaction between ULK1 and the mitogen-activated protein kinase kinase kinase MLK3 (mixed lineage kinase 3) was necessary for MLK3 phosphorylation and downstream activation of the kinase ERK5. This autophagy-independent activity of ULK1 promoted the transcription of key antiviral IFN-stimulated genes (ISGs) and was essential for IFN-γ-dependent antiviral effects. These findings define a previously unknown IFN-γ pathway that appears to be a key element of the antiviral response.

17 β-Estradiol Oxidative Stress Attenuation and Autophagy-Induced Dopaminergic Neuroprotection.

Objective Degeneration of dopaminergic neurons in the substantia nigra of the brain stem is the main pathological aspect of Parkinson’s disease (PD). 17 β-estradiol (E2) has neuroprotective effects on substantia nigra, however, the underlined mechanism is not well-known. In this study, we evaluated the neuroprotective effects of E2 in the ovariectomized 6-hydroxydopamine- (6-OHDA) rat model of PD. Materials and Methods In this experimental study, all animals were ovariectomized to avoid any further bias in E2 levels and then these ovariectomized rats were randomly assigned into three experimental groups (10 rats in each group): ovariectomized control group (OCG), ovariectomized degeneration group receiving 25 μg of 6-OHDA into the left corpus striatum (ODG), and ovariectomized E2 pretreatment group pretreated with 0.1 mgkg-1of 17 β-estradiol for three days prior to the destruction of corpus striatum with 6-OHDA (OE2PTG). The apomorphine behavioral test and Nissl staining were performed in all experimental groups. The expressions of Sequestosome-1 (P62), Unc- 51 like autophagy activating kinase (Ulk1), and microtubule-associated proteins 1A/1B light chain 3B (Lc3) genes were evaluated using reverse transcription- polymerase chain reaction (RT-PCR). Results E2 administration reduced the damages to the dopaminergic neurons of the substantia nigra. The motor behavior, the number of rotations, and histological tests in the treatment group showed the cell survival improvement in comparison with the control groups indicating that E2 can inhibit the neurodegeneration. P62 and Lc3 were expressed in all experimental groups while Ulk1 was not expressed in ODG group. Moreover, Ulk1 was expressed after the treatment with E2 in OE2PTG group. ConclusionE2 prevents neurodegeneration in dopaminergic neurons of the midbrain by over-expression of Ulk1 gene and augmenting the induction of autophagy.