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Pharmacological inhibition of ULK1 blocks mTOR-dependent autophagy

Katy J. Petherick1, Owen J. L. Conway1, Chido Mpamhanga2, Simon A. Osborne2, Ahmad

Kamal2, Barbara Saxty2 and Ian G. Ganley1 1From the MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences,

University of Dundee, Dundee, DD1 5EH, UK 2From MRC Technology, Centre for Therapeutics Discovery, 1-3 Burtonhole Lane, Mill Hill,

London, NW7 1AD, UK

Running title: ULK1 inhibitor

To whom correspondence should be addressed: Ian G. Ganley, MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dundee, DD1 5EH, UK. Tel.: +44(0)1382-388905; E-mail: i.ganley@dundee.ac.uk Keywords: Autophagy, ULK1, Protein kinase, inhibitor, cancer

Background: Autophagy is an intracellular lysosomal degradation pathway implicated in many diseases, but there are currently no specific autophagy inhibitors. Results: Small molecule inhibition of ULK1, the upstream autophagy initiating kinase, blocks autophagosome initiation and maturation. Conclusion: ULK1 plays a role in autophagosome maturation as well as initiation. Significance: ULK1 can be targeted to block autophagy for disease therapy. ABSTRACT Autophagy is a cell protective and degradative process that recycles damaged and long-lived cellular components. Cancer cells are thought to take advantage of autophagy to help them to cope with the stress of tumorigenesis; thus targeting autophagy is an attractive therapeutic approach. However, there are currently no specific inhibitors of autophagy. ULK1, a serine/threonine protein kinase, is essential for the initial stages of autophagy and here we report that two compounds, MRT67307 and MRT68921, potently inhibit ULK1 and 2 in vitro and block autophagy in cells. Using a drug-resistant ULK1 mutant, we show that the autophagy inhibiting capacity of the compounds is specifically through ULK1. ULK1 inhibition results in

accumulation of stalled early autophagosomal structures, indicating a role for ULK1 in the maturation of autophagosomes as well as initiation. INTRODUCTION

Macroautophagy, called autophagy in this instance, is a membrane-driven process that traffics intracellular components to the lysosome for recycling (1). It is essentially a cell survival mechanism and is upregulated under conditions of stress, allowing cells to supply building blocks and energy to maintain function during periods of starvation. Autophagy also eliminates impaired or foreign components that if left to persist could cause damage. Due to these functions, autophagy has been linked to many diseases from neurodegeneration to cancer (2). It is therefore an attractive therapeutic target, especially in cancer where it acts as a survival mechanism allowing the tumor to cope with an increased metabolic demand and damage caused by chemotherapeutics (3,4). Hence, autophagy inhibition is thought to be a viable treatment approach. However, to date there are no specific small molecule autophagy inhibitors and this has hampered validation of autophagy as a potential target in cancer. Hydroxychloroquine has entered clinical trials as an autophagy inhibitor (5-11), but it is far from specific given that it disrupts lysosomal function. The lysosome is the end point of

http://www.jbc.org/cgi/doi/10.1074/jbc.C114.627778The latest version is at JBC Papers in Press. Published on April 1, 2015 as Manuscript C114.627778

Copyright 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

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autophagy as well as most other intracellular transport pathways including endocytosis.

The most understood pathway of autophagy activation occurs upon inhibition of mTOR, either by amino acid withdrawal or a direct pharmacological block. This leads to activation of the ULK1 protein kinase complex followed by the VPS34 lipid kinase complex (12-15). This in turn leads to the recruitment of a unique ubiquitin-like conjugation system resulting in the conjugation of LC3, and its family members, to the lipid phosphatidylethanolamine in the forming autophagosome, or phagophore. This lipidated form of LC3, termed LC3-II, is thought to drive elongation and cargo incorporation into the autophagosome (16). Thus there are many potential targets to inhibit autophagy. Kinase inhibitors have proved successful in the clinic, making ULK1 and VPS34 attractive candidates for inhibition. Specific VPS34 inhibitors have recently been published and these do indeed inhibit autophagy (17-19). However, VPS34 is also essential for sorting in the endocytic pathway, meaning that like hydroxychloroquine, VPS34 inhibition also affects endocytosis and lysosomal turnover in general. ULK1 on the other hand, is believed to be specific for autophagy, although other roles cannot be ruled out at this stage (20). We therefore set out to identify compounds that could inhibit ULK1 activity, not only to act as tool reagents to aid research but importantly to provide a proof-of-principle that targeting ULK1 could block autophagy.

EXPERIMENTAL PROCEDURES Reagents and antibodies-MRT compounds were synthesized as described (21). AZD8055 was from Selleck Chemicals, Bafilomycin A1 from Enzo Life Sciences. Rabbit anti-ULK1, ATG5 and ATG13 were from Sigma. Rabbit anti-phospho S757 ULK was from Cell Signalling Technology, rabbit anti-phospho S318 ATG13 was from Abnova. Mouse anti α-tubulin was from Merck-Millipore. Mouse anti-LC3 for immunofluoresence was from MBL and sheep anti-LC3, used for immunoblotting, was generated by the Division of Signal Transduction Therapy,

University of Dundee, from recombinant full length human GST-LC3b and affinity purified. Recombinant DNA-Procedures were performed using standard protocols and mutagenesis was performed using QuikChange site-directed mutagenesis (Stratagene). Constructs are described and available on the reagents website (https://mrcppureagents.dundee.ac.uk/). Cell culture-Immortalized wild-type MEFs have been described previously (22). LKB1 MEFs were a kind gift from Tomi Mäkelä (University of Helsinki, Finland) and previously described (23). ULK1/2 double knockout MEFs were a kind gift from Craig Thompson (Memorial Sloan-Kettering Cancer Center, NY) and previously described (24). TBK1/IKKε knockout and matched MEFs were a kind gift from Shizuo Akira (Osaka University, Japan) and previously described (25). MEFs and 293T cells were grown in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% fetal bovine serum and penicillin/streptomycin and cultured at 37 °C, 5% CO2. For induction of autophagy, cells were typically grown to 75% confluency and washed twice and incubated in Earle’s balanced salt solution (EBSS) for 1h (or complete medium as a control) unless indicated. MRT67307 (10µM), MRT68921 (1µM), AZD8055 (1µM) or bafilomycin A1 (50nM) was included where indicated. Transfection and transduction was as described (15). Cell lysis, immunoprecipitation-Carried out as described (15). Immunoblots were quantified by densitometry, using ImageJ. Immunofluorescence-Carried out as described (26). Slides were visualized on a Nikon TiS inverted microscope and images processed using NIS Elements (Nikon) and Adobe Photoshop. Live cell microscopy-Cells were washed and incubated in phenol red-free media and imaged on a Nikon TiE inverted microscope with an Okolab environmental chamber at

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37°C, 5% CO2. Images were obtained and processed using NIS Elements (Nikon). Kinase assays-Initial ULK1 kinase assays were performed with GST-ULK1, produced in Sf9 cells, which is described on the reagents website (https://mrcppureagents.dundee.ac.uk/). For other experiments, recombinant GST-ULK1 (wild-type, kinase dead (K46I) and M92T and M92Q) was expressed in 293T cells, purified and eluted from a glutathione-sepharose column. Kinase assays were carried out in 50mM Tris-HCl, pH 7.4, 10mM Mg acetate, 0.1mM EGTA, and 0.1% β-mercaptoethanol, containing 30µM cold ATP, and 0.5µCi of [γ32-P]ATP for 5min at 25°C. Prior to ATP addition, reaction mixes were prewarmed to 25°C for 5min. Reactions were stopped by the addition of sample buffer, followed by SDS-PAGE, transfer to nitrocellulose, and analysis by autoradiography and immunoblot. For IC50 curve measurements, kinase assays were performed as described (27), using myelin basic protein (Sigma) as a substrate. Kinase profiling was as described (28), performed by the International Centre for Kinase Profiling (http://www.kinase-screen.mrc.ac.uk). Statistical analysis-Experiments were carried out a minimum of three times and error bars represent standard error of the mean (SEM). Significance in related samples was determined by paired student’s T-test. A p-value of 0.05 or less was deemed significant. RESULTS AND DISCUSSION

In vitro screening of known kinase inhibitors led to the discovery that the TBK1 inhibitor, MRT67307 (29) also targeted ULK1 and ULK2 with high potency (IC50 45 and 38nM respectively, Fig. 1A). It is of no surprise that a compound would target both ULK1 and 2, given the high degree of similarity between the kinase domains; it is also desirable given the degree of redundancy observed in knockout mouse models (20). In order to find more potent inhibitors, we re-analysed a closely related series of analogues generated during the original TBK1 screen. This led to the discovery of MRT68921 as the

most potent inhibitor of both ULK1 and 2, with over a 15-fold reduction in the IC50 for ULK1 (2.9nM) and 30-fold for ULK2 (1.1nM), see Fig. 1A. To see if in vitro activity of the compounds could translate to inhibition in cells, we treated mouse embryonic fibroblasts (MEFs) with either MRT67307 or MRT68921 in combination with amino acid withdrawal, to inhibit mTOR and activate ULK1. Bafilomycin A1 was also included to inhibit lysosomal turnover and enable autophagic flux measurement (30) (Fig. 1B). Loss of mTOR-mediated ULK1 phosphorylation occurred upon incubation of cells in Earle’s balanced salt solution (EBSS) and this was not prevented by treatment with either of the MRT compounds. ATG13 phosphorylation at serine 318 was used as a measure of ULK1 activity (31), and this correlated well with EBSS treatment and loss of the ULK1 mTOR phosphorylation, showing greater than a 5-fold increase over basal levels (Fig. 1B). 10µM MRT67307 was sufficient to reduce phospho-ATG13 to control levels and in-line with the in vitro IC50s, 10-fold less MRT68921 (1µM) resulted in a similar reduction. Therefore these compounds can reduce ULK1 activity in cells. Importantly, these compounds also block autophagy as indicated by LC3-II levels. Basal autophagy was low under the experimental conditions used, however EBSS treatment resulted in a 5-fold increase in bafilomycin-sensitive LC3-II levels, indicating strong mTOR-dependent autophagy induction. Both compounds blocked any bafilomycin-induced increase, demonstrating inhibition of autophagic flux. Both compounds behaved similarly and though there was no LC3-II flux, a small 2-fold increase in LC3-II levels was observed.

ULK1 is part of a multi-protein complex and at least two of these proteins, ATG13 and FIP200, are required for maximal ULK activity (14,15,32); it is possible that the MRT compounds inhibit ULK by disrupting these interactions. To look at this we stably expressed FLAG-tagged ULK1 in ULK1 and ULK2 double-knockout cells (dKO) and immunoprecipitated ULK in the presence or absence of autophagy inducing conditions. As can be seen in Figure 1C, the presence of

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inhibitor did not reduce the amounts of ATG13 and FIP200 co-precipitating with ULK1, but did reduce the level of phosphorylated ATG13 in this complex. To further assess autophagy, we looked at endogenous LC3 by immunofluorescence (Fig. 1D). Under control conditions, MRT68921 caused a slight reduction in basal LC3 puncta, indicating the compound can block basal autophagy. However, the changes were not statistically significant, which could be due to low basal level of autophagy during the treatment. In contrast and as with the western blot data, EBSS caused a large increase in bafilomycin-sensitive LC3 puncta accumulation. Likewise, this was blocked by both MRT compounds (though only MRT68921 is shown in Fig. 1D), confirming that these compounds do indeed block autophagy in cells. Also, like the western blot data (Fig. 1B), a small two-fold increase in distinct LC3 puncta was observed with the inhibitors, which may represent stalled phagophores (see below).

Both MRT compounds inhibit ULK1 in vitro and in cells, and also block autophagy. However, they could be targeting autophagy through another kinase. The in vitro kinase profiling of MRT67307 revealed that it is a relatively specific kinase inhibitor, targeting TBK1/IKKε but also hitting the AMPK-related kinases (33). To assess the specificity of MRT68921, we profiled the inhibitor at 1µM against a broad panel of 80 protein kinases representing all areas of the human kinome (Fig. 2A). As with MRT67307, MRT68921 was relatively specific, but still inhibited a number of kinases by over 80% (see dark shaded bars on Fig. 2A). Most notably, TBK1/IKKε and the AMPK-related kinases were still targeted. TBK1 and AMPK have been implicated in autophagy (34-40), so it was important to rule these out as autophagy-inhibiting targets of MRT68921. To analyse the role of TBK1, we took knockout MEFs and asked if autophagy could still be induced and inhibited by MRT68921 (Fig. 2B). To induce autophagy we utilized the mTOR inhibitor AZD8055 (41) and found in TBK1-containing cells, a large increase in LC3-II flux, which was inhibited by

MRT68921. In TBK1 knockout cells, the pattern of LC3-II accumulation was almost identical, indicating that TBK1 is not essential for mTOR-mediated autophagic flux and is not the autophagy-inhibiting target of MRT68921. Of course we do not rule out TBK1’s involvement in more specific forms of autophagy such as basal autophagy or xenophagy. However, this result highlights the caution that is needed when using TBK1 inhibitors to validate a role for TBK1 in autophagy.

To analyse AMPK-related kinase involvement we took advantage of the fact that these kinases, with the exception of AMPK (which can also be activated by CaMKK2), require LKB1-mediated phosphorylation for activation (42). Using LKB1 knockout MEFs, we found that LC3 flux was comparable to matched, wild-type MEFs and was inhibited to the same extent with MRT68921 (Fig. 2C). Therefore, these kinases are not the target of MRT68921 in blocking autophagy. Interestingly, LC3 flux was slightly higher under basal and autophagy-inducing conditions in the LKB1 knockout MEFs, which is similar to that seen in AMPK knockout cells (36). This supports the idea that AMPK, or one of the related kinases, though not essential for autophagy does play a regulatory role.

Both the MRT compounds are competitive ATP-binding site inhibitors and may inhibit autophagy through other kinases, or even non-kinase ATP-binding proteins. To rule this out and confirm ULK1 as the target, we generated a drug-resistant ULK1 mutant. The gatekeeper residue is a bulky hydrophobic amino acid present at the base of the ATP-binding pocket in all typical protein kinases. When this residue is mutated it can change the ATP-binding properties of the kinase. In order to identify residues that might result in loss of inhibitor binding, but retain ATP binding, we mutated the gatekeeper residue of ULK1 (methionine 92) to various amino acids. Mutation to alanine or glycine resulted in loss of kinase activity, but a change to threonine or glutamine resulted in an ULK1 construct that retained in vitro activity in a simple auto-phosphorylation kinase assay (Fig. 3A).

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Satisfyingly, mutation of methionine 92 to threonine (M92T) allowed ULK1 to retain kinase activity in the presence of MRT67307, at a concentration that inhibited wild-type (WT) and the glutamine mutant. To analyse the resistance of ULK1 M92T to the MRT compounds in greater detail, we characterised the IC50 for this mutant and found over a 70-fold decrease in sensitivity, confirming the drug-resistance (Fig. 3B).

We next confirmed the M92T mutant could rescue the MRT68921-mediated autophagy inhibition. For this, either WT or M92T FLAG-ULK1 was stably expressed in ULK1/2 dKO MEFs (Fig. 3C). Re-expression of WT or M92T ULK1 restored ATG13 phosphorylation and autophagy (as observed by LC3-II flux), caused by the loss of endogenous ULK1 and 2. In these cells it appears that ULK1 is sufficient to rescue autophagy, highlighting the redundancy of ULK2. Importantly, MRT68921 was able to inhibit the WT-restored ATG13 phosphorylation and autophagy similarly to cells expressing endogenous ULK1 (compare Fig. 1B with 3C). However, in cells expressing a similar level of M92T ULK1, MRT68921 failed to reduce neither ATG13 phosphorylation nor LC3 flux (Fig. 3C). This indicates that MRT68921 is indeed blocking autophagy through ULK1 kinase inhibition. To our knowledge, this is the first example of specifically inhibiting ULK1 with a small molecule to disrupt autophagy.

To confirm inhibition is through ULK1, we used the ULK1 rescue MEFs to analyse LC3 immunofluorescence (Fig. 3D). Cells expressing both WT and M92T ULK1 displayed a comparable increase in the number of bafilomycin-sensitive LC3 puncta in response to EBSS treatment. Similarly, the LC3 puncta flux was blocked by MRT68921 treatment only in the WT ULK1 expressing cells, not the M92T ULK1 cells, confirming the MRT compound blocks autophagy through ULK1 inhibition. As with the immunofluorescence analysis in cells endogenously expressing ULK (see Fig. 1D), we did see a small increase in the formation of distinct and bright LC3 puncta upon MRT68921 treatment in the WT-expressing

cells and given that no LC3 flux was observed, these structures could possibly represent stalled phagophores or immature autophagosomes. These bright puncta were not readily observed under control conditions with inhibitor indicating that stimulated autophagy differs somewhat from basal autophagy, or that the equivalent puncta that accumulate are smaller and harder to distinguish from the background staining. To gain further insight into these aberrant structures we carried out live-cell microscopy in GFP-LC3 expressing ULK1-rescue cells (see Movie1 and 2). Incubation of both WT- and M92T-ULK1 expressing cells in EBSS resulted in rapid formation of GFP-LC3 autophagosomes after as little as 15 minutes. In contrast, in WT but not M92T cells, MRT68921 treatment delayed the formation of GFP-LC3 structures, which only started to accumulate following 30 minutes of treatment. Additionally, these structures appeared brighter and morphologically distinct to those formed in the untreated WT cells (or treated/untreated M92T ULK1 cells). The reader is encouraged to watch the movies, but representative images are shown in Fig. 4A. The distinct appearance, timing and lack of flux of the inhibitor-induced LC3 structures suggested that they are not regular autophagosomes. We hypothesized that ULK1 inhibition primarily blocks autophagy induction, however some residual ULK activity remains to allow autophagosome initiation but not maturation. To analyse this further, we co-stained cells for LC3 and the phagophore markers ULK1 and ATG5 (Fig. 4B and C). We found that the majority of the inhibitor-induced LC3 structures, over 70%, contained ULK1 and ATG5, which is in contrast to structures found in the absence of inhibitor that showed less than a third co-localising with ULK1 or ATG5. Additionally, inhibitor treatment did not appear to reduce the number of ULK1 or ATG5 puncta forming upon autophagy stimulation. Firstly, this suggests that ULK activity is not essential for its recruitment to autophagosomal structures (a similar observation has been made with kinase-dead ULK1 constructs (43)). Secondly, given that the LC3 structures contain ULK1

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and ATG5, both phagophore markers (44,45), this suggests ULK1 plays a role in phagophore maturation into complete autophagosomes. This is not an unreasonable proposal as a similar phenomenon has been observed in yeast, whereby expression of a kinase dead form of Atg1 (yeast ULK) did indeed block autophagy, but also resulted in increased recruitment of Atg8 (yeast LC3 homolog) and Atg17 to the pre-autophagosomal structure (PAS) (46). Additionally, in mammalian cells, dissociation of ULK1 from the omegasome precedes autophagosome maturation (47). Our data fits well with these observations suggesting a function for Atg1/ULK1 activity in disassembly of the PAS/phagophore to allow autophagosome maturation. However, further work in characterising these structures is needed to clarify this. We therefore suggest that ULK regulates autophagy through at least two mechanisms: i) Regulation of the VPS34 complex, by AMBRA1/Beclin1 phosphorylation, upstream of LC3 conjugation (12,48); and ii) controlling maturation of the phagophore and transition to the mature

autophagosome, downstream of LC3 conjugation.

In summary, we have shown that MRT67307, and especially MRT68921, potently inhibit ULK1 and 2 in vitro and ULK1 in cells. Importantly, this was sufficient to block autophagic flux. We demonstrated specificity in the autophagy block through the generation of the drug-resistant M92T ULK1 mutant. Although it might be expected that pharmacologically inhibiting ULK1 would block autophagy, this has not been previously shown and highlights the importance of ULK kinase activity in autophagy induction. It also acts as a proof-of-principle in developing small molecule inhibitors to block autophagy for therapy in diseases such as cancer. Finally, we have developed a system to enable molecular analysis of ULK1 function in autophagy and revealed a role for ULK1 in the maturation of autophagosomes. Future work will hopefully identify the targets of ULK1 that enable the phagophore to autophagosome switch.

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A. L., Harris, J., Harris, S. D., Hashimoto, M., Haspel, J. A., Hayashi, S., Hazelhurst, L. A., He, C., He, Y. W., Hebert, M. J., Heidenreich, K. A., Helfrich, M. H., Helgason, G. V., Henske, E. P., Herman, B., Herman, P. K., Hetz, C., Hilfiker, S., Hill, J. A., Hocking, L. J., Hofman, P., Hofmann, T. G., Hohfeld, J., Holyoake, T. L., Hong, M. H., Hood, D. A., Hotamisligil, G. S., Houwerzijl, E. J., Hoyer-Hansen, M., Hu, B., Hu, C. A., Hu, H. M., Hua, Y., Huang, C., Huang, J., Huang, S., Huang, W. P., Huber, T. B., Huh, W. K., Hung, T. H., Hupp, T. R., Hur, G. M., Hurley, J. B., Hussain, S. N., Hussey, P. J., Hwang, J. J., Hwang, S., Ichihara, A., Ilkhanizadeh, S., Inoki, K., Into, T., Iovane, V., Iovanna, J. L., Ip, N. Y., Isaka, Y., Ishida, H., Isidoro, C., Isobe, K., Iwasaki, A., Izquierdo, M., Izumi, Y., Jaakkola, P. M., Jaattela, M., Jackson, G. R., Jackson, W. T., Janji, B., Jendrach, M., Jeon, J. H., Jeung, E. B., Jiang, H., Jiang, H., Jiang, J. X., Jiang, M., Jiang, Q., Jiang, X., Jiang, X., Jimenez, A., Jin, M., Jin, S., Joe, C. O., Johansen, T., Johnson, D. E., Johnson, G. V., Jones, N. L., Joseph, B., Joseph, S. K., Joubert, A. M., Juhasz, G., Juillerat-Jeanneret, L., Jung, C. H., Jung, Y. K., Kaarniranta, K., Kaasik, A., Kabuta, T., Kadowaki, M., Kagedal, K., Kamada, Y., Kaminskyy, V. O., Kampinga, H. H., Kanamori, H., Kang, C., Kang, K. B., Kang, K. I., Kang, R., Kang, Y. A., Kanki, T., Kanneganti, T. D., Kanno, H., Kanthasamy, A. G., Kanthasamy, A., Karantza, V., Kaushal, G. P., Kaushik, S., Kawazoe, Y., Ke, P. Y., Kehrl, J. H., Kelekar, A., Kerkhoff, C., Kessel, D. H., Khalil, H., Kiel, J. A., Kiger, A. A., Kihara, A., Kim, D. R., Kim, D. H., Kim, D. H., Kim, E. K., Kim, H. R., Kim, J. S., Kim, J. H., Kim, J. C., Kim, J. K., Kim, P. K., Kim, S. W., Kim, Y. S., Kim, Y., Kimchi, A., Kimmelman, A. C., King, J. S., Kinsella, T. J., Kirkin, V., Kirshenbaum, L. A., Kitamoto, K., Kitazato, K., Klein, L., Klimecki, W. T., Klucken, J., Knecht, E., Ko, B. C., Koch, J. C., Koga, H., Koh, J. Y., Koh, Y. H., Koike, M., Komatsu, M., Kominami, E., Kong, H. J., Kong, W. J., Korolchuk, V. I., Kotake, Y., Koukourakis, M. I., Kouri Flores, J. B., Kovacs, A. L., Kraft, C., Krainc, D., Kramer, H., Kretz-Remy, C., Krichevsky, A. M., Kroemer, G., Kruger, R., Krut, O., Ktistakis, N. T., Kuan, C. Y., Kucharczyk, R., Kumar, A., Kumar, R., Kumar, S., Kundu, M., Kung, H. J., Kurz, T., Kwon, H. J., La Spada, A. R., Lafont, F., Lamark, T., Landry, J., Lane, J. D., Lapaquette, P., Laporte, J. F., Laszlo, L., Lavandero, S., Lavoie, J. N., Layfield, R., Lazo, P. A., Le, W., Le Cam, L., Ledbetter, D. J., Lee, A. J., Lee, B. W., Lee, G. M., Lee, J., Lee, J. H., Lee, M., Lee, M. S., Lee, S. H., Leeuwenburgh, C., Legembre, P., Legouis, R., Lehmann, M., Lei, H. Y., Lei, Q. Y., Leib, D. A., Leiro, J., Lemasters, J. J., Lemoine, A., Lesniak, M. S., Lev, D., Levenson, V. V., Levine, B., Levy, E., Li, F., Li, J. L., Li, L., Li, S., Li, W., Li, X. J., Li, Y. B., Li, Y. P., Liang, C., Liang, Q., Liao, Y. F., Liberski, P. P., Lieberman, A., Lim, H. J., Lim, K. L., Lim, K., Lin, C. F., Lin, F. C., Lin, J., Lin, J. D., Lin, K., Lin, W. W., Lin, W. C., Lin, Y. L., Linden, R., Lingor, P., Lippincott-Schwartz, J., Lisanti, M. P., Liton, P. B., Liu, B., Liu, C. F., Liu, K., Liu, L., Liu, Q. A., Liu, W., Liu, Y. C., Liu, Y., Lockshin, R. A., Lok, C. N., Lonial, S., Loos, B., Lopez-Berestein, G., Lopez-Otin, C., Lossi, L., Lotze, M. T., Low, P., Lu, B., Lu, B., Lu, B., Lu, Z., Luciano, F., Lukacs, N. W., Lund, A. H., Lynch-Day, M. A., Ma, Y., Macian, F., MacKeigan, J. P., Macleod, K. F., Madeo, F., Maiuri, L., Maiuri, M. C., Malagoli, D., Malicdan, M. C., Malorni, W., Man, N., Mandelkow, E. M., Manon, S., Manov, I., Mao, K., Mao, X., Mao, Z., Marambaud, P., Marazziti, D., Marcel, Y. L., Marchbank, K., Marchetti, P., Marciniak, S. J., Marcondes, M., Mardi, M., Marfe, G., Marino, G., Markaki, M., Marten, M. R., Martin, S. J., Martinand-Mari, C., Martinet, W., Martinez-Vicente, M., Masini, M., Matarrese, P., Matsuo, S., Matteoni, R., Mayer, A., Mazure, N. M., McConkey, D. J., McConnell, M. J., McDermott, C., McDonald, C., McInerney, G. M., McKenna, S. L., McLaughlin, B., McLean, P. J., McMaster, C. R., McQuibban, G. A., Meijer, A. J., Meisler, M. H., Melendez, A., Melia, T. J., Melino, G., Mena, M. A., Menendez, J. A., Menna-Barreto, R. F., Menon, M. B., Menzies, F. M., Mercer, C. A., Merighi, A., Merry, D. E., Meschini, S., Meyer, C. G., Meyer, T. F., Miao,

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Acknowledgements-We thank the Ganley lab for discussions and manuscript reading. This work was supported by the Medical Research Council and by the Division of Signal Transduction Therapy Unit (including AstraZeneca, Boehringer-Ingelheim, GlaxoSmithKline, Merck KGaA, Janssen Pharmaceutica and Pfizer). FOOTNOTES The Abbreviations used are: DMEM, Dulbecco’s modified Eagles medium; EBSS, Earle’s balanced salt solution; MEF, mouse embryonic fibroblast; dKO, double-knockout; ULK, Unc51-like kinase, TBK1, TANK-binding kinase 1, LKB1, Liver kinase B1, mTOR, mammalian target of rapamycin; AMPK, AMP-activated protein kinase. FIGURE LEGENDS FIGURE 1. MRT67307 and MRT68921 inhibit ULK and block autophagy in cells. A. Summary of MRT67307 and 68921 structures and in vitro ULK1 and 2 IC50s. B. MEF cells were incubated in EBSS for 1h in the presence or absence of either 10µM MRT67307, 1µM MRT68921 or 50nM Bafilomycin A1, followed by lysis and immunoblotting with the indicated antibodies (* indicates non-specific staining). Quantitation of phospho-serine 318 of ATG13, normalised to total ATG13, is shown below next to quantitation of LC3-II levels, normalised to tubulin. C. Representative blot of a FLAG immunoprecipitation from ULK1/2 double knockout MEFs (ULK dKO) expressing FLAG-ULK1. D. Representative micrographs of endogenous LC3 immunofluorescence in MEFs incubated in complete media (control) or EBSS for 1h in the presence or absence of 1µM MRT68921 and 50nM Bafilomycin A1. Quantitation of LC3 puncta is shown on the right. All quantitation represent means ±SEM from at least three independent experiments. *P<0.05. Scale bar, 10µm. FIGURE 2. Kinase selectivity of MRT68921. A. Activity of 80 recombinant kinases measured in the presence of 1µM MRT68921. Data represent percent activity remaining relative to activity in the absence of inhibitor. Darker shaded bars to the right indicate kinases that were inhibited by over 80%. B. TBK1 knockout (KO) and matched wild type (WT) MEFs were incubated with/without 1µM AZD8055 in the presence or absence of 50nM bafilomycin (baf) and 1µM MRT68921. Representative immunoblot is shown with quantitation of LC3-II levels normalised to tubulin above. C. Similar to panel B, but LKB1 knockout and matched wild type MEFs were used. All quantitations are from three independent experiments ±SEM. *P<0.05, ***P<0.001. FIGURE 3. MRT68921 specifically blocks autophagic flux through ULK1 inhibition. A. In vitro ULK1 autophosphorylation kinase assay in the presence of 1µM MRT67307 with GST-tagged wild type (WT), kinase-dead (KD (K46I)), M92T and M92Q ULK1. B. In vitro IC50 plot with GST-tagged WT and M92T ULK1 in the presence of MRT68921. C. ULK1/2 double knockout (dKO) MEFs stably expressing FLAG-tagged WT or M92T ULK1, were treated with EBSS for 1h in the presence or absence of 1µM MRT68921 and 50nM bafilomycin. Lysates were subject to immunoblotting with the indicated antibodies. Quantitation of LC3-II levels, normalised to tubulin is shown on the right. D. Endogenous LC3 immunofluorescence in ULK dKO MEFs rescued with the indicated FLAG-tagged ULK1 construct. Cells were incubated in complete media (control) or EBSS for 1h in the presence or absence of 1µM MRT68921 and 50nM bafilomycin (baf). Quantitation of LC3 puncta is shown on the right. All quantitation represent means ±SEM from at least three independent experiments. *P<0.05. Scale bar, 10µm. FIGURE 4. ULK inhibition also disrupts autophagosome maturation. A. ULK1-rescue MEFs stably expressing GFP-LC3 were washed and placed in complete media or EBSS with/without 1µM MRT68921 and images taken every 30 sec for 90 min. Representative images are shown from EBSS-treated samples, derived from Movie1 (WT ULK, top panels) or Movie2 (M92T ULK1, bottom panels). B. MEF cells were incubated in EBSS for 1h with/without 1µM

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MRT68921 followed by fixing and staining with antibodies to LC3 (red) or ULK1 (green). Quantitation of the degree of co-localisation is shown to the right. C. Cells, treated as in B, stained for LC3 (red) and ATG5 (green). All quantitation represent means ±SEM from at least three independent experiments. *P<0.05, n/s not significant. Scale bars, 10µm. Movie1. GFP-LC3 expressing WT-ULK1 rescue MEFs were incubated in complete media (ctrl) or EBSS with/without 1µM MRT68921 and imaged every 30sec. Scale bar, 10µm. Movie2. GFP-LC3 expressing M92T-ULK1 rescue MEFs were incubated in complete media (ctrl) or EBSS with/without 1µM MRT68921 and imaged every 30sec. Scale bar, 10µm.

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Kamal, Barbara Saxty and Ian G. GanleyKaty J. Petherick, Owen J. L. Conway, Chido Mpamhanga, Simon A. Osborne, Ahmad

Pharmacological Inhibition of ULK1 Blocks mTOR-Dependent Autophagy

published online April 1, 2015J. Biol. Chem. 

  10.1074/jbc.C114.627778Access the most updated version of this article at doi:

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VOLUME 290 (2015) PAGES 11376 –11383DOI 10.1074/jbc.A114.627778

Pharmacological inhibition of ULK1 kinase blocks mammalian target of rapamycin (mTOR)-dependent autophagy.Katy J. Petherick, Owen J. L. Conway, Chido Mpamhanga, Simon A. Osborne, Ahmad Kamal, Barbara Saxty, and Ian G. Ganley

PAGE 11378:

In Fig. 1A, the molecular structures of the compounds for MRT67307 and MRT68921 were mistakenly switched. The corrected figure is shownbelow. This correction does not affect the interpretation of the results or conclusions of this work.

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THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 290, NO. 48, p. 28726, November 27, 2015© 2015 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

28726 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290 • NUMBER 48 • NOVEMBER 27, 2015

ADDITIONS AND CORRECTIONS

Authors are urged to introduce these corrections into any reprints they distribute. Secondary (abstract) services are urged to carry notice ofthese corrections as prominently as they carried the original abstracts.