Autophagy-Dependent Metabolic Reprogramming Sensitizes TSC2-Deﬁcient Cells to the Antimetabolite 6-Aminonicotinamide

Published OnlineFirst December 2, 2013; DOI: 10.1158/1541-7786.MCR-13-0258-T

Molecular Cancer Cell Cycle and Senescence Research

Autophagy-Dependent Metabolic Reprogramming Sensitizes TSC2-Deﬁcient Cells to the Antimetabolite 6-Aminonicotinamide

Andrey A. Parkhitko1,4,7, Carmen Priolo1,4, Jonathan L. Coloff3,4, Jihye Yun4,5, Julia J. Wu6, Kenji Mizumura1,4, Wenping Xu1,4, Izabela A. Malinowska2,4, Jane Yu1,4, David J. Kwiatkowski2,4, Jason W. Locasale8, John M. Asara4,5, Augustine M.K. Choi1,4, Toren Finkel6, and Elizabeth P. Henske1,4

Abstract The mammalian target of rapamycin complex 1 (mTORC1) is hyperactive in many human cancers and in tuberous sclerosis complex (TSC). Autophagy, a key mTORC1-targeted process, is a critical determinant of metabolic homeostasis. Metabolomic profiling was performed to elucidate the cellular consequences of autophagy dysregulation under conditions of hyperactive mTORC1. It was discovered that TSC2-null cells have distinctive autophagy-dependent pentose phosphate pathway (PPP) alterations. This was accompanied by enhanced glucose uptake and utilization, decreased mitochondrial oxygen consumption, and increased mitochondrial reactive oxygen species (ROS) production. Importantly, these findings revealed that the PPP is a key autophagy-dependent compensatory metabolic mechanism. Furthermore, PPP inhibition with 6-aminonicotinamide (6-AN) in combination with autophagy inhibition suppressed proliferation and prompted the activation of NF-kB and CASP1 in TSC2-deficient, but not TSC2-proficient cells. These data demonstrate that TSC2-deficient cells can be therapeutically targeted, without mTORC1 inhibitors, by focusing on their metabolic vulnerabilities. Implications: This study provides proof-of-concept that therapeutic targeting of diseases with hyperactive mTORC1 can be achieved without the application of mTORC1 inhibitors. Mol Cancer Res; 12(1); 48–57. 2013 AACR.

Introduction Tuberous sclerosis complex (TSC) is an autosomal dom- inant syndrome caused by germline mutations of the TSC1 Autophagy is a catabolic process leading to the degrada- – tion of cytoplasmic content in lysosomal compartments, and TSC2 tumor suppressor genes (3). Patients with TSC thereby providing an endogenous source of nutrients for develop histologically benign but life-threatening proliferative energy production under stress conditions and allowing lesions in multiple organs, including the brain, heart, lung, and dysfunctional organelles, including mitochondria, to be kidney. The TSC1 and TSC2 gene products form a complex "recycled" (1). Tumor cells often upregulate autophagy to that suppresses mammalian target of rapamycin complex 1 promote survival and drug resistance (2). The role of (mTORC1). mTORC1 is a major regulator of protein trans- autophagy in cancer therapy is an area of active clinical lation, autophagy, and metabolism. Hyperactive mTORC1 investigation. occurs in the tumors that develop in patients with TSC and in many human cancers, leading to differential metabolic requirements in the setting of low autophagy levels (4, 5). Autophagy inhibition has been shown to suppress the Authors' Afﬁliations: 1Division of Pulmonary and Critical Care Medicine; 2Division of Translational Medicine, Brigham and Women's Hospital; growth of tumor cells (6). We previously found that con- 3Department of Cell Biology, 4Harvard Medical School; 5Division of Signal stitutive activation of mTORC1 in TSC2-null cells leads to Transduction, Beth Israel Deaconess Medical Center, Boston, Massachu- setts; 6National Heart Lung and Blood Institute; 7Russian State Medical decreased levels of basal and stress-induced autophagy, and University, Moscow, Russia; and 8Division of Nutritional Sciences, Cornell increases their vulnerability to further autophagy inhibition University, New York, New York (7), yet the metabolic consequences of autophagy inhibition Note: Supplementary data for this article are available at Molecular Cancer in the context of hyperactive mTORC1 are not fully under- Research Online (http://mcr.aacrjournals.org/). stood. Moreover, the metabolic pathways used by tumor A.A. Parkhitko and C. Priolo contributed equally to this work. cells to maintain growth and survival when autophagy is inhibited are poorly characterized. We report here that the Corresponding Authors: Carmen Priolo, Brigham and Women's Hospital, Harvard Medical School, 1 Blackfan Circle, Boston, MA 02115. Phone: 617- metabolic reprogramming induced by autophagy inhibition 355-9012; Fax: 617-355-9016; E-mail: [email protected]; and Elizabeth leads to pentose phosphate pathway (PPP) "addiction" P. Henske; E-mail: [email protected] selectively in Tsc2-deﬁcient cells. We also provide proof- doi: 10.1158/1541-7786.MCR-13-0258-T of-concept that the metabolic consequences of mTORC1 2013 American Association for Cancer Research. hyperactivation can be therapeutically targeted without

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Autophagy and Glucose Metabolism

inhibiting mTORC1 itself, thereby revealing novel thera- ion mode. The dwell time was 3 ms per SRM transition and peutic strategies for TSC. the total cycle time was 1.55 seconds. Approximately 10 to 14 data points were acquired per detected metabolite. Experimental Procedures Samples were delivered to the Mass Spectrometry (MS) via Cell lines normal phase chromatography using a 4.6 mm i.d. 10 cm þ þ m Tsc2 / p53 / and Tsc2 / p53 / mouse embryonic Amide Xbridge HILIC column (Waters Corp.) at 350 L/ fibroblasts (MEF) were the gift of D.J. Kwiatkowski (Harvard min. Gradients were run starting from 85% buffer B (HPLC Medical School, Boston, MA) and were confirmed to exhibit grade acetonitrile) to 42% B from 0 to 5 minutes; 42% B to Tsc2 deficiency and constitutive activation of mTORC1 by 0% B from 5 to 16 minutes; 0% B was held from 16 to 24 immunoblotting of tuberin. Tsc2 deficiency and constitutive minutes; 0% B to 85% B from 24 to 25 minutes; 85% B was activation of mTORC1 were confirmed by immunoblotting held for 7 minutes to reequilibrate the column. Buffer A was of tuberin and phospho-S6, respectively. The novel Tsc2-null comprised of 20 mmol/L of ammonium hydroxide/20 þ ¼ cystadenoma cell line, 105K, was derived from a Tsc2 / mmol/L of ammonium acetate (pH 9.0) in a water: C57Bl/6 mouse renal tumor and isolated in the laboratory acetonitrile ratio of 95:5. Peak areas from the total ion of Dr. Elizabeth Henske. These cells were confirmed to current for each metabolite SRM transition were integrated have loss of the second allele of Tsc2 by PCR, loss of tuberin using the MultiQuant v2.0 software (AB/SCIEX). expression, and increased phospho-S6 levels by immunoblot- The resulting raw data from the MultiQuant software ting. All cells were cultured in Dulbecco's Modified Eagle were normalized by total proteins and then uploaded in Medium (DMEM) supplemented with 10% FBS, 100 metaboanalyst (http://www.metaboanalyst.ca/MetaboAna- mg/mL of penicillin, and 100 mg/mL of streptomycin. lyst/) for subsequent data processing and analyses. Raw and normalized/processed data are reported in Supplementary Drugs and shRNA Table S1. Metabolites that were not detected in at least one Chloroquine diphosphate salt, bafilomycin A, and 6-ami- sample were excluded from the analysis. Data were normal- nonicotinamide (6-AN) were obtained from Sigma-Aldrich. ized to the median (per sample) and processed through log Spautin-1 was obtained from Cellagen Technology. Short transformation. Heat map and hierarchical clustering was hairpin RNAs (shRNA) against Atg5 (TRCN0000099434 generated using Pearson correlations and the Ward method. and TRCN0000099431), LAMP2a (TRCN0000086974), Metabolite Set Enrichment Analysis (MSEA) was performed Beclin1 (TRCN0000087290), or control (shGFP) were using Metaboanalyst that exploits the Kyoto Encyclopedia of obtained from the RNAi Consortium (TRC). Genes and Genome (KEGG) (http://www.genome.jp/kegg/ pathway.html) pathway database. Fifty-eight metabolites Mice significantly increased in Tsc2 / MEF with chloroquine þ Tsc2 / mice were studied in the A/J genetic background. treatment compared with vehicle (P < 0.05, calculated by þ The severity of renal lesions in the Tsc2 / mice was scored the Student t test with univariate analysis and equal group using a quantitative index as previously described (8), variation) were used for MSEA analysis. For reference meta- incorporating standardized gross inspection and microscopic bolites, a total of 235 metabolites were used. Metabolite sets histologic parameters. The animal studies were approved containing at least 5 compounds were used in the analysis. by the Children's Hospital Boston Animal Care and Use MSEA calculates hypergeometrics test score based on cumu- Committee. lative binominal distribution. Only 3 metabolites were found þ þ increased in Tsc2 / MEFs upon chloroquine treatment. Metabolite profiling Cells were harvested in quadruplicate, and intracellular Nutrient consumption metabolites extracted using 4 mL of cold (80C) 80% Metabolite levels in the medium were measured using the volume for volume (v/v) aqueous methanol. Cells and the Yellow Springs Instruments (YSI) 7100 as previously metabolite-containing supernatants were collected into con- described (9). ical tubes. Insoluble material in lysates was centrifuged at 2,000 g for 15 minutes, and the resulting supernatant was Glucose and fatty acid oxidation evaporated using a refrigerated speed vac. Samples were Cells were seeded in 12-well plates, treated for 24 hours, resuspended using 20 mL high-performance liquid chroma- and incubated for 3 hours at 37C with either 1 mCi/mL of tography (HPLC) grade water for mass spectrometry. Then, [14C(U)]-labeled D-glucose or [14C(U)]-labeled palmitate 10 mL was injected and analyzed using a 5500 QTRAP triple (American Radiolabelled Chemicals Inc.). Then, 3 mol/L of quadrupole mass spectrometer (AB/SCIEX) coupled to a perchloric acid was added to the culture media and the dishes Prominence UFLC HPLC system (Shimadzu) via selected were sealed with a phenylethylamine (Sigma-Aldrich)-satu- fi 14 reaction monitoring (SRM) of a total of 254 endogenous rated Whatman lter paper to capture C-CO2, as previ- water-soluble metabolites for steady-state analyses of sam- ously described (10). Following 3-hour incubation at room ples. Some metabolites were targeted in both positive and temperature on a gentle shaker, the filter paper was removed, negative ion mode for a total of 287 SRM transitions using placed into Ultima Gold F Scintillation Fluid (PerkinElmer pos/neg polarity switching. Electrospray (ESI) voltage was Inc.), and radioactivity counts were read in a 1600 liquid þ4,900 V in positive ion mode and 4,500 V in negative scintillation analyzer (Packard).

www.aacrjournals.org Mol Cancer Res; 12(1) January 2014 49

Downloaded from mcr.aacrjournals.org on September 30, 2021. © 2014 American Association for Cancer Research. 50 o acrRs 21 aur 2014 January 12(1) Res; Cancer Mol akik tal. et Parkhitko Downloaded from D C A Log2 (fold change) Cell proliferation (%) 100 120 –1.5 –1.0 –0.5 20 40 60 80 1.5 0.0 0.5 1.0 0 Coenzyme A N-Carbamoy UDP-N-acet Pyrophosph Glyceropho aminoimida 4-aminobut asparagine Methionine phenylalan Carbamoyl acetylphos ribose-pho 2-Isopropy Methylcyst Glutathion cystathion 2,3-dihydr L-arginino Indole-3-c glutathion glutathion O8P-O1P Imidazole shikimate threonine glycerate Uric acid oto CQ Control cytosine histidine tyrosine proline lactate purine dTDP dATP Octulose-1,8- UMP OBP uricil IMP biphosphate 0 123 4 31247568 Published OnlineFirstDecember2,2013;DOI:10.1158/1541-7786.MCR-13-0258-T Phospho-S6 mcr.aacrjournals.org (235/236) * β-Actin Tsc2 Tsc2 Tsc2 Tsc2 CQ +/+ –/– –/– +/+ CQ CQ –1.5 –1.0 –0.5 –+ 0.0 0.5 1.0 1.5 Days oto CQ Control Deoxyribose- phosphate on September 30, 2021. © 2014American Association for Cancer Research. CQ Control * –1.5 –1.0 –0.5 0.0 0.5 1.0 1.5 D-sedoheptulose-1,7- oto CQ Control biphosphate B Pentose phosphatepathway Amino sugarmetabolism * Pyrimidine metabolism Aspartate metabolism

Pyruvate metabolism Microscopic Macroscopic Protein biosynthesis tumor score tumor score 10 20 30 40 Rna transcription 0 0 1 2 3 4 5

–1.5 –1.0 –0.5 Control 1.0 1.5 0.0 0.5 oto CQ Control phosphate Ribose-5- 0

** CQ CQ . . . . 1.21.0 1.4 0.8 0.6 0.4 0.2 oeua acrResearch Cancer Molecular -log10 (Pvalue) –1.5 –1.0 –0.5 CQ Control 1.5 0.0 0.5 1.0 oto CQ Control Fructose-1,6- biphosphate * Published OnlineFirst December 2, 2013; DOI: 10.1158/1541-7786.MCR-13-0258-T

Autophagy and Glucose Metabolism

Oxygen consumption version 5.04 for Windows; GraphPad Software, www.graph- Oxygen consumption was measured under basal condi- pad.com). Significance was defined as P < 0.05. Data are tions or in the presence of the Trifluorocarbonylcyanide shown as means SD. phenylhydrazone (FCCP) using the Seahorse Bioscience XF24 analyzer as previously described (7). Levels of oxygen Results consumption were normalized to cell number. Autophagy inhibition suppresses proliferation and tumorigenesis of Tsc2-null cells and leads to metabolic ROS production reprogramming Mitochondrial reactive oxygen species (ROS) were mea- To investigate the impact of autophagy suppression on the sured by using MitoSOX (Invitrogen) staining (5 mmol/L proliferation and metabolism of cells with hyperactive for 15 minutes at 37 C). Cells were washed with PBS, mTORC1 signaling, we used chloroquine, which raises trypsinized and resuspended in PBS containing 1% FBS. lysosomal pH and inhibits the fusion between autophago- fl Data were acquired with a BD FACSCanto II ow cytometer somes and lysosomes, to treat Tsc2-deficient MEFs in (BD Biosciences) and analyzed with FlowJo analytical soft- monolayer culture. Chloroquine partially suppressed cell ware (Treestar; ref. 11). proliferation (38% after 96 hours of treatment, P < 0.05; Fig. 1A and Supplementary Fig. S1A) in a TSC2-dependent Crystal violet staining þ/ manner (Fig. 1A). Next, we treated Tsc2 mice, which Cells were plated into 96-well plates (1,000 cells/well). fi develop spontaneous renal cystadenomas, with chloroquine After treatment, cells were xed with 10% formalin for 5 (50 mg/kg/day, intraperitoneally, 5 days/week) for 4 minutes, stained with 0.05% crystal violet in distilled months. Chloroquine decreased the number of macroscopic water for 30 minutes, washed 2 times with tap water, and microscopic renal lesions by approximately 50% (Fig. and drained. Crystal violet was solubilized with 100 mLof 1B). Chloroquine treatment did not affect body weight methanol and the plate was read with a BioTek plate (Supplementary Fig. S1B). reader [optical density (OD) 540]. To identify compensatory metabolic mechanisms activated upon suppression of autophagy, metabolite profiling of Antibodies and immunoblot analysis m Phospho-S6 (Ser235/236), phospho-S6K (Thr 389), total Tsc2-null MEFs treated with chloroquine (5 mol/L) was S6, total S6K, phospho-4E-BP (Thr 37/46), total p65, performed. Chloroquine induced extensive alterations, b-actin, and phospho-p65 antibodies were obtained from including changes in the glucose, amino acid, and nucleotide Cell Signaling Technology, tuberin antibody from Abcam, utilization/biosynthesis pathways (Fig. 1C and Supplemen- Beclin-1 and caspase-1 antibodies from Santa Cruz, LC3 tary Table S1). Importantly, mTORC1 activity was not antibody from Novus Biologicals, Atg5 antibody from affected by chloroquine as assessed by the phosphorylation of Sigma, and LAMP2A antibody from Invitrogen. For immu- ribosomal protein S6 (Fig. 1C). MSEA applied to metabolites whose levels increased in chloroquine-treated Tsc2-null noblot analyses, cells were washed with PBS and harvested in fi a lysis buffer containing Nonidet P-40. Whole-cell lysates cells identi ed the PPP as the most enriched metabolic were resolved by electrophoresis, and proteins were trans- pathway (Fig. 1C and D). In contrast, metabolites of the fl PPP were either unchanged or decreased upon treatment ferred onto polyvinylidene di uoride membrane (Immobi- þ/þ lon P; Millipore), blocked in Tris-buffered saline Tween-20 with chloroquine in Tsc2 cells (Supplementary Fig. S2 buffer (Cell Signaling Technology), and probed with the and Supplementary Table S1). indicated antibodies in this buffer. Autophagy inhibition enhances glucose uptake and IL-6 measurement glucose oxidative metabolism via non-mitochondrial IL-6 levels were measured in the conditioned media using pathways We next measured uptake of glucose and secretion of a Luminex LX100 instrument. þ þ lactate in Tsc2 / and Tsc2 / MEFs treated for 24 hours Statistical analysis with chloroquine. Interestingly, glucose uptake was signif- – icantly increased by chloroquine in Tsc2-null cells (P < One- and two-sample t tests and the Mann Whitney test þ þ were used for in vitro and in vivo studies (GraphPad Prism 0.05; Fig. 2A) but not Tsc2 / cells. The amount of lactate

Figure 1. Chloroquine (CQ) inhibits the proliferation and tumorigenicity of Tsc2-deficient cells and reprograms their metabolism. A, proliferation of Tsc2 / and þ þ Tsc2 / MEFs treated with chloroquine (5 mmol/L) for 4 days (crystal violet staining). Time points represent average of three independent experiments SD. Comparison of Tsc2 / treated with chloroquine versus control: , P < 0.05. B, macroscopic (top) and microscopic (bottom) score of spontaneous renal þ tumors in Tsc2 / mice (n ¼ 15 for macroscopic and n ¼ 8 for microscopic score) following 4 months of intraperitoneal treatment with chloroquine (50 mg/kg/day, 5 days/week) or placebo (n ¼ 14 for macroscopic and n ¼ 14 for microscopic score). Photographs show representative kidneys of a control mouse and a chloroquine-treated mouse. Arrows, macroscopic lesions. , P < 0.01. C, heat map showing the top 40 metabolites significantly changed in Tsc2 / MEFs treated with chloroquine (5 mmol/L) for 24 hours (n ¼ 4 samples) versus control (n ¼ 4 samples; left). Immunoblot analysis of phospho-S6 Ser 235/236 in Tsc2 / MEFs treated with chloroquine (5 mmol/L) for 24 hours. Metabolic Set Enrichment Analysis (MSEA) of the Tsc2 / MEFs treated with chloroquine versus control (right). D, box plots of individual pentose phosphate pathway metabolites that were significantly changed in Tsc2 / MEFs treated with chloroquine (5 mmol/L) for 24 hours.

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Parkhitko et al.

A Glucose uptake Lactate secretion B Control CQ 3.0 4.5 140 2.5 4.0 3.5 120 2.0 3.0 100 1.5 2.5 80 2.0 60 1.0 1.5 Arbitrary units Arbitrary units 40 1.0 0.5 Arbitrary units 0.5 20 0 0 0 Control CQ Control CQ Control Atg5-1 Atg5-2 Beclin1 Basal FCCP-induced Tsc2–/– Tsc2+/+ shRNA O2 consumption

Figure 2. Autophagy inhibition enhances glucose uptake and suppresses oxygen consumption. A, glucose uptake and lactate secretion measured by YSI in þ þ Tsc2 / and Tsc2 / MEFs treated with chloroquine (CQ, 5 mmol/L) or control for 24 hours (left) or infected with shRNA against the autophagy genes Atg5 (Atg5-1 and Atg5-2) or Beclin1 (right). Bars, average of 4 independent samples SD. , P < 0.05; , P < 0.01. B, intact cellular respiration measured using the Seahorse Bioscience XF24 analyzer, under basal conditions or in the presence of FCCP in Tsc2 / MEFs treated with chloroquine (5 mmol/L) for 24 hours. Levels of oxygen consumption were normalized to cell number. Bars, average of 3 independent experiments SD. , P < 0.05.

in the media was unchanged, indicating that the chloro- inhibited by shRNA also exhibited signiﬁcantly higher quine-induced glucose uptake in Tsc2 / cells is not asso- glucose consumption relative to control shRNA, with no ciated with increased aerobic glycolysis. Tsc2 / MEFs in increase in lactate secretion (Fig. 2A and Supplementary Fig. which the autophagy gene Atg5 or Beclin 1 (Atg6) was S3), consistent with the chloroquine results.

A D[U-14C]glucose B [14C]palmitate 1.4 1.4

1.2 2

2 1.2 1.0 1.0 * C-CO C-CO 0.8 0.8 14 14 0.6 0.6 0.4 0.4 Relative Relative 0.2 0.2 0 0.0 Control CQ Control CQ Tsc2–/– Tsc2–/– C D D[U-14C]glucose D[U-14C]glucose [14C]palmitate 1.4 1.4 4.0

3.5 2 1.2 2 1.2 2 3.0 1.0 1.0

C-CO C-CO C-CO 2.5 0.8 0.8 14 14 14 2.0 0.6 0.6 *** 1.5 0.4 0.4 1.0 Relative Relative Relative 0.2 0.2 0.5 0.0 0.0 0 Control CQ Rapa CQ+Rapa Control CQ Control CQ Tsc2–/– Tsc2+/+ Tsc2+/+

14 Figure 3. Autophagy inhibition enhances glucose oxidative metabolism in mTORC1-dependent manner. A, glucose oxidation measured by C-CO2 production in Tsc2 / MEFs following 24-hour treatment with chloroquine (CQ, 5 mmol/L) and 3-hour labeling with D[U-14C]glucose (left). Bars, average of 5 14 / independent experiments SD. , P < 0.01. B, fatty acid oxidation measured by C-CO2 production in Tsc2 MEFs following 24-hour treatment with chloroquine (5 mmol/L) and 3-hour labeling with [U-14C]palmitate (right). Bars, average of 4 independent experiments SD. , P < 0.05. C, glucose oxidation 14 / measured by C-CO2 production in Tsc2 MEFs following 24-hour treatment with chloroquine (5 mmol/L), rapamycin (20 nmol/L), or both and 3-hour labeling with D[U-14C]glucose. Bars, average of 3 independent experiments SD. , P < 0.05; , P < 0.01. D, glucose and fatty acid oxidation measured 14 þ/þ by C-CO2 production in Tsc2 MEFs following 24-hour treatment with chloroquine (5 mmol/L) and 3-hour labeling with D[U-14C]glucose (left) or [U-14C]palmitate (right). Bars, average of 3 independent experiments SD.

52 Mol Cancer Res; 12(1) January 2014 Molecular Cancer Research

Autophagy and Glucose Metabolism

A B

Tsc2–/– Control CQ 6-AN Both 120 Tsc2+/+ 100 –/–

80 Tsc2

Figure 4. Autophagy inhibition and 60 6-AN act synergistically to suppress the growth of Tsc2-null 40 cells. A and B, proliferation of / þ/þ +/+ Tsc2 and Tsc2 MEFs treated Cell proliferation (%) 20 m

with chloroquine (CQ, 5 mol/L), 6- Tsc2 AN (10 mmol/L), or both for 4 days, 0 measured by crystal violet staining. Control CQ 6-AN Both Right: crystal violet staining. MEFs , P < 0.01. C, proliferation of C D þ þ Tsc2 / and Tsc2 / MEFs treated Tsc2–/– 120 120 with the autophagy inhibitor Tsc2+/+ m spautin-1 (5 mol/L), 6-AN (10 100 100 mmol/L), or both for 4 days (crystal violet staining). , P < 0.01. 80 80 D, proliferation of Tsc2 / MEFs treated with chloroquine (5 mmol/L) 60 60 plus 6-AN (10 mmol/L), NADPH (0.1 mmol/L) alone, or all three 40 40 for 4 days (crystal violet staining). Cell proliferation (%) 20 Cell proliferation (%) 20

0 0 Control Spautin 6-AN Both

Control NADPH CQ + 6-AN

CQ + 6-AN + NADPH

To deﬁne the metabolic routes to which glucose is directed Together, these results suggest that autophagy inhibition when autophagy is inhibited, we assayed oxidative metab- in Tsc2-null cells leads to increased glucose utilization via the olism. First, we measured the oxygen consumption rate of PPP, coupled with decreased oxygen consumption, in an cells treated with chloroquine. After 24 hours treatment with mTORC1-dependent manner. chloroquine, Tsc2 / MEFs displayed a signiﬁcant suppression of both basal and maximal (FCCP-induced) oxygen Autophagy inhibition sensitizes Tsc2-null cells to 6-AN consumption rate (Fig. 2B), consistent with our previous On the basis of the above results, we hypothesized that results in Tsc2-null rat leiomyoma ELT3 cells (7). A con- Tsc2-null cells become dependent on the PPP under autop- comitant increase in mitochondrial ROS was observed in hagy inhibition. To test this hypothesis, we used the anti- Tsc2 / MEFs treated with chloroquine for 24 hours metabolite 6-AN, a competitive inhibitor of glucose-6- (Supplementary Fig. S4), suggesting progressive accumula- phosphate dehydrogenase (G6PD) and 6-phosphogluconate tion of damaged mitochondria. dehydrogenase (6PGD), the two key enzymes of the oxida- Next, we dissected changes in oxidative utilization of tive branch of the PPP. G6PD catalyzes the conversion of 14 individual nutrients by measuring C-CO2 release from glucose-6-phosphate into 6-phosphogluconolactone, which cells labeled for 3 hours with D[U-14C]glucose or is the rate-limiting step of the pentose phosphate shunt, and [U-14C]palmitate. Consistent with the metabolomic results, 6PGD the subsequent oxidation of 6-phosphogluconolac- chloroquine induced an increase in glucose oxidation selec- tone. Cells were treated with chloroquine, 6-AN, or both tively in Tsc2-null cells (P < 0.05; Fig. 3A and D). In drugs for 96 hours. Strikingly, the chloroquine–6-AN com- addition, fatty acid oxidation decreased in chloroquine- bination selectively inhibited the proliferation of Tsc2 / treated Tsc2-null cells (P < 0.05; Fig. 3B and D), consistent cells (Fig. 4A and B). No consistent effect on mTORC1 with the reduction in oxygen consumption rate. Treatment signaling was noted (Supplementary Fig. S5A). To validate with rapamycin markedly reduced glucose oxidation even in these results, we used a mechanistically distinct inhibitor of the presence of chloroquine (P < 0.05; Fig. 3C), suggesting autophagy, spautin-1, which promotes the proteasomal mTORC1 dependence. degradation of Beclin 1 (12). Consistent with the

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A 120 100 80 * 60 ** ** 40 Figure 5. Genetic inhibition of 20 Cell proliferation (%) macro- or chaperone mediated- 0 autophagy sensitize Tsc2-null cells Control 6-AN +6-AN +6-AN +6-AN to 6-AN. A, proliferation of Tsc2 / Atg5-1 Atg5-2 Lamp2A MEFs infected with control shRNA or shRNA against Atg5 (Atg5-1 and B Atg5-2) or Lamp2A and treated with 6-AN (10 mmol/L) for 4 days shRNA Control Atg5-1 Atg5-2 (crystal violet staining). Time points represent average of 3 BafilomycinA + ++shRNA Control Lamp2A independent experiments SD. , P < 0.05; , P < 0.01. B, Atg5 Lamp2A immunoblot analysis of Atg5 (left) and Lamp2A (right) in shRNA- infected Tsc2 / MEFs. C, β β / -Actin -Actin autophagy ﬂux analysis in Tsc2 MEFs infected with control shRNA or shRNA against Atg5 (Atg5-1 and Atg5-2) or Lamp2A. Immunoblot analysis of LC3 following 6 hour- C treatment with BaﬁlomycinA 20 shRNA Control Atg5-1 Atg5-2 Lamp2A nmol/L. BafilomycinA ++++

LC3-II

β-Actin

chloroquine results, treatment with the combination of Combined autophagy and PPP inhibition leads to spautin-1 and 6-AN selectively inhibited the proliferation activation of NF-kB and inflammasome pathways of Tsc2 / cells (Fig. 4C). In addition, 6-AN treatment of We found that chloroquine treatment led to an increase in cells infected with either Atg5 or LAMP2a (a chaperone- ROS levels in Tsc2 / MEFs (Supplementary Fig. S4), mediated autophagy gene) shRNA decreased proliferation of which, together with cytosolic release of mitochondrial Tsc2 / cells compared with control shRNA (Fig. 5). The DNA due to impaired mitophagy (14), may activate NF- addition of rapamycin did not further affect proliferation in kB/Rel activity (15). Consistent with this reasoning, phos- the presence of chloroquine and 6-AN (Supplementary Fig. phorylation of p65/RelA was induced at 96 hours by the S5B). The PPP generates ribose-5-phosphate and NADPH. combination of chloroquine and 6-AN (Fig. 6A), associated Interestingly, supplementation with 0.1 mmol/L of with increased secretion of IL-6, a known transcriptional NADPH (13) was sufficient to sustain the proliferation of target of NF-kB (Supplementary Fig. S7). IL-6 was not þ þ Tsc2 / cells during treatment with the combination of detectable in Tsc2 / control cells at baseline or after chloroquine and 6-AN (Fig. 4D), suggesting that produc- treatment with chloroquine/6-AN (Supplementary Fig. tion of NADPH via PPP is essential to the proliferation of S7). Furthermore, we observed accumulation of cleaved Tsc2 / cells. caspase-1, a marker of inflammasome activation, under the The combination of chloroquine and 6-AN suppressed combined chloroquine/6-AN treatment at 96 hours (Fig. the proliferation of a novel Tsc2-null cystadenoma cell line, 6B). Supplementation with 0.1 mmol/L of NADPH was named 105K (Supplementary Fig. S6), similarly to the sufficient to prevent accumulation of cleaved caspase-1 þ Tsc2 / MEFs. These cells were derived from a Tsc2 / under chloroquine/6-AN treatment for 96 hours (Fig. C57Bl/6 mouse renal tumor, and confirmed to have loss of 6C), consistent with the rescue of cell proliferation by the second allele of Tsc2 gene (Supplementary Fig. S6), loss NADPH (Fig. 4D). of tuberin expression, increased phospho-S6 levels, and To test the hypothesis that activation of NF-kB is nec- decreased levels of autophagy (measured by p62 accumula- essary for the inhibition of cell proliferation by chloroquine tion; Supplementary Fig. S6). and 6-AN, we treated the cells with parthenolide, which

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Figure 6. Combined autophagy and pentose phosphate pathway AB inhibition lead to activation of NF-kB and the inflammasome. A, immunoblot analysis of phospho- p65 (RelA, ser 536), total RelA, phospho-S6 (ser 235/236), and þ þ total S6 in Tsc2 / and Tsc2 / MEFs treated with chloroquine (CQ, 5 mmol/L), 6-AN (10 mmol/L), or both for 4 days. B, immunoblot analysis of caspase-1 (pro- and cleaved-form) in Tsc2 / and þ þ Tsc2 / MEFs treated with CD chloroquine (5 mmol/L) and 6-AN (10 mmol/L) for 4 days. C, immunoblot analysis of caspase-1 (pro- and cleaved-form) in Tsc2 / MEFs treated with chloroquine (5 mmol/L) and 6-AN (10 mmol/L), NADPH (0.1 mmol/L) alone, or all three for 4 days. D, working model: autophagy inhibition enhances ROS production, leading to PPP dependency and inflammasome activation. G6P, glucose-6- phosphate; 6P-G, 6-phosphogluconate; R-5P, ribose-5-phosphate. inhibits both p65/RelA and caspase-1 via alkylation (16). In parallel with the PPP upregulation, we found that Parthenolide restored proliferation in cells treated with chloroquine induced ROS production at a higher rate in chloroquine and 6-AN (Supplementary Fig. S8). These data Tsc2-null cells than in cells with intact TSC2. This could be suggest that the combination of chloroquine and 6-AN due to impaired mitophagy and subsequent accumulation of inhibits the proliferation of Tsc2-null cells via ROS-depen- dysfunctional mitochondria, which would increase the cell dent activation of NF-kB and caspase-1. requirement for reducing power to buffer-elevated ROS production. TSC2-deficient cells may be particularly vul- nerable to loss of reducing power because they are known to Discussion produce elevated amounts of ROS at baseline (17) and to be Chloroquine and its analog hydroxychloroquine, which highly sensitive to increased ROS (18). block lysosome–autophagosome fusion and lysosomal func- Therefore, these data suggest that the PPP becomes a tion thus suppressing macroautophagy and chaperone- crucial metabolic survival mechanism in TSC2-null cells mediated autophagy, can inhibit cancer cell survival under when they encounter the increase in oxidative stress due to stress conditions (1). We report here that prolonged (4 further autophagy suppression. To test this hypothesis, we months) treatment with chloroquine suppressed the number used the antimetabolite 6-AN, which inhibits the PPP- þ and size of renal tumors in Tsc2 / mice, consistent with the dependent NADPH supply (19), in combination with decreased spontaneous renal tumors we previously observed chloroquine. The chloroquine/6-AN combination dramat- in Tsc2/Beclin1 double heterozygous mice (7). We hypoth- ically suppressed the proliferation of Tsc2-null cells, which esized that metabolic compensatory mechanisms are acti- was rescued by supplementation of NADPH. Similar results vated when autophagy is suppressed by chloroquine in Tsc2- were seen with spautin-1 (12), a recently reported inhibitor deficient cells, thereby preventing a more complete thera- of Beclin-1, and with downregulation of Atg5 and Lamp2a peutic response. Metabolite profiling revealed striking genes, supporting the hypothesis that the effect of chloro- changes in the metabolome of Tsc2-null cells treated with quine in these experiments is autophagy-mediated. Spautin- chloroquine, including increased levels of five PPP interme- 1 has been found to induce cell death under glucose diate metabolites. Isotope labeling experiments revealed an deprivation conditions in breast cancer cells (12), further increase in glucose oxidation under chloroquine treatment, underscoring the links between the metabolic status of which may reflect activation of the oxidative branch of the tumor cells and their response to autophagy inhibition. PPP. The pentose phosphate shunt, which is often upregu- Notably, the combination of chloroquine and 6-AN lated in cancer, has both biosynthetic and oxidative func- triggered activation of the NF-kB and inflammasome path- tions, representing the main source of NADPH via its ways in a TSC2-dependent manner, and parthenolide (16), oxidative branch and supplying ribose-5 phosphate for which inhibits both NF-kB and caspase-1, rescued prolif- nucleotide synthesis. eration under these conditions. Inflammasomes are

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Parkhitko et al.

multiprotein complexes that mediate the cleavage and acti- approaches targeting the metabolism of tumors with vation of caspase-1, thereby stimulating an inflammatory mTORC1 activation may have broad clinical impact. response and/or programmed cell death (20). Multiple factors can activate the inflammasome, including autophagy Disclosure of Potential Conflicts of Interest D.J. Kwiatkowski is a consultant/advisory board member of Novartis. No potential inhibition, mitochondrial DNA release, increased ROS, and conflicts of interest were disclosed by the other authors. activation of NF-kB (15, 21), thus affecting cell survival/ proliferation. Recent work has linked the NF-kB pathway to Authors' Contributions the immune response and cellular senescence and has sug- Conception and design: A.A. Parkhitko, C. Priolo, J.J. Yu, D.J. Kwiatkowski, k J.W. Locasale, A.M.K. Choi, E.P. Henske gested that NF- B may serve a tumor-suppressor function in Development of methodology: A.A. Parkhitko, C. Priolo, K. Mizumura, W. Xu, some circumstances. Together, our results lead us to the J.W. Locasale, J.M. Asara, E.P. Henske conclusion that the inhibition of autophagy and the PPP Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): A.A. Parkhitko, J.L. Coloff, J.J. Wu, W. Xu, I.A. Malinowska, leads to depletion of NADPH, increased ROS production, T. Finkel, E.P. Henske and activation of NF-kB/Rel. We propose that this ulti- Analysis and interpretation of data (e.g., statistical analysis, biostatistics, compu- tational analysis): A.A. Parkhitko, C. Priolo, J. Yun, J.J. Wu, I.A. Malinowska, mately suppresses cell proliferation via activation of the D.J. Kwiatkowski, J.W. Locasale, J.M. Asara, A.M.K. Choi, E.P. Henske inflammasome (Fig. 6D). Writing, review, and/or revision of the manuscript: A.A. Parkhitko, C. Priolo, Our results point toward novel therapeutic strategies for I.A. Malinowska, J.J. Yu, D.J. Kwiatkowski, J.W. Locasale, J.M. Asara, E.P. Henske neoplasms with mTORC1 activation by targeting their Study supervision: C. Priolo, J.W. Locasale, A.M.K. Choi, E.P. Henske intrinsic dependence on autophagy and the metabolic com- Acknowledgments pensatory mechanisms triggered by autophagy inhibition. The authors thank Min Yuan and Susanne Breitkopf for help with mass spec- Thus, in TSC and related diseases, including lymphangio- trometry experiments. This research was conducted in partial fulfillment of A.A. fi Parkhitko's doctoral degree requirements at the Department of Molecular Biology, leiomyomatosis (LAM), our data provide a rst proof-of- Russian State Medical University, under the supervision of Dr. Olga Favorova. concept that therapeutic targeting of cells with hyperactive mTORC1 can be accomplished by targeting their metabolic Grant Support vulnerabilities without the utilization of mTORC1 inhibi- This work was supported by the LAM Foundation, the Adler Foundation, the Tuberous Sclerosis Alliance, the National Institute of Diabetes and Digestive and tors. We note that the autophagy inhibitors chloroquine and Kidney Diseases (to E.P. Henske); grants NIH 5P01CA120964-04 and NIH hydroxychloroquine (Plaquenil) are currently being tested in DF/HCC, Cancer Center Support Grant P30CA006516-46 (to J.M. Asara); the National Heart, Lung, and Blood Institute (to J.J. Yu); and the National Cancer clinical trials for multiple types of cancer and for LAM, and a Institute (to D.J. Kwiatkowski). novel derivative of chloroquine with higher efficacy in vitro The costs of publication of this article were defrayed in part by the payment and in vivo (22) has been recently synthesized. Furthermore, of page charges. This article must therefore be hereby marked advertisement in more than 100 cancer trials using "Rapalogs" and other accordance with 18 U.S.C. Section 1734 solely to indicate this fact. mTORC1 inhibitors are currently enrolling patients, yet Received May 24, 2013; revised October 25, 2013; accepted November 15, 2013; durable clinical responses are uncommon. Therefore, novel published OnlineFirst December 2, 2013.

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www.aacrjournals.org Mol Cancer Res; 12(1) January 2014 57

Autophagy-Dependent Metabolic Reprogramming Sensitizes TSC2-Deficient Cells to the Antimetabolite 6-Aminonicotinamide

Andrey A. Parkhitko, Carmen Priolo, Jonathan L. Coloff, et al.

Mol Cancer Res 2014;12:48-57. Published OnlineFirst December 2, 2013.

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