TSC2 Deﬁciency Unmasks a Novel Necrosis Pathway That Is ...tumors (6, 8). Expression of TSC1 and...

Molecular and Cellular Pathobiology

TSC2 Deficiency Unmasks a NovelNecrosis Pathway That Is Suppressed by theRIP1/RIP3/MLKL Signaling CascadePiotr T. Filipczak1, Cindy Thomas1,Wenshu Chen1, Andrew Salzman2,Jacob D. McDonald1, Yong Lin1, and Steven A. Belinsky1

Abstract

Tuberous sclerosis complex (TSC) is a genetic multiorgandisorder characterized by the development of neoplastic lesionsin kidney, lung, brain, heart, and skin. It is caused by aninactivating mutation in tumor suppressor genes coding theTSC1/TSC2 complex, resulting in the hyperactivation of mTOR-and Raf/MEK/MAPK–dependent signaling that stimulatestumor cell proliferation and metastasis. Despite its oncogeniceffect, cells with TSC deficiency were more sensitive to oxidativestress and dependent on mitochondrial metabolism, providinga rationale for a new therapeutic approach. The current studyshows that simultaneous inhibition of two major pathwaysregulating redox homeostasis using L-buthionine-sulfoximine(BSO, glutathione synthesis inhibitor) and auranofin (thiore-doxin reductase inhibitor) induces oxidative burst, mitochon-drial damage, and necrotic cell death in TSC-deficient cells in ahighly synergistic and cell context–specific manner. Further-

more, blocking RIP1/RIP3/MLKL–dependent signaling usingchemical inhibitors necrostatin-1 (Nec-1) and necrosulfona-mide (NSA) synergizes with BSO and auranofin in killing TSC-deficient cells. Expression analysis demonstrated that RIP1,RIP3, and MLKL protein levels are elevated in cells with TSC2deficiency, and their inactivation enhances mitochondrial dys-function in a glutaminolysis-dependent and autophagy-inde-pendent manner. Finally, supplementation with the mitochon-drial metabolite a-ketoglutarate, whose synthesis is regulatedby RIP1/RIP3/MLKL, rescues cells from the sensitizing effect ofNec-1 and NSA. Together, this study identifies a previouslyunrecognized novel regulated necrotic death pathway thatinvolves mitochondrial homeostasis, is suppressed by theRIP1/RIP3/MLKL signaling in TSC-deficient cells, and couldbe a promising therapeutic target for TSC-associated tumors.Cancer Res; 76(24); 7130–9. �2016 AACR.

IntroductionTuberous sclerosis complex (TSC) is an autosomal-dominant

multiorgan disorder caused by a germline-inactivating muta-tion in the tumor suppressor genes TSC1 or TSC2 (1). Theincidence of TSC has been estimated as 1 in 6,000 personsworldwide, with the average age at diagnosis being 7.5 years (2,3). Clinical manifestation of TSC is development of tumors andneoplastic lesions in kidney, lung, brain, heart and skin, amongwhich renal cell carcinoma (RCC), renal angiomyolipoma(AML), pulmonary lymphangioleiomyomatosis (LAM), andbrain tumors constitute the most common cause of TSC-asso-ciated deaths (3–6). In addition, sporadic forms of AML andLAM characterized by mutation in TSC1 or TSC2 genes maydevelop in patients who do not carry germline mutation inthese genes (5). Despite well-defined etiology, effective thera-peutic options for TSC-associated tumors are limited mainly to

invasive surgery and in the case of LAM to lung transplant, withno pharmaceutical treatment capable of inducing long-termremission of tumor growth (5–7).

Loss-of-function mutation in the TSC1 or TSC2 gene has beendefined as the main factor driving malignancy of TSC-associatedtumors (6, 8). Expression of TSC1 and TSC2 leads to synthesis oftheir protein products hemartin and tuberin, respectively, whichform heterodimeric complex inhibiting the small GTPase Rashomolog enriched in brain (Rheb), that is necessary for theactivationofmTORcomplex1 (mTORC1; ref. 9).While innormalcells, mTOR-dependent signaling is tightly regulated by the avail-ability of nutrition and growth factors, the lack of a functionalTSC1/TSC2 complex in TSC-deficient cells results in constitutivehyperactivation of mTORC1 and its downstream targets to stim-ulate protein translation, cell growth, and metabolic reprogram-ming (9, 10). In addition, TSC1/TSC2 complex deficiency leads toRheb-dependent inhibition of Raf/MEK/MAPK signaling andsensitizes cells to estrogen stimulation (11). In vivo studies alsodemonstrated a critical role for this pathway in the regulation ofmetastasis of TSC-associated tumor cells. Activation of Raf/MEK/MAPK signaling with estrogen elevated the number of cellscirculating in blood, conferred resistance to matrix depriva-tion–induced apoptosis (anoikis), and promoted TSC-deficientcells' colonization in the lung, consistent with the higher inci-dence of LAM in the lungs of women (11, 12). Together, disrup-tion of TSC1/TSC2 complex promotes the malignant phenotypeof tumor cells via complexmechanisms involvingmultiple down-stream targets and signaling pathways.

1Lovelace Respiratory Research Institute, Albuquerque, New Mexico. 2RadicalTherapeutics, West Tisbury, Massachusetts.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Authors: Yong Lin, Lovelace Respiratory Research Institute, 2425Ridgecrest Drive, SE, Albuquerque, NewMexico 87108-5127. Phone: 505-348-9645;Fax: 505-348-9465; E-mail: [email protected]; and StevenA. Belinsky, [email protected]

doi: 10.1158/0008-5472.CAN-16-1052

�2016 American Association for Cancer Research.

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Dependence onmTOR hyperactivity for proliferation has beenthe target for therapies approved for AML and LAM treatment.Rapalogs (analogues of rapamycin) called sirolimus and ever-olimus that block mTORC1 activity show clinical efficacy inpreventing LAM and AML progression and stabilizing respiratoryand renal functions through cytostatic rather than cytotoxic effect(13–16). Consistent with this mechanism, when treatment isdiscontinued due to side effects associated with long-term drugadministration (which include stomatitis, rash, fatigue, hypergly-cemia, hyperlipidemia, and myelosuppression) tumor growthand further deterioration of renal and respiratory functions occurs(5, 17). Moreover, blocking mTOR activity triggers cytoprotectiveautophagy that confers resistance to cellular stress. Animal studiessupport this hypothesis by demonstrating that simultaneouslyblocking mTOR and autophagy is superior to targeting mTORactivity alone for preventing TSC-associated tumorigenesis (18,19). The hypersensitivity of TSC2-null cells to oxidative stress wasfurther substantiated by treatment with the alkaloid chelerythrinechloride that induces cell death in a reactive oxygen speciesmanner (20). In addition, cell death caused by chelerythrinechloride was inhibited by necrostatin-1 (Nec-1), an inhibitor ofRIP1 kinase activity, suggesting an important role for RIP1-depen-dent signaling in the regulation of TSC2-deficient cell survival.RIP1 kinase–dependent signaling leads to the activation of itsdownstream target RIP3 that later phosphorylatesMLKL, resultingin formation of the necrosome and induction of programednecrosis called necroptosis (21).

On the basis of the findings described above, we hypothesizedthat simultaneous targeting of two major mechanisms responsi-ble for redox homeostasis, glutathione- and thioredoxin-depen-dent pathways, may synergistically induce cell death in TSC2-deficient cells due to their hypersensitivity to oxidative stress. Totest this hypothesis, toxicity induced by the glutathione biosyn-thesis inhibitor L-buthionine-sulfoximine (BSO) and thioredoxinreductase inhibitor auranofinwas assessed in vitro and in vivousingtwo different cellular models of TSC2 deficiency. The role of theRIP1/RIP3/MLKL-dependent pathway in modulating sensitivityof TSC2-deficient cells to oxidative and mitochondrial damage–inducing treatment was also investigated.

Materials and MethodsCell lines and reagents

TSC2-wild-type and TSC2-deficient cell lines originated from amurine embryonic fibroblast (MEF) and human angiomyoli-poma (621) used and described in previous reports (18–20) werea gift from Dr. Elizabeth Henske (Brigham and Women's Hospi-tal, Boston, MA). Cell line authentication was performed for 621-derived cell lines using FTA Sample Collection Kit for HumanCellAuthentication (ATCC) and for MEF-derived cell lines usingmultiplex PCR assay targeting short tandem repeat (STR)markersdeveloped by Almeida and colleagues (22). In addition, cellstested negative for mycoplasma contamination using MycoProbeDetection Kit (R&D Systems)were confirmed for TSC2 expressionstatus using Western blot analysis (primary antibody from CellSignaling Technology). Cells were cultured in high-glucoseDMEM supplemented with 10% FBS, 2 mmol/L glutamine, and1% penicillin/streptomycin (Life Technologies) at 37�C in ahumidified 5% CO2 atmosphere and subcultured for up to 20passages. BSO, auranofin, Nec-1, N-benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethyl ketone (z-VAD-FMK), a-ketoglutarate

(a-KG),N-acetyl-L-cysteine (NAC), and chloroquine diphosphatesalt (CQ) were obtained from Sigma-Aldrich. Necrosulfonamide(NSA) was obtained from Toronto Research Chemicals Inc.,MHY1485 was obtained from EMD Millipore, and TNFa wasobtained from R&D Systems.

Animal studyThe study was performed under a protocol approved by Insti-

tutional Animal Care and Use Committee at Lovelace RespiratoryResearch Institute (Albuquerque, NM). MEF-TSC2–deficient cells(3 � 106) were inoculated bilaterally into the posterior flanks of7-week-old athymic nude mice (Charles River Laboratories). Thetreatment began 10 days after the inoculation, when the tumorsize was approximately 75 mm3. Combinations of BSO, aurano-fin, andNec-1 dissolved in PBSwere injected intraperitoneally for5 consecutive days for 2 weeks. Control animals were injectedwith PBS according to the same schedule. The size of tumors wasmeasured twice a week using a caliper, and the tumor volumewascalculated according to the formula volume¼ (length�width2)/2. Necropsy of the animals followed by harvesting and weighingof tumors was performed 3 days after the last injection of thetested compounds.

Proliferation and viability assaysFor the crystal violet assay, cells were plated in a 48-well format

(3–10 � 103/well) and subjected to treatment with BSO andauranofin combinations 24 hours later. After 72 hours of treat-ment, cells were fixed with methanol at �20�C and stained with0.1% crystal violet for 30 minutes. Cells were washed twice withwater and solubilized by the addition of 10% acetic acid followedby shaking for 10 minutes. Absorbance was read using a platereader at 595 nm. For propidium iodide (PI) exclusion test, cellswere plated in a 24-well format (10–30� 103/well) and subjectedto treatment with BSO, auranofin, NAC, Nec-1, and NSA combi-nations 24 hours later. After 24 hours of treatment, cells (bothattached to the surface and floating in the culture medium) werecollected by trypsinization, washed with PBS, and suspended in10 mg/mL PI PBS solution. The relative number of living cells (lowfluorescence–emitting population) and dead cells (high fluores-cence–emitting population) was assessed by flow cytometricanalysis at 535 nm.

ROS and mitochondrial potential analysesCells were plated in a 24-well format (10–30 � 103/well) and

subjected to treatment with BSO, auranofin, NAC, Nec-1, andNSA combinations 24 hours later. For ROS measurement, 16hours after treatment, cells were incubated with 2 mmol/L 20,70-dichlorofluorescin diacetate (DCFDA, Sigma-Aldrich) diluted incomplete culture medium for 15 minutes at 37�C. Next, the cellswere collected from the culture dish, washed with PBS, andanalyzed for green fluorescence intensity using a flow cytometerat 485 nm. To assess mitochondrial potential, 16 hours aftertreatment with BSO, auranofin, Nec-1, and NSA combinations,cells were incubated with 2 mmol/L JC-1 probe (Sigma-Aldrich)diluted in complete culturemedium for 15minutes at 37�C.Next,the cells were collected from the culture dish, washed with PBS,and analyzed for red fluorescence (535 nm) to green fluorescence(485 nm) ratio using flow cytometry. Alternatively, cells stainedwith JC-1 probe and counterstained for nuclei using 10 mg/mL ofHoechst 33342 were subjected to microscopic analysis at 535 nmand 485 nm using a fluorescent microscope.

Novel Necrosis Pathway Suppressed by RIP/MLKL Signaling

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TransfectionsCells were plated in a 6-well format (1 � 105/well), cultured

overnight, and transfected with siRNA using Lipofectamine 3000according to the manufacturer's protocol. Cells were reseededonto a 24-well plate format 24 hours after transfection, subjectedto treatment with BSO, auranofin, and a-ketoglutarate 48 hoursafter transfection, and analyzed for cell death 24 hours later usingPI exclusion test. Twodifferent control siRNAs (Life Technologies)and MLKL siRNAs (Life Technologies and GE Dharmacon) wereused and gave identical results.

Western blottingCells were lysed using RIPA buffer (Cell Signaling Technology)

supplemented with proteinase/phosphatase inhibitors cocktail(Cell Signaling Technology) and 1 mmol/L of phenylmethane-sulfonylfluoride (PMSF; Sigma-Aldrich). After incubation on ice(20minutes), lysateswere centrifuged (15,000� g, 10minutes) at4�C and supernatants were aliquoted and stored at �80�C. Totalprotein content was determined using Pierce BCA Protein AssayKit (Thermo Scientific), and 70 mg of protein was electrophoret-ically fractionated on 4%–15% Mini-PROTEAN TGX Precast10-well gel (Bio-Rad), blotted onto a nitrocellulose 0.45-mmmembrane (Bio-Rad), blocked for 60 minutes in 5% nonfat milkin TTBS (TBS fromBoston Bioproducts supplementedwith 0.05%of Tween-20 from Sigma-Aldrich), and incubated overnight withprimary antibody in 4�C. Polyclonal antibodies for activatedcaspase-3 (made in rabbit; Cell Signaling Technology), RIP1(made in mouse; Becton Dickinson), RIP3 (made in rabbit;Abgent), p62 (made in mouse; BD Biosciences), LC3B (made inrabbit; Abgent), and b-actin (made in rabbit, Cell SignalingTechnology) were used for primary detection. Primary antibodieswere detected with goat anti-rabbit or anti-mouse horseradishperoxidase–conjugated secondary antibodies (Cell SignalingTechnology) and visualized using SuperSignal West Pico Chemi-luminescent Substrate kit (Thermo Scientific). Quantitative anal-ysis of band intensity was performed using ImageJ.

TUNEL stainingTissue sections were fixed with 10% neutral buffered formalin

solution, sequentially incubated with 30% and 70% histologicgrade ethanol, and processed for paraffin embedding. Sections (5mm) were cut, immersed in several changes of xylene, rehydratedin graded alcohols, and washed in PBS. Antigen retrieval was thenperformed by incubating cells with 2 mg/mL proteinase K (Sigma-Aldrich) at 37�C for 15 minutes. Slides were washed with PBS,incubated with premixed TUNEL reagents (Roche Applied Sci-ence) in accordance with the manufacturer's protocol, washedwith PBS, and immunostained for activated caspase-3 asdescribed in the section "Immunostaining" (see below).

ImmunostainingTumor sections werefixed, paraffin embedded, rehydrated, and

antigen retrieved according to the protocol described in "TUNEL"section (see above). For immunostaining of cells from in vitroculture, cells were collected from the culture dish via trypsiniza-tion, placed onto glass slides using a cytocentrifuge Cytospin 3(Shandon), fixed with methanol at �20�C for 20 minutes, andwashed with PBS. Tumor sections and cells were blocked in 1%BSA and0.1%TritonX-100 in PBS. The slideswere incubatedwiththe primary antibody at 4�C overnight. Polyclonal antibodiesmade in rabbit for HMGB1 (Sigma-Aldrich) and activated cas-

pase-3 (Cell Signaling Technology) were used for primary detec-tion. Slides were next incubated with goat anti-rabbit FITC- orAlexa 647–conjugated secondary antibody for 1 hour in roomtemperature, counterstained for nuclei using 1 mg/mL DAPI(Sigma-Aldrich) solution in PBS, and coverslipped using Vecta-shield mounting medium (Vector Laboratories). Microscopicanalysis was performed using a Zeiss fluorescent microscopeequipped with Slidebook 5.0 software.

Statistical analysisThe results for each treatment group are summarized as the

mean value� SEM. Comparisons of results between groups wereperformed using Student t test, and among groups using one-wayANOVA test followed by Dunnett posttest (GraphPad Prism5.04). Statistical significance was defined as a P value <0.05 (�),<0.01 (��), or <0.001 (��).

ResultsBSO and auranofin synergize to induce necrotic cell death inTSC2-deficient cells

Two cellular models of TSC2 deficiency MEFs, and 621 cellsisolated from ahuman angiomyolipoma, were used to investigatethe role of TSC2 status in BSO- and auranofin-induced toxicity.Crystal violet assay demonstrated that cotreatment with BSO andauranofin reduced the number of MEF and 621 cells with TSC2deficiency in a dose-dependent manner (53%, 80%, and 98%reduction for MEF-TSC2�/� and 82%, 86%, and 91% reductionfor 621-TSC2�/� after 1 mmol/L/0.3 mmol/L, 1 mmol/L/1 mmol/L,and 1 mmol/L/3 mmol/L auranofin/BSO treatment, respectively).In contrast, wild-type cells were significantly less affected (19%,38%, and 77% reduction for MEF-TSC2þ/þ and 20%, 39%, and78% reduction for 621-TSC2þ/þ after 1 mmol/L/0.3 mmol/L, 1mmol/L/1 mmol/L, and 1 mmol/L/3 mmol/L auranofin/BSO treat-ment, respectively; Fig. 1A). To determine whether reduction ofTSC2-deficient cell number is caused by inhibition of prolifera-tion or by cell killing, PI exclusion test was applied for directdetection of dead cells. While no increase in cell death was foundafter individual treatment with BSO or auranofin, the combina-tion of these compounds increased themortality from 5% to 21%and from 4% to 27% in MEF-TSC2�/� and 621-TSC2�/� cells,respectively, without affecting the viability of TSC2þ/þ cell lines(Fig. 1B). The mechanism of cell death induced in TSC2-deficientcells byBSOandauranofinwas investigated.Western blot analysisrevealed no activation of caspase-3, themajor enzyme involved inapoptosis, while cotreatment with the pan-caspase inhibitorzVAD also failed to rescue cells from BSO/auranofin-induced celldeath (Fig. 1C; Supplementary Fig. S1). In contrast, immunoflu-orescent staining demonstrated characteristics for necrosis as themain mechanism of cell killing as evident by nucleus-to-cyto-plasm exclusion of DNA-binding protein HMGB1 after BSO/auranofin treatment (Fig. 1D).

The combination treatments in TSC2þ/þ cells reduced andincreased protein levels of the autophagy biomarkers p62 andLC3B-II, respectively (Supplementary Fig. S2A). In contrast, noeffect on these biomarkers was seen in TSC2�/� cells (Supple-mentary Fig. S2A). The autophagy inhibitor chloroquine poten-tiated mortality of TSC2�/� cells induced with BSO and aurano-fin, indicating that autophagy does not contribute to cell killing bythe treatment. In contrast, themTOR inhibitor rapamycin reducedcell death (Supplementary Fig. S2B). No significant effects by

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these agents on cell death were seen in TSC2þ/þ cells (Supple-mentary Fig. S2B).

BSO/auranofin combination induces oxidative burst andmitochondrial damage in TSC2-deficient cells

Chemicals tested in this investigation have well definedrole as free radical (BSO and auranofin) and mitochondrialdamage (auranofin) inducing agents (23–26), therefore thesecellular stress responses were evaluated. Combined treatmentwith BSO and auranofin increased ROS levels 2.7- and 2.0-fold in MEF-TSC2�/� and 621-TSC2�/� cells, respectively. Nosignificant effects were observed in wild-type cells (Fig. 2A).The addition of the free-radical scavenger N-acetyl cysteine(NAC) prevented the elevation of ROS levels and cell deathconsistent with a ROS-dependent mechanism of cell killing(Fig. 2A and B). Measurement of mitochondrial electricpotential using the JC-1 probe demonstrated that the relativenumber of functionalmitochondria was reduced 23% and 26% inMEF-TSC2�/� and 621-TSC2�/� cells, respectively, with no reduc-tion found in the wild-type cells (Fig. 2C). Fluorescent imaging ofMEF-TSC2�/� and 621-TSC2�/� cells stained with JC-1 probefurther revealed a decrease of red fluorescent signal associatedwith functional mitochondria and increase of green fluorescenceassociated with cytoplasm caused by BSO/auranofin treatment(Fig. 2D).

Suppression of RIP1/RIP3/MLKL signaling sensitizesTSC2-deficient cells to BSO/auranofin toxicity in anmTOR-dependent manner

Medvetz and colleagues showed that cell death inMEF-TSC2�/�

cells caused by the ROS inducer chelerythrine chloride was

mediated by RIP1-dependent signaling (20). Thus, the RIP1kinase activity inhibitor Nec-1 and the RIP3/MLKL interactioninhibitor necrosulfonamide (NSA) were used to study theinvolvement of this pathway in BSO/auranofin-induced celldeath. Unexpectedly, PI exclusion showed that Nec-1 and NSAsignificantly synergize with BSO and auranofin to induce killingin TSC2-deficient cells (increase of cell death up to 43%–

73%, Fig. 3A). No significant increase in the number of deadcells was detected when Nec-1 or NSA were applied individuallyor combined with BSO or auranofin alone (not shown). Amodest toxicity of BSO/auranofin/Nec-1 and BSO/auranofin/NSA combinations was also found in TSC2þ/þ cells; however, itdid not exceed 16% of mortality in any of the experimentalcombinations (Fig. 3A). Similar to BSO/auranofin cotreatment,mortality induced by BSO/auranofin/Nec-1 and BSO/auranofin/NSA was not reduced by addition of the caspase inhibitor zVAD,substantiating necrosis as the mechanism of cell death (Supple-mentary Fig. S1). However, consistent with a role for Nec-1 andNSA in blocking necroptosis, these drugs abrogated the effect ofTNFa combined with zVAD for induced cell death in TSC2-wild-type cells (Supplementary Fig. S3). Further studies revealed thatNec-1 and NSA potentiated BSO/auranofin-induced free-radicalformation up to 4.3–5.1-fold in TSC2�/� cell lines (Fig. 3B), andloss of mitochondrial potential was elevated up to 29%–55%(Fig. 3C). While the addition of Nec-1 or NSA further inducedautophagy in TSC2-wild-type cells, no effect was detected inTSC2-deficient cells (Supplementary Fig. S2A), and chemicalmTOR stimulation (rapamycin) and autophagy inhibition(chloroquine) further confirmed the BSO/auranofin/Nec-1/NSA-induced cell death is mTOR-dependent but autophagy-independent (Supplementary Fig. S2B).

Figure 1.

Treatment with BSO and auranofin combination synergizes to induce necrotic cell death specifically in TSC2-deficient cells. MEF- and 621-derived cell lineswere treated with BSO (0.3–3 mmol/L) and auranofin (1 mmol/L). Total number of cells was assessed after 72-hour incubation using crystal violet assay (A),and the number of dead cells was assessed after 24-hour incubation using PI exclusion test (B). The results represent a mean of three independentexperiments � SEM (�� , P < 0.01; �� , P < 0.001). C, The levels of activated caspase-3 in TSC2-deficient cells incubated for 24 hours with 1 mg/mL adriamycin(positive control) versus 1 mmol/L BSO þ 1 mmol/L auranofin were analyzed using Western blot analysis. D, The localization of HMGB1 protein in untreated TSC2-deficient cells versus cells incubated with 1 mmol/L BSO þ 1 mmol/L auranofin for 24 hours was analyzed with immunofluorescence (scale bar, 5 mm).






RNA interference technique was used to test whether knock-down of MLKL, a downstreammediator of RIP1/RIP3-dependentsignaling, affects the sensitivity of TSC2-deficient cells to BSO/auranofin. Downregulation of MLKL protein level by RNA inter-ference, confirmed with Western blot analysis, increased cellmortality up to 33% in MEF-TSC2�/� cells with BSO/auranofintreatment, indicating that the signaling downstream from RIP1/

RIP3 kinase activity is critical for TSC2-deficient cell sensitization(Fig. 4A). Knockdown experiments using 621-TSC�/�were incon-clusive because of high toxicity induced in this cell line bytransfection (not shown). Finally, to test whether elevated sensi-tivity to test compounds is a result of mTOR hyperactivity, MEF-and 621-wild-type cells were treated with mTOR activatorHMY1485. Chemical stimulation of mTOR sensitized MEF- and

Figure 2.

BSO/auranofin combination inducesoxidative burst and mitochondrialdamage in TSC2-deficient cells.MEF- and 621–derived cell lines wereincubated with 1 mmol/L BSOþ 1 mmol/L auranofin for 16 hours,stained with DCFDA for free-radicaldetection (A) or with JC-1 probe formitochondrial potential measurement(C and D), and analyzed with flowcytometry or fluorescent microscopy(in D, representative pictures made in621-TSC2�/� cells shown; scale bar,5 mm). B, The mortality of cellsincubated with 1 mmol/L BSO þ1 mmol/L auranofin for 24 hours withversus without 2 mmol/L NAC wasassessed using PI exclusion test. Theresults represent a mean of threeindependent experiments � SEM(� , P < 0.05; �� , P < 0.01; ��, P < 0.001).

Figure 3.

Inhibition of RIP1/RIP3/MLKL–dependent pathway sensitizes TSC2-deficient cells to BSO/auranofin combination in a highly synergistic manner. MEF- and621–derived cell lines were incubated with 1 mmol/L BSO, 1 mmol/L auranofin, 30 mmol/L Nec-1, or 3 mmol/L NSA for 24 hours for the assessment of cell deathusing PI exclusion test (A), or for 16 hours for free-radical detection with DCFDA (B) and mitochondrial potential measurement with JC-1 probe (D). The resultsrepresent a mean of three independent experiments � SEM (� , P < 0.05; �� , P < 0.01; �� , P < 0.001).

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621-wild-type cells, increasing mortality induced with BSO/aur-anofin, BSO/auranofin/Nec-1, BSO/auranofin/NSAup to 14.6%–

16.9%, 16.2%–34.0% and 24.5%–52.7%, respectively (Fig. 4B).

Elevated RIP1/RIP3/MLKL signaling contributes to glutaminemetabolism dependence of TSC2-deficient cells

Among other functions, RIP1/RIP3/MLKL activity is known tostimulate glutaminolysis, which is indispensable for TSC2-defi-cient cells to survive stress conditions as described previously (27,28). Thus, the hypothesis that inhibition of RIP1/RIP3/MLKLsignaling sensitizes TSC2-deficient cells to BSO/auranofin viainhibition of glutaminolysis was tested. Treatment of MEF- and621-TSC2-deficient cells with Nec-1 or NSA caused 18%–48%reduction in levels of a-ketoglutarate, a critical product of gluta-mine conversion (Fig. 5A). Furthermore, supplementation ofculturemediumwithexogenousa-ketoglutarate reduced cell deathinduced with BSO/auranofin/Nec-1 and BSO/auranofin/NSA by19%–40% and 13%–27%, respectively (Fig. 5B), and abrogatedthe sensitizing effect of MLKL knockdown by BSO/auranofintreatment (Fig. 5C). Recent reports demonstrated that a-ketoglu-tarate may regulate autophagy via various mechanisms includingAkt/mTOR signaling (29, 30); however, no changes in p62 andLC3B-II protein levels were found in our experimental setting(Fig. 5D). Finally, expression analysis performed using Westernblot analysis revealed that RIP1, RIP3, andMLKL protein levels areelevated 1.9–7.6-fold in the 621-TSC2–deficient cells comparedwith the wild-type cells, with the similar trend in RIP1 and RIP3expression changes observed in the MEF model (Fig. 5E).

BSO/auranofin/Nec-1 treatment synergistically inhibits growthof TSC2-deficient tumors and induces necrosis in vivo

Mouse xenograft model of MEF-TSC2�/� cells, which form fastgrowing tumors in contrast to low tumorigenic 621 cells, was used

to test the antitumor efficacy of BSO, auranofin, andNec-1. Whiletherewas no therapeutic effect of any of these compounds appliedalone, treatment with BSO/auranofin and BSO/auranofin/Nec-1significantly reduced the growth of TSC2-deficient tumors asexpressed by a reduction of tumor volume and mass by 37%–

78% compared with sham-treated animals (Fig. 6A and B).TUNEL/caspase-3 double staining was performed in tumor tissuesections to evaluate the extent and the mechanism of cell death.While no significant increase in the number of TUNEL-positivecells was observed in tumors subjected to BSO/auranofin treat-ment (not shown), BSO/auranofin/Nec-1 increased positivity by5-fold compared with sham (Fig. 6C and D). While 72% of deadcells in sham tumors stained positive for activated caspase-3, themajority (79%) of TUNEL-positive cells detected in BSO/aura-nofin/Nec-1–treated tumorswerenegative for activated caspase-3,further substantiating the dominant role for necrosis versusapoptosis induced by this combined treatment (Fig. 6C and E).

DiscussionThese studies have identified a targeted multidrug approach

that alters redox homeostasis and induces mitochondrial damageto synergistically and potently inhibit the growth of TSC2-defi-cient tumors in an mTOR-dependent manner. Furthermore, anovel TSC context–dependent function for RIP1/RIP3/MLKL sig-naling through activation of glutaminolysis that sustains theviability of TSC2-deficient cells subjected to oxidative and mito-chondrial stress was identified. Inhibition of RIP1/RIP3/MLKL-regulated glutaminolysis disrupts mitochondrial homeostasis inTSC2-deficient cells constituting a new form of regulated necrosis(Fig. 7). Autophagy does not contribute to killing TSC2-deficientcell by this new regulated cell death mechanism; rather the basalautophagy activity protects TSC2-deficient cells against the toxic

Figure 4.

MLKL knockdown and mTOR stimulationsensitizes cells to BSO/auranofin/Nec-1/NSA–induced cell death. A, MEF-TSC2–deficient cellswere transfected with control- versus MLKL-siRNA and analyzed for knockdown efficacyusing Western blot analysis 72 hours later (left),subjected to 1 mmol/L BSO þ 1 mmol/L auranofintreatment 48 hours after transfection, andanalyzed for number of dead cells 24 hours later(right). B, TSC2 wild-type cells were incubatedwith 1mmol/L BSO, 1mmol/L auranofin, 30mmol/LNec-1, or 3 mmol/L NSA in the absence versuspresence of MHY1485 for 24 hours and analyzedfor the number of cell death using PI exclusiontest. The results represent a mean of threeindependent experiments � SEM (� , P < 0.05;�� , P < 0.01; �� , P < 0.001).






effects of the tested drugs. In contrast, mTOR hyperactivationassociated with TSC2 deficiency is required to induce necrosis byBSO, auranofin, and RIP1/MLKL inhibitors in TSC2-deficientcells.

The contribution of TSC2 deficiency to sensitivity to oxidativestress has been demonstrated in variety of malignant and non-malignant cellular models. Elevated oxidative stress, accompa-nied by increased expression of stress markers such as CHOP andHO-1, was found in hippocampal neurons isolated from TSC2-deficient rat embryos at increased vulnerability of cells todeath viathemitochondrial death pathway (31). This effect was reversed byrapamycin, indicating a critical role for mTOR hyperactivation inthis process. Furthermore, whole organism overexpression ofRheb, which mimics TSC-null phenotype, sensitized Drosophilamelanogaster to oxidative stress inducedwithH2O2 viamTOR/S6Khyperactivation–dependent mechanism and promoted earlysenescence of their locomotor behavior (32). Clinical validationof this phenomenon was demonstrated through detection ofelevated levels of oxidized DNA in angiomyolipoma tissue sam-ples isolated from TSC patients (33). Moreover, human TSC–associated brain neoplastic lesions showed that elevated expres-sion of the gene coding for glutamate-cysteine ligase catalyticsubunit (GCLC) is increased in TSC2-deficient cells in response toelevated free-radical levels, and its knockdown results in growtharrest and cell death of brain tumor–derived cells (34). Themechanism of increased ROS production in TSC2-deficient cellsis not fully understood, but has been associated with the accu-mulation of impaired mitochondria, the main source of free-radical species. In TSC-deficient cells removal of dysfunctional

mitochondria via mitophagy is disabled because of mTOR hyper-activity and its inhibitory effect on autophagosome assembly(35, 36). In addition, mTOR activates transcription of genes formitochondrial biogenesis including peroxisome proliferator-acti-vated receptor g coactivator-1a (PGC-1a; ref. 37). Thus, the factthat elevated levels of free radicals block the ability of TSC2-deficient cells to accommodate additional oxidative stressinduced by exogenous factors led to the identification of cheler-ythrine chloride (20). Chelerythrine chloride–induced cell killingwas ROS-dependent, and was rescued by the free-radical scaven-ger N-acetyl-cysteine (NAC) in TSC2-deficient cells. Our studiesgreatly expand this approach by demonstrating that simultaneoustargeting of redox homeostasis via glutathione depletion (BSO)and mitochondrial integrity via thioredoxin reductase inhibition(auranofin) is highly synergistic in TSC2-deficient cell killing atrelatively low doses of both drugs. The combination of BSO andauranofin reduces viability and clonogenic survival of head andneck cancer cells, and sensitizes lung and head and neck cancercells to cisplatin and erlotinib, respectively (38–39). However, ashigh as 100 mmol/L BSO in combination with 0.5-5 mmol/Lauranofin was used to reduce viability while 1 mmol/L BSOcombinedwith 1 mmol/L auranofinwas sufficient to induce deathof TSC2-deficient cells.

One pathway recently described to regulate redox and mito-chondrial homeostasis is the RIP1/RIP3/MLKL pathway. Whileinitially defined as responsible for the induction of programmednecrosis (necroptosis) after stimulation with TNFa and underhypoxia–reperfusion stress (40–42), it was further found toplay an oncogenic function. We recently reported that RIP1

Figure 5.

RIP1/RIP3/MLKL contributes to TSC2-deficient cell resistance by activation of glutaminolysis. A, Untreated TSC2-deficient cells and cells incubated with 30 mmol/LNec-1 or 3 mmol/L NSA for 6 hours were analyzed for the levels of a-ketoglutarate. B, Cells incubated with 1 mmol/L BSO, 1 mmol/L auranofin, 30 mmol/LNec-1, or 3 mmol/L NSA for 24 hours in the absence versus presence of 2 mmol/L a-ketoglutarate were analyzed for number of cell death using PI exclusiontest. C, Cells transfected with control versus MLKL siRNA and incubated with 1 mmol/L BSO þ 1 mmol/L auranofin in the absence versus presenceof 2mmol/La-ketoglutaratewere analyzed for number of cell death usingPI exclusion test.D,The levels of p62andLC3B-II proteins in TSC2-deficient cells cultured inthe absence versus presence of 2 mmol/L a-ketoglutarate were analyzed using Western blot analysis. E, The levels of RIP1, RIP3, and MLKL proteins inTSC2-wild type versus TSC2-deficient cells were analyzed usingWestern blot analysis (left) and assessed using ImageJ (right). The results represent amean of threeindependent experiments � SEM (� , P < 0.05; �� , P < 0.01; �� , P < 0.001).

Filipczak et al.





overexpression is found in human lung tumors and mouse lungexposed to cigarette smoke, and that RIP1 potentiates neoplastictransformation of lung epithelial cells by suppressing carcinogen-induced oxidative stress (43). Depletion of RIP1 in lung cancercells decreased mitochondrial oxidative phosphorylation by sup-pressing the expression of PGC-1a and promoted excessive aer-obic glycolysis, which in turn accelerated DNA damage andinhibited cell proliferation (44). RIP1 has also been shown tosuppress basal autophagy (45); however, this does not occur inthe context of TSC deficiency. Reports regarding TSC2-deficientcells are limited to the observation that inhibition of RIP1 activityusing Nec-1 protects from cell death induced with chelerythrinechloride (20). However, an opposite effect was shown in ourcurrent study where inhibition of RIP1 kinase activity, disruptionof RIP3/MLKL binding, or MLKL knockdown sensitizes cells toBSO and auranofin in a TSC2-dependent manner. The differencein outcome between studies may relate to other effects by cheler-ythrine chloride that include inhibition of protein kinase C (46),making the nature of cellular stress induced with this agent highlypleiotropic. In contrast, BSO and auranofin induce oxidative andmitochondrial damage in a highly specificmanner by glutathionedepletion and thioredoxin reductase inhibition. In addition, theconcentration of Nec-1 used by Medvetz and colleagues was3-fold higher than used in our study, which may contribute tooff-target effects such as inhibition of indoleamine-pyrrole

2,3-dioxygenase (IDO) by high dose of Nec-1 (47). RIP1 andRIP3 proteins are involved in regulation of multiple processes(including NF-kB signaling, activation of inflammasome viaNLRP3, activation of caspase-8, and secretion of IL8) based onthe mechanisms independent from their kinase activity (21, 48).Therefore, targeting of kinase-dependent signaling rather thantheir "scaffold" functions provides a therapeutic benefit againstTSC2-deficient cells. Although the mechanism of the sensitizingeffect of RIP1/RIP3/MLKL inhibition remains to be defined,experiments using the a-ketoglutarate supplementation and themeasurement of its endogenous levels described in this studysupport a critical role for glutaminemetabolism regulation for cellsurvival. Previously, Choo and colleagues found that TSC2-defi-cient cells are highly dependent on glutaminolysis for survival instress conditions induced by glucose deprivation (28). Simulta-neously, RIP3/MLKL complex was demonstrated to positivelyregulate glutamine conversion by activating glutamate-ammonialigase (GLUL) and glutamate dehydrogenase-1 (GLUD1; ref. 27).Knowing that glutaminolysis is the process that exclusivelyoccurs in and contributes to mitochondria function, it is likelythat the synergism in cell killing induced by RIP1/RIP3/MLKLinhibition and BSO/auranofin treatment is the result of targetingmitochondrial homeostasis via three distinct mechanisms: redox-,structural-, and metabolic-based. This finding is of high clinicalimportance for developing new treatment strategies for TSC

Figure 6.

BSO/auranofin/Nec-1 combination synergistically inhibits growth of TSC2-deficient tumors and induces necrosis in vivo. MEF-TSC2–deficient tumorsgrowing in athymic nude mice were subjected to treatment with auranofin (1 mg/kg), BSO (450 mg/kg), and Nec-1 (10 mg/kg). Tumor volume was assessedusing a caliper (A) and tumor mass was assessed by weighing (B). C–E, Tumor sections were double-stained with TUNEL (green fluorescence) and for activatedcaspase-3 (red fluorescence), and the number of positive cells was assessed using fluorescent microscopy. The results represent a mean of two independentexperiments � SEM (� , P < 0.05; �� , P < 0.01; �� , P < 0.001). In C, scale bar, 20 mm.






patients, as numerous studies have identified small-moleculedrugs targeting glutamine metabolism. One inhibitor of gluta-minase (GLS) called CB-839 is currently under evaluation in aphase I clinical trial in patients with advanced lung, breast, andrenal tumors (49). In addition, combining the CB-839 analogueBPTES with a HSP90 inhibitor induced cell death selectively inmTOR-driven tumors, supporting clinical benefit of targetingglutaminolysis specifically in this type of malignancy. Thus, ourstudy provides a novel target, distinct from mTOR but associatedwith its hyperactivation, that may be used to develop moreeffective alternative therapies against TSC-associated tumors, andthat extendbeyond tuberous sclerosis to include cancerswith highincidence of spontaneous TSC1/2 gene mutations (50), likebladder, endometrial, and skin malignancies.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: P.T. Filipczak, A. Salzman, Y. Lin, S.A. BelinskyDevelopment of methodology: A. Salzman

Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): P.T. Filipczak, W. ChenAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): P.T. Filipczak, W. Chen, A. Salzman, Y. LinWriting, review, and/or revision of the manuscript: P.T. Filipczak, W. Chen,A. Salzman, Y. Lin, S.A. BelinskyAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): C.L. Thomas, J.D. McDonaldStudy supervision: S.A. Belinsky

AcknowledgmentsWe thank Ms. Elise Calvillo of LRRI for the graphics artwork.

Grant SupportThis work was supported by Lovelace Respiratory Research Institute internal

funding.The costs of publication of this articlewere defrayed inpart by the payment of

page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received April 15, 2016; revised September 9, 2016; accepted October 7,2016; published OnlineFirst October 18, 2016.

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2016;76:7130-7139. Published OnlineFirst October 18, 2016.Cancer Res Piotr T. Filipczak, Cindy Thomas, Wenshu Chen, et al. Suppressed by the RIP1/RIP3/MLKL Signaling CascadeTSC2 Deficiency Unmasks a Novel Necrosis Pathway That Is

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