Therapeutic Targeting of the Secreted Lysophospholipase D Autotaxin Suppresses Tuberous Sclerosis Complex-Associated Tumorigenesis You Feng1,2, William J

Home , ACADS, Alkaline phosphatase, Lysophospholipase

Published OnlineFirst May 11, 2020; DOI: 10.1158/0008-5472.CAN-19-2884

CANCER RESEARCH | METABOLISM AND CHEMICAL BIOLOGY

Therapeutic Targeting of the Secreted Lysophospholipase D Autotaxin Suppresses Tuberous Sclerosis Complex-Associated Tumorigenesis You Feng1,2, William J. Mischler1,2, Ashish C. Gurung1,2, Taylor R. Kavanagh1,2, Grigoriy Androsov1,2, Peter M. Sadow2,3, Zachary T. Herbert2,4, and Carmen Priolo1,2

ABSTRACT ◥ Tuberous sclerosis complex (TSC) is an autosomal dominant foundly impacted the transcriptomeofthesecellswhileinducing disease characterized by multiorgan hamartomas, including renal minor gene expression changes in TSC2 add-back cells. RNA- angiomyolipomas and pulmonary lymphangioleiomyomatosis sequencing studies revealed transcriptomic signatures of LPA (LAM). TSC2 deficiency leads to hyperactivation of mTOR and S1P, suggesting an LPA/S1P-mediated reprogramming of the Complex 1 (mTORC1), a master regulator of cell growth and TSC lipidome. In addition, supplementation of LPA or S1P metabolism. Phospholipid metabolism is dysregulated upon rescued proliferation and viability, neutral lipid content, and TSC2 loss, causing enhanced production of lysophosphatidylcho- AKT or ERK1/2 signaling in human TSC2-deficient cells treated line (LPC) species by TSC2-deficient tumor cells. LPC is the with GLPG1690. Importantly, TSC-associated renal angiomyo- major substrate of the secreted lysophospholipase D autotaxin lipomas have higher expression of LPA receptor 1 and S1P (ATX), which generates two bioactive lipids, lysophosphatidic receptor 3 compared with normal kidney. These studies increase acid (LPA) and sphingosine-1-phosphate(S1P).Wereporthere our understanding of TSC2-deficient cell metabolism, leading to that ATX expression is upregulated in human renal angiomyo- novel potential therapeutic opportunities for TSC and LAM. lipoma-derived TSC2-deficient cells compared with TSC2 add- back cells. Inhibition of ATX via the clinically developed com- Significance: This study identifies activation of the ATX–LPA/ pound GLPG1690 suppressed TSC2-loss associated oncogenicity S1P pathway as a novel mode of metabolic dysregulation upon in vitro and in vivo and induced apoptosis in TSC2-deficient cells. TSC2 loss, highlighting critical roles for ATX in TSC2-deficient GLPG1690 suppressed AKT and ERK1/2 signaling and pro- cell fitness and in TSC tumorigenesis.

Introduction TSC genes leads to hyperactivation of mTOR Complex 1 (mTORC1), which integrates growth factor and nutrient signaling to stimulate cell Tuberous sclerosis complex (TSC), an autosomal dominant disease growth, proliferation, and metabolism (3–8). Clinical trials of TSC and characterized by multisystem hamartomas, including benign tumors LAM with the mTORC1 inhibitor rapamycin showed heterogeneous of the brain, kidney, heart, and lung, affects one in 8,000 live births. response of tumor lesions and stabilization of pulmonary function; About 30% of women with TSC develop lymphangioleiomyomatosis however, tumor growth and pulmonary function decline resumed (LAM), a cystic lung destruction associated with diffuse proliferation when treatment was stopped (9, 10). Similarly, in laboratory studies, of smooth muscle actin-positive cells that can progress to pulmonary rapamycin exerts a cytostatic effect in TSC2-deficient cells. These failure requiring oxygen supplementation and lung transplant. Spo- studies highlight the need for additional therapeutic regimens in TSC radic LAM can also occur, characterized by somatic mutations in the and LAM. TSC1 or TSC2 gene and frequently associated with renal angiomyo- Choline phospholipid metabolism is dysregulated in TSC2-defi- lipomas (1, 2). TSC2 deficiency due to inactivating mutations in the cient cells, and distinct lysophosphatidylcholine (LPC) species are significantly increased in LAM patient plasma (6) and suppressed by treatment with rapamycin and chloroquine (11), supporting the hypothesis that circulating LPC may participate in TSC/LAM path- 1Pulmonary and Critical Care Medicine, Department of Medicine, Brigham and ogenesis. LPC is the major substrate of autotaxin (ATX), a secreted Women's Hospital, Boston, Massachusetts. 2Harvard Medical School, Boston, Massachusetts. 3Department of Pathology, Massachusetts General Hospital, lysophospholipase D that degrades LPC to lysophosphatidic acid Boston, Massachusetts. 4Molecular Biology Core Facilities, Dana-Farber Cancer (LPA), a bioactive lipid known to play roles in cell proliferation, Institute, Boston, Massachusetts. angiogenesis, and tumor metastases via specific G protein–coupled Note: Supplementary data for this article are available at Cancer Research receptors (GPCR; ref. 12). ATX also degrades sphingosylphosphor- Online (http://cancerres.aacrjournals.org/). ylcholine (SPC), converting it into sphingosine-1-phosphate (S1P), a W.J. Mischler and A.C. Gurung contributed equally to this article. metabolite regulating cell motility (13). ATX is involved in wound healing, inflammation, and angiogenesis, and was identified among the Corresponding Author: Carmen Priolo, Brigham and Women's Hospital top 40 upregulated genes in a model of metastatic mammary and Harvard Medical School, 20 Shattuck Street, Boston, MA 02115. Phone: 857- 307-0783; Fax: 617-732-7421; E-mail: [email protected] carcinoma (14). Here, we show the impact of inhibiting the ATX pathway on the Cancer Res 2020;80:2751–63 biology of TSC2-deficient cells in vitro and in vivo. GLPG1690 doi: 10.1158/0008-5472.CAN-19-2884 (developed by Galapagos NV) is a compound that specifically targets 2020 American Association for Cancer Research. ATX and has progressed to phase III clinical trial for idiopathic

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pulmonary fibrosis (IPF; ClinicalTrials.gov Identifier: NCT03711162). 30 minutes prior to each experiment. Rapamycin (LC Laboratories), We found that ATX is upregulated in TSC2-deficient cells, and that MK2206 (Selleckchem), and SCH772984 (Cayman Chemical) were GLPG1690 inhibits the oncogenic potential of TSC2-deficient cells dissolved in DMSO. in vitro and in vivo. Short-term treatment with GLPG1690 inhibits the phosphorylation of AKT and ERK1/2 in TSC2-deficient cells, whereas Cell proliferation assay long-term treatment suppresses lipid synthesis and promotes fatty acid Cells were plated on 12-well plates and treated with increasing doses oxidation, leading to lower neutral lipid content in TSC2-deficient of GLPG1690 or rapamycin (20 nmol/L) in medium supplemented cells. TSC-associated renal angiomyolipomas express significantly with 10% FBS unless otherwise specified. After 68 to 96 hours of higher levels of LPA receptor 1 (LPAR1) and S1P receptor 3 (S1PR3) incubation, cells were fixed with formalin and stained with crystal compared with normal kidney. Consistent with these results, ATX violet, then dissolved in methanol and read on a Synergy HT BioTek products LPA and S1P rescue the proliferation, survival, and tran- plate reader. scriptome of human renal angiomyolipoma-derived TSC2-deficient cells treated with GLPG1690. Migration assay In summary, our data support a role for the ATX–LPA/S1P Oris assays (Catalog no. CMA5.101; Platypus Technologies) pathway in TSC-associated tumorigenesis with potential therapeu- use a stopper to create a cell-free detection zone in the center of tic implications. each well of a 96-well plate. Assays were performed according to the manufacturer's instructions. Briefly, 30,000 cells were seeded in DMEM containing 2% FBS per well around the stoppers. After Materials and Methods cells attached overnight, the stoppers were removed (except for Cell lines, plasmids, CRISPR gene editing, and treatments 0 hour control wells) and GLPG1690 (3 mmol/L for Tsc2 / MEFs The following cell lines were used: (i) isogenic derivatives of and 6 mmol/L for the human TSC2-deficient cells) or DMSO LAM patient renal angiomyolipoma-derived TSC2-deficient vehicle was added. Cells were allowed to migrate to the center 621-101 cells (gift of Dr. Elizabeth Henske). These cells were of wells for 18 hours before the 96-well plate was scanned on a derived from a LAM patient renal angiomyolipoma (15) and carry Celigo imager. Migration was quantified by measuring the – the same somatic bi-allelic TSC2 gene inactivating mutations as %woundhealing(tend t0, 40% well mask) and normalized to the patient's LAM cells (G1832A missense mutation of one allele, vehicle control. and loss of the other allele) (16). The isogenic derivative pair includes empty vector 621-102 cells and TSC2 add-back 621-103 Soft agar colony formation assay þ þ cells(SupplementaryFig.S1);and(ii)Tsc2 / and Tsc2 / mouse Cells (10,000/well) were mixed in a layer of 0.4% Noble agar embryonic fibroblasts (MEF, gift of Dr. David Kwiatkowski; (BD Biosciences) in DMEM with 10% FBS (1 mL) and plated ref. 17). ontopofalayerof0.6%agarinDMEMwith10%FBS(3mL) All cell lines were grown in DMEM supplemented with 10% FBS, in 6-well plates. After agar solidified, cells were treated with 100 IU/mL of penicillin, and 100 mg/mL of streptomycin, unless GLPG1690 (6 mmol/L) or DMSO control (0.06%) in 1 mL of specified otherwise. 621-102 and 621-103 cells were grown under medium, twice a week for 6 weeks. Images of the entire wells were antibiotic selection pressure with zeocin (30 mg/mL). Zeocin was taken with an Olympus SZH10 Research Stereo Microscope and removed before each experiment. colonies were counted. TSC2 deficiency, constitutive activation of mTORC1, and rapamycin sensitivity were validated after each thawing by immunoblotting RNA-sequencing analysis for tuberin/TSC2 and phospho-S6 kinase or phospho-S6 ribosomal Human TSC2-deficient or TSC2 add-back cells were plated on protein in the presence or absence of FBS. Mycoplasma testing 10 cm dishes and treated with vehicle or GLPG1690 (6 mmol/L) in (MycoAlert Mycoplasma Detection Kit; Lonza) was conducted after DMEM with 2% FBS, 0.18% DMSO, and 0.1% BSA. LPA (6 mmol/L) or each thawing and at least monthly. Cells were no longer used in S1P (6 mmol/L) was supplemented to human TSC2-deficient cells. experiments after reaching passage 40. After 24-hour treatment, cells were washed with cold PBS (6 mL) and Tsc2 / MEFs were infected with pBabe-Puro-Myr-Flag-AKT1 (18) RNA was collected with PureLink RNA Mini Kit (Invitrogen) follow- and/or transfected with pCMV-myc-ERK2-L4A-MEK1_fusion (gift ing the manufacturer's instructions. The concentration of purified from Melanie Cobb; Addgene plasmid #39197; http://n2t.net/ RNA was measured using Nanodrop. Two micrograms of RNA were addgene:39197; RRID:Addgene_39197) using Fugene HD (Promega). submitted for Illumina RNA-sequencing (RNA-seq), which was con- For CRISPR gene editing, Tsc2 / MEFs were transfected with a ducted by the Molecular Biology Core Facilities, Dana-Farber Cancer predesigned TrueGuide sgRNA targeting Enpp2 (assay ID Institute. CRISPR480928_SGM) or TrueGuide sgRNA Negative Control non- Libraries were prepared using Kapa strandedmRNA Hyper targeting 1 and TrueCut Cas9 v2 (Invitrogen) following the manu- Prep sample preparation kits from 100 ng of purified total facturer's recommendations. Because of low transfection efficiency, RNA according to the manufacturer's protocol. The finished single cell clones were grown and screened for on-target genome dsDNA libraries were quantified by Qubit fluorometer, Agilent editing using the Alt-R Genome Editing Detection Kit (IDT). T7EI TapeStation 2200, and RT-qPCR using the Kapa Biosystems assay results were analyzed by visualizing the cleavage products and Library Quantification Kit according to manufacturer's proto- the full-length amplicon (forward primer: 50-GAATCTCTCCGAT- cols. Uniquely indexed libraries were pooled in equimolar ratios CACTACCATTT; reverse primer: 50-AGGCAGGTGGTGTTTCA- and sequenced on an Illumina NextSeq500 with single-end 75 bp TAG) on a 2% agarose gel. reads. GLPG1690 was obtained from Medkoo Biosciences and dissolved in Sequenced reads were aligned to the UCSC hg19 reference DMSO. LPA and S1P were obtained from Sigma and Avanti Polar genome assembly and gene counts were quantified using Lipids and preconjugated with 2% fatty acid-free BSA at 37C for 20 to STAR (v2.5.1b; ref. 19). Differential gene expression testing was

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performed by DESeq2 (v1.10.1; ref. 20) and normalized read Immunoblotting counts (FPKM) were calculated using cufflinks (v2.2.1; ref. 21). Total proteins were extracted through 30-minute incubation on RNA-seq analysis was performed using the VIPER snakemake ice with Nonidet P-40 lysis buffer containing protease and phos- pipeline (22). phatase inhibitors, and resolved on Bolt Bis-Tris Plus polyacryl- Gene set enrichment analysis (GSEA) was performed using the R amide gels (Life Technologies). Antibodies against PARP (Catalog package GSEABase (23). Entrez IDs ranked by decreasing fold no. 9532S), phospho-AKT (S473; Catalog no. 4060S), AKT (Catalog changes from DESeq2 results table were used as input and evaluated no. 4685S), phospho-ERK (T202/Y204; Catalog no. 9101S), ERK1/2 against the mdsig database v6.2 (24–26). Gene ontology enrichment (Catalog no. 9102S), phospho-S6 ribosomal protein (S235/236; analysis was performed by VIPER using on genes selected from the Catalog no. 2211S), total S6 ribosomal protein (Catalog no. DESeq2 results table that had a fold change >2orfoldchange<2 2317S), phospho-S6 kinase (Catalog no. 9234S), total S6-kinase and an adjusted P value <0.05 against a background of all genes (Catalog no. 2708S), tuberin/TSC2 (Catalog no. 4308S), phospho- detected in the dataset. RSK (S380; Catalog no. 9335), total RSK (Catalog no. 9355), fatty Published RNA-seq data (27) were obtained through dbGap. Dif- acid synthase (Catalog no. 3180S), acetyl-CoA carboxylase (Catalog ferential gene expression (DESeq) analysis was performed for 12 renal no. 3676S), Stearoyl-CoA desaturase 1 (Catalog no. 2794S), CCTa angiomyolipomas and 4 normal kidney tissue samples using R. (Catalog no. 6931S), and BrdUrd (5292S) were obtained from Cell Transcripts per million (TPM) values for LPA and S1P receptors Signaling Technology. Anti-b-actin antibody (Catalog no. A5316) were obtained and plotted. was obtained from Millipore Sigma and anti-CPT1A antibody (Catalog no. ab128568) from Abcam. qPCR analysis Two micrograms of total RNA (PureLink RNA Mini Kit; Fatty acid oxidation Invitrogen) were retrotranscribed with the SuperScript IV TSC2 add-back (300,000/well for 24 hours and 150,000/well for First-Strand Synthesis System (Invitrogen). The following TaqMan 72 hours) and TSC2-deficient cells (200,000/well for 24 hours and probes (Applied Biosystems) were used: ENPP2 (HS00905125_m1), 100,000 for 72 hours) were seeded in 12-well plates and treated Enpp2 (mouse Mm00516572_m1), FASN (Hs01005622_m1), with GLPG1690 (6 mmol/L) or control (0.06% DMSO) in DMEM PCYT1A (Hs00192339_m1), ACACA (Hs01046047_m1), SCD with 10% FBS for 24 or 72 hours. Cells were then incubated for (Hs01682761_m1), LPAR1 (HS00173500_m1, LPAR2 (HS01109356_m1), 3hoursat37Cwith1mCi/mL of [U-14C]palmitate (PerkinElmer LPAR3 (HS00173857_m1), LPAR4 (Hs01099908_m1), LPAR5 Inc.). 3M perchloric acid was added to the cell culture medium and (Hs01054871_m1), LPAR6 (Hs05006584_m1), S1PR3 (Hs01019574_m1), the wells were sealed with Whatman filter paper saturated with 14 and S1PR5 (Hs00924881_m1). phenethylamine (Sigma-Aldrich) to capture C-CO2.Theplates were gently shaken for 3 hours at room temperature and the filter Flow cytometry analyses paper was removed and placed into Ultima Gold F Scintillation þ þ For BrdUrd incorporation, Tsc2 / and Tsc2 / MEFs cells were Fluid (PerkinElmer Inc.). Radioactivity was counted on a Packard plated on 10 cm plates and incubated with GLPG1690 (3 mmol/L) or Tri-Carb Liquid Scintillation Analyzer. Data were normalized DMSO control for 68 hours in DMEM supplemented with 10% FBS. against the protein mass (total micrograms from three indepen- Two hours before collecting the cells, 10 mmol/L BrdUrd (Upstate) dent wells). was added. Adherent cells were trypsinized and combined with floating cells. Cells were pelleted (1,000 rpm, 5 minutes), resus- De novo lipid synthesis pended in 50 mLofPBS,andfixed with 6 mL of precooled 70% Human TSC2-deficient cells (400,000/well for 24 hours and ethanol at room temperature for 30 minutes. Cells were pelleted, 200,000 for 72 hours) and TSC2 add-back cells (600,000/well for washed with 1 mL of 0.5% BSA in PBS, pelleted again, and 24 hours and 300,000/well for 72 hours) were seeded in six-well resuspended with 500 mL of 2M HCl in PBS. After 20-minute plates and treated with GLPG1690 (6 mmol/L) or control (0.06% incubation at room temperature, 1 mL of 0.5% BSA in PBS was DMSO) in DMEM with 10% FBS for 24 or 72 hours. Cells were then added immediately to each sample. Cells were pelleted, resuspended labeled with [1-14C]acetic acid (0.5 mCi/mL; Perkin-Elmer) for in 50 mL of BrdUrd-FITC Ab (BD-Biosciences; Catalog no. 556028) 4 hours, washed two times with PBS and collected for lipid or mouse IgG negative control, and incubated in the dark at room extraction using isopropanol (500 mL). Radioactivity from 20 mL temperature for 30 minutes. One milliliter of 0.5% BSA in PBS was of the lipid extract was counted on a Packard Tri-Carb Liquid added to each sample. Cells were again pelleted and resuspended Scintillation Analyzer. Data were normalized against the protein in 500 mLofPI(10mg/mL in distilled H2O) with 10 mLofRNaseA mass (total micrograms from three independent wells). (10 mg/mL). After 30-minute incubation at room temperature, cells were kept on ice and analyzed on a flow cytometer. Animal studies For BODIPY493/503 staining, 621-102 and 621-103 cells were Subcutaneous tumors were generated by injecting Tsc2 / MEFs seeded on six-well plates (200,000 cells per well) in DMEM with in female NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice (The Jack- 2% FBS. After attachment, cells were treated with vehicle control son Laboratory, JAX stock no. 005557). Three independent trials (0.18% DMSO þ 0.1% BSA), GLPG1690 (6 mmol/L), GLPG1690 were conducted. 2.5 106 cells/mouse were resuspended in 100 mL (6 mmol/L) þ LPA (6 mmol/L), LPA (6 mmol/L), GLPG1690 (6 of PBS and injected with matrigel (1:1) in a single flank. When mmol/L) þ S1P (6 mmol/L) or S1P (6 mmol/L) for 70 hours. Cells tumors reached a palpable size, mice were treated with GLPG1690 were then washed with PBS, incubated with 2 mL of 4 mmol/L BODIPY (60 mg/kg/day) or control (DMSO) diluted in sterile vehicle (0.25% 493/503 (Catalog no. D3922; Invitrogen) in PBS at 37C in the dark for Tween 80/0.25% PEG 200 in distilled water) through intraperito- 30 minutes, rinsed with PBS, trypsinized, and resuspended in 300 mLof neal injection using a 27G needle for 30 days. Tumors were PBS. Flow cytometry was performed to obtain a minimum of 10,000 harvested 4 hours after BrdUrd injection (1 mg/mouse, i.p.) and events per condition. the last treatment, and submitted for histopathologic analyses.

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The animal studies were conducted under a protocol approved by This result was validated in Tsc2 / MEFs, which showed 30-fold þ þ the Institutional Animal Care and Use Committee at the Brigham and increase in ATX expression compared with Tsc2 / MEFs (Supple- Women's Hospital. mentary Fig. S2A). Treatment with rapamycin (20 nmol/L, 24 hours) significantly reduced ATX expression selectively in TSC2-deficient Histology and IHC 621-102 cells (Fig. 1A), suggesting that ATX expression may be Dissected tumors were fixed in formalin for 24 hours. Hematoxylin regulated downstream of mTORC1 in this patient-derived cell line. and eosin and IHC staining were performed on 5-mm sections of formalin-fixed and paraffin-embedded (FFPE) samples. IHC was ATX inhibition halts proliferation of TSC2-deficient cells performed by InvivoEx Inc. Next, to determine the role of ATX in the proliferation and survival Paraffin sections of tissue were dewaxed using xylene and rehy- of TSC2-deficient cells, we used a specific ATX inhibitor, GLPG1690, drated in graded ethanol. DNA hydrolysis was performed using HCl which is currently being tested in phase III clinical trial for IPF and neutralized with sodium borate buffer. Sections were then incu- (NCT03711162). First, using a synthetic ATX substrate, FS-3 (LPC bated in a 1:200 dilution of the mouse monoclonal anti-BrdUrd analogue), in a fluorogenic activity assay, we confirmed that primary antibody (Bu20a; Cell Signaling Technology) at 4C over- GLPG1690 inhibits the enzymatic activity of recombinant ATX night, and then incubated in a 1:200 dilution of biotinylated goat anti- in vitro with an IC50 of 64 nmol/L (Supplementary Fig. S2B), mouse IgG secondary antibody (Vector Laboratories) for 1 hour at validating its potency. Second, we tested GLPG1690 in both room temperature. Immunoreactivity was visualized using streptavi- TSC2-deficient models, the human renal angiomyolipoma cells and din-alkaline phosphatase followed with substrate Vector blue, which Tsc2 / MEFs, in dose-dependent experiments. GLPG1690 inhibited fi fi resulted in a blue immunoreactive signal; sections were then counter- the proliferation of TSC2-de cient cells at a signi cantly lower IC50 stained with nuclear fast red and mounted. (5.46 0.24 mmol/L for human TSC2-deficient cells; 2.84 0.22 mmol/L for Tsc2 / MEFs) than TSC2-expressing cells (7.34 0.15 Transmission electron microscopy mmol/L for human TSC2 add-back cells, Fig. 1B; 4.71 1.17 mmol/L þ þ Small tumor fragments (1–2mm3) were fixed in formaldehyde- for Tsc2 / MEFs; Supplementary Fig. S2C) by crystal violet staining. glutaraldehyde-picric acid fixative (2.5% glutaraldehyde, 1.25% para- Consistent with these results, inhibition of ATX via CRISPR sgRNA formaldehyde, and 0.03% picric acid in 0.1M sodium cacodylate buffer, suppressed TSC2-deficient cell proliferation by 75% (Supplementary pH 7.4) at 4C. Three tumor samples from each group (drug or Fig. S2D). On-target genome editing was confirmed using T7 endo- control) were analyzed at the Electron Microscopy Core Facility nuclease I (T7EI), which recognizes and cleaves mismatched DNA (Harvard Medical School). heteroduplexes. T7EI assay results were analyzed by visualizing the fi Tissue samples were post xed with 1% osmium tetroxide (OsO4)/ cleavage products and the full-length amplicon on a 2% agarose gel 1.5% potassium ferrocyanide (KFeCN6) for 1 hour, washed in water (Supplementary Fig. S2D). two times, one time in 50 mmol/L maleate buffer (pH 5.15, MB) and In addition, GLPG1690 induced moderate apoptosis levels selec- incubated in 1% uranyl acetate in MB for 1 hour followed by one wash tively in the TSC2-deficient cells, as shown by PARP cleavage (an in MB, two washes in water and subsequent dehydration in grades of apoptosis marker) in the presence of the drug (6 mmol/L, 6 or 72 hours) alcohol (10 minutes each; 50%, 70%, 90%, 2 10 minutes 100%). The in immunoblotting analysis (Fig. 1C). As expected, rapamycin alone samples were left in propyleneoxide for 1 hour and infiltrated over- did not induce apoptosis. night in a 1:1 mixture of propyleneoxide and TAAB Epon (TAAB Laboratories Equipment Ltd., https://taab.co.uk). The following day ATX inhibitor GLPG1690 inhibits the migration and anchorage- the samples were embedded in TAAB Epon and polymerized at 60C independent growth of TSC2-deficient cells for 48 hours. Ultrathin sections (about 80 nm) were cut on a Reichert We tested the impact of GLPG1690 on other oncogenic properties Ultracut-S microtome, picked up on to copper grids stained with lead of TSC2-deficient cells. GLPG1690 inhibited the migration of citrate, and examined in a JEOL 1200EX Transmission electron LAM patient renal angiomyolipoma-derived TSC2-deficient cells by microscope or a TecnaiG2 Spirit BioTWIN. Images were recorded 73% (Fig. 1D) and that of Tsc2 / MEFs by 37% (Supplementary with an AMT 2k CCD camera. Fig. S2E) in the presence of 10% FBS, as shown by 18-hour Oris migration assays. Statistical analysis Next, we found that GLPG1690 inhibited the anchorage-independent Statistical analyses were performed using GraphPad Prism 5. Data growth of TSC2-deficient cells (621-102) by 82% (Fig. 1E). TSC2 add- are reported as median 95% CI unless otherwise noted. Statistical back cells (621-103) formed 74% less colonies in soft agar than TSC2- significance was defined as P < 0.05. deficient cells at baseline, and were not affected by the drug (Fig. 1E).

GLPG1690 impacts the transcriptome of TSC2-deficient cells Results To understand the mechanisms through which inhibition of the ATX gene expression is significantly upregulated in TSC2- ATX pathway by GLPG1690 suppresses TSC2-loss associated onco- deficient cells genicity, we performed RNA-seq analysis on human TSC2-deficient TSC2-deficient cells produce increased levels of LPC compared and TSC2 add-back cells treated with the drug or vehicle, in the with TSC2-expressing cells (6). Because LPC is a preferential presence of LPA or S1P, the two lipid products of ATX. Treatment with substrate of the secreted lysophospholipase D ATX, we assayed GLPG1690 induced substantial gene expression changes in the TSC2- fi fi < the expression of this enzyme in TSC2-de cient and TSC2-expres- de cient cells: 5,116 genes were differentially expressed (Padj 0.05), sing cells. ATX mRNA expression was nearly four-fold higher in including 294 genes with absolute log2 (fold change) > 1.0 (205 LAM patient renal angiomyolipoma-derived TSC2-deficient cells upregulated genes, and 89 downregulated genes; Fig. 2A; Supplemen- < (621-102) compared with the isogenic TSC2 add-back control cells tary File S1). Only 280 differentially expressed genes (Padj 0.05) (621-103; Fig. 1A). including one gene with absolute log2 (fold change) > 1.0 were found in

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Figure 1. Impact of ATX inhibition on the oncogenicity of TSC2-deficient cells. A, ATX mRNA expression is downregulated by TSC2 add-back or rapamycin treatment in LAM patient renal angiomyolipoma-derived TSC2-deficient cells. Human TSC2-deficient cells (TSC2) or TSC2 add-back cells (TSC2þ) were treated with vehicle (DMSO) or rapamycin (20 nmol/L) for 24 hours. Two-way ANOVA with Tukey multiple comparisons test was applied. B, TSC2 cells are more sensitive to GLPG1690 than TSC2þ cells. Cells were treated with GLPG1690 in the presence of 10% FBS for 96 hours and cell proliferation was quantified with crystal violet staining. Multiple

t tests was applied. IC50 was 5.46 0.24 mmol/L for TSC2 and 7.34 0.15 mmol/L (mean SEM) for TSC2þ cells. C, GLPG1690 induces apoptosis in human TSC2 cells but not in TSC2þ cells. Cells were treated with GLPG1690 (6 mmol/L), rapamycin (20 nmol/L), or the combination for 6 or 72 hours. The red arrow points to the cleaved PARP, which indicates cell apoptosis. D, GLPG1690 inhibits the migration of human TSC2 cells. Cells were treated with GLPG1690 (6 mmol/L) for 18 hours. Images were obtained on a Celigo imager. Mann–Whitney test was applied. E, GLPG1690 inhibits the anchorage-independent growth of TSC2 cells. Cells were treated with GLPG1690 (6 mmol/L) or control (0.06% DMSO) twice a week for 6 weeks. Two-way ANOVA with Tukey multiple comparisons test was applied. , P < 0.01; , P < 0.001; , P < 0.0001; NS, nonsigniﬁcant.

the TSC2 add-back cells (Fig. 2A; Supplementary File S1). Interest- reversed by adding back S1P, and expression of 15 genes was reversed ingly, ATX mRNA levels were reduced by GLPG1690, selectively in by both LPA and S1P. The rescue was defined as a significant change TSC2-deficient cells, suggesting that the drug suppresses not only the (with opposite sign) in gene expression in GLPG1690 þ LPA or S1P- activity but also the transcription of ATX (Fig. 2B). To identify treated cells versus GLPG1690-treated cells (Fig. 3A; Supplementary transcriptional changes at the pathway level, GSEA was conducted, Fig. S3A; Supplementary File S1). These results suggest that LPA and revealing 50 significantly enriched KEGG gene sets and 36 significantly S1P drive nonredundant transcriptional programs in TSC2-deficient enriched Hallmark gene sets in the TSC2-deficient cells. These includ- cells, differentially contributing to ATX signaling. Gene Ontology ed cell cycle (KEGG), focal adhesion (KEGG), oxidative phosphory- (GO) term enrichment analysis of the RNA-seq data revealed that lation (Hallmark), adipogenesis (Hallmark), apoptosis (Hallmark), LPA mainly regulated inflammatory-associated pathways and adhe- and fatty acid metabolism (Hallmark; Fig. 2C; Supplementary File S1). sion-associated genes, whereas S1P regulated lipid metabolism- associated genes (Supplementary File S1). ATX products LPA and S1P reverse the transcriptomic changes To validate the role of LPA and S1P in mediating the effects of induced by GLPG1690 inTSC2-deficient cells GLPG1690 on the biology of TSC2-deficient cells, these cells were To test whether the effects of GLPG1690 were mediated by the ATX treated with GLPG1690 (6 mmol/L), LPA (6 mmol/L), or S1P lipid products, we supplemented the culture media of drug-treated (6 mmol/L), or the combination of both in the presence of 2% FBS human renal angiomyolipoma-derived TSC2-deficient cells with LPA for 72 hours. Crystal violet staining showed that either LPA or S1P or S1P and tested these conditions in RNA-seq, proliferation, and could partially rescue the proliferation of TSC2-deficient cells upon survival experiments. treatment with GLPG1690 (Fig. 3B). In line with the RNA-seq data, In the RNA-seq analysis, of the 294 genes impacted by GLPG1690 in supplementation of both LPA and S1P (3 mmol/L þ 3 mmol/L) fully < > 621-102 cells [Padj 0.05, log2 (fold change) 1.0], expression of rescued proliferation under the same conditions (Supplementary 147 genes was reversed by adding back LPA, expression of 64 genes was Fig. S3B). Immunoblotting revealed that supplementation of

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Figure 2. GLPG1690 induces marked changes in gene expression selectively in human TSC2-deﬁcient cells. A, Volcano plots showing GLPG1690-induced gene

expression change [Padjusted < 0.05, absolute log2(fold change) > 1] in human TSC2-deficient cells (top) and TSC2 add-back cells (bottom). Cells were treated with GLPG1690 (6 mmol/L) or vehicle (DMSO) in DMEM with 2% FBS for 24 hours. B, ATX transcription is reduced by GLPG1690 in human TSC2-deficient cells. TPM values are shown. Two-way ANOVA with Tukey multiple comparisons test was applied. C, Selected gene sets enriched upon GLPG1690 treatment in human TSC2-deficient cells. , P < 0.001; , P < 0.0001; NS, nonsignificant.

either LPA or S1P could prevent PARP cleavage (apoptosis) in the Importantly, LPAR1 and S1PR3 were also significantly overex- TSC2-deficient cells treated with GLPG1690 (Fig. 3C). pressed in TSC-associated renal angiomyolipomas, as tested in a published RNA-seq dataset (Fig. 3F). TSC2-deficient cells and TSC-associated renal angiomyolipomas overexpress LPA and S1P receptors GLPG1690 treatment suppresses AKT and ERK1/2 RNA-seq experiments corroborated a role for the ATX products, phosphorylation in TSC2-deficient cells LPA and S1P, in the response to treatment with GLP1690. These lipids To assess GLPG1690-induced cell signaling changes, we screened act through specific GPCRs, LPARs and S1PRs. We tested the expres- 43 P-kinase sites and 2 related proteins in the LAM patient-derived sion of these receptors in human TSC2-deficient and TSC2 add-back TSC2-deficient cells and the TSC2 add-back control cells treated cells in the RNA-seq database and by qPCR. LPAR1 and S1PR3 were with GLPG1690 (6 mmol/L,6hours)orDMSO.Twenty-fourof significantly overexpressed in TSC2-deficient cells (Fig. 3D and E). these P-kinase sites (or proteins) showed greater than 25% sup- Treatment with GLPG1690 led to an increase in the expression of pression by GLPG1690 treatment specifically in the TSC2-deficient LPAR1 and a decrease in the expression of S1PR3 in these cells cells; eight of them showed greater than 50% change with the (Fig. 3E, top). LPAR1 expression was also enhanced by treatment inhibitor, including ERK1/2 (T202/Y204, T185/Y187) and AKT1/ with rapamycin, whereas S1PR3 expression was not affected (Fig. 3E, 2/3 (S473; Supplementary Fig. S5A), which are known to mediate bottom). Lower or unchanged expression levels in TSC2-deficient signaling downstream of LPAR/S1PR (28–34). We confirmed the versus TSC2 add-back cells were found for S1PR5, LPAR2, and LPAR3 effect of GLPG1690 on AKT and ERK phosphorylation by immu- (Supplementary Fig. S4). noblotting: 6-hour treatment with GLPG1690 (6 mmol/L) led to a

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Figure 3. LPA and S1P reverse GLPG1690 effects on the transcriptome and proliferation/apoptosis in human TSC2-deficient cells. A, TSC2-deficient cells were incubated for 24 hours with LPA (6 mmol/L), S1P (6 mmol/L), GLPG1690 (6 mmol/L), either lipid in combination with the drug, or vehicle (DMSO þ 0.1% BSA) in media with 2% FBS. TSC2 add-back cells were incubated with GLPG1690 or vehicle. Differentially expressed genes in human TSC2-deficient cells (TSC2) treated with GLPG1690 versus

vehicle [Padjusted < 0.05, absolute log2(fold change) > 1] are depicted in the heat map. Each row represents a gene and each column a sample. Samples are color- coded. Unsupervised clustering enables visualization of groups of genes rescued by LPA or S1P. One differential gene was identified in TSC2 add-back cells (TSC2) treated with GLPG1690 versus vehicle. B, Cells were treated with GLPG1690 (6 mmol/L) or vehicle (0.18% DMSO and 0.1% fatty acid-free BSA) with or without the presence of LPA (6 mmol/L; left) or S1P (6 mmol/L; right) in DMEM with 2% FBS for 72 hours. One-way ANOVA with Tukey multiple comparisons test was applied. C, LPA and S1P rescue human TSC2-deficient cells from GLPG1690-induced apoptosis (cleaved PARP). Cells were treated as in B. D, Expression of LPARs and S1PRs in human TSC2-deficient versus TSC2 add-back cells (TPM values). Two-way ANOVA with Sidak multiple comparisons test was applied. E, LPAR1 and S1PR3 expression in human TSC2-deficient cells and TSC2 add-back cells treated with GLPG1690 (6 mmol/L, 2% FBS; TPM values, top) or rapamycin (20 nmol/L, 10% FBS; RT-qPCR analysis, bottom) for 24 hours. Two-way ANOVA with Tukey multiple comparisons test was applied. F, LPAR1 and S1PR3 have higher expression in renal angiomyolipomas compared with normal kidney. Mann–Whitney test was applied. , P < 0.05; , P < 0.01; , P < 0.001; , P < 0.0001.

decrease in P-AKT (S473) by 68 10% and in P-ERK (T202/Y204) supplementation of LPA, whereas P-ERK levels were rescued by by 56 12% in the human TSC2-deficient cells (Fig. 4A and B). supplementation of S1P (Fig. 4C). P-S6 (S235/236), a direct target of mTORC1, was not affected under Next, to ask whether suppression of AKT and ERK signaling plays a this condition. Consistent results were obtained in Tsc2 / MEFs role in GLPG1690 proapoptotic and antiproliferative effects, we used (Supplementary Fig. S5B). two approaches. First, we treated cells with a specific AKT or ERK Intriguingly, a differential effect of LPA and S1P supplementation inhibitor in combination with GLPG1690. The human TSC2-deficient on AKT/ERK activation was found. P-AKT levels were rescued by cells were pretreated for 30 minutes with AKT inhibitor MK2206

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Figure 4. GLPG1690 impacts AKT and ERK signaling in human TSC2-deficient cells. A, Human TSC2-deficient cells and TSC2 add-back cells were treated with GLPG1690 (6 mmol/L), rapamycin (20 nmol/L), the combination of the two, or vehicle (DMSO) in DMEM with 10% FBS for 6 hours. B, P-AKT (S473) and P-ERK1/2 (T202/Y204) densitom- etry analysis from three biological replicates using ImageJ. C, Human TSC2-deficient cells and TSC2 add- back cells were incubated with LPA (6 mmol/L), S1P (6 mmol/L), GLPG1690 (6 mmol/L), the combination of each lipid with the drug, or vehicle (DMSO) in DMEM with 2% FBS for 72 hours. D, Human TSC2-deficient cells were pretreated with AKT inhibitor MK2206 (4 mmol/L) or ERK inhibitor SCH772984 (2 mmol/L) for 30 minutes, and then incubated with GLPG1690 (6 mmol/L) for 18 hours in DMEM with 10% FBS. E, Constitutively active ERK, myristoylated (Myr) AKT, or both were expressed in Tsc2/ MEFs (top). Cells were treated with vehicle (DMSO) or GLPG1690 (3 mmol/L) in DMEM with 10% FBS for 92 hours before crystal violet staining (bottom). One-way ANOVA with Dunnett's multiple comparisons test was applied and data are shown as mean SD. , P < 0.05.

(4 mmol/L) or ERK inhibitor SCH772984 (2 mmol/L), and then ThesedatasupportaroleforAKTandERKsignalinginTSC2- incubated with GLPG1690 (6 mmol/L) for 18 hours in the presence deficient cell proliferation, including effects downstream of the of 10% FBS. Immunoblotting was performed to detect cleaved PARP. ATX/LPA/S1P axes. Either inhibitor induced low levels of apoptosis as single agent and worked synergistically in combination with GLPG1690 to enhance Inhibition of the ATX–LPA/S1P pathway by GLPG1690 induces apoptosis (Fig. 4D). Second, to test whether constitutive activation of reprogramming of lipid metabolism in TSC2-deficient cells AKT or ERK would prevent the impact of GLPG1690 treatment on The RNA-seq analysis revealed substantial changes in genes of fatty TSC cell proliferation, we expressed myristoylated-AKT (myr-AKT) acid metabolism in 621-102 cells treated with GLPG1690. Specifically, or a fusion of ERK2 with the low activity form of its upstream regulator, in the gene sets of fatty acid metabolism and adipogenesis, 63 of the MAP kinase MEK1 (35), in Tsc2 / MEFs (Fig. 4E). Cells were 146 genes and 86 of 186 genes were significantly altered transcrip- treated with GLPG1690 (3 mmol/L) or vehicle for 92 hours. The tionally. Four enzymes involved in fatty acid oxidation, including acyl- proliferation rate upon drug treatment (drug/DMSO, each normalized CoA dehydrogenase short chain (ACADS), acyl-CoA thioesterase 8 to its own baseline) was significantly higher in the presence of (ACOT8), and malonyl-CoA decarboxylase (MLYCD), were upregu- coexpression of myr-AKT and constitutively active ERK compared lated, whereas seven enzymes involved in lipid synthesis, including with the empty vector control (Fig. 4E). fatty acid synthase (FASN), acetyl-CoA carboxylase alpha (ACACA),

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Figure 5. Impact of GLPG1690 treatment on TSC2-deficient cell lipid metabolism. A, GLPG1690 treatment reduces the neutral lipid content in human TSC2-deficient cells. Cells were treated with control (0.18% DMSO þ 0.1% BSA), GLPG1690 (6 mmol/L), GLPG1690 (6 mmol/L) þ LPA (6 mmol/L), LPA (6 mmol/L), GLPG1690 (6 mmol/L) þS1P (6 mmol/L) or S1P (6 mmol/L) in the presence of 2% FBS for 70 hours. Cells were stained with BODIPY493/503 (4 mmol/L, 30 minutes) and analyzed by flow cytometry. One sample t test was used and data are shown as mean SD. B, GLPG1690 treatment upregulates fatty acid oxidation and downregulates lipid synthesis. TSC2-deficient cells and TSC2 add-back cells were treated with GLPG1690 (6 mmol/L) or vehicle (0.06% DMSO) in DMEM with 10% FBS for 24 or 72 hours. Cells were incubated with [U-14C]palmitate (1 mCi/mL) for 3 hours (top) or [1-14C]acetic acid (0.5 mCi/mL) for 4 hours (bottom). Counts per minute (CPM) were normalized against protein mass. Two-way ANOVA with Tukey multiple comparisons test was applied. C, Immunoblotting analysis of lipogenic enzymes. Cells were treated with GLPG1690 (6 mmol/L), rapamycin (20 nmol/L), or the combination of the two for 72 hours. D, RT-qPCR analysis of genes involved in lipid synthesis. Cells were treated with vehicle (DMSO) or GLPG1690 (6 mmol/L) for 24 or 72 hours. One-way ANOVA with Tukey multiple comparisons test was applied. , P < 0.05; , P < 0.01; , P < 0.001; , P < 0.0001.

and acyl-CoA synthetase long chain family member 1 (ACSL1), were the decrease in neutral lipid content was rescued by adding back either downregulated (Supplementary File S1). LPA or S1P (Fig. 5A). To validate the metabolic reprogramming suggested by the tran- To further determine the causes of these changes, we performed scriptome changes, we used flow cytometry-based neutral lipid quan- 14C-palmitate oxidation (fatty acid oxidation) and 14C-acetate lipid tification and 14C labeling experiments to trace fatty acid oxidation and incorporation (de novo lipid synthesis) assays upon ATX inhibition for de novo lipid synthesis. TSC2-deficient cells were treated with 24 or 72 hours in TSC2-deficient and TSC2 add-back cells. GLPG1690 GLPG1690 (6 mmol/L), LPA (6 mmol/L), S1P (6 mmol/L), the com- significantly promoted b-oxidation selectively in TSC2-deficient cells bination of GLPG1690 with either lipid, or vehicle (DMSO), in the at 24 hours and in both cell lines at 72 hours (Fig. 5B). The drug also presence of 2% FBS for 70 hours. Cellular neutral lipids were stained significantly downregulated de novo lipid synthesis in both cell lines at with BODIPY493/503. GLPG1690 decreased neutral lipid content by 72 hours (Fig. 5B). 29% (P < 0.01) in TSC2-deficient cells, which have higher neutral lipid Consistent with these results, the protein expression of the lipogenic content than TSC2 add-back cells (P < 0.01), as expected. Intriguingly, enzyme CCTa (CTP:phosphocholine cytidylyltransferase a), which is

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Figure 6. GLPG1690 inhibits the proliferation of Tsc2/ MEFs in vivo. A, GLPG1690 reduces TSC tumor burden. Mice were treated with GLPG1690 (60 mg/kg/day; n ¼ 15) or vehicle (n ¼ 18) intraperitoneally for 30 days. Treatment reduced tumor volume by 40% (Mann–Whitney test was applied). B, GLPG1690 did not affect mouse body weight. C, Hematoxylin and eosin (H&E) of representative tumors treated with vehicle (top) or GLPG1690 (bottom). D, GLPG1690 decreased lipid droplets and induced endoplasmic reticulum inflation in Tsc2/ tumors shown by electron microscopy. N, nucleus; LD, lipid droplet; ER, endoplasmic reticulum. E, GLPG1690 selectively decreased BrdUrd incorporation in Tsc2/ MEFs in vitro. Cells were incubated with GLPG1690 (3 mmol/L) or vehicle (DMSO) for 68 hours in DMEM with 10% FBS and flow cytometry was performed. Multiple t tests was applied. F, GLPG1690 suppressed BrdUrd incorporation in Tsc2/ tumors. BrdUrd was injected intraperitoneally 4 hours before harvesting the tumors. IHC with BrdUrd antibody showed 38% reduction in BrdUrdþ cells with GLPG1690 treatment (Mann–Whitney test was applied). G, Working model of the impact of ATX inhibition on TSC tumor cells. , P < 0.05; NS, nonsignificant.

involved in lipid droplet biogenesis (36) was suppressed by 50% (Fig. 5D). Expression of the mitochondrial fatty acid oxidation rate- following 72-hour treatment with GLPG1690, whereas no change in limiting enzyme carnitine palmitoyltransferase 1A (CPT1A) was not CCTa protein expression was found in TSC2 add-back cells (Fig. 5C; affected by drug treatment. Supplementary Fig. S6). Minor changes in the expression (30%) of two enzymes involved in de novo fatty acid synthesis, fatty acid GLPG1690 suppresses TSC tumorigenesis in vivo synthase (FASN) and acetyl-CoA carboxylase a (ACCa), occurred Treatment with GLPG1690 led to a reduction in tumor burden by in TSC2-deficient cells (Fig. 5C; Supplementary Fig. S6), and expres- 40% (P ¼ 0.016; Fig. 6A). Mouse body weight was not affected by sion of the desaturase stearoyl-CoA desaturase 1 (SCD1) was sup- GLPG1690 treatment (Fig. 6B) and no drug toxicity was found. pressed in both TSC2-deficient and TSC2 add-back cells (Fig. 5C). Pathologic analysis revealed clusters of more differentiated, fibro- FASN and SCD1 were confirmed to be regulated transcriptionally blast-like cells. As an observation, subcutaneous fat around the

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tumors appeared to be less abundant in the drug-treated mice and biosynthesis. This enzyme participates in nuclear membrane, tumors seemed to infiltrate less into the muscle (Fig. 6C). Inter- nucleoplasmic reticulum, and lipid droplet biogenesis, and con- estingly, electron microscopy images revealed inflated endoplasmic tributes to phospholipid homeostasis. ACC, FASN, and SCD1 reticulum and confirmed a reduction in lipid droplets in the tumors mediate fatty acid synthesis. Interestingly, TSC2-deficient cells treated with GLPG1690 (Fig. 6D), consistent with the BODIPY493/ have the ability to upregulate expression of the lipogenic enzyme 503 staining and fatty acid oxidation/ de novo lipid synthesis assays FASN over time in culture (72 hours compared with 24 hours), results. Finally, consistent with the effect of GLPG1690 on Tsc2 / likely to enhance fatty acid synthesis when exogenous availability MEFs in vitro (Fig. 6E), we found a decrease in BrdUrd incorpo- decreases; however, treatment with GLPG1690 prevented this ration in tumor cells by 38% (P ¼ 0.034; Fig. 6F). increase, potentially making these cells more vulnerable to nutrient depletion. Another mechanism involves lipid catabolic processes. Treatment with GLPG1690 led to enhanced fatty acid oxidation Discussion selectively in the TSC2-deficient cells at 24 hours and in both This study identifies a novel mode of metabolic dysregulation in TSC2-deficient and TSC2 add-back human renal angiomyolipoma the TSC tumor microenvironment, the ATX–LPA/S1P pathway cells at 72 hours. (Fig. 6G). These data suggest a role for the ATX pathway in the regulation of ATX regulates availability of two bioactive lipids, LPA and S1P, the intracellular lipidome of TSC2-deficient cells. for activating specificmembraneGPCRs.ATXgeneratesLPAand Surprisingly, although suppression of LPA and S1P levels through S1P through its lysophospholipase D activity and binds and ATX inhibition would be expected to upregulate ATX expression in delivers these lipids to their receptors, protecting them from tissues due to feedback regulation (50), we found that treatment with phosphatase degradation (37). The ATX pathway has been asso- GLPG1690 suppressed the expression of ATX in human TSC2-defi- ciated with cancer progression and metastasis (38, 39). LPA and cient cells, suggesting that this compound acts through multiple S1P regulate several physiological processes, including cell pro- mechanisms to suppress ATX activity. liferation, cell migration/invasion, angiogenesis, and inflamma- In summary, our studies suggest that dysregulated ATX–LPA/S1P tion. These lipids activate a series of GPCRs, at least six for LPA pathways are critical players in TSC2-deficient cell fitness and in TSC (LPAR1-6) and five for S1P (S1PR1-5), stimulating a wide variety tumorigenesis, and that ATX could be tackled for novel therapeutic of downstream signaling including PI3K/AKT and RAS/ERK modalities in TSC and LAM. pathways (40–47). Importantly, two of these GPCRs, LPAR1 and S1PR3, are upregu- Disclosure of Potential Conflicts of Interest lated in TSC-associated renal angiomyolipomas (Fig. 3F), consistent No potential conflicts of interest were disclosed. with TSC2-deficient human cells (Fig. 3D and E). GLPG1690 is a potent and specific ATX inhibitor currently in Authors’ Contributions phase III clinical trials for IPF. Its safety and target engagement was Conception and design: Y. Feng, C. Priolo shown in phase I and II trials (48, 49). We found that inhibition of Development of methodology: Y. Feng, C. Priolo Acquisition of data (provided animals, acquired and managed patients, provided the ATX pathway using GLPG1690 suppresses the oncogenicity of fi facilities, etc.): Y. Feng, W.J. Mischler, A.C. Gurung, T.R. Kavanagh, G. Androsov, TSC2-de cient cells, including cell proliferation, cell migration, P.M. Sadow, Z.T. Herbert, C. Priolo anchorage-independent growth, and tumor growth in vivo.Con- Analysis and interpretation of data (e.g., statistical analysis, biostatistics, sistent with GLPG1690 effects, ATX gene editing via CRISPR computational analysis): Y. Feng, W.J. Mischler, A.C. Gurung, T.R. Kavanagh, sgRNA dramatically suppressed the proliferation of Tsc2 / MEFs. G. Androsov, P.M. Sadow, Z.T. Herbert, C. Priolo Taken together, these data suggest a substantial role for ATX–LPA/ Writing, review, and/or revision of the manuscript: Y. Feng, A.C. Gurung, T.R. Kavanagh, P.M. Sadow, C. Priolo S1P signaling pathway in TSC tumorigenesis. Mechanistically, AKT Administrative, technical, or material support (i.e., reporting or organizing data, and ERK signaling were affected in cells treated with GLPG1690 and constructing databases): A.C. Gurung, C. Priolo combination of the ATX inhibitor with either AKT or ERK1/2 specific Study supervision: C. Priolo inhibitors led to enhanced apoptosis in TSC2-deficient cells; on the contrary, expression of constitutively active AKT and ERK rendered Acknowledgments the cells significantly less sensitive to the antiproliferative effect of We thank Maria Ericsson (Electron Microscopy Facility, Harvard Medical GLPG1690. School) for providing assistance with electron microscopy, and Victor Barrera Moreover, ATX inhibition ledtoLPAandS1P-dependent Burgos of the Harvard Chan Bioinformatics Core, Harvard T.H. Chan School of PublicHealth,Boston,MA,forassistance with the analysis of RNA-seq datasets transcriptomic and metabolic reprogramming. Our RNA-seq obtained through dbGap. This work was supported through NIH R01HL130336 experiments uncovered specific roles for LPA and S1P in the and funds from the Department of Defense (W81XWH-16-1-0165) and the context of TSC2 loss. These bioactive lipids reversed differential Tuberous Sclerosis Alliance (50K Crowdfunded Research Challenge) to C. Priolo. changes in the transcriptome of TSC2-deficient cells treated with Y. Feng was supported by a Postdoctoral Fellowship co-funded by the Tuberous GLPG1690, with major involvement of LPA in cell adhesion/ Sclerosis Alliance and The LAM Foundation and a microgrant from the Brigham motility and inflammatory processes, and of S1P in sterol and Research Institute. Work by Victor Barrera Burgos was partially funded by the “HSCI Center for Stem Cell Bioinformatics.” We are grateful to the Engles lipid biosynthesis. Program in TSC and LAM Research for supporting publication of this work. We found that inhibition of ATX by GLPG1690 led to a reprogramming of lipid metabolism via multiple mechanisms. One The costs of publication of this article were defrayed in part by the payment mechanism included reduction in the mRNA and/or protein of page charges. This article must therefore be hereby marked advertisement in expression of lipogenic enzymes,CCT,ACC,FASN,andSCD1, accordance with 18 U.S.C. Section 1734 solely to indicate this fact. with associated decrease in lipid droplet content and de novo lipid synthesis in cells treated with the drug. CCT is the rate-limiting Received September 16, 2019; revised March 25, 2020; accepted May 5, 2020; enzyme in the CDP-choline pathway for phosphatidylcholine published first May 11, 2020.

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Autotaxin Pathway Inhibition Halts TSC Tumorigenesis

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AACRJournals.org Cancer Res; 80(13) July 1, 2020 2763

Therapeutic Targeting of the Secreted Lysophospholipase D Autotaxin Suppresses Tuberous Sclerosis Complex-Associated Tumorigenesis

You Feng, William J. Mischler, Ashish C. Gurung, et al.

Cancer Res 2020;80:2751-2763. Published OnlineFirst May 11, 2020.

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