Therapeutic Targeting of TFE3/IRS-1/PI3K/mTOR Axis in ......2018/09/24 · ASPL–TFE3 (8),...

Cancer Therapy: Preclinical

Therapeutic Targeting of TFE3/IRS-1/PI3K/mTORAxis in Translocation Renal Cell CarcinomaNur P. Damayanti1, Justin A. Budka2, Heba W.Z Khella3, Mary W. Ferris2,Sheng Yu Ku4, Eric Kauffman5, Anthony C.Wood1, Khunsha Ahmed1,Venkata Nithinsai Chintala1, Remi Adelaiye-Ogala1, May Elbanna1, Ashley Orillion1,Sreenivasulu Chintala1, Chinghai Kao1,W. Marston Linehan6, George M.Yousef3,Peter C. Hollenhorst2, and Roberto Pili1

Abstract

Purpose: Translocation renal cell carcinoma (tRCC) repre-sents a rare subtype of kidney cancer associated with variousTFE3, TFEB, or MITF gene fusions that are not responsive tostandard treatments for RCC. Therefore, the identification ofnew therapeutic targets represents an unmet need for thisdisease.

Experimental design: We have established and character-ized a tRCC patient-derived xenograft, RP-R07, as a novelpreclinical model for drug development by using next-gener-ation sequencing and bioinformatics analysis. We thenassessed the therapeutic potential of inhibiting the identifiedpathway using in vitro and in vivo models.

Results: The presence of a SFPQ-TFE3 fusion [t(X;1)(p11.2; p34)] with chromosomal break-points was identi-fied by RNA-seq and validated by RT-PCR. TFE3 chromatinimmunoprecipitation followed by deep sequencing analysisindicated a strong enrichment for the PI3K/AKT/mTOR

pathway. Consistently, miRNA microarray analysis alsoidentified PI3K/AKT/mTOR as a highly enriched pathwayin RP-R07. Upregulation of PI3/AKT/mTOR pathway inadditional TFE3–tRCC models was confirmed by signifi-cantly higher expression of phospho-S6 (P < 0.0001) andphospho-4EBP1 (P < 0.0001) in established tRCC cell linescompared with clear cell RCC cells. Simultaneous verticaltargeting of both PI3K/AKT and mTOR axis provided agreater antiproliferative effect both in vitro (P < 0.0001)and in vivo (P < 0.01) compared with single-node inhibition.Knockdown of TFE3 in RP-R07 resulted in decreased expres-sion of IRS-1 and inhibited cell proliferation.

Conclusions: These results identify TFE3/IRS-1/PI3K/AKT/mTORas apotential dysregulated pathway in TFE3–tRCC, andsuggest a therapeutic potential of vertical inhibition of thisaxis by using a dual PI3K/mTOR inhibitor for patients withTFE3–tRCC. Clin Cancer Res; 1–13. �2018 AACR.

IntroductionMicropthalmia transcription factor (MiT) family translocation

renal cell carcinoma (tRCC) is a distinct subtype of kidney cancercharacterized by gene fusions, resulting from translocationsinvolving TFE3 [Xp11.2 locus (1) or TFEB (6p21 locus; ref. 2]with various partner gene (3). Since its introduction as a separateclinical entity in the 2004 World Health Organization classifica-

tion of renal tumors, tRCC has gained increasing recognition inclinical practice. It is estimated that one third of pediatric RCCs,15% of RCCs in patients �45 years of age (4), and up to 4% ofadult RCCs overall may have MiT family translocations (5).However, despite the clinical burden that tRCC presents, thereis a paucity of data regarding effective management (6).

Despite the identification of multiple TFE3 gene fusionsin tRCC including PSF–TFE3, NONO–TFE3, PRCC–TFE3 (7),ASPL–TFE3 (8), CLTC–TFE3 (9), and recent novel fusionTFE3–DVL-2 (10) and TFE3–RBM10 (11), themolecularmechan-isms underpinning tRCC oncogenesis are not well understood(3). Moreover, the heterogeneity of the dysregulated signalingpathways resulting from the variety of TFE3 gene fusions, com-bined with the lack of drugs targeting the chimeric oncoproteins,poses additional challenges to establish effective treatments.Genetically engineered cell lines (12), as well as mouse models(13), have been generated to study the biology of various tumorsharboring TFE3 fusions. However,more researchers are turning topatient-derived xenograft (PDX) models, which maintain thefidelity of the original tumor, including genomic integrity, tumorheterogeneity, and potential therapeutic responsiveness (14).Therefore, a PDXmodel can provide a translatable representationof tRCC in the laboratory setting that allows improving ourunderstanding of tumorigenesis and the real-world applicabilityof treatment options.

Identifying the target genes and DNA-binding landscape ofTFE3 is critical to characterize its functional role as a transcription

1Genitourinary Program, Division of Hematology & Oncology, Indiana UniversityMelvin and Bren Simon Cancer Center, Indianapolis, Indiana. 2Medical Sciences,Indiana University School of Medicine, Bloomington, Indiana. 3Department ofLaboratory Medicine and the Keenan Research Centre for Biomedical Science atthe Li KaShing Knowledge Institute, St. Michael's Hospital, Toronto, Canada.4Department of Pharmacology & Therapeutics, Roswell Park Cancer Institute,Buffalo, NewYork. 5Department of Urology andDepartment of Cancer Genetics,Roswell Park Cancer Institute, Buffalo, New York. 6Urologic Oncology Branch,NCI, NIH, Bethesda, Maryland.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Prior presentation: This study was in part previously presented at the 2017American Association for Cancer Research Annual Meeting.

Corresponding Author: Roberto Pili, Indiana University School of Medicine, 535Barnhill drive, Indianapolis, IN 46202. Phone: 317-278-7776; Fax: 317-278-7776;E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-18-0269

�2018 American Association for Cancer Research.

ClinicalCancerResearch

www.aacrjournals.org OF1

Research. on June 24, 2021. © 2018 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

Published OnlineFirst July 30, 2018; DOI: 10.1158/1078-0432.CCR-18-0269

http://crossmark.crossref.org/dialog/?doi=10.1158/1078-0432.CCR-18-0269&domain=pdf&date_stamp=2018-9-25http://clincancerres.aacrjournals.org/

factor in a complex gene regulatory network. To date, studiesutilizing next-generation sequencing (NGS) technology havereported TFE3 target genes andDNAbinding profile in embryonicstem cells (15), mouse embryonic fibroblasts (16), and melano-ma cells (17). However, to the best of our knowledge, the targetgenes and DNA binding profile of TFE3 in tRCC cells have not yetbeen reported. Chromatin immunoprecipitation followed bydeep sequencing (ChIP-Seq), an application of next NGS, pro-vides an efficient method for global profiling of DNA-bindingproteins and identification of their target sites on a genome widescale. Therefore, ChIP-Seq is a valuable tool that could be used togain novel biological insight of TFE3 gene regulatory networksand oncogenic pathways in tRCC.

In addition to transcription factors, miRNAs also play anintegral role in a tightly controlled genetic regulatory system.miRNAs are short, noncoding RNA molecules that posttranscrip-tionally targetmRNA tomodulate gene expression (18). In highlyinterconnected network, both transcription factors and miRNAwork to orchestrate cascade and/or combined regulatory func-tions to facilitate cellular physiology (19). Therefore, analysis ofboth the miRNA and transcription factor regulatory network arepertinent in the identification of key genomic elements and theirassociated pathways. Furthermore, dysregulated miRNAs havebeen frequently implicated in carcinogenesis (20). Thus, theirexpression profile is of particular importance in oncology to aid inbiomarker selection (21), cancer classification, and moleculartarget identification (22).

In this study, we established a novel tRCC PDX preclinicalmodel to serve as a platform for improving our understanding ofthis disease. We performed molecular characterization studies,including RNA sequencing (RNA-seq), ChIP-seq, miRNA expres-sion profiling by RT-PCR, and immunodetection techniques. Weapplied the molecular signatures of our tRCC PDX model togenerate hypothesis regarding potentially targetable pathwaysinvolved in oncogenesis using bioinformatic pathway analysistools. We subsequently assessed the therapeutic potential ofinhibiting an identified dysregulated pathway in tRCC using bothin vitro and in vivo studies.

Materials and MethodsMethods PDX RP-R07t generation

The studies presented were conducted in accordance with theDeclaration of Helsinki, and after approval by the RPCI Institu-tional Review Board and obtaining written consent from thesubject. Nonnecrotic areas of lymphoid metastatic nodule from

a patient with tRCC were sectioned into approximately 4 mm3

pieces. Fragments of the tumor containing both malignant cellsand supportive stromal components were implanted subcutane-ously into the flanks implanted subcutaneously into anaesthe-tized 5-week to 6-week-old female NSG mice (The Jackson Lab-oratory). During the engraftment phase, tumors were allowed toestablish and grow and then were harvested upon reaching a sizeof 1,500 mm3. Harvested tumor was divided for three purposes:(i) patient-derived cell line; (ii) subsequent expansion throughserial passaging in NSG mice; and (iii) biological assays forhistologic and molecular characterization of established PDX.The mice (P1 generation) were maintained under pathogen-freeconditions and a 12-hour light/dark cycle. When P1 tumorsreached an approximate size of 1,800 mm3, they were harvested,fragmented, and reimplanted into additional mice (P2 genera-tion) while maintained as a live bank according to approvedInstitutional Animal Care and Use Committee protocols. Whenenough P2 reached a volume greater than 200 mm3, the animalswere divided into four groups (vehicle, rapamycin, MLN0128,and BEZ-235).

Patient-derived cells RP-R07RP-R07 tumor pieces (�4mm2) were placed in a 6-well culture

plate and removed after being cultured for 24 hours in supple-mented DMEM high glucose media (10% FBS; 1% penicillin/streptomycin). Adherent cells were a mixed population of tumorcells and fibroblasts. These cells were cultivated with feeder cellsand supplemented with ROCK inhibitor until approximately80% confluent. Serial passaging of these heterogeneous cultureswas performed, until a homogeneous monolayer of RP-R07 cellswas present. RP-R07 cell were subsequently cultured in DMEMmedium (Gibco) supplementedwith 10%FBS and1%penicillin/streptomycin at 37�C, 5% CO2. The UOK109 and UOK146 celllines were established by Dr. Linehan's laboratory at the NCI(Rockville, MD; ref. 3).

Fusion detection by RNA-seqThe RNeasy Mini Kit (Qiagen) was used for the isolation of

RNA according to the manufacturer's instructions. Site-specificreverse transcription was performed with the reverse transcriptaseSuperscript III (Invitrogen) and five 30 primers spaced throughoutthe TFE3 transcript. Following reverse transcription and subse-quent second strand synthesis, the sequencing library for fusiondetection was generated using an Illumina TruSeq sample prep-aration protocol for single-end reads. Reads were aligned usingTopHat with Bowtie1 and the fusion search option selected.Finally, tophat fusion post was used to identify putative fusiontranscripts with a minimum of three supporting fusion reads.

Fusion validation by RT-PCRTotal RNA was extracted by TRIzol (Invitrogen) according to

manufactures instructions. Two microgram of RNA was used toperform cDNA synthesis by iScript cDNA Synthesis Kit (Bio-Rad)and then subjected to PCR reactions. To detect hybrid transcriptsthe resulting cDNA was subject to amplification with the SFPQexon 7 primer 50-CGTCAACGTGAGATGGAAGA-30 (forwardprimer) and the exon 6 TFE3 primer 50-GCAGGAGTTGCTGA-CAGTGA-30 (reverse primer) for SFPQ-TFE3, PRCC exon1primer,50-AGGAAAGAGCCCGTGAAGAT-30 (forward primer) andTFE3 exon 6 primer, 50-GTTCTCCAGATGGGTCTGC-30 (reverseprimer) to detect PRCC-TFE3 and NONO exon 9 primer,

Translational Relevance

Despite the significant progress achieved by targeted ther-apies in renal cell carcinoma, patients with translocation RCCcontinue tohave a poor outcome. The lack of understandingofthe biology of this aggressive subtype remains a major hurdlefor the development of effective therapies. Thus, we havediscovered a key signaling pathway activated by the transcrip-tional factor TFE3 as the result of the pathognomonic genomicalteration in translocation RCC. Therefore, we have identifiedan effective combination strategy that can be readily translatedto patients with this orphan, deadly disease.

Damayanti et al.

Clin Cancer Res; 2018 Clinical Cancer ResearchOF2



http://clincancerres.aacrjournals.org/

50-ATCAAGGAGGCTCGTGAGAA-30 (forward primer), and TFE3exon 6 primer, 50-GTTCTCCAGATGGGTCTGC-30 (reverse primer)to detect NONO-TFE3. In these analyses, all reverse-transcribedsamples gave a b-actin PCR product of the expected size. Theamplification conditions were 93�C for 20 seconds, 58�C for 40seconds, and 72�C for 40 seconds for 35 cycles in a final volume of25 mL. The products were separated by electrophoresis in agarosegels followed by staining with ethidium bromide.

ChIP-seq and analysisChIP was performed as described previously (23). Cells were

crosslinked using 1% (v/v) Formaldehyde (Thermo Fisher Scien-tific) for 15 minutes before quenching with 2 mol/L glycine for 5minutes. Cells were sonicated for 3 minutes (30 seconds on, 30seconds off) using a Diagenode Bioruptor Pico water bath soni-cator. Following sonication, the lysate was rotated with the TFE3antibody (P16: sc-5958) for 4 hours at 4�C before subsequentwashing andDNA isolation by phenol–chloroform. Library prep-aration was performed as described previously (24). Reads werealigned to the genome with Bowtie before duplications and hg19blacklisted readswere removed. Peak calling was performed usingMacs v1.4.2, and following peak calling, nearest neighboringgenes were determined using the Useq platform (http://useq.sourceforge.net/). Enriched motifs were determined using theMEME-ChIP software within the MEME Suite online package forall called TFE3-binding sites.

Total RNA extraction for miRNA expression screeningTotal RNA isolationwas done using themiRNeasy Kit (Qiagen)

according to the manufacturer's protocol. RNA quality and con-centration were determined spectrophotometrically (NanoDrop1000 Spectrophotometer; NanoDrop Technologies Inc.). Sam-ples optimal for analysis were stored at �80�C.

miRNA expression screening by TaqMan low-density arraycards

Five hundred nanograms of total RNA from each sample werereverse transcribed using aMegaplex Primer PoolHuman Set AþB(Life Technologies) with a TaqManmiRNA Reverse-TranscriptionKit as suggested by themanufacturer. cDNA samples of individualpatients were analyzed by a TaqMan low-density array humanmiRNA card set AþB. Relative expression was determined usingtheDDCTmethod and expression valueswere normalized to smallnuclear RNA, U6 snRNA, RNU48, and RNU44.

MTT cell proliferation assayRP-R07, UOK-109, and UOK-146 cells (3,000 cells/well) were

seeded in 96-well plates and incubated for 24 hours at 37�C and5% CO2. The following day, cells were treated with drugs withdefined concentrations. All drugs for in vitro study sunitinib(LC Laboratories), gemcitabine (LC Laboratories), doxorubicin(LC Laboratories), crizotinib (LC Laboratories), BKM-120(Novartis), MLN0128 (Millenium), and BEZ-235 (Novartis) weredissolved in DMSO for the preparation of stock solutions(10 mmol/L). Cell viability was determined by measuring dehy-drogenase activity. We changed the medium and applied 100mLof serum-free mediumwith 25mL of MTT (5mg/mL) to each welland incubated the cells for 4 hours at 37�C and 5% CO2 to allowthe formation of a purple formazan salt. The medium wasreplaced with 100mL of methanol to dissolve the formazancrystals and the plates were incubated for a further 15minutes

at room temperature before the absorbance was measured at l ¼570nm using a Micro Plate Reader (BioTek Synergy HTX).

Histology/IHCMice were sacrificed by CO2 asphyxiation at defined time

points. Collected tumor tissuewas fixed in 10%buffered formalinovernight followed by an additional 24 hours in 70% ethanol.Formalin-fixed embedded tissue was cut using microtome with10-mm thickness. Tissue slides were dried overnight and subjectedto deparaffinization in xylene. For antigen retrieval, slides wereboiled for 10minutes in 10mmol/L sodium citrate pH 6 solutionfor all antibodies. ImmPRESS detection system (Vector Labora-tories) was used for detection of TFE3 [TFE3 (P-16): sc-5958,Santa Cruz Biotechnology]. Staining was visualized using 3,30-diaminobenzidine (DAB; Sigma; FAST DAB) and slides werecounterstained with hematoxylin.

Immunofluorescence microscopyCells grown on glass coverslips with and without drug treat-

mentwerewashedwith PBS and fixedwith 4%paraformaldehydeon ice for 15 minutes. After fixation, cells were washed with PBSand then permeabilized with 0.1% Triton X-100 in PBS at roomtemperature for 20 minutes followed by blocking with 2.5% BSAin PBS for 90 minutes. Cells were then incubated with theindicated primary antibodies; rabbit anti–phospho-ribosomalS6 Ser235/236 (Cell Signaling Technology, #2211, at 10 mg/mL),rabbit anti–phospho-4EBP-1 (Cell Signaling Technology; #2855and#9451, at 20mg/mL), rabbit anti–phosphor-AKT (ser473;CellSignaling Technology #4060 at 10 mg/mL), mouse anti-IRS-2(Millipore #MAB S15 at 10 mg/mL), rabbit anti-LDH (Santa CruzBiotechnology; #sc-33781 at 10 mg/mL), rabbit anti-TFEB (BethylLaboratories; #A303-673Aat 10mg/mL), rabbit anti-TFEC (Sigma;#AV32279), rabbit anti-N-terminal TFE3 (Santa Cruz Biotechnol-ogy; #sc-33781), and IR in IF buffer (PBS containing 2.5% BSAand 0.1%Triton X-100) overnight at 4�C. Cells were washed threetimes with PBS and incubated with the corresponding secondaryantibodies conjugated to Alexa Fluor 633-conjugated goat anti-mouse IgG or Alexa Fluor 633–conjugated goat anti-rabbit IgG(1:2,000; Life Technologies) in IF buffer for 30 minutes at roomtemperature. PBS washed coverslips were mounted onto glassslides with VECTASHIELD antifade mounting medium (VectorLabs). Images were acquired on EVOS-FLc AMEFC4300 fluores-cence imaging system (Thermo Fisher Scientific) with the sameacquisition parameters for each group. Images taken were pro-cessed and quantifiedwith ImageJ software (NIH, Bethesda,MD).

siRNA-mediated TFE3 silencingCells were transfected with siRNAs targeting TFE3 (Silencer

Select siRNAs; Sigma; #s14032) or a scrambled siRNA (SilencerSelect Negative Control No. 1 siRNA; Sigma; #4390843). RP-R07cells were cultured in 6-well plates until 50% to 60% confluence,transfected with TFE3-siRNA or scramble with a final concentra-tion 100 nmol/L using Lipofectamine RNAiMAX TransfectionReagent (Invitrogen; #13778075) according to themanufacturer'sinstructions. At 72hours after transfection, cellswereharvested forqRT-PCR or immunofluorescence analyses.

In vivo animal treatmentsThe Institute Animal Care and Use Committee at Indiana

University approved all mouse protocols used in this study. Micewere housed in a BSL-2 level animal facility maintained on a

TFE3 Translocation Renal Cell Carcinoma and PI3K/mTOR

www.aacrjournals.org Clin Cancer Res; 2018 OF3



http://useq.sourceforge.net/http://useq.sourceforge.net/http://clincancerres.aacrjournals.org/

12-hour light/dark cycle, at a constant temperature (22 � 2�C)and relative humidity (55�15%).NSGmice for in vivo studywerepurchased from an in-house colony maintained at Indiana Uni-versity. Approximately 6-week-old NSG mice were implantedsubcutaneously with �3 mm3 pieces of RP-R07 tumor andallowed to grow until tumors reached 200 mm3 in volume priorto treatment with either vehicle, Rapamycin (EMD Chemicals),MLN0128 (kindly provided by Millenium Pharmaceuticals), orBEZ-235 (kindly provided by Novartis Pharmaceuticals). Tumorbearing mice received therapy with Rapamycin (2 mg/kg daily;intraperitoneal injection). MLN0128 (3 mg/kg, three times aweek; intraperitoneal injection), BEZ-235 (25 mg/kg daily-5days/week; oral gavage), or vehicle (daily-5 days/week; oralgavage). Throughout the study, all mice receiving therapy wereweighed twiceweekly tomonitor for toxicities. Tumor growthwasassessed by serial caliper measurements twice weekly.

Statistical analysisStatistical analysis was performed using GraphPad prism soft-

ware. Differences among experimental groups were tested by Stu-dent t test or for variances by ANOVA followed by Tukey posttest.P values less than0.05were considered tobe statistically significant.

ResultsEstablishment of RP-R-07t PDX model

A24-year-oldCaucasianmale patientwithnopreviousmedicalhistory presented with a symptomatic, large tumor mass in theright kidney. The patient underwent nephrectomywhich revealeda high-grade, mixed clear cell/papillary RCC. Further analysisdriven by his young age led to the diagnosis of MiT family tRCCassociated with an Xp11.2 translocation/TFE3 gene fusion byFISH analysis. The patient developed rapidly growing metastases,initially in the lymph nodes and lungs. Therapies with a VEGFRtyrosine kinase inhibitor (sunitinib), an mTOR inhibitor (ever-olimus), and chemotherapeutic drugs (doxorubicin and gemci-tabine) had no effect on tumor progression and eventually thepatient deceased within 1 year from diagnosis.

During the course of the disease, we obtained a lymph nodebiopsy (Fig. 1A). The resected tissue was partitioned into approx-imately 3 to 5 mm3 pieces, processed, and implanted subcutane-ously into 6-week-old female NOD/SCID gamma (NSG) mice.We allowed tumors to grow to a size of approximately 1,500mm3

during the engraftment phase, at which point they were harvestedfor the following purposes: (i) establishment of a cell line, (ii)further expansion through serial passaging in NSGmice (Fig. 1B),and (iii) histologic and molecular characterization. To developthe RP-R07 cell line, we adopted a conditional reprogrammingmethod utilizing Rho-associated, coiled-coil containing proteinkinase (ROCK) inhibitor and irradiated NIH-3T3 murine fibro-blasts. We evaluated whether our PDX preclinical model main-tained histologic features of the original lymph node metastasis.The cellular architecture in our PDX tumor model remainedremarkably faithful to the tumor original morphology (Fig.1C), exhibiting similar mixed papillary and clear cells character-istics. Furthermore, our PDXmodel demonstrated the same strongTFE3 nuclear immunoreactivity observed in the original biopsy.

Identification of SFPQ–TFE3 fusion in RP-R07 by RNA-seqTo identify the specific TFE3 fusion gene and chromosomal

breakpoint in our preclinical model, we sequenced RNA isolatedfrom RP-R07 cells. Reverse transcription using multiple oligonu-

cleotides complementary to TFE3 was followed by NGS to char-acterize TFE3 fusion transcripts. A fusion transcript was identifiedspanning the SFPQ geneon chromosome1pand theTFE3 geneonchromosome Xp. The genomic coordinates of the RNA fusionjunction localized to specific chromosomal break-points(Chr1:35652601; ChrX:48895638; Fig. 1D). This location corre-sponds to the end of SFPQ exon 9 and the beginning of TFE3 exon5. The expression of the SFPQ–TFE3 fusion transcript was con-firmed by subjecting cDNA fromRP-R07 to RT-PCR amplificationwith 50-SFPQ and30-TFE3primers and, as a control, primer sets forthe NONO-TFE3 (25) and PRCC-TFE3 fusions (26). Only theSFPQ–TFE3 hybrid transcript with a predicted size of 375bpwas observed in RP-R07 (Fig. 1E), whereas the NONO–TFE3and PRCC–TFE3 fusions were not detected. Using the sameSFPQ–TFE3 primers, we did not detect the presence of theSFPQ–TFE3 transcript in the NONO–TFE3 fusion-bearingUOK-109 cell line (Fig. 1F; ref. 25), the PRCC–TFE3 fusion-bearing UOK-146 cell line (26), the UMR-C2 ccRCC cell line, orthe HK-2 human renal cell tubule cell line.

TFE3 nuclear expression is characteristic of tRCCXp11.2 tRCC contains fusion genes that encode chimeric

proteins consisting of the N-terminal portion of different trans-location or inversion partners fused to the C-terminal portion ofTFE3 (3). Therefore, chromosomal rearrangements involving theTFE3 gene at Xp11.2 are characterized by strong nuclear immu-noreactivity for the C-terminal portion of TFE3 regardless of TFE3fusion gene partner (27). We identified enhanced nuclear immu-noreactivity of C-terminal TFE3 in three different tRCC models:RP-R07 (SFPQ–TFE3), UOK-109 (NONO–TFE3), and UOK-146(PRCC–TFE3). In contrast, nuclear immunoreactivity was low inthe ccRCC cell line, UMR-C2 (Fig. 1G). However, despite thecommon presence of C-terminal TFE3 immunoreactivity, eachtRCC model demonstrated distinct expression levels and distri-bution patterns. Colocalization analysis (Fig. 1H) represents thelevel of C-terminal TFE3 nuclear localization. The UOK-109model showed the highest C-terminal TFE3 nuclear immunore-activity with a dense expression pattern (R ¼ 0.78, UOK-109-UMR-C2; P < 0.0001). RP-R07 demonstrated moderateC-terminal TFE3 nuclear immunoreactivity with a diffuseexpression profile (R ¼ 0.69, RP-R07-UMR-C2; P < 0.0001);whereas, the UOK-146 model demonstrated the lowestC-terminal TFE3 nuclear immunoreactivity with a mixedexpression pattern involving diffuse and speckle components(R ¼ 0.62, UOK-146-UMR-C2; P < 0.0001).

ChIP-seq–based TFE3-DNA binding profiling reveals TFE3occupancy of genomic regions with consensus E-box motifsboth in vitro and in vivo

WeperformedChIP-seq to explore theDNA-binding landscapeof TFE3 in RP-R07 (patient-derived cells) and RP-R07t (PDX) andUOK-146 cells. TheWestern blot analysis result demonstrates thespecificity of the antibody recognizing TFE3 in all three cellsRP-R07, UOK-146, and UOK-109 (Supplementary Fig. S1).ChIP-seq in RP-R07 cells identified 3032 significant TFE3 peaks.ChIP-seq from RP-R07t identified 856 significant TFE3 peaks(Supplementary Tables S1 and S2). A comparison of TFE3ChIP-seq signal across all bound regions revealed a strong overlapbetween RP-R07 and RP-R07t (Fig. 2A), indicating that the in vitroand in vivo model have similar TFE3-binding profiles. Further-more, we have also observed identical binding profile of TFE3 in

Damayanti et al.





UOK-146 cells (Supplementary Fig. S2). An unbiased search foroverrepresented sequencemotifs inTFE3-bound regionsbyMEME-ChIP identified the known TFE3 target E-boxmotif (CACGTGA) asthe most enriched motif in both cell line and tumor samples (Fig.2B). Notably, AP-1 (TGACTCA) and ETS (AGGAA) binding motifsare the second and third most enriched motifs in TFE3 boundregions in the cell line. The identified TFE3peaksdistribution inRP-R07 and RP-R07t are presented in Supplementary Figs. S3 and S4,respectively.

TFE3 target genes are associated with the PI3K/AKT/mTORsignaling pathway

We applied our ChIP-seq results to study the molecular path-ways targeted by the SFPQ–TFE3 fusion gene product usingpathway analysis bioinformatics tools, including KEGG,

PANTHER, and WIKI. Comprehensive panels of 287 KEGG path-ways, 96 PANTHER pathways, and 403 WIKI tools pathwaysassociated with the SFPQ–TFE3 fusion gene product are listed inSupplementary Tables S3, S4, and S5, respectively. Based on theseresults, we noted that the PI3K/AKT/mTOR axis was consistentlyranked as a top significantly influenced pathway in all threeanalysis methods (Fig. 2C–E). When looking closely at ourChIP-seq results, we were able to identify of SFPQ–TFE3 targetedgenes related to the PI3K/AKT/mTOR pathway, such as PI3KCA,TSC1, AKT3, PTEN, 14-3-3, ITGB1, IGFR1, and IRS-1 (Fig. 2F).

miRNAome landscape profiling reveals molecular pathwaysignatures of RP-R-07

After profiling the TFE3 transcriptional architecture in our tRCCmodel, we further studied its posttranscriptional miRNAome

Figure 1.

Generation of a PDX RP-R07t and RP-R07 cells from a patient with tRCC. A, Schematic diagram of development of PDX as a model for therapeutic strategiesin the treatment of patients with tRCC. B, PDX model demonstrates its ability to self-propagate. B, Growth of primary tumor graft is represented as tumor volumeversus time after implantation. Different color indicated different passages. At least four mice were included in each group. C, Faithful resemblance of cellularcomplexity, architecture, and molecular signature of PDX tumor to the patient tumor. Hematoxylin–eosin staining revealed that the PDX model (bottom)recapitulates the histologic appearance of patient tumor, showing characteristic phenotype of mixed papillary architecture and clear or eosinophilic cytoplasm,nested alveolar pattern, voluminous cytoplasm, prominent nucleoli, and the presence of psammoma bodies. Molecular signature of X11p translocation,immunoreactivity of nuclear TFE3, is observed in patient biopsy sample (top right) and is preserved in PDX model (bottom right). D, TFE3 fusion architecture byRNA-seq. In-frame fusion transcripts were identified with the chromosomal coordinates corresponding to the fusion position indicated in red (GRCh37/hg19)and the fusion sequence in grey. E, PCR fusion validation in RP-R07 using SFPQ–TFE3, NONO–TFE3, and PRCC–TFE3 primers, F, PCR validation in RP-R07,UOK-109, UOK-146, UMR-C2, and HK2 using SFPQ–TFE3 primer. G, Nuclear immunoreactivity of TFE3 is exclusive for tRCC. Immunofluorescence profile of patient-derived cells and cell line stained with the same TFE3 (internal epitope sc-4784) antibody shows positive nuclear immunoreactivity of TFE3, identified withcolocalization (grey white) of nuclear TFE3 (cyan) with DNA stain Hoechst (red) in tRCC cells; RP-R07, UOK-146, and UOK-109 and lower expression (P < 0.005)of nuclear TFE3 in RCC cell line UMR-C2 H. Colocalization analysis by measuring Pearson correlation coefficient (R) between green channel (TFE3) and redchannel (Hoechst), indicating strong nuclear localization of TFE3 in tRCC (R >0.5), and significantly lower expression of nuclear TFE3 in UMR-C2 (R < 0.5; P < 0.005).H, Quantitative analysis.






landscape. Expression profile analysis of thewholemiRNAome intRCC (RP-R07t), ccRCC, and pRCCPDXmodels (also establishedin our lab) was performed using TaqMan low-density arrayhuman microRNA card set AþB. These are preloaded arrays withTaqMan Gene Expression Assays for mature miRNAs. Despiteoverlapped histologic features of ccRCC and pRCC PDX in ourtRCC PDX (Fig. 3A), several miRNAs showed variable expressionamong the three varied subtypes (Fig. 3B). Unsupervised hierar-chical clustering using one minus Pearson correlation with aver-age linkage further classified tRCC, ccRCC, and pRCC into threewell-defined clusters and differentially expressed miRNA intonine well-defined clusters (Fig. 3C). Cluster 3 had the greatestdeviation of miRNA expression, with a more than two-foldchange of expression in our tRCCmodel as compared with ccRCCand pRCC PDX models (Fig. 3D). To further understand thebiological impact of differentially expressed miRNA in our tRCCmodel,weusedDIANA-miRPath to performapathway analysis ofthe miRNA in cluster 3. Hierarchical clustering heatmap revealedsignificantly targeted pathways by the miRNA signature in cluster3 (Fig. 3E). The top significantly enriched pathways based on thenumber of miRNA-targeted genes associated with each pathway(Fig. 3F) were "Pathways in cancer" (P ¼ 1.11E�16), "PI3K-Akt

signaling pathway" (P ¼ 4.91E�09), "Proteoglycans in cancer"(1.11E-16), "Focal adhesion" (P ¼ 6.26E�10), "Viral carcino-genesis" (P ¼ 1.11E�16), "MAPK signaling pathway" (P ¼0.000121), and "Hippo signaling pathway" (P ¼ 1.11E�16). Acomplete list of the statistically enriched pathways targeted bydifferential expression of miRNA in cluster 3 is available inSupplementary Table S6. Enriched KEGG PI3K/AKT signalingpathway visualization (Supplementary Fig. S5) shows thatalmost all predicted genes in this pathway are targeted byaberrantly expressed miRNA in cluster 3, including PI3KCA,AKT1, IRS1, RPS6, TSC1, eIF4BP1, and mTOR among others. Acomplete list of targeted genes in the PI3K/AKT signalingpathway with their associated miRNA from cluster 3 is reportedin Supplementary Table S7.

Upregulation of PI3K/AKT/mTOR pathway in tRCCThe PI3K/AKT (28) and mTOR (29) signaling pathways func-

tion interdependently to regulate cellular growth, proliferation,angiogenesis, and survival. Their roles are so intertwined suchthat they are often unified into one unique signaling axis, PI3K/AKT/mTOR. Oncogenic dysregulation of this pathway has beenimplicated in a variety of tumors, including ccRCC (30). Thus, we

Figure 2.

Profiling of TFE3-DNA binding landscape in RP-R07 and RP-R07t by ChIP-seq. A, Heatmap comparison of TFE3 binding in RP-R07 and RP-R07t at all sitescalled as bound in either sample. B, The three most enriched sequence motifs identified by the MEME-ChIP program for TFE3 bound regions in RP-R07 orRP-R07t. C–E, Top 12 pathways associated with TFE3 target genes identified by KEGG (C), PANTHER (D), and WIKI tools (E). F, Selected TFE3-targeted genesassociated with the PI3K/AKT/mTOR pathway.

Damayanti et al.





were interested in testing whether this pathway is also involved intRCC, starting with the examination of P13K/AKT/mTOR activityin our tRCC panel. Phosphorylation of S6 ribosomal protein(S6rp) and 4E-BP1 occurs at the end of the P13K/AKT/mTORsignaling cascade to facilitate translation. Thus, by measuring theimmunoreactivity of phosphorylated S6rp (31) and 4E-BP1, wecan gauge the level of P13K/AKT/mTOR activation. Using aquantifiable immunofluorescence technique, our results suggesta higher level of phospho-S6rp (Fig. 4A and C) expression in thetRCC cell lines as compared with the ccRCC cell line. The expres-sion levels of phospho-4E-BP1 in the tRCC cell lines were alsohigher than those observed in the ccRCC cell line as well (Fig. 4BandD). These results suggest that the PI3K/AKT/mTORpathway isdisproportionately upregulated in tRCC regardless of TFE3 genefusion partner.

Effective in vitro multinodal P13K/AKT/mTOR inhibition inRP-R07

On the basis of the dysregulation of the P13K/AKT/mTORpathway, we tested the antitumor effect of inhibiting this pathwayin tRCC cell lines, as a potential therapeutic strategy. We designedthree vertical inhibition schemas to target the PI3K/AKT/mTORaxis at different points within the pathway: (i) PI3K/AKT axis

inhibition with the P13K inhibitor BKM-120, (ii) m-TOR axisinhibition with the pan-mTOR inhibitor MLN0128, and (iii)simultaneous inhibition of the PI3K/AKT and mTOR axis withthe dual P13K/mTOR inhibitor BEZ-235. We first examinedwhether the drug response profiles of our tRCC panel werereflective of the lack of response to antineoplastic agents that thepatients experienced. RP-R07, UOK 109, and UOK-146 cells wereexposed to increasing concentrations of sunitinib, doxorubicin,and gemcitabine for 96 hours (Fig. 4E–G). The tRCC celllines were relative insensitive to these anti-neoplastic agentsexcept for gemcitabine. Thus, we evaluated our multinodalPI3K/AKT/mTOR inhibition strategy in vitro. An MTT assay wasperformed after cells were treated with different concentrations ofBKM-120, MLN0128, rapamycin, or BEZ-235 for 96 hours toassess the antitumor activity of these agents (Fig. 4H–J; Supple-mentary Fig. S6). BKM-120 treatment inhibited cellular prolifer-ation in a concentration-dependent manner with IC50 values of420, 373.6, and 714 nmol/L for RP-R07, UOK-109, and UOK-146, respectively (Fig. 4K). The dual TORC1/TORC2 inhibitorMLN0128 demonstrated greater antiproliferative effect than thePI3K inhibitor BKM-120 with 10-fold lower IC50 values (RP-R07:IC50 ¼ 49.4 nmol/L, UOK-109: IC50 ¼ 24.3 nmol/L, andUOK-146: IC50¼8.18nmol/L). Treatmentwith dual PI3K/mTOR

Figure 3.

miRNA profiling of RP-R07t. A, IHC staining of RP-R07, pRCC, and ccRCC PDX. B, Heatmap showing 786 differential miRNA expression profile. C, Hierarchicalclustering of miRNA expression separate tRCC subtype from pRCC and tRCC.D, Number of differentially expressed RP-R07t miRNAwith >2-fold change comparedwith pRCC and or ccRCC in each cluster in C. E, miRNAs versus pathways heatmap (clustering based on significance levels). F, Top significant KEGG pathwayassociated with differentially expressed miRNA based on the number of associated miRNA target genes.






inhibitor BEZ-235 had the lowest IC50s in our tRCC panel(RP-R07: IC50 ¼ 12.2 nmol/L, UOK-109: IC50 ¼ 13.41 nmol/L,and UOK-146: IC50 ¼ 7.03 nmol/L). Taken together, theseresults suggest that simultaneous vertical inhibition of thePI3K/AKT/mTOR axis with a dual PI3K/mTOR inhibitorprovides a greater antiproliferative effect in vitro as compared withP13K/AKT or mTOR inhibition alone for the treatment of tRCC.

Attenuation of PI3K/AKT/mTOR downstream targets byBEZ-235

To validate whether the antiproliferative effect of BEZ-235 inRP-R07 cells was associated with biochemical attenuation of thePI3K/AKT/mTOR pathway, we assessed the phosphorylation andexpression levels of selected key nodes by immunofluorescence.BEZ-235 treatment inhibited phosphorylation of 4EBP-1 atSerine-65 (78% inhibition; P < 0.0001) and Threonine-37(58% inhibition: P < 0.0001; Fig. 5A and B). Correspondingsignal reduction of phosphorylated AKT at Serine-473 (27%inhibition; P < 0.001) and phosphorylated S6rp at Serine 240/244 (63.3% inhibition; P < 0.0001) was also observed. In addi-tion, we observed decreased expression of PI3K/AKT/mTOR pos-sible downstream target such as lactate dehydrogenase (54.4%

inhibition; P < 0.0001), IRS-1 (33.9% inhibition; P < 0.0001),TFE3 (24.1% inhibition; P < 0.0001), TFEB (53% inhibition; P <0.0001), and TFEC (41% inhibition; P < 0.0001).

TFE3 silencing attenuates PI3K/AKT/mTOR signaling andsuppresses IRS-1 expression and cell proliferation in RP-R07

We have shown that wild-type TFE3 is a potential downstreamtarget of PI3K/AKT/mTOR axis as PI3K/mTOR inhibition sup-presses TFE3 expression. Therefore, to further determine whetherTFE3 inhibition by PI3K/AKT/mTOR directly affects cellular bio-logical function, we assessed the effect of siRNA-mediated TFE3silencing on RP-R07 cell proliferation. First, we confirmed theefficacy of our siRNA treatment by assessing TFE3 endogenousmRNA transcript level and protein expression after cells treatmentwith TFE3-siRNA and negative control-siRNA, scramble RNA(Fig. 6A). Further, decrease of protein expression levels of TFE3following TFE3-siRNA treatment was observed by immunofluo-rescence (Fig. 6B and C). Next, we assessed whether inhibition ofTFE3 provides regulatory feedback on PI3K/AKT/mTOR signalingaxis. Decrease expression of phospho-4EBP1 (Fig. 6D and F)and phospho-S6 ribosomal (Fig. 6E and G) was observed inTFE3-siRNA treated cells compared with scramble RNA treated

Figure 4.

Simultaneous vertical inhibition of PI3K/AKT andmTORpathways in tRCC.A,PI3K/AKT/m-TORpathway upregulation in tRCC. Representative immunofluorescenceimages of fixed tRCC cells; RP-R07, UOK-109, UOK-146 and ccRCC cell, UMR-C2, stained with anti-phospho S6 (red) and anti F-actin (green), and Hoechst(A; blue); anti-phospho 4EBP-1 (red) and Hoechst (B; blue) and corresponding quantification (C andD). E–J,RP-R07 exhibits similar chemoresistance as observed inthe patient. Three different tRCC cells, RP-R07, UOK-109, and UOK-146, were treated for 96 hours with indicated concentrations of different drugs. Cell growth wasassessed by MTT assay and absorbance was measured at 589 nm. Each dot and error bar on the curves represents mean� SD (n ¼ 8). All experiments wererepeated at least three times. K, IC50 value of agent in H to J. BEZ-235 has the lowest IC50 compared with other agents.

Damayanti et al.





cells. This result suggests the possible regulatory role TFE3 onPI3K/AKT/mTOR pathway via a feedback loop mechanism inwhich TFE3 is not only downstream target of PI3K/AKT/mTORpathway, but also directly regulates this signaling pathway. As faras the regulatory mechanism of TFE3 on PI3K/AKT/mTOR path-way, we propose that this may be mediated through TFE3 targetgenes which are upstream effectors of this signaling axis. On thebasis of our ChIP-seq results (Fig. 2F; Supplementary Table S2),TFE3 binds to IRS-1, an upstream effector of PI3K/AKT/mTOR(32). Therefore, we further validated the transcriptional regula-tory role of TFE3 on IRS-1 by assessing the effect of TFE3 inhi-bition on IRS-1 expression. SiRNA mediated silencing of TFE3decreased endogenous expression of IRS-1mRNA transcript com-pared with scramble RNA treatment (P < 0.01; Fig. 6H). Next, theanalysis of immunofluorescence images further demonstrated thedecrease of IRS-1 protein expression following TFE3-siRNA treat-ment compared with scramble RNA treatment (P < 0.001; Fig. 6Iand J). Finally, TFE3 silencing inhibited cell proliferationof RP-R07 in a time-dependent fashion (Fig. 6K). FollowingTFE3-siRNA treatment at 96 and 110 hours, the cell number wassignificantly reduced (96 hours P¼ 0.04, 110 hours P < 0.00001)in RP-R07 compared with the nonsilencing control siRNA trans-fected cells and nontreated cells.

Effective in vivo multinodal P13K/AKT/mTOR inhibition inRP-R07

Next, we investigated the antiproliferative effect of MLN0128and BEZ-235 in vivo. We also wanted to assess the efficacyof rapamycin, an mTOR complex-1 (mTORC1) inhibitor, as acomparator because the mTOR inhibitors that are FDA approvedin the treatment of RCC, everolimus and temsirolimus, have asimilar mechanism of action (33). Everolimus and temsirolimushave shown limited clinical activity in RCC (34–37), in part dueresistance mechanism(s) via mTORC2-mediated rebound AKThyperphosphorylation. Therefore, we postulated that MLN0128and BEZ-235 could provide superior antitumor effects as com-pared with rapamycin given their ability to impact this resistancepathway. To test these drugs, NSG mice bearing subcutaneouslyimplanted RP-R07 xenografts were treated with MLN0128(3 mg/kg), rapamycin (2 mg/kg), or BEZ-235 (25 mg/kg) for28 days. The vehicle and treatment group mice (5 mice/group)maintained their body weight throughout, incurring in modestweight loss (Supplementary Fig. S7). Treatment of RP-R07 xeno-grafts with MLN0128, rapamycin, and BEZ-235 resulted indecreased tumor weight (Fig. 6L) compared with the vehiclecontrol. However, only treatment with BEZ-235 resulted in astatistically significant lower tumor weight when compared with

Figure 5.

BEZ-235 treatment associates with attenuation of PI3K/AKT/mTOR pathway. A, Immunofluorescence images of p-4EBP-1(ser 65), p-4EBP-1 (Thr37), pS6, p-AKT(S473), LDH, IRS-1, TFE3. TFEB, TFEC in BEZ-235 treated RP-R07 cells. B, Immunofluorescence quantification and Student t test analysis.






vehicle control (P < 0.01). RP-R07t cells of the tumor in theBEZ-235–treated group appeared smaller than those in the vehicle(Fig. 6M), indicating inhibition of mTOR pathway (38). Theproliferation marker Ki67 was reduced in the BEZ-235–treatedgroup as compared with the untreated control (Fig. 6M and N),indicating inhibition of cell cycle (90% inhibition, P < 0.0001).The in vivo immunostaining replicated the in vitro results demon-strating that BEZ-235 significantly reduced the phosphorylationlevel of S6 ribosomal protein (83% inhibition, P < 0.0001; Fig. 6MandO). The analysis ofmicrovessel density, as measured by CD31staining, revealed significant inhibition of tumor angiogenesis inBEZ-235–treated RP-R07 tumors (56% inhibition at day 28; P <0.0001) comparedwith vehicle (Fig. 6MandP).Overall, these datasuggest the therapeutic efficacy of a multinodal P13K/AKT/mTORinhibition strategy for TFE3–tRCC (Supplementary Fig. S8).

DiscussionTherapeutic strategies to effectively treat MiT family transloca-

tion renal cell carcinoma have yet to be established. More impor-tantly, there is a clinical need for evidence-based treatments, as asignificant number of patients, likely underestimated by histo-logical misclassification, may be afflicted by this subtype of RCC.In our study, we have characterized theDNAbinding landscape ofa TFE3 gene fusion product by ChIP-seq using our recentlyestablished PDX model bearing a SFPQ–TFE3 fusion. TFE3 bind-ing to genomic regions containing E-box motif was confirmed,and 3032 TFE3 binding sites were associated with 2213 putativeTFE3 target genes. Interestingly, our ChIP-seq data also indicateTFE3 binding on ETS andAP-1 binding sites. TFE3 binding on ETSbinding motif is consistent with previous reports (3), whereas

Figure 6.

TFE3 transcriptionally regulates IRS-1, an upstream effector of PI3K/AKT/mTOR, and its downregulation inhibits RP-R07 cells proliferation. Evaluation of TFE3knockdown by siRNA by qRT-PCR and by immunofluorescence.A, TFE3mRNA expression level in RP-R07. B, Immunofluorescence quantification and Student t testanalysis. C, Immunofluorescence images of N-terminal TFE3 in RP-R07. siRNA-mediated silencing of TFE3 downregulates PI3K/AKT/mTOR downstreameffectors 4EBP-1 (D and F) and S6 ribosomal (E and G) activity. Immunofluorescence quantification, Student t test analysis and representative immunofluorescenceimages. siRNA-mediated silencing of TFE3 downregulates IRS-1 at the RNA-level (H) and protein level (I and J). Immunofluorescence quantification, Studentt test analysis, and representative immunofluorescence images. K, TFE3-siRNA inhibited RP-R07 cell proliferation. Graph represents cells number concentration(cells/mL) after treatment with no siRNA, siRNA control (scramble), and TFE3-siRNA (siTFE3) at 50 nmol/L for 24 hours (siTFE3 vs. scramble P ¼ 0.74),48 hours (siTFE3 vs. scramble P ¼ 0.38), 72 hours (siTFE3 vs. scramble P ¼ 0.13), 96 hours (siTFE3 vs. scramble P ¼ 0.04), and 110 hours (siTFE3 vs. scrambleP < 0.0001). L–P, Dual inhibition of PI3K-mTOR by BEZ-235 results in antitumor activity in vivo. RP-R07 cells (5 � 106) were injected subcutaneously into theflank of male SCID mice. Mice bearing RP-R07 xenograft tumors (n ¼ 5 for each group) were randomized into four groups: (i) vehicle, (ii) MLN0128 (3 mg/kg), (iii)rapamycin (2 mg/kg), and (iv) BEZ235 (25 mg/kg). No apparent drug toxicity was observed for each treatment. All mice were weighed twice weekly tomonitor drug toxicities (i.e., �20% bodyweight loss). Dual inhibition PI3–mTOR therapy significantly inhibits tumor growth. L, Average tumor weight (gram) inSCID mice treated with indicated single agent. Only tumors from mice treated with BEZ-235 was significantly smaller (P < 0.001, one-way ANOVA, multiplecomparison) than tumor from mice treated with vehicle. M, Dual inhibition of PI3–mTOR by single-agent BEZ-235 inhibits angiogenesis and attenuatesPI3K–mTOR signaling in vivo. Representative immunofluorescence images of paraffin-embedded RP-R07 tumor slice (8 mm) treatedwith vehicle (top) and BEZ-235(bottom) stained with anti-Ki67 (left), anti-phosphoS6 ribosomal (middle), and angiogenesis marker CD31 (right). N–P, Quantitative analyses.

Damayanti et al.





TFE3 binding on AP-1 binding site has not been previouslyreported. ETS and AP-1 binding sites are known to be enrichedat enhancers of genes that promote epithelial–mesenchymaltransition and cellular migration and invasion (24). Pathwayanalysis using KEGG, PANTHER, and Wiki tools identified thePI3K/AKT/mTOR axis as the top significantly influenced pathway.Specific TFE3 target genes were also associated with this pathway,such as PI3KCA, AKT3, IRS-1, TSC-1, EIF4B, and VEGFR-2, sug-gesting that the PI3K/AKT/mTOR axis represents a rational ther-apeutic target for this disease.

miRNAs are small RNA molecules with 19 o t23 nucleotidelength which act as either tumor promoters or tumor suppressorsby targeting the transcription and translation of specific genes. Thedifferential miRNA signature in ccRCC compared with normalkidney tissues has been well established, and specific miRNAsdifferences have been identified inmetastatic ccRCC (20, 39). Weused microarray technology to evaluate the miRNA expressionprofile of our tRCC model, and observed a distinct miRNAexpression profile as compared with pRCC and ccRCC models,despite the presence of mixed papillary/clear cell histologic fea-tures. Our results are consistent with recent miRNA profiling ofXp11 tumors bearing SFPQ–TFE3 and ASPSCR1–TFE3 (40),demonstrating distinct miRNA profiles against published dataset of ccRCCandpRCC(41). Interestingly, despite different tumorpanels and slight difference in our clustering algorithm andmethod, we also found that tRCC miRNA expression profile iscloser to ccRCC compared with pRCC (40). Moreover, our bio-informatic tools indicated that the differential expression ofmiRNAs could be linked to several targets genes and pathways.Consistent with our ChiP-seq data, PI3K/AKT/mTOR was iden-tified once again as a pathway with significant (P ¼ 4.91E�9)association with the miRNA signatures in RP-R07. Similar pre-dicted miRNAs target genes associated with the PI3K/AKT/mTORpathway, as seen in ChIP-seq, were identified as well. It isnoteworthy that some pathways associated with differentialmiRNA expression identified in our study are the same miRNA-associated pathways identified in previous work (40) using largerpanel of tRCC tumors. These miRNAs associated pathwaysinclude PI3K pathway, cell cycle, p53, lysine degradation, erbBsignaling, and wnt signaling pathway. However, different path-ways identified in our analysis may due to the fact that the tRCCtumor panels involved in previous work consisted of SFPQ–TFE3and ASPSCR1–TFE3 tumors while we focused on a SFPQ–TFE3model. Aberrant activation of PI3K/AKT/mTORpathway itself hasbeen reported in RCC (42). Although previous studies havedemonstrated the association of AKT/mTOR pathway (3) andupregulated phosphor-S6 (43) with tRCC, our results furthersupport the role of PI3K/AKT/mTOR pathway in tRCC as apotential target for therapeutic interventions.

Data integration of tRCC molecular signatures is a valuableresource to generate new hypotheses regarding therapeutic tar-geted pathways. Therefore, we used a panel of tRCCmodels to testthe hypothesis whether inhibition of the PI3K/AKT/mTORaxis would lead to antitumor response. First, we verified thatthe P13K/AKT/mTOR signaling is overexpressed in our tRCCpanel by upregulation of phospo-S6rp and phospho-4E-BP1protein expression. Then, we enacted a variable, multinodalP13K/AKT/mTOR inhibition strategy using three treatment armsto examine the effects of blocking this pathway at different pointsin vitro and in vivo: (i) PI3K inhibition with BKM-120, (ii) pan-mTOR inhibition with MLN0128, and (iii) simultaneous vertical

inhibition of PI3K and mTOR with BEZ-235. Although all threetreatment arms had a greater antiproliferative effect as comparedwith the MET inhibitor crizotinib, BKM-120 had a modest effect,which is possibly due to inadequate inhibition by targeting PI3Kaxis alone. In contrast, MLN0128 and BEZ-235 potently inhibitedproliferation of all tRCC cells models tested in a concentration-dependent fashion, with BEZ-235 exerting the greatest effect.Although the three therapeutic agents had similar treatmenttrends across our tRCC panel, there were differential IC50 valuesamong the tRCC models bearing distinct TFE3 gene fusions. ThetRCC models included in our study did not show a significantresponse to the MET inhibitor crizotinib. These results seem to bein contrast with previous work (44) that suggest an inhibitoryeffect ofMET inhibition in a tRCCmodel with ASLP–TFE3 fusion.One possible explanation for this difference is thatMET upregula-tion may be specific for ASPL–TFE3 fusion and our tRCC panelsdo not have ASPL–TFE3 fusion. These results may also implydifferential regulatory pathways in a fusion partner-dependentfashion and support previous report with differential cathepsin-kexpression in tRCC (45). Thorough discussion and analysis on therole ofMET inhibition strategy in tRCChas been recently reported(3). BEZ-235 was the only treatment that resulted in significanttumor reduction in vivo compared with the modest tumor growthinhibition by rapamycin or MLN0128 alone. Even though dualmTOR inhibition with MLN0128 conferred greater efficacy oftumor growth inhibition compared with partial mTOR inhibi-tion, possibly due to attenuation of the mTORC2–AKT reactiva-tion mechanism (46), our results suggest that neither form ofmTORblockade in isolation is sufficient to elicit significant tumorcontrol in TFE3–tRCC. These results corroborate ourfinding in theclinic where the patient did not benefit from single-agent treat-ment with everolimus, a mTOR inhibitor, suggesting the need ofalternative therapeutic strategy such as simultaneous PI3K andmTOR inhibition.

Interestingly, tRCC does not present a highmutational burden,as the clinical aggressiveness might suggest (47). As previouslyreported in tRCC, RP-R-07 tumor did not carry mutations incanonical genes such as TP53, VHL, PIK3CA, RAS, PTEN, as perthe clinical report (48). The absence of subtype-specific chromo-somal abnormalities, besides the fusion genes, suggests a poten-tial "driver" role of TFE3 in the oncogenesis and response totherapies of tRCC. By using siRNA-mediated TFE3 silencingstrategy, we showed that attenuated wild-type TFE3 expressionexerts inhibitory effect on RP-R07 cell proliferation. These dataalso suggest that dimerization with wild-type TFE3 is probablyrequired for the biological effects of chimeric TFE3. Next,we also showed possible feedback regulatory mechanismof TFE3 on PI3K/AKT/mTOR by demonstrating inhibition ofPI3K/AKT/mTOR downstream effectors following TFE3-siRNAtreatment. To further investigate TFE3 feedback loop regulatorymechanism on PI3K/AKT/mTOR, we examined TFE3 target genesbased onourChIP-seq results.Oneof TFE3 target geneswas IRS-1,an upstream modulator of PI3K/AKT/mTOR axis (3). Using thesame TFE3 silencing strategy, we confirmed our ChIP-seq resultthat TFE3 transcriptionally regulates IRS-1. Furthermore, weshowed that TFE3 silencing inhibits IRS-1 expression. IRS-1 isone of upstream effectors of PI3K/AKT/mTOR pathway, which isnegatively controlled by p70S6 kinase (49). However, our studysuggests an alternative positive regulation of IRS-1 by TFE3 thatlikely bypasses P70S6K regulation, and ultimately results in PI3K/AKT/mTOR aberrant activation. Collectively, our results suggest






that targeting PI3K/AKT/mTOR results in TFE3 inhibition, and,ultimately, attenuates its feedback loop activation by downregu-lating its transcriptional target, IRS-1, one of upstream modula-tors of the pathway. Although, there is possibility that TFE3feedback loop mechanism on PI3K/AKT/mTOR may be achievedthrough other upstream nodes of this signaling axis.

In summary, TFE3 tRCC remains a therapeutic challenge (3).Despite the commonmixed clear cell and papillary cell morphol-ogy, the reported clinical response to targeted therapies, includingVEGF receptor tyrosine kinase and mTOR inhibitors, is modest(50). The results from the use of immune checkpoint inhibitorsare still not available. Overall, our results suggest the therapeuticpotential of PI3K/AKT/mTOR inhibition in patients with tRCC.We identified that simultaneous vertical inhibition targeting PI3KandmTOR had greater antitumor response than single node PI3Kor mTOR inhibition. However, due to reported toxicity ofBEZ-235 (51), further investigation of the safety and therapeuticpotential of PI3K/AKT/mTOR inhibition in patients with tRCC aswell as efforts to develop new PI3K/AKT/mTOR inhibitors withlower toxicities are need.

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

Authors' ContributionsConception anddesign:N.P.Damayanti, K.Ahmed, S. Chintala, P.C.Hollenhorst,R. Pili

Development of methodology: N.P. Damayanti, H.W.Z Khella, S.Y. Ku,E. Kauffman, R. Adelaiye-Ogala, S. Chintala, C. Kao, W.M. Linehan,G.M. Yousef, R. PiliAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): N.P. Damayanti, J.A. Budka, H.W.Z Khella,M.W. Ferris, S.Y. Ku, V.N. Chintala, R. Adelaiye-Ogala, M. Elbanna,W.M. Linehan, G.M. Yousef, R. PiliAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): N.P. Damayanti, J.A. Budka, H.W.Z Khella,V.N. Chintala, R. Adelaiye-Ogala, M. Elbanna, G.M. Yousef, R. PiliWriting, review, and/or revision of the manuscript: N.P. Damayanti,J.A. Budka, H.W.Z Khella, E. Kauffman, A.C. Wood, S. Chintala, W.M. Linehan,G.M. Yousef, P.C. Hollenhorst, R. PiliAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): N.P. Damayanti, H.W.Z Khella, R. Adelaiye-Ogala, A. Orillion, P.C. Hollenhorst, R. PiliStudy supervision: G.M. Yousef, P.C. Hollenhorst, R. Pili

AcknowledgmentsThis study was in part supported by the NCI P30 CA016056 (to R. Pili) and a

research donation from Richard and Deidre Turner and family. We would alsolike to thank the MTMR and Pathology Core Facilities at Roswell Park CancerInstitute.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received January 30, 2018; revised June 19, 2018; accepted July 23, 2018;published first July 30, 2018.

References1. Argani P. MiT family translocation renal cell carcinoma. Semin Diagn

Pathol 2015;32:103–13.2. Argani P, Lae M, Hutchinson B, Reuter VE, Collins MH, Perentesis J, et al.

Renal carcinomas with the t(6;11)(p21;q12): clinicopathologic featuresand demonstration of the specific alpha-TFEB gene fusion by immuno-histochemistry, RT-PCR, andDNAPCR.Am J Surg Pathol 2005;29:230–40.

3. Kauffman EC, Ricketts CJ, Rais-Bahrami S, Yang Y, Merino MJ, Bottaro DP,et al. Molecular genetics and cellular features of TFE3 and TFEB fusionkidney cancers. Nat Rev Urol 2014;11:465–75.

4. Komai Y, Fujiwara M, Fujii Y, Mukai H, Yonese J, Kawakami S, et al. AdultXp11 translocation renal cell carcinoma diagnosed by cytogenetics andimmunohistochemistry. Clin Cancer Res 2009;15:1170–6.

5. Zhong M, De Angelo P, Osborne L, Keane-Tarchichi M, Goldfischer M,Edelmann L, et al. Dual-color, break-apart FISH assay on paraffin-embed-ded tissues as an adjunct to diagnosis of Xp11 translocation renal cellcarcinoma and alveolar soft part sarcoma. Am J Surg Pathol 2010;34:757–66.

6. Kakoki K, Miyata Y, Mochizuki Y, Iwata T, Obatake M, Abe K, et al. Long-term treatment with sequential molecular targeted therapy for Xp11.2translocation renal cell carcinoma: a case report and review of the literature.Clin Genit Cancer 2017;15:e503–e6.

7. WetermanMA,WilbrinkM,Geurts vanKessel A. Fusion of the transcriptionfactor TFE3 gene to a novel gene, PRCC, in t(X;1)(p11;q21)-positivepapillary renal cell carcinomas. PNAS 1996;93:15294–8.

8. Argani P, Antonescu CR, Illei PB, Lui MY, Timmons CF, Newbury R, et al.Primary renal neoplasms with the ASPL-TFE3 gene fusion of alveolar softpart sarcoma: a distinctive tumor entity previously included among renalcell carcinomas of children and adolescents. Am J Pathol 2001;159:179–92.

9. Argani P, Lui MY, Couturier J, Bouvier R, Fournet JC, Ladanyi M. A novelCLTC-TFE3 gene fusion in pediatric renal adenocarcinoma with t(X;17)(p11.2;q23). Oncogene 2003;22:5374–8.

10. Argani P, Zhong M, Reuter VE, Fallon JT, Epstein JI, Netto GJ, et al. TFE3-fusion variant analysis defines specific clinicopathologic associationsamong Xp11 translocation cancers. Am J Surg Pathol 2016;40:723–37.

11. Argani P, Zhang L, Reuter VE, Tickoo SK, Antonescu CR. RBM10-TFE3 renalcell carcinoma: a potential diagnostic pitfall due to cryptic intrachromo-somal Xp11.2 inversion resulting in false-negative TFE3 FISH. Am J SurgPathol 2017;41:655–62.

12. Argani P, AntonescuCR, Couturier J, Fournet JC, Sciot R,Debiec-RychterM,et al. PRCC-TFE3 renal carcinomas: morphologic, immunohistochemical,ultrastructural, and molecular analysis of an entity associated with thet(X;1)(p11.2;q21). Am J Surg Pathol 2002;26:1553–66.

13. Goodwin Matthew L, Jin H, Straessler K, Smith-Fry K, Zhu J-F, MonumentMichael J, et al. Modeling alveolar soft part sarcomagenesis in the mouse:a role for lactate in the tumor microenvironment. Cancer Cell 2014;26:851–62.

14. Williams SA, Anderson WC, Santaguida MT, Dylla SJ. Patient-derivedxenografts, the cancer stem cell paradigm, and cancer pathobiology in the21st century. Lab Invest 2013;93:970–82.

15. Betschinger J, Nichols J, Dietmann S, Corrin PD, Paddison PJ, Smith A. Exitfrom pluripotency is gated by intracellular redistribution of the bHLHtranscription factor Tfe3. Cell 2013;153:335–47.

16. Martina JA, Diab HI, Brady OA, Puertollano R. TFEB and TFE3 are novelcomponents of the integrated stress response. EMBO J 2016;35:479–95.

17. Strub T, Giuliano S, Ye T, Bonet C, Keime C, Kobi D, et al. Essential role ofmicrophthalmia transcription factor for DNA replication, mitosis andgenomic stability in melanoma. Oncogene 2011;30:2319–32.

18. Jackson RJ, Standart N. How Do MicroRNAs regulate gene expression?Sci STKE 2007;2007:re1.

19. Butz H, Szabo PM, Khella HW, Nofech-Mozes R, Patocs A, Yousef GM.miRNA-target network reveals miR-124as a key miRNA contributing toclear cell renal cell carcinoma aggressive behaviour by targeting CAV1 andFLOT1. Oncotarget 2015;6:12543–57.

20. Khella HW, BakhetM, Allo G, Jewett MA, Girgis AH, Latif A, et al. miR-192,miR-194 and miR-215: a convergent microRNA network suppressingtumor progression in renal cell carcinoma. Carcinogenesis 2013;34:2231–9.

21. Zheng B, Jeong S, Zhu Y, Chen L, Xia Q.miRNA and lncRNA as biomarkersin cholangiocarcinoma(CCA). Oncotarget 2017;8:100819–30.

Damayanti et al.





22. Zhang X, Zhang M, Cheng J, Lv Z, Wang F, Cai Z. MiR-411 functions as atumor suppressor in renal cell cancer. Int J Biol Markers 2017;32:e454–60

23. Hollenhorst PC, Shah AA, Hopkins C, Graves BJ. Genome-wide analysesreveal properties of redundant and specific promoter occupancy within theETS gene family. Genes Develop 2007;21:1882–94.

24. Plotnik JP, Budka JA, Ferris MW, Hollenhorst PC. ETS1 is a genome-wideeffector of RAS/ERK signaling in epithelial cells. Nucleic Acids Res2014;42:11928–40.

25. Clark J, Lu YJ, Sidhar SK, Parker C,Gill S, SmedleyD, et al. Fusion of splicingfactor genes PSF and NonO (p54nrb) to the TFE3 gene in papillary renalcell carcinoma. Oncogene 1997;15:2233–9.

26. Sidhar SK, Clark J, Gill S, Hamoudi R, CrewAJ, Gwilliam R, et al. The t(X;1)(p11.2;q21.2) translocation in papillary renal cell carcinoma fuses a novelgene PRCC to the TFE3 transcription factor gene. Hum Mol Genet1996;5:1333–8.

27. Argani P, Lal P, Hutchinson B, Lui MY, Reuter VE, Ladanyi M. Aberrantnuclear immunoreactivity for TFE3 in neoplasmswith TFE3 gene fusions: asensitive and specific immunohistochemical assay. Am J Surg Pathol2003;27:750–61.

28. Fresno Vara JA, Casado E, de Castro J, Cejas P, Belda-Iniesta C, Gonzalez-Baron M. PI3K/Akt signalling pathway and cancer. Cancer Treat Rev2004;30:193–204.

29. Tee AR, Blenis J. mTOR, translational control and human disease. SeminCell Develop Biol 2005;16:29–37.

30. Lin F, ZhangPL, YangXJ, Prichard JW, LunM,BrownRE.Morphoproteomicand molecular concomitants of an overexpressed and activated mTORpathway in renal cell carcinomas. Ann Clin Lab Sci 2006;36:283–93.

31. Pantuck AJ, Seligson DB, Klatte T, Yu H, Leppert JT, Moore L, et al.Prognostic relevance of the mTOR pathway in renal cell carcinoma:implications for molecular patient selection for targeted therapy. Cancer2007;109:2257–67.

32. Shaw LM. The insulin receptor substrate (IRS) proteins: at the intersectionof metabolism and cancer. Cell Cycle 2011;10:1750–6.

33. Battelli C, Cho DC. mTOR inhibitors in renal cell carcinoma. Therapy(London, England: 2004) 2011;8:359–67.

34. Motzer RJ, Escudier B, Oudard S, Hutson TE, Porta C, Bracarda S, et al.Phase 3 trial of everolimus for metastatic renal cell carcinoma: final resultsand analysis of prognostic factors. Cancer 2010;116:4256–65.

35. MotzerRJ, BarriosCH,KimTM,FalconS,Cosgriff T,HarkerWG, et al. Phase IIrandomized trial comparing sequential first-line everolimus and second-linesunitinib versus first-line sunitinib and second-line everolimus in patientswith metastatic renal cell carcinoma. J Clin Oncol 2014;32:2765–72.

36. Motzer RJ, Escudier B, McDermott DF, George S, Hammers HJ, Srinivas S,et al. Nivolumab versus everolimus in advanced renal-cell carcinoma.N Engl J Med 2015;373:1803–13.

37. Choueiri TK, Escudier B, Powles T, Mainwaring PN, Rini BI, Donskov F,et al. Cabozantinib versus everolimus in advanced renal-cell carcinoma.N Engl J Med 2015;373:1814–23.

38. Huang S, Bjornsti MA, Houghton PJ. Rapamycins: mechanism of actionand cellular resistance. Cancer Biol Ther 2003;2:222–32.

39. Butz H, Nofech-Mozes R, Ding Q, Khella HWZ, Szabo PM, Jewett M,et al. Exosomal MicroRNAs are diagnostic biomarkers and can mediatecell-cell communication in renal cell carcinoma. Eur Urol Focus2016;2:210–8.

40. Marchionni L, Hayashi M, Guida E, Ooki A, Munari E, Jabboure FJ, et al.MicroRNA expression profiling of Xp11 renal cell carcinoma. Hum Pathol2017;67:18–29.

41. Munari E, Marchionni L, Chitre A, Hayashi M, Martignoni G, Brunelli M,et al. Clear cell papillary renal cell carcinoma: micro-RNA expressionprofiling and comparison with clear cell renal cell carcinoma and papillaryrenal cell carcinoma. Hum Pathol 2014;45:1130–8.

42. Guo H, German P, Bai S, Barnes S, Guo W, Qi X, et al. The PI3K/AKTpathway and renal cell carcinoma. J Genet Genom 2015;42:343–53.

43. Argani P,Hicks J, DeMarzo AM, Albadine R, Illei PB, LadanyiM, et al. Xp11translocation renal cell carcinoma (RCC): extended immunohistochemicalprofile emphasizing novel RCC markers. Am J Surg Pathol 2010;34:1295–303.

44. Tsuda M, Davis IJ, Argani P, Shukla N, McGill GG, Nagai M, et al. TFE3fusions activate MET signaling by transcriptional up-regulation, defininganother class of tumors as candidates for therapeutic MET inhibition.Cancer Res 2007;67:919–29.

45. Martignoni G, Gobbo S, Camparo P, Brunelli M, Munari E, Segala D, et al.Differential expression of cathepsin K in neoplasms harboring TFE3 genefusions. Mod Pathol 2011;24:1313–9.

46. Choo AY, Yoon SO, Kim SG, Roux PP, Blenis J. Rapamycin differentiallyinhibits S6Ks and 4E-BP1 tomediate cell-type-specific repression ofmRNAtranslation. PNAS 2008;105:17414–9.

47. Classe M, Malouf GG, Su X, Yao H, Thompson EJ, Doss DJ, et al.Incidence, clinicopathological features and fusion transcript landscapeof translocation renal cell carcinomas. Histopathology 2017;70:1089–97.

48. Malouf GG, Su X, Yao H, Gao J, Xiong L, He Q, et al. Next-generationsequencing of translocation renal cell carcinoma reveals novel RNA splic-ing partners and frequent mutations of chromatin-remodeling genes. ClinCancer Res 2014;20:4129–40.

49. Zhang J, Gao Z, Yin J, QuonMJ, Ye J. S6K directly phosphorylates IRS-1 onSer-270 to promote insulin resistance in response to TNF-(alpha) signalingthrough IKK2. J Biol Chem 2008;283:35375–82.

50. Tannir NM, Jonasch E, Albiges L, Altinmakas E, Ng CS, Matin SF, et al.Everolimus versus sunitinib prospective evaluation inmetastatic non-clearcell renal cell carcinoma (ESPN): a randomized multicenter phase 2 trial.Eur Urol 2016;69:866–74.51.

51. CarloMI,Molina AM, LakhmanY, Patil S,WooK,DeLuca J, et al. A Phase Ibstudy of BEZ235, dual inhibitor of phosphatidylinositol 3-Kinase (PI3K)and mammalian target of rapamycin (mTOR), in patients with advancedrenal cell carcinoma. Oncologist 2016;21:787–8.






Published OnlineFirst July 30, 2018.Clin Cancer Res Nur P. Damayanti, Justin A. Budka, Heba W.Z Khella, et al. Translocation Renal Cell CarcinomaTherapeutic Targeting of TFE3/IRS-1/PI3K/mTOR Axis in

Updated version

10.1158/1078-0432.CCR-18-0269doi:

Access the most recent version of this article at:

Material

Supplementary

http://clincancerres.aacrjournals.org/content/suppl/2018/08/01/1078-0432.CCR-18-0269.DC1Access the most recent supplemental material at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. (CCC)Click on "Request Permissions" which will take you to the Copyright Clearance Center's

.http://clincancerres.aacrjournals.org/content/early/2018/09/24/1078-0432.CCR-18-0269To request permission to re-use all or part of this article, use this link



http://clincancerres.aacrjournals.org/lookup/doi/10.1158/1078-0432.CCR-18-0269http://clincancerres.aacrjournals.org/content/suppl/2018/08/01/1078-0432.CCR-18-0269.DC1http://clincancerres.aacrjournals.org/cgi/alertsmailto:[email protected]://clincancerres.aacrjournals.org/content/early/2018/09/24/1078-0432.CCR-18-0269http://clincancerres.aacrjournals.org/

/ColorImageDict > /JPEG2000ColorACSImageDict > /JPEG2000ColorImageDict > /AntiAliasGrayImages false /CropGrayImages false /GrayImageMinResolution 200 /GrayImageMinResolutionPolicy /Warning /DownsampleGrayImages true /GrayImageDownsampleType /Bicubic /GrayImageResolution 300 /GrayImageDepth -1 /GrayImageMinDownsampleDepth 2 /GrayImageDownsampleThreshold 1.50000 /EncodeGrayImages true /GrayImageFilter /DCTEncode /AutoFilterGrayImages true /GrayImageAutoFilterStrategy /JPEG /GrayACSImageDict > /GrayImageDict > /JPEG2000GrayACSImageDict > /JPEG2000GrayImageDict > /AntiAliasMonoImages false /CropMonoImages false /MonoImageMinResolution 600 /MonoImageMinResolutionPolicy /Warning /DownsampleMonoImages true /MonoImageDownsampleType /Bicubic /MonoImageResolution 900 /MonoImageDepth -1 /MonoImageDownsampleThreshold 1.50000 /EncodeMonoImages true /MonoImageFilter /CCITTFaxEncode /MonoImageDict > /AllowPSXObjects false /CheckCompliance [ /None ] /PDFX1aCheck false /PDFX3Check false /PDFXCompliantPDFOnly false /PDFXNoTrimBoxError true /PDFXTrimBoxToMediaBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXSetBleedBoxToMediaBox true /PDFXBleedBoxToTrimBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXOutputIntentProfile (None) /PDFXOutputConditionIdentifier () /PDFXOutputCondition () /PDFXRegistryName () /PDFXTrapped /False

/CreateJDFFile false /Description > /Namespace [ (Adobe) (Common) (1.0) ] /OtherNamespaces [ > /FormElements false /GenerateStructure false /IncludeBookmarks false /IncludeHyperlinks false /IncludeInteractive false /IncludeLayers false /IncludeProfiles false /MarksOffset 18 /MarksWeight 0.250000 /MultimediaHandling /UseObjectSettings /Namespace [ (Adobe) (CreativeSuite) (2.0) ] /PDFXOutputIntentProfileSelector /NA /PageMarksFile /RomanDefault /PreserveEditing true /UntaggedCMYKHandling /LeaveUntagged /UntaggedRGBHandling /LeaveUntagged /UseDocumentBleed false >> > ]>> setdistillerparams> setpagedevice

Therapeutic Targeting of TFE3/IRS-1/PI3K/mTOR Axis in ......2018/09/24 · ASPL–TFE3 (8),...

Documents

Transcript of Therapeutic Targeting of TFE3/IRS-1/PI3K/mTOR Axis in ......2018/09/24 · ASPL–TFE3 (8),...