© 2016 Nature America, Inc. All rights reserved. J.J.Z. J.J.Z. ( Boston, USA. Massachusetts, 9 7 Biology, Dana-Farber Cancer Institute, Boston, USA. Massachusetts, USA. Massachusetts, Harvard Medical School, Boston, USA. Massachusetts, 1 ( expression HER2 and (PR) receptor progesterone (ER), receptor estrogen their in well as histologically, BCBMs parental the of roughly 2–3 months ( were grafted directly into the brains of SCID mice patients, with Dana-Farber Brain Metastasis (DF-BM)354 a and DF-BM355, median survival ( PDXs orthotopic of panel a lished To develop therapeutic strategies for HER2-positive BCBM, we estab for patients with HER2-positive BCBM. combination with an mTOR inhibitor should be conducted that a biomarker-driven clinical trial of PI3K inhibitor in instability with therapeutic resistance. These findings suggest in DNA-repair , which suggests an association of genomic showed hypermutated genomes with enrichment of mutations of 4EBP1, an mTORC1 effector. The two nonresponding PDXs response was correlated with a reduction in the phosphorylation durable tumor regressions in three of five PDXs, and therapeutic therapies. Combined inhibition of PI3K and mTOR resulted in and their use for the identification of targeted combination of HER2-expressing breast cancer brain metastases (BCBM), development of orthotopic patient-derived xenografts (PDXs) in treating HER2-positive breast cancer. We report the Brain metastases represent the greatest clinical challenge Keith L Ligon Thomas M Roberts Zhigang C Wang B Elizabeth Claus Marika Hayashi Eugenio Marco Shom Goel Jing Ni breast cancer brain metastases xenografts of HER2-positive orthotopic patient-derived remissions in mice bearing and mTORC1 yields durable Combination inhibition of PI3K nature medicine nature Received 2 February; accepted 9 May; published online 6 June 2016; Department Department of Radiation Oncology, Dana-Farber Cancer Institute, Boston, USA. Massachusetts, Department of Neurosurgery, Brigham and Women’s Hospital, Boston, USA. Massachusetts, Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, USA. Massachusetts, [email protected] 1 , 2 , 11 1 , , Shakti H Ramkissoon , 3 , , Daniel G Stover 3 , 4 5 3 & Jean J Zhao , , Lori A Ramkissoon 1 , , Quang-De Nguyen 7 , 4 3

, Department Department of Pathology, Brigham and Women’s Hospital, Boston, USA. Massachusetts, 8 , , J Dirk Iglehart 1 , , Brian M Alexander , 2 , , Eric P Winer Supplementary Fig. Supplementary 1 11 These These authors contributed equally to this work. ), ), K.L.L. ( 1 3 , , , Hanbing Guo 2 , 12 3 1 Fig. 1 Fig. , 3 , 4 3 , , 3 6 12 , , Ian E Krop 11 [email protected] , , Yun Jee Kang , , Azra H Ligon , , Nancy U Lin , , Shaozhen Xie 9 a , 10 ). Fresh BCBMs from two two from BCBMs Fresh ). , , Guo-Cheng Yuan ). ). The PDXs resembled 3 1 Department Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, , 2 , , Victor Luu 3 ,

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These These authors jointly directed this work. should Correspondence be addressed to 8 / n (BBB) BKM120, a pan-PI3K inhibitor that penetrates the blood–brain barrier ulator of the PI3K pathway—we tested the combination of ( lapatinib with tentwith the resistance of the donor’s tumor consisto HER2-directed is therapy whichlapatinib, to response no showed PDXsDF-BM355 inhibitorlapatinib. HER2 a with PDXsDF-BM355treated we BCBMs in widespread is PTEN of loss the that confirms ( staining no PTEN showed 67% BCBMs, tive 1 Table Supplementary ( PTEN suppressor tumor of the levels detectable patients with NoneBCBM. HER2-positive of the five PDXs expressed We PDXsestablished subsequently by using from BCBMs three other 7 cytokeratin and (CK7) of the absence markers (GFAPglial marker and OLIG2) ( epithelial of expression the verified also We group quickly reached the study endpoint (developing systemic systemic (developing endpoint study the reached quickly group ( results these firmed con treatment after and before (MRI) imaging resonance Magnetic ( time over regressed also tumors large BKM120 The RAD001. introduced and and tumors, large bearing group control the from mice two removed we response, this of nature unprecedented ( bioluminescence by measured as regression, istration of BKM120 and together RAD001 resulted in marked tumor nation of lapatinib and RAD001 ( BBB the penetrates either lapatinib or BKM120 with RAD001, an mTORC1 inhibitor that combined we this, Toexplore BCBMs. in resistance PI3K-inhibitor mTORovercome might it is this, whether not with known inhibition therapies that In are metastases. against keeping effective extracranial systemic to refractory notoriously are metastases brain and unique, mTORC1inhibi with tion (ref. by overcome be can this and inhibition, PI3K to resistance model. DF-BM355 the in activity mTORC1 suppress not did completely therefore and inhibition PI3K ( translation ates we observed little change in p-4EBP1, an mTORC1 effector that medi mTOR,and of PI3K respectively— effectors downstream S6RP—the and AKT of phosphorlyation the reduced treatments these control, in signaling PI3K-pathway tumor as to compared the Although, BKM120. and/or to lapatinib response assessed we PI3K, and HER2 m Supplementary Fig. 2a . To assess the response of HER2-positive BCBMs to targeted therapy, Whereas DF-BM355 PDXs showed limited response to the combi response PDXs limited DF-BM355 showed Whereas mediate can cancers breast in activity mTOR persistent Notably, To understand the lack of response to combined inhibition with with inhibition combined to response of lack the Tounderstand 4 [email protected] 1 2 3– 0 2 8 Department Department of Biological Chemistry and Molecular Pharmacology, School School of Public Health, Yale University, New Haven, Connecticut, USA. 5 . Again, no response was observed ( 10 Department Department of Radiation Oncology, Brigham and Women’s Hospital, Supplementary Fig. 2c Fig. Supplementary o tions n o i at c i n u m com f e i r B 7 , 8 . 5 Fig. 2 Fig. , Department Department of and Biostatistics Computational b 6 ). Of 27 clinical specimens of HER2-posi of specimens clinical 27 Of ). ). Because DF-BM355 lacks PTEN—a key reg ). However, the brain microenvironment is ). However, brain microenvironment the ). b ). The remaining mice in the control control the in mice remaining The ). Supplementary Supplementary Fig. 2e , d Supplementary Fig. 2a ). Even combined HER2 HER2 combined Even ). Fig. 1 Fig. Fig. 2 Fig. c a ), which further further ), which ). Owing to the the to Owing ). ), ), the admin 1 Fig. 1 Fig. , 2 . Fig. 2 Fig. Fig. Fig. 1 b and , b b a ). ). ). ). ).  ------­

© 2016 Nature America, Inc. All rights reserved. i. 5a Fig. Ki67 staining, as compared to the control ( nificant reductions in p-S6RP and p-4EBP1 and significant decreases in whereas the combination therapy led to durable tumor regression, sig RAD001 monotherapy had nor meaningful BKM120 effectsneither in DF-BM355, thein DF-BM354findings our model,with Consistently DF-BM590). and DF-BM507 DF-BM463, (DF-BM354, BCBM of els we BCBMs, tested the same therapy in the four remaining PDX mod control ( and the to monotherapies relative staining), pase-3 (as measured by Ki67 and staining), apoptosis increased (cleaved cas proliferation decreased significantly also and p-4EBP1 reduced edly resistance rapamycin to contributes p-4EBP1 of re-emergence rapid a that and lived, short is p-S6RP, p-4EBP1 on inhibits stably effect its inhibitor) sistent with reports indicating that although rapamycin (an mTORC1 con are observations These inhibited. mTORC1completely not was drug neither control, to suppressed ( p-4EBP1 alone significantly compared as p-S6RP, reduced therapies mono both Although assessment. pharmacodynamic for treatment of d 4 after mice from tumors harvested we RAD001, and BKM120 ( efficacy to owing chosen BET bromodomain the inhibitor JQ1, with which downregulates BKM120 MYC of expression— combination a tumors—nor PDX the in levels p-ERK high of because MEK162—chosen inhibitor MEK the neither a from combination experiments; additional of BKM120 with old. d 270–280 were mice recipient the point at which d, 210 around toms after from aging-associated disease phenotypes, died we stopped treatment group control the in d 90 ( approximately mice all whereas observation, of 2e Fig. Supplementary and for weeks after ( treatment cessation healthy luciferase-signal-free remained mice and weeks, 14 of period treatment the over level undetectable a to nearly declined tumors RAD001-treated ferase ( signals luci high with phenotypes) neurologic or morbidity of symptoms cells. tumor of >75% in staining strong 2+, cells; tumor of >75% in staining weak 1+, cells; tumor of >90% in staining no 0, samples. BCBM HER2-positive human 27 on performed scores (IHC) immunohistochemistry 25 bars, Scale PDXs. corresponding the and biopsies surgical patient two of analyses immunophenotypic and 500 (left); 5 cm bars, Scale mice. in number passage P1–P5, graft; primary P0, mice. of cohorts new into re-injected then and , a luciferase with transduced and dissociated explanted, were brain the in xenografts The mice. SCID female of brains the into directly grafted were BCBM with individuals from tissues metastatic brain Fresh studies. preclinical in use for models BCBM PDX orthotopic generating of process the depicting ( PDXs. BCBM HER2-positive 1 Figure s n o i at c i n u m com f e i r B  ( statuses ER To determine whether these results could be replicated in other other in replicated be could results these whether determine To To understand the mechanism underlying the synergy between between synergy the underlying mechanism the understand To results by underscored is combination this of efficacy unique The µ m (middle). ( m (middle).

, b Establishment of orthotopic orthotopic of Establishment Supplementary Figs. 3 Figs. Supplementary ). Notably, DF-BM354 and DF-BM355 show disparate disparate show DF-BM355 and DF-BM354 Notably, ). 9 . Notably, combined BKM120–RAD001 treatment mark treatment BKM120–RAD001 Notably,combined . Fig. 1 Fig. µ Fig. Fig. 2 m. ( m. MYC b c ) Representative histologic histologic ) Representative b ) Compiled result of PTEN PTEN of result ) Compiled c Fig. 2 Fig. ). ). Notably, the in signal luciferase BKM120- and ), which suggests that the BMK120–RAD001 BMK120–RAD001 the that suggests which ), amplifications in the PDX tumors—showed tumors—showed PDX the in amplifications ). All mice treated survived the 210-d period period 210-d the survived treated mice All ). d a ). To avoid potential confounding symp To ). confounding potential avoid ) Schematic ) Schematic and and

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n PDXs and their matched patient specimens harbored mutations mutations harbored specimens patient matched their and PDXs genes DNA-repair in mutations to ( derived radiation and/or PDXs were responding the whom from chemotherapy patients the did than therapy of cycles more had from who derived were patients PDXs nonresponding two the that knowledge ( Mb per mutations ~60–70 was contrast, by BCBMs, nonresponsive in rate Mb per mutations ten roughly is BCBMs positive HER2- in rate mutation the that data reported recently with line in nonsynonymous of tumors Mb was rate per mutations ~7–8 in ( somatic responding The PDXs. these in alterations genetic of ( CNVs identical 6 Fig. variations Copy-number (CNVs) were frequent DF-BM355). in all five of for the tumor models ( unavailable was blood tumors and matched samples from the donor patients’ (patient blood on AKT–mTOR the depend BCBMs pathway. models ( AKT–mTOR-dependent signature genes The three responding models showedmice. significantlyuntreated from higher tumors expression on of analyses transcriptome performed we models, between responses therapeutic differential the for basis els ( the amount of p-S6RP and p-4EBP1 was also not reduced in these mod on the survival of mice bearing DF-BM507 and DF-BM590 PDXs, and 5c Fig. Supplementary ( levels caspase-3 cleaved and Ki67 p-4EBP1, in changes similar with along therapy, BKM120–RAD001 to responses durable of hormone-receptor expression. The DF-BM463 model also exhibited combination might be effective for HER2-positive regardless BCBMs, We also performed whole-exome sequencing (WES) of all five PDX By contrast, BKM120–RAD001 combination therapy had little effect Fig. 2 ER ), and each PDX and its matched patient tumor shared almost almost shared tumor patient matched its and PDX each and ), DF-BM355 DF-BM354 Fig. 2 Fig. 2 Fig. Supplementary Table 2 Table Supplementary f and c P0–P1 g PR Supplementary Fig. 5e h ), ), which suggests that some, but not all, HER2-positive (25.9% ). Relevant to this hypermutation phenotype is the the is phenotype hypermutation this to Relevant ). Supplementary Fig. 7a Fig. Supplementary PTEN IHCscore 1+,

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© 2016 Nature America, Inc. All rights reserved. advances precision medicine for cancer. for medicine precision advances and analyses, genotypic and phenotypic of integration the facilitates models PDX brain-metastasis-specific of use the that demonstrates study More broadly, our BCBM. HER2-positive with individuals for clinic the into fast-tracked be could findings preclinical our of tion under evaluation in clinical the advanced transla solid malignancies, the from (RAD001) everolimus to resistance with samples cancer correlated is breast instability genomic high that revealed trial metastatic BOLERO-2 of analysis recent a Tables 3 Supplementary ( genes DNA-repair several in t ( range interquartile the 1.5× within is that value farthest the to extending whiskers lower and upper with quartiles, third and first the to correspond plots Box mice. untreated from tissues tumor xenograft brain from genes signature AKT–mTOR-dependent ( indicated as compound, or control vehicle with DF-BM590 and DF-BM507 DF-BM463, DF-BM354, bearing * group). per images 25 bars, Scale treatments. indicated 4 d with for treated tumors DF-BM355 on caspase-3 cleaved p-S6RP, and Ki67 p-4EBP1, of ( RAD001 and BKM120 or control vehicle with treated mice DF-BM355-bearing ( mouse. one represents line Each ( point time imaging each at determined (ROI) interest of regions ( 5 mm. ( RAD001 with 90% PEG300) or BKM120 combined and NMP (10% control vehicle with treated mice DF-BM355-bearing of ( 1 cm. bars, ( mg/kg) (7.5 RAD001 and mg/kg) (30 BKM120 with treatment combined after and before tumor DF-BM355 bearing mice of analysis bioluminescence-imaging ( RAD001. and BKM120 of combination the to PDXs BCBM HER2-positive of 2 Figure nature medicine nature e test). ( test). d a ) Kaplan–Meier survival curve of curve survival ) Kaplan–Meier

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© 2016 Nature America, Inc. All rights reserved. Q.-D.N., A.H.L., R.D., E.B.C., B.M.A. and Z.C.W. provided administrative, technical K.L.L. and J.J.Z. wrote and/or revised the manuscript. J.N., S.H.R., S.X., L.A.R., interpreted data. J.N., S.H.R., S.X., S.G., J.D.I., I.E.K., G.-C.Y., T.M.R., E.P.W., experiments.N.U.L., J.N., S.H.R., S.X., S.G., D.G.S., E.M., K.L.L. and J.J.Z. analyzed and S.X., H.G., V.L., Y.J.K. and M.H. performed the surgeries and the experiments. J.N., S.H.R., S.X., K.L.L. and J.J.Z. developed methodology. J.N., S.H.R., J.N., S.H.R., S.X., E.P.W., N.U.L., K.L.L. and J.J.Z. conceived and designed CA142536 (J.J.Z.) and 1P50CA168504 (T.M.R., I.E.K., E.P.W., N.U.L., and J.J.Z.).(J.J.Z.), 1K08NS087118 (S.H.R.), P50 CA165962 (T.M.R., K.L.L. and J.J.Z.), P01 Institutes of Health (NIH) grants R01 CA187918 (T.M.R. and J.J.Z.), CA172461 J.J.Z.); Breast Cancer Alliance (J.J.Z.); Komen scholar grant (E.P.W.); and FoundationUS National (N.U.L., E.P.W., Z.W. and J.J.Z.); Aid for Cancerdata-analysis Research (E.P.W.program. This work was supported and by the Breast Cancer Research (VigeneTech) for quantification of pS6RP and p4EBP IHC data by the Cellvigene with the Ion Torrent sequencing system. We thank J. Ruan and M. Ruan Technologies, Thermo Fisher) for assistance with WES and transcriptome analyses Core for histopathological analyses. We thank F. Pan, D. Light and R. Qi (Life R. Bronson and the Dana-Farber/Harvard Cancer Center Rodent Histoplathology G. Dai at the Dana-Farber Lurie Family Image Center for MRI imaging. We thank We thank D. Livingston for reading the manuscript. We thank R. Modiste and s n o i at c i n u m com f e i r B  AUTH A ckn o O wledgments R R C O NTRIBUTI O NS in vitro and in vivo

13. 12. 11. 10. 9. 8. 7. 6. 5. 4. 3. 2. 1. r at online available is information permissions and Reprints v The authors declare competing interests:financial details are available in the E.P.W., N.U.L., K.L.L. and J.J.Z. supervised and coordinated all aspects of the work. or material support. H.G., V.L., Y.J.K. and M.H. provided technical assistance. C e e O p

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© 2016 Nature America, Inc. All rights reserved. P-S6RP and p-4EBP indexes were calculated as a percentage of positive areas in reactivity were performed using the Image J software with ImmunoRatio plugin. 60× magnification, and quantifications of Ki67 and cleaved caspase-3 immuno cells in four or five random areas of each sample. Images were captured at 40× or Antibody validation is provided on the manufacturers’ website. and xylene washes. Coverslips were mounted with Permount (Fisher Scientific). dehydration graded by followed Mayer’sstain, using hematoxylin performed Signal was visualized by the HRP–DAB reaction. Counterstaining for nuclei was pre made solution/slide) for 2 h or from Vector Laboratories (1:200)(HRP) conjugated forsecondary antibody from 30 EnVision+mins). System (Dako) (100- room temperature with corresponding horseradish species-specific peroxidase at incubated were slides solution, (TBST) 20 Tween and saline Tris-buffered or VectorMIB-1,1:200 multiple After 1× with washes K-451,1:1,000). #VP lab (Dako Ki67 and 1:500) #AB9610, (Chemicon OLIG2 1:500), #z0334, (Dako PgR 636, 1:150), HER2 (ThermoScientific SP3, 1:50), CK7 (Dako, 1:100), GFAP estrogen receptor (ER) 1:400), SP1, (ThermoScientific 1:40), progesterone receptor #9664, (PR) (Dako Signaling (Cell caspase-3 cleaved 1:1,000), or 1:400 #2855, Signaling (Cell p-4EBP1-Thr37/46 1:1,000), or 1:400 #2211, Signaling mary antibodies: PTEN (Cell Signaling #9559, 1:400), p-S6RP-Ser235/236 (Cell (Dako) for 10 min, and then incubated overnight at 4 °C with the following pri (pH 6.0) for 20 min. Subsequently, sections were treated with peroxidase block alcohol before performing heat antigen retrieval in 10 mM sodium citrate buffer with xylene, and this was followed by gradation washes in 100%, 95%, 80% ethyl Briefly,5- sections. paraffin on System (Dako) EnVision+ DAB using protocols standard to according performed, Immunohistochemistry. compliance in Committee Use guidelines. animal NIH with and Care Animal Institute Cancer Farber animal Dana- the by approved All protocols to according evaluated. performed were then experiments were tumors lesions the tumor and generated, with were samples sections tissue H&E-stained embedding. brain paraffin for methods the standard by processed and were 7.0), pH (PBS, phosphate-buffered in saline (PFA) paraformaldehyde 4% of injection racardiac int by perfused were xenografts bearing animals all euthanized tumors, for brain of used evaluation were neuropathological lower) for Alternatively, or 5 experiments. therapeutic passage (at PDXs low-passage and grafts (P0–1), primary from cells tumor into introduced was expression Luciferase and re-injected intracranially into additional animals (serial passaging dissociated dissection, by collected were animals symptomatic from tumors brain euthanasia, After euthanasia. water necessitating or endpoints humane other or food obtain to inability posture, hunched loss, body-weight rapid ment of systemic symptoms of morbidity or including phenotypes, neurologic were housed under standard conditions and monitored closely for the develop closed with 9-mm autoclips (BD Diagnostic Systems). Mice bearing xenografts ( striatum right the into cells tumor viable 100,000 coordinates z to made was identify the incision bregma, which served scalp as the zero 1-cm coordinates A ( maintained. was anesthesia deep while bars ear from pressure by gentle secured was head the whereby frame, otactic stere a into positioned xylazine and ketamine or isoflurane oxygen-diluted with anesthetized were (IcrTac:ICR-Prkdcscid) Taconic from acquired mice intracranial in injections. 8–10-week-old use female, severe combined before immunodeficiency (SCID) ice on incubated temporarily and microliter, per of suspension A cells viable of 100,000 at a concentration Biotech). prepared was cells breast metastatic (Miltenyi methods enzymatic using and Tubes C gentleMACS mechanical in dissociated was tissue tumor fresh models, breast metastatic patient-derived Toestablish program. Bank Tissue Living 93-085 and 10-417) within the Dana-Farber/Harvard Cancer Center (DF/HCC) IRB approved (DFCI (IRB) protocol Board Review Institutional Dana-Farber at and the Brigham Women’sneurosurgery undergoing Hospital as of part the were from with BCBMs patients acquired and patients, brain fresh metastases xenografts. Patient-derived ONLINE METHOD doi: = 0 mm). A burr hole was created in the skull in the right hemisphere at hemisphere right the in skull the in created was hole burr A mm). 0 = Ki67 and cleaved caspase-3 indexes were calculated as a percentage of positive 10.1038/nm.4120 x = 0 mm, S y = 2 mm, Diaminobenzidine (DAB), brightfield staining was was staining brightfield (DAB), Diaminobenzidine Written informed consent was obtained from from obtained was consent informed Written z = 0 mm, and each animal was with injected µ m-thick sections were deparaffinized deparaffinized were sections m-thick z = 2 mm). The scalp was was scalp The mm). 2 = x = 0 mm, y = 0 mm, in vivo µ ). ). - - - - - l

factor (EGF, 20 ng/ml), basic fibroblast growth factor (bFGF, 20 ng/ml), and ng/ml), (bFGF, 20 factor growth fibroblast basic ng/ml), (EGF, 20 factor (Stemcell Technologies) media supplemented with heparin NS-A sulfate (2 NeuroCult mg/ml), epidermal growth in cells expressing blasticidin-Luciferase 2 blasticidin with selection antibiotic 8 polybrene with overnight suspension in 5 ~MOI at (pLenti-blasticidin-Luciferase) Luciferase encoding lentivirus a qPCR Lentivirus titration Kit (Applied Biological Materials, Inc.) by determined were Viraltiters use. future for °C −80 atstored and aliquoted trifugation (SW28, 16,600 rpm, 2 h). Viral pellets were resuspended in PBS and were filtered through a 0.45- and the viral supernatants were collected 1 d and 2 d later. The viral supernatants(1 PEI by cells HEK293T into 4:3:1 of ratio the at pMD.G and pCMV-delta8.9 test was done. The pLenti-blasticidin-Luciferase vector was co-transfected with at wereNo early passages. used authentication or mycoplasma-contamination and 100 Manassas, VA). The cells were maintained in DMEM293T cell supplementedline was obtained fromwith the American10% TypeFBS Culture Collection transduction. (ATCC, and production Lentiviral data analyses were done by two investigators blinded to the group allocation. formed using the Cellvigene software (VigeneTech). The IHC experiments and fication, and quantification of p-S6RP and p-4EBP immunoreactivity was per random3–5 areas of sample.each Images were captured at or40× magni 60× gating-system model 1025T. temperature and respiration Animal were monitored and air. regulated (Stony by SAII the monitoring Brook, NY) and supplied medically into flurane iso 1.5% of mixture a of inhalation the through procedure imaging the out through anesthetized were Animals positioning. animal precise for used was laser with AutoPac receiving. and excitation RF both for used was coil (RF) radiofrequency volume birdcage of ID rate 23-mm Bruker-made slew The a T/m/s. and 3440 mT/m 440 of amplitude gradient maximum a which provides system, shim room-temperature second-order to up with integrated bore horizontal and gradient B-GA12S2 the with Refrigerated) equipped System, Magnet Superconducting Shielded (Ultra USR bore clear cm 30 Tesla imaging. MRI the group allocations. measurements and the data analysis were done by two investigators blinded to outcome the with treatments drug group. The the within s.d. and means, two probability of type 1 error ( pilot studies in laboratoriesthe and StatsToDothe program on ofbasis the the of the control mice. The sample size for the experiments was determined by the endpoints the than longer 50–100% point a controlto groups, the in mice the followed to endpointstheir d) (30–100 or, if longertreated mice survived than groups, and five or six mice were used for each experimental cohort. Mice were tumor lesion. Tumor-bearing mice were randomized into control and10 × treatment 0.2–1.0 ROI with signals luciferase developed mice gavage. All compounds were purchased from Haoyuan ChemExpress, Co. Tween80 0.1% daybyweightoralonce a body mg/kg andadministeredat 100 hydroxypropyl 0.5% dissolved Lapatinibin was with (HPMC) methylcellulose 10% NMP with 90% PEG300, and delivered orally each day to mice at 7.5 mg/kg.emulsion pre-concentrate dilution, in ordissolved glucose (Novartis) 5% with given orally once per day at 30 mg/kg. RAD001 Treatmentwas freshly prepared from a micro CareStream MI Software. with analyzed were signals The (DF-BM354). min 5 or (DF-BM355) min 20 for 4000MM Station Image Kodak with obtained were images and recorded was expression luciferase later, min 10 mg/kg). (7 xylazine and mg/kg) (100 luciferin (Promega) (~60 mg/kg), together with anesthetized reagents ketamine imaging. Bioluminescence hydrocortisone (0.5 µ Tumor cells were isolated from passage 0 or 1 PDXs and transduced with with transduced and PDXs 1 or 0 passage from isolated were Tumor cells Treatment was started at 3–6 weeks after the injection of tumor cells, once cells, tumor of injection the after weeks 3–6 at Treatment started was g/ µ l) (4:1 to DNA). The culture medium was replaced 1 d after transfection, µ g/ml g/ml penicillin–streptomycin. The cells were frozen after receipt and in vivo MRI experiments were performed on a Bruker BioSpec 7.0 7.0 BioSpec Bruker a on performed were experiments MRI . BKM120 was dissolved in 10% NMP with 90% PEG300 and µ g/ml). g/ml). These tumor cells were then propagated in mice. α ) at 0.05, power at 0.8, expected difference between µ For imaging, mice were injected i.p. with with i.p. injected were mice imaging, For m m filter and were then concentrated by ultracen µ µ /l o nih h pplto of population the enrich to g/ml Human embryonic kidney (HEK) (HEK) kidney embryonic Human g/ml, and then subjected to 3-d 3-d to subjected then and g/ml, nature medicine nature 6 as baseline levels of levels baseline as d ------

© 2016 Nature America, Inc. All rights reserved. structed and sequenced in technical duplicate using the Ion Protonplatform,Ion the using duplicate technical in sequenced and structed Transcriptomeanalysis. number accession the with dbGaP NCBI the in deposited been have data sequencing CNV alterationsto visualize package Depth formodified R was further read the from tool segment-plotting The data. sequencing the visualize and 20 reads were excluded. R and Bioconductor packages than fewer somatic with mutationsmutationtumors,DNA-repaircalls of the in of genes analyses the For removed. were samples tumor for reads 20 and samples normal for reads ten than fewer with Variantcalls Fisher). Thermo tumor–normal pair workflow by using ion reporter software (Life Technologies, ants were carried out by AmpliSeq Exome single-sample (Somatic) workflowTorrent and Server. Further data analysis, variant calling and the annotation of vari alignment of sequencing reads was performed using Torrent Suite Software and Torrent Proton using one PI chip kit V2 (Life Technologies, SystemThermo (Life Fisher).Technologies, The Thermo Fisher), and were then sequenced on an Ion IonOneTouch the using by2 particles ion-spheretemplate-positive obtain to Thermo Fisher). Two or three libraries were multiplexed and clonally amplified Technologies, (Life KitQuantitation IonquantitatedbyLibrary were libraries exome final The genome.human the of exons coding the of 97% than greater Exome kit (Life Technologies, Thermo Fisher) that provides targeted regions of DNA libraries were constructed from 100 ng of gDNA using the Ion(Qiagen). AmpliSeq kit tissue & blood DNeasy using tumors PDX or blood peripheral patient’s the from facturer’s Briefly, DNAextracted genomic was instructions. manu the to according Fisher), Technologies, Thermo (Life Protonplatform sequencing. Whole-exome were obtained using OsiriX software. slice thickness = 1.0 mm; and number of slices = 12. 3D of volume reconstructions (field FOV s; 40 view) = 20 min × 20 mm 2 = time acquisition total 2; = averages of number eters: = 33 ms;TE (echo time) = ms; 2500 TR (repetition time) rareparam factor = 8; following the and sequence suppression fat with (RARE) echo spin then acquire the reference images. T2 weighted images were obtained from and fast gain receive gain, RF reference shim, autoautomatic frequency, the center run matic to it enabled which light, traffic the with protocol run and imaging loaded scout was orthogonal three a magnet, the in positioned were nature medicine nature Bruker Paravision 5.1 was used for MRI data acquisition. Once animals animals Once acquisition. data MRI for used was 5.1 Paravision Bruker p h s 0 0 1 0 6 3 . 2 v ; ; matrix size = 256 × 256; spatial resolution = 78 × 78 1 . p 1 AmpliSeq human-transcriptome libraries were con were human-transcriptomelibraries AmpliSeq . h eoe a sqecd n h In Torrent Ion the on sequenced was exome The 1 4 were used to prioritize 1 5 . . The µ m 2 ------;

transcriptome data have been deposited in the NCBI GEO with the accession accession the with GEO NCBI the in deposited been have data transcriptome that is within 1.5× of the interquartile range. Code is available upon request. The 75 CORO1B performed performed in the laboratory. No animals were excluded from the analysis. numbers were chosen on the basis of power calculations of 0.8 and pilot studies are considered to be significant when Student’s analysis. Statistical number TJP3 PVRL2 HNRPAB published study ( study published previously a in RAD001 (mTOR)inhibitor rapamycin of target mechanistic) induced by AKT in a transgenic mouse model and sensitive to mammalian (or genes upregulated AKT–mTOR35 the of mean representsthe score signature the 35 ‘up’ genes in the original manuscript as the ‘AKT–mTOR’ signature. The derived experimentally was pathway using terms the ‘AKT AND mTOR.’ Of results,the only one signature b PI3K–AKT–mTOR the target pathway, ( MSigDB searched we normalized by gene to enable comparison. Given that the two agents(ref. DESeq2 R-bioconductorin packages this study Thermo Fisher) to generate count data. Count data were transformed using the analyzed by Torrent Suite and ampliSeqRNA analysis plugin (Life Technologies, Fisher), andon sequenced then an Ion Torrent Proton machine. Data was first Thermo Technologies, (Life System 2 OneTouchIon the using by amplified clonally and multiplexed were libraries Eight preparation. library cDNA for Briefly, 10 ng of total RNAdescribed samples from previously brain xenograft as tumor tissue was and used instructions manufacturer’s the to according 17. 16. 15. 14. r o th

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