Downloaded by guest on September 25, 2021 a Chen new Wentao creates immunotherapy cells for prostate opportunities and cells B surface in cell proteome Myc-induced the of remodeling Large PNAS oncogenes or combina- metabolite or functionally cytotoxic for targets but candidates surface immunotherapy. type-dependent possible as identify cell thereof and tions a how fashion in demonstrate remodeling convergent surfaceome studies bidirectional the dramatic Our to of (BiTEs). MYC leads bis- in engagers overexpression to MYC up-regulation cell sensitive were their T antibodies they verified showed pecific recombinant and NCR3LG1, and lines generated ligand targets cell overexpression We activating two cell -H6. these killer sig- to as TNFRSF10B, attacking natural known cell as the immune also such and as 5, turnover such receptor nals cell death as driving known also surface can- expression hematological of increased also treating analogs overexpression for nucleoside MYC Paradoxically, used toxic cers. of commonly to cytarabine, sensitive sets We more like distinct fashion. cells in making type-dependent trans- nutrient porters increases cell and transporters large nucleoside a including overexpressiontransporters and in MYC systematic surfaceome that found cell down-regulation) have found and the to up- We engineered (both of remodeling levels. origin dramatic cell MYC in prostate cell resulted low or model over- or cell isogenic MYC in B three high of and of in impact proteins either fashion the surface lines autologous 800 investigated cell on we a expression types, in cell proteome different remodels surface overexpression but onco- cell MYC to how the cancers due identify change To attack that expression. proteins to Antibody surface cancers. of opportunities knowledge prostate many require exciting and in are cell overexpressed B for therapeutics factor (received including 2020 2, cancers December powerful approved human and a CA, Angeles, is Los California, MYC of University at Medicine of 2020) School 8, Geffen September David review Witte, N. Owen by Edited and 94158; CA Francisco, 91320; CA Oaks, Thousand ies.Fclapicto ftergo pnigMCcre- MYC spanning metastatic region and and the amplification primary of MYC amplification both PC, Focal in In disease. happen (13). overexpression cancers MYC (PC), per- lung high cancer a of in prostate and centage in carcinoma hepatocellular found and MYC cancers, been ovarian and also chromosomal amplification have by overexpression MYC altered is (10–12). expression translocation MYC where lymphoma pathways 9). NF-κB (8, and cas- RAS/RAF/MAPK, transduction promoter WNT, signal MYC including cancer-associated cades, the various as by cancers deregula- targeted human its is in However, frequently (8). occurs processing tion polymerase mRNA number RNA and a activity, the by promoter elongation, tissues, regulated control tightly pro- normal that is mechanisms and In protein of genesis (3–7). MYC the the cancers of to human expression contributes many MYC that of Overexpressed oncogene gression 2). driving (1, a processes is biosynthesis in involved M eateto hraetclCeity nvriyo aiona a rnic,C 94158; CA Francisco, San California, of University Chemistry, Pharmaceutical of Department Y’ oei ua acrwsfis icvrdi Burkitt’s in discovered first was cancer human in role MYC’s 01Vl 1 o e2018861118 4 No. 118 Vol. 2021 xrsinof expression Ci oefltasrpinfco otoln the controlling factor transcription powerful a is YC | glycoproteomics a,b,1 utYnMou Yun Kurt , ,0 ifrn ee,mn fwihare which of many , different >1,000 e eateto ellradMlclrPamclg,Uiest fClfri,SnFacso A94158 CA Francisco, San California, of University Pharmacology, Molecular and Cellular of Department c nttt fBoeia cecs cdmaSnc,Tie,Tia 11529; Taiwan Taipei, Sinica, Academia Sciences, Biomedical of Institute | surfaceome a,c,1 | MYC ag Solomon Paige , | antibody a au Aggarwal Rahul , >80 ulse aur 2 2021. 22, January Published doi:10.1073/pnas.2018861118/-/DCSupplemental at online information supporting contains article This 2 1 the under Published Submission.y Direct PNAS a is article equity.y This or gain financial Celgene personal from no funding but research received Corporation J.A.W. and K.K.L. statement: interest Competing the wrote J.A.W. and K.K.L., W.C., R.A., K.Y.M., tools; W.C., paper.y and reagents/analytic data; K.Y.M., new analyzed W.C., contributed K.K.L. and R.A. research; P.S., K.Y.M., research; designed performed J.A.W. K.K.L. and and K.K.L., P.S., K.Y.M., W.C., contributions: Author rti ietya ti nipratfco o omltissue normal for factor approaches important molecule small an Other (26). is small homeostasis and it binding renewal MYC as the for targeting directly over compatible concerns protein also features are There surface struc- (25). molecules helical few is heterodimeric with MYC forms tures tar- that and given at challenging disordered aimed proved intrinsically development cancer has drug treating directly in However, MYC useful geting 24). be 23, may (4, activity inhibition patients or that expression suggesting MYC regression, inactivat- tumor of that cause can demonstrate Several MYC models (22). ing mouse number proliferation transgenic and a in growth of cell studies expression in the involved factors up-regulating stim- of and by PC growth in Mech- ulates (18). oncogenic tandem MYC promotes of a MYC upstream anistically, epi- (21), loci regulatory and fusion the genetic TMPRSS2-ERG of metastatic duplication other as lethal by such triggered defects in genetic be MYC frequently also 18–20). can more (14, hap- overexpression (mCRPC) times event cancer This five prostate PC (14–17). castrate-resistant to primary overexpression four of MYC 8% pens in in observed results commonly that defect genetic a ates owo orsodnemyb drse.Eal [email protected] Email: addressed. be may correspondence whom To work.y this to equally contributed K.Y.M. and W.C. ain hro spsil addtsfrimnteayor combi- immunotherapy or for metabolites. cytotoxic candidates targets possible more surface as thereof also identify nations studies are nucleoside up-regulated Our cells two transporters. to These nucleosides cells. toxic to overexpressing two sensitive a MYC (BiTEs) these engagers and kill cell targeted member T to We bispecific family ligand. recombinant receptor using activating receptors TNF cell a in killer as involved natural receptors such two turnover of cell proliferation up-regulation cell of paradoxical for up-regulation and important broad transporters through using metabolite surfaceome >80 cells bidirectional the the prostate large in characterized and induces changes overexpression cells broadly overex- MYC B We when proteomics. in and cancers. surfaceome factor drive MYC-driven transcription can powerful pressed a is MYC Significance d b eateto hrpui icvr,AgnResearch, Amgen Discovery, Therapeutic of Department ei .Leung K. Kevin , NSlicense.y PNAS d eateto eiie nvriyo aiona San California, of University Medicine, of Department https://doi.org/10.1073/pnas.2018861118 a n ae .Wells A. James and , . y https://www.pnas.org/lookup/suppl/ a,e,2 | f11 of 1

BIOCHEMISTRY have indirectly targeted consequences of MYC by interfering Results with the MYC transcriptional program [BET bromodomain Elevated MYC Expression Level Results in Dramatic and Bidirectional inhibitors (27, 28)] or targeting downstream cellular events (3, Surfaceome Remodeling in Three Isogenic Cell Lines from B Cells and 29, 30). Another approach is targeting cell surface proteins up- Prostate Cells. To broadly profile the impact of MYC overexpres- regulated by MYC in cancer cells. Antibody-based therapeutics sion on the surfaceome, we adopted a mass spectrometry-based could be a fruitful approach, as antibodies are highly selec- proteomic approach to quantitatively analyze the cell surface gly- tive and now represent 8 of the top 10 selling drugs. There coprotein expression. We began by analyzing the surfaceome of are currently only about 24 surface targets for which there are P493-6 cells (34), an engineered B cell model of Burkitt’s lym- approved antibody therapeutics, despite there being more that phoma in which MYC is overexpressed (high MYC) but can 4,000 human genes that encode membrane proteins (31, 32). be suppressed by a tetracycline-inducible promoter by >20-fold Thus, discovering cell surface antigens as targets for therapies (low MYC) (Fig. 1A). To quantify the changes in surface protein to treat MYC-driven cancers is of high interest. expression we employed stable isotope labeling by amino acids Here, we broadly characterize the MYC-driven surfaceome in in (SILAC) (35) in which P493-6 cells were grown in both B cells and prostate cells using a mass spectrometry-based media containing a mixture of heavy or lysine/arginine with proteomic approach, called cell surface capture (CSC) (33). We and without tetracycline to generate low-MYC and high-MYC find MYC overexpression induces large bidirectional changes states for 1, 2, or 3 d to follow the time course. for 20 to 30% of the 500 to 800 different membrane proteins We were able to quantitatively compare the levels of about quantified. Despite differences in expression patterns among 500 membrane glycoproteins in both the low- and high-MYC the cell types, gene set enrichment analysis reveals commonly samples taken at 1, 2, and 3 d. There was a strong correlation up-regulated functions, especially those involving transporter (r > 0.85) between the distribution of proteins and expression activities that are important for cell proliferation, and paradox- levels found over the 3-d time course (SI Appendix, Fig. S1); this ically cell turnover such as TNF receptor family members and suggests that the impact of MYC expression on the surfaceome natural killer cell activating ligands. We validated the functional had reached a near-steady state even after 1 d of induction and relevance of two up-regulated nucleoside transporters for sen- reflects the consistency of the mass spectrometry (MS) data. sitizing cells to toxic nucleosides. Furthermore, we generated Rather than averaging all of the datasets, which did not differ high-affinity recombinant antibodies to some of the up-regulated substantially, we chose to focus on the day 3 dataset containing targets and showed these can selectively induce activa- 494 membrane proteins detected in both the low- and high-MYC tion with high-MYC B cell lymphoma cells and prostate cells samples. A standard volcano plot (Fig. 1B and Dataset S1) shows using bispecific T cell engagers (BiTEs). Our studies reveal large changes in expression of 2- to 10-fold up or down for how simple overexpression of MYC can substantially remodel 206 proteins representing about two-fifths of the detected sur- the surfaceome in a cell type and cell autologous fashion and faceome. These changes are bidirectional where 131 membrane provide a resource for potential drug targets. proteins were up-regulated and 75 down-regulated.

A C

B D

Fig. 1. Stable overexpression of MYC in an engineered B cell line (P493-6) and two prostate cell lines (RWPE-1 and PrEC-LHS) leads to large and bidirectional changes in the cell surfaceome. (A) Induction of MYC in P493-6 cells upon removal of tetracycline leads to >20-fold induction of MYC by Western blotting. A total of 20 µg cell lysate was loaded to each lane. GAPDH was used as the loading control. (B) Volcano plot showing bidirectional remodeling of cell surface

proteome of high versus low MYC in P493-6 cells. Surface proteins with log2 enrichment ratio (ER) < 1, P value < 0.05 and log2 ER > 1, P value < 0.05 are shown as red and green dots, respectively. The 20 commonly up-regulated proteins are labeled and shown as purple (P < 0.05) and blue (P > 0.05) dots in each plot. (C) Stable overexpression of MYC in RWPE-1 and PrEC-LHS cells leads to greater than fourfold increase in MYC expression by Western blot as processed above. (D) Volcano plots processed as in B showing bidirectional remodeling of surface proteins for high versus low MYC for RWPE-1 cells (Left) and PrEC-LHS cells (Right).

2 of 11 | PNAS Chen et al. https://doi.org/10.1073/pnas.2018861118 Large remodeling of the Myc-induced cell surface proteome in B cells and prostate cells creates new opportunities for immunotherapy Downloaded by guest on September 25, 2021 Downloaded by guest on September 25, 2021 iial matu tt hne swsse o h P493-6 the for seen was reflecting as surfaceomes change, observed cells. expres- state B the impactful in of similarly 35% changes a than twofold more than of sion greater showing bidirectional similar to remodeling led cells epithelial these in overexpression ag eoeigo h y-nue elsraepoem nBclsadpott el rae new creates cells prostate and cells B in proteome immunotherapy surface for cell opportunities Myc-induced the of the remodeling Large of (E each al. MYC. et for of Chen (Spearman) levels changes coefficients low surfaceome correlation experiments. of versus surfaceomic clustering The high MYC Hierarchal experiments. low expressing (D) versus surfaceomic three. lines high all MYC three cell between the low three correlation in modest versus proteins among a changed significant differences high suggest shows and and the experiments squares similarities in corresponding MS revealed detected the surfaceomics in lines (C low shown proteins. cell versus are up-regulated comparisons three high commonly three the 20 the in and in remodeling proteins proteins bidirectional surface shows (B experiments observed similarities. surfaceomic the as of well diagram as Venn differences (A) PrEC-LHS). and (RWPE-1 2. Fig. 813 and 861 1D identified (Fig. we For respectively analyzed. cells entire proteins, was RWPE-1 the surface digestion and that on-bead PrEC-LHS except the the above from as fraction iso- processed tryptic the were SILAC postculturing introduced Samples obtaining (ReDiMe) dimethylation chemically (38). of reductive we by expense labels changes cells the topic RWPE-1 substantial to for suggesting Due medium adherent, surfaceomes. of the less S2 impact in were Fig. the cells reflecting Appendix, significantly Myc lines, (SI varied cell overexpression blot- also control MYC Western morphologies isogenic than a by cell their more assessed provide The from to as 1C). to led levels (Fig. lines able MYC ting cell studies. in are two isogenic increase lines these our fourfold in for cell overexpression is background the papil- MYC RWPE-1 genetic with of simple (S). immortalized subunit Both relatively antigen line catalytic T 18. cell telomerase epithelial small lomavirus prostate SV40 the human the (L), a the and epithelial antigen encoding (H), prostate retroviruses T hTERT human lentivi- amphotropic large with with a by SV40 infected lines generated is line cell were PrEC-LHS cell stable overexpression transduction. Isogenic MYC ral (37). without RWPE-1 or and PrEC- lines, (36) cell LHS epithelial prostate immortalized of surfaceome enx nlzdhwMCoeepeso eoesthe remodels overexpression MYC how analyzed next We oprn h lbladseicMCidcdcagsi h ufcoefrteegnee elln P9-)adtetopott ellines cell prostate two the and (P493-6) line cell B engineered the for surfaceome the in changes MYC-induced specific and global the Comparing BD ACE Mda odcag 2) > change fold (Median 0 0 100 0 100 200 262 oa in Total l sets all 439 365 144 e Size Set 75 pe lto h prgltd(e)o onrgltd(le ufc rtisi h he ihvru o MYC low versus high three the in proteins surface (blue) down-regulated or (red) up-regulated the of plot UpSet ) P493-6 177 131 down-regulated 141 188 up-regulated 25 PrEC-LHS RWPE-1 P493-6 Intersection Size RWPE-1 250 176 and 43 200 100 100 Total 0 PrEC-LHS 363 .MYC S1). Dataset ;tePrEC-LHS- the ); 223 122 345 206 9 4 92 012 20 26 59 148 194 0 93 3 0 3 36 69 105 97 32 09 20 23 23 79 89 NRF0,DCBLD2 TNFRSF10A, ROBO1, TOMM22, NCR3LG1, SLC5A6, SLC29A2, LRRC8D, LRRC8A, PTPRG, SLC38A5, SLC43A3, SLC30A1, RRP12, TNFRSF8, SCARB1, IGF1R, SLC29A1, SLC1A5, FASN, P493-6 0.377 0.31 pamncorrelation Spearman otdaaial naycl ie(i.2E change (Fig. that proteins 2 D line top (Fig. cell of proteins any set total in the the dramatically for for those most and maps S1) heat Dataset the in viewed Spear- with in ratios, and quantified SILAC be the correlation of can man plots lines correlation overall cell pairwise The three the respectively. the P493-6, between and comparisons RWPE-1, PrEC-LHS, quanti- down-regulated 40% in and the up-regulated Specifically, of uniquely were (148/495) features. surfaceome 29.9% fied distinct and very (194/814), 23.8% exhibits (345/862), lines, line cell three cell the among each proteins up-regulated common 20 are onrgltdb oeta wfl nteetrecl lines or cell three up-regulated these generated proteins in was surface twofold 2B; than plot (Fig. of more upset number by an down-regulated the lines, visualize cell to the the between enriched RWPE-1 with significantly proteins for compare those proteins To 293 respectively). of and PrEC-LHS, either and (275 MYC to much proteins) overexpressing compared is cells total overexpress- proteins) B there lines 1,240 (613 surprisingly, cell of MYC prostate Not two 206 ing line. the to between cell overlap (176 each greater 16% to three to unique the 14 were between and common cell lines, in three cell were all proteins between surface identified PrEC- identified were 2A in proteins (Fig. and identified 1,240 lines respectively, were of surfaceomes, proteins total cell 495 a P493-6 and and RWPE-1, 2). 814, (Fig. LHS, 862, surfaceomes their total, to observed In we changes and proteins 0 log 2024 2 0 -2 PrEC -LHS 0.321 enx oprdtetrecl ie ntrso h total the of terms in lines cell three the compared next We 2 0.5 IA nihetratio enrichment SILAC log .Teesmlrte n ifrne a lobe also can differences and similarities These S1). 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-2 P493-6 IA nihetratio enrichment SILAC 1 RWPE-1 -2024 -1 PrEC-LHS and S3; Fig. Appendix, SI 0

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IA nihetratio enrichment SILAC PrEC-LHS -1 RWPE-1 0 https://doi.org/10.1073/pnas.2018861118 1 GALNT2 SEL1L NSDHL GOLM1 TAPBP ANXA2 M6PR CLPTM1L NT5E SEMA7A TMX3 SLC26A6 APP SEMA4D TSPAN14 TTYH3 IFNGR1 MFAP3 SLC9A1 PTPRA PTPRS FAS PLXNB1 CNNM4 ATP1B3 ICOSLG SLC43A3 SLC4A2 RHBDF2 TNFRSF10B DSG2 PVRL1 SLC52A2 SLC2A1 SYPL1 RER1 CD63 CPNE1 SRPRB SLC1A4 TFRC CXADR SLC4A7 ICAM5 SLC39A14 SLC31A1 SLC7A1 SLC46A1 NCR3LG1 2 .Atog there Although S1). Dataset and 2 Rfrtesurface the for ER PNAS aae S1). Dataset | f11 of 3 and

BIOCHEMISTRY Thus, although MYC-induced changes do show some overlap porters of nucleosides also known as equilibrative nucleoside at the individual target level, there are significant cell line- tansporter-1 and -2, ENT1 and ENT2, that are elevated system- dependent differences suggesting that context is an important atically from two- to fourfold in the MYC high versus low states factor in determining the exact nature of the MYC-driven in all three cell lines (SI Appendix, Fig. S4C. Resistance to the surfaceome. cytotoxic nucleoside cytarabine (Ara-C), often used in treatment of acute myeloid , can be caused by down-regulation Common Functional Impact of MYC Overexpression on the Sur- of ENT1 (39). Thus, we tested the sensitivity to cytarabine faceome. Despite differences between the surfaceomes of the treatment for the high- and low-MYC expressing cells (Fig. 4). three MYC overexpressing cell lines, GeneSet enrichment anal- Indeed, two of the cell lines overexpressing MYC, the B cell line ysis (GSEA) shows the changes harmonize remarkably well for P493-6 (Fig. 4A) and the prostate cell line PrEC-LHS (Fig. 4B), the functions of the up-regulated proteins among the three cell showed >10-fold higher sensitivity to cytarabine in the high- lines, suggesting functional redundancy (Fig. 3A and Dataset MYC state (half-maximal inhibitory concentration (IC50) 3 µM S2). The dominant category by far is the broad up-regulation in high MYC vs. >30 µM in low MYC). This higher sensitiv- of solute and nutrient carrier proteins across all three cell lines. ity to Ara-C rendered by MYC overexpression was substantially Small molecule metabolite and ion transporters represent over blunted by adding an ENT-specific inhibitor S-(4-Nitrobenzyl)- half of the functional categories that are up-regulated. For exam- 6-thioinosine. We also tested RWPE cells and found them to be ple, about 40 surface proteins in the organic acid transmembrane 10-fold more sensitive to cytarabine (IC50 300 nM) compared to category were detected in our data, and more than half were the P493-6 or PrEC-LHS cells either in low- or in high-MYC significantly up-regulated in one or more cell lines (Fig. 3B). states, and the S-(4-Nitrobenzyl)-6-thioinosine inhibitor did not Similar results were seen for the sodium ion transmembrane alleviate the cytotoxicity (Fig. 4C). These data suggest another transporter set (Fig. 3C) and the amino acid transporter set transporter other than ENT1/2 may be at play for cytarabine (Fig. 3D). uptake or that the RWPE cells lack efflux pumps of cytarabine GeneSet analysis also identified a global enrichment of trans- that may be present in the other two cell lines. membrane transporter ( [GO]: 0055085), which The next category of proteins that had significantly up- further enforces that transporters are highly up-regulated in regulated expression patterns were ones broadly associated with MYC-overexpressing cell lines. Of the 199 proteins identified and in particular eight receptors from the TNF receptor with such annotation, 82 of these proteins were found to be family (Fig. 5A), including TNFRSF10A, -B, and -D; TNFRSF8; up-regulated in at least one of the cell lines (SI Appendix, Fig. TNFRSF11A; TNFRSF1B; and TNFRSF4. These receptors rep- S4A). These conclusions were reinforced by GeneSet analysis resent 7 of the top 10 up-regulated proteins in the apoptotic signal- using REACTOME showing the changes were dominated by ing pathway among the three cell lines, especially for the prostate the small molecule transporter class of proteins, summarized epithelial derived cells. To test whether the overexpression of in SI Appendix, Fig. S4B. Moreover, 9 of the 20 commonly these receptors in the MYC-expressing prostate cells has func- up-regulated proteins among the three cell lines were small tional effects, we treated both the MYC-overexpressing cells and molecule or ion solute carrier proteins including LRRC8A, their isogenic controls with the soluble agonist, TRAIL. Indeed, LRRC8D, SLC1A5, SLC29A1, SLC30A1, SLC43A3, SLC38A5, for the PrEC-LHS (Fig. 5B) and RWPE-1 cells (Fig. 5C) the SLC29A2, and SLC5A6. Two of these commonly expressed MYC-overexpressing cells showed greater than 10-fold increased transporters, SLC29A1 and SLC29A2, are well-known trans- sensitivity by TRAIL-induced apoptosis. The sensitivity of the

ABCD

Fig. 3. Functional similarities revealed by GSEA among the high- versus low-MYC surfaceomic datasets for the P493-6 B cells and the RWPE-1 and PrEC-LHS prostate cells. (A) Top 50 enriched gene sets identified by GSEA of the surfaceomic dataset using Gene Ontology terms show clustering among high- versus low-MYC surfaceomes for the three cell lines. Positive normalized effect size (up-regulation) is shown in red, and negative (down-regulation) normalized effect size is shown in blue. Proteins were preranked by median SILAC peptide ratio and GSEA was performed using MySigDB C5 GO gene set collection. (B–D) Surface proteins involved in organic acid transmembrane transport (GO: 1903825), sodium ion transmembrane transporter activity (GO: 0015081), and amino acid transport (GO: 0006865) that are up-regulated in the each of the three high- versus low-MYC surfaceomes with a distinct set of transporters.

4 of 11 | PNAS Chen et al. https://doi.org/10.1073/pnas.2018861118 Large remodeling of the Myc-induced cell surface proteome in B cells and prostate cells creates new opportunities for immunotherapy Downloaded by guest on September 25, 2021 Downloaded by guest on September 25, 2021 ino NS1Buigflwctmtywt recombi- a Appendix, with (SI cytometry below described flow S5D). TNFRSF10B Fig. using to Appendix, antibody (SI TNFSF10B nant MYC of of sion of knockdown S5 plot sub- by Fig. was correlation TNFSF10B suppressed particular the in stantially and in sensitive, especially seen were in also knockdown is S5C versus smaller This much overexpression ear- 1E). had MYC seen cells knockdown PrEC- (Fig. MYC-overexpressing basal MYC lier those the the to in compared thus low effects and were pro- cells levels 458 MYC LHS the starting among the bidirectional Appendix expression because mild (SI protein a detected surface had teins deter- This cell and cells. on untreated knockdown S5A) effect the MYC between Fig. surfaceome the cell Appendix , and the (SI on impact 42) repres- the mined gene (41, MYC CRISPR-mediated (CRISPRi) basal the using sion down threefold knocked affected by so this and how expression surfaceome determine cell to basal sought the We S5A). Fig. Appendix, in that to (40). lines comparable cell is cancer prostate in TRAIL some cytarabine to to sensitive cells sensitizes extremely overexpression are MYC-overexpressing MYC cells Titer-Glo. RWPE Cell the using whereas inhibitor, death ENT cell the for by assayed blocked and be h can states. which 48 low-MYC cytarabine for and to high- nM) both cells 650 PrEC-LHS up and (ranging B-cells cytarabine P493-6 of concentrations increasing with (Left S-(4-Nitrobenzyl)-6-thioinosine 4. Fig. ag eoeigo h y-nue elsraepoem nBclsadpott el rae new creates cells prostate and cells B in proteome immunotherapy surface for cell opportunities Myc-induced the of remodeling Large al. et Chen h rCLSclshv aa xrsino Y (SI MYC of expression basal have cells PrEC-LHS The (r estvt fhg-vru o-Y xrsigclst h yooi uloie yaaie(rC ( (AraC) cytarabine nucleotide, cytotoxic the to cells expressing low-MYC versus high- of Sensitivity = B .) neetnl,teewr oetresthat targets some were there Interestingly, −0.5). and .W ute aiae h erae expres- decreased the validated further We C).

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Viability Viability Viability eune obnit nqecoe n nlzdfrkinetic for analyzed and were clones test unique this passing into Clones bin S7A). Fig. to but Appendix , sequenced ectodomain (SI target Fc ELISA the to by to not binding evaluated high-affinity were for rounds the clones tested four and After phage recently S6). individual Fig. more selection, overex- Appendix, are of (SI and (48) discovered KRAS we (47) by that pressed proteins proteins to surface human antibodies of recombinant ectodomains 500 high-quality than previously produce more has antigen to that antibod- an used platform employed renewable been display We and Fab-phage NCR3LG1. and recombinant and expression generate TNFRSF10B to to ies out set we Cell T Bispecific for Potential NCR3LG1 Engagement. and and Overexpression TNFRSF10B Validate Further to to Antibodies Recombinant thus of and Generation killing NK too. to survival their susceptible to MYC- counter make degree more would lesser This cells a 2E). to (Fig. overexpressing cells and epithelial cells prostate cell B the (NK) the in the killer in observed natural especially also in a we ligand, apoptosis 46), vein activating (45, same to NCR3LG1 this susceptible In of 44). overexpression cells (43, make TRAIL of would presence it as doxical 0.0 0.5 1.0 1.5 0.0 0.5 1.0 1.5 0.0 0.5 1.0 1.5 .10111 0 1000 100 10 1 0.1 0.01 .10111 0 1000 100 10 1 0.1 0.01 .10111 0 1000 100 10 1 0.1 0.01 h vrxrsino NRF0 mrse sa para- as us impressed TNFRSF10B of overexpression The C rsaeclsepesn ihMC(e)wr rae nduplicate in treated were (red) MYC high expressing cells prostate ) 436+ET/ inhibitor ENT1/2 + P493-6 WE+ET/ inhibitor ENT1/2 + RWPE H N12inhibitor ENT1/2 + LHS Cytarabine, µM Cytarabine, µM Cytarabine, µM ofrhrvldt u ufc rtoisresults proteomics surface our validate further To Right n rtcinafre yET/ inhibitor ENT1/2 by afforded protection and ) MYC MYC . Minhibitor µM 5.7 + MYC . Minhibitor µM 5.7 + MYC ihMYC High . Minhibitor µM 5.7 + MYC High https://doi.org/10.1073/pnas.2018861118 PNAS | f11 of 5

BIOCHEMISTRY Apoptotic Signaling Pathway eightfold increase in expression of NCR3LG1 in the high-MYC ABPrEC-LHS Cells TRAIL Treatment TNFRSF10A P493-6 cells (Figs. 1B and 2E). In addition to the high-MYC TNFRSF10B 1.0 P493-6 cells, we examined the surface NCR3LG1 expression of TNFRSF10D TNFRSF8 two Burkitt’s lymphoma cell lines, Raji (55) and Ramos (56) ITGAV cells, known to have high levels of MYC and one low-MYC TNFRSF11A EBV-immortalized B cell line from a healthy doner (57) by anti- TM2D1 0.5

TNFRSF1B Viability NCR3LG1 Fab staining (Fig. 7C). Indeed, all of the high-MYC TNFRSF4 PrEC-LHS-EV cell lines showed up to fivefold higher levels of staining relative CD14 PrEC-LHS-Myc CD40 to the low-MYC EBV-immortalized B cells. We further con- CD70 0.0 -10 -9 -8 -7 structed an anti-NCR3LG1-BiTE and found it activated Jurkat EPHA2 much better when mixed with the high-MYC PrEC-LHS and FAS TRAIL concentration (g/mL) SPN RWPE prostate cells relative to the low-MYC isogenic controls PDCD6 RWPE-1 Cells TRAIL Treatment CD38 C (Fig. 7B). We expanded the study to the P493-6, Raji, and Ramos TLR3 cells compared to the low-MYC B cells. The anti-NCR3LG1- ARL6IP5 1.0 CD27 BiTE activation assay in the high-MYC B cells showed signifi- TMEM109 cant activation (Fig. 7D) that was about threefold higher than P2RX4 in the low-MYC controls. Collectively, the recombinant anti- LAMP1 0.5 NGFR body studies corroborate that these targets are overexpressed Viability SORT1 RWPE-1-EV in these cell lines and amenable to targeted killing using BiTE SGPL1 RWPE-1-Myc technology. 0.0 -10 -9 -8 -7 log SILAC enrichment ratio P493-6 2 Discussion RWPE-1 PrEC-LHS -2 -1 0 1 2 TRAIL concentration (g/mL) MYC is one of the most dominant transcription factors affecting Fig. 5. Some prominent members of the apoptotic signaling pathways are expression of 10 to 15% of genes and is often highly up-regulated generally up-regulated in high-MYC conditions and sensitize cells to TRAIL- in B cell and prostate cancer cells as well as others (58). To induced apoptosis. (A) Proteins involved in apoptotic signaling pathways systematically understand how MYC overexpression affects the (GO: 0097190), including TNFRSF10A, TNFRSF10B, and TNFRSF8, are up- cell surfaceome based on cell types in a cell autologous fash- regulated in each of the three high- versus low-MYC surfaceomes. (B and C) ion, we took a reductionist approach where we generated or PrEC-LHS and RWPE-1 cells in high- and low-MYC states were treated with employed isogenic cell lines having either low or high levels of recombinant TRAIL. MYC-overexpressing PrEC-LHS and RWPE-1 cells show MYC expression. These isogenic models are certainly oversim- much higher sensitivity to TRAIL-induced apoptosis. plified because no single cell type or context can recapitulate real tumors. However, we believe this is an important and pragmatic approach to investigate the surface proteome regulated by MYC

and equilibrium binding properties by biolayer interferometry (SI Appendix, Figs. S7B and S8). Data are shown for two of the PrEC-LHS cells RWPE-1 cells high-affinity Fabs obtained for TNFRSF10B and NCR3LG1 with A dissociation constant values of 9.6 and 0.3 nM, respectively. We further validated the binding specificity on cells using CRISPRi knockdown cells (SI Appendix, Fig. S7C). We observed reduction in binding in the NCR3LG1 and TNFRSF10B knockdown cell lines relative to the isotype control as assessed by flow cytom- etry. These data validate that these high-affinity antibodies are specific for their target proteins on cells. We next tested the ability of the antibodies to detect the over- expression of their respective target on the MYC overexpression SA Alexa647 SA Alexa647 cell lines relative to the low-MYC control cell lines. We first -Myc cells SA stain only -EV cells SA stain only tested the anti-TNFRSF10B recombinant biotinylated Fab and -Myc cells TNFRSF10B-E2 / SA stain -EV cells TNFRSF10B-E2 / SA stain found higher cell binding in both the high-MYC PrEC-LHS and high-MYC RWPE-1 cells (Fig. 6A) relative to the low-MYC B isogenic cell controls. This further validates the mass spectrome- 400000 try data showing similar increases in expression of TNFRSF10B Jurkat-only on the high-MYC cells. We then cloned the anti-TNFRSF10B PrEC-LHS-EV 300000 PrEC-LHS-Myc Fab into a BiTE format (49–51) by fusing the well-established RWPE-1-EV anti-CD3 scFv (52, 53) to the C terminus of Fab heavy chain RWPE-1-Myc containing a His6-tag for purification. The anti-TNFRSF10B- 200000 BiTE was tested for T cell activation by coculturing low- or high-MYC cell lines with engineered Jurkat T cells containing an 100000 NFAT-GFP reporter (54). Indeed, the high-MYC cells showed significantly greater T cell activation than the low-MYC cells at 0.1 nM concentration of anti-TNFRSF10B-BiTE (Fig. 6B). 0 Using the same experimental platform as above we tested blank 0.1 nM 1 nM our high-affinity antibodies to NCR3LG1 for binding and T Fig. 6. Anti-TNFRSF10B antibody E2 shows preferred binding to PrEC-LHS cell activation to high- and low-MYC cells (Fig. 7). Using the and RWPE-1 cells with MYC overexpression. (A) The TNFRSF10B BiTE acti- anti-NCR3LG1 recombinant biotinylated Fab, we were able vates Jurkat cells significantly better in cells with MYC overexpression than to observe higher binding signal to high-MYC PrEC-LHS and in cells without MYC overexpression when treated with 0.1 nM BiTE. (B) high-MYC RWPE-1 cells relative to the low-MYC isogenic con- Treatment with the TNFRSF10B BiTE alone resulted in no significant T cell trols (Fig. 7A). The mass spectrometry data showed an almost activation.

6 of 11 | PNAS Chen et al. https://doi.org/10.1073/pnas.2018861118 Large remodeling of the Myc-induced cell surface proteome in B cells and prostate cells creates new opportunities for immunotherapy Downloaded by guest on September 25, 2021 Downloaded by guest on September 25, 2021 ag eoeigo h y-nue elsraepoem nBclsadpott el rae new creates cells prostate and cells B in proteome immunotherapy surface for cell opportunities Myc-induced the of remodeling Large al. et many Chen so individu- drives line, MYC cell that B given other surprising the be each from not from differed may they This differed ally. as lines Even much cell lines. as cell epithelial almost three prostate half the than two among more vary the as changes dependent expression type precise the cell morphologi- of The highly are overexpression. lines, see MYC we cell for changes observed is RWPE-1 are overexpression and changes MYC no cal PrEC-LHS by While in growth cells. cell B observed of in alteration overexpression rate MYC significant proliferation with increased correlated as that corresponding such are observe there consequences, find we We move- phenotypic another. changes a to state reflecting The one bidirectional, from 62). ment and studies 61, symmetric previous remarkably (48, our are proteins between in membrane correlation data at poor sequencing on correlate observed RNA strongly have and We surfaceome not level. might protein global transcription the that gene that possible of is over- it increase Myc 60), (59, globally though transcription) transcription Even of gene 32). amplify (amplifier to (31, anno- shown time been 4,000 has given of expression any genome total surfaceome the at of a expressed half total about are is that the and there genes of protein that membrane one-third expectation tated we the to estimate on we up based Nonetheless sampled abundance. have low our would of realize ones We pro- and proteins. of nonglycosylated miss teins surface 30% will we observed to because incomplete 800 20 is sampling to for 500 changes more total 10-fold the to to 2- induces compared overexpression can samples one reproducible where tumors. setting and primary complex controlled pure highly nM obtain 1 a and low-MYC 0.3 in with at coculturing cells overexpression when Jurkat minimum activates a BiTE to NCR3LG1 cells (D) Jurkat line. of cell activation the B keeping immortalized while EBV when cells low-MYC minimum Ramos a a and cells. to to cells Raji, B binding EBV-immortalized Jurkat P493-6, visible of high-MYC activation no with (C the but coculturing keeping overexpression. ( (purple), when while MYC overexpression. MYC Ramos MYC without overexpressing and with cells cells (red), RWPE-1 cells RWPE-1 Raji and RWPE-1 and PrEC-LHS and with PrEC-LHS PrEC-LHS coculturing with when to coculturing nM binding 1 higher and shows 0.3 H3 at cells antibody NCR3LG1 (A) cells. MYC i.7. Fig. h ufcoisdt hwdaai hne hr MYC where changes dramatic show data surfaceomics The niNRL1atbd 3sospeerdbnigt ihMCcl ie n h C3G iEatvtsJra el hnccluigwt high- with coculturing when cells Jurkat activates BiTE NCR3LG1 the and lines cell high-MYC to binding preferred shows H3 antibody Anti-NCR3LG1 CD AB -Myc cells -Myc cells PrEC-LHS cells SA NCR3LG1-H3/SA stain SA stainonly Aea4 SA Alexa647 SA Alexa647 Ramos Raji Ramos EBV Raji P493-6 EBV P493-6 NCR3LG1- SA stainonly NCR3LG1- SA stainonly -EV cells -EV cells NCR3LG1- SA stainonly NCR3LG1- SA stainonly RWPE-1 cells H3 Alexa647 H3 NCR3LG1-H3/SA stain SA stainonly /SA stain H3 H3 /SA stain /SA stain /SA stain C3G nioyH hw togbnigt ihMCP9- (orange), P493-6 high-MYC to binding strong shows H3 antibody NCR3LG1 ) nti ls.MCi nw odiepoieainadhsits has and proliferation drive to of known quarter is are a MYC overexpression class. roughly MYC this by surfaceome, in induced the proteins 10% encoding up-regulated about (31, the represent genes proteins genes membrane the carrier class carrier solute encoding of solute annotated genes while Thus, 415 4,000 32). are roughly there lines cur- the the proteins, cell Of in (GO: annotation study). Myc-overexpression transport this with transmembrane rent the identified to or proteins of connected twofold 199 are 0055085, one by 82 up-regulated least 2B), carriers seen (Fig. at ion targets in 365 metal systematically the and greater is Of metabolite, that 3A). solute, (Fig. class cel- the dominant different is most in up-regulated The oncogenesis contexts. MYC lular of the and convergence proteins functional up-regulated the the highlights among This redundancy functions. functional protein in coherence remarkable is transmembrane of enrichment consistent geneset is transport. of and amplification observation 64). MYC our (63, harbor with to subtype known (NEPC) is cancer in NEPC up-regulated prostate to be neuroendocrine to coupled identi- transport the lines transmembrane analysis ion cell geneset expression cancer fied transcriptome prostate cell of published prostate pro- variety previously the surface a cell For of from previously. analysis teomes systematic here used recent lines line a cell comparison, the cell line between TALL differences S9 ). the the Fig. to and Appendix, system- due them (SI be observe could overexpression not This We MYC did by (6). but altered (TALL) proteins atically leukemia two lymphoblastic these acute detect did cell T and PD- in of expression CD47 induces MYC found have Others genes. ept h ifrne nidvda rti xrsinthere expression protein individual in differences the Despite

MFI MFI 100000 200000 300000 400000 500000 600000 700000 250000 300000 350000 400000 100000 150000 200000 50000 0 0 ln . M 1nM 0.3nM blank ln . M 1nM 0.3nM blank Ramos Raji P493-6 EBV Jurkat-only RWPE-1-EV PrEC-LHS-Myc PrEC-LHS-EV Jurkat-only RWPE-1-Myc h C3G iEatvtsJurkat activates BiTE NCR3LG1 The B) https://doi.org/10.1073/pnas.2018861118 PNAS | f11 of 7

BIOCHEMISTRY greatest effect on increasing translation, an anabolic process with overexpressed and common in all three surfaceomic datasets. It tremendous nutrient requirement (7, 26). We have recently per- is not clear whether enhanced TNFRSF10B transcription upon formed surfaceomic studies on a common isogenic breast epithe- MYC overexpression is responsible for this finding. It is also lial cell line containing oncogenes such as oncogenic KRAS, possible that differential protein degradation and trafficking are EGFR, HER2, BRAF, MEK, and Akt that drive prolifera- responsible for higher TNFRSF10B abundance on the cell sur- tion through the MAPK or Akt pathways. In these studies we face upon MYC overexpression. We found that the whole-cell observed a total of 375 proteins upregulated and only 44 anno- TNFRSF10B levels in PrEC-LHS-Myc and RWPE-1-Myc cells tated with transmembrane transport activity (GO: 0055085), are unchanged or decreased, respectively, in comparison to their representing 11.7% (62). Thus, compared to other oncogenes, corresponding EV cells (SI Appendix, Fig. S11). A recombi- MYC seems to have a particularly strong and differential effect nant antibody we generated to the ectodomain of TNFRSF10B in promoting the solute carriers. further demonstrated overexpression of TNFRSF10B in the There are at least 20 targets that are commonly up-regulated Myc-overexpressed cell lines. Moreover, the antibody could in all three cell lines, and 7 belong to the solute carrier family activate T cells in a BiTE format justifying further preclin- including SLC1A5, SLC29A1, SLC30A1, SLC43A3, SLC38A5, ical investigation of this target antibody. In this same vein SLC29A2, and SLC5A6. Two of these, SLC29A1 and SLC29A2, we identified the NK activating ligand, also called B7-H6, for also known as ENT1 and ENT2, are nucleoside transporters binding NK30p on NK cells (45, 81). NCR3LG1 was com- that have been found to be overexpressed in colon cancer. It monly up-regulated on all three MYC-overexpression cell lines. is not surprising to find that 6 of these 7 SLC transporters The high-affinity recombinant antibodies to NCR3LG1 we pro- do not appear to be essential to most cell lines, whereas 170 duced showed increased binding to cells overexpressing MYC, of 808 cell lines in CRISPR screens (CRISPR [Avana] Pub- further validating the surfaceomics data. Moreover, an anti- lic 20Q4) and 5 of 710 cell lines in RNAi screens (combined NCR3LG1 BiTE construct was found to activate T cells to RNAi) are found to be dependent on SLC1A5 (SI Appendix, a greater degree for the MYC-overexpression cells as well as Fig. S10) (65–67). MYC regulation of these transporters in Raji and Ramos cells also found to overexpress MYC and combination with dysregulation of efflux pumps determines NCR3LG1. overall sensitivity vs. resistance to toxic nucleosides like cytara- The expression of these TNF receptor family members as bine (68, 69). We found the MYC-induced overexpression well as the NK cell activating ligand is paradoxical to cell sur- of ENT1/2 leads to increased sensitivity to cytarabine for at vival but there are other examples. It has long been known least two of the three cell lines, the P493-6 B cells and the that constitutive expression of MYC suppresses cell cycle arrest PrEC-LHS prostate cells, but that RWPE cells were highly sen- and accelerates intrinsic apoptosis often in a p53-dependent sitive independent of MYC expression and did not depend on fashion through the mitochondria by elevation of proapoptotic ENT1/2. It is possible that cytarabine is getting into RWPE BH3 family members (82). More recently it has been discovered cells via another transporter and/or that these cells lack ABC that MYC expression can induce apoptosis through the extrin- transporters that efflux the drug (70, 71). MYC levels have sic pathway, such as the (83). Others have shown been correlated to both up- and down-regulation of ABC MYC can up-regulate the DR5 receptor for TRAIL (44). To efflux pumps (71). ABCC4 has been demonstrated to partic- rationalize these findings it has been proposed that MYC over- ipate in cytarabine efflux, and likely other members of the expression leads to sensitization to apoptosis that may serve as ABCC family also mediate its export (72). Three of the other a cell surface fail-safe mechanism like p53 to remove cells with SLCs (SLC1A5, SLC38A5, and SLC43A3) also found commonly inappropriate MYC activation prior to complete development of up-regulated on the three cell lines are organic acid and amino the cancer phenotype (43). Our findings are consistent with these acid transporters. While SLC43A3 is an orphan transporter published reports. whose function is less known (73), SLC1A5 and SLC38A5 In summary, we believe the surfaceomics datasets compar- are glutamine transporters that are often up-regulated in can- ing low-MYC and high-MYC expressing cells provide a useful cers for enhanced glutaminolysis (74–77). The last two are resource for understanding how MYC dramatically remodels SLC5A6, a sodium/vitamin transporter (78), and SLC30A1, the surfaceome in a cell autologous fashion. This work adds to a Zn2+ transporter known to be highly expressed in nons- our understanding of MYC biology and provides focused sets mall cell lung cancer (79). The three cell lines used in this of surface targets and combinations to validate in more complex study are not suitable for elucidating the dependency on SLC settings. These studies provide a useful data resource for gener- transporters for Myc-driven cancer cell growth because the ating recombinant antibodies to further validate potential cancer growth of both PrEC-LHS and RWPE-1 is not dependent on drug targets. Myc overexpression and P493-6 is an engineered cell line with Myc placing under an artificial tet-off system. Nonetheless, Materials and Methods Yue et al. (80) found that SLC7A5 loss by RNA interfer- Cell Line Generation for Surfaceomic Experiments. Parental P493-6, RWPE-1, ence resulted in diminished Myc expression and lower growth and PrEC-LHS were a gift from Michael J. Evans at University of California, for Daudi cells consistent with its importance. Our data high- San Francisco. The lentiviral transfer plasmid pCDH-puro-cMyc was pur- chased from Addgene (Addgene: 46970) (84). pCDH-puro-cMyc was modi- light the possibility of using cytotoxic metabolites to target the fied to create pCDH-zeo-cMyc in which the puromycin resistance gene was up-regulated transporters, recognizing that the balance of trans- replaced by a zeocin resistance gene (Ble). The Myc gene in pCDH-zeo-cMyc port and efflux can be complex and pleotropic with redundant was then replaced by a piece of short decoy sequence (ATCGATCGATC- functions. GATCG) to create the empty vector plasmid pCDH-zeo-EV. RWPE-1-EV and Another class of proteins that are systematically up-regulated PrEC-LHS-EV were generated by lentiviral transduction of pCDH-zeo-EV into is TNF receptor superfamily members, including TNFRSF10A, the parental RWPE-1 and PrEC-LHS cells, respectively. RWPE-1-Myc and PrEC- -B, and -D; TNFRSF8; TNFRSF11A; TNFRSF1B; and LHS-Myc were generated by lentiviral transduction of pCDH-zeo-Myc into TNFRSF4; which paradoxically sensitize cells to killing by TNF the parental RWPE-1 and PrEC-LHS cells, respectively. After transduction, family members. Other studies have shown that MYC sensitizes cells were allowed to recover for 2 d prior to zeocin resistance selection (200 µg/mL) for 1 wk. P493-6 and PrEC-LHS cells were grown in RPMI 1640 media cells to apoptosis and to TRAIL (43, 44). Indeed, we find that (GE Healthcare Life Sciences) supplemented with 10% fetal bovine serum. the MYC overexpression in the prostate epithelial cells was 10- RWPE-1 cells were grown in Keratinocyte Serum Free Medium (Gibco). The to 100-fold more sensitive to TRAIL killing than the non-MYC transduced stable cell lines are maintained in media supplemented with overexpression control. In particular, TNFRSF10B was highly zeocin (50 µg/mL).

8 of 11 | PNAS Chen et al. https://doi.org/10.1073/pnas.2018861118 Large remodeling of the Myc-induced cell surface proteome in B cells and prostate cells creates new opportunities for immunotherapy Downloaded by guest on September 25, 2021 Downloaded by guest on September 25, 2021 ag eoeigo h y-nue elsraepoem nBclsadpott el rae new creates cells prostate and cells B in proteome immunotherapy surface for cell opportunities Myc-induced the of remodeling Large al. et Chen cthadrlae eetoswt elvldtdadhg-iest Fab- high-diversity of and rounds well-validated (3 library four a phage mag- with conducted selections on We release” immobilized streptavidin. and “catch and bound chromatography containing affinity beads were netic A and proteins Fc-fusion protein biotinylated glycosylation The by (48). and purified al. cells, et pro- secretion Martinko in in biotinylation Fc-fusion described facilitate C-terminal as site-specific as to perform expressed to cells engineered were further expiHEK293T NCR3LG1 in and teins TNFRSF10B ELISA. single-pass Phage and on I based Procedure sorting Panning cell Phage to prior d 2 for recover PrEC-LHS- expression. to lentiviral expression. GFP Trans- allowed by cells. BFP were PrEC-LHS-dCas9-KRAB obtained cells on the duced into are based plasmids constructs sgRNA d sorting by the sgRNA 2 cell transduction for expressing into of recover cells rounds to dCas9-BFP-KRAB dCas9-KRAB allowed two of were to cells transduction prior Transduced cells. lentiviral PrEC-LHS Experiments. by parental CRISPRi obtained for were vector Generation Line lentiviral Cell pHR-SFFV a 46914) from (Addgene: expressed (42). vector 46911) lentiviral (Addgene: was pU6 dCas9-BFP-KRAB a into and cloned were TTCAGAGCGT (NCR3LG1 NCR3LG1 GACGCACCAGTCGT; CGTAGGC Y (MYC MYC GCTGGTCACGCGCC; L-[ containing (Gibco) media CSC. SILAC and Labeling, ReDiMe SILAC, 8.Peiul ecie etr eeue o RSR xeiet 4,48). (TNFRSF10B (47, (42, TNFRSF10B experiments BiTEs CRISPRi sgRNAs and for Fabs Individual used of (48). were expression vectors described described for Previously vector previously adapted 48). described as and previously a vector cloning used (InvivoGen) Gibson We pFUSE-hIgG1-Fc by generated the was from NCR3LG1 and Construction. TNFRSF10B and Design Vector MSV000085930. identifier MassIVE deposited and deposition PXD020799 were ProteomeXchange number data the proteomics with (http://massive.ucsd.edu) The MassIVE (87). com- in proteins, Skyline surface by cell quantified for filtered and were bined, Results previously 86). 62, as 61, acquisition (48, data-dependent described with proteomics separately analyzed bottom–up then with were the peptides tryptic quench and To peptides cleaved shaking. PNGase while temperature 32 room add at reaction, h 1 for incubated were 8 and 8 bicarbon- Sigma-Aldrich) triethylammonium or mM 200 label) 100 (per (light urea, sample digested M each (2 To buffer ate). on-bead urea digested in then trypsin were by samples H The The with reduced min. bicarbonate). 30 washed were triethylammonium for then samples temperature room were The at beads 7.5). iodoacetamide mM pH 55 10 PBS at with tris(2-carboxyethyl)phosphine alkylated urea, mM M 5 (2 with buffer on-bead to pH (PBS) urea binding saline phosphate-buffered and and NaCl, 7.5), lysis M (1 radioimmunoprecipitation cell buffer with high-salt after buffer, washed ReD- Briefly, assay were by beads protocol. labels the modified isotopic beads, light a through neutravidin and went with heavy cells (38) the PrEC-LHS introduce iMe and to step RWPE-1 additional for PNGase an with samples bond The N-glycan the (48). of treatment hydrolysis by of recovery peptide and tryptic digestion, N-linked tryptic the on-bead binding by by (85). followed enriched beads, were glycoproteins neutravidin glycoproteins surface to surface cell the the of lysis gly- cell purification on after subsequent Briefly, handles the biotin for install units to can treatment biotin-hydrazide labeling with periodate CSC reaction mild the by by employed followed glycoproteins We surface protocol. captures CSC that the technique to subjected were and lines 20 ratio cell fluence and PrEC-LHS count media and cell regular RWPE-1 1:1 The with at protocol. cultured mixed CSC and the 20 to harvested respectively. subjected were and cells treatment, culturing P493-6 tetracycline by light cells ng/mL cells or P493-6 heavy P493-6 100 light low-Myc with and and or heavy high-Myc The without generating cells. for on used labeling were isotope the of L-[ poration or Laboratories) Isotope L-[ and Cambridge label; (heavy nine .M Muhar M. 2. Lorenzin F. 1. axis. regulation. gene dependent 12 Science C 6 , 14 LMsqdfie ietgn-euaoyfntoso h BRD4-MYC the of functions gene-regulatory direct defines SLAM-seq al., et ifrn rmtrafiiisacutfrseict nMYC- in specificity for account affinities promoter Different al., et 0–0 (2018). 800–805 360, N 4 × riie(ih ae)frfiepsae oesr ulincor- full ensure to passages five for label) (light arginine ] f1 NH 1% of µL 10 f4 formaldehyde- 4% of µL 9 f06Msdu ynbrhdie h samples The cyanoborohydride. sodium M 0.6 of µL ains otiigsnhtcvratCR o CDRL3, for CDRs variant synthetic containing variants) eLife 4 Hfloe y16 by followed OH 111(2016). e15161 5, h etrue oepesteED of ECDs the express to used vector The 2 436clswr utrdi PI1640 RPMI in cultured were cells P493-6 n rabfe 2Mue,10mM 100 urea, M (2 buffer urea and O 13 + × C 2770.3PP) GGGGCGCGCG- 128747801.23-P1P2): 6 10 , ) d 8 add µL), 15 6 N el eehretda 0 con- 80% at harvested were cells - + 2 77372-12:GGTGGG- 17373307.23-P1P2): yieadL-[ and lysine ] 13 h cooan ftetype the of ectodomains The 22112-12:GGGCAA- 22926121.23-P1P2): ,d C, rCLSda9KA cells PrEC-LHS-dCas9-KRAB f4 formaldehyde 4% of µL 2 ffri cd The acid. formic of µL ouin(ev label; (heavy solution ◦ o 0mnand min 30 for C 12 13 C C 6 , 6 14 , 15 N N 2 lysine ] 4 × argi- ] 10 6 ietosAad,Ntoa acrIsiueP1A926 n h Harry (J.A.W.). Sciences the Pharmaceutical New and in Program Professorship P41CA196276, Cancer this Hind Institute Dianna of Prostate Cancer and Much UCSF National Research. from Health Awards, funding of Directions by Institutes supported Canadian was and the sup- work was Zhou by K.K.L. Xin part discussions. useful Dr. in the for thank ported laboratory providing Wells We the for line. of (UCSF) members cell other Kadlecek Jurkat Theresa reporter and We GFP NFAT-dependent discussions. (UCSF) helpful Weiss Professor the and Arthur for Ruggero lines thank Davide cell Professor providing and kindly Evans for Michael (UCSF) Francisco San California, ACKNOWLEDGMENTS. 37 at D-Biotin mg/mL 1 OD with supplemented to pro- Broth Terrific described in previously grown a were following Briefly, expressed (48). were tocol scFv) anti-CD3 an BiTEs. to and Fabs of Expression antiphage Lifesciences). (HRP)-conjugated (GE peroxidase were antibody plates horseradish 0.05% monoclonal the a 7.4, to bound using pH were (PBS, detected that buffer Phages then min. ELISA 30 in addi- for BSA) 1:5 the 0.2% h by diluted Tween-20, followed supernatants 1 min phage for 30 of for wt/vol) tion wells (2% NeutrAvidin-coated the (BSA) 4 on albumin captured at serum overnight bovine mg/mL) 20 with at (10 blocked NeutrAvidin were with and coated Max- were Ninety-six-well protocol. plates described nega- isorp ELISAs previously for Phage a antibodies. used using Fc-binding conducted was unwanted were alone any Fc-domain remove to The selection (88). tive CDRH3 and CDRH2, CDRH1, ihtePoemXhnedpsto number deposition ProteomeXchange the PXD020799 with massive.ucsd.edu) Availability. 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Cytotoxicity Cytarabine CytoFLEX a using cytometry flow Coulter). h by (Beckman 20 quantified was After expression control. reporter blank GFP nM a 1 as of 37 total used at A is nM. incubation (anti- 1 Fab or (BiTEs) anti-NCR3LG1 0.3 of antibody or concentration final engaging anti-TNFRSF10B a cell at mixtures and T cell medium to Bispecic in added diluted 1:1. was of anti-NCR3LG1-anti-CD3) or ratio TNFRSF10B-anti-CD3 a at cells Myc assay. recovery. BiTE of before d expression 2 sgRNA for for puromycin select without to medium d to taken 3 being for cells to RWPE-1 puromycin and with (42) PrEC-LHS treated previously infection, were after described days Two NCR3LG1 particles. and viral generate TNFRSF10B Lines. for Cell plasmids sgRNA CRISPRi of puri- were Preparation chromatography, Fabs Ni-NTA and and sonication chromatography by affinity lysed A respectively. and Protein harvested by were fied Cells 30 h. to 18 reduced isopropyl subsequently 20 nM and was 0.1 temperature and Incubation mM respectively. 1 of addition the .L Soucek L. 4. .D Horiuchi D. 3. /Lttayln o 6h rhg y,PE-H el xrsigEV expressing cells PrEC-LHS Myc, high or h) 36 for tetracycline µg/mL (2008). rattmr iheeae Y expression. MYC elevated with tumors breast ◦ o 8h h elvaiiyi hnotie yWT8asy(Cayman assay WST-8 by obtained then is viability cell The h. 18 for C yaaie(im) ouiie ndmtysloie(MO,and (DMSO), dimethylsulfoxide in solubilized (Sigma), cytarabine µM ◦ 600 .Attlo 0n fboiyae C-cfso rF-oanwas Fc-domain or ECD-Fc-fusion biotinylated of nM 20 of total A C. ◦ ,rsetvl,adteclue eealwdt hk o 6to 16 for shake to allowed were cultures the and respectively, C, f06t . n hnFbadBT xrsinwsidcdby induced was expression BiTE and Fab then and 0.8 to 0.6 of oeigMcihbto sacne therapy. cancer a as inhibition Myc Modeling al., et n asV identifier MassIVE and uktNA-eotrclswr ie ihhg-y rlow- or high-Myc with mixed were cells NFAT-reporter Jurkat I1kns niiina agtdteayaanttriple-negative against therapy targeted a as inhibition kinase PIM1 al., et ◦ l rtoisdtst r eoie nMsIE(http:// MassIVE in deposited are datasets proteomics All shrci coli Escherichia ,clswr eltdb etiuain NFAT-dependent centrifugation. by pelleted were cells C, etakPoesrMcalEasa nvriyof University at Evans Michael Professor thank We -4Ntoezl--honsn Sga,a (Sigma), S-(4-Nitrobenzyl)-6-thioinosine µM v-agdFb n iE lgtcanfused (light-chain BiTEs and Fabs Avi-tagged 436clsepesn o tetdwith (treated low expressing cells P493-6 rCLSadRP- el iho without or with cells RWPE-1 and PrEC-LHS 4 D3 otiigepeso plasmids expression containing (DE3) C43 MSV000085930. E23 el eetasetdwith transfected were cells HEK293T https://doi.org/10.1073/pnas.2018861118 a.Med. Nat. β- D -1-thiogalactopyranoside, 3112 (2016). 1321–1329 22, Nature PNAS 679–683 455, | f11 of 9 ◦ ◦ ◦ C C C

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