Interferon-γ induces tumor resistance to anti-PD-1 immunotherapy by promoting YAP phase separation

•IFN-γ promotes tumor cell YAP condensation after anti-PD-1 therapy•Hydrophobic interactions mediated by the coiled-coil domain initiate condensation•YAP condensates form a transcription hub to maximize target gene expression•YAP phase separation mediates immunotherapy resistance Interferon-γ (IFN-γ)-mediated adaptive is one major barrier improving in solid tumors. However, mechanisms are not completely understood. Here, we report that IFN-γ nuclear translocation and of therapy cells. Hydrophobic mediate droplet initiation, weak intrinsically disordered region C terminus promote formation. partitions with factor TEAD4, histone acetyltransferase EP300, Mediator1 forms transcriptional hubs for maximizing transcriptions, independent canonical STAT1-IRF1 program. Disruption reduced growth, enhanced immune response, sensitized cells therapy. activity negatively correlated patient outcome. Our study indicates pro-tumor effect through its suggests can be used as predictive biomarker combination Anti-PD-1/PD-L1 has shown promising clinical outcomes treatment many cancer types (Brahmer et al., 2015Brahmer J. Reckamp K.L. Baas P. Crinò L. Eberhardt W.E. Poddubskaya E. Antonia S. Pluzanski A. Vokes E.E. Holgado al.Nivolumab versus docetaxel advanced squamous-cell non-small-cell lung cancer.N. Engl. Med. 2015; 373: 123-135Crossref PubMed Scopus (6394) Google Scholar; Ferris 2016Ferris R.L. Blumenschein Jr., G. Fayette Guigay Colevas A.D. Licitra Harrington K. Kasper Even C. recurrent carcinoma head neck.N. 2016; 375: 1856-1867Crossref (3051) Motzer 2015Motzer R.J. Escudier B. McDermott D.F. George Hammers H.J. Srinivas Tykodi S.S. Sosman J.A. Procopio Plimack E.R. al.CheckMate 025 InvestigatorsNivolumab everolimus renal-cell carcinoma.N. 1803-1813Crossref (4221) Robert 2015Robert Long G.V. Brady Dutriaux Maio M. Mortier Hassel J.C. Rutkowski McNeil Kalinka-Warzocha previously untreated melanoma without BRAF mutation.N. 372: 320-330Crossref (4130) Topalian 2012Topalian S.L. Hodi F.S. Brahmer J.R. Gettinger S.N. Smith D.C. Powderly J.D. Carvajal R.D. Atkins M.B. al.Safety, activity, correlates antibody 2012; 366: 2443-2454Crossref (9454) Zou 2016Zou W. Wolchok Chen PD-L1 (B7-H1) PD-1 pathway blockade therapy: Mechanisms, response biomarkers, combinations.Sci. Transl. 8: 328rv4Crossref (1520) Scholar), whereas resistance, including primary, adapted, acquired, common tumors (Sharma 2017Sharma Hu-Lieskovan Wargo Ribas Primary, adaptive, acquired immunotherapy.Cell. 2017; 168: 707-723Abstract Full Text PDF (2626) 2015Topalian Drake C.G. Pardoll D.M. Immune checkpoint blockade: denominator approach therapy.Cancer Cell. 27: 450-461Abstract (2700) Scholar). Blockade PD-1/PD-L1 activates T accompanied interferon-γ (IFN-γ) secretion, which been proposed both pro- anti-tumoral activities. dependent (Spranger 2013Spranger Spaapen R.M. Zha Y. Williams Meng Ha T.T. Gajewski T.F. Up-regulation PD-L1, IDO, T(regs) microenvironment driven CD8(+) cells.Sci. 2013; 5: 200ra116Crossref (1263) Taube 2012Taube J.M. Anders R.A. Young G.D. Xu H. Sharma R. McMiller T.L. Klein A.P. Colocalization inflammatory B7-h1 expression human melanocytic lesions supports an mechanism escape.Sci. 4: 127ra37Crossref (1684) becoming key issue broaden (Alspach 2019Alspach Lussier Schreiber Interferon γ important roles promoting inhibiting spontaneous therapeutic immunity.Cold Spring Harb. Perspect. Biol. 2019; 11: a028480Crossref (197) Benci 2016Benci J.L. Qiu Wu T.J. Dada Twyman-Saint Victor Cucolo Lee D.S.M. Pauken K.E. Huang A.C. al.Tumor interferon signaling regulates multigenic program blockade.Cell. 167: 1540-1554.e1512Abstract (621) Thus, fully elucidating how helps resist will benefit use (Minn Wherry, 2016Minn A.J. Wherry E.J. Combination therapies convergence on signaling.Cell. 165: 272-275Abstract (183) The Hippo downstream effector Yes-associated protein (YAP) sense versatile upstream signals control fate, progression (Halder 2012Halder Dupont Piccolo Transduction mechanical cytoskeletal cues TAZ.Nat. Rev. Mol. Cell 13: 591-600Crossref (672) 2016Meng Z. Moroishi T. Guan K.-L. Mechanisms regulation.Genes Dev. 30: 1-17Crossref (965) Misra Irvine, 2018Misra Irvine K.D. network biological functions.Annu. Genet. 2018; 52: 65-87Crossref (221) Totaro 2018Totaro Panciera YAP/TAZ responses.Nat. 20: 888-899Crossref (464) Zanconato 2016Zanconato F. Cordenonsi at roots cancer.Cancer 29: 783-803Abstract (1082) Zheng Pan, 2019Zheng Pan D. development disease.Dev. 50: 264-282Abstract (354) complex partner, TEA (TEAD) family members, associates promoters thousands functional enhancers genes (Stein 2015Stein Bardet A.F. Roma Bergling Clay I. Ruchti Agarinis Schmelzle Bouwmeester Schübeler Bauer YAP1 exerts via TEAD-mediated activation enhancers.PLoS e1005465Crossref (231) 2015Zanconato Forcato Battilana Azzolin Quaranta Bodega Rosato Bicciato Genome-wide association between YAP/TAZ/TEAD AP-1 drives oncogenic growth.Nat. 17: 1218-1227Crossref (647) In cells, required addiction interaction BRD4 (Cordenonsi Piccolo, 2018Cordenonsi Transcriptional BRD4.Nat. 24: 1599-1610Crossref (72) bind chromatin remodeling factors (Chang 2018Chang Di Biagio Lucon Xiccato Aragona Giulitti Gandin al.The SWI/SNF mechanoregulated inhibitor TAZ.Nature. 563: 265-269Crossref (159) Skibinski 2014Skibinski Breindel Prat Galván Rolfs Gupta P.B. LaBaer Kuperwasser transducer TAZ interacts regulate breast epithelial lineage commitment.Cell Rep. 2014; 6: 1059-1072Abstract (122) modifiers (Oh 2014Oh Slattery Ma White K.P. Mann R.S. Yorkie recruiting methyltransferase complex.Cell 449-459Abstract (55) Stein Mediators (Galli 2015Galli G.G. Carrara Yuan W.C. Valdes-Quezada Gurung Pepe-Mooney Zhang Geeven Gray N.S. de Laat al.YAP growth controlling pause release from dynamic enhancers.Mol. 60: 328-337Abstract (174) Scholar) regulation. Recently, evidence accumulating playing initiation (Boija 2018Boija I.A. Sabari B.R. Dall’Agnese Coffey E.L. Zamudio A.V. Li C.H. Shrinivas Manteiga Hannett N.M. al.Transcription activate phase-separation capacity their domains.Cell. 175: 1842-1855 e1816Abstract (755) Hnisz 2017Hnisz Chakraborty A.K. Sharp P.A. A model control.Cell. 169: 13-23Abstract (901) 2018Sabari Boija Abraham B.J. al.Coactivator super-enhancers links control.Science. 361: eaar3958Crossref Whether integrates DNA binding, modification, maintains these processes spatiotemporally support remains unknown. also suppression directly or indirectly regulating functions (White 2019White S.M. Murakami Yi entanglement Hippo-Yap/Taz immunity.Oncogene. 38: 2899-2909Crossref (38) number secreted cytokines, chemokines, extracellular matrix (ECM) molecules recruit multiple subtypes, tumor-associated macrophages, myeloid-derived suppressor regulatory (Murakami 2017Murakami Shahbazian Surana Graham G.T. Weiner L.M. reprogramming pancreatic ductal adenocarcinoma.Oncogene. 36: 1232-1244Crossref (135) Ni 2018Ni X. Tao Barbi Q. Park B.V. N. Lebid Ramaswamy Wei essential treg-mediated antitumor immunity.Cancer Discov. 1026-1043Crossref (112) Wang 2016Wang Lu Dey Deng C.C. Jiang Fang Zhao Konaparthi Hua al.Targeting YAP-dependent MDSC infiltration impairs progression.Cancer 80-95Crossref (327) cancer-associated fibroblasts (Calvo 2013Calvo Ege Grande-Garcia Hooper Jenkins R.P. Chaudhry S.I. Williamson Moeendarbary Charras Sahai Mechanotransduction remodelling generation maintenance fibroblasts.Nat. 15: 637-646Crossref (858) establish immunosuppressive microenvironment. Although combinational verteporfin showed significant inhibition compared either monotherapy mice, function regulation clear (Ni utilize genetically engineered subcutaneous allograft mice adenocarcinoma models investigate immunotherapy. We describe molecular mediating IFN-γ-dependent resistance. stimulates leading survival IFN-stimulated (ISGs). represents new circuit To dissect vivo, KrasLox-STOP-Lox(LSL)-G12D;Trp53flox/flox; ZsGreenLox-STOP-Lox (KPZ) (Madisen 2010Madisen Zwingman T.A. Sunkin Oh S.W. Zariwala H.A. Gu Ng L.L. Palmiter Hawrylycz M.J. Jones A.R. al.A robust high-throughput Cre reporting characterization system whole mouse brain.Nat. Neurosci. 2010; 133-140Crossref (3979) DuPage 2011DuPage Cheung Mazumdar Winslow M.M. Bronson Schmidt Crowley Jacks Endogenous responses antigens expressed adenocarcinomas delay malignant 2011; 19: 72-85Abstract (164) (Figure S1A). evaluate effect, burden groups found no difference size S1B). monitor isolated KrasLox-STOP-Lox(LSL)-G12D;Trp53flox/flox (KP) (a tumor-model-derived line [TDCL]) implanted them subcutaneously into C57B/6 1A). Tumors TDCL exhibited day 12 day18 developed 21 (Figures 1B 1C), consistent subset patients who respond initially eventually (Borghaei 2015Borghaei Paz-Ares Horn Spigel D.R. Steins Ready N.E. Chow L.Q. Felip nonsquamous 1627-1639Crossref (6937) Herbst 2016Herbst Kim D.W. Pérez-Gracia Han J.Y. Molina J.H. Arvis C.D. Ahn al.Pembrolizumab treated, PD-L1-positive, (KEYNOTE-010): randomised controlled trial.Lancet. 387: 1540-1550Abstract (4679) analyzed compositions cytotoxic lymphocytes (CTLs) responding resistant stages, was represented percentages granzyme B (GZMB)-positive flow cytometry. Consistently, CTL upregulated stage but comparable group 1D 1E), indicating were activated become unresponsive eventually. histopathological analysis, hematoxylin eosin (H&E) staining, did show necrosis S1E). examine during therapy, visualized immunofluorescence (IF) using well-validated antibody(Mao 2017Mao Sun Role Yap KrasG12D-induced cancer.Oncotarget. 110877-110889Crossref (13) stage, cytoplasm bright tight junction 1F S1J) (Zhou 2018Zhou Flodby Luo Castillo Liu Yu F.-X. McConnell Varghese Chimge N.-O. al.Claudin-18-mediated stem progenitor homeostasis tumorigenesis.J. Clin. Invest. 128: 970-984Crossref (95) CD8 IF sections translocated nucleus, punctate distribution immune-cell-infiltrated area. contrast, non-infiltrated areas group, 1G). confirm this observation, KP samples another Indeed, structure stages S1C S1F–S1H). whether puncta other anti-PD-1/PD-L1 stained renal (RCC) transplanted BALB/c model, endpoint (Zhang 2019Zhang Duan Xia Dong TFEB evasion mTOR induction PD-L1.Clin. Cancer Res. 25: 6827-6838Crossref Anti-PD-L1 led well 1H). characterize properties puncta, identified measured 3D reconstructions. They colocalized heterochromatin, dense DAPI staining 1I S1I) calculating Pearson’s correlation coefficient (r = −0.17), suggesting localize euchromatin activation. radial ranged 0.2 0.6 μm S1D). summary, exhibiting micron distribution, colocalizes heterochromatin CD8+ T-cell-infiltrated regions different tissues. This occurs only within tumors, might require derived system. Interleukin-12 (IL-12) (Garris 2018Garris C.S. Arlauckas S.P. Kohler R.H. Trefny M.P. Garren Piot Engblom Pfirschke Siwicki Gungabeesoon al.Successful requires cell-dendritic crosstalk involving cytokines IL-12.Immunity. 49: 1148-1161.e7Abstract (474) (Ayers 2017Ayers Lunceford Nebozhyn Murphy Loboda Kaufman Albright Cheng Kang Shankaran V. al.IFN-γ-related mRNA profile predicts blockade.J. 127: 2930-2940Crossref (1867) frequently (TME) Through stimulation 4-h incubation IFN-γ, IL-12 2A S2A). Moreover, accumulation formation nucleus dose time Lewis (LLC) 2B 2C). It observed A549 understand YAP, detected levels western blot. increased 2D). YAP-positive occur independently IF, TDCLs labeled endogenously monomeric green fluorescent (mEGFP) CRISPR-Cas9 genome editing S2B S2C). Upon stimulation, mEGFP-YAP revealed seemingly observations vivo cultured 2E) generated IFNGR1 knockout lines S2D S2E). disappeared IFNGR1−/− 2A). Next, examined INFGR1−/− injecting mice. absence signaling, could 2F). this, when mutant unable 2G). Recent studies have plays (Hnisz prompted us detect condensates. bears two (IDRs) located N (1–164) (266–504) termini S3A). fused mEGFP IDRs purified bacterial S3B S3C). mEGFP, terminal-IDR (N-IDR), (C-IDR) added 25% polyethylene glycol-6000 (PEG-6000) solution, increases macromolecular crowding separation. Both IDR recombinant proteins rendered solution opaque, contrast remained 3A). solutions mEGFP-positive micron-sized puncta. upon 1/2 dilution 3B), reversible. addition, live imaging tracing fluorescence recovery photobleaching (FRAP) fuse recovered seconds S3F S2G; Video S1). wetting experiments droplets wet cover-slide glass touching surface (Videos S2 S3). These results indicate C-terminal phase-separated PEG solution. comparison, C-IDR stronger droplet-forming ability than N-IDR S3D, S3E, S3H). eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiI1MzIzYjE1ZWI3MzI2ZDFlMThlOGRlYzY4MGM2NDA0NyIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjg1MTk1NDU5fQ.kjy7825Tts83hjHbkFbkea7rGi0jDPvMvzIGdO-GX2esNgqFJy47R3nY6Pqh3I9_eQfQb9KS4mJ4slM_Dp4gW61RT2Nw4_Bmrwiess-gQb_vgPyQAq-rAaxXOSNPN0Q-X_JoPCBt1gIltkcWdOTeEDQ3mYD779IZO6ft-Rrs4Vg57LmlM7uF5Gl8gqOuTj0lf68UK7viin9fwlzhm5PjQTHhu1fnx85GvA_uZS6sr_7WZ5yd3lBv_mwvwME4-hScyPkVXDZslC_xgd19f60cYbWhZBJCtr5sgGuRAmxonVLEyfBJd0IiikxHOsDHOv9KrZLJBRbrAT3Ev32wq9f_qw Download .mp4 (0.24 MB) Help files S1. YAP-C-IDR fusion vitro, related Figure 3 eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiIzNjgzMzI5YzE5Mjc4MGI0MmEwMWM2ZDcxYzk0ZDAyZiIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjg1MTk1NDU5fQ.cd8YTQj88enr8NEENo0EHxLzLnkbAtpA4FyeRYUbkhOneLbSrPBrsS8Dt00LD2szD8AuI0s2pe9ig064e51v6hbgzlYaJYGy7h74gHnLS9-yzKa-zl6ay1liZpu9eqqxTyysmAQXb0DLmDv7fUpbdgsNYQX9dYR0O1u6GGMhl_kxFTCEZOv5aJJzZ50v6VHsvw4TXJnS3T0ZdN8W1jnigSbvH9VPIXs0pBepkQORnh60xoKVb755TR8PvC9qdyFok4W1NTAgQdjnNHZP_2k6ufRVH8TG2SRKdXGLKhMg6655FfSooAdb1Mhw2m45MBiN4TLd_fidI10gqYR8aWtX-Q (0.54 S2. Wetting assay YAP-N-IDR YAP-C-IDR, eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiJmM2UwNDRiZGZiNGQxNDk0NWU3NzE3Zjc2OGE2MGE3NSIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjg1MTk1NDU5fQ.rw5tbPRmQZKjVzadhrW9A9F7-Ftt9DaLohXx4J8wWz-H-892kV9xCtHyMRvrjWhWjIIq_N46-Uj2Dc-h47PLlN407lx1IIVoSJbca-mVg9AIuqVd9NuNhqo2Y9gBWB2Qd9N65Xudz3jf2XtgGhDMsG3tOGuXCcrvd69sLLluscf4Z9JKkQjl26-N7fJhk9-LnVz2YQFH5E5hFoDAcfAFOJpAB8T074Kn6t3yzYE_WAKV5eYGr92tlWiXvK8anbA5O5kCxIBbdiaeQLG8GUlAEd7kYkCvwNq02NMpuhUzUw-ny2u1fSV_M67XzuzHW5SYqKtrSum7zFhLLI8RTLNxww (0.46 S3. determine separately optoDroplets (Shin 2017Shin Berry Pannucci Haataja Toettcher J.E. Brangwynne C.P. Spatiotemporal intracellular transitions light-activated optoDroplets.Cell. 159-171.e14Abstract (438) C-IDR, control, blue-light 3C; S4). liquid-like detecting FRAP behavior S3I S3J). result indicated eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiI2M2QxZDg1NDExYTI1ZWY3YTM0YWRkM2I5ODE2OTQ1NSIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjg1MTk1NDU5fQ.sQCGRR87hWWRFhQ77KDgBiSBoTn71Z7FYi-fERL-BOxl_kHXu0EZn--6XJKpPdLxgGmnB0cfGMQQ276erSyZpcazPnjmGACSdO6stLZac4GTS-J759OkNRsNAfQH8sWKqgoYMB3IAB6FDtMWjU8Yb6RZi1MCptXog3ehIxTeYxoapX4oGIRnHe_obIIilrEfllkxlCXZHJTwp4WzKklYCsXgK3KM2RbMO5qSx4L4T-EAvuBTR8CPyX-NHoM27tMerMFJkdQ514reLhBdM7JEwUjukv3rlYdijAcX0BQwKs51uL_YOHY-IGycMaHrD46lUXfDJz2WsU3IwRqlOSY8mw (6.57 S4. Opto-IDR Since increasing localization, reasoned expressing wild-type (WT) (S127A) cause WT YAPS127A AD293 Intriguingly, distribution. As expected, transfected more larger 3D), localization. mEGFP-YAPS127A 10 s photobleaching, previous description (Nott 2015Nott Petsalaki Farber Jervis Fussner Plochowietz Craggs T.D. Bazett-Jones D.P. Pawson Forman-Kay Bal