Author Manuscript Published OnlineFirst on August 30, 2021; DOI: 10.1158/2326-6066.CIR-21-0543 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Targeting the Atf7ip–Setdb1 complex augments antitumor immunity by boosting tumor immunogenicity Hai Hu1, Alireza Khodadadi-Jamayran2, Igor Dolgalev2,3, Hyunwoo Cho2,3,4 , Sana Badri3, Luis A Chiriboga3, Briana Zeck3, Miguel Lopez De Rodas Gregorio5, Catríona M. Dowling1,6, Kristen Labbe1, Jiehui Deng1, Ting Chen1, Hua Zhang1, Paul Zappile7, Ze Chen8, Beatrix Ueberheide9, Angeliki Karatza1, Han Han1, Michela Ranieri1, Sittinon Tang1, George Jour3, Iman Osman10, Antje Sucker11, Dirk Schadendorf11, Aristotelis Tsirigos2,3, Kurt A Schalper5, Vamsidhar Velcheti1, Hsin-yi Huang1, Yujuan Jin12, Hongbin Ji12,13, John T. Poirier1, Fei Li14,*, Kwok-Kin Wong1,15, * 1Division of Hematology & Medical Oncology, Laura and Isaac Perlmutter Cancer Center, NYU School of Medicine, New York, NY, USA. 2Applied Bioinformatics Laboratories and Genome Technology Center, Division of Advanced Research Technologies, NYU School of Medicine, New York, NY, USA. 3Department of Pathology, NYU School of Medicine, New York, NY, USA. 4Department of Radiation Oncology, NYU School of Medicine, New York, NY, USA. 5Department of Pathology, Yale University School of Medicine, New Haven, CT, USA 6School of Medicine, University of Limerick, Limerick, Ireland. 7Genome Technology Center, Division of Advanced Research Technologies, NYU School of Medicine, New York, NY, USA. 8Department of Medicine, NYU School of Medicine, New York, NY, USA. 9Proteomics Laboratory, NYU School of Medicine, New York, NY, USA. Downloaded from cancerimmunolres.aacrjournals.org on September 26, 2021. © 2021 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 30, 2021; DOI: 10.1158/2326-6066.CIR-21-0543 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 10Department of Dermatology, NYU School of Medicine, New York, NY, USA. 11Department of Dermatology, University Hospital, Essen, Germany. 12State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai, China. 13School of Life Science, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou, China. 14Department of Pathology, School of Basic Medical Sciences, Fudan University, Shanghai, China. 15Lead Contact *Corresponding Authors: Fei Li, Ph.D. Mailing address: Room 208, East Building 1, 131 Dong’an Road, Xuhui District, Fudan University, Shanghai 200032, China Phone: +86 15202100163 Email: [email protected] Kwok-Kin Wong, M.D., Ph.D. Mailing address: 550 1st Avenue, Smilow 1011, NYU School of Medicine, New York, NY10016, USA Phone: +1 2122639203 Email: [email protected]. Downloaded from cancerimmunolres.aacrjournals.org on September 26, 2021. © 2021 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 30, 2021; DOI: 10.1158/2326-6066.CIR-21-0543 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Running title: Atf7ip/Setdb1 ablation stimulates antitumor immunity Keywords: immune escape; epigenetic CRISPR screen; Atf7ip/Setdb1; tumor antigen; antitumor immunity Authors’ Disclosures K.K.W. is a founder and equity holder of G1 Therapeutics, and he has consulting/sponsored research agreements with the following: (consulting & sponsored research) AstraZeneca, Janssen, Pfizer, Novartis, Merck, Ono, and Array; (sponsored research only) MedImmune, Takeda, TargImmune, and BMS. K.K.W., H.H. and F.L. have ownership interest in a patent application. No potential conflicts of interest were disclosed by the other authors. Funding Information K.K.W is supported by NIH Research Project Grant Program (RO1CA248896, RO1CA219670 and RO1CA215471). Synopsis: Using a CRISPR-based suppressor screen, the authors identify Atf7ip and Setdb1 as epigenetic regulators of tumor antigen expression and presentation. Genetic deficiency in these regulators enhances antitumor immunity, suggesting a new immunotherapeutic strategy for overcoming tumor immune evasion. Downloaded from cancerimmunolres.aacrjournals.org on September 26, 2021. © 2021 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 30, 2021; DOI: 10.1158/2326-6066.CIR-21-0543 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Abstract Substantial progress has been made in understanding how tumors escape immune surveillance. However, few measures to counteract tumor immune evasion have been developed. Suppression of tumor antigen expression is a common adaptive mechanism that cancers use to evade detection and destruction by the immune system. Epigenetic modifications play a critical role in various aspects of immune invasion, including the regulation of tumor antigen expression. To identify epigenetic regulators of tumor antigen expression, we established a transplantable syngeneic tumor model of immune escape with silenced antigen expression and used this system as a platform for a CRISPR–Cas9 suppressor screen for genes encoding epigenetic modifiers. We found that disruption of the genes encoding either of the chromatin modifiers activating transcription factor 7 interacting protein (Atf7ip) or its interacting partner SET domain bifurcated histone lysine methyltransferase 1 (Setdb1) in tumor cells restored tumor antigen expression. This resulted in augmented tumor immunogenicity concomitant with elevated endogenous retroviral (ERV) antigens and mRNA intron retention. ERV disinhibition was associated with a robust type I interferon response and increased T- cell infiltration, leading to rejection of cells lacking intact Atf7ip or Setdb1. ATF7IP or SETDB1 expression inversely correlated with antigen processing and presentation pathways, interferon signaling, and T-cell infiltration and cytotoxicity in human cancers. Our results provide a rationale for targeting Atf7ip or Setdb1 in cancer immunotherapy. Downloaded from cancerimmunolres.aacrjournals.org on September 26, 2021. © 2021 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 30, 2021; DOI: 10.1158/2326-6066.CIR-21-0543 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Introduction The immune system plays a key role in preventing carcinogenesis in immunocompetent hosts, a process known as immune surveillance (1). However, cancer cells can escape immune surveillance during the cancer immunoediting process (2). Although considerable knowledge has accumulated on how tumors avoid immune surveillance, the development of effective therapies to overcome tumor immune evasion remains a challenge. Tumor antigens are processed and presented to CD4+ helper T cells and circulating CD8+ cytotoxic T cells, which leads to activation of adaptive immunity by CD4+ helper T cells or the direct killing of tumor cells by infiltrated CD8+ cytotoxic T cells (3). One common mechanism of tumor cell immune evasion is loss of antigen expression (4). In an autochthonous murine model of sarcomagenesis, chronic T-cell attack on tumor cells can silence the expression of tumor-specific antigens (5). In human lung cancer evolution, promoter hypermethylation of genes encoding neoantigenic mutations is an epigenetic mechanism of immune escape (6). Thus, searching for candidate therapeutic targets that can promote tumor antigen expression and boost antitumor immunity when inhibited may lead to novel strategies for cancer immunotherapy. Targeting epigenetic modifiers shows potential in enhancing antitumor immune response (7). For example, inhibition of polycomb repressive complex 2 (PRC2) substituents and histone deacetylases can restore expression of MHC-I antigen processing and presentation machinery and T-cell mediated antitumor immunity (8,9). Downloaded from cancerimmunolres.aacrjournals.org on September 26, 2021. © 2021 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 30, 2021; DOI: 10.1158/2326-6066.CIR-21-0543 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Other studies show that DNA methyltransferase and histone deacetylase inhibitors, or histone demethylase inhibition increases tumor immunogenicity through upregulation of tumor antigen expression, including antigens derived from endogenous retroviruses (ERV) (10-13). These findings suggest that targeting epigenetic regulators offers an attractive avenue for restoring immune surveillance. Here, we performed an CRISPR–Cas9 suppressor screen in an antigen silenced immune escaped lung adenocarcinoma tumor model. We identified Atf7ip and its interacting partner Setdb1 as potential therapeutic targets to augment tumor immunogenicity. Functional and mechanistic studies demonstrated that Atf7ip–Setdb1 deficiency stimulated antitumor immunity in multiple tumor models. Thus, our findings provide a rationale for nominating ATF7IP and SETDB1 as potential novel immunotherapeutic targets in cancer patients. Downloaded from cancerimmunolres.aacrjournals.org on September 26, 2021. © 2021 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 30, 2021; DOI: 10.1158/2326-6066.CIR-21-0543 Author manuscripts