O. Dufva, J. Klievink, K. Saeed, M. Kankainen, Mette Ilander, Tiiina Hannunen, S. Lagström, P. Ellonen, Dean Anthony Lee, S. Mustjoki
{"title":"Abstract A065: Genome-scale CRISPR screens identify essential genes for tumor sensitivity to NK cells","authors":"O. Dufva, J. Klievink, K. Saeed, M. Kankainen, Mette Ilander, Tiiina Hannunen, S. Lagström, P. Ellonen, Dean Anthony Lee, S. Mustjoki","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-A065","DOIUrl":null,"url":null,"abstract":"Harnessing natural killer (NK) cells to attack tumors could improve immune-based cancer treatment strategies. However, mechanisms regulating sensitivity or resistance of cancer cells to the effector function of NK cells are incompletely understood. Here, we performed genome-scale CRISPR-Cas9 loss-of-function screens in human cancer cells to discover genes that influence susceptibility to primary human NK cells. To screen for genes essential for the interaction between NK cells and cancer cells, we infected human cancer cells expressing Cas9 with a genome-scale lentiviral guide RNA library. The resulting pool of knockout cells was exposed to NK cells expanded from peripheral blood of healthy donors. Enriched and depleted knockout clones were detected by next-generation sequencing of the integrated sgRNA cassettes, enabling identification of genes conferring resistance or susceptibility to NK cell-mediated lysis. The screens were performed in cell lines from diverse cancer types, including chronic myeloid leukemia (CML), B cell acute lymphoblastic leukemia, diffuse large B-cell lymphoma (DLBCL), and multiple myeloma. We recovered several known mechanisms of NK cell/cancer cell interactions, demonstrating feasibility of the screening approach. Loss of genes encoding components of the MHC class I complex (B2M, HLA-A, HLA-C, HLA-E) sensitized multiple cancer cell lines to NK cell-mediated lysis. This is consistent with missing-self recognition as a fundamental mechanism of NK cell activation. Furthermore, knockout of IFN-JAK-STAT signaling mediators led to increased tumor cell lysis, suggesting that MHC class I induction in response to NK cell-derived IFN gamma enables NK cell evasion by tumor cells. We also identified genes essential for effective NK cell-mediated lysis. NCR3LG1, encoding the B7-H6 ligand for the NKp30 activating NK cell receptor, was essential for NK cell lysis of CML cells. In contrast, knockout of apoptotic mediators and TRAIL pathway components conferred resistance to NK cell cytotoxicity in DLBCL cells, indicating heterogeneity in NK cell/cancer cell interactions between cancer types. Our data support a view that distinct mechanisms regulate sensitivity to NK cell cytotoxicity in different cancers. Importantly, our results indicate that loss-of-function mutations in the antigen-presenting machinery and the IFN-JAK-STAT pathway sensitize tumors to NK cell effector function. As alterations in these genes are associated with resistance to T-cell immunotherapies such as PD-1 blockade, NK cell-based therapies could be employed to overcome resistance in these patients. In summary, we suggest that systematic identification of mechanisms governing tumor immune susceptibility has the potential to uncover novel immunotherapy targets. Citation Format: Olli Dufva, Jay Klievink, Khalid Saeed, Matti Kankainen, Mette Ilander, Tiiina Hannunen, Sonja Lagstrom, Pekka Ellonen, Dean A Lee, Satu Mustjoki. Genome-scale CRISPR screens identify essential genes for tumor sensitivity to NK cells [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A065.","PeriodicalId":22141,"journal":{"name":"Tackling the Tumor Microenvironment: Beyond T-cells","volume":null,"pages":null},"PeriodicalIF":0.0000,"publicationDate":"2019-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Tackling the Tumor Microenvironment: Beyond T-cells","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-A065","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
引用次数: 0
Abstract
Harnessing natural killer (NK) cells to attack tumors could improve immune-based cancer treatment strategies. However, mechanisms regulating sensitivity or resistance of cancer cells to the effector function of NK cells are incompletely understood. Here, we performed genome-scale CRISPR-Cas9 loss-of-function screens in human cancer cells to discover genes that influence susceptibility to primary human NK cells. To screen for genes essential for the interaction between NK cells and cancer cells, we infected human cancer cells expressing Cas9 with a genome-scale lentiviral guide RNA library. The resulting pool of knockout cells was exposed to NK cells expanded from peripheral blood of healthy donors. Enriched and depleted knockout clones were detected by next-generation sequencing of the integrated sgRNA cassettes, enabling identification of genes conferring resistance or susceptibility to NK cell-mediated lysis. The screens were performed in cell lines from diverse cancer types, including chronic myeloid leukemia (CML), B cell acute lymphoblastic leukemia, diffuse large B-cell lymphoma (DLBCL), and multiple myeloma. We recovered several known mechanisms of NK cell/cancer cell interactions, demonstrating feasibility of the screening approach. Loss of genes encoding components of the MHC class I complex (B2M, HLA-A, HLA-C, HLA-E) sensitized multiple cancer cell lines to NK cell-mediated lysis. This is consistent with missing-self recognition as a fundamental mechanism of NK cell activation. Furthermore, knockout of IFN-JAK-STAT signaling mediators led to increased tumor cell lysis, suggesting that MHC class I induction in response to NK cell-derived IFN gamma enables NK cell evasion by tumor cells. We also identified genes essential for effective NK cell-mediated lysis. NCR3LG1, encoding the B7-H6 ligand for the NKp30 activating NK cell receptor, was essential for NK cell lysis of CML cells. In contrast, knockout of apoptotic mediators and TRAIL pathway components conferred resistance to NK cell cytotoxicity in DLBCL cells, indicating heterogeneity in NK cell/cancer cell interactions between cancer types. Our data support a view that distinct mechanisms regulate sensitivity to NK cell cytotoxicity in different cancers. Importantly, our results indicate that loss-of-function mutations in the antigen-presenting machinery and the IFN-JAK-STAT pathway sensitize tumors to NK cell effector function. As alterations in these genes are associated with resistance to T-cell immunotherapies such as PD-1 blockade, NK cell-based therapies could be employed to overcome resistance in these patients. In summary, we suggest that systematic identification of mechanisms governing tumor immune susceptibility has the potential to uncover novel immunotherapy targets. Citation Format: Olli Dufva, Jay Klievink, Khalid Saeed, Matti Kankainen, Mette Ilander, Tiiina Hannunen, Sonja Lagstrom, Pekka Ellonen, Dean A Lee, Satu Mustjoki. Genome-scale CRISPR screens identify essential genes for tumor sensitivity to NK cells [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A065.
利用自然杀伤(NK)细胞攻击肿瘤可以改善基于免疫的癌症治疗策略。然而,调节癌细胞对NK细胞效应功能的敏感性或抗性的机制尚不完全清楚。在这里,我们在人类癌细胞中进行了基因组规模的CRISPR-Cas9功能丧失筛选,以发现影响对原代人类NK细胞易感性的基因。为了筛选NK细胞与癌细胞相互作用所必需的基因,我们用基因组级慢病毒引导RNA文库感染表达Cas9的人类癌细胞。由此产生的敲除细胞池暴露于从健康供者外周血扩增的NK细胞。通过对整合的sgRNA磁带进行下一代测序,可以检测到富集和缺失的敲除克隆,从而鉴定出对NK细胞介导的裂解具有抗性或易感性的基因。筛选来自不同癌症类型的细胞系,包括慢性髓性白血病(CML)、B细胞急性淋巴细胞白血病、弥漫性大B细胞淋巴瘤(DLBCL)和多发性骨髓瘤。我们恢复了NK细胞/癌细胞相互作用的几个已知机制,证明了筛选方法的可行性。编码MHC I类复合体(B2M, HLA-A, HLA-C, HLA-E)成分的基因缺失使多种癌细胞对NK细胞介导的裂解敏感。这与缺失自我识别作为NK细胞激活的基本机制是一致的。此外,IFN- jak - stat信号介质的敲除导致肿瘤细胞裂解增加,这表明MHC I类诱导对NK细胞来源的IFN γ的反应使NK细胞逃避肿瘤细胞。我们还确定了有效NK细胞介导的裂解所必需的基因。NCR3LG1编码NKp30激活NK细胞受体的B7-H6配体,是CML细胞NK细胞裂解所必需的。相比之下,敲除凋亡介质和TRAIL通路成分使DLBCL细胞对NK细胞的细胞毒性产生抗性,这表明NK细胞/癌细胞在不同癌症类型之间的相互作用存在异质性。我们的数据支持一种观点,即在不同的癌症中,不同的机制调节对NK细胞毒性的敏感性。重要的是,我们的研究结果表明,抗原呈递机制和IFN-JAK-STAT通路的功能缺失突变使肿瘤对NK细胞效应物功能敏感。由于这些基因的改变与对t细胞免疫疗法(如PD-1阻断)的耐药性有关,因此可以采用基于NK细胞的疗法来克服这些患者的耐药性。总之,我们认为系统地识别肿瘤免疫易感性的机制有可能发现新的免疫治疗靶点。引文格式:Olli Dufva, Jay Klievink, Khalid Saeed, Matti Kankainen, Mette Ilander, Tiiina Hannunen, Sonja Lagstrom, Pekka Ellonen, Dean A Lee, Satu Mustjoki。基因组级CRISPR筛选鉴定肿瘤对NK细胞敏感性的必要基因[摘要]。第四届CRI-CIMT-EATI-AACR国际癌症免疫治疗会议:将科学转化为生存;2018年9月30日至10月3日;纽约,纽约。费城(PA): AACR;癌症免疫学杂志2019;7(2增刊):摘要nr A065。