Functional genomic landscape of cancer-intrinsic immune evasion to cytotoxic T lymphocyte killing [CRISPRseq]

The genetic circuits that allow cancer cells to evade destruction by the host immune system remain poorly understood. To identify a phenotypically robust core set of genes and pathways that facilitate cancer cell-intrinsic evasion to cytotoxic T lymphocyte (CTL)-mediated killing, we performed genome-wide CRISPR screens across a panel of genetically diverse cancer models cultured in the presence of CTLs. We uncovered a core set of 182 genes whose individual perturbation leads to either cancer cell sensitivity or resistance to CTL toxicity. Systematic exploration of our dataset using genetic co-similarity reveals the hierarchical and coordinated nature by which genes and pathways act to orchestrate intrinsic CTL evasion, with discrete functional modules controlling the interferon response and tumor necrosis factor alpha (TNFa)-induced cytotoxicity emerging as dominant sub-phenotypes. Our data establish a central role for previously identified negative regulators of the Type II interferon response (e.g. Ptpn2, Socs1, Adar1) in mediating intrinsic CTL evasion and demonstrate a requirement for the lipid droplet related gene Fitm2 for maintaining cell fitness upon exposure to interferon gamma (IFNg). Additionally, we identify the autophagy pathway as a conserved mediator of cancer intrinsic CTL evasion, required to resist cytokine-mediated cytotoxicity caused by IFNg and TNFa. By mapping cytokine- and CTL-based genetic interactions, as well as in vivo CRISPR screens, we illuminate the pleiotropic nature by which autophagy acts to orchestrate intrinsic CTL evasion and highlight the importance of our observed effects within the tumor microenvironment. Collectively, our data expands our appreciation of the genetic circuits that contribute to cancer intrinsic immune evasion, highlighting the importance of leveraging systematic functional genomics approaches for furthering our understanding of this biology. CRISPR screens in Renca (wild-type vs. Atg12-KO vs. Fitm2-KO), Renca-HA (wild-type vs. Atg12-KO vs. Fitm2-KO), EMT6-HA, CT26-HA, 4T1-HA, B16-Ova, MC38-Ova cultured in the presence or absence of IFNg, TNFa or cytotoxic T-lymphocytes

目前人们对癌细胞逃避免疫系统清除的遗传调控通路仍缺乏充分认知。为筛选出可调控癌细胞固有免疫逃逸、对抗细胞毒性T淋巴细胞（cytotoxic T lymphocyte, CTL）介导杀伤的表型稳健核心基因集与通路，我们针对一组遗传背景各异的癌细胞模型开展全基因组CRISPR筛选，这些模型均在与CTL共培养的条件下培育。

我们最终鉴定得到182个核心基因，单个基因的扰动即可使癌细胞对CTL杀伤呈现敏感性或耐药性。通过遗传共相似性分析本数据集，我们揭示了基因与通路协同调控癌细胞固有CTL逃逸的层级式调控模式；其中调控干扰素应答与肿瘤坏死因子α（tumor necrosis factor alpha, TNF-α）诱导的细胞毒性的独立功能模块，成为主导性亚表型类群。

本研究证实了此前报道的II型干扰素应答负调控因子（如Ptpn2、Socs1、Adar1）在介导癌细胞固有CTL逃逸中的核心作用，并证明脂滴相关基因Fitm2是癌细胞暴露于γ干扰素（interferon gamma, IFN-γ）后维持细胞存活所必需的基因。此外，我们发现细胞自噬（autophagy）通路是癌细胞固有CTL逃逸的保守调控因子，可帮助癌细胞抵抗由IFN-γ与TNF-α介导的细胞毒性杀伤。

通过构建细胞因子与CTL介导的遗传互作网络，结合体内CRISPR筛选实验，我们阐明了细胞自噬调控癌细胞固有CTL逃逸的多效性机制，并凸显了本研究发现的效应在肿瘤微环境中的重要意义。综上，本研究拓展了人们对癌细胞固有免疫逃逸相关遗传调控通路的认知，同时凸显了借助系统性功能基因组学方法深入解析该生物学过程的重要价值。

本研究涉及的CRISPR筛选模型包括：Renca（野生型 vs Atg12敲除型 vs Fitm2敲除型）、Renca-HA（野生型 vs Atg12敲除型 vs Fitm2敲除型）、EMT6-HA、CT26-HA、4T1-HA、B16-Ova、MC38-Ova，上述模型均在添加或不添加IFN-γ、TNF-α或CTL的条件下培养。