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

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. Overall design: 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

目前学界对癌细胞逃避免疫系统杀伤的遗传调控网络仍知之甚少。为筛选出一组表型稳定的核心基因与通路，以介导癌细胞固有免疫逃逸、对抗细胞毒性T淋巴细胞（cytotoxic T lymphocyte, CTL）介导的杀伤，我们针对一组遗传背景各异的癌细胞模型开展了全基因组CRISPR筛选，上述细胞均在添加CTL的培养基中培养。本研究鉴定出182个核心基因，单个基因的扰动即可使癌细胞对CTL杀伤产生敏感性或抗性。通过遗传共相似性分析对本数据集进行系统探索，我们揭示了基因与通路协同调控癌细胞固有CTL逃逸的层级化机制：控制干扰素应答以及肿瘤坏死因子α（tumor necrosis factor alpha, TNFα）诱导的细胞毒性的独立功能模块，成为主要的亚表型类群。本研究证实，此前已报道的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逃逸中发挥核心作用；同时证明脂滴相关基因Fitm2是癌细胞暴露于干扰素γ（interferon gamma, IFNγ）后维持细胞适合度所必需的。此外，我们发现自噬通路是癌细胞固有CTL逃逸的保守调控介质，可帮助癌细胞抵抗由IFNγ与TNFα介导的细胞毒性。通过绘制细胞因子与CTL相关的遗传互作图谱，并结合体内CRISPR筛选实验，我们阐明了自噬调控癌细胞固有CTL逃逸的多效性机制，并强调了本研究观测到的效应在肿瘤微环境中的重要性。综上，本研究拓展了学界对癌细胞固有免疫逃逸相关遗传调控网络的认知，并强调了利用系统功能基因组学方法深入解析该生物学过程的重要性。实验设计概述：在Renca（野生型 vs. Atg12基因敲除型 vs. Fitm2基因敲除型）、Renca-HA（野生型 vs. Atg12基因敲除型 vs. Fitm2基因敲除型）、EMT6-HA、CT26-HA、4T1-HA、B16-Ova、MC38-Ova细胞模型中开展CRISPR筛选，上述细胞均分别在添加或不添加IFNγ、TNFα或CTL的条件下培养。