三陰性乳腺癌的雌激素受體,、孕激素受體,、人類表皮生長因子受體HER2均為陰性，故對(duì)內(nèi)分泌治療和HER2靶向治療無效,，目前主要依靠手術(shù)和放化療,。近年來，免疫細(xì)胞程序性死亡受體PD-1及其配體PD-L1等免疫檢查點(diǎn)（腫瘤細(xì)胞逃避人體免疫細(xì)胞追殺的關(guān)鍵）抑制劑雖然對(duì)其他癌癥已經(jīng)獲得極大成功,，但是僅對(duì)不到五分之一的三陰性乳腺癌患者有效,。

　　2021年9月27日，全球自然科學(xué)三大旗艦期刊之一,、美國《細(xì)胞》正刊發(fā)表哈佛大學(xué)醫(yī)學(xué)院達(dá)納法伯癌癥研究所,、哈佛大學(xué)陳曾熙公共衛(wèi)生學(xué)院的研究報(bào)告，發(fā)現(xiàn)了三陰性乳腺癌免疫治療瓶頸的關(guān)鍵靶點(diǎn),。

　　該研究首先利用基因編輯技術(shù)建立了超過4500個(gè)基因的基因剔除篩選庫,，隨后將其轉(zhuǎn)染小鼠三陰性乳癌4T1細(xì)胞，注入免疫功能缺陷小鼠和免疫功能正常小鼠的乳腺脂肪墊內(nèi)形成腫瘤,，16天后收集小鼠三陰性乳腺癌組織,，對(duì)超過4500個(gè)基因進(jìn)行大規(guī)模并行測(cè)序篩選。

　　結(jié)果發(fā)現(xiàn),，腫瘤細(xì)胞的泛素連接酶COP1編碼基因被剔除以后,，可減少巨噬細(xì)胞相關(guān)趨化因子分泌和腫瘤巨噬細(xì)胞浸潤、防止抗腫瘤免疫細(xì)胞被腫瘤巨噬細(xì)胞吞噬,、增強(qiáng)抗腫瘤免疫和免疫檢查點(diǎn)PD-1或PD-L1抑制劑療效,、顯著縮小腫瘤體積并延長小鼠生存時(shí)間。

　　轉(zhuǎn)錄組學(xué),、表觀基因組學(xué)和蛋白質(zhì)組學(xué)分析表明,，COP1主要通過蛋白酶體降解趨化因子轉(zhuǎn)錄蛋白C/EBPδ而發(fā)揮作用，COP1底物TRIB2可充當(dāng)連接COP1和C/EBPδ的支架,，導(dǎo)致C/EBPδ多聚泛素化而被蛋白酶體降解,。此外，利用泛素連接酶抑制劑MLN4924或蛋白酶體抑制劑MG132可防止C/EBPδ泛素化或降解,，從而抑制巨噬細(xì)胞趨化基因表達(dá),，防止腫瘤細(xì)胞逃避人體免疫細(xì)胞追殺。

　　因此,，該研究結(jié)果表明COP1可調(diào)節(jié)腫瘤免疫微環(huán)境趨化因子分泌和巨噬細(xì)胞浸潤,，為突破三陰性乳腺癌免疫治療瓶頸奠定了基礎(chǔ),。

Cell. 2021 Sep 27. Online ahead of print.

In vivo CRISPR screens identify the E3 ligase Cop1 as a modulator of macrophage infiltration and cancer immunotherapy target.

Xiaoqing Wang, Collin Tokheim, Shengqing Stan Gu, Binbin Wang, Qin Tang, Yihao Li, Nicole Traugh, Zexian Zeng, Yi Zhang, Ziyi Li, Boning Zhang, Jingxin Fu, Tengfei Xiao, Wei Li, Clifford A. Meyer, Jun Chu, Peng Jiang, Paloma Cejas, Klothilda Lim, Henry Long, Myles Brown, X. Shirley Liu.

Dana-Farber Cancer Institute, Boston, MA, USA; Harvard T.H. Chan School of Public Health, Boston, MA, USA; Anhui University of Chinese Medicine, Hefei, Anhui, China.

HIGHLIGHTS

• In vivo CRISPR screens identify new immune targets regulating the tumor microenvironment

• Cop1 knockout in cancer cells enhances anti-tumor immunity

• Cop1 modulates chemokine secretion and macrophage infiltration into tumors

• Cop1 targets C/ebpδ degradation via Trib2 and influences ICB response

Despite remarkable clinical efficacy of immune checkpoint blockade (ICB) in cancer treatment, ICB benefits for triple-negative breast cancer (TNBC) remain limited. Through pooled in vivo CRISPR knockout (KO) screens in syngeneic TNBC mouse models, we found that deletion of the E3 ubiquitin ligase Cop1 in cancer cells decreases secretion of macrophage-associated chemokines, reduces tumor macrophage infiltration, enhances anti-tumor immunity, and strengthens ICB response. Transcriptomics, epigenomics, and proteomics analyses revealed that Cop1 functions through proteasomal degradation of the C/ebpδ protein. The Cop1 substrate Trib2 functions as a scaffold linking Cop1 and C/ebpδ, which leads to polyubiquitination of C/ebpδ. In addition, deletion of the E3 ubiquitin ligase Cop1 in cancer cells stabilizes C/ebpδ to suppress expression of macrophage chemoattractant genes. Our integrated approach implicates Cop1 as a target for improving cancer immunotherapy efficacy in TNBC by regulating chemokine secretion and macrophage infiltration in the tumor microenvironment.

KEYWORDS: triple-negative breast cancer; CRISPR screening; immunotherapy; E3 ubiquitin ligase; Cop1; C/ebpδ

DOI: 10.1016/j.cell.2021.09.006