Loss of tumor suppressors promotes a non-cell-autonomous inflammatory tumor microenvironment and enhances LAG3+T cell mediated immune suppression [ATAC_seq]

Low response rate, treatment relapse, and resistance remain key challenges in the application of immune checkpoint blockade (ICB). Here we report that loss of specific tumor suppressors (TSs) induces an inflammatory response and promotes an immune suppressive tumor microenvironment. Importantly, low expression of these TSs is associated with a higher expression of immune checkpoint inhibitory mediators. Using CRIPSR/Cas9 in vivo loss-of-function screening, we identified NF1, TSC1, and TGF-b RII as TSs that regulate immune composition. Loss of each of these three TSs leads to alterations in chromatin accessibility and enhances IL6-JAK3-STAT3/6 inflammatory pathways. This results in an immune suppressive landscape characterized by increased LAG3+ CD8 and CD4 T cells. ICB targeting LAG3 in combination with PD-L1 decreased metastatic progression in preclinical triple negative breast cancer (TNBC) mouse models of NF1-, TSC1- or TGF-b RII- deficient tumors. Together, our studies identify LAG3 as a new ICB target for patients whose cancer displays inactivation of these TSs. In addition, our studies reveal an unexpected role for TSs in the regulation of metastatic spread via non-cell-autonomous modulation of the immune compartment. To investigate epigenetic alterations of downstream molecules by knockout of Nf1, Tsc1, Tgfbr2 in 4T1 cell in vitro.

免疫检查点阻断（immune checkpoint blockade, ICB）的临床应用仍面临应答率低、治疗复发与耐药三大核心难题。本研究发现，特定肿瘤抑制因子（tumor suppressors, TSs）的缺失可诱发炎症反应，并促进免疫抑制性肿瘤微环境的形成。值得注意的是，此类肿瘤抑制因子的低表达与免疫检查点抑制性介质的高表达呈显著正相关。本研究通过CRISPR/Cas9体内功能缺失筛选，鉴定出NF1、TSC1及TGF-β RII为调控肿瘤免疫组分的肿瘤抑制因子。这三类肿瘤抑制因子的缺失均可改变染色质开放状态，并激活IL6-JAK3-STAT3/6炎症通路，进而形成以LAG3阳性CD8 T细胞及CD4 T细胞浸润增多为特征的免疫抑制微环境。在NF1、TSC1或TGF-β RII缺陷型三阴性乳腺癌（triple negative breast cancer, TNBC）临床前小鼠模型中，靶向LAG3联合PD-L1的免疫检查点阻断治疗可显著抑制肿瘤转移进展。综上，本研究确定LAG3可作为针对上述肿瘤抑制因子失活患者的新型免疫检查点阻断治疗靶点。此外，本研究揭示了肿瘤抑制因子通过非细胞自主方式调控免疫组分，进而影响肿瘤转移的全新功能。本研究还体外构建了4T1细胞中Nf1、Tsc1及Tgfbr2敲除模型，以探究下游分子的表观遗传调控异常。