Therapeutic decoupling of MHC-I and PD-L1 expression increases the efficacy of immune checkpoint blockade (koTRAF3_ATACseq). Therapeutic decoupling of MHC-I and PD-L1 expression increases the efficacy of immune checkpoint blockade (koTRAF3_ATACseq)

Immune checkpoint blockade (ICB) therapy has revolutionized the treatment of multiple cancers, but the majority of patients remain refractory due to impaired MHC-I expression. Although the combination of immunotherapy with existing anti-cancer treatments has shown synergy in multiple scenarios, the immunomodulatory effects of conventional therapies remain poorly understood. Here, we integrated CRISPR-mediated genetic screens and data mining of perturbation associated gene expression data to identify drugs that can specifically up-regulate MHC-I without inducing PD-L1. Using FACS-based genome-wide CRISPR screens, we identified Traf3, a critical suppressor of the NF-κB pathway, as a suppressor of MHC-I, but not PD-L1. A Traf3-knockout gene expression signature is associated with better survival in ICB-naive cancer patients and better response to ICB in published clinical trials. From publicly available drug treatment data, we identified SMAC mimetics as the top candidates of having similar transcriptional effects as Traf3-knockout. We experimentally validated that the SMAC mimetic birinapant specifically up-regulated MHC-I, sensitized cancer cells to T-cell-dependent killing, and synergized with ICB in the treatment of established tumors. Our findings provide a strong preclinical rationale for treating MHC-I-low tumors with SMAC mimetics to enhance sensitivity to existing immunotherapy. Moreover, the integrated approach used in this study can be generalized to identify drugs with immunomodulatory effects to enhance immunotherapy efficacy. Overall design: ATAC-seq on TRAF3-replete or -deficient B16F10 cells that were treated with vehicle or 1ng/ml IFNγ.

免疫检查点阻断（immune checkpoint blockade, ICB）治疗已彻底重塑多种癌症的治疗范式，但多数患者因主要组织相容性复合体I类分子（MHC-I）表达受损仍呈难治性状态。尽管免疫治疗与现有抗癌疗法的联合方案在多场景中展现出协同效应，传统疗法的免疫调节作用仍未得到充分阐释。本研究整合CRISPR介导的遗传筛选与扰动相关基因表达数据挖掘，旨在筛选可特异性上调MHC-I且不诱导程序性死亡受体配体1（PD-L1）表达的药物。通过基于流式细胞术的全基因组CRISPR筛选，我们鉴定出肿瘤坏死因子受体相关因子3（Traf3）——核因子κB（NF-κB）通路的关键抑制因子——作为MHC-I而非PD-L1的负调控因子。Traf3基因敲除的基因表达特征，与未接受过ICB治疗的癌症患者更优的生存结局，以及已发表临床试验中更佳的ICB应答水平显著相关。从公开可用的药物处理数据中，我们筛选得到SMAC模拟物（SMAC mimetics）为与Traf3基因敲除具有相似转录效应的顶级候选药物。我们通过实验验证，SMAC模拟物比里纳潘特（birinapant）可特异性上调MHC-I表达，使癌细胞对T细胞依赖性杀伤敏感，并在已形成肿瘤的治疗中与ICB产生协同作用。本研究结果为使用SMAC模拟物治疗MHC-I低表达肿瘤以增强其对现有免疫治疗的敏感性提供了坚实的临床前依据。此外，本研究采用的整合策略可推广至筛选具有免疫调节作用的药物，以提升免疫治疗疗效。整体实验设计：对经溶媒或1ng/ml干扰素γ（IFNγ）处理的TRAF3充足或缺陷的B16F10细胞进行转座酶可及性测序（ATAC-seq）。