Targeting the mevalonate pathway potentiates NUAK1 inhibition-induced immunogenic cell death and antitumor immunity

The induction of immunogenic cell death (ICD) impedes tumor progression via both tumor cell-intrinsic and -extrinsic mechanisms, representing a robust therapeutic strategy. However, ICD-targeted therapy remains to be explored and optimized. Through kinome-wide CRISPR-Cas9 screen, NUAK1 is identified as a potential target. The ICD-provoking effect of NUAK1 inhibition depends on the production of reactive oxygen species (ROS), consequent to the downregulation of NRF2-mediated antioxidant gene expression. Moreover, the mevalonate pathway/cholesterol biosynthesis, activated by XBP1s downstream of ICD-induced endoplasmic reticulum stress, functions as a negative feedback mechanism. Targeting the mevalonate pathway with CRISPR knockout or HMGCR inhibitor simvastatin amplifies NUAK1 inhibition-mediated ICD and antitumor activity, while cholesterol dampers ROS, ICD and therefore tumor suppression. The combination of NUAK1 inhibitor and statin enhances the efficacy of anti-PD-1 therapy. Collectively, our study unveils the promise of blocking the mevalonate-cholesterol pathway in conjunction with ICD-targeted immunotherapy. Overall design: Mouse tumors (each set containing three independent samples) were washed with ice-cold PBS and cut into small pieces, which were then subjected to enzymatic digestion with collagenase D and DNase I (Roche) using a gentleMACS Tissue Dissociator (Miltenyi Biotec). Cells were filtered through a 70-µm filter and washed with PBS. To extract immune cells, cells were stained with biotin anti-mouse CD45 antibody (Biolegend) and separated by anti-biotin MicroBeads (Miltenyi Biotec) according to the manufacturer's protocol. Sorted immune cells with a purity greater than 95% and viability higher than 85% were subjected to 10x Genomics scRNA-seq. To control the final number of cells captured, approximately 30,000 cells per sample were loaded onto 10X Chromium Single Cell Platform (10X Genomics) at a concentration of 1,000 cells per µl (Single Cell 3' library and Gel Bead Kit v.3) as described in the manufacturer's protocol. Finally, the library pool was prepared, and sequencing was performed on an Illumina NovaSeq 6000 platform.

免疫原性细胞死亡（immunogenic cell death, ICD）可通过肿瘤细胞内在与外在双重机制阻滞肿瘤进展，是一种强效的治疗策略。然而，靶向ICD的治疗仍有待探索与优化。通过全激酶组CRISPR-Cas9筛选，本研究鉴定出NUAK1为潜在治疗靶点。NUAK1抑制所引发的ICD效应依赖于活性氧簇（reactive oxygen species, ROS）的产生，该过程源于核因子E2相关因子2（nuclear factor erythroid 2-related factor 2, NRF2）介导的抗氧化基因表达下调。此外，由ICD诱导的内质网应激下游剪接型X盒结合蛋白1（XBP1s）激活的甲羟戊酸通路/胆固醇生物合成，发挥负反馈调控作用。通过CRISPR敲除或3-羟基-3-甲基戊二酰辅酶A还原酶（3-hydroxy-3-methylglutaryl-CoA reductase, HMGCR）抑制剂辛伐他汀靶向甲羟戊酸通路，可增强NUAK1抑制介导的ICD与抗肿瘤活性；而胆固醇则会抑制ROS产生、ICD效应及后续的肿瘤抑制作用。联合使用NUAK1抑制剂与他汀类药物可提升抗PD-1治疗的疗效。综上，本研究揭示了阻断甲羟戊酸-胆固醇通路联合靶向ICD的免疫治疗的应用前景。

实验整体设计：将小鼠肿瘤（每组包含3份独立样本）用预冷磷酸盐缓冲液（PBS）洗涤后切成小块，使用胶原酶D与脱氧核糖核酸酶I（DNase I，Roche），结合gentleMACS组织解离仪（Miltenyi Biotec）进行温和酶解。将细胞通过70μm滤膜过滤后，用PBS洗涤。为提取免疫细胞，使用生物素标记的抗小鼠CD45抗体（Biolegend）对细胞进行染色，随后按照产品说明书使用抗生物素微珠（Miltenyi Biotec）进行分选。将纯度≥95%、存活率≥85%的分选免疫细胞进行10x Genomics单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）。为控制最终捕获的细胞数量，按照制造商的操作流程，以每微升1000个细胞的浓度，将每份样本约30000个细胞上样至10X Chromium单细胞平台（10x Genomics），所用试剂为单细胞3'文库及凝胶珠试剂盒v.3。最终完成文库混合制备，并在Illumina NovaSeq 6000测序平台上完成测序。