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. 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.