Targeting LOXL4-driven matrix stiffening with acetyldigoxin restores CD8⁺ T cell function in lung cancer

Extracellular matrix (ECM) stiffness is known to impair T cell function, yet the underpinning molecular cascade remains undefined. This paper investigates the role of lysyl oxidase-like 4 (LOXL4) in ECM stiffening and CD8⁺ T cell function in lung cancer. Loxl4 knockout and mouse recombinant LOXL4 protein systems, along with Piezo1, Ybx1, Acly, and Kat2a conditional knockout mouse models were established. ECM stiffness was measured by atomic force microscopy, and T cell exhaustion markers were analyzed using flow cytometry. RNA sequencing, ATAC-seq, CUT&Tag, chromatin immunoprecipitation-quantitative polymerase chain reaction, and luciferase assays were used to explore the underlying molecular mechanisms. Molecular docking was performed to explore Food and Drug Administration-approved agents targeting LOXL4. The results demonstrated that tumor-derived LOXL4 stiffened the ECM, which activates the mechanosensor Piezo1 in CD8⁺ T cells, triggering Ca²⁺ influx and downstream FAK1-YAP1 signaling. Nuclear YAP1 transactivated YBX1, which recruited the metabolic enzyme ACLY and the histone acetyltransferase KAT2A to exhaustion gene loci, epigenetically reinforcing terminal exhaustion. Conditional knockout of Piezo1, Ybx1, Acly, and Kat2a in murine CD8⁺ T cells abolished stiffness-induced exhaustion and suppressed tumor growth. Acetyldigoxin was identified as a high-affinity LOXL4 inhibitor. It softened the ECM, disrupted the mechanosignaling-epigenetic axis, reversed CD8⁺ T cell exhaustion, and synergized with anti-PD-1 blockade to achieve durable tumor regression. In conclusion, this study uncovers a mechanotransduction-to-epigenetic pathway where LOXL4-driven matrix stiffening induces CD8⁺ T cell exhaustion. Repurposing acetyldigoxin as a LOXL4-targeted therapy offers a promising clinical strategy to overcome ECM-mediated immunotherapy resistance in lung cancer.