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

Abstract

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. <i>Loxl4</i> knockout and mouse recombinant LOXL4 protein systems, along with <i>Piezo1</i>, <i>Ybx1</i>, <i>Acly</i>, and <i>Kat2a</i> 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 <i>Piezo1</i>, <i>Ybx1</i>, <i>Acly</i>, and <i>Kat2a</i> 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.