Entrectinib binds to HMGB1 and activates cardiomyocyte autophagy by inhibiting OTUD5-MTORC1 signaling to induce cardiotoxicity

Entrectinib stands unparalleled as the sole neurotrophic tyrosine receptor kinase (NTRK) inhibitor that has demonstrated clinical efficacy in treating brain metastases across various cancer types. However, its potential to induce severe cardiotoxicity, compounded by the current lack of effective intervention strategies, poses a substantial risk of treatment failure, underscoring the critical need for in-depth research on the molecular mechanism. Here, we utilized proteomics analysis and a murine model with cardiomyocyte-specific <i>atg7</i> deletion to reveal that entrectinib activated autophagy in cardiomyocytes, subsequently triggering apoptosis and leading to cardiac dysfunction. Mechanistically, entrectinib directly bound to the HMGB1 protein at the 103rd phenylalanine residue, enhancing its nuclear localization. In the nucleus, HMGB1 suppressed the transcription of the deubiquitinating enzyme OTUD5, a vital regulator of the MTORC1 pathway, which subsequently inhibited the MTORC1 pathway, culminating in the activation of macroautophagy/autophagy. Furthermore, our research demonstrated that HMGB1 inhibition could prevent the cardiotoxicity induced by entrectinib in both <i>in vivo</i> and <i>in vitro</i> models. Specifically, we found that tanshinone IIA could mitigate the cardiotoxic effects of entrectinib by reducing HMGB1 protein levels. Taken together, our findings elucidated the mechanism underlying entrectinib-induced cardiotoxicity, offering a theoretical foundation for the safer clinical application of this targeted therapy.<b>Abbreviations and Acronyms</b>: ATG7: autophagy related 7; CHX: cycloheximide; CKMB: creatine kinase myocardial band; CQ: chloroquine; c-PARP: cleaved poly (ADP-ribose) polymerase family; DAPI: 4'6-diamidino-2-phenylindole; EF: ejection fraction; FS: fractional shortening; GSEA: gene set enrichment analysis; H&E: hematoxylin and eosin; HW:TL: ratio of heart weight to tibia length; KEGG: Kyoto Encyclopedia of Genes and Genomes; MYH6: myosin heavy polypeptide 6, cardiac muscle, alpha; MYH7: myosin, heavy polypeptide 7, cardiac muscle, beta; NPPA: natriuretic peptide type A; NPPB: natriuretic peptide type B; PI: propidium iodide; qPCR: quantitative real-time PCR; SD: standard deviation; SRB: sulforhodamine B; WGA: wheat germ agglutinin.