Integrating Network Pharmacology and Experimental Validation Reveals EGFR/PI3K/Akt Axis Mediates Ibrutinib-Induced...

This study aims to systematically explore the molecular mechanism underlying ibrutinib-induced cardiotoxicity.

Network toxicology was applied using DrugBank, PharmMapper, GeneCards, OMIM, and TTD databases to identify overlapping targets between ibrutinib and myocardial injury, followed by PPI network construction (STRING; confidence ≥0.7) and GO/KEGG enrichment analyses (Metascape). Molecular docking (AutoDock Vina) verified ibrutinib's binding affinity to core targets. In vitro, H9C2 cardiomyocytes were exposed to ibrutinib (0, 10, 20, 40, and 80 μM) for 24 h; CCK-8, DCFH-DA, EdU, and Calcein-AM/PI assays were performed to assess cell viability, ROS levels, proliferation, and cell death, respectively. Western blot analyzed phosphorylation of EGFR/PI3K/Akt/mTOR pathway proteins.

A total of 42 overlapping targets were screened from 337 ibrutinib-related targets and 471 myocardial injury-related genes. PPI network analysis identified EGFR, AKT1, SRC, ESR1, and CASP3 as hub genes, and KEGG enrichment analysis identified the PI3K-Akt pathway as the most significantly enriched cascade. Molecular docking confirmed that ibrutinib stably bound BTK and EGFR with a binding energy of -30.2 kcal/mol and formed multiple hydrogen bonds and salt bridges (2.2-3.2 Å). In H9C2 cells, ibrutinib dose-dependently reduced viability (IC50 = 119.49 μM), increased ROS production (40 and 80 μM), inhibited proliferation (10 and 20 μM), and promoted cell death (5 and 10 μM). Western blot confirmed that ibrutinib significantly downregulated phosphorylation of PI3K p85, Akt (Ser473), mTOR (Ser2448), and p70S6K (Thr389) without altering total protein expression.

Ibrutinib induces cardiotoxicity by targeting BTK/EGFR and inhibiting the downstream EGFR/PI3K/Akt/mTOR pathway, providing a mechanistic framework for clinical safety management of BTK inhibitors.