Indirubin Targets PI3K–AKT Signaling in Acute Lymphoblastic Leukemia: Integrated Computational and Experimental Evidence

The present work sought to clarify both the therapeutic activity and the mechanistic basis of indirubin in acute lymphoblastic leukemia (ALL) by integrating network pharmacology approaches with experimental validation. Genes associated with indirubin and ALL were collected from publicly accessible databases to identify potential targets. Protein–protein interaction (PPI) networks were constructed and analyzed in Cytoscape to isolate core genes. Functional enrichment analyses, including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG), were performed to characterize the biological roles and pathways linked to indirubin’s activity against ALL. A multi-layered network incorporating drug–disease relationships, functional annotations, and signaling pathways was established. Molecular docking of indirubin with selected core proteins was executed using AutoDock Vina software. To confirm computational predictions, both cell-based in vitro assays and animal-based in vivo experiments were subsequently performed. Analysis of the PPI network identified eight central targets of indirubin in ALL, among which AKT1, CASP3, and the mammalian target of rapamycin were included. GO and KEGG analyses indicated that indirubin may influence ALL progression through diverse biological processes and multiple signaling cascades, with the PI3K–AKT pathway emerging as a key regulatory mechanism. Docking simulations revealed stable, favorable binding interactions between indirubin and the core protein targets. Experimental validation confirmed that indirubin suppressed the proliferation of ALL cells and triggered both cell cycle arrest and apoptosis, with the PI3K–AKT signaling axis likely involved in these effects. By combining network pharmacology, molecular docking, and experimental verification in both in vivo and in vitro models, this study elucidated the biological effects and underlying mechanisms of indirubin in ALL, providing potential directions for future therapeutic development.