Alisertib impairs the stemness of hepatocellular carcinoma by inhibiting purine synthesis.

Hepatocellular carcinoma tumor-repopulating cells (HCC-TRCs) drive disease progression, yet their purine metabolism mechanisms remain poorly understood. This study revealed that the stemness index, strongly linked to poor HCC prognosis, exhibited a robust positive correlation with purine metabolism through single-sample gene set enrichment analysis. Integrated drug screening across CTRP, GDSC, and PRISM databases identified alisertib, an aurora kinase A (AURKA) inhibitor, as a potent agent targeting stemness. Using fibrin gel-based 3D-cultured HCC-TRCs, mechanistic studies demonstrated that alisertib suppresses xanthine and hypoxanthine production by inhibiting the AURKA-AKT signaling axis. This disruption markedly impaired tumor spheroid formation, migration, and invasion in vitro, while significantly suppressed tumor growth in vivo, which could be rescued by the AKT agonist SC79. Our findings revealed a novel therapeutic strategy targeting purine metabolism through AURKA-AKT axis inhibition, effectively eliminating HCC-TRCs.