LM2I leads to CAD ubiquitination and liver cancer suppression through activation of ASS1

• LM2I, a specific activator of ASS1, suppresses liver cancer by promoting CAD ubiquitination and degradation, thereby inhibiting pyrimidine synthesis. • ASS1 and CAD expression are negatively correlated in liver cancer, and LM2I is more effective in low-ASS1-expressing cells. • LM2I exhibits antitumor activity in vivo and in vitro with no observed toxicity in mice, highlighting its therapeutic potential. • Pyrimidine supplementation rescues LM2I-induced proliferation inhibition, confirming the mechanistic link between ASS1 activation and pyrimidine metabolism.