Pyrimidine deficiency activates RAP1 lactylation-induced macropinocytosis

Macropinocytosis is a crucial survival strategy for tumor cells. While external factors are known to induce macropinocytosis, the role of intrinsic cellular signals remains unexplored. Through high-throughput compound screening, we identified endogenous pyrimidine deficiency as a potent inducer of macropinocytosis in cancer cells. Mechanistically, DHODH inhibition leads to metabolic reprogramming, characterized by increased glycolysis and lactate accumulation. Lactylation proteomics analysis via mass spectrometry revealed RAP1 K208 lactylation as a pivotal event that enhances RAP1-IKK interaction, leading to the activation of p65. This cascade upregulates EREG, which in turn stimulates EGFR-PAK1 signaling and macropinocytosis. We demonstrate that this adaptive response contributes to resistance against DHODH inhibitors in various cancer models. Combining DHODH inhibitors with macropinocytosis inhibitors, including EGFR tyrosine kinase inhibitors, exhibits synergistic anti-tumor effects both in vitro and in vivo. Our findings unveil a previously unknown link between nucleotide metabolism and macropinocytosis, offering new insights into cancer cell adaptation and potential therapeutic strategies.