Plasma membrane-associated ARAF condensates fuel RAS-related cancer drug resistance

Raf protein kinases are major Ras effectors that function by phosphorylating MEK. Although all three Raf isoforms share a conserved Ras binding domain and bind to GTP-loaded Ras, only ARAF uniquely enhances Ras activity. Here we uncovered the molecular basis of ARAF in regulating Ras activation. The disordered N-terminal sequence of ARAF drives self-assembly, forming ARAF-RAS condensates tethered to the plasma membrane. These structures concentrate active Ras locally, impeding NF1-mediated negative regulation of Ras, thereby fostering receptor tyrosine kinase (RTK)-triggered Ras activation. In RAS-mutant tumors, loss of the ARAF N terminus sensitizes tumor cells to pan-RAF inhibition. In hormone-sensitive cancers, increased ARAF condensates drive endocrine therapy resistance, whereas ARAF depletion reverses RTK-dependent resistance. Our findings delineate ARAF-RAS protein condensates as distinct subcellular structures sustaining Ras activity and facilitating oncogenic Ras signaling. Targeting ARAF-RAS condensation may offer a strategy to overcome drug resistance in both wild-type and mutant ARAF-mediated scenarios.