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

Abstract

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. ARAF uniquely enhances RAS activity via N-terminal disordered sequence, forming membrane ARAF–RAS condensates that shield RAS from negative regulation and drive cancer drug resistance.