Oncogenic RAS induces a distinctive form of non-canonical autophagy mediated by the P38-ULK1-PI4KB axis.

Cancer cells with RAS mutations exhibit enhanced autophagy, essential for their proliferation and survival, making it a potential target for therapeutic intervention. However, the regulatory differences between RAS-induced autophagy and physiological autophagy remain poorly understood, complicating the development of cancer-specific anti-autophagy treatments. In this study, we identified a form of non-canonical autophagy induced by oncogenic KRAS expression, termed RAS-induced non-canonical autophagy via ATG8ylation (RINCAA). RINCAA involves distinct autophagic factors compared to those in starvation-induced autophagy and incorporates non-autophagic components, resulting in the formation of non-canonical autophagosomes with multivesicular/multilaminar structures labeled by ATG8 family proteins (e.g., LC3 and GABARAP). We have designated these structures as RAS-induced multivesicular/multilaminar bodies of ATG8ylation (RIMMBA). A notable feature of RINCAA is the substitution of the class III PI3K in canonical autophagy with PI4KB in RINCAA. We identified a regulatory P38-ULK1-PI4KB-WIPI2 signaling cascade governing this process, where ULK1 triggers PI4KB phosphorylation at S256 and T263, initiating PI4P production, ATG8ylation, and non-canonical autophagy. Importantly, elevated PI4KB phosphorylation at S256 and T263 was observed in RAS-mutated cancer cells and colorectal cancer specimens. Inhibition of PI4KB S256 and T263 phosphorylation led to a reduction in RINCAA activity and tumor growth in both xenograft and KPC models of pancreatic cancer, suggesting that targeting ULK1-mediated PI4KB phosphorylation could represent a promising therapeutic strategy for RAS-mutated cancers.