Mitochondria are the powerhouses of cells. Mitochondrial C-Raf is a potential cancer therapeutic target, as it regulates mitochondrial function and is localized to the mitochondria by its N-terminal domain. However, Raf inhibitor monotherapy can induce S338 phosphorylation of C-Raf (pC-RafS338) and impede therapy. This study identified the interaction of C-Raf with S308 phosphorylated DAPK (pDAPKS308), which together became co-localized in the mitochondria to facilitate mitochondrial remodelling. Combined use of the Raf inhibitors sorafenib and GW5074 had synergistic anti-cancer effects in vitro and in vivo, but targeted mitochondrial function, rather than the canonical Raf signaling pathway. C-Raf depletion in knock-out MEFC-Raf-/- or siRNA knock-down ACHN renal cancer cells abrogated the cytotoxicity of combination therapy. Crystal structure simulation showed that GW5074 bound to C-Raf and induced a C-Raf conformational change that enhanced sorafenib binding affinity. In the presence of pDAPKS308, this drug-target interaction compromised the mitochondrial targeting effect of the N-terminal domain of C-Raf, which induced two-hit damages to cancer cells. First, combination therapy facilitated pC-RafS338 and pDAPKS308 translocation from mitochondria to cytoplasm, leading to mitochondrial dysfunction and ROS generation. Second, ROS facilitated PP2A-mediated de-phosphorylation of pDAPKS308 to DAPK. PP2A then dissociated from the C-Raf-DAPK complex and induced profound cancer cell death. Increased pDAPKS308 modification was also observed in renal cancer tissues, which correlated with poor disease-free survival and poor overall survival in renal cancer patients. Besides mediating the anti-cancer effect, pDAPKS308 may serve as a predictive biomarker for Raf inhibitors combination therapy, suggesting an ideal pre-clinical model that is worthy of clinical translation.
