Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy with an exceptionally poor prognosis, as reflected in a 5-year survival rate of less than 8%. The complex molecular landscape underlying PDAC aggressiveness, which is characterized by genetic alterations, hyperactivation of receptor tyrosine kinases (RTKs), and dysregulated embryonic developmental signaling, poses a significant challenge to single-targeted therapies. Targeting key regulators of tumor signaling networks is essential for effective PDAC therapy.

Cell viability was assessed using the MTT assay. A phospho-receptor tyrosine kinase (p-RTK) array assay was conducted to profile basal RTK activity and evaluate drug effects in pancreatic cancer cells. Co-immunoprecipitation assays were performed to examine the interaction between receptor-like tyrosine kinase (RYK) and EGFR. Small interfering RNA (siRNA) transfection was utilized to achieve RYK knockdown. The therapeutic efficacy of the treatment was evaluated in a pancreatic cancer xenograft mouse model.

In the PANC-1 PDAC cell line, p-RTK array revealed that phosphorylated EGFR exhibited the highest basal activity, followed by IGF-1R and RYK. Co-immunoprecipitation assays demonstrated that RYK was cross-phosphorylated by EGFR. RYK knockdown led to increased phosphorylation of glycogen synthase kinase-3 beta (GSK3β) at serine 9 (S9), an inhibitory modification, without altering phosphorylated β-catenin levels. In addition, RYK silencing promoted nuclear translocation of GLI1, a key effector of the Sonic Hedgehog (Shh) signaling. Targeting the RYK-GSK3β axis with a 2,4-dimethylpyridin-3-ol analog of the multi-RTK inhibitor sunitinib induced cancer cell-selective cytotoxicity and exhibited potent antitumor efficacy in PDAC xenograft mouse model. Notably, unlike other multi-RTK inhibitors such as sunitinib and gefitinib, especially the latter of which inhibited both EGFR and RYK, the sunitinib analog only suppressed Sonic Hedgehog (Shh)/GLI1 signaling. Moreover, this modulation reprogramed macrophage polarization toward M1 phenotype, thereby enhancing cancer cell death in the tumor microenvironment.

Our study identifies RYK as a novel EGFR cross-phosphorylation target and functions as an upstream regulator of GSK3β. The RYK-GSK3β axis plays a critical role in counterbalancing both canonical Wnt signaling and RTK-mediated non-canonical Wnt/β-catenin signaling, as well as canonical and KRAS-driven non-canonical GLI1 activity. Pharmacological inhibition of the RYK-GSK3β using a 2,4-dimethylpyridin-3-ol analog of sunitinib effectively suppressed tumor growth, reprogrammed macrophage phenotypes, and inhibited metastasis, underscoring its potential as a promising therapeutic strategy against PDAC.