LLC-Luc & PANC-1-Luc | Novel Sunitinib Analogue Against Pancreatic Ductal Adenocarcinoma

Br J Pharmacol: Novel Sunitinib Analogue BJ-2412 Targets the RYK-GSK-3β Axis Against Pancreatic Ductal Adenocarcinoma

This study focuses on pancreatic ductal adenocarcinoma (PDAC), a malignancy with extremely high aggressiveness and limited treatment options. It reveals, for the first time, the pro-tumorigenic mechanism of the atypical receptor tyrosine kinase RYK in PDAC, confirming RYK as a novel activator of GSK-3β. The study systematically elucidates that BJ-2412, a novel sunitinib analogue developed by the research team, exerts multi-faceted anti-PDAC effects—including inhibiting tumor growth, reversing chemoresistance, blocking metastasis, and remodeling the tumor immune microenvironment—by targeting the RYK-GSK-3β axis.

This study employed a comprehensive preclinical research system encompassing in vitro cellular assays, molecular mechanism studies, and in vivo animal experiments. Key methods are as follows:

1. Cell Model Construction and Phenotypic Assays: Human PDAC cell lines PANC-1 (gemcitabine-resistant), MIAPaCa-2, Capan-1, Capan-2, normal pancreatic ductal epithelial cells H6c7, and human acute monocytic leukemia cell line THP-1 (differentiated into macrophages) were used to establish tumor-macrophage co-culture systems. Cell viability/proliferation was assessed by MTT assay, apoptosis by flow cytometry, cancer stem cell (CSC) stemness by sphere formation assay, and cell invasion by Transwell assay.

2. Kinase Activity and Molecular Interaction Assays: The KINOMEscan platform was used for kinome-wide inhibition profiling across 468 kinases. The Proteome Profiler Human Phospho-RTK Array and Phospho-Kinase Array were used to screen differentially regulated kinases. Western blotting detected expression and phosphorylation of key signaling molecules; subcellular fractionation assessed nucleocytoplasmic distribution. Stable RYK knockdown was achieved via siRNA, and co-immunoprecipitation (Co-IP) validated protein-protein interactions between RYK and EGFR.

3. Transcriptomic and Bioinformatics Analysis: The NanoString nCounter PanCancer Pathways panel was used for transcriptomic profiling of BJ-2412-treated cells to screen differentially expressed genes. Kaplan-Meier survival analysis, based on TCGA and GEO datasets, validated the clinical prognostic value of key target genes, complemented by qPCR for mRNA validation.

4. Immune Cell Phenotyping Assays: Flow cytometry (FACS) assessed the M1 (CD86+)/M2 (CD206+) macrophage phenotype ratio. Fluorescence live-cell imaging and Annexin V staining visualized macrophage-tumor cell interactions and tumor cell apoptosis in the co-culture system.

5. In Vivo Animal Studies: A BALB/c nude mouse xenograft model using PANC-1-Luc cells evaluated in vivo anti-tumor efficacy. The chick chorioallantoic membrane (CAM) model and a C57BL/6 mouse LLC-Luc allograft model evaluated anti-metastatic effects. Bioluminescence imaging (BLI) monitored tumor growth and metastasis in real-time. Immunohistochemistry (IHC) and flow cytometry analyzed macrophage infiltration and phenotype in tumor tissues.

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Core Conclusions and Clinical Significance

1 Core Research Conclusions

1. First revelation of a novel biological function for RYK in PDAC: It confirms that the atypical RTK family member RYK is a positive activator of GSK-3β in PDAC, contrary to previous understanding. RYK is activated via tyrosine phosphorylation through heterodimerization with EGFR, functioning as a key driver of PDAC pathogenesis and representing a novel therapeutic vulnerability.
2. Elucidation of the RYK-GSK-3β axis as a central node integrating Shh and WNT signaling in PDAC: RYK activates GSK-3β to enhance the pro-tumorigenic Shh-GLI1 pathway while suppressing non-canonical WNT5A signaling. Targeting RYK can simultaneously intervene in these two key pathways that co-operate in PDAC tumorigenesis, overcoming the limitations of single-pathway targeted therapies.
3. Identification of the RYK-Shh axis as a critical pathway for PDAC tumor-immune microenvironment crosstalk: RYK activation in tumor cells upregulates Shh paracrine signaling, inducing TAM polarization toward the immunosuppressive M2 type. Targeting RYK blocks this process, reprogramming TAMs to the anti-tumor M1 type and effectively remodeling the immunosuppressive PDAC microenvironment.
4. Confirmation of the superior anti-PDAC activity of the novel sunitinib analogue BJ-2412: BJ-2412 possesses a unique RYK inhibitory activity distinct from its parent drug sunitinib. It exhibits multi-faceted effects including selective PDAC cell killing, cancer stem cell elimination, reversal of gemcitabine resistance, anti-metastasis, and immune microenvironment remodeling. Its in vivo tumor inhibition and anti-metastatic efficacy are significantly superior to sunitinib and the first-line drug gemcitabine, positioning it as a highly promising candidate for PDAC targeted therapy with clinical translation potential.

2 Clinical Significance and Research Value

PDAC remains one of the most difficult solid tumors to treat clinically. Existing targeted therapies have limited efficacy, with chemotherapy resistance and the immunosuppressive microenvironment representing two core clinical bottlenecks. This study not only elucidates the oncogenic mechanism of RYK in PDAC for the first time, providing a novel therapeutic target, but also develops BJ-2412, a small-molecule drug candidate targeting RYK. By intervening in a single target, this drug achieves dual regulation of tumor cell-intrinsic survival signals and the tumor immune microenvironment, overcoming the limitations of traditional TKI inhibitors (e.g., susceptibility to resistance, inability to modulate the microenvironment). This offers a novel strategy for PDAC targeted therapy and provides a new direction for combination therapy in gemcitabine-resistant PDAC patients, holding significant theoretical innovation value and clinical translational potential.

Reference

Yadav K, Chaudhary P, Ghimire K, Mishra S, Lee H, Bae D, Chang JH, Jeong BS, Kim JA. Therapeutic targeting of GSK-3β activation by the tyrosine kinase RYK in pancreatic ductal adenocarcinoma with BJ-2412, a novel sunitinib analogue. British Journal of Pharmacology. 2026. doi: 10.1111/bph.70455