WEE1 reinforces C-MYC driven oncogenic programs through GSK3ß inhibition

Background: Esophageal adenocarcinoma (EAC) remains a lethal malignancy with a 5-year survival rate below 20%. The nuclear kinase WEE1 is a key regulator of the G2/M checkpoint, whereas the oncogenic transcription factor c-MYC, dysregulated in ~70% of human cancers, is challenging to target directly. Identifying upstream regulators that control MYC stability offers an alternative therapeutic strategy. Here, we discovered that WEE1 reinforces MYC-driven oncogenic programs by inhibiting GSK3beta, and that inhibition of WEE1 promotes proteasome-mediated MYC degradation. Methods and Results: Gene set enrichment analysis across TCGA and GEO datasets showed consistent enrichment of MYC target gene signatures in WEE1-high EAC tumors. Immunofluorescence in normal esophagus and EAC tissues demonstrated strong overexpression and positive correlation between WEE1 and C-MYC, which was also validated in EAC cell lines compared to non-cancerous and Barrett's esophagus cells. Genetic knockdown or pharmacologic inhibition of WEE1 reduced MYC protein levels, transcriptional activity, and downstream gene expression, as confirmed by reporter assays, qRT-PCR, and RNA sequencing. Cycloheximide chase assays revealed a shortened MYC half-life upon WEE1 inhibition, whereas the proteasome inhibitor MG132 rescued MYC degradation. Mechanistically, WEE1 inhibition activated GSK3beta, a kinase required for MYC ubiquitination and proteasomal turnover. Conversely, WEE1 overexpression stabilized MYC by elevating inhibitory GSK3beta-S9 phosphorylation. A kinase-dead WEE1 mutant failed to stabilize MYC, indicating a catalytic-activity-dependent mechanism. Proximity ligation assays further demonstrated increased GSK3beta-MYC interaction following WEE1 inhibition. A high-throughput screen of 892 FDA-approved drugs identified Panobinostat as a synergistic partner of the WEE1 inhibitor MK1775. The combination significantly suppressed the growth of human EAC PDX-derived organoids and inhibited tumor progression in EAC PDX models in vivo. Conclusion: These findings define a WEE1-GSK3beta-MYC signaling axis in which WEE1 stabilizes MYC and sustains MYC-driven oncogenic programs. WEE1 inhibition activates GSK3beta, promoting proteasome-mediated MYC degradation. The combination of WEE1 inhibition and Panobinostat represents a promising therapeutic approach for MYC-driven EAC.