Introduction
Ameloblastoma (AM) frequently develops resistance to the BRAF inhibitor vemurafenib, primarily mediated by ameloblastoma-associated fibroblasts (AMFs). However, the potential contribution of the circadian clock in this resistance has not been explored.

Objectives
This study aimed to elucidate the role of the BMAL1-LHX8 axis during tumor-stroma crosstalk, and to investigate the therapeutic potential of targeting this axis to augment vemurafenib sensitivity for AM.

Methods
Patient-derived AM cells and AMFs were utilized to reconstruct the stroma-rich AM tumoroid in-vitro for recapitulating the tumor-stroma interplay and assessing the pharmacological effect of vemurafenib. Time-series RNA-sequencing, luciferase assays, and CRISPR/Cas9 gene editing were used to define the transcriptional landscape and the BMAL1-LHX8 regulatory network. The efficacy of combining the clock modulator GSK4112 with vemurafenib was assessed in stroma-rich AM tumoroids and cell line-based xenograft models.

Results
Patient-derived AMFs exhibit enhanced secretory activity, and elevated metabolic and contractile functions contribute to increased stromal stiffness through ECM remodeling in AM. Tumor-stroma crosstalk was recapitulated in stroma-rich AM tumoroid, which is evidenced by the dynamic alteration of normal fibroblasts (NFs) to AMF-like state. Transcriptomic profiling of stroma-rich tumoroids revealed severe disruption of the molecular clock and LHX8 in AMFs. Functional studies demonstrated that BMAL1-driven LHX8 expression promotes pro-tumorigenic AMF activities, including growth factor secretion and ECM remodeling. Critically, pharmacological inhibition of the BMAL1-LHX8 axis with GSK4112 potently sensitized AM to vemurafenib in both stroma-rich tumoroid and xenograft models.

Conclusion
Our findings reveal the role of the BMAL1-LHX8 axis in underlying AMF-mediated drug resistance in AM, and propose that the molecular clock modulation in tumor-stroma crosstalk represents a potential therapeutic avenue for ameloblastoma.