Abstract 49: The PER3-RORB axis defines a tumor-suppressive circadian state associated with improved survival in head and neck squamous cell carcinoma.

Abstract Background: The circadian clock governs DNA repair, cell-cycle progression, metabolism, and stress responses. Circadian disruption has been linked to tumor progression and therapy resistance, yet the clock components influencing prognosis in head and neck squamous cell carcinoma (HNSCC) remain undefined. We sought to characterize circadian dysregulation in HNSCC and identify circadian signatures that predict overall survival (OS). Methods: RNA-seq, clinical, and survival data from TCGA-HNSCC (n=565) were analyzed using a circadian gene panel comprising core positive regulators (BMAL1, CLOCK, NPAS2, RORA, RORB, RORC) and negative regulators (PER1/2/3, CRY1/2, NR1D1/2). Log2-normalized expression was z-standardized per gene, and composite positive- and negative-arm scores were generated as mean standardized expression within each panel. Prognostic performance was evaluated using time-dependent ROC curves and multivariable Cox models. Circadian network integrity was assessed using the clock coherence disruption (ΔCCD) metric, comparing clock-gene correlation structure in tumors against a GTEx esophageal mucosa reference across 1,000 permutations. Differential expression between circadian-defined subgroups was evaluated with limma’s empirical Bayes-moderated models. Results: Circadian gene expression was heterogeneous across primary sites and AJCC stages, indicating circadian disruption independent of clinical factors. High RORB (HR= 0.75, p=0.015), and PER3 (HR=0.88, p=0.05) were associated with reduced mortality. In ROC analysis, PER3 and RORB showed modest prognostic discrimination individually (AUC 0.39/0.43 and 0.41/0.47 at 3/5 years, respectively). In contrast, the PER3-RORB composite score (0.52/0.56) outperformed all other circadian metrics. Clock coherence analysis demonstrated preserved circadian correlation structure in PER3-RORB-High tumors (CCD = 0.44) and marked disruption in PER3-RORB-Low tumors (CCD = 0.74), yielding a ΔCCD of 0.31. No permutation replicate exceeded this value (empirical p < 0.001), indicating highly non-random network breakdown associated with PER3-RORB loss. PER3-RORB-high tumors showed coordinated upregulation of NPAS2, metabolic and stress-response genes (DHCR24, ABCA1, LRP1, ATP6V1A) and modulation of mitochondrial components (NDUFB6), linking this axis to metabolic and stress adaptation programs. Conclusion: PER3-RORB defines a biologically coherent circadian state linked to improved survival in HNSCC. While these findings support a tumor-suppressive role for PER3-RORB, independent external validation is required to confirm robustness and clinical generalizability. This work highlights circadian regulation as a mechanistic modifier of tumor behavior and a promising avenue for biomarker development. Citation Format: Harold Nathan Tan, Oriol Mirallas, Paul Selvadurai, Michael Wotman, Luana Sousa, Neal Akhave, Khaled Sanber, Renata Ferrarotto, George R. Blumenschein, Maura L. Gillison, Faye M. Johnson, Timothy A. Yap. The PER3-RORB axis defines a tumor-suppressive circadian state associated with improved survival in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 49.