Divergent Roles of Circadian Regulators CLOCK and CRY1 in Driving Pro-Tumoral Stemness and Immunoevasion in Osteosarcoma

The circadian clock is a cell-autonomous regulatory system that influences diverse cancer-related processes, including cell proliferation, metabolism, and immune regulation. While core clock regulators are known to affect tumor biology, their distinct tumor-intrinsic and microenvironmental roles in osteosarcoma (OS) remain poorly defined. Here, we report that the expression of CLOCK and CRY1, but not their canonical partners BMAL1 and CRY2, is significantly associated with poor survival in OS and linked to oncogenic programs. Integrative transcriptomic and immune analyses reveal that CLOCK and CRY1 are positively correlated with cancer stem cell (CSC) markers, epithelial-mesenchymal transition (EMT) drivers, metabolic and metastatic genes, and immunosuppressive factors such as (e.g., MYC, SLC16A1, HK1, TNC, CD276, ITGA4, WISP1, POSTN, VEGFA). Knockdown of CLOCK or CRY1 in 143B OS stem-like cells significantly reduces the expression of these genes, supporting a functional role in maintaining tumor-promoting phenotypes. Moreover, high CLOCK and CRY1 expression correlates with reduced infiltration of CD4⁺ T cells and dendritic cells, elevated cancer-associated fibroblasts (CAFs) and myeloid-derived suppressor cells (MDSCs), and increased markers of immune exclusion and dysfunction. In contrast, BMAL1 and CRY2 show minimal or inverse associations with these parameters. These findings uncover an unexpected divergence among circadian regulators, positioning CLOCK and CRY1 as potential drivers of OS aggressiveness via both tumor-intrinsic and immune-evasive mechanisms, and suggest their therapeutic targeting as a promising strategy for disrupting circadian-linked oncogenic circuits in OS.