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Abstract

Colorectal cancer (CRC) is the second leading cause of cancer-related death, and responses to immune checkpoint therapies remain limited due to the immuno-suppressive tumor microenvironment. Tumor-associated macrophages (TAMs) are playing a crucial role in shaping this environment, with the poliovirus receptor (PVR/CD155)-TIGIT axis representing an immune checkpoint system which suppresses T/NK cell activities. However, the transcriptional mechanisms regulating PVR expression in macrophages still remains poorly understood. This thesis investigates the role of the circadian nuclear receptor REVERBa (NR1D1) and its regulatory function in macrophage-mediated anti-tumor responses in CRC. Using human CRISPR/Cas9-modified THP1-derived macrophages, PBMC-derived primary macrophages with HT29 cells or patient-derived tumor organoids (PDOs) from patients with CRC in co-culture, we characterized REVERBa expression and its functional impact on macrophage-tumor interactions. Chromatin-immunoprecipitation revealed that REVERBa directly binds to the human PVR gene promoter. Furthermore, pharmacological modulation showed that REVERB SR9009 agonist reduced PVR expression and promoter binding, whereas SR8278 antagonist increased both, highlighting REVERBa as a fine-tunable repressor of immune checkpoint regulation. Notably, CRISPR/Cas9-modification of the human REVERB gene disrupted ligand responsiveness, suggesting an altered or misfolded protein conformation and loss of function. Functional assays further demonstrated that REVERB modulation by gene editing or ligands altered macrophage efferocytosis, phagocytosis as well as cytokine expression. Importantly, combining PVR blocking with macrophages enhanced tumor cell killing in both HT29 and PDO co-cultures, underscoring the translational relevance of these findings. Altogether, these findings identify REVERBa as a novel regulator of PVR expression and macrophage function in CRC. By linking circadian biology, transcriptional repression and immune checkpoint regulation, this work highlights REVERB as a potential target, supporting future investigations of its modulation in combination with immune checkpoint blockade as a macrophage-focused immunotherapeutic strategy against CRC.