<h3>Purpose/Objective(s)</h3> We and others have shown that Chk-1/2 inhibition (Chk-i) in combination with ionizing radiation (IR) has significant preclinical and clinical antitumor effects in head and neck squamous cell carcinoma (HNSCC). However, biomarkers of resistance and maximizing response to combination Chk-i and IR have been elusive. Here, utilizing TCGA and clinical trial data as well as preclinical models of Chk-i resistance, we report tumor intrinsic immune signatures of Chk-1/2 activity and inhibitor sensitivity. <h3>Materials/Methods</h3> Gene Ontology (GO) enrichment analysis of HNSCC samples with CHK1/2 gene amplification or overexpression was performed utilizing RNAseq data extracted from TCGA. Using human HNSCC cells, UM-SCC1, secondary Chk-i resistance was generated via serial Chk-i exposure, whereby tolerance of resistant SCC-1 (R-SCC1) cells exceeded the IC50 by 10-fold. Tumor cell-directed immune regulators were quantified by qPCR +/- Chk-i and/or IR. Multiplex gene profiling was performed on pre-treatment tumor samples from patients enrolled in a phase 1b clinical trial of a Chk-i and IR. <h3>Results</h3> TCGA analysis revealed CHK1/2 activity negatively correlates with innate immunity, inflammation and granulocyte chemotaxis, as these GO scores were significantly quelled in CHK1/2 amplified or overexpressed tumor samples, signifying a suppressive interplay between Chk-1/2 signals and anti-tumor immunity. qPCR revealed enhanced immune signatures related to recruitment of tumor associated macrophages (TAMs) in R-SCC1 as compared to SCC1, which was proportionally enhanced by Chk-i. The addition of IR differentially altered key facilitators of TAMs in SCC1 versus R-SCC1, such as increased CSF1 in R-SCC1 cells only, and decreased CXCL14 with a greater effect observed in SCC1. Further supporting a role of Chk-1/2 inhibitor sensitivity in immune suppression, baseline and Chk-i induced STAT6 expression, which may inhibit immune tolerogenic M2-macrophage polarization, was decreased in R-SCC1 as compared to SCC1. Baseline STAT1 gene expression was also elevated in SCC1 versus R-SCC1. Consistent with TCGA and preclinical data, clinical trial analyses support a link between tumor-directed immune signatures and Chk-i sensitivity, as tumors from HNSCC patients responsive to Chk-i were enriched in Th1 subsets and poorly responsive patient samples exhibited enhanced M2-macrophage activity. <h3>Conclusion</h3> Our study reveals a link between CHK1/2 activity and immune surveillance, as well as identifies key mediators of immune activation and macrophage polarization specific to Chk-i resistance, altogether implicating a mechanism of immune suppression that impedes clinical response to Chk-i + IR. Future studies validating tumor-directed immune signatures as biomarkers of efficacy of Chk-1/2 inhibition and identifying targetable mediators to enhance responsiveness are warranted.