A PRRX1 Signature Identifies TIM-3 and VISTA as Potential Immune Checkpoint Targets in a Subgroup of Microsatellite Stable Colorectal Cancer Liver Metastases

Disease recurrence and drug resistance are major challenges in the clinical management of patients with colorectal cancer livermetastases (CLM), and because tumors are generally microsatellite stable (MSS), responses to immune therapies are poor. The mesenchymal phenotype is overrepresented in treatment-resistant cancers and is associatedwith an immunosuppressed microenvironment. The aim of this work was to molecularly identify and characterize a mesenchymal subgroup of MSS CLM to identify novel therapeutic approaches. We here generated a mesenchymal gene expression signature by analysis of resection specimens from 38 patients with CLM using ranked expression level of the epithelial-to-mesenchymal transitionrelated transcription factor PRRX.. Downstream pathway analysis based on the resulting gene signature was performed and independent, publicly available datasets were used to validate the findings. A subgroup comprising 16% of the analyzed CLM samples were classified as mesenchymal, or belonging to the PRRX1(high) group. Analysis of the PRRX. signature genes revealed a distinct immunosuppressive phenotype with high expression of immune checkpoints HAVCR2/TIM-. and VISTA, in addition to the M2 macrophage marker CD163 The findings were convincingly validated in datasets from three external CLMcohorts. Upregulation of immune checkpoints HAVCR2/TIM-3 and VISTA in the PRRX.high subgroup is a novel finding, and suggests immune evasion beyond the PD-1/PD-L1 axis, which may contribute to poor response to PD-1/PD-L1-directed immune therapy in MSS colorectal cancer. Importantly, these checkpoints represent potential novel opportunities for immune-based therapy approaches in a subset of MSS CLM.