Distinct biological and immune microenvironment features of MSI-H BRAF ^V600E colorectal cancer: Potential for combined PD-1, BRAF, and EGFR inhibition.

Abstract

e14598 Background: Immune checkpoint inhibitors (ICIs) have shown remarkable efficacy in microsatellite instability-high (MSI-H) colorectal cancer (CRC). However, a substantial subset of patients fails to achieve durable clinical benefit. BRAF mutations—particularly BRAF V600E —are associated with aggressive tumor behavior and poor clinical outcomes, and targeted therapies provide effective treatment options. Epidemiological studies reveal that BRAF mutations frequently coexist with MSI-H status. Nevertheless, the optimal treatment strategy for MSI-H BRAF V600E CRC remains unclear, including whether these patients benefit most from ICIs alone, targeted therapy alone, or combined ICIs and targeted therapy therapy. This study aims to comprehensively characterize the biological properties and tumor immune microenvironment (TME) in MSI-H BRAF V600E CRC, with the goal of improving therapeutic strategies for MSI-H BRAF V600E CRC. Methods: MSI-H BRAF WT and MSI-H BRAF V600E cell lines were generated from the MC38 background. The biological behaviors of cells were evaluated using colony formation, CCK-8, transwell, and Western bloting. Flow cytometry and multiplex immunofluorescence were performed to evaluate immune cell infiltration. RNA sequencing was conducted to explore underlying molecular mechanisms. Therapeutic efficacy was assessed in vivo across four groups: vehicle, anti-PD-1, BRAFi+EGFRi, and anti-PD-1+BRAFi+EGFRi. Results: MSI-H BRAF V600E cells exhibited significantly enhanced malignancy. In vivo, MSI-H BRAF V600E tumors displayed profound alterations in the immune microenvironment, including reduced infiltration of CD8⁺ T cells, along with decreased IFN-γ and GZMB levels. Conversely, M2 macrophages were significantly increased. Multiplex immunofluorescence confirmed reduced CD8⁺ T cell infiltration, and elevated PD-1⁺ cell frequencies. Notably, MSI-H BRAF V600E tumors exhibited an increased proportion of PD-1⁺CD8⁺ T cells and greater spatial separation between CD8⁺ T cells and PD-L1⁺ tumor cells. In vivo, combined PD-1, BRAF, and EGFR inhibition resulted in the strongest tumor suppression in MSI-H BRAF V600E tumors, demonstrating a marked therapeutic advantage, including decreased infiltration of CD8⁺ T cells, along with decreased IL-2 and GZMB levels. Conclusions: MSI-H BRAFV600E CRC exhibits distinct molecular and immunological characteristics. Our findings demonstrate that triple-combination therapy targeting PD-1, BRAF, and EGFR substantially enhances antitumor activity in MSI-H BRAFV600E tumors, outperforming ICIs alone or targeted therapy alone. Collectively, these results support a refined molecular subclassification of MSI-H CRC and provide valuable insights for optimizing personalized immunotherapeutic strategies for CRC patients with MSI-H BRAFV600E.