BRAF D594A mutation defines a unique biological and immuno-modulatory subgroup associated with functional CD8+ T cell infiltration in colorectal cancer

Abstract

Background: BRAF non-V600 mutation occupies a relatively small but critical subset in colorectal cancer (CRC). However, little is known about the biological functions and impacts of BRAF class III mutation in CRC. Here, we aim to explore how D594A mutation impacts on biological behaviors and immune related signatures in murine CRC cells.

Methods: BRAF V600E (class I), G469V (class II) and D594A (class III) mutant cell lines were established based on MC38 cells. The biological behaviors of cells were evaluated in respect of cell growth, cell proliferation, cell apoptosis, cell migration and invasion by the methods of colony-forming assay, CCK-8 assay, Annexin V/PI staining and transwell assay. The concentrations of soluble cytokines were detected by ELISA. The membrane expression of immuno-modulatory molecules and the pattern of tumor infiltrating lymphocyte were evaluated by flow cytometry. The molecular mechanism was explored by RNA sequencing. Immunohistochemistry (IHC) staining was used for the detection of CD8α in tumor tissues. qRT-PCR and western blot were performed to assess the mRNA and protein expression. Anti-PD-L1 treatment and cytokines neutralization experiments were conducted in in vivo models.

Results: D594A mutant cells displayed lower grade malignancy characteristics than V600E (class I) and G469V (class II) mutant cells. Meanwhile, D594A mutation led to evident immuno-modulatory features including upregulation of MHC Class I and PD-L1. In vivo experiments displayed that the frequency of infiltrated CD8⁺ T cells was significantly high within D594A mutant tumors, which may provide potential response to anti-PD-L1 therapy. RNA sequencing analysis showed that D594A mutation led to enhanced expression of ATF3 and THBS1, which thus facilitated CXCL9 and CXCL10 production upon IFN-γ treatment. In addition, CXCL9 or CXCL10 neutralization reduced the infiltration of CD8⁺ T cells into THBS1-overexpressing tumors.

Conclusions: D594A mutant CRC exhibited lower aggressiveness and immune-activated phenotype. ATF3-THBS1-CXCL9/CXCL10 axis mediated functional CD8⁺ T cells infiltration into the microenvironment of D594A mutant CRC. Our present study is helpful to define this mutation in CRC and provide important insights in designing effective immunotherapeutic strategies in clinic.

Keywords: BRAF; CXCL9/CXCL10; Colorectal cancer; D594A; THBS1.