Increased CCL2/CCR2 axis in MYC/BCL2 double-expressor DLBCL promotes tumor progression by increasing M2 macrophages

The pathogenesis of c-MYC and BCL2 double-expressor diffuse large B cell lymphoma (DE DLBCL) remains unclear, particularly in terms of the tumor microenvironment. We aimed to analyze the immune microenvironment of DE DLBCLs to understand the basis of DE-DLBCL's aggressiveness and identify potential therapeutic targets. To discover the key features of DE-DLBCL, fourteen non-DE and 14 DE DLBCL samples underwent RNA-seq analysis. For validation, formalin-fixed paraffin-embedded tissues from 126 patients were immunostained for CD3, CD4, CD8, FOXP3, CD68 and CD163. Two c-MYClow/BCL2low DLBCL (SU-DHL-10 and HT) and 2 c-MYChigh/BCL2high DLBCL cell lines (DOHH2 and OCI-LY8) were used for in vitro assays. The prognostic impact of differential chemokine secretion and immune profiles was evaluated in a mouse tumor model. RNA-seq analysis revealed significantly higher CCL2 and CCR2 mRNA in DE DLBCLs (Padj < 0.05). Public datasets showed higher M2/macrophage ratio and lower T cell infiltration in DE DLBCLs (P < 0.05). Immunohistochemistry confirmed higher M2 macrophages and M2/total macrophage ratio in DE DLBCL (P=0.010 and P=0.098, respectively). In vitro, c-MYChigh/BCL2high cell lines exhibited increased CCL2 secretion, as seen in RNA-seq analysis. Knockdown and overexpression studies identified that MYC and BCL2 overexpression increased CCL2 secretion by upregulation of NF-κB p65 subunit, and the increased CCL2 secretion was crucial for M2 polarization. In mouse tumor models, CCL2 was related to aggressive tumor growth and an immunosuppressive tumor microenvironment. In conclusion, the increased CCL2/CCR2 axis confers aggressiveness of DE DLBCLs by increasing M2 polarization.