Epstein−Barr virus-encoded EBNA2 alters immune checkpoint PD-L1 expression by downregulating miR-34a in B-cell lymphomas

Abstract

Cancer cells subvert host immune surveillance by altering immune checkpoint (IC) proteins. Some Epstein−Barr virus (EBV)-associated tumors have higher Programmed Cell Death Ligand, PD-L1 expression. However, it is not known how EBV alters ICs in the context of its preferred host, the B lymphocyte and in derived lymphomas. Here, we found that latency III-expressing Burkitt lymphoma (BL), diffuse large B-cell lymphomas (DLBCL) or their EBNA2-transfected derivatives express high PD-L1. In a DLBCL model, EBNA2 but not LMP1 is sufficient to induce PD-L1. Latency III-expressing DLBCL biopsies showed high levels of PD-L1. The PD-L1 targeting oncosuppressor microRNA miR-34a was downregulated in EBNA2-transfected lymphoma cells. We identified early B-cell factor 1 (EBF1) as a repressor of miR34a transcription. Short hairpin RNA (shRNA)-mediated knockdown of EBF1 was sufficient to induce miR-34a transcription, which in turn reduced PD-L1. MiR-34a reconstitution in EBNA2-transfected DLBCL reduced PD-L1 expression and increased its immunogenicity in mixed lymphocyte reactions (MLR) and in three-dimensional biomimetic microfluidic chips. Given the importance of PD-L1 inhibition in immunotherapy and miR-34a dysregulation in cancers, our findings may have important implications for combinatorial immunotherapy, which include IC inhibiting antibodies and miR-34a, for EBV-associated cancers.

The role of cancer-derived microRNAs in cancer immune escape

“…Increased immune checkpoint ligands especially PD-L1 is closely related with cancerassociated miRNA expression pattern. To be more specific, previous studies have indicated that the loss of miR-3609, miR-195-5p, miR-148a-3p, miR-873, miRNA-497-5p, miR-191-5p, miR-34a, and miR-138 closely correlated with the increased PD-L1 expression on numerous cancer cells [86][87][88][89][90][91][92][93][94]. In addition, Dong et al found that decreased miR-140, miR-142, miR-340, and miR-383 enormously elevated PD-L1 expression on cervical cancer cells [95].…”

EBV microRNA-BHRF1-2-5p targets the 3′UTR of immune checkpoint ligands PD-L1 and PD-L2

“…13 The EBV latent membrane protein-1 (LMP1) is known to induce PD-L1/L2 in malignant B cells. 14,15 EBNA2 also increases PD-L1, which it achieves indirectly via regulation of the PD-L1 targeting microRNA-34a (miR-34a). 15 By contrast, a role by which EBV might regulate PD-L1 and PD-L2 expression has to our knowledge not been described.…”

Immunomodulatory MicroRNAs in cancer: targeting immune checkpoints and the tumor microenvironment

“…Epstein-Barr virus (EBV) miR-BART cluster, which includes miR-BART-2, -4, -5, -18 and -22, was found to be expressed by solid tumors like CRC and gastric cancer and was associated with poor prognosis [63]. For example, miR-34a is downregulated by EBV, which enhances the PD-L1 expression and so the immune evasion [64]. For example, miR-34a is downregulated by EBV, which enhances the PD-L1 expression and so the immune evasion [64].…”

“…It is noteworthy that solid tumors with positive expression of miR-BART cluster show a prominent upregulation of PD-1 and PD-L1 immune checkpoint proteins [63]. For example, miR-34a is downregulated by EBV, which enhances the PD-L1 expression and so the immune evasion [64]. Interestingly, patients with high levels of EBV-miRNA have also parallel high expressions of PD-1, PD-L1, transforming growth factor beta 1 (TGFb1), IL-10, IFN-c, and TGFb2, which make them good candidates for immune checkpoint therapy [65].…”