Examination of the Functional Relationship between PD-L1 DNA Methylation and mRNA Expression in Non-Small-Cell Lung Cancer

SIMPLE SUMMARY: For non-small-cell lung cancer (NSCLC) patients undergoing immunotherapy blocking the PD-1/PD-L1 interaction, the cancer cell PD-L1 expression level is a determinant of treatment efficiency. This study was conducted to determine whether PD-L1 DNA methylation regulates and can predict PD-L1 expression in NSCLC. Tumor biopsies and cell lines were analyzed for PD-L1 DNA methylation, mRNA expression, and protein expression. CRISPR-Cas9 and dCas9 fusions with TET1 and DNMT3A were used to change the PD-L1 DNA methylation levels. In this study, we found that the PD-L1 DNA methylation status functionally influences its expression. However, although methylation can be inversely correlated with the expression of PD-L1 in NSCLC lines, this association is not strong in NSCLC tumor samples. The fact that PD-L1 DNA methylation status does not inevitably mirror the expression level is important for future attempts to improve the effectiveness of PD-1/PD-L1 immunotherapy in NSCLC. ABSTRACT: Immunotherapy targeting the interaction between programmed cell death protein 1 (PD-1) and programmed death-ligand 1 (PD-L1) is a treatment option for patients with non-small-cell lung cancer (NSCLC). The expression of PD-L1 by the NSCLC cells determines treatment effectiveness, but the relationship between PD-L1 DNA methylation and expression has not been clearly described. We investigated PD-L1 DNA methylation, mRNA expression, and protein expression in NSCLC cell lines and tumor biopsies. We used clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR-Cas9) to modify PD-L1 genetic contexts and endonuclease deficient Cas9 (dCas9) fusions with ten-eleven translocation methylcytosine dioxygenase 1 (TET1) and DNA (cytosine-5)-methyltransferase 3A (DNMT3A) to manipulate PD-L1 DNA methylation. In NSCLC cell lines, we identified specific PD-L1 CpG sites with methylation levels inversely correlated with PD-L1 mRNA expression. However, inducing PD-L1 mRNA expression with interferon-γ did not decrease the methylation level for these CpG sites, and using CRISPR-Cas9, we found that the CpG sites did not directly confer a negative regulation. dCas9-TET1 and dCas9-DNMT3A could induce PD-L1 hypo- and hyper-methylation, respectively, with the latter conferring a decrease in expression showing the functional impact of methylation. In NSCLC biopsies, the inverse correlation between the methylation and expression of PD-L1 was weak. We conclude that there is a regulatory link between PD-L1 DNA methylation and expression. However, since these measures are weakly associated, this study highlights the need for further research before PD-L1 DNA methylation can be implemented as a biomarker and drug target for measures to improve the effectiveness of PD-1/PD-L1 immunotherapy in NSCLC.