Novel Mechanism of Post-Transcriptional Regulation of PD-L1 Expression By 3'-UTR Binding Proteins

Programmed cell death ligand 1 (PD-L1) plays a key role in tumor immune escape by negatively regulating cytotoxic T-cells (CTLs) via PD-1 receptors. Thus, those tumors with high PD-L1 expression are thought to be sensitive to PD-1/PD-L1 blockade, reactivating CTL reactions to tumors. However, the mechanism that regulates PD-L1 expression in tumor cells has not been fully elucidated, understanding of which would help to develop effective anti-tumor immunotherapy. Recently, we reported that disruption of 39-UTR of PD-L1 led to a remarkably enhanced expression of PD-L1 in a wide variety of human cancers, particularly adult T-cell leukemia/lymphoma (ATL) and diffuse large B-cell lymphoma. In these cancers, stability of PD-L1 transcripts is negatively regulated via their 39-UTR sequence, whose disruption thus, results in markedly elevated PD-L1 expression and immune evasion (Kataoka et al., Nature, 2016). It has been well established that non-coding regions (i.e., 59- and 39-untranslated regions (UTRs)) play important roles in the regulation of mRNA expression, which is accomplished by several transacting factors that bind to cis-regulatory elements within the UTR. Based on this knowledge, we investigated the mechanism that controls PD-L1 expression through 39-UTR sequence, primarily focusing on those transacting RNA-binding proteins (RNA-BPs). To determine the relevant regions within the 39-UTR which are capable of repressing PD-L1, several cell lines were transfected with luciferase reporter vectors, in which a luciferase coding sequence was concatenated to various deletion mutants of the PD-L1 39-UTR (Panel A). We identified two critical sequences, segment 5 and 2 within the PD-L1 39-UTR, whose existence significantly decreased luciferase expression (Panel B; 293T data, mean ± SD, * denotes t-test p PD-L1 39-UTR. The library was virally infected into cells and those with high expression of PD-L1 were concentrated by FACS, and the enrichment of each sgRNA was evaluated by high-throughput sequencer. The enriched sgRNAs localized to positions compatible with the luciferase assay, confirming that the two regions are actually responsible for the regulation of PD-L1 expression. Next, we searched RNA-BPs that bind to these regions in mass spectrometry, utilizing flag-peptide-tagged RNA pull-down method (Panel C). In brief, we synthesized PD-L1 39-UTR RNA segments along with β-actin mRNA as control in vitro, and conjugated a flag-peptide to their 39-ends. Using these RNA-baits, an immunoprecipitation experiment was performed in 293T cells and the co-immunoprecipitated proteins were analyzed by mass spectrometry. The data obtained from different segments were compared to each other. In addition to those proteins binding to multiple regions within the PD-L1 39-UTR, we found proteins that specifically interacted with the repressive segments (segment 5 and 2) identified through luciferase assays. Finally, to confirm that the proteins detected by mass spectrometry actually suppress PD-L1 expression, we performed knock-down experiments using siRNA designed for the RNA-BPs that are presumed to interact with the segment 5 and 2. Three siRNA constructs per gene were transfected to 293T cells and their effect on PD-L1 expression was evaluated by RQ-PCR. Panel D shows relative PD-L1 expressions for each siRNA targets with median line, which are grouped according to relevant PD-L1 39-UTR segments. The negative regulatory effect of these RNA-PBs on PD-L1 expression was largely confirmed. PD-L1 protein level was also increased, when these genes were knocked out by CRISPR/Cas9 system. Expression of these genes in ATL and other lymphomas was also evaluated. In summary, we identified critical sequences within the PD-L1 39-UTR and RNA-BPs that bind to these sequences and negatively regulate PD-L1 expression. Our findings should not only provide novel insight into the molecular mechanisms by which PD-L1 expression in tumor cell is regulated but also help to identify potential targets for immune therapy. Disclosures No relevant conflicts of interest to declare.