Regulation of PD-L1: Emerging Routes for Targeting Tumor Immune Evasion

1Over the past decades, a novel therapy that utilizes human immune system to treat cancer is increasingly popular, which is known as cancer immunotherapy. The immunosuppressive microenvironment of tumor is one of the six distinct biological properties that enable tumor growth and metastasis. Human tumors typically harbor genomic instability, which induce somatic mutations. Accumulation of mutations may facilitate tumor growth and metastasis, while some non-synonymous mutations, leading to replacement of amino acid residual, create new T cell epitopes (neoepitopes), offering opportunities for immune system to recognize and eliminate cancer cells. It has been reported that the number of non-synonymous mutations, defined as mutational load, is closely related with the efficacy of immunotherapy. However, cancer cells collaborate with immune cells to dodge the immune destruction, and the anti-cancer pathway is intervened in this microenvironment. The depressed immunology of T cells, if appropriately empowered, may be an efficient and powerful weapon against cancer. Specifically, active vaccination, adoptive cell transfer therapy and immune checkpoint blockade are the three major approaches that could turn on T cell-based anti-cancer immune reaction. In recent years, immune checkpoint blockade therapy (ICBT) has exhibited momentous clinical benefits, placing tumor immunotherapy under the spotlight. PD-L1, a type I transmembrane protein with an extracellular N-terminal domain, inhibits the immune response through interaction with receptor PD-1 expressed on T cells. Under physiological conditions, PD-L1 is expressed in a wide range of cell types and tissues and shown to be overexpressed with immune activation, such as inflammations. The PD-L1/PD-1 axis maintains the balance between tolerance and autoimmunity and thus deficiency or excess function of it can lead to a variety of disease. Many auto-immune diseases have been found to be associated with PD-L1/PD-1 disruption including arthritis and lupus. PD-L1 expression has been found positive in 5–40% tumor cells, helping them to dodge the immune elimination through interaction of PD-L1 on the surface of cancer cells with PD-1 on T cells. Thus, blockade of PD-L1/PD-1 axis assists the recognition and elimination of cancer cells. PD-L1 expression on tumor cells has been reasonably detected as a biomarker of ICBT. Further investigation revealed that the inducible but not continuous expression of PD-L1 is associated with activated CD8+ T cells in hepatocellular carcinoma, although the expression of PD-L1 is not independently prognostic.

2The binding of immune checkpoint inhibitors and optimal targets is the core idea of ICBT. By inhibiting the immune-suppressive pathways, ICBT allows the clearance of cancer cells by the immune system. Several immune checkpoints are discovered to be optimal targets for immune blockade, including the cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell-death protein 1 (PD-1)/programmed cell-death 1 ligand 1 (PD-L1) pathways. Drugs targeting these two pathways have nourished recently and many of them have been approved by FDA. Drugs that target PD-1 like Pembrolizumab (Keytruda) and Nivolumab (Opdivo) were approved in 2014. Some PD-L1 inhibitors were also approved including Atezolizumab (Tecentriq) (2016), Avelumab (Bavencio) (2017) and Durvalumab (Imfinzi) (2017). Ipilimumab (Yervoy) is a monoclonal antibody targeting CTLA-4 that gained approval in 2011. Information comes from the official website of United States Food and Drug Administration. Notably, inhibitors targeting PD-1 or PD-L1 have been found to be especially advantageous in the treatment of many kind of cancer, including non-small cell lung carcinoma (NSCLC), renal cell carcinoma (RCC), bladder cancer, breast cancer, melanoma and Hodgkin’s lymphoma. The landscape of cancer therapy is evolving with deeper and wider acknowledgment of Immunotherapy with PD-1 or PD-L1 blockade.

3Despite of the promising laboratory results and many positive clinical applications, there seems to be a discount on its overall clinical benefits due to intrinsic and/or acquired resistance to this therapy. In certain cancer patients, the significant clinical response and enduring tumor retardation achieved by ICBT have improved patient progress-free survival (PFS) and overall survival (OS). However, the efficacy rate and profits of usage in general patients remain at a modest level, impeding the widespread application of ICBT. The tumor immunogenicity is a multi-level and delicately modulated process. Therefore, accumulation of mutations may lead to dysregulation of immunogenicity and create an immunosuppressive microenvironment, causing intrinsic resistance to ICBT. Among them is the insufficiency of T cell infiltration. On the other hand, after the significant retardation and durable response of tumor when initially treated with anti-PD-1 therapy, relapses in the long term were observed even after continuous therapy. The acquired resistance to ICBT in melanoma was reported to be associated to antigen presentation deficiency, in which the interferon signal pathway was involved. Alternative checkpoints were discovered to be adaptively upregulated after PD-L1 targeting treatment. Moreover, PD-L1 upregulation after chemotherapy and nivolumab treatment was reported as a potential cause of acquired resistance. In these tumors, immune evasion involves PD-L1/PD-1 interaction, which is the reason why the therapy initially worked. But the aftereffect of increased PD-L1 may have partially restored PD-L1/PD-1 function by providing more PD-L1 sites that were not neutralized by injected antibodies. Nonetheless, not enough investigations have been done to clarify the adaptive upregulation of PD-L1. In this scenario, understanding the mechanisms of PD-L1 regulation in cancer cells would certainly benefit the development of more effective and durable ICBTs.

4While the PD-1/PD-L1 pathway has been proven both theoretically and clinically a mature and efficient target for immunotherapy, it is of urgent need to develop more effective approaches to target PD-L1. Firstly, many disadvantages of PD-L1 targeted antibodies are unneglectable. The relatively large size of Mono-antibodies (MAbs) may prohibit its penetration into the complex tumor microenvironment, and thus limiting the therapeutic efficacy. It is crucial to develop new drugs with smaller sizes and to improve the specificity of tumor PD-L1 targeting, even though existing drugs and research are flourishing.

5Secondly, the primary and acquired resistance to ICBT in many tumors highlights a crucial requirement for developing alternative PD-L1/PD-1-targeting approaches. Several cancer mutations have been suggested to be the cause of PD-L1 suppression and therefore primary resistance to PD-L1 blockade drugs. Inactivation mutations of JAK1/2 is an example. Thirdly, As a protector of host tissue and regulator of inflammation, PD-1/PD-L1 is located not only on tumor cells but also on normal cells, including anti-tumor T cells and tumor associated macrophages. The blockage of physiological PD-1/PD-L1 functions inevitably brings about unfavored results- the depletion of cells which are meant to be activated and functioning. Lastly, the activation of oncogenic pathways, including RAS/RAF/MAPK and PI3K signaling, combined with the complexity of tumor microenvironment, may desensitize anti-tumor immunity. The main components of tumor microenvironment, including infiltrated T cells, metabolites (will be further discussed) and oxidative stress, have been reported to be disruptors of anti-tumor immunity. Our understanding on the mechanisms of ICBT resistance and PD-L1 regulation remains rather limited, proposing an urgency to decode the multifaceted roles and complex control of PD-L1 in cancer.

6The enthusiastic devotion from both clinical and biological investigators have brought the PD-1/PD-L1 biology into a new era in cancer research. Translational studies targeting the PD-1/PD-L1 pathway have boosted dramatically in recent years. Some progresses in the research of PD-L1 expression in cancer, especially at transcriptional and epigenetic levels, have been forged into a regulatory model for unified explanation. However, more recent findings that shed light into the multifaceted control of PD-L1 as a membranous protein has not been systematically discussed. In this review, we will summarize the exciting progresses in PD-L1 research in a more comprehensive manner, aiming to facilitate future basic and translational studies in the field of cancer immunotherapy.

7Enhanced PD-L1 expression was detected in a wide range of cancers but the prognostic and predictive value of it is controversial. It’s also a sign of efficacy of ICBT targeting PD-1/PD-L1, as reported in B-cell lymphomas, breast cancer, small-cell lung cancer and pancreatic cancer. Given that many oncogenes are upregulated by gained copy number alterations (CNAs), efforts have been made to clarify the relationship between PD-L1 expression and CNA. As the main form of CNA, PD-L1 copy number amplification directly leads to PD-L1 mRNA upregulation. Tumors harboring PD-L1 amplification presents significantly higher load of mutation, comparing to non-amplified subjects. Increased copy number of chromosome 9p24, predominant amplification of focal gene CD274 (which resides on chromosome 9p24.1, as shown in Figure 1), together with abundant PD-L1 expression were observed in a subset of small-cell lung cancer (SCLC). The Janus kinase 2 (JAK2) amplification was documented to be simultaneously activated with 9p24.1 chromosome copy number amplification and upregulated PD-L1 expression in primary cancers (Figure 2), suggesting a possible transactivation between JAK2 and PD-L1 genes. What’s more, PD-L1/PD-L2 alterations were defined as a feature of Classical Hodgkin lymphomas (cHLs). Specifically, amplification of 9p24.1 was reported to be associated with patients’ advanced stage disease and poor prognosis in cHL and in Epstein-Barr virus-associated gastric cancer (EBVaGC). These findings collectively suggest that CD274 gene amplification is a crucial factor that drives PD-L1 expression in cancer, and thus targeting PD-L1 at genetic level may be a rationalized strategy in PD-L1 positive tumors. Considering the rapid development of gene therapies, such prospect won’t be infeasible.

8Structural variations may also be responsible for elevated transcription of PD-L1. For example, truncation of its 3′UTR was reported to be associated with aberrant PD-L1 expression in multiple cancers.

9Epigenetic regulation was revealed to be involved in PD-L1 expression in cancer cells. Micro RNAs (miRNAs), defined as 22–24 nucleotides non-coding single-stranded RNAs, have been implicated in the regulation of PD-L1 expression. The binding of some miRNAs to the PD-L1 mRNA causes the latter one to degrade and thus PD-L1expression is suppressed. Specifically, the abundance of miR-513, miR-570, miR-34a, and miR-200 were reported to have an inverse correlation with PD-L1 expression, as described in Figure 1. Among them is miR-513 which inhibits PD-L1 protein translation by binding to 3′ untranslated regions (UTRs) of PD-L1 RNA as complement. Supportively, IFN-γ-induced PD-L1 expression was diminished by introducing miR-513 into Jurkat cells, while anti-miR-513 enhanced PD-L1 expression in cholangiocytes. Similar function was found with miR-570. Research has shown that mutation of the PD-L1 3′ UTR which disrupts the association with miR-570, correlated with overexpression of PD-L1. P53 was reported to regulate PD-L1 through miR-34. In the case of miR-200, the process of epithelial-to-mesenchymal transition (EMT) is found to be mediated by the regulation of PD-L1 expression by miR-200. Moreover, MiR-197 was reported to repress STAT3, a regulator of PD-L1, to decrease PD-L1 expression, as demonstrated in Figure 1. Other miRs reported to regulate PD-L1 includes miR-424, miR-138, miR-17 and cluster miR-25-93-106b. Most recently, a mechanism that stabilizes PD-L1 mRNA was reported through modulation of the AU-rich element-binding protein tristetraprolin (TTP).

10Recent studies have also focused on the promoter methylation of PD-L1 (mPD-L1), which was suggested to be a biomarker for prediction of response to PD-1/PD-L1 targeted ICBT. Significant inverse correlations between mPD-L1 and patient age was reported. The correlation between mPD-L1 and PD-L1 mRNA expression shares similar pattern, indicating a potential interaction between patient age and methylation of PD-L1 gene and that promoter methylation suppresses PD-L1 expression in colorectal cancer (CRC). Correlation between PD-L1 promoter methylation and clinical outcomes was also revealed in other cancers including NSCLC and prostate cancer. Moreover, in patients treated with PD-1/PD-L1 targeting drugs, enhanced mPD-L1 is associated with worse overall survival and recurrence-free survival. Epigenetic therapy has also been suggested to sensitize tumor response to PD-L1 targeting drugs. Interestingly, results proved no meaningful correlation between PD-L1 mRNA expression and patients’ outcome.

11Several transcriptional factors have been found to control PD-L1 transcriptional activation (Figure 2). As an example, PTEN represses PD-L1 transcription and expression in breast cancer cells, suggesting a new tumor suppressive function of PTEN. In addition, PD-L1 expression decreased after inhibition of phosphoinositide 3-kinase (PI3K) pathway using the AKT inhibitors, further emphasizing the role of PTEN and PI3K signaling in PD-L1 regulation. Transcription activity, demonstrated by the level of PD-L1 mRNA expression, was promoted through JAK2/STAT1 pathway, as was shown in pancreatic cancer cells treated with anticancer agents (5-fluorouracil, gemcitabine, or paclitaxel). Notably, when treated with chemotherapeutic drugs, the MAPK pathway was also reported to upregulate PD-L1 in cancer cells. While distinct signaling pathways share the ability to control PD-L1 expression by regulating its transcription, the exact mechanisms involved may vary considerably.

12Hypoxia inducible factor 1α (HIF-1α) is a major cancer driver and a potential therapeutic target. The binding of HIF-1α to PD-L1 promoter, a hypoxia response element (HRE), stimulates the transcription of PD-L1. Research has revealed the co-existence of HIF-1α overexpression, increased PD-L1 level, and repression of T-cell function. It was also reported that PD-L1 works predominantly in lactate-enriched tumor microenvironments. Meanwhile, T cell autophagy is induced in a microenvironment lack of amino acids tryptophan and arginine as well as glucose. In this nutrients-deprived situation, glucose metabolism shrinks while the lactate accumulates, creating an optimal environment for PD-1/PD-L1 interaction and resistance to cancer therapies consequently. In other words, Lactate, as a major metabolite under hypoxia condition, may protect tumor cells from cytotoxic T-cell targeting. Accordingly, tumor cell metabolic reprograming was found to correlate with immune suppression. Taken together, it is suggested that hypoxic environments, which induce activation of HIF-1α and accumulation of lactate, contribute to evasion of tumor cells from immune system. The transactivation of PD-L1 by HIF-1 represents a crucial step in the above-mentioned process, and may be a promising target to combat the immune suppression of tumor cells.

13STAT3 is another important transcriptional factor that upregulates PD-L1 expression by binding to PD-L1 promoter. Mutations of oncogene chimeric nucleophosmin/anaplastic lymphoma kinase (ALK) have been found to upregulate PD-L1 expression, and this effect could be abolished by silencing STAT3. Furthermore, Latent membrane protein-1 (LMP1) of Epstein-Barr virus was found to increase both PD-L1 expression and STAT3 phosphorylation (p-STAT3) (Figure 2). Consistently, the JAK3 inhibitor CP-690550 blocked the above process through suppressing p-STAT3. NF-κB, as a transcriptional factor mediating inflammation-associated tumorigenesis, has been reported to boost PD-L1 expression. However, the exact mechanisms remain unclear. NF-κB is required for LMP1-induced PD-L1 expression, which is evidenced by decreased PD-L1 induction caused by NF-κB inhibitors. Notably, the NF-κB inhibitor abolished INF-induced PD-L1 expression, while MAPK, PI3K and STAT3 inhibitors did not. Thus NF-κB also seems to be involved in INF-γ-induced PD-L1 expression.

14N-glycosylation is a crucial protein modification that determines protein structure and function, especially the function of membrane proteins. By altering protein conformation, glycosylation may modulate protein activities and protein–protein interactions, such as those between ligands and receptors. In Western Blot assays, the majority of PD-L1 is detected at 45 kDa representing the glycosylated species, while the non-glycosylated form is detected at 33 kDa. By bioinformatics prediction, mass spectrometry and mutagenesis, PD-L1 was found to be exclusively N-glycosylated at N35, N192, N200, and N219.

15The PD-L1 molecule containing N192, N200, and N219 residues forms a region that is the prerequisite for PD-L1 binding to GSK3β, and N-glycosylation on these sites buries the necessary residues and disrupts the interaction between PD-L1 and GSK3β. Glycogen synthase kinase 3beta (GSK3β), a serine/threonine protein kinase, was originally identified as a regulator of glycogen metabolism. When bound to non-glycosylated PD-L1, GSK3β leads to phosphorylation and consequent ubiquitination of PD-L1 (Figure 3). In addition, it was further elucidated that inactivation of GSK3β by activating EGFR enhanced PD-L1 expression by preventing it from being ubiquitinated. Significantly, a small molecular inhibitor of glycosylation, tunicamycin, was found to efficiently decrease PD-L1 expression in cancer cells. Latest results have provided evidence that targeting glycosylated PD-L1 promotes PD-L1 internalization and degradation, leading to eradication of triple-negative breast cancer cells.

16Phosphorylation involves in a widespread of regulatory mechanisms in cellular signaling, and may affect the conformation, activity, and interactions of proteins. Although one protein may contain multiple phosphorylation sites, the phosphorylation of PD-L1 has been sparsely reported. As mentioned above in the glycosylation part, GSK3β is a multifunctional switch that mediates the direct phosphorylation of a wide range of substrates, including e IF2B, cyclin D1, c-Jun, c-myc, NFAT, MCl-1, and Snail. It also contributes to the phosphorylation of PD-L1 through an evolutionarily conserved GSK3β phosphorylation motif on PD-L1 (Figure 3). Furthermore, the phosphorylation mediated by GSK3β has been found to initiate the interaction with E3 ligase, which targets proteins to proteasomal degradation.

17Meanwhile, it was reported that treatment of the epidermal growth factor (EGF) would induce tyrosine phosphorylation, together with acetylation and ubiquitination of PD-L1. These provide evidential hypothesis for the effects of Gefitinib, an inhibitor of EGFR, in promoting the immune response against breast cancer. Gefitinib was found to cut down on PD-L1 expression and limit its oncogenic potential, therefore promoting T cell immunity. These findings suggest that targeting EGFR by Gefitinib not only suppresses MAPK-dependent tumor proliferation, but also blocks PD-L1-dependent immune suppression. Based on the predicted isoelectric points corresponding to different modifications, the PhosphoSite database has listed potential phosphorylation sites of PD-L1 (basal Isoelectric point = 6.76). However, no systematic experimental characterization of PD-L1 phosphorylation has been carried out. It also deserves in-depth study how PD-L1 phosphorylation varies and fluctuates in response to distinct microenvironments, therapeutic stresses and interaction with its partner proteins.

18Ubiquitination-dependent proteasomal degradation controls the metabolism of many proteins, including membrane proteins like PD-L1. As mentioned above, the EGF treatment may induce tyrosine phosphorylation, acetylation, and ubiquitination of PD-L1. The increased PD-L1 mono- and multi-ubiquitination induced by EGF were blocked by gefitinib treatment. Recent study further revealed that ubiquitin E3 is involved in PD-L1 downregulation in EGFR wild-type NSCLC. In a recent study, cyclin D-CDK4 kinase was reported to destabilize PD-L1 via cullin 3-SPOP, which was proved to be involved in Pd-L1 ubiquitination. Surprisingly, the EGF-stimulated PD-L1 mono-ubiquitination not only coexisted with PD-L1 overexpression, but also seemed to occur ahead of its upregulation. Inhibition of the ubiquitin E1 by blocking its activating enzyme decreased PD-L1 mono- and multi-ubiquitination and total PD-L1 protein expression at the same time, suggesting a possible causal relationship between ubiquitination and overexpression of PD-L1.

19CMTM6, a type-3 transmembrane protein was recently identified as a positive regulator of PD-L1. Decrease of CMTM6 expression downregulated PD-L1 protein level in a wide range of human tumor cells and in primary human dendritic cells. Apart from CMTM6, its closest family member, CMTM4, was confirmed to share similar function (Figure 3). Of note, the enhancement of PD-L1 protein pool stimulated by CMTM6 was not associated with any variation in PD-L1 transcription. Instead, CMTM6 was found to interact with PD-L1 on cell surface, interfering its ubiquitination to prolong its half-life. It was also functionally confirmed that by enhancing PD-L1 protein pool, CMTM6 improves the evasion ability of PD-L1positive tumor cells to immune elimination.

20On the contrary to ubiquitination, deubiquitination of PD-L1 stabilizes the protein from degradation. The deubiquitination and stabilization of PD-L1 significantly affect the inflammatory response or so-called ‘inflammation-mediated anti-tumor immunity’. Recently, COP9 signalosome 5 (CSN5) was identified as a crucial protein that promotes the deubiquitination of PD-L1 (Figure 3). It was reported that tumor necrosis factor alpha (TNF-α), as one of the major inflammatory cytokines secreted by macrophages, plays an important role in maintaining cancer cell evasion from immune system. Mechanistically, TNF-α may activate NF-κB and induce CSN5 expression, leading to PD-L1 stabilization. Consistently, CSN5 has been found to be indispensable for TNF-α-mediated PD-L1 stabilization because of its function in deubiquitinating PD-L1. With potential translational significance, the authors found that destabilization of PD-L1 by curcumin, an inhibitor for CSN5, may benefit immunotherapy.

21PD-L1 functions on the membrane surface, but it may also translocate into the cytoplasm. Many membrane proteins are shuttled between the recycling endosomes and cell surface, and PD-L1 has been tracked in recycling endosomes. Furthermore, inhibition of endocytic recycling by primaquine caused vast depletion of membrane PD-L1 protein level in wild-type cells. These results suggest that: first, a large proportion of membrane PD-L1 undergoes metabolism and internalization continuously; second, the dynamic recycling and releasing of PD-L1 maintains the amount of PD-L1 located on cell membrane. Notably, CMTM6, recognized as a PD-L1 regulator, is predominantly identified in recycling endosomes together with TFRC and RAB11, factors that define the endocytic recycling compartment. What’s more, CMTM6 co-localizes with PD-L1 both on the plasma membrane and in recycling endosomes, so that CMTM6 functions as a protector of PD-L1 that prevents it from being targeted for lysosome-mediated degradation and increases its protein pool (Figure 3).

22Interestingly, membrane and cytoplasmic PD-L1 expression is more significant in macrophage cells than in cancer cells. Studies have been done to test PD-L1 molecule in peripheral blood mononuclear cells (PBMC) and surprisingly revealed a novel human PD-L1 splice variant in activated PBMC. Further studies compared the conventional isoform with the novel isoform and found distinct localization patterns between both proteins. Specifically, the conventional isoform is predominantly expressed on the plasma surface, while the novel isoform is distributed mainly on intracellular membrane. The alternative splicing of PD-L1 may be a posttranscriptional regulator that modulates PD-L1 expression as well as its function in determining the outcome of specific immune responses in the peripheral tissues.

23In addition to its cellular distribution, PD-L1 has also been detected outside the cells, proposing its potential role as a semi-invasive biomarker. An A/C polymorphism at position 8923 was detected together with increased level of plasma soluble PD-L1 (sPD-L1) in NSCLC patients, especially those with adenocarcinoma. Investigation is now undergoing to define the value of plasma PD-L1 protein levels as a predictive biomarker of prognosis in NSCLC and also as a reliable companion diagnostics for individualized treatment with ICBT.

24Unlike cytosolic proteins, many membrane proteins are mainly degraded through the lysosomal pathway. As mentioned in the ubiquitination part, CMTM6 reduces PD-L1 ubiquitination and increases its stability. Interestingly, different opinion presents another explanation about the stabilization of membrane PD-L1 by CMTM6. In addition to its expression at the plasma membrane, CMTM6 is predominantly identified in recycling endosomes. Although CMTM6 is not required for PD-L1 maturation, it functions in protecting PD-L1 from lysosome-mediated degradation. Thus, CMTM6 depletion, via the reduction of PD-L1, significantly alleviates the suppression of tumor-specific T cell activity in vitro and in vivo. Although there is no doubt that CMTM6 suppresses PD-L1 degradation, the effect still seems to be indirect, requiring the competitive transportation to the recycling endosome. It remains unclear which protein may directly interact with CMTM6 and transport it to lysosome for degradation (Figure 3). Future efforts to clarify this crucial node would benefit the development of alternative PD-L1-targeting approaches.

25Some mutations of PD-L1 gene may impede the protein level of PD-1/PD-L1 but others may cause disturbance on protein folding, and therefore disrupt the interaction of PD-1 and PD-L1. PD-1 and PD-L1 bind through the conserved front and side of their Ig variable (Ig V) domains, representing the structural basis for the design of intervention molecules. By locating the loops at the ends of the IgV domains on the same side of the PD-1/PD-L1 complex, a surface is formed, being similar to the antigen-binding surface of antibodies and T-cell receptors. Several residues have been identified to play important roles in folding and forming the PD-1/PD-L1 interface. The immune receptor-like loops provide a new surface for further study and potentially the design of molecules that would affect PD-1/PD-L1 binding and thereby regulate the immune system. Multiple peptides and small-molecular compounds have been evaluated in preclinical models, in order to develop novel PD-1/PD-L1 inhibitors.

26In addition to directly block the interaction between PD-1 and PD-L1, methods have also been developed to inhibit the dimerization of PD-L1, and hence the PD-1/PD-L1 interaction. Particularly, this effect could be achieved by small molecular compounds such as BMS-202 and BMS-8, with considerable translational significance. Since small molecules behold advantages in terms of production scale, quality standardization, pharmacological kinetics and tissue distribution, it is of enormous interest to discover small molecular drugs targeting the PD-L1/PD-1 axis. Despite the structural insights provided by recent crystallographic research, it is still unclear how the reported PTMs, e.g., glycosylation, phosphorylation, ubiquitination, etc., may affect the conformation and molecular interactions of PD-L1/PD-1. Understanding these detailed processes would also improve the confidence of structure-based drug design targeting this crucial immune suppression signaling pathway.

27PD-L1-targeted ICBT is a promising breakthrough in the field of cancer immunotherapy, but primary and acquired resistances have presented enormous challenges in this fast-evolving area. It has been suggested that the post-treatment positive conversion of PD-L1 expression may be a cause of resistance. The regulatory pathways of PD-L1 are of meaningful potential to be translated into therapeutic approaches for tackling the resistance to ICBT. The significant PD-L1 overexpression found in multiple cancer types may be an output of interconnected regulatory network, which involves molecular alterations at genetic, epigenetic, transcriptional, translational, post-translational, and structural levels. In fact, several key regulators of PD-L1 have long been established as cancer-related genes, such as JAK2, PTEN, MAPK, PI3K, HIF-1α, STAT3, TNFα, NF-κB, and INF-γ, etc. Existing small molecular compounds targeting these genes/pathways may be repurposed for modulating PD-L1, thus providing readily tools to improve T cell-dependent anticancer immunity. Likewise, the discovery of key post-transcriptional modifications (PTMs) that control PD-L1 stability such as glycosylation, phosphorylation, and ubiquitination also provide alternative strategies for targeting PD-L1. It is worthy to further analyze the function of curcumin (CSN5 inhibitor) and tunicamycin (glycolysis inhibitor) in suppressing PD-1/PD-L1 signaling in vivo and in preclinical models. The inhibitors o In addition, the connection between cancer metabolism and resistance to immunotherapy suggests potential benefit for combined targeting of tumor glycolysis and PD-1/PD-L1 axis. Apart from controlling the abundance of PD-L1 in cells, the mechanisms underlying PD-L1 transportation and structural modulation may also provide novel strategies to optimize the blockage of PD-L1. With the multifaceted regulation of PD-L1 being revealed, it would be more feasible to develop complementary therapies to sustain the response once cancer cells acquire resistance to the initial treatment.

28The prosperity and challenges of immunotherapies targeting the PD-1/PD-L1 axis warrant increasing attentions by biological and pharmaceutical scientists. In our opinion, several research directions would be especially beneficial to a sustained improvement of ICBT.

29Firstly, the regulation of PD-L1 should be further clarified in more specified conditions, considering the variations in tumor regions and developmental stages. It has been suggested that PD-L1 expression may differ considerably on the tumor boundary. Cells located here have higher accessibility where immune cells encounter the tumor cells. Thus, tissue sampling by traditional methods may not robustly capture such alterations and result in low fidelity in different assays such as Western Blot, qPCR and microarray tests. On the other hand, hypoxia-related induction of PD-L1 is more likely to occur in the center of solid tumors where oxygen is less accessible. Moreover, our recent study found that PD-L1 is significantly upregulated in metastatic CRCs while compared to primary tumors. Thus, the regulation of PD-L1 during metastasis and its corresponding biomarker significance should be considered differentially from those in the primary tumors. To investigate the regulation of PD-L1 in tumors, it is essential to precisely mark the region and stage (e.g., primary vs. metastatic, pre-treatment vs. post-treatment, etc.) of a particular patient, because these variations are associated with the indicated mechanisms.

30Secondly, the link between PD-L1 expression and cancer subtyping has been investigated based on genomic and transcriptomic characterizations of tumors. In many tumors, the microsatellite instability (MSI) subtype is linked to PD-L1 positivity and considered as a key factor indicating the suitability for checkpoint blockade therapy. Even though, more comprehensive understanding on the implications of PD-L1 in cancer subtyping should also be founded by insights into the epigenetic and metabolic reprograming of cancer cells. As described previously, epigenetic and metabolic alterations in tumors are emerging as crucial factors affecting the abundance of PD-L1. In a translational perspective, significant and functional alterations at these facets may also present novel biomarkers and intervention opportunities.

31Thirdly, it deserves tremendous efforts to clarify the overlaps and differences between PD-L1 and its homolog PD-L2 in their functions and regulations in various tumors. Although PD-L2 was initially considered to be mainly expressed in immune cells, recent studies have revealed its positive expression in different tumor cells with potential prognostic significance. As an example, we found that PD-L2 is expressed in a considerable subset of CRC cells, with independent association with poor patient survival. It is thus of interest to clarify the relative importance of PD-L1 and PD-L2 in a specific tumor type. Will one protein compensate the function of the other, or be upregulated when its homolog is blocked in immunotherapy? Which ligand of PD-1 may play a predominant role in suppressing T-cell immunity in a given cancer type of patient, and should this be considered when optimizing the strategy for immunotherapy? These questions should be addressed, in order to understand and improve the effectiveness and sustainability of ICBT.

32Finally, the structure-based drug design targeting PD-L1 may not be limited in the binding surface to PD-1 or the site mediating its dimerization. If allosteric control of PD-L1 activity could be identified, additional approaches targeting PD-L1 would be feasible. Moreover, the protein interactions between PD-L1 and its reported regulators (e.g., CSN5, CMTM6, etc.) could be characterized in and enough resolution, rational design of blocking peptides or compounds may also be developed. In other words, basic research about the structural dynamics and detailed interaction sites of PD-L1 may provide additional resources for the development of de novo PD-L1 targeting approaches.

33Immune checkpoint blockade therapy represents a breakthrough in cancer treatment, but the primary and acquired resistance to immunotherapy warrant further efforts to understand the multifaceted regulation of PD-L1 in cancer. As a cell surface protein that responds to microenvironment stimuli, PD-L1 reacts promptly to balance the outside stresses and inside requirements of cells, representing a key node in the cancer signaling network. In this scenario, the effective and sustained targeting of PD-L1 has to take the complexity of its regulation into account. Identification of the exact causes of PD-L1 upregulation and responsive functional compensations in a broader range of molecular events would improve the targeting specificity and efficiency. A chasm is yet to be crossed by obtaining small molecular inhibitors of PD-L1 in addition to antibody drugs, to improve the cancer distribution and metabolic kinetics of immunotherapeutic medicines. Current approaches for targeting PD-L1 could also affect its normal functions in immune cells, with expected unwanted effects. In these scenarios, targeting PD-L1 effectively and specifically in cancer cells remains a Gordian knot.

34YW and JX wrote the manuscript. HW, HY, CL, and J-YF contributed to revisions of the manuscript.

35The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.