To explore whether a cross-talk exists between PARP inhibition and PD-L1/PD-1 immune checkpoint axis, and determine whether blockade of PD-L1/PD-1 potentiates PARP inhibitor (PARPi) in tumor ...suppression.
Breast cancer cell lines, xenograft tumors, and syngeneic tumors treated with PARPi were assessed for PD-L1 expression by immunoblotting, IHC, and FACS analyses. The phospho-kinase antibody array screen was used to explore the underlying mechanism of PARPi-induced PD-L1 upregulation. The therapeutic efficacy of PARPi alone, PD-L1 blockade alone, or their combination was tested in a syngeneic tumor model. The tumor-infiltrating lymphocytes and tumor cells isolated from syngeneic tumors were analyzed by CyTOF and FACS to evaluate the activity of antitumor immunity in the tumor microenvironment.
PARPi upregulated PD-L1 expression in breast cancer cell lines and animal models. Mechanistically, PARPi inactivated GSK3β, which in turn enhanced PARPi-mediated PD-L1 upregulation. PARPi attenuated anticancer immunity via upregulation of PD-L1, and blockade of PD-L1 resensitized PARPi-treated cancer cells to T-cell killing. The combination of PARPi and anti-PD-L1 therapy compared with each agent alone significantly increased the therapeutic efficacy
Our study demonstrates a cross-talk between PARPi and tumor-associated immunosuppression and provides evidence to support the combination of PARPi and PD-L1 or PD-1 immune checkpoint blockade as a potential therapeutic approach to treat breast cancer.
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4‐1BB tumor necrosis factor receptor superfamily (TNFRSF9), CD137) is a critical immune stimulator that sustains T cell activity and antitumor immune response. The strategy to eliminate cancers by ...agonistically targeting 4‐1BB is under clinical investigation. As a protein expressed in an inducible manner, 4‐1BB is under tight control on both transcription and translation levels to maintain its homeostasis. So far, the mechanisms underlying the transcriptional activation of 4‐1BB have been well‐interpreted; however, it remains inexplicit how 4‐1BB is regulated on the protein level. In this study, we presented experimental evidence supporting that 4‐1BB, especially the heavily N‐glycosylated (mature) form, is polyubiquitinated and subjected to the ubiquitin‐proteasomal system for degradation. By performing proximity‐dependent biotin identification screening coupled with biochemical assays, we identified that F‐box/LRR‐repeat protein 20 acts as the E3 ligase that promotes the polyubiquitination of 4‐1BB at the intracellular domain. Our data provided mechanistic insight into 4‐1BB regulation on the protein level by unmasking, for the first time, a posttranslational mechanism governing 4‐1BB abundance in cells. The findings of this study could potentially guide the development of 4‐1BB–targeted therapy for cancers as well as other immune disorders.
4‐1BB is a critical immune stimulator that sustains T cell activity and antitumor immune response. In this study, we present experimental evidence supporting that 4‐1BB is polyubiquitinated and subjected to the ubiquitin‐proteasomal system for degradation. Proteomics analysis identified that F‐box/LRR‐repeat protein 20 serves as one of the E3 ligases to ubiquitinate and destabilize 4‐1BB. Our findings could guide the development of 4‐1BB‐targeted therapy for cancer as well as other immune disorders.
Immune checkpoint inhibitors (ICIs) are novel class of anti-cancer drugs that exhibit significant therapeutic effects even in patients with advanced-stage cancer. However, the efficacy of ICIs is ...limited due to resistance. Therefore, appropriate biomarkers to select patients who are likely to respond to these drugs as well as combination therapy to overcome the resistance are urgently necessary. Cancer is caused by various genetic alterations that lead to abnormalities in oncogenic signaling pathways. The aberrant oncogenic signaling pathways serve as not only prognostic and predictive biomarkers, but also targets for molecularly targeted therapy. Growing evidence shows that the aberrant oncogenic signaling pathways in cancer cells facilitate the resistance to ICIs by modulating the regulation of immune checkpoint and cancer immune surveillance. Indeed, it has been demonstrated that some molecular targeted therapies significantly improve the efficacy of ICIs in preclinical and clinical studies. In this review, we highlighted several oncogenic signaling pathways including receptor tyrosine kinases (RTKs), MAPK, PI3K-AKT-mTOR, JAK-STAT, Hippo, and Wnt pathways, and summarized the recent findings of the mechanisms underlying the regulation of cancer immunity and the ICI resistance induced by these aberrant oncogenic signaling pathways in cancer cells. Moreover, we discussed potential combination therapies with ICIs and molecularly targeted drugs to overcome the resistance and increase the efficacy of ICIs.
ObjectiveIn the tumour microenvironment, critical drivers of immune escape include the oncogenic activity of the tumour cell-intrinsic osteopontin (OPN), the expression of programmed death ligand 1 ...(PD-L1) and the expansion of tumour-associated macrophages (TAMs). We investigated the feasibility of targeting these pathways as a therapeutic option in hepatocellular carcinoma (HCC) mouse models.DesignWe analysed the number of tumour-infiltrating immune cells and the inflammatory immune profiles in chemically induced liver tumour isolated from wild-type and OPNknockout (KO) mice. In vitro cell cocultures were further conducted to investigate the crosstalk between TAMs and HCC cells mediated by OPN, colony stimulating factor-1 (CSF1) and CSF1 receptor (CSF1R). The in vivo efficacy of anti-PD-L1 and CSF1/CSF1R inhibition was evaluated in OPN overexpressing subcutaneous or orthotopic mouse model of HCC.ResultsThe numbers of TAMs, as well as the expression levels of M2 macrophage markers and PD-L1 were significantly decreased, but the levels of cytokines produced by T-helper 1 (Th1) cells were upregulated in tumour tissues from OPN KO mice compared with that from the controls. In addition, we observed a positive association between the OPN and PD-L1 expression, and OPN expression and TAM infiltration in tumour tissues from patients with HCC. We further demonstrated that OPN facilitates chemotactic migration, and alternative activation of macrophages, and promotes the PD-L1 expression in HCC via activation of the CSF1-CSF1R pathway in macrophages. Combining anti-PD-L1 and CSF1R inhibition elicited potent antitumour activity and prolonged survival of OPNhigh tumour-bearing mice. Histological, flow cytometric and ELISA revealed increased CD8+ T cell infiltration, reduced TAMs and enhanced Th1/Th2 cytokine balance in multiple mouse models of HCC.ConclusionsOPN/CSF1/CSF1R axis plays a critical role in the immunosuppressive nature of the HCC microenvironment. Blocking CSF1/CSF1R prevents TAM trafficking and thereby enhances the efficacy of immune checkpoint inhibitors for the treatment of HCC.
Immune checkpoint blockade therapy has demonstrated promising clinical outcomes for multiple cancer types. However, the emergence of resistance as well as inadequate biomarkers for patient ...stratification have largely limited the clinical benefits. Here, we showed that tumors with high TYRO3 expression exhibited anti-programmed cell death protein 1/programmed death ligand 1 (anti-PD-1/PD-L1) resistance in a syngeneic mouse model and in patients who received anti-PD-1/PD-L1 therapy. Mechanistically, TYRO3 inhibited tumor cell ferroptosis triggered by anti-PD-1/PD-L1 and facilitated the development of a protumor microenvironment by reducing the M1/M2 macrophage ratio, resulting in resistance to anti-PD-1/PD-L1 therapy. Inhibition of TYRO3 promoted tumor ferroptosis and sensitized resistant tumors to anti-PD-1 therapy. Collectively, our findings suggest that TYRO3 could serve as a predictive biomarker for patient selection and a promising therapeutic target to overcome anti-PD-1/PD-L1 resistance.
Pro-inflammatory cytokines produced in the tumor microenvironment lead to eradication of anti-tumor immunity and enhanced tumor cell survival. In the current study, we identified tumor necrosis ...factor alpha (TNF-α) as a major factor triggering cancer cell immunosuppression against T cell surveillance via stabilization of programmed cell death-ligand 1 (PD-L1). We demonstrated that COP9 signalosome 5 (CSN5), induced by NF-κB p65, is required for TNF-α-mediated PD-L1 stabilization in cancer cells. CSN5 inhibits the ubiquitination and degradation of PD-L1. Inhibition of CSN5 by curcumin diminished cancer cell PD-L1 expression and sensitized cancer cells to anti-CTLA4 therapy.
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•TNF-α stabilizes cancer cell PD-L1 in response to chronic inflammation•Activation of NF-κB by TNF-α induces CSN5 expression leading to PD-L1 stabilization•CSN5 enzyme activity controls T cell suppression via PD-L1 deubiquitination•Destabilization of PD-L1 by CSN5 inhibitor curcumin benefits anti-CTLA4 therapy
Lim et al. show that inflammation increases PD-L1 expression in tumors through TNF-α-mediated activation of NF-κB, leading to transactivation of CSN5. CSN5 reduces PD-L1 ubiquitination and stabilizes it. Inhibition of CSN5 cooperates with anti-CTLA4 to enhance anti-tumor T cell function and reduce tumor growth.
Metformin has been reported to possess antitumor activity and maintain high cytotoxic T lymphocyte (CTL) immune surveillance. However, the functions and detailed mechanisms of metformin’s role in ...cancer immunity are not fully understood. Here, we show that metformin increases CTL activity by reducing the stability and membrane localization of programmed death ligand-1 (PD-L1). Furthermore, we discover that AMP-activated protein kinase (AMPK) activated by metformin directly phosphorylates S195 of PD-L1. S195 phosphorylation induces abnormal PD-L1 glycosylation, resulting in its ER accumulation and ER-associated protein degradation (ERAD). Consistently, tumor tissues from metformin-treated breast cancer patients exhibit reduced PD-L1 levels with AMPK activation. Blocking the inhibitory signal of PD-L1 by metformin enhances CTL activity against cancer cells. Our findings identify a new regulatory mechanism of PD-L1 expression through the ERAD pathway and suggest that the metformin-CTLA4 blockade combination has the potential to increase the efficacy of immunotherapy.
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•Metformin enhances antitumor CTL immunity by blocking PD-L1/PD-1 axis•Metformin-activated AMPK directly binds to and phosphorylates PD-L1 at S195•Abnormal PD-L1 glycosylation induced by pS195 leads to PD-L1 degradation by ERAD•Combination therapy with metformin and anti-CTLA4 has a synergistic antitumor effect
Cha et al. elucidated a mechanism to show that metformin-activated AMPK phosphorylates PD-L1 at S195 to induce abnormal glycosylation and degrades PD-L1 through an ERAD pathway. This study suggests the potential to use metformin as an adjuvant with various non-PD-L1/PD-1-targeting immune therapies.
Triple‐negative breast cancer (TNBC) lacks a well‐defined molecular target and is associated with poorer outcomes compared to other breast cancer subtypes. Programmed cell death protein 1 ...(PD‐1)/programmed death‐ligand 1 (PD‐L1) blockade therapy shows a 10% to 20% response rate in TNBC patients. Our previous studies show that PD‐L1 proteins are heavily glycosylated in TNBC, and the glycosylation plays an important role in the PD‐L1 protein's stability and immunosuppressive function. However, a strategy for PD‐L1 deglycosylation in TNBC is poorly defined. Here we found that a saccharide analog, 2‐deoxy‐
d‐glucose (2‐DG), inhibits glycosylation of PD‐L1 and its immunosuppressive function by combining with EGFR inhibitor, gefitinib. Interestingly, 2‐DG/gefitinib‐induced deglycosylation of PD‐L1 decreased the expression level of PD‐L1 protein as well as its binding with PD‐1. However, there was no significant decrease in 4‐1BB expression and its binding with 4‐1BBL by 2‐DG/gefitinib. Furthermore, we demonstrated that the combination treatment of 2‐DG/gefitinib and 4‐1BB antibody enhances antitumor immunity in TNBC syngeneic murine models. Together, our results suggest a new immunotherapeutic strategy to enhance antitumor immunity by PD‐L1 deglycosylation and 4‐1BB stimulation in TNBC.
Protein glycosylation provides proteomic diversity in regulating protein localization, stability, and activity; it remains largely unknown whether the sugar moiety contributes to immunosuppression. ...In the study of immune receptor glycosylation, we showed that EGF induces programmed death ligand 1 (PD-L1) and receptor programmed cell death protein 1 (PD-1) interaction, requiring β-1,3-N-acetylglucosaminyl transferase (B3GNT3) expression in triple-negative breast cancer. Downregulation of B3GNT3 enhances cytotoxic T cell-mediated anti-tumor immunity. A monoclonal antibody targeting glycosylated PD-L1 (gPD-L1) blocks PD-L1/PD-1 interaction and promotes PD-L1 internalization and degradation. In addition to immune reactivation, drug-conjugated gPD-L1 antibody induces a potent cell-killing effect as well as a bystander-killing effect on adjacent cancer cells lacking PD-L1 expression without any detectable toxicity. Our work suggests targeting protein glycosylation as a potential strategy to enhance immune checkpoint therapy.
•N-linked glycosylation is required for physical contact between PD-L1 and PD-1•EGF/EGFR stimulates PD-L1 glycosylation via B3GNT3 glycosyltransferase•Glycosylated-PD-L1 antibody induces PD-L1 internalization•Glycosylated-PD-L1-ADC possesses potent toxicity as well as bystander effects
Li et al. show that glycosylation of PD-L1 is essential for PD-L1/PD-1 interaction and immunosuppression in triple-negative breast cancer (TNBC). They generate a glycosylation-specific antibody that induces PD-L1 internalization and an antibody-drug conjugate with potent anti-tumor activities in TNBC models.
Extracellular interaction between programmed death ligand-1 (PD-L1) and programmed cell death protein-1 (PD-1) leads to tumour-associated immune escape. Here we show that the immunosuppression ...activity of PD-L1 is stringently modulated by ubiquitination and N-glycosylation. We show that glycogen synthase kinase 3β (GSK3β) interacts with PD-L1 and induces phosphorylation-dependent proteasome degradation of PD-L1 by β-TrCP. In-depth analysis of PD-L1 N192, N200 and N219 glycosylation suggests that glycosylation antagonizes GSK3β binding. In this regard, only non-glycosylated PD-L1 forms a complex with GSK3β and β-TrCP. We also demonstrate that epidermal growth factor (EGF) stabilizes PD-L1 via GSK3β inactivation in basal-like breast cancer. Inhibition of EGF signalling by gefitinib destabilizes PD-L1, enhances antitumour T-cell immunity and therapeutic efficacy of PD-1 blockade in syngeneic mouse models. Together, our results link ubiquitination and glycosylation pathways to the stringent regulation of PD-L1, which could lead to potential therapeutic strategies to enhance cancer immune therapy efficacy.
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