Development of an Anti-canine PD-L1 Antibody and Caninized PD-L1 Mouse Model as Translational Research Tools for the Study of Immunotherapy in Humans.

Immune checkpoint blockade therapy, one of the most promising cancer immunotherapies, has shown remarkable clinical impact in multiple cancer types. Despite the recent success of immune checkpoint blockade therapy, however, the response rates in patients with cancer are limited (∼20%-40%). To improve the success of immune checkpoint blockade therapy, relevant preclinical animal models are essential for the development and testing of multiple combination approaches and strategies. Companion dogs naturally develop several types of cancer that in many respects resemble clinical cancer in human patients. Therefore, the canine studies of immuno-oncology drugs can generate knowledge that informs and prioritizes new immuno-oncology therapy in humans. The challenge has been, however, that immunotherapeutic antibodies targeting canine immune checkpoint molecules such as canine PD-L1 (cPD-L1) have not been commercially available. Here, we developed a new cPD-L1 antibody as an immuno-oncology drug and characterized its functional and biological properties in multiple assays. We also evaluated the therapeutic efficacy of cPD-L1 antibodies in our unique caninized PD-L1 mice. Together, these in vitro and in vivo data, which include an initial safety profile in laboratory dogs, support development of this cPD-L1 antibody as an immune checkpoint inhibitor for studies in dogs with naturally occurring cancer for translational research. Our new therapeutic antibody and caninized PD-L1 mouse model will be essential translational research tools in raising the success rate of immunotherapy in both dogs and humans. Significance: Our cPD-L1 antibody and unique caninized mouse model will be critical research tools to improve the efficacy of immune checkpoint blockade therapy in both dogs and humans. Furthermore, these tools will open new perspectives for immunotherapy applications in cancer as well as other autoimmune diseases that could benefit a diverse and broader patient population.

Nuclear export signal mutation of epidermal growth factor receptor enhances malignant phenotypes of cancer cells.

Nuclear epidermal growth factor receptor (EGFR) has been shown to be correlated with drug resistance and a poor prognosis in patients with cancer. Previously, we have identified a tripartite nuclear localization signal (NLS) within EGFR. To comprehensively determine the functions and underlying mechanism of nuclear EGFR and its clinical implications, we aimed to explore the nuclear export signal (NES) sequence of EGFR that is responsible for interacting with the exportins. We combined in silico prediction with site-directed mutagenesis approaches and identified a putative NES motif of EGFR, which is located in amino acid residues 736-749. Mutation at leucine 747 (L747) in the EGFR NES led to increased nuclear accumulation of the protein via a less efficient release of the exportin CRM1. Interestingly, L747 with serine (L747S) and with proline (L747P) mutations were found in both tyrosine kinase inhibitor (TKI)-treated and -naïve patients with lung cancer who had acquired or de novo TKI resistance and a poor outcome. Reconstituted expression of the single NES mutant EGFRL747P or EGFRL747S, but not the dual mutant along with the internalization-defective or NLS mutation, in lung cancer cells promoted malignant phenotypes, including cell migration, invasiveness, TKI resistance, and tumor initiation, supporting an oncogenic role of nuclear EGFR. Intriguingly, cells with germline expression of the NES L747 mutant developed into B cell lymphoma. Mechanistically, nuclear EGFR signaling is required for sustaining nuclear activated STAT3, but not for Erk. These findings suggest that EGFR functions are compartmentalized and that nuclear EGFR signaling plays a crucial role in tumor malignant phenotypes, leading to tumorigenesis in human cancer.

4-1BB: A promising target for cancer immunotherapy.

Immunotherapy, powered by its relative efficacy and safety, has become a prominent therapeutic strategy utilized in the treatment of a wide range of diseases, including cancer. Within this class of therapeutics, there is a variety of drug types such as immune checkpoint blockade therapies, vaccines, and T cell transfer therapies that serve the purpose of harnessing the body's immune system to combat disease. Of these different types, immune checkpoint blockades that target coinhibitory receptors, which dampen the body's immune response, have been widely studied and established in clinic. In contrast, however, there remains room for the development and improvement of therapeutics that target costimulatory receptors and enhance the immune response against tumors, one of which being the 4-1BB (CD137/ILA/TNFRSF9) receptor. 4-1BB has been garnering attention as a promising therapeutic target in the setting of cancer, amongst other diseases, due to its broad expression profile and ability to stimulate various signaling pathways involved in the generation of a potent immune response. Since its discovery and demonstration of potential as a clinical target, major progress has been made in the knowledge of 4-1BB and the development of clinical therapeutics that target it. Thus, we seek to summarize and provide a comprehensive update and outlook on those advancements in the context of cancer and immunotherapy.

Ephrin receptor A10 monoclonal antibodies and the derived chimeric antigen receptor T cells exert an antitumor response in mouse models of triple-negative breast cancer.

Expression of the receptor tyrosine kinase ephrin receptor A10 (EphA10), which is undetectable in most normal tissues except for the male testis, has been shown to correlate with tumor progression and poor prognosis in several malignancies, including triple-negative breast cancer (TNBC). Therefore, EphA10 could be a potential therapeutic target, likely with minimal adverse effects. However, no effective clinical drugs against EphA10 are currently available. Here, we report high expression levels of EphA10 in tumor regions of breast, lung, and ovarian cancers as well as in immunosuppressive myeloid cells in the tumor microenvironment. Furthermore, we developed anti-EphA10 monoclonal antibodies (mAbs) that specifically recognize cell surface EphA10, but not other EphA family isoforms, and target tumor regions precisely in vivo with no apparent accumulation in other organs. In syngeneic TNBC mouse models, we found that anti-EphA10 mAb clone #4 enhanced tumor regression, therapeutic response rate, and T cell-mediated antitumor immunity. Notably, the chimeric antigen receptor T cells derived from clone #4 significantly inhibited TNBC cell viability in vitro and tumor growth in vivo. Together, our findings suggest that targeting EphA10 via EphA10 mAbs and EphA10-specific chimeric antigen receptor-T cell therapy may represent a promising strategy for patients with EphA10-positive tumors.

FBXL20-mediated ubiquitination triggers the proteasomal degradation of 4-1BB.

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.

N-Glycosylation Facilitates 4-1BB Membrane Localization by Avoiding Its Multimerization.

Leveraging the T cell immunity against tumors represents a revolutionary type of cancer therapy. 4-1BB is a well-characterized costimulatory immune receptor existing on activated T cells and mediating their proliferation and cytotoxicity under infectious diseases and cancers. Despite the accumulating interest in implementing 4-1BB as a therapeutic target for immune-related disorders, less is known about the pattern of its intracellular behaviors and regulations. It has been previously demonstrated that 4-1BB is heavily modified by N-glycosylation; however, the biological importance of this modification lacks detailed elucidation. Through biochemical, biophysical, and cell-biological approaches, we systematically evaluated the impact of N-glycosylation on the ligand interaction, stability, and localization of 4-1BB. We hereby highlighted that N-glycan functions by preventing the oligomerization of 4-1BB, thus permitting its membrane transportation and fast turn-over. Without N-glycosylation, 4-1BB could be aberrantly accumulated intracellularly and fail to be sufficiently inserted in the membrane. The N-glycosylation-guided intracellular processing of 4-1BB serves as the potential mechanism explicitly modulating the "on" and "off" of 4-1BB through the control of protein abundance. Our study will further solidify the understanding of the biological properties of 4-1BB and facilitate the clinical practice against this promising therapeutic target.

Glycosylation of Immune Receptors in Cancer.

Evading host immune surveillance is one of the hallmarks of cancer. Immune checkpoint therapy, which aims to eliminate cancer progression by reprogramming the antitumor immune response, currently occupies a solid position in the rapidly expanding arsenal of cancer therapy. As most immune checkpoints are membrane glycoproteins, mounting attention is drawn to asking how protein glycosylation affects immune function. The answers to this fundamental question will stimulate the rational development of future cancer diagnostics and therapeutic strategies.

TYRO3 induces anti-PD-1/PD-L1 therapy resistance by limiting innate immunity and tumoral ferroptosis.

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.

Saccharide analog, 2-deoxy-d-glucose enhances 4-1BB-mediated antitumor immunity via PD-L1 deglycosylation.

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.

Oncogenic signaling pathways associated with immune evasion and resistance to immune checkpoint inhibitors in cancer.

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.

IL-6/JAK1 pathway drives PD-L1 Y112 phosphorylation to promote cancer immune evasion.

Glycosylation of immune receptors and ligands, such as T cell receptor and coinhibitory molecules, regulates immune signaling activation and immune surveillance. However, how oncogenic signaling initiates glycosylation of coinhibitory molecules to induce immunosuppression remains unclear. Here we show that IL-6-activated JAK1 phosphorylates programmed death-ligand 1 (PD-L1) Tyr112, which recruits the endoplasmic reticulum-associated N-glycosyltransferase STT3A to catalyze PD-L1 glycosylation and maintain PD-L1 stability. Targeting of IL-6 by IL-6 antibody induced synergistic T cell killing effects when combined with anti-T cell immunoglobulin mucin-3 (anti-Tim-3) therapy in animal models. A positive correlation between IL-6 and PD-L1 expression was also observed in hepatocellular carcinoma patient tumor tissues. These results identify a mechanism regulating PD-L1 glycosylation initiation and suggest the combination of anti-IL-6 and anti-Tim-3 as an effective marker-guided therapeutic strategy.

Disruption of tumour-associated macrophage trafficking by the osteopontin-induced colony-stimulating factor-1 signalling sensitises hepatocellular carcinoma to anti-PD-L1 blockade.

OBJECTIVE: In 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. DESIGN: We 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. RESULTS: The 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. CONCLUSIONS: OPN/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.

Deglycosylation of PD-L1 by 2-deoxyglucose reverses PARP inhibitor-induced immunosuppression in triple-negative breast cancer.

Triple-negative breast cancer (TNBC), the most difficult-to-treat breast cancer subtype, lacks well-defined molecular targets. TNBC has increased programmed death-ligand 1 (PD-L1) expression, and its immunosuppressive nature makes it suitable for immune checkpoint blockade therapy. However, the response rate of TNBC to anti-PD-L1 or anti-programmed cell death protein 1 (PD-1) therapy remains unsatisfactory, as only 10-20% of TNBC patients have a partial response. Glycosylated PD-L1, the functional form of PD-L1, is required for PD-L1-PD-1 interaction. TNBC cells have significantly higher levels of glycosylated PD-L1 than non-TNBC cells do. In a screening of glucose analogs to block PD-L1 glycosylation, we found that 2-deoxyglucose (2-DG) can act as a glucose analog to decrease PD-L1 glycosylation. Because PARP inhibition upregulates PD-L1, 2-DG reduced PARP inhibition-mediated expression of glycosylated PD-L1. The combination of PARP inhibition and 2-DG had potent anti-tumor activity. Together, our results provide a strong rationale for investigating the targeting of PD-L1 glycosylation in TNBC further.

Metformin Promotes Antitumor Immunity via Endoplasmic-Reticulum-Associated Degradation of PD-L1.

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.

STT3-dependent PD-L1 accumulation on cancer stem cells promotes immune evasion.

Enriched PD-L1 expression in cancer stem-like cells (CSCs) contributes to CSC immune evasion. However, the mechanisms underlying PD-L1 enrichment in CSCs remain unclear. Here, we demonstrate that epithelial-mesenchymal transition (EMT) enriches PD-L1 in CSCs by the EMT/ß-catenin/STT3/PD-L1 signaling axis, in which EMT transcriptionally induces N-glycosyltransferase STT3 through ß-catenin, and subsequent STT3-dependent PD-L1 N-glycosylation stabilizes and upregulates PD-L1. The axis is also utilized by the general cancer cell population, but it has much more profound effect on CSCs as EMT induces more STT3 in CSCs than in non-CSCs. We further identify a non-canonical mesenchymal-epithelial transition (MET) activity of etoposide, which suppresses the EMT/ß-catenin/STT3/PD-L1 axis through TOP2B degradation-dependent nuclear ß-catenin reduction, leading to PD-L1 downregulation of CSCs and non-CSCs and sensitization of cancer cells to anti-Tim-3 therapy. Together, our results link MET to PD-L1 stabilization through glycosylation regulation and reveal it as a potential strategy to enhance cancer immunotherapy efficacy.

Angiogenin/Ribonuclease 5 Is an EGFR Ligand and a Serum Biomarker for Erlotinib Sensitivity in Pancreatic Cancer.

Pancreatic ribonuclease (RNase) is a secreted enzyme critical for host defense. We discover an intrinsic RNase function, serving as a ligand for epidermal growth factor receptor (EGFR), a member of receptor tyrosine kinase (RTK), in pancreatic ductal adenocarcinoma (PDAC). The closely related bovine RNase A and human RNase 5 (angiogenin [ANG]) can trigger oncogenic transformation independently of their catalytic activities via direct association with EGFR. Notably, high plasma ANG level in PDAC patients is positively associated with response to EGFR inhibitor erlotinib treatment. These results identify a role of ANG as a serum biomarker that may be used to stratify patients for EGFR-targeted therapies, and offer insights into the ligand-receptor relationship between RNase and RTK families.

Eradication of Triple-Negative Breast Cancer Cells by Targeting Glycosylated PD-L1.

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 ß-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.

Stress hormones promote EGFR inhibitor resistance in NSCLC: Implications for combinations with ß-blockers.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) resistance mediated by T790M-independent mechanisms remains a major challenge in the treatment of non-small cell lung cancer (NSCLC). We identified a targetable mechanism of EGFR inhibitor resistance whereby stress hormones activate ß2-adrenergic receptors (ß2-ARs) on NSCLC cells, which cooperatively signal with mutant EGFR, resulting in the inactivation of the tumor suppressor, liver kinase B1 (LKB1), and subsequently induce interleukin-6 (IL-6) expression. We show that stress and ß2-AR activation promote tumor growth and EGFR inhibitor resistance, which can be abrogated with ß-blockers or IL-6 inhibition. IL-6 was associated with a worse outcome in EGFR TKI-treated NSCLC patients, and ß-blocker use was associated with lower IL-6 concentrations and improved benefit from EGFR inhibitors. These findings provide evidence that chronic stress hormones promote EGFR TKI resistance via ß2-AR signaling by an LKB1/CREB (cyclic adenosine 3',5'-monophosphate response element-binding protein)/IL-6-dependent mechanism and suggest that combinations of ß-blockers with EGFR TKIs merit further investigation as a strategy to abrogate resistance.

Rational combination of immunotherapy for triple negative breast cancer treatment.

Recent evidence indicates that tumor infiltrating lymphocytes (TILs), including cytotoxic T cells, are present in the tumor microenvironment of triple-negative breast cancers (TNBC). Despite the presence of cytotoxic T cells, these tumors still develop, progress, and metastasize, suggesting evasion of immune response. One mechanism of immunosuppression is the presence of the T cell inhibitory molecule, programmed death protein 1 (PD-1), on infiltrating T cells and its cognate ligand programmed death ligand 1 (PD-L1) on tumor cells, myeloid dendritic cells (DCs), and macrophages, in the tumor microenvironment. Because TNBC is immunologically insensitive, combinatorial strategies may be ideal to increase both anti-proliferation activity and cytotoxic T cells activity in TNBC. On the basis of two recently discovered regulatory mechanisms of PD-L1, we discuss the potential interactions to boost anti-tumor immunity against TNBC in this review and propose therapeutic strategies that could reduce PD-L1 expression by chemotherapeutic drugs or targeted therapies and sensitize TNBC to immunotherapies.

PARP Inhibitor Upregulates PD-L1 Expression and Enhances Cancer-Associated Immunosuppression.

Purpose: 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.Experimental Design: 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.Results: PARPi upregulated PD-L1 expression in breast cancer cell lines and animal models. Mechanistically, PARPi inactivated GSK3ß, 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 in vivoConclusions: 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. Clin Cancer Res; 23(14); 3711-20. ©2017 AACR.