PD-L1 Expression In Cancer Cells Research Articles

Immune-related signature identifies IL1R2 as an immunological and prognostic biomarker in pancreatic cancer

Objective: Pancreatic cancer is one of the most aggressive malignancies, a robust prognostic signature and novel biomarkers are urgently needed for accurate stratification of the patients and optimization of clinical decision-making. Methods: A list of bioinformatic analysis were applied in public dataset to construct an immune-related signature. Furthermore, the most pivotal gene in the signature was identified. The potential mechanism of the core gene function was revealed through GSEA, CIBERSORT, ESTIMATE, immunophenoscore algorithm, single cell analysis and functional experiment. Results: An immune-related prognostic signature and associated nomogram were constructed and validated. Among the genes constituting the signature, IL1R2 was identified as the gene occupying the most paramount position in the risk signature. Meanwhile, Knockdown of IL1R2 significantly inhibited the proliferation, invasion and migration ability of pancreatic cancer cells. Additionally, high IL1R2 expression was associated with reduced CD8+ T cell infiltration in pancreatic cancer microenvironment, which may be due to high PD-L1 expression in cancer cells. Finally, the IPS algorithm proved that patients with high IL1R2 expression possessed a higher tumor mutation burden and a higher probability of benefiting from immunotherapy. Conclusion: In conclusion, our study constructed an efficient immune-related prognostic signature and identified the key role of IL1R2 in the development of pancreatic cancer, as well as its potential to serve as a biomarker for immunotherapy efficacy prediction for pancreatic cancer.

Abstract PR009: SMARCAL1 is a dual regulator of innate immune signaling and PD-L1 expression that promotes tumor immune evasion

Abstract Genomic instability can trigger cancer-intrinsic innate immune responses that promote tumor rejection. However, cancer cells often evade these responses by overexpressing immune checkpoint regulators, such as PD-L1. Here, we identify the SNF2-family DNA translocase SMARCAL1 as a factor that favors tumor immune evasion by a dual mechanism involving both the suppression of innate immune signaling and the induction of PD-L1-mediated immune checkpoint responses. Mechanistically, SMARCAL1 relieves endogenous DNA damage and suppresses cGAS-STING-dependent immune signaling during cancer cell growth. Simultaneously, it cooperates with the AP-1 family member JUN to maintain chromatin accessibility at a transcriptional regulatory element in the PD-L1 gene, thereby promoting PD-L1 expression in cancer cells. Loss of SMARCAL1 enhances anti-tumor immune responses and sensitizes tumors to immune checkpoint blockade in a mouse melanoma model. Collectively, these studies uncover SMARCAL1 as a valuable target for cancer immunotherapy. Citation Format: Giuseppe Leuzzi, Alessandro Vasciaveo, Angelo Taglialatela, Xiao Chen, Tessa M. Firestone, Allison R. Hickman, Wendy Mao, Tanay Thakar, Alina Vaitsiankova, Jen-Wei Huang, Raquel Cuella-Martin, Samuel B. Hayward, Jordan S. Kesner, Ali Ghasemzadeh, Tarun S. Nambiar, Patricia Ho, Alexander Rialdi, Maxime Hebrard, Yinglu Li, Jinmei Gao, Saarang Gopinath, Oluwatobi A. Adeleke, Bryan Venters, Charles G. Drake, Richard Baer, Benjamin Izar, Ernesto Guccione, Michael-Christopher Keogh, Raphael Guerois, Lu Sun, Chao Lu, Andrea Califano, Alberto Ciccia. SMARCAL1 is a dual regulator of innate immune signaling and PD-L1 expression that promotes tumor immune evasion [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr PR009.

Role of sex and sex hormones in PD-L1 expression in NSCLC: clinical and therapeutic implications.

Currently, immunotherapy based on PD-1/PD-L1 pathway blockade has improved survival of non-small cell lung cancer (NSCLC) patients. However, differential responses have been observed by sex, where men appear to respond better than women. Additionally, adverse effects of immunotherapy are mainly observed in women. Studies in some types of hormone-dependent cancer have revealed a role of sex hormones in anti-tumor response, tumor microenvironment and immune evasion. Estrogens mainly promote immune tolerance regulating T-cell function and modifying tumor microenvironment, while androgens attenuate anti-tumor immune responses. The precise mechanism by which sex and sex hormones may modulate immune response to tumor, modify PD-L1 expression in cancer cells and promote immune escape in NSCLC is still unclear, but current data show how sexual differences affect immune therapy response and prognosis. This review provides update information regarding anti-PD-1/PD-L immunotherapeutic efficacy in NSCLC by sex, analyzing potential roles for sex hormones on PD-L1 expression, and discussing a plausible of sex and sex hormones as predictive response factors to immunotherapy.

Tumor-infiltrating lymphocytes, PD-L1, and MMR-deficiency combined characterization may identify subgroups of rectal cancer patients who would benefit from immunotherapy

IntroductionMismatch repair deficiency, immunological fertility, and PD-L1 expression status are key histopathological and molecular features defining tumor responsiveness to immunotherapy and, eventually, prognosis. These were investigated in a series of locally advanced rectal cancer patients treated with postoperative chemotherapy and radiotherapy. Materials and methodsTumor-infiltrating lymphocyte (TIL) density was assessed in hematoxylin-eosin tissue sections. PD-L1 expression and the expression of MMR proteins (MLH1, PSM2, MSH2, and MSH6) were assessed with immunohistochemistry. Their association with histopathological variables (node involvement and tumor budding) and prognosis was assessed. ResultsThe TIL-density was significantly higher in the invading tumor front and was inversely related to tumor budding and directly with better overall survival (OS) and distant metastasis-free survival (DMFS) (p = 0.02 and 0.02, respectively). Cancer cell PD-L1 expression was related to high TIL-density (p < 0.01) but not to prognosis, although its overexpression defined a trend for poorer OS in patients with high TIL-density. High PD-L1 expression by stroma infiltrating immune cells was linked with better OS and DMFS (p = 0.007 and 0.001, respectively. MMR deficiency was recorded in 26.2 % of cases, and this was linked with higher TIL-density, but not with prognosis. ConclusionsDense intratumoral lymphocytic infiltration relates to a better prognosis in rectal cancer, although it is also linked with PD-L1 expression that may adversely modulate the anti-tumor effects of TILs. This latter subgroup of patients (high TIL-density/high cancer cell PD-L1 expression) could be an additional target for anti-PD-1/PD-L1 immunotherapy, along with the established subgroup of MMR deficient patients.

Significance of Lipopolysaccharides in Gastric Cancer and Their Potential as a Biomarker for Nivolumab Sensitivity.

Lipopolysaccharides are a type of polysaccharide mainly present in the bacterial outer membrane of Gram-negative bacteria. Recent studies have revealed that lipopolysaccharides contribute to the immune response of the host by functioning as a cancer antigen. We retrospectively recruited 198 patients with gastric cancer who underwent surgery. The presence of lipopolysaccharides was determined using immunohistochemical staining, with the intensity score indicating positivity. The relationship between lipopolysaccharides and CD8, PD-L1, TGFBI (a representative downstream gene of TGF-β signaling), wnt3a, and E-cadherin (epithelial-mesenchymal transition marker) was also investigated. Thereafter, we identified 20 patients with advanced gastric cancer receiving nivolumab and investigated the relationship between lipopolysaccharides and nivolumab sensitivity. After staining for lipopolysaccharides in the nucleus of cancer cells, 150 negative (75.8%) and 48 positive cases (24.2%) were found. The lipopolysaccharide-positive group showed increased cancer stromal TGFBI expression (p < 0.0001) and PD-L1 expression in cancer cells (p = 0.0029). Lipopolysaccharide positivity was significantly correlated with increased wnt3a signaling (p = 0.0028) and decreased E-cadherin expression (p = 0.0055); however, no significant correlation was found between lipopolysaccharide expression and overall survival rate (p = 0.71). In contrast, high TGFBI expression in the presence of LPS was associated with a worse prognosis than that in the absence of LPS (p = 0.049). Among cases receiving nivolumab, the lipopolysaccharide-negative and -positive groups had disease control rates of 66.7% and 11.8%, respectively (p = 0.088). Lipopolysaccharide positivity was associated with wnt3a, TGF-β signaling, and epithelial-mesenchymal transition and was considered to tend to promote therapeutic resistance to nivolumab.

Enhanced bypass of PD-L1 translation reduces the therapeutic response to mTOR kinase inhibitors

Increased PD-L1 expression in cancer cells is known to enhance immunosuppression, but the mechanism underlying PD-L1 upregulation is incompletely characterized. We show that PD-L1 expression is upregulated through internal ribosomal entry site (IRES)-mediated translation upon mTORC1 inhibition. We identify an IRES element in the PD-L1 5'-UTR that permits cap-independent translation and promotes continuous production of PD-L1 protein despite effective inhibition of mTORC1. eIF4A is found to be a key PD-L1 IRES-binding protein that enhances PD-L1 IRES activity and protein production in tumor cells treated with mTOR kinase inhibitors (mTORkis). Notably, treatment with mTORkis invivo elevates PD-L1 levels and reduces the number of tumor-infiltrating lymphocytes in immunogenic tumors, but anti-PD-L1 immunotherapy restores antitumor immunity and enhances the therapeutic efficacy of mTORkis. These findings report a molecular mechanism for regulating PD-L1 expression through bypassing mTORC1-mediated cap-dependent translation and provide a rationale for targeting PD-L1 immune checkpoint to improve mTOR-targeted therapy.

Abstract 6636: Metformin suppresses tumor immune evasion through downregulation of PD-L1 expression in cancer cells and PD-1 expression in NK cells and T cells

Abstract Up-regulation of programmed cell death 1 (PD-1) receptor on T cells and its ligand (PD-L1) on cancer cells is reported in various tumor microenvironment, and the interaction between PD-L1 and PD-1 inhibits the cytotoxic activity of NK cells and T cells, allowing cancer cells to evade immune destruction. Extensive studies suggested that metformin, the most widely used first-line drug for treatment of type 2 diabetes, had a potential efficacy of enhancing anti-tumor immune response. However, the detail mechanisms underlying the efficacy are unknown. Here, we showed that metformin decreases AKT-mediated β-catenin S552 phosphorylation and β-catenin transactivation in an AMPK activation-dependent manner, resulting in reduced CD274 transcription in cancer cells. In the cocultures of cancer cells with NK cells or T cells, cancer cells induced PD-1 protein stability and expression in the NK cells and T cells, these PD-1 upregulation was also observed in infiltrated NK cells and T cells in vivo mouse tumors. Metformin reduced the half-life and expression of PD-1 in an AMPK activation-dependent manner in vitro and in vivo. These inhibitory roles of metformin in PD-L1 expression in cancer cells and in cancer cell-induced PD-1 expression in NK cells and T cells led to ameliorated the cancer cell-reduced cytotoxic activity of NK cells and T cells and inhibited tumor growth in vivo models. These findings suggest that metformin may act as both PD-L1 and PD-1 blockades within tumor microenvironment, providing a mechanistic insight for the efficacy of metformin to improve immunotherapy in human cancer. Citation Format: Su Hwan Park, Jong-Ho Lee. Metformin suppresses tumor immune evasion through downregulation of PD-L1 expression in cancer cells and PD-1 expression in NK cells and T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6636.

Abstract 5119: Nano-immunotherapy targeting PD-L1, PLK1, and TLR9 for treatment of non-small cell lung cancer

Abstract Given the complexity of cancer, it is becoming apparent that combination therapy - in which complementary biological pathways are simultaneously targeted - is required to achieve clinically meaningful responses. To that end, PDX Pharma, in collaboration with Oregon Health &amp; Science University, has developed a proprietary nanoparticle platform (Pdx-NP™) that can effectively co-deliver a plethora of therapeutic modalities while maintaining a small size in saline (100 nm), suitable for infusion and tumor accumulation. This enables the targeting of complementary cancer and immune pathways, leading to synergistic clinical benefits. Herein, we report on the second generation ARAC-02 (Antigen Release Agent and Checkpoint Inhibitor) which is designed to improve the efficacy of immune checkpoint inhibitors (ICIs) in non-small cell lung cancer (NSCLC). While ICIs have improved survival in a subset of patients with advanced NSCLC, only a minority of patients respond to ICIs, highlighting the need for combination immunotherapy. ARAC-02 co-delivers a polo-like kinase 1 (PLK1)-targeted therapy (volasertib), a PD-L1 antibody, and the immune-stimulant CpG. These three agents were carefully selected based on their roles in cancer and immune pathways. Volasertib 1) selectively kills cancer cells, 2) modulates the immune-suppressive tumor microenvironment, and 3) upregulates PD-L1 expression in cancer cells, providing opportunity for targeted delivery with PD-L1 antibody on the nanoparticles. CpG is a TLR9 agonist that enhances antigen presentation to generate tumor-specific T cells. PD-L1 antibodies serve as tumor targeting agent for nanoparticle delivery as well as an ICI (i.e., PD-L1 degradation upon internalization with the nanoparticles), releasing the brakes and allowing T cells to attack the cancer. We found that the optimized ARAC-02 is preferentially internalized in PD-L1 expressing NSCLC cells, demonstrating a 10-fold greater uptake to PD-L1(high) NSCLC cells than PD-L1(low) normal cells. ARAC-02 effectively kills NSCLC cells regardless of their mutational status, while sparing normal cells. In a metastatic lung tumor model, ARAC-02 treatments reduced the tumor burden by 30-fold vs. saline control and 8-fold vs. the first generation ARAC (not containing CpG), whose effect was mainly mediated by CD8+ T cells. ARAC-02 was found to be safe to mice as assessed by body weight, serum biomarkers, and histology. Importantly, intravenous infusions of the platform was also found to be safe in a preliminary toxicology study in non-human primates. Due to its unique ability to catalyze various steps of the adaptive immune response, ARAC-02 is anticipated to provide superior outcomes in NSCLC and a broad range of tumor types regardless of baseline PD-L1 expression. Citation Format: Moataz Reda, Worapol Ngamcherdtrakul, Ruijie Wang, Noah Crumrine, Cole Baker, Alyssa Wallstrum, Jeremy Saito, Natalie White, Wassana Yantasee. Nano-immunotherapy targeting PD-L1, PLK1, and TLR9 for treatment of non-small cell lung cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5119.

Abstract 4438: Bypassing immune evasion in colorectal cancer by integrin inhibition-mediated downregulation of PD-L1

Abstract Integrin receptors have long posed a potentially attractive target for disrupting cancer hallmarks. Promising preliminary findings with integrin inhibition as an adjuvant to chemotherapy have not translated to clinical success. However, the effect of integrin inhibition on tumor-immune cell interactions remains largely unexplored. Further investigation could shed light on a connection between integrin signaling and immune checkpoint expression, opening the path for using integrin inhibitors to sensitize otherwise resistant tumors to immunotherapy. Fluorescently labeled wild-type HCT-116 colorectal cancer cells and TALL-104 T-cells were co-cultured and treated with GLPG0187, a small molecule integrin inhibitor, at various doses. Co-cultures were observed at different time points using fluorescence microscopy and cell counts were quantified using ImageJ software. The co-culture included the cell viability marker ethidium homodimer so the number of viable cells of each type could be determined using colocalization. This assay revealed synergistic cancer cell killing, indicating that integrin inhibition may be sensitizing cancer cells to immune cells. The hypothesized mechanism involves TGF-beta-mediated PD-L1 expression in cancer cells. GLPG0187 has been shown to inhibit the ɑvβ6 integrin receptor, which is responsible for the activation of latent-TGFβ into the active form. By blocking this receptor, there would potentially be a reduction of TGFβ signaling and therefore a reduced expression of PD-L1 leading to an enhanced immune response. To investigate this mechanism, both WT and p53-/- HCT-116 cells were pre-treated with GLPG0187 and subsequently with latent-TGFβ. Western blot analysis demonstrated that the addition of latent-TGFβ increased the expression of PD-L1 in cancer cells. Additionally, a low dose of integrin inhibitor rescued these effects, returning PD-L1 expression back to control levels. However, the higher dose of the drug did not reduce PD-L1 expression. This could potentially be due to off-target effects conflicting with the proposed pathway, however, these findings are still under active investigation. Ongoing proteomic experiments include a larger range of both drug and latent-TGFβ doses. Probing for additional downstream markers of TGFβ and up-stream markers of PD-L1 will help to further elucidate the mechanism. Further co-culture experiments include anti-PD-L1 and anti-PD-1 therapy to investigate the viability of integrin inhibition as an adjuvant to immune checkpoint blockade. Citation Format: William J. MacDonald, Brooke Verschleiser, Lindsey Carlsen, Kelsey E. Huntington, Wafik S. El-Deiry. Bypassing immune evasion in colorectal cancer by integrin inhibition-mediated downregulation of PD-L1. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4438.

Role of PARP Inhibitors in Cancer Immunotherapy: Potential Friends to Immune Activating Molecules and Foes to Immune Checkpoints.

Poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi) induce cytotoxic effects as single agents in tumors characterized by defective repair of DNA double-strand breaks deriving from BRCA1/2 mutations or other abnormalities in genes associated with homologous recombination. Preclinical studies have shown that PARPi-induced DNA damage may affect the tumor immune microenvironment and immune-mediated anti-tumor response through several mechanisms. In particular, increased DNA damage has been shown to induce the activation of type I interferon pathway and up-regulation of PD-L1 expression in cancer cells, which can both enhance sensitivity to Immune Checkpoint Inhibitors (ICIs). Despite the recent approval of ICIs for a number of advanced cancer types based on their ability to reinvigorate T-cell-mediated antitumor immune responses, a consistent percentage of treated patients fail to respond, strongly encouraging the identification of combination therapies to overcome resistance. In the present review, we analyzed both established and unexplored mechanisms that may be elicited by PARPi, supporting immune reactivation and their potential synergism with currently used ICIs. This analysis may indicate novel and possibly patient-specific immune features that might represent new pharmacological targets of PARPi, potentially leading to the identification of predictive biomarkers of response to their combination with ICIs.

Evaluation of PD-1 and PD-L1 expression in cancer cells and tumor-infiltrating lymphocytes in endometrial carcinoma: A single-institution experience

Evaluation of PD-1 and PD-L1 expression in cancer cells and tumor-infiltrating lymphocytes in endometrial carcinoma: A single-institution experience

Dynamic host immunity and PD-L1/PD-1 blockade efficacy: developments after "IFN-γ from lymphocytes induces PD-L1 expression and promotes progression of ovarian cancer".

In the article titled "IFN-γ from lymphocytes induces PD-L1 expression and promotes progression of ovarian cancer" in 2015, we showed that PD-L1 expression is induced by IFN-γ from lymphocytes in the tumour microenvironment. This article proposed that PD-L1 expression in cancer cells is not stable but varies among cases, or even within a case, which is influenced by the stromal infiltration of cytotoxic lymphocytes. Immune-checkpoint inhibitors, especially anti-PD-1/PD-L1 therapies, are now widely used to treat various types of cancer. Predictive biomarkers for the efficacy of immune-checkpoint inhibitors include PD-L1 expression, MSI/mismatch repair deficiency and high tumour mutation burden. However, clinical trials have proven that their use in ovarian cancer is still challenging. Reliable biomarkers and new treatment strategies may be sought by elucidating the complex immune microenvironment of ovarian cancer. Although the interaction between cytotoxic lymphocytes and PD-1/PD-L1 on tumour cells is at the centre of therapeutic targets, other immune checkpoints and various immunosuppressive cells also play important roles in ovarian cancer. Targeting these role players in combination with PD-1/PD-L1 blockade may be a promising therapeutic strategy.

Computational design of PD-L1 small molecule inhibitors for cancer therapy.

Drug repurposing opens new avenues in cancer therapy. Drug repurposing, or finding new uses for existing drugs, can substantially reduce drug discovery time and costs. Cheminformatics, genetics, and systems biology advances enable repositioning drugs. Clinical usage of PD-1/PD-L1 blocking has been approved because of its efficacy in improving prognosis in select groups. The PD-1/PD-L1 axis was considered to represent a mechanism for tumour evasion of host tumour antigen-specific T-cell immunity in early preclinical research. The expression of PD-L1 in cancer cells causes T lymphocytes to become exhausted by transmitting a co-inhibitory signal. A better understanding of how PD-L1 is regulated in cancer cells could lead to new therapeutic options. In this view, the study was aimed to repurpose the existing FDA-approved drugs as a potential PD-L1 inhibitor through e-Pharmacophore modelling, molecular docking and dynamic simulation. e-Pharmacophore screening retrieved 324 FDA-approved medications with the fitness score ≥ 1. The top 10-docked FDA candidates were compared with IN-35 (Clinical trial candidate) for its interaction pattern with critical amino acid residues. Mirabegron and Indacaterol exhibited a greater affinity for PD-L1 with docking scores of - 9.213kcalmol-1 and - 8.023kcalmol-1, respectively. Mirabegron retain interactions at all three major hotspots in the PD-L1 dimer interface similar to IN-35. MM-GBSA analyses indicated that Mirabegron uses less energy to create a more stable complex and retains all of the inhibitor's positive interactions found in clinical trial ligand IN-35. Molecular dynamics simulation analysis of the Mirabegron complex showed a similar pattern of deviation in correlation with IN-35, and it retains the interaction with the active key amino acids throughout the simulation time. Our present study has shown Mirabegron as a powerful inhibitor of PD-L1 expression in cancer cells using a drug-repurposing screen.

EGFR-Mutated Non-Small Cell Lung Cancer and Resistance to Immunotherapy: Role of the Tumor Microenvironment.

Lung cancer is a leading cause of cancer-related deaths worldwide. About 10–30% of patients with non-small cell lung cancer (NSCLC) harbor mutations of the EGFR gene. The Tumor Microenvironment (TME) of patients with NSCLC harboring EGFR mutations displays peculiar characteristics and may modulate the antitumor immune response. EGFR activation increases PD-L1 expression in tumor cells, inducing T cell apoptosis and immune escape. EGFR-Tyrosine Kinase Inhibitors (TKIs) strengthen MHC class I and II antigen presentation in response to IFN-γ, boost CD8+ T-cells levels and DCs, eliminate FOXP3+ Tregs, inhibit macrophage polarization into the M2 phenotype, and decrease PD-L1 expression in cancer cells. Thus, targeted therapy blocks specific signaling pathways, whereas immunotherapy stimulates the immune system to attack tumor cells evading immune surveillance. A combination of TKIs and immunotherapy may have suboptimal synergistic effects. However, data are controversial because activated EGFR signaling allows NSCLC cells to use multiple strategies to create an immunosuppressive TME, including recruitment of Tumor-Associated Macrophages and Tregs and the production of inhibitory cytokines and metabolites. Therefore, these mechanisms should be characterized and targeted by a combined pharmacological approach that also concerns disease stage, cancer-related inflammation with related systemic symptoms, and the general status of the patients to overcome the single-drug resistance development.

Sensitization of Resistant Breast Cancer Cells with a Jumonji Family Histone Demethylase Inhibitor.

Simple SummaryUsing a cell culture model of resistant breast cancer cells with the phenotype that is often responsible for the early relapse of triple-negative breast cancer, namely, the persistence of these cells in reversible quiescence under a variety of challenges, we found that reprogramming the epigenome by treatment with JIB-04, a small-molecule inhibitor of Jumonji-family histone demethylases, sensitized resistant cells. We used this model of deep intrinsic resistance featuring many molecular mechanisms of achieving this phenotype to perform lengthy evaluations of less cytotoxic doses of JIB-04. We found that resistant cells derived from triple-negative inflammatory breast cancer cell lines were either much more sensitive to JIB-04 than the parental cell line or altered by the treatment such that they became sensitive to the chemotherapeutic drugs paclitaxel and doxorubicin. Notably, JIB-04 exposure increased PD-L1 expression in cancer cells, which means that JIB-04 may have clinical applications in improving the responses of triple-negative breast cancer to anti-PD-L1 therapy.In the present study, we evaluated JIB-04, a small-molecule epigenetic inhibitor initially discovered to inhibit cancer growth, to determine its ability to affect deep intrinsic resistance in a breast cancer model. The model was based on a function-based approach to the selection of cancer cells in a cell culture that can survive a variety of challenges in prolonged, but reversible, quiescence. These resistant cancer cells possessed a variety of mechanisms, including modifications of the epigenome and transcriptome, for generating a high degree of cellular heterogeneity. We found that long pretreatment with JIB-04 sensitized resistant triple-negative inflammatory breast cancer cells and their parental cell line SUM149 to the chemotherapeutic drugs doxorubicin and paclitaxel. Resistant cancer cells derived from another inflammatory breast cancer cell line, FC-IBC02, were considerably more sensitive to JIB-04 than the parental cell line. Investigating a mechanism of sensitization, we found that JIB-04 exposure increased the expression of PD-L1 in resistant cells, suggesting that JIB-04 may also sensitize resistant breast cancer cells to anti-PD-L1 immune therapy. Finally, these results support the usefulness of a cell culture-based experimental strategy for evaluating anticancer agents, such as JIB-04, that may halt cancer evolution and prevent the development of cancer resistance to currently used therapies.

Genome editing of PD-L1 mediated by nucleobase-modified polyamidoamine for cancer immunotherapy.

Immune checkpoint blockade therapy against programmed death protein-1 and its ligand (PD-1/PD-L1) has been accepted as a promising approach to activate the immune system's anti-tumor response. Although small interfering RNA (siRNA) or antibodies can block the PD-1/PD-L1 pathway, the effect of this blockade is temporary and reversible. Here, we developed a nano-delivery system to achieve permanent disruption of the PD-L1 gene based on Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) gene editing technology. In this system, the CRISPR/Cas9 plasmid was delivered into melanoma B16F10 cells using a nucleobase-modified polyamidoamine (PAMAM) derivative namely AP-PAMAM, which was constructed through the modification with 2-amino-6-chloropurine. Meanwhile, the carrier could efficiently facilitate the endosomal escape of CRISPR/Cas9 plasmid and thereby inhibit PD-L1 expression in cancer cells. Moreover, the intravenous injection of AP-PAMAM/plasmid nanoparticles could recruit and activate CD8+ T cells at the tumor site, promoting the secretion of cytokines and the killing of tumor cells. Overall, this nano-delivery system for genome editing provided a promising strategy to block the PD-1/PD-L1 pathway and obtain effective tumor immunotherapy.

Mithramycin suppresses tumor growth by regulating CD47 and PD-L1 expression

Mithramycin A (MIT) has reacquired extensive research attention due to its anti-solid tumor activity and improved pharmacological production. Mechanismly, MIT was broadly used as a c-Myc inhibitor, and c-Myc regulated CD47 and PD-L1 expression which has been demonstrated. However, how MIT affects immune check-point molecules remains unknown. In this study, we found CD47 expression was higher in melanoma of pan-tissue array. MIT inhibited CD47 expression both in mRNA and protein level in melanoma cells (SK-MEL-28 and B16). MIT inhibited c-Myc, Sp-1 and CD47 expression in a concentration-dependent way. MIT inhibited the surface CD47 expression and promoted the phagocytosis of SK-MEL-28 cells by THP-1 cells. We found MIT inhibited tumor growth in melanoma allograft mice and CD47 expression in tumor mass. We also found MIT upregulated PD-L1 expression in cancer cells possibly via inhibiting PD-L1 ubiquitination, increasing ROS and IFN-γ. Combination of MIT and anti-PD-1 antibody showed enhanced antitumor activity compared to MIT and anti-PD-1 antibody alone in MC38 allograft mice. Using immune checkpoint array we found MIT inhibited expression of FasL and Galectin3. These results suggest that MIT inhibits CD47 expression, while improves PD-L1 expression. Furthermore, the combination of MIT and anti-PD-1 antibody exerts potent antitumor effect.

Expression of non-homologous end joining factor, Ku80, is negatively correlated with PD-L1 expression in cancer cells after X-ray irradiation.

The growing importance of antitumour immunity by cancer immunotherapy has prompted studies on radiotherapy-induced immune response. Previous studies have indicated that programmed cell death-1 ligand (PD-L1) expression is regulated by DNA damage signalling. However, PD-L1 up-regulation after radiotherapy has not been fully investigated at the clinical level, particularly in the context of expression of DNA repair factors. The present study examined the correlation of mRNA expression between PD-L1 and non-homologous end joining (NHEJ) factors using The Cancer Genome Atlas database analysis. Among NHEJ factors, Ku80 mRNA expression was negatively correlated with PD-L1 mRNA expression levels in several types of cancer (colon adenocarcinoma, breast invasive carcinoma, skin cutaneous melanoma, lung adenocarcinoma, head and neck squamous cell carcinoma, uterine corpus endometrial carcinoma, cervical squamous cell carcinoma and endocervical adenocarcinoma). To verify the negative correlation in clinical samples, the present study analysed whether Ku80 expression levels affected PD-L1 up-regulation after radiotherapy using cervical squamous cell carcinoma samples. Quantitative evaluation using software analysis of immunohistochemically stained slides revealed that patients with low Ku80 positivity in biopsy specimens demonstrated increased PD-L1 expression levels after 10 Gy irradiation (Spearman's rank correlation coefficient=−0.274; P=0.017). Furthermore, PD-L1 induction levels in tumour cells after 10 Gy of irradiation were significantly inversely correlated with Ku80 expression levels (Spearman's rank correlation coefficient=−0.379; P<0.001). The present study also confirmed that short interfering RNA-mediated Ku80 depletion was associated with greater X-ray-induced PD-L1 up-regulation in HeLa cells. These results indicated that radiotherapy could enhance PD-L1 induction in tumour cells with low Ku80 expression in a clinical setting. Furthermore, these data highlighted Ku80 as a potential predictive biomarker for immune checkpoint therapy combined with radiotherapy.

Super-enhancer receives signals from the extracellular matrix to induce PD-L1-mediated immune evasion via integrin/BRAF/TAK1/ERK/ETV4 signaling.

Objective:PD-L1 and PD-L2 expression levels determine immune evasion and the therapeutic efficacy of immune checkpoint blockade. The factors that drive inducible PD-L1 expression have been extensively studied, but mechanisms that result in constitutive PD-L1 expression in cancer cells are largely unknown.Methods:DNA elements were deleted in cells by CRISPR/Cas9-mediated knockout. Protein function was inhibited by chemical inhibitors. Protein levels were examined by Western blot, mRNA levels were examined by real-time RT-PCR, and surface protein expression was determined by cellular immunofluorescence and flow cytometry. Immune evasion was examined by in vitro T cell-mediated killing.Results:We determined the core regions (chr9: 5, 496, 378–5, 499, 663) of a previously identified PD-L1L2-super-enhancer (SE). Through systematic analysis, we found that the E26 transformation-specific (ETS) variant transcription factor (ETV4) bound to this core DNA region but not to DNA surrounding PD-L1L2SE. Genetic knockout of ETV4 dramatically reduced the expressions of both PD-L1 and PD-L2. ETV4 transcription was dependent on ERK activation, and BRAF/TAK1-induced ERK activation was dependent on extracellular signaling from αvβ3 integrin, which profoundly affected ETV4 transcription and PD-L1/L2 expression. Genetic silencing or pharmacological inhibition of components of the PD-L1L2-SE-associated pathway rendered cancer cells susceptible to T cell-mediated killing.Conclusions:We identified a pathway originating from the extracellular matrix that signaled via integrin/BRAF/TAK1/ERK/ETV4 to PD-L1L2-SE to induce PD-L1-mediated immune evasion. These results provided new insights into PD-L1L2-SE activation and pathways associated with immune checkpoint regulation in cancer.

Narrative review of emerging roles for AKT-mTOR signaling in cancer radioimmunotherapy.

ObjectiveTo summarize the roles of AKT-mTOR signaling in the regulation of the DNA damage response and PD-L1 expression in cancer cells, and propose a novel strategy of targeting AKT-mTOR signaling in combination with radioimmunotherapy in the era of cancer immunotherapyBackgroundImmunotherapy has greatly improved the clinical outcomes of many cancer patients and has changed the landscape of cancer patient management. However, only a small subgroup of cancer patients (~20–30%) benefit from immune checkpoint blockade-based immunotherapy. The current challenge is to find biomarkers to predict the response of patients to immunotherapy and strategies to sensitize patients to immunotherapy.MethodsSearch and review the literature which were published in PUBMED from 2000–2021 with the key words mTOR, AKT, drug resistance, DNA damage response, immunotherapy, PD-L1, DNA repair, radioimmunotherapy.ConclusionsMore than 50% of cancer patients receive radiotherapy during their course of treatment. Radiotherapy has been shown to reduce the growth of locally irradiated tumors as well as metastatic non-irradiated tumors (abscopal effects) by affecting systemic immunity. Consistently, immunotherapy has been demonstrated to enhance radiotherapy with more than one hundred clinical trials of radiation in combination with immunotherapy (radioimmunotherapy) across cancer types. Nevertheless, current available data have shown limited efficacy of trials testing radioimmunotherapy. AKT-mTOR signaling is a major tumor growth-promoting pathway and is upregulated in most cancers. AKT-mTOR signaling is activated by growth factors as well as genotoxic stresses including radiotherapy. Importantly, recent advances have shown that AKT-mTOR is one of the main signaling pathways that regulate DNA damage repair as well as PD-L1 levels in cancers. These recent advances clearly suggest a novel cancer therapy strategy by targeting AKT-mTOR signaling in combination with radioimmunotherapy.