Sensitizing tumors to anti-PD-1 therapy by promoting NK and CD8+ T cells via pharmacological activation of FOXO3

BackgroundStimulating antitumor immunity by blocking programmed death-1 (PD-1) or its ligand (programmed death-ligand 1 (PD-L1) is a promising antitumor therapy. However, numerous patients respond poorly to PD-1/PD-L1 blockade. Unresponsiveness to immune-checkpoint blockade (ICB) can cast significant challenges to the therapeutic options for patients with hard-to-treat tumors. There is an unmet clinical need to establish new therapeutic approaches for mitigating ICB unresponsiveness in patients. In this study, we investigated the efficacy and role of low-dose antineoplastic agent SN-38 or metformin in sensitizing unresponsive tumors to respond to ICB therapy.MethodsWe assessed the significant pathological relationships between PD-L1 and FOXO3 expression and between PD-L1 and c-Myc or STAT3 expression in patients with various tumors. We determined the efficacy of low-dose SN-38 or metformin in sensitizing unresponsive tumors to respond to anti-PD-1 therapy in a syngeneic tumor system. We deciphered novel therapeutic mechanisms underlying the SN-38 and anti-PD-1 therapy-mediated engagement of natural killer (NK) or CD8+ T cells to infiltrate tumors and boost antitumor immunity.ResultsWe showed that PD-L1 protein level was inversely associated with FOXO3 protein level in patients with ovarian, breast, and hepatocellular tumors. Low-dose SN-38 or metformin abrogated PD-L1 protein expression, promoted FOXO3 protein level, and significantly increased the animal survival rate in syngeneic mouse tumor models. SN-38 or metformin sensitized unresponsive tumors responding to anti-PD-1 therapy by engaging NK or CD8+ T cells to infiltrate the tumor microenvironment (TME) and secret interferon-γ and granzyme B to kill tumors. SN-38 suppressed the levels of c-Myc and STAT3 proteins, which controlled PD-L1 expression. FOXO3 was essential for SN38-mediated PD-L1 suppression. The expression of PD-L1 was compellingly linked to that of c-Myc or STAT3 in patients with the indicated tumors.ConclusionWe show that SN-38 or metformin can boost antitumor immunity in the TME by inhibiting c-Myc and STAT3 through FOXO3 activation. These results may provide novel insight into ameliorating patient response to overarching immunotherapy for tumors.

The effect of hypoxia on PD-L1 expression in bladder cancer

Introduction Recent data has demonstrated that hypoxia drives an immunosuppressive tumour microenvironment (TME) via various mechanisms including hypoxia inducible factor (HIF)-dependent upregulation of programmed death ligand 1 (PD-L1). Both hypoxia and an immunosuppressive TME are targetable independent negative prognostic factors for bladder cancer. Therefore we sought to investigate whether hypoxia is associated with upregulation of PD-L1 in the disease. Materials and methods Three human muscle-invasive bladder cancer cell lines (T24, J82, UMUC3) were cultured in normoxia (20% oxygen) or hypoxia (1 and 0.1% oxygen) for 24 h. Differences in PD-L1 expression were measured using Western blotting, quantitative polymerase chain reaction (qPCR) and flow cytometry (≥3 independent experiments). Statistical tests performed were unpaired t tests and ANOVA. For in silico work an hypoxia signature was used to apply hypoxia scores to muscle-invasive bladder cancers from a clinical trial (BCON; n = 142) and TCGA (n = 404). Analyses were carried out using R and RStudio and statistical tests performed were linear models and one-way ANOVA. Results When T24 cells were seeded at < 70% confluence, there was decreased PD-L1 protein (p = 0.009) and mRNA (p < 0.001) expression after culture in 0.1% oxygen. PD-L1 protein expression decreased in both 0.1% oxygen and 1% oxygen in a panel of muscle-invasive bladder cancer cells: T24 (p = 0.009 and 0.001), J82 (p = 0.008 and 0.013) and UMUC3 (p = 0.003 and 0.289). Increasing seeding density decreased PD-L1 protein (p < 0.001) and mRNA (p = 0.001) expression in T24 cells grown in both 20 and 1% oxygen. Only when cells were 100% confluent, were PD-L1 protein and mRNA levels higher in 1% versus 20% oxygen (p = 0.056 and p = 0.037). In silico analyses showed a positive correlation between hypoxia signature scores and PD-L1 expression in both BCON (p = 0.003) and TCGA (p < 0.001) cohorts, and between hypoxia and IFNγ signature scores (p < 0.001 for both). Conclusion Tumour hypoxia correlates with increased PD-L1 expression in patient derived bladder cancer tumours. In vitro PD-L1 expression was affected by cell density and decreased PD-L1 expression was observed after culture in hypoxia in muscle-invasive bladder cancer cell lines. As cell density has such an important effect on PD-L1 expression, it should be considered when investigating PD-L1 expression in vitro.

Pan-Cancer Analysis Reveals Alternative Splicing Characteristics Associated With Immune-Related Adverse Events Elicited by Checkpoint Immunotherapy

Immune-related adverse events (irAEs) can impair the effectiveness and safety of immune checkpoint inhibitors (ICIs) and restrict the clinical applications of ICIs in oncology. The predictive biomarkers of irAE are urgently required for early diagnosis and subsequent management. The exact mechanism underlying irAEs remains to be fully elucidated, and the availability of predictive biomarkers is limited. Herein, we performed data mining by combining pharmacovigilance data and pan-cancer transcriptomic information to illustrate the relationships between alternative splicing characteristics and irAE risk of ICIs. Four distinct classes of splicing characteristics considered were associated with splicing factors, neoantigens, splicing isoforms, and splicing levels. Correlation analysis confirmed that expression levels of splicing factors were predictive of irAE risk. Adding DHX16 expression to the bivariate PD-L1 protein expression-fPD1 model markedly enhanced the prediction for irAE. Furthermore, we identified 668 and 1,131 potential predictors based on the correlation of the incidence of irAEs with splicing frequency and isoform expression, respectively. The functional analysis revealed that alternative splicing might contribute to irAE pathogenesis via coordinating innate and adaptive immunity. Remarkably, autoimmune-related genes and autoantigens were preferentially over-represented in these predictors for irAE, suggesting a close link between autoimmunity and irAE occurrence. In addition, we established a trivariate model composed of CDC42EP3-206, TMEM138-211, and IRX3-202, that could better predict the risk of irAE across various cancer types, indicating a potential application as promising biomarkers for irAE. Our study not only highlights the clinical relevance of alternative splicing for irAE development during checkpoint immunotherapy but also sheds new light on the mechanisms underlying irAEs.

Posttranslational Modifications in PD-L1 Turnover and Function: From Cradle to Grave

Programmed death-ligand 1 (PD-L1) is one of the most classic immune checkpoint molecules. Cancer cells express PD-L1 to inhibit the activity of effector T cells’ cytotoxicity through programmed death 1 (PD-1) engagement in exposure to inflammatory cytokines. PD-L1 expression levels on cancer cells might affect the clinical response to anti-PD-1/PD-L1 therapies. Hence, understanding molecular mechanisms for regulating PD-L1 expression is essential for improving the clinical response rate and efficacy of PD-1/PD-L1 blockade. Posttranslational modifications (PTMs), including phosphorylation, glycosylation, ubiquitination, and acetylation, regulate PD-L1 stability, cellular translocation, and interaction with its receptor. A coordinated positive and negative regulation via PTMs is required to ensure the balance and function of the PD-L1 protein. In this review, we primarily focus on the roles of PTMs in PD-L1 expression, trafficking, and antitumor immune response. We also discuss the implication of PTMs in anti-PD-1/PD-L1 therapies.

Apoptosis-Independent Reduction of Programmed Death Ligand-1 Levels by Boningmycin Through AMPK-Mediated Endoplasmic Reticulum-Associated Protein Degradation in Human Tumor Cells

Boningmycin (BON), a new member of the bleomycin family, exhibits highly potent activity against tumor cells in vitro and in vivo. It remains unclear if BON can affect the protein levels of programmed death ligand-1 (PD-L1) in a manner similar to that of other antitumor agents. Potent inhibition of cell survival by BON was observed in non-small-cell lung cancer NCI-H460 cells and sarcoma HT1080 cells. Apoptosis-independent reduction of PD-L1 was observed after exposure to BON. Furthermore, BON-treatment increased AMP-activated protein kinase phosphorylation, however, this increase was suppressed by treatment with specific inhibitor (compound C) or RNAi-mediated knockdown of AMPKα. BON-induced PD-L1 reduction is mediated by the endoplasmic reticulum-associated degradation pathway. Its mode of action is similar to that of metformin on the PD-L1 protein. In conclusion, it is firstly reported that BON can decrease PD-L1 protein levels through the AMPK activated endoplasmic reticulum- associated degradation pathway.

Identification and Evolutionary Characterization of Papillomavirus Sequences in New World Monkeys (Genera Sapajus and Alouatta) from Argentina

Objective: In this study, we investigated the occurrence of papillomavirus (PV) infection in non-human primates (NHP, Platyrrhine) of northeastern Argentina by using broad-spectrum PCR primers at the L1 gene. In addition, we conducted a phylogenetic and coalescence analysis of viral sequences to explore their evolutionary history and evaluate the co-speciation hypothesis in the context of primate evolution. Methods: We obtained samples of 57 individuals from wild and captive populations of Alouatta caraya, Sapajus nigritus and Sapajus cay. We assessed PV infection by PCR amplification with the CUT primer system and sequencing of 337 bp (112 amino acids) of the L1 protein. The viral sequences were analyzed by phylogenetic and Bayesian coalescence methods to estimate the age of the most common recent ancestor (tMCRA) with BEAST, v1.4.8 software. We evaluated viral/host tree congruence with TreeMap v3.0. Results: We identified two novel putative PV sequences of the genus Gamma- PV in Sapajus sp and Alouatta caraya (SPV1 and AcPV1, respectively). The tMRCA of SPV1 was estimated at 11,941,682 years before present (ybp) and that of AcPV1 at 46,638,071 ybp, both predating the coalescence times of their hosts: 6.4 million years (MYA) and 6.8 MYA, respectively. Based on the comparison of primate and viral phylogenies, we could not reject the null hypothesis that the PV tree is no more congruent with the host tree than a random tree would be (P>0.05). Thus, a model of virus-host coevolution was rejected. Conclusion: This study presents the first report of PV infection in Platyrrhine species from Argentina, expands the range of described hosts for these viruses, and proposes new scenarios for their origin and dispersal.

Crizotinib efficacy in advanced non-squamous NSCLC patients with ALK or ROS1 rearrangement

In patients with advanced non-small cell lung cancer (NSCLC), comprehensive genetic diagnostics is currently carried out in order to qualify for molecularly targeted therapies and immunotherapy. The aim of the study was to assess the usefulness of the reverse transcriptase (RT-PCR) method in the diagnosis of gene rearrangements, the effectiveness of EGFR, ALK, ROS1, and PD-L1 inhibitors in first-line treatment in NSCLC patients. We enrolled 95 non-squamous NSCLC patients with known status of EGFR, ALK, ROS1, MET and RET genes and PD-L1 protein expression. We used the real time PCR, fluorescence in situ hybridization (FISH), immunohistochemistry (IHC) and RT-PCR techniques for determination of predictive factors. In patients with ALK and ROS1 genes alteration, the median overall survival was 34 months in crizotinib treated patients and 6 months in patients who received chemotherapy (HR = 0.266, p = 0.0056). The risk of death was lower in patients treated with molecularly targeted therapies or immunotherapy compared to patients with predictive factors without personalized treatment (HR = 0.265, 95% CI 0.116–0.606) and to patient without predictive factors who received chemotherapy (HR = 0.42, 95% CI 0.162–1.09). Diagnosis of predictive factors and implementation of personalized treatment are key to prolonging the survival in advanced NSCLC patients.

The Function and Oncological Significance of PD-L1 and its Antibodies

The PD-L1 protein, also known as programmed death-ligand 1 is a protein encoded in the CD274 gene. Specifically, PD-L1 belongs to the immunoglobulin superfamily of proteins, and it is a transmembrane protein that allows nutrients across the cell membrane 11. PD-L1 works in close connection with T cells (thymus cells) and B cells (bone marrow or bursa-derived cells)1. PD-L1 binds to the PD-L receptor on T cells to regulate and sometimes inhibit (in the case of cancer) the activated T cells, B cells, and myeloid cells. Once their activation is inhibited by PD-L1, the T-cells are unable to fight foreign substances in the body like infections, diseases, and cancers, thus allowing cancerous tumors to grow without check3. Elevated levels of PD-L1 have been found in a variety of cancers, including melanoma, non-small cell lung cancer (NSCLC), Hodgkin’s lymphoma, bladder cancer, renal cell carcinoma (RCC), head and neck squamous cell carcinoma (HNSCC), breast cancer, Merkel cell carcinoma, hepatocellular carcinoma (HCC) and gastric cancer (GC)4. Among other information, this study also examines the protein sequence of PD-L1, alignments of the sequence, the structure, functional domains, gene expression, copy number, and mutation profiles.

Discordance of PD-L1 Expression at the Protein and RNA Levels in Early Breast Cancer

We aimed to assess if the discrepant prognostic information between Programmed Death Ligand 1 (PD-L1) protein versus mRNA expression in early breast cancer (BC) could be attributed to heterogeneity in its expression. PD-L1 protein and mRNA expression in BC tissue microarrays from two clinical patient cohorts were evaluated (105 patients; cohort 1: untreated; cohort 2: neoadjuvant chemotherapy-treated). Immunohistochemistry (IHC) with SP142, SP263 was performed. PD-L1 mRNA was evaluated using bulk gene expression and RNA-FISH RNAscope®, the latter scored in a semi-quantitative manner and combined with immunofluorescence (IF) staining for the simultaneous detection of PD-L1 protein expression. PD-L1 expression was assessed in cores as a whole and in two regions of interest (ROI) from the same core. The cell origin of PD-L1 expression was evaluated using multiplex fluorescent IHC. IHC PD-L1 expression between SP142 and SP263 was concordant in 86.7% of cores (p < 0.001). PD-L1 IF/IHC was weakly correlated with spatial mRNA expression (concordance 54.6–71.2%). PD-L1 was mostly expressed by lymphocytes intra-tumorally, while its stromal expression was mostly observed in macrophages. Our results demonstrate only moderate concordance between the various methods of assessing PD-L1 expression at the protein and mRNA levels, which may be attributed to both analytical performance and spatial heterogeneity.

Mouse Papillomavirus L1 and L2 Are Dispensable for Viral Infection and Persistence at Both Cutaneous and Mucosal Tissues

Papillomavirus L1 and L2, the major and minor capsid proteins, play significant roles in viral assembly, entry, and propagation. In the current study, we investigate the impact of L1 and L2 on viral life cycle and tumor growth with a newly established mouse papillomavirus (MmuPV1) infection model. MmuPV1 L1 knockout, L2 knockout, and L1 plus L2 knockout mutant genomes (designated as L1ATGko-4m, L2ATGko, and L1-L2ATGko respectively) were generated. The mutants were examined for their ability to generate lesions in athymic nude mice. Viral activities were examined by qPCR, immunohistochemistry (IHC), in situ hybridization (ISH), and transmission electron microscopy (TEM) analyses. We demonstrated that viral DNA replication and tumor growth occurred at both cutaneous and mucosal sites infected with each of the mutants. Infections involving L1ATGko-4m, L2ATGko, and L1-L2ATGko mutant genomes generally resulted in smaller tumor sizes compared to infection with the wild type. The L1 protein was absent in L1ATGko-4m and L1-L2ATGko mutant-treated tissues, even though viral transcripts and E4 protein expression were robust. Therefore, L1 is not essential for MmuPV1-induced tumor growth, and this finding parallels our previous observations in the rabbit papillomavirus model. Very few viral particles were detected in L2ATGko mutant-infected tissues. Interestingly, the localization of L1 in lesions induced by L2ATGko was primarily cytoplasmic rather than nuclear. The findings support the hypothesis that the L2 gene influences the expression, location, transport, and assembly of the L1 protein in vivo.