Programmed Cell Death Ligand 1 Gene Research Articles

Stimuli-responsive magnetic silica-poly-lactic-co-glycolic acid hybrid nanoparticles for targeted cancer chemo-immunotherapy.

Chemotherapy and immunotherapy are two important modalities in cancer management. However, due to multiple reasons, a monotherapy is only partially effective. Hence, if used concurrently in targeted and stimuli-responsive manner, it could have been superior therapeutically. To facilitate co-delivery of chemotherapeutic and immunotherapeutic agent to the target cancer cells, engineered nanoparticles, i.e., a pH-responsive polymer PLGA-coated magnetic silica nanoparticles (Fe3O4-SiO2-PLGA-PDA-PTX-siRNA NPs) encapsulating paclitaxel (PTX) and siRNA against programmed cell death ligand-1 (PD-L1) are synthesized and characterized. Developed nanoparticles demonstrated pH-sensitive sustained drug release up to 10 days. In vitro 4T1 cell line studies showed efficient cellular uptake, PD-L1 gene downregulation, and apoptosis. Further, in vivo efficacy studies carried out in the mice model demonstrated a significant reduction of tumor growth following treatment with dual-Fe3O4-SiO2-PLGA-PDA-PTX-siRNA NPs as compared with monotherapy with Fe3O4-SiO2-PLGA-PDA-PTX NPs. The high therapeutic efficacy observed with dual-Fe3O4-SiO2-PLGA-PDA-PTX-siRNA NPs was mainly due to the cytotoxic effect of PTX combined with targeted silencing of the gene of interest, i.e., PD-L1, which in turn improve CD8+ T cell-mediated cancer cell death as evident with increased proliferation of CD8+ T cells in co-culture experiments. Thereby, dual-Fe3O4-SiO2-PLGA-PDA-PTX-siRNA NPs may have a promising anti-cancer treatment potential against breast cancer; however, the beneficial effects of dual loading of PTX + PD-L1 siRNA may be corroborated against other cancer models such as lung and colorectal cancer models as well as in clinical trials.

Tislelizumab plus nimotuzumab is effective against recurrent or metastatic oral squamous cell carcinoma among patients with a performance status score ≥ 2: a retrospective study.

The efficacy of treatments targeting recurrent or metastatic head and neck squamous cell carcinoma are unsatisfactory in practice for patients with a ECOG PS score ≥ 2. Thus, this study retrospectively evaluated the safety and efficacy of a programmed cell death 1 inhibitor (tislelizumab) combined with an epidermal growth factor receptor inhibitor (nimotuzumab) in treating patients with a PS score ≥ 2 who suffer from recurrent or metastatic oral squamous cell carcinoma (OSCC). Fifteen patients were treated with tislelizumab (200 mg IV Q3W) and nimotuzumab (200 mg IV Q3W). Programmed cell death-ligand 1 (PD-L1) expression in tumor biopsies was assessed with immunohistochemistry. Whole-exome sequencing was used to evaluate treatment efficacy based on PD-L1 expression and gene mutation. At a median follow-up of 9.6 months, median overall survival was 10.1 months, and median progression-free survival was 4.0 months. Overall response rate was 40%, with 6/15 patients achieving partial response. Eight patients exhibited nine adverse events, eight out of nine being grade 2 and the remaining being grade 3. Whole-exome sequencing showed that DYNC1I2, THSD7A, and FAT1 mutations were associated with patient prognosis. Combination therapy involving tislelizumab plus nimotuzumab is a promising, low-toxicity treatment for recurrent or metastatic OSCC in patients with a PS score ≥ 2.

Elevated programmed cell death-1 protein/ligand (PD-1/PD-L1) and variants are associated with susceptibility to multiple myeloma: a case-control study in the Chinese cohort

Multiple myeloma (MM) is a malignant disorder characterised by progressive immune dysregulation. The importance of programmed cell death protein 1 (PD-1)/programmed cell death ligand 1 (PD-L1) in MM has been documented in various populations, but studies have been limited to the Chinese cohort. In the present study, we examined the role of PD-1/PDL-1 in large cohorts of Chinese patients with MM and healthy controls to reveal a possible association with MM. Three hundred thirty-four MM patients and 202 healthy age-sex-matched subjects were enrolled in the present study. Serum levels of PD-1 and PD-L1 were quantified by ELISA. Percentages of T cells (CD4+ and CD8+ T cells) expressing PD-1 receptor were assessed by flow cytometry. Variants in PD-L1 (rs4143815) and PD-1 gene (rs2227981, rs2227982, rs7421861 and rs11568821) were genotyped by PCR-RFLP method. Patients with multiple myeloma had higher levels of PD-1 and PDL-1 than healthy controls, indicating an important role for programmed cell death protein-1 and its ligand in the pathogenesis of MM. T cells expressing PD-1 receptors were also significantly higher in MM patients than in controls. Mutants for PD-L1 (rs4143815) and PD-1 (rs2227982 and rs7421861) polymorphisms were significantly more common in MM than in HC. Interestingly, PD-L1 (rs4143815) and PD-1 (rs2227982 and rs7421861) variants were linked to higher sPD-L1 and sPD-1 levels, respectively. PD-1/PD-L1 levels are significantly higher in MM patients and could be a promising biomarker for the disease. Variants of PD-L1 and PD-1 are linked to serum-soluble proteins and are associated with the development of MM.

Regulation of PD-L1 expression in non-small cell lung cancer by interleukin-1β.

Programmed cell death-ligand 1 (PD-L1) is a biomarker for prediction of the clinical efficacy of immune checkpoint inhibitors in various cancer types. The role of cytokines in regulation of PD-L1 expression in tumor cells has not been fully characterized, however. Here we show that interleukin-1β (IL-1β) plays a key role in regulation of PD-L1 expression in non-small cell lung cancer (NSCLC). We performed comprehensive screening of cytokine gene expression in NSCLC tissue using available single-cell RNA-Sequence data. Then we examined the role of IL-1β in vitro to elucidate its induction of PD-L1 on NSCLC cells. The IL-1β gene is highly expressed in the tumor microenvironment, particularly in macrophages. The combination of IL-1β and interferon-γ (IFN-γ) induced a synergistic increase in PD-L1 expression in NSCLC cell lines. IL-1β and IFN-γ also cooperatively activated mitogen-activated protein kinase (MAPK) signaling and promoted the binding of downstream transcription factors to the PD-L1 gene promoter. Furthermore, inhibitors of MAPK signaling blocked upregulation of PD-L1 by IL-1β and IFN-γ. Our study reports high levels of IL-1β in the tumor microenvironment may cooperate with IFN-γ to induce maximal PD-L1 expression in tumor cells via activation of MAPK signaling, with the IL-1β-MAPK axis being a promising therapeutic target for attenuation of PD-L1-mediated suppression of antitumor immunity.

Tailored modulation of stemness and drug resistance marker characteristics in K-Ras mutant lung cancer cells via PD-L1 gene suppression

AimsWe aimed to analyze the association of tumor cell-specific Programmed Cell Death Ligand 1 (PD-L1) expression with stemness markers and multi-drug resistance genes in non-small cell lung cancer. Main methodsATP Binding Cassette Subfamily G Member 2 (ABCG2), Aldehyde dehydrogenase 1 family, member A1 (ALDH1A1), CD44, Epithelial cell adhesion molecule (EPCAM), Kruppel-like factor 4 (KLF4), MYC Proto-Oncogene (MYC), Nanog Homeobox (NANOG), SRY-Box Transcription Factor 2 (SOX2) as well as ATP Binding Cassette Subfamily C Member (ABCC) 1–6, ABCC10–12 with ATP Binding Cassette Subfamily B Member (ABCB) 1 and ABCB4–9 belongs to ABC transporters family were analyzed and their correlation with PD-L1 was evaluated using Cancer Cell Line Encyclopedia and The Cancer Genome Atlas data sets. We validated potential lung cancer stemness markers harboring ABCG2, CD44, ALDH1A1 and SOX-2, which are affected as a result of siRNA-mediated suppression of PD-L1 via flow cytometry. K-Ras downstream signaling proteins as well as multidrug resistance proteins were also investigated. Key findingsPD-L1 was found to be positively correlated with CD44, whereas negatively correlated with ALDH1A1 (Pearson r = 0.44, r = −0.48; respectively) in 45 K-Ras mutated NSCLC cells based on CCLE database. While ABCC5 was dominantly decreased in K-Ras mutant lung cancer cells affected by PD-L1 gene suppression, expression changes were observed in the activation of distinct signaling pathways in a cell line-dependent manner. SignificanceThe evaluation of markers in lung adenocarcinoma with different types of K-Ras mutations in terms of both stemness and drug resistance markers will contribute to the development of personalized immunotherapy regimens.

Cyclic adenosine monophosphate/phosphodiesterase 4 pathway associated with immune infiltration and PD-L1 expression in lung adenocarcinoma cells.

BackgroundThe cyclic adenosine monophosphate/phosphodiesterase 4 (cAMP/PDE4) pathway is involved in inflammation and immune regulation; however, the effect of cAMP/PDE4 on immune infiltration and immune evasion in lung adenocarcinoma (LUAD) remains unclear.MethodsCBioPortal, which is the The Cancer Genome Atlas (TCGA) online database, and the Kaplan Meier plotter were used to analyze the association between genes and the prognosis of TCGA-LUAD. Tumor Immune Estimation Resource (TIMER) was used to analyze the association between gene expression and immune infiltration. The Genecards database was used to identify the transcription factors of related genes. The lung adenocarcinoma cell line H1299 and A549 were treated with cAMP pathway drugs. Flow cytometry and qRT-PCR were used to detect the PD-L1 protein and gene expression, respectively. A one-way analysis of variance with Tukey’s post-hoc test or a Student’s t-test were used.ResultsIt was found that PDE4B and CREB1, which are downstream genes of the cAMP/PDE4 axis, were differentially expressed in LUAD and adjacent tissues and are correlated with the prognosis and immune infiltration of LUAD. In the CBioPortal database, cAMP pathway genes are closely related to programmed cell death-ligand 1 (PD-L1) expression in TCGA-LUAD. The protein-protein interaction revealed that there was a direct interaction between CREB1/CREBBP, which are the downstream molecules of the cAMP/PDE4 axis, and MYC; additionally, MYC was predicted to bind to the PD-L1 transcription site and regulate PD-L1 expression. CREB1 was also predicted to transcriptionally bind to both MYC and PD-L1. These results predicted the interaction network of cAMP/PDE4/CREB1/CREBP/MYC/PD-L1, and the core factor may be related to MYC. In the cell experiment, forskolin (an adenylate cyclase activator) and zardaverine (a PDE4 inhibitor) enhance the cAMP pathway and decrease PD-L1 expression, while SQ2253 (an adenylate cyclase inhibitor) inhibits the cAMP pathway and increases PD-L1 expression of the LUAD cell lines H1299 and A549, and MYC regulation by these drugs was positively correlated with PD-L1 regulation, which verified the regulation of the cAMP/PDE4 pathway on MYC and PD-L1.ConclusionsThis study showed that the cAMP/PDE4 pathway may play an important role in PD-L1 regulation and immune infiltration in LUAD.

Genetic Variation of PD-L1 Gene Affects its Expression and Is Related to Clinical Outcome in Epithelial Ovarian Cancer.

ObjectiveThis study aims to investigate the effect of polymorphisms of programmed cell death-ligand 1 (PD-L1) on the risk and patient’s outcomes of epithelial ovarian cancer (EOC).MethodsTotally, 568 patients and 532 healthy women were included. Three polymorphisms in the PD-L1 gene, rs2297136, rs4143815 and rs4742098, were genotyped by the polymerase chain reaction/ligase detection reaction (PCR-LDR). Survival analysis was performed in 234 patients (received primary debulking surgery followed by platinum-based chemotherapy).ResultsPatients with the rs2297136 AG + GG genotypes had shorter progression-free survival (PFS) (hazard ratio (HR)=1.44, 95% CI=1.03-2.01) and overall survival (OS) (HR=1.55, 95% CI=1.06-2.27) than those with the AA genotype. Moreover, the mRNA and protein expression levels of PD-L1 in EOC tissues with the rs2297136 AG + GG genotypes were remarkably higher than those with the AA genotype (P=0.032 and P=0.047, respectively). Survival analysis showed that high expression of PD-L1 mRNA was remarkably associated with worse 10-year PFS (HR=1.55, 95% CI=1.28-1.88) and OS (HR=1.51, 95% CI=1.00-2.28) in EOC patients.ConclusionsThe rs2297136 may not only effectively influence the expression of PD-L1, but also is significantly associated with EOC patients’ outcomes.

Implication of ARID1A Undercurrents and PDL1, TP53 Overexpression in Advanced Gastric Cancer

AT-rich interactive domain-containing protein 1A (ARID1A), TP53 and programmed cell death-ligand 1 (PDL1) are involved in several protein interactions that regulate the expression of various cancer-related genes involved in the progression of the cell cycle, cell proliferation, DNA repair, and apoptosis. In addition, gene expression analysis identified some common downstream targets of ARID1A and TP53. It has been established that tumors formed by ARID1A-deficient cancer cells exhibited elevated PDL1 expression. However, the aberrations in these molecules have not been studied in this population especially in Gastric Cancer (GC). In this backdrop we aimed to investigate the role of the ARID1A mutation and expression of ARID1A, TP53 and PDL1 genes in the etiopathogenesis of Gastric Cancer (GC) in the ethnic Kashmiri population (North India). The study included 103 histologically confirmed GC cases. The mutations, if any, in exon-9 of ARID1A gene was analysed by Polymerase Chain Reaction (PCR) followed by Sanger sequencing. The mRNA expression of the ARID1A, TP53 and PDL1 genes was analysed by Quantitative real time-PCR (qRT-PCR). We identified a nonsense mutation (c.3219; C > T) in exon-9 among two GC patients (∼2.0%), which introduces a premature stop codon at protein position 1073. The mRNA expression of the ARID1A, TP53 and PDL1 gene was significantly reduced in 25.3% and elevated in 47.6 and 39.8% of GC cases respectively with a mean fold change of 0.63, 2.93 and 2.43. The data revealed that reduced mRNA expression of ARID1A and elevated mRNA expression of TP53 and PDL1 was significantly associated with the high-grade and advanced stage of cancer. Our study proposes that ARAD1A under-expression and overexpression of TP53 and PDL1 might be crucial for tumor progression with TP53 and PDL1 acting synergistically.

Expression of miRNA-601 and PD-L1 among Iranian Patients with Lung Cancer and Their Relationship with Smoking and Mycoplasma Infection.

Objective microRNAs (miRNAs) are highly conserved noncoding RNA molecules that mainly function to regulate gene expressions, and have a significant role in tumourigenesis. Programmed cell death-ligand 1 (PD-L1) is a major co-inhibitory checkpoint signal that controls T cell activities, maintains peripheral tolerance and is contribute to the development of cancer. The aim of this study is to examine miRNA-601 and PD-L1 gene expression in patients with non-small-cell lung cancer (NSCLC) and its relation with Mycoplasma infection. Materials and Methods In this case-control study, respiratory secretions and blood samples were collected from 80 healthy people and 80 NSCLC patients. The expression levels of miRNA-601 and PD-L1 were evaluated using real-time polymerase chain reaction (qRT-PCR). The presence of Mycoplasma species in respiratory secretions was detected by biochemical assays and PCR. Results There was no significant difference in the expression level of miRNA-601 between control and patients with tumour stage I, but miRNA-601 expression was significantly downregulated in patients with tumour stages II, III, and IV (P<0.05). A significant, negative relationship was found between miRNA-601 expression and tumour stage (P<0.001). Overexpression of PD-L1 was found in all of the disease stages. PCR results showed the presence of Mycoplasma pneumoniae (M. pneumoniae) in respiratory secretions from patients with stages III and IV NSCLC. We observed that 72% of patients with stages III and IV NSCLC had a positive smoking history and 65.3% were positive for Mycoplasma. ConclusionSerum miRNA-601 may act as a potential noninvasive biomarker for lung cancer and Mycoplasma infection prognosis.

The Prognostic Significance of PD1 and PDL1 Gene Expression in Lung Cancer: A Meta-Analysis.

BackgroundImmune checkpoint blockade therapy represents an extraordinary advance in lung cancer treatment. It is important to determine the expression of immune checkpoint genes, such as programmed cell death 1 (PD1) and programmed cell death-ligand 1 (PDL1), to develop immunotherapeutic strategies. The aim of this study was to explore the association between PD1 and PDL1 gene expression and prognoses and outcomes in lung cancer.MethodsThis meta-analysis analyzed 1,251 patients from eight different microarray gene expression datasets and were evaluated for their prognostic implications and verified using another independent research.ResultsThe mean expression levels of PDL1 in adenocarcinoma (AD) and squamous cell carcinoma (SC) were significantly higher in patients who died than in patients who did not. There was a trend toward incremental increases in PD1 and PDL1 expression significantly decreasing the risk of relapse and death among AD patients (HR = 0.69; 95% CI = 0.53 ~ 0.91; HR = 0.68; 95% CI = 0.54 ~ 0.84, respectively) and SC patients (HR = 0.53; 95% CI = 0.32 ~ 0.89; HR = 0.78; 95% CI = 0.57 ~ 1.00 respectively), as early-stage patients in this study were more likely to have high expression of both PD1 and PDL1 than late-stage patients (P-trend < 0.05). In contrast, late-stage SC patients expressing one or more of the genes at a high level had a significantly elevated risk of relapse (HR = 1.51; 95% CI = 1.07 ~ 2.11) and death (HR = 1.41; 95% CI = 1.08 ~ 1.84). This result was consistent with the validation data set.ConclusionThese findings indicate that high expression of PD1 and PDL1 is associated with superior outcome in early-stage lung cancer but an adverse outcome in late-stage lung cancer. The expression levels of PD1 and PDL1 individually or jointly are potential prognostic factors for predicting patient outcomes in lung cancer.

PD-L1 Expression Fluctuates Concurrently with Cyclin D in Glioblastoma Cells.

Despite Glioblastoma (GBM) frequently expressing programmed cell death ligand-1 (PD-L1), treatment with anti-programmed cell death-1 (PD1) has not yielded brilliant results. Intratumor variability of PD-L1 can impact determination accuracy. A previous study on mouse embryonic fibroblasts (MEFs) reported a role for cyclin-D in control of PD-L1 expression. Because tumor-cell growth within a cancer is highly heterogeneous, we looked at whether PD-L1 and its cochaperone FKBP51s were influenced by cell proliferation, using U251 and SF767 GBM-cell-lines. PD-L1 was measured by Western blot, flow cytometry, confocal-microscopy, quantitative PCR (qPCR), CCND1 by qPCR, FKBP51s by Western blot and confocal-microscopy. Chromatin-Immunoprecipitation assay (xChIp) served to assess the DNA-binding of FKBP51 isoforms. In the course of cell culture, PD-L1 appeared to increase concomitantly to cyclin-D on G1/S transition, to decrease during exponential cell growth progressively. We calculated a correlation between CCND1 and PD-L1 gene expression levels. In the temporal window of PD-L1 and CCND1 peak, FKBP51s localized in ER. When cyclin-D declined, FKBP51s went nuclear. XChIp showed that FKBP51s binds CCND1 gene in a closed-chromatin configuration. Our finding suggests that the dynamism of PD-L1 expression in GBM follows cyclin-D fluctuation and raises the hypothesis that FKBP51s might participate in the events that govern cyclin-D oscillation.

Fit-for-purpose quantitative liquid biopsy based droplet digital PCR assay development for detection of programmed cell death ligand-1 (PD-L1) RNA expression in PAXgene blood samples.

Development of a clinically applicable liquid biopsy-based test for PD-L1 mRNA expression would be beneficial in providing complementary evidence to current immunohistochemistry assays. Hence, we report the development of a fit-for-purpose assay for detection of blood PD-L1 mRNA expression using droplet digital polymerase chain reaction (ddPCR). TaqMan® assays were selected based on coverage of the PD-L1 gene and were tested for linearity and efficiency using real-time quantitative PCR. Four reference genes were analyzed in positive control cell line (A549 treated with interferon gamma, [IFN γ]) genomic DNA. The PD-L1 primer/probe sets were also evaluated in ddPCR for limit of blank, limit of detection, and precision. Finally, thirty-five healthy volunteer samples were evaluated to establish a baseline level of PD-L1 expression. In ddPCR, the limit of blank was determined to be 0 copies and the limit of detection was determined to be less than or equal to 19 copies of PD-L1. The average intra-run coefficient of variation in the ddPCR assay was 7.44% and average inter-run CV was 7.70%. Treatment of A549 cells with IFN γ resulted in a 6.7-fold increase in PD-L1 expression (21,580 copies in untreated cDNA versus 145,000 copies in treated cDNA). Analysis of healthy human samples yielded a median value of 1659 PD-L1 copies/μL with a range of 768-7510 copies/μL. The assay was transferred to an external service provider and results from our in-house experiments and those conducted externally shows a correlation of 0.994. In conclusion, a fit-for-purpose liquid biopsy-based, purely quantitative ddPCR assay for the detection of PD-L1 mRNA expression was developed and validated using PAXgene RNA blood samples. Linearity, reproducibility, limit of blank and limit of detection were measured and deemed suitable for clinical application. This ultra-sensitive liquid biopsy ddPCR assay has promising clinical potential in screening, longitudinal monitoring and disease progression detection.

Interleukin 6 regulates the expression of programmed cell death ligand 1 in thyroid cancer.

Programmed cell death ligand 1 (PD‐L1), inducing T cell exhaustion to facilitate immune escape of tumor cells, is upregulated by interleukin 6 (IL‐6) in T cell lymphoma and ovarian cancer. The purpose of this study is to investigate the expression of IL‐6 and PD‐L1 in thyroid cancer, and whether IL‐6 regulates PD‐L1 expression. As a result, IL‐6 and PD‐L1 were highly expressed in thyroid cancer tissues. Multivariate logistic analysis showed that tumor size, distant metastasis, and risk stratification were significantly associated with IL‐6 expression (P < .05), and multifocality, lymph node metastasis, distant metastasis, risk stratification, and IL‐6 expression were identified as the independent predictors of PD‐L1 expression (P < .05). The invasiveness of thyroid cancer was significantly enhanced after IL‐6 treatment or PD‐L1 overexpression. PD‐L1 positive rate correlated with IL‐6 expression in cancer tissues (P < .001), and after IL‐6 treatment, the PD‐L1 expression in TPC‐1 and BCPAP significantly increased. The mitogen‐activated protein kinase pathway (MAPK) and the Janus‐activated kinase (JAK)–signal transducers and activators of transcription 3 (STAT3) signaling pathways were activated by IL‐6, and the IL‐6–induced PD‐L1 expression decreased after treatment with these two signaling pathway inhibitors. Knockdown of transcription factors c‐Jun and stat3 suppressed the expression of PD‐L1 induced by IL‐6, and these two factors could bind to PD‐L1 gene promoter directly and promote its transcription. It is concluded that IL‐6 and PD‐L1 are overexpressed in thyroid cancer and are related to tumor invasiveness. IL‐6 upregulates PD‐L1 expression through the MAPK and JAK‐STAT3 signaling pathways, which function via transcription factors c‐Jun and stat3.

RNAseq analysis identifies involvement of EBNA2 in PD-L1 induction during Epstein-Barr virus infection of primary B cells

Epstein-Barr virus (EBV) is a causative agent of infectious mononucleosis and several types of malignancy. RNAseq of peripheral blood primary B cell samples infected with wild-type EBV revealed that expression of programmed cell death ligand-1 (PD-L1) is markedly induced by infection. This induction of PD-L1 was alleviated by knockout of the EBNA2 gene, but knockout of LMP1 had little effect. ChIPseq, ChIA-PET, and reporter assays further confirmed that EBNA2-binding sites in the promoter region and at 130 kb downstream of the PD-L1 gene played important roles in PD-L1 induction. Our results indicate that EBV mainly utilizes the EBNA2 gene for induction of PD-L1 and to evade host immunity on infection of primary B cells. Furthermore, pathway analysis revealed that genes involved in the cell cycle, metabolic processes, membrane morphogenesis, and vesicle regulation were induced by EBNA2, and that EBNA2 suppressed genes related to immune signaling.

Frequent amplification of HDAC genes and efficacy of HDAC inhibitor chidamide and PD-1 blockade combination in soft tissue sarcoma

BackgroundThe advent of immune checkpoint therapy has been a tremendous advance in cancer treatment. However, the responses are still insufficient in patients with soft tissue sarcoma (STS). We aimed to...

Ruxolitinib reverses checkpoint inhibition by reducing programmed cell death ligand-1 (PD-L1) expression and increases anti-tumour effects of T cells in multiple myeloma.

Multiple myeloma (MM) tumour cells evade host immunity through a variety of mechanisms, which may potentially include the programmed cell death ligand-1 (PD-L1):programmed cell death protein-1 (PD-1) axis. This interaction contributes to the immunosuppressive bone marrow (BM) microenvironment, ultimately leading to reduced effector cell function. PD-L1 is overexpressed in MMBM and is associated with the resistance to immune-based approaches for treating MM. Ruxolitinib (RUX), an inhibitor of the Janus kinase (JAK) family of protein tyrosine kinases, is approved for myeloproliferative diseases. We investigated the effects of RUX alone or in combination with anti-MM agents on the expression of PD-L1 and T-cell cytotoxicity in MM. We showed that the expression of the PD-L1 gene was markedly increased in BM mononuclear cells from patients with MM with progressive disease versus those in complete remission. Furthermore, RUX treatment resulted in a concentration-dependent reduction of PD-L1 gene expression in the MM tumour cells cultured alone or co-cultured with stromal cells compared with untreated cells. The results also demonstrated that RUX increased MM cell apoptosis in the presence of interleukin-2-stimulated T cells to a similar degree as the treatment with anti-PD-1 or anti-PD-L1 antibodies. In summary, these results indicate that RUX can block PD-L1 expression resulting in augmentation of anti-MM effects of T cells.

Abstract 3132: Single nucleotide polymorphisms (SNPs) in PD-L1 as predictive biomarkers for checkpoint inhibitor based-immunotherapy in caucasian patients with advanced NSCLC

Abstract Effective predictive biomarkers are needed for checkpoint inhibitors (ICI) based-immunotherapy in non-small-cell lung cancer (NSCLC). This study aims to evaluate whether single nucleotide polymorphisms (SNPs) in programmed cell death-ligand 1 (PD-L1) might effectively predict tumor response to ICI in Caucasian patients with advanced NSCLC. Peripheral blood samples and clinico-pathological data were prospectively collected from advanced NSCLC patients treated with anti-Programmed cell death protein 1 (PD-1) inhibitor nivolumab. rs822336, rs2282055 and rs4143815 PD-L1 SNPs were analysed based on previous reports available in the literature. PD-L1 SNPs were analysed following DNA extraction from stored peripheral blood lymphocytes by Taqman Real-Time PCR assay. Presence of PD-L1 SNPs were correlated with clinico-pathological data. Mechanisms underlying the effect of PD-L1 SNPs on PD-L1 expression were analysed in vitro utilizing five NSCLC cell lines, RNA expression and flow cytometry analysis as well as PROMO bioinformatic tool. PD-L1 SNPs localization were analysed utilizing Ensembl genome browser. Differences were considered statistically significant with p value&amp;lt; 0.05. Twenty-eight patients were included in the study. rs2282055 was present as wild type (wt) TT (rs2282055&amp;gt;T), GT and GG genotype in 56.25%, 34.38% and 9.38%, respectively, of patients. On the other hand, rs822336 was present as wt GG (rs822336&amp;gt;G), GC and CC genotype in 28.13%, 37.50% and 34.38%, respectively, of patients. Progression free survival (PFS) was significantly correlated with presence of both PD-L1 SNPs rs2282055 and rs822336 but not with rs4143815. Patients carrying GG either for rs2282055 or rs822336 had a significantly (p=0.0434 and p=0.0131, respectively) shorter PFS as compared to those carrying other PD-L1 SNP genotypes. NSCLC cell lines carrying either GG for rs2282055 or rs822336 significantly (p&amp;lt;0.05) expressed a higher level of PD-L1 expression as compared to cells with other PD-L1 SNP genotypes. Interferon gamma treatment induced PD-L1 expression regardless of PD-L1 SNPs. rs822336 and rs2282055 mapped in enhancer-promoter and intronic regions of PD-L1 gene, respectively. rs822336 was further investigated because of its highest clinical significance and gene localization. Twelve transcription factors were identified to potentially bind rs822336. Two of them discriminated between alternative alleles at the SNP locus and are further under investigation. Previous studies have shown the role of SNPs in cancer patient outcome as well as in mRNA and protein expression changing. In the present study we demonstrated that some of PD-L1 SNPs can affect the basal expression of PD-L1. These results have clinical significance since PD-L1 SNPs predict tumor response to nivolumab-based immunotherapy in Caucasian patients with advanced NSCLC. Citation Format: Francesco Sabbatino, Giovanna Polcaro, Luigi Liguori, Valentina Manzo, Valeria Conti, Jessica Dal Col, Anita Vergatti, Cristiana Stellato, Vincenzo Casolaro, Amelia Filippelli, Soldano Ferrone, Stefano Pepe. Single nucleotide polymorphisms (SNPs) in PD-L1 as predictive biomarkers for checkpoint inhibitor based-immunotherapy in caucasian patients with advanced NSCLC [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3132.

Programmed Cell Death-1: Programmed Cell Death-Ligand 1 Interaction Protects Human Cardiomyocytes Against T-Cell Mediated Inflammation and Apoptosis Response In Vitro

Aim: Immunological checkpoint therapy is considered a powerful method for cancer therapy and acts by re-activating autologous T cells to kill the cancer cell. Myocarditis cases have been reported in cancer patients after immunological therapy; for example, nivolumab treatment is a monoclonal antibody that blocks programmed cell death-1/programmed cell death ligand-1 ligand interaction. This project provided insight into the inflammatory response as a benchmark to investigate the potential cardiotoxic effect of T cell response to the programmed cell death-1 (PD-1)/programmed cell death ligand-1 (PD-L1) axis in regulating cardiomyocyte injury in vitro. Methods and Results: We investigated cardiomyopathy resulted from the PD-1/PD-L1 axis blockade using the anti-PD-1 antibody in Rockefeller University embryonic stem cells-derived cardiomyocytes (RUES2-CMs) and a melanoma tumor-bearing murine model. We found that nivolumab alone did not induce inflammatory-related proteins, including PD-L1 expression, and did not induce apoptosis, which was contrary to doxorubicin, a cardiotoxic chemotherapy drug. However, nivolumab was able to exacerbate the immune response by increasing cytokine and inflammatory gene expression in RUES2-CMs when co-cultured with CD4+ T lymphocytes and induced apoptosis. This effect was not observed when RUES2-CMs were co-cultured with CD8+ T lymphocytes. The in vivo model showed that the heart function of tumor-bearing mice was decreased after treatment with anti-PD-1 antibody and demonstrated a dilated left ventricle histological examination. The dilated left ventricle was associated with an infiltration of CD4+ and CD8+ T lymphocytes into the myocardium. PD-L1 and inflammatory-associated gene expression were significantly increased in anti-PD-1-treated tumor-bearing mice. Cleaved caspase-3 and mouse plasma cardiac troponin I expressions were increased significantly. Conclusion: PD-L1 expression on cardiomyocytes suppressed T-cell function. Blockade of PD-1 by nivolumab enhanced cardiomyocyte inflammation and apoptosis through the enhancement of T-cell response towards cardiomyocytes.

Phage display-identified PD-L1-binding peptides reinvigorate T-cell activity and inhibit tumor progression

Blockade of programmed cell death ligand-1 (PD-L1) restores T-cell activity and enhances anti-tumor immunity. Screening a phage-displayed peptide library for peptides that selectively bind to PD-L1-overexpressing cells identified two peptides, CLQKTPKQC and CVRARTR (PD-L1Pep-1 and PD-L1Pep-2, respectively) that appeared to block PD-L1. PD-L1Pep-1 and PD-L1Pep-2 preferentially bound to high PD-L1-expressing cells over low PD-L1-expressing cells; binding was further enhanced by interferon-γ, an inducer of PD-L1 expression. Binding affinities of PD-L1Pep-1 and PD-L1Pep-2 were approximately 373 and 281 nM, respectively. Cellular binding of the PD-L1-binding peptides was reduced by silencing PD-L1 gene expression or competition with anti-PD-L1 antibody. PD-L1Pep-1 and PD-L1Pep-2 induced the internalization and downregulated cell surface levels of PD-L1. The PD-L1-binding peptides restored cytokine secretion and T-cell proliferation to cells inhibited by co-culture with tumor cells or culture on PD-L1-coated plates. Intravenously injected PD-L1Pep-1 and PD-L1Pep-2 efficiently homed to tumor tissues, inhibited tumor growth, and increased CD8+/FoxP3+ ratio in mice. The PD-L1-binding peptides in combination with doxorubicin or PD-L1-targeted liposomal doxorubicin inhibited tumor growth and increased CD8+/FoxP3+ ratio more efficiently than doxorubicin alone and untargeted liposomal doxorubicin, respectively. These results suggest that PD-L1Pep-1 and PD-L1Pep-2 block PD-L1 and reinvigorate T-cell activity, inhibiting tumor growth by enhancing anti-tumor immunity.

Optimizing PD-L1 evaluation on cytological samples from advanced non-small-cell lung cancer.

Aim: Programmed cell death-ligand 1 (PD-L1) predicts response to immune checkpoint inhibitors in non-small-cell lung cancer (NSCLC) patients. Most NSCLCs are diagnosed at an advanced stage and using minimally invasive diagnostic procedures that yield small biopsies or cytological samples. Methods: Cytological smears and paired histological samples from 52 advanced NSCLC patients were tested for PD-L1 expression by immunocyto/histochemistry (ICC/IHC) and for PD-L1 gene status by FISH. Results:PD-L1 was overexpressed in 9/52 (17%) cytological samples and in seven (13.5%) matched biopsies. The concordance between immunocytochemistry and IHC was 92.3% (48/52;p<0.001). The concordance between PD-L1 gene status on cytology and histology was 69.2% (18/26;p<0.001). No correlation between IHC and fluorescence in situ hybridization results was found. Conclusion: Our data support the feasibility and reliability of PD-L1 protein and PD-L1 gene assessment on direct cytological smears from NSCLC patients whenever histological sample are inadequate.