Expression Of Immune Checkpoint Proteins Research Articles

Integrated analyses for identification of a three-gene signature associated with Chaihu Shugan San formula for hepatocellular carcinoma treatment.

Chaihu Shugan San (CSS) is a well-known traditional herbal formula that has the potential to ameliorate hepatocellular carcinoma (HCC); however, its mechanism of action remains unknown. Here, we identified the key targets of CSS against HCC and developed a prognostic model to predict the survival of patients with HCC. The effect of CSS plus sorafenib on HCC cell proliferation was evaluated using the MTT assay. LASSO-Cox regression was used to establish a three-gene signature model targeting CSS. Correlations between immune cells, immune checkpoints and risk score were determined to evaluate the immune-related effects of CSS. The interactions between the components and targets were validated using molecular docking and Surface Plasmon Resonance (SPR) assays. CSS and sorafenib synergistically inhibited HCC cell proliferation. Ten core compounds and 224 targets were identified using a drug compound-target network. The prognostic model of the three CSS targets (AKT1, MAPK3 and CASP3) showed predictive ability. Risk scores positively correlated with cancer-promoting immune cells and high expression of immune checkpoint proteins. Molecular docking and SPR analyses confirmed the strong binding affinities of the active components and the target genes. Western blot analysis confirmed the synergistic effect of CSS and sorafenib in inhibiting the expression of these three targets. In conclusion, CSS may regulate the activity of immune-related factors in the tumour microenvironment, reverse immune escape, enhance immune responses through AKT1, MAPK3, and CASP3, and synergistically alleviate HCC. The co-administration of sorafenib with CSS has a strong clinical outlook against HCC.

ARID1A deficiency promotes progression and potentiates therapeutic antitumour immunity in hepatitis B virus-related hepatocellular carcinoma.

Exploring predictive biomarkers and therapeutic strategies of ICBs has become an urgent need in clinical practice. Increasing evidence has shown that ARID1A deficiency might play a critical role in sculpting tumor environments in various tumors and might be used as pan-cancer biomarkers for immunotherapy outcomes. The current study aims to explored the immune-modulating role of ARID1A deficiency in Hepatitis B virus (HBV) related hepatocellular carcinoma (HBV-HCC) and its potential immunotherapeutic implications. In the current study, we performed a comprehensive analysis using bioinformatics approaches and pre-clinical experiments to evaluate the ARID1A regulatory role on the biological behavior, and immune landscape of Hepatitis B virus (HBV) related hepatocellular carcinoma (HBV-HCC). A total of 425 HBV-related hepatocellular carcinoma patients from TCGA-LIHC, AMC and CHCC-HBV cohort were enrolled in bioinformatics analysis.Immunohistochemical staining of HBV-HCC specimens and ARID1A deficiency cellular models were used to validate the results of the analysis. Our results have shown that ARID1A deficiency promoted tumor proliferation and metastasis. More importantly, ARID1A deficiency in HBV-HCC was associated with the higher TMB, elevated immune activity, and up-regulated expression of immune checkpoint proteins, especially TIM-3 in HBV-HCC. Further, the expression of Galectin-9, which is the ligand of TIM-3, was elevated in the ARID1A knockout HBV positive cell line. To conclude, we have shown that the ARID1A deficiency was correlated with more active immune signatures and higher expression of immune checkpoints in HBV-HCC. Additionally, the present study provides insights to explore the possibility of the predictive role of ARID1A in HBV-HCC patients responsive to immunotherapy.

SAMHD1 Inhibitor Combined with Immune Checkpoint Inhibitor Elicits an Anti-AML Immunity

Despite advances in targeted therapy and use of molecular/cytogenetic risk stratification to guide treatment, clinical outcomes for patients with acute myeloid leukemia (AML) remain unfavorable. Harnessing the immune system with CAR-T or immune checkpoint inhibitors (ICIs) has been proven successful in other hematological malignancies and solid tumors. The use of these immunotherapies for the AML treatment remains challenging due to lack of selective target antigens or low expression of immune checkpoint proteins. Recently, one strategy proposed to transform AML from immune-cold to immune-hot is to trigger a type-I interferon (IFN-I) response via a dsDNA related cGAS/STING signaling or dsDNA related RIG-I/MDA5 signaling. We recently reported that the dNTP and Ara-CTP (active form of cytarabine [AraC]) hydrolase SAMHD1, whose expression levels are elevated in AML patients relative to normal donors, limits IFN-I and T cell responses in murine AML model ( Blood, 2022, 140 [Supplement 1]: 679-680). We also found that innate immune activation seen following SAMHD1 knockdown is associated with its catalytic activity inhibition in cancer cells, providing a rationale for the development of SAMHD1 inhibitor. To identify SAMHD1 inhibitor, we conducted a virtual screen on 8,000,000 compounds based on SAMHD1 crystal structure (PBD 6TXC). The top 500 hits were then assessed in SAMHD1-WT- or H233A (SAMHD1 loss-of-function mutant) -expressing Molm13 cells using a phenotypic screen for AraC sensitization. Specifically, a SAMHD1 inhibitor should sensitize SAMHD1-WT (S-WT) but not H233A (S-H233A) cells to AraC treatment. Among the top 20 hits from phenotypic screen, our HPLC-based cell-free and cell-based SAMHD1 enzymatic assay revealed Samin27 as the most potent hit in blocking SAMHD1 hydrolysis and promoting AraCTP accumulation. Therefore, we assessed the efficacy of Samin27 combined with AraC in vivo. Samin27 administration (25mg/kg i.p. qd, starting on day 8 for 7 days) enhanced AraC efficacy (50mg/kg i.p. qd, starting 8th day for 5 days) in NSG mice implanted with Molm13 cells, as evidenced by extended survival relative to AraC alone. Moreover, the physical interaction between Samin27 and SAMHD1 protein were confirmed in a Carr-Purcell-Meiboom-Gill (CPMG) nuclear magnetic resonance (NMR) titration assay. RNA-seq results revealed Samin27 ex-vivo treatment remarkably upregulated IFN-I response genes in Molm13 cells, consistent with transcriptome results of SAMHD1-knockdown (S-KD) Molm13 cells. SAMHD1 activity is known to restrict replication of LINE-1 (L1), which is the only autonomously active transposable elements (TE) in mammals; elevated retrotransposition induces DNA damage and promotes cytoplasmic DNA accumulation, resulting in an IFN-I response via cGAS signaling. Next, to explore the mechanism underlying SAMHD1-inhibition induced immunity, we asked if SAMHD1 loss promoted retrotransposition and upregulated TE transcripts in leukemia cells. Through re-analysis of RNA-seq data in Molm13 cells, we found SAMHD1 inhibition upregulated most L1 elements and LTR-containing endogenous retrovirus (ERV) subfamilies. To determine if Samin27 induced immunity via L1 upregulation, we employed 3TC treatment (3TC, the nucleoside reverse transcriptase inhibitor) to block L1 upregulation. 3TC treatment blocked Samin27-mediated IFN-I response genes upregulation. Given that IFN-I response can stimulate innate immune cells (macrophage and T cell) cross-priming, potentially rendering AML cells more susceptible to anti-CD47 monoclonal antibody (aCD47) treatment, we then assessed whether combining a SAMHD1 inhibitor with an aCD47 act synergistically. We first implanted murine MLL-AF9 (MA9) leukemia cells into WT B6 mice. Then we treated leukemic mice with isotype control (Ctrl), anti-CD47 (BE0283 [BioXCell], 10mg/kg, i.p. qd, 3 weeks), Samin27 (25mg/kg i.p. qd, 3 weeks) or combination. Notably, pharmacological inhibition of SAMHD1 remarkably inhibited leukemia progression, decreasing leukemia burden (Fig. A.B). In another cohort, combination treatment extended mouse survival (Fig. C). Collectively, our results may provide a rationale for combining Samin27 with aCD47 against AML population, where single anti-CD47 treatment shows little effects.

Integrating bulk and single-cell RNA sequencing data reveals epithelial-mesenchymal transition molecular subtype and signature to predict prognosis, immunotherapy efficacy, and drug candidates in low-grade gliomas.

Objective: Epithelial-mesenchymal transition (EMT) is a tightly regulated and dynamic process occurring in both embryonic development and tumor progression. Our study aimed to comprehensively explore the molecular subtypes, immune landscape, and prognostic signature based on EMT-related genes in low-grade gliomas (LGG) in order to facilitate treatment decision-making and drug discovery. Methods: We curated EMT-related genes and performed molecular subtyping with consensus clustering algorithm to determine EMT expression patterns in LGG. The infiltration level of diverse immune cell subsets was evaluated by implementing the single-sample gene set enrichment analysis (ssGSEA) and ESTIMATE algorithms. The distinctions in clinical characteristics, mutation landscape, and immune tumor microenvironment (TME) among the subtypes were subjected to further investigation. Gene Set Variation Analysis (GSVA) was performed to explore the biological pathways that were involved in subtypes. The chemo drug sensitivity and immunotherapy of subtypes were estimated through GDSC database and NTP algorithm. To detect EMT subtype-related prognostic gene modules, the analysis of weighted gene co-expression network (WGCNA) was performed. The LASSO algorithm was utilized to construct a prognostic risk model, and its efficacy was verified through an independent CGGA dataset. Finally, the expression of the hub genes from the prognostic model was evaluated through the single-cell dataset and in-vitro experiment. Results: The TCGA-LGG dataset revealed the creation of two molecular subtypes that presented different prognoses, clinical implications, TME, mutation landscapes, chemotherapy, and immunotherapy. A three-gene signature (SLC39A1, CTSA and CLIC1) based on EMT expression pattern were established through WGCNA analysis. Low-risk patients showed a positive outlook, increased immune cell presence, and higher expression of immune checkpoint proteins. In addition, several promising drugs, including birinapant, fluvastatin, clofarabine, dasatinib, tanespimycin, TAK-733, GDC-0152, AZD8330, trametinib and ingenol-mebutate had great potential to the treatment of high risk patients. Finally, CTSA and CLIC1 were highly expressed in monocyte cell through single-cell RNA sequencing analysis. Conclusion: Our research revealed non-negligible role of EMT in the TME diversity and complexity of LGG. A prognostic signature may contribute to the personalized treatment and prognostic determination.

IL-1β mediates the induction of immune checkpoint regulators IDO1 and PD-L1 in lung adenocarcinoma cells

IntroductionInflammation plays a significant role in various cancers, including lung cancer, where the inflammatory cytokine IL-1β is often elevated in the tumor microenvironment. Patients with lung adenocarcinoma show higher levels of serum IL-1β compared to healthy individual. Moreover, IL-1β blockade reduces the incidence and mortality of lung cancer. Our prior studies revealed that alveolar type-II cells, the precursors for lung adenocarcinoma, display an induction in the expression of the enzyme tryptophan 2,3-dioxygenase (TDO2) during normal lung development. This induction of TDO2 coincides with an increase in IL-1β levels and is likely caused by IL-1β. Given that cancer cells can co-opt developmentally regulated pathways, we hypothesized that IL-1β may exert its pro-tumoral function by stimulating TDO2 and indoleamine 2, 3-dioxygenase-1 (IDO1), parallel enzymes involved in the conversion of tryptophan (Trp) into the immune-suppressive oncometabolite kynurenine (Kyn). Our goal was to determine whether IL-1β is a common upstream regulator of immune checkpoint regulators.MethodsTo determine whether IL-1β regulates IDO1, TDO2, PD-L1, and PD-L2, we measured mRNA and protein levels in lung adenocarcinoma cells lines (A549, H1792, H1838, H2347, H2228, HCC364 and HCC827) grown in 2D or 3D and in immortalized normal lung epithelial cells (HBEC3-KT and HSAEC1-KT). To determine the importance of the NFκB pathway in mediating IL-1β -regulated cellular effects, we used siRNA to knockdown RelA/p65 in IL-1β treated cells. The levels of Trp and Kyn in the IL-1β-treated cells and media were measured by mass spectrometry.ResultsUpon IL-1β stimulation, lung adenocarcinoma cells exhibited significant increases in IDO1 mRNA and protein levels, a response that depended on the NFκB pathway. Interestingly, this induction was more pronounced in 3D spheroid cultures compared to monolayer cultures and was not observed in normal immortalized lung epithelial cells. Furthermore, the conversion of Trp to Kyn increased in cells exposed to IL-1β, aligning with the heightened IDO1 expression. Remarkably, IL-1β also upregulated the expression of programmed death ligand-1 (PD-L1) and PD-L2 in multiple cell lines, indicating that IL-1β triggers parallel immune-suppressive mechanisms in lung adenocarcinoma cells.ConclusionsOur studies demonstrate that lung adenocarcinoma cells, but not normal immortalized lung epithelial cells, respond to IL-1β signaling by inducing the expression of parallel immune checkpoint proteins that have the potential to promote immune evasion.AWNWNjEXZEagXxNY6iY8VPVideo

Activation of immune evasion machinery is a part of the process of malignant transformation of human cells

Malignant transformation of human cells is associated with their re-programming which results in uncontrolled proliferation and in the same time biochemical activation of immunosuppressive pathways which form cancer immune evasion machinery. However, there is no conceptual understanding of whether immune evasion machinery pathways and expression of immune checkpoint proteins form a part of the process of malignant transformation or if they are triggered by T lymphocytes and natural killers (NK) attempting to attack cells which are undergoing or already underwent malignant transformation. To address this fundamental question, we performed experimental malignant transformation of BEAS-2B human bronchial epithelium cells and RC-124 non-malignant human kidney epithelial cells using bracken extracts containing carcinogenic alkaloid called ptaquiloside. This transformation led to a significant upregulation of cell proliferation velocity and in the same time led to a significant upregulation in expression of key immune checkpoint proteins – galectin-9, programmed death ligand 1 (PD-L1), indoleamine 2,3-dioxygenase (IDO1). Their increased expression levels were in line with upregulation of the levels and activities of HIF-1 transcription complex and transforming growth factor beta type 1 (TGF-β)-Smad3 signalling pathway. When co-cultured with T cells, transformed epithelial cells displayed much higher and more efficient immune evasion activity compared to original non-transformed cells. Therefore, this work resolved a very important scientific and clinical question and suggested that cancer immune evasion machinery is activated during malignant transformation of human cells regardless the presence of immune cells in microenvironment.

Skin infiltrating NK cells in cutaneous T-cell lymphoma are increased in number and display phenotypic alterations partially driven by the tumor.

Cutaneous T-cell lymphomas (CTCL) are characterized by focal infiltration of malignant T cell clones in solitary skin lesions. Many CTCL patients experience an indolent disease, but some progress to advanced disease with high fatality. We hypothesized that natural killer (NK) cells participate in local control of tumor growth in CTCL skin. Immunohistochemistry and flow cytometry analysis of the density, localization, phenotype and function of NK cells in twenty-nine fresh or formalin-fixed skin biopsies from twenty-four CTCL patients and twenty-three biopsies from twenty healthy controls highlighted higher numbers of CD56+CD3- NK cells in CTCL skin. A reduced fraction of CTCL skin NK cells expressed the maturation marker CD57, the cytotoxic protein granzyme B and the activation marker CD69, indicating reduced tumor-killing abilities of the NK cells. Retained expression of immune checkpoint proteins or inhibitory proteins including PD1, TIM3, LAG3, CD73 and NKG2A and the activating receptors CD16 and NKp46 indicated maintained effector functions. Indeed, the capacity of NK cells to produce anti-tumor acting IFNγ upon PMA+ionomycin stimulation was similar in cells from CTCL and healthy skin. Co-cultures of primary human NK cells or the NK cell line NKL with CTCL cells resulted in reduced levels of granzyme B and CD69, indicating that close cellular interactions with CTCL cells induced the impaired functional NK cell phenotype. In conclusion, increased numbers of NK cells in CTCL skin exhibit a partially impaired phenotype in terms of activity. Enhancing NK cell activity with NK cell activating cytokines such as IL-15 or immune checkpoint blockade therefore represents a potential immunotherapeutic approach in CTCL.

Role of Mismatch Repair Deficiency Status and Microsatellite Instability in Relation to the Expression of Immune Checkpoint Proteins in Colorectal Cancer.

Introduction Colorectal cancer (CRC) is the third most common cancer in the world among men and second among women worldwide. One of the major molecular pathways responsible for the development of colorectal cancer (CRC) is the microsatellite instability (MSI) pathway. During carcinogenesis, the tumor cellsexpress programmed death ligand-1 (PD-L1), which reduces the immunogenicity leading to the escape of immune attack. Anti-PD-L1 interactionis an upcoming line of research for the treatment of colorectal carcinoma patients. Materials and methods The present study was an ambispective study where the mismatch repair deficiency status (MMR) and programmed death ligand-1 (PD-L1) expression were studied using immunohistochemistry and then later analyzed and compared with the clinicopathological parameters and MSI status in relation to the expression of programmed death ligand-1 (PD-L1) in neoplastic and immune cells in a total of 55 biopsy specimen. MMR expression was reported as retained or loss of nuclear staining, and PD-L1 expression was taken as positive with a cut-off of more than or equal to 5% membranous positivity in both tumor cells and immune cells. Results The analysis showed a significant correlation of microsatellite instability (MSI) status with two of the clinicopathological parameters, which were the site of the tumor (p-value<0.001) and M stage (p-value<0.001). PD-L1 expression in neoplastic cells showed no significant correlation with the clinicopathological parameters, whereas PD-L1 expression in immune cells showed a significant association with gender (p-value=0.043). Also, MSI status showed a significant association with PD-L1 expression in tumor cells (p-value <0.001).

Full spectrum flow cytometry-powered comprehensive analysis of PBMC as biomarkers for immunotherapy in NSCLC with EGFR-TKI resistance.

Clinical studies suggest that immune checkpoint inhibitor (ICI) monotherapy has limited benefits in non-small cell lung cancer (NSCLC) patients after epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) failure. However, data about efficacy of ICI plus chemotherapy remain controversial, probably attributed to the heterogeneity among such population, and robust efficacy biomarkers are urgent to explore. A total of 60 eligible patients who received ICI plus chemotherapy after EGFR-TKI treatment failure were enrolled, 24 of whom peripheral blood mononuclear cell (PBMC) samples were collected at baseline and after 2 cycles oftreatment. We have designed a 23-color-antibody panel to detect PBMC by full spectrum flow cytometry. For EGFR-TKI resistant NSCLC patients: 1) ICI plus chemotherapy achieved an objective response rate (ORR) of 21.7% and a median progression-free survival (PFS) of 6.4months. 2) clinical characteristics associated with worse efficacy included liver metastasis and platelet-to-lymphocyte ratio (PLR) > 200. 3) the proportion of immune cell subset associated with better efficacy was higher baseline effective CD4+T cells (E4). 4) the baseline expression of immune checkpoint proteins (ICPs) on cell subsets associated with better efficacy included: higher expression of CD25 on dendritic cells (DC) and central memory CD8+T cells (CM8), and higher expression of Lymphocyte activation gene 3 (LAG-3) on effective memory CD8+T cells (EM8). 5) the expression of ICPs after 2 cycles of treatment associated with better efficacy included: higher expression of CD25 on CD8+T/EM8 /natural killer (NK) cells. 6) the dynamic changes of ICPs expression associated with worse efficacy included: significantly decrease of T cell immunoglobulin and ITIM domain (TIGIT) expression on regular T cells (Tregs) and decrease of V-domain immunoglobulin suppressor of T cell activation (VISTA) expression on Th1. 7) a prediction model for the efficacy of ICI plus chemotherapy was successfully constructed with a sensitivity of 62.5%, specificity of 100%, and area under curve (AUC) = 0.817. Some EGFR-TKI-resistant NSCLC patients could indeed benefit from ICI plus chemotherapy, but most patients are primary resistant to immunotherapy. Comprehensive analysis of peripheral immune cells using full spectrum flow cytometry showed that compared to the proportion of cell subsets, the expression type and level of ICPs on immune cells, especially CD25, were significantly correlated with the efficacy of immunotherapy.

Identification of age- and immune-related gene signatures for clinical outcome prediction in lung adenocarcinoma.

The understanding of the factors causing decreased overall survival (OS) in older patients compared to younger patients in lung adenocarcinoma (LUAD) remains. Gene expression profiles of LUAD were obtained from publicly available databases by Kaplan-Meier analysis was performed to determine whether age was associated with patient OS. The immune cell composition in the tumor microenvironment (TME) was evaluated using CIBERSORT. The fraction of stromal and immune cells in tumor samples were also using assessed using multiple tools including ESTIMATE, EPIC, and TIMER. Differentially expressed genes (DEGs) from the RNA-Seq data that were associated with age and immune cell composition were identified using the R package DEGseq. A 22-gene signature composed of DEGs associated with age and immune cell composition that predicted OS were constructed using Least Absolute Shrinkage and Selection Operator (LASSO). In The Cancer Genome Atlas (TCGA)-LUAD dataset, we found that younger patients (≤70) had a significant better OS compared to older patients (>70). In addition, older patients had significantly higher expression of immune checkpoint proteins including inhibitory T cell receptors and their ligands. Moreover, analyses using multiple bioinformatics tools showed increased immune infiltration, including CD4+ T cells, in older patients compared to younger patients. We identified a panel of genes differentially expressed between patients >70 years compared to those ≤70 years, as well as between patients with high or low immune scores and selected 84 common genes to construct a prognostic gene signature. A risk score calculated based on 22 genes selected by LASSO predicted 1, 3, and 5-year OS, with an area under the curve (AUC) of 0.72, 0.72, 0.69, receptively, in TCGA-LUAD dataset and an independent validation dataset available from the European Genome-phenome Archive (EGA). Our results demonstrate that age contributes to OS of LUAD patients atleast in part through its association with immune infiltration in the TME.

The effect of blocking immune checkpoints LAG-3 and PD-1 on human invariant Natural Killer T cell function

Invariant Natural Killer T (iNKT) cells undergo immune exhaustion during chronic activation caused by cancer and viral infections, such as HIV. Exhaustion is marked by cell dysfunction and increased expression of immune checkpoint proteins programmed cell-death-1 (PD-1) and lymphocyte-activation-gene-3 (LAG-3). We hypothesize that blockade of PD-1 and/or LAG-3 will enhance iNKT cell function. Utilizing peripheral blood mononuclear cells from healthy donors, LAG-3 and PD-1 expression on iNKT cells was assessed using flow cytometry following in vitro stimulation with iNKT-specific stimulant α-galactosylceramide (n = 4). Efficacy of anti-LAG-3 and/or anti-PD-1 antibody blockades in enhancing iNKT cell function was assessed by determining proliferative capacity and IFN-γ production (n = 9). LAG-3 and PD-1 expression on iNKT cells peaked at Day 4 (98.8%; p ≤ 0.0001 and 98.8%; p = 0.005, respectively), followed by steep decrease by Day 10, coinciding with peak iNKT cell proliferation. In a 10-day blocking assay, both the anti-PD-1 alone and dual anti-PD-1 and anti-LAG-3 significantly increased iNKT proliferation (6 and 6.29 log2 fold-change respectively) compared to the no blockade control (ANOVA-p = 0.0005) with the dual blockade system being more effective (t-test-p = 0.013). This provides proof-of-concept for LAG-3 and PD-1 as immunotherapeutic targets to enhance human iNKT cell function, with the long-term goal of addressing immune exhaustion.

A fluorene derivative inhibits human hepatocellular carcinoma cells by ROS-mediated apoptosis, anoikis and autophagy

Fluorene was previously reported to have anticancer activity against human cancer cells. In this study, we examined the in vitro function of 9-methanesulfonylmethylene-2, 3-dimethoxy-9 H -fluorene (MSDF), a novel fluorene derivative, its anticancer potential in human hepatocellular carcinoma (HCC) cells and its underlying molecular mechanism. The disruption of cellular homeostasis caused by MSDF was found to promote reactive oxygen species (ROS) generation, leading to the activation of cellular apoptosis. As a survival strategy, cells undergo autophagy during oxidative stress. MSDF-induced apoptosis occurred through both receptor-mediated extrinsic and mitochondrial-mediated intrinsic routes. The development of acidic vesicular organelles and the accumulation of LC3-II protein suggest an increase in the autophagic process. Apoptosis was detected by double staining. The MAPK/ERK and PI3K/Akt signaling pathways were indeed suppressed during treatment. Along with elevated ROS generation and apoptosis, MSDF also caused anoikis and cell death by causing cells to lose contact with their extracellular matrix. ROS production was induced by MSDF and sustained by an NAC scavenger. MSDF-induced apoptosis led to increased autophagy, as shown by the suppression of apoptosis by Z-VAD-FMK. However, inhibition of autophagy by inhibitor 3-MA increased MSDF-induced apoptosis. More evidence shows that MSDF downregulated the expression of immune checkpoint proteins, suggesting that MSDF could be used in the future as an adjuvant to improve the effectiveness of HCC immunotherapy. Altogether, our results highlight the potential of MSDF as a multitarget drug for the treatment of HCC.

AKT inhibition interferes with the expression of immune checkpoint proteins and increases NK-induced killing of HL60-AML cells.

To determine the role of the AKT pathway in the regulating of natural Killer-induced apoptosis of acute myeloid leukemia cells and to characterize the associated molecular mechanisms. BALB/c nude mice were injected with HL60 cells to induce a xenogenic model of subcutaneous leukemic tumors. Mice were treated with perifosine, and their spleens were analyzed using biometry, histopathology, and immunohistochemistry. Gene expression analysis in leukemia cells was performed by real-time PCR. Protein analysis of leukemia and natural Killer cells was performed by flow cytometry. AKT inhibition in HL60 cells, followed by co-culture with natural Killer cells was performed to assess cytotoxicity. Apoptosis rate was quantified using flow cytometry. Perifosine treatment caused a reduction in leukemic infiltration in the spleens of BALB/c nude mice. In vitro , AKT inhibition reduced HL60 resistance to natural Killer-induced apoptosis. AKT inhibition suppressed the immune checkpoint proteins PD-L1, galectin-9, and CD122 in HL60 cells, but did not change the expression of their co-receptors PD1, Tim3, and CD96 on the natural Killer cell surface. In addition, the death receptors DR4, TNFR1, and FAS were overexpressed by AKT inhibition, thus increasing the susceptibility of HL60 cells to the extrinsic pathway of apoptosis. The AKT pathway is involved in resistance to natural Killer-induced apoptosis in HL60 cells by regulating the expression of immune suppressor receptors. These findings highlight the importance of AKT in contributing to immune evasion mechanisms in acute myeloid leukemia and suggests the potential of AKT inhibition as an adjunct to immunotherapy.

Implications of GITR expression profile for precision immunotherapeutics.

e14561 Background: Glucocorticoid-induced TNFR-related protein (GITR) is a type I transmembrane protein expressed mostly on CD25+CD4+ T-regs and upregulated on all T-cells upon activation. It serves as a T-cell co-stimulatory receptor and its role in cancer treatment is being explored. While pre-clinical data using combination therapies involving GITR agonism showed promise, early clinical trials have not shown improved outcomes to date. Methods: RNA expression levels of 397 genes in 514 patients with a variety of cancer types were assessed and analyzed at OmniSeq. Transcript levels were normalized to internal housekeeping gene profiles and ranked as percentiles using a reference population. High expression was defined as ≥ 75th percentile, and low-moderate as &lt;75th percentile. Univariate p-values were calculated using Welch t-test or Fischer’s exact test. Multivariate p-values were calculated using logistical regression. Results: A total of 99 patients (19.3%) had GITR high expression. Cancers with the highest proportion of GITR high expression were lung (7/20, 35%) and breast (19/49, 39%), and both these cancers showed a statistically significant association with GITR high expression on multivariate analysis (lung: OR 3.5, p = 0.04, 95% confidence interval (CI) 1.06 – 10.06 CI and breast: OR 4.59, p &lt;0.001, 95% CI: 2.11 – 9.85). 30% of lung and 30.6% of breast cancer patients had a GITR high with ligand low-moderate expression profile; these cancers had the highest proportion of patients with this profile. GITR high expression showed significant association with high RNA expression of multiple known immune checkpoint proteins (PD-1, PD-L1, CTLA4, LAG3, OX40, ICOS and CD137, but not TIM3) as well as PD-L1 IHC ≥1 on univariate analysis, but statistically significant association was only seen with PD-L1 IHC ≥1 (p = 0.007) and OX40 high expression (p = 0.001) on multivariate analysis. Conclusions: While clinical trials with GITR agonism are ongoing, our data suggests selection of certain types of malignancies depending on GITR status may be required. Moreover, there is a correlation between high GITR and high OX40 RNA expression, which could imply increased efficacy using this targeted combination. Further clinical development based on this immune-profiling is warranted. [Table: see text]

Abstract 1423: Regulation of SKP2 expression by methyltransferase-independent function of EZH2 in tumor growth and progression

Abstract Skp2, an F box protein and a substrate-binding component of the SCFSkp2 ubiquitin E3 ligase complex, promotes ubiquitination and degradation of cyclin-dependent kinase inhibitors (CKIs), including p21, p27, and p57. Aberrant Skp2 expression is associated with many human cancers. EZH2 (Enhancer of Zeste Homologue 2) is a core catalytic subunit of the polycomb repressive complex 2 (PRC2) that can regulate gene expression by mediating the methylation of lysine 27 of histone H3 (H3K27me3). Accumulating evidence shows that EZH2 could bind to transcription factors and activate gene expression independently of its methyltransferase activity, leading to the activation of oncogenic signaling pathways and promoting the proliferation of tumor cells. Although the methyltransferase-independent function of EZH2 is important in cell proliferation, its mechanism in cell cycle regulation is still unclear. In this study, we demonstrate that EZH2 positively regulates Skp2 gene expression. We show that suppression of EZH2 expression resulted in a significant decrease of Skp2 protein levels, while the EZH2 enzymatic inhibitors, EPZ6438, had no impact on protein levels of Skp2, which suggests that EZH2 regulates Skp2 expression independently of its methyltransferase activity. Consistently, EZH2 depletion reduced the mRNA levels of Skp2, while EPZ6438 inhibited EZH2 methyltransferase activity without affecting the mRNA levels of Skp2. Importantly, EZH2 expression is positively correlated with the expression of EMT (Epithelial-mesenchymal transition) regulators (YAP1/TAZ, ZEB1, and Snail) and immune checkpoint proteins (FGL1 and Siglec-15) in cervical and pancreatic cancer cells, but reversely correlated with the expression of CKIs (p21, p27, and p57). Taken together, our findings suggest a novel mechanism by which EZH2 regulates tumorigenesis through its methyltransferase-independent function by regulating Skp2 expression. Targeting the methyltransferase-independent function of EZH2 may have therapeutic efficacy for cancer patients. Citation Format: Tania Marlyn Colon, Wei Dai, Byeong Hyeok Choi. Regulation of SKP2 expression by methyltransferase-independent function of EZH2 in tumor growth and progression [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 1423.

Soluble immune checkpoints as correlates for HIV persistence and T cell function in people with HIV on antiretroviral therapy.

In people with HIV (PWH) both off and on antiretroviral therapy (ART), the expression of immune checkpoint (IC) proteins is elevated on the surface of total and HIV-specific T-cells, indicating T-cell exhaustion. Soluble IC proteins and their ligands can also be detected in plasma, but have not been systematically examined in PWH. Since T-cell exhaustion is associated with HIV persistence on ART, we aimed to determine if soluble IC proteins and their ligands also correlated with the size of the HIV reservoir and HIV-specific T-cell function. We used multiplex bead-based immunoassay to quantify soluble programmed cell death protein 1 (PD-1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), lymphocyte activation gene-3 (LAG-3), T cell immunoglobulin domain and mucin domain 3 (TIM-3), PD-1 Ligand 1 (PD-L1) and PD-1 Ligand 2 (PD-L2) in plasma from PWH off ART (n=20), on suppressive ART (n=75) and uninfected controls (n=20). We also quantified expression of membrane-bound IC and frequencies of functional T-cells to Gag and Nef peptide stimulation on CD4+ and CD8+ T-cells using flow cytometry. The HIV reservoir was quantified in circulating CD4+ T-cells using qPCR for total and integrated HIV DNA, cell-associated unspliced HIV RNA and 2LTR circles. Soluble (s) PD-L2 level was higher in PWH off and on ART compared to uninfected controls. Higher levels of sPD-L2 correlated with lower levels of HIV total DNA and higher frequencies of gag-specific CD8+ T-cells expressing CD107a, IFNγ or TNFα. In contrast, the concentration of sLAG-3 was similar in uninfected individuals and PWH on ART, but was significantly elevated in PWH off ART. Higher levels of sLAG-3 correlated with higher levels of HIV total and integrated DNA, and lower frequency of gag-specific CD4+ T cells expressing CD107a. Similar to sLAG-3, levels of sPD-1 were elevated in PWH off ART and normalized in PWH on ART. sPD-1 was positively correlated with the frequency of gag-specific CD4+ T cells expressing TNF-a and the expression of membrane-bound PD-1 on total CD8+ T-cells in PWH on ART. Plasma soluble IC proteins and their ligands correlate with markers of the HIV reservoir and HIV-specific T-cell function and should be investigated further in in large population-based studies of the HIV reservoir or cure interventions in PWH on ART.

Characterization of Viscum album L. Effect on Immune Escape Proteins PD-L1, PD-L2, and MHC-I in the Prostate, Colon, Lung, and Breast Cancer Cells

Background: Viscum album L. (VA) preparations possess immunomodulatory properties and are used in complementary medicine to support cancer therapy. It is unclear if there is an impact of VA on the expression of immune checkpoint proteins on the surface of cancer cells. This study was designed to investigate the role of commercially available VA preparations on checkpoint programmed death ligand 1, 2 (PD-L1, PD-L2) and on major histocompatibility complex class I (MHC-I). Methods: Four human cancer cell lines (prostate, colon, lung, and breast) were assayed for their PD-L1, PD-L2, and MHC-I level after stimulation with interferon-gamma (IFN-γ). The toxicity of mistletoe preparations for the cells was analysed. Afterwards, the effect of mistletoe preparations on the PD ligands and MHC-I was investigated. Results: Surface protein analysis demonstrated that all tested tumour cell lines increased the PD-L1, PD-L2, and MHC-I-expression, but to different extents, after IFN-γ stimulation. Treatment with VA extracts did not influence the viability of the cells. The expression of PD ligands and MHC-I was not affected by incubation with the VA preparations. Conclusion: Our investigation concludes that VA treatment does not interfere with the expression of PD ligands or MHC-I among selected cancer cells.

Na, K-ATPase α1 cooperates with its endogenous ligand to reprogram immune microenvironment of lung carcinoma and promotes immune escape.

Dysregulated endocrine hormones (EHs) contribute to tumorigenesis, but how EHs affect the tumor immune microenvironment (TIM) and the immunotherapy of non-small cell lung cancer (NSCLC) is still unclear. Here, endogenous ouabain (EO), an adrenergic hormone, is elevated in patients with NSCLC and closely related to tumor pathological stage, metastasis, and survival. EO promotes the suppression of TIM in vivo by modulating the expression of immune checkpoint proteins, in which programmed cell death protein ligand 1 (PD-L1) plays a major role. EO increases PD-L1 transcription; however, the EO receptor Na- and K-dependent adenosine triphosphatase (Na, K-ATPase) α1 interacts with PD-L1 to trigger the endocytic degradation of PD-L1. This seemingly contradictory result led us to discover the mechanism whereby EO cooperates with Na, K-ATPase α1 to finely control PD-L1 expression and dampen tumoral immunity. In conclusion, the Na, K-ATPase α1/EO signaling facilitates immune escape in lung cancer, and manipulation of this signaling shows great promise in improving immunotherapy for lung adenocarcinoma.

A novel necroptosis-related lncRNA based signature predicts prognosis and response to treatment in cervical cancer.

Background: Necroptosis has been demonstrated to play a crucial role in the prognosis prediction and assessment of treatment outcome in cancers, including cervical cancer. The purpose of this study was to explore the potential prognostic value of necroptosis-related lncRNAs and their relationship with immune microenvironment and response to treatment in cervical cancer. Methods: Data from The Cancer Genome Atlas (TCGA) were collected to obtain synthetic data matrices. Necroptosis-related lncRNAs were identified by Pearson Correlation analysis. Univariate Cox and multivariate Cox regression analysis and Lasso regression were used to construct a necroptosis-related LncRNAs signature. Kaplan-Meier analysis, univariate and multivariate Cox regression analyses, receiver operating characteristic (ROC) curve, nomogram, and calibration curves analysis were performed to validate this signature. Gene set enrichment analyses (GSEA), immunoassays, and the half-maximal inhibitory concentration (IC50) were also analyzed. Results: Initially, 119 necroptosis-related lncRNAs were identified based on necroptosis-related genes and differentially expressed lncRNAs between normal and cervical cancer samples. Then, a prognostic risk signature consisting of five necroptosis-related lncRNAs (DDN-AS1, DLEU1, RGS5, RUSC1-AS1, TMPO-AS1) was established by Cox regression analysis, and LASSO regression techniques. Based on this signature, patients with cervical cancer were classified into a low- or high-risk group. Cox regression confirmed this signature as an independent prognostic predictor with an AUC value of 0.789 for predicting 1-year OS. A nomogram including signature, age, and TNM stage grade was then established, and showed an AUC of 0.82 for predicting 1-year OS. Moreover, GSEA analysis showed that immune-related pathways were enriched in the low-risk group; immunoassays showed that most immune cells, ESTIMAT scores and immune scores were negatively correlated with risk score and that the expression of immune checkpoint-proteins (CD27, CD48, CD200, and TNFRSF14) were higher in the low-risk group. In addition, patients in the low-risk group were more sensitive to Rucaparib, Navitoclax and Crizotinib than those in the high-risk group. Conclusion: We established a novel necroptosis-related lncRNA based signature to predict prognosis, tumor microenvironment and response to treatment in cervical cancer. Our study provides clues to tailor prognosis prediction and individualized immunization/targeted therapy strategies.

Targeting PRMT9 Elicits Cgas-Mediated Anti-Leukemia Immunity

Immunotherapy has improved outcomes for patients with blood cancers such as B-ALL. However, for patients with immune-cold cancers such as AML, currently available immunotherapies are less effective, likely due to lack of functional T cells and/or low expression of immune checkpoint proteins like PD-L1 . For these patients, development of an effective leukemia-targeting drug with immune-stimulating activity is required. We previously reported elevated PRMT9 levels in AML patient specimens, which enhanced leukemia cell proliferation by promoting RNA translation (Blood, 2021,138 [Supplement 1]: 358). Recently, our transcriptome profiling of AML lines revealed increased expression of interferon-stimulated genes (ISGs) after PRMT9-knockdown (KD), suggesting activation of innate immunity. To evaluate a potential PRMT9 function in anti-AML immunity, we transplanted either WT immunocompetent or Rag2-/- immunodeficient recipient mice with inducible Prmt9-KD or control MLL-AF9 (MA9) AML donor cells and monitored leukemia development by in-vivo bioluminescence Imaging. Unlike Rag2-/- recipients (overall survival <60 days), 5 of 7 WT recipients harboring Prmt9-KD AML survived the observation period (>120 days) and MA9 cells disappeared over time. To assess immune responses upon Prmt9 inhibition, we performed single-cell RNA-sequencing (scRNAseq) in BM and spleen cells from Prmt9-KD or control MA9-transplanted WT recipients (Fig. A). ScRNAseq analysis revealed that, relative to controls, Prmt9-KD induced T cell activation, based on increased T cell expression of Cd69, Gzmb and Ifng. After reanalyzing T cells at higher granularity based on levels of classical markers (e.g., Cd44, Sell, Fig. B, C), we observed reduced numbers of naïve T cells as well as increased effector/memory T cell subsets; increased numbers of cytotoxic T lymphocytes (CTLs) as well as decreased regulatory T cells (Tregs) subset (Fig. D). To validate T cell activation upon Prmt9 KD, we assessed an MA9/OVA transplant model (engineered with OVA to track antigen-specific T cells) and found that mice harboring Prmt9-KD MA9/OVA cells showed increased numbers of OVA-specific CD8+ T cells in BM tracked by flow cytometry after H-2Kb-OVA257-264 tetramer staining (Fig. E). Notably, scRNAseq analysis of multiple immune lineages in BM engrafted with Prmt9-KD MA9 cells revealed upregulation of Isg15, Cxcl10 and other ISGs. To confirm that type-I IFN responses mediate these effects, we implanted Prmt9-KD MA9 cells into WT or type-I IFN receptor KO (Ifnar1-/-) recipients and found that the survival advantage conferred by Prmt9-KD was lost in Ifnar1-/- recipients (Fig. F). Interestingly, Prmt9-KD in MA9 cells also increased expression of Isg15, Cxcl10 and other ISGs by Q-PCR assay, and the effect was blocked by cGAS deletion in MA9 cells, suggesting that Prmt9-KD activates cGAS-STING signaling. Finally, to determine whether Prmt9-KD phenotypes require tumor-intrinsic cGAS activity, we knocked out cGAS in Prmt9-KD MA9/OVA cells and implanted them into WT recipients. Relative to cGAS-WT controls, cGAS-KO in the presence of Prmt9-KD abolished survival advantages and cancer-specific T cell responses seen following Prmt9 KD in MA9/OVA cells (Fig. E). We also developed a potent new PRMT9 inhibitor (PRMT9i) based on our identified lead compound (Blood, 2021,138: 358). CyTOF analysis revealed that PRMT9i treatment ablated primary AML cells (CD34+CD45dim) and relatively increased T cell frequencies in co-cultures of immune cells with leukemia cells from primary AML specimens (n=3). However, PRMT9i anti-AML effects were significantly impaired by T cell depletion. Notably, CyTOF analysis revealed significantly upregulated PD-L1 levels in 2 of 3 (CD34+CD45dim) AML co-cultures after PRMT9i treatment (Fig. G), suggesting activation of the adaptive PD-1/PD-L1 axis, in agreement with the” unwanted negative effects” of cGAS-STING activation as others depicted. Accordingly, PRMT9i treatment of these 2 co-cultures combined with immune checkpoint inhibition (ICI, anti-PD1) synergistically eradicated primary AML cells (Fig. H) and promoted expansion of CD69+ and IFNγ+ CD8+ T cell subsets. Overall, we demonstrate that AML cell-derived PRMT9 regulates AML immune evasion, and that combining PRMT9i with ICIs could constitute a therapeutic strategy against PRMT9-proficient cancers like AML, in which single ICIs show limited efficacy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal