Gene Expression In Cell Lines Research Articles

Modulation of the CGAS-Sting Anti-Tumor Immune Response Pathway in an NF-KB-Dependent or -Independent Manner As a Potential Therapeutic Target in Classical Hodgkin Lymphoma (cHL)

Introduction: Cytosolic DNA of exogenous or endogenous origin triggers activation of cyclic GMP-AMP (cGAMP) synthase (cGAS), a cytosolic DNA sensor, that activates innate immune responses through production of the second messenger cGAMP and subsequently activation of the adaptor protein STING. The latter activates TBK1 and IKK kinases that, in turn, activate IRF3 and NF-κB transcription factors, which induce expression of interferons (IFNs), chemokines and cytokines involved in anti-tumor immune responses. The potential role of cGAS-STING pathway in anti-tumor immune responses in classical Hodgkin lymphoma (cHL) remains unknown to date. In this study, we hypothesized that NF-kB pathway, a critical player in cHL oncogenesis, may regulate anti-tumor immune responses through modulation of the cGAS-STING pathway activity and gene expression of the relevant interferons and chemokines. Methods: STING expression was immunohistochemically analyzed in a pilot study group of 32 untreated patients with cHL and available tissue from diagnostic lymph node biopsies using a previously validated monoclonal antibody and standard methods. In addition, control lymphoid tissue (2 reactive lymph nodes, 2 tonsils) were used. At least 500 neoplastic Hodgkin & Reed-Sternberg (HRS) cells were counted and an arbitrary 10% cutoff was used for positivity. The in vitro system included 6 cHL cell lines (MDA-V, L-1236, L-428, L-540, HDLM-2, KM-H2). Gene expression (mRNA level) and protein expression and activation (phosphorylation) of cGAS-STING pathway components at baseline and experimental conditions were analysed by quantitative RT-PCR (RT-qPCR) and Western blot analysis, respectively. The cHL cell lines were treated with a STING agonist and TBK1/IKK inhibitor (Amlexanox) alone or in combination with other agents. Silencing of STING and NF-kB genes was performed in all cHL cell lines using transient transfection (Nucleofector system, Amaxa, Lonza) with specific STING and NF-kB (RELA) siRNA constructs. The cGAS-STING-associated anti-tumor immune responses were evaluated by assessing the RNA levels of IFN-β, CXCL10, IFN-γ, and a control gene (GAPDH), with RT-qPCR. Results: In reactive lymphoid tissue, STING expression was restricted to dendritic cells and a subset of T-lymphocytes with a cytoplasmic staining pattern, which served as internal positive controls in all immunostainings. Using an arbitrary 10% cutoff, STING was positive in the neoplastic HRS cells of cHL in 20 of 32 (63%) patients with a predominant cytoplasmic pattern. STING expression at the mRNA and protein level was substantially higher in L-1236, L-428 and HDLM-2 compared to other cHL cell lines. Treatment with STING agonist alone stimulated gene expression of IFN-β and/or CXCL10 at a variable level depending on the cell line indicating functional cGAS-STING anti-tumor immune response pathway in cHL. Knocking down STING gene resulted in dramatic increase in CXCL10 gene expression in cHL cells. Silencing of NF-kB (RELA) resulted in activation of cGAS-STING pathway as shown by IRF3 activation (phosphorylation) and increased IRF3 protein levels in L-428 cells, which was associated with increased CXCL10 mRNA levels. Treatment with Amlexanox resulted in downregulation of IFN-β and/or CXCL10 gene expression in cHL cell lines. Conclusion: The cGAS-STING pathway is activated and, at least in part, regulated by the oncogenic NF-kB pathway. STING agonists and Amlexanox modulate gene expression of type 1 IFNs in cHL with direct therapeutic implications. A large cohort of cHL patients is currently being analyzed for the expression and prognostic significance of STING and the final results will be available at the time of the meeting.

Effects of Thymoquinone and Cisplatin on C-MYC, KRAS, p53 and EGFR Gene Expression in Lung Cancer Cell Lines

Lung cancer is one of the most common causes of death.It is known that genetic reasons in its etiology.Lung cancer has been shown to be associated with the EGFR,P53,KRAS and c-MYC genes.Thymoquinone is an antitumoral and antineoplastic bioactive substance procured from Nigella sativa plant.Cisplatin is a frequently used chemotherapeutic agent in the treatment of lung cancer.Our study has been conducted to examine the effects of Tq and Cis on gene expressions on lung cancer cell lines.Potential effects of Tq and Cis on A549,HTB54, CRL5820 and BEAS2B cell lines and cell viability using MTT has been evaluated.Cell culture has been effectuated with RPMI supplemented with 10% FBS,1% antibiotic and DMEM(37°C, %5 CO2).Cells were cultured for 24 h in 96 well plates(2500/ml cells) 10% FBS RPMI appropriate medium.The cells have been exposured 100 μM Tq and 200 μM Cis for 4h under incubation conditions.DMSO has been used for negative control.RT PCR has been conducted using SYBR Green qPCR Master Mix(reference gene GAPDH).As a result, p53 gene suppression has been shown in lung adenocarcinoma with Tq and Cis and epidermoid carcinoma with Cis only.EGFR gene suppression has been shown in lung adenocarsinoma with Tq only and epidermoid carcinoma with Cis only.C-MYC gene suppression has been shown in lung adenocarsinoma with both substances(more at Tq).It has been shown that KRAS gene suppression does not occur in any cell line.In addition, it has been shown that no gene expression is suppressed after Tq and cis exposure in the mesothelioma cell line.

The ERα-NRF2 signalling axis promotes bicalutamide resistance in prostate cancer

BackgroundBicalutamide is a nonsteroidal antiandrogen widely used as a first-line clinical treatment for advanced prostate cancer (PCa). Although patients initially show effective responses to bicalutamide treatment, resistance to bicalutamide frequently occurs and leads to the development of castration-resistant PCa (CRPC). This research investigated the roles of the oestrogen receptor α (ERα)-nuclear factor E2-related factor 2 (NRF2) signalling pathway in bicalutamide resistance in PCa cells.MethodsWe performed bioinformatic analysis and immunohistochemical staining on normal and cancerous prostate tissue to evaluate ERα and NRF2 expression and their correlation. Gene expression and localization in PCa cell lines were further investigated using real-time reverse transcription PCR/Western blotting and immunofluorescence staining. We treated PCa cells with the ER inhibitor tamoxifen and performed luciferase reporter assays and chromatin immunoprecipitation (ChIP) assays to understand ERα-dependent NRF2 expression. Overexpression and knockdown of ERα and NRF2 were used to explore the potential role of the ERα-NRF2 signalling axis in bicalutamide resistance in PCa cells.ResultsWe found that the expression of ERα and NRF2 was positively correlated and was higher in human CRPC tissues than in primary PCa tissues. Treatment with oestrogen or bicalutamide increased the expression of ERα and NRF2 as well as NRF2 target genes in PCa cell lines. These effects were blocked by pretreatment with tamoxifen. ChIP assays demonstrated that ERα directly binds to the oestrogen response element (ERE) in the NRF2 promoter. This binding led to increased transcriptional activity of NRF2 in a luciferase reporter assay. Activation of the ERα-NRF2 signalling axis increased the expression of bicalutamide resistance-related genes. Inhibition of this signalling axis by knockdown of ERα or NRF2 downregulated the expression of bicalutamide resistance-related genes and inhibited the proliferation and migration of PCa cells.ConclusionsWe demonstrated the transcriptional interaction between ERα and NRF2 in CRPC tissues and cell lines by showing the direct binding of ERα to the ERE in the NRF2 promoter under oestrogen treatment. Activation of the ERα-NRF2 signalling axis contributes to bicalutamide resistance in PCa cells, suggesting that the ERα-NRF2 signalling axis is a potential therapeutic target for CRPC.CxbrQyr1MPyN3X1_gKxrKxVideo

α-ketoglutarate suppresses immediate early gene expression in cancer cells

Cancer cells exhibit increased glutamine consumption compared to normal cells, supporting cell survival and proliferation. Glutamine is converted to α-ketoglutarate (αKG), which then enters the tricarboxylic acid cycle to generate ATP. Recently, therapeutic modulation of glutamine metabolism has become an attractive metabolic anti-cancer strategy. However, how synergistic combination therapy is required to overcome glutamine metabolism drug resistance remains elusive. To address this issue, we first investigated the role of αKG in regulating gene expression in several cancer cell lines. Using RNA-seq analysis and histone modification screening, we demonstrated that αKG reduced the expression of the immediate early gene (IEG) in cancer cells in an H3K27 acetylation-dependent manner. Conversely, glutaminase (GLS) inhibitors induce IEG expression in cancer cells. Furthermore, we showed that siRNA knockdown of orphan nuclear receptor subfamily 4 group A member 1 (NR4A1) induces IEG expression. Notably, the NR4A1 agonist cytosporone B sensitizes GLS inhibitor resistance to cancer cell death. Together, these findings indicate that therapeutic targeting of IEG dysregulation by αKG can be a potentially effective anti-cancer therapeutic strategy for glutamine metabolism inhibitors.

Effects of glabridin on cell proliferation and long non-coding RNA expression in HEC-1B cells

Purpose: Endometrial cancer is one of the most common gynecological cancers in the world. Glabridin is a main isoflavone in Glycyrrhiza glabra (licorice) root. It has several therapeutic effects such as anti-proliferative and anti-inflammatory. Long non-coding RNAs (LncRNAs) play a role in a variety of cellular processes, and their abnormal expression may contribute to tumor development and progression. In this study, the effects of glabridin on LncRNAs gene expression and viability of HEC-1B human endometrial cancer cell lines have been investigated. 
 Materials and methods: Glabridin was applied to HEC-1B cells in concentrations of 1 μM, 10 μM, 20 μM, 40 μM, 60 μM, and 80 μM. Glabridin's effect on HEC-1B cell proliferation was also evaluated using MTS assay. Expression profiles of LncRNAs such as H19, RNU43, LNC-MYC-3:1 and ABCC5-AS1:1 were determined by real-time PCR. 
 Results: Glabridin reduced the viability of HEC-1B cells in a time- and dose-dependent manner. The half maximal inhibitory concentration (IC50) dose in HEC-1B cells was detected to be 21.32 μM and 13.5 μM at the 24th and 48 hours, respectively. Glabridin has been observed to cause a significant decrease in the expression of H19 and RNU43 while increasing in the expression of LNC-MYC-3:1 and ABCC5-AS1:1.
 Conclusion: Glabridin could induce HEC-1B cell death by regulating LncRNAs expression. As a result, glabridin is a potential candidate for a more effective therapeutic agent against human endometrial cancer.

Single-cell profiling of peripheral neuroblastic tumors identifies an aggressive transitional state that bridges an adrenergic-mesenchymal trajectory.

Peripheral neuroblastic tumors (PNTs) represent a spectrum of neural-crest-derived tumors, including neuroblastoma, ganglioneuroblastoma, and ganglioneuroma. Malignant cells in PNTs are theorized to interconvert between adrenergic/noradrenergic and mesenchymal/neural crest cell states. Here, single-cell RNA-sequencing analysis of 10 PNTs demonstrates extensive transcriptomic heterogeneity. Trajectory modeling suggests that malignant neuroblasts move between adrenergic and mesenchymal cell states via an intermediate state that we term "transitional." Transitional cells express programs linked to a sympathoadrenal development and aggressive tumor phenotypes such as rapid proliferation and tumor dissemination. Among primary bulk tumor patient cohorts, high expression of the transitional gene signature is predictive of poor prognosis compared with adrenergic and mesenchymal expression patterns. High transitional gene expression in neuroblastoma cell lines identifies a similar transitional H3K27-acetylation super-enhancer landscape. Collectively, our study supports the concept that PNTs have phenotypic plasticity and uncovers potential biomarkers and therapeutic targets.

273 (PB053) - Comparison of biomarker selection methods in high-dimensional genomic data

Background: High-dimensional genomic data are indispensable in drug research and development. Genomic data-based biomarker discovery provides comprehensive insights to drug mechanism of action and efficacy prediction. However, it faces the difficulty of selecting suitable signature genes from several thousand or more genes with highly fluctuating and often tightly correlated expression patterns. This study aims to compare the performance of three recent signature gene selection algorithms in several large in vitro cell assays. Methods: Drug response datasets of irinotecan, cetuximab, pelitinib, gefitinib, erlotinib and KRAS (G12C) inhibitor-12 were obtained from the Genomics of Drug Sensitivity in Cancer (GDSC). Cell line gene expression data were gathered from our internal dataset of 20,432 genes, with 1,068 cell lines, across 23 tissues. The three algorithms evaluated were Stable Iterative Variable Selection (SIVS), Precision Lasso (PL) and Whitening Lasso (WL). Efficacy variance between different cancer types (ANOVA p-value) and prediction accuracy of drug efficacy were calculated to evaluate the results. Results: The accuracy of PL predictions was more positively correlated with the number of genes than SIVS and WL. The number of signature genes was limited from 5 to 50 and the most precise results were obtained for all drugs with 30 to 50 genes composite biomarkers. The PL, WL and SIVS methods separately selected 30, 17 and 6 genes as composite biomarkers for irinotecan with an accuracy of 85.7%, 78.2% and 87.3% respectively, in the training set. In validation set, the prediction accuracy of the SIVS panel was the highest at 83.1%. For erlotinib, SIVS, WL and PL picked out 22, 23 and 32 genes as the best composite biomarker with an accuracy of 73%, 83% and 76.8%, respectively. Overall, composite biomarkers selected by SIVS was the superior choice in four of six drugs. In most cases, these three algorithms took less time than 100 iterations of Boruta under the same conditions. Conclusions: Compared with PL and WL, the SIVS method appears to obtain a higher prediction accuracy with a relatively smaller number of genes in this research. PL tended to select many more signature genes than SIVS and WL to yield comparable performance. Overlapping signature genes between the three algorithms was generally low, including for genes belonging to same molecular pathways. The cost and practicality of in vivo experiments would have made it difficult to select a large number of cancer types, drug response, and gene expression data. Therefore, selecting the best predictive composite biomarker in vitro screening for in vivo validation can significantly reduce costs and accelerate the early drug development process. No conflict of interest.

Formal Meta-Analysis of Hypoxic Gene Expression Profiles Reveals a Universal Gene Signature

Integrating transcriptional profiles results in identifying gene expression signatures that are more robust than those obtained for individual datasets. However, a direct comparison of datasets derived from heterogeneous experimental conditions is problematic, hence their integration requires applying of specific meta-analysis techniques. The transcriptional response to hypoxia has been the focus of intense research due to its central role in tissue homeostasis and prevalent diseases. Accordingly, many studies have determined the gene expression profile of hypoxic cells. Yet, despite this wealth of information, little effort has been made to integrate these datasets to produce a robust hypoxic signature. We applied a formal meta-analysis procedure to datasets comprising 430 RNA-seq samples from 43 individual studies including 34 different cell types, to derive a pooled estimate of the effect of hypoxia on gene expression in human cell lines grown ingin vitro. This approach revealed that a large proportion of the transcriptome is significantly regulated by hypoxia (8556 out of 20,888 genes identified across studies). However, only a small fraction of the differentially expressed genes (1265 genes, 15%) show an effect size that, according to comparisons to gene pathways known to be regulated by hypoxia, is likely to be biologically relevant. By focusing on genes ubiquitously expressed, we identified a signature of 291 genes robustly and consistently regulated by hypoxia. Overall, we have developed a robust gene signature that characterizes the transcriptomic response of human cell lines exposed to hypoxia in vitro by applying a formal meta-analysis to gene expression profiles.

Clinical significance and potential mechanism of heat shock factor 1 in acute myeloid leukemia.

Background: Heat shock factor 1 (HSF1) is now considered to have the potential to be used as a prognostic biomarker in cancers. However, its clinical significance and potential function in acute myeloid leukemia (AML) remain underexplored.Methods: In this study, the expression pattern and clinical significance of HSF1 in AML were examined by integrating data from databases including The Cancer Genome Atlas (TCGA), The Genotype–Tissue Expression (GTEx), Vizome, Cancer Cell Line Encyclopedia (CCLE) and Gene Expression Omnibus (GEO). Linkedomics was applied to collect HSF1–related genes in AML. GeneMANIA was applied to outline HSF1–related functional networks. CancerSEA analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and Gene Set Enrichment Analysis (GSEA) were performed to mine the potential mechanism of HSF1 in leukemogenesis. Single–sample Gene Set Enrichment Analysis (ssGSEA) was applied to explore the correlation between HSF1 and infiltrating immune cells in AML.Results: HSF1 expression was elevated in AML compared to healthy controls and indicate a poor overall survival. HSF1 expression was significantly correlated with patients age, associated with patient survival in subgroup of bone marrow blasts (%) >20. Functional analyses indicated that HSF1 plays a role in the metastatic status of AML, and is involved in inflammation–related pathways and biological processes. HSF1 expression was significantly correlated with the immune infiltration of nature killer cells and T cell population.Conclusion: HSF1 plays a vital role in the molecular network of AML pathogenesis, and has the potential to be a biomarker for prognosis prediction.

Predicting cancer drug response using parallel heterogeneous graph convolutional networks with neighborhood interactions.

Due to cancer heterogeneity, the therapeutic effect may not be the same when a cohort of patients of the same cancer type receive the same treatment. The anticancer drug response prediction may help develop personalized therapy regimens to increase survival and reduce patients' expenses. Recently, graph neural network-based methods have aroused widespread interest and achieved impressive results on the drug response prediction task. However, most of them apply graph convolution to process cell line-drug bipartite graphs while ignoring the intrinsic differences between cell lines and drug nodes. Moreover, most of these methods aggregate node-wise neighbor features but fail to consider the element-wise interaction between cell lines and drugs. This work proposes a neighborhood interaction (NI)-based heterogeneous graph convolution network method, namely NIHGCN, for anticancer drug response prediction in an end-to-end way. Firstly, it constructs a heterogeneous network consisting of drugs, cell lines and the known drug response information. Cell line gene expression and drug molecular fingerprints are linearly transformed and input as node attributes into an interaction model. The interaction module consists of a parallel graph convolution network layer and a NI layer, which aggregates node-level features from their neighbors through graph convolution operation and considers the element-level of interactions with their neighbors in the NI layer. Finally, the drug response predictions are made by calculating the linear correlation coefficients of feature representations of cell lines and drugs. We have conducted extensive experiments to assess the effectiveness of our model on Cancer Drug Sensitivity Data (GDSC) and Cancer Cell Line Encyclopedia (CCLE) datasets. It has achieved the best performance compared with the state-of-the-art algorithms, especially in predicting drug responses for new cell lines, new drugs and targeted drugs. Furthermore, our model that was well trained on the GDSC dataset can be successfully applied to predict samples of PDX and TCGA, which verified the transferability of our model from cell line in vitro to the datasets in vivo. The source code can be obtained from https://github.com/weiba/NIHGCN. Supplementary data are available at Bioinformatics online.

The A to I editing landscape in melanoma and its relation to clinical outcome

ABSTRACT RNA editing refers to non-transient RNA modifications that occur after transcription and prior to translation by the ribosomes. RNA editing is more widespread in cancer cells than in non-transformed cells and is associated with tumorigenesis of various cancer tissues. However, RNA editing can also generate neo-antigens that expose tumour cells to host immunosurveillance. Global RNA editing in melanoma and its relevance to clinical outcome currently remain poorly characterized. The present study compared RNA editing as well as gene expression in tumour cell lines from melanoma patients of short or long metastasis-free survival, patients relapsing or not after immuno- and targeted therapy and tumours harbouring BRAF or NRAS mutations. Overall, our results showed that NTRK gene expression can be a marker of resistance to BRAF and MEK inhibition and gives some insights of candidate genes as potential biomarkers. In addition, this study revealed an increase in Adenosine-to-Inosine editing in Alu regions and in non-repetitive regions, including the hyperediting of the MOK and DZIP3 genes in relapsed tumour samples during targeted therapy and of the ZBTB11 gene in NRAS mutated melanoma cells. Therefore, RNA editing could be a promising tool for identifying predictive markers, tumour neoantigens and targetable pathways that could help in preventing relapses during immuno- or targeted therapies.

DTSyn: a dual-transformer-based neural network to predict synergistic drug combinations.

Drug combination therapies are superior to monotherapy for cancer treatment in many ways. Identifying novel drug combinations by screening is challenging for the wet-lab experiments due to the time-consuming process of the enormous search space of possible drug pairs. Thus, computational methods have been developed to predict drug pairs with potential synergistic functions. Notwithstanding the success of current models, understanding the mechanism of drug synergy from a chemical-gene-tissue interaction perspective lacks study, hindering current algorithms from drug mechanism study. Here, we proposed a deep neural network model termed DTSyn (Dual Transformer encoder model for drug pair Synergy prediction) based on a multi-head attention mechanism to identify novel drug combinations. We designed a fine-granularity transformer encoder to capture chemical substructure-gene and gene-gene associations and a coarse-granularity transformer encoder to extract chemical-chemical and chemical-cell line interactions. DTSyn achieved the highest receiver operating characteristic area under the curve of 0.73, 0.78. 0.82 and 0.81 on four different cross-validation tasks, outperforming all competing methods. Further, DTSyn achieved the best True Positive Rate (TPR) over five independent data sets. The ablation study showed that both transformer encoder blocks contributed to the performance of DTSyn. In addition, DTSyn can extract interactions among chemicals and cell lines, representing the potential mechanisms of drug action. By leveraging the attention mechanism and pretrained gene embeddings, DTSyn shows improved interpretability ability. Thus, we envision our model as a valuable tool to prioritize synergistic drug pairs with chemical and cell line gene expression profile.

Cytotoxic Effects of Rheum khorasanicum Extract on MCF-7 Human Breast Cancer Cell Line and ODC1 Gene Expression

Background: Polyamine metabolism has a critical role in cancer initiation and progression. The abnormal overexpression of the ornithine decarboxylase (ODC1) enzyme gene could be a characteristic of cancer cells. Objectives: The current study evaluated the cytotoxic effects of Rheum khorasanicum extract in three different solvents, including distilled water, ethanol, and n-hexane, on the MCF-7 breast cancer cell line and assessed the expression level of the ODC1 gene upon the abovementioned treatments. Methods: We cultured MCF-7 cancer cells in complete cell culture media and subsequently subjected them to increasing concentrations (0 - 200 μg/mL) of the extract. The cytotoxic activity of the extract was investigated using an MTT assay. The quantitative analysis of ODC1 gene expression was performed using real-time PCR. Results: The cell viability was significantly decreased in concentration- and time-dependent manners. The mRNA expression of the ODC1 gene declined gradually with the increasing concentrations of extracts. Conclusions: Our findings indicated that R. khorasanicum extract could potentially influence ODC1 gene expression in breast cancer cells and exert inhibitory effects on cancer cell proliferation.

Abstract 100: IL-1β pathway promotes cell proliferation and 5-FU resistance in African American colon cancer cell lines

Abstract Introduction: Colorectal Cancer (CRC) is the third most common cancer and third cause of cancer-related death in the US. When taking race and ethnicity into consideration, African Americans (AA) present with the highest incidence and mortality rates for CRC. Studies have found that AA patients, when compared to Caucasian American (CA) patients, have a lower response to the standard of care chemotherapeutic agent 5-Fluorouracil (5-FU) as well as lower frequency of MSI tumors, making them also less likely to respond to conventional immunotherapies. Our recent findings have shown that tumors from AA patients present higher expression of genes involved in pro-inflammatory processes as well as lower expression of genes promoting anti-tumoral activities. One of the genes that was found upregulated in AA tumors is IL1B, which encodes for the pro-inflammatory cytokine Interleukin-1β (IL-1β). Therefore, we investigated the role of the IL-1β pathway in novel AA colon cancer cell lines. Here, we evaluated changes in cell proliferation, apoptosis, 5-FU response, and activation of inflammatory pathways. Methods: Our approach includes analysis of gene expression in both AA and CA colon cancer cell lines by using RNA sequencing to evaluate pro-inflammatory genes differentially expressed among the cell lines. Furthermore, we used an MTS assay to detect viable cells following treatment with IL-1β, alone or in combination with different concentrations of 5-FU, as well as analyzing changes in apoptosis via Flow Cytometry. To better understand the mechanisms of IL-1β-induced effects, we used IL-1 Receptor Antagonist (IL-1Ra) to block the IL-1β pathway and evaluated changes in cell viability and 5-FU response via MTS assay, as well as activation of NF-kB pathway, via detection of phospho-IкB-α protein using Western Blot analysis. Results: Results from MTS assays indicated that cell proliferation in response to IL-1β differs between the AA and the CA colon cancer cell lines, with the AA colon cancer cells being more sensitive to lower concentrations of the cytokine when compared to CA cell lines. Further, we saw an increased expression of phospho-IкB-α following treatment with IL-1β. This expression was reduced when the cells were treated in combination with IL-1β and the IL-1Ra. The IL-1Ra also appears to reduce the pro-proliferative effects induced by IL-1β. Importantly, our results show that 5-FU sensitivity is drastically reduced in the presence of IL-1β for both AA and CA colon cancer cell lines, suggesting that IL-1β may play a role in defining resistance to the chemotherapeutic drug. Conclusions: Taken together, our results demonstrated a differential response to IL-1β for the AA colon cancer cell lines, suggesting a probable role played by the cytokine in driving inflammation-related cancer progression and reveals a possible new target to exploit in immunotherapy for this population. Citation Format: Marzia Spagnardi, Jenny Paredes, Jone Garai, Jovanny Zabaleta, Jennie Williams, Laura Martello-Rooney. IL-1β pathway promotes cell proliferation and 5-FU resistance in African American colon cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 100.

Abstract 3886: 3D genome conformation analysis in ST-EPN-ZFTA ependymoma identifies RCOR2 as a potential target

Abstract Ependymoma (EPN) is a tumor of the central nervous system that occurs in children and adults. Childhood EPNs are one of the most aggressive pediatric brain tumors, arising both in infratentorial and supratentorial (ST) regions of the brain. Molecular profiling has identified distinct subgroups of ependymomas, including ST-EPN-ZFTA and PF-EPN-A as the most common and most aggressive ones in children. To this date, the only curative and indispensable treatment option is surgical resection followed by radiation. Unfortunately, there are no targeted therapies in clinical use. However, EPN cells have been extremely difficult to culture and expand in vitro, which explains why molecular studies on EPN cells have been hampered. This shortcoming has recently been overcome by establishing EPN models including human cell lines and organoids as well as patient derived xenografts (PDX). 3D genome conformation analysis has emerged as a powerful approach to understand tumor development and to identify novel drug targets for the treatment of different tumors. In this project we have used genome-wide chromosome conformation capture (Hi-C), complemented with H3K27ac (active enhancers) as well as gene expression in primary ependymoma tumors and cell lines to identify regulatory mechanisms underlying aberrant expression of genes that are essential for ependymoma tumorigenesis. Results of this study showed that the fusion between ZFTA and RELA leads to the formation of new regulatory environments that are recurrently associated with aberrant overexpression of RCOR2 only in ST-EPN-ZFTA ependymoma cells. Indeed, compared to all other ependymoma subgroups as well as to normal brain, RCOR2 is significantly upregulated only in ST-EPN-ZFTA patient samples. To test oncogenic relevance of RCOR2 for growth and maintenance, we depleted RCOR2 by RNAi and observed a strongly reduced cell survival in several ST-EPN-ZFTA cell lines but not or to a lesser extent in PF-EPN-A cells. Since RCOR2 is an adaptor protein lacking enzymatic activity, we looked at the expression of the proteins known as an interacting partner of RCOR2. Lysine-histone demethylase 1 (LSD1) is one of them and its expression in ST-EPN-ZFTA significantly correlates with RCOR2 expression. We therefore hypothesized that ST-EPN-ZFTA cells may be sensitive to LSD1 depletion, too. Indeed, depletion of LSD1 by RNAi resulted in a strong growth inhibition in ST-EPN-ZFTA cells, but not in PF-EPN-A cells. However, none of the tested LSD1 inhibitors exerted anti-proliferative activity at clinically reachable doses in the ST-EPN-ZFTA cells indicating a potential LSD1 non-enzymatic function in this particular tumor type. In summary, our study nominates RCOR2 as a oncogenic gene specific for ST-EPN-ZFTA ependymomas. Current studies focus on the identification of other interacting partners of the RCOR2-LSD1 complex and how to target these for therapeutic approaches. Citation Format: Aylin Camgöz, Konstantin Okonechnikov, Owen Chapman, Monika Maurmann, Frank Buchholz, Stefan Pfister, Lukas Chavez, Marcel Kool. 3D genome conformation analysis in ST-EPN-ZFTA ependymoma identifies RCOR2 as a potential target [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3886.

Abstract 2087: AXL inhibition enhances Type 1 interferon (IFN) response and potentiates chemo-immunotherapy

Abstract Chemotherapy elicits anti-tumor immune responses by inducing immunogenic tumor cell death, targeting suppressive immune cells and activating Type 1 interferon (IFN) responses (1, 2, 3). Chemotherapeutic agents lead to cGAS - STING cytosolic DNA sensing pathway activation that results in Type 1 IFN release (4). IFN receptor (IFNAR1, IFNAR2) signaling mimics viral immune responses, is associated with clinical benefit (5, 6) and is exploited by chemo-immunotherapies (7, 8). The receptor tyrosine kinase AXL is associated with immune evasion and immunotherapy resistance (8). AXL serves as a critical regulatory checkpoint for TLR-induced IFN responses in dendritic cells, macrophages and natural killer cells (9, 10). IFN receptor signaling induces AXL expression (11) and AXL activation on dendritic cells is targeted by viruses (e.g. Zika) to abrogate IFN responses and inhibit anti-viral immunity. AXL serves as an IFN-response checkpoint by blocking IFNAR1 and IFNAR2 signaling. We hypothesized that tumor cells exploit AXL signaling to abrogate Type 1 IFN responses and inhibit antitumor immunity. We evaluated whether AXL inhibition promotes Type 1 IFN signalling and enhances immune checkpoint inhibitor efficacy. AXL kinase inhibition (bemcentinib) in combination with chemotherapy (doxorubicin) increased IFN response gene expression in mammary carcinoma and melanoma cell lines. In vivo, bemcentinib treatment potentiated the efficacy of immune checkpoint inhibitor treatment in combination with intratumoral doxorubicin injection (i.t) in the syngeneic myeloid derived suppressor cell (MDSC)- rich refractory mammary adenocarcinoma 4T1 model. In addition, bemcentinib treatment in conjunction with i.t. doxorubicin enhanced Type 1 IFN response, reduced cancer stemness and epithelial to mesenchymal (EMT) gene expression in this model. Furthermore, this combination treatment regimen sensitized the immune checkpoint inhibitor refractory Braf-mutant melanoma (YUMM1.7) by enhancing the type 1 IFN response resulting in significantly improved median overall survival. In conclusion, this study provides evidence that bemcentinib potentiates chemo-immunotherapy by enhancing tumor Type 1 IFN response and dampening EMT.

Abstract 215: MDM2 inhibition as a non-hormone dependent radiosensitizing strategy in p53 wild-type breast cancer models

Abstract Background: Radiation therapy (RT) is standard in the treatment of most women with breast cancer (BC), though efficacy of RT remains inadequate for women with locally advanced BC. Thus, more effective radiosensitization strategies are needed. We performed a radiosensitizer screen paired with transcriptomic and proteomic data to identify potential mediators of RT resistance and identify novel therapeutic targets. Methods: Microarray and RPPA data were used for gene/protein expression and pathway analysis. Clonogenic survival assays were performed to assess radiosensitization and calculate relative enhancement ratios (rERs) with MDM2 inhibitors navtemadlin (AMG-232) and alrizomadlin (APG-115) in p53 wild-type (WT) and mutant (MT) models of estrogen receptor positive (p53 WT: MCF-7, p53 MT: T47D) and triple-negative BC (p53 WT: CAL-51, p53 MT: MDA-MB-231). AlamarBlue was used to determine IC50 concentrations of MDM2 inhibitors. Clonogenic and IC50 experiments performed in parallel with p53 CRISPR knockout models of p53 WT cell lines. DNA damage response was assessed with γ-H2AX Immunofluorescence microscopy. Flow Cytometry with Annexin V staining was used to evaluate apoptosis and Propidium Iodide staining for cell cycle progression. Light microscopy with β-gal staining was used to evaluate senescence. In vivo efficacy of combination therapy was evaluated with CAL-51 and CAL-51 p53 CRISPR xenograft models. Results: An MDM2 inhibitor (JNJ-26854165) was nominated as an effective drug in treatment for RT-resistant BC cell lines (R2 = 0.43, p value <0.01) in our novel radiosensitizer screen. Differential gene expression and pathway analysis in non-overlapping p53 WT BC cell lines treated +/-RT identified apoptosis, cell cycle, and p53 signaling as the top pathways induced in p53 WT cell lines by RT. MDM2 was significantly overexpressed after RT compared to no RT in p53 WT cells. Cell growth was decreased by MDM2 inhibition with navtemadlin and alrizomadlin in p53 WT cells (IC50s: 264-592nM) and not in p53 MT cells (IC50s >10μm). MDM2 inhibition radiosensitized p53 WT cells (rERs: 1.81-2.85) but not p53 MT cells (rERs: 1.00-1.03). This phenotype was reversed in p53 CRISPR knockout cells (rERs: 1.06-1.12). MDM2 inhibition in combination with RT demonstrated delayed DNA damage repair compared to RT alone. p53 WT cells demonstrated increased G1 cell cycle arrest with RT and combination therapy. Combination MDM2 inhibition and RT resulted in increased apoptosis and senescence in p53 WT cell lines. In vivo xenograft studies are ongoing. Conclusions: These results demonstrate the combination of RT and MDM2 inhibition may be an effective therapeutic strategy in patients with p53-wild type breast cancer, regardless of hormone receptor status. Citation Format: Charles A. Nino, Cassandra L. Ritter, Andrea M. Pesch, Anna R. Michmerhuizen, Benjamin C. Chandler, Yashmeet Kaur, Maria Fields, Lynn Lerner, Lori J. Pierce, Corey W. Speers. MDM2 inhibition as a non-hormone dependent radiosensitizing strategy in p53 wild-type breast cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 215.

Abstract 1030: IX2X2

Abstract Introduction: Prostate cancer (PCa) is the second leading cause of cancer-related mortality in men. More than 85% of PCa and advanced aggressive castration-resistant prostate cancer (CRPC) depend on the androgen receptor (AR) for growth. AR-targeted therapeutics are the mainstay treatment option for PCa. Majority of PCa and CRPC respond to AR-LBD-binding antagonists such as enzalutamide and become refractory after a brief period of response. One of the main resistance mechanisms is the expression of AR splice variants (AR-SVs). AR-SVs lack LBD and are constitutively active, causing aggressive phenotype. Description: Rationale-based drug design was employed to synthesize selective AR irreversible covalent antagonists (SARICA). SARICAs were evaluated in AR transactivation to determine the antagonistic activity. SARICAs were incubated with purified recombinant AR activation function-1 (AF-1) protein and mass spectrometry was performed to determine their covalent binding. SARICAs were evaluated in AR-SV transactivation assay and in a Schild’s plot to determine the nature of inhibition (reversible Vs irreversible). SARICAs were tested in PCa cell line gene expression and proliferation assays and in tumor xenograft experiments. Summary: SARICAs inhibited AR transactivation in submicromolar range. Mass spectrometry with purified AF-1 protein demonstrated covalent binding of SARICAs and identified C406 and C327 as SARICA-interacting nucleophiles. Both amino acids are important for AR-V7 function and stability, making them appropriate drug targets. SARICAs inhibited the activity of AR-V7 and irreversibly inhibited AR transactivation, indicating that these covalent binders are irreversible antagonists. SARICAs selectively inhibit the proliferation and growth of AR-positive prostate cancer cells and tumors, but not AR-negative cells and tumors. Conclusion: These first-in-class AR-selective covalent molecules thus serve as chemical probes to advance our understanding of the AR-AF-1 and they potentially offer an opportunity to treat refractory cancers. Disclosure: This work was supported by an NCI grant (CA229164 and CA229164S1), a DOD grant (W81XWH2110055), and by Muirhead endowment. The SARICAs are licensed to Oncternal therapeutics, San Diego, CA. RN is a consultant to Oncternal therapeutics. Citation Format: Thamarai U. Ponnusamy, Thirumagal T. Thiyagarajan, Ramesh Narayanan. IX2X2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1030.

Study on the effect and mechanisms of SIGLEC-15 on laryngeal squamous cell carcinoma

Objective: To investigate the effect of sialic acid combined with immunoglobulin-like lectin 15 (SIGLEC-15) on laryngeal squamous cell carcinoma (LSCC) and underlying mechanisms. Methods: The Cancer Genome Atlas (TCGA), Cancer Cell Line Encyclopedia (CCLE) and Gene Expression Profiling Interactive Analysis (GEPIA2) databases were used for bioinformatics analysis. Cell Counting Kit-8 (CCK8), flow cytometry, and Transwell method were used respectively to detect proliferation, apoptosis, cell cycle, metastasis and invasion behaviors of the cells. Gene chip method was used for detecting up-regulated and down-regulated genes and performing enrichment analysis of differential genes. Western Blotting (WB) and Quantitative Real-time PCR (qPCR) were used to detect the expressions of proteins. Tumor formation experiments in nude mice were used to detect the effect of SIGLEC-15 on the growth of transplanted tumors. Wilcoxon rank sum test, t-test and Log-rank test were used for statistical analysis. Results: SIGLEC-15 was highly expressed in laryngeal squamous cell carcinoma and closely related to life in being. TU686SIGLEC-15-with low expression of SIGLEC-15 was constructed. Compared to TU686SIGLEC-15+, TU686SIGLEC-15-showed significantly reduced activities of proliferation (48 h: 1.32±0.23 vs. 2.56±0.37, t=6.59, P<0.05), migration (1 036.52±51.22 vs. 1 819.62±180.24, t=7.22, P<0.05) and invasion (469.21±112.25 vs. 961.45±102.03, t=7.85, P<0.05); early increased apoptosis ((23.27±1.12)% vs. (5.64±1.61)%, t=11.32, P<0.05); blocked cell cycle at G0/G1 ((59.32±3.65)% vs. (35.46±3.57)%, t=9.85, P<0.05); the knockdown of SIGLEC-15 resulted in up-regulation of 864 genes, down-regulation of 357 genes, with significant changes in molecules of cell cycle, apoptosis and JAK/STAT signal pathways, and the expressions of p-JAK2, p-STAT3, Caspase-3, Bad, Bcl-2, and Cyclin d1 proteins. Tumor formation experiments in nude mice showed that at 8 weeks after the tumors were implanted, the growth transplanted tumors of TU686 SIGLEC-15-cell group was slower than that of TU686 SIGLEC-15+cell group, with significant difference in the mean tumor weights between two groups ((0.382±0.054) g vs. (1.277±0.126) g, t=8.44, P<0.05), while the expression of SIGLEC-15 was lower in the transplanted tumors of SIGLEC-15 knockdown group compared to control group, with significant difference(11.29±2.17 vs. 36.25±7.56, t=9.28, P<0.05). Conclusion: SIGLEC-15 is highly expressed in LSCC and can promote the progression of LSCC through the JAK2-STAT3 pathway.

EMT-Related Gene Signature: A New Method for Personalized Risk Assessment in Patients with Hepatocellular Carcinoma

Background: Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer-related death worldwide, accounting for more than 700,000 deaths annually. Epithelial-mesenchymal transition (EMT) is posited to contribute to HCC progression. We, therefore, aimed to elucidate the relationship between EMT-related gene (ERG) expression and prognosis in patients with HCC. Methods: Univariate and multivariate Cox regression analyses were performed to identify prognostic-related differentially expressed EMT-related genes (DE-ERGs). Time-dependent receiver operating characteristic and Kaplan-Meier curves were used to assess the prognostic performance of the EMT-related signature. pRRophetic R package was used to evaluate sorafenib sensitivity in the GSE14520 cohort. Gene expression in Huh7 and L02 cell lines was detected by real-time PCR and Western blotting. Differential expression of the three genes between HCC tissues and normal tissues was validated using immunohistochemical analyses. Results: Of the 76 identified DE-ERGs, 29 were associated with overall survival. Three prognosis-related ERGs (GOLM1, SOX4, and CD14) were screened out by multivariate Cox regression. A gene signature was identified based on the three prognostic-related ERGs. HCC patients with a low-risk score had a better prognosis and were more sensitive to sorafenib compared to those with a high-risk score. Moreover, we further confirmed increased expression of GOLM1 and SOX4, and decreased expression of CD14, in liver cancer cell line and HCC tissue. Conclusions: The results of the present study demonstrate the utility of an ERG signature as a potential biomarker informing prognosis in patients with HCC, which may contribute to the implementation of personalized therapies.