Gene Expression In Cell Lines Research Articles

Abstract A015: Cancer co-dependency mapping identifies a functional interplay between PRC2 and MLL-MEN1 complex in lymphoma

Abstract Interplays between chromatin-associated complexes and modifications critically contribute to the partitioning of epigenome into stable and functionally distinct domains. Yet there is a lack of systematic identification of chromatin crosstalk mechanisms, limiting our understanding of the dynamic transition between chromatin states during development and disease. Here we perform co-dependency mapping of genes encoding nuclear proteins using CRISPR-Cas9-mediated fitness screens in pan-cancer cell lines to quantify gene-gene functional relationships. We identify 84 co-dependency modules and further define the molecular context underlying the essentiality of these modules by incorporating mutational, epigenome, gene expression and drug sensitivity profiles of cell lines. These analyses assign new protein complex functions and biochemical interactions, and predict new chromatin crosstalk, including an unexpected co-dependency between two transcriptionally counteracting chromatin complexes - polycomb repressive complex 2 (PRC2) and MLL-MEN1 complex. We show that PRC2-mediated H3K27 tri-methylation regulates the genome-wide distribution of MLL1 and MEN1. In lymphoma cells with EZH2 gain-of-function mutations, the re-localization of MLL-MEN1 complex drives oncogenic gene expression and results in a hypersensitivity to pharmacologic inhibition of MEN1. Together, our findings provide a resource for discovery of trans-regulatory interactions as mechanisms of chromatin regulation and potential targets of synthetic lethality. Citation Format: Xiao Chen, Yinglu Li, Chao Lu. Cancer co-dependency mapping identifies a functional interplay between PRC2 and MLL-MEN1 complex in lymphoma. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr A015.

Uptake of Flaxseed Dietary Linusorbs Modulates Regulatory Genes Including Induction of Heat Shock Proteins and Apoptosis.

Flaxseed (Linum usitatissimum L.) is gaining popularity as a superfood due to its health-promoting properties. Mature flax grain includes an array of biologically active cyclic peptides or linusorbs (LOs, also known as cyclolinopeptides) that are synthesized from three or more ribosome-derived precursors. Two flaxseed orbitides, [1-9-NαC]-linusorb B3 and [1-9-NαC]-linusorb B2, suppress immunity, induce apoptosis in a cell line derived from human epithelial cancer cells (Calu-3), and inhibit T-cell proliferation, but the mechanism of LO action is unknown. LO-induced changes in gene expression in both nematode cultures and human cancer cell lines indicate that LOs promoted apoptosis. Specific evidence of LO bioactivity included: (1) distribution of LOs throughout the organism after flaxseed consumption; (2) induction of heat shock protein (HSP) 70A, an indicator of stress; (3) induction of apoptosis in Calu-3 cells; and (4) modulation of regulatory genes (determined by microarray analysis). In specific cancer cells, LOs induced apoptosis as well as HSPs in nematodes. The uptake of LOs from dietary sources indicates that these compounds might be suitable as delivery platforms for a variety of biologically active molecules for cancer therapy.

Landscape of immune-related signatures induced by targeting of different epigenetic regulators in melanoma: implications for immunotherapy

BackgroundImprovement of efficacy of immune checkpoint blockade (ICB) remains a major clinical goal. Association of ICB with immunomodulatory epigenetic drugs is an option. However, epigenetic inhibitors show a heterogeneous landscape of activities. Analysis of transcriptional programs induced in neoplastic cells by distinct classes of epigenetic drugs may foster identification of the most promising agents.MethodsMelanoma cell lines, characterized for mutational and differentiation profile, were treated with inhibitors of DNA methyltransferases (guadecitabine), histone deacetylases (givinostat), BET proteins (JQ1 and OTX-015), and enhancer of zeste homolog 2 (GSK126). Modulatory effects of epigenetic drugs were evaluated at the gene and protein levels. Master molecules explaining changes in gene expression were identified by Upstream Regulator (UR) analysis. Gene set enrichment and IPA were used respectively to test modulation of guadecitabine-specific gene and UR signatures in baseline and on-treatment tumor biopsies from melanoma patients in the Phase Ib NIBIT-M4 Guadecitabine + Ipilimumab Trial. Prognostic significance of drug-specific immune-related genes was tested with Timer 2.0 in TCGA tumor datasets.ResultsEpigenetic drugs induced different profiles of gene expression in melanoma cell lines. Immune-related genes were frequently upregulated by guadecitabine, irrespective of the mutational and differentiation profiles of the melanoma cell lines, to a lesser extent by givinostat, but mostly downregulated by JQ1 and OTX-015. GSK126 was the least active drug. Quantitative western blot analysis confirmed drug-specific modulatory profiles. Most of the guadecitabine-specific signature genes were upregulated in on-treatment NIBIT-M4 tumor biopsies, but not in on-treatment lesions of patients treated only with ipilimumab. A guadecitabine-specific UR signature, containing activated molecules of the TLR, NF-kB, and IFN innate immunity pathways, was induced in drug-treated melanoma, mesothelioma and hepatocarcinoma cell lines and in a human melanoma xenograft model. Activation of guadecitabine-specific UR signature molecules in on-treatment tumor biopsies discriminated responding from non-responding NIBIT-M4 patients. Sixty-five % of the immune-related genes upregulated by guadecitabine were prognostically significant and conferred a reduced risk in the TCGA cutaneous melanoma dataset.ConclusionsThe DNMT inhibitor guadecitabine emerged as the most promising immunomodulatory agent among those tested, supporting the rationale for usage of this class of epigenetic drugs in combinatorial immunotherapy approaches.

Synergistic Anti-Tumor Effect on Novel Combination of Selinexor with Azacitidine in AML By Targeting XPO1/eIF4E/C-MYC Signaling

Background: Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy with high mortality and poor outcome. Highly effective therapy options are always needed for AML. Selinexor (KPT-330) is an oral bioactive selective CRM1 (XPO1) inhibitor, and the clinical trial showed the combination of KPT-330 over standard induction therapy benefits much for AML patients. Azacitidine (AZA), a DNA hypomethylation agent, can induce cell apoptosis and exhibit potential therapeutic efficacy against AML. However, it has not yet determined the anti-tumor effect of KPT-330 with AZA in AML. Here we examined the anti-leukemia effect and potential mechanism of KPT-330 combined with AZA in AML cells to provide evidence for further clinical trials. Methods: A total of 64 AML patients’ BM samples and 64 normal control from healthy volunteers were obtained from Zhongda Hospital Southeast University, in which the 2 patients’ samples were further treated with indicated drugs in vitro. Cell Counting Kit-8 (CCK-8) was used for cell viability assay, Annexin-V/PI staining following flow cytometry analysis for apoptosis, and qPCR and Western Blot for gene expression in AML cell lines and patient samples. CalcuSyn analysis was performed for a synergistic effect. RNA-seq was performed in U937 cells treated with 0.7uM AZA for 48 hrs, and the significantly differentially expressed genes (DEGs) were identified and gene set enrichment analysis (GSEA) was performed. Results: Cell proliferation arrest and apoptosis were significantly observed in U937 and MV4-11 cells treated with the combination of KPT-330 with AZA compared to single drug control (data not shown). Then, we further explored the synergistic effect of the combination in patients’ samples. Results showed that cell growth arrest and apoptosis significantly increased in primary samples from two de novo AML patients compared to the single drug controls, and CalcuSyn analysis showed a synergistic effect of the combination on cell growth arrest (Fig. 1A &1B). To understand the mechanisms underlying the synergy, RNA-seq analysis was performed in U937 cells treated by 0.7uM AZA and 4655 DEGs were identified. XPO1, eIF4E, and MYC are in the top DEGs upon AZA treatment, and GSEA analysis indicated MYC_TARGETS and apoptosis pathway are enriched in DEGs (Fig. 1C &1D ). Moreover, the mRNA level of XPO1 and eIF4E was increased in our AML patient cohort compared with healthy controls (Fig. 1E), and the high expression of XPO1 or elF4E are associated with poor outcome (Fig. 1F). XPO1 is the key component of nuclear-exporting machinery; elF4E, the cargo of XPO1 is translation initiation factor and plays a critical role in translational machinery, which regulates the C-MYC translation. It is also reported that AZA regulates the transcription of C-MYC by affecting methylation. We observed a combination of KPT-330 with AZA significantly down-regulated the mRNA and protein level of oncogene eIF4E and C-MYC in two of the patients’ samples compared to single drug controls (Fig. 1G &1H ). Also, C-MYC knock-down was not only significantly increased the apoptosis in U937 cells compared to scramble shRNA (shCTL) control, but also significantly enhanced the combination-mediated apoptosis compared to that of single drug control in U937 cells (Fig. 1I). these data indicated that XPO1/eIF4E/C-MYC signaling plays critical roles in the combination-mediated anti-leukemia, and the mechanistic model is summarized in Fig.1J. Conclusions: Our data for the first time showed the synergistic effect of a novel combination of KPT-330 with AZA on cell growth arrest and apoptosis in AML patients and also identified the underlying mechanism by targeting XPO1/eIF4E/C-MYC signaling. Our results also highlighted the feasibility of the clinical trial for the combination in the therapy of AML patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract C038: Repeat RNA dysregulation of cellular states in the pancreatic cancer microenvironment

Abstract Aberrant transcription of the repeat RNAs is a common feature in epithelial cancers including PDAC, but the function of these non-coding RNAs in cancer development is relatively unexplored. We have found that these repeat RNAs are sensed and replicate like retroviruses, and now have identified the ability of these viral-like elements to be transmitted from cancer cells through extracellular vesicles (EVs). PDAC-derived EVs applied to cancer-associated fibroblasts (CAFs) activates interferon-stimulated genes (ISGs) and is able to drive CAFs towards an inflammatory CAF (iCAF) phenotype with concordant loss of myofibroblast CAF (myCAF) marker genes. Using in-vitro transcription, we demonstrate that individual repeat RNAs (HSATII, HERVK (env), LINE-1 5’UTR and LINE-1 3’UTR) are sufficient to induce ISG response in CAFs with HSATII and HERVK (env) having the most potent ISG response. In contrast, PDAC cells were found to induce epithelial-mesenchymal transition (EMT) with loss of epithelial gene expression. To determine the potential sensor of HSATII repeat RNAs, we utilized CRISPR/Cas9 knockout of the viral RNA sensors RIG-I, MDA5, and MAVS in PDAC and CAF cells. Notably, these sensors were important for PDAC repeat RNA sensing and response, but not in CAF cells. Evaluating the innate immune pathway further downstream, we used genetic knockout of IRF3 with CRISPR/Cas9 knockout and find significant downregulation of key EMT genes that are shared with myCAF markers (ACTA2, FN1, SERPINE1). Interestingly, HSATII RNA activated IRF3 dependent EMT genes in PDAC cells, but induced IRF3 degradation in CAF cells that results in loss of myCAF gene expression. Furthermore, we found that conditioned media from HSATII transfected CAF activates EMT-related gene expression (ACTA2, FN1, SERPINE1) in PDAC cell lines, which indicates an cell extrinsic mechanism to augment EMT induction in PDAC cells. We utilized next generation spatial transcriptomic platforms NanoString GeoMx and CosMx to understand the spatial distribution of repeat RNAs in human PDAC tumors. We find that repeat RNAs can be found as a gradient from PDAC cells to the surround tumor microenvironment consistent with delivery of these RNA species. Analysis of over 300,000 individual cells in 3 PDAC tumor specimens, we find that high repeat PDAC cells have lost epithelial gene expression and high repeat CAFs have lost myCAF gene expression. Altogether, these findings support the “infection” of repeat RNAs disrupts cellular identity in both tumor cells and the CAF microenvironment as a mechanism for tumor progression. Citation Format: Eunae You, Luli Zou, Patrick Danaher, Ildiko E. Phillips, Michael J. Raabe, Bidish Patel, Amaya Pankaj, Khrystyna North, Sean Kim, Youngmi Kim, Martin Aryee, David T. Ting. Repeat RNA dysregulation of cellular states in the pancreatic cancer microenvironment [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C038.

Synergistic Sensitivity of Selinexor with Chidamide By Targeting NF-Κb /c-FLIP Signaling Via Suppressing HDAC1/XPO1 Activity in T-ALL

Background T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease with a poor prognosis. Exportin-1 (XPO1) overexpression is observed in ALL and is associated with a poor prognosis for the patients. Recently, XPO1 inhibitor is reported to show anti-tumor effects in T-ALL. HDAC inhibitors (HDACi) are also reported to inhibit T-cell leukemia, and Chidamide is a new selective HDACi. Here, we explored the effect of Chidamide combined with the XPO1 inhibitor Selinexor in T-ALL and the underlying mechanisms. Methods Cell Counting Kit-8 (CCK-8) was used for cell viability assay, Annexin-V/PI staining following flow cytometry analysis for apoptosis, and qPCR and Western Blot for gene expression in CEM and Molt4 T-ALL cell lines. CalcuSyn analysis was performed for a synergistic effect. Cellular fractionation combined with western blot was used to detect the distribution of observed proteins. The co-immunoprecipitation(Co-IP) assay was utilized for the protein-protein interaction. Results We first observed that both Chidamide and Selinexor induce cell proliferation arrest of T-ALL cells in a dose-dependent manner. Then we found that Selinexor significantly increased the Chidamide-mediated cell proliferation arrest in both CEM and Molt4 cells and CalcuSyn analysis showed they have a synergistic effect (Fig. 1A). Moreover, the combination significantly increased the apoptosis (Fig.1B) and cell cycle arrest at G0/G1 phase in CEM cells (Fig. IC) and Molt-4 cells compared to the single drug controls. Consistently, the combination significantly up-regulated the expression of pro-apoptotic factors Cleaved caspase-3 and BAX, but down-regulated that of anti-apoptotic effectors BcL2 and MCL1, and the cell cycle regulators CDK2 and CyclinE2 compared to that of the single drug controls in the cells (Fig.1D). These data together indicate the combination of Chidamide with Selinexor has synergistic anti-leukemia effects in T-ALL. To understand the underlying mechanism of the synergy, we observed the apoptosis regulatory proteins Cellular FLICE inhibitory protein(c-FLIP) is significantly down-regulated upon the combination treatment compared to the single drug controls (Fig. 1E). c-FLIP is reported to be transcriptionally regulated by NF-kB. Interestingly, we observed that Chidamide significantly down-regulates the expression of HDAC1 and NF-κB p50 in T-ALL cells (Fig. 1F). Moreover, IκB-α and NF-κB p65 are XPO1 cargoes, and the interaction of IκB-α with NF-κB p65 prevents NF-κB binding to target DNA. We also observed the accumulation of IκB-α, and NF-κB p65 in the nuclear fraction in CEM (Fig. 1G) and Molt4 cells upon the Selinexor treatment. The co-IP assay showed the IκB-α bound to NF-κB p65 subunit in the cells and this interaction is enhanced in nuclear faction upon Selinexor treatment (Fig.1H), which might prevent NF-κB dependent transcription of target genes such as c-FLIP. Furthermore, the combination of Selinexor with Chidamide showed significant effects on cell growth arrest, the expression of CDK2, CyclinE2, Cleaved caspase3, and BCL2 in primary cells from T-ALL patients (Fig.1I). Finally, qPCR results revealed c-FLIP was overexpressed in T-ALL patients compared to the normal controls in our T-ALL patient cohort (Fig.1J). These data together indicated that the combination exerts its anti-tumor effect by targeting XPO1/HDAC1/NF-kB/c-FLIP signaling. The mechanism model is shown in Fig.1K. Conclusions Our results indicated that the combination of Selinexor with Chidamide has synergistic sensitivity on cell proliferation arrest, apoptosis, and cell cycle arrest in T-ALL by targeting NF-kB/c-FLIP oncogenic signaling through suppressing XPO1/HDAC1 activity. Our data also provide experimental evidence for the further clinical trial of the new potential combination in therapy of T-ALL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

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.