The histone lysine methyltransferase EZH2 is frequently altered in lymphoid tumors. Its overexpression or mutation is associated with tumor progression and resistance to chemotherapy. This makes it an attractive target for inhibition especially in combination treatments with established chemotherapeutics with the goal of overcoming chemotherapy resistance. However, the impact of antagonistic effects in rationally designed drug combinations remains poorly understood necessitating thorough investigation. In the current study, we show that the combinational treatment with SAM-competitive EZH2 inhibitors leads to platinum resistance due to increased platinum efflux. On a molecular level, we have discovered off-target effects leading to the upregulation of proteins that are associated with chemotherapy resistance. Our findings underline the need for detailed studies of combination therapies in order to rule out adverse effects of rational therapeutic approaches.S-adenosylmethionine (SAM) binding site in combination with the common second-line chemotherapeutic oxaliplatin. The change in cytotoxic effects under pharmacological EZH2 inhibition was evaluated revealing a drastic increase in oxaliplatin resistance after 72 h and longer periods of combinational incubation. This outcome was independent of cell type but associated to reduced intracellular platinum. Pharmacological EZH2 inhibition revealed increased expression in SRE binding proteins, SREBP1/2 and ATP binding cassette subfamily G transporters ABCG1/2. The latter are associated with chemotherapy resistance due to increased platinum efflux. Knockdown experiments revealed that this was independent of the EZH2 functional state. The EZH2 inhibition effect on oxaliplatin resistance and efflux was reduced by additional inhibition of the regulated target proteins. In conclusion, pharmacological EZH2 inhibition is not suitable in combination with the common chemotherapeutic oxaliplatin in T-cell lymphomas revealing an EZH2-independent off-target effect.T-cell lymphomas are heterogeneous and rare lymphatic malignancies with unfavorable prognosis. Consequently, new therapeutic strategies are needed. The enhancer of zeste homologue 2 (EZH2) is the catalytic subunit of the polycomb repressive complex 2 and responsible for lysine 27 trimethylation of histone 3. EZH2 is overexpressed in several tumor entities including T-cell neoplasms leading to epigenetic and consecutive oncogenic dysregulation. Thus, pharmacological EZH2 inhibition is a promising target and its clinical evaluation in T-cell lymphomas shows favorable results. We have investigated EZH2 expression in two cohorts of T-cell lymphomas by mRNA-profiling and immunohistochemistry, both revealing overexpression to have a negative impact on patients’ prognosis. Furthermore, we have evaluated EZH2 inhibition in a panel of leukemia and lymphoma cell lines with a focus on T-cell lymphomas characterized for canonical EZH2 signaling components. The cell lines were treated with the inhibitors GSK126 or EPZ6438 that inhibit EZH2 specifically by competitive binding at the  Enhancer of zeste homologue 2 (EZH2) is part of the polycomb repressive complex 2 (PRC2) and canonically responsible for the inactivation of gene transcription by trimethylation of lysine 27 of histone 3 (H3K27me3) . AdditioPeripheral mature T-cell Non-Hodgkin’s Lymphomas (MTCL) are a rare group of tumor entities with an unfavorable prognosis . Due to As we and others have shown before, T-cell lymphomas overexpress EZH2, which is correlated to worse survival rates ,12. ThisS-adenosylmethionine (SAM) inhibitors that can be subdivided into EZH2-specific and EZH1/2 dual inhibitors [Coherently, different types of EZH2 inhibitors were developed. Roughly, there are three main types of EZH2 inhibitors with the first-in-line being the S-adenosylhomocysteine (SAH) hydrolase inhibitor 3-Deazaneplanocin A (DZNep) that inhibits the activity of methyl transferases globally ,15. Secohibitors ,18. It whibitors .Efficacy of EZH2 inhibitor monotherapy in relapsed and refractory MTCL is moderate with overall response rates of the EZH1/2 inhibitor DS-3201 of 55.6% and complete response rates of 24.4% in a first-in-human study . On the In the current study, we have assessed EZH2 expression in MTCL and evaluated EZH2 inhibition as therapy in T-cell neoplasm cell lines in combination with established chemotherapeutics. Furthermore, we have investigated the effects on a random basis in a broad panel of cell lines from different backgrounds to elucidate possible generalized sensitizations to commonly used chemotherapeutics. Unexpectedly, we have found a drastic increase in oxaliplatin resistance through pharmacological EZH2 inhibition. We have revealed that SAM-competitive EZH2-inhibitors led to an increase in SRE binding factor (SREBF) expression in an EZH2-independent manner. We have shown that an increased ATP binding cassette subfamily G (ABCG) transporter expression is responsible for an enhanced oxaliplatin efflux.In conclusion, while EZH2 inhibitors show promising results as well as synergistic effects with other chemotherapeutics, our data do not support the combination with platinum derivates. We have shown that pharmacological EZH2 inhibition can induce an increase in chemotherapy resistance, most prominent in oxaliplatin, which is commonly used as the second-line treatment of T-cell lymphomas. Our study underlines the importance of thorough investigations of rational drug combinations.The study population of the gene expression profiling cohort was described before . The stuIn a second clinically annotated cohort, tissue from 50 patients with MTCL was used to create tissue microarrays (TMA), as reported by Schümann et al. . The stuThe gene expression profiling was performed as described before using HG-U133-plus2.0 arrays . Detaile2 and passaging did not exceed 10 times after thawing. Mycoplasma negativity was confirmed repeatedly and after the respective last experiment using MycoSPY® Master Mix .The cell lines Jurkat , SUPT11 , HH , and Daudi  were obtained from the German Collection of Microorganisms and Cell Cultures   and cultivated in RPMI-1640 Medium  supplemented with 10% fetal bovine serum  and 1% penicillin/streptomycin . Loucy  was obtained from American Type Culture Collection  and cultivated in RPMI-1640 Medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Oci-Ly1  was obtained from DSMZ  (9 September 2020) and cultivated in RPMI-1640 Medium with 20% fetal bovine serum and 1% penicillin/streptomycin. DERL-2  was obtained from DSMZ  (18 January 2017) and cultivated in RPMI-1640 Medium with 20% fetal bovine serum, 1% penicillin/streptomycin, and 0.02 ng/µL recombinant IL-2 . The cell line SR786  was cultivated in RPMI-1640 Medium with 10% fetal bovine serum and 1% penicillin/streptomycin. All cells were cultured in a humidified atmosphere at 37 °C and 5% CO® multimode microplate reader  after 2 h of Resazurin treatment . Experiments were performed in triplicates. Graphpad Prism software v8.4.3 (RRID:SCR_002798) was used for the calculation of IC50 values and SynergyFinder (RRID:SCR_019318) was used for the modulation and calculation of synergy scores [Cells were seeded at appropriate densities/100 µL/well in 96-well plates in 8 replicates. Detailed densities are listed in y scores . In combCells were seeded at 500 k cells/mL and incubated with 4 µM GSK126, oxaliplatin at half maximal reduction in viability or both for 24, 48, or 72 h before being collected by centrifugation and cleared from supernatant. The pellets were washed with PBS and stained using FITC Annexin V Apoptosis Detection Kit with 7-AAD  according to the manufacturer’s instructions. Cells were analyzed by flow cytometry using BD LSR Fortessa™ Cell Analyzer  and evaluated by BD Life Sciences FlowJo™ v10.6.1. Experiments were performed thrice and the exemplary gating strategy is shown in Cells were seeded at 500 k cells/mL and incubated with 4 µM GSK126, oxaliplatin at half maximal reduction in viability or both for 24, 48, or 72 h before being collected by centrifugation and cleared from supernatant. The pellets were washed with PBS and fixed with 70% ice-cold ethanol for at least 24 h at 4 °C. The ethanol was removed and cells were washed with PBS. The cells were incubated in 1 mg/mL RNase  at 37 °C for 30 min. The pellets were stained with DAPI , directly analyzed by flow cytometry using BD LSR Fortessa™ Cell Analyzer , and evaluated by BD Life Sciences FlowJo™ v10.6.1. Experiments were performed in triplicates and the exemplary gating strategy is shown in 3 at 70 °C over night. Platinum content was measured by inductively coupled plasma mass spectrometry using Bruker 820-MS ICP Mass Spectrometer  with Agilent ICP-MS Expert software v2.1 and expressed as ng of platinum per 106 cells. A single ICP-MS measurement represents the average of 20 scans per replicate within 5 replicates from the same liquid sample, with an error <5%. The platinum concentrations that we report at each time point were averaged across four series of cultures, ensuring that the values are correctly scaled to account for cell population differences and dilutions. Standard curves were generated by using aqueous serial dilutions of stock solutions traceable back to standard reference material from the National Institute of Standards and Technology. For comparison, the values were normed to the respective oxaliplatin monotherapy control at each given time point. Experiments were performed thrice.ICP-MS is based on the vaporization, atomization, and ionization of a sample in plasma for subsequent mass spectrometric analysis and was applied as described before ,29. The g for 45 min. After 24 h, media were changed to RPMI-1640 Medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin and after 48 h selection by puromycin  was applied.Cells were seeded at 200 k cells/500 µL/well in 24-well plates in RPMI-1640 Medium supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, and polybrene . EZH2 knockdown virus particles were obtained from Sigma-Aldrich   and Peroxidase-conjugated AffiniPure F(ab’)2 Fragment goat-anti-mouse IgG  were used. Primary antibodies were diluted in skim milk or bovine serum albumin solution according to the manufacturer’s instruction, incubated over night at 4 °C, and afterwards washed in PBST. Horse radish peroxidase-coupled secondary antibodies were diluted in skim milk and incubated for 1 h at room temperature. The membrane was washed in PBST before being incubated with ECL™ Prime Western Blotting Detection Reagent  and signals were detected on the ChemiDoc Imager detecting system . Western blots were performed from at least 3 individual cell lysates per sample and a representative blot is shown. Quantitative analyses were performed using National Institutes of Health ImageJ v1.51 (RRID:SCR_003070) for band intensity calculation. Values were normed to the respective housekeeping control before being normed to the control cell line. Whole uncropped Western blots can be found in Protein isolation and Western blot analysis was carried out as described before with some modifications . Briefly−ΔΔCT method as described [RNA was isolated using peqGOLD TriFast™  according to the manufacturer’s instructions. cDNA was synthesized from 2 µg RNA using MMLVRT protocol . Target gene expression was analyzed by RT-qPCR using SYBR™ Select Master Mix for CFX  according to the manufacturer’s instructions using 60 ng cDNA and a primer concentration of 250 nM. Data were acquired using CFX384 Touch Real-Time PCR detection system  with Bio-Rad CFX Manager Software v3.1 (RRID:SCR_017251) and quantified using the comparative 2escribed . The houHomo sapiens reference genome available on ENSEMBL using the STAR aligner v2.5.2b (RRID:SCR_004463). Unique gene hit counts were calculated using the feature Counts from the Subread package v1.5.2 (RRID:SCR_009803). Only unique reads that fall within exon regions were counted. Raw counts of all samples were combined to a matrix, gene names were annotated using biomaRt (RRID:SCR_019214) [RNA was isolated using peqGOLD TriFast™ according to the manufacturer’s instructions and analyzed by Genewiz  from two independent experiments of the cell lines HH, DERL2, Jurkat, and Loucy. Quality control, sequencing, and basic bioinformatics were executed by Genewiz as service. RNA samples were quantified using Qubit 4.0 Fluorometer  and RNA integrity was checked with RNA Kit  on Agilent 5300 Fragment Analyzer  with a mean quality score of 35.95. RNA-sequencing library preparation was prepared using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina following the manufacturer’s instructions . Sequencing libraries were validated using the NGS Kit on the Agilent 5300 Fragment Analyzer and quantified by Qubit 4.0 Fluorometer. The sequencing libraries were multiplexed and loaded on the flowcell on the Illumina  NovaSeq 6000 instrument according to the manufacturer’s instructions. The samples were sequenced using a 2 × 150 Pair-End (PE) configuration v1.5 with 93.95% of bases having a read depth >30. Image analysis and base calling were conducted by the NovaSeq Control Software v1.7 on the NovaSeq instrument. Raw sequence data were converted into fastq files and de-multiplexed using Illumina bcl2fastq v2.20 (RRID:SCR_015058). One mismatch was allowed for index sequence identification. Sequence reads were trimmed to remove possible adapter sequences and nucleotides with poor quality using Trimmomatic v0.36 (RRID:SCR_011848). The trimmed reads were mapped to the _019214) . We omit_019214) . Differe_019214) . All anat-test if not stated otherwise. For normed analyses, Welch’s correction was applied. For the comparisons of expression data between samples, two-sided, unpaired Student’s t-test (2 groups) or one-way ANOVA test (>2 groups) were applied. Survival data were illustrated by Kaplan–Meier-plots and group comparisons were performed by log-rank statistics.Basic statistics were performed using Graphpad Prism software v8.4.3 applying two-sided, unpaired Student’s n = 381), EZH2 gene expression profiling was analyzed and compared to normal lymphoid control tissue (n = 14). Tumor tissues were found to have significantly higher EZH2 expression than healthy control (tumor vs. control: median EZH2 expression 11.07 (IQR = 1.02) vs. 10.24 (IQR = 1.13); p < 0.0001)   A. In the 0.0096) B.n = 50), EZH2 protein expression was compared to healthy lymph node samples (n = 10) and found to be significantly elevated in tumor tissue (tumor vs. control: median EZH2 H-score 85.0 (IQR = 115.0) vs. 30.0 (IQR = 28.0); p = 0.0063)  in univariate analysis  C. Furtheanalysis D.The validated overexpression of EZH2 in MTCL provides a rationale for EZH2 inhibition as a possible targeted therapy in these entities. While others could show first promising results by EZH2 inhibition, we could not validate a significant reduction in cell viability by GSK126 and EPZ6438 at clinically relevant concentrations in a well-characterized panel of T-cell neoplasm cell lines, each with known PRC2 mutation status and H3K27 trimethylation level . The panTherefore, we combined the commonly used chemotherapeutics doxorubicin, oxaliplatin, and cytarabin, as well as the cytotoxic and hypomethylating agent 5-azacytidin with pharmacological EZH2 inhibition by GSK126 in the same panel as before in a screening approach. The comprised maximal-found sensitivity modulation of each cell line and chemotherapeutic in combination with GSK126 is shown in As the sensitivity modulation with the highest shift and the only modulation with independence from cellular background was found for oxaliplatin in the screening approach, we aimed to investigate the mechanistic changes leading to the increase in oxaliplatin resistance. Therefore, we broadened the cell line panel with one additional T-ALL cell line and one cell line with ALCL, ALK+ background. The additionally used cell lines were also well-characterized with known PRC2 mutation status and H3K27 trimethylation level . FurtherFor further studies, we focused on the cell line HH. In addition, we analyzed Jurkat in order to validate effects that might be generalized independently from the cellular background. Concentration-dependent synergy matrices of either GSK126 or EPZ6438 D were coIn order to clearly evaluate EZH2 dependence on the induction of oxaliplatin resistance in mature T-cell lymphomas, we established an EZH2 knockdown in HH. Furthermore, the EZH2 knockdown was established in Jurkat to rule out cell line-specific effects. On the protein level, EZH2 and H3K27 trimethylation downregulation was proven E and addIn order to elucidate the mechanism of resistance, different pathways that might be involved including induction of apoptosis, cell cycle deregulation, and drug transporter activity were analyzed. Data are shown in the exemplary cell lines HH and Jurkat  and all In all tested cell lines, oxaliplatin led to a progressive reduction in viable cells consistent with an induction of apoptotic and necrotic cells . The eff0G1-phase was induced by oxaliplatin without any arrest in other phases. In Jurkat, however, G2M-arrest was induced which led to a shift to Sub-G0G1-phase. Again, the oxaliplatin effects were lowered by additional GSK126 mirroring the resistance. Once more, deregulation was first observed after 48 h of incubation and more prominent in T-cell lymphoma cell lines.Oxaliplatin was able to induce cell cycle deregulation in all tested cell lines . The patp < 0.006 and p < 0.004, respectively). The time point was consistent with the arising of differences between oxaliplatin as single agent or in combination with GSK126 from apoptosis and cell cycle progression analysis. Intriguingly, the intracellular platinum content was not reduced by EZH2 knockdown alone  protein due to the induction of multi drug resistance . Accordingly, the EZH2-specific inhibitor EPZ6438 was already approved for the treatment of epithelioid sarcoma by FDA [Pharmacological EZH2 inhibition was proven to be effective in the treatment of entities with activating EZH2 mutations such as Diffuse Large Cell B-Cell as well as Follicular Lymphoma in phase II clinical trials ,42. Howea by FDA . As EZH2a by FDA . This shTherefore, in clinical therapies, new drugs are often combined with established regimen in search of synergistic effects. However, antagonistic modulations of drug sensitivity also need to be taken into consideration. In the present study, we have shown that EZH2 inhibitors have moderate effects on the sensitivity modulation of the commonly used chemotherapeutics doxorubicin, cytarabin, and 5-azacytidin. Moreover, we have proven that pharmacological EZH2 inhibition by SAM-competitive small molecule drugs increase oxaliplatin resistance drastically in mature T-cell lymphoma, T-cell leukemia, and B-cell lymphoma cell lines. We have shown for HH, DERL-2, Jurkat, and Loucy that secondary readouts such as cell cycle deregulation and induction of apoptosis by oxaliplatin were reduced by additional pharmacological inhibition. While the initial response to oxaliplatin varied between the cell lines, the reduction in oxaliplatin activity seems to be universal indicating a mechanism of resistance independent from the primary oxaliplatin effect.Additionally, the intracellular platinum content was lowered in HH and Jurkat by pharmacological EZH2 inhibition. This indicates that an increase in oxaliplatin efflux might be one of the mechanisms involved in the increase in resistance.The effect was not correlated to the cellular background or EZH2 expression which demonstrates a universal mechanism independent from EZH2 inhibition. This was further underlined by the lack of induced oxaliplatin resistance by non-SAM-competitive EZH2 inhibitor DZNep and EZH2 knockdown or the lack of reduced intracellular platinum under EZH2 knockdown. SAM-competitive EZH2 inhibitors might also impede the activity of the related protein EZH1 that therefore needs to be taken into consideration as a possible off-target. In the setting of EZH2 knockdown, EZH1 would not be affected thereby potentially compensating for the induction of resistance. It was shown that there is an overlap of targets between the two proteins and EZH1 might act as a backup protein in case of EZH2 depletion ,45.However, off-target effects independent from PRC2 signaling also need to be taken into consideration. Lately, EZH2 independent effects of SAM-competitive EZH2 inhibitors have been shown in hepatocellular carcinoma cell lines and correlated with an induction of expression of the transcription factor SRE binding protein . FurtherAs the induction of oxaliplatin resistance was found independent of EZH1, EZH2, or H3K27 trimethylation status of the different cell lines, a PRC2-dependent, or non-canonical EZH2-signaling pathway is unlikely. We hypothesize that the block of oxaliplatin activity is indeed an EZH2-independent off-target effect of SAM-competitive EZH2-inhibitors. We propose that the SREBP1/2-mediated upregulation of ABCG1/2 leads to an increased oxaliplatin efflux which results in resistance. We have provided proof by the reduction in GSK126-induced oxaliplatin resistance and efflux by additional pharmacological SRE binding protein or ABC transporter inhibition. As neither Fatostatin nor Glibenclamide as monotherapy were able to alter oxaliplatin sensitivity or the intracellular platinum content, we hypothesize that the induction of SRE binding proteins or ABC transporters by pharmacological EZH2 inhibition is needed for the consecutive inhibitors to show effects.However, the complexity of the investigated system needs to be taken into consideration. The shown data provide insight into the regulated mechanisms by pharmacological EZH2 inhibition and their effects on oxaliplatin sensitivity. We could prove that EZH2-independent SREBP1/2 and ABCG1/2 deregulation are involved in increasing oxaliplatin resistance. EZH2-dependent effects as well as other modulations were not investigated further and go beyond the scope of this paper.While EZH2 inhibitors show promising however moderate results as single agents in T-cell lymphomas as well as synergistic effects with other chemotherapeutics, our data do not support the combination with platinum derivates. This study demonstrates that pharmacological EZH2 inhibition can lead to an increase in chemotherapy resistance, here shown for oxaliplatin, a common second-line treatment drug in T-cell lymphomas. This underlines the necessity of encompassing investigations of rational drug combinations.


Abnormal hyaluronic acid (HA) metabolism is a major factor in tumor progression, and the metabolic regulation of HA mainly includes HA biosynthesis and catabolism. In glioma, abnormal HA biosynthesis is intimately involved in glioma malignant biological properties and the formation of immunosuppressive microenvironment; however, the role of abnormal HA catabolism in glioma remains unclear.HA catabolism is dependent on hyaluronidase. In TCGA and GEPIA databases, we found that among the 6 human hyaluronidases , only HYAL2 expression was highest in glioma. Next, TCGA and CGGA database were further used to explore the correlation of HYAL2 expression with glioma prognosis. Then, the mRNA expression and protein level of HYAL2 was determined by qRT-PCR, Western blot and Immunohistochemical staining in glioma cells and glioma tissues, respectively. The MTT, EdU and Colony formation assay were used to measure the effect of HYAL2 knockdown on glioma. The GSEA enrichment analysis was performed to explore the potential pathway regulated by HYAL2 in glioma, in addition, the HYAL2-regulated signaling pathways were detected by flow cytometry and Western blot. Finally, small molecule compounds targeting HYAL2 in glioma were screened by Cmap analysis.In the present study, we confirmed that Hyaluronidase 2 (HYAL2) is abnormally overexpressed in glioma. Moreover, we found that HYAL2 overexpression is associated with multiple glioma clinical traits and acts as a key indicator for glioma prognosis. Targeting HYAL2 could inhibit glioma progression by inducing glioma cell apoptosis and cell cycle arrest.Collectively, these observations suggest that HYAL2 overexpression could promote glioma progression. Thus, treatments that disrupt HA catabolism by altering HYAL2 expression may serve as effective strategies for glioma treatment.The online version contains supplementary material available at 10.1186/s12935-023-02998-4. Glioma has a very high mortality and disability rate, with an annual incidence of 4.67 to 5.73 per 100,000, and it is also the most common primary intracranial malignant tumor, posing a serious hazard to human health and bringing enormous social pressure . ConsequAn increasing number of studies have shown that tumor microenvironment remodeling is conducive to unrestricted tumor proliferation, and leads to further modification of malignant tumor behaviors, which are critical for tumor progression . ConceivThe extracellular matrix (ECM) acts as an important barrier that prevents normal tissue from becoming cancerous. However, the crosstalk of tumor cells with healthy tissues can reshape the extracellular matrix, recruit immune cells, and promote angiogenesis, which converts this environment into a supportive microenvironment for tumor progression . As a maNevertheless, in our previous reports, we mainly discussed the effect of abnormal HA biosynthesis on malignant glioma behaviors. In the present study, we wanted to elaborate the role of HA catabolism in glioma progression. And HA degradation is mainly mediated by hyaluronidase. Furthermore, some researchers reported that hyaluronidase is associated with tumor malignant progression, which has been demonstrated in prostate cancer, urinary bladder cancers, laryngeal cancer and breast cancer , 13. TheIn the present study, our results confirmed that among the six hyaluronidases , only HYAL2 was overexpressed in glioma patients, and HYAL2 overexpression was negatively correlated with the survival time of glioma patients. Moreover, high HYAL2 expression can be used as a key indicator for glioma patient prognosis. HYAL2 knockdown reduced glioma cell viability and induced glioma cell apoptosis and cell cycle arrest. Therefore, these results support that targeting HYAL2 in glioma may provide a new approach to eliminate glioma cells and improve the prognosis of glioma patients with further research.2.Glioma cell lines  and HUVECs were obtained from the Neurosurgery Laboratory of the First Affiliated Hospital of Harbin Medical University. Glioma cell lines were cultured in Dulbecco’s modified Eagle’s medium  with 10% fetal bovine serum , and Roswell Park Memorial Institute (RPMI)-1640 medium  containing 10% FBS was utilized to culture the HUVEC cell line. Finally, these cell lines were placed in a 37 °C incubator with 5% COGlioma tissues and traumatic brain tissues were derived from surgical resection at the Department of Neurosurgery, First Affiliated Hospital of Harbin Medical University. The Ethics Committee of the First Affiliated Hospital of Harbin Medical University agreed to carry out this research.HYAL2 siRNA was used to treat U251 and LN229 glioma cell lines for 48 h. After 10 µl MTT (5 mg/ml) was added to each cell culture well, the cell culture dish was cultured in a 37 °C incubator for another 4 h. Then, 150 µl DMSO was used to replace the original medium. A BioTek ELx800 (USA) microplate reader was used to evaluate cell viability at 490 nm.The U251 and LN229 glioma cell lines were cultured with siHYAL2 for 48 h. Then, an EdU assay kit  was used to measure the effect of siHYAL2 on glioma proliferation viability according to the instructions.U251 glioma cells were routinely digested, and a cell suspension was prepared. Then, 1000 cells per well were seeded in a 6-well cell culture dish. Next, siHYAL2 was used to transfect glioma cells. Then, the cells were cultured for 10 days. Next, 4% paraformaldehyde was added to fix the cells for 15 min, and the cells were stained with crystal violet solution.U251 and LN229 cell lines were treated with siHYAL2 for 48 h. Then, the glioma cells were lysed in RIPA solution on ice for 30 min. Next, total protein was extracted from glioma cell lines. 12.5% SDS‒PAGE gels were used to separate protein samples, and then the protein samples were transferred to PVDF membranes. After routinely blocking the membranes and incubating them with the primary and secondary antibodies, the GeneGnome XRQ Imaging System  was used to observe the immunoreactions. The primary antibodies were as follows: anti-HYAL2 ; anti-CCND1 ; anti-CCNB1 ; anti-Bcl-2 ; anti-BAX ; and anti-β-actin .Lipofectamine 2000 reagent  and siRNA were formulated into a transfection working solution according to the proportion, and then it was used to transfect glioma cell lines according to the manufacturer’s instructions. HYAL2 siRNAs were obtained from GENERAL BIOSYSTEMS (China).Total RNA was collected with TRIzol reagent  from glioma cell lines (U251 and LN229). The Roche Transcriptor cDNA Synthesis Kit  was used to obtain cDNA. Next, the cDNA was mixed well with the SYBR Green PCR Master Mix Kit  according to the instructions. Finally, an ABI Prism 7500 fast thermocycler  was used to measure the expression levels of target genes. The primer sequences for HYAL2 and GAPDH are shown in Table The collected glioma tissues were embedded in paraffin. The subsequent processes were performed as reported previously. The primary antibody used for immunohistochemical staining was anti-HYAL2 .Glioma cells were treated with siHYAL2 for 48 h. Next, the collected cells were stained with the Cell Cycle and Apoptosis Analysis Kit  or the Annexin V-FITC Apoptosis Detection Kit  according to the instructions. Finally, the observations were obtained by flow cytometry.http://bioconductor.org/. The different numbers of asterisks indicate p < 0.05, < 0.01, and < 0.001 in pictures.The pancancer transcriptome dataset that included 33 kinds of cancer transcriptome data was downloaded from The Cancer Genome Atlas (TCGA) database. The glioma transcriptome and clinical data were derived from the TCGA and CGGA databases. The online database GEPIA was used to obtain the expression data of HYAL1, HYAL2, HYAL3, HYAL4, HYALP1 and SPAM1 in glioma. Prism software version 7.0 was used to analyze the differences between groups. Bioinformatics analysis was completed by R software (version-4.2.0). The R packages used for bioinformatics analysis were derived from In a previous study, we found that abnormal HA metabolism is associated with glioma malignant progression, and interfering with HA synthesis has an inhibitory effect on glioma . In the Due to HYAL2 is overexpressed in multiple cancers including glioma, we aimed to further demonstrate the correlation of HYAL2 with glioma. First, the differentially expressed genes (DEGs) in glioma tissues relative to normal tissues in the TCGA database were assessed. Then, these DEGs were intersected with HAase genes , HYAL2 was the only overlapping gene . In the Chinese Glioma Genome Atlas (CGGA) database, survival filtration analysis was performed to find the DEGs in mRNA-array_301, mRNA-seq_325 and mRNA-seq_693; next, intersection analysis was used to identify overlapping genes between DEGs screened in mRNA-array_301, mRNA-seq_325 and mRNA-seq_693 and HAase genes , and again, HYAL2 was the only overlapping gene . These evidences supported that HYAL2 was abnormally overexpressed in glioma. Therefore, we assessed the relative mRNA expression level of HYAL2 in four glioma cell lines  and HUVECs, which showed that the expression of HYAL2 in glioma cell lines was increased significantly compared with that in HUVECs  Fig. A. MoreovBecause HYAL2 was overexpressed in glioma and closely related to a poor prognosis in glioma patients, we further investigated the correlation of HYAL2 expression with the clinical characteristics of glioma patients. In the CGGA  database, the data regarding HYAL2 mRNA expression and the patients’ clinical information, including age, histology and IDH mutation status and 1p19q codeletion status, were extracted. The correlation analysis demonstrated that the expression of HYAL2 was closely associated with the IDH mutation status and 1p19q codeletion status Fig. A-B. As sGiven that HYAL2 might act as a risk factor for glioma prognosis, we wanted to determine whether disturbing HYAL2 expression could exhibit a therapeutic outcome in glioma. Subsequently, siRNA was used to knockdown HYAL2 in U251 and LN229 glioma cell lines . The MTT assay results showed that the viability of U251 and LN229 glioma cells was decreased significantly by siHYAL2 , the GSEA results showed that high HYAL2 expression resulted in significant enrichment of the cell cycle and apoptosis pathways in glioma , and ultimately, four small molecule compounds that may target HYAL2 in gliomas were discovered: sphingosine, D-609, NVP-BGJ398 and itopride. The 2D and 3D structures of four small molecule compounds were obtained from PubChem . Then, these 20 genes were uploaded to CMap  have been identified in humans . IncreasTo assess the potential therapeutic value of targeting HYAL2, HYAL2 siRNA was used to treat glioma, and it was found that HYAL2 knockdown reduced glioma cell viability and proliferation. We further explored the possible signaling pathway by which HYAL2 affects glioma by GSEA, meanwhile, found that HYAL2 could significantly affect apoptosis and the cell cycle in glioma. Subsequent studies demonstrated that targeting HYAL2 can induce glioma cell apoptosis and arrest the cell cycle in the G1 phase. In addition, it has been reported that cell cycle signals can modulate cell proliferation . ConsideHA is a relatively simple polysaccharide, and its family mainly includes high molecular weight HA (HMW-HA), low molecular weight HA (LMW-HA), and oligomeric HA (o-HA), which are mainly achieved by the tight coordination of HA biosynthesis and degradation processes . MoreoveTumor-derived HAase may be an attractive therapeutic target for tumors. Many synthetic or natural products, including high molecular mass poly(styrene-4-sulfonate), fenoprofen, glycerrhizic acid, and plant-derived compounds, have been used as HAase inhibitors . In the In conclusion, our observations revealed that HYAL2 overexpression in gliomas is associated with prognosis. Together with our previous studies, all of these results suggest that there are metabolic abnormalities related to HA in gliomas, including abnormalities in HA biosynthesis and catabolism. Therefore, the results of this study further enhance the understanding of the role of HA in glioma pathogenesis and provide a novel target for glioma treatment.Below is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2


Triple Negative Breast Cancer (TNBC), a subtype of breast cancer, has fewer successful therapeutic therapies than other types of breast cancer. Insulin-like growth factor receptor 1 (IGF1R) and the Insulin receptor (IR) are associated with poor outcomes in TNBC. Targeting IGF1R has failed clinically. We aimed to test if inhibiting both IR/IGF1R was a rationale therapeutic approach to treat TNBC. We showed that despite IGF1R and IR being expressed in TNBC, their expression is not associated with a negative survival outcome. Furthermore, targeting both IR/IGF1R with inhibitors in multiple TNBC cell lines did not inhibit cell growth. Linsitinib, a small molecule inhibitor of both IGF1R and IR, did not block tumour formation and had no effect on tumour growth in vivo. Cumulatively these data suggest that while IGF1R and IR are expressed in TNBC, they are not good therapeutic targets. A potential reason for the limited anti-cancer impact when IR/IGF1R was targeted may be because multiple signalling pathways are altered in TNBC. Therefore, targeting individual signalling pathways may not be sufficient to inhibit cancer growth. Triple negative breast cancer (TNBC) is an aggressive disease with a high risk of recurrence, and a lack of recognised molecular targets for therapy . CurrentInsulin-like growth factor receptor 1 (IGF1R) and its signalling has been extensively studied in breast cancer and it has been implicated in resistance to hormone therapies, HER2 targeted therapies, and chemotherapy . PreviouBoth the IR and the IGF1R belong to the same tyrosine kinase receptor subfamily and share structural homology in the tyrosine kinase domain . BecauseOur hypothesis was that inhibition of IR in addition to IGF1R may overcome poor efficacy issues related to previously tested IGF1R inhibitors. We aimed to evaluate whether inhibition of both IR and IGF1R was a rationale therapeutic approach to treat TNBC.2. Linsitinib and NVP-BEZ-235 were obtained from Carbosynth whilst xentuzumab was obtained under Material Transfer Agreement from Boehringer Ingleheim. Cisplatin was obtained from St Vincent’s University Hospital, Dublin Ireland. All drugs, not already in clinical formulation, were reconstituted in DMSO and stocks of 10 mM were stored at -20°C. Cell lines used in these experiments were confirmed as mycoplasma free by regular testing. Cell lines were fingerprinted using STR profiling.MDA-MB-468, HCC1143, HCC1937, HCC70, CAL-851, HDQ-P1, HCC1806, CAL120, CAL51, BT549, Hs578T, MDA-MB-231, MDA-MB-157, HCC1187, BT20 cells were acquired from collaborators in UCLA. All cell lines were grown in RPMI-1640 media supplemented with 10% FCS and grown in a 37°C incubator maintained with 5% COBreastMark is a database based on an algorithm developed by Molecular Therapeutics for Cancer, Ireland (MTCI) . Using tThe expression status of IR-A, IR-B, IGF1R and IGF-II mRNA was analysed in a panel of triple negative breast cancer cell lines  and MCF10A which represented a non-cancer breast control. RNA was extracted using the mirVana™ miRNA Isolation Kit  according to the manufacturer’s instructions. Triplicate RNA samples were isolated from the cell lines at 70–80% confluency. cDNA was prepared from 2 μg of total RNA using a high-capacity RNA to cDNA kit , master mix solution and reverse transcription conditions described as per 3, whilst Hs578T, HCC70, MDA-MB-468, HCC1143, HCC1937, CAL-851, BT549, MDA-MB-231, HCC1806, and BT20 were seed at 3 × 103 cells/well, whilst CAL120, HCC1187, MDA-MB-157, and HDQ-P1 were seeded at 5 × 103 cells/well in 96-well plates. Plates were incubated overnight at 37°C followed by addition of drugs at the appropriate concentrations (<10 μM) and incubated for a further 5 days until wells were 80% to 90% confluent. Two types of combination assays were used. Fixed combination, where a single concentration of each drug was combined and fixed ratio proliferation, where drugs are combined over a range of concentrations at a fixed relative ratio. Following five days of incubation with drug, the media was removed, and the wells were washed once with PBS. Paranitrophenol phosphate (PNP) (ThermoFischer) substrate  was added to each well and incubated at 37°C for 1–2 hours. 50 μL of 1 M NaOH was added and the absorbance was read at 405 nM (reference– 620 nM).Proliferation was measured using an acid phosphatase assay. CAL51 were seeded at 1 x 103 cells/well (except BT549 which were seeded at 1.5 x103 cells/well) in 10% FCS with Matrigel™  and incubated overnight at 37°C. Complete media (30 μL) with/without serial dilutions of drug, was added to the wells and incubated at 37°C for a further 5 days. 12 μL of PrestoBlue® Cell Viability reagent  was added to the wells and incubated at 37°C for 2–8 hrs  and fluorescently measured at 535/590 nm excitation/emission wavelength on Biotek plate reader using KC4 software. A blank consisting of media and 2–4% Matrigel™ as appropriate was used to eliminate background. Percentage viability was calculated relative to untreated control. Each assay was performed in biological triplicate.96 well plates were coated with 50 μL polyhema (Sigma Aldrich S3932 5 mg/mL w/v in 96% ethanol) and baked at 50°C for two days. Plates are stored at room temperature until required, for up to 6 months. Cells were seeded at a density of 3 x 10Protein was extracted using RIPA buffer with 1× protease inhibitors, 2 mM PMSF and 1 mM sodium orthovanadate (Sigma-Aldrich) was added to cells and incubated on ice for 20 min. Following centrifugation at 10,000 rpm for 10 min at 4°C, the resulting lysate was stored at −80°C. Protein quantification was performed using the bicinchoninic acid (BCA) assay (Pierce).Magnetic bead assays were performed on the Luminex® MagPix® System  using Milliplex Map Phospho Mitogensis RTK Magnetic Bead 7-Plex kit  and Milliplex Map phosphor Human SRc Family Kinase Magnetic Bead 8-Plex kit (Merck Millipore 48-650Mag).Protein (1–10 μg) was diluted in appropriate volume of assay buffer  and the assay was performed as per the manufacturer’s instructions.scid/lcrCrl) mice were obtained from Charles Rivers (UK), maintained in pathogen-free conditions and fed a standard diet. Mice were kept at 25°C, with a 12 hour light-dark schedule and free access to food and water. All experiments were approved by DCU Research Ethics Committee (DCUREC/2015/208) and the Health Products Regulatory Authority (HPRA) under project authorization AE19115/P009, were carried out in accordance with the relevant guidelines and regulations and in compliance with the ARRIVE guidelines.Female SCID  arm.HCC1143 cells were grown as cell line–derived xenografts models to test the efficacy of linsitinib on tumour formation and tumour growth. HCC1143 cells were grown as described above and were prepared for implantation in serum free RPMI 1640 media and injected sub-cutaneously on the left flank of the mice at 5 x10Linsitinib (25 mg/kg) or vehicle (25 mM L-tartaric acid) were administered daily by oral gavage to determine the impact on tumour formation. Treatment commenced on day 4 following tumour induction and continued for 78 days. Treatment commenced prior to tumours becoming palpable.3 nor any tumour dimension exceeding 15 mm. Tumour measurements were blinded to treatment group. Treatment regime was 5 days on with 2 days off. A one week drug holiday was given following three weeks on treatment. The mice were sacrificed by cervical dislocation after the last linsitinib treatment or at a point when a humane end point was reached, such as a loss of body weight exceeding 20% of weight at start of treatment.Mice were monitored daily for signs of toxicity. Animal body weights were measured three times/week and tumour volumes were measured twice weekly, using calliper measurements. Tumour volume was calculated as ((L*H*D)/1.9). Maximum allowable tumour was no greater than 1600 mmTumours were dissected on sacrifice and collected in 10% formalin or snap frozen in liquid nitrogen.To assess the impact of IGF1R and IR expression on DFS, OS and DDFS, survival curves are based on Kaplan-Meier estimates and the log-rank P-value is shown for difference in survival. Cox regression analysis was used to calculate hazard ratios. The R package ’survival’ was used to calculate and plot the Kaplan-Meier survival curve. All calculations were carried out in the R statistical environment. IGF-II mRNA expression was compared between basal and non-basal TNBC cell lines using the Fisher’s exact test. The combination of xentuzumab and cisplatin or docetaxel in TNBC cells was compared using a Student’s T-test. Statistical significance for tumour growth and body weight changes was determined by two-way ANOVA with Tukey’s multiple comparison test using GraphPad Prism v8.Using the BreastMark platform and the ssp2006 classifier, we examined the differences in mRNA expression of IGF1R and IR in 2185 breast cancers, including 255 basal-like breast cancers, and found that neither expression of IGF1R nor IR was elevated in a specific subtype . NeitherIR is alternatively spliced to form two isoforms A and B. IR (IR-A and IR-B) mRNA was detected in 10 of the 11 TNBC cell lines tested, whilst IGF1R mRNA was detected in the 11 TNBC cell lines and insulin like growth factor 2 (IGF-II) mRNA was detected in 6 of the 11 cell lines . No sign50 value was achieved in 3/7 basal like cell lines, whilst an IC50 value was only reached in 1/4 non-basal-like cell lines, at concentrations up to 10 μM  were exposed to increasing concentrations of both agents alone and in combination . HoweverWe aimed to determine if xentuzumab could enhance response to treatment with either chemotherapy or PI3K inhibition in the CAL-51 and HCC1143 cell lines which are PIK3CA mutant. Xentuzumab was tested in combination with cisplatin, docetaxel and the PI3K inhibitor NVP-BEZ-235. The addition of xentuzumab to cisplatin did not significantly increase the anti-proliferative effect of cisplatin, however addition of xentuzumab to docetaxel significantly increased the anti-proliferative effects of docetaxel in both CAL-51 and HCC1143 cells . FinallyThe basal-like cell-line HCC1143 was selected for further in vivo analysis as the in vitro results showed linsitinib inhibited both ligand dependent and ligand independent growth in this cell line. HCC1143 cell-line derived xenografts were used to assess linsitinib growth inhibition in vivo. The rate of tumour formation was analysed by monitoring tumour detection rates. At 19 days post implantation 100% (9/9) untreated mice, 33.3% (4/12) of vehicle treated mice and 58.3% (7/12) of the linsitinib treated mice had palpable tumours. At 35 days after implantation 9/9 untreated mice, 9/12 vehicle treated mice and 11/12 linsitinib treated mice had palpable tumours . Linsiti3 (n = 9); 24.6 +/- 10.0 mm3 (n = 11) and 23.4 +/- 10.2 mm3 (n = 8) for the vehicle, linsitinib and control arms respectively. Tumour volumes were normalised to day 33 volumes and fold increase in tumour volume compared. No significant difference in tumour volume was detected between the treatment, control and vehicle arms  using in-silico analysis. These findings raise questions regarding the oncogenic impact of IGF1R or IR expression in TNBC.Several pre-clinical studies have indicated that IR signalling may play a role in resistance to IGF1R therapy. A study of 438 breast tumours found that IR was expressed in 49% of breast tumours compared to 32% for IGF1R . IR is aTo date, therapeutic strategies aimed at disrupting IGF signalling have largely focused on antibodies that target IGF1R. However, clinical studies of IGF1R antagonism in breast cancer have been disappointing. Further supporting the idea of dual targeting IGF1R/IR, the IR has been implicated in resistance to IGF1R monoclonal antibody therapy. Downregulation of IR by shRNA sensitised pancreatic and breast cancer cell lines, and pancreatic xenograft tumours, to IGF1R inhibition . In the To test targeting of the IGF1R/IR axis, we tested the fully human monoclonal antibody xentuzumab which inhibits IGF signalling by selectively neutralising IGF ligands. Thus, it could be an effective and tolerable approach for inhibiting IGF-I and IGF-II signalling, without interfering with insulin/IR signalling. However, xentuzumab alone did not significantly inhibit proliferation in 6 of the 8 TNBC cell lines tested. Despite xentuzumab inhibiting IGF-I ligand stimulated proliferation in HCC1143 cells, xentuzumab failed to further inhibit proliferation relative to xentuzumab alone. Furthermore, in serum starved TNBC cells, stimulation with IGF ligands had no effect on proliferation rates in 2/3 cell lines tested. Only the HCC1143 cell line showed enhanced growth when treated with IGF ligands. These results possibly indicate a lack of dependence on IGF signalling in some TNBC cells. Identification of IGF-dependent tumours may be of relevance to xentuzumab efficacy in the clinic.Linsitinib is a potent and highly selective tyrosine kinase inhibitor with similar biochemical potency against IGF1R and IR. We demonstrated that linsitinib inhibited growth of TNBC cell lines in both 2D and 3D assays, with potential preference for basal-like TNBCs, which correlates with work from Rigiracciolo et al., (2020) . ConsistTo assess the impact of combined targeting of IGF ligands and the IGF/INS receptors, we tested the combination of linsitinib and xentuzumab in TNBC, but the combination failed to improve growth inhibition in vitro. Previous studies have demonstrated the benefit of combining xentuzumab with paclitaxel in xenograft and orthotopic implanted mouse studies. These studies used the 4T1 mouse model, which can also be used to study metastasis in vivo . Our stuTo determine if greater anti-tumour activity would be observed in vivo, we tested the effect of targeting IR/IGF1R in a cell-line derived xenograft model of TNBC, using the HCC1143 cell line. In our study, linsitinib at 25 mg/kg did not prevent or delay tumour formation, but it did impact animal body weight. Additionally, continued dosing beyond tumour formation did not restrict tumour growth. Previously, linsitinib has been assessed in vivo to examine the effect on glucose uptake in non-small cell lung carcinoma xenografts, showing that at 60 mg/kg, linsitinib inhibited growth in IGF1R and IR positive tumours . In IGF1Linsitinib was investigated in a clinical trial with patients with advanced cancers, in combination with irinotecan; though no breast cancer patients were evaluated. Patients were treated with 400–450 mg linsitinib with irinotecan once daily on days 1–3, 8–10 and 15–17. Grade 1/2 weight loss was reported in 30% of patients . In a PhTo date the search for targeted treatments to improve the outcomes of TNBC patients have failed to provide benefit in the clinic. Both IR and IGF1R are associated with poor outcomes in TNBC, whilst IGF1R signalling has been implicated in resistance to both hormone and targeted therapy, as well as chemotherapy. In our study, we have shown that despite IGF1R and IR being expressed in all breast cancer subtypes including TNBC, in silico analysis demonstrated that neither IGFR1 nor IR expression are associated with outcome in this subtype. Furthermore, targeting the IR/IGF1R axis in TNBC failed to produce robust inhibition of cancer cell lines in vitro. Supporting the limited role of IGF1R and IR as a target in TNBC, linsitinib, an IGF1R/IR small molecule inhibitor, failed to block tumour formation in vivo and additionally, had no significant effect on tumour growth. The in vivo study utilised a cell line model that did respond to linsitinib and xentuzumab under ligand-stimulated conditions in vitro. Cumulatively these data suggest that while IGF1R and IR are expressed in TNBC, they do not represent good therapeutic targets. The issues may align with the fact that TNBC is a grouping of breast cancers that are negative for expression of specific receptors, unlike HER2 or ER positive tumours where a dominant signalling pathway is identified. TNBC is a grouping of breast cancers that are negative for expression of specific targetable receptors unlike HER2 or ER positive breast cancer, where a dominant pathway is identified. The cumulative results of our study suggest that while IGF1R and IR are expressed in TNBC, they do not represent a dominant signalling pathway and are therefore not good therapeutic targets. Bearing in mind that TNBC is a very heterogenous disease, with distinct molecular subtypes , future S1 Fig(DOCX)Click here for additional data file.S2 FigCells were serum starved (2% FCS) for 24 hours followed by addition of IGF-I or IGF-II (50 ng/mL), with /without linsitinib 5 μM or xentuzumab (40 μg/mL) (treated with 2% FCS). Standard errors represent the average results from triplicate independent results.(DOCX)Click here for additional data file.S1 TableTarget quantity expressed as either + ≤100, ++ ≤200, +++ ≤500 or ++++ >500. ‘-‘not tested.(DOCX)Click here for additional data file.S2 TablePCR conditions with (A) detailing the components of master mix solution for reverse transcription reaction and (B) detailing Thermo-cycler steps, temperature and duration for RT-PCR experiment.(DOCX)Click here for additional data file.S1 Dataset(XLSX)Click here for additional data file.


The screening of experimental and repurposing drugs has enormous potential for rapid clinical impact, particularly when new therapeutic strategies are sought. By screening two drug libraries, nicotinamide phosphoribosyltransferase (NAMPT) and histone deacetylase (HDAC) inhibitors were identified as agents with the highest activity against neuroendocrine tumor-derived cell lines. An evaluation of different NAMPT inhibitors revealed a reduction in basal oxidative phosphorylation and energy production to be partially but not completely responsible for cell death following the exposure to the drug, with the yes-associated protein 1 (YAP1) playing a pivotal role in the sensitivity to the NAMPT inhibitors. Insight into the contribution of YAP1 guided the evaluation of combinations of HDAC and NAMPT inhibitors for the treatment of neuroendocrine neoplasms.Background: Over the last few decades of treatment, the outcomes for at least some subsets of neuroendocrine neoplasms (NENs) have improved. However, the identification of new vulnerabilities for this heterogeneous group of cancers remains a priority. Methods: Using two libraries of compounds selected for potential repurposing, we identified the inhibitors of nicotinamide phosphoribosyltransferase (NAMPT) and histone deacetylases (HDAC) as the agents with the highest activity. We validated the hits in an expanded set of neuroendocrine cell lines and examined the mechanisms of action. Results: In Kelly, NH-6, and NCI-H82, which are two neuroblastoma and one small cell lung cancer cell lines, respectively, metabolic studies suggested that cell death following NAMPT inhibition is the result of a reduction in basal oxidative phosphorylation and energy production. NAMPT is the rate-limiting enzyme in the production of NAD+, and in the three cell lines, NAMPT inhibition led to a marked reduction in the ATP and NAD+ levels and the catalytic activity of the citric acid cycle. Moreover, comparative analysis of the mRNA expression in drug-sensitive and -insensitive cell lines found less dependency of the latter on oxidative phosphorylation for their energy requirement. Further, the analysis of HDAC and NAMPT inhibitors administered in combination found marked activity using low sub-lethal concentrations of both agents, suggesting a synergistic effect. Conclusion: These data suggest NAMPT inhibitors alone or in combination with HDAC inhibitors could be particularly effective in the treatment of neuroendocrine neoplasms. Neuroendocrine neoplasms (NENs) constitute a heterogeneous group of malignancies, arising from the cells of the endocrine and nervous system and classified via the originating site of the primary tumor . They reThe primary treatment of patients with localized neuroendocrine neoplasms is surgical, but with metastatic progression, prognosis is poor and therapeutic options are limited . CurrentWith a goal of identifying novel drug classes active against NEN, we explored the activity of available, approved, and experimental drugs against NEN cell lines. Using three human neuroendocrine cell lines, NCI-H69, DMS79, and NCI-H72, we screened two small molecule drug libraries selected for potential repurposing, the National Center for Advancing Translational Sciences (NCATS) Pharmaceutical Collection (NPC), and the Mechanism Interrogation PlatE (MIPE) library. Two classes of agents emerged from this screen: inhibitors of nicotinamide phosphoribosyltransferase (NAMPT), and histone deacetylases (HDAC). We demonstrated both classes of inhibitors to be highly active as single agents and synergistic when combined even at low sub-lethal concentrations. Since both previous and current studies have indicated that the inhibitors of both nicotinamide phosphoribosyltransferase (NAMPT), and histone deacetylases (HDAC) have a profound impact on the synthesis of critical tricarboxylic acid cycle (TCA) intermediates, we hypothesized that their use in combination would result in marked metabolic stress that could lead to cell death, and thus decided to explore their activity in combination.GMX-1778 and STF-118804 were purchased from APExBIO . Romidepsin (depsipeptide) was purchased from Sigma-Aldrich . CA3 (CIL56) was purchased from Selleckchem . pBABE-puro (1764) and pBABE YAP1 (15682) were from Addgene .The initial screen included three cell lines from the NCI-60; the validation panel included 13 cell lines of neuroendocrine origin, including two of neuroblastoma origin. Neuroblastoma cell lines, whose expression profile resembles pheochromocytoma [6) were seeded in 100 mm dishes in a complete medium containing 2 μg/mL puromycin. After 2 weeks, the selection-isolated colonies were propagated for further analysis.Plasmids were transfected into the Kelly cells using the Fugene HD transfection reagent  according to the manufacturer’s protocol. At 48 h post-transfection, the cells  the NCATS Pharmaceutical Collection (NPC), a library of ~2750 small molecule drugs approved for use by the USFDA or related agencies in foreign countries, and (2) the NCATS Mechanism Interrogation PlatE (MIPE) library, which includes 1920 mechanistically annotated agents prioritized for clinical relevance.p-Values were computed using two-way ANOVA.The determination of the ATP and NAD+ levels and the metabolic profiling of the Kelly, NCI-H82, and UOK-269 cells were performed by the Metabolomics Core at the AdventHealth Research Institute . Briefly, the cells were treated with GMX1778 and STF-118804 at the described concentration for 48 h. The cells were pelleted, frozen, and sent for analysis. The bars represent the standard deviation of three independent experiments. The data were analyzed using GraphPad Prism 8 software . ® 680RD Goat anti-Mouse and IRDye® 800CW Goat anti-Rabbit . The signal was quantitated using the Odyssey CLx Imaging System, and the exposures within the linear range were captured using ImageStudio software V3.1 .Western blotting was carried out according to the manufacturer’s instructions. The membranes were incubated with primary antibodies, SLC52A3 , YAP1, PARP, beta-actin , and secondary antibodies, IRDye5) were seeded in 35 mm dishes containing 7 mL of complete medium and exposed to increasing concentrations of GMX-1778 and STF-118804 for 48 hr. Apoptosis was measured as previously described (36) using the annexin V fluorescein isothiocyanate (annexin V-FITC) Apoptosis Detection Kit  according to the manufacturer’s instructions. Following the treatment, the annexin positive cells were quantitated via FACS analysis  using FCS Express Cytometry Software . The percentage of annexin positive cells was calculated and the delta between the treated and untreated control was determined.The cells  under the accession number GSE216537.Total RNA from the NCI-H82, Kelly, NH6, UOK-269, OE-19, NCI-H2342, GCIY, and SNU-119 cell lines was isolated using the RNeasy Mini Kit  according to the manufacturer’s instructions. RNA quality was assessed on the Agilent Bioanalyzer . RNA libraries were prepared from poly-A pull-down-enriched mRNAs from the total RNA samples  and were sequenced at the Columbia Genome Center using NovaSeq6000 . Data were visualized in Qlucore Omics Explorer v. 3.4 , including principal component analysis (PCA), heat maps, and unsupervised hierarchical clustering. Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway analysis (QIAGEN IPA) were used for network- and pathway-focused analyses of transcriptomics data. The expression data are deposited in the Gene Expression Omnibus (GEO) (3/well) were seeded in white 96-well plates  in 0.1 mL of complete medium. The cells were exposed to the indicated concentration of romidepsin for 6 h, then washed and grown for 42 h in the presence of increasing concentrations of GMX-1778 and STF-118804. Cell viability was assessed using the CellTiter-Glo assay  and the microplate reader PHERAstar FS . The Excess of Bliss (EOB) score was used for synergy evaluation.For the initial screen, the CellTiter-Glo assay was used to quantitate the viability of three human neuroendocrine cell lines, NCI-H69, DMS79, and NCI-H727 , seeded 4 cells per well) in a growth medium supplemented with 10% inactivated fetal bovine serum (FBS). After a 24 h incubation at 37 °C without/with GMX1778 (10 nM) or STF11804 (20 nM), the cells were washed twice with a pre-warmed assay medium . The oxygen-consumption rate (OCR) and extra-cellular acidification rate (ECAR) values were simultaneously measured following the sequential injections of inhibitors of mitochondrial oxidative phosphorylation: (I) oligomycin (2 µM), an ATP synthase inhibitor; (II) antimycin A (0.5 µM), an inhibitor of complex III; and (III) rotenone (0.5 µM), an inhibitor of complex I, inhibiting uncoupled respiration. The ATP production rates were analyzed using the Agilent Seahorse XF Real-Time ATP Rate Assay Report Generator that automatically calculates the XF Real-Time ATP Rate Assay Parameters . The data were normalized to cell numbers by measuring the staining of nuclei (DNA content) using the CyQUANT® Cell Proliferation Assay Kit  following the manufacturer’s recommendations. p-Values were computed using a paired t-test result analysis for statistical significance.To evaluate the ATP production, a real-time ATP rate assay was performed using a Seahorse XF96 Cell Analyzer according to the manufacturer’s recommendations . Briefly, the cells  were seeded in Seahorse XF96 cell culture microplates . The initial screen for the novel small molecules that could inhibit neuroendocrine cell growth used the NCATS NPC and the MIPE libraries and three human neuroendocrine cell lines, NCI-H69, DMS79, and NCI-H727, and tested 4670 compounds . From thFollowing the validation in the extended neuroendocrine cell line panel, we next examined the sensitivity to NAMPT inhibition using a panel comprised of different solid tumor cell lines. The sensitivity to either GMX-1778 or STF-118804 was assessed via FACS analysis after a 48 h drug exposure A,B. Withmax > 0.1, Log2 fold change > 1, p value < 0.05). Amongst these, the genes comprising the Broad Institute Hallmark pathways of hypoxia (48/196), glycolysis (36/195), and cholesterol (15/73) were found to be differentially expressed using the Gene Set Enrichment Analysis  from those shown to be insensitive  A,B. A toAnalysis . The redAnalysis C.Not surprisingly, the genes in the neuroendocrine-related Pancreas Beta Hallmark pathway were over-expressed in the sensitive cells. Similarly, we also found that 557 genes annotated as neuroendocrine via the Ingenuity Pathway Analysis (IPA) clearly separated the two groups of cells, which was also shown in the genes encoding proteins expressed in the adrenal medulla as annotated in The Human Protein Atlas .+-depletion, we assessed ATP production and the level of TCA intermediates via LC-MS/MS and Seahorse analyses in the cells with a range of sensitivity to the NAMPT inhibitors, GMX-1778 and STF-118804. Marked reductions in the intracellular ATP and NAD+ levels were observed in the Kelly cells after 48 h of exposure to 2 nM GMX-1778 and 10 nM STF-118804 and in NCI-H82 to 30 nM of either drug , and the Yes-Associated Protein 1 (YAP1) amongst the genes expressed at lower levels in the drug-sensitive group . SLC52A We then turned to examine YAP1, a transcriptional co-activator  that assGiven the reduced expression and previous studies suggesting a role for YAP1 in the inhibition of apoptosis  and drugPrevious work in our laboratory has demonstrated that treatment of the neuroendocrine cancer cells with romidepsin for six hours can reduce the basal OCR by 25–40% and reduce acetyl-CoA levels with subsequent cell death. This reduction in the OCR and acetyl-CoA levels can be rescued via exogenous citrate, acetate, and acetyl-CoA [We also theorized that HDAC inhibition could suppress the YAP expression, as previously reported . TreatmeFinally, when the NH-6, NCI-H82, and Kelly cells were exposed for 6 h to romidepsin and then to the increasing concentrations of GMX-1778 or STF-118804 for 48 h, clear synergy was observed for both the two-drug combinations even with the combination of sub-lethal concentrations of the HDAC and NAMPT inhibitors C.Neuroendocrine neoplasms (NEN) constitute a diverse group of malignancies whose heterogeneity has hindered the identification of effective clinical therapies. Therefore, the discovery of vulnerabilities in NEN and the small molecules targeting them remains a priority. In this study, we have identified two classes of inhibitors, those targeting nicotinamide phosphoribosyltransferase (NAMPT) and histone deacetylases (HDAC), with NAMPT inhibitors as a drug class being particularly active against some neuroendocrine cells. We identify a preferential dependence of the NEN cells on oxidative phosphorylation for energy, and the reduced expression of both SLC52A3, a riboflavin protein transporter, and the Yes-Associated Protein, YAP1, a core component of the Hippo pathway involved in the regulation of metabolism, promoting glycolysis, and glutaminolysis, as identifiable factors that could guide treatment decisions in a precision oncology strategy. Finally, we provide compelling evidence of synergy when both the NAMPT and HDAC inhibitors are used together in what would be a novel therapeutic strategy.The screening of two libraries of compounds using three neuroendocrine tumor derived cell lines identified NAMPT and HDAC inhibitors as two classes of compounds particularly effective in reducing neuroendocrine tumor cell viability and proliferation. In this study, we have investigated the effect of NAMPT and HDAC inhibitors on energy metabolism, ATP production, and ultimately cell death. From the initial screen and its subsequent validation/confirmation, neuroendocrine neoplasms were identified as sensitive to the effect of the NAMPT inhibitors (GMX-1178 and STF-118804). When two representative cell lines, Kelly (neuroblastoma) and NCI-H82  were exposed to GMX-1778 and STF-118804, a decrease in the ATP and NAD+ levels were observed when compared to the levels in the untreated cells. NADH, the reduced form of NAD, drives the generation of ATP via oxidative phosphorylation (OXPHOS). As an essential coenzyme, NAD gains two electrons and a proton from the substrates at multiple steps of the TCA cycle, being consequently reduced to NADH. The TCA catalytic activity analysis in the two sensitive cell lines, together with the gene expression profiling analysis and the ATP production analysis between the NAMPT-sensitive and -insensitive cells, suggest that drug insensitivity in the latter can be attributed at least in part to their ability to leverage both mitochondrial respiration and glycolysis for the energy requirement, while the sensitive cell lines derive their energy primarily from mitochondrial respiration and undergo oxidative stress after NAMPT inhibition. Indeed, the increased expression of genes involved in hypoxia and glycolysis in the NAMPT-insensitive cell lines supports this hypothesis.Riboflavin, known as vitamin B2, is involved in a variety of metabolic pathways and performs key metabolic functions by mediating the transfer of electrons in a biological oxidation–reduction reaction . RiboflaFinally, the epigenetic and metabolic vulnerabilities in the neuroblastoma and small cell lung cancer cell lines serving as representative NENs were evident in the drug–drug combination analysis where the combination of HDAC and NAMPT inhibitors demonstrated synergy even when both agents were combined at low sub-lethal concentrations. HDAC inhibitors, including five approved by regulatory agencies worldwide with excellent toxicity profiles, are both unique in their mechanism of action and not fully explored in rational combinations. Our data demonstrating the synergy in leading to oxidative stress and cellular toxicity at sub-lethal concentrations with comparably low concentrations of NAMPT inhibitors could, in combination, reduce the amount of NAMPT inhibitor needed, improving their tolerability.Gradually, the intersection of metabolism and epigenetics has become manifest in oncology. The state of nutrient availability has been shown to impact the state of differentiation, gene expression, and alterations in the epigenome . Thus, tIn conclusion, our results have identified the simultaneous inhibition of HDACs and NAMPT as a novel approach for the treatment of neuroendocrine neoplasms. The role played by YAP1 in the sensitivity to the NAMPT inhibitors and its reduction via romidepsin aggravates the depletion of NAD+ and the reduction in the ATP production that occurs with the inhibition of NAMPT, leading to enhanced cellular cytotoxicity. Combinations of HDAC and metabolic inhibitors should be evaluated with the aim of identifying new therapeutic options for the treatment of neuroendocrine neoplasms.


The COVID-19 pandemic caused by SARS-CoV-2 has resulted in millions of confirmed cases and deaths worldwide. Understanding the biological mechanisms of SARS-CoV-2 infection is crucial for the development of effective therapies. This study conducts differential expression (DE) analysis, pathway analysis, and differential network (DN) analysis on RNA-seq data of four lung cell lines, NHBE, A549, A549.ACE2, and Calu3, to identify their common and unique biological features in response to SARS-CoV-2 infection. DE analysis shows that cell line A549.ACE2 has the highest number of DE genes, while cell line NHBE has the lowest. Among the DE genes identified for the four cell lines, 12 genes are overlapped, associated with various health conditions. The most significant signaling pathways varied among the four cell lines. Only one pathway, “cytokine-cytokine receptor interaction”, is found to be significant among all four cell lines and is related to inflammation and immune response. The DN analysis reveals considerable variation in the differential connectivity of the most significant pathway shared among the four lung cell lines. These findings help to elucidate the mechanisms of SARS-CoV-2 infection and potential therapeutic targets. As of 30 April 2023, the coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to more than 765 million confirmed cases and nearly 7 million deaths worldwide, reported to the World Health Organization (WHO). It’s reported that SARS-CoV-2 infection is similar to other respiratory infections such as SARS-CoV and MERS-CoV, which caused outbreaks in Southern China in 2003 and Saudi Arabia in 2012, respectively . This siin vitro lung model for assessing the fundamental events that contribute to respiratory diseases. Our research of interest lies in comparing these four distinct lung cell lines: normal human bronchial epithelial cell line (NHBE), A549 lung adenocarcinoma cell line (A549), A549 expressing angiotensin-converting enzyme 2 receptors (A549.ACE2), and Calu-3 lung epithelial cell line . This comparison is driven by the RNA-seq data , providiRecent studies related to COVID-19 disease have shed light on various aspects of the disease, including its pathogenesis, mechanisms of action, and potential therapeutic interventions. Here are some key findings. Although many studies have investigated COVID-19 using RNA-seq data from different cell lines, few have examined the common and unique biological characteristics among NHBE, A549, A549.ACE2, and Calu3 cell lines based on DE analysis, pathway analysis, and differential network (DN) analysis. Additionally, most of these studies have used only one approach for DE analysis, which could lead to biased results. In this study, we conduct a comparative analysis of the four different lung cell lines, namely, NHBE, A549, A549.ACE2, and Calu3. Our approaches involve DE analysis, pathway analysis, and DN analysis to identify both unique and common disease mechanisms among these cell lines. The structure of this paper is as follows. In Our research project falls within the scope of the Disease Maps to Modeling COVID-19 research topic at the Critical Assessment of Massive Data Analysis (CAMDA) Annual Conference 2022.2 transformed counts per gene per million mapped reads (CPM). After removing genes with a median log2(CPM) below −1, only 13111 genes are kept for the subsequent analysis. The right panel in 2(CPM) after removing the lowly expressed genes, which is in contrast to the highly left-skewed histogram before the filtering process, that is, shown in the left panel of The RNA-seq data was obtained from the Gene Expression Omnibus (GEO) under the accession number GSE147507 . To be sThe study is designed to compare the mock-treated group and SARS-CoV-2-infected group in four different lung cell lines  with the aim of identifying singular and common DE genes, influenced pathways, and DN in these cell lines infected with the SARS-CoV-2 virus.For A549.ACE2, the independent biological triplicates are also mock-treated or SARS-CoV-2-infected with or without Ruxolitinib (Rux) pre-treatment. Rux is verified as an effective and safe treatment for COVID-19 patients . In our A549.ACE2 is a modified version of A549 cells that expresses ACE2, the receptor for SARS-CoV-2 viral entry . In our The objective of this study is to identify the singular and common DE genes, influenced pathways, and DN in the four lung cell lines  infected with the SARS-CoV-2 virus. All DE analysis, Pathway analysis, and DN analysis are performed based on the four pairs of comparison between the mock-treated group and SARS-CoV-2-infected group in each of the four cell lines . Below, we outline the statistical methods used for the analysis of DE genes, pathways, and DN. All statistical analyses are conducted using R version 4.0.2 .DESeq2  infected with the SARS-CoV-2 virus. To prevent bias and validate the results of DE genes, we use multiple methods rather than relying on a single approach. Three commonly used R packages for DE analysis are utilized in our analysis, which are DESeq2 , edgeR , which adjusts for differences in library size and composition by scaling the counts for each sample to ensure that the average log-fold change between samples is zero. Finally, limma employs a method called quantile normalization, which adjusts for differences in library size and composition by matching the distribution of expression values across samples. As for statistical modeling techniques employed in this study, DESeq2 utilizes the negative binomial (a.k.a. gamma-poisson) distribution. Similarly, edgeR employs negative binomial-based models, with the addition of an empirical Bayes procedure to shrink the dispersions. Limma, on the other hand, utilizes a linear modeling-based method and incorporates an empirical Bayes approach to borrow information between genes. To adjust p-values for multiple comparisons, the Benjamini–Hochberg correction , the R package RankAggreg  by different DE methods  of a given pathway under a specific condition is measured. Here, perturbation denotes the disturbance that induces changes in the gene-gene association. Human signaling pathways from the Kyoto Encyclopedia of Genes and Genomes (KEGG) database  by comparing networks between different sample groups . In the dnapath  to perfodatabase , which iterature  to invesDESeq2, edgeR, and limma, for identifying DE genes among a total of 13111 genes in the four lung cell lines . The percentage of DE genes for each cell line is presented in the parenthesis in each entry of the table. It is worth noting that these methods exhibit minor differences, except for the limma method, which can detect almost 1.5 times as many DE genes for A549 in comparison with DESeq2 or edgeR. Among the four cell lines, NHBE has the lowest percentage of DE genes, which is approximately 0.2%, whereas A549.ACE2 has the highest percentage of DE genes, around 60%, which is consistent with the fact that ACE2 serves as the receptor for SARS-CoV-2 viral entry  cutoff of 0.05, only three pathways are identified as significant in the NHBE lung cell type cf., , “CytokiThe lung cell type A549 is analyzed to identify the top 10 significant signaling pathways cf., . Among tFor the lung cell type A549.ACE2 cf., , the topAmong the top 10 significant signaling pathways identified for the lung cell type Calu3 cf., , the mosFurthermore, DESeq2, and DN analysis is conducted on two sets of genes: 1) a total of 13,111 genes and 2) DE genes identified for each lung cell line. The DE genes are obtained using p-values of differential connectivity score for each pathway, which could be attributed to the small sample size of these two cell lines, as each group only has three replicates (mock vs. SARS-CoV-2 infected). By examining the significant DC pathways for both sets of genes, we can gain insight into the specific biological processes that are influenced by these genes in each lung cell line. This information can help us better understand the molecular mechanisms underlying the COVID-19 disease. Based on the outcomes of the DN analysis conducted on 13,111 genes, the subsequent analysis is performed.To determine the DC pathways that are significantly affected by these genes, we analyze the DN results for both sets of genes separately. For the entire set of 13,111 genes, we identify the top ten significant DC pathways for each lung cell line and these results are presented in The lung cell line NHBE infected with SARS-CoV-2 demonstrates three important characteristics cr., . First, For the lung cell line A549 cr., , the twoFor the lung cell line A549.ACE2 cr., , similarFor the lung cell line Calu3 cr., , the topThe network plots cr.  are geneUnfortunately, Reactome does not currently have an exact mapping file to KEGG. The purpose of this study is to analyze the RNA-seq data of the four lung cell lines NHBE, A549, A549.ACE2, and Calu3, and identify their common and unique biological features by conducting the DE analysis, pathway analysis, and DN analysis on the data from each cell line.DESeq2, edgeR, and limma. The cell line A549.ACE2 exhibits the highest number of DE genes, while the cell line NHBE has the lowest, which is consistent with the finding in the literature  in the future would enable us to establish connections between our results of pathway analysis and DN analysis. Furthermore, we can also apply similar DE analysis, pathway analysis, and DN analysis to investigate other lung-related diseases using these lung cell lines. Furthermore, we can investigate the distinct and shared mechanisms of the SARS-CoV-2 virus and other comparable viruses, which can provide more insights into the pathogenesis of COVID-19.Our study aims to investigate the unique and shared mechanisms underlying COVID-19 disease in the four distinct lung cell lines NHBE, A549, A549.ACE2, and Calu3 using DE analysis, pathway analysis, and DN analysis. Following the application of the various statistical techniques to the RNA-Seq data, we successfully 1) generate the ordered lists of DE genes for individual cell lines through a rank aggregation method, identify overlapping DE genes across four distinct cell lines, 2) discover significant signaling pathways unique to each cell line, recognized shared significant signaling pathways among all cell lines, 3) unveil significant DN pathways for each cell line and identify common significant DC pathways among them. The outcomes of these analyses, which are previously demonstrated, reveal promising results. We eagerly anticipate the validation of these findings by experts in the fields of biology, medicine, and pharmacology. However, our study has limitations, including a small sample size and the absence of covariates in the analysis. Additionally, the current versions of the R packages


To screen potential tumor antigens for melanoma vaccine development and identify different immune subtypes.http://xena.ucsc.edu/). Subsequently, transcriptome data and clinical information of 210 melanoma cohort GSE65904 were downloaded from Gene Expression Omnibus (GEO), a large global public database. All the transcriptome expression data matrices were log2 transformed for subsequent analysis. GEPIA, TIMER, and IMMPORT databases are also used for analysis. Cell function experiments were performed to validate the role of the IDO1 gene in melanoma cell line A375.Transcriptional data (HTSEQ-FPKM) and clinical information of a 472 Melanoma cohort GDC TCGA Melanoma (SKCM) were downloaded from the UCSC XENA website (Our study provides potential tumor antigens for vaccine development in melanoma patients: GZMB, GBP4, CD79A, APOBEC3F, IDO1, JCHAIN, LAG3, PLA2G2D, XCL2. In addition, we divide melanoma patients into two immune subtypes that have significant differences in tumor immunity and may have different responses to vaccination. In view of the unclear role of IDO1 in melanoma, we selected IDO1 for cell assay validation. Cell function assay showed that IDO1 was significantly overexpressed in the melanoma A375 cell line. After IDO1 knockdown, the activity, invasion, migration and healing ability of A375 cell lines were significantly decreased.Our study could provide a reference for the development of vaccines for melanoma patients. The discovery of vaccines was a milestone in the history of human medicine . It origin vitro and in vivo experiments [Melanoma is a kind of malignant tumor highly related to immunity . Currenteriments . TherefoIn this study, mutation analysis, immune cell infiltration analysis, survival analysis, single-cell analysis, weighted co-expression network analysis, and immune microenvironment analysis were performed by combining melanoma data from multiple tumor databases. Our study may provide some new ideas for the diagnosis and treatment of melanoma.http://xena.ucsc.edu/). Subsequently, transcriptome data and clinical information of 210 melanoma cohort GSE65904 were downloaded from Gene Expression Omnibus (GEO), a large global public database. All the transcriptome expression data matrices were log2 transformed for subsequent analysis.First, transcriptional data (HTSEQ-FPKM) and clinical information of a melanoma cohort of 472 patients called GDC TCGA Melanoma (SKCM) were downloaded from the UCSC XENA website  is an open and comprehensive web tool that be used to explore the tumors data [cBioportal website  between the two groups were evaluated using log-rank test, and p < 0.05 was defined as statistically significant differences.GEPIA2 is a comprehensive analysis website based mainly on TCGA data . In thishttps://cistrome.shinyapps.io/timer/) is a web site can be used to explore immune cells infiltration. In this study, the site’s Gene module was used to explore the association of genes with immune cell infiltration in melanoma by setting the cancer type to SKCM.The TIMER database  for each sample was then calculated using the TMB function.We first reviewed the literature to summarize 47 immune checkpoint genes and 25 immunogenic cell death (ICD) regulatory genes. Then the expression of these genes in different immune subtypes was obtained by differential analysis. Subsequently, 28 marker genes of immune cells were collected through literature review, and the immune cell score of each sample was calculated by R package “ssGSEA”. In order to explore the differences in the distribution of immune cells in different immune subtypes, heat maps and bar charts were used to show the results respectively. We then calculated the immune score, stromal score, total score, and tumor purity score for each sample using the R package “Essclause”, and compared the differences in these scores among different immune subtypes. Finally, we used the R package “ImmuneSubtypeClassifier” to divide melanoma patients into different subtypes (C1–C6) and explore the correspondence between different immune subtypes.We used the reduceDimension function of R package “Monocle” to conduct dimension reduction analysis, set max_components to 2, and use the method “DDRTree” to get different states of cells. Then the plot_cell_trajectory function is used to display the results of different immune subtypes.WGCNA  is an analysis method designed to find co-expressed gene modules by calculating the weight between genes. In this study, by WGCNA analysis of the previously obtained immune genes, the optimal soft threshold value was set as 5, the minimum number of module genes was set as 30, deepSplit was set as 3, and modules with MEDissThres lower than 0.3 were merged, finally, 5 modules were obtained. The differences in the characteristics of these modules in different immune subtypes were then explored.2 with saturated humidity. Transfection of cells with siRNAs . Human immortalized keratinocytes (HaCaT) were purchased from the Cell Resource Center of Peking Union Medical College, China. All cells were cultured in high-glucose DMEM (Gibco), containing 10% fetal bovine serum  and 1% penicillin-streptomycin solution (Gibco) at 37°C with 95% air and 5% COh siRNAs  was carr−ΔΔCt method.The total RNA was extracted from cultured cells with Trizol reagent (Invitrogen) and reverse-transcribed with PrimeScript RT Reagent Kit with gDNA Eraser (TaKaRa). qRT-PCR was implemented utilizing AceQ Universal SYBR qPCR Master Mix  on an ABI Stepone plus PCR system . The sequences of primers were listed in The Cell Counting Kit-8 (CCK8) method was used to assess cell proliferation. The cells were seeded in a 96-well cell culture plate. Subsequently, 24 hours after siRNA transfection, each day of the following 4 days. 10 μL of CCK-8 solution  were added to each well of the plate. After incubating for 2 h at 37°C away from light, Finally, the absorbance of each well was detected with a microplate reader at 450 nm.The transfected A375 cells were kept in 6-well plates for approximately 14 days. Thereafter, the clones were stained with crystal violet and then imaged and counted.Wound healing, transwell migration, and invasion assays were performed as previously reported by us .Experimental data were processed using the SPSS software (version 26.0). All data were illustrated employing mean ± SD. One-way Analysis of Variance (ANOVA) and Turkey’s multiple comparisons of the means were employed in the analysis of multiple sets of data unless otherwise specified.Copy-number analysis of genes in melanoma showed that there were more copy-number variations in melanoma patients, which may be a factor for the poor prognosis of melanoma . Furtherp < 0.05). At the same time. Their upregulation was associated with a better prognosis.Finally, nine antigen genes were screened out of 1489 possible tumor antigens, which were APOBEC3F, CD79A, GBP4, GZMB, IDO1, JCHAIN, LAG3, PLA2G2D, and XCL2 –2i. Theyp < 0.05, In addition, we found that the nine genes were all correlated with macrophage cells, dendritic cells, and B cells –3i. Thenp < 0.001  and the total number of mutations per melanoma patient using the “mafTools” package. No significant difference was found between immune subtypes IS1 and IS2  and 5b. p < 0.05), including common CD274, CTLA4, PDCD1G2, etc. The distribution of immunogenic cell death (ICD) regulation genes was also different in the two immune subtypes  regulation genes between the two immune subtypes. The results indicated that, as shown in subtypes  and 6d, Each patient’s immune cell score was quantified by ssGSEA analysis. To specifically distinguish which express differences between the two subtypes of immune cells are statistically significant, we explored the specific differences between them in immune subtype analysis, as shown in p < 0.001), and the tumor purity of immune subtype IS2 was lower than that of immune subtype IS1 , while there was no statistical difference between the survival analysis of Green and Meblue modules (p < 0.05). The correlation coefficients were 0.81 and 0.17, respectively . Subsequently, the A375 cell line and WM-115 cell lines were transfected with three siRNAs and PCR results showed that all three siRNAs significantly knocked down IDO1 expression, with higher knockdown efficiency in si-IDO1-2 and si-IDO1-3 . So subsequent functional experiments were performed in the SI-IDO1-2 and SI-IDO1-3 groups. CCK-8 showed a significant decrease in cell viability of A375 cell lines and WM-115 cell lines after IDO1 knockdown (p < 0.01). Clone formation experiments showed that the proliferation ability of A375 cells and WM-115 cell lines decreased significantly after IDO1 knockdown (p < 0.01). A scratch experiment showed that migration rate of A375 cell line and WM-115 cell lines decreased significantly after IDO1 knockdown (p < 0.01). Transwell experiments showed that the migration and invasion abilities of A375 cell lines and WM-115 cell lines were significantly reduced after IDO1 knockdown . Considering that our survival analysis shows that IDO1 is highly expressed in human melanoma with a good prognosis, the role of IDO1 in vivo may have a more complex mechanism, such as activation of the immune system. All of our experiments were repeated three times.First, PCR results showed that IDO1 expression was upregulated in melanoma A375 cell lines and WM-115 cell lines compared with normal epidermal HaCaT cell lines  and tumor-associated antigen (TAA) . TSA is Immune reprogramming is widely found in various tumors, especially melanoma, which is highly immune-related . Due to GSE65904 is the dataset used in our study. In the original paper on this dataset, the authors used gene-expression profiling to molecularly stratify metastatic melanoma and found an increased risk of developing distant metastases in the pigmentation and proliferative groups compared with the hyperimmunoreactive group . In our The tumor antigens and immunological subtypes of various malignancies have currently been identified by numerous research. By examining GEO and TCGA data, Huang et al. discovered three tumor antigens in cholangiocarcinoma, namely CD247, FCGR1A, and TRRAP. Additionally, they separated cholangiocarcinoma patients into immune “hot” and immune “cool” kinds, which had noticeably different results . A totalWe identified potential tumor antigens for cutaneous melanoma using a combination of multiple assayed methods. In addition, we divided melanoma patients into two immune subtypes, which have significant differences in prognosis and immunity, which may provide reference for the appropriate population for vaccination.