However, most studies that make use of EOC cell lines fail to recognize the importance of subtype. Furthermore, the similarity of cell lines to their cognate primary tumors is often ignored. Identification of cell lines with high molecular similarity to primary tumors is needed in order to better guide pre-clinical EOC research and to improve development of targeted therapeutics and diagnostics for each distinctive subtype. This study aims to generate a reference dataset of cell lines representative of the major EOC subtypes. We found that non-negative matrix factorization (NMF) optimally clustered fifty-six cell lines into five groups, putatively corresponding to each of the five EOC subtypes. These clusters validated previous histological groupings, while also classifying other previously unannotated cell lines. We analysed the mutational and copy number landscapes of these lines to investigate whether they harboured the characteristic genomic alterations of each subtype. Finally we compared the gene expression profiles of cell lines with 93 primary tumor samples stratified by subtype, to identify lines with the highest molecular similarity to HGSOC, CCOC, ENOC, and MOC. In summary, we examined the molecular features of both EOC cell lines and primary tumors of multiple subtypes. We recommend a reference set of cell lines most suited to represent four different subtypes of EOC for both in silico and in vitro studies. We also identify lines displaying poor overall molecular similarity to EOC tumors, which we argue should be avoided in pre-clinical studies. Ultimately, our work emphasizes the importance of choosing suitable cell line models to maximise clinical relevance of experiments.Epithelial ovarian cancer (EOC) is the most fatal gynaecological malignancy, accounting for over 200,000 deaths worldwide per year. EOC is a highly heterogeneous disease, classified into five major histological subtypes–high-grade serous (HGSOC), clear cell (CCOC), endometrioid (ENOC), mucinous (MOC) and low-grade serous (LGSOC) ovarian carcinomas. Classification of EOCs is clinically beneficial, as the various subtypes respond differently to chemotherapy and have distinct prognoses. Cell lines are often used as  BRCA1/2 mutations and hormone receptor (HR) deficiencies in non-HGSOC/high grade ENOC subtypes limits the use of poly (ADP-ribose) polymerase (PARP) inhibitors in disease management (Ovarian cancer (OC) is the most deadly cancer of gynaecological origin, with over 300,000 new cases and 200,000 deaths occurring in 2020 . In the nagement . Targetenagement  but haveCell lines are often utilized to study cancer biology, as a cheaper and less time-consuming alternative to patient derived explants or animal models. There exists over 100 OC cell lines, with around 70 commercially available through the American Type Culture Collection (ATCC), European Collection of Authenticated Cell Cultures (ECACC), Rikagaku Kenkyusho , Leibniz Institute DSMZ, CellBank Australia (CBA) and/or the Japanese Collection of Research Biosources Cell Bank (JCRB) . Cancer Since cell lines do not possess the morphological traits necessary for histological subtyping, current studies utilize molecular characteristics in order to determine the subtypes of these EOC models. A seminal study by in vitro models to study different subtypes of ovarian cancer. However, there exists no study that compares the molecular profiles of EOC cell lines to primary tumor tissue of multiple EOC subtypes. Here, we integrate and analyse EOC cell line molecular data from multiple different sources, with the aim of generating a reference dataset of lines representative of the major EOC subtypes. We apply non-negative matrix factorisation (NMF) to a panel of 56 EOC cell lines. This resulted in five stable clusters that largely validated results obtained in previous studies, while also suggesting new subtype annotations for five previously unannotated cell lines. Additionally, genetic profiles of EOC cell lines were compared to 93 EOC primary tumor samples, from multiple independent datasets, stratified by subtype. We investigate the molecular similarity of cell lines to not only HGSOC primary tumors, but also CCOC, ENOC, and MOC. Ultimately, we identify the most and least representative cell lines to represent these EOC subtypes, generating a reference dataset for future in silico and in vitro studies. Consequently, our work highlights the need for the generation of additional, subtype specific datasets, in particular for the LGSOC and ENOC subtypes.These studies have made remarkable progress in identifying the most suitable An overview of the methodology followed is displayed in Ovarian cancer cell line RNA sequencing (RNA-seq) data was obtained from three sources: n = 11, TP53 mutational status and treatment for each cell line and is detailed in OC cell lines that were misclassified, engineered or non-epithelial in origin were removed from analysis n = 11, . Two celn = 5), HGSOC (n = 5), ENOC (n = 4) and MOC (n = 5). RNA-seq data for these 19 primary tumors were analyzed in this study. n = 11), HGSOC (n = 8) and ENOC (n = 4). There is no publicly available RNA-seq data for LGSOC primary tumors . In total, transcriptomic data for 16 CCOCs, 64 HGSOCs, 8 ENOCs and 5 MOCs were used.Ovarian primary tumor tissue data was obtained from three independent datasets . Corona Quality control checks were performed on all FASTQ files using the FastQC package . Forwardr) was carried out by running NMF for values of r from 2 to 8, using 50 random initiation points. Quality measures including the cophenetic correlation coefficient, residual sum of squares and dispersion were used to select the most suitable value of r. In the presented data, clustering for r values of 2 and 5 displayed high quality metrics . A variance stabilizing transform was applied to read counts using the DESeq2 package . In orde metrics . After sFor cell lines present in the CCLE study, mutational and copy number landscape was originally determined by The uniformly processed EOC cell line and tumor tissue counts were upper quartile normalized. ComBat  was thenW and H. The basis matrix W delineates the contribution of a small subset of genes to what are termed ‘metagenes’. This is essentially a decomposition of genes into those whose co-expression influences cluster assignment . Seeding is used to initialize the starting point for the NMF algorithm . The NMF package contains a number of built in seeding methods. Here the ‘random’ method is used, whereby these initial values are obtained from a uniform distribution with the same range as the input matrix. For reproducibility, a numerical value is passed into the seed function to seed the random number generator. In order to achieve a stable result from this random seeding method, multiple runs of NMF are required.Non-negative matrix factorization is a method of unsupervised learning often applied to gene expression data to extract biologically relevant information . It funcsignment . The coer), a critical parameter that is selected by the user-ideally, r should be small enough to reduce noise but large enough to preserve biologically relevant information. r for which the cophenetic correlation coefficient starts decreasing. The cophenetic correlation coefficient indicates the robustness of clusters for a given choice of r. r is only relevant if the information captured by factorization is greater than that derived from permuted data, otherwise this increase will lead to overfitting. They suggest using the smallest value of r where the decrease in residual sum of squares (RSS) is lower than the decrease observed in randomized data. By comparing the residual error of NMF from the original data to that of permuted data, a solution is identified that is more information than noise, while preventing overfitting.The number of metagenes and sample clusters are defined by the factorization rank  and ENOC/CCOC/MOC (ARID1A)  have not been investigated in terms of EOC subtype since their creation, with original annotation data unavailable for four of these lines . Additionally, these cell lines are rarely cited in literature and as such, mutational and copy number data was largely unavailable . TYKNUCP(ARID1A) . Ultimatin vitro models. Some progress has been made in identifying suitable cell lines to utilize as HGSOC models. Both As discussed previously, subtype assignment of certain cell lines can prove difficult to resolve using transcriptional data, especially when mutational or copy number data is unavailable. Comparison of cell lines to tumor tissue is required to determine the suitability of these Publicly available transcriptomic data was available for primary tumors of HGSOC, CCOC, ENOC and MOC origins  were within the top 20 most correlated cell lines to HGSOC primary tumors, with Caov4, COV413A, OVCA432, OVCAR3 and OVKATE representing the most highly correlated cell lines . This stBRCA2, MYC amplifications), as well as those that are generally not observed in HGSOC   . This hiin vitro EOC studies should be avoided, and importantly, there is a striking need to develop additional ENOC cell lines. We observe a similar pattern for other lines classified as ENOC. Cell lines classified as HSGOC and CCOC may represent the best available models of this subtype, as they possess the highest median correlations to ENOC primary tumors (None of the six lines classified as ENOC ranked within the top 20 most correlated lines to ENOC tumors . In facty tumors . Indeed y tumors  and CCOCy tumors .All lines classified as MOC (apart from JHOM2B) fell within the top 20 most highly correlated lines to MOC primary tumors . COV644,Most cell lines classified as LGSOC  were poorly correlated to primary tumors of HGSOC, CCOC, ENOC, and MOC origin –5. AdditOverall, we show that correlation of gene expression patterns between EOC cell lines and primary tumors can identify models with high molecular similarity to specific subtypes and aid in subtype identification, with the use of techniques such as NMF. In summary, we generated a reference dataset of cell lines most representative of HGSOC, CCOC, and MOC . We alsoin vitro models, at least for gene expression-based studies. Potential cell line models of LGSOC were also reported. Finally, we highlight the need for generation of additional subtype-specific datasets to help identify suitable models of less studied EOC subtypes, including LGSOC.In this study, we utilized publicly available transcriptomic, mutational and copy number data to investigate the molecular characteristics of 56 EOC cell lines. We demonstrated that cell lines optimally clustered into five stable groups that likely represent the five histological subtypes of EOC. We also compared the gene expression profiles of these cell lines to primary tumors of four EOC subtypes, generally observing that HGSOC, CCOC and MOC cell lines were highly correlated to their respective primary tumors. We noted that ENOC cell lines, especially A2780, were poorly correlated overall to not only ENOC tumors but EOC overall. We identified cell lines that feature the major genomic and transcriptomic features of each particular subtype and are therefore the most suitable in silico investigation that lacks experimental validation. However, we note that our results are in agreement with other studies of this nature , genes involved in homologous repair , or genes found to be associated with an increased risk of developing EOC overall   .in vitro models such as spheroids, multicellular organoid systems and tumor-on-a-chip models (It is possible that the use of two-dimensional, monocultures of individual cell lines may become greatly reduced or outdated in cancer research, due to the rising popularity of three dimensional p models . Cell lip models . In any In conclusion, although major technological progress and healthcare improvements have facilitated more effective cancer treatments, the last 20 years have only seen modest improvements in OC survival rates . Indeed,


To determine whether the width of the shoulder and the size of the bystander effect arecorrelated using clonal lineages derived from a cultured cell line.HCT 116 (p53 wildtype) cells were grown at cloning density and individual viablecolonies were picked off and grown to establish a series of cell lines from bothunirradiated and irradiated progenitors. These cell lines were then irradiated togenerate full survival curves. Highly variant clones were then tested to determine thelevel of the bystander effect using a medium transfer protocol.n is assessed in terms of radiosensitivity. The parent cell line hasan n value of 1.1 while the most variant clones have nvalues of 0.88 (Clone G) and 5.5 (Clone A). Clonal lines subject to irradiation prior toisolation differed in bystander signal strength in comparison to clonal lines which werenot initially irradiated (P = .055).The multi-target model gave the best fit in these experiments and size of the shoulderBased on these experiments we suggest there may be a link between shoulder size of amammalian cell line and the strength of a bystander effect produced in vitro. This mayhave implications for radiotherapy related to out-of-field effects. Recently, the effects of low dose IR have gained attention due both to the increasinguse of IR in medical diagnostics, the use of novel protocols in radiotherapy such as FLASHand MRT, and the interest in small modular reactors as energy sources in remoteenvironments.2 High dose direct IRgenerally leads to significant cell death through processes such as reproductive death orapoptosis35 while non-targeted and low dose radiationappears to involve other mechanisms.7 Radiation-induced bystander effects (RIBE)are of particular interest since they involve cell killing, transformation and initiation ofcell signaling pathways in cells that have not been directly exposed to IR but have receivedsignals from directly exposed cells.814 RIBE have been widely studied both in vivo and in vitro and they appearto be associated with low dose radiosensitivity1518 with somesuggestion that they require wildtype p53 to be expressed. This is relevant since many tumors have compromised p53 function,2022 meaning that additional killing due to RIBE would predominantly affectnormal cells around the tumor rather than the tumor itself. However, the research in thisarea is quite controversial with contradictory reports about RIBE even in laboratories usingthe same protocols and cells. A possible explanation for this is “drift” within cultured cell lines leading toclonal heterogeneity in populations of genetically identical cells. To test whether thismight be a factor, we decided to revisit clonal heterogeneity with respect to clonalsensitivity. Through investigation of clonal populations we aim to approach in a moresystematic way the often heterogenous nature of malignancies.25 The literature often refers to clonalheterogeneity within a tumor as a “fuel for resistance” and studying this key challenge inoptimizing individual therapies is necessary to advance cancer treatment.25 Since radiotherapy can lead to secondmalignancies,2628 some cell lines were derived fromcultures of cells exposed to 1 Gy to determine whether there was greater variability interms of radiosensitivity in these lines. With a better understanding of the heterogeneityof response in clonal sub-populations we may gain a new perspective which could improveradiation treatment.The impacts of ionizing radiation (IR) on human cells are important for radiationprotection, environmental risk assessment, and radiation therapy.2 Falcontissue culture flasks at 37°C and 5% CO2. Subcultures were conducted using0.25% phenol red-free trypsin solution with 0.192 mM EDTA every 6-7 days. Trypsinizedcells were neutralized using a greater volume of growth media. Cell cultures were 70–80%confluent upon culture. Cell concentrations were determined using Bio-Rad TC20 automatedcell counter . All reagents were purchasedfrom Gibco, ThermoFisher Scientific.The immortalized human epithelial HCT116 (p53 wildtype) cell line derived from a largeintestine/colon carcinoma was used in this study. Clonal cell lines were isolated fromthis parent cell line. These cells were routinely cultured in Roswell Park MemorialInstitute (RPMI) 1640 growth medium supplemented with 10% fetal bovine serum (FBS), 100U/mL penicillin, 100 ug/mL streptomycin, and 2.05 mM L-Glutamine. This growth medium wasalso used in the bystander effect assays. Cells were grown in 75 cmPetri dishes were seeded with 200–300 cells. These were allowed to form viable coloniesof at least 50 cells. After 7 days, individual clones of various sizes were chosen forclonal expansion. These colonies were scraped off the dish and resuspended in smallmultiwall plates . The cloneswere passaged into T25 flasks when confluent and grown to produce a sufficient supply ofcells for the experiments. Some clones were isolated from plates where cells had beenexposed to 1 Gy radiation after cells had adhered to the culture plate in order to examinethe effect of this dose on subsequent clonal heterogeneity .Figure 1All irradiations were performed using a Cesium-137 gamma-emitting source with a dose rateof 198.4 mGy/min and flasks were placed 30 cm away from the source . Direct irradiations for survival curve data generation wereconducted 15–20 hours post seeding. Irradiations to generate medium for bystander effectassays were also completed in this manner. Cell seeding densities were determined using the plating efficiencies (PE)determined for each clonal cell line. Clonogenic assays were conducted to develop fullsurvival curves upon irradiation of cells at the following dose points: 0, 0.5, 1.0, 3.0,5.0, 7.0, 10.0, and 15.0 Gy. This wide range in dose points provided an overall assessmentat cell survival across doses. Flasks were irradiated at the appropriate dose and returnedto the incubator immediately following irradiation and grown for nine days at 37°C in anatmosphere of 5% CO2 in air. On day nine, all flasks were stained with 15%Carbol Fuchsin solution . Colonies were counted manuallyto determine the surviving fraction for each dose. The data were entered into GraphPadPrism 8 software , to generate survival curvegraphs.Flasks containing approximately 70% confluent cultures were used for clonogenic survivalassays. Cells were removed from the flask using a Trypsin-EDTA working solution describedabove. Detached cells were neutralized with growth medium and mixed to form a single cellsuspension. These cells were counted and plated to perform a clonogenic assay using themethod described by Puck and Marcus.2) were seeded with cells in 5 mL growthmedia for the following treatments and incubated for 6 hours at 37°C and 5%CO2: plating efficiency, direct irradiation (2.0 Gy), bystander effect donorand recipient, and sham donor and recipient . Least square error linear regression analyses were performed ondata to produce the multi-target and linear-quadratic models using GraphPad Prism 8. Datafor bystander effect assays were also collected as mean of three replicates in threeindependent trials. Standard error of the mean error bars are used in all figures. Todetermine variance between groups in bystander effect experiments, t-tests were conductedbetween the sham and bystander groups and the sham and direct irradiation groups for eachcell line. These post hoc analyses were performed using Welch’s t-test. Significance wasdetermined at the 95% confidence interval.+/+ line alongside all clonal cell lines derived from either anirradiated or non-irradiated population prior to clone isolation. Data were not fitted toany established model for this figure. Clonogenic survival over a dose range of 0–15 Gyafter direct exposure to a cesium-137 gamma source shows variation between each clonalline. Plating efficiency variations were also observed between clonal lines and arepresented in n and D0 areobserved where n is a common indicator for low dose radiosensitivity orthe size of the cell survival curve shoulder. In comparison to the parent cell line, CloneA had the largest n value of 5.5 while Clone G had the smallestn value of 0.88 . There is a trendfor n value to correlate with bystander induced reduction in survivalhowever other additional factors could be involved. Also, while to our knowledge no previousexperiments were set up to examine this relationship, there is anecdotal evidence in theliterature that shoulder size and bystander signal strength are related inversely. The paperby Mothersill et al (2002) examined parent cell lines and radiosensitive lines with variousDNA repair defects derived from these parents. Irrespective of the nature of the repair defect, all radiosensitive lines were moreradiosensitive than their parent line. Also many radioresistant cell lines such as PC3 donot show bystander associated cell death while radiosensitive lines such as SW48 do showstrong bystander effects.When all clonal populations were tested for the presence of bystander signals following thebystander medium transfer assay, it was demonstrated that most clonal populations regardlessof their origin from a non-irradiated or irradiated population, displayed a significantreduction in cell survival following receipt of ICCM . Howeverder size . Unfortuder size  does sugIn certain clonal populations, bystander signals were not produced even though they wereradiosensitive. Consistent with previous findings this could suggest the presence of a lowdose hyperradiosensitivity with increased radioresistance as the dose increased (HRS/IRR)mechanism. In instances of hyperradiosensitivity a generally greater than expected responseto radiation is observed. However, various studies have shown that certain cell lines onlyrespond to bystander signals in the lower dose region where HRS is seen.Apart from the findings in relation to RIBE, the data in this paper suggest that themathematical expressions based on classical target theory predictions do not provide goodfits to these results. This is important to note because many of the classical experimentswere done using a few cell lines such as CHO or V79 cells. These have high platingefficiencies of the order of 80-90% but limited expression of tissue of origincharacteristics. Most modern radiobiology is done using lines which express importantparameters related to epithelial cell or tumor function, but which have plating efficienciesbelow 50%. High plating efficiencies are necessary to derive meaningful target theory basedconclusions. This is because of the statistical probability that radiation is the cause of acell not forming a colony if the PE is high. With a low plating efficiency, the cause of notforming a colony need not be the radiation effect. Nowadays the focus is on moleculareffects so that plating efficiency is not such an issue.In conclusion, the data presented show marked clonal heterogeneity in cell lines derivedfrom both irradiated and unirradiated progenitors. This manifests as differences in doublingtime, plating efficiency and radiosensitivity. The data also reveal a weak correlationbetween shoulder size (a surrogate for low dose radioresistence) and the ability of ICCM toreduce the plating efficiency of unirradiated cells. The data also suggest that the commonlyused mathematical expressions traditionally used to fit survival curve data provide poorfits to the data in this paper, possibly due to the low plating efficiency of the celllines. In conclusion, the results may have implications for tumor radiotherapy where clonalheterogeneity is an important limitation for treatment.


B-cell lymphomas represent the majority of non-Hodgkin lymphomas and are the most common lymphoid malignancies in the Western world. Genetic alterations or epigenetic modulations can lead to tumor initiation and tumor progression. Aside from standard care, targeted, individualized therapies can be highly effective. Here, we evaluated the impact of simultaneous specific inhibition of two key regulators involved in B lymphoid tumor progression. Spleen tyrosine kinase (SYK) is a B-cell receptor-associated kinase acting as a proto-oncogene in B-cell malignancies, while bromodomain and extra-terminal domain (BET) proteins are epigenetic reader proteins involved in histone recognition and transcription regulation. The simultaneous inhibition of SYK and BET showed enhanced anti-proliferative effects, as well as inducing a distinct combination-specific gene expression profile, suggesting SYK and BET inhibition as a promising combination in the treatment of B-cell lymphoma.Background: Both bromodomain and extra-terminal domain (BET) proteins and spleen tyrosine kinase (SYK) represent promising targets in diffuse large B-cell (DLBCL) and Burkitt’s lymphoma (BL). We evaluated the anti-lymphoma activity of the isoform-specific bivalent BET inhibitor AZD5153 (AZD) and the pan-BET inhibitor I-BET151 (I-BET) as single agents and in combination with SYK inhibitor Entospletinib (Ento) in vitro. Methods: The effect of the single agents on cell proliferation and metabolic activity was evaluated in two DLBCL and two BL cell lines. Proliferation, metabolic activity, apoptosis, cell cycle and morphology were further investigated after a combined treatment of AZD or I-BET and Ento. RNAseq profiling of combined AZD+Ento treatment was performed in SU-DHL-4 cells. Results: Both BET inhibitors reduced cell proliferation and metabolic activity in a dose- and time-dependent manner. Combined BET and SYK inhibition enhanced the anti-proliferative effect and induced a G0/G1 cell cycle arrest. SU-DHL-4 demonstrated a pronounced modulation of gene expression by AZD, which was markedly increased by additional SYK inhibition. Functional enrichment analyses identified combination-specific GO terms related to DNA replication and cell division. Genes such as ADGRA2, MYB, TNFRSF11A, S100A10, PLEKHH3, DHRS2 and FOXP1-AS1 were identified as possible key regulators. Conclusion: Simultaneous inhibition of BET and SYK enhanced the anti-proliferative effects, and induced a combination-specific gene expression signature. Targeted therapies by small molecule inhibitors and their combinations have been shown to efficiently affect tumor growth and survival in hematological neoplasias. B-cell receptor-associated kinases (BAKs) like spleen tyrosine kinase (SYK), as well as the bromodomain (BRD)-containing proteins such as the bromodomain and extra-terminal domain (BET) proteins play a critical role in signaling pathway transduction. The specific inhibition of BET and SYK represents a promising tool in hematological neoplasias.BET co-activators represent a group of epigenetic readers involved in histone recognition and transcription regulation ,2. BET pEarly BET inhibitors JQ1 and I-BET762 (GSK525762A) were designed to target the BRD4-NUT (nuclear protein in testis) fusion protein in aggressive NUT midline carcinoma. The specific binding of these inhibitors to BRD4, induces the displacement of BRD4 fusion from chromatin and leads to tumor regression in vivo ,11. FollBased on the promising results of targeting BET proteins in hematological and solid tumors, several BET inhibitors have been developed and are under clinical investigation, although currently without FDA approval .Most prominent BET inhibitors are pan-BET inhibitors, with specificity to more than one representative BET family protein. Representative pan-BET inhibitors are I-BET151 GSK1210151A) , ABBV-07151A , ABHere, we aimed to compare the anti-tumorigenic effect of a simultaneous SYK inhibition by Entospletinib with pan-BET inhibition by I-BET151 (I-BET) or isoform-specific bivalent BET inhibition by AZD5153 (AZD) in a DLBCL and Burkitt’s lymphoma in vitro approach. We investigated the synergistic potential of additional specific SYK inhibition and the resulting specific gene expression modulation patterns in both B-lymphoma subtypes. Inhibitor-based transcriptome modulation was evaluated by RNA sequencing and supported by microarray analyses.We uncovered additional anti-tumorigenic effects in B-lymphoma cell lines induced by simultaneous SYK and BET inhibition, which was investigated by analyzing proliferation, metabolic activity, apoptosis and cell cycle analyses. Cellular processes such as DNA replication and cell cycle were identified as key modulating pathways by high-throughput RNA sequencing.Human Burkitt’s lymphoma (BL) cell lines DG-75 and RAJI and diffuse large B-cell lymphoma (DLBCL) cell lines SU-DHL-4 (GCB) and U-2946 (ABC) were purchased from the German Collection of Microorganisms and Cell Cultures , cultured as recommended by the manufacturer in RPMI 1640 media  supplemented with 10% heat-inactivated FCS  and 100 µg/mL penicillin and streptomycin . Cultured cells were regularly tested for mycoplasma contamination and authenticity (cell surface markers by flow cytometry).AZD5153 (AZD), I-BET 151 (GSK1210151A) (I-BET) and Entospletinib (GS-9973) (Ento) were obtained from Selleck Chemicals  and prepared according to the manufacturer’s instructions to a 10 mM stock and stored at −80 °C until ready for use.5 cells/mL) were exposed to AZD, I-BET, Ento or DMSO (control) as mono substance (0.001 µM–10 µM) or in combination for up to 72 h Cell lines . Viable cells were determined by trypan blue staining and cell count with a hemocy-tometer . Metabolic activity was determined via tetrazolium compound WST-1  according to the manufacturer’s protocol and previously described in .TM  and BD FACS Suite Software .Early and late apoptosis was analyzed by Annexin V FITC  and Propidium Iodide (PI)  staining according to the manufacturer’s protocol and previously described in . MeasureTM, BD Biosciences, Heidelberg, Germany). Cell cycle distribution was examined by FlowJo Software .Protocol was previously described in . Briefly4 cells per slide  in a Cytospin 3 centrifuge . Slides were stained for 6 min with May–Grunwald working solution , rinsed with microscopy buffer according to WEISE (pH 7.2) , stained 20 min in Giemsa working solution  and rinsed thoroughly with microscopy buffer again. The slides were air dried at room temperature before analysis. Morphological changes were analyzed by the EVOS® XL Core Imaging System .Coated cytoslides  were prepared with 5 × 10Cell pellets were resuspended in QIAzol Lysis Reagent  and RNA isolation was performed with miRNeasy Mini Kit  according to the manufacturer’s instructions and previously described in .Poly-A RNA was enriched from 1 µg total RNA using the NEBNext Poly (A) mRNA Magnetic Isolation Module  and sequencing libraries were prepared using the NEBNext Ultra II RNA Library Prep Kit . Sequencing was conducted on an Illumina NextSeq500  as single reads with 75 bp length with a total of 480 million reads.Gene expression analyses were also performed by microarray, using the Human Clariom D Array  according to manufacturer’s instructions and previously described in . In brieaffycoretools with probe mappings drawn from clariomhumantranscriptcluster.db. and a linear model was fitted of the form:ijY is the intensity for gene j in sample I, iTreatment is the treatment which sample i was subjected to, iLine is the cell line from which sample i was drawn, Batchi is the batch which sample i belongs to and ij is the error term, with the coefficients β0, β1 and β2 estimated by least squares. All contrasts were tested using the eBayes method of limma to perform a moderated t-test for each gene and the results were corrected for multiplicity using the Benjamini–Hochberg correction.Microarray data were RMA normalized using limma and probes were mapped to genes using Rsubread [edgeR was used to filter low read counts using the filterByExpr function, normalized using the TMM method, and a differential expression analysis was performed using the GLM method to fit a generalized linear model, specifying no intercept and with treatment group and batch as covariates. All contrasts were tested using the glmQLFTest function of edgeR, which performs an empirical Bayes quasi-likelihood F-test for each gene [p-value of <0.05 and a fold-change > 2.The RNAseq data were processed as follows: The raw reads from different lanes were concatenated and then trimming and quality control were performed using CutAdapt via TrimGalore! . TrimmedRsubread . edgeR wach gene , and thePrincipal component analysis (PCA) of the microarray data  and the RNAseq data  was performed to identify trends in the data. For the PCA plots, the cell batch effect was controlled by first using the removeBatchEffect function of limma.p-value of <0.05 being taken as significant. When considering the importance of GO terms, terms were ranked by Enrichr’s combined score. This is calculated by multiplying the log of the p-value for a given term by the z-score of the deviation of that term from its expected rank, given the size of the gene set. In this way the combined score aims to better account for biases introduced into enrichment analysis by variation in gene set size.Gene ontology (GO) enrichment analysis of biological process terms was performed using Enrichr  with a BTo compare the two platforms, the overlaps between all like gene lists were obtained and can be seen in p < 0.05, ** p < 0.01, *** p < 0.001.Statistical data are presented as mean  ±  standard deviation (SD) from at least three biologically independent samples. Statistical differences between multiple comparisons were analyzed using one-way ANOVA followed by Tukey’s test as post-hoc analysis for normally-distributed data. The Kruskal–Wallis test was applied to non-parametric data. Differences were considered statistically significant for * Synergy was evaluated by a mathematical Bliss independence model . Bliss sBurkitt’s lymphoma (BL) cell lines DG-75 and RAJI and diffuse large B-cell lymphoma (DLBCL) cell lines SU-DHL-4 and U-2946 were exposed to dose ranges of isoform-specific bivalent BET inhibitor AZD5153 (AZD) or pan-BET inhibitor I-BET 151 (I-BET) (0.001–10 µM) for 24 h, 48 h and 72 h . Both inp < 0.05) and 72 h (p < 0.01) Entospletinib (Ento) exposure. Metabolic activity was significantly reduced within 48 h but not after 72 h of substance application .Both combinations  induced a significant reduction of cell proliferation after 72 h exposure compared to the DMSO control in DG-75, SU-DHL-4 and U-2946 a,b. CellHowever, compared to the Ento single agent, the combinations induced an additional anti-proliferative effect in DG-75, RAJI and U-2946. Further, in SU-DHL-4 the combination induced a significant reduction of proliferation and metabolic activity compared to both BET inhibitors as single agents.Furthermore, both combinations reduced metabolic activity significantly in the DLBCL cell line SU-DHL-4 compared to the DMSO control and AZD or I-BET single agents, respectively b. HoweveThe synergistic potential of BET and SYK inhibition in BL and DLBCL cell lines was evaluated by a Bliss independence model  staining at 72 h a,b. The Moreover, we investigated if simultaneous inhibition of BET and SYK is able to boost the effect on cell cycle blockade after 72 h exposure, compared to the single agents.AZD as a mono application significantly increased the percentage of cells in G0/G1 phase in the BL cell line DG-75 and the DLBCL cell line SU-DHL-4 a,b. The Application of I-BET as a single agent did not induce cell cycle changes, while the I-BET+Ento combination significantly intensified the G0/G1 blockade compared to control as well as to the single agents in DG-75, SU-DHL-4.However, the cell cycle of BL cell line RAJI was not significantly affected a. RepresTo evaluate if Ento, AZD, I-BET as single agent or in combination induce apoptosis in BL or DLBCL cell lines after 72 h exposure, Annexin V/PI double staining was performed and analyzed by flow cytometry. During the tested conditions, no significant apoptosis induction could be observed for any tested lymphoma cell lines after 72 h c,d.RNA sequencing was performed in order to evaluate the underlying molecular mechanism of simultaneous BET and SYK inhibition in the DLBCL cell line SU-DHL-4. Principal component analysis (PCA) of exposed SU-DHL-4 cells (after batch correction) showed a distinct clustering of the treatment groups a.Volcano plots of the differential gene expression analysis highlight the significant deregulated genes by each respective treatment. In particular, Ento+AZD combined exposure .Ento and AZD single agent exposures vs. DMSO control . The comFurthermore, comparing Ento+AZD combined exposure to the single agents Ento –j.To validate the RNAseq data, Human Clariom D Array analyses  were carried out for the DLBCL cell line SU-DHL-4 and the BL cell line DG-75  affirminFOXP1-AS1) and solute carrier family 30 member 4 (SLC30A4) highly overexpressed by both procedures.Within the Ento+AZD combination, we found the genes of the long non-coding RNA (lncRNA) FOXP1 antisense RNA 1, E2F transcription factor 8 (E2F8) and chromatin assembly factor 1 subunit B (CHAF1B) were identified as highly downregulated by both tools and in both cell lines.Furthermore, MYB proto-oncogene, transcription factor AICDA) and BTB domain and CNC homolog 2 (BACH2) genes as significantly overexpressed, affirmed by both the RNAseq and microarray analyses. In DG-75, AICDA was also significantly upregulated by Ento single agent exposure. However, SPARC-like 1 (SPARCL1) was identified as significantly downregulated in SU-DHL-4.Ento single agent exposure revealed activation-induced cytidine deaminase (COBLL1), Ras-association domain family member 6 (RASSF6) and Rhotekin 2 (RTKN2) were identified as significantly downregulated, while COBLL1 and RTKN2 were also found to be significantly downregulated in DG-75.AZD single agent only showed overlaps in downregulated genes. Here, Cordon-Bleu WH2 repeat protein-like 1  were unique to this treatment.Gene ontology (GO) enrichment analyses by Enrichr identified several GO terms for the respective condition in the DLBCL cell line SU-DHL-4.The combination of Ento and AZD induced a significant downregulation of several genes, allocated to GO terms, which are mostly related to DNA replication and cell cycle processes . Howeverp < 0.05 level. The Ento+AZD combination revealed no enriched terms for the overexpressed differentially expressed genes but a total of 120 enriched terms for the underexpressed genes , centromer (CEN), cell division cycle (CDC), kinesin family (KIF), GINS complex subunit (GINS), having more than one gene allocated to the top 10 GO terms. Furthermore, GO term intersections between the conditions are summarized in A further approach was to identify which of the most strongly downregulated DEGs were allocated to the top 10 significant GO terms . This anFurthermore, GO terms were condensed using REVIGO and subsequently visualized by CirGO identifying GO clusters. Three big clusters were identified revealing DNA replication, kinetochore organization and cell cycle as the most affected biological processes . Most GOEpigenetic changes and deregulated BCR signaling by molecular lesions are able to promote B-lymphomagenesis ,38,39. THere, we comparatively evaluated the anti-tumorigenic effect of isoform-specific bivalent BET inhibitor AZD5153 and pan-BET inhibitor I-BET151 in a DLBCL and Burkitt’s lymphoma in vitro approach and further investigated the cell- and molecular effect of simultaneous SYK inhibition by Entospletinib.Both, AZD5153 and I-BET151 as single agents efficiently reduced proliferation and metabolic activity at low dosage in all tested BL and DLBCL cell lines, with slightly lower IC50 values for AZD compared to I-BET. Compared to Rhyasen et al., our evaluated IC50 values were slightly higher for AZD . FurtherFollowing on from this, the simultaneous exposure of BET inhibitor AZD or I-BET with SYK inhibitor Entospletinib to the BL and DLBCL cell lines further affects proliferation. A pronounced effect was observed in DLBCL cell line SU-DHL-4 by Ento addition, revealing additionally reduced proliferation and metabolic activity. However, the corresponding Bliss values of both combinations showed no synergistic effect, due to the good response to Ento single agent exposure by SU-DHL-4 cells. Ento addition in all other tested cell lines improved anti-proliferative activity moderately, while respective positive Bliss values indicate slight synergy due to the low response to Ento single agent exposure. In a recent study by Kim et al., it was shown that BET inhibition by GS-5829 in combination with Ibrutinib (BTK inhibitor) or Entospletinib synergistically increased anti-leukemia activity and induced apoptosis in primary co-cultured CLL cells . CombinaMoreover, we identified a significant G0/G1 cell cycle arrest induced by both combinations in BL cell lines DG-75 and DLBCL cell line SU-DHL-4 compared to control. In DLBCL U-2946 only Ento+AZD induced a significant cell cycle arrest. However, the comparison of both combinations to the single agents only slightly increased the cell cycle blockade.Similar findings were observed for BET inhibition as a single agent with BRD4 inhibitor JQ1, inducing a G1 cell cycle arrest in DLBCL cell lines . JQ1 wasIn order to clarify the underlying molecular mechanism, we further investigated the gene expression modulation and functional enrichment of single agents Ento and AZD and the specific gene set modulation by the Ento+AZD combination.By RNAseq analyses we identified that both single agents induced gene expression changes, while the combination was capable of highly increasing the gene expression modulation compared to control, as well as to both single agents. The fold change range of the combination likewise increased compared to the single agents. Rhyasen et al. investigated the effect of AZD5153 in xenograft leukemia and lymphoma models and also performed RNAseq on a hematologic cell line panel revealing 174 differentially expressed genes (DEGs) across this panel . CompareADGRA2) as the most significantly underexpressed gene in SU-DHL-4 cells. ADGRA2 or TEM5/GPR124  was identified as an orphan G protein-coupled receptor in endothelial cells with roles in brain angiogenesis and Wnt signaling [ADGRA2 had a worse overall survival [ADGRA2 in glioblastoma significantly decreased the proliferation of the cancer cells by disrupting the mitotic progression and chromosome segregation [ADGRA2 and its role in lymphoma is unknown. However, the DLBCL cell line SU-DHL-4 exhibits an ADGRA2 missense mutation inducing a protein change p.A765T [ADGRA2 expression remains to be clarified.Within the Ento+AZD combination, we identified the adhesion G protein-coupled receptor A2 . This receptor regulates various biological processes such as apoptosis, cell survival and differentiation through the activation of several signal transduction pathways such as nuclear factor κB (NF-κB), Jun N-terminal kinase (JNK), p38, extracellular signal-related kinase (ERK) and phosphoinositide 3-kinase (PI3K) [TNFRSF11A (RANK) expression has been linked to several cancers. Its overexpression is correlated to breast cancer and prostate cancer with high metastatic potential and to gliomas [TNFRSF11A induces cancer cell proliferation, migration and invasiveness in cervical cancer [KLF5 was not significantly affected by any of the treatments. Finally, TNFRSF11A induces survival and proliferation pathways in B cells in vitro with progression to B-cell malignancies [TNFRSF11A could be one reason for the effective anti-proliferative effect by the single agents and the intensification by Ento+AZD combination in SU-DHL-4 cells.A further significant downregulation has been observed in the gene TNF receptor superfamily member 11a (e (PI3K) ,52. Aber gliomas ,54,55. I gliomas ,57,58. Tgnancies . The sigS100A10). S100A10 is involved in various cellular processes, regulates plasminogen activation [S100A10 expression is correlated with tumor development, invasion, poor prognosis and further can act as a biomarker due to its expression in various tumor cells [The Ento+AZD combination was exclusively able to significantly downregulate the S100 calcium binding protein A10 (tivation  and has tivation ). Furtheor cells ,62,63,64MYB  as highly underexpressed. The transcription regulator is known to be aberrantly expressed or rearranged in leukemias, lymphomas and also solid tumors [MYB [MYB downregulation inhibits cell proliferation and invasion in solid tumors [The combined exposure additionally revealed d tumors ,66. SU-Dors [MYB , which wd tumors ,69.PLEKHH3) were also identified as significantly downregulated. The function of PLEKHH3 is not yet understood, while pleckstrin homology (PH) domains are known to be part of several proteins, e.g., cytoskeletal proteins, function in signal transduction, mediating protein–protein interactions and membrane association [PLEKHH3 was identified in blood immune cells associated with obstructive sleep apnea [PLEKHH3 in cancer has not been described until now.Pleckstrin homology, MyTH4 and FERM domain containing H3  was highly and significantly upregulated. High DHRS2 expression was observed in BET inhibitor resistant liver cancer cell lines. The specific BET inhibition in liver cancer cells induced an increase of DHRS2 expression in sensitive (Hep3B) and resistant cells (HepG2). This could be assumed to be related to drug escape mechanisms [DHRS2 was further related to invasion, lymph node metastasis and is associated with worse prognosis in esophageal squamous cell carcinoma. The overexpression of DHRS2 leads to decreased tumor proliferation and tumor volume both in vitro and in vivo, while the DHRS2 knockdown increases it [DHRS2 levels, which was linked to HDACi sensitivity [DHRS2 needs to be clarified in DLBCL.However, within the Ento+AZD combination we identified that the gene dehydrogenase/reductase 2  was significantly upregulated after combined Ento+AZD exposure in SU-DHL-4 cells. Currently, detailed knowledge about the actual function of COLCA2 is limited. However, it is assumed to have tumor suppressor function and low expression has been correlated to colorectal cancer [Furthermore, the colorectal cancer-associated 2 gene  enrichment analyses identified the main biological processes which were affected after combined or single agent exposure in DLBCL cell line SU-DHL-4. For the Ento+AZD combination, GO term enrichment analysis identified combination-specific GO terms compared to the single agents. Furthermore, only the underexpressed results of Ento+AZD combined exposure revealed several highly significantly modified GO terms. All other conditions showed no significance, but sometimes a high combined score.CDK2, CDCA5, CDC7, WEE1, E2F8, CCNA2, CDC6, CDC25A and CDC25C.The identified downregulation of general biological processes is related to DNA replication and cell division processes which may be caused by cell death induction due to combined BET and SYK inhibition. This assumption can be affirmed by the observed significant downregulation of cell cycle-related genes such as MCM), centromer (CEN), cell division cycle (CDC), kinesin family (KIF), GINS complex subunit (GINS). Several top GO terms contain significantly modulated genes related to the minichromosome maintenance complex (MCM). MCM proteins are DNA helicases regulating and initiating DNA replication processes by unwinding duplex DNA, thus playing a pivotal role in cell division [MCMs have been shown to be involved in tumorigenesis. MCM3 for example, has been identified being overexpressed in several cancers including leukemia and lymphoma [MCM2 expression was also suggested as a negative prognostic marker in DLBCL [MCM5 by JQ1 BET inhibitor was already identified in thyroid cancer cells [MCM genes in our study. Further, the cell division cycle gene CDC7 is highly expressed in a variety of tumors, suggesting a role as a biomarker [CDC7 and MCM2 have been also suggested as prognostic markers in DLBCL [Furthermore, the relation of the top 10 GO terms  and significant DEGs identified various gene sets with crucial roles in DNA replication processes such as mini chromosome maintenance . CDC7 ain DLBCL .GINS1 and GINS2 are likewise involved in DNA replication initiation and are upregulated in a variety of tumors, indicating potential as a prognostic marker [c marker ,91. CDC4c marker ,93. The c marker ). The siSeveral of the identified genes related to the top 10 GO terms are involved in DNA replication processes. We further identified their functions in tumorigenesis or high expression in cancers, suggesting biomarker and prognostic marker potential.Furthermore, GO terms were condensed using REVIGO, summarizing the GO terms based on semantic similarities and they were then visualized using CirGO, which highlighted changes in GO clusters. In particular, the arising GO clusters enriched in the underexpressed DEGs revealed that more than 50% of identified GO terms are related to DNA replication and cell cycle processes, as with the top GO terms when simply sorted by combined score. The emerging GO clusters match with the biological data of the cell cycle blockade as well as the mode of action of BET inhibition by AZD and its interference in histone recognition.The intervention in the biological processes is suggested to be mainly caused by the BET inhibition process, due to its major effects on biological and gene expression levels. However, the Ento addition leads to the specific gene and pathway deregulation caused by the combined exposure.The combination-specific emerging GO terms as well as gene modulation are, for the most part, exclusive and therefore they provide an indication for a combination-specific gene set modulation by Entospletinib and AZD5153 combined exposure. Both the enhanced biological response as well as the strongly enhanced gene modulation by the combination suggest an advantageous intervention for DLBCL subtypes, which requires further investigation.Here, we identified a moderate synergy of simultaneous BET and SYK inhibition in a BL and DLBCL in vitro approach affecting cell cycle modulation but not apoptosis. Expression analysis characterized a distinct gene expression modulation, while the combination strongly increased this modulation. The combination-specific emerging DEGs and GO terms are mostly exclusive and therefore provide an indication for a combination-specific gene set modulation. Although the changes in the main processes are mainly provoked by AZD exposure, Ento addition leads to specifically modified gene and pathway deregulation. The main affected biological processes can be related to DNA replication and cell division.ADGRA2, MYB, TNFRSF11A, S100A10, PLEKHH3, DHRS2 and FOXP1-AS1 presumably play a pivotal role of the Ento+AZD acting mode in SU-DHL-4 cells.The significantly differentially expressed genes Future work remains to fully understand the underlying mechanism of the Ento+AZD combination. Prospectively, it remains to clarify which modulated genes are key regulators by including functional analyses and extending the work to other B-lymphoma cell lines. Proteomic analyses as well as STRING analyses could further validate the affected biological processes investigated by RNAseq. A full understanding of the Ento+AZD combination-induced effects will also need to involve 3D and in vivo models. This is especially important for a better transferability and investigation of clinical value.


The presence of all these data in a review allows in one look to advance the research on CT using cell cultures by facilitating the selection of the most appropriate methods, conditions, and cell lines for future toxicological, pharmacological, and physiological studies.Cyanotoxins (CTs) are a large and diverse group of toxins produced by the peculiar photosynthetic prokaryotes of the domain Cyanoprokaryota. Toxin-producing aquatic cyanoprokaryotes can develop in mass, causing “water blooms” or “cyanoblooms,” which may lead to environmental disaster—water poisoning, extinction of aquatic life, and even to human death. CT studies on single cells and cells in culture are an important stage of toxicological studies with increasing impact for their further use for scientific and clinical purposes, and for policies of environmental protection. The higher cost of animal use and continuous resistance to the use of animals for scientific and toxicological studies lead to a progressive increase of cell lines use. This review aims to present (1) the important results of the effects of CT on human and animal cell lines, (2) the methods and concentrations used to obtain these results, (3) the studied cell lines and their tissues of origin, and (4) the intracellular targets of CT. CTs reviewed are presented in alphabetical order as follows: aeruginosins, anatoxins, BMAA ( Cyanotoxins (CTs) are a large and diverse group of toxins produced by the peculiar photosynthetic prokaryotes of the domain Eubacteria, commonly known as cyanobacteria or blue-green algae, and since 1999 named Cyanoprokaryota . Some aqDifferent approaches have been applied to classify CT, two of which are the most common: by the target of their action, or by chemical composition. By target, CT are classified as hepatotoxins, neurotoxins, dermatoxins, and cytotoxins, whereas chemically they are divided in peptides, alkaloids, phosphorylated cyclic N-hydroxyguanine, diaminoacids, and lipopolysaccharides, the last widely recognized as endotoxins. Prolonged use of drinking water, contaminated with low-doses CTs, may have also carcinogenic effect . Thus, mCell cultures are very convenient for toxicological studies. They allow to reveal the mechanisms of cytotoxic effects, the affected tissues, intracellular targets, and ways to minimize cytotoxicity . The useAlthough different methods are developed for testing of toxins in cell- and animal-based studies, during the last years, the trials on the use of animals have significantly decreased. This is caused by the high cost of these types of clinical trials and increasing resistance to the use of animals for scientific studies. Therefore, the significance and use of cell lines is gradually increasing.This review aims to present (1) the important results of the effects of CT on human and animal cell lines; (2) the methods and concentrations used to obtain these results, (3) the studied cell lines, and (4) the intracellular targets of CT. The presence of all these data in a review allows in one look to advance the toxicological and pharmacological studies of CT using cell cultures by facilitating the selection of the most appropriate methods, conditions, and cell lines.Aeruginosin CT contains as a basic structure 2-carboxy-6-hydroxyoctahydroindol that are serine protease inhibitors . They inAnatoxins-a are two types of low molecular bicyclic amino alkaloids: anatoxin-a (ANTX) and homoanatoxin-a (hANTX). The best known of them is ANTX, which was the first to be identified as a low molecular alkaloid (165 Da). hANTX is a homologue of anatoxin-a with molecular weight 179 Da and has propionyl instead of an acetyl group at C-2. ANTX and anatoxin-a (S) (ANTX(S)) are neurotoxins. ANTX binds competitively to acetylcholine receptors, while anatoxin-a (S) inhibits acetylcholine esterase [β-N-methylamino-L-alanine (BMAA) is an environmental nonprotein and toxic amino acid that may harm nervous system via oxidative stress, binding to neuromelanin, forming high toxic metabolites like formaldehyde or inhibiting enzyme activity of glutathione reductase, β-amilase, catalase, and RNase H, and in this way to provoke sporadic neurodegenerative development, such as Alzheimer's disease and amyotrophic lateral sclerosis [clerosis , 27, 28.clerosis  and refeCYN is a cyclic quinidine alkaloid combined with hydroxymethyl uracil . It has D (AT D), coibamide A (CoA), ichthyopeptins A (Ich A) and B (Ich B), kahalalide F (KF), 4-Fluoro-3-methyl-benzylamino-KF (KF2), morpholin-4-yl-benzylamino-KF (KF4), homodolastatin 16 (HD16), lagunamide C–Lag C, pitipeptolides–Pit A-F, aurilides and wewakpeptins A-D. Depsipeptides show cytotoxic activity and are protease inhibitors selective for chymotrypsin, leukocyte, and pancreatic elastases. They negatively influence the metabolism of human astrocytes [Depsipeptides are palmyramide A , apratoxin trocytes .LPS consist of lipid A, the core polysaccharides (mainly glucosamine) and an outer polysaccharide chain, and are common compounds of the cell walls of cyanoprokaryotes and Gram-negative bacteria . They haLyngbyatoxins were first identified from Moorea producens (formerly Lyngbya majuscula). They are tumor-promoting agents which bound eucaryotic protein kinase C (PKC) isozymes .β-amino acid 3-amino-9-methoxy-2,6,8-trimethyldeca-4,6-dienoic acid (ADDA). MC are lipophilic toxins very resistant to hydrolysis, oxidation, and high temperatures. The main route of human exposure is the ingestion of contaminated drinking water, consumption of contaminated food or algal dietary supplements, and body contact, while more occasional routes are hemodialysis and inhalation. MC are classified mainly as hepatotoxins because they block eucaryotic PP  [MC are cyclic nonribosomal heptapeptides with low molecular weight (800–1100 Da), which contain several uncommon nonproteinogenic amino acids such as N-methyldehydroalanin (MDHA) derivatives and the uncommon 4, PPP5) . Chronicßβ-amino acid -3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid (ADDA). Ten variants have been discovered with nodularin-R being the predominant toxin variant. NODs are relatively stable compounds, with low sensitivity to light or temperature. NOD affects hepatocytes binding their PPs by noncovalent bonds, which increases the rate of phosphorylation. They are often attributed to gastroenteritis, allergic irritation reactions, and liver diseases. Nodularin-R is the most notorious as a potent hepatotoxin that may cause serious damage to the liver of humans and other animals. NODs have similar effects as microcystins and weak carcinogenicity [Nodularins (NODs) are cyclic nonribosomal pentapeptides and contain several unusual nonproteinogenic amino acids such as N-methyl-didehydroaminobutyric acid and the Retinol, a novel retinoic acid (RA) analogue 7-hydroxy RA, 4-oxo-RA, and several analogues were identified in cyanobacterial blooms . They acSaxitoxin (SXT) is a collective name for a group of more than 20 cyclic nonribosomal peptide molecules, formed by sulphation at different sites of two basic molecules: SXT and neo-SXT. Based on their toxicology, SXT are grouped in three classes—carbamate derivatives, gonyautoxins, N-sulfocarbomoyl derivatives, and decarbomoyl derivatives—decarbamoylsaxitoxin. They have a neurotoxic effect by blocking voltage-gated sodium channels .50 or ID50 are different for cells in culture and human body.Studies on cell cultures cannot reveal all possible effects of toxins on the human body. This is due to the following reasons: (1) no matter how many cultures are tested, they will not cover the whole variety of cells in the body; (2) there are often significant differences between the cells in culture, the primary cell lines and the cells in the body tissues in the quantity and quality of expressed proteins (genes expression), metabolic pathways and cell function –115. Thein vitro formed organ-like structures such as artificial neuronal network [The use of cell cultures in toxicological studies will remain the main approach due to its speed, relatively low cost, reproducibility, precision with respect to the studied intracellular components, and ethical acceptability. The use of cell cocultures –118 and  network , cardiom network , and org network , which aThe presence of all these data on the cytotoxicity of aeruginosins, anatoxins, cylindrospermopsin, depsipeptides, lipopolysaccharides, lyngbyatoxins, microcystins, nodularins, cyanobacterial retinoids, and saxitoxins in a review is a great advantage. It allows the advancement of research on CT using cell cultures by facilitating the selection of the most appropriate methods, conditions, and cell lines for toxicological and pharmacological studies. In addition, it could increase the use of CT in functional studies of their intracellular targets. Therefore, this review allows in one look to advance the toxicological, physiological, and pharmacological studies of CT by the knowledge of their harmful effects with a focus on human and animal health as well as on environmental protection.


T-cell redirecting bispecific antibodies hold high promise for treatment of B-cell malignancies. B-cell maturation antigen (BCMA) exhibits high expression on normal and malignant mature B cells including plasma cells, which can be enhanced by inhibition of γ-secretase. BCMA is considered a validated target in multiple myeloma but whether mature B-cell lymphomas can be targeted by the BCMAxCD3 T-cell redirector teclistamab is currently unknown. BCMA expression on B-cell non–Hodgkin lymphoma and primary chronic lymphocytic leukemia (CLL) cells was assessed by flow cytometry and/or IHC. To assess teclistamab efficacy, cells were treated with teclistamab in presence of effector cells with/without γ-secretase inhibition. BCMA could be detected on all tested mature B-cell malignancy cell lines, while expression levels varied per tumor type. γ-secretase inhibition universally increased BCMA surface expression. These data were corroborated in primary samples from patients with Waldenstrom's macroglobulinemia, CLL, and diffuse large B-cell lymphoma. Functional studies with the B-cell lymphoma cell lines revealed teclistamab-mediated T-cell activation, proliferation, and cytotoxicity. This was independent of the level of BCMA expression, but generally lower in mature B-cell malignancies compared with multiple myeloma. Despite low BCMA levels, healthy donor T cells and CLL-derived T cells induced lysis of (autologous) CLL cells upon addition of teclistamab. These data show that BCMA is expressed on various B-cell malignancies and that lymphoma cell lines and primary CLL can be targeted using teclistamab. Further studies to understand the determinants of response to teclistamab are required to identify which other diseases might be suitable for teclistamab targeting.Besides reported BCMA expression on multiple myeloma, we demonstrate BCMA can be detected and enhanced using γ-secretase inhibition on cell lines and primary material of various B-cell malignancies. Furthermore, using CLL we demonstrate that low BCMA-expressing tumors can be targeted efficiently using the BCMAxCD3 DuoBody teclistamab. Treatment of B-cell lymphoproliferative disorders such as B-cell non–Hodgkin lymphoma (B-NHL) and chronic lymphocytic leukemia (CLL) has improved significantly over the last years. This is mostly due to a rapidly expanding treatment armamentarium of targeted agents such as inhibitors of pivotal B-cell receptor pathway kinases BTK and PI3K, and the Bcl-2 inhibitor venetoclax . NeverthFrom stem cell transplantations (SCT), it has become clear that long-lived T cell–mediated anticancer responses are feasible . HoweverFor hematologic malignancies, the most prominent TAA has been CD19. The first BsAb to be approved by the FDA for relapsed/refractory acute lymphoblastic leukemia was the CD19-targeting bispecific T-cell engager (BiTE) blinatumomab . The shoIn line with the expression of BCMA on PBs and PCs, BCMA is highly expressed on multiple myeloma cells, and is considered a validated target, based on approval of the BCMA-targeted therapies belantamab mafodotin and idecabtagene vicleucel, for treatment of advanced multiple myeloma . ComparaTo assess whether BCMA can be used as a target in B-NHL and CLL, we studied whether BCMAxCD3 BsAb  could mediate T-cell activation as well as killing of the lymphoma cell lines. This BsAb has been developed with Genmab DuoBody technology, resulting in enhanced stability compared with conventional BsAb formats. As a proof of concept that BCMA can evoke an autologous T-cell response, CLL was used as a target.+CD19+ of at least 85%. Paraffin‐embedded bone marrow (BM) and lymph node (LN) tissue  was obtained from the pathology department of the Amsterdam University Medical Centers, location AMC. BM mononuclear cells  were isolated from peripheral blood of patients with CLL  or buffy BM-MNC;  from BM  BM-MNC; .Written Full BCMAxCD3 BsAb (teclistamab) and controls BCMAxnull (BC3B4) and nullxCD3 (CNTO7008) were provided by Janssen Pharmaceuticals.Mycoplasma by PCR. Cell lines were authenticated by short tandem repeat analysis and were thawed at least 1 week prior to starting experiments. Cell lines were acquired in the following years: RPMI-8226 in 2015, MWCL-1 in 2018, BCWM.1 in 2018, Mec-1 in 2005, Ramos in 1999, OCI-Ly7 in 2016, Jurkat in 2002, U266 in 2015, CII in 2012, PGA-1 in 2012, Daudi in 2018, OCI-Ly3 in 2016, and JeKo-1 in 2013.Primary CLL cells, RPMI-8226, MWCL-1, BCWM.1, Mec-1, Ramos, OCI-Ly7, and Jurkat cells were cultured in Iscove's Modified Dulbecco's Medium . HD-derived PBMCs, U266, CII, PGA-1, Daudi, OCI-Ly3, and JeKo-1 cells were cultured in RPMI1640 medium (Thermo Fisher Scientific). Medium was supplemented with 10% FCS and 1% penicillin/streptomycin. MWCL-1 and BCWM.1 cell lines were kindly provided to M. Spaargaren by Dr. S.P. Treon, OCI-LY3 by Dr. R. Kuppers and OCI-LY7 by Dr. U. Klein, RPMI-8226 was obtained from ATCC and all other cell lines from DSMZ. Cell lines were tested every 2 months for PBMCs were washed with PBA  and stained using fluorescently labeled antibodies for 20 minutes on ice. The following antibodies were used: BCMA APC, BCMA PE (BioLegend), IgG2a kappa isotype PE (BD Biosciences), CD3 V500 (BD Biosciences), CD4 BV605 (BD Biosciences), CD4 PerCPefl710 (eBioscience), CD5 PE (eBioscience), CD5 PerCPCy5.5 (BioLegend), CD8 BV510 (BioLegend), CD8 PECy7 (eBioscience), CD19 APC (BD Biosciences), CD19 FITC (BD Biosciences), CD20 FITC (BD Biosciences), CD25 APC (BD Biosciences), CD25 BV786 (BD Biosciences), CD27 PerCPefl710 (eBioscience), CD38 PE (BD Biosciences), CD38 BV421 (Sony), CD45RA BV650 (BioLegend), CD107a PECy7 (BD Biosciences), CD138 FITC (Molecular Probes), CCR7 BUV395 (BD Biosciences), IgD PE-CF594 (BD Biosciences), IFNγ BV421 (BD Biosciences), IL2 PE-Dazzle594 (BioLegend), TNFα AF700 (BD Biosciences). To exclude dead cells, Fixable Viability Dye eFluor 780 was used according to manufacturer's instructions. For staining of intracellular cytokines, cells were fixed and permeabilized using the Fixation/Permeabilization Solution Kit (BD Biosciences). After antibody staining, samples were washed using PBA and acquired on BD FACS Canto or LSR Fortessa flow cytometer and analyzed with FlowJo v10. To quantify number of cells using flow cytometry, 123count eBeads Counting Beads (Thermo Fisher Scientific) according to manufacturer's instructions. BCMA molecules per cell were determined by usage of PE Fluorescence Quantitation Kit (BD Biosciences).Cell lines or CLL cells were cultured either in medium or in presence of 100 nmol/L γ-secretase inhibitor . After 24 or 48 hours, BCMA was detected by flow cytometry as described above. Relative expression was calculated compared with isotype controls. For qPCR, total RNA was isolated using the RNeasy mini kit (Qiagen) and cDNA was transcribed by RevertAid (Fermentas), using random hexamer primers (Promega). qPCRs were performed using SYBR Green master mix (Applied Biosystems) and measured on a Quantstudio 3 (Applied Biosystems). Expression of BCMA was normalized to GAPDH. Linear regression software was used for analysis.+ and CD8+ T cells were isolated using MACS beads (Miltenyi Biotec), according to manufacturer's instructions. Cocultures were incubated in the presence of 100 ng/mL BCMAxCD3, BCMAxnull, or nullxCD3. A total of 100 nmol/L γ-secretase inhibitor (Ly411575) was added where indicated. Viability of the target cells was assessed using TO-PRO-3 (Invitrogen) and MitoTracker Orange (Invitrogen) using flow cytometry. Specific lysis of target cells was calculated as (% target cell death in treated sample − % cell death target cells in medium control)/(100 − % cell death target cells in medium control) *100%. Samples were excluded when cell death in medium controls exceeded 50%.Cell lines or primary CLL samples were labeled with Cell Trace Violet  or carboxyfluorescein diacetate succinimidyl ester (Thermo Fisher Scientific) according to manufacturer's instructions and cocultured with HD PBMCs or CLL-derived (autologous) T cells in different effector-target (E:T) ratios. Where indicated prior to co-culture CD4+ multiple myeloma cells in the BM-MNCs was determined by flow cytometry, as described previously (+ cells in the presence of Flow-Count Fluorospheres (Beckman Coulter) and LIVE/DEAD Fixable Dead Cell Stain Near-IR fluorescent reactive dye (Invitrogen). The percentage of lysis induced by teclistamab was calculated using the following formula: % lysis multiple myeloma cells = 1 − (absolute number of surviving CD138+ cells in treated wells/absolute number of surviving CD138+ cells in untreated wells) × 100%.BM-MNCs derived from 6 patients with multiple myeloma, containing 2%–65% tumor cells, as well as autologous effector cells and immune suppressive cells, were used in flow cytometry–based lysis assays. Sample viability at start of the assays, assessed using 7-AAD (BD Biosciences), was more than 95%. BM-MNCs were incubated in RPMI1640 + 10% FBS (Integro) with teclistamab (0.16 μg/mL) in 96-well U-bottom plates for 48 hours. The survival of primary CD138eviously . BrieflyPBMCs from HD patients were labeled with CTV and cultured alone or in 1:1 E:T ratio with RPMI-8226, JeKo-1, CII or BCWM1. PBMCs were incubated in the presence of 100 ng/mL BCMAxCD3, BCMAxnull, or nullxCD3 or stimulated with CD3 (clone 1XE) and CD28 (clone 15E8) antibodies. A total of 100 nmol/L γ-secretase inhibitor (Ly411575) was added where indicated. After 4 days, proliferation was measured by flow cytometry as described above.PBMCs from HD or CLL patients were incubated in the presence of 100 ng/mL BCMAxCD3, BCMAxnull, or nullxCD3 or stimulated with CD3 (clone 1XE) and CD28 (clone 15E8) antibodies for 2 days. Where indicated HD PBMCs were cocultured in a 1:1 E:T ratio with RPMI-8226, JeKo-1, CII, or BCWM1. A total of 100 nmol/L γ-secretase inhibitor (Ly411575) was added where indicated. Brefeldin A , GolgiStop (BD Biosciences), and anti-CD107a PE-Cy7 were added 4–6 hours before assessment of activation, degranulation, and cytokine production by flow cytometry as described above.Cell lines or CLL cells were cultured either in medium or in presence of 100 nmol/L γ-secretase inhibitor . After 24 or 48 hours, supernatants were harvested and stored at −20°C. sBCMA in supernatants was measured by ELISA using antibody pairs for BCMA.IHC staining for BCMA , CD138, Pax-5, cyclin D1, and IgM were performed on paraffin‐embedded BM and LN tissue. Staining was performed by PhenoPath Laboratories on a Dako Autostainer EQ240 system. Results were assessed by two independent pathologists (C.J.M. van Noesel).n < 5 a Shapiro–Wilk test. P values were calculated by using two-sided paired or unpaired t tests, Wilcoxon matched-pairs signed-rank test, Mann–Whitney test, repeated measures or ordinary one-way ANOVA (followed by Bonferroni post hoc test) or Kruskal–Wallis test (followed by Dunn post hoc test). Correlations were determined by simple linear regression. Statistical analysis was performed using Graphpad PRISM version 8.3.0 with significance set at P < 0.05.Data were checked for normality by a D'Agostino–Pearson test or if The data generated in this study are available upon request from the corresponding author.B-cell malignancy cell lines were tested for BCMA expression by flow cytometry. As expected, the multiple myeloma cell lines U266  and RPMI-8226 (gMFI 4621) expressed high levels of BCMA . AlthougR2 = 0.92). Together, these data indicate that besides multiple myeloma, cell lines derived from other B-cell malignancies express BCMA which can be enhanced by γ-secretase inhibition.Because BCMA is cleaved off by γ-secretase, the different B-cell malignancy lines were incubated for 24–48 hours with 100 nmol/L γ-secretase inhibitor (Ly411575). All lymphoma cell lines showed an increased BCMA level after γ-secretase inhibition . ViabiliThe results regarding BCMA expression on different B-cell lymphoma cell lines prompted us to explore primary material of patients with CLL and B-cell lymphoma. Primary CLL cells showed marginal expression of BCMA protein and mRNA  and B. IBCMA expression by IHC was assessed on primary tissue of BM and/or LNs infiltrated by multiple myeloma, LPL (WM), DLBCL, CLL, or MCL. To determine the fraction of tumor cells expressing BCMA, tissues were also stained for CD138 (multiple myeloma), IgM (LPL), cyclin D1 (MCL), and Pax-5  after 2 days of coculture with the different cell lines in the presence of either teclistamab or anti-CD3/CD28 stimulation , BCWM.1 , CII , and JeKo-1 . Cell lines and age-matched HD PBMCs were cultured in the presence of 100 ng/mL teclistamab or the control BsAbs (BCMAxnull or nullxCD3) with or without 100 nmol/L γ-secretase inhibitor. As a positive control, anti-CD3/CD28 antibodies were added to the cocultures. Both CD4mulation . No T-cemulation . Similarmulation . Activatmulation . In addimulation . Again, mulation . It ther+ or CD8+ or CD4+ and CD8+ together (1:1 ratio) in the presence of teclistamab for 96 hours. Surprisingly, CD4+ T cells were not able to induce killing, in sharp contrast to CD8+ T cells, which induced lysis up to 60% , CLL and DLBCL primary samples. Despite low BCMA expression on CLL, teclistamab could efficiently lyse CLL cells, using both HD T cells or autologous-derived T cells.Our data indicate that BCMA expression below a certain threshold level of BCMA is needed for γ-secretase inhibition to be additive/synergistic in the context of teclistamab. Above that level of BCMA, using γ-secretase inhibition does not enhance lysis, proliferation, or secretion of cytokines by T cells. This is in line with an earlier study of teclistamab in multiple myeloma , but in Nevertheless, γ-secretase inhibition could be useful for patients that express BCMA just below threshold level. γ-secretase inhibitors have been used in clinical trials for treatment of Alzheimer's disease , as wellDespite high BCMA expression on BCWM.1 and CII, coculture with HD T cells and teclistamab led to less efficient killing of these cell lines as compared with the low BCMA-expressing cell line JeKo-1. Because also anti-CD3/CD28 stimulation led to lower responses upon coculture of these cell lines, the T-cell suppressive effect is likely caused by the tumor cells. For CLL and DLBCL, evidence has accumulated for an immunosuppressive tumor microenvironment, that favors tumor growth. Furthermore, in many of these diseases T-cell dysfunction has been described in terms of upregulation of exhaustion markers , defectiIn conclusion, our data show that BCMA expression is not only limited to multiple myeloma, but can also be detected to various levels on other B-NHL subtypes and CLL. Treatment of B-cell lines using the BCMAxCD3 BsAb teclistamab led to lysis of these cells as well as to robust T-cell activation, independent of BCMA expression level. CLL cells, which expressed low levels of BCMA could be efficiently targeted by teclistamab, providing proof of principle that BCMA-directed therapies might be beneficial for other B-cell malignancy patients besides multiple myeloma.Supplementary Tables 1-3, Figures 1-4Supplementary Table 1. CLL Patient characteristics. Supplementary Table 2. Healthy donor characteristics. Supplementary Table 3. MM patient characteristics. Supplementary Figure 1. γ-secretase inhibition increases BCMA levels but does not affect viability of B-cell malignancy cell lines. Supplementary Figure 2. γ-secretase inhibition does not affect viability CLL cells. Supplementary Figure 3. BCMAxCD3 DuoBody® induces activation, degranulation, and cytokine secretion by T cells in the presence of B cell malignancy cell lines. Supplementary Figure 4. BCMAxCD3 DuoBody® induces autologous killing of MM cells.Click here for additional data file.


Middle East respiratory syndrome coronavirus (MERS-CoV) is caused by a well-known coronavirus first identified in a hospitalized patient in the Kingdom of Saudi Arabia. MERS-CoV is a serious pathogen affecting both human and camel health globally, with camels being known carriers of viruses that spread to humans. In this work, MERS-CoV genomic sequences were retrieved and analyzed by multiple sequence alignment to design and predict siRNAs with online software. The siRNAs were designed from the orf1ab region of the virus genome because of its high sequence conservation and vital role in virus replication. The designed siRNAs were used for experimental evaluation in selected cell lines: Vero cells, HEK-293-T, and Huh-7. Virus inhibition was assessed according to the cycle threshold value during a quantitative real-time polymerase chain reaction. Out of 462 potential siRNAs, we filtered out 21 based on specific selection criteria without off-target effect. The selected siRNAs did not show any cellular toxicity in the tested cell lines at various concentrations. Based on our results, it was obvious that the combined use of siRNAs exhibited a reduction in MERS-CoV replication in the Vero, HEK-293-T, and Huh-7 cell lines, with the highest efficacy displayed in the Vero cells.  In 2012, a new coronavirus was identified in an infected butcher in the Kingdom of Saudi Arabia, who developed a respiratory illness and died 11 days after hospital admission [l health . Camels l health ,4. The ml health ,6. Coronaviridae. The size of the viral genome is approximately 25–32 kb. MERS-CoV falls under lineage C betacoronaviruses (βCoVs). To date, only four coronavirus groups have been reported—designated as Alpha, Beta, Gamma, and Delta—and they have high genetic diversity, which favors the emergence of new strains [The MERS-CoV belongs to the family  strains . Genetic strains . Current strains ,10, bact strains , fungal  strains ,13, para strains ,15, vira strains . Several strains . Additio strains . The det strains ,20,21,22 strains ,27,28,29 strains  against  strains , dengue  strains , influen strains ,35, hepa strains , human p strains , SARS co strains , and MERThis present work gains significance because of the lack of antiviral therapy or vaccines for MERS-CoV, and the generated data will provide valuable information about the advancement and further use of oligonucleotide-based antivirals as an alternative therapy. The main objectives of this work were to design potential siRNAs by using a computational approach and to experimentally evaluate the reduction of viral load in Vero, HEK-293-T, and Huh-7 cell lines. Based on the results, a significant reduction in viral RNA was observed after using the combined siRNAs.The MERS-CoV genomic sequences were retrieved from GenBank  and aligned by using BioEdit software (Version 7.2). The orf1ab region was identified as a potential target during siRNA design. The siRNAs were designed and selected by siDirect (Version 2.0) ,29,39,40http://rna.tbi.univie.ac.at accessed on 1 May 2022) was used to predict the secondary structure of the siRNAs. We have provided figures of the predicted siRNAs in our manuscript. An online bioinformatics tool (RNAfold server—4) with 60–80% confluency. All the experiments were performed in triplicate. The antiviral potency of the siRNAs was accessed 48 h post-transfection. We selected these three cell lines  based on their differential cell line susceptibility, replicative capacity, better cell growth and multiplication, and their cytopathic effects in in vitro assays [Dulbecco’s modified Eagle’s medium (DMEM) was used to grow the cells at 37 °C. The growth and quantification of MERS-CoV infection as well as viral RNA products of replication were performed by using standard published protocols ,41. The o assays ,43,44. ® and further grown in a standard culture plate at defined conditions in DMEM. The standard siRNA dilutions were prepared at various concentrations  from 50 μM stock solutions by adding 100 μL of Opti-MEM and Lipofectamine 2000 and further incubated at room temperature for 30 min. The complex mixture was delivered slowly to the grown cells, which were allowed to grow further for 24 h at 37 °C. The cellular toxicity of the siRNAs in different cell lines was analyzed by using an MTT assay kit (Invitrogen) following the manufacturer’s instructions. The absorbance was measured at 570 nm using a SpectraMax i3x imaging cytometer, and the standard formula was applied for the cytotoxicity calculation. The detailed protocol for the cytotoxicity assay has been described previously [The siRNAs were delivered to the grown cells by using reverse transfection with Lipofectamine 2000  following the manufacturer’s instructions. The cells were procured from ATCCeviously .To evaluate the inhibitory effect of siRNAs against MERS-CoV experimentally, we used multiple combinations of siRNAs with various doses  on selected grown cells. In our earlier study, we evaluated the siRNAs at various concentrations ranging from 0.1, 0.5, 1.0, 2.5, 5.0, 10, 25, and 50 nM. Here, the concentrations of siRNAs  were chosen because they produced better results than other concentrations in our previous study ,45. The The multiple sequence alignment results showed high conservation at various locations among the human and camel isolates. The conserved region of the ORF1ab gene is presented in p-value of <0.0001. The cytotoxicity results of each cell line are provided in The cellular toxicities of the designed siRNAs were evaluated in the three selected cell lines and were observed to be concentration-dependent. None of the tested siRNAs displayed significant toxicity in any cell lines. The data were statistically analyzed by using the GraphPad Prism (9.3.0) two-way ANOVA software and considered significant with a The virus inhibition and the level of viral RNA in the cell lines were determined by the Ct value of qPCR for all combinations of siRNAs in the cell supernatant as well as the cell lysate. The analysis was performed by using only four different concentrations  of siRNAs, as per previous works ,29. The The RNAfold server was used to predict the secondary structure of the designed siRNAs. The minimum free energy (MFE) and thermodynamics ensemble (%) data for all siRNAs are provided in In 2012, MERS-CoV was identified in a hospitalized patient in the Kingdom of Saudi Arabia, where the highest fatality rate of the disease has been reported. Tremendous efforts have been made in MERS-CoV research using varied approaches that have contributed significantly towards disease management. Despite this enormous global research effort, no vaccines or antivirals are currently available against MERS-CoV, but various strategies are being used for their development, including oligonucleotide (siRNAs/miRNAs)-based therapy ,49,50,51In our study, the software produced 462 siRNAs from the orf1ab region. We selected and evaluated only 21 siRNAs following the standard criteria for filtration ,26. The We evaluated only certain siRNAs for combined use in the Vero, Huh-7, and HEK-293-T cell lines. The cellular toxicity of each designed siRNA was evaluated in the three cell lines before inducing virus infection and qPCR. The results showed no cytotoxicity of the evaluated siRNAs in the tested cell lines. The experimental evaluation results of each siRNA combination and their antiviral potency were significantly variable across different cell lines, even at the same concentration, which could be due to the growth and multiplication properties of the cell lines after siRNA delivery and virus infection. Many siRNA combinations showed significant and strong inhibitory effects toward viral replication in Vero cells. The Ct values from the qPCR data indicated a significant inhibition of replication and a reduction in viral RNA in both the cell supernatant and lysate of the Vero, Huh-7, and HEK-293-T cell lines. We evaluated all 21 combinations of siRNAs in the selected cell lines. Better siRNA efficacy and reduction of viral load were observed in Vero cells due to the better growth and multiplication of the virus in this cell line. In silico approaches are commonly used to screen possible strategies for overcoming infections, with successful examples in similar studies that emphasize bioinformatic approaches and their utility . One limBased on our computational approach combined with the experimental evaluation of selected siRNAs in terms of cytotoxicity and qPCR, we concluded that the in silico designing and filtration of siRNAs can be an effective approach to alternative oligonucleotide-based therapeutics for MERS-CoV. The experimental evaluation results of the antiviral potency of siRNAs provided valuable information about the selection of siRNAs tested in various cell lines at selected concentrations with multiple combinations. Recently, after many barriers and challenges were overcome, the FDA approved several siRNA-based therapeutics. However, many other challenges must still be addressed, such as enzymatic degradation, rapid renal clearance, endosomal trapping, plasma protein sequestration, and activation of the immune system. To overcome these barriers, many strategies and techniques are being used to modify the backbone, bases, sugars, conjugation of aptamers, and exosome-based delivery of siRNAs. We observed that the Vero cells were better than other cells because of virus multiplication and cell growth for testing the siRNAs against MERS-CoV. Finally, the overall results proved that the computational approach of this study can be used to design siRNAs as potential therapeutics against MERS-CoV, and our experimental evaluation provided information about the selection of these siRNAs.


Malignant pleural mesothelioma (MPM) is a deadly cancer primarily of the lung pleura, strongly associated with asbestos exposure. According to the ESMO Clinical Practice Guidelines, MPM patients may be suitable for best supportive care only, active  or multimodality treatments. Surgery, although controversial, is an option in some centres, but the recognised standard of care is systemic chemotherapy (cisplatin-pemetrexed) while other therapies, including immunotherapy  or combination therapies  are being investigated . DespiteRobinson et al. review their use of different mouse models to investigate therapeutic approaches to treat MPM. They developed some of the first human and mouse mesothelioma cell lines using human tumours in immunocompromised mice and intraperitoneal injection of asbestos respectively. They also developed the MexTAg mouse, an asbestos-induced mesothelioma mouse model in which expression of the Simian Virus 40 large T antigen (SV40 TAg) is controlled by a mesothelin tissue-specific promoter. The advantage with this model is it generates 100% mesotheliomas in response to asbestos. More recently, they also developed a bilateral tumour model which was developed to analyse the difference between immune checkpoint blockade responsive and non-responsive tumours against a homogeneous background.The development of novel treatments for MPM requires substantial preclinical data before drugs can enter a clinical trial. Mesothelioma cell lines are extensively used to screen individual or combinations of drugs to determine cytotoxicity, anti-cancer mechanisms and drug resistance, and to identify potentially clinically relevant biomarkers. However, 2D cell culture has limitations as lack of heterogeneity and a tumour microenvironment and acquisition of mutations limit their usefulness . Three-dPezzicoli et al. discussed current and potentially upcoming standards of care for MPM and reviewed several novel therapeutic agents recently in clinical trial. Lurbinectedin is a transcriptional inhibitor and blocks the function of tumour associated macrophages. Mesothelioma cell lines were highly sensitive to lurbinectedin and in the SAKK 17/16 phase II clinical trial, lurbinectedin demonstrated activity following immune checkpoint inhibitor therapy, indicating a phase III trial is warranted. Pezzicoli et al. also discussed the outcome pf several tyrosine kinase inhibitor trials. Of particular interest, were results from preclinical studies using CDK4/6 inhibitors, that demonstrated cell toxicity and inhibition of tumour growth in mouse models. The MiST2 study  was recently announced. The median progression free survival and overall survival was 128 and 217 days respectively. The authors plan to further investigate CDK4/6 inhibition in another randomised study  in MPM which have not yet entered clinical trial. STAT3 is important for cell survival, proliferation, self-renewal and chemoresistance. KDM4A demethylates the tumor suppressor SETD2 products H3K36me3 and H3K9me3, which are essential in cell growth and DNA damage repair. Heparanase promotes tumor growth, angiogenesis, metastasis and chemoresistance. All molecules reviewed impact some of the hallmarks of cancers but their role in MPM and the effect of targeting these molecules on MPM tumour growth require considerably more preclinical studies.Oien et al. to have anti-cancer effects in cisplatin-resistant and pemetrexed-resistant MPM cell lines. Interestingly, NF2-mutated cell lines were more sensitive to QC. Inhibition of the hippo pathway was identified using RNA-seq and closely-associated transcription factors YAP1, CDK4, and KRAS were downregulated. However, the effect of QC in vivo has not been investigated.Repurposing of existing drugs has also been proposed for MPM. Quinacrine (QC), which is an antimalarial drug and an antibiotic, was shown by Precision medicine approaches to treat MPM have been proposed  and the in vitro and in vivo models to test gene and variant function. Together, scientists and clinicians can utilise their specific expertise to collaborate and plan better animal studies and clinical trials to maximise effective drug design to treat diseases like MPM which have shown limited therapeutic improvement.Developing new approaches for treating MPM are urgently needed and it has been suggested that a circular pathway of bench to bedside and back again approach may be key to clinical breakthroughs . Many pr