These mutations cause an increase in the levels of the oncometabolite D-2-hydroxyglutarate (D-2-HG), which leads to widespread changes in several cellular processes, including the epigenetic landscape. The aim of our study was to explore whether the methylome of IDH mutant cartilage tumors is altered upon tumor progression and whether these underlying epigenetic vulnerabilities could be used as a target for therapy in both IDH wildtype and IDH mutant high-grade chondrosarcomas. As surgery is nowadays the only treatment option for chondrosarcoma patients, the identification of novel therapeutic strategies remains an important endeavor. The findings in this study show that histone deacetylase (HDAC) inhibition may represent a promising therapeutic strategy for all chondrosarcoma patients, since sensitivity towards this therapeutic option seems independent of the IDH mutation status and the chondrosarcoma subtype.Cartilage tumors frequently harbor mutations in the isocitrate dehydrogenase IDH1 or IDH2) genes are common in enchondromas and chondrosarcomas, and lead to elevated levels of the oncometabolite D-2-hydroxyglutarate causing widespread changes in the epigenetic landscape of these tumors. With the use of a DNA methylation array, we explored whether the methylome is altered upon progression from IDH mutant enchondroma towards high-grade chondrosarcoma. High-grade tumors show an overall increase in the number of highly methylated genes, indicating that remodeling of the methylome is associated with tumor progression. Therefore, an epigenetics compound screen was performed in five chondrosarcoma cell lines to therapeutically explore these underlying epigenetic vulnerabilities. Chondrosarcomas demonstrated high sensitivity to histone deacetylase (HDAC) inhibition in both 2D and 3D in vitro models, independent of the IDH mutation status or the chondrosarcoma subtype. siRNA knockdown and RNA expression data showed that chondrosarcomas rely on the expression of multiple HDACs, especially class I subtypes. Furthermore, class I HDAC inhibition sensitized chondrosarcoma to glutaminolysis and Bcl-2 family member inhibitors, suggesting that HDACs define the metabolic state and apoptotic threshold in chondrosarcoma. Taken together, HDAC inhibition may represent a promising targeted therapeutic strategy for chondrosarcoma patients, either as monotherapy or as part of combination treatment regimens.Mutations in the isocitrate dehydrogenase ( Chondrosarcomas are bone malignancies characterized by the production of cartilage and account for 20% of all malignant bone tumors ,2. BasedIDH1 or IDH2, collectively referred to as IDH) genes , occurring in ~50% of the cases [2 in the tricarboxylic acid cycle, while the IDH1 enzyme is located in the cytosol where it performs a similar reaction. The hotspot mutations induce a gain-of-function of these enzymes, resulting into abnormal cellular concentrations of the oncometabolite D-2-hydroxyglutarate (D-2-HG) [IDH1R132C mutation, which is one of the most efficient D-2-HG producers [The most common genetic alteration in central conventional chondrosarcoma is the hotspot mutation affecting the arginine residues encoded by the isocitrate dehydrogenase 1 and -2  and histone demethylases (family of Jumonji enzymes) [IDH mutations interfere with the dynamic processes that regulate the accessibility of the DNA and could thereby permanently change gene expression, which could ultimately lead to tumor formation and growth. Indeed, the introduction of an IDH mutation induces the formation of benign cartilage tumors  in mice [IDH mutated enchondromas, which are considered as the benign pre-cursor lesions of chondrosarcoma, are characterized by a CpG island methylator phenotype (CIMP)-positive status [IDH mutation is an early genetic event which highly alters the epigenetic landscape in early phases of cartilage tumor formation. The characteristic hypermethylation phenotype is also observed in primary IDH mutant chondrosarcomas [IDH mutations might be beneficial for chondrosarcoma patients, and could potentially help to overcome the intrinsic chemotherapy resistance in chondrosarcoma. Due to the high structural similarity between α-KG and its antagonist D-2-HG, α-KG dependent enzymes are inhibited by the high levels of the oncometabolite ,11, causenzymes) ,13, lead in mice . IDH mute status . The mute status ,16. Togesarcomas . TherefoIDH mutant enchondroma towards chondrosarcoma using a methylation array on primary tumor samples. In fact, high grade tumors demonstrated an increased number of hypermethylated genes as compared to low grade tumors, suggesting that epigenetic mechanisms play an important role in chondrosarcoma progression. Therefore, a broad compound screen containing 128 compounds which target different epigenetic key players (including histone deacetylases (HDACs), sirtuins (SIRTs), histone demethylases (HDMs), histone acetyltransferases (HATs), histone methyltransferases (HMTs), and DNA methyltransferases (DNMTs)) was performed on IDH wild type and mutant chondrosarcoma cell lines to explore whether these epigenetic changes can be used as a target for novel anti-cancer therapy. No synthetic lethal interactions with IDH mutations were identified, but several general interesting targets were determined, and interesting compound classes included HDAC and bromodomain and extra-terminal motif (BET) protein inhibitors. Additionally, we explored if one of the most promising hits, the HDAC inhibitor romidepsin, could help to sensitize chondrosarcoma cells to chemotherapy or small molecule inhibitors. To address this question, a drug screen with a panel of non-epigenetic drugs was designed to explore if a combination treatment could be a promising therapeutic strategy for chondrosarcoma patients.In this study, we confirmed that the hypermethylation phenotype is retained upon progression from IDH mutant cartilage tumors were CIMP-positive, except for one enchondroma and one low-grade chondrosarcoma   . Hierarcsarcomas A. Thus, 89 genes . Of note89 genes . Biologi89 genes . Hence, The primary epigenetics compound screen was performed as described in IDH mutation status, as only one potential synthetic lethal interaction was observed in JJ012 cells   . Howevertubacin) . Hence, The human genome encodes for eleven subtypes of HDAC enzymes, which can be divided into four different classes: class I , class IIA , class IIB (HDAC6 and HDAC10), and class IV (HDAC11). To identify the most potent and specific HDAC inhibitor for follow-up studies, all HDAC inhibitors were screened at a lower concentration  and ordered based on HDAC class specificity . As show50 values in the range of 0.89 to 1.96 nM after 72 h of treatment  were treated with a high dose of romidepsin  for 24 h or 48 h. Caspase 3/7 activity was significantly induced in SW1353 cells after 24 h, and in all cell lines after 48 h C. A concAs HDAC inhibitors induce the acetylation of histones, the level of acetylated histone 3 (acH3K9) was determined after 24 h and 48 h treatment with romidepsin. An induction of acH3K9 was observed in all three chondrosarcoma cell lines D. HoweveTwenty non-epigenetic drugs were selected for the HDAC inhibitor combination drug screen . These dThe general Bcl-2 family member inhibitor, ABT-737, and the specific Bcl-2 inhibitor, venetoclax, showed the highest synergistic effect when combined with romidepsin, especially at higher drug concentrations . InductiIDH mutant enchondroma towards chondrosarcoma. We showed that the CIMP-positive status is retained in high-grade IDH mutant chondrosarcoma. Moreover, the number of highly methylated genes seemed to increase upon tumor progression. This suggests that methylation patterns change upon chondrosarcoma progression, and that an increased number of methylated genes is associated with aggressiveness of the disease. Since we chose to include a homogeneous IDH mutant subgroup of chondrosarcomas, it remains to be established whether methylation is equally important in IDH wildtype chondrosarcomas. The epigenetics compound screen results, indicating sensitivity to the same epigenetic modulators, suggest that the epigenetic landscape is comparable between IDH mutant and wildtype chondrosarcomas. This is further supported by the lack of a synthetic lethal interaction between the IDH mutation and a broad spectrum of epigenetic regulators, even though these kinds of synthetic lethal interactions  were previously described in IDH mutant acute myeloid leukemia (AML) and glioma [IDH mutation on the methylome. In this study, we explored whether the methylome is altered upon progression from d glioma ,26,27. TIDH mutant chondrosarcoma tumors, we expected that targeting of DNMTs by for instance decitabine and azacytidine would be effective in chondrosarcoma. However, none of these DNMT inhibitors had a pronounced effect on chondrosarcoma cell viability. Nevertheless, the epigenetics compound screens identified several other classes of interesting drug targets for both IDH wildtype and IDH mutant chondrosarcoma, among which inhibitors against Aurora kinases, FLT3, HDAC, and JAK showed the most pronounced effect on chondrosarcoma cell growth. Most of these targets play a major role in growth signaling pathways (FLT3 and JAK) or cell cycle control (Aurora kinases). We have previously shown that targeting cell cycle regulators, especially Checkpoint Kinase 1 (CHK1), could be a promising therapeutic strategy for chondrosarcoma patients [Based on the retained CIMP-positive status and the increasing amount of hypermethylated genes in high-grade patients . Hence, COL2A1), leading to the disruption of normal cartilage development [The HDAC enzymes play a prominent role in skeletal development and aberrant expression is associated with a wide variety of bone-related abnormalities. HDAC1 and HDAC3 are most abundantly expressed in healthy human cartilage , and we elopment , while iIDH mutation status and the chondrosarcoma subtype , indicating that HDAC inhibition could be a promising therapeutic strategy for all chondrosarcoma patients. Romidepsin is clinically approved for the treatment of cutaneous T-cell lymphoma and other peripheral T-cell lymphomas. In vitro studies of romidepsin treatment in T-cell lymphomas report IC50 values in the low nanomolar range (1 to 11 nM) [50 and IC50 values in our 2D and 3D chondrosarcoma cell culture models. The currently approved dosing regimen of romidepsin in patients with T-cell lymphomas reaches maximum plasma concentrations of 700 nM [IDH mutation status independent manner. As in vitro and in vivo models differ in nutrient availability, metabolism and cell types involved in tumor growth, the downstream effects of IDH mutations on the epigenetic landscape and the metabolism might also vary between these two types of models.Previously, it was shown by other groups that the HDAC inhibitors romidepsin, trichostatin A, and sodium valproate affect cell proliferation in 2D in vitro models of chondrosarcoma ,32. In to 11 nM) ,34, whicf 700 nM , which ff 700 nM . Additiof 700 nM  should bWe also explored if sub-optimal concentrations of romidepsin could be used in a combination treatment strategy, to either sensitize chondrosarcoma to non-epigenetic therapies or to reduce HDAC inhibitor toxicity. However, HDAC inhibition could not sensitize chondrosarcoma cells to four different types of chemotherapy , although this has been extensively described in other tumor types . Only thIDH mutation status  and of all twenty compounds included in the custom designed compound library for the HDAC inhibitor combination drug screen are available in the IDH wild type (IDHWT)) [IDH1R132G) [IDH2R172S) [IDHWT) [IDH1R132C) [IDHWT) [IDH1R132C) [IDH2R172W) [IDHWT) [IDHWT) [2 at 37 °C. PCR-based mycoplasma tests and STR profiling  were performed regularly.The central conventional chondrosarcoma cell lines CH2879 ((IDHWT)) ,46, JJ01H1R132G) , SW1353  [IDHWT)  and L835H1R132C) ; the ded [IDHWT) , HT1080 H1R132C) ,51, L297H2R172W)  and L325 [IDHWT) ; and the [IDHWT)  were cul [IDHWT) . Long-te [IDHWT)  were culThe chondrosarcoma cell lines CH2879, JJ012, and SW1353 were seeded in optimized cell densities  in black 96-well plates. After overnight attachment, cells were treated with the epigenetics compound library at a concentration of 2 µM. After 72 h of treatment, cells were fixed with 4% formaldehyde and stained with 2 µg/mL Hoechst 33342 . Plates were imaged using a BD Pathway 855 microscope  and the number of Hoechst 33342 positive nuclei was quantified using the Image-Pro software . All plates were included in the analysis and data were normalized to the negative controls  to obtain percent of control values. All compounds that reduced the nuclei count ≥50% in at least one of the cell lines were selected for the secondary epigenetics compound screen. The screen was performed in triplicate. A schematic overview of the primary epigenetics compound screen can be found in IDH1 mutant cell lines  were pre-exposed to 10 µM AGI-5198 for 72 h [IDH1 mutant cell lines were seeded in black 96-well plates  in normal growth medium supplemented with 10µM AGI-5198 or 0.1% DMSO as a control. To mimic D-2-HG production, CH2879 cells were seeded (5000/well) in normal growth medium supplemented with 250 µM D-2-HG or 1% PBS as a control. After overnight attachment, cells were treated with the selected compounds (n = 61) from the primary screen at a concentration of 2 µM. Compounds were diluted in medium supplemented with 0.1% DMSO, 10µM AGI-5198, 1% PBS or 250 µM D-2-HG. After 72 h of treatment, a nuclei count was performed as described under the methods section of the primary epigenetics compound screen. All plates were included in the analysis and data were normalized to the negative controls  to obtain percent of control values. Compounds that reduced the nuclei count ≥50% in all cell lines were considered as potential therapeutic options for both IDH wildtype and IDH mutant chondrosarcomas. A ≥75% difference in nuclei count between cells treated with or without 10 µM AGI-5198 or cells treated with or without 250 µM D-2-HG was considered as a potential synthetic lethal interaction with the IDH mutation. The screen was performed in triplicate. A schematic overview of the secondary epigenetics compound screen can be found in To reduce the D-2-HG levels, CH2879 cells (7000/well), JJ012 cells (3000/well), and SW1353 cells (3000/well) were seeded in 96-well plates in normal growth medium supplemented with 0.1% DMSO or romidepsin (1 nM (JJ012 cells) and 0.75 nM (CH2879 and SW1353 cells)). After overnight attachment, cells were treated with the custom designed compound library. For each library compound, five concentrations were tested, which were based on previous experience . CompounFor the 2D cell cultures, the cell viability assays, nuclei count assays and data analysis were performed as previously described . Cells wFor the 3D cell cultures, the cell viability assays and the immunohistochemical stains (Ki-67 and cleaved caspase 3) were performed as previously described . Cells w(GAPDH) (Dharmacon) and an siKinasePool  were used as negative controls. siRNA SMARTpools against Kinesin Family Member 11 (KIF11) and Polo Like Kinase 1 (PLK1) (Dharmacon) were used as positive controls. To reduce toxicity, the medium was refreshed 24 h after the transfection. Three days after medium refreshment, cells were fixed, stained with Hoechst 33342 and imaged with the Cellomics ArrayScan VTI HCS 700. Data were normalized to the negative controls to obtain percent of control values. The experiment was performed in triplicate. Reverse siRNA transfection was performed for CH2879 cells  and JJ012 cells (5000/well) in 96-well plates. To achieve transient knockdown of all HDAC subtypes, cells were transfected with 0.1 µL/well DharmaFECT 3  and 50 nM siRNA SMARTpools (Dharmacon), which consist of four individual siRNAs targeting a gene of interest. The mock condition, the siRNA SMARTpool against Glyceraldehyde-3-Phosphate Dehydrogenase RNA expression of all HDAC subtypes in chondrosarcoma cell lines was extracted from a next generation RNA sequencing dataset we previously described . Reads pThe apoptosis and cell cycle assays were performed and analyzed as previously described . Cells wSample preparation, western blotting and quantification were performed as previously described . Cells wTo determine significant changes between experimental groups, a nonparametric Kruskal–Wallis test followed by a Dunn’s post-hoc test was performed. A Grubbs’ test was performed to detect outlier values in the dose–response curve datasets. All statistical tests were performed in GraphPad Prism 8. The Z’-factor was used as a measure for compound screen quality and was calculated to determine the effect size between negative and positive controls . To calcIDH mutant cartilage tumors are associated with tumor progression and this study shows that targeting of epigenetic regulators could be a potential therapeutic strategy for both IDH wildtype and IDH mutant chondrosarcoma patients. Our study establishes that HDAC enzymes play a prominent role in the epigenetic landscape and survival of chondrosarcoma cells, especially class I HDACs. Chondrosarcoma cell lines are highly sensitive to the class I HDAC inhibitor romidepsin, irrespective of the chondrosarcoma subtype or the IDH mutation status. In addition, our study identifies glutaminolysis and Bcl-2 family member inhibitors as potential candidates to be used in combination with HDAC inhibition, suggesting that romidepsin influences the metabolic state and apoptotic threshold of chondrosarcoma cells. Further studies are needed to elucidate the exact mechanisms underlying HDAC inhibitor sensitivity in chondrosarcoma and to confirm romidepsin efficacy in an orthotopic chondrosarcoma mouse model. Taken together, pharmacological inhibition of HDAC enzymes may represent a promising targeted therapeutic strategy for chondrosarcoma patients. In spite of the fact that the mechanisms underlying HDAC inhibitor sensitivity are currently unknown, the identification of novel treatment strategies for chondrosarcoma remains an important objective, especially for patients with unresectable or high-grade tumors.In summary, alterations in the methylome of


CEN‐tools), a website and python package, in which users can interrogate the essentiality of a gene from large‐scale genome‐scale CRISPR screens in a number of biological contexts including tissue of origin, mutation profiles, expression levels and drug responses. We show that CEN‐tools is suitable for the systematic identification of genetic dependencies and for more targeted queries. The associations between genes and a given context are represented as dependency networks (CENs), and we demonstrate the utility of these networks in elucidating novel gene functions. In addition, we integrate the dependency networks with existing protein–protein interaction networks to reveal context‐dependent essential cellular pathways in cancer cells. Together, we demonstrate the applicability of CEN‐tools in aiding the current efforts to define the human cellular dependency map.An emerging theme from large‐scale genetic screens that identify genes essential for cell fitness is that essentiality of a given gene is highly context‐specific. Identification of such contexts could be the key to defining gene function and also to develop novel therapeutic interventions. Here, we present Context‐specific Essentiality Network‐tools ( CEN‐tools allows users to navigate public large‐scale gene essentiality datasets and extract context‐specific essentiality networks, where context refers to the genetic background, gene expression, CNVs and other cell line features. Genetic perturbation experiments enable identification of genes that are essential for the survival and fitness of the cell. It is now widely accepted that the binary nature of gene essentiality as defined in classical genetics is too simplistic and gene essentiality is highly context‐specific. There are genes, often termed as core fitness genes, which are required for core functioning and housekeeping of the cells and their knockout causes loss of fitness, in principle, in all cell types in all conditions. However, a large number of genes, termed context‐specific essential genes, play important roles for cell fitness only in a particular genetic or environmental context . CEN‐tools is an integrated database and set of computational tools to explore context‐dependent gene essentiality from pooled CRISPR data sets, obtained from the two largest publicly available essentiality screening projects: the DepMap project ‐tools , both in pancancer and within tissue/cancer type, and represent them in Context‐specific Essentiality Networks or CENs. Each edge in this network represents the type of association used to annotate the underlying contexts. To demonstrate the value of CENs, we collected the co‐essentiality networks from the PICKLES database  TP53. This is an observation often made in pooled CRISPR screens as cells harboring the WT tumor suppressor genes are often dependent on their repressors for proliferation  promoter containing multiple binding sites for SRF  and SHOC2  become available to ultimately aid designing better therapeutics.Using CEN‐tools for group‐wise testing for significant association we identified many previously described molecular markers that are associated with essentiality, including, but not limited to mutational dependence on major cancer drivers and tissue‐specific lineage dependency markers ).Tissue annotations were obtained from both CCLE website and Cell Model Passports. In case of four cell lines , discrepancies between the two websites were found in terms of their annotations, hence their expression profiles were examined on a t‐sne plot and based on these cells were manually annotated as “Bone” tissue. In case of discrepancies in cancer type annotations, the Cellosaurus Bairoch,  under thet al, 2 fold change of 1 and non‐essential genes a median log2 fold change of 0.Corrected log fold changes from Project Score and DepMap projects were retrieved from Project Score, in which the same preprocessing pipeline was applied. “Essentiality scores” were calculated by computing the scaled log2 fold change. For this, gene‐level fold changes were first quantile normalised per sample and then median scaled according to BAGEL essential and non‐essential genes  matrix calculated using the MAGeCK : Tissue refers to the tissue of origin of the cell lines. Cancer refers to the further classification of the cell lines into the cancer type within each tissue. 2et al, et al, et al, Mutation (discrete context): For mutation analysis, two different mutational annotations obtained from CCLE database : For expression analysis, cell line expression values were obtained from the Cell Line Passport and CCLE database as normalised FPKM values : Drug responses for the cell lines were obtained as normalised Z‐scores from the CancerRxGene database (Yang U (two‐samples Wilcoxon) tests were used with default parameters  in R  to only include the genes represented in the PICKLES database as BRAF co‐essential genes . Further subsetting was then performed on the network file as follows:Project = ‘BROAD’comparision_in = ‘Skin’effector = ‘expression’ or effector = ‘tissue’ or effector = ‘hotspot_mutation’ or effector = ‘hotspot_co_mutation’.higher = ‘high_in_group’ or is.na(higher)median_group > 0 | is.na(median_group)width ≥ 2dashes = FALSEet al, The resulting subnetwork was imported in Cytoscape Advanced edge filter optionsFor Expression context correlations show edges corresponding to: Positive correlationsFor Tissue/Cancer context comparisons show edges corresponding to: Increase in essentiality/expressionFor Mutation context comparisons show edges corresponding to: Both Increase and decrease in essentiality/expressionSelect mutation annotations: CCLE: hotspot mutationsOnly show tissue/cancer edges in which the median essentiality score of the essential context is higher than: 0.3Only show mutation edges in which the median essentiality score of the essential context is higher than: 0.4Perform context comparisons: Within a tissue of origin/cancer type.Confidence level of association (Tissue/Cancer type): 1:LowConfidence level of association (Mutation): 2:MediumSelect: Group APress “Initialise network”et al, Download the network and the node attributes by clicking the download buttons in the left sidebar. Open the network file in Cytoscape Advanced edge filter optionsFor Expression context correlations show edges corresponding to: Positive correlationsFor Tissue/Cancer context comparisons show edges corresponding to: Increase in essentiality/expressionFor Mutation context comparisons show edges corresponding to: Increase in essentiality/expressionSelect mutation annotations: CCLE: hotspot mutationsOnly show tissue/cancer edges in which the median essentiality score of the essential context is higher than: 0.2Only show mutation edges in which the median essentiality score of the essential context is higher than: 0.4Perform context comparisons: Within a tissue of origin/cancer type.Confidence level of association (Tissue/Cancer type): 1: LowConfidence level of association (Mutation): 2: MediumSelect: Group APress “Initialise network”. The network will have too many nodes to be displayed but will still be generated (you will see a warning).Download the network and the node attributes by clicking the download buttons in the left sidebar. Open the network file in Cytoscape.For visualisation, only display nodes with “TF” attribute and any other nodes directly associated with these “TF” nodes.et al, To elucidate the cellular essential processes of the NRAS‐mutant skin cancer cells, we integrated the NRAS‐mutant skin CEN, comprising the statistical associations between the gene dependencies, with the STRING protein interaction network, comprising biological associations in the human cells Toggle below to display edges corresponding to either Tissue or Cancer subtype: TissueAdvanced edge filter optionsFor Expression context correlations show edges corresponding to: Positive correlationsFor Tissue/Cancer context comparisons show edges corresponding to: Increase in essentiality/expressionFor Mutation context comparisons show edges corresponding to: Increase in essentiality/expressionSelect mutation annotations: CCLE: hotspot mutationsOnly show tissue/cancer edges in which the median essentiality score of the essential context is higher than: 0.2Only show mutation edges in which the median essentiality score of the essential context is higher than: 0.4Perform context comparisons: Within a tissue of origin/cancer type.Confidence level of association (Tissue/Cancer type): 1: LowConfidence level of association (Mutation): 1: LowSelect: Group APress “Initialise network”. A network will appear.On the main panel, press the toggle button “Filter 1: Hide edges corresponding to change only in expression”. This will update the networkOn the side panel, press the “Load tissues represented in the current network”.Upon clicking the button in step 5 a dropdown menu with “Filter to only show edges corresponding to following tissues (press ‘refresh’ to update the list if filtering parameters are changed)” will appear. Select “Skin” on from this dropdown menu.Download the networks and the node attribute files for the shown networks. These files will be used later for network annotation.Press “Map the current nodes to a PPI network” button. This will automatically trigger switching to a new tab “Integration with PPI network”.On the Integration with PPI network tab, select the following options: Display Network from STRING: Map CEN onto STRING network.Select both output options: Show CEN‐mapped STRING network: Upon clicking this button a purple button “Retrieve PPI network of CEN from STRING” will appear. Press this button to visualise the network on the main panel. Use, adjust STRING interaction score: 400.Perform enrichment analysis and further choose the following options. Choose the enrichment category to display: KEGGChoose enrichments FDR cut‐off: 0.05et al, Download the network and the node attributes by clicking the download buttons in the left sidebar. Open the network file in Cytoscape , growth property, microsatellite stability, tissue and cancer subtype. Only cell lines for which we had essentiality data, mutation information and expression data are available for selection.Selected cell lines are presented in a table and highlighted in a T‐distributed Stochastic Neighbour Embedding (t‐SNE) plot for further interactive selection. In the t‐SNE plot, each cell line is represented by a dot. Cell lines with similar gene expression profiles will be closer together. The t‐SNE plot was generated with the following steps:Rtsne package  in R (version: 3.6.1). Default parameters were used except for perplexity, which was set to 70 and the number of maximal iterations, which was changed to 500.The gene expression matrix of each project  containing FPKM values with genes as columns and cell lines as rows was reduced to two dimensions using the ggplot2 package using the scatterplot function geom_point (version: 3.3.0)  or “Add points to selection” (add the choice to previously selected cell lines) button. More information on the selected cell lines is shown in the corresponding table.To support the decision, the dots can be highlighted by tissue of origin or subtype.The choice of selectable dots can be restricted by using the above‐described dropdown menus for mutations, CNV's etc. on the left panel of the app. Cell lines that do not adhere to the chosen criteria will appear as faint grey dots in the t‐SNE plot and cannot be clicked. Cell lines that adhere to the chosen criteria but are not part of the selection appear as grey dots and can be clicked to add them to the selection.As soon as the user is satisfied, the selected cell lines can be submitted for further Context analysis to CEN‐tools by hitting the “Click here to confirm your selection” button. The user should also give their selection a name that will be later on used as a label in plots of subsequent analysis.Advanced python tool users of CEN‐tools are also able to restrict the analysis to their choice of cell lines. For detailed guidance on the python tool refer to the CEN‐tool python documentation.RPL22 mutation and the essentiality of its paralog RPL22L1 was investigated in the “BROAD” project. The following steps were taken:The association of Navigate to the “Context analysis” tab and “Tissue/Coessentiality” subtab.On the left menubar select “Advanced selection” and then “Interactive Cell Line Selector”Hit the “Launch the interactive Cell Line Selector” button. You will be redirected to a new tab. Wait a little while until the interface is fully loaded.On the left menubar choose: Show cell lines with mutations from: Input genesType “RPL22” in the appearing text boxHit the “Submit” buttonShould cell lines contain a mutation in all (‘AND’) or at least one (‘OR’) of the above chosen genes?: ORSelection based on Copy Number Variation (CNV)?: No selection based on CNVCell culture growth properties: AllGenome stability: AllTissue of origin : AllCancer type : AllColour cell lines by: TissueChoose a name for your selection: e.g. “RPL22 non‐silent mutation”Hit the “Click here to confirm your selection” button. You will be redirected to the “Context analysis” tabOn the left menubar select: Start typing to select gene: RPL22L1Cells not chosen will be labelled as “Pancancer”, do you wish to subset Pancancer list by tissue type?: NoERBB2 amplification with its essentiality in breast and oesophagus cell lines was investigated in the “BROAD” project. The following steps were taken:The association of Navigate to the “Context analysis” tab and “Tissue/Coessentiality” subtab.On the left menubar select “Advanced selection” and then “Interactive Cell Line Selector”Hit the “Launch the interactive Cell Line Selector” button. You will be redirected to a new tab. Wait a little while until the interface is fully loaded.On the left menubar choose: Show cell lines with mutations from: No selection based on mutationSelection based on Copy Number Variation (CNV)?: YesWait until you see a black box below the slider. This might take a while.Choose a range of relative copy numbers to restrict the choice of genes with CNV: 1.1‐7.22Pick 1 or more genes, whose relative CN lies within the selected range: ERBB2Should cell lines contain a CNV in all (‘AND’) or at least one (‘OR’) of the above chosen genes?: ORCell culture growth properties: AllGenome stability: AllTissue of origin : Breast, OesophagusCancer type : AllColour cell lines by: TissueChoose a name for your selection: e.g. “ERBB2 amplification”Hit the “Click here to confirm your selection” button. You will be redirected to the “Context analysis” tab.On the left menubar select: Start typing to select gene: ERBB2Cells not chosen will be labelled as “Pancancer”, do you wish to subset Pancancer list by tissue type?: YesSubset Pancancer to a specific tissue of origin: Breast, OesophagusWRN helicase in colorectal cell lines was investigated in the “SANGER” project. The following steps were taken:1Navigate to the “Context analysis” tab and “Tissue/Coessentiality” subtab.2On the left menubar select “Advanced selection” and then “Interactive Cell Line Selector”.3Hit the “Launch the interactive Cell Line Selector” button. You will be redirected to a new tab. Wait a little while until the interface is fully loaded.4On the left menubar choose: aShow cell lines with mutations from: No selection based on mutationbSelection based on Copy Number Variation (CNV)?: No selection based on CNVcCell culture growth properties: AlldGenome stability: MSIeTissue of origin : Colon/RectumfCancer type : AllgColour cell lines by: TissuehChoose a name for your selection: e.g. “MSI in colorectal”5Hit the “Click here to confirm your selection” button. You will be redirected to the “Context analysis” tab.6On the left menubar select: aStart typing to select gene: WRNbCells not chosen will be labelled as “Pancancer”, do you wish to subset Pancancer list by tissue type?: YescSubset Pancancer to a specific tissue of origin: Colon/RectumThe association of microsatellite instability (MSI) with the essentiality of the P < 0.01). It is possible to add these edges; however, users should avoid adding confidence 1 edges  for tissues that also have low median score to avoid including high number of false‐positive edges.Lenient threshold  is generally applied for “Tissue” context in which there is likelihood of a large number of confounding factors , therefore a higher dynamic range of essentiality score distributions. In some cases, tissue contexts will have a very high dynamic range and median essentiality score can be less than 0.2 even though the association is of high statistical confidence  is generally applied for “Cancer type” contexts .Stringent threshold  is applied when the tested context is a single mutation in a single gene in a single tissue/cancer type.CENs that are used in consort with PPI integration are generated with a medium threshold to identify all possible enriched complexes.The network tab of CEN‐tools offers advanced options to users, which can be used to apply a range of thresholds to identify the most CENs for their study. In this study, we have used a multiple thresholding system depending on how many confounding factors one would expect within the group being tested and the purpose of the study as detailed below:Users should note that these thresholds are recommendations and they may choose to use a different thresholding method depending on the requirement.https://cancer.sanger.ac.uk/cell_lines). The cell line was cultured in DMEM/F12 media (Life Technologies) supplemented with 10% heat‐inactivated (50°C for 20 min) FBS, 20 μg/ml blasticidin and penicillin–streptomycin at 37°C with 5% CO2. Logarithmic growth phase of the cells was maintained by passing the cells every 2–3 days. Cells were tested and found to be mycoplasma free.The Cas9‐expressing A375 cell line was obtained from the Sanger Institute Cancer Cell Line Panel  Kit: CLS‐010G). To establish a clonal reporter line, 8 days post‐transduction, cells were individually sorted into 96‐well plates using FACS (MoFlo XDP). The clonal cells were further cultured for 3 weeks, and 48 individual clones were tested for their expression of GFP. Five clones exhibiting different levels of GFP were selected for expansion. The selected clone exhibited the highest constitutive expression of GFP when grown in culture supplemented with 10% serum and the highest loss of GFP signal when transduced with a single gRNA targeting 6 cells/well. Treated cells were detached from culture plates using EDTA, washed twice with PBS supplemented with divalent ions and analysed using flow cytometry. All flow cytometry was performed on a Cytoflex flow cytometer. 10,000 events per sample was acquired for each sample. Live cells were gated using forward and side scatter. GFP was excited at a wavelength of 488 nm and emission detected using a 530/30 band pass filter; BFP was excited at 405 nm and the emission detected using a 450/50 band pass filter. Analysis was performed using FlowJo software (Treestar).Drug treatments and gRNA transductions were performed on six‐well culture plates with 1 × 10Conceptualisation: SS, EP; Data curation, methodology, formal analysis, investigation, validation: SS, CD; Visualisation, software: SS, PW; Software: CD; Writing—original draft: SS, CD, PW; Writing—review & editing: EP, GJW. Supervision: SS, EP, GJW; Resources and funding acquisition: GJW, EP; Project administration: SS, EP.The authors declare that they have no conflict of interest.AppendixClick here for additional data file.Table EV1Click here for additional data file.Table EV2Click here for additional data file.Table EV3Click here for additional data file.Table EV4Click here for additional data file.Review Process FileClick here for additional data file.


Natural products are the ideal basis for the design of novel efficient molecular entities. Podophyllotoxin, a naturally occurring cyclolignan, is an example of natural product which displays a high versatility from a biological activity point of view. Based on its unique chemical structure, different derivatives have been synthesized presenting the original antitumoral properties associated with the compound, i.e., the tubulin polymerization inhibition and arising anti-topoisomerase II activity from structural modifications on the cyclolignan skeleton. In this report, we present a novel conjugate or hybrid which chemically combines both biological activities in one single molecule. Chemical design has been planned based in our lead compound, podophyllic aldehyde, as an inhibitor of tubulin polymerization, and in etoposide, an approved antitumoral drug targeting topoisomerase II. The cytotoxicity and selectivity of the novel synthetized hybrid has been evaluated in several cell lines of different solid tumors. In addition, these dual functional effects of the novel compound have been also evaluated by molecular docking approaches. Podophyllum, presents a tubulin interaction at the colchicine binding site that inhibits its polymerization, destabilizing and preventing the formation of microtubules required for cell division [In recent decades, the potential bioactivity of natural products has become well-known and well-characterized. Hence, the singularity of these compounds on the mechanisms of actions is based on three main characteristics: chemical skeletons, chiral structures and molecular complexity ,2. Moreodivision . The effdivision .As occurs with most natural products, structure–activity relationship (SAR) studies on the cyclolignan skeleton have been previously reported. From these reports, etoposide emerged as a new drug derived from podophyllotoxin that presented a particularity in its activity. The structural changes performed on the cyclolignan skeleton to obtain etoposide modified its mechanism of action, with it becoming an inhibitor of DNA-topoisomerase II (Topo-II) . The cheHowever, it is not only these structural changes that attribute novel properties to podophyllotoxin derivatives. The chemical skeleton of this cyclolignan has been widely modified, including different functional groups and heterocycles ,9. From Exploring the properties of the inversion of the configuration at position C-7 of podophyllotoxin structure, a huge structure–activity relationship has been carried out, taking hybridization as a tool and combining the cyclolignan skeleton with other compounds with different pharmacological activities, and obtaining interesting results on the bioactivity ,25,26,27Drug discovery must be firstly supported by rational design, but also by a complete subsequent evaluation of the activity. In this context, cytomic and proteomic techniques are essential to confirm the success of the design ,29,30 anAccording to the previous experience of the group with cyclolignans and taking into account that protein–drug interaction requires a difficult optimization, we have planned a new family of hybrids, named biscyclolignans, that combine, in a single molecule, both structure requirements to directly target tubulin and Topo-II, taking advantage of the duality of the skeleton of podophyllotoxin.O-demethylepipodophyllotoxin as Topo-II inhibitor and the other from podophyllic aldehyde as tubulin polymerization inhibitor  T.S.Ying, family Berberidaceae) by a previous procedure optimized by us [1, was purified (40%), both cyclolignan fragments could be obtained.Podophyllotoxin, the natural product, is the starting material of both cyclolignan fragments present in the final conjugate. The first step in the synthesis is the isolation of podophyllotoxin from commercial resin of ed by us . Once th2 [On one side, for the podophyllic aldehyde rest, the γ-lactone ring of podophyllotoxin was opened in basic conditions and the carboxylic acid was esterified with a methyl group. Oxidation under Swern conditions led to the podophyllic aldehyde, 2 ,34.O-demethylation at the C-4′ and epimerization of the C-7 position of podophyllotoxin, 1, followed by replacement by an aromatic diamine-type linker, gave rise to the other fragment of the hybrid, 3, in a one-pot reaction [2 was condensed with the 7β-cyclolignan 3 through an imine-type bond, obtaining the final 7β,9-biscyclolignan 4 , colorectal (HT-29) and breast cancer (MCF-7) cell lines. The cytotoxicity against them was evaluated at different concentrations. Several observations about the behavior of the compounds can already be highlighted in this assay, as shown in For this study, the podophyllic aldehyde, 2, was more potent in MG-63 cell line, while the MCF-7 cell line was slightly affected by this compound, and HT-29 was practically unaffected after 24 h. These results of our lead compound are in accordance with previous works and confirm the selectivity of this compound in comparison with the natural product podophyllotoxin, 1 [3, there were two cell lines very sensitive to this compound, HT-29 and MCF-7, while the osteosarcoma cell line MG-63 presented a reduced affectation compared with the other two. These differences in behavior show that both precursors are well-differentiated in their activity, as expected from the structural–activity relationship of the cyclolignans [4. This compound showed remarkable activity in the osteosarcoma cell line, being more potent than its precursors in this cell line. With these initial results, we observed the efficiency of the hybridization in cyclolignans and the potential of the new conjugate 4, which improved the activity of the precursors in the osteosarcoma cell line.The evaluation of cytotoxicity across the three human tumoral cell lines displayed differences at 24 h incubation for all the tested compounds. Podophyllic aldehyde, toxin, 1 . Regardiolignans . MoreoveApoptosis triggering is an important approach for determining the pattern of cell death mediated by the compounds. In this case, annexin V was used as a marker in flow cytometry for the determination of the cells in apoptosis stage, in combination with propidium iodide (PI), which detects late apoptosis and cell necrosis . The ana2, showed a modest apoptosis effect, which is in accordance with our previously reported studies where we observed that the initiation of inhibition of tubulin polymerization in this compound and podophyllotoxin, 1, did not show total cell death until longer incubation times [These analyses gave interesting results for the tested compounds. The first compound, podophyllic aldehyde, on times .+ cells. The other cell lines tested only increased apoptotic cells by approximately 10% compared to the control, although dot-plots with different cell distributions were observed on the HT-29 and MCF-7 cell lines.The most affected cell line by this precursor was MG-63 with a 63.85% of annexin3, since a strong variation compared to the control was observed, as shown in 2. Finally, the most outstanding results were obtained by the final conjugate 4. MCF-7 cell line, whose viability was not affected by compound 4, showed a cell distribution related to that of compound 3 by flow cytometry. This fact indicates that activity related to topoisomerase inhibition is present in this conjugate. Although this result is important, the values obtained for the MG-63 cell line draw much more attention where almost all the cells are in apoptosis (94.68% annexin+ cells), with a behavior closer to that of aldehyde 2. This particular value shows that the combination of both cyclolignan moieties in the same structure triggers an intense cellular response that could be mediated by the addition of the two mechanisms of action involved in cyclolignan structures.Interesting results were obtained for the 7β-cyclolignan precursor Since the inhibition of tubulin polymerization and the inhibition of Topo-II have a critical role in the cell cycle, we evaluated this to get more insights about the potency of the compounds tested.Bearing in mind previously reported studies, the cell cycle arrest by podophyllotoxin and non-lactonic cyclolignans such as podophyllic aldehyde, is reached in the range of µM and sub-µM concentrations ,12. The As can be observed in 2, and this tendency is confirmed in this study. The final conjugate also shows increased biological activity in this colorectal cancer cell line, which may be attributed to tubulin inhibition activity.HT-29 cell line was usually the most affected by other compounds synthetized by our group ,13,31, l4 was the most potent in arresting the cycle at the G2/M phase. These results suggest that the final conjugate 4 is able to act simultaneously in both targeted proteins, reaching our objective of merging both mechanisms of action in a single molecule.Finally, the MG-63 cell line seems to present a higher disruption of the cell cycle even under µM range. In this cell line, both precursors have shown activity below μM range and the final hybrid 4 can act as an inhibitor for the targeted proteins. As described in the methods section  and docking calculations have been carried out in order to obtain structural evidence that compound  section , we set ock-VINA .Podophyllotoxin has been described to occupy the colchicine binding site. This pocket is embedded in the β-subunit, close to the interface with α−tubulin and GTP binding site. It is thought that ligands that occupy the colchicine binding site inhibit tubulin dimerization via dimer destabilization, thus preventing the required contacts of the protomers from forming the microtubule ,38.4 for which ΔG = −12.5 kcal/mol. Interestingly, our results predict that the binding energy of 4 is even higher than that of podophyllotoxin, the reference compound.In 4 occupies the colchicine site. These results suggest that the compound 4 is able to hinder the formation of the dimer and the subsequent polymerization of the tubulin to form microtubules better than the precursors. This is a consequence of the larger size of compound 4, which fits in the dimerization interface, interacting with both α-tubulin and β-tubulin  fits in the β-tubulin strand placing the trimethoxyphenyl residue in the inner part of the pocket while the four fused rings are located closer to the interface of the two protomers , thus justifying the decreased dimerization energy where 4 is bound to tubulin, and also its higher binding energy.Podophyllotoxin .Additionally, DNA-topoisomerase αII isoform has been studied to further explore the complementary activity of the new conjugate  studies , two eto4 was highly similar to the one of etoposide. Likewise, for these compounds, 1 and 2, lower values were obtained for the interaction with the DNA and, especially, with the protein. When the simulation of etoposide was carried out, a higher value for the interaction with the DNA was obtained. This result agreed with previously reported studies of this compound, where it was described that etoposide presents a high affinity to the nicked DNA strand [As expected, podophyllotoxin and podophilic aldehyde have obtained lower energy values (ΔG) than etoposide, since the inhibition of Topo-II is not their pharmacological target; however, the energy found for the new hybrid A strand . The strTaking etoposide as the reference for Topo-II inhibition, it can be observed that the molecule is located in the middle of the DNA break. In addition, the dimethoxyphenyl ring interacts with the protein through a hydrogen bond between the main-chain of S462 and the hydroxyl at C-4 position. This interaction seems to be responsible for the interaction with the protein. As expected, podophyllotoxin and podophyllic aldehyde do not present an optimal disposition for interaction either with DNA or with protein, which justifies the lower values obtained in the calculations carried out . In addi4, whose binding free energy is equivalent to that for etoposide, adopts an optimal disposition for interaction with DNA, remaining perfectly intercalated between the two strands resulting from the break. Regarding the interactions with the protein, it interacts with two basic residues of the protein , thus leading to a stabilization of the complex. This fact justifies the considerable increase in the energy of the ligand–protein interaction that compound 4 presents with respect to the other compounds.Conjugate O-demethylation in C-4′ [2 and 3 integrated in the structure of the final hybrid 4, the new conjugate presented the best values in viability, improving the precursors’ results on the osteosarcoma cell line. This is an important result that displays how the two structures may contribute to the final activity of the conjugate and shows the potential of the novel dual conjugate. The extensive flow cytometry analysis confirms the expectations of the viability results.A combination of two different podophyllotoxin derivatives in a single entity has originated a novel hybrid compound which shows, simultaneously, the dual mechanisms of action associated with cyclolignans. The chemical requirements for tubulin and Topo-II inhibition were successfully included in the same molecule. This novel hybrid combines the best modifications carried out in the non-lactonic podophyllotoxin derivatives  with the in C-4′ . The che+), the anti-topoisomerase activity is represented by an alteration in the cellular distribution in the cytometer graphic. Final compound 4 presented a total distortion in the distribution of cells in the dot plot according to the dual behavior implied in the conjugate, reaching the highest annexin+ value among the tested compounds. Furthermore, cell cycle inhibition was an important parameter to determine the activity of the compounds and the dual behavior of conjugate 4, showing the highest potency in cell cycle arrest with a disruption mediated by the addition of two mechanisms: tubulin and topoisomerase inhibition. From the in c evaluation, the remarkable selectivity found in the tumor lines tested should be noted, where the best results were obtained in the osteosarcoma line compared to the other solid tumor lines tested. These results are interesting because osteosarcoma is particularly aggressive, presenting a high risk of metastasis [The evaluation of cell death effect reveals two different behaviors for these compounds. While tubulin inhibition is reflected by an increment in apoptotic cells  spectrometers in CDCl3 using the residual solvent signal as reference. Chemical shift (δ) values are expressed in ppm, followed by multiplicity and coupling constants (J) in Hz.1) was isolated from commercial resin of P. emodi following the protocol previous described by us [Podophyllotoxin (ed by us .2) was synthesized from podophyllotoxin as described in our previous works [Podophyllic aldehyde  in dry CH2Cl2 (2.5 mL) stirring with NaI  for 15 min at room temperature (rt) and N2 atmosphere. MeSO3H  was dropped while the reaction was maintained in an ice bath. The mixture was stirred at rt for 5 h, then N2 was bubbled and the solvent was evaporated. The raw reaction was dissolved in dry THF (6 mL) and p-phenylenediamine  and Et3N  were added, stirring the mixture for 19 h at rt. The crude product was diluted with EtOAc and the solution was washed with saturated aqueous NaCl, dried over Na2SO4, filtered, and the solvent was removed under vacuum. The crude product was chromatographed on silica gel, eluting with 30% EtOAc/CH2Cl2 to yield compound 3 .7β-Cyclolignan 4: A mixture of aldehyde 2 , anhydrous MgSO4  and amine 3  was dissolved in CH2Cl2 (3 mL) and stirred at rt for 3 d. The reaction mixture was diluted with CH2Cl2, filtered and concentrated under vacuum. The crude product was chromatographed on silica gel, eluting with 20% EtOAc/CH2Cl2 to yield compound 4 . See 7β,9-Biscyclolignan 2, 37 °C). All media and supplements were purchased from Sigma .The human colorectal adenocarcinoma , the human breast cancer  and the human osteosarcoma  cell lines were grown in adherent monolayer culture in Dulbecco’s Minimum Essential Medium (DMEM) medium for HT29 and MCF7 cells and RPMI medium for MG-63, and both were supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% L-glutamine and 1% penicillin/streptomycin in a humidified atmosphere  and 5 μL of the propidium iodide and incubated for 15 min at room temperature in the dark. After this time, 400 μL of 1X annexin-binding buffer was added. For data analysis of the annexin+/− and PI+/− ratios, the Infinicyt™ software  was used. By representing viability (% annexin− cells) on the vertical and concentrations of the compound on the horizontal one, curves of viability were obtained.To determine cell growth inhibition, early and late apoptosis, as well as cell death, the annexin V/propidium iodide assay  was applied. A total of 200,000 cells were seeded in 35-mm cell culture dishes  and allowed to settle for 24 h. Then, serial dilutions of the chemical compounds  were added to expose the cells for 24 h. After the incubation period, cells were detached by trypsinization and collected into flow cytometry tubes together with the supernatants. After three washing steps with PBS , 1X annexin-binding buffer was added to re-suspend the cells at a 1 × 10To study the cell cycle of HT-29, MCF-7 and MG-63 cells, a solution containing ribonuclease (RNase), propidium iodide (PI) and detergent was added to the cells. As described for the apoptosis assay, cells were collected into flow cytometry tubes and washed three times with PBS . Then, 200 μL of the solution mentioned above was added to each tube and incubated for 10 min at room temperature in the dark.In both flow cytometry assays, data acquisition was performed in a FACS Canto II flow cytometer  using the FACS Diva software . For data analysis, the Infinicyt™ software  was used.4 in the pockets occupied by podophyllotoxin and etoposide in 1SA1 and 5GWK, respectively. For the latter case, two ligand molecules were included in the simulation, replacing the two molecules of etoposide resolved in 5GWK.Our models for tubulin dimer and Topo-II were based on the PDB 1SA1 and 5GWK ,38, resp+ and Cl- were added to the system to account for a 0.15 mol/L KCl concentration. Coordinates of the Hydrogen atoms were generated with CHARMM [Once the complexes were prepared, they were placed in the center of a cubic water box large enough to contain the protein and at least 10 Å of solvent on all sides. To mimic the intracellular conditions, Kh CHARMM  using sth CHARMM , the CHAh CHARMM ,47, and h CHARMM  to accouh CHARMM  algorith4 were assigned using ParamChem [To calculate the docking energy corresponding to each of the ligands, the coordinates of the protein and DNA chains of last frames of the MD simulations were extracted for a subsequent docking stage and carried out using Autodock-Vina . As expearamChem ,51. The aramChem .4, was synthesized and evaluated. Using this natural product as a starting material for preparing the two fragments of the compound, we obtained a novel conjugate that combines the structural requirements for both anti-tubulin polymerization and inhibition of Topo-II activities. The extensive biological analysis carried out with the compounds showed a different behavior for each precursor, and those different behaviors were also observed in the conjugate. The final hybrid 4 achieved the best profile in the viability studies and the highest values in apoptosis induction and in cell cycle arrest at G2/M phase, improving the precursors’ properties. Additionally, the final conjugate had a cytotoxic selectivity in osteosarcoma cells compared to other tumor cell lines. These assays were complemented with molecular docking studies to confirm that the two protein inhibition activities can be involved in the mechanisms of action of the novel conjugate. These results are promising, showing the potential of this 7β,9-biscyclolignan 4. Further studies that include more structure–activity relationships are currently in progress.In this study, a new conjugate derived from podophyllotoxin, the 7β,9-biscyclolignan


Rhopalurus junceus is a scorpion belonging to the Buthidae family that finds its habitat in Cuba. This scorpion is known by the common name of “Blue Scorpion”. The venom is used on the island of Cuba as an alternative cure for cancer and, more recently, in the research of active components for biomedicine. Recently, the venom has been tested in several studies to investigate its effects on cancer cell lines, and the initial results of in vitro studies demonstrated how this poison can be effective on certain carcinoma cell lines . The aim of this review is, therefore, to describe the effects of the venom on carcinoma lines and to investigate all anti-cancer properties studied in the literature. The research was conducted using four databases, Pub Med, Scopus, EBSCO, and Web of Science, through the use of keywords, by two independent reviewers following the PRISMA protocol, identifying 57 records. The results led to a total of 13 articles that met the eligibility criteria. The data extracted for the purpose of meta-analysis included the IC50 of the venom on carcinoma cell lines. The results of the meta-analysis provided a pooled mean of the IC50 of 0.645 mg/mL , with a standard error (SE) = 0.045, p < 0.001. The analysis of the subgroups, differentiated by the type of cell line used, provided insight regarding how the scorpion venom was effective on the cell lines of lung origin  with a pooled mean of IC50 0.460 mg/mL  SE (0.087) p < 0.001. The results described in the literature for in vitro studies are encouraging, and further investigations should be carried out and deepened.The  Rhopalurus junceus is a scorpion belonging to the Buthidae family that finds its habitat in Cuba. The venom is used in the Cuban islands as an alternative cure for cancer and, more recently, in the search for active components in biomedicine. This scorpion is known by the common name of “Blue Scorpion”, and Escozul is a diluted solution of its venom.+- and K+-ion channels in neuroblastoma cells. Other studies from the same research group investigated the toxic potential of the venom aimed at cancerous cell lines, noting an efficacy directed toward epithelial cancerous cell lines [The first article present in the international scientific literature in the medical field, which investigated the anti-tumor properties, is a communication by Garcia-Gomez et al. 2011 [ll lines ,4.Rhopalurus junceus (blue scorpion).There are currently no literature reviews on the systemic effects of this poison. In light of these discoveries and in view of the ever increasing need to identify chemotherapies in the neoplastic field, in this review we aimed to summarize the results of the studies of the last decade, inherent in the anticancer effects, of the venom of The study was conducted with reference to the guidelines described by the PRISMA  . The matAfter an initial selection phase, in which the records were identified in the databases, potentially suitable articles were qualitatively assessed with the aim of identifying any useful information that described the effects and potential of the blue scorpion venom.Scientific studies that investigated the therapeutic effects of scorpion venom extracts were considered potentially eligible—in particular, all studies that investigated the effects of the poison on normal and carcinomatous cell lines. Secondly, we also researched those articles that could have any information on the composition of the poison, on its bioavailability, and on potential in vivo effects or in animal models, conducted in recent years (10 years), published in English, and indexed in international databases of medicine.Rhopalurus junceus, starting in 2011. The potentially eligible articles were finally subjected to a full-text analysis in order to verify their use for a qualitative and quantitative analysis. The inclusion and exclusion criteria applied in the full-text analysis are as follows:We included all those studies that reported data on the effects on cancer cell lines; the composition of the extracted venom; the bioavailability; the teratogenic, toxic, and therapeutic effects.The studies were excluded if there was no reported relevant data in the biomedical field, if they were not written in English, or if they were not published in international medical journals.We decided to choose articles published in the last 10 years because researchers began to investigate the possible use of the poison of The articles were identified using the electronic databases, Pub Med, Scopus, Web of Science, and EBSCO, and the bibliographical references of the articles were consulted to potentially identify further studies to include.The research of the studies was conducted between 15 July 2020 and 30 July 2020.The following search terms were used in Pub Med, Scopus, EBSCO, and Web of Science searches: Rhopalurus junceus , Vidatox  . To compThe reviewers responsible for the research and screening of the records were two reviewers (M.D. and D.S.). The search was conducted independently, and the keywords and databases to be used were preliminarily agreed upon. A third reviewer, to the end of the search for potentially eligible studies, decided on any doubtful situations (G.T.).Rhopalurus junceus venom on cancer line cells  was researched.The screening was performed by reading the title and the abstract to eliminate any records not related to the subject of the review. In the case of the doubt of the individual reviewer, the full text of the article was read. The articles obtained were subjected to full-text analysis by the two reviewers (13 articles), from which those eligible for the qualitative analysis and inclusion in the meta-analysis for the result were identified. The results sought by the two reviewers were the following: Primary outcome—the effects of 50 values were calculated for each individual study in order to estimate the pooled mean of the venom’s effect on the cancer cell line was calculated for the meta-analysis.For the quantitative analysis, following the extraction of the data of the included studies, a statistical analysis of the data was conducted, which included a meta-analysis of the effects of the venom on the cell lines on which it was effective (primary outcome). A subgroup analysis was also conducted (the groups differed by the type of cell line tested). The subgroups analyzed were as follows: cervix, lung mammary, gland/breast, and colorectal adenocarcinoma. The mean and 95% confidence intervals of the ICI2), using the Open Meta-Analyst software. If the measurement turned out to be greater than 50%, the heterogeneity rate was considered high. The results of the meta-analysis were represented by a forest plot.The protocol with which the meta-analysis was performed was based on the recommendations of the Cochrane Manual for Systematic Reviews of Interventions. We decided to use Open Meta-Analyst version 10  as the meta-analysis software. The presence of heterogeneity was evaluated by calculating the Higgins index , we identified 57 records. With the use of the EndNote software, the duplicates were removed, resulting in 19 records. With the inclusion and exclusion criteria applied, we maintained 13 articles, of which only 7 dealt with the effects of scorpion venom on cancer cell lines and only 4 studies reported the data used in the meta-analysis. All selection and screening procedures are described in the flow chart, shown in The studies included for qualitative analysis were the following: Garcia-Gomez et al. 2011 , Diaz-GaWe included seven studies in the quantitative analysis: Diaz-Garcia et al. 2013 , Giovann50 of the venom (mean and standard deviation), the number of times the test was repeated, the method of extraction of the poison, any other results obtained, and information on the article . The results are summarized in The extracted data included the type of cell lines tested, the ICFrom The method of collecting the poison was identical in all four studies, performed with electrical stimulation, filtered and centrifuged, and separated from the supernatant before finally being stored at a temperature of −20 degrees centigrade. In Diaz-Garcia et al. 2015 , the ven50 without the standard deviation, which was, therefore, not usable for the purpose of meta-analysis [The studies presented in analysis .I2, equal to 99.02%. The reasons for this heterogeneity are found in the different cell lines tested with the poison and, in some studies, the fraction of the poison used.The risk of bias within the studies was assessed by the first reviewer (M.D.) and for the four studies they were relatively low; the risk of bias between studies was also low as the four studies were performed by the same research group with a similar study design. Despite everything, there were differences that make the results of the studies heterogeneous with each other; in fact, the heterogeneity between the results stands at I2 equal to 99.35%, lung I2 equal to 99.3%, and breast I2 equal to 93.54%. In addition, the high heterogeneity was confirmed by the graphic analysis of the confidence intervals of the forest plot, which demonstrates the low overlap in all the subgroups.To minimize the heterogeneity in the results, we decided to perform a subgroup analysis , the subgroup analysis demonstrated a very slight reduction in heterogeneity with values for the subgroup: cervix Statistical data analysis was performed using Open Meta-Analyst version 10 software .I2 equal to 99.702% with Q = 5030.957 df 15. p <0.001, and a random effect model was applied. The results shown in 50 was obtained at 0.645 mg/mL , standard error (SE) = 0.045, p < 0.001.The meta-analysis showed an absence of heterogeneity with 50 0.917 mg/mL  SE (0.060) p < 0.001 I2 99.35%;Subgroup Cervix: pooled mean of IC50 0.460 mg/mL  SE (0.087) p < 0.001 I2 99.3%;Subgroup Lung: pooled mean of IC50 0.699 mg/mL  SE 0.030 p < 0.001 I2 93.54%;Subgroup mammary gland/breast: pooled mean of IC50 mg/mL 0.890  SD (Standard Deviation) 0.020.Subgroup colorectal, adenocarcinoma: ICThe analysis of the subgroups reports the following results :SubgroupDiaz-Garcia et al. 2013 [The effects on cell lines of pulmonary origin were confirmed in a 2015 study in which the venom, fractionated into its components (five fractions according to their weight), showed different efficacies. The FI fraction induced a decrease in the cell viability in MRC-5 more than in A549. The FII and FIII did not show differences between cancer and normal cells. The FIV and FV differences were significantly lower .Rhopalurus junceus scorpion venom was able to induce apoptotic cell death against murine breast cancer cells in vitro and effectively inhibited mammary tumor progression in mice [The effects on the breast cell line also came from a 2017 study by Diaz-Garcia et al. , who rep in mice .Another consideration is to be made on the ineffectiveness described by Giovannini et al. 2017 , for theSynergistic effects were found instead between blue scorpion venom (0.5 mg/mL) in a HeLa cancer cell line when combined with 5-fluorouracil (0.5–500 µM), cisplatin (3.13–25 µM), or doxorubicin (3.13–25 µM) in an experiment conducted by Yglesias-Rivera et al. 2019 .A recent study published in 2020 also reported the effects on glioblastoma cell lines, indicating that a venom concentration of 50 μg/mL was capable of decreasing the viability of T98G cells at levels significantly lower than the previous studies conducted on different cell lines .Studies on the molecular components of the venom conducted by Rodriguez-Ravelo et al. investigated the different composition of scorpion venom in different geographical areas of Cuba from five sites , identifying no substantial difference in the composition . A subseThis poison causes few adverse symptoms on humans. Diaz-Garcia et al. 2015 found caseinolytic, gelactinase, and hemolytic activity with a toxic effect on the gastrocnemius muscles with the release of CK and blood LDHA only at high concentrations . Again, in this study they identified that low molecular weight fractions (<4 kDa) induced significant cytotoxicity in A549 cells (lung cancer) while high molecular weight proteins (45–60 kDa) were responsible for the hyaluronidase activity and the toxic effect against MRC-5 .Through a pharmacokinetic study on mice carrying tumors, we identified that the venom was rapidly eliminated from the body and had a greater localization for tumor tissues; moreover, the intravenous route compared to the oral one appeared to increase the bioavailability of the drug . LagartoRhopalurus junceus venom as an antitumor agent have not been published and are not currently present in international medical scientific journals. Di Lorenzo et al. 2012 reported a case report on a patient with gastric cancer who felt the benefits of the venom, with an improvement in the quality of life and a reduction in pain as well as an unexpected reduction in prostate-specific antigen (PSA) [Clinical trials on the use of en (PSA) .The venom’s mechanism of action may be associated with peptides that recognize sodium and potassium channels, and the venom’s selective cytotoxic effect could also be associated with its effect on inducing apoptosis via the mitochondrial pathway . There aRhopalurus junceus venom by mechanisms associated with the increased expression of apoptotic genes, such as Bax, Noxa, and Puma; the mechanism of action involved the upregulation of the bax, p53, and caspase 3  genes; the downregulation of the bcl-2 genes likely as a result of the upregulation of p53. This included the activation of caspases, chromatin condensation, and the formation of apoptotic nuclei, in Diaz 2013 [On the other hand, research demonstrated that cell lines, such as Hela and MDA-MB-231, were prone to cell death when treated with The increase in p53 led to an upregulation of bax and caspase 3 and to a downregulation of bcl-2. The increase in the bax/bcl-2 ratio activated the mitochondrial pathway of apoptosis with the release of cytochrome c (Cyt c) and the activation of caspase cascades , which led to the proteolytic breakdown of important cytoplasmic and nuclear substrates .− and K+ ions, while variations in the concentration of Ca+ varied the shape and volume, allowing the neoplastic cell to invade the surrounding tissues [The role of ion channels in the tumor adhesion, proliferation, and invasion processes is known . The int tissues .There was a confirmation that the venom of the blue scorpion led to a reduction in the expression of ki-67 and CD31, whose overexpression was associated with tumor angiogenesis.− and K+, such as KCl and water, which is mediated by an increase in intracellular Ca2+ ions, which leads to cell shrinkage and allows the cell to invade brain tissue [These processes occur through the interaction of ion channels with changes in the cell morphology and volume. In particular, this was demonstrated to be the mechanism of the growth and invasion of glioma cells, mainly through the electrochemical efflux of Cln tissue .R. junceus could act against neoplasms could be through three mechanisms: a mitochondrial apoptotic action through the intrinsic pathway with an increase in the expression of p53 and an increase in the bax-BCL-2 ratio; the blocking action of the Na K and Cl canal, which would act on the concentration of intracellular calcium with a reduction in the capacity of variation of the shape and volume and, therefore, of tissue invasion; an influence on the CD 31- and Ki-67-mediated neoangiogenesis processes [Therefore, the mechanism of action with which the poison of rocesses .The histological examination of tumor mass in studies on animal models also highlighted how the scorpion venom induced an extensive necrosis in the tumor compared to the tissue from the evidence of the untreated control group .There was also an antimicrobial effect carried out by the presence of phospholipase and hyaluronidase in the venom with antibacterial action .The analysis of the articles included in the quantitative assessment confirmed that the blue scorpion venom was more effective on cell lines of epithelial glandular origin with the exception of the latest study reported by Lozano-Trujillo on glioblastoma lines.50 (venom concentration that causes 50% reduction in cells) of 0.645 mg/mL. The analysis of the subgroups showed that the venom had a greater efficacy in IC50 against cell lines of lung origin  with a pooled mean of IC50 0.460 mg/mL compared to mammary glandular cell lines  with a pooled mean of IC50 0.699 mg/mL. All subsequent studies tended to confirm the first cell line study conducted by Diaz-Garcia et al. 2013 [The meta-analysis of the results provided an average pooled ICThe cell lines that were not affected by the venom were those of hematopoietic origin: U937 (histiocytic lymphoma), K562 (chronic myelogenous leukemia), Raji (Burkitt’s lymphoma), and certain non-neoplastic lines: MDCK , Vero , and BALB/3T3 .50 75 μg/mL) [The next steps to be taken in light of the results obtained must be directed toward a greater confirmation of the efficacy on cell lines in which the preliminary data reported an efficacy at very low concentrations  , and theRhopalurus junceus is cytotoxic toward cancer cell lines of epithelial origin at a concentration given by the meta-analysis equal to 0.645 mg/mL. There were no data in the scientific literature showing that the homeopathic alcoholic dilution version (Escozul) was effective for both in vitro and animal models. Blue scorpion venom could represent an interesting source of chemotherapy drugs and its properties should be further investigated.The evidence from the literature demonstrated how the venom of the scorpion