Cancer cell lines are a defined population of cells, originally sourced from tumour tissue, that can be maintained in culture for an extended period of time. They are a critical laboratory-based model, and are frequently used to unravel mechanisms of cancer cell biology. In all cells, gene activity is in part regulated by DNA methylation, the epigenetic process by which methyl groups are added to DNA. In this study, we demonstrate that at a global level, DNA methylation profiles are globally well conserved, but we identify specific sites that are consistently more methylated in tumour-derived cell lines compared to the original tumour tissue. The genes associated with these common differentially methylated regions are involved in important cellular processes and are strongly enriched for epigenetic mechanisms associated with suppression of gene activity. This study provides a valuable resource for identifying false positives due to cell culture and for better interpretation of future cancer epigenetics studies.DNA methylation is a heritable epigenetic mark that is fundamental to mammalian development. Aberrant DNA methylation is an epigenetic hallmark of cancer cells. Cell lines are a critical in vitro model and very widely used to unravel mechanisms of cancer cell biology. However, limited data are available to assess whether DNA methylation patterns in tissues are retained when cell lines are established. Here, we provide the first genome-scale sequencing-based methylation map of metastatic melanoma tumour tissues and their derivative cell lines. We show that DNA methylation profiles are globally conserved in vitro compared to the tumour tissue of origin. However, we identify sites that are consistently hypermethylated in cell lines compared to their tumour tissue of origin. The genes associated with these common differentially methylated regions are involved in cell metabolism, cell cycle and apoptosis and are also strongly enriched for the H3K27me3 histone mark and PRC2 complex-related genes. Our data indicate that although global methylation patterns are similar between tissues and cell lines, there are site-specific epigenomic differences that could potentially impact gene expression. Our work provides a valuable resource for identifying false positives due to cell culture and for better interpretation of cancer epigenetics studies in the future. DNA methylation is a stable and somatically heritable epigenetic modification. In mammals, DNA methylation involves attachment of a methyl group to a cytosine, usually in the context of a CpG dinucleotide. Methylation patterns are established during early embryogenesis and play a key role in cellular differentiation, lineage commitment and maintenance of cell identity ,2. In soAlthough cancer cell lines have been an indispensable and a very widely used model for cancer biology studies for decades, they often do not fully represent the epigenetic state that exists in vivo ,17,18. IHere, we provide the first genome-scale methylation profiles of metastatic melanoma tumour tissues and the corresponding cell lines derived from these tissues using reduced representation bisulfite sequencing (RRBS) . RRBS isRRBS was used to establish genome-wide DNA methylation maps of melanoma tissues (53T and 60T) and their derived cell lines , producing a total of 101.4 million 100 bp sequenced reads that were then mapped to the GRCh37 reference human genome . We analr = 0.96, r = 0.87, As expected from previous analyses of human cell lines and somatic tissues , the metWe observed that both the cell lines were globally hypermethylated as compared to their corresponding tumour tissue. In both cases, we identified 10% higher median methylation in cell lines compared to tumour tissues  in the comparison between 53T and 53C and 13,089 DMFs in the comparison between 60T and 60C . All DMFConsistent with the global methylation patterns, we found that a much higher proportion of DMFs were hypermethylated in the melanoma cell lines. Compared to 53T and 60T, 88% and 83% of the DMFs were significantly hypermethylated in the cell lines, respectively . The metp-value < 0.01, SUZ12 and EZH2 . However, this is consistent with known inter-tumour heterogeneity of methylation levels. A recent whole genome bisulfite sequencing study which profiled nine cell lines from different tumours reported global methylation levels in those lines that ranged from 41.8% to 65.4% .In this study, we identified 783 regions that showed common differential methylation between both the analysed pairs of cell lines compared to their tumour tissue of origin and 94.1% of these regions were hypermethylated in the cell lines. In a previous study, RRBS analysis was performed between embryonic stem cells (ES) and ES cells differentiated in vitro into neural precursor cells (NPC). The global methylome was significantly correlated between the ES cells and in vitro derived NPCs (rho = 0.81). However, a much higher number of CpGs (~8%), that were unmethylated in ES cells, became methylated in NPCs, and a relatively smaller proportion of CpGs (2%) that were methylated in ES cells, became unmethylated in NPCs . FurtherPrevious studies have identified common hypermethylated loci in multiple cell lines, when compared to tumour tissue . Here, wDNA methylation establishes and stabilises cellular phenotypes by maintaining gene expression states, which means that specific DNA methylation patterns are characteristic of particular cell types, lineages and phenotypes . AlthougWe discovered that the genes associated with the common site-specific methylation differences between cell lines and their corresponding tumour tissues are very strongly enriched for the repressive H3K27me3 mark. Polycomb Group (PcG) proteins including Polycomb Repressive Complexes, PRC1 and PRC2 are widely recognized to mediate gene silencing of developmental genes and also play a role in repressing genes in cancer ,58. One It was reported that for a set of genes highly enriched with H3K27me3, CGI promoters were aberrantly hypermethylated in cancer cells but in not in normal cells . AlthougWe have analysed two pairs of tissues and their corresponding derivative cell lines, and it could be argued that this is a low sample number. However, such materials  are very rare, and to our knowledge, this study provides an RRBS map of tissues and matched derivative cell lines for the first time, for any tumour. Importantly, in both of our pairs independently, we observed consistent patterns of methylation, and overall conservation of global methylation, consistent with site-specific hypermethylation in the cell lines compared to the tissues. These results further indicate a common pattern of methylation in cell lines, as compared to tumour tissues in this study. A potential limitation is the absence of gene expression data for these samples. However, our hypothesis was that gene expression is a consequential event to genetic and epigenetic alterations, and we primarily wanted to investigate the nature of conservation and changes in DNA methylation, due to the establishment of cells in in vitro culture. Future studies with gene expression profiles of matched tissues and derivative cell lines will provide additional insight into the changes in expression program, which are due to establishment of cells in in vitro culture.The New Zealand melanoma (NZM) tissue samples and their derivative cell lines used in this study were from surgical samples of two patients diagnosed with metastatic melanoma, coded as NZM53 and NZM60. For this paper, the NZM53 and NZM60 cell lines are designated 53C and 60C, respectively, to distinguish them from their tumour tissues of origin, 53T and 60T, respectively. The original tumour samples were obtained with written consent from each patient under the guidelines and specific approval of the Auckland Area Health Board Ethics Committee, New Zealand.According to comprehensive molecular profiling of NZM cell lines , SequenoDNA was extracted from freshly cut sections of formalin-fixed paraffin-embedded (FFPE) melanoma tissue with the QIAamp DNA FFPE tissue kit, using xylene to dissolve the paraffin . The twoMelanoma cells were isolated from pathologically-confirmed metastatic malignant melanomas and long-term cell cultures were established as previously described . For thiWe used reduced representation bisulfite sequencing (RRBS) to map genome-wide DNA methylation as we have described previously ,69,70,71As previously described, the sequenced RRBS reads were quality checked using FastQC (RRID:SCR_014583) and processed using bioinformatics tools developed in house ,30. The identgeneloc program of the DMAP tool [https://genecards.weizmann.ac.il/geneloc/index.shtml, accessed on 7 April 2017), super-enhancers , CTCF binding sites  and specific repeat classes .We used our in-house Differential Methylation Analysis Package (DMAP) to identify RRBS fragments that were differentially methylated between the tissues and their derived cell lines . BrieflyMAP tool . We usedWe used Metascape (RRID:SCR_016620)  and EnriIn conclusion, our sequencing-based genome-scale analysis demonstrates a global conservation of DNA methylation patterns following the establishment of in vitro cell cultures, which had been derived from matching human metastatic melanoma tissues, and therefore provides supporting evidence in favour of using cell lines as in vitro models for methylation studies in cancer epigenetic research and drug discovery. Our results also provide an impetus for conducting similar studies in other cancer types to assess methylation patterns. We also demonstrate region-specific epigenomic differences between the cell lines and the corresponding matching tumour tissues, and reveal novel genomic features of these regions, thus providing a valuable resource for cancer methylome studies to reduce false-positive candidates from the epigenetic effects of cell culture. This resource will allow better interpretation of cancer epigenetics studies in future.


Graft-versus-host disease (GvHD) of the skin is a severe allo-immune reaction and complication following allogeneic stem cell transplantation. Over the past years, intensive pre-clinical research has led to an improved understanding of the pathophysiology of acute and to a lesser extend chronic GvHD. This has translated into the approval of several new agents for the treatment of both forms of GvHD. This review summarizes the most recent advances in underlying pathomechanisms, clinical trials and newly approved agents for GvHD, with a special focus on skin involvement. Graft-versus-host disease (GvHD) remains a frequent complication following allogeneic stem cell transplantation (HSCT). Despite better pretransplant conditioning, prophylaxes and graft manipulation, treatment refractory GvHD accounts for 5-30% . Skin isWhile up to 50% of patients after allogeneic HSCT are affected by any type of GvHD , cutaneoPathomechanisms driving GvHD need to be discussed in light of host and donor characteristics. Host predisposing factors refer to the inflammatory milieu at the time of HSCT. A certain degree of skin toxicity, i.e. inflammation or damage, will inevitably follow treatment with chemotherapeutic agents. Upon infusion of donor stem cells, the presence of chronic viral infection and an altered host microbiome may further contribute to a favorable host environment facilitating GvHD.via pathogen-associated molecular patterns (PAMP) and danger-associated molecular patterns  released from injured cells  classification system, acute GvHD may occur as classic aGvHD within the first 100 days post HSCT, or persistent/recurrent or late-onset aGvHD, reflecting clinical signs of acute GvHD beyond 100 days following HSCT. The latter is typically observed after reduction of immunosuppressive therapy. Chronic GvHD can be divided into the classic form, without signs of aGvHD and the overlap syndrome, combining features of chronic and acute GvHD .In aGvHD the extent or severity of organ involvement falls into 4 stages, and categories are defined for skin, gut and liver , 11. ForWhile highly desired, there is currently no available tool to predict the occurrence of GvHD safely. A standard GvHD prophylaxis for patients following transplantation consists of a combination of immunosuppressive agents . For manThe choice of treatment greatly depends on the grade and localization of GvHD. For locally restricted forms of grade I acute skin GvHD, the initial treatment approach will consist of a topical steroid . Low-potA well-established therapeutic option is extracorporeal photopheresis (ECP) . ParticuAntithymocyte globulin (ATG) is also commonly used in patients with steroid-refractory aGvHD. While some older studies have presented data that may indicate a less than expected effect and a higher rate of infections, more recent studies have shown better outcomes [summarized and discussed in ]. RecentThe lack of an internationally accepted primary endpoint in clinical trials of cGvHD makes the comparability of previously published trials difficult and the chosen primary endpoints may not automatically translate into a clinical benefit. While this issue has been increasingly addressed in the past years and we are now starting to obtain data from RCTs and discussions on defining a gold standard for the endpoint of these trials . FurtherFirst-line therapy in the case of a mild cGvHD are topical steroids, mostly higher potency steroids . Cutaneous atrophy or local infections may occur during long-term use of topical steroids; topical calcineurin inhibitors (tacrolimus and pimecrolimus) may spare steroids in some of these patients .Systemic steroids alone or in combination with calcineurin inhibitors are required for severe cGvHD . HoweverIn a more recent randomized control trial in cGvHD, ECP was added to the standard of care in a first line setting of patients newly diagnosed with moderate or severe cGvHD . At 28 wIn an extensive review of 27 studies, where ECP was used in steroid-resistant or dependent cGvHD, the mean response rate for cutaneous cGvHD was 74% . A compaNotably, a recent systematic review of adverse events in second- and third-line treatments for acute and chronic GvHD revealed a lower incidence of infectious adverse events in acute GvHD and lower number of grade 3-5 adverse events in cGvHD in patients on ECP compared to any pharmaceutical management highlighting the safety aspect of this form of treatment .Another therapeutic approach is the use of Rituximab. The anti-CD20 monoclonal antibody is widely used especially in sclerosing cGvHD of the skin, fasciitis, musculoskeletal cGvHD. Clinical responses up to 70% have been described . In addiIn the past years a number of novel agents have been tested in advanced clinical trials in acute or chronic GvHD and some of these drugs are now available for clinical use. While none of these trials was specifically designed only to assess the effect on skin GvHD, a large percentage of subjects included in these trials were affected by skin GvHD Table 1.Ruxolitinib, an inhibitor of the Janus kinase (JAK) 1/2, was approved for the treatment of steroid-refractory acute GvHD in 2019. JAK signaling plays an important role in regulating the underlying immune cells relevant for GvHD such as dendritic cells, macrophages as well as B- and T cells. Most importantly, there is preclinical evidence to suggest that inhibition of the JAK pathway does not negatively impact the GvL effect [as summarized in ].Approval followed a successful open-label, single arm, phase 2 trial (NCT02953678) . Of the A number of IL-2R antibodies, namely daclizumab, basiliximab and inolimomab, have been tested in clinical trials for the treatment of aGvHD. These agents generally showed some level of clinical activity in treating aGvHD, especially in cases where skin or gut was involved, , 47. HowThe TNFa antibodies infliximab and etanercept have both been investigated for the treatment of aGvHD. Studies with infliximab have delivered mixed results with a modest effect but a generally high rate of infectious complications, , 51. TheCombining TNFa with IL-2R blocking may be attractive. A prospective trial assessing patients treated with basiliximab in combination with etanercept had an ORR at day 28 of 91% and a two-year OS rate of 55% . in patients with steroid refractory GvHD , 56. Sev. In a pr. Most re. ORR at In addition to the well-established first-line treatment with prednisolone and CNIs, several new agents have recently been approved for the treatment of cGvHD.In 2017 ibrutinib was approved as a second-line treatment for steroid-resistant cGvHD. Approval came following a successful phase I/II trial (NCT02195869). Forty-two patients with active cGvHD were enrolled who had previously failed 1 to 3 prior treatments. After a median follow-up of 13.9 months the best OR was 67% . NotablyIn addition to its positive clinical activity in aGvHD, there are data from a retrospective survey, which indicates that ruxolitinib leads to a high response and 6-month survival rates in heavily pretreated patients afflicted with chronic GvHD . Based oBelumosudil is a serine/threonine kinase inhibitor blocking the activity of Rho-associated coiled-coil kinase 2 (ROCK2). The efficacy of belumosudil was evaluated in a phase 2 randomized multicentre trial in cGvHD-patients who had receive 2-5 prior lines of therapy (NCT03640481). Primary endpoint of this study of the best ORR, which was 74% and 77% for belumosudil at 200 mg daily and at 200 mg twice daily, respectively . Of noteAn innovative cellular approach in the treatment of GvHD is the adoptive transfer of regulatory T cells (Tregs). Tregs are a subset of CD4 T cells with an immunosuppressive and immunoregulatory function , 66. TheMild acute GvHD of the skin can usually be controlled with standardized treatment protocols. Severe acute skin GvHD requires more aggressive conventional treatments, but newer approaches including cellular therapies are now available. Chronic forms of skin GvHD are the transplant physician’s enemies. Functional impairment and decreased quality of life need to be avoided strenuously. Marked improvement may be obtained with extracorporal photopheresis. Drugs targeting either B cells or the JAK-STAT pathway have opened new treatment perspectives. Regulatory T cells may reverse cGvHD of the skin, but data are still preliminary. It is encouraging that patients who have overcome cGVHD, experience a health status and quality of life similar to those HSCT patients without a history of cGvHD .All authors have contributed equally in designing the review article, writing and revising. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Compounds were more toxic to melanoma cells in vitro than commonly used anti-melanoma agent dacarbazine as measured by TUNEL assay. They induced both caspase-independent apoptosis evident by colocalization of TUNEL with endonuclease G (EndoG) and caspase-mediated apoptosis measured by colocalization of TUNEL with caspase-activated DNase (CAD). In addition, compounds 3 and 5 strongly induced oxidative injury to melanoma cells as measured by TUNEL colocalization with heme oxygenase-1 (HO1). Dacarbazine induced only caspase-independent apoptosis, which may explain why it is less cytotoxic to melanoma cells than compounds 3, 4 and 5.The discovery of chimeric anti-melanoma agents is reported. These molecules are potent growth suppressors of melanoma cells  Malignant melanoma (MM), also known as cutaneous melanoma, is a cancer that originates from melanocytes . MelanomThiazole-derived compounds are well-known among drugs, natural products and pharmacologically active synthetic molecules . Severalhttps://dtp.cancer.gov/). These compounds were tested for cytotoxicity at a single dose of 10 µM. Several compounds showed potent activity against several cancer cell lines, and some of them were particularly potent against melanoma cell lines. Eight of these chimeric compounds (50), total growth inhibition (TGI) and 50% lethal concentration (LC50) values. Based on these data, we found a very promising structure–activity relationship of these molecules against melanoma cell lines. Overall, amino-thiazole derivatives (1–7) [8) [50 values as low as 3.22 µM. Phenyl ethyl derivative (1) inhibited the growth of all the cell lines except SK-MEL-2. Phenyl substituted derivative (2) showed good activity against LOX IMVI and MDA-MB-435 cell lines with GI50 values of 3.22 and 3.49 µM, respectively. This molecule (2) showed moderate growth inhibition against M14 and UACC-62 cell lines with GI50 values less than 10 µM. Monosubstituted phenyl derivatives 3, 4 and 5 showed very significant activities against all the tested cell lines with GI50 values as low as 1.32 µM for SK-MEL-28. 2,4-Difluoro substituted phenyl derivative (6) is found to be a good growth inhibitor of LOX IMVI and SK-MEL-5 cell lines with GI50 values of 1.71 and 1.88 µM, respectively, while it failed to show any significant activity against other cell lines. 2,4-Dimethyl substituted derivative (7) is a potent growth inhibitor of all the tested cell lines of the melanoma panel. Among all the reported thiobenzamide-derived thiazoles [8) showed significant activity against the melanoma panel.In our effort to develop new methodologies to synthesize bioactive compounds –14, we hompounds  were selivatives , 1–7 [151–7) [8) . Fluoro hiazoles , only 3-2) showed weak TGI values (greater than 48 µM) but compound (6) did not show any useful TGI values against the cell lines . N-Phenylethyl derivative (1) had the TGI values 3.17 and 6.82 µM against LOX IMVI and MALME-3M cell lines, respectively, but did not show any significant TGI against any other cell lines (3) substituted phenyl derivative (3) showed good TGI value against LOX IMVI cell line and moderate values against M14, SK-MEL-28 and SK-MEL-5 cell lines. A similar pattern was observed for other compounds .Compound  showed potent LC50 values against LOX IMVI and MALME-3M cell lines. Based on the GI50, TGI and LC50 values, compounds 3 and 5 are the most promising agents in the series. Although our compounds were most potent against LOX IMVI cell line, these cells are semi-adherent, which limited our ability to fix these cells. Hence, for the proof of concept studies below, we used adherent cell lines SK-MEL-5 and SK-MEL-28.These potent compounds also showed significant LCntration . CompounN-Aryl compounds with a small hydrophobic substituent such as fluoro, methyl and trifluoromethoxy groups showed significant activities. 3-Hydroxy substituted phenyl group is an exception.Based on the anti-melanoma potency, we can deduce a structure activity relationship (SAR) of the compounds. Aliphatic substituted compounds did not show any significant anti-melanoma activity. 2) inhibited the growth of HFF-1 cells with an IC50 value of 21 µM, which was much higher than the GI50 values against some of the melanoma cell lines. Trifluoromethoxy-substituted compound (3) did not show any notable toxicity against the tested healthy cell line, which is very important for the further drug development of this compound. 3-Fluorophenyl derivative (5) was somewhat toxic to HFF1 cells. Compound 7 showed good tolerance to healthy cell lines with the IC50 value of 40 µM. Like compound 3, the positive control, dacarbazine, also did not show any notable toxicity against HFF1 cells. IC50 values of compounds 1 and 8 were not determined against the HFF-1 cell line for their low solubility in the growth media.To assess the potential use of lead compounds as anti-melanoma agents, we tested their toxicity for the normal human foreskin fibroblast (HFF-1) cell line taking into consideration that MM is usually surrounded by the cells of this kind. The effects of some of the potent compounds on this cell line were very encouraging . Phenyl-3 caused 23% apoptosis at 6 µM and 54% apoptosis at 12 µM (5) was a very potent apoptosis-inducing agent that caused 100% cell death at 12 µM concentration. Dacarbazine, the positive control, caused 22% apoptotic cell death at 200 µM concentration. This very high concentration effected 22% apoptosis, which shows the high potency of our novel compounds against melanoma cell line.After determining the selective toxicity of potent molecules, we determined their mode of action against the melanoma cell line. Apoptosis or programmed cell death is a physiological process to remove unwanted or damaged mammalian cells. We detected apoptosis mode of cell death by our potent compounds using flow cytometry-based Annexin V assay. Compound at 12 µM . 3-Fluor3, 4 and 5) were cytotoxic to melanoma cells , and the cytotoxicity seemed to be dose-dependent. Surprisingly, the cytotoxicity of dacarbazine was negligent, and at lower concentration, it even seemed to be cytoprotective. In all three compounds as well as in dacarbazine, the cytotoxicity at 10 µM was higher than at 5 µM concentration. Interestingly, the cytotoxicity of the two test compounds was higher than dacarbazine (our positive control). It did not reach statistical significance for compound 3 at 10 µM versus dacarbazine at 10 µM, but it was significant in two other compounds (4 and 5) versus dacarbazine at 10 µM. Based on these data, and taking them together with the selectivity towards melanoma versus non-melanoma cells, we concluded that our new compounds have promise as new anti-melanoma drugs. Immunostaining for major marker of oxidative injury, heme oxygenase-1 (HO1) was done to determine whether the test compounds induce oxidative injury. The measurement of mean intensity of colour, which illustrates the level of protein expression, showed it was induced by the exposure with the two out of three compounds . Contrary to HO1, no induction of CAD or EndoG was observed . Apparently, none of these compounds induced cell death endonucleases. Activation of endonucleases and their translocation to the nucleus is actually the last step when cell death becomes irreversible. It is often preceded by an oxidative cell injury that activates or aggravates the cell death mechanism. To measure whether our compounds induce oxidative injury, we chose to measure HO1 which is a well-established mechanistic marker of oxidative injury [a). Similar to the previous experiments, we observed strong elevation of HO1 in TUNEL-positive cells after exposure to the compounds (b). The elevation was dose-dependent. However, dacarbazine killed melanoma cells without induction of HO1. We next examined whether caspase-activated DNase (CAD), a known mechanistic marker of caspase-mediated apoptosis [3, 4 and 5). These compounds induced the colocalization of CAD with TUNEL indicating that these chemicals were capable of inducing caspase-mediated apoptosis in addition to the caspase-independent one (c). For some reason, no dose dependency was observed, the colocalization was at the same level at 5 and 10 µM. Apparently, the CAD/TUNEL association had an upper limit and reached a plateau. Unlike our compounds, dacarbazine did not induce any association of CAD with TUNEL. This suggested dacarbazine did not induce caspase-mediated apoptosis at the used concentrations. Thus, our compounds had two mechanisms while dacarbazine had only one mechanism of cell death in this model. To determine whether there may be an association between DNA fragmentation measured by TUNEL assay, and immunocytochemistry (ICC) of EndoG, the marker of caspase-independent apoptosis [d). The increase was dose-dependent, and dacarbazine at 5 µM did not show any effect, while our compounds even at 5 µM showed the increase of EndoG-TUNEL association. Overall, the effect of our compounds in terms of EndoG involvement seemed to be similar to that of dacarbazine; all three compounds and dacarbazine induced caspase-independent apoptosis.Apoptosis is usually associated with profound DNA fragmentation that makes the cell death irreversible. DNA fragmentation could be due to the increasing of nuclear DNA access to apoptotic endonucleases and the induction of these enzymes ,19. Thisma cells a,c, and ompounds b,c. Contobserved d,e. Appae injury ,21. Becaompounds b. The elpoptosis ,23, is cdent one c. For sopoptosis , and thepoptosis ,26 knownpoptosis . The colin vitro, we applied the fluorescent TUNEL assay combined with quantitative ICC for three markers: HO1 for oxidative cell injury, CAD for caspase-mediated apoptosis and EndoG for caspase-independent apoptosis. Our TUNEL data showed that compounds are more toxic to melanoma cells than dacarbazine. Two of three compounds (4 and 5) induced oxidative injury as measured by the increase mean intensity of HO-1. However, none of the compounds or dacarbazine induced apoptotic endonucleases. This is not surprising because apoptotic endonucleases are commonly present in the cell in sufficient amounts to be able to destroy DNA upon cell death without induction (which most likely would not be possible in a dead cell).Thus, to determine the mechanism of action of our compounds on melanoma cells Because TUNEL assay measures DNA fragmentation by endonucleases, we use ICC for two endonucleases, EndoG and CAD, to determine the mode of cell death. Our experiments showed that our compounds induce both caspase-independent and caspase-dependent apoptosis, as well as oxidative injury to melanoma cells. Contrary to our compounds, the currently used anti-melanoma drug dacarbazine induced only caspase-independent apoptosis. No caspase-dependent apoptosis or oxidative injury was observed after exposure of melanoma cells to the drug. The fact that compounds use three cytotoxic mechanisms instead of one explains why our thiazole compounds are more effective.50 values at low single digit µM concentration. Some of these compounds are non-toxic or less toxic to healthy HFF1 cell lines. Mode of action has been determined by flow cytometry and TUNEL assay. Potent molecules were found to be apoptotic and DNA-fragmenting agents. The mode of action studies determined that our new compounds simultaneously use three cytotoxic mechanisms to kill melanoma cells. In vivo anti-melanoma and ADMET studies will be reported in due course.In conclusion, we have discovered potent anti-melanoma agents by screening a panel of 50 compounds and eight of these compounds showed potent anti-melanoma properties with GIClick here for additional data file.


Cyclopeptides or cyclic peptides are polypeptides formed by ring closing of terminal amino acids. A large number of natural cyclopeptides have been reported to be highly effective against different cancer cells, some of which are renowned for their clinical uses. Compared to linear peptides, cyclopeptides have absolute advantages of structural rigidity, biochemical stability, binding affinity as well as membrane permeability, which contribute greatly to their anticancer potency. Therefore, the discovery and development of natural cyclopeptides as anticancer agents remains attractive to academic researchers and pharmaceutical companies. Herein, we provide an overview of anticancer cyclopeptides that were discovered in the past 20 years. The present review mainly focuses on the anticancer efficacies, mechanisms of action and chemical structures of cyclopeptides with natural origins. Additionally, studies of the structure–activity relationship, total synthetic strategies as well as bioactivities of natural cyclopeptides are also included in this article. In conclusion, due to their characteristic structural features, natural cyclopeptides have great potential to be developed as anticancer agents. Indeed, they can also serve as excellent scaffolds for the synthesis of novel derivatives for combating cancerous pathologies. Cancer has become an enormous burden in society and a leading cause of death in the world. Apart from aging, the risk factors of modern daily life such as smoking, unhealthy diet and mental stress also play significant roles in the growing incidence of cancer . AccordiFrom ancient times, taking Chinese Medicine as an example, people have been familiar with medicinal properties of natural resources and the benefits of using them to treat diseases . Hence, Escherichia coli) . According to the SAR assessment results, the N-methyl alanine residue was not the key functional group for the cytotoxicity of apratoxins [Apratoxins are a novel family of cytotoxic cyclodepsipeptides, and they were isolated from cyanobacterium e models . Mechanie models . Liu et e models  discoverl Sec61α . Furtherl Sec61α  found thyl group . Likewisry claim . This SAtivities . Apratoxratoxins . Overall66) isolated from sea hare, aurilides B (150) and C (151) were obtained from cyanobacterium L. majuscula. Both aurilides B and C were cyclic depsipeptides with potent in vitro cytotoxic activities. In H460 cells and mouse neuroblastoma (neuro-2a) cells, the LC50 values of aurilide B were found to be 0.04 and 0.01 µM, respectively whereas the LC50 values of aurilide C were found to be 0.13 and 0.05 µM, respectively. Moreover, aurilide B was demonstrated to induce microfilament network disruption against A-10 smooth muscle cells with an IC50 value of 2.9 µM. In an in vivo experiment, aurilide B was shown to deliver net tumor cell killing activity in the NCI’s hollow fiber assay [Unlike aurilide (er assay . A quanter assay . Collect152–154) were cyclic depsipeptides isolated from L. majuscula with anti-proliferative effects against several cancer cell lines [50 values of lagunamides A were found ranging from 1.64 to 6.4 nM. Lagunamide B was only tested for its cytotoxicities against P388 and HCT8 cell lines; its IC50 values were determined to be 20.5 and 5.2 nM, respectively. Subsequent studies revealed that the cytotoxic effects of lagunamides A and B was highly associated with the induction of mitochondrial apoptosis [50 values varied from 2.1 nM to 24.2 nM against a panel of cancer cell lines such as P388, A549, PC3, HCT8 and SK-OV3 [Lagunamides A-C (ll lines . Tripathpoptosis . As for d SK-OV3 .L. majuscula was homodolastatin 16 (155), which was a homologue of the potent anticancer agent dolastatin 16. Compared to dolastatin 16, homodolastatin 16 only exhibited moderate activities against esophageal (WHCO1 and WHCO6) and cervical (ME180) cancer cell lines with IC50 values of 4.19, 9.85 and 8.09 µM, respectively [Another cyclic depsipeptide identified from ectively .156) was another cytotoxic cyclic depsipeptide derived from Guamanian strain of L. majuscula. Other than cytotoxic activities against KB cells (IC50 = 0.04 µM) and LoVo cells (IC50 = 0.72 µM), lyngbyabellin A also exhibited specific cytoskeletal-disrupting effects to microfilaments in A-10 cells at concentrations ranging from 0.01 to 7.24 µM [157), F (158) and H (159) were also isolated from L. majuscula. They exhibited cytotoxicity against H460 and KB cancer cells with IC50 or LC50 values ranging from 0.1 to 0.4 µM [160) from Moorea bouillonii, a rather new cytotoxic member of the lyngbyabellin class. This compound showed a potent anticancer potential against the CRC HCT116 cells with IC50 value of 40.9 nM [Lyngbyabellin A ( 7.24 µM . To furt 7.24 µM . Similaro 0.4 µM . In the  40.9 nM . Althoug161), another cyclic depsipeptide, was first isolated from L. majuscula. In the H460 cell line, palmyramide A displayed merely a weak cytotoxicity (IC50 = 39.7 µM). However, in neuro-2a cells, palmyramide A led to the blockage of the voltage-gated sodium channel with an IC50 value of 17.2 µM [Palmyramide A ( 17.2 µM . As a re162) and B (163) were two cyclic depsipeptides isolated from two distinct collections of marine cyanobacteria obtained from Papua New Guinea. Hoiamide A was a potent bioactive cyclic depsipeptide found in the assemblage of Lyngbya majuscula and Phormidium gracile [+ influx in mouse neocortical neurons [N-terminal kinase (JNK) and caspase signaling pathways [50 values of 11.2 and 8.3 µM, respectively [Hoiamides A  was a cyclic dodecapeptide derived from L. majuscula in Papua New Guinea [165), another novel cyclic peptide congener of wewakazole, from cyanobacterium Moorea peoducens. As cyanobactins, the most prominent bioactivity of the two compounds is cytotoxicity. When testing against H460 cells, wewakazole B (IC50 = 1.0 µM) was more cytotoxic than wewakazole (IC50 = 10 µM). Besides, wewakazole B also exhibited cytotoxicity against MCF7 cells with an IC50 value of 0.58 µM. Different from wewakazole, wewakazole B was inactive in siderophore assay [Wewakazole  was isolated from L. majuscula. It is a cyclic lipopeptide with moderate cytotoxicity against several cancer cell lines. Hectochlorin showed an average GI50 value of 5.1 µM in the NCI-60 screening assay; the colon, melanoma, ovarian and renal cell lines were the most sensitive to this compound [50 values of 0.86 and 1.20 µM, respectively [Hectochlorin , a cyclic depsipeptide found in the process of a cytotoxicity-guided fractionation, was isolated from L. majuscula. The cyclic DMMC exhibited potent selectivity and cytotoxicity against the cancer cell lines HCT116, H460 and MDA-MB-435 with IC50 values of 0.02, 0.063 and 0.22 µM, respectively. Furthermore, a ring-opened form of DMMC was produced by means of base hydrolysis. Remarkably, the linear form of DMMC shared the same potency of cytotoxicity against the same panel of cancer cell lines . When acting against A10 cells, both cyclic and linear DMMCs disrupted the cellular microfilament networks and caused dramatic alterations in cell morphology, which were shown as the result of cellular apoptosis. Overall, these biological data indicated the potential anticancer efficacy of both cyclic and linear forms of DMMC [Desmethoxymajusculamide C (DMMC) ( of DMMC . The sig168), another cyclodepsipeptide isolated from L. majuscula, was first discovered in Singapore. The compound displayed in vitro cytotoxicities against human lymphoblastic leukemia (MOLT-4) and MCF7 cells with IC50 values of 32 nM and 4.0 µM, respectively [169) and C (170). Both compounds exhibited cytotoxicities when tested against MOLT-4  and MCF7  cells [Hantupeptin A (ectively . A subsey) cells . Furthery) cells ,250.L. majuscula were designated as laxaphycins A (171) and B (172). Laxaphycin B showed significant cytotoxicities against the CCRF-CEM drug-sensitive human leukemic lymphoblasts (IC50 = 1.11 µM), as well as the vinblastine-resistant subline overexpressing Pgp CEM/VLB100 (IC50 = 1.02 µM) and CEM/VM-1 subline with DNA topoisomerase II alteration (IC50 = 1.37 µM). However, laxaphycin A was found to be inactive towards these cell lines at 20 µM [50 values ranging from 0.19 to 1.0 µM. Furthermore, obvious biological synergism was observed on DLD1  and MDR leukemic cells in the presence of laxaphycins A and B. Similar to the compounds mentioned previously, laxaphycins B2 (173) and B3 (174) were also isolated from the same assemblage of the cyanobacteria. These two novel cyclic peptides, displayed merely moderate cytotoxicities against the three leukemic cell lines above [175) was a new member of B-type laxaphycins, and it was discovered from the marine cyanobacterium Hormothamnion enteromorphoides. This compound yielded growth-inhibitory activity against HCT116 (IC50 = 1.7 µM); synergistic effect was obtained when it was used in combination with laxaphycin A [Two cyclic peptides isolated from at 20 µM . Subseques above . Laxaphyphycin A .176) was a cytotoxic cyclic depsipeptide isolated in L. confervoides. From the MTT assay results, we noticed that this compound exhibited cytotoxicity against KB and LoVo cells with IC50 values of 0.97 and 5.24 µM, respectively [β-amino acid was an essential moiety for the bioactivities of obyanamide and its analogs [Obyanamide  was a macrocyclic depsipeptide with anticancer potential isolated from L. confervoides. Anti-proliferative activities of grasssypeptolide A were investigated in the U2OS (IC50 = 2.2 µM), HeLa (IC50 = 1.0 µM), HT29 (IC50 = 1.5 µM) and IMR-32 (IC50 = 4.2µM) cancer cell lines [177–179). The in vitro anti-proliferative effects of these compounds were evaluated in HT29  and HeLa  cells. According to the cell viability assay results, grassypeptolide C was 16-23 times more potent than grassypeptolide A. Moreover, both grasspeptolides A and C could induce cell cycle arrest at the G1 phase at relatively low concentrations ; they could also induce the G2/M phase arrest at higher concentrations . These two compounds have the capability to bind to metal ions such as Cu2+ and Zn2+, which may denote a partial mechanism of their cytotoxicities [180) and E (181), two novel members of the family, were isolated from the marine cyanobacterium Leptolyngbya sp. These two compounds were cytotoxic to HeLa cells  and mouse neuro-2a blastoma cells  [Grassypeptolide A (ll lines . Subsequxicities . The stuxicities . Grassypctively) . To prom182), a cyclic depsipeptide extracted from a bioassay-guided fractionation, was confirmed as a potent cytotoxin isolated from Lyngbya sp. in Palau. When cultured with KB cells, palauamide yielded significant cytotoxicity with an IC50 value of 13 nM [Palauamide  was a cyclic depsipeptide that was first isolated from the marine filamentous cyanobacterium Leptolyngbya sp. in Coiba National Park, Panama. The preliminary biological activity screening showed that this compound displayed potent cytotoxicity against NCI-H460 and mouse neuro-2a cells with LC50 < 17.9 nM. The evaluation against the NCI’s in vitro 60 cancer cell line panel highlighted its remarkable anti-proliferative response. From the flow cytometric analysis, coibamide A was shown to induce cell cycle arrest at the G1 phase [3 to 200-300 mm3. However, different degrees of weight loss were observed in the animal experiment. Such result implicated that targeted delivery and toxicity profile should be improved for drug development [Coibamide A (G1 phase . SubsequG1 phase  demonstrelopment .184–188) were five β-amino acid-containing cyclopeptides derived from the marine cyanobacterium Lyngbya sp. in Palau. These ulongamides exhibited moderate in vitro cytotoxicity against KB and LoVo cells with IC50 values around 1 and 5 µM [Ulongamides A-E , another cytotoxic cyclopeptide, was isolated from Lyngbya sp. in Palau. This compound yielded cytotoxicity against KB cells with an IC50 value of 0.63 µM [Ulongapeptin ( 0.63 µM . No furtOkeania sp. is the origin of the cytotoxic cyclodepsipeptide odoamide (190). As another new member of aurilide class, odoamide has been reported with a significant cytotoxicity against HeLa S3 cells (IC50 = 26.3 nM) and a moderate toxicity to brine shrimp [The marine cyanobacterium e shrimp . From the shrimp .191), a cyclic depsipeptide isolated from cyanobacterium Nostoc sp., was first discovered as an antifungal agent two decades ago. Subsequent studies indicated that cryptophycin 1 was also an anti-proliferative agent with significant antitumor activities [50 = 4 pM), SKOV3 (IC50 = 7 pM) and MCF7 (IC50 = 16 pM). Some recent reports demonstrated that cryptophycin 1 provided an anticancer potency ~100-fold higher than the mainstay anticancer drug paclitaxel; therefore, a great interest was immediately prompted to investigate the mechanism of action of this compound [Cryptophycin 1  is a cyclic depsipeptide isolated from the cyanobacterium of Symploca sp. This compound was found to have good selectivity against different types of cancer cells. For instance, largazole was less cytotoxicy to the nontransformed murine mammary epithelial MDA-MB-231 cells , but it was more susceptible to the invasive transformed human mammary epithelial (NmuMG) cells . Similarly, this cyclopeptide displayed higher inhibitory effect against U2OS transformed fibroblastic osteosarcoma  than NIH3T3 nontransformed fibroblasts  [50 values of 0.08 µM and 0.32 µM, respectively [Kip1 and telomeric repeat-binding factor 1 (TRF1) in vitro [Largazole ( > 8 µM) . Based o > 8 µM) . In the ectively . The in ectively . It is win vitro . In addiin vitro . While sSymploca sp. From Papua New Guinea led to the isolation of a novel cyclopeptide—symplocamide A (193). This compound showed potent cytotoxicity against H460 and neuro-2a cells with IC50 values of 40 nM and 29 nM, respectively. In fact, symplocamide A shares a structure very similar to protease inhibitors; thus, it can be served as a potent chymotrypsin inhibitor (IC50 = 0.38 µM). The role of protease inhibition in cancer therapy is worth to be studied. On the other hand, symplocamide A was reported to show moderate anti-parasite activities against malaria, Chagas disease and leishmaniasis [Studies on a cyanobacterium of maniasis . Solid pmaniasis .Symploca sp. in Palau, and they were named tasipeptins A (194) and B (195). These two compounds exhibited cytotoxicities against KB cells with IC50 values of 0.93 and 0.82 µM, respectively [Two other cyclic depsipeptides had been isolated from ectively . Due to ectively . It is bSymploca cf. hydnoides from Guam led to the isolation of a series of cyclic depsipeptides, veraguamides A-G (196–202). The results of several cell viability assays showed that veraguamides A-G displayed weak to moderate cytotoxicities against HT29 and HeLa cells with IC50 values varying from 0.84 to 49 µM and 0.54 to 49 µM, respectively. From the SAR assessments, the seven semi-synthetic analogs of veraguamide G explicitly denoted the sensitive positions of veraguamides that were responsible for their cytotoxic activities [Cytotoxicity-directed fractionation of cyanobacterium tivities .203), a novel cyclic depsipeptide containing four amino acid units, was discovered in a bioassay-guided investigation of Moorea producens, a species of filamentous cyanobacteria collected from Florida, USA. This compound displayed cytotoxic activities against H460 and mouse neuro-2a neuroblastoma cells with the EC50 values of around 1.89 × 10−5 µM [Floridamide ( 10−5 µM .204) was a highly toxic cyclic depsipeptide found in the cyanobacterium Rivularia sp. near the island of Vieques. It was found to be strongly cytotoxic against H460 cells with an IC50 value of 60 nM. As a member of the “kulolide superfamily”, the biological properties of viequeamied A were reported to be highly divergent [Viequeamide A (ivergent . The totivergent .Fungi are eukaryotic organisms with worldwide distribution and different habitats. It is estimated that biodiversity of fungi is between 2.2 to 3.8 million species around the world . Moreove205) was a cyclic tetrapeptide isolated from the fungi of Tolypocladium sp. obtained from the Great Lakes. The compound displayed predominant anti-proliferative and cytotoxic activities toward MIA PaCa-2 cells with GI50 and LC50 values of 5.3 and 22 nM, respectively. Moreover, as a potent HDAC inhibitor, 1-alaninechlamydocin induced apoptosis and cell cycle arrest at phase G2/M as well. HDAC inhibitory activity also primarily contributed to in vitro bioactivities of the compound [1-Alaninechlamydocin  showed moderate cytotoxicity. The compound exhibited anti-proliferative effect to K562 cells with the GI50 value of 6.8 µM and cytotoxicity to HeLa cells with CC50 value of 16.68 µM. However, human umbilical vein endothelial cells (HUVEC) was also sensitive to pseudoxylallemycin C (GI50 = 6.96 µM), which demonstrated low specificity of the compound [compound .207) and G (208) were two new members of cycloaspeptides—the ABA-containing cyclic pentapeptides discovered in different fungi. These two compounds were derived from the fungus Isaria farinosa, an inhabitant of Cordyceps sinensis obtained from Linzhi, Tibet. They showed inhibitory activities toward MCF7 cells with respective GI50 values of 18.7 and 15.2 µM respectively, which were comparable to the positive control 5FU (GI50 = 15 µM) [Cycloaspeptides F (= 15 µM) .1 (MA1) (209), a cyclopentapeptide with versatile biological activities, was originally isolated from the terrestrial plant fungus Aspergillus niger. Liu et al. investigated its anticancer mechanism of action in human prostate cancer PC3 (IC50 = 130 nM) aand LNCaP (IC50 = 90 nM) cell lines. Coexistence of apoptosis, necrosis and autophagy were all induced by mitochondrial damage, which illustrated the mechanism of MA1-mediated cell death in prostate cancer cells. In addition, stimulation of the AMP-activated serine/threonine protein kinase/mammalian target-of-rapamycin (AMPK/mTOR) pathway by adding oxidative stress or decreasing ATP could lead to MA1-mediated autophagy [A. tubingenesis also yielded MA1 with significant cytotoxicity against HeLa (IC50 = 9.48 nM) and P388 (IC50 = 132.9 nM) cell lines [1 is an early-discovered cyclic peptide with diverse biological properties and sufficient studies have demonstrated its bioactivities as well as mechanism of action. However, for further promotion to clinical stage, more supporting data in animal models shall be carried out. Another new member of the malformin class, designated as malformin E (210), was isolated from the endophytic fungus A. tamarii collected from the roots of Ficus carica. The compound also showed in vitro cytotoxic effects on MCF7 and A549 cells, with IC50 values of 0.65 and 2.42 µM, respectively. Moreover, malformin E also exhibited antimicrobial activities against several strains of bacteria and fungi [Malformin Autophagy . In humautophagy . Recentlll lines . Malformnd fungi .228), neo-N-methylsansalvamide (211), was isolated from the fungus Fusarium solani KCCM90040 derived from the potato in Korea. The new cyclic pentadepsipeptide was able to inhibit in vitro cell growth of A549, SK-OV-3, SK-MEL-2 (human melanoma) and MES-SA (human uterine sarcoma) cells with EC50 values ranging from 10.0 to 14.74 µM [N-methylsansalvamide and paclitaxel were also observed when acting against MES-SA, HCT15  and two multidrug resistance sublines (MES-SA/DX5 (human uterine corpus sarcoma) and HCT15/CL05). The EC50 values of paclitaxel added with 3 µM neo-N-methylsansalvamide were significantly decreased by several orders of magnitude [Another new analog of sansalvamide A (14.74 µM . Moreoveagnitude . As a re212), a cyclic nonapeptide, was isolated from Cyclindrocarpon sp. strain A101-96 during the period of a screening of new metabolites with cytotoxic effects from fungal extracts. Six cancer cell lines were assayed for its anticancer potential, while the compound only showed moderate cytotoxicity to colorectal adenocarcinoma (COLO-320), human leukemia (HL-60), L1210 and human T lymphocyte Jurkat cell lines with IC50 value around 11 µM. Neither antimicrobial or nematicidal effect was observed for cylindrocyclin A [Cylindrocyclin A  and B (214). Both compounds are octadecacyclopeptides with natural cyclic β hairpins. Gymnopeptides A and B displayed significant anti-proliferative activities against HeLa, A431 (human epidermoid carcinoma), T47D, MCF7 and MDA-MB-231 cells with IC50 values ranging from 18.0 to 88.4 nM and 14.0 to 44.3 nM, respectively [Studies on the mushroom ectively . Due to ectively . As a re215), a cyclic hexapeptide derived from the fungus Diaporthe toxica, was also an N-methylated derivative of the mycotoxin phomopsin A. The initial in vitro screening of cytotoxicity revealed its impact on viability and vitability of HepG2 cells (IC50 = 6.1 µM) [Phomopsin F ( 6.1 µM) .216–219), were purified from a basidiomycete identified as Pterula species. Pterides I and II showed remarkable cytotoxicity against P388 cell line with IC50 values of 41 and 40 nM, respectively. Nevertheless, compared to the first two compounds, pteratides III and IV were much less active  due to their much different structural discrepancy to pteratides I and II [Four cytotoxic cyclodepsipeptides, pteratides I-IV (I and II . Practic220), with entire composition of non-protein amino acids, was first isolated from the marine-derived fungus Aspergillus terreus SCSGAF0162  and B (222) were two bioactive cycloheptapeptides previously isolated from two different insect pathogenic fungi Cordyceps sp. BCC 1788 and 16173, respectively. The two cyclic peptides displayed anti-proliferative activities against KB, BC (human lymphoma), NCI-H187 and Vero  cells with IC50 values of cordyheptapeptide A measured as 0.78, 0.2, 0.18 and 14 µM, respectively, the IC50 values of cordyheptapeptide B as 2.0, 0.66, 3.1 and 1.6 µM, respectively. In addition, cordyheptapeptide A also exhibited moderate antimalarial activities [50 = 10.6 and 7.4 µM, respectively) and Ehrlich’s ascites carcinoma (EAC)  cell lines [223) and E (224), from a marine-derived fungus Acremonium persicinum in 2012. Cordyheptapeptide C displayed cytotoxicity against SF-268 and MCF7 with IC50 values of 3.7 and 3.0 μM, respectively. And the other one showed similar cytotoxicity against SF-268, MCF7 and NCI-H460 cell lines  [Cordyheptapeptides A  . However225) and B (226) were two cyclic heptapeptides extracted from the culture broth of the marine fungus Scytalidium sp. from the Bahamas. When incubated with HCT116 cell line, the two compounds displayed in vitro cytotoxicity with IC50 values of 2.7 and 11.0 µM, respectively. Tested in the NCI 60 cell line panel, scytalidamides A and B exhibited moderate cytotoxicity with mean GI50 values of 7.9 and 4.1 µM, respectively. The screening test also demonstrated the most sensitive cell lines were MOLT-4 (IC50 = 3.0 µM) for scytalidamide A and UACC-257 (IC50 = 3.0 µM) for scytalidamide B [Scytalidamides A (damide B .227), a cyclic pentadepsipeptide produced by the marine-derived fungus Zygosporium masonii, was shown to possess potent anticancer properties. Evaluated in the NCI cell line assays (median GI50 = 9.1 µM), the compound yielded significant in vitro cytotoxicity against central nervous system (CNS) cancer cell line SF-268 (IC50 = 6.5 nM) and renal cancer cell line RXF393 (IC50 ≤ 5.0 nM) with high selectivity [50 values between the natural and synthetic cyclic peptides when acting against SF-268 [Zygosporamide (ectivity . The tott SF-268 .228), a natural cyclic depsipeptide, was isolated first from a marine fungus of Fusarium sp. living on the seagrass Halodule wrightii. The compound showed cytotoxicity against several cancer cells lines, including HCT116, COLO 205 and SK-MEL-2 cells with IC50 values of 9.7, 3.46 and 5.84 μM, respectively [Sansalvamide A  and B (230) were two novel cyclodepsipeptides derived from the fungus Aspergillus clavatus. Xenograpsus testudinatus, a kind of hydrothermal vent crab, is the habitat where they found the fungus. Jiang et al. determined that both compounds suppressed the proliferation of HCC (hepatocellular carcinoma) cell lines  in a dose-dependent as well as time-dependent manner, with IC50 values around 32 μM. Compared with human normal hepatocyte, HCC cell lines, especially HepG2 cells, were more sensitive in clavatustides-induced cell proliferation suppression. Their proliferation inhibitory property was due to suppression of G1/S phase transition [Clavatustides A  was isolated from the fungus er 20 μM .232) and B (233), were isolated from the fungus Scopulariopsis brevicaulis, which was found in the marine sponge Tethya aurantium. Both compounds significantly inhibited viability of several cancer cell lines including Colo357 (pancreatic adenosquamous carcinoma), Panc89 (pancreatic tumor) and HT29 cells at a concentration of 14.87 μM [Two cyclodepsipeptides, scopularides A (14.87 μM . However234) was first isolated from the marine fungus Clonostachys sp. ESNA-A009. It was highly active against several cancer cell lines including LNCaP, SKBR3, HT29 and HeLa with the GI50 values on the order of 10−8 M [Leishmania [N-methylation on the amino acids of IB-01212 were found to enhance its cytotoxic effect, which were corroborating evidence for further discovery and synthesis of new analogs for anticancer drug development [The cytotoxic cyclodepsipeptide IB-01212  and B (236) were cyclopeptides isolated from the marine fungus Microsporum cf. gypseum in bryozoan Bugula sp. from Virgin Islands in the US. Both were shown to be potent HDAC inhibitors and demonstrated cytotoxicity especially against HCT116 cell line, with IC50 values of 1.12 and 15.82 μM, respectively [Microsporins A (ectively . To exteectively ,334.This review included the recently discovered natural anticancer cyclopeptides, which have been summarized and categorized according to their origins. An abundant number of cyclopeptides with diverging chemical and biological characteristics can be obtained from the aqueous and terrestrial portions of our nature. Cyclopeptides derived from natural sources have drawn great attention due to their high therapeutic potential for drug development. Today, many studies have shown that natural cyclopeptides are effective in the treatment and preventive measures of cancerous pathologies. Other biological activities such as antiviral, antibacterial and antimalarial effects have also been extensively observed whilst some are considered promising. Hence, detailed mechanism studies and structural modification of cyclopeptides are desperately needed for the development of cyclopeptide as therapeutic drug candidates. Nonetheless, the current research studies on the anticancer effects of natural cyclopeptides are just the tip of the iceberg. The natural cyclopeptides are undoubtedly a rich source as bioactive compound leads gifted from nature to benefit human welfare.


Very high risk neuroblastoma is characterised by increased MAPK signalling, and targeting MAPK signalling is a promising therapeutic strategy. We used a deeply characterised panel of neuroblastoma cell lines and found that the sensitivity to MEK inhibitors varied drastically between these cell lines. By generating quantitative perturbation data and mathematical modelling, we determined potential resistance mechanisms. We found that negative feedbacks within MAPK signalling and via the IGF receptor mediate re-activation of MAPK signalling upon treatment in resistant cell lines. By using cell-line specific models, we predict that combinations of MEK inhibitors with RAF or IGFR inhibitors can overcome resistance, and tested these predictions experimentally. In addition, phospho-proteomic profiling confirmed the cell-specific feedback effects and synergy of MEK and IGFR targeted treatment. Our study shows that a quantitative understanding of signalling and feedback mechanisms facilitated by models can help to develop and optimise therapeutic strategies. Our findings should be considered for the planning of future clinical trials introducing MEKi in the treatment of neuroblastoma. Only few targeted therapies are currently available to treat high-risk neuroblastoma. To address this issue we characterized the drug response of high risk neuroblastoma cell lines and correlated it with genomic and transcriptomic data. Particularly for MEK inhibition, we saw that our panel could be nicely separated into two groups of resistant and sensitive cell lines. Genomic and transcriptomic markers alone did not help to discriminate between responders and non-responders. We used signalling perturbation data to build cell line specific signalling models. Our models suggest that negative feedbacks within MAPK signalling lead to a stronger reactivation of MEK in MEKi resistant cell lines after MEK inhibition. Model analysis suggested that combining MEK inhibition with IGF1R or RAF inhibition could be an effective treatment and we characterised this combination using phosphoproteomics by mass-spectrometry and growth assays. Our study confirms the importance of quantitative understanding of signalling and may help plan future clinical trials involving MEK inhibition for the treatment of neuroblastoma. Neuroblastoma is the most common and devastating extracranial childhood solid tumour, accounting for 15% of all childhood cancer deaths. The 5-year survival rate is 75% overall, but it is below 45% for so-called high-risk neuroblastoma that represent about 40% of patients . TelomerThis makes RAS/MAPK pathway inhibition a promising treatment option for neuroblastoma, and ALK and MEK inhibitors are already being tested in early clinical trials . HoweverResistance to targeted therapies of signalling pathways are often mediated by feedbacks that re-wire or re-activate signalling. For example, resistance to PI3K/mTOR inhibition in breast cancer is often mediated by feedbacks that lead to activation of JAK/STAT signalling . SimilarWe report here how a more quantitative understanding of feedback mechanisms might help to optimise combinatorial treatment. We used a neuroblastoma cell line panel representing the class of very high-risk neuroblastoma, which we profiled for drug sensitivity, genomic and transcriptomic alterations. We observed strong differences in the sensitivity to MEK inhibition. To arrive at a mechanistic understanding of resistance to MEK inhibition, we generated systematic perturbation data and quantified signalling using data-driven models. By this we described qualitative and quantitative differences in feedback structures that might confer the observed robustness to MEK inhibition. We then identified potential combinations capable of sensitising highly resistant cell lines to MEK inhibition, and tested these combinations systematically.We collected a panel of 9 neuroblastoma cell lines  and performed molecular profiling of these cells , using live cell imaging and computing growth rates from confluency measurements . In thisTo get insights into the underlying mechanisms of resistance to the MEK inhibitor AZD6244, we selected 6 neuroblastoma cells lines that represented the spectrum of sensitivity to MEK inhibition . Using these cell lines, we performed perturbation experiments, in which we stimulated the cells by growth factors for 30 minutes, and additionally inhibited specific pathways for 90 minutes . After pWe designed the experiments such that they probe the AKT/mTOR and MAPK signalling pathways . SpecifiOverall, the perturbation experiments yielded 240 data points per cell line, which are visualised in a heatmap in To get further insights into this high-dimensional data set, we performed principal component analysis (PCA) on the perturbation data . The PCAWhen we applied hierarchical clustering on the cell line panel, SKNSH was clustered separately, suggesting that it has a very atypical response to the perturbations, with a generally very high response to all ligands, and an especially strong response to PDGF  bottom. To get further, more mechanistic, insights into potential resistance mechanisms, we used the perturbation data to parameterise signalling models. We applied our previously developed method that has been derived from Modular Response Analysis  to fit When starting with a canonical literature network see , we obtaAll extended models had similar, but different, parameters for the inhibitor strength. However, there is a strong interdependence of the inhibitor strength and link strength downstream of the inhibitor which render comparison between those link strengths in different cells difficult see . As all When inspecting the parameters for ligand-induced pathway activation, we noticed that they reflected a strong heterogeneity in ligand response between the cell lines. Reassuringly, they matched the expression of the corresponding receptors in many cases . The parIn contrast to the receptor-associated parameters, the strength of intra-cellular kinase paths are less variable, and most paths are comparable between cell lines . The mosApart from the RAF-mediated feedback, MAPK signalling is also controlled by receptor-mediated feedbacks. In the KELLY cell line, our modelling procedure extended the model by a negative feedback from S6K to IGFR that could then explain the strong accumulation of pMEK by IGF following AZD6244 treatment . ReceptoIn summary, the signalling parameters derived from the perturbation data by our models show that cell lines diverge in receptor expression and feedback regulation, with strong multi-layered feedbacks for some of the resistant cell lines.χ2 p<0.05), but did not improve the IMR32 model  that is more pronounced in the cell lines IMR32 and KELLY compared to the other cell lines modelled, including the most sensitive cell lines CHP212 and LAN6 . We also32 model . Interes32 model . This hiTo gain a more systematic understanding of the effect of MEK and IGFR inhibition on the signalling states of the cells, we generated deep (phospho-)proteomics profiles using tandem mass-tag (TMT) based mass spectrometry , 29. We When we investigated the phosphorylation of components of the MAPK pathway more closely, we found many RAF negative feedback/crosstalk sites to be down-regulated after MEK inhibition  in both cell lines . MEK1 S2Taken together, the proteomics data is coherent with the model that MAPK signalling in N206 is controlled by a dual feedback structure involving RAF and IGFR, whereas it is mainly controlled by a RAF-mediated feedback in IMR32. It furthermore supports the notion that treatment with MEK and IGFR inhibitors would show synergy in N206.Feedback regulation is often a central aspect for drug resistance that could be overcome by a vertical inhibition strategy, where an inhibition of an upstream node prevents pathway reactivation. Based on our models, we tested if the additional application of an inhibitor targeting the feedback nodes (RAF and IGFR) would sensitise resistant cells toward MEK inhibition . We quanWhen trying to overcome the model-derived strong ERK-RAF feedback found in all three cell lines with a combination of MEK and RAF inhibition we only found a synergistic effect for two of the three cell lines (N206 and KELLY), whereas IMR32 remained resistant and no synergy could be detected. We hypothesised that this observed resistance in IMR32 might be either because the vertical inhibition by MEKi and RAFi was molecularly not effective or that IMR32 might no longer depend on ERK signalling for survival and growth. To distinguish the former from the latter we decided to compare model simulation and measurements for perturbation effects of selected inhibitor combinations on pMEK and pERK in IMR32 and KELLY cells.Based on the model simulations, in both cell lines the vertical inhibition of MEK + RAF inhibitor was predicted to suppress MAPK signalling much stronger than MEK inhibitor alone or in combination with an ERK inhibitor. Moreover, the suppressive effect was predicted to be even more profound in IMR32 than in KELLY  left. WeAs both KELLY and N206 have strong multi-layered feedbacks , we alsoNeuroblastoma is a complex disease with distinct subtypes that display radically different outcomes, ranging from spontaneous regression in low-risk groups to only 50% survival of patients in the high risk neuroblastoma group. Mutations in RAS/MAPK signalling are a hallmark of high risk neuroblastoma, and also define a subgroup of patients with ultra-high-risk neuroblastoma and an even worse survival. Therefore targeted treatment might be a valid strategy to treat those patients. However, response to MEK inhibitors are very variable, and it is thus important to understand mechanisms of resistance and how to circumvent these.In this work, we explored how a more quantitative understanding of signalling can be used to design combinatorial treatments to counteract drug resistance. We used a panel of deeply profiled cell lines representing high risk neuroblastoma and showed that the response to MEK inhibitors is variable, with some cell lines responding at low doses in the nM range, whereas others are highly resistant. By using signalling perturbation-response data, we characterised the signalling network surrounding MAPK. Analysis of that perturbation data with the modelling framework of modular response analysis unveiled that MAPK signalling is controlled by a multi-layered feedback with variable strength. A central finding was that MEK-inhibitor sensitive cells are controlled by low feedbacks within the MAPK cascade, whereas a subset of resistant cell lines shows strong multi-layered feedbacks that may be causal for resistance. Simulation of cell-line specific models suggested that different combinations of inhibitors can be used to overcome resistance, and experiments could confirm these predictions in two out of three cell lines.Our work highlights that systematic perturbation data are a powerful source to probe intracellular signalling pathways. The connectivity of signalling pathways implies that minor quantitative alterations of the network can lead to many changes in response, not all of which alter the phenotype. In this work, we saw that multivariate analysis of the perturbation data alone was not fruitful to separate cell lines with respect to their drug sensitivity. In contrast, integration of data by models highlighted that variations of only a few links is enough to explain the differences between those cell lines. Modelling was therefore key to integrate the data and to unveil feedback loops as potential sources of resistance.In our work we used a maximum likelihood version of MRA, but there are multiple other methods that might be suited to reconstruct semi-quantitative signalling networks from perturbation data.  proposedDrug resistance to targeted therapies have been attributed to negative feedback loops in multiple tumours. Most importantly, sensitivity to MEK inhibitors is strongly influenced by a pathway-intrinsic feedback, where ERK phosphorylates RAF at multiple sites –17. ThisOur modelling analysis suggested that some neuroblastoma cell lines possess two major layers of feedback in MAPK signalling. One of these feedbacks is pathway-intrinsic (from ERK to RAF) and one is a feedback to the IGF receptor. Interestingly, different cell lines show different relative strength of feedbacks from ERK to RAF and IGFR, and simulations show that those require different strategies for vertical inhibition. For the cell line KELLY, modelling unveiled an extremely strong negative feedback from ERK to RAF. This suggests that a combination of MEK and RAF inhibitor will be more potent than a combination of MEK and IGFR inhibitor. In contrast, in the cell line N206, both feedbacks have similar strength, suggesting that both combinations might be potent. In line with these predictions, experiments showed that in KELLY indeed the combination of MEK and RAF inhibitors is much more potent to reduce growth compared to the combination of MEK and IGFR. In contrast, in N206 both combinations reduce growth.Our phospho-proteomics analysis shows that the combination of MEK and IFGR also has different effects in the two cell lines: Whereas it shows clearly synergistic effects of the combination in N206, there is no sign of synergy in IMR32. By aggregating the phosphoproteome to kinase activities using kinase enrichment scores, one can also get insights into the re-wiring of signalling after perturbation. In our case, it clearly shows how the re-activation of RAF after MEK inhibition is inhibited by the treatment with IGFR inhibitors, and IGFR and MEK inhibitors synergize in reducing AKT activity in N206. The phosphoproteome also showed that the dual treatment of IGFR and MEK manifests itself in synergistic downregulation of important proteins that are regulated by convergent signalling of MEK and AKT, such as MYCN and EIF4B.Interestingly, a third resistant cell line, IMR32, showed no response in growth to MEK inhibitor in vertical combination with either RAF and/or IGFR inhibitor on growth, even though it’s cellular ERK signalling was strongly responsive. This highlights that cancer cells might lose ERK-mediated cell cycle control, suggesting that coupling of cellular phenotype to signalling pathways is not necessarily strict , 41. To in vivo and whether they could explain relapses. Our description of the wiring of the RAS/MAPK pathway in neuroblastoma will support the design of clinical trials using combinatorial treatments to prevent or overcome therapy resistance. In addition, the framework described here could be used to analyse signalling in tumours of individual patients While it will be technically challenging to assess signalling network responses in tumour patients, ex vivo cultures—so-called avatars—could be an option createModrylation , i.e acthttps://www.genome.jp/kegg/pathway/hsa/hsa04010.html and https://www.genome.jp/kegg-bin/show_pathway?hsa04151) with intermediate nodes suppressed, the addition of the well documented ERK->RAF feedback and all receptors corresponding to RTK. Each cell line was fitted first on the literature network, then we extended the networks independently using a greedy hill climbing approach, until no significant link could be added. We then performed successive rounds of reduction to identify the non-significant links. Most removed links relate to receptor connections. Only three connections not related to receptors were removed during this procedure, each for one cell line only. To facilitate model comparison, these links were ultimately retained in the model, as otherwise the model parameters would not be comparable. Those models with final topology yielded similar values for the inhibition parameters so we generated new models with those parameters fixed to the mean value across all 6 models and re-fitted each cell line with inhibitor values fixed. With this fitting strategy the links between models became directly comparable as the non identifiability induced by the inhibitor parameters was removed  and blocked for 30 minutes in 1:1 PBS:Odyssey blocking buffer. The primary antibodies were incubated overnight at 4C one at a time and the corresponding secondary during the following day for 2h at room temperature in 1:1 PBST/Odyssey. We used the following primary antibodies: pIGF1R betaY1135/Y1136 1:1000 (CST 3024), pAKT S473 1:2000 (CST 4060), total MYCN 1:200 (Santa Cruz sc-53993) and pMEKS217/S221 1:1000 (CST 9154).Cells were grown to confluency for 3 days in full medium and treated with AEW541 10μM and/or AZD6244 10μM or control DMSO for 4h.For the proteomics and phosphoproteomics cells were grown to confluency for 3 days in full medium and treated with AEW541 106. The top 20 most intense precursor ions with charges from +2 to +6 were selected for fragmentation with an isolation window of 0.7 m/z. Fragmentation was done in an HCD cell with a normalised collision energy of 30% and analysed in the detector with a resolution of 45 000 (200 m/z), AGC target value of 105, maximum IT of 86 ms. We used the same parameters for phosphoproteome analysis with the exception of MS2 maximum IT that was set to 240 ms.We used an adapted version of the TMT workflow : samplesThe acquired raw files were analysed using MaxQuant v1.6.10.43 , with TMlimma package  A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. 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If you believe any reviews to be missing, please contact [LINK]Reviewer's Responses to QuestionsComments to the Authors:Please note here if the review is uploaded as an attachment.Reviewer #1: In this paper the authors investigate mechanisms of drug resistance in a panel of high risk neuroblastoma cells focusing on the ERK pathway that is frequently altered in high risk neuroblastoma. They use a combination of experimental perturbation data and an elegant mathematical method to reconstruct signaling networks in order to decipher molecular mechanisms of drug resistance. Surprisingly, rather that linked to genetic mutations drug resistance seems mediated by differential network topologies in particular different feedback loops. While not entirely new, this finding is an important contribution to the growing case that network adaptations can be responsible for drug resistance in cancer and that we need to look beyond genetic mutations. Overall, the paper is well conceived, and the combination of experimental work and mathematical modelling is convincing. I am happy to recommend publication, but have a few comments that should be addressed.Major commentsAs negative feedback loops are ultimately made responsible for the differences in drug resistance, it would strengthen these conclusions by showing more evidence of their direct involvement. For instance, one would expect phosphorylation sites to change that mediate these feedback in a manner that correlates with the feedback strengths. These data seem to be there, but summarizing them in a figure would make the message clearer. Also, mutants that break the feedback could be used to show that is really the feedback that mediates drug resistance.Fig. 6B. The authors claim a “strong synergistic effect of the combination of MEK and IGFR inhibitions on growth 291 in N206 but little in KELLY or IMR32.” This is not apparent from the figure. A formal measure of synergism should be used, such as the Chou-Talalay method or Loewe isoboles.Fig. 6D. These data are not very convincing. The triple drug combination does not seem to be more potent than the dual combinations. This figure need error bars and a proper statistical comparison.Minor commentsThe MYN amplification status of the cell lines should be reported.“A strong RAF-mediated feedback is a known resistance mechanism against MEK inhibitors , …” should also cite Sturm et al. 2010.“… a known nexus between AKT and MAPK signalling (?).” Please supply the citation.https://doi.org/10.1016/j.ccr.2014.07.015). However, here the authors associate the de-phosphorylation of this residue with lower MYCN protein levels. Would be worth discussing or mitigating this claim as it is only based on a correlation.MYCN phosphorylation on S62 has been linked to enhanced degradation using phosphosite mutants , at least for MEKi.- Supplementary Figure2: Which method was used to calculate the correlation? As mutation data are qualitative (or at least Boolean), Pearson’s correlation is maybe not the best method to apply here. The authors should add more details about this and perhaps try other methods, like non parametric tests, to infer association between IC50 and mutations.- Line 94-96: “To get insights into the underlying mechanisms of resistance to the MEK inhibitor AZD6244, we selected 6 neuroblastoma cells lines that represented the spectrum of sensitivity to MEK inhibition ”, the period at the end of the sentence is missing.- Page 9, line249: What does the (?) stand for?- In figure 5 part C legend: there is a typo on phosphopeptides- Supplementary Figure 3: Is it really adjusted p>0.95? In the text it is written 0.93 and 0.94 for 1000 most variable genes and GO signal transduction genes respectively. Please double check.- Supplementary Figure 4,5,9: color code is missing-Supplementary Figure 17: Gene-sets should be re-ordered according to the GeneRatio to better see the ranking.**********Have the authors made all data and (if applicable) computational code underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data and code underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data and code should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. 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Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript.If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. 27 Sep 2021AttachmentReviewer_response-2.pdfSubmitted filename: Click here for additional data file. 1 Oct 2021Dear Dr. Blüthgen,We are pleased to inform you that your manuscript 'Neuroblastoma signalling models unveil combination therapies targeting feedback-mediated resistance' has been provisionally accepted for publication in PLOS Computational Biology.Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. 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