Canine mammary gland tumors (CMGTs) are quite common in intact female dogs. In diagnosed cases, approximately 50% of mammary gland tumors metastasized. Chemotherapy is a practical treatment to increase the median survival time, but it has severe side effects and impacts on resistance. BmKn-2, an antimicrobial peptide derived from scorpion venom, has displayed anticancer effects in oral and colon human cancer cell lines. Consequently, BmKn-2 may be useful in the molecular treatment of CMGTs. This study investigated the effect of BmKn-2 on the proliferation inhibition, apoptotic induction and the related mechanisms of those actions in CMGT cell lines, metastatic (CHMp-5b) and non-metastatic (CHMp-13a). The experimental results showed that BmKn-2 effectively inhibited proliferation and induced apoptosis in both CMGT cell lines via Bcl-2 (B-cell lymphoma-2) down-regulation and Bax (Bcl2 associated X) up-regulation gene expressions. Therefore, BmKn-2 could be used as a candidate molecular treatment for CMGTs in the future.50) was 30 µg/mL for CHMp-5b cell line and 54 µg/mL for CHMp-13a cell line. The induction of apoptosis was mediated through Bcl-2 and Bax expression after BmKn-2 treatment. In conclusion, BmKn-2 inhibited proliferation and induced apoptosis in both CHMp-5b and CHMp-13a cell lines via down-regulation of Bcl-2 and up-regulation of Bax relative mRNA expression. Therefore, BmKn-2 could be feasible as candidate treatment for CMGTs.The most common neoplasms in intact female dogs are CMGTs. BmKn-2, an antimicrobial peptide, is derived from scorpion venom and has published anticancer effects in oral and colon human cancer cell lines. Thus, it is highly likely that BmKn-2 could inhibit CMGT cell lines which has not been previously reported. This study investigated the proliferation and apoptotic properties of BmKn-2 via Bax and Bcl-2 relative gene expression in two CMGT cell lines, metastatic (CHMp-5b) and non-metastatic (CHMp-13a). The results showed that BmKn-2 inhibited proliferation and induced apoptosis in the CMGT cell lines. The cell morphology clearly changed and increased apoptosis in a dose dependent of manner. The half maximum inhibitory concentration (IC Mesobuthus martensii Karsch [The most common group of neoplasms in intact female dogs is canine mammary gland tumors (CMGTs) ,2. The mi Karsch . Previoui Karsch ,15. BmKni Karsch ,17. Theri Karsch . In the i Karsch  and conti Karsch . The Bcli Karsch . Especiai Karsch . Thus, ti Karsch ,24. The BmKn-2 peptide was derived from China Peptides Corporation . The molecular weight of the peptide was 1448.81 Daltons, and its polymer unit was 13. The net charge of BmKn-2 was +2 and its hydrophobicity was 56.23% [2 before use [The CMGT cell lines were obtained from the Laboratory of Veterinary Surgery, Graduate School of Agricultural and Life Sciences, The University of Tokyo. Two canine mammary gland tumor cell lines; CHMp-5b (metastatic cell line) and CHMp-13a (non-metastatic cell line) were chosen to use in this research . Both cefore use .2. After that, MTT  labeling reagent 10 µL  was added into each well. The cell lines were incubated in microplates for 4 h at 37 °C with 5% CO2 in humidified incubator. Next, the cell lines were added 100 µL the solubilization solution (10% sodium dodecyl sulfate in 0.01 M HCl) into each well. A solvent control was made of cells treated with 0.1% DMSO, while the optical density (OD) control was untreated cells. The microplates were incubated overnight before OD measurement at 580 nm using a microplate reader.MTT assay was used to assess cell viability. The CHMp-5b and CHMp-13a cell lines were cultured overnight in 96 well-plates with 5000 cells per well. Treatments were in triplicate with various doses  of BmKn-2, and they were incubated for 24 h at 37 °C with 5% COThe CHMp-5b and CHMp-13a cell lines were cultured overnight in 6-well plates at 200,000 cells per well and were treated with BmKn-2 at 0, 15, 30, 60 µg/mL in triplicate for 24 h. The cell lines were stained using FITC-labeled-Annexin V and PI  and incubated for 15 min at room temperature in the dark, according to the manufacturer’s protocols. The number of apoptotic cells was evaluated using flow cytometry . The cells were collected and evaluated using the CellQuestPro software  and apoptotic cells were indicated using a FACS Calibur with the CellQuest Pro Software . The experiments were carried out in triplicate.TM software . The reference genes, GAPDH and RPS-19, were used to normalize the expression of the apoptotic genes. The relative mRNA expression level was calculated using the 2−ΔΔCT method [The total RNA extraction was carried out using TRIZOL reagent  for all cell lines, treated with BmKn-2 peptide at 0, 15, 30, 60 µg/mL. The extracted RNA concentration of each treatment was 1 µg/µL. The cDNA was synthesized from purified RNA using iScript Reverse Transciption Supermixed for Real-time PCR . Real-time PCR was performed using iTaq Universal SYBR Green Supermix  on Bio-Rad CFX Connect Real-Time system. Primer sequences used for real-time PCR were Bax, Bcl-2, RPS-19  and GAPDH   Table 1Table 1. T method  and the p-values less than 0.05 were considered significantly different.The experiments were performed in triplicate and the data presented as mean ± standard deviation. The significance of differences was evaluated using one-way ANOVA followed by Scheffé’s multiple comparison test as post-hoc analysis for which groups were fixed factors using the R-statistic program  and under R Studio ver. 1.4.1103 with the Rcmdr package. The normal distribution and homogeneity of variance were confirmed using the Shapiro-Wilk test and Levene’s test, respectively. 50) values at 24 h of CHMp-5b and CHMp-13a were 30 ± 1.32 µg/mL and 54 ± 2.03 µg/mL, respectively (The CHMp-5b and CHMp-13a cell lines were treated with different concentrations of BmKn-2 (0–100 µg/mL) for 24 h. Cell viability was determined based on MTT assay. The half maximum inhibitory concentration  in to 1.47 and 1.80 by adding BmKn-2 at 30 and 60 µg/mL a. With C50 of BmKn-2 in CHMp-5b was 30 µg/mL which was nearly the IC 50 of this peptide in human oral squamous carcinoma (HSC4) and mouth epidermoid carcinoma (KB) cells were 26 µg/mL and 34 µg/mL [50 of CHMp-13a was 54 µg/mL which was close to the value reported in a study of the peptide on colon cancer cell lines (SW620) was 40 µM [In this study, the MTT assay results demonstrated the cytotoxicity of BmKn-2 in CMGT cell lines, CHMp-5b and CHMp-13a. The IC34 µg/mL . Furtheras 40 µM . BmKn-2 as 40 µM  while BmApoptosis is an effective mechanism to control malignant cells, inhibit hyperplasia, stop tumor progression and terminate carcinogenesis ,30. ManiThis research also studied BmKn-2 apoptosis-induction mechanisms in CMGT cell lines by observing the expression of Bax and Bcl-2 mRNA, which are generally approved apoptosis indicators . The BclThe experimental results demonstrated that BmKn-2 effectively inhibited proliferation and induced apoptosis in CMGT cell lines, metastatic (CHMp-5b) and non-metastatic (CHMp-13a) via the Bax up-regulation and Bcl-2 down-regulation mRNA expression pathways. BmKn-2 increased more apoptosis in metastatic CHMp-5b CMGT cells when compared to non-metastatic CHMp-13a CMGT cells. However, there were few necrotic cells in both CMGT cell lines. These results suggested that BmKn-2 induced apoptotic cell death with less inflammatory response. Therefore, BmKn-2 has potential for use as a novel candidate treatment for CMGTs. Further study should investigate in vivo experiments using BmKn-2 and the development of practical treatments.


Lipofilling may constitute a technique to assist reconstruction of breasts following prophylactic mastectomy for patients with mutated BRCA1 or BRCA2 genes. However, to date it is not clear whether adipose-derived stromal cells (ADSCs) increase the risk of tumor initiation and progression in this situation. Therefore, the interactions of BRCA1 mutated breast cancer cell lines with normal ADSCs were investigated in the present study. Characteristics of MDA-MB-436 (BRCA1 c.5277 + 1G > A) and HCC1937 (BRCA1 p.Gln1756.Profs*74) were compared to MDA-MB-231 and T47D BRCA1/2 wild-type breast cancer cell lines. ADSCs were cultivated from lipoaspirates of a panel of BRCA1/2- wildtype patients. Interactions of conditioned medium (CM) of these cells with the breast cancer lines were studied using proliferation and migration assays as well as adipokine expression western blot arrays. CM of ADSCs exhibit a dose-dependent stimulation of the proliferation of the breast cancer cell lines. However, of the ADSC preparations tested, only 1 out of 18 samples showed a significant higher stimulation of BRCA1-mutated MDA-MB-436 versus wildtype MDA-MB-231 cells, and all CM revealed lower stimulatory activity for BRCA1-mutated HCC1937 versus wildtype T47D cells. Additionally, migration of breast cancer cells in response to CM of ADSCs proved to be equivalent or slower for BRCA1/2 mutated versus nonmutated cancer cells and, with exception of angiopoietin-like 2, induced expression of adipokines showed no major difference. Effects of media conditioned by normal ADSCs showed largely comparable effects on BRCA1-mutated and wildtype breast cancer cell lines thus advocating lipofilling, preferentially employing allogeneic non-mutated ADSCs. The most common cancer among women is breast cancer (BC) and around 5–10% of BC cases are hereditary. A quarter of all these cases are linked to germline mutations such as breast cancer genes (BRCA) 1 and 2 which function as tumor suppressor genes . PatientBilateral prophylactic mastectomy reduces the risk of BC by more than 90% in patients bearing BRCA1/2 mutations. In the US over 100.000 women undergo this form of operation each year. Within 4 years the rate of bilateral prophylactic mastectomy increased by approximately 35.7% only in the US. This operation is recommended for women between the age of 25–30 years . The benADSC have the ability of multilineage differentiation into bone, cartilage, several muscle types, blood vessels, nerves as well as skin. They are also associated with adipogenesis in the transplanted tissue and revascularization via paracrine effects. These cells can be easily harvested by standard liposuction without the need for further cultivation . The fatCurrently, little is known about the interaction between ADSCs and BRCA-mutated normal and cancer cells. It is assumed that growth factors and cytokines secreted by ADSCs have a crucial impact on cancer initiation, progression and metastasis . SeveralIn the present study we compared the BRCA1/2 wildtype BC cell lines MDA-MB-231 and T47D with two BRCA1-mutated cell lines, namely MDA-MB-436 and HCC1937. Cell lines were tested for their alterations of proliferation and migration as well as for changes in the expression of selected adipokines in response to CM derived from a panel of ADSCs. For this investigation we used normal ADSCs due to the nonavailability of BRCA1-mutated ADSCs, questionable validity of the phenotype of genetically modified ADSCs and the putative use of allogeneic normal ADSCs that exhibit low immunogenicity.ADSCs were recovered from BRCA1/2 wildtype female patients following liposuction with the written consent of the patients according to the Ethics Approval 366/2003 of the Ethics Committee of the Medical University of Vienna, Vienna, Austria. Fat particles resulting from aspiration through the 12 gauge cannulas were incubated for 5 days in RPMI-1640 medium  supplemented with 30% fetal bovine serum (Seromed) and antibiotics , thereafter fat tissue was discarded and the ADSCs that became attached to the tissue culture flasks were further cultivated and expanded using 10% FBS. The ADSCs were characterized by flow cytometry by testing the expression of CD73, CD90, and CD105 and negative reactivity for CD34  using a Cytomics FC500 FACS  as described previously [ The BRCA1-mutated breast cancer cell lines MDA-MB-436 and MDA-MB-231 as well as the control lines T47D and HCC1937 were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum  and on confluence cells were detached with trypsin/EDTA (Sigma-Aldrich) and cell numbers counted with a LUNA cell counter .Adipokine markers were analyzed using the ARY024 Proteome Profiler Array  according to manufacturer’s instructions. Experiments were done in duplicate and individual membranes calibrated using the included protein controls. Arrays were evaluated using ImageJ and Origin 9.1 software .4 cells in 100 µl medium were distributed to wells of 96-well microtiter plates  and ten twofold dilutions of CM were added in triplicate starting with a 1:1 ratio of CM to culture medium. Assays were at least performed in triplicate. The plates were incubated for four days and viable cells detected using a modified 3--2,5-diphenyltetrazoliumbromid (MTT) assay . Test results were calculated using Origin 9.1 software . 1 × 10Cell lines MDA-MB-436, MDA-MB-231 and HCC1937 were kept in 6-well plates (TPP) in 3 ml medium until confluency was reached. T47D showed no migratory capacity under these conditions and was not considered for these experiments. Then, 2 perpendicular scratches were set to remove cells using a plastic tip and wells were supplemented with 1 ml of control medium or respective ADSC-CMs and further incubated under tissue culture conditions. Pictures were taken using a light microscope (magnification 40x) for 3 successive days and areas not covered by cells calculated by ImageJ software (imagej.net) for several positions. Migratory capacity is presented as area newly covered by the breast cancer cells.P < 0.05 regarded as significant difference.Statistical significance was determined by t-tests and ADSCs were cultivated from lipoaspirates and this cell population was tested for purity by flow cytometric detection of the specific markers CD73, CD90, CD105 and absence of CD34 as described previously [The results show that the CM of ADSCs result in a dose-dependent stimulation of the proliferation of the cancer cell lines and that, in case of the Fat3A CM, this effect is more pronounced for the nonmutated cell lines MDA-MB-231 and T47D versus the BRCA1/2-mutated cell lines MDA-MB-436 and HCC1937, respectively Fig.  and 2B. The migration of the cancer cell lines was investigated in migration assays supplemented with control medium and ADSC-CM, as described previously . T47D ceA quantitative determination of the migratory capacity was carried out using image analysis. For MDA-MB-231, addition of F13 and HAB media showed a trend of lower migration which was significant for F13 and day 1 Fig. . For theThe four breast cancer cell lines were preincubated with 50% of the respective ADSC-CM and after further incubation analyzed for the expression of proteins using an adipokine proteome profiler array. Of the 58 adipokines analyzed, 12 proteins are shown for the MDA-MB-231/MDA-MB-436 pair and 10 proteins for the HCC1937/T47D pair, respectively  on wildtype and BRCA1-mutated breast cancer cell lines and found no increased protumor effects of CM of ADSCs.


Although radiation therapy represents a core cancer treatment modality, its efficacy is hampered by radioresistance. The effect of ionizing radiations (IRs) is well known regarding their ability to induce genetic alterations; however, their impact on the epigenome landscape in cancer, notably at the CpG dinucleotide resolution, remains to be further deciphered. In addition, no evidence is available regarding the effect of IRs on the DNA methylome profile according to the methionine dependency phenotype, which represents a hallmark of metabolic adaptation in cancer.We used a case–control study design with a fractionated irradiation regimen on four cancerous cell lines representative of HCC (HepG2), melanoma , and glioblastoma (U251). We performed high-resolution genome-wide DNA methylome profiling using the MethylationEPIC BeadChip on baseline conditions, irradiated cell lines (cumulative dose of 10 Gy), and non-irradiated counterparts. We performed epigenome-wide association studies to assess the effect of IRs and methionine-dependency-oriented analysis by carrying out epigenome-wide conditional logistic regression. We looked for epigenome signatures at the locus and single-probe (CpG dinucleotide) levels and through enrichment analyses of gene ontologies (GO). The EpiMet project was registered under the ID#AAP-BMS_003_211.EWASs revealed shared GO annotation pathways associated with increased methylation signatures for several biological processes in response to IRs, including blood circulation, plasma membrane-bounded cell projection organization, cell projection organization, multicellular organismal process, developmental process, and animal organ morphogenesis. Epigenome-wide conditional logistic regression analysis on the methionine dependency phenotype highlighted several epigenome signatures related to cell cycle and division and responses to IR and ultraviolet light.IRs generated a variation in the methylation level of a high number of CpG probes with shared biological pathways, including those associated with cell cycle and division, responses to IRs, sustained angiogenesis, tissue invasion, and metastasis. These results provide insight on shared adaptive mechanisms of the epigenome in cancerous cell lines in response to IR. Future experiments should focus on the tryptic association between IRs, the initiation of a radioresistance phenotype, and their interaction with methionine dependency as a hallmark of metabolic adaptation in cancer.The online version contains supplementary material available at 10.1186/s13148-021-01199-y. Epigenome alterations, including modifications of DNA methylation, represent a hallmark of cancer initiation, progression, metastasis, and recurrence –5. CanceRadiation therapy represents a core modality of cancer treatment . For insThe effect of IRs is well known regarding their ability to induce genetic alterations . BesidesTo date, data are sparse regarding the effect of IRs on the epigenomic landscape of established cancerous cell lines, notably following a fractionated irradiation regimen. Moreover, no work has systematically evaluated the impact of IRs at a nucleotide resolution level using a standardized protocol of fractionated irradiation using multiple cancerous cell lines. In addition, no data have been reported regarding specific epigenome signatures, considering the methionine dependency phenotype, which represents a hallmark of metabolic adaptation in cancer –8.To address this knowledge gap, we used four cancerous cell lines to infer potential epigenomic alterations that IRs could induce in HCC, glioblastoma, and melanoma. HCC and glioblastoma were used as an example of cancer treatable by IRs and melanoma as an example of cancer with no indication of IR according to current guidelines. We were able to identify shared annotation pathways for epigenomic signatures associated with and exposure to IR. Furthermore, we performed epigenome-wide conditional analyses according to methionine dependency phenotype and highlighted several epigenome signatures related to cell division and response to IR.The EpiMet project was designed to assess the effect of IR, as a cellular stress model, on the epigenome landscape of cancerous cell lines using an EWAS approach. We also looked for potentially contrasted epigenome signatures considering the methionine dependency phenotype using well-phenotyped cell lines. For this purpose, we used four cell lines and three cancer models: HCC, melanoma, and glioblastoma. The EpiMet project was declared at the University of Lorraine  and registered under the ID #AAP-BMS_003_211.2 at 37 °C.We cultivated the human HCC HepG2 (ATCC®) cell lines in Dulbecco’s modified Eagle’s medium (DMEM)  supplemented with 10% volume per volume of heat-inactivated fetal bovine serum (FBS) (Sigma–Aldrich), 1% glutamine (Sigma–Aldrich), and 1% penicillin–streptomycin (P/S) (Sigma–Aldrich). We cultivated the human melanoma cell lines  in DMEM supplemented with 10% volume per volume of FBS, 2% glutamine, and 1% P/S. We cultivated the human glioblastoma cell line U251 (European Collection of Authenticated Cell Cultures [ECACC]) in DMEM supplemented with 10% volume per volume of heat-inactivated FBS, 1% non-essential amino acid (NEAA) (Sigma-Aldrich), 1% glutamine, 1% pyruvate (Sigma–Aldrich), and 1% P/S. All the cells were incubated in a humidified atmosphere with 5% COWe used a case–control study design with a fractionated irradiation regimen on four cancerous cell lines. In the irradiation arm, the cells were exposed to four IR doses at 2.5 Gy per irradiation at a one-week interval, totaling a cumulative dose of 10 Gy. In the control arm, cell lines were not irradiated and were maintained in similar conditions for the same study period. All cells were maintained in standard culture conditions with a renewal of culture media two times a week. To perform DNA methylome profiling, we prepared flash-frozen cell pellets from irradiated cell lines (‘End_Irradiation’) one week after the fourth irradiation and their non-irradiated counterparts (‘End_No irradiation’) on the same day. We also prepared flash-frozen cell pellets from baseline conditions for each cell line (‘Baseline’) before the initiation of the study. All experiments were performed in biological triplicate, totaling 36 cell flasks . We performed cell line irradiation at the Department of Radiation Oncology of the Lorraine Institute of Oncology . The cells were irradiated in the plateau growth phase to avoid any artifacts resulting from the cell cycle. Irradiation was performed on a 6-MeV X-ray linear accelerator  .β value, ranging between 0 (fully unmethylated CpG probe) and 1 (fully methylated CpG probe). Background correction and normalization were implemented using the SWAN method (R Package Minfi) [We carried out bisulfite conversion of 600 ng of DNA extracted from cell lines using the EZ DNA Methylation kit . The genome-wide profiling of DNA methylome was determined using the Infinium MethylationEPIC BeadChip array  at the Functional Genomics Facility of the INSERM unit UMR_S 1256 . The Infinium MethylationEPIC BeadChip provides coverage of > 850,000 CpG probes in enhancer regions, gene bodies, promoters, and CpG islands. The arrays were scanned on an Illumina iScan® system, and raw methylation data were extracted using Illumina’s Genome Studio methylation module. For each CpG probe, the methylation level was described as a e Minfi) . Probe aβ value. We performed primary component analysis (PCA) to assess the clustering of methylation profiles according to the whole methylation landscape of the studied cell lines. The top ten principal components  were calculated with their respective eigenvalue. PCA plots were used to report on the three top eigenvalues. We performed our analytical approach using a sequential approach. In step #1, we compared the epigenome landscape between ‘Baseline’ and ‘End_No irradiation’ conditions to assess whether the maintenance of cell lines affected DNA methylation. In step #2, we performed an EWAS that pooled ‘Baseline’ and ‘End_No irradiation’ conditions to compare them with the ‘End_Irradiation’ condition. The aim of step #2 was to assess whether the exposure to a cumulative dose of 10 Gy affected the DNA methylation landscape of the studied cell lines. In this step, we performed a sensitivity analysis EWAS by removing the ‘Baseline’ condition from the control group to compare the ‘End_Irradiation’ versus ‘End_No irradiation’ condition. In step #3, we performed the same analysis described in step #2 on each of the four cell lines. Finally, in step #4, we assessed using enrichment analyses the gene ontology (GO) pathways significantly associated with exposure to IRs and looked for shared GO annotations between the studied cell lines. In each EWAS (steps #1–3), we compared the mean β values of each CpG probe between the two subgroups using the t-test with Bonferroni correction. Output data included the mean β values in each subgroup, the difference of β values, the nominal P-value, and the Bonferroni corrected P-value. To assess the effect of IRs on the epigenome landscape with respect to the methionine dependency phenotype, we performed conditional logistic regression EWAS using the methylome profiles from methionine-dependent cell lines (MeWo-LC1) and their methionine-independent counterpart (MeWo) [β values > 0.1 or <  − 0.1, respectively) using the GO Enrichment Analysis tool [We visually inspected the whole-genome distribution of the CpG probes according to their t (MeWo) . We usedsis tool . For thesis tool . We idenβ value density distribution  for each of the four studied cell lines. All methylome profiles passed quality checks and exhibited a valid β values > 0.1  among HepG2, MeWo-LC1, and U251 cell lines  for the following main annotations: blood circulation, plasma membrane-bounded cell projection organization, and cell projection organization (Table P < 0.0001)  Fig. B, includIn conditional logistic regression EWAS that compared methionine-dependent MeWo-LC1 cells to their non-methionine dependent mother cells MeWo, the enrichment analyses performed on the CpG probes with beta regression coefficient values > 200 highlighted several pathways, including cell division and responses to IR and ultraviolet light  in association with exposure to IRs that were consistent with epigenomic adaptive mechanisms involving sustained angiogenesis, tissue invasion, and metastasis. The enrichment folds for the shared epigenomic signatures were found to be highly correlated across cell lines. In addition, we highlighted several epigenome signatures related to cell division and response to IRs in association with the methionine dependency phenotype that we addressed using two melanoma cell lines with opposite methionine dependency phenotypes.Using an original approach through a case–control study design and four cancerous cell lines , we provided better insight at a high-resolution scale on the DNA methylome variations in association with IRs. While we did not find monogenic epigenome signatures in association with exposure to IRs, we identified significant enrichments in CpG probes exhibiting increased or decreased methylation levels  . InteresThe present study has several strengths. First, we used an original case–control study design on several cancerous cell lines, including two cell lines known for their opposed methionine dependency phenotypes. Second, we assessed DNA epigenome signatures using the high-resolution Infinium MethylationEPIC on more than 850 k CpG probes. Third, we assessed the effect of a fractionated regimen of IRs with a cumulated dose of 10 Gy, and we assessed both baseline and end of irradiation conditions in the control arm to control for the risk of bias. Our study aimed to investigate the effect of moderate hypofractionation without generating an excess of mitotic cell death. Hypofractionation is a new standard applied in the management of prostate and breast cancers but also for radioresistant tumors such as the three cancer models investigated in the EpiMet protocol. In the irradiation protocol regimen that we applied in the present study, we aimed to model the radiation boost delivered at the end of the treatment schedule. Fourth, we report the first evidence on the association of epigenome landscape alterations with IRs, considering the methionine dependency phenotype. Fifth, we performed several bioinformatics approaches that unraveled shared enriched annotation pathways in relation to IRs exposure. Nevertheless, we acknowledge several potential limitations of the study that should be considered in interpreting our results. First, even if a fractionated regimen of IRs seems to be the more adapted experimental design to assess the effect of adaptive epigenome alterations to radiation-induced cellular stress, it remains that a higher cumulative dose (> 10 Gy) could induce a more striking epigenome signature associated with IRs and should be investigated in future experiments. Second, we did not assess and quantify the effect of IRs at the cellular level by using a surrogate biomarker to estimate DNA damage . Third, In our study, we did not perform methylation profiles on human cancer samples before and after IR exposure for several reasons. First, the EpiMet project received regulatory authorizations from the ethics committee of the University of Lorraine to perform analyses on four cell lines representative of three cancer models . Second, we developed the EpiMet project to assess the influence of the methionine dependency phenotype on the effect of IRs on the DNA methylome landscape. Methionine dependency is defined as the ‘inability of cells to grow when methionine is replaced in culture medium by its metabolic precursor homocysteine’ . AccordiIn conclusion, the present study unraveled shared epigenomic adaptive mechanisms in response to IRs using in vitro models of epidemiologically leading cancerous diseases. These mechanisms are related to cell cycle and division, responses to IR, sustained angiogenesis, tissue invasion, and metastasis. Our results pave the way toward a future research agenda regarding the epigenomic correlates of radioresistance, the development of a clonal selection of radiation-resistant cells, and tumor relapse. Future experimental designs will help better understand the long-term effect of chronic cellular stress on initiating and maintaining epigenetic modifications that could initiate and perpetuate a radioresistance phenotype and its interaction with methionine dependency as a hallmark of metabolic adaptation in cancer.Additional file 1: Figure S1. Quality control regarding the genome-wide distribution of the CpG probes according to their β value. All DNA methylome profiles had a beta distribution and passed the quality criteria.Additional file 2: Figure S2. 3-D plot using the three top eigenvectors  derived from the primary component analysis on the genome-wide methylome landscape of the studied cell lines, according to study conditions: ‘Baseline’ and ‘End_No irradiation.’Additional file 3: Figure S3. 2-D plot using the two top eigenvectors  derived from the primary component analysis on the genome-wide methylome landscape of the studied cell lines. Cell lines are indicated using elliptical shapes. Study conditions are indicated using the color code .Additional file 4:Table S1a. Results from the epigenome-wide association study that assessed the effect of ionizing radiations on the DNA methylation profile comparing “End_Irradiation” vs. “End_No irradiation or Baseline” conditions.Additional file 5:Table S1b. Results from the epigenome-wide association study that assessed the effect of ionizing radiations on the DNA methylation profile comparing End_Irradiation” vs. “End_No irradiation” conditions.Additional file 6:Table S2. Results from the epigenome-wide association study that assessed the effect of ionizing radiations on the DNA methylation profile comparing “End_Irradiation” vs. “End_No irradiation or Baseline” conditions in the HepG2 cell line.Additional file 7:Table S3. Results from the epigenome-wide association study that assessed the effect of ionizing radiations on the DNA methylation profile comparing “End_Irradiation” vs. “End_No irradiation or Baseline” conditions in the MeWo-LC1 cell line.Additional file 8: Table S4. Results from the epigenome-wide association study that assessed the effect of ionizing radiations on the DNA methylation profile comparing “End_Irradiation” vs. “End_No irradiation or Baseline” conditions in the MeWo cell line.Additional file 9: Table S5. Results from the epigenome-wide association study that assessed the effect of ionizing radiations on the DNA methylation profile comparing “End_Irradiation” vs. “End_No irradiation or Baseline” conditions in the U251 cell line.Additional file 10: Figure S4. Epi-Manhattan plot reporting the results of the conditional logistic regression epigenome-wide association study that compared ‘End_Irradiation’ vs. (Baseline/End_No irradiation) according to the methionine dependency phenotype.Additional file 11:Tables S6. Results from enrichment analyses that used the CpG probes exhibiting a difference of β values >0.1 (a) or <-0.1 (b), considering all cell lines. Results from enrichment analyses that used the CpG probes exhibiting a difference of β values >0.1 in HepG2 (c), MeWo (d), MeWo-LC1 (e), and U251 (f). Results from enrichment analyses that used the CpG probes exhibiting a difference of β values <-0.1 in HepG2 (g), MeWo (h), MeWo-LC1 (i), and U251 (j).Additional file 12: Figure S5. Venn diagram illustrating the shared gene ontology annotation pathways between HepG2 and MeWo-LC1 cell lines associated with CpG probes with an increased methylation level and HepG2, MeWo-LC1, and U251 cell lines associated with CpG probes with a decreased methylation level.Additional file 13: Table S7. Results from enrichment analyses that used the CpG probes with beta regression coefficient values > 200 (a) and <-200 (b) in conditional logistic regression EWAS that compared methionine-dependent MeWo-LC1 cells to their non-methionine dependent mother cells MeWo.


Invadopodia, actin-rich structures that release metallo-proteases at the interface with extra-cellular matrix, in a punctate manner are thought to be important drivers of tumour invasion. Invadopodia formation has been observed in-vitro and in-vivo in numerous metastatic cell lines derived from multiple tumour types. However, prostate cancer cell lines have not been routinely reported to generate invadopodia and the few instances have always required external stimulation.In this study, the invasive potential of primary prostate adenocarcinoma cell lines, which have never been fully characterised before, was investigated both in-vitro invadopodia assays and in-vivo zebrafish dissemination assay. Subsequently, circulating tumour cells from prostate cancer patients were isolated and tested in the invadopodia assay.Retention of E-cadherin and N-cadherin expression indicated a transitional state of EMT progression, consistent with the idea of partial EMT that has been frequently observed in aggressive prostate cancer. All cell lines tested were capable of spontaneous invadopodia formation and possess a significant degradative ability in-vitro under basal conditions. These cell lines were invasive in-vivo and produced visible metastasis in the zebrafish dissemination assay. Importantly we have proceeded to demonstrate that circulating tumour cells isolated from prostate cancer patients exhibit invadopodia-like structures and degrade matrix with visible puncta. This work supports a role for invadopodia activity as one of the mechanisms of dissemination employed by prostate cancer cells.The combination of studies presented here provide clear evidence that invadopodia activity can play a role in prostate cancer progression.The online version contains supplementary material available at 10.1186/s12885-022-09424-4. Cells were seeded in triplicate in four 96-well plates at a density of 6 × 10Cell lysates were separated by acrylamide gel electrophoresis. Proteins were then transferred onto a nitrocellulose membrane followed by overnight incubation at 4 °C with the following primary antibodies: antimouse E-cadherin , antimouse GAPDH . Membranes were washed in TBST and incubated with the respective HRP-conjugated secondary antibody . Proteins were visualised using Pierce enhance chemiluminescence (ECL) western blotting substrate and quantified by densitometric analysis using ImageJ software.Coverslips were coated with a thin layer of Cy3-conjugated gelatine (EMD Millipore’s QCM Gelatin) according to the manufacturers protocol. Seeded cells were fixed in 4% paraformaldehyde (PFA) and stained.PFA fixed cells on coverslips were permeabilised in 0.2% Triton X-100 and blocked with 5% BSA. Coverslips were incubated with the following primary antibodies antimouse E-cadherin , antimouse Cortactin , Alexa Fluor 647 anti-human CD45  followed by goat antimouse Alexa Fluor 488 IgG secondary antibody . Cells were imagined on an Olympus IX71 microscope.Zebrafish experiments were conducted under the UK Home Office project licence PPL 70/7912 and had been approved by the King’s College Ethical Review Committee. Approximately 500 GFP-tagged PCa cells or AsPC-1 cells were injected into the yolk sac using a Nikon SMZ-U zoom 1:10 Picospritzer II microinjection station. The embryos were checked at 24 h post injection to ensure that GFP signal was restricted to the yolk sac xenograft. 3 days post-injection the percentage of embryos with cancer cell tail invasion was calculated.Blood samples for CTC isolation were drawn from patients diagnosed with PCa with a Gleason score equal to 7 or more. For each patient, 7.5 ml of blood was collected in a 10 ml vacutainer EDTA tube, maintained at room temperature and processed within 4 h of collection. The study was conducted upon the obtainment of written informed consent and under the King’s Health Partners’ Prostate Cancer Biobank (KHP PCaBB)  blindingRoutinely used PCa cell lines are not amenable to the study of spontaneous invadopodia formation. We sought to identify whether alternative PCa cell lines could be utilised. Three malignant cell lines, CT-1532, CT-1535 and CT-1542 had been previously isolated from radical prostatectomy specimens and immortalised using a recombinant retrovirus encoding the E6 and E7 transforming proteins of human papilloma virus serotype . These tLoss of E-cadherin has been linked to epithelial-mesenchymal transition (EMT) in PCa progression and metastasis . HoweverPCa cell lines were subsequently tested for their ability to form invadopodia. Invadopodia were defined as actin-enriched puncta co-localizing with degraded matrix. Cells were additionally stained for cortactin a specific marker of invadopodia . Remarkain-vitro, we tested their metastatic potential in-vivo using the zebrafish yolk sac invasion assay [in-vivo metastatic behaviour of the PCa cell lines.Having evaluated the ability of our PCa cell lines to invade on assay . Fluoreson assay , while nOur findings support a role for invadopodia activity in the dissemination of PCa. To validate this hypothesis, we tested circulating tumour cells (CTCs) isolated from the peripheral blood of 17 PCa patients (minimum Gleason grade 7) (Supplementary table in-vitro study models. The data presented here suggest that human prostate autologous 1532, 1535 and 1542 cancer cell lines are a suitable research model for invadopodia studies in PCa.Invadopodia represents a promising therapeutic target to prevent cancer metastasis . HoweverPrevious attempts to identify invadopodia activity in PCa cells have had limited success . We hypoin-vitro and in-vivo [in-vivo are able to switch to an alternative metalloproteases-independent mechanism of invasion that does not rely on invadopodia activity. PCa cells are reported to be capable of amoeboid cell migration [As the ability of cells to form invadopodia and degrade the matrix is thought to correlate with their invasive and metastatic capacity  in-vivo , it is rigration , 35, a migration . Alternaigration .Circulating tumour cells can be thought to represent a snapshot of the cancer cells that have escaped the primary tumour. In the recent years, some evidence has emerged for an association between invadopodia and CTCs. CTCs isolated from breast cancer patients were able to generate invadopodia-like structures and invade into a 3D ECM . We now Taken together our study supports further exploration of invadopodia activity in the PCa setting with the potential to develop novel therapeutics. Moreover, we propose that the PCa cell lines characterised here offer a robust and valid model for such future studies.Additional file 1. Additional file 2.


N,N disubstituted selenoureas were synthesized, characterized, and their antioxidant ability was tested using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS) assays. Additionally, their cytotoxic activity was tested in vitro in a panel of three different cancer  and two normal cell lines. Each selenourea entity contains a para-substituted phenyl ring with different electron-withdrawing and electron-donating groups, and different aliphatic and aromatic nuclei. All of the synthesized selenoureas present antioxidant capacity at high concentrations in the DPPH assay, and three of them  showed greater radical scavenging capacity than ascorbic acid at lower concentrations. These results were confirmed by the ABTS assay, where these novel selenoureas present even higher antioxidant capacity than the reference compound Trolox. On the other hand, 10 selenoureas present IC50 values below 10 µM in at least one cancer cell line, resulting in the adamantyl nucleus (6a–6e), the most interesting in terms of activity and selectivity. Outstanding results were found for selenourea 6c, tested in the NCI60 cell line panel and showing an average GI50 of 1.49 µM for the 60 cell lines, and LC50 values ranging from 9.33 µM to 4.27 µM against 10 of these cancer cell lines. To gain insight into its anticancer activity mechanism, we investigated the cell cycle progression of the promising compound 6c, as well as the type of programmed-cell death in a colon cancer cell line it provokes (HT-29). Compound 6c provoked S phase cell cycle arrest and the induction of cell death was independent of caspase activation, suggesting autophagy, though this assertion requires additional studies. Overall, we envision that this compound can be further developed for the potential treatment of colon cancer.A series of 30 novel  Cancer is a group of complex and multifactorial diseases and is considered the third leading cause of death globally. The World Health Organization (WHO) estimates that one-sixth of deaths can be attributed to cancer . Lung (1Selenium (Se) is an essential micronutrient; trace amounts are necessary for the function of the human body and it is mainly obtained through diet and/or nutritional supplements . Se is aThe anticancer effect of selenium compounds may be asserted through various pathways in the cell . They haSeveral organoselenium agents have been developed for the inhibition of different types of cancer cell growth. Among them, selenoureas represent a group of simple but well-studied molecules with anticancer and/or antioxidant properties ,18,19,20With the above facts in mind, and using the fragment-based approach, we designed the compounds presented in this paper. Herein, we pursue the development of new molecules, with the selenourea functioning as cytotoxic agents decorated in the nitrogen atoms with a small set of fragment hits. Therefore, we select nucleus as coumarin , furan , thiazolH-tetrazolium bromide (MTT). Ten of these selenoureas were chosen by considering their cell growth inhibitory activity, and their IC50 values were determined towards breast (MCF-7), colon , and lung (HTB-54) cancer cells, along with a breast non-malignant derived cell line (184B5) and a lung non-malignant derived cell line (BEAS-2B) to determine their selectivity. The radical scavenging activity for all the analogs was assessed in vitro using colorimetric assays of 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) at different times and concentrations.The cytotoxic activity of these novel selenoureas was screened in vitro at two doses (10 and 50 μM) against a panel of cancer cell lines derived from breast (MCF-7), lung (HTB-54), and colon (HT-29), using a colorimetric assay of 3--2,5-diphenyl-21H-, 13C- and 77Se-NMR spectra were registered on a Bruker Avance Neo 400 MHz in CDCl3 and DMSO-d6, operating at 400, 100 and 76 MHz, respectively, using TMS as the internal standard. Chemical shifts are reported in δ values (ppm) and J values are reported in hertz (Hz). The IR spectra were obtained on a Thermo Nicolet FT-IR Nexus spectrophotometer with KBr pellets.All the chemical reagents for the synthesis were purchased from E. Merck , Scharlau , Panreac Química S.A. , Sigma-Aldrich Química, S.A.  and Acros Organics . TLCs were performed on aluminum pre-coated sheets (E. Merck Silica gel 60 F254). Silica gel 60 (0.040–0.063 mm) 1.09385.2500  was used for Column Chromatography. Melting points were determined using a Mettler FP82 + FP80 apparatus  and was not corrected. Ia–e), followed by the treatment with triphosgene and elemental selenium in methylene chloride under reflux in presence of triethylamine to obtain isoselenocyanates (IIa–e) -2,5-diphenyltetrazolium bromide (MTT) assay . Firstly1d, 1e, 2a, 2d, 3a, 6a, 6b, 6c, 6d, and 6e) passed this threshold and four of them  in all three cancer cell lines. The data are expressed as IC50 (mean ± SD) in a panel of four cancer cell lines , and are shown in 50 values of a well-known antitumor selenocompound, i.e., sodium selenite (Na2SeO3), were used as reference for comparision with the selected selenoureas. The only IC50 reported value found in the literature for sodium selenite was 17 µM in MCF-7 cells after 72 h of treatment [1e, 2a, 6a, 6b, 6c, 6d, and 6e) showed greater cytotoxic activity than sodium selenite towards this cancer cell line, even at shorter timepoints. The highest cytotoxic activity among the selenourea derivatives was exhibited by adamantyl compounds in all the cell lines with IC50 values below 10 µM for compounds 6b, 6c, 6d, and 6e. No correlation was observed with the insertion of electron-withdrawing or -donating groups.Those derivatives that reduced the growth of the cell lines below 50% at 10 µM in at least one cell line were selected for being evaluated at five concentrations between 0.01 and 100 µM. Ten compounds  and lung (BEAS-2B), respectively. According to the SI results, most of the compounds exhibited SI values below 1, with the exception of 2a, 6a, 6b, 6c, 6d, and 6e in breast cancer, and only 6d in lung cancer. Remarkably, the most effective analog (6c) presents similar or slightly better cytotoxic activity than many of the most recent urea [The selectivity index (SI) was calculated as the ratio between ICent urea  and thioent urea  derivatient urea ,46.6c, against the four cell lines was selected for testing in the NCI-60 cell line panel. This compound shows an average cell growth inhibition of −9.58% at 10 µM, meaning that compound 6c is able to kill cells in most of the cell lines. The dose–response was also studied in the same 60 cancer cell lines, with outstanding results. Average GI50 of 1.49 µM and TGI of 9.17 µM were found. 50, TGI, and LC50 values of representative cell lines. Full data mean GI50 graphs are provided in the To further explore the potential of these selenoureas, the most effective analog, The arrest of cell cycle progression caused by some anticancer drugs entails the inhibition of cancer cell proliferation ,50. More6c, we investigated the effect of this compound on cell cycle progression using flow cytometry analysis, with propidium iodide as a DNA staining agent. We analyzed the effect at 48 h treatment with four different concentrations  of compound 6c on HT-29 cells. Camptothecin (10 μM) was used as a positive control. As shown in 6c caused cell cycle arrest at the S phase in a concentration-dependent manner. The cell cycle modulation of 6c is very similar to camptothecin, the reference compound used in the study.To gain further knowledge about the mechanism of action of 6c to induce cell death, we performed Annexin V-APC/7AAD assays in HT-29 cells cultured either in the presence or absence of this compound for 48 h. Assays were conducted at different 6c concentrations ranging from 0.1 to 10 µM. Camptothecin (10 µM) was used as positive control. As shown in 6c increased the number of dead cells (7AAD positive cells). Interestingly, no significant differences in the number of dead cells were found between HT-29 cells treated with 6c and HT-29 cells treated with 6c after pre-incubation with the pancaspase inhibitor z-VAD-fmk  were the least cytotoxic. No specific correlation in the cytotoxic activity between selenoureas containing different electron-withdrawing and electron-donating groups at the 4-phenyl position was found. An MTT assay demonstrated that five compounds  exhibited IC50 values below 10 µM against the four cancer cells lines tested , showing greater cytotoxic activity than the reference cisplatin. Adamantyl-containing selenourea 6c was selected as the most potent cytotoxic agent, and was tested in the NCI60 cell line panel, with outstanding results. Average GI50 of 1.49 µM and TGI of 9.17 µM were found, showing more potent antiproliferative activity than several anticancer drugs, including 5-fluorouracil, gefitinib, or oxaliplatin. To gain insight into its anticancer activity mechanisms, we investigated the cell cycle progression of this promising 6c compound, as well as the type of programmed-cell death in a colon cancer cell line it causes (HT-29). Compound 6c provoked S phase cell cycle arrest with a decrease in cells in the G1 phase, and the induction of cell death was independent of caspase activation, suggesting autophagy, though this assertion requires additional studies. Overall, we envision that this compound can be further developed for the potential treatment of colon cancer.In the present work, 30 novel 1–6e and coumarin-derived selenoureas 3a–e present fast kinetics, reaching the maximal antioxidant activity at time 0 in a DPPH assay. On average and in both experiments, thiazol-containing selenourea 2c was found to be the most potent antioxidant compound at low concentrations.The radical scavenging ability of the new selenoureas was evaluated using DPPH and ABTS assays. All these selenoureas present important antioxidant activity in DPPH and ABTS assays, presenting, in some cases, higher or similar radical scavenging activity than the reference substances (ascorbic acid and Trolox). Nitrile para-substituted selenoureas 6c demonstrated an outstanding cytotoxic activity, along with potent radical scavenging activity. These results provide an excellent starting point for the development of new selenium-based medication with both antioxidant and chemotherapeutic potential.Although no overall correlation between cytotoxicity and antioxidant activity was found, compound


Our screen identified several compounds with strong synergistic and antagonistic effects, which we further investigated using multiple irradiation doses. Navitoclax, which emerged as the most promising radiosensitizer, exhibited synergy with irradiation regardless of the p53 mutation status in all 13 HNSCC cell lines. We performed a live cell apoptosis assay for two representative HNSCC cell lines to examine the effects of navitoclax and irradiation. As a single agent, navitoclax reduced proliferation and induced apoptosis in a dose-dependent manner, whereas the navitoclax–irradiation combination arrested cell cycle progression and resulted in substantially elevated apoptosis. Overall, we demonstrated that combining navitoclax with irradiation resulted in synergistic in vitro antitumor effects in HNSCC cell lines, possibly indicating the therapeutic potential for HNSCC patients.Conventional chemotherapeutic agents are nonselective, often resulting in severe side effects and the development of resistance. Therefore, new molecular-targeted therapies are urgently needed to be integrated into existing treatment regimens. Here, we performed a high-throughput compound screen to identify a synergistic interaction between ionizing radiation and 396 anticancer compounds. The assay was run using five human papillomavirus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) cell lines cultured on the human tumor-derived matrix Myogel Treatment approaches include surgery combined with radio-, chemo-, immuno- or targeted therapy relying on the epidermal growth factor receptor (EGFR) inhibitor cetuximab (Erbitux). Chemoradiotherapy is a standard treatment for locoregional advanced or unresectable HNSCC. Despite improved treatment options, the 5-year survival rate for HNSCC has remained stagnant, at approximately 50%2. Furthermore, current chemotherapeutic agents are nonselective and accompanied by severe side effects. Combinations of cetuximab or chemotherapy with irradiation appear to improve patient survival, although response rates to cetuximab remain low3. Therefore, new molecular-targeted therapies, which can be integrated into existing treatment regimens, are urgently needed.Head and neck squamous cell carcinoma (HNSCC) represents the eighth most common malignancy globally4. These tests are often conducted on a two-dimensional (2D) plastic surface or using animal-derived extracellular matrices, such as Matrigel, thereby overlooking the important interaction between cancer cells and the human tumor microenvironment (TME). To overcome this problem, we have developed a human tumor leiomyoma-derived matrix, Myogel, to more effectively mimic the human TME6. Previously, we demonstrated that using Myogel-coated wells improved the predictability of in vitro anticancer compound testing compared to conventional plastic and animal-derived matrix-coated wells7.Current in vitro anticancer compound testing carries a low predictive value, since only 5% of compounds demonstrating efficacy in preclinical tests have been approved following clinical trials8, both critical in carcinogenesis and representing a major obstacle to effective treatment9. One suggested mechanism of resistance to anticancer treatment lies in the altered expression of B-cell lymphoma 2 (Bcl-2) family members9. The Bcl-2 family proteins, such as Bcl-2 and Bcl-xL, control cell death by regulating the mitochondrial outer membrane permeability, allowing for the release of intermembrane proteins to the cytoplasm and caspase activation leading to apoptosis11. Inactivating the mutation of tumor suppressor p53 is the most frequent genomic alteration in HNSCC12. Functional p53 directly and indirectly inhibits Bcl-213, rendering Bcl-2 a potential therapeutic target in HNSCC. Navitoclax (ABT-263), a potent and selective inhibitor of Bcl-2/Bcl-xL, has demonstrated in vitro and in vivo activity against a large panel of cancer cell lines, such as small-cell lung cancer and hematologic malignancies15. However, only a few in vitro studies exist for HNSCC17. To our knowledge, in vitro studies regarding the presence of the extracellular matrix combining multiple irradiation doses and navitoclax remain unreported.Ionizing radiation induces double-strand DNA breaks, while unsuccessful repair halts the cell cycle or leads to apoptosis. One cancer hallmark is the ability of tumor cells to resist cell deathThis study aims to identify new anticancer compounds with radiosensitizing properties for HNSCC. We performed high-throughput compound screening (HTS) on five HPV-negative HNSCC cell lines cultured in the human tumor–derived matrix Myogel. Interestingly, several anticancer compounds demonstrated strong synergistic or antagonistic effects when combined with ionizing radiation. To further investigate the synergistic interaction among the best-performing hits, we combined the anticancer compounds and ionizing radiation in a pairwise dose–response manner and tested the combinations in 6 × 5 dose–response matrices. Navitoclax, as the most promising radiosensitizer, was further examined using an additional eight HPV-negative HNSCC cell lines. The live cell apoptosis assay on two HNSCC cell lines confirmed that navitoclax substantially increased irradiation-induced apoptosis.18.High-throughput compound screening (HTS) is a widely used method for identifying effective drug candidates targeting cancer cells. We used a compound library of 396 FDA-approved drugs as well as experimental drug candidates and probes together with ionizing radiation to investigate potential synergistic and antagonistic combinations in five locally established HNSCC cell lines with previously characterized mutation profilesThe compound screening revealed a remarkable variation across the five HPV-negative HNSCC cell lines in their response to most compounds and compound–irradiation combinations Fig. . However21. Based on the obtained synergy scores, we classified the compound–irradiation combinations as additive, antagonistic or synergistic. Combinations with a synergy score > 10 were considered strongly synergistic and those < − 10 were considered strongly antagonistic, while synergy scores between − 5 and 5 were classified as additive. Other synergy scores represented borderline synergistic/antagonistic drug pairs.To validate the most promising combinations, we performed a comprehensive drug combination screen of 15 compounds and multiple irradiation doses measured in 6 × 5 dose–response matrices using the same HTS protocol used in the initial screen. To test whether the combinations acted synergistically or antagonistically, we compared the observed responses to the expected combination responses based on the ZIP reference model using the SynergyFinder web application (version 2)The dose–response analysis verified that, among seven tested epigenetic modifiers, only two  exhibited a promising synergy Fig. a. Only oNavitoclax exhibited the highest synergy when combined with ionizing radiation among the tested compounds Fig. a. A stro18. We observed a strong combination synergy across ten cell lines and a moderate synergistic effect in three other cell lines  is a useful method for investigating effective cancer targeting compounds. We performed HTS using a library of 396 FDA-approved and investigational compounds and ionizing radiation as an initial screen to identify synergistic and antagonistic combinations for HPV-negative HNSCC cells cultured on a human tumor–derived Myogel matrix. Our screen revealed wide variation among the HNSCC cell lines tested in their responses to the majority of compounds and compound–irradiation combinations. Most compounds did not interact with irradiation , while some exhibited a strong synergy or antagonism. Following the initial screen, the most prominent compound–irradiation combinations were further investigated using five tenfold compound concentrations and four irradiation doses in the dose–response matrix analysis. We further investigated the most synergistic compound, navitoclax, using eight additional cell lines and a live cell apoptosis assay.Multiple studies confirmed that radiosensitivity and radioresistance can depend on the schedule pattern. Although our study features only one type of schedule, we designed it to mimic the typical HNSCC treatment schedule, whereby radiotherapy begins simultaneously or shortly after drug administration. Furthermore, we chose to irradiate cells 24 h after cell seeding and drug administration to ensure complete cell attachment before irradiation.14. However, only three in vitro studies exist for navitoclax in HNSCC17, only one of which included navitoclax combined with irradiation in HNSCC cell lines22. In that study, Ow et al. found that this combination did not significantly improve the response and yielded only a modest benefit in two of eight cell lines22. Experiments were performed using a clonogenic survival assay with only one irradiation dose and two navitoclax concentrations. Several clinical trials have been conducted or are ongoing on navitoclax as a single agent or in combination with other anticancer compounds to treat leukemia and solid tumors. However, the navitoclax–radiotherapy combination remains clinically unexplored. Additionally, to our knowledge, no clinical trials exist for HNSCC. Thus, we report here for the first time a strong synergy between navitoclax combined with ionizing radiation in HNSCC cell lines  is the most frequent genomic alteration in HNSCC, accounting for approximately 50% of casestus Fig. .14. Navitoclax induces intrinsic apoptosis in human tumor cells15. Our findings confirm that this phenomenon also occurs in HNSCC. Using a live cell apoptosis assay, we demonstrated that navitoclax triggers apoptosis in HNSCC cells  apoptosis in damaged cells by releasing a mitochondrial protein to cytosol, leading to the activation of caspase proteases25. The first clinical trial to study afatinib in combination with radiation therapy in HNSCC is ongoing (NCT01783587). Furthermore, tretinoin exhibited a moderate synergy with irradiation in our screen, similar to a previous study in which tretinoin modulated radiosensitivity in HNSCC cell lines26. We also observed a radiosensitization of talazoparib, a PARP1/2 inhibitor, in two of our HNSCC cell lines, similar to findings among small cell lung cancer (SCLC) and some other solid tumors27. Several clinical trials combining talazoparib and radiotherapy are now recruiting lung, gynecological and breast cancer patients .In addition to navitoclax, our screen identified other promising radiosensitizers, including afatinib. Previous studies, which reported the radiosensitization of afatinib in HNSCC cell lines, support this finding28. To date, no in vitro or clinical studies for HNSCC combined with BI 2536 and irradiation exist. Clinical trials for BI 2536 primarily focus on leukemia and solid tumors, such as breast, pancreatic, prostate and lung cancers. A phase II clinical trial for BI 2536 was completed for a panel of solid tumors, including HNSCC (NCT00526149). GSK-461364, an experimental compound, lacks in vitro studies for HNSCC. The only existing clinical trial for GSK-461364 was completed for non-Hodgkin's lymphoma (NCT00536835). Pemetrexed, a dihydrofolate reductase inhibitor, serves as treatment for pleural mesothelioma and non-small cell lung cancer (NSCLC). The pemetrexed–irradiation combination appears to carry schedule-dependent interactions in NSCLC and HNSCC cells29. In this study, 24-h incubation with pemetrexed in the HNSCC cell line (CAL-27) followed by irradiation (0–8 Gy) resulted in moderate antagonism29. Furthermore, pemetrexed was evaluated as a novel treatment for HNSCC patients. Although several clinical studies have been completed, the benefit of pemetrexed in HNSCC treatment remains unclear30. Surprisingly, based on our data, all cell lines sensitive to pemetrexed as a single agent exhibited a strong antagonism when combined with irradiation. This raises concerns regarding concomitant pemetrexed and radiotherapy in HNSCC.We also identified radioresistant properties among several drugs in HNSCC cell lines. Interestingly, two PLK1 inhibitors (BI 2536 and GSK-461364) showed strong antagonism when administered 24 h before irradiation. One study reported PLK1 inhibition causing radiosensitization or radioresistance depending on the treatment schedule in osteosarcoma and colorectal cancer cell lines using a clonogenic assayTaken together, our HTS screen revealed both radiosensitive and radioresistant compounds for HNSCC cells. Pemetrexed and PLK1 inhibitors (BI 2536 and GSK-461364) exhibited a strong antagonism when combined with irradiation, whereas the combinations of irradiation with afatinib, tretinoin or talazoparib exhibited a promising synergy, in agreement with previous studies. The most promising finding—and, to our knowledge, the first such report—was that navitoclax combined with irradiation exhibited the strongest synergy, triggered apoptosis and halted the proliferation of HNSCC cells. Our findings, thus, encourage clinical trials using navitoclax combined with irradiation as treatment for HPV-negative HNSCC.31. Among these, 12 UT-SCC cell lines were established in the Department of Otorhinolaryngology-Head and Neck Surgery at Turku University Hospital  and one cell line (OU-SCC-9B) was established at Oulu University Hospital . The cells were cultured in a minimal essential medium  supplemented with l-glutamine (2 mmol/l), 10% fetal bovine serum, a nonessential amino acid solution, penicillin (100 U/ml), streptomycin (100 μg/ml) and 250-ng/ml fungizone  at 37 °C in an atmosphere of 5% CO2. Cell lines were mycoplasma-free and tested using the PCR Mycoplasma Test Kit I/C . Cell line authentication was not performed.We used 13 HPV-negative locally established HNSCC cell lines taken from primary and metastatic sites  approved the study protocol, and all methods were performed in accordance with the relevant guidelines and regulations. To establish the metastatic oral tongue squamous cell carcinoma cell line , and all research was performed in accordance with relevant regulations. All prospective liquid handling was performed using an automated reagent dispenser  and a HighRes Biosystems automation platform. Before screening, we performed irradiation dose optimization to determine the suitable dose  for each cell line. Next, we screened using the compound library applying a predefined irradiation dose (EC20). Compounds with the most synergistic and antagonistic effects were identified and further investigated using multiple doses of irradiation .We used drug sensitivity and resistance testing (DSRT) adapted from a platform for leukemia cellsWe used a compound library of 396 FDA-approved drugs as well as investigational compounds and probes , and then placed in an automated incubator overnight. On the following day, cells were counted using the Scepter 2.0 Cell Counter  and suspended to the desired density. The number of cells per well (500–750 cells/well) was optimized in our previous study7. Cells were seeded using a reagent dispenser  with a plate stacker, and the cells were left to adhere for 24 h in an automated incubator. After 24 h, plates were irradiated by gamma irradiator OB29/4  using EC20 irradiation doses and placed back in an automated incubator. Control plates were also transferred to the gamma irradiator room to ensure similar handling for all plates. After 48 h, the CellTiter-Glo 2.0 (CTG) luminescent cell viability assay  was used to determine the amount of ATP in the assay wells. Plates were handled using the ACell platform allowing plates to cool (to RT) for 15 min, followed by dispensing 25-µl CTG to the assay wells using a Certus Flex dispenser . The plates were centrifuged for 3 min at 1000 rpm at RT  and the luminescence signal was detected using the PHERAstar FS HT reader .To conduct drug sensitivity testing at the High Throughput Biomedicine Unit (HTB) at the Institute for Molecular Medicine Finland (FIMM), we used an automated system including ACell automation platform , a benchtop pipetting robot , an ambient storage hotel  and an automated incubator . On the first day, Myogel was thawed on ice (4 °C) and diluted with a serum-free cell culture medium to 500 µg/ml. Diluted Myogel (500 µg/ml) was added to 384-well compound plates using a reagent dispenser . Plates were subsequently centrifuged for 20 s at 173×https://breeze.fimm.fi) 34. DSS was described in several previous studies33, combining several parameters  into a single metric. The compound effect was normalized against positive  and negative (0.1% DMSO) controls to calculate the dose–response curves for each compound in each cell line and condition (with and without irradiation) separately. Each cell line was screened using two parallel compound sets. One set served as a control (single agent) and the other set was irradiated with an optimized EC20 irradiation dose (compound–irradiation combination). Otherwise, plates were handled identically. To determine the synergistic and antagonistic effects of the compounds and irradiation, we calculated the delta DSS (ΔDSS) for each compound and cell line by subtracting the DSS for the compound–irradiation combination from the single agent DSS  using the Breeze software . Otherwise, we used the same DSRT workflow as described above. To test whether the compound–irradiation combinations acted synergistically or antagonistically, we compared the observed responses to the expected combination responses. We then calculated the responses, and generated the dose–response matrices using the ZIP reference model with the SynergyFinder web application . The suspension of two cell lines (UT-SCC-42A and UT-SCC-24B) was labeled according to the manufacturer's instructions using CellTrace Far Red . One million cells were mixed with 1-ml PBS containing 1-µl CellTrace Far Red. Cells were placed in an incubator for 20 min at + 37 °C. Following incubation, a 5-ml culture medium was added, followed by 5-min incubation. Labeled cells were centrifuged for 5 min at 173×g at RT and suspended in a fresh culture medium. Cell suspension was diluted at a density of 100 000 cells/ml and divided into four groups: control with 0.1% DMSO and navitoclax concentrations  in the presence of the IncuCyte Caspase-3/7 Apoptosis Assay Reagent . Cell suspension was pipetted onto the wells . Plates were placed in the Incucyte S3 live cell analysis system (Sartorius) and imaged every 2 h at objective 20× (9 images per well). Six replicates were used for each condition. After 24 h, plates were transferred to an irradiator room and one plate was irradiated with 8 Gy. After irradiation, the plates were placed back in Incucyte and imaged for 48 h. Cancer cell proliferation (red object count) and apoptotic cells (green object count) were quantified using the Incucyte analysis software. The apoptotic index (percentage of apoptotic cells) was determined by selecting apoptotic cells (green and red objects) divided by the total cell number (red object) multiplied by 100.Two 96-well plates  were coated with 500-µg/ml Myogel (50-µl per well). Plates were then placed in an incubator overnight. On the following day, we prepared the cell suspension for the apoptosis assay using trypsin/EDTA to detach cells from the flaskSupplementary Figure S1.Supplementary Figure S2.Supplementary Figure S3.Supplementary Tables.


V600EBRAF mutations, while the response to PI3k/mTOR inhibition was better in the presence of the Q61LNRAS mutation. Our study uncovers a large panel of new vulnerabilities in conjunctival melanoma and establishes the background for the expansion of therapeutic options in the management of this tumor.We determined the sensitivity of several conjunctival melanoma cell lines to kinase inhibitors as well as a few other inhibitors using an image-based high throughput drug screening assay with 542 compounds. All cell lines demonstrated sensitivity to cell cycle inhibition, especially with polo-like inhibitors. The response to MAPK pathway inhibition was better in the presence of V600EBRAF  mutations were more sensitive to BRAF/MEK inhibition, while CRMM2 bearing the Q61LNRAS mutation was more sensitive to PI3k/mTOR inhibition. All cell lines demonstrated sensitivity to cell cycle inhibition, being more pronounced in CRMM2, especially with polo-like inhibitors. Our data also revealed new vulnerabilities to Hsp90 and Src inhibition. This study demonstrates that the genomic background partially influences the response to targeted therapy and uncovers a large panel of potential vulnerabilities in conjunctival melanoma that may expand available options for the management of this tumor.Recent evidence suggests that numerous similarities exist between the genomic landscapes of both conjunctival and cutaneous melanoma. Since alterations of several components of the MAP kinases, PI3K/mTOR, and cell cycle pathways have been reported in conjunctival melanoma, we decided to assess the sensitivity of conjunctival melanoma to targeted inhibition mostly of kinase inhibitors. A high content drug screening assay based on automated fluorescence microscopy was performed in three conjunctival melanoma cell lines with different genomic backgrounds with 489 kinase inhibitors and 53 other inhibitors. IC50 and apoptosis induction were respectively assessed for 53 and 48 compounds. The genomic background influenced the response to MAK and PI3K/mTOR inhibition, more specifically cell lines with  Conjunctival melanoma (CJM) most frequently arises from proliferation of atypical melanocytes localized in the conjunctiva . A smaller proportion of conjunctival melanoma develops from preexisting conjunctival nevi or de novo. CJM is associated with a high recurrence rate and a significant mortality, affecting 25–35% of the patients at 10 years ,5,6.NF1 predominate (50–33%), followed by mutations in BRAF (46–29%) and NRAS (26–11%) [mutBRAF, mutNRAS, mutNF1, Triple WT) [ATRX, often co-occurring with mutation in NF1 in tumors arising in the tarsal conjunctiva have been reported in 17 out of 68 conjunctival melanomas [TERT promoter mutations identified in up to 54% of the cases and NRAS mutations have been associated with metastatic disease [CTNNB1, coding for β-Catenin, were recently identified in 17% of the cases [RET, TP53, C-KIT, ARID2, TET2, CDKN2A, MAPK2, RAC1, MET and even in genes mutated in uveal melanoma such as SF3B1, GNAQ, and GNA11 [CCND1 gene coding for cyclin D1), 12p, 13q, 14p, and 17q gains, while losses have been described in chromosomes 1p, 3q, 4q, 6q, 8p, 9p (encompassing the CDKN2A gene), 12p,14p, 17q, and 19 [V600EBRAF or Q61LNRAS mutations [CJM not only shares clinical characteristics with cutaneous melanoma such as lymphatic metastatic spread, but the genomic background also shares numerous similarities with cutaneous melanoma. The genomic landscape is characterized by a high tumor somatic mutational burden and a predominance of C > T transitions, suggesting a role for UV light in the development of this tumor ,8,9. Our NRAS 26–% [11,12,(26–11%) ,14. Thesiple WT)  exist inelanomas . Both TE disease . Mutatiohe cases . Rare mund GNA11 ,14,16. T, and 19 ,19. In vutations ,19.Probably due to the rarity of CJM, reports documenting the use of BRAF inhibitors or MEK inhibitors in the management of this tumor are limited to less than 10 cases, with variable success . In CJM Based upon our identification of alterations of signaling pathways in CJM, we aimed to assess the response of CJM cell lines with different genomic backgrounds to inhibition of these pathways. We, therefore, designed a high content drug screen of more than 500 compounds predominantly including tyrosine kinase inhibitors. Our study revealed previously unknown susceptibilities to multiple targets, and a genotype-dependent response to MAPK, PI3K/mTOR, as well as cell-cycle inhibitors, expanding the therapeutic options for the management of advanced CJM.2 in a medium composed of Ham’s F-12 (Kaighn’s) without phenol red , supplemented with 10% Fetal Bovine Serum FBS , antibiotic antimycotic 100× solution (cat. n° A5955 Sigma-Aldrich) as previously described [Conjunctival Recurrent Malignant Melanoma-1 cells , Conjunctival Recurrent Malignant Melanoma-2 cells , and CM2005.1 (RRID: CVCL_M592) were grown at 37 °C, with 5% COescribed . CRMM1, https://broadinstitute.github.io/picard/, accessed on 27 February 2022; Broad Institute, v.2.23.8). Base quality score recalibration was performed on the processed BAM files with GATK (v4.1) [Whole exome sequencing (WES) was performed in all three cell lines by using the Twist Comprehensive Exome Panel  on a HiSeq 4000 sequencer  raw reads were aligned to the UCSC hg19 genome reference  with BWAK (v4.1) , BaseRechttps://variantvalidator.org/, accessed on 15 February 2022). Presence of variants in different cancer types was checked in COSMIC v95 .Variants were annotated with RefSeq , gnomAD 2) using a 4×/0.2 NA objective, allowing imaging of the full well surface in a single image, while still allowing precise segmentation of individual cells, nuclei, or regions of interest.The compounds screened were selected from different Ecole Polytechnique Fédérale de Lausanne (EPFL)’s Biomolecular Screening Facility (BSF) compounds libraries, generating a sub-collection of 489 kinase inhibitors and 53 other selected inhibitors . ChemicaDose–response curves were established for 53 compounds with concentrations ranging from 3000 to 0.1 nM using Echo acoustic dispensing, in the same conditions as for the primary screen.We acknowledge the NCCR Chemical Biology supported by the SNSF for purchasing the BSF-EPFL Collections.Apoptosis induction was assessed using Annexin V assay  with concentrations ≥ IC50 previously obtained grouped in three categories  using Echo acoustic dispensing. Annexin V, Hoechst, and ethidium homodimer-1 stainings were performed in 96-well plates  after 48 h of incubation and automated imaging was performed in the same conditions as for the primary screen, but using a 10×/0.45 objective.For the entire screening assay, the dose–response curve and apoptosis assays were performed in duplicate. A preliminary validation for each assay and for each cell line was performed using the statistical Z’ approach , using phttps://cellprofiler.org/citations/, accessed on 22 June 2021) [https://www.knime.com/, accessed on 20 January 2022) with the assessment of size, shape, and intensity features of all segmented objects. Each plate contained replicates of positive and negative controls , and all results were normalized according to the controls of the corresponding plate. Screen hit assignment was based upon the normalized values, a hit being defined as such if the value was 3 SD superior to the corresponding negative control for all replicates.All the images were analyzed using either pipelines in CellProfiler v4.2.1 (ne 2021)  or custone 2021)  and extrDepending on the assay, different features and calculated values were considered as readouts: for the primary screen and the dose responses, total cell counts (based on Hoechst staining) and dead-cell ratios  were utilized. For the apoptosis assay, total cell counts (based on Hoechst staining) and percentages of Annexin V-positive cells were considered (based on Annexin V intensity).V600EBRAF mutation as well as missense variants with uncertain significance in NSD1 and BRCA1; however, no other clear-cut mutation was detected.Mutations identified in all cell lines are summarized in V600EBRAF mutation as previously published [ROS1), ROS1P2161S, was also discovered, together with a frameshift mutation in TET2 Methylcytosine Dioxygenase 2 (p.Glu1459LeufsTer)TET2). P2161SROS1 is predicted to be pathogenic by in silico tools. CRMM1 contained as well a CDKN2A c.193 + 5G > A mutation, affecting p14ARF, and a probably damaging mutation in the retinoblastoma gene, RB1S648L.CRMM1 harbored the ublished . InteresQ61NRASL mutation as previously described [G936EALK, previously reported in basal cell carcinoma [P287LCCND1. In vitro, P287L impairs the interaction between CCND1 and the AMBRA1 complex, which is responsible for ubiquitination and subsequent degradation of CCND1 [CRMM2 contained a escribed , as wellarcinoma  and skinof CCND1 .The Z’ scores obtained for each plate  were very good, validating each of the screening plates, and providing excellent screening windows .In total, 60 hits (40 with score ≥ 0.1) were identified for CM2005.1, 49 hits (34 with score ≥ 0.1) for CRMM1, and 58 hits (48 with score ≥ 0.1) for CRMM2 . For allDose–response curves were established for 53 selected compounds (with a hit score ≥ 0.05 for at least one of the cell lines). All Z’ scores obtained for each cell line  allowed a good validation of the plates, providing results that could be further analyzed and fitted for IC50 determination. The results are summarized in V600EBRAF mutation than in CRMM2, bearing the Q61LNRAS mutation. CM2005.1 was the most sensitive cell line to MAPK pathway inhibition with IC50 below 50 nM for six MEK inhibitors and Dafrafenib. CRMM1 was also responsive with IC50 below 50 nM for five MEK inhibitors and PLX4720, while CRMM2 was less responsive with one IC50 below 50 nM for AZD8330 and for PLX4720. Among the MEK inhibitors, AZD8330 was associated with a stronger inhibition in all cell lines. Interestingly, downstream inhibition of ERK with Ravoxertinib was relatively potent in CRMM1, but inefficient in CRMM2. C-MET inhibitors were not as efficient as BRAF or MEK inhibitors in all cell lines.Globally, the response to MAP kinase inhibition was better and stronger in cell lines with the The inhibition of the PI3K/mTOR axis was more efficient in CRMM2 than in CM2005.1 and CRMM1. More specifically, IC50s were lower in CRMM2 for five mTOR inhibitors, three PI3K inhibitors, and a dual PI3K/mTOR inhibitor, Omipalisib. In CRMM2, this dual inhibition of PI3K and mTOR was the most efficient. A response to Sirolimus was only observed in CRMM1.Cell cycle inhibition was assessed with cyclin dependent kinase (CDK) inhibitors and with antimitotic polo-like kinase inhibitors and aurora kinase inhibitors. Overall, the inhibition was stronger in CRMM2. Dinaciclib, an inhibitor of CDK2, 9, 7, and 5, was associated with the lowest IC50 in all the cell lines. Importantly, no inhibitors of cyclin dependent kinase 4/6  were identified as hits in the three cell lines. RGB-286638 was effective in all cell lines, but its effects might also be attributed to the possible multikinase inhibition of GSK-3β, JAK2, and MEK1. Polo-like inhibitors were more effective in CRMM2 and CRMM1: in CRMM2, 4 out of 5 polo-like inhibitors were associated with IC50 below 100 nM and 2 out of 3 in CRMM1. Two aurora kinase inhibitors were effective in CRMM2 and Alisertib was associated with an IC50 of 32.7 nM.EGFR, KRAS, or ALK mutations, was effective in all cell lines with a stronger inhibition in CRMM2.Ganetespib, a heat shock protein 90 inhibitor, used in clinical trials for the treatment of non-small cell lung carcinoma with The Z’ scores obtained for each plate  were again excellent and allowed a good discrimination of the Annexin V positive hits .C-Met Inhibition by Tivantinib was not associated with induction of apoptosis in CM2005.1 and CRMM1. Apoptosis induction was observed in CM2005.1 with all MEK inhibitors tested and in CRMM2 with the six MEK inhibitors efficient in this line. In CRMM1, only Cobimetinib, AS703026, AZD8330, and TAK-733 triggered apoptosis, but Selumetinib, PD184352, and GDC-0094, despite low IC50, did not induce apoptosis.Inhibition of the PI3K/mTOR pathway and cell cycle inhibition with CDKI was systematically associated with induction of conventional apoptosis when the cell lines were sensitive to the drugs, as demonstrated by the Annexin V assay.Except for Rigosertib inducing apoptosis in all cell lines, apoptosis was not seen with polo-like kinase inhibitors in CM2005.1. Apoptosis induction was more frequently observed with polo-like kinase in CRMM2 and CRMM1.Among the three checkpoints kinase inhibitors studied, only CHIR-24 was effective in all three cell lines and associated with apoptosis induction.Based upon our previous studies combining genomic and transcriptomic data as well as pathway activation in CJM, we assessed, in vitro, the response to kinase inhibition in the largest image-based high content drug screening study performed so far in this tumor. Our results revealed previously unknown susceptibilities to multiple drugs, establishing a framework for the selection of future targeted therapy in the management of advanced CJM.V600EBRAF mutations and the lower IC50 identified with Dabrafenib for CM2005.1 (44.16 nM) lays within the same range as previously reported for this drug [Q61LNRAS mutation with PLX4720 is, however, surprising and might possibly be due to a non-specific toxic effect.Upstream inhibition of the MAPK kinase pathway with BRAF inhibitors PLX470 and Dabrafenib was, as expected, more efficient in cell lines with his drug . PLX4720V600EBRAF mutations. Among the 10 MEK inhibitors assessed, AZD8330, an ATP non-competitive MEK1/MEK2 inhibitor, appears to be the most efficient with the lowest IC50 and apoptosis induction in all the cell lines. However, in a phase 1 clinical study with AZD8330 in 82 patients including 18 cutaneous melanomas [NRAS mutation, an objective response in 12.4% of the patients, and stable disease in 55% of the patients [V600EBRAF mutations [Our study confirms that MEK inhibitors are a treatment of choice for cell lines with elanomas , only onpatients . Althougpatients , in a phpatients . Severalutations ,50,51 reV600EBRAF mutations [Downstream MAPK inhibition with ERK inhibitors was only effective in CRMM1 with low IC50 identified both for PD184352 and Ravoxertinib, without apoptosis induction. Antitumor activity has been demonstrated with Ravoxertinib in a phase I study in 47 patients with advanced solid tumors with stable disease in 34% and a partial response in two colon carcinomas with Q61LNRAS mutation and Omipalisib, a dual inhibitor of the p110 subunit of Pi3K and the mTOR inhibitor, was associated with the lowest IC50 in our study. Our results are in line with previous data in NRAS-mutated skin melanoma cell lines where Omipalisib was the most potent inhibitor of the PI3K/mTOR pathway [NRAS-mutated skin melanoma cell lines [Inhibition of the PI3K/mTOR axis was more efficient in CRMM2 with  pathway . In a la pathway . The comll lines . This coll lines . The effll lines , bladderll lines , and renll lines . Recentlll lines .RB1S648L mutation and CRMM2 contains a P287LCCND1 mutation that impairs Cyclin D1 degradation. Among cyclin dependent kinase inhibitor (CDK), Dinaciclib, a second-generation CDK inhibitor of CDK2, CDK9, CDK5, and CDK1, was the most potent with the lowest IC50. Our results are similar to those observed in vitro with several cell lines including skin melanoma cell lines where caspase 3 activation was noted in most of the cases [BRAS or NRAS mutations, nor with CDK2 levels. In lung carcinoma cell lines, apoptosis induction by Dinaciclib was, however, stronger in the presence of a KRAS mutation [Inhibition of cellular division was more effective in CRMM1 and CRMM2 which grow faster than CM2005.1. Of note, CRMM1 harbors a he cases . In cutahe cases . The goohe cases . In cutahe cases , the effmutation . Clinicamutation , clinicamutation . Clinicamutation , it is pmutation , suggestNRAS mutation with Volasertib and other polo-like inhibitors can be correlated with the increased mitotic stress identified in RAS mutated cells and increased sensitivity to PLK inhibition [Polo-like kinases are important regulators of cell division and Polo-Like kinase 1 has been involved in centrosome maturation, spindle assembly, mitosis entry, chromatin condensation, and cytokinesis . Plk1 ovhibition . In a phhibition .Aurora kinases play important roles during mitosis. More specifically, aurora kinase A (AKA) is involved in spindle assembly, centrosome maturation, and separation and cytokinesis . AKA expBRAF- and NRAS mutated cell lines [BRAF resistant melanoma cell lines [BRAF-mutated melanoma assessing the combination of XL888 and Vemurafenib and/or Cobimetinib are ongoing.In our study Ganetespib, a heat shock protein 90 inhibitor was associated with IC50 below 100 nM in two cell lines and apoptosis induction in all cell lines. Heat shock protein 90 (Hsp90) has been implicated in the assembly of multiple chaperone complexes, regulating the stability and function of client proteins such as wild type c-RAF , mutant ll lines . Ganetesll lines . Evidencll lines . In thisKX2-391, Tirbanibulin, was also very efficient in CRMM2. Tirbanibulin reversibly binds β-tubulin and inhibits tubulin polymerization , growth The use of 2D cultures of established conjunctival melanoma cell lines is a limitation of our study. Despite several attempts, patient-derived short-term 3D cultures in spheroids from conjunctival melanoma have, so far, not been successful in our research. We nevertheless believe that the results presented in our study are valid as the genomic background of the cell lines used reflects the oncogenic drives observed in conjunctival melanoma.In the largest drug screening performed so far in CJM, we assessed the response of CJM cell lines with different genomic background to kinase inhibitors targeting multiple dysregulated pathways. We believe that the identification of new vulnerabilities not only establishes new mechanistic research perspectives, but also uncovers new opportunities for the management of either locally advanced primary CJM or metastatic CJM. The combination of cycle cell inhibitors with either checkpoints inhibitors or MEK inhibitors appears notably as a promising option of overcoming tumor resistance and increasing tumor response. Our manuscript also highlights the necessity of fully characterizing the genomic background of all tumor cells, in order to optimize the therapeutic response.