BAP1 to be associated with higher resistance to MEK inhibition. However, reconstitution of BAP1 in a BAP1-deficient UM cell line was unable to restore sensitivity to MEK inhibition. We then compared UM tumors from The Cancer Genome Atlas (TCGA) with mutations in BAP1 with tumors with wild-type BAP1. Principal component analysis (PCA) clearly differentiated both groups of tumors, which displayed disparate overall and progression-free survival data. Further analysis provided insight into differential expression of genes involved in signaling pathways, suggesting that the downregulation of the eukaryotic translation initiation factor 2A (EIF2A) observed in UM tumors with BAP1 mutations and M3 UM cell lines might lead to a decrease in ribosome biogenesis while inducing an adaptive response to stress. Taken together, our study links loss of chromosome 3 with decreased sensitivity to MEK inhibition and gives insight into possible related mechanisms, whose understanding is fundamental to overcome resistance in this aggressive tumor.The use of MEK inhibitors in the therapy of uveal melanoma (UM) has been investigated widely but has failed to show benefits in clinical trials due to fast acquisition of resistance. In this study, we investigated a variety of therapeutic compounds in primary-derived uveal melanoma cell lines and found monosomy of chromosome 3 (M3) and mutations in  EGFR)-mutated non-small cell lung cancer (NSCLC) with EGFR inhibitors, treatment of breast cancer gene (BRCA)-mutated ovarian cancer with poly-ADP (adenosine diphosphate)-ribose polymerase (PARP) inhibitors and treatment of BRAF-mutated cutaneous melanoma and NSCLC with BRAF inhibitors or MEK inhibitors [Uveal melanoma (UM) is the most common malignancy of the eye, affecting 5 to 7 individuals per million people per year . While phibitors ,9,10,11.hibitors .GNAQ and GNA11, which are considered to be early events and key drivers of UM development and lead to a constitutive activation of the mitogen-activated protein kinase (MAPK)/ERK pathway [BRAFV600E-mutated cutaneous melanoma has been shown to be successful, MEK inhibitors have also been investigated as potential therapies in UM [Around 90% of UM tumors harbor mutually exclusive mutations in two homologous G-protein alpha subunits,  pathway ,14,15. A pathway ). Since es in UM ,18. Despes in UM ,20.(BAP1) have been found in more than 80% of metastasizing UM [BAP1 is a nuclear deubiquitinase which, besides its capacity of deubiquinating proteins, also functions as a central binding partner in complexes with BRCA1, HCF1 or ASXL1, among others, thereby affecting cell proliferation, DNA damage response and differentiation processes through influencing chromatin remodeling [BAP1 have been shown to promote a stem cell-like state and loss of protein expression has been associated with increased metastatic risk [BAP1 gene is encoded on chromosome 3p.21, it is likely that M3 in UM unmasks recessive mutations in BAP1 [One of the most important prognostic factors in UM is monosomy of chromosome 3. Monosomy 3 (M3) is strongly correlated with metastatic risk, and gene clusters on chromosome 3 as well as on chromosome 8p build a basis for classification of UM tumors in a high or low risk class using microarray-based gene expression profiling ,22,23. Mizing UM . BAP1 ismodeling ,26,27. Bmodeling ,29,30. Itic risk ,32. Sinc in BAP1 .BAP1 mutational status. However, reconstitution of wild-type BAP1 in a BAP1-deficient M3 cell line was unable to sensitize cells to MEK inhibitors, suggesting that other mechanisms associated with loss of chromosome 3 might affect sensitivity to MEK inhibition in combination or independently of BAP1 loss. To address this, we performed an analysis of The Cancer Genome Atlas (TCGA) for UM to compare the pathways that are overrepresented in UM tumors with BAP1 mutations and M3 compared to UM tumors with wild-type BAP1 and D3. We identified eIF2 signaling and ERK/MAPK signaling as important pathways. These findings can help to build a path for understanding resistance mechanisms in UM, which is critical for learning how to effectively target this lethal cancer.To the best of our knowledge, this study reports for the first time that patient-derived UM cell lines with M3 show increased resistance to inhibitors targeting MEK compared to disomy 3 (D3) UM cell lines. We placed these findings in the context of We evaluated the sensitivity of three D3  and three M3  UM cell lines to a variety of chemotherapeutic and molecularly targeted compounds at nine different concentrations to obtain comprehensive dose-response curves throughout different inhibitor classes. Treatment of UM cell lines with MEK inhibitors for 5 days resulted in a significantly higher cell viability for M3 cell lines than D3 cell lines after treatment, even after exposure to high concentrations (>20 µM) . TrametiBAP1 mutations are occurring with high frequency in monosomy 3 UM tumors [BAP1 mutations leading to protein loss in all M3 cell lines. These results are in agreement with other studies [BAP1. We first reconstituted wild-type BAP1 or an inactive p.C91S mutant BAP1 in the UPMM2 cell line and confirmed re-expression of BAP1 at the protein level by Western blot  control  where BAP1 gene is located, resulting in complete inactivation of BAP1. However, UM tumors with wild-type BAP1 showed neutral copy numbers in chromosome 3 . Six tumors without mutations in BAP1 but with deletions in chromosome 3 were considered unclear for the inactivation of BAP1 and discarded from further analyses. The remaining 74 tumors where then split into 35 samples with BAP1 mutations and 39 without BAP1 mutations.To get clinical significance of the differences in resistance to MEK inhibitors observed in UM cell lines, we analyzed 80 UM tumors from The Cancer Genome Atlas (TCGA) dataset . We comperations  showed tBAP1 exhibited a much shorter overall survival (p = 4 × 10−6) and a higher incidence of metastases, indicated by a shorter progression-free survival (p = 2 × 10−6) than patients with wild-type BAP1  for overall survival and 7.6 (95% CI 2.8–20.4) for progression-free survival. This indicates the risk of dying or progressing for UM patients with tumors with BAP1 mutations is about 10 and 8 times higher, respectively, than for patients with wild-type BAP1.UM patients from TCGA that had tumors with mutations in ype BAP1 . The hazBAP1 mutations. Principal component analysis (PCA) of RNA-Seq data showed that they constitute completely different entities  correction method. That lead to the identification of 7130 differentially expressed genes after an FDR q < 0.05 (3988 upregulated genes and 3142 downregulated genes). The top 50 differentially expressed genes are displayed in We then compared the gene expression of tumors with BAP1 mutations, we performed gene expression network analysis with Qiagen Ingenuity Pathway Analysis (IPA) software using a stringent list of differentially expressed genes (an FDR q < 10−10 and a fold change > 2 in both directions). Surprisingly, the most significant network showed an indirect link between BAP1 and ERK  and a stringent criterion (an FDR q < 10−10 and a fold change > 2 in both directions). We performed IPA for both gene lists and identified their overrepresented canonical pathways. The top 35 enriched canonical pathways significant in both gene lists are displayed in p = 0.001) (BAP1-mutant versus wild-type BAP1 tumors.To find the overrepresented pathways caused by mutations in = 0.001) , indicatBAP1 UM tumors (p = 4 × 10−10) (p = 4 × 10−7) and mTOR signaling (p = 7 × 10−4) (The eukaryotic initiation factor 2 (eIF2) signaling pathway is the most significant pathway identified by IPA comparing wild-type to mutant × 10−10) . Similar × 10−4) .BAP1 mutations displayed a downregulation in EIF2A and in the expression of ribosomal protein genes for the large and small subunits in a coordinated manner  by qPCR, which showed lower gene expression levels in the M3 cell lines than in the D3 cell lines  showed no significant differences  cell line led to decreased cell proliferation, cell cycle progression and invasion, while knockdown of BAP1 in a BAP1-wild type ICC cell line showed the opposite effect [BAP1 in BAP1-wild type UM cell lines resulted in no changes or even in a decrease of in vitro tumorigenicity, respectively, defined by proliferation rate, migration and invasion ability [BAP1 as a tumor suppressor is limited, and that effects of BAP1 loss on tumor aggressiveness are highly plastic and probably regulated by a variety of other factors that need to be elucidated. Thus, reconstitution of the BAP1-deficient UPMM2 cell line with wild-type BAP1 was unable to sensitize cells to MEK inhibitors, suggesting that BAP1 expression is not sufficient to confer sensitivity in the context of chromosome 3 loss.As henotype ,32, we piewed in ). Similae effect . Interes ability . Due to To further understand the implications of the ERK/MAPK pathway in the mechanism of resistance of the M3 cell lines to MEK inhibitors, we compared the phosphorylation state of MEK and ERK for the D3 and M3 cell lines at basal levels and upon treatment with trametinib. However, no significant differences were observed in our experimental conditions, suggesting that other pathways might be implicated in the mechanism of resistance to MEKi in UM cell lines.BAP1-mutations is highly associated with M3, as has previously been shown, supporting the hypothesis of M3 unmasking recessive BAP1-mutations in UM [EIF1AX and SRSF2/SF3B1, which showed good prognosis, and two groups with M3 and high incidence of BAP1 mutations, whose patients displayed poor prognosis. However, there was no direct comparison of tumors with BAP1 mutations with those with wild-type BAP1. Here, we removed samples with unclear BAP1 mutation/inactivation and compared UM tumors based on their BAP1 mutational status. We show that UM tumors can be classified in two distinct groups according to their BAP1 mutational status, with differential gene expression profiles and BAP1 mutations associated with poor prognosis.To evaluate other potential pathways implicated in the mechanism of resistance to MEKi, we analyzed clinical and genomic data from TCGA of UM patients. Our analysis of UVM-TCGA data confirms that the occurrence of ns in UM . The orins in UM  classifiBAP1 mutations with M3 in UM, differences in gene expression and prognosis could be a result of differential chromosome 3 status independent of BAP1. The options for investigating the exact role of BAP1 independent of M3 as a driver of tumor aggressiveness in UM using genomic and transcriptomic patient data are limited because of the lack of data from M3 tumors without BAP1 mutations. This tight bond of M3 and mutations in BAP1 is by itself a strong indicator of the presence of both factors being necessary for a metastasizing, aggressive UM phenotype. It has been shown previously that loss of BAP1 expression can be utilized as a prognostic marker in RCC and breast cancer [BAP1 mutations and loss of BAP1 expression in UM patient tissue and showed a significant correlation between BAP1 loss and poor survival [BAP1 being seemingly necessary but not sufficient to confer sensitivity to MEK inhibition in our setting, we detected an indirect link between BAP1 and ERK in the most significant network based on gene expression network analysis of UVM-TCGA data when analyzing BAP1 mutant compared to wild-type BAP1 (representative for M3 compared to D3) tumors. In a mouse model of mesothelioma, conditional knockout of Bap1 increased phosphorylation of ERK and accelerated tumor development, especially in the presence of accompanying Nf2 and Cdkn2ab deletions [Due to the strong association of t cancer ,37. Shahsurvival , whose fsurvival ,39,40 aneletions , emphasiBAP1 in UPMM2, when reconstitution at the same time caused a decrease in proliferation. Since we could not detect the observed resistance pattern when cells were treated with other compounds, we hypothesize that, if at all, replication/proliferation differences selectively play a role in altering sensitivity to MEK inhibitors, perhaps by cell cycle-dependent differences in MEK/ERK activation, which has been reported for myeloid leukemia cell lines [BAP1-reconstituted UM cells will need to be addressed in future work.In general, all our UM cell lines display low proliferation rates, and the M3 cell lines we used have shown significantly lower replicative potential compared to the D3 cell lines. While lower proliferation rates in M3 compared to D3 cell lines were observed, the differences were not significant . Similarll lines . FurtherBAP1-mutant (M3) tumors with potential relevance in affecting sensitivity to MEK inhibition is eIF2 Signaling. eIF2 signaling is critical for the integrated stress response (ISR), which can be induced by different sources of cellular stress. One important source of cellular stress is endoplasmic reticulum (ER) stress, characterized by an accumulation of unfolded proteins in the ER and an activation of the unfolded protein response (UPR) pathway. One major effector activated in the UPR is double-stranded RNA-activated protein kinase (PKR)-like endoplasmic reticulum kinase (PERK). PERK, also known as eIF2AK3, is activated by stress-induced molecular chaperones, e.g., GRP78, and in turn phosphorylates eukaryotic initiation factor 2α (eIF2α). Active peIF2α mediates translational attenuation and expression of a variety of ISR-related genes, resulting in two possible outcomes that depend on the intensity and duration of ER stress: survival or apoptosis . iewed in ,48. DiffBAP1-mutant UM tumors are more aggressive than those with wild-type BAP1, it would be expected that they have a higher capacity of synthesizing proteins. Conversely, we observed a systematic downregulation of the ribosomal protein genes along with downregulation of EIF2A, which we validated by qPCR, and might result in a coordinated decrease of ribosome biogenesis in BAP1-mutant tumors. In fact, under certain stress conditions in the tumor microenvironment such as hypoxia, nutrient deprivation, and oxidative and genotoxic stress, inhibition of eIF2 can trigger coordinated decreases in protein biosynthesis to preserve energy and prevent the accumulation of damaged and misfolded proteins. This in turn allows for the selective translation of mRNAs critical in the adaptive response to stress . Thus, BAP1. Despite seemingly not having a direct effect on susceptibility to MEK inhibition, we show that UM tumors can be classified in two tumor subgroups with different clinical outcomes based on their BAP1 mutation status by analyzing UVM-TCGA data. BAP1-mutant tumors display differential expression of key genes involved in canonical pathways that could play critical roles in modulating the response to MEK inhibition, such as downregulation of the eIF2 signaling to decrease ribosome biogenesis. Since M3-UM tumors define a highly aggressive cancer entity resistant to common therapies, understanding the implications of differentially regulated pathways and resulting resistance mechanisms is indispensable for identifying new ways of targeting this lethal disease. Further investigation of the possible relevance of the aforementioned signaling pathways in therapy resistance is highly needed.Taken together, our study reveals decreased sensitivity to MEK inhibition in M3-UM cell lines that was not restored by reconstitution of wild-type v/v) fetal bovine serum  and 1% (v/v) Penicillin-Streptomycin . Cell line 92-1 was cultured in RPMI 1640 medium  supplemented with 10% FBS and 1% Penicillin-Streptomycin. All cell lines were routinely tested for mycoplasma by PCR.Uveal melanoma cell lines UPMD1, UPMD2, UPMM2, UPMM3 and UPMM4 were kindly provided as early passages by the Department of Human Genetics, University Hospital Essen (Dr. M. Zeschnigk) and were derived from untreated primary uveal melanomas and characterized regarding their chromosome 3 status . PrimaryBAP1 or inactive mutant p.C91S BAP1 in the BAP1-deficient M3 cell line UPMM2, retroviruses were generated in HEK-293GP cells cultured in DMEM medium  supplemented with 10% FBS and 1% Penicillin-Streptomycin. HEK-293GP cells were transfected with pVSV-G envelope plasmid [For stable expression of wild-type  plasmid  (Addgene plasmid  (Addgene plasmid  (Addgene2 for 5 days (120 h).Trametinib  and Refametinib  were dissolved to 10 mM in DMSO and printed at nine concentrations from 5 nM to 25 µM in triplicates in white-bottom sterile polystyrene 384-well plates  using a Tecan D300e Digital Dispenser  and the corresponding software D300e Control . DMSO was printed as a negative control. Pre-printed plates were sealed and stored at −80 °C until used. Prepared assay plates were equilibrated at room temperature and 500 cells per well were seeded in 30 µL culture medium with a Multidrop Combi dispenser (Thermo Fisher Scientific). Assay plates were incubated at 37 °C and 5% COCellTiter Glo reagent  was prepared according to the manufacturer’s protocol. Assay plates were equilibrated at room temperature for 30 min and 30 µL of a 1:4 dilution of CellTiter Glo reagent in Dulbecco’s phosphate-buffered saline  were added to each well. Plates were shaken for 2 min and incubated for 8 min at room temperature protected from light. Luminescence was measured at 500 milliseconds using a Tecan Spark 10 M microplate reader (Tecan) and the corresponding software Spark Control . Cell viability was normalized to non-treated DMSO controls and data was analyzed in Microsoft Excel  and GraphPad Prism 9 .BAP1-reconstituted UPMM2 cells per well were seeded in 30 µL culture medium in triplicates in white-bottom sterile polystyrene 384-well plates  in seven replicate plates per experiment and incubated at 37 °C and 5% CO2. Every 24 h, cell viability was assessed via CellTiter Glo viability assay (see Five hundred cells of ssay see  as a surBAP1-reconstituted UPMM2 cells (3000) per well were seeded in 2 mL culture medium in duplicates in 6-well cell culture plates. Even distribution of single cells was ensured via microscopy. Assay plates were incubated for 5 weeks at 37 °C and 5% CO2. Culture medium was refreshed every 3–5 days. After 5 weeks, culture medium was aspirated, and cells were washed 1× with cold DPBS  and placed on ice. For fixation, 1 mL of cold methanol  was added per well and incubated for 10 min on ice under gentle agitation. Methanol was removed and 1 mL of 0.1% crystal violet  in 25% (v/v) methanol was added and incubated for 30 min at room temperature under mild agitation. Staining solution was removed, wells were washed thoroughly with tap water and plates were air-dried upside-down on tissue paper. Plates were scanned using an Epson Perfection V850 Pro Scanner  and the corresponding software Epson Scan . Confluency was quantified as previously described using the ImageJ-Plugin “ColonyArea” [onyArea” . Data waWestern blot experiments were performed as described previously . In briehttps://portal.gdc.cancer.gov). BAP1 mutation status was obtained from Robertson and colleagues [BAP1 but with monosomy in chromosome 3 were considered uncertain for BAP1 inactivation and were discarded from further analyses.RNA-Sequencing (RNA-Seq) and segmented DNA copy numbers (GRCh37/hg19) data from 80 uveal melanoma (UVM) patients from The Cancer Genome Atlas (TCGA) were downloaded from the Genomic Data Commons (GDC) data portal on 19 December 2020   and analSegmented DNA copy numbers (GRCh37/hg19) were interrogated every 0.5 Mb in chromosome 3 to display the paired copy number for each UVM-TCGA tumor, which were sorted by their averaged copy number in chromosome 3.t tests were performed between tumors with mutations in BAP1 and those without mutations in BAP1 considering the group variances. All calculated p values were corrected using the Benjamini and Hochberg false discovery rate (FDR) method to discard false positives by the fact of performing multiple tests. Principal Component Analysis (PCA) was computed with Partek Genomics Suite  using all transcripts expressed in the 74 UM tumors . Significant genes with an FDR q < 10−3 and a fold change (FC) > 1.5, as well as genes with FDR q < 10−10 and FC > 2 were further analyzed with Ingenuity Pathway Analysis (IPA) software Spring Release (March 2021), v62089861  software to identify overrepresented canonical pathways for BAP1 mutations.Gene expression levels were estimated by the RNA-Seq Expectation-Maximization (RSEM) normalization method and log2 transformed. Gene expression data were analyzed as previously reported . BrieflyPPIB), also known as cyclophilin B, and plotted as relative values. Primer sequences are shown in RNA was extracted using the AllPrep DNA/RNA Kit  according to the manufacturer’s protocol. RNA was quantified using a Nanodrop 2000c spectrophotometer (Thermo Fisher Scientific) and 1 µg or 2 µg of RNA were reverse transcribed to cDNA using the High Capacity cDNA Reverse Transcription Kit  according to the manufacturer’s instructions. Conventional PCR was performed with cDNA samples obtained from 2 µg RNA using the Hot Star Taq Plus Master Mix Kit  according to the manufacturer’s protocol. PCR products were evaluated on a 2% agarose gel to assess primer specificity and gel-purified using the QIAquick Gel Extraction Kit  and quantified. Serial dilutions of each amplicon were generated to serve as standards of known concentrations during quantitative reverse-transcribed PCR (qRT-PCR). qRT-PCR was performed on diluted cDNA samples or standards of known concentrations using LightCycler 480 SYBR Green I Master Mix  on a LightCycler 480 (Roche) with the following conditions: 95 °C for 5 min and 45 cycles of 95 °C for 10 s, 60 °C for 10 s and 72 °C for 15 s. Melting curves were generated for each sample. Gene expression was analyzed in Microsoft Excel (v16.0.13801.20288) and GraphPad Prism 9 (v9.0.0) by generating standard curves from known amplicon dilutions and interpolating sample Cp values for absolute quantification. Data was normalized to the housekeeping gene peptidylprolyl isomerase B (


Tumor recurrence is one of the major challenges in clinical management of chordoma. Despite R0-resection, approximately 50% of chordomas recur within ten years after initial surgery. The underlying molecular processes are poorly understood resulting in the lack of associated therapeutic options. This is not least due to the absence of appropriate cell culture models of this orphan disease.n = 6) were examined to validate whether the new model is representative. Cell viability after HOX/PBX complex inhibition with small peptides was determined by MTS assays.The intra-personal progression model cell lines U-CH11 and U-CH11R were compared using array comparative genomic hybridization, expression arrays, RNA-seq, and immunocytochemistry. Cell line origin was confirmed by short tandem repeat analysis. Inter-personal cell culture models  and the corresponding recurrent tumor (U-CH11R). Array comparative genomic hybridization and RNA-sequencing analyses revealed profound genetic similarities between both cell lines pointing to transcriptomic reprogramming as a key mechanism of chordoma progression. Network analysis of the recurrence specific genes highlighted HOX/PBX signaling as a common dysregulated event. Hence, HOX/PBX complexes were used as so far unknown therapeutic targets in recurrent chordomas. Treating chordoma cell lines with the complex formation inhibiting peptide HXR9 induced cFOS mediated apoptosis in all chordoma cell lines tested. This effect was significantly stronger in cell lines established from chordoma relapses.Clearly differing gene expression patterns and vulnerabilities to HOX/PBX complex inhibition in highly therapy resistant chordoma relapses were identified using the first intra-personal loco-regional and further inter-personal chordoma progression models. For the first time, HOX/PBX interference was used to induce cell death in chordoma and might serve as the basic concept of an upcoming targeted therapy for chordomas of all progression stages.The online version contains supplementary material available at 10.1186/s13046-021-02037-y. Chordomas are slow growing but locally invasive tumors of the axial skeleton . MicroscTBXT gene, appear to significantly contribute to tumorigenesis. Thus, shRNA- and sgRNA-mediated TBXT repression in chordoma cell lines have been shown to suppress cell growth and induce apoptosis [TBXT is associated with chordoma risk in rare familiar cases, whereas the nonsynonymous SNP rs2305089 can be also found in sporadic chordomas and has prognostic significance in overall survival [PBRM1, SMARCB1), PI3K signaling genes  and the LYST gene [EGFR and CDKN2A gene loci, respectively [en-bloc resection is the recommended treatment for primary tumors whenever feasible. For incompletely resected chordomas an adjuvant high-dose radiotherapy should be considered [en-bloc resection with or without subsequent radiation is high. Loco-regional recurrence rates of > 50% were published [Chordoma cells are thought to originate from cellular remnants of the notochord, as the most consistent feature is the sustained expression of the developmental transcription factor brachyury. Beyond being a sensitive chordoma marker, high levels of nuclear brachyury, encoded by the poptosis , 4. Moresurvival . FurtherYST gene . Somaticectively . Based onsidered . Though,ublished . Notablyublished . Althougublished . Completublished .Despite the high propensity to relapse, little is known about the underlying molecular processes accounting for chordoma progression. This is probably due to the rareness of the disease (incidence< 0.1:100000) and the deficiency of appropriate cell culture models .Here, we investigated the molecular differences in primary and recurrent chordomas using the cell lines U-CH11 and U-CH11R, which were established from the primary and the recurrent sacral chordoma of the same patient. We identified targetable genetic features of U-CH11R and inhibited the dimerization of PBX and the HOX transcription factors to induce apoptosis. This approach was extended to further chordoma cell lines.The establishment and maintenance of chordoma cell lines has been previously described . The cho2 and 15,000 cells/cm2 in biological and technical triplicates. Cell viability was examined at different time points up to 336 h. PDT was calculated by GraphPad Prism v.5  using a best fit exponential growth equation.Population doubling time (PDT) and cell viability were assessed by MTS assays  according to the manufacturer’s recommendations. To determine the PDT of U-CH11 and U-CH11R, cells were seeded in 96-well culture plates at 7500 cells/cm50 values were determined using GraphPad Prism v.5. All cell lines were tested in biological and technical triplicates.For cell viability assays in response to HXR9 and CXR9, cells were seeded in 96-well culture plates and allowed to adhere overnight, following the incubation of the peptides for 24 h. The amino acid sequences of HXR9 and CXR9 have been previously published by Morgan et al. , 21. TheThe colony formation assay was performed as previously described . Plated Array comparative genomic hybridization (aCGH) was performed with the SurePrint G3 Human CGH Microarray 8 × 60 (Agilent Technologies) as described previously . Array-Chttps://www.chordomafoundation.org). The GATK workflow for calling single nucleotide polymorphisms (SNPs) and indels in RNA-sequencing experiments was used . Raw reads were processed with Trimmomatic (v0.39) to remove adapter sequences and aligned with the STAR (v2.7) 2-pass method against the human genome build37 (hg19) [RNA-sequencing data of the primary chordoma tissue as well as U-CH11 and U-CH11R cell lines was obtained from the Chordoma Foundation (7 (hg19) –24. VariThe functional annotation of the variants was performed using ANNOVAR . Only exp < 0.05, FC > 2). Each cell line was tested at least in duplicates. Basal expression levels of selected genes are given . Total RNA was reverse transcribed to cDNA using the SuperScript IV Reverse Transcriptase Kit . Gene expression was quantified by qRT-PCR utilizing the QuantiTect SYBR Green Master Mix (Qiagen) and uniquely designed gene expression primers  including GAPDH and ACTB reference genes. Analyses were performed in technical triplicates and in at least biological duplicates.Gene expression analysis of -△△Ct).miRNA was extracted using the miRNeasy Mini Kit (Qiagen) according to the manufacturer’s handbook. For mature miRNA expression analysis, cDNA synthesis of 1 μg total RNA was carried out using the miScript Reverse Transcription Kit (Qiagen) with HighFlex buffer. qRT-PCR was performed using the QuantiNova SYBR Green PCR Kit and the Light-cycler Rotor Gene Q (Qiagen). The miScript Primer Assay for the mature Hs-miR-196a_2, Hs-miR-196b_2 and mirScript Universal Primer  were used according to manufacturer’s protocol. Relative miRNA levels were normalized to RNU6_2 snRNA. All measurements were performed in technical and biological triplicates. Relative fold changes were calculated using the comparative cycle threshold (Ct) eq. . AdherChordoma cell lines were grown in 10% FCS supplemented IMDM to 90% confluency in 6-well plates and allowed to adhere overnight. Cells were then transfected with 100 nM miR-196a-5p mimic (Dharmacon) or scrambled sequence  by using Lipofectamine 2000 (ThermoFisher Scientific) according to the manufacturer’s protocol and harvested after 6 h for Western blot and qRT-PCR analyses.Immunostaining of formalin-fixed and paraffin-embedded cell blocks and tissue sections (4 μm) were carried out by the avidin-biotin-complex method applying the K005 AP/RED Detection System . The antibodies used are given in Additional file Cell pellets were resuspended in RIPA lysis buffer containing protease and phosphatase inhibitor (ThermoFisher Scientific). Lysates were incubated in liquid nitrogen for 3 min, thawed and then centrifuged at 14000 rpm and 4 °C to remove debris. Protein quantification of the supernatants was performed using the Pierce BCA Protein Assay Kit (ThermoFisher Scientific). Prior Western blot analyses samples were equalized for protein amount, reduced with DTT and denatured at 90 °C for 5 min. Protein samples were resolved using 4–12% Bis-Tris gels (ThermoFisher Scientific) and then transferred to nitrocellulose membranes using a wet transfer device. Membranes were probed with primary antibodies at 4 °C overnight.To assess apoptosis in chordoma cell lines following a 24 h treatment with HXR9 and CXR9, cleaved PARP (Asp214), cleaved Caspase-7  and a cocktail of primary antibodies to detect apoptosis biomarkers pro/p17-Caspase-3, cleaved Caspase-3, along with ß-Actin loading control  were used.Protein lysates of miRNA mimic transfected cells were analyzed using an antibody against HOXA7  and ß-Actin .Membranes were incubated with secondary antibodies (Goat anti-Mouse IgG (H + L) 1:10000, ThermoFisher Scientific; Goat anti-Rabbit-IgG (whole molecule) 1:2000, Sigma-Aldrich, St. Louis, USA) and detected with the WesternSure Chemiluminescent Substrate . Immunoblots were quantified by densitometry using the ImageJ software (NIH).Caspase 3/7 activation in cells was assessed using the EarlyTox Caspase3/7 NucView 488 kit  following the manufacture’s instructions. Chordoma cells were plated at approx. 2500 cells/chamber in an 8-well Nunc Lab-Tek II CC2 chamber slide system (ThermoFisher Scienfic) and allowed to adhere overnight. HXR9 and CXR9 peptides (30 μM) were added to the cells and incubated for 24 h. The Caspase-3/7 NucView was then added directly to the cells at a final concentration of 5 μM and incubated at room temperature for 30 min, protected from light. Imaging was performed on the PAULA Cell imager .https://systems.crump.ucla.edu/rankrank/rankranksimple.php). Genes were ranked by their log10-transformed t-test p-values and effect size direction; the Benjamini and Yekutieli method was applied as a multiple hypothesis correction factor. A rank-rank overlap heatmap was generated to visualize the range of gene expression overlap between two signatures. The strength of enrichment is indicated as -log10 transformed hypergeometric p-value [The overlap trend between two complete, continuous gene-expression signatures was identified and visualized using the rank-rank hypergeometric overlap (RRHO) algorithm  were performed by computing overlaps between identified class-specific gene signatures and gene sets derived from the Molecular Signature Database (MSigDB) v7.1 . For stahttp://www.targetscan.org/vert_72/), miRDB v6.0 (www.mirdb.org) and mirDIP v4.1 (http://ophid.utoronto.ca/mirDIP/).Micro-RNA target genes of the miR-196a-5p were predicted using TargetScan v7.2  according to the WHO criteria. The cell line U-CH11 was characterized previously . The celThe immunocytochemical chordoma panel of the cell line largely represents the panel of the fixed tumor tissue   of U-CH11R was significantly shorter  of the exonic SNPs detected in the primary chordoma tissue were conserved in the derived primary chordoma cell line U-CH11 . Furthermore, a number of variants  in the tissue that were not detected in U-CH11 were found in U-CH11R supporting the hypothesis that a cell clone within the primary chordoma gave rise to the recurrent tumor. All samples harbored the brachyury Gly177Asp SNP (rs2305089) which is strongly associated with chordoma in the European population .EHD3 (rs772124528) was detected, which was also found in U-CH11R. Two additional mutations in ABCC1 (rs201020041) and MCCC1 (rs150862707) were exclusively found in U-CH11R. Moreover, a number of pathogenic mutations, such as in ABCF3, were detected in both, U-CH11 and U-CH11R, but not in the tumor tissue.Exonic alterations were further filtered by allele frequency and genomic consequence. The mean rankscore of various SNP analysis tools was calculated to predict the deleteriousness of any amino acid change. The score ranges from 0 to 1 and variants with higher risks are predicted to be more likely pathogenic. A cut-off of 0.8 was used to filter the most likely deleterious substitutions. Figure Expression differences between U-CH11 and U-CH11R were identified by microarray gene expression analysis. More than 3000 genes were found to be significantly differently expressed , COL1A2 (3054-fold), and POSTN (2995-fold) are the top overexpressed genes in U-CH11R versus U-CH11. Also, several oncogenes such as BCL2 (11.5-fold), BRAF (9.8-fold), KRAS (4.9-fold), and SRC (2.8-fold) are significantly stronger expressed in the recurrent chordoma cell line  were applied to the overexpressed genes in U-CH11R. GSEA revealed that these genes are associated with extracellular matrix structural constituent, extracellular structure organization and cell matrix adhesion Fig. b, bone mGABRA2 (− 1533-fold), lnc-TPTE3 (− 1159-fold) and GLYAT (− 1075.2-fold)  Fig. . Therefon = 3) and recurrent (n = 3) sacral chordoma cell lines.To further confirm the identified expression differences between the matched primary and recurrent chordoma cell lines U-CH11 and U-CH11R, the analysis was extended to an additional microarray gene expression data set including unmatched primary  10− 37). GSEA of these genes revealed an association to the GO-terms extracellular structure organization, cell junction organization and cell junction assembly  and recurrent (n = 3) sacral chordoma cell lines  compared to U-CH11  and 44% (p < 0.01), respectively. In the primary chordoma cell lines (U-CH19 and U-CH11) this reduction could only be observed in trend  compared to the primary chordoma cell line U-CH19 (22.5%) and in U-CH11R 69%) compared to the corresponding line U-CH11 (21%), fitting their lower sensitivity to the peptide  treated cell lines. Strong signals of cleaved PARP, cleaved Caspase-3 and cleaved Caspase-7 were detected in the recurrent chordoma cell lines U-CH1 and U-CH11R subsequent to HXR9 treatment confirming the previous results observed in the NucView Caspase-3/7 assay, whereas the signals were weak in the primary cell lines U-CH19 and U-CH11. Additionally, HXR9 induced cleavage of poly (ADP-ribose) polymerase (PARP) in all cell lines compared to CXR9 control further substantiating induction of apoptosis Fig. d.CFOS expression analyses in response to a four-hour treatment with 30 μM of the peptides and a vehicle control (DMSO) were conducted and normalized against GAPDH and ACTB  of CFOS mRNA levels was observed in U-CH11R compared to U-CH11  in U-CH1 compared to U-CH19, which served as representative examples of a primary and recurrent chordoma cell line  and following loco-regional recurrence (U-CH11R) serve as a unique cell line progression model to overcome this deficiency. Notably, the cell lines were established from patient samples that were not exposed to chemotherapeutic drugs and radiation. Therefore, an influence on the tumor biology in response to a systemic tumor treatment is excluded.Both cell lines exhibited a very similar morphology and immunocytochemical profile that largely coincided with the findings in the corresponding tissues. However, the recurrent chordoma and derived cell line U-CH11R had a higher Ki-67 index and a shorter PDT in cell culture than the primary tumor and its corresponding cell line U-CH11. These findings implicate substantial differences between the primary and the recurrent tumor detectable in the tissues and being reflected in the corresponding cell lines.CASP9, EPH2A, and DVL1, located on 1p36.32–36.11, to contribute to oncogenesis in chordoma [The aCGH profile of both cell lines was almost congruently altered, including losses of chromosomes 3p, 22q and on chromosome 10, which are frequently reported in chordomas . The prochordoma . In a sechordoma . TherefoERCC2, encoding the Xeroderma Pigmentosum Protein, is frequently altered in different types of cancer . ABBC1 efficacy .To explore how the genomic features contribute to differential gene expression in U-CH11 and U-CH11R, gene expression patterns were compared. GSEA of the downregulated genes in U-CH11R revealed an association to RNA-Splicing. Although pathologically altered splicing events and enhanced dependency on the spliceosome in chordoma are yet barely investigated, gene expression alterations affecting components of the splicing machinery have been recently detected in a widespread of other cancer types and are suggested to promote cancer development and treatment resistance. Promising strategies by which pathologic splicing events may be modulated for cancer therapy are currently under investigation and may be transferred to chordoma in the future .CASP9 and DVL1. The upregulated genes in U-CH11R, however, could not be explicitly linked to the chromosomal alterations, suggesting further key regulatory mechanisms. The highest upregulation was observed for genes referring to the structural proteins caldesmon (CALD) and collagen type I alpha 2 (COL1A2). Caldesmon and COL1A2 are found at high levels in various gastrointestinal tumors carcinoma [HOX genes appeared to be highly upregulated in recurrent chordoma cell lines. Shah et al. recently suggested that aberrant HOX gene expression might contribute to oncogenesis by promoting the activation of anti-apoptotic pathways and being involved in cell invasion, EMT, DNA damage repair and proliferation [HOX genes to be overexpressed in recurrent glioblastomas [HOXA cluster have been reported to be super-enhancer associated in vitro and in vivo [HOXC8 overexpression has been shown to promote proliferation, colony formation, and cell invasion in the recurrent chordoma derived cell lines U-CH1 and U-CH2 [HOXB7, HOXB13, and HOXA-AS3 were detected among the upregulated genes in recurrent chordomas. Their overexpression has frequently been observed in a broad range of solid tumors, such as gastric-, prostate-, and small cell lung cancer and is associated with poor prognosis [HOX genes in recurrent chordoma cell lines, we assume the same underlying key regulator. We therefore assessed the expression of the miRNA gene family MIR196, located within the HOX cluster [HOX mRNA levels during embryonic development and to function as either oncogenes or tumor suppressors in a variety of tumor entities. A tumor suppressive role was suggested in osteosarcoma, melanoma, and breast cancer [HOX mRNA levels, we observed significantly lower miR-196a levels in recurrent chordoma cell lines. By STRING analysis, we identified an interaction network between HOX and PBX factors in recurrent chordomas, potentially regulated by the miR-196a. The mechanism of HOX-PBX dimerization and its inhibition using the small peptide HXR9 have previously been described in several tumor entities [CFOS expression following HXR9 treatment was detected in recurrent chordoma cell lines [Moreover, a high proportion of genes that appeared to be downregulated in U-CH11R mapped to 1p, such as the above-mentioned genes arcinoma –47. To parcinoma . Thus, warcinoma . Interesferation . Moreovelastomas . In chor in vivo . Furthernd U-CH2 . We therrognosis –53. Due  cluster . The miRt cancer –56. Compentities . Therefoll lines .Taken together, our results suggest that transcriptomic reprogramming occurs during chordoma recurrence, which does not derive from genomic events. The upregulation of the HOX-PBX network is, at least in parts, regulated by the miR-196a in recurrent chordoma and can be disrupted using the peptide HXR9 in order to induce apoptosis.Additional file 1: Figure 1. Results of mycoplasma PCR of cell lines included in the analyses. (−) negative control; (+) two different mycoplasma positive controls showing the expected amplicon (~ 500 bp). All cell lines tested were negative. Figure 2. Immunohistochemistry (IHC) of the primary and the relapsed sacral chordoma in comparison to the derived cell lines. IHC results were interpreted as follows: “no immunoreactivity detected” (−), “immunoreactivity ≤ 30%” (+), “immunoreactivity > 30% and < 70%” (++), “immunoreactivity in ≥ 70%” (+++) of the total number of chordoma cells analyzed. HE: hematoxylin/eosin; EMA: epithelial membrane antigen. NOS: not otherwise specified. Figure 3. Validation of gene expression differences between U-CH11 and U-CH11R. Expression levels of selected genes were quantified by qRT-PCR analyses and normalized against GAPDH. Experiments were performed in technical triplicates and at least in biological duplicates and Student’s t-tests were performed to determine statistical significance . Figure 4. Dual Luciferase reporter assay of miR-196a-5p in HEK293T cells. Firefly luciferase reporters containing either the complementary site of HOXA7 and HOXB8 or the perfect antisense sequence of miR-196a were co-transfected with a miR-196a mimic or a scrambled control, respectively. Firefly luciferase activity was normalized to Renilla luciferase activity. Experiments were performed in technical and biological triplicates and Student’s t-tests were performed to determine statistical significance . Figure 5. Activation of Caspase-3/7 in U-CH19 and U-CH1. Activation of cleaved Caspase-3/7 (green channel) in U-CH19 (a) and U-CH1 (b) treated for 24 h with 30 μM CXR9 or HXR9 investigated by EarlyTox Caspase-3/7 NucView 488 assays. Figure 6. Induction of apoptosis in HXR9 treated chordoma cell lines assessed by immunocytochemistry of cleaved caspase-3 (red staining). Cell lines were treated for 4 h with 30 μM HXR9 or the control peptide CXR9. No cleaved caspase-3 positivity was observed in response to CXR9 treatment. Figure 7. Expression levels of cFOS normalized against ACTB. CFOS expression levels in response to treatment with HXR9 or two control compounds (CXR9 and DMSO) in U-CH11 versus U-CH11R (a) and U-CH19 compared to U-CH1 (b) cell lines quantified by qRT-PCR and normalized against ACTB. Gene expression experiments were performed in technical and biological triplicates. Statistical differences were determined by Student’s t-tests . Table 1. Antibodies used for immunostainings of chordoma cell lines and tissue. Table 2. STR profiles of U-CH11R and U-CH11. Table 8. Base-level expression of selected genes based on microarray gene expression data. Mean raw intensity values and standard deviations are given.Additional file 2: Table 3. Filtered exonic variants in the primary tumor tissue (11PT) and the cell lines U-CH11 and U-CH11R.Additional file 3: Table 4. List of up- and downregulated genes in U-CH11R versus U-CH11 based on the microarray gene expression analysis.Additional file 4: Table 5. GSEA of differentially expressed genes in U-CH11R and U-CH11Additional file 5: Table 6. GSEA of RRHO Plot Quadrant A.Additional file 6: Table 7. List of potential miR-196a-5p target genes in recurrent chordoma cell lines.


KRAS mutations are one of the most common oncogenic drivers in non-small cell lung cancer (NSCLC) and in lung adenocarcinomas in particular. Development of therapeutics targeting KRAS has been incredibly challenging, prompting indirect inhibition of downstream targets such as MEK and ERK. Such inhibitors, unfortunately, come with limited clinical efficacy, and therefore the demand for developing novel therapeutic strategies remains an urgent need for these patients. Exploring the influence of wild-type (WT) KRAS on druggable targets can uncover new vulnerabilities for the treatment of KRAS mutant lung adenocarcinomas. Using commercially available KRAS mutant lung adenocarcinoma cell lines, we explored the influence of WT KRAS on signaling networks and druggable targets. Expression and/or activation of 183 signaling proteins, most of which are targets of FDA-approved drugs, were captured by reverse-phase protein microarray (RPPA). Selected findings were validated on a cohort of 23 surgical biospecimens using the RPPA. Kinase-driven signatures associated with the presence of the KRAS WT allele were detected along the MAPK and AKT/mTOR signaling pathway and alterations of cell cycle regulators. FoxM1 emerged as a potential vulnerability of tumors retaining the KRAS WT allele both in cell lines and in the clinical samples. Our findings suggest that loss of WT KRAS impacts on signaling events and druggable targets in KRAS mutant lung adenocarcinomas. KRAS is the most frequently mutated, making up to 85% of all RAS mutations, followed by NRAS (11%) and HRAS (4%). KRAS mutations are frequent genomic events in lung cancer, especially in Non-Small Cell Lung Cancers (NSCLC). Within NSCLC, KRAS mutations are almost exclusively seen in adenocarcinomas, with up to 40% of adenocarcinoma harboring oncogenic mutations of the KRAS gene. These mutations are rarely detected in the other lung cancer subtypes. Across the forty-four oncogenic KRAS mutations identified, 90% occur at codon 12, with G12C (40%) and G12V (22%) mutations being the most frequently observed in lung adenocarcinomas collectively [Mutational activation of the RAS superfamily of small guanosine triphosphate (GTP)-binding proteins is one of the first described and most common oncogenic events in human cancer ,2. Of thectively ,4.EGFR mutant lung adenocarcinomas [KRAS-mutant-specific inhibitors have opened new therapeutic opportunities for treating KRAS mutant NSCLC. According to the ongoing CodeBreak 100 study, treatment with the small G12C inhibitor Sotorasib (or AMG510) in combination with standard chemotherapy has shown objective remission in 33% of enrolled patients [KRAS mutations. Another promising G12C inhibitor, Adagrasib, is also showing clinical activity in patients with advanced and previously treated NSCLC. According to the KRYSTAL-1 phase 1/2 trial results, in patients with advanced NSCLC G12C KRAS mutant tumors previously treated with chemotherapy and anti-PDL1 therapy, Adagrasib yielded a 96% disease control rate with a 45% objective response rate [KRAS-mutated cancers heavily relies on further understanding the context in which KRAS mutant signaling occurs.Undoubtedly, the introduction of precision medicine has revolutionized treatment for lung cancer patients. For example, small kinase inhibitors able to modulate the activity of aberrantly activated epidermal growth factor receptors (EGFRs) are routinely used as first-line treatment options for rcinomas . These trcinomas . Direct rcinomas ,7,8,9. Ircinomas ,11,12. Trcinomas . Recentlpatients ,14,15. Gnse rate . Howevernse rate ,14,15,16KRAS point mutations, the role of wild-type (WT) KRAS in the carcinogenesis of mutant tumors has only been partially explored. Previous research has indicated that the WT KRAS protein can either promote or inhibit tumor progression in KRAS mutant tumors in a context-dependent manner [KRAS mutant cancer cells is achieved through continuously tuning the mutant and WT allele product in a context-specific manner. From a therapeutic perspective, previous studies focusing on KRAS mutant colorectal cancer and acute myeloid leukemia have indicated that the WT copy of the KRAS gene is associated with resistance to treatment with MEK inhibition [KRAS mutant lung adenocarcinomas lacking or retaining the WT copy of the KRAS gene. Such approach provided us the unique opportunity to capture the effects of KRAS homo- and heterozygous mutations on druggable signal transduction molecules. Exploring the impact different genotypes can have on cell signaling events will help identify distinct druggable vulnerabilities and potentially advance precision medicine for KRAS mutant lung adenocarcinoma patients.While most research efforts have been directed toward dissecting specific t manner . In facthibition ,18. Howe2 at 37 °C. Cell lines were sub-cultured using trypsin/EDTA  at a ratio ranging between 1:3 and 1:6 based on cell lines’ proliferation rates. Based on the COSMIC database , the A549, H2122 and H1373 cell lines harbor KRAS homozygous mutations; the A427, H1734, H23, H358 and SK-LU-1 have heterozygous KRAS mutations; and the Calu-3, H1838 and H522 were established from KRAS WT tumors. Mutational status of mutant models was confirmed by polymerase chain reaction (PCR). A panel of eleven human adenocarcinoma cell lines including the A427, A549, Calu-3, H1373, H1734, H1838, H2122, H23, H358, H522 and SK-LU-1 models was obtained from the American Type Culture Collection . Following manufacturer’s instructions, cell cultures were maintained in media  supplemented with 10% fetal bovine serum  in a humidified atmosphere with 5% COn = 4) were collected for each cell line. After 72 h of incubation with the compounds, plates were brought to room temperature for 30 min. Media were replaced with a 1:1 solution of CellTiter-Glo and fresh media, and cells were lysed on an orbital shaker at room temperature for 5 min. Luminescence signal was measured using a Beckman Coulter DTX 880 microplate reader  [Cell viability assay was performed using CellTiter-Glo Luminescent Cell Viability Assay  following manufacturer’s instructions. In brief, for each cell line, a cell suspension containing 5000 to 7500 viable cells was seeded in 96-well plates 24 h before treatment with either the small kinase MEK inhibitor Selumetinib , the Cdk 4/6 inhibitor Palbociclib  or the thiazole antibiotic known to modulate FoxM1 expression and transcriptional activity Siomycin . Number of plated cells was selected based on each line’s proliferation rate. Compounds were dissolved in dimethyl sulfoxide (DMSO) (Selumetinib and Siomycin) or phosphate-buffered saline (PBS) , and cells were treated in a 2-fold serial dilution curve ranging from 0.15 μM to 150 μM for Selumetinib, from 1.12 μM to 10 μM for Palbociclib and from 0.25 μM to 1.25 μM for Siomycin. Matched PBS and DMSO control data were collected for each dilution point. Independent biological replicates  .n = 6) in 6-well plates and cultured until 80% confluent. Cells were washed twice with PBS  and lysed in Tissue Protein Extraction Reagent (T-PER)  supplemented with 300 mM sodium chloride and a mixture of protease and phosphatase inhibitors to prevent protein degradation and preserve the integrity of the phosphoproteome, as previously described [Cell lines profiled for signaling transduction analysis were seeded in technical replicates . The local Institutional Review Board approved the protocol for this study, and written voluntary informed consent was collected from each patient before surgical removal of the tumor. Specimens were snap-frozen within 30 min upon surgical resection, embedded in optimal cutting temperature compound (OCT) and stored at −80 °C. EGFR (Exons 18–21) and KRAS (Exons 1–2) mutation status was analyzed by the enrolling institution using Sanger sequencing as previously described [A total of 23 retrospective biospecimens obtained from surgically treated For each biospecimen, pure tumor epithelia (>95%) were isolated for downstream DNA and protein analysis using laser capture microdissection (LCM). Each sample was cut into 8μm sections, mounted on uncharged glass slides, and stored at −80 °C until microdissected. A representative section stained with Hematoxylin  and Eosin  was evaluated to confirm the presence and amount of tumor in each specimen. LCM-dedicated slides were stained with the HistoGene LCM Frozen Section Staining Kit  for PCR analysis and as previously described for RPPA analysis ,22. IsolDNA extraction was performed using the Arcturus PicoPure DNA Extraction Kit  following manufacturing instructions. In brief, ten microliters of buffer were added to each cap, and samples were incubated at 65 °C for 3 h followed by 10 min at 95 °C. DNA concentration was measured using the Qubit fluorometer ; an average of 40 ng/uL of DNA was recovered for each sample. The genomic DNA was subjected to PCR amplification using GoTag Master Mix  and the following primers:The annealing temperature was adjusted at 60 °C, targeting 35 cycles to generate sufficient amplicon. Sanger sequencing of the amplicons in the region spanning codons 12 and 13 was performed by GENEWIZ, Inc , using the forward primer sequence 5′-CGATACACGTCTGCAGTCAACT-3′. For samples returning a high background signal, additional Sanger sequencing was performed using the reverse primer sequence 5′-CCTCATCTGCTTGGGATGGAAG-3′ to ensure appropriate interpretation of the results.n = 3).Cell lysates and microdissected material were immobilized in technical triplicates onto nitrocellulose-coated glass slides  using an Aushon 2470 arrayer  equipped with 185 μm pins in two independent sets of arrays. Reference standards were printed along with the experimental samples for internal quality control. Selected arrays were stained with Sypro Ruby Protein Blot Stain  following the manufacturer’s directions to quantify the total protein amount of each sample and used for normalization. Each array was probed with one polyclonal or monoclonal primary antibody targeting a protein of interest using an automated system . Primary antibodies were validated for their specificity to the target protein by Western blotting, as previously described . Arrays Clonogenic assay was performed using soft agar colony formation method as previously described . In briehttps://cancer.sanger.ac.uk/cell_lines, accessed on 4 September 2018) and the NCI RAS initiative website  and cell lines were filtered for KRAS mutation status.To examine frequency and KRAS zygosity in commercially available cell lines across tumor types, KRAS mutation status for all available cell lines was retrieved from the Catalogue of Somatic Mutations in Cancer database v94 .For drug sensitivity testing performed in-house, IC50s were calculated using a linear regression curve fit method after normalization on the vehicle controls using GraphPad v9. IC50 were also retrieved for four MEK and ERK inhibitors, namely Trametinib, ERK-6604, ERK-2440 and Ulixertinib, from the Genomics of Drug Sensitivity in Cancer (GDSC) database (KRAS mutant lines retaining (KRASm/WT+) or lacking (KRASm/WT−) the WT alleles and between KRASm/WT+, KRASm/WT−, and WT cell lines. p values < 0.05 were considered significant. Significant findings were displayed using bar graphs created in GraphPad v.6.07. Within each cell line, RPPA values were normalized to baseline values; bar graphs visualize mean and standard error of each group.Spearman rank-order correlation coefficients of RPPA-based continuous values were calculated in JMP v5.1  across the 183 measured analytes. Interactions with a correlation coefficient greater than 0.90 were displayed using network maps generated in Gephi v0.9.2. Non-parametric tests were computed to compare expression/activation of the 183 signaling molecules between KRAS WT allele in KRAS mutant tumors has emerged as a factor that modulates resistance to MEK and ERK inhibition in preclinical studies [KRAS allele in lung cancers and its clinical implications. To answer this question, we first explored the frequency of KRAS WT allele in 116 KRAS mutant cell lines profiled by the COSMIC database and by the NCI-funded RAS initiative. KRAS mutant tumors lacking the WT allele (KRASm/WT−) were more prevalent in lung cancer cell lines compared to other tumor types. Indeed, of the 36 lung cancer models retrieved from the COSMIC database, 47.2% were KRASm/WT−, compared to 30.3% in the pancreatic group and 20% in the large intestine lines , and a relatively large number of these cell lines  did not retain a copy of the WT allele compared to other codon-specific alterations.While retention of the  studies ,17,25, lne lines . WT copyKRAS oncogenic mutations is affected by the presence of the WT allele as previously described for different tumor types [KRAS mutant adenocarcinoma cell lines, of which four  retained a copy of the WT allele. Presence of the WT allele in these models was first retrieved from the COSMIC database and subsequently confirmed by PCR analysis. Cell lines were treated with Selumetinib in an 11-point dilution curve with concentrations ranging from 0.15 μM to 150 μM. As shown in KRAS allele were more sensitive to the compound compared to KRASm/WT+ and to KRAS WT cells. Specifically, IC50 ranged between 1.5 and 6.3 μM for KRASm/WT− cells compared to 11.3–71 μM for KRASm/WT+ and 30–390 μM for the KRAS WT models. Based on the Genomics of Drug Sensitivity in Cancer (GDSC) data, similar trends were also observed, for the same KRAS mutant cell lines, with the MEK and ERK inhibitors Trametinib, ERK-6604, ERK-2440 and Ulixertinib , the expression and activation level of 183 signaling proteins by reverse-phase protein microarray (RPPA). The antibody panel selected for this analysis mainly captured expression or activation of targets and downstream substrates of FDA-approved anti-cancer drugs or investigational agents. To increase the biological relevance of our findings, we focused on changes occurring at the pathway level more than variations of individual proteins.To dissect kinase-driven signaling events associated with the WT KRASm/WT+ and KRASm/WT− cell lines. Pair-wise correlation coefficients were calculated for each protein, and interconnections with Spearman Rho correlation coefficients greater than 0.90 were used to generate network maps where the dimension of each node is proportional to the number of interconnections of each protein. Spearman Rho correlation coefficients were greater than 0.90 for 354 and 767 interconnections in the KRASm/WT+ and KRASm/WT− cell lines, respectively . On the contrary, KRASm/WT+ had overall more sparse interconnections with two main subgroups of proteins clustering together (green and turquoise clusters). Of interest, the Ras protein-specific guanine nucleotide-releasing factor 1 (Ras-GRF1), a regulator of RAS activity, emerged as a central node in both networks. However, in the KRASm/WT+ lines, the network including Ras-GRF1 S916 also contained a number of receptor tyrosine kinases and downstream signaling molecules such as EGFR Y1173, c-Met, Her2, IRS-1 S612 and B-Raf S445, along with different members of the PKC family, including PKCδ T505, PKCα S652 and PKCα/BII T638/641. In the KRASm/WT- cell lines, on the other hand, Ras-GRF1 was interconnected with the cell cycle regulators Ki67, Rb S780, p27Kip, pan-methylated Histone H3 proteins and a number of apoptotic molecules including cleaved Caspases 6, 7 and 9 and BAD S155 (We first looked at interconnected proteins within the ectively . SignaliBAD S155 .KRAS WT allele in our models, we then compared the expression or activation of signaling molecules in KRASm/WT+ and KRASm/WT− cells (KRASm/WT− cells compared to KRASm/WT+ as well as to WT cell lines (KRASm/WT− models. The KRASm/WT− cells also presented with increased activation of signaling molecules involved in anchorage-independent growth including members of the Src family, FAK Y576/577, CrkL Y207, and Paxillin Y118 (KRASm/WT− cells, we performed a clonogenic assay. A total of 5,000 cells were seeded in soft agar and the number and colony dimensions were compared between KRASm/WT+ and KRASm/WT− lines after 21 days. As expected, KRASm/WT− cells formed larger and more abundant colonies compared to the KRASm/WT+ (To further characterize signaling events associated with the T− cells . Expressll lines . Along wlin Y118 A. To expRASm/WT+ B.KRASm/WT+ cells presented with higher activation of several members of the MAPK pathway compared to KRASm/WT- models. For example, Raf activity, measured as phosphorylation levels of B-Raf S445, C-Raf S338 and MEK 1/2 S217/221, was greater in KRASm/WT+ cell lines compared to KRASm/WT− cell lines (KRAS WT lines. ERK 1/2 activation was also enhanced in WT and KRASm/WT+ cell lines compared to KRASm/WT− along with increased phosphorylation on the downstream transcription factor Elk-1 (KRASm/WT+ and cell lines not harboring a KRAS mutation. Similarly, phosphorylation levels of members of the AKT/mTOR signaling axis were higher in KRASm/WT+ models compared to KRASm/WT− cells including mTOR S2448, the downstream substrates p70S6 kinase phosphorylated on residues T389 and T412 and the transcription factors FoxO1 S256 and FoxO1 T24/FoxO3 T32 (KRASm/WT+ lines (ll lines B. These or Elk-1 B. Of intoxO3 T32 A. Of intT+ lines .KRAS WT allele strongly affects signal transduction events in KRAS mutant NSCLC cell lines. KRAS WT and KRASm/WT+ cells present with increased activation of the MAPK and AKT/mTOR signaling axis compared to the KRASm/WT− lines. In addition, the presence of a KRAS WT allele may modulate downstream signaling activity through aberrant cytosol sequestration of apoptotic signaling molecules.Taken together, these data indicate that the presence of a KRAS mutant lesions, in KRASm/WT− models we identified distinctive alterations in proteins involved in cell cycle progression compared to KRASm/WT+ cell lines (KRASm/WT− lines. On the contrary, proteins involved in cell cycle progression were overexpressed/activated in the KRASm/WT+. A few examples include increased expression of Cyclin A2 and B1, deactivation of the tumor suppressor Rb through phosphorylation of the S780 residue and expression and activation of the transcription factor FoxM1 (KRAS mutant and WT cell lines, its activation was not significantly different between the WT and KRASm/WT+ lines (While changes in the activation of the MAPK and AKT/mTOR pathways have already been tested as potential therapeutic targets for ll lines A. For exor FoxM1 C. Of intT+ lines C.KRASm/WT+ cells, we examined if the Cdk4/6 inhibitor Palbociclib affects proliferation of KRASm/WT+ and KRASm/WT− cell lines differently. Incubation with Palbociclib had a very minimal effect across all cell lines (KRASm/WT+ and KRASm/WT− lines (KRASm/WT+ lines (KRASm/WT− models were minimally affected by the compound and response to Siomycin was independent from the TP53 mutational status. As expected, incubation with Siomycin reduced activation of FoxM1 in sensitive models. Taken together these data indicate the FoxM1 and its transcriptional activity may represent a therapeutic target for KRASm/WT+ lung adenocarcinomas.Because FoxM1 and Rb, two downstream targets of Cdk4/6 activity, were hyperphosphorylated in the ll lines A. We theT− lines B. IncubaT+ lines C. The KRKRAS mutant adenocarcinoma samples collected from patients undergoing surgical resection. Pure tumor epithelia were isolated from the surrounding tumor microenvironment using LCM, and the presence/absence of the WT copy of the KRAS gene was determined by PCR (KRAS allele; FoxM1 activation levels were higher in the KRASm/WT+ tumors compared to the KRASm/WT− lesions (KRAS mutant lung adenocarcinomas retaining the WT KRAS allele and that downregulation of its transcriptional activity may represent a therapeutic target for this group of patients.To test the clinical significance of FoxM1 activation, we analyzed a cohort of d by PCR . Of the  lesions C. Taken KRAS mutant lung adenocarcinomas with a focus on druggable targets and downstream substrates of FDA-approved or investigational agents. In line with previous findings, our study suggests that the presence of the WT KRAS allele significantly alters the biology and potential response to treatment for KRAS mutant tumors [KRAS mutant lung cancer models lack the WT copy of the gene more frequently than other tumor types and such homozygous alterations are mostly seen in cell lines harboring G12C and G12V mutations. Interestingly, these mutations with the least percentage of cell lines retaining a WT allele are frequently associated with a worse prognosis [KRAS mutant NSCLC, has suggested a slight increase in progression-free and overall survival in patients with G12V and G12C KRAS mutant tumors compared to those harboring other KRAS oncogenic mutations [KRAS mutant tumors are more frequently associated with the loss of the WT KRAS allele, the increased sensitivity to Selumetinib in combination with docetaxel observed clinically may be due to the absence, in a number of these lesions, of the WT allele of the KRAS gene.In this study, we used a translational approach to characterize signal transduction events in homo- and heterozygous t tumors . From a rognosis . Howeverutations ,27. AlthKRASm/WT− and KRASm/WT+ cell lines [KRASm/WT+ are overall less sensitive to agents targeting KRAS downstream substrates. Similar trends were also observed in a study by Ambrogio et al., in which inhibition of downstream KRAS activity with Selumetinib, Trametinib and the dual MEK/CRAF inhibitor CH5126766 was only seen in the absence of a WT KRAS [KRAS WT allele presented with decreased activation of several members of the MAPK pathway, not only compared to the heterozygous lines but also to models without oncogenic KRAS mutations. While this observation somewhat contradicts the “buffering” hypothesis, our data confirmed a higher degree of interconnections and cross-talks in KRASm/WT− cell lines compared to KRASm/WT+ models suggesting the loss of the KRAS WT allele has profound implications on the signaling network of these tumors.As previously reported, our data confirmed a significant difference in response to MEK 1/2 and ERK 1/2 inhibitors in ll lines . Specifi WT KRAS . Accordi WT KRAS . In our KRASm/WT− presented with decreased activation of several members of the MAPK pathway, not only compared to the heterozygous lines, but also to models without oncogenic KRAS mutations. Downregulation of MAPK signaling in KRASm/WT− was detected along with increased activation of several molecules involved in anchorage-independent growth, suggesting some protection against anoikis is gained when the WT KRAS allele is lost. Thus, loss of the WT allele in KRAS mutant lung adenocarcinomas and its effect on tumor invasiveness and metastasization should be evaluated in future investigations.Because KRAS is a key regulator of many signaling molecules, we next compared signal transduction events across our models. From a signaling perspective, KRAS mutant lesions, cell lines retaining a KRAS WT allele presented with distinct expression and activation of proteins involved in the regulation of the cell cycle [KRAS WT+ cell lines. While activation of FoxM1 and deactivation of Rb in our model appeared to be independent from the G1/S regulators Cdk4/6, it is well known that the expression of the transcription factor FoxM1 increases throughout the cell cycle and regulates expression of key proteins involved in its progression. For example, numerous reports have previously indicated that knockdown of FoxM1 is associated with a marked decrease in Cyclin A and B activity, suggesting a prominent role of FoxM1 in modulating the expression of proteins involved in G2/M transition, as also confirmed by our data [From a therapeutic perspective, while changes in activation of the MAPK and AKT/mTOR pathways were expected and have already been tested as potential therapeutic targets for ll cycle ,18,25. Nour data ,31,32,33our data , the comKRAS mutant tumors lacking or retaining the WT allele, we sought to test the effect of Siomycin, a thiazole antibiotic known to inhibit FoxM1 mRNA and protein expression [KRASm/WT+ and KRASm/WT− models. As expected, because expression and activation of FoxM1 were significantly different across comparison groups, inhibition of this node led to a significant reduction in cell viability in the KRASm/WT+ lines, while it only marginally affected KRASm/WT− cells. To validate the clinical relevance of our findings, FoxM1 activation in KRASm/WT+ was also confirmed in surgical biospecimens collected from NSCLC patients affected by lung adenocarcinoma.Prompted by the changes in FoxM1 expression and activity between pression , on KRASKRAS zygosity can easily be explored in pre-clinical in vitro models and biochemical assays, validating these findings in animal models and clinical samples can be challenging from a technical perspective. The heterogeneous composition of the tissue, where cancer cells are commingled with non-malignant cells, renders capturing zygosity of oncogenic mutations in whole-tissue-based analysis unattainable [While assessing the role of tainable ,37. To oKRAS zygosity may profoundly affect cancers’ phenotypes and responses to targeted compounds. For example, a recent paper by Liu and colleagues suggested that loss of the WT copy of the KRAS allele is not a rare event in lung adenocarcinomas and is associated with shorter survival [KRAS mutant lung adenocarcinomas. These observations have important clinical implications as they suggest that KRAS zygosity may represent an underestimated pathological marker of KRAS mutant lung cancers. Understanding the roles and effects of zygosity on signal transduction networks may open new opportunities for allocating patients to targeted treatments, understating mechanisms of resistance, and devising new single agent and combination treatments.Although the identification of druggable genomic alternations has revolutionized cancer treatment and management, predicting response to treatment still remains an unmet need in oncology. Zygosity and the presence of a wild-type copy of a mutant oncogene, in particular, are rarely accounted for in the therapeutic decision-making process for cancer patients. However, emerging data suggest that survival . In addiKRAS gene in mutant lung adenocarcinomas modulates signaling events and response to treatment, a few study limitations must be addressed. First, our observations are merely descriptive and based on a limited number of cell lines generated from lung adenocarcinomas. Given the highly variable genetic background of these models and different degree of dependency on mutant KRAS, generalizing our observations is premature and needs to be validated on larger datasets. Second, because we used cell lines harboring diverse KRAS point mutations, signaling events captured by this analysis may represent a heterogeneous mixture of networks. The possibility that specific KRAS mutants can present distinct shifts in signaling under the presence of WT protein should be further investigated. Nevertheless, our findings strongly encourage the notion that different genotypes, specifically the presence of the KRAS WT allele in KRAS mutant lung adenocarcinomas, prominently affect signaling events and response to treatment. Understanding the underlying mechanisms of these changes can potentially uncover new druggable targets and fulfill the unmet therapeutic needs of patients with KRAS mutant lung adenocarcinoma.While our data support the hypothesis that retention of the WT copy of the


Secondary xenograft by percutaneous injection mimicked most of the features of SS with dermal infiltration, epidermotropism, and blood spreading. These models also allowed assessing the intra-individual heterogeneity of patient SC. Subclones sharing the same TCR gene rearrangement evolved independently according to culture conditions and/or after xenografting. This clonal selection was associated with some immunophenotypic plasticity and limited genomic evolution both in vitro and in vivo. The long-term amplification of SC allowed us to develop eight new SC lines derived from four different patients. These lines represent the cell of origin diversity of SC and provide new tools to evaluate their functional hallmarks and response to therapy.Sézary Syndrome (SS) is a rare aggressive epidermotropic cutaneous T-cell lymphoma (CTCL) defined by erythroderma, pruritis, and a circulating atypical CD4 + T-cell clonal population. The diversity of Sézary cell (SC) phenotype and genotype may reflect either plasticity or heterogeneity, which was difficult to evaluate dynamically until the achievement of long-term SC expansion. Therefore, we developed six defined culture conditions allowing for the expansion of SC defined by their phenotype and monoclonality in four of seven SS cases. Engraftment of SC through the intrafemoral route into immunodeficient NOD.Cg-Prkdc(scid)Il2rg( SCM), central memory (TCM), effector memory (TEM), and naive or transitional memory (TTM) T cells [Sézary Syndrome (SS) is a rare and aggressive cutaneous T-cell lymphoma (CTCL) defined by erythroderma with a circulating atypical CD4 + T-cell clone. By definition, blood involvement is high with B2 stage  . However T cells . Their h T cells . Their dSo far, the biological characterization of SCs has been impaired by the lack of reproducible in vivo and in vitro models. A murine model was developed after injection of MBL2 T lymphoma cells followed by inflammatory peptide inoculation into the ears, resulting in a local development of lymphoma . More reIn this study, we established new in vitro culture conditions and a PDX model to achieve SC expansion from fresh patient cells. These models confirmed genomic and immunophenotypic interindividual diversity and revealed different subpopulations among original clonal T cells within the same patient. Establishing eight new SC lines corresponding to several SC differentiation stages also provides novel tools to characterize SC hallmarks.Blood samples from 14 adult patients with SS T4NxMxB2 stage (at one time of illness) were collected at the Oncodermatology Department   in immunodeficient NOD.Cg-Prkdc(scid)Il2rg(tm1Wjll)/SzJ (NSG) mice (from the Jackson Laboratory). The monitoring of mice engraftment and tissue analyses are described in the PBMCs from primary patient samples were injected intrafemorally or percutaneously (1 × 10SCs purified by FACS were co-cultured with or without MS5 or MS5-Delta Like 1 (MS5-DL1) stromal cells, as described previously . MS5 andOne million cells from SC lines  were plated per six-plate well and treated for 48 h with romidepsin (10 nM), doxorubicin (20 nM), and vorinostat (3 µM) , 26. ForImmunohistochemistry and Hematoxylin–Eosin–Saffron staining were performed as described previously  and in tCultured cells were harvested using the cytogenetic standard protocol for metaphase lymphocytes and mFISH experiments were performed as described previously –29.Oligonucleotide array-based CGH was conducted as described previously .ARID1A, CARD11, CCR4, CD28, DNMT3A, FAS, FASN, IDH2, JAK3, KDM6B, MLL3/KMT2C, PLCG1, RHOA, SETD1B, STAT3, STAT5B, TET2, TP53, ZEB1) [Lymphopanel sequencing was performed on seven patients and eight SC lines using the IonS5 (ThermoFisher Scientific). This lymphopanel was designed with Ion Ampliseq technology  to identify mutations within frequently altered genes in SS and peripheral T-cell lymphoma  , 31. DatStatistical significance of compared measurements was determined using the Mann–Whitney nonparametric test using GraphPad Prism .P values < 0.05 were considered significant.In seven patients, SCs were identified by their CD3 + CD4 + CD8- phenotype and the monotypic expression of a TCRVβ variant. The proportion of tumor cells was already dominant in unsorted blood samples (88 ± 6%) and purity of the tumor population reached 99 ± 2% after sorting in all samples Fig. . SCs wern = 5 minimum) and the percentage of TCRVβ characteristic of tumor cells was analyzed from bone marrow (BM) aspiration. Only 14.3% (patients #2 and #10 out of 14 cases) of Sézary samples engrafted, even after 6 months of monitoring   were obtained through xenografting.via targeted lymphopanel analyses  karyotyping and aCGH analyses. For patient #2, #6, and #10, two or three major tumor subclones were detected in the original sample. Long-term culture of PDC or PDX cells achieved selection of one or two of these original subclones Fig. , which wses Fig. . Other tses Fig. . The mutSCs Fig. .Fig. 6ClWhile underscoring interindividual heterogeneity in the cell of origin and phenotype of SCs, our results support restricted intraindividual heterogeneity. PDC and PDX models and the derived SC lines revealed sub-clones already present in the original SC bulk, limited phenotypic plasticity or genomic changes after expansion.To evaluate whether SC lines could be relevant for therapeutic screening, five  were treated with therapies currently used to treat patients with SS  –40. L1 aDuring the last several decades, very few CTCL cell lines or experimental models have been developed , 41, 42.Standard short-term culture methods used to expand CTCL cells involve adding different cytokines such as IL-2, IL-4, IL-7, and IL-15 and unspecific mitogen as PHA in the medium. Starting from skin samples generally favors the proliferation of benign infiltrating T cells and is not suitable for growing tumor T cells . In fourCM phenotype but were able to grow with or without MS5 stromal cells. In vivo, the SCs derived from these patients also showed different speeds of engraftment  and involved different tissues or organs depending on the patient. Recently, γ-secretase inhibitors inhibiting NOTCH signaling were found to induce apoptosis in SC lines and primary patient cells, supporting the importance of the NOTCH signaling pathway as a new target to treat patients with SS [Our results also support the need to use different defined cytokines and stromal cells for SC expansion and maintenance as permissive culture conditions were patient-dependent and not predicted by the SC differentiation stage. Indeed, SCs from patient #2 and #10 displayed the same naive T cells/T with SS , 45. In  with SS  and may Pilot studies in immunodeficient mice have primarily used the percutaneous injection of CTCL cell lines to effectively monitor tumorigenesis at a theoretically orthotopic site , 21. HowCM/TEM phenotypes that evolved independently during culture and/or xenografting providing two clonally related cell lines (L1 and L2). For patient #10, limited clonal evolution was observed after expansion in culture (loss of der(4)t chromosome). Such intratumoral clonal heterogeneity has been reported in a transformed mycosis fungoides case after large cell transformation by comparing skin and blood compartments [Genetic characterization of fresh samples confirmed heterogeneity between patients and to a lesser extent within the same individual. Interestingly, original tumor cells from patient #2 contained two major subclones with naive and Tartments . TherefoThe lack of preclinical models of CTCL has also limited the validation of new therapeutic agents , 50. TwoThis study provides an original methodology to amplify SCs and obtain new SC lines, which better represent SS diversity according to cell of origin phenotype or differentiation stage. The models confirmed that SCs mostly displayed interindividual heterogeneity with no or very little plasticity in long-term expansion. Different responses to therapies may depend on the cell of origin of SCs, as well as some biological differences regarding proliferation, tumorigenesis, and migration. Using percutaneous injection, we also obtained an avatar of SS mimicking both skin and blood involvement, which may be used to test the effects of therapeutic agents at both levels, as well as the mechanisms regulating the balance between blood and skin compartments.Supplementary TablesSupplementary FiguresSupplementary materials and methods


DNA methyltransferase inhibitors (DNMTIs), such as zebularine, play a significant effect on the demethylation and reactivation of TSGs. This study aimed to investigate the effect of zebularine on p16INK4a, p14ARF, p15INK4b, and DNA methyltransferase 1 gene expression, cell growth inhibition, and apoptosis induction in HCC PLC/PRF5 and pancreatic cancer PA-TU-8902 cell lines. Both cell lines were cultured and treated with zebularine at different times. The MTT assay, real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR), and flow cytometry were used to determine cell viability, gene expression, and apoptotic cells, respectively. The result indicated that zebularine inhibited cell growth of both cell lines significantly as time- and dose-dependent manner (P < 0.007). The agent induced significant down-regulation of DNMT1 and up-regulation of p16INK4a, p14ARF, p15INK4b (P < 0.028). Besides, it had a significant apoptosis effect on both cell lines (P < 0.001). This compound had a strong significant effect on PLC/PRF5 in comparison to PA-TU-8902 cells. Concluding, zebularine inhibited PLC/PRF5 and PA-TU-8902 cell growth and induced apoptosis in these cell lines. The most likely mechanism underlying the zebularine played its role involves down-regulation of DNMT1 and up-regulation of p16INK4a, p14ARF, and p15INK4b genes.Tumorigenesis must be understood as a summary of altered genetic and genomic changes resulting in the inactivation of tumor suppressor genes (TSGs). One of the characterizations of epigenetic alterations is DNA methylation. Epigenetic alteration of the p16INK4a, p14ARF, p15INK4b In contrast to genetic events, epigenetic alteration is a reversible change characterized by three mechanisms, including DNA hypermethylation, DNA hypomethylation, and histone modifications affecting chromatin conformation without any change in the structure of DNA sequences  activity. An experimental study has demonstrated that this agent incorporates into DNA and exhibits cell growth inhibition by DNMT1 inhibition in human cancer cell lines, including T24 bladder cancer, SW48, HCT15, and HT-29 colon cancer, PC3 prostate cancer, CFPAC-1 pancreatic cancer, and CALU-1 lung cancer  and pancreatic cancers are lethal human cancers with a wide geographical variation , 2. As fequences . Epigeneequences -6. In adequences -10. DNA equences , 13, andequences -16 has bg cancer . A preclma cells . Other rectively , 20. In ectively , 22. Preectively . The resMaterialsHepatocellular carcinoma PLC/PRF5 and pancreatic cancer PA-TU-8902 cell lines were purchased from the National Cell Bank of Iran-Pasteur Institute and cultured and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with fetal bovine serum 10% and antibiotics in a humidified atmosphere of 5% CO2 in air at 37 ◦C. Zebularin was provided from Sigma  and dissolved in distilled water as a stock solution. All other experimental solutions were obtained by diluting the provided stock solution. Other agents including, DMSO, antibiotics, 3--2, 5-diphenyl-2H-tetrazolium bromide (MTT), trypsin-EDTA, Phosphate-buffered saline (PBS), Annexin-V-(FITC), propidium iodide (PI), DMEM were purchased from Sigma. Total RNA extraction kit (TRIZOL reagent) and real-time polymerase chain reaction (PCR) kits (qPCR MasterMix Plus for SYBR Green I dNTP) were obtained from Applied Biosystems Inc. . This work was approved by the Ethics Committee of Jahrom University of Medical Science with a code number of IR.JUMS.REC.1398.022. Cell culture and cell viability5 cells per well were treated with various concentrations of zebularine . After 24 and 48 h of incubation, MTT (0.5 mg/mL PBS) was added to each well and incubated at 37 °C for 3 h to determine the number of living cells. Then the formed formazan crystals were dissolved in DMSO and shaken for 10 min to dissolve all of the crystals. Finally, the optical density was detected by a microplate reader at a wavelength of 570 nM. Each experiment was repeated three times (triplicates).The effect of zebularine on PLC/PRF5 and PA-TU-8902 cell viability was investigated using MTT assay, based on the previously described method . BrieflyCell apoptosis assay5 in 24-well plates were seeded in triplicate and incubated overnight. Then, the PLC/PRF5 and PA-TU-8902 cells were treated with zebularine in indicated concentration (74.65 and 98.82 μM respectively), according to IC50 values, for 24 and 48 h, the control groups were incubated with medium + distilled water, distilled water equivalent to the drug solvent was used. Following the staining of the samples using annexin V-FITC and PI, they were analyzed by FACScan flow cytometry .An apoptosis assay was performed to obtain the apoptotic effect of zebularine on PLC/PRF5 and PA-TU-8902 cells, based on the previously described method . BrieflyReal-time quantitative reverse-transcription polymerase chain reaction(qRT-PCR) analysisTo determine whether zebularine could affect p16INK4a, p14ARF, p15INK4b, and DNA methyltransferase 1 gene expression, qRT-PCR was performed. After treatment times (24 and 48 h), total RNA was isolated from PLC/PRF5 and PA-TU-8902 cells treated with zebularine (74.65 and 98.82 μM respectively) using Trizol reagent (Invitrogen), and cDNA was synthesized from total RNA with Superscript III reverse transcriptase (Invitrogen). The expression of mRNAs was measured by quantitative real-time PCR using StepOnePlus  instrument and SYBER green PCR kit (TaKaRa Bio). The thermocycling condition and the amplification reactions were performed as mentioned previously . The priStatistical analysisP < 0.05.The database was set up with the SPSS 16.0 software package  for analysis. The data were acquired from three tests and are shown as means ± standard deviations. Statistical comparisons between groups were performed with ANOVA (oneway ANOVA) and Turkey test. A significant difference was considered as Cell viability P < 0.003). IC50 values were obtained with approximately 74.65 and 98.82 μM, as a mean of 24 and 48 h, for PLC/PRF5 and PA-TU-8902 cell lines, respectively. The viability of Hepatocellular carcinoma PLC/PRF5 and pancreatic cancer PA-TU-8902 cell lines treated with zebularine  was determined by MTT assay as mentioned in the method section. The inhibitory effect of zebularine on both PLC/PRF5 and PA-TU-8902 cell lines was dependent on the dose and incubation time, as shown in Cell apoptosis To determine whether zebularine could induce apoptosis, the PLC/PRF5 and PA-TU-8902 cells were stained using annexin-V-(FITC), as mentioned in the method section. As depicted in Gene expression The effect of zebularine on p16INK4a, p14ARF, p15INK4b, and DNA methyltransferase 1 gene expression was evaluated by quantitative real-time RT-PCR analysis. The result indicated that treatment with zebularine (24 and 48 h) upregulated p16INK4a, p14ARF, p15INK4b, and down-regulate DNMT1 gene expression significantly in both cell lines, the PLC/PRF5 and PA-TU-8902, In-vitro studies have indicated that DNA demethylating agents can reactivate silenced TSGs such as p14ARF, p16INK4A, and p53 (In-vitro study has been shown that DNA methyltransferase inhibitor 5-aza-CdR can induce G2/M cell cycle arrest in leukemia cells by p21WAF1/CIP1 up-regulation (Cell cycle progression and regulation is a highly-regulated process that involves multiple mechanisms and checkpoints. The molecular mechanism of various stages of the cell cycle has been evaluated during the past decade. The CDKs are a family of enzymes that form the heart of the regulatory machine during the cell cycle progression. The active form of these enzymes includes a complex of two proteins, a kinase, and a cyclin, which form positive regulators and induce cell cycle progression. Whereas CKIs account for the important negative regulators which stop cell cycle progression in response to multiple regulatory signals. The deregulation of the cell cycle regulatory genes such as CDKs is one of the most frequent alterations during tumorigenesis and cancer development . The hyp and p53 , 35. In  and p53 . Another and p53 . In acut and p53 . Increas and p53 . In esop and p53 . An impo and p53 . In addi and p53 . In-vitrgulation . Overexpgulation . Besidesgulation .In summary, DNA methyltransferase inhibitors can inhibit cell growth and induce apoptosis by reactivation of cell cycle regulatory genes, INK4, and the Cip/Kip family. We did not evaluate the effect of 5-aza-CdR on the Cip/Kip family in HCC and pancreatic cancer. Therefore, this evaluation is recommended.In conclusion, our finding demonstrated that zebularine inhibited PLC/PRF5 and PA-TU-8902 cell growth and induced apoptosis in these cell lines. The most likely mechanism underlying the zebularine inhibited cell growth and induced apoptosis involves down-regulation of DNMT1 and up-regulation of p16INK4a, p14ARF, and p15INK4b genes. This result suggests that zebularine may have wide therapeutic applications in hepatocellular carcinoma and pancreatic cancer.This study is a research work that reports the Effect of Zebularine on p16INK4a, p14ARF, p15INK4b, and DNA Methyltransferase 1 Gene Expression, Cell Growth Inhibition, and Apoptosis Induction in Human Hepatocellular Carcinoma PLC/PRF5 and Pancreatic Cancer PA-TU-8902 Cell Lines. It has not been previously submitted by any other journal, and all of the authors read and approved the final version of the manuscript. All authors generated the ideas and contributed to the writing of the manuscript, acquisition of data, analysis, and interpretation of data, and revised the article. All authors approved the final revision.


Despite extensive research and the recent introduction of innovative therapeutics, lung cancer remains the first cause of cancer-related death, with a 5 year survival of only 17% . RecentlIn this work, we performed an integrative functional genomic analysis, combining in vitro dependency data within a large collection of cancer cell lines, gene druggability information and patients’ transcriptomics and mutational data. Through this approach, we identified and validated the EGLN1 gene as a novel druggable dependency, preferentially associated with KRAS-mutated lung AD.To identify dependency genes that can be used as new therapeutic targets, we performed a CRISPR/Cas9 screening in the A549 cell line, derived from KRAS-mutated lung AD [To validate the results of our screening, we performed a data integration-based analysis of different and complementary -omics sources Fig. a. First,To identify dependency genes that were preferentially associated with the KRAS-mutated genetic background, we mined the dependency profiles of KRAS-mutated and KRAS wild-type lung cancer cell lines, extracting 1374 genes having a significantly different CERES score  in KRAS-mutated AD cell lines with two sgRNAs Fig. a. As shoFinally, to provide a proof of principle of the possibility to pharmacologically target EGLN1, we selected a panel of lung cancer cell lines and treated them with the EGLN1 inhibitor molidustat. All tested cell lines were sensitive to molidustat, with EC50 in the range of micromolar. Notably, KRAS-mutated cell lines displayed the highest sensitivity Fig. c-e.To gain further insights into the pro-oncogenic mechanism controlled by EGLN1, we relied on the notion that co-dependencies can be used to identify genes that have a similar function . We founOverall, our results uncover a previously unknown pro-oncogenic function of the EGLN1 gene in KRAS-mutated lung AD. EGLN1 inhibitors are currently in clinical trials for anemia, demonstrating good tolerability and safety . Our fin