We found tumor specific TNC gene and protein overexpression that directly correlated with higher tumor grade , H3K27 M mutation (p = 0.012), shorter progression free survival (p = 0.034), and poorer overall survival (0.041) in association with these factors. TNC knockdown via lentiviral shRNA transfection of HGG (n = 1) and DIPG (n = 3) cell lines resulted in decreased cell proliferation, migration, and invasion in vitro (p < 0.01), while TNC cDNA transfection resulted in increased cell migration, invasion and proliferation (p < 0.01) as well as altered cell morphology in H3K27 M mutant DIPG lines. Whole transcriptome sequencing analysis (RNA-Seq) on DIPG (n = 3) and HGG (n = 2) cell lines after TNC cDNA, shRNA, and empty vector control transfection revealed the effects of TNC expression level on global gene expression profiles. Together, our findings reveal TNC expression in DIPG in association with H3K27 M mutation and VEGF signaling, and suggest that TNC may contribute to DIPG tumor phenotype, and serve as a clinically detectable biomarker for DIPG.Diffuse intrinsic pontine glioma (DIPG), an infiltrative, high grade glioma (HGG) affecting young children, has the highest mortality rate of all pediatric cancers. Despite treatment, average survival is less than twelve months, and five-year survival under 5%. We previously detected increased expression of Tenascin-C (TNC) protein in DIPG cerebrospinal fluid and tumor tissue relative to normal specimens. TNC is an extracellular matrix (ECM) glycoprotein that mediates cell-matrix interactions, guides migrating neurons during normal brain development and is thought to maintain the periventricular stem cell niche in the developing brain. Tumor TNC expression is reported in adult glioma and other cancers. However, the pattern and effects of TNC expression in DIPG has not been previously explored. Here, we characterize TNC expression in patient derived pediatric supratentorial HGG (The online version of this article (10.1186/s40478-019-0727-1) contains supplementary material, which is available to authorized users. Brain tumors are the most common solid cancer in children. Approximately 15% of pediatric brain tumors arise in the brainstem, of which up to 80% are a subtype known as diffuse intrinsic pontine glioma (DIPG), an infiltrative, high grade glioma affecting young children . DIPG tyH3F3A and HIST1H3B, respectively) . O. O54]. Oresponse , 54, 61.response : measuriIn conclusion, we characterized TNC expression in pediatric glioma tissue and cell lines, including DIPG, with higher endogenous expression levels in tumors harboring the H3K27 M mutation. TNC overexpression is associated with more malignant cell behavior, including increased cell proliferation and migration, and patterns of gene expression consistent with a pro-angiogenic, mesenchymal tumor phenotype. Our findings suggest TNC overexpression in pediatric high grade and diffuse midline glioma is clinically detectable and may significantly contribute to tumor biology. Given this, we are currently investigating the effects of TNC expression in vivo on tumor growth and animal survival in an animal xenograft model of pediatric glioma, and evaluating changes in TNC expression by DIPG cells in vitro in response to novel epigenetic therapies. Our results suggest TNC may serve as a feasible disease biomarker and novel therapeutic target for this challenging disease.Additional file 1:Figure S1. TNC expression in pediatric glioma cell lines modified via shRNA and cDNA transfection in vitro. Pediatric supratentorial high-grade glioma (HGG n = 1) and brainstem glioma (DIPG n = 3) cell lines were modified via lentiviral transfection of TNC cDNA and shRNA constructs. Two distinct shRNA constructs, 232 and 234, were generated. a)TNC transcript level after transfection, measured via qPCR, demonstrating TNC shRNA knockdown (left) and cDNA amplification (right), relative to empty vector controls . Y-axis: Normalized TNC mRNA expression level (percent GAPDH). b) TNC protein level after transfection, measured via western blot, demonstrating decreased protein expression after shRNA knockdown (upper panels) and increased expression cDNA amplification (lower panels), relative to empty vector controls. Densitometry analysis of western blot signal intensity confirms statistical significance of TNC expression patterns between transfected lines and empty vector controls . Y-axis: TNC protein band intensity . Error bars represent standard error of the mean. c) Fluorescent microscopy images showed transfection efficiency of > 95% in both cDNA and shRNA. Scale bar = 200 μm. (TIF 138230 kb)Additional file 2:Figure S2. Targeted gene expression analysis in pediatric glioma cell lines. Levels of TNC and related transcripts were measured via qPCR in pediatric supratentorial highgrade glioma (HGG n = 3) and brainstem glioma (DIPG n = 5) cell lines. a) Expression levels of TNC, Notch 1, Notch 2, MYCN and PDGFRA across cell lines studied. Values are normalized to GAPDH expression levels in each cell line. Y-axis: Relative gene expression to GAPDH. Error bars represent standard error of the mean. b) Correlation between TNC expression each gene of interest across cell lines, as measured by qPCR. Positive correlation was found between TNC and Notch 2, and between TNC and PDGFRA, but did not reach statistical significance. c) Correlation between TNC transcription and genes of interest  across cell lines, as measured by RNA-Seq. TNC expression positively correlated with Notch2 (R2 = 0.817) and PDGFRA (R2 = 0.820). Each data point represents one glioma cell line. (TIF 101751 kb)Additional file 3:Figure S3. Unsupervised hierarchical clustering of cell line gene expression profiles RNA-Seq analysis was performed in triplicate on DIPG (n = 3) and HGG (n = 2) cell lines after TNC cDNA, shRNA, and empty vector control transfection. Differentially expressed genes  were identified between cDNA and shRNA lines. a) Unsupervised analysis of resulting gene expression profiles by cell line (Y-axis) demonstrates clustering by technical replicate, followed by experimental condition. b) Scatterplot depicting differential TNC expression levels across shRNA and cDNA cell lines holds when adjusted for cell culture conditions (with or without serum FBS). c) Explicit modeling for differentially expressed genes both as means and as sums across technical replicates results in minimal change in data sets when adjusted for culture conditions. Together, these data demonstrate that observed transcriptomes and resulting cell functions are driven by induced differences in TNC expression. (TIF 136432 kb)Additional file 4:Figure S4. Relative TNC expression differs across single-cell subpopulations. Single cell RNA-Seq of H3K27 M mutant pediatric glioma was performed by Filbin et al. [p = 0.0002). Y-axis: mean gene expression (in log2 TPM/10). Error bars represent standard error of the mean. Relative gene expression of the three DIPG subpopulations was normalized to all DIPG cells. Published data analyzed and presented with permission of the Dr. Mariella Filbin. (TIF 17718 kb)n et al. , revealiAdditional file 5:Online Resource 1. Whole transcriptome (RNA-Seq) analysis of pediatric glioma cell lines. Whole transcriptome sequencing analysis (RNA-Seq) was performed in triplicate on DIPG (n = 3) and HGG (n = 2) cell lines after TNC cDNA, shRNA, and empty vector control transfection. (PDF 28227 kb)Additional file 6:Online Resource 2. Differential gene expression analysis of RNA-Seq data Resulting transcriptomes of cell lines transfected with TNC cDNA were compared to TNC knockdown (shRNA) and controls. Gene count tables from HTSeq were used as input for EBSeq version 1.16.0 [n 1.16.0 . Genes w


This article has been corrected: For clarity in describing the experiments, the authors have included an additional sentence at the end of the second-to-last paragraph of the Discussions section. The updated paragraph is shown below. The authors declare that these corrections do not change the results or conclusions of this paper.in vitro and in vivo data using three types of GEP-NEC cell lines would be underpowered for drawing some conclusion about a clinical position of each regimen. Actually, reports about the establishment of GEP-NEC cell lines are limited, and GEP-NEC cell lines are almost commercially unavailable. Second, CDDP resistance is caused by multiple mechanisms such as increased inactivation by reactive oxygen species, mismatch repair deficiency, increased nucleotide excision repair, increased homologous recombination proficiency and over expression of antiapoptotic BCL-2 as well as ABC transporters . Hence, comprehensive approaches including genomic analysis are required, and our hypothesis might explain only a part of potential mechanisms. Finally, although CPT-11 exhibited a certain in vitro antitumor effect for GEP-NEC cell lines, in vivo experiments should also be conducted for 7-ethyl-10-hydroxy-CPT (SN-38) as active metabolite of CPT-11.Some limitations should be clarified in this study. First, 21086-21099. https://doi.org/10.18632/oncotarget.24930Original article: Oncotarget. 2018; 9:21086–21099.


For over half a century, the carbazole skeleton has been the key structural motif of many biologically active compounds including natural and synthetic products. Carbazoles have taken an important part in all the existing anti-cancer drugs because of their discovery from a large variety of organisms, including bacteria, fungi, plants, and animals. In this article, we specifically explored the literature from 2012 to 2018 on the anti-tumour activities reported to carbazole derivatives and we have critically collected the most significant data. The most described carbazole anti-tumour agents were classified according to their structure, starting from the tricyclic–carbazole motif to fused tetra-, penta-, hexa- and heptacyclic carbazoles. To date, three derivatives are available on the market and approved in cancer therapy. Hyella caespitosa) and fungi (e.g. Aspergillus species). The parent compound 9H-carbazole was isolated from coal tar in 1872 by Graebe and GlazerMurraya koenigii Spreng in 1962Ochrosia elliptica (Apocynacae) before being entirely synthesized, could be considered as the first initial lead compound of carbazole analogues. Thereafter, an ellipticine analogue named N-methyl-9-hydroxyellipticinium acetate (Celiptium®) has been developed. Since 1982, Celiptium® is still currently used in the treatment of metastatic breast cancer,N-methyl-9-hydroxyellipticinium acetate acted as a topoisomerase II inhibitor and an intercalating agent, stabilizing the cleavable complex of topoisomerase II and inducing DNA breakages, thereby inhibiting DNA replication and RNA and protein synthesis. New N-thioalkylcarbazole derivatives were synthesized and evaluated in comparison to ellipticine. Among the bioactive carbazole-type derivatives, 7--heptane-1-thiol -one scaffold   . Alectin®) (Novartis)  , describCompared to the previously recent published reviewsThe increased interest in the use of carbazole derivatives for the cancer therapy can also be expressed in the research of patents. In 2.2.1.1  with low dark toxicity (IC50>60 µM)Ferrocenyl platinum(II) complex [Pt(Fc-tpy)(NPC)]Cl (HNPC = N-propargylcarbazole) was synthesized and evaluated for its anti-proliferative properties in visible light against HaCaT (human keratinocyte) cell lines. Compound 1  exhibite2.2.N-Acylated carbazoles were synthesized and evaluated for their anti-proliferative activities against CAL 27 (squamous cell carcinoma) cell lines. The IC50 values of the most active compounds 2a and 2b , pancreas carcinoma (Panc1), lung carcinoma , non-small-cell lung carcinoma (H460), human colon carcinoma (HCT116) and normal breast epithelium (MCF10A) cell lines. Carbazole derivatives demonstrated moderate to good activities and among them, compound 3  was foun2.4.4a, 4c, 4d, 4e, 4g, 4i and 4j) inhibited XO with IC50 values comprised between 4.3 and 5.6 µM. Furthermore, these derivatives showed a better activity than the standard drug allopurinol . Interestingly, compound 4a bearing a cyclopropyl ring was found to be the most potent inhibitor of XO with an IC50 of 4.3 µM. Compounds 4b, 4d, 4f, 4h and 4j were found to be potent inhibitors of tyrosinase . These results suggest the possible use of these compounds for the design and development of novel XO and tyrosinase inhibitorsThese derivatives  were syn2.5.5  and TCC-SUP (urinary bladder cell carcinoma) cell lines, and their mechanism of action was investigated by means of molecular docking studies. Every benzosporalen and carbazole derivative showed interesting anti-proliferative activities, with GImpound 6  had very2.7.MHY407 , 13 carbazole alkaloids were evaluated against HL-60 leukaemia cell line. Murrayafoline A Extracted from zoline-I  showed e2.11.N-alkylcarbazole derivatives 9a–c (Synthesized ves 9a–c  showed i2.12.Clausena excavata BURM. f. (Rutaceae) and its cytotoxic activities against A549 lung carcinoma and HeLa cervix adenocarcinoma cell lines were assessed, showing IC50 values of 17.77 and 6.47 µM, respectivelyA carbazole alkaloid, excavatine A , was iso2.13.Clausena wallichii (Rutaceae) and tested for both anti-bacterial and cytotoxicity activities. After evaluations, clausenawalline F  but showed less activity against A549 . Compounds 10 and 11  was demonstrated in vivo by using a mouse model of breast cancer. The carbazole group contributed to Rac1 inhibitory activity and then new compounds  and 13b  were des2.16.14a and 14b  and interaction analysis demonstrated that compounds  and 14b  had the 2.17.50 = 12 nM) and MCF-7 (IC50 = 14 nM) breast cancer cell lines. The IC50 of the positive controls podophyllotoxin and combretastatin CA-450 = 20 and 5 nM, respectively). Niu et al. described further investigations on the anti-oesophageal squamous cell carcinoma (ESCC) activity and mechanisms of SL-3–19 in vitro and in vivo. Mechanistically, SL-3–19 inhibited ESCC cell growth by inducing cell apoptosis and arresting the cell cycle at G2/M phase in a dose-dependent manner. In addition to microtubule assembly inhibition, this compound showed a significant disruption of the vascular structure by obstructing the formation of capillary-like tubes in vitro and the growth of ESCC xenografts and microvessel density in vivoThe carbazole sulfonamide IG-1052.18.15 and trimethoxyphenylurea 16, were active against both HL60 (acute promyelocytic leukaemia) and KB (a subline of HeLa) cell lines. These compounds are potent cell proliferation inhibitors, especially 15 that showed the best activities with IC50 values of 5.3 µM against HL60 cells and 6.7 µM against KB cells. The anti-proliferative activity was correlated with the inhibition of tubulin polymerization, which ranged from 20 to 50% inhibitionTwo 1,4-dimethylcarbazole derivatives , trimeth2.19.17a-c  against HL60 acute promyelocytic leukaemia cell line. Compound 17a, which was found to be the most active, also demonstrated a high inhibition at 10−5 M against MCF-7, HCT116, PC3 (prostate cancer) and MRC5  cell lines. Furthermore, fluorescence measurements were carried out and showed that 17a had some DNA binding propertiesSeveral alkylguanidines derived from carbazole were prepared and tested for their anti-cancer activity. Three compounds 17a-c  were tesIn summary, various pharmacomodulations in the series of tricyclic carbazoles were performed to obtain new anti-tumour compounds and to investigate the structure–activity relationships. Several substituents were essentially introduced on the nitrogen atom of the indole moiety or on the benzene moiety. 3.3.1.3.1.1.c]carbazoles were investigated as a p53 activator using two cell lines, HT1080 (lung fibrosarcoma) and RCC45  cell lines. The highest activity  was observed with compound 18 (A synthesized series of cyclopentacarbazole-3-carbaldehyde20a and 20b (50 from 0.009 to 0.05 µM). In parallel, compounds 20a and 20b with a longer alkyl N1–N10 bridge exhibited apoptosis-inducing activity toward IPC-81 (acute myeloid leukaemia) cells, but not toward normal fibroblastsDerivatives of N and 20b . Both moa]carbazole derivatives with substituent on position 4 were synthesized and their biological activities were evaluated as Pim kinase inhibitors and in vitro anti-proliferative agents. Compound 21  and on PC3 cells (IC50 around 6 µM)Other pyrrolocarbazole-1,3-dione derivatives were tested as Checkpoint kinase 1 (Chk1) inhibitors. Compounds 22 and 23  and also to be active against proliferative activities of prostatic cancer cells PC3 (IC50 was around 3 µM). A 1,6-dihydropyrazolocarbazole, compound 25 (50 around 0.09 µM)A series of pyrazolocarbazoles carbazole .A series of carbazole analogues comprising pyrido, isoxazolo, pyrimido and pyrazolo templates were synthesized and evaluated for their cytotoxicity against AGS (gastric cancer) and HeLa cell lines. Among the tested derivatives, 3–-9-methyl-4,5-dihydro-10Briefly, when comparing the mentioned tetracyclic carbazoles containing a five-membered ring, SAR study can be correlated with substitutions at the carbazole ring .3.2.3.2.1.27a, 5H-benzo[b]carbazol-11(6H)-one structural scaffold which presented a high selective ALK inhibition at a nanomolar scale NPM)-ALK fusion gene with an IC50 value of 3.0 nM. In vivo studies in mice, using ALK fusion gene-positive NSCLC xenograft model, showed that orally administrated compound 27a significantly regressed tumours. Currently, this compound is being evaluated in phase I/II clinical trials for the treatment of ALK-positive NSCLCAlectinib/CH5424802  cell lines. Compounds 28, 29a,b -58, 29a,b  exhibite3.2.2.M. koenigii, had a strong anti-tumour promoting activity. The expression of the Epstein–Barr Virus Early Antigen (EA-EBV) in Raji cells was inhibited by more than 90% when tested at 16 µg/mL (50% inhibition at 22.8 µM). The compound did not alter Raji cell’s viability due to very low cytotoxicity. Girinimbine showed strong anti-oxidant properties comparable to α-tocopherol (vitamin E) and inhibited the superoxide generation in the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced differentiated HL-60 cellsGirinimbine , a carbac]carbazole derivatives 30a–d , HeLa (IC50=24.3 µM) and AGS (IC50=33.8 µM) cancer cell lines were employed. It showed a growth inhibitory effect with IC50 in the micromolar range (from 12.6 to 33.8 µM). Mahanine is also a DNA intercalative cytotoxic molecule, which presented anti-oxidant capacities. On human cancer cell lines, it indicated excellent radical scavenging of the 2,2-diphenyl-1-picrylhydrazyl radical , 2,2′-azino-bis, OH• (12.0 µM) and nitric oxide radical ]. Mahanine showed as well alpha-glucosidase inhibitory activity with IC50 value of 21.4 µMMahanine -3,11-dihydropyranocarbazoles was evaluated by SRB (sulfo-rhodamine B) assay against MCF-7, HeLa and A549 cell lines. Among these derivatives, compound 34 . All the designed compounds demonstrated a higher potency against HeLa than against the other tested cell linesThe in vitro cytotoxicity of the pyridocarbazole derivatives and their analogues were tested against A549 and HT29 cell lines with IC50 values ranging from 0.07 µM to 4.45 µM. For example, compound 35 . Compounds 36a–c also exhibited stronger cytotoxic activity against MCF-7 cell linesAmong pyrimidocarbazole derivatives 36a–d , 2-amino3.2.5.37a–c  cells. Results showed that two analogues, compounds 42a and 42b A series of 3-substituted-pyrrolocarbazole analogues (3-aroyl-derived pyrrolocarbazoles) was synthesized and evaluated as PARP-1 inhibitors. They were also tested in a cell-based assay that evaluated their ability to attenuate the depletion of NAD and 42b , display4.2.4.2.1.b]carbazoles were synthesized to undergo a one dose screening at 10−5 M, followed by a five dose screening for the best compounds using the NCI 60 cell lines list. Compound 43 Several inert metal complexes such as pyridocarbazole-rhodium (III) were synthesized and characterized by X-ray crystallography. Stability studies were carried out including evaluation of Pim-1 kinase inhibitory activity. Compound 44  was foun45a and 45 b , benzo[a]pyrene (B[a]P) (7 (B[a]P)  and seve4.3.2.M. euchrestifolia (Rutaceae) and was evaluated against HL-60 cell line. Murrayazolinine displayed a significant interaction with the caspase-9/caspase-3 pathway, leading to the cellular apoptosisMurrayazolinine  was isol4.3.3.bc]carbazole-amonafide derivatives possessed a good cytotoxic activity with IC50 values in the sub-micromolar to micromolar range against HTC116 cell line, and were also selective for cancer cells when compared to a HEK293 (non-cancerous human embryonic kidney) cell line. Compound 46  (11–(2-Methylpropyl)-12,13-dihydro-2-methyl-8-(pyrimidin-2-ylamino)-4P-11981)  is a pot5.3.Among the carbazole derivatives, four synthetic indolopyrrolocarbazoles  are curr5.3.1.1 . Streptocarbazole B was only active against P388 and HeLa cells, demonstrating IC50 values of 12.8 and 22.5 µM, respectivelyIsolated from the marine-derived actinomycetes strain  A and B  were tes5.3.3.a]carbazole derivatives The biological activity of indolopyrazolocarbazole 50 and 12-(1-deoxy-β-d-glucopyranos-1-yl)-12,13-dihydro-5H-indolopyrazolocarbazole 51 (50 and 51 were active toward HCT116 (human colon carcinoma) cell line with similar IC50 values of 12 and 11 µM, respectively.Glycosylated indolopyrazolocarbazole analogues of K252c  have beeazole 51 97. Inter5.5.a]pyrimidocarbazoles and azaindolopyrimidocarbazoles was synthesized and their anti-cancer activity was evaluated through topoisomerase II inhibition and in cellulo assay using the NCI-60 cell line. Although no topoisomerase II inhibition was observed, compound 52a  using the MTT colorimetric method. From the results, compounds 53a–f  displaye7.Cancer is a very complex disease and the increase of the biological targets can be synergistically coordinated to relieve patients from cancer burden. Many new cancer therapies have been developed in the last years, but this research field still presents many challenges. Among the natural products, the carbazole alkaloids have shown several biological activities . Since 2In a recent study, Iman et al.


Notch1 and Ikaros were most common in T-ALL, whereas ETS transcription factors (Erg and Ets1) were targeted in most ELs. Cyclin E status did not impact leukemogenesis or oncogene activations. Whereas most SB insertions were lost during culture of EL cell lines, Erg insertions were retained, indicating Erg’s key role in these neoplasms. Surprisingly, cyclin ET74AT393A conferred growth factor independence and altered Erg-dependent differentiation in EL cell lines. These studies provide new molecular insights into erythroid leukemia and suggest potential therapeutic targets for human leukemia.Insertional mutagenesis is a powerful means of identifying cancer drivers in animal models. We used the Sleeping Beauty (SB) transposon/transposase system to identify activated oncogenes in hematologic cancers in wild-type mice and mice that express a stabilized cyclin E protein (termed cyclin ET74AT393A). Cyclin E governs cell division and is misregulated in human cancers. Cyclin ET74AT393A mice develop ineffective erythropoiesis that resembles early-stage human myelodysplastic syndrome, and we sought to identify oncogenes that might cooperate with cyclin E hyperactivity in leukemogenesis. SB activation in hematopoietic precursors caused T-cell leukemia/lymphomas (T-ALL) and pure red blood cell erythroleukemias (EL). Analysis of >12,000 SB integration sites revealed markedly different oncogene activations in EL and T-ALL:  By combining conditional expression of the SB transposase with the T2Onc transposon in various genetic backgrounds, SB screens have been used extensively to identify\cancer genes and how they cooperate with one another in wild type and cancer-sensitizing backgrounds, and across many cancer types6.Insertional mutagenesis is a powerful means of identifying the molecular drivers of cancer initiation and progression in animal models. Sleeping Beauty (SB) is a transposon/transposase insertional mutagenesis system that is designed to either overexpress nearby genes or inactivate genes, depending on the transposon’s integration site and orientation7. One important means of cyclin E regulation is phosphorylation-dependent degradation by the SCFFbw7 ubiquitin ligase12. To study the physiologic consequences of abnormal cyclin E degradation, we previously created a knock-in mouse model that ablated two cyclin E phosphorylation sites (T74 and T393) that trigger its degradation14. The cyclin ET74AT393A mutation caused increased cyclin E abundance and epithelial cell hyperproliferation. However, these mice did not spontaneously develop epithelial dysplasia or tumors, suggesting that compensatory mechanisms maintain tissue architecture and suppressed tumorigenesis. Cyclin ET74AT393A expression also caused ineffective erythropoiesis with marked expansion of immature erythroid precursors in the spleen and bone marrow, impaired erythroid differentiation, and mild anemia. These features resemble the early stages of human refractory anemia/myelodysplastic syndrome (MDS).In this study, we employed SB to identify oncogenes that might promote multi-step carcinogenesis in a mouse model engineered to express a stabilized version of the cyclin E protein. Cyclin E, in conjunction with its catalytic partner CDK2, has crucial roles in cell division, and cyclin E-CDK2 deregulation causes genome instability and contributes to cancer development and progression16, we used two different T2/Onc2 strains that contained the transposon array on different chromosomes. The most common malignancies were immature T-cell leukemia/lymphomas (T-ALL) and pure red blood cell erythroleukemias (EL), and there was a non-significant trend towards more EL in the cyclin ET74AT393A mice.Because MDS can evolve to leukemia in humans, we speculated that cyclin ET74AT393A mice may provide a sensitized background to identify genetic events that cooperate with abnormal cyclin E regulation to promote leukemia. We thus used interferon-inducible Mx-Cre to activate the SB transposase in hematopoietic precursors to identify genes that might cooperate with abnormal cyclin E regulation to promote leukemia. The stabilized cyclin E allele neither predisposed mice to hematologic cancers nor altered gene activations by SB. Strikingly however, Mx-Cre-induced SB activation caused highly penetrant hematologic cancers within 8–13 weeks after Cre induction. To control for biases in transposon integrations that frequently occur proximal to the T2Onc arrayErg and Ets1) were the most commonly activated genes in ELs and were activated in the almost all of these tumors.To identify activated oncogenes in these neoplasms, we determined the transposon insertion sites in all ELs and T-ALLs. Transposon insertions that are shared by multiple independent tumors, termed common insertion sites (CIS), often occur in the vicinity of cancer-associated genes, which provides the selective pressure for these shared insertions. We identified CIS using two different statistical methods and found that the CIS profile of ELs and T-ALLs differed markedly. Whereas Notch and Ikaros insertions were most common in T-ALL, ETS family transcription factors , the cyclin E mutant cell lines did not and proliferated in the absence of GFs. Next, Erg knockdown resulted in erythroid differentiation of the WT but not the cyclin ET74AT380A cell lines. These findings suggest that properly regulated cyclin E activity influences both growth factor requirements and differentiation in EL cells.While T-ALL is common in SB screens performed in blood cells, EL has not, and we thus examined EL in more detail by developing 5 transplantable EL cell lines. Transposon analyses indicated that the vast majority of CISs found in primary ELs were lost during culture of EL cell lines, suggesting that they were not required for their proliferation and maintenance 18 Mice (7–10 weeks old) were injected with poly I:C to activate Cre recombinase expression and subsequent transposase expression. Mx1-Cre induction and SB expression was monitored by loss of GFP expression in peripheral blood leukocytes from a subset of study mice, which demonstrated 71–86% loss of GFP-expressing cells following induction  Fig. . The maton Table .Figure 1 contained mixed tumors of both lymphoid and erythroid lineages identified by parallel flow cytometry studies  mice developed EL with intermixed megakaryocytic differentiation, which caused prominent splenomegaly and involved the bone marrow and sometimes the liver  and 53 T-ALLs (thymus or lymph node), amplified the transposon integration sites via ligation-mediated PCR, and identified the transposon integration sites by next generation sequencing. We mapped 5002 EL insertion sites and 7012 T-ALL sites Table . Each tuNotch1, Ikzf1, Rasgrp1, and Akt26. The presence of the cyclin E mutation did not impact the spectrum of CIS observed. Since T-ALLs have been well studied in insertional mutagenesis studies, we focused on the ELs for the remainder of our study24.gCIS identified 74 total CIS in T-ALLs Table . The chrErg and Ets1 in 65% and 44% of ELs, respectively. In fact, only 4 of the 40 ELs lacked insertions targeting either Erg or Ets1 . While CIS within donor chromosomes were filtered from our analyses, some of the CIS found within donor chromosomes have been reported in other transposon-mediated insertion studies targeting hematologic neoplasms, and thus may represent physiologically significant gene alterations rather than local hopping. Examples of these types of CIS (on chromosome 4) that are likely to have contributed to EL in our study include Csf3r (35% of ELs), Bach2 (41% of ELs) and Cdkn2a/Arf (5% of ELs). Because we found each of these CIS in both T2Onc strains, these insertions were not restricted to potential local hops. TAPDANCE, an alternate statistical method that eliminates donor chromosome insertions, was also used to annotate CIS and gene associations in T-ALL and EL. The most frequent CIS found by TAPDANCE in T-ALL included Notch1, Ikzf1, Rasgrp1, Akt2, Runx1, FoxP1, whereas the most frequent CIS present in EL were Erg and Ets1, followed by Epo, Gata1, Pik3ca and Fli1  expression and increased Ter119 (glycophorin B), consistent with erythroid maturation  is a receptor tyrosine kinase that is mutated in AML and confers a poor clinical prognosis34. We identified three Flt3 insertions in ELs, all of which were located downstream of exon 9 . Compared with the other cell lines, line 4489 (with the Flt3 insertion) was exquisitely sensitive to all three inhibitors, showing that this EL cell line remained dependent on FLT3 activity for proliferation . Because cyclin E has non-catalytic activities (e.g. replication origin licensing), one possibility is that mutations that differentially impact cyclin E abundance versus activity may lead to different biologic outcomes38. However, we do not have evidence to directly support this speculation from this study.Based on our previous work showing that mice with a stabilized cyclin E protein develop proliferative and differentiation anomalies in hematopoietic cells, we undertook this study to use SB to identify putative cyclin E-cooperating oncogenes. In light of the phenotypic consequences of cyclin ET74AT393A expression in mice, as well as the roles for cyclin E in human cancer and murine cancer models40. Our findings that cyclin ET74AT393A rendered two EL cell lines growth factor independent supports this notion, although these studies involved a limited number of cell lines. Of note, our attempts to convert WT-EL cell lines to GF independence through ectopic cyclin ET74AT393A expression were unsuccessful because ectopic cyclin ET74AT393A expression was toxic.While cyclin ET74AT393A expression did not alter carcinogenesis, it did impact growth factor requirements and differentiation of EL cell lines. Cyclin E-CDK2 activation has previously been proposed to be a crucial feature of the G1 restriction point, which is defined at the point in the G1 phase of the cell cycle after which cells no longer require extracellular mitogens for S-phase entry22, EL is much less common25. In humans, acute erythroleukemia (EL) is a rare and relatively poorly characterized form of acute myeloid leukemia19. Until recently, the diagnostic criteria for EL encompassed a diverse set of hematopoietic malignancies that share some degree of erythroid hyperplasia, ranging from an expanded abnormal myeloid blast population with erythroid hyperplasia to a pure erythroid leukemia with erythroid blasts encompassing greater than 80% of the marrow cellularity . The prior diagnostic criteria for acute erythroleukemia thus included both erythroleukemia (erythroid/myeloid) and pure erythroid leukemia, which occurs less commonly. Because erythroleukemia (erythroid/myeloid) frequently arises from pre-existing MDS , the WHO recently modified the diagnostic criteria for EL: the more common erythroleukemia (erythroid/myeloid) is now classified as MDS or AML based on the percentage of myeloid blast population, irrespective of the erythroid component42. Therefore, many cases that had been previously diagnosed as EL are now reclassified as low grade MDS. Pure erythroid leukemia (PEL) remains a distinct diagnostic entity per WHO criteria and is characterized by malignant immature erythroid cells that represent greater than 80% of the marrow. Because of its rare occurrence and previously imprecise diagnostic criteria, our understanding of PEL remains limited. A few small-scale clinical studies on PEL have demonstrated that the leukemic cells usually have complex chromosomal abnormalities, frequent p53 mutations, and extremely poor overall survival44.SB activation by Mx-Cre caused two distinct hematologic neoplasms, T-ALL and EL, that differed morphologically, immunophenotypically, and with respect to the identified CIS associated with each disease. While T-ALL has been extensively described in SB models29, our studies support a crucial role for ETS transcription factors in the development and maintenance of EL. Most of the ELs that we studied (90%) had inserts in one of the ETS transcription factors  and it is tempting to speculate that the few ELs without these insertions may have activated these pathways through other mechanisms. However, we do not have direct evidence to support this speculation. Strikingly, ERG-associated SB insertions were retained in each of the five EL cell lines that we derived from primary ELs, while the vast majority of other CIS found in the primary tumors were not  associated with peripheral cytopenia, circulating erythroid blasts and frequent extramedullary involvement of the liver and spleen. We identified many insertion sites in ELs that target genes involved in myeloproliferative disorders, growth factor signaling, and signal transduction. In accordance with previous work on murine and human ELnot Fig. . This sunot Fig. . Our stuBach2 , a transcriptional repressor that regulates antibody class switching and has been implicated in B-cell neoplasm46. Bach1 and Bach2 are highly regulated and homologous proteins with key functions in iron hemostasis, erythropoiesis, megakaryopoiesis, and autoimmunity47. Our finding that Bach2 insertions were retained in EL cell lines suggests a biologic selection for persistent Bach2 deregulation and perhaps a role for Bach2 in EL development and/or maintenance. During erythropoiesis, elevated levels of free heme bind to and inhibit Bach1, thereby activating the expression of Beta-globin and other genes involved in erythroid differentiation48. Although Bach2 functions as a transcriptional repressor during B-cell maturation and has been implicated in B-cell neoplasms, other studies have shown the Bach2 is also regulated by heme binding and that overexpression of Bach2 in hematopoietic stem cells promotes erythroid commitment, suggesting it may also play an early role in directing erythroid lineage fate decisions49.The other CIS that was retained in multiple EL cell lines was Finally, pure erythroid leukemia is an aggressive poorly characterized form of AML. Now that the WHO has redefined PEL as a distinct form of AML, new clinical studies are needed to identify the molecular pathways that promote PEL. The functional studies presented here provide candidate genes and pathways that should serve as a resource for future studies of human PEL and help define altered pathways that promote this rare, but highly aggressive, leukemia.13. The Cre inducible RosaSBase-LSL transposase and the T2/Onc2 transposon (Lines 6113 and 6070) mice were obtained from N. Copeland18. Mx1-Cre transgenic mice were obtained from Jackson Laboratories . The breeding schemes to generate study mice are shown in Fig. in vitro prior to injection. Recipient C57/Bl6 mice were sublethally irradiated (700 Gy) 24 hours prior to transplantation and infused with 1 × 106 cultured erythroleukemia cells. Each cell line was transplanted into five recipient mice. Mice were monitored daily using institutional standard procedures and euthanized when indicated. Animal studies and all animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) and were carried out at the Fred Hutchinson Cancer Research Center (FHCRC). All methods were performed in accordance with the guidelines and regulation established by FHCRC IACUC, Institutional Review, and Institutional Biosafety Committees.All mice used in these studies were previously described. Homozygous cyclin ET74AT393A mice were developed and maintained as described51.Mice were euthanized via carbon dioxide inhalation and necropsies were performed. All tissues were harvested and fixed in 10% formalin for 5–7 days and stored in 70% ethanol until submitted for pathological evaluation. Sternums for bone marrow analysis were decalcified in Formical-4  for 24 hrs. after fixation in formalin. After fixation, all samples were submitted to the Experimental Histopathology Core Facility at FHCRC where tissues were processed, embedded in paraffin, cut into slides, and stained with H&E according to standard techniques. Tissues were reviewed by a board-certified hematopathologist (KRL) and a board-certified veterinary pathologist (SEK). Peripheral blood was harvested and analyzed via Siemens Advia 2120i with multispecies software and a manual differential analysis. Lymphomas and leukemias were classified using the Bethesda recommendations for mice4CL, 12 mM NaHCO3, 0.13 mM EDTA) at room temperature and washed twice in PBS + 2% FBS. Flow cytometry analysis included a 4-color T-cell panel ) and a 6-color erythroid/myeloid panel -PerCP-Cy5.5, and B220-FITC (BD Biosciences) and CD3e Alexa Fluor 488 (BioLegend). Cells were also stained with DAPI to determine cell viability. Flow cytometry was performed on a Canto 2 (Becton Dickinson) or a custom designed LSR2  and analyzed using either FlowJo Software  or Woodlist, a noncommercial software program developed in our clinical laboratory52.Primary splenocytes and thymocytes were recovered by crushing excised spleen or thymic tissue between glass microscope slides, washing with PBS + 2% FBS, and passing through an 80 µm mesh filter . Cells were washed once in PBS + 2% FBS and were resuspended in PBS + 2% FBS and kept on ice until analysis. For analysis of bone marrow, marrow was extracted from femurs using a mortar and pestle and processed as above. For analysis of peripheral blood, red blood cells were lysed , and submitted for direct sequencing on an Illumina HiSeq 2000. Sequencing reads were analyzed using gene-centric common insertion site analysis (gCIS) or TAPDANCE21.Genomic DNA was extracted from frozen tissue using the Puregene Core Kit A (Qiagen) according to manufacturer’s instructions. Five μg of genomic DNA in a volume of 100 μl of ddH2O was sheared to 300 bp lengths using the Covaris E-series sonicator according to the manufacturer’s protocol. One μg of DNA (20 μl) was end-repaired in a reaction containing T4 DNA polymerase and polynucleotide kinase and incubated at 20−5 M monothioglycerol, 2 U/ml erythropoietin , and 50 ng/ml mouse stem cell factor. Cell lines were then maintained in culture as indicated in the text. Lentivirus infection with Erg-specific shRNA, and retroviral infection with cyclin ET74AT393A and wildtype cyclin E were performed as previously described53.Primary tumor cell lines were derived by plating splenocytes in IMDM media containing 20% FBS, 4.5 × 1011. Anti-ERG antibody (CM421AC) was from Biocare Medical  and anti-FLT3 antibody (8F2) was from Cell Signaling .Western blotting was performed as describedCell lines were incubated with the indicated concentrations of drugs and incubated for 72 hours. Viability was measured using a resazurin-based assay and % viability was normalized to the untreated control.Supplemental InformationTable S2Table S3Table S4Table S8


Scientific Reports 10.1038/s41598-019-49390-9, published online 09 September 2019Correction to: This Article contains errors in the Introduction.“TRAF3-deficient B cells were more susceptible to biochemical Pim2 inhibition and more resistant to inhibitors of the phosphatidyl inositol-3 kinase/protein kinase B (PI3K/Akt) pathway compared to wild type (WT) B cells. Low TRAF3 expression in human BCL and MM cell lines correlated with higher Pim2 protein levels and decreased susceptibility to Pim2 inhibitor-mediated cell death.”should read:“TRAF3-deficient B cells were less susceptible to biochemical Pim2 inhibition compared to wild type (WT) B cells. Low TRAF3 expression in human BCL and MM cell lines correlated with higher Pim2 protein levels and increased susceptibility to Pim2 inhibitor-mediated cell death.”


This study focuses on the adaptation of natural Newcastle disease virus (NDV)strains isolated from wild birds to human tumor cells. Many candidates forvirotherapy are viruses pathogenic for human. During recombination of geneticmaterial, there always exists a risk of getting a virus with an unstablegenome. This problem can be solved by using natural apathogenic viruses asoncolytic agents. The Newcastle disease virus is the causative agent ofcontagious avian diseases. Its natural strains exhibit an antitumor effect andare considered safe for humans. As shown in earlier studies, the oncolyticproperties of natural strains can be enhanced during adaptation to cellcultures, without interference in the virus genome. This study demonstratesthat serial passaging increases the viral infectious titer in cancer cells.Moreover, the viability of tumor cells decreases post-infection when Newcastledisease virus strains are adapted to these cell cultures. The findings of thisstudy complement the well-known data on the adaptation of the Newcastle diseasevirus to human cancer cells. Hence, it is possible to obtain a NDV strain witha more pronounced oncolytic potential during adaptation. This should be takeninto account when choosing a strategy for designing anticancer drugs based onthis virus. Avulavirus), for selectivedestruction of tumor cells [Virotherapy is an experimental method of targeted therapy of malignant diseasesthat employs oncolytic viruses, including the Newcastle disease virus (NDV) exhibits in vivo, 17.Mea in vivo,17, 18.Investigation of the mechanism of viral antitumor cytotoxic action allowedresearchers to put forward a hypothesis that a strain with more pronouncedlytic properties can be obtained during viral adaptation to a cell culture. During in vivo model. The overall weekly survival rate of mice withascites tumors after treatment with the late-passage virus increased from 0 to63.6%. Moreover, the virus actively replicated in tumor cells and exhibited acytotoxic effect faster than the wild-type strain[in vitro followedby 13 passages in vivo. Later, a number of successful clinicaltrials of allogeneic and autologous vaccines based on the NDV 73-T strainagainst metastatic melanoma were conducted in the USA[Thus, the strain based on natural MD20Z  was obtained by serial (n =38) passaging in Ehrlich ascites carcinoma cells and was tested on ane strain. Cassel e strain confirme the USA.Therefore, taking into account the data confirming successful adaptation ofwild-type strains with an enhanced antitumor effect during sequentialpassaging, we studied the adaptation of wild-type strains of various pathotypeson lines of human tumor cells in order to verify the effectiveness of thecytotoxic properties acquired after passaging of the strains.Cell lines We studied transplantable cell lines: Vero – kidney epithelial cellsextracted from the African green monkey; HCT116 – human colon carcinomacell line ; Hela – human epithelioid cervix carcinoma; MCF7– human breast adenocarcinoma cell line ; and A549 – humannon-small lung carcinoma cell line .2) at 37°C in an incubator with 5% CO2.Vero and tumor cell lines were grown in Dulbecco’s modified Eagle’smedia (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS)and 50 mg/ml gentamicin. Cells were maintained in cultural flasks (25cmNewcastle disease virus strains 777/2010[Our study was performed using four NDV strains: two virulent mesogenic NDVstrains isolated from rock doves in Russia (NDV/Altai/pigeon/770/2011 andNDV/777/2010) and twoPreparation of Newcastle disease virus strains The viral strains were amplified in the allantoic fluid of 10-day-oldembryonated eggs. The presence of the virus was determined by hemagglutinationassay (HA) with rooster erythrocytes. The hemagglutinin titers of each viralstrain were determined according to the standard procedure recommended by theWHO .Titration of the Newcastle disease virus on Vero cell line (TCID50)Titration of the Newcastle disease virus on the Vero cell line, which issensitive to the cytopathic effect of NDV, was performed according to theprocedure described in . The celThe next day, the cell monolayer was washed with Hanks’ solution and10-fold viral dilutions per well were added. The dilutions were prepared usingMEM maintenance medium supplemented with 1% heat-inactivated FBS. The plateswere incubated under standard conditions for 1 h to adsorb the virus; thevirus-containing liquid (VCL) was then replaced with the maintenance medium.Every day, the cell monolayer was inspected under a microscope to detect thepresence of CTE. The final titration was carried out on day 4. The infectioustiters of the virus on the Vero cell line were calculated according to theCerberus method in the Ashmarin’s modification and presented aslgTCID50/ml .Serial passages of NDV strains in transplantable Vero cell line and tumorhuman cell lines Preparation of the cells and infection with 10-fold dilutions of the virus wereperformed using the same procedure as the one described in the titrationmethod. On day 4 post-infection, the infectious titer was counted and VCL ofthe extreme dilutions was collected. The next passage was carried out withinfection of Vero cells with the virus collected in extreme dilution using thesame method. Hence, we conducted a total of eleven passages and calculated theinfectious titer of each passage.Colorimetric analysis of cell viability after infection with NDV strains(the MTT assay) The viability of human tumor cell lines was analyzed using the MTT assay 4 dayspost-infection with viral strains. The one-day-old monolayer of tumor cells onthe 96-well plate was washed with Hanks’ solution and incubated withviruses  for 1 h under standard conditions. Thecells were then washed with Hanks’ solution and incubated in 200 μlof the maintenance medium for 4 days under standard conditions. Control tumorcells without the virus were incubated in MEM maintenance medium.540-V630)/(K540-K630) ×100%, where V and K denote the values for virus-infected and control wells,respectively.On day 4, the cells were washed with Hanks’ solution. The MTT workingsolution at a concentration of 5 mg/ml was prepared using the MEM maintenancemedium. The MTT solution (100 μl) was added to each well. The plate wasincubated for 4 h under standard conditions. The MTT solution was then replacedwith DMSO (150 μl per well), and the cells were incubated for 1 h in thedark at room temperature. The optical density was measured at wavelengths of540 nm and 630 nm on a LonzaBiotek ELX808 absorbance microplate reader (USA).Cell viability was evaluated according to the ratio between the relativepercentage of living and control uninfected cells using the formula: under standard conditions. Theone-day-old cell monolayer was washed twice with Hanks’ solution andincubated with viruses in a 1-ml volume  withadsorption for 45 min under standard conditions. The cells were then washedtwice with Hanks’ solution, and a fresh 10 ml of the MEM maintenancemedium was added. The flasks were incubated under standard conditions. Theinfectious titer of the culture medium was determined 3, 6, 24, 48, and 72 hpost-infection with NDV strains by titration on the respective tumor and modelcell lines using the conventional method.Human tumor cell lines and control Vero cells were cultured in a growth mediumin culture flasks .A high titer of the mesogenic strains isolated from rock doves wasshown in hemagglutination assay and during titration on Vero cells.Newcastle disease virus strains were propagated in the allantoic fluid of10-day-old embryonated eggs. The presence of the virus in allantoic fluid wasconfirmed by hemagglutination assay with rooster erythrocytes; infectious viraltiter was determined in the Vero cell lineafter 11 passages in the Vero cell line. The titer of these strainswas initially higher than those of lentogenic strains and was 6.7 lgTCID50/mlafter the first passage. We failed to reach values as high as the titers of thelentogenic strains NDV/mallard/Amur/264/2009 andNDV/teal/Novosibirsk_region/320/2010 for mesogenic strains in late passages,despite the fact that the titers increased from 3.6 to 5.0 lgTCID50/ml and from3.6 to 4.3 lgTCID50/ml, respectively. The presence of a “virulent”sequence at the F protein cleavage site is probably affected not only by thevirus’ ability to efficiently replicate in cells with the formation ofCTE. It is quite possible that belonging to a certain pathotype, which isdirectly associated with the cytolytic capacity, also plays a role, since allthe studied strains contained a “virulent” sequence at the cleavagesite, but only the NDV/Altai/pigeon/770/2011 and NDV/Altai/pigeon/777/2010belonged to the mesogenic type [According to the first scheme, the viral titers of the mesogenic strainsNDV/Altai/pigeon/770/2011 and NDV/Altai/pigeon/777/2010 increased to 7.5 and7.7 lgTCID50/ml, respectively. Syncytia-like structuresappeared by the 7th–10th passages (on days2–3), which are characteristic of CTE upon infection with paramyxoviruses.Despite the difference in CTE in different passages, the maximum lesion of themonolayer amounted to about 90%, regardless of passage; the destructive changeswere usually most pronounced on day 4 post-infection.During adaptation to the Vero cell line, the titer of all studied viral strainsincreased and remained unchanged after seven or eight passages. In addition tothe increase in infectious titer in the TCID50 assay, the morphological changesin the cell monolayer became more pronounced. After infection with mesogenicstrains, a cytotoxic effect was observed in the1th passage in Vero cellsand the NDV/mallard/ Amur/264/2009 strain in the 10th passage ondays 2 and 3, respectively. Syncytia formation was not observed during primarypassaging of these viruses in Vero cells.Syncytia formation, which is typical in cultivation of differentparamyxoviruses, took place only in a few cases: the NDV/Altai/pigeon/770/2011strain in the 7No morphological changes in the monolayer were observed during passaging of theNDV/mallard/ Amur/264/2009 and NDV/teal/Novosibirsk_region/320/2010 strains,except for a single case of syncytium formation in the monolayer.th passage were used to perform a comparative evaluation oftheir anticancer properties against tumor cell lines .The adapted mesogenic strains NDV/Altai/pigeon/770/2011,NDV/Altai/pigeon/777/2010, and lentogenic strain NDV/mallard/Amur/264/2009 inthe 10Fig. 3A).The multiplicity of infection of tumor cell lines was 8 HAU/10,000 cells. Cellsurvival after infection was assessed by MTT assay on day 4.Titration of VCL of NDV/Altai/pigeon/770/2011collected after 3, 6, 24, 48 and 72 h showed the dynamics of viral titergrowth. The highest titer on day 3 was observed for Vero cells; the lowesttiter, for the HCT116 cell line. Similar data were obtained for the NDV/Altai/pigeon/777/2010 strain. The lowest titer was obtained on the HCT116 and MCF 7cell lines.The dynamics of the replication activity of mesogenic strains was determinedfor all tumor cell lines and for the Vero cell line as a positive control.The NDV/Altai/pigeon/770/2011 and NDV/Altai/ pigeon/777/2010 strains aresimilar in their characteristics and replication activity, so one strain, NDV/Altai/pigeon/770/2011, was selected for further study focused on virusadaptation to tumor cell lines with a view to increasing the oncolyticpotential. This strain was cultured on tumor cell lines until the 11thpassage as described above.The titer increased after virus passage (passages 1–11) in the celllines: in HCT116, by 1.9 lgTCID50/ ml; in HeLa, by 0.6 lgTCID50/ml; in A549, by0.9 lgTCID50/ml; and in MCF7, by 0.6 lgTCID50/ml. However, an increase in viraltiter by almost two orders of magnitude was observed only in viral adaptationto HCT116 cells. The viability of tumor cell lines after infection with10in vitro.A different technique might be required for the adaptation to tumor cells,which would include culturing in higher passages or passaging in the allantoicfluid of ECE and in its combination with ECE Therefore, there probably is no direct correlation between the dynamics ofvirus development in tumor cell lines and the effectiveness of antitumor actionvia direct oncolysis of these cells. This fact can be used to confirm thedifferences in the sensitivity of each specific cell line to the oncolyticactivity of the virus. Thus, we managed to reduce the viability of HCT116 cellsby 5 and 10% after they had been infected with the NDV/Altai/pigeon/770/2011and NDV/Altai/pigeon/777/2010 strains, which were passaged in transplantableVero cells. The HCT116 cell line is perhaps more sensitive to infection withadapted strains. We assume that this effect can be enhanced by longer passagingof strains .Virus adaptation can be employed as a method to increase the infectious titerin order to subsequently use the virus as an antitumor agent exhibiting morepronounced oncolytic properties. The sensitive Vero cell line can be used todevelop wild-type NDV strains. Adaptation of NDV strains to Vero cells (11passages) statistically significantly increases the viral titer regardless ofvirulence and initial titer: the average infectious titer was increased by 0.8and 1.1 lgTCID50/ml for mesogenic strains and by 0.7 and 1.4 lgTCID50/ml forlentogenic strains.st passagedecreased after infection with the adapted strain NDV/Altai/pigeon/770/2011 inthe 11th passage. MCF7 cells remained insusceptible to both NDV in the1st and 11th passages. This result indicates that it is possible toenhance oncolytic properties by strains adaptation and optimization of thepassaging procedure.The viability of tumor cells sensitive to NDV in the 1


Basal-like breast cancer (BLBC) is an aggressive breast cancer subtype with features similar to the basal cells surrounding the mammary ducts. Treatment of patients with BLBC has been challenging due to the lack of well-defined molecular targets. Due to the clinical and pathological similarities of BLBC with BRCA-deficient breast cancers, the effectiveness of Poly (ADP-ribose) polymerase inhibitors (PARPi) has been tested in early phase clinical trials for patients with advanced BLBC, with limited clinical responses. Recently, it was reported that HORMAD1 overexpression sensitizes BLBC to HR-targeting agents by suppressing homologous recombination. Our independent analysis suggests that HORMAD1 is aberrantly overexpressed in about 80% of BLBC, and its expression in normal tissues is restricted to testis. Our experimental data suggests that HORMAD1 overexpression correlates with focal hypomethylation in BLBC. On the other hand, investigation of the Genomics of Drug Sensitivity in Cancer dataset revealed significantly reduced sensitivity of HORMAD1-overexpressing BLBC cell lines to Rucaparib, a commonly used PARPi. To further assess the role of HORMAD1 in PARPi sensitivity, we generated three HORMAD1-overexpressing xenograft models using the HORMAD1-low BLBC cell lines HCC1954, HCC1806, and BT20; we then subjected these xenograft models to Rucaparib treatment. Ectopic expression of HORMAD1 enhances tumor formations in two of these models, and significantly reduces sensitivity to Rucaparib in the HCC1954 model. Taken together, our data suggest that epigenetic activation of HORMAD1 by hypomethylation in BLBC may endow reduced sensitivity to Rucaparib treatment in some tumor models. Basal-like breast cancer (BLBC) possesses a particularly aggressive clinical phenotype and a molecular subtype defined by an array of genes that are expressed by normal basal epithelial cells –3. Most HORMAD1 encodes a HORMA domain-containing protein which binds to DNA double-strand breaks created during meiosis, promotes synapsis formation, and activates homologous recombination . It was To examine HORMAD1 expression in normal human tissues, we analyzed Affymetrix U133 plus 2.0 microarray data for 34 normal tissue types from the GEO Human Body Index dataset (GSEA7307) . This reTo examine the signaling alterations characteristic of HORMAD1 overexpression, we analyzed the Reverse Phase Protein Array (RPPA) data for TCGA tumors . The sigR = 0.214, To investigate the causes of HORMAD1 overexpression, we correlated HORMAD1 expression with copy number and DNA methylation at this locus using matched Affymetrix SNP 6.0 copy number data and Illumina Human DNA Methylation 450 data available from TCGA. Copy number data analysis suggests that HORMAD1 expression does not correlate with copy number . We therefore examined the DNA methylation status of the HORMAD1 CpG island region by bisulfate pyrosequencing. HORMAD1 expression levels across breast cancer tissues were determined by qPCR Figure , and HORTo further verify that HORMAD1 expression is regulated by DNA methylation, we examined whether HORMAD1 expression could be induced by the DNA demethylation agent 5-azacytidine (5-aza). We treated the MCF10A benign basal epithelial cell line and the HCC1806 basal breast cancer cell line, neither of which overexpress HORMAD1, with 5-aza. HORMAD1 demethylation and overexpression was substantially induced in both cell lines after 5-aza treatment, suggesting that regulation of DNA methylation could be one of the main mechanisms by which this gene is regulated . Taken tPARP inhibitors (PARPi) have shown promising clinical effect on BRCA-mutant ovarian and breast cancer . And mosin vivo, we generated several xenograft mouse models using BLBC cell lines engineered to ectopically overexpress HORMAD1 and assessed the tumors’ responses to Rucaparib treatment. We selected three HORMAD1-low BLBC cell lines, HCC1954, HCC1806, and BT20, then engineered these cell lines to ectopically express either HORMAD1 or yellow fluorescent protein (YFP) as a control. The engineered lines were inoculated into fat pads of female athymic nude mice. Upon tumor establishment, mice bearing either HORMAD1-overexpressing tumors or YFP-expressing tumors were randomized and treated with Rucaparib (10 mg/kg) or the vehicle phosphate-buffered saline (PBS). HORMAD1 protein levels in the xenograft tumors were verified by western blots . All breast tumor patient tissues were obtained from the Tumor Bank of Lester and Sue Smith Breast Center at Baylor College of Medicine. 293FT cells used for lentivirus packaging were purchased from Invitrogen. HCC1143, HCC1937, HCC1954, and HCC1806 cells were cultured in RPMI 1640 (Cellgro) with 10% fetal bovine serum (Thermo Fisher Scientific). BT20 cell was cultured in DMEM (Thermo Fisher Scientific) with 10% fetal bovine serum. Sum102PT was cultured in Ham’s F-12 supplemented with 5 ug/ml insulin, 1 ug/ml Hydrocortisone, 10 ng/ml EGF, 5 mM Ethanolamine, 10 mM HEPES, 5 ug/ml Transferrin, 10 nM Triiodothyronine, 50 nM Sodium Selenite, 1 g/L Bovine Serum Albumin.5-azadC was purchased from Sigma. 5-azadC was dissolved in 1× phosphate-buffered saline . In methylation inhibition experiments, cells were treated with 0.5–5 uM for 24 hours. Cells were then harvested and expression levels of HORMAD1 were analyzed by qPCR. Complementary DNA was generated from 1 ug of total RNA using the Transcriptor First Strand cDNA synthesis Kit (Roche) in the presence of both oligo (dT) and random primers. RT-PCR of HORMAD1 was performed with Platinum Taq High Fidelity (Invitrogen) and primers . Detail about primers can be found in DNA methylation of HORMAD1 was determined by bisulfite pyrosequencing. Bisulfite conversion of genomic DNA (1 ug each) of 45 Triple Negative tissues was performed using the EZ DNA methylation kit (Zymo D5001) according the manufacturer’s instructions. A CpG island around the transcription start site was amplified from bisulfite-converted DNA . The methylation status of 11 CpG sites within this CpG island were examined in a Qiagen pyrosequencing machine at MD Anderson DNA Methylation Analysis Core . The relative expression level of HORMAD1 in each of the tissues was then determined by qPCR . Detail of primers can be find in HORMAD1  was designed by Nanostring Technologies based on the gene sequence. Expression of this gene was quantified from 500 ng total RNA using the Nanostring Counter Assay System following the manufacturer’s instructions. Raw counts were normalized to the mRNA levels of the house-keeping genes TFRC, TBP, and PUM1.The code set (Version.2) for https://portal.gdc.cancer.gov/). In addition, we also analyzed the Molecular Taxonomy of Breast Cancer International Consortium  [https://genome-cancer.ucsc.edu/) [Affymetrix U133 plus 2.0 microarray data for 34 normal tissue types were obtained from the Human Body Index dataset HBI, GSE7307). Gene expression values were extracted with the MAS5 algorithm and were scaled to a reference sample using a house-keeping gene probe set provided by Affymetrix as previously described . RNA-seqT-12 V3) . HORMAD1T-12 V3) . MAD is sc.edu/) , 30. HOR7. Gene esc.edu/) . The TCGsc.edu/) .https://www.cancerrxgene.org/downloads). The included PARP inhibitors are Olaparib (Drug ID: 1017 & 1495), Veliparib (Drug ID: 1018), Rucaparib (Drug ID: 1175), and Talazoparib (Drug ID: 1259). Gene expression data of human cancer cell lines were downloaded from the GDSC web portal, from which HORMAD1 expression data were extracted. Cell lines with both gene expression data and drug sensitivity data were selected to study the relationship between HORMAD1 overexpression and sensitivity to PARP inhibitors. The threshold for HORMAD1 overexpression was calculated by median + MAD (default constant = 1.4826). MAD is calculated by mad  function in R with the default constant. For pan-cancer analysis, the threshold was set up based on all cell lines in the dataset. For the analysis of all breast cancer cell lines or basal-like breast cancer cell lines, the threshold was set up based on all breast cancer cell lines. After that, cell lines were divided into two groups based on the status of HORMAD1 expression (high or other). IC50 value was plotted against HORMAD1-expression status. T-test was adopted to evaluate the differences of IC50 values between two groups. P-value was calculated on log-transformed values. p < 0.05 was regarded as statistical significant. Boxplots represent 1st, 2nd, and 3rd quantiles.IC50 values of 1074 human pan-cancer cell lines against 265 compounds were obtained from Genomics of Drug Sensitivity in Cancer (GDSC)15 website (https://xenabrowser.net) [p-value.To assess the cell signaling alterations characteristics of HORMAD1 overexpression in BLBC tumors, we downloaded the RBN  normalized reverse phase protein array (RPPA) data for 747 TCGA breast cancer tumors from Xena Functional Genomics Explorer (ser.net) . Using tser.net)  to ident™ Lentiviral Support Kit (Invitrogen). Cells with high GFP reporter expression were selected using flow cytometry.Human HORMAD1 sequence verified cDNA was obtained from Dharmacon (MHS1010-9204021). The cDNAs for HORMAD1 and YFP control were then cloned into the pcDNA3 vector (Invitrogen). These lentiviral constructs verified by sequencing before being infected into selected cell lines using the ViraPower7 transduced HCC1954, HCC1806, or BT20 cell lines, with either HORMAD1 overexpression or YFP vectors, were re-suspended in 20% Matrigel (VWR) solution. Cells were injected bilaterally in 4–6-week-old female athymic nude mice (Harlan Sprague-Dawley). For in vivo therapeutic efficacy evaluation, the mice bearing engrafted tumor were randomized into four treatment arms: YFP/PARPi+, YFP/PARPi-, HORMAD1 /PARPi+, and HORMAD1/PARPi-. Rucaparib used in animal studies was obtained from Selleck Chemicals. 10 mg/kg Rucaparib or vehicle was administered to mice by oral gavage using a blunt needle. The growth of xenograft tumors was monitored daily and tumor volume was measured using the formula 1/2(length × width2). Mice were terminated and tumors were collected when tumors reach 1500 mm3 or at the designated endpoint of the experiment.All animal studies were performed in accordance with protocols approved by BCM Institutional Animal Care and Use Committee. 1 × 10Xenograft tumor tissues were extracted in RIPA lysis buffer with complete protease inhibitor cocktail supplements (Roche). Protein samples were separated in SDS-PAGE gel and then transferred to 0.2 uM nitrocellulose membrane. The primary HORMAD1 antibody (SIGMA HPA037850) was diluted by 1:1000 ratio in 5% BSA-PBST buffer for blotting. The primary GAPDH antibody was purchase from Santa Cruz (#4970) and works under 1:2000 dilution. The western blots were visualized in Bio-Rad ChemiDoc imaging system and processed by ImageJ software .in vitro experiment data were analyzed by unpaired Student’s t-tests, and all data are shown as mean ± standard deviation. Analysis of in vivo tumor growth curves was carried out using ANOVA. Kaplan–Meier survival analysis was carried out using the R survival package to estimate the function of Rucaparib on progression-free survival in BLBC with different HORMAD1 levels. Tumors were considered “progression-free” until the measured tumor volume was more than double the tumor volume at the beginning of the experiment, at which point the tumors were deemed to have “progressed.” Comparisons between survival curves were carried out by generalized Wilcoxon test.The


The dataset showed that the inhibitory activity of kaempferol was comparatively stronger than gallic acid. Thereby, kaempferol is offered as a potent anticancer agent for further investigation and beneficial as a dietary supplement. The data within this article relates to the research article entitled “Screening phytochemical content, antioxidant, antimicrobial and cytotoxic activities of Catharanthus roseus (L.) G. Don stem extract and its fractions” .This data article indicates the  Specifications tableValue of the data•The data reveal the inhibitory activity of kaempferol and gallic acid against various cancer cell lines  and 2.•in vitro anticancer activity than gallic acid, in particular against SMA (Glioblastoma murine), SJ-G2 (Glioblastoma) and A2780 (Ovarian) (It is clearly shown that kaempferol possessed greater Ovarian)  and 2.•50 values confirm the strong cytotoxicity of kaempferol  G. Don stem extract and its fractions” 50 values of kaempferol and gallic acid across a panel of cancer cell lines, which inhibit the cell growth by 50%, were also determined  in a 96-well plate at a density of 2500–4000 cells per well. When cells were at logarithmic growth after 24 h, they were treated with the working solutions of kaempferol and gallic acid to give a final concentration of 50–0.01 µM. After 72 h of incubation, the proportion of live cell growth was determined using the MTT -2,5-diphenyltetrazolium bromide) assay.2.2Kaempferol, gallic acid and dimethyl sulphoxide (DMSO) were purchased from Sigma-Aldrich Co. Human cancer cell lines were obtained from the American Type Culture Collection . 3--2,5-diphenyltetrazolium bromide (MTT) and Dulbecco׳s Modified Eagle׳s Medium (DMEM) were products of Gibco by Life Technologies .2.32 at 37 °C. All cancer cell lines were cultured in DMEM supplemented with 10% foetal bovine serum, 100 IU/mL penicillin, 100 µg/mL streptomycin and 2 mM l-glutamine. The non-tumour derived MCF10A cells were cultured in DMEM:F12 (1:1) cell culture media, 5% heat inactivated horse serum, supplemented with penicillin (100 IU/mL), streptomycin (100 µg/mL), 20 mM Hepes, l-glutamine (2 mM), epidermal growth factor (20 ng/mL), hydrocortisone (500 ng/mL), cholera toxin (100 ng/mL) and insulin (10 µg/mL). Cytotoxic activity of kaempferol and gallic acid on various cancer cell lines was assessed using MTT assay as described in a previous study All tested cancer lines and one non-tumour derived normal breast cell line (MCF10A) were maintained in a humidified atmosphere 5% CO


Listeria monocytogenes (Lmat-LLO) represents a valuable anticancer vaccine and drug delivery platform. Here we show that in vitro Lmat-LLO causes ROS production and, in turn, apoptotic killing of a wide variety of melanoma cells, irrespectively of their stage, mutational status, sensitivity to BRAF inhibitors or degree of stemness. We also show that, when administered in the therapeutic setting to Braf/Pten genetically engineered mice, Lmat-LLO causes a strong decrease in the size and volume of primary melanoma tumors, as well as a reduction of the metastatic burden. At the molecular level, we confirm that the anti-melanoma activity exerted in vivo by Lmat-LLO depends also on its ability to potentiate the immune response of the organism against the infected tumor. Our data pave the way to the preclinical testing of listeria-based immunotherapeutic strategies against metastatic melanoma, using a genetically engineered mouse rather than xenograft models.Attenuated  Melanoma is the most aggressive form of skin cancer and remains a therapeutic challenge, especially when it reaches the metastatic stage . Since oListeria monocytogenes (Lmat). Among the microorganisms exploited as anticancer vaccines, Lmat was chosen in light of the strong and multifaceted immune response that it triggers [Spurred by these successful applications of systemic immunotherapy, we have decided to assess the antitumoral effects elicited by an attenuated and non-pathogenic version of triggers , 7, of itriggers , as welltriggers .at-LLO strain [Here we show that the LmO strain  very effO strain , it greaat-LLO [at-LLO infection rate over time  attests that the biological effects observed with Lmat-LLO are consequences of the bacterial life cycle rather than of a general toxicity phenomenon.The ability of Lmat-LLO   and of different BRAF, NRAS, and NF1 mutational status  in the tumor area was followed by 14 intraperitoneal (IP) injections of a lower Lmat-LLO dose (105 or 106 CFU), one per day  was chosen on the basis of IC50 calculation and absence of toxic side effects . Consistently with the results obtained in vitro, the impairment in tumor growth caused by Lmat-LLO is associated with an increase in the number of apoptotic Cleaved Caspase 3-positive cells  [at-LLO potentiates the response of the immune system against the tumor and, therefore, that its antitumoral effects are at least in part non-cell autonomous [Lmors Fig. a–c, so tors Fig. , left. Clls Fig. . Howeverlls Fig. , right [. right) , as well. right) , 15, 17.tonomous .Fig. 2Lmat-LLO treatment to impair the metastatic burden. Indeed, the measurements obtained by ultrasound imaging indicate a decrease in the volume of regional LN ) are described in Supplementary Fig. The characteristics of the strains of at-LLO, the XFL-7 strain was electroporated with pGG-34 plasmid as reported in [To obtain Lmorted in .214-386 plasmid. Both strains were subsequently grown in BHI medium with 34 μg/ml chloramphenicol at 37 °C.Analogously, to obtain Lm(ct), the XFL-7 strain was electroporated with the pGG-34-OVAtm1Mmcm Ptentm1Hwu Tg(Tyr-cre/ERT2)13Bos/BosJ mice were purchased from the Jackson Laboratory (013590). For in vivo experiments, Tyr::CreER+ (heterozygous for CreER), BrafCA/+ (heterozygous for BrafV600E), and Ptenlox/lox (homozygous for Pten loss) mice were used, as described in Supplementary Fig. B6.Cg-Braft-test or Chi-square test, as needed. Values of p < 0.05 were considered statistically significant .Statistical analyses were performed using unpaired and two-tailed Student Supplementary Figures S1-18Supplementary MethodsSupplementary Table S1