scholarly journals CBMS-10 FUNCTIONAL ROLE OF MYCN IN SHH TYPE TP53 MUTATED MB’S METABOLISM

2019 ◽  
Vol 1 (Supplement_2) ◽  
pp. ii6-ii6
Author(s):  
Kiyotaka Yokogami ◽  
Takashi Watanabe ◽  
Shinji Yamashita ◽  
Asako Mizuguchi ◽  
Hideo Takeshima
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Functional Role ◽  
Ex Vivo ◽  
Therapeutic Option ◽  
In Vitro Models ◽  
Metabolic Phenotype ◽  
Matched Pair

Abstract BACKGROUND Medulloblastoma is classified in 4 subgroups. Prognosis and therapeutic option were different from each subgroups. Thus, we need subgroup-specific in vitro models for investigating new therapeutic targets. Little established medulloblastoma cell-lines, which have been subgrouped is available. Especially, commercially available SHH type TP53 mutated cell-line is only DAOY. We established new cell lines 505CSC / 507FBS from the patient with SHH type with TP53 mutated MB. This matched pair cell line showed high expression of MYCN in serum free conditioned medium. To know the functional role of N-MYC in MB, we used 507CSC and DAOY. MATERIAL AND METHODS Using chemical inhibitor of MYCN in 507CSC and DAOY, proliferation assay, mRNA expression and measurements of ex-vivo metabolic phenotype were performed. RESULTS MYCN inhibition leads to cell death in both cell lines. MYCN regulated glucose, glutamine and methionine metabolism. Especially the targets were PKM2, GLS2, MAT2A, DNMT1 and 3A. CONCLUSION MYCN is a target of therapy in a patient with SHH type TP53 mutated medulloblastoma.

scholarly journals P07.01 Functional role of N-Myc in SHH type TP53 mutated MB’s metabolism

2019 ◽  
Vol 21 (Supplement_3) ◽  
pp. iii36-iii36
Author(s):  
K Yokogami
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Functional Role ◽  
Ex Vivo ◽  
Therapeutic Option ◽  
In Vitro Models ◽  
Metabolic Phenotype ◽  
Matched Pair

Abstract BACKGROUND Medulloblastoma is classified in 4 subgroups. Prognosis and therapeutic option was different from each subgroups. Thus, we need subgroup-specific in vitro models for investigating new therapeutic targets. Little established medulloblastoma cell-lines, which have been subgrouped is available. Especially, commercially available SHH type TP53 mutated cell-line is only DAOY. We established new cell lines 505CSC / 507FBS from the patient with SHH type with TP53 mutated MB. This matched pair cell line showed high expression of N-MYC in serum free conditioned medium. To know the functional role of N-MYC in MB, we used 507CSC and DAOY. MATERIAL AND METHODS Using chemical inhibitor of N-Myc in 507CSC and DAOY, proliferation assay, mRNA expression and measurements of ex-vivo metabolic phenotype were performed. RESULTS N-MYC inhibition leads to cell death in both cell lines. N-MYC regulated glucose, glutamine and methionine metabolism. Especially the targets were PKM2, GLS2, MAT2A, DNMT1 and 3A. CONCLUSION N-MYC is a target of therapy in a patient with SHH type TP53 mutated medulloblastoma.

scholarly journals Sa1923 The Role of Histone Deacetylase Inhibition in Ex Vivo and In Vitro Models of Inflammatory Bowel Diseases

2012 ◽  
Vol 142 (5) ◽  
pp. S-360
Author(s):  
Alexander J. Edwards ◽  
Markus Gwiggner ◽  
Andrew Claridge ◽  
Rebecca Morgan-Walsh ◽  
Annette L. Hayden ◽  
...  
Keyword(s):  
Histone Deacetylase ◽  
Inflammatory Bowel Diseases ◽  
Ex Vivo ◽  
In Vitro Models ◽  
Histone Deacetylase Inhibition ◽  
Bowel Diseases ◽  
Inflammatory Bowel

scholarly journals Micro-RNA Profiling Reveals the Key Role of miR-206 in the Hematopoietic Potential of Human Embryonic and Induced Pluripotent Stem Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1275-1275
Author(s):  
Stephane Flamant ◽  
Jean-Claude Chomel ◽  
Christophe Desterke ◽  
Olivier Feraud ◽  
Emilie Gobbo ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Pluripotent Stem Cells ◽  
Target Genes ◽  
Hematopoietic Differentiation ◽  
Induced Pluripotent

Abstract Although human pluripotent stem cells (hPSCs) can theoretically be differentiated into any cell type, their ability to generate hematopoietic cells shows a major variability from one cell line to another. The reasons of this variable differentiation potential, which is constant and reproducible in a given hPSC line, are not clearly established. In order to study this phenomenon, we comparatively studied 4 human embryonic stem cell lines (hESC) and 11 human induced pluripotent stem cell (hiPSC) lines using transcriptome assays. These cell lines exhibited a significant variability to generate in vitro hematopoiesis as evaluated by day-16 embryoid body (EB) formation followed by clonogenic (CFC) assays. Four out of 11 iPSC lines (PB6, PB9, PB12.1, and PB14.3) were found to lack any hematopoietic differentiation ability whereas 7 cell lines showed variable hematopoietic potential. Among hESC lines, H9 and CL0 had low H1 and SA01 exhibited high hematopoietic potential using the above assays. Among hESC and hIPSC displaying hematopoietic potential, two sub-groups were further defined based on their hematopoietic CFC efficiency: a group of poor (generation of less than 100 CFC/105 cells, PB4 / PB10 /H9 /CL01), and high hematopoietic competency (more than 120 CFC/105 cells, PB3/ PB6.1 /PB7 /PB13 /PB17 /SA01/H1). Using global miRNome analysis performed at the pluripotency stage, the expression of 754 individual miRNAs was analyzed from 15 hPSC lines in order to explore a potential predictive marker between both sub-groups of pluripotent cells according to their hematopoietic potency. Using this approach, 27 miRNAs out of 754 appeared differentially expressed allowing the identification of a miRNA signature associated with hematopoietic-competency. The hematopoietic competency was associated with down-regulation of miR-206, miR-135b, miR-105, miR-492, miR-622 and upregulation of miR-520a, miR-296, miR-122, miR-515, miR-335. Amongst these, miR-206 harbored the most significant variation (0.04-Fold change). To explore the role of miRNA-206 in this phenomenon, we have generated a miR-206-eFGP-Puro lentiviral vector which was transfected in hESC line H1 followed by puromycin selection. As a control, H1 cell line was transfected with a Arabidopsis thaliana microRNA sequence (ath-miR-159a), which has no specific targets in mammalian cells. The correct expression of the transgenes were evaluated by flow cytometry (using GFP) and q-RT-PCR for miR-206 expression. The hematopoietic potential of H1 cell line and its miR-206-overexpressing counterpart was then tested using standard in vitro assays via d16-EB generation. We found that both CFC numbers and percentage of CD34+ were significantly lower in H1-mir-206-derived day-16 EB cells than in H1-ath- derived day-16 EB cells (p < 0.05). Thus, over-expression of miR-206 in this blood-competent hESC appeared to repress its hematopoietic potential at very early stage, since a similar lower CFC efficiency was observed in day-3 EB cells derived from miR-206 overexpressing H1 cell line. We then conducted an integrative bioinformatics analysis on miR-206 predicted target genes. To this end, 773 mRNA target transcripts of the broadly conserved (across vertebrates) miR-1-3p/206 family were identified in the TargetScan database and were integrated into the global transcriptomic analysis performed by microarray on day-16 EB cells. Using supervised ranking product analysis, 62 predicted gene targets of the miR-1-3p/206 family were found to be significantly up-regulated in hematopoietic-competent EB samples including the transcription factors RUNX1 and TAL1. Hierarchical unsupervised clustering, based on this subset of 62 predicted mir-206 target genes, fully discriminated hematopoietic-deficient from hematopoietic-competent cells. In conclusion, miRNA profiling performed at pluripotency stage could be useful to predict the ability to human iPSC to give rise to blood cell progenitors. This work emphasizes for the first time the critical role of the muscle-specific miR-206 in hematopoietic differentiation. Finally, these results suggest that genetic manipulation of hESC/iPSC could be used to enhance their hematopoietic potential and to design protocols for generation of hPSC-derived hematopoietic stem cells with long-term reconstitution ability. Disclosures No relevant conflicts of interest to declare.

scholarly journals The Pro-Tumorigenic Vascular Niche Sustains the T-Cell Acute Lymphoblastic Leukemia Phenotype and Fosters Resistance to Therapy

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 279-279
Author(s):  
Filomena Di Giacomo ◽  
Xujun Wang ◽  
Danilo Fiore ◽  
Lorena Consolino ◽  
Jude Phillip ◽  
...  
Keyword(s):  
High Risk ◽  
Cell Lines ◽  
Ex Vivo ◽  
Lymphoblastic Leukemia ◽  
Serum Cytokine ◽  
Nsg Mice ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Free Media

Abstract Introduction. T-cell acute lymphoblastic leukemia (T-ALL) is a genetically heterogeneous malignancy associated with a high risk of treatment failure. Efforts to improve outcomes have focused on underlying genetic defects. However, new evidence suggests that the microenvironment can foster drug resistance/relapses. Identification of factors that contribute to microenvironment-mediated chemo-refractoriness remains an important challenge. Here, we sought to construct an in vitro platform to dissect tumor-host interactions and to optimize drug treatments using Patient-Derived Tumor Xenograft models (PDTX) of high risk adult T-ALL and engineered human endothelial cells. Methods. T-ALL PDTX were established and serially passaged in NSG mice. Engraftment was monitored by flow cytometry of peripheral blood and/or MRI. Mice were sacrificed and leukemic cells were harvested from the spleen/bone marrow. To determine the ex vivo growing conditions, we first cultured a panel of 8 "bona fide" T-ALL cell lines and 11 PDTX cells alone in complete RPMI 20% FCS supplemented with IL2, IL12, IL15 and IL7; or co-cultured with human E4-ORF1 endothelial cells (ECs) without ILs in complete RPMI 20% FCS or serum/cytokine-free media. CDK4/6, MEK, PI3K and JAK inhibitors were used at 0.1 and 1 µM alone and in combination. Cell titer glo, cell titer blue, Annexin-V and S-cell cycle analysis were used as readouts. Total RNA from cells before and after co-culture was extracted for paired-end RNA sequencing on an Illumina HiSeq2500. Results. To study the supporting role of ECs, we first co-cultured ECs with T-ALL cell lines in vitro (serum/cytokine free co-culture) and showed that ECs could reproducibly sustain the viability of 3/8 cell lines (Loucy, KOPTK1, P12 Ichikawa) serum/cytokine-free media. A partial rescue was seen with 3 additional lines (HPB-ALL, CCRF-CEM, CUTLL1), while 2 (KE37, DND41) underwent massive cell death. We next tested whether either ILs or CXCL12 could provide anti-apoptotic signals and demonstrated that KOPTK1 and Loucy were only partially rescued by IL15 or CXCL12. Conversely, IL7, although capable of inducing a robust upregulation of pSTAT5, had no effect (CCRF-CEM and CUTLL1). We then characterized 11 PDTX from 15 high-risk adult T-ALL patients. All PDTX were serially propagated and caused T-ALL in subsequent NSG mice (massive spleen and bone marrow infiltration with extensive paravertebral mass associated with paralysis and multi-organ involvement). Genomic analysis (RNA-seq) demonstrated a high concordance between primary (pre-implant) and PDTX samples. All of them were extensively studied ex vivo, demonstratingthat T-ALL PDTX cells could only survive in ILs supplemented media, even better if enriched of growth factors and supplements for the expansion of human hematopoietic cells. However, when PDTX cells were treated with targeting compounds they all underwent massive apoptosis. Conversely, individual PDTX T-ALL could be selectively rescued by ECs, allowing the construction of individual drug response profile. To extend these data, 7 PDX T-ALL samples were screened against a 430-targeted compound library in supplemented RPMI or Stem Span media. Results indicated differential cell killing and gain (NFKB, BTK) and loss (TP-53, IGF-1R) of targets. Conclusions. These data clearly demonstrate a key role of aberrantly activated vascular niche in T-ALL cell maintenance and drug resistance. We envisage that drug screening of EC+T-ALL will lead to the identification of actionable targets in each individual patient. Our report supports the potential for future personalized curative strategies aimed at targeting both tumor cells and host tissue supporting niche elements disrupting pro-tumorigenic signals within leukemia cell niches. Disclosures Foà: Roche: Consultancy, Speakers Bureau; Genentech: Consultancy; Janssen: Consultancy, Speakers Bureau; Gilead: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; BMS: Consultancy; Pfizer: Speakers Bureau; Ariad: Speakers Bureau. Rafii:Angiocrine Bioscience: Equity Ownership, Other: Non-paid consultant.

scholarly journals Functional properties of edible insects: a systematic review

2021 ◽  
pp. 1-54
Author(s):  
V. D’Antonio ◽  
N. Battista ◽  
G. Sacchetti ◽  
C. Di Mattia ◽  
M. Serafini
Keyword(s):  
Glucose Metabolism ◽  
Functional Role ◽  
Dietary Intervention ◽  
Ex Vivo ◽  
Edible Insects ◽  
Cellular Models ◽  
Pubmed Database

Abstract Consumption of edible insects has been widely suggested as an environmentally sustainable substitute for meat to reduce GHG emissions. However, the novel research field for edible insects rely on the content of bioactive ingredients and on the ability to induce a functional effect in humans. The goal of this manuscript was to review the available body of evidence on the properties of edible insects in modulating oxidative and inflammatory stress, platelet aggregation, lipid and glucose metabolism and weight control. A search for literature investigating the functional role of edible insects was carried out in the PUBMED database using specific keywords. A total of 55 studies, meeting inclusion criteria after screening, were divided on the basis of the experimental approach: in vitro studies, cellular models/ex vivo studies or in vivo studies. In the majority of the studies, insects demonstrated the ability to reduce oxidative stress, modulate antioxidant status, restore the impaired activity of antioxidant enzymes and reduce markers of oxidative damage. Edible insects displayed anti-inflammatory activity reducing cytokines and modulating specific transcription factors. Results from animal studies suggest that edible insects can modulate lipid and glucose metabolism. The limited number of studies focused on the assessment of anticoagulation activity of edible insects make it difficult to draw conclusions. More evidence from dietary intervention studies in humans is needed to support the promising evidence from in vitro and animal models about the functional role of edible insects consumption.

scholarly journals MiR-624-5p enhances cell resistance against cisplatin via PDGFRA/Stat3/PI3K axis in ovarian cancer

2020 ◽  
Vol 19 (4) ◽  
pp. 691-698
Author(s):  
Lin I-Ju ◽  
Tian YongJie
Keyword(s):  
Ovarian Cancer ◽  
Cell Viability ◽  
Cell Line ◽  
Cell Lines ◽  
Clinical Stage ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Reporter Assay

Purpose: The purpose of this study was to evaluate the role of miR-624-5p in ovarian cancer.Methods: MiR-624-5p expression in ovarian cancer {OC) cell lines and normal cells (NCs) was evaluated and compared the differential miR-624-5p in OC A2780 cells and cisplatin-resistant OC cell line (A2780/DDP). CCK-8 was used to evaluate changes in cell viability of the A2780 and A2780/DDP cell lines as well as silenced miR-624-5p. Western Blot examined the Stat3 and phosphorylated Pi3k. The binding between PDGFRA and miR-624-5p was predicted on Targetscan and verified through Luciferase Reporter Assay. The role of PDGFRA in A2780/DDP by overexpressing PDGFRA was evaluated by RT-qPCR and CCK-8 assays. RT-qPCR assay also measured miR-624-5p expression responsive to different dosages of cisplatin and CCK8 examined viability levels correspondingly. In addition, the interplay of PDGFRA and miR-624-5p by combined downregulation of both miR-624-5pand PDGFRA were evaluated.Results: OC cells had higher miR-624-5p expression than NCs but lower compared to cisplatinresistant A2780/DDP cells. A2780/DDP cells had higher viability than OC cell line A2780. Stat3 and phosphorylated PI3K were activated in A2780/DDP cells. Silencing miR-624-5p led to lower viability inA2780/DDP cells. miR-624-5p expression dropped as the cisplatin concentration increased, resulting in decreasing viability respectively. Luciferase Reporter assay validated the binding of miR-624-5p and PDGFRA in A2780/DDP cells. Overexpressed PDGFRA induced lower cell viability in A2780/DDP cells. Downregulation of PDGFRA partially restored the lowered viability and inhibited Stat3 as well as phosphorylated Pi3k induced by miR-624-5p inhibitor.Conclusion: MiR-624-5p could add to the cellular resistance to cisplatin in OC in-vitro model, which indicated that it might help unveil the mystery of drug-resistance in clinical stage of ovarian cancer. Keywords: MiR-624-5p, resistance, cisplatin, PDGFRA/Stat3/PI3K, ovarian cancer

Organoids as ex vivo culture system to investigate infection-host interaction in gastric pre-carcinogenesis.

2022 ◽  
Vol 16 ◽  
Author(s):  
Cristina Di Giorgio ◽  
Rosalinda Roselli ◽  
Michele Biagioli ◽  
Silvia Marchianò ◽  
Eleonora Distrutti ◽  
...  
Keyword(s):  
Gastric Mucosa ◽  
Cell Lines ◽  
Ex Vivo ◽  
Three Dimensional ◽  
Mucosal Injury ◽  
In Vitro Models ◽  
World Population ◽  
Barr Virus

Abstract: Advancements in stem cell research have enabled the establishment of three-dimensional (3D) primary cell cultures, known as organoids. These culture systems follow the organization of an in vivo organ, as they enclose the different epithelial cell lines of which it is normally composed. Generation of these 3D cultures has bridged the gap between in vitro models, made up by two-dimensional (2D) cancer cell lines cultures, and in vivo animal models, that have major differences with human diseases. Organoids are increasingly used as a model to study colonization of gastric mucosa by infectious agents and to better understand host-microbe interactions and the molecular events that lead to infection, pathogen-epithelial cells interactions and mechanisms of gastric mucosal injury. In this review we will focus on the role of organoids as a tool to investigate molecular interactions of Helicobacter (H.) pylori and Epstein Barr Virus (EBV) and gastric mucosa and how these infections, that affect ≈ 45% of the world population, might progress to gastric cancer, a highly prevalent cancer and the third leading cause of cancer death.

Bcr-Abl Directly Activates Stat5 Independent of Jak2

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 511-511
Author(s):  
Oliver D. Hantschel ◽  
Eva Eckelhart ◽  
Ines Kaupe ◽  
Florian Grebien ◽  
Kay-Uwe Wagner ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Cell Lines ◽  
Tyrosine Kinases ◽  
Functional Role ◽  
Kinase Inhibitors ◽  
Knock Out ◽  
Kinase Substrate ◽  
Knock Out Mice

Abstract Abstract 511 Persistent activation of the transcription factor Stat5 is a signaling hallmark of Chronic Myelogenous Leukemia (CML). In mouse models, Stat5 was required for initial myeloid and lymphoid transformation (by Bcr-Abl p210 or p185 and v-Abl). Most importantly, we and others recently showed that Stat5 was also required for maintenance of Bcr-Abl-dependent leukemia in vivo and for engraftment and reconstitution of Bcr-Abl p210-positive leukemia in secondary recipients. Therefore, Stat5 is of central functional importance in the Bcr-Abl signaling network and represents a possible critical vulnerable node in CML. In contrast to the well-studied functional role of Stat5 in disease models, the molecular mechanism of Bcr-Abl dependent Stat5 activation, including the tyrosine kinase(s) that phosphorylate and activate Stat5, remain only partially understood. In particular, conflicting views on the involvement of the Jak2 kinase and its potential role as a drug target in CML exist. We used conditional Jak2 knock-out mice to study the contribution of Jak2 in Bcr-Abl-dependent transformation and leukemogenicity. Jak2 ablation did not compromise the Bcr-Abl p210-mediated transforming capability in primary murine bone marrow- or fetal liver-derived hematopoietic cells in vitro. In contrast, initial lymphoid transformation by v-abl and Bcr-Abl p185 was abolished in Jak2 knock-out mice. Jak2 deletion did not have an effect on maintenance of lymphoid leukemia cells in vitro, whereas deletion of Stat5 induced a G1 arrest and subsequent apoptosis. In line with this, ablation of Jak2 expression after leukemia induction did not alter disease latency or disease phenotype. Consistently, we did not observe a decrease in Stat5 activation upon siRNA-mediated knock-down of Jak2 alone or all four Jak kinases (Jak1, Jak2, Jak3 and Tyk2) in CML cell lines. Using a panel of pharmacological inhibitors, we found that neither Jak2-selective, nor pan-Jak kinase inhibitors or Src family kinase-selective inhibitors led to a decrease in Stat5 phosphorylation, while the highly selective Bcr-Abl inhibitor nilotinib completely abrogated Stat5 phosphorylation. To study possible contributions of other tyrosine kinases in the Bcr-Abl dependent activation of Stat5, we used Ba/F3 cells expressing the TKI-resistant Bcr-Abl mutant T315I in combination with different broad-specificity tyrosine kinase inhibitors, like dasatinib. At dasatinib concentrations that inhibited several dozens of tyrosine kinases, Stat5 phosphorylation in Ba/F3 Bcr-Abl T315I cells was unaffected, excluding a role for most tyrosine kinases other than Bcr-Abl in Stat5 activation and pointing towards a direct phosphorylation of Stat5 by Bcr-Abl. Together, this data excludes a role of Jak and Src kinases in the activation of Stat5 in Bcr-Abl positive cell lines. Finally, in comprehensive enzyme kinetic analysis experiments using recombinant kinase, Stat5 had a similar KM value for Bcr-Abl as the canonical direct Bcr-Abl substrate CrkL and displayed only mildly lower kinase substrate parameters (vmax, kcat) than CrkL, fully compatible with direct phosphorylation of Stat5 by Bcr-Abl. Together with our earlier data on the pivotal role of Stat5 in the transcriptional and signaling network of Bcr-Abl, we propose a hypersensitive switch-like behavior of the Bcr-Abl-Stat5 kinase substrate pair that mechanistically rationalizes the central functional role of Stat5 in the signaling of CML cells. In summary, we provide compelling evidence that activation of Stat5 by Bcr-Abl is likely to be direct and that targeting of Jak2 in CML may not be of therapeutic benefit, as Jak2 is not required for CML initiation or maintenance. Disclosures: Hantschel: Novartis: Honoraria; Bristol-Myers Squibb: Honoraria.

scholarly journals Cflar Splicing SNP As Predictor of Chemo-Sensitivity to Anticancer Drugs

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2056-2056
Author(s):  
Lata Chauhan ◽  
Emilie J Bergsma ◽  
Jatinder K Lamba
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Anticancer Drugs ◽  
Cancer Cell ◽  
Cancer Cell Line ◽  
Chemotherapeutic Agents ◽  
Extrinsic Pathway ◽  
Wide Range

Abstract Background: Anticancer therapeutics leverages activation of apoptosis signal transduction pathways (extrinsic and intrinsic apoptotic pathways) in cancer cells. Apoptosis induced by the extrinsic pathway complements that induced by the intrinsic pathway, so targeting extrinsic pathway is considered a useful new therapeutic approach. Preclinical data suggests TNF related apoptosis inducing ligand (TRAIL) as a promising approach as apoptosis of tumor cells is achievable in vivo without lethal toxicities. CASP8 and FADD-like apoptosis regulator (CFLAR) is an inhibitor of death receptor signaling that inhibits TRAIL-mediated caspase 8 auto-activation and subsequent apoptosis. We recently identified a splicing single nucleotide polymorphism (SNP) rs10190751 G>A in CFLAR, where presence of the variant allele (A) was associated with alternate splicing as well as with chemo-sensitivity to chemotherapeutic agent triptolide. However role of CFLAR and the splicing SNP on chemo-sensitivity to wide array of anticancer drugs is not known. Objective: Given the central role of CFLAR in apoptotic pathway, the goal of this study was to investigate impact of CFLAR and its splicing SNP on cytotoxicity of wide range of chemotherapeutic drugs including the ones extensively used in hematological malignancies. Methods: We selected chemotherapeutic agents with wide range of mechanisms of action as blocking DNA biosynthesis, interfering with structure or function of DNA or protein synthesis, interfering with DNA transcription or replication as well as drugs that are cell cycle specific or not. We selected nine Epstein-Barr-virus transformed lymphoblastoid cell lines (LCLs) that are part of International HapMap project representing different genotype for rs10190751 (CFLAR splicing polymorphism; 3 in each genotype category) with twelve different chemotherapeutic agents. Further validation of CFLAR's role in in vitro chemosensitivity was evaluated using CFLAR knockdown and overexpression studies in pancreatic and leukemic cell lines such as Panc-1 and THP1. Results: CFLAR splicing SNP rs10190751, was associated with in vitro cytotoxicity of several chemotherapeutic agents (Bortezomib, SAHA, doxorubicin, sorafenib). The results of screening of 122 FDA approved drugs and their relation with CFLAR as well as its splicing SNP will be presented at the annual meeting. As an example we show below that knock down of CFLAR isoforms have a significant impact on in vitro chemosensitivity to bortezomib and SAHA (Figure 1) Conclusion: Our results suggest critical role of CFLAR in anticancer drug mediated cell death. Additionally splicing SNP in CFLAR seems to play an important role in drug sensitivity/resistance. Therapeutic strategies to directly or indirectly inhibit the expression and/or function of CFLAR might be an attractive option to overcome resistance to wide range of chemotherapeutic agents. Figure 1. Impact of siRNA mediated knockdown or of CFLAR on Bortezomib and SAHA sensitivity in THP1 and Panc-1 cancer cell line. Figure 1. Impact of siRNA mediated knockdown or of CFLAR on Bortezomib and SAHA sensitivity in THP1 and Panc-1 cancer cell line. Disclosures No relevant conflicts of interest to declare.

scholarly journals Genomic and Transcriptomic Characterization of Canine Osteosarcoma Cell Lines: A Valuable Resource in Translational Medicine

2021 ◽  
Vol 8 ◽  
Author(s):  
Cecilia Gola ◽  
Diana Giannuzzi ◽  
Andrea Rinaldi ◽  
Selina Iussich ◽  
Paola Modesto ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Driver Mutations ◽  
Matched Pair ◽  
Osteosarcoma Cell ◽  
Driver Genes ◽  
Primary Bone Tumor ◽  
Wall Cell

Osteosarcoma (OSA) represents the most common primary bone tumor in dogs and is characterized by a highly aggressive behavior. Cell lines represent one of the most suitable and reproducible pre-clinical models, and therefore the knowledge of their molecular landscape is mandatory to investigate oncogenic mechanisms and drug response. The present study aims at determining variants, putative driver genes, and gene expression aberrations by integrating whole-exome and RNA sequencing. For this purpose, eight canine OSA cell lines and one matched pair of primary tumor and normal tissue were analyzed. Overall, cell lines revealed a mean tumor mutational burden of 9.6 mutations/Mb (range 3.9–16.8). Several known oncogenes and tumor suppressor genes, such as ALK, MYC, and MET, were prioritized as having a likely role in canine OSA. Mutations in eight genes, previously described as human OSA drivers and including TP53, PTCH1, MED12, and PI3KCA, were retrieved in our cell lines. When variants were cross-referenced with human OSA driver mutations, the E273K mutation of TP53 was identified in the Wall cell line and tumor sample. The transcriptome profiling detected two possible p53 inactivation mechanisms in the Wall cell line on the one hand, and in D17 and D22 on the other. Moreover, MET overexpression, potentially leading to MAPK/ERK pathway activation, was observed in D17 and D22 cell lines. In conclusion, our data provide the molecular characterization of a large number of canine OSA cell lines, allowing future investigations on potential therapeutic targets and associated biomarkers. Notably, the Wall cell line represents a valuable model to empower prospective in vitro studies both in human and in dogs, since the TP53 driver mutation was maintained during cell line establishment and was widely reported as a mutation hotspot in several human cancers.

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