Articles de revues : « SET-2 cell lines »

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Santamaria, P., T. Utsugi, B. J. Park, N. Averill, S. Kawazu et J. W. Yoon. « Beta-cell-cytotoxic CD8+ T cells from nonobese diabetic mice use highly homologous T cell receptor alpha-chain CDR3 sequences. » Journal of Immunology 154, n^o 5 (1 mars 1995) : 2494–503. http://dx.doi.org/10.4049/jimmunol.154.5.2494.

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Abstract Insulin-dependent diabetes mellitus (IDDM) in nonobese diabetic (NOD) mice results from a cell-mediated autoimmune process against pancreatic beta-cells. We have shown that beta-cell-cytotoxic CD8+ T cell clones can transfer IDDM to irradiated NOD mice if co-injected with nondiabetogenic CD4+ spleen T cells. To determine whether CTLs recruited to pancreatic islets recognize a restricted set of local Ags, we sequenced TCR-alpha and TCR-beta cDNA generated by anchor PCR from CD8+ CTL lines and clones derived from islets of 10 different NOD mice. These CTL lines were oligoclonal, but did not show skewed V alpha, V beta, J alpha, or J beta gene usage when compared with CD8+ spleen T cells. However, of the 26 different CTL-derived TCR-alpha sequences from all of these CTL lines and clones, 17 (65%) used one of three highly related, N region-encoded, CDR3 motifs. Motifs 1 and 2 (7 clonotypes each) contained a hydrophobic amino acid followed by Arg and a negatively charged or a polar residue (Asn or Gly), respectively. Motif 3 (3 clonotypes) was x-Arg-Gly. In 12 of these 17 rearrangements, the core sequence was followed by Tyr or Ser. By contrast, none of 31 different TCR-alpha rearrangements used by CD8+ spleen T cells encoded motifs 1 or 2, and only one encoded motif 3. Different TCR-beta rearrangements within individual lines also used homologous CDR3 sequences, but these sequences varied between lines. Skewed TCR-alpha-CDR3 usage by islet-derived CTLs was substantiated further by isolation of CTL clones transcribing highly homologous TCR-alpha, but different TCR-beta, rearrangements. These data suggest that CTLs recruited to pancreatic islets during spontaneous IDDM recognize a restricted set of beta-cell autoantigenic determinants.

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Quentmeier, Hilmar, Claudia Pommerenke et Hans G. Drexler. « Epigenetic Modifier Mutations in the LL-100 Panel ». Blood 132, Supplement 1 (29 novembre 2018) : 5271. http://dx.doi.org/10.1182/blood-2018-99-110060.

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Abstract The NCI-60 human cell line panel, developed for use in drug development comprises sixty human cancer cell lines derived from nine different tissues. Only six cell lines of the NCI-60 were derived from blood cancers. Therefore, most forms and subtypes of leukemia and lymphoma are not represented in the NCI-60 panel. To respond to this apparent gap, we suggest the novel LL-100 panel, 100 leukemia and lymphoma cell lines representing the major leukemia/lymphoma entities, for basic research and drug development. Whole exome sequencing and RNA sequencing were performed to identify mutations in 100 cell lines. Here we list the 100 cell lines, ordered by subtype and show mutations in epigenetic modifier genes. We found cell lines with mutations in ASXL1, EZH2, IDH1, TET2 and in DNMT3A. Hitherto, cell line OCI-AML3 was the only human cell line described with a DNMT3A mutation. Twenty-two percent of patients with acute myeloid leukemia contain DNMT3A mutations and the median overall survival with DNMT3A mutations is shorter than without. Most DNMT3A mutations are heterozygous and alter amino acid R882, R882H being the most common DNMT3A mutation in AML. Exogenously mutant murine R878H (equivalent to human R882H) inhibits DNMT3A activity in a dominant negative manner. We describe here that the AML cell line SET-2 carries a heterozygous G to A transition at chr2_25234373 (hg38) which leads to the DNMT3A R882H amino acid substitution. Chip-based methylation analysis revealed that the described DNMT3A targets IRF8, KLF2, HOXA11 and HOXB2 are hypomethylated in cell lines OCI-AML3 (DNMT3A R882C) and in SET-2 (DNMT3A R882H). These data suggest that SET-2 is a novel model cell line for functional analysis of the DNMT3A R882 mutation and a first gain in knowledge through data mining the LL-100 panel. Disclosures No relevant conflicts of interest to declare.

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Moudgil, Tarsem, Bernard Fox et Hong-Ming Hu. « 85 Detection of human angiotensin-converting enzyme 2 receptor (hACE2R) on human cancer cell lines ». Journal for ImmunoTherapy of Cancer 9, Suppl 2 (novembre 2021) : A93. http://dx.doi.org/10.1136/jitc-2021-sitc2021.085.

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BackgroundSARS-CoV-2 infections have delayed administration of treatments for some patients with cancer, increasing the number of avoidable deaths. However, we hypothesized that infection of cancer cells with SARS-CoV-2 might increase the immunogenicity of those cancer cells. Here we sought to determine whether non-small cell lung cancer (NSCLC) and head and neck squamous cell cancer (HNSCC) cell lines could be a potential target of SARS-CoV-2, which binds and infects host cells via interactions between the viral spike glycoprotein and the human angiotensin-converting enzyme 2 receptor (hACE2) receptor. Through an institutional research tissue protocol, our lab has established a panel of cancer cell lines of various histologies. Here we set out to identify whether HNSCC and NSCLC cell lines expressed the hACE2R. We also investigated the expression of neuropilin-1, a molecule reported to facilitate SARS-CoV-2 cell entry.MethodsEstablished cell lines were phenotyped by flow cytometric analysis utilizing the anti-hACE2R antibody from Novus Biologicals. Cell lines were also stained with mIgG1 and anti-CD3 antibodies as negative staining controls.ResultsWe identified that three of eight NSCLC cell lines expressed the hACE2R and two of these had strong expression of neuropilin-1. Evaluation of HNSCC cell lines identified seven of seven cell lines expressed detectable levels of hACE2R but only one of seven HNSCC celllines expressed substantial levels of neuropilin-1. Preliminary evaluation of a renal cell carcinoma (RCC) cell line revealed strong staining for hACE2R.ConclusionsOur study found that a majority of HNSCC cell lines (100% n=7) and approximately a third of the NSCLC cell lines (37.5%, n=8) tested express the hACE2R. Some cell lines express both hACE2R and neuropilin-1, potentially increasing their susceptibility for infection with SARS-C0V-2. While these studies were performed with cultured cell lines that may have modulated expression of hACE2R, it is possible that in vivo these tumors express the ACE2R and could serve as a target and possible reservoir for SARS-CoV-2.AcknowledgementsSupportThe Chiles foundation, Nancy Lematta

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Nagai, T., H. Harigae, H. Ishihara, H. Motohashi, N. Minegishi, S. Tsuchiya, N. Hayashi, L. Gu, B. Andres et JD Engel. « Transcription factor GATA-2 is expressed in erythroid, early myeloid, and CD34+ human leukemia-derived cell lines ». Blood 84, n^o 4 (15 août 1994) : 1074–84. http://dx.doi.org/10.1182/blood.v84.4.1074.1074.

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Abstract To understand the functional roles that the GATA factors may play during hematopoietic cell differentiation, we examined the expression of GATA factor mRNAs and protein products in various human cell lines. Blot hybridization analyses demonstrated that GATA-1 and GATA-2 mRNAs are expressed abundantly in a set of cell lines established from human myelogenous leukemia cells, but the expression pattern of each factor is distinct. GATA-2 mRNA is expressed in all cell lines tested that express erythroid markers, and, in addition, the mRNA is also expressed in three CD34+ cell lines and two early myeloid cell lines. In contrast, the expression of GATA-1 mRNA showed tight correlation to that of the erythroid/megakaryocytic lineage markers. We also found that the GATA-2 probe identifies two types of mRNA. Structural analysis of genomic DNA clones encoding human GATA-2 coupled with RNA blot analysis demonstrated that there exists an alternative use of polyadenylation consensus sequences in a single exon and this causes the molecular heterogeneity among GATA-2 mRNAs. Through immunochemical and immunohistochemical analyses using anti-GATA-1- and anti-GATA-2- specific antibodies, GATA-2 protein was clearly shown to be present in the nuclei of leukemia-derived early myeloid and CD34+ cell lines, whereas both GATA-1 and GATA-2 proteins are expressed in erythroid/megakaryocytic cell lines. Thus, the expression profile of GATA-2 is consistent with the hypothesis that GATA-2 plays unique roles for the transcriptional activation of genes in cells at an early stage of hematopoietic differentiation and in developing cells of the erythroid and myeloid lineages.

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Nagai, T., H. Harigae, H. Ishihara, H. Motohashi, N. Minegishi, S. Tsuchiya, N. Hayashi, L. Gu, B. Andres et JD Engel. « Transcription factor GATA-2 is expressed in erythroid, early myeloid, and CD34+ human leukemia-derived cell lines ». Blood 84, n^o 4 (15 août 1994) : 1074–84. http://dx.doi.org/10.1182/blood.v84.4.1074.bloodjournal8441074.

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To understand the functional roles that the GATA factors may play during hematopoietic cell differentiation, we examined the expression of GATA factor mRNAs and protein products in various human cell lines. Blot hybridization analyses demonstrated that GATA-1 and GATA-2 mRNAs are expressed abundantly in a set of cell lines established from human myelogenous leukemia cells, but the expression pattern of each factor is distinct. GATA-2 mRNA is expressed in all cell lines tested that express erythroid markers, and, in addition, the mRNA is also expressed in three CD34+ cell lines and two early myeloid cell lines. In contrast, the expression of GATA-1 mRNA showed tight correlation to that of the erythroid/megakaryocytic lineage markers. We also found that the GATA-2 probe identifies two types of mRNA. Structural analysis of genomic DNA clones encoding human GATA-2 coupled with RNA blot analysis demonstrated that there exists an alternative use of polyadenylation consensus sequences in a single exon and this causes the molecular heterogeneity among GATA-2 mRNAs. Through immunochemical and immunohistochemical analyses using anti-GATA-1- and anti-GATA-2- specific antibodies, GATA-2 protein was clearly shown to be present in the nuclei of leukemia-derived early myeloid and CD34+ cell lines, whereas both GATA-1 and GATA-2 proteins are expressed in erythroid/megakaryocytic cell lines. Thus, the expression profile of GATA-2 is consistent with the hypothesis that GATA-2 plays unique roles for the transcriptional activation of genes in cells at an early stage of hematopoietic differentiation and in developing cells of the erythroid and myeloid lineages.

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Resar, Linda, Donna Marie Williams, Lingling Xian, Wenyan Lu, Briyana Chisholm, Li Luo, Zhizhuang Joe Zhao, Ophelia Rogers, Jerry L. Spivak et Alison R. Moliterno. « High Mobility Group A1 Chromatin Remodeling Proteins Amplify Inflammatory Networks to Drive Leukemic Transformation in Chronic Myeloproliferative Neoplasia in Humans and JAK2V617F Transgenic Mouse Models ». Blood 132, Supplement 1 (29 novembre 2018) : 102. http://dx.doi.org/10.1182/blood-2018-99-119549.

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Abstract Introduction: Myeloproliferative neoplasms (MPN) are clonal hematopoietic stem cell (HSC) disorders characterized by overproduction of mature blood cells and increased risk of transformation to myelofibrosis (MF) and acute myeloid leukemia (AML), although molecular mechanisms driving disease progression remain elusive. While most patients who acquire a JAK2V617F mutation in CD34+ cells present with chronic, indolent Polycythemia Vera (PV), ~25% will progress to MF or AML. High Mobility Group A1/2 (HMGA1/2) genes encode oncogenic chromatin remodeling proteins which are overexpressed in aggressive leukemia where they portend adverse outcomes. In murine models, Hmga1/2 overexpression drives clonal expansion and uncontrolled proliferation. HMGA1/2 genes are also overexpressed in MPN with disease progression. We therefore sought to: 1) test the hypothesis that HMGA proteins are required for leukemic transformation and rational therapeutic targets in MPN progression, and, 2) identify mechanisms mediated by HMGA1/2 during disease progression. Methods: We measured HMGA1/2 in JAK2V617F mutant human AML cell lines from MPN patients (DAMI, SET-2), CD34+ cells from PV patients during chronic and transformation phases, and JAK2V617F transgenic murine models of PV (transgenic JAK2V617F) and PV-AML (transgenic JAK2V617F/MPLSV; Blood 2015;126:484). To elucidate HMGA1/2 function, we silenced HMGA1 or HMGA2 via short hairpin RNA in human MPN-AML cell lines (DAMI, SET-2) and assessed proliferation, colony formation, and leukemic engraftment in immunodeficient mice. To further assess Hmga1 function in vivo, we crossed mice with heterozygous Hmga1 deficiency onto murine models of PV and PV-AML. Finally, to dissect molecular mechanisms underlying HMGA1, we compared RNA-Seq from MPN-AML cell lines (DAMI, SET-2) after silencing HMGA1/2 to that of controls and applied Ingenuity Pathway Analysis. Results: HMGA1/2 mRNA are up-regulated in all JAK2V617F-positive contexts, including primary human PV CD34+ cells and total bone marrow from JAK2V617F mouse models for PV compared to controls. Further, there is a marked up-regulation in both HMGA1/2 in CD34+ cells from PV patients after transformation to MF or AML and in leukemic blasts from our PV-AML mouse model compared to PV mice. Overexpression of HMGA1/2 also correlates with clonal dominance of human JAK2V617F-homozygous stem cells and additional mutations of epigenetic regulators (EZH2, SETBP1). Silencing HMGA1 or HMGA2 in human MPN-AML cell lines (DAMI, SET-2) dramatically halts proliferation, disrupts clonogenicity, and prevents leukemic engraftment in mice. Further, heterozygous Hmga1 deficiency decreases splenic enlargement in PV mouse models with advancing age. Moreover, heterozygous Hmga1 deficiency prolongs survival in the transgenic PV-AML murine model with fulminant leukemia and early mortality. PV-AML mice survived a median of 5 weeks whereas PV-AML mice with heterozygous Hmga1 deficiency survive a median of 12 weeks (P< 0.002). The leukemic burden was also decreased in mice with Hmga1 deficiency. Preliminary RNA-Seq analyses from DAMI and SET-2 cells show that HMGA1 drives pathways involved in Th1/Th2 activation, chemotaxis, cell-cell signaling, myeloid cell accumulation and other immune cell trafficking, inflammation, and injury, suggesting that HMGA1 co-opts immune and inflammatory networks to drive tumor progression. Surprisingly, atherosclerosis pathways are also induced by HMGA1. Conclusions: HMGA1/2 genes are overexpressed in MPN with highest levels in more advanced disease (MF, AML) both in primary human tumors and murine models. Strikingly, silencing HMGA1 or HMGA2 halts proliferation and clonogenicity in vitro and prevents leukemic engraftment in vivo. Further, heterozygous Hmga1 deficiency prolongs survival in a murine model of fulminant MPN AML and decreases tumor burdens. Finally, preliminary RNA-Seq analyses suggest that HMGA1 amplifies transcriptional networks involved in immune cell trafficking and inflammation to drive tumor progression. Unexpectedly, HMGA1 also regulates pathways involved in atherosclerosis, implicating HMGA1 as a novel link between clonal hematopoiesis and cardiovascular disease. Our findings further highlight HMGA1/2 as a key molecular switch for leukemic transformation in MPN and opens the door to novel therapeutic approaches to prevent disease progression. Disclosures No relevant conflicts of interest to declare.

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Lindhout, E., A. Lakeman, M. L. Mevissen et C. de Groot. « Functionally active Epstein-Barr virus-transformed follicular dendritic cell-like cell lines. » Journal of Experimental Medicine 179, n^o 4 (1 avril 1994) : 1173–84. http://dx.doi.org/10.1084/jem.179.4.1173.

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Follicular dendritic cells (FDC) are unique nonlymphoid cells found only in germinal centers. FDC can be distinguished from other accessory cells based on a characteristic set of cell surface markers. It is known that FDC are able to rescue germinal center B cells from apoptosis. To investigate the role of FDC in the process of selection and maturation of B cells during germinal center reactions, we tried to establish factor-independent immortalized FDC-like cell lines. Because freshly isolated FDC express the Epstein-Barr Virus (EBV) receptor CD21, we attempted EBV transformation on isolated FDC. After incubation of FDC-enriched cell populations with EBV, cell lines were obtained consisting of slowly duplicating very large cells. These cell lines have a fibroblast-like morphology but could be clearly distinguished from several human fibroblast cell lines by displaying a different phenotype including intercellular adhesion molecule 1, CD40, and CD75 expression. Detection of the EBV-encoded proteins latent membrane protein 1 and Epstein-Barr virus nuclear antigen 2 in our FDC-like cell lines implicated successful EBV transformation. FDC-like cells are able to bind nonautologous B cells and preserve the latter from apoptosis. The binding of B cells to FDC-like cells is dependent on adhesion via lymphocyte function-associated antigen 1/intercellular adhesion molecule 1 and closely resembles the pattern of emperipolesis as described by others. These data demonstrate that FDC can be successfully infected by EBV, and that the cell lines obtained share phenotypic and functional characteristics with freshly isolated FDC.

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Troeger, Anja, Pascal-David Johann, Mumine Senturk, Michael D. Milsom et David A. Williams. « Intact Rac Signaling Is Important for Leukemia Cell Survival ». Blood 116, n^o 21 (19 novembre 2010) : 2885. http://dx.doi.org/10.1182/blood.v116.21.2885.2885.

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Abstract Abstract 2885 Rho GTPases, Ras-related small G proteins, regulate multiple cell processes in hematopoietic cells. There is growing evidence that acute myeloid leukemia (AML) blasts and particularly MLL-rearranged AML blasts, rely on Rac activity (Mulloy JC et al, Blood, 2010). However, little is known about the role of these GTPases in acute lymphoblastic leukemia (ALL) and particularly precursor B cell ALL. To investigate the role of Rac and potential compensation by other GTPases in ALL, we first assessed the protein expression and activation of Rac in a number of B-ALL cell lines (SEM; RS4,11; REH; Nalm 6; Raji), compared with a T-ALL cell line (Jurkat) and several AML cell lines (ML2; MV4,11). Of these cell lines SEM; RS4,11; ML2 and MV4,11 are characterized by MLL-fusion genes. Jurkat and MLL-rearranged AML cell lines show higher expression of Rac proteins compared to B cell leukemia lines (Table 1). Overall, B-ALL cell lines exhibit highly variable levels of Rac expression and activity with no obvious correlation to the presence of MLL-fusion proteins. We then investigated proliferation and apoptosis in cell lines treated with the small molecule inhibitor NSC23766 (NSC), which blocks interaction of a subset of guanine exchange factors (GEFs) with Rac and thus inhibits its activation. Treatment with NSC led to ∼2-fold increase in cells arrested at G0/G1 and induced apoptosis in a dose-dependent fashion at NSC concentrations previously demonstrated to be non-toxic in normal hematopoietic cells (Muller LUW et al., Leukemia, 2008) (Table 2). The lymphoid cell lines Jurkat, Raji and SEM appeared less responsive to NSC with no increased apoptosis at 40μM NSC. There was no correlation between NSC response and baseline expression or activation status of Rac. However, cell lines resistant to NSC exhibited a paradoxical and transient early increase in Rac activation, suggesting the existence of compensatory activation mechanisms. To determine if the relative resistance observed in some cell lines was related to dependence on GEFs not targeted by NSC and to validate that the inhibitory effect of NSC was specifically due to Rac inhibition in sensitive cells, shRNAs were utilized to knock-down different members of the Rac subfamily. Effective shRNA-mediated knockdown was validated by western blot. Knockdown of Rac1 or Rac2 consistently induced apoptosis compared to non-targeting vector controls in NSC sensitive cell lines ML2 and Nalm6, with ML2 cells appearing slightly more sensitive to knock-down of Rac2 (Table 3). Knock-down of either Rac1 or Rac2 had little effect upon Jurkat cells which are resistant to NSC treatment. These data suggest that Jurkat cells are not dependent upon Rac signaling for survival; however we cannot discount the possibility that some compensation may occur between Rac1 and Rac2. These experiments demonstrate the importance of intact Rac signaling pathways for the survival of the majority of leukemia cell lines tested and demonstrate that dependence on Rac signaling is not restricted to leukemias characterized by MLL-rearrangements. Our observations also suggest that activation of different Rac isoforms may influence sensitivity towards pharmacological Rac inhibition. Table 1: Baseline Expression of Rac assessed by Western blot Cell line Jurkat ML-2 MV-4,11 RS-4,11 SEM Nalm 6 REH Raji Rac/b-actin expression* 1.6 2.5 1.7 0.5 0.7 0.8 1.0 1.0 (*arbitrary units, italics indicate cell lines carrying MLL-rearrangements) Table 2: % AnnexinV+ cells after treatment of the different cell lines with increasing doses the Rac-specific inhibitor NSC Cell line Jurkat ML-2 MV-4,11 RS-4,11 SEM Nalm 6 REH Raji control 6%+1.4 6%+1.3 9%+0.3 12%+3.6 9%+1.9 7%+1.5 9%+2 13%+2.3 20uM NSC 6%+1.4 9%+1.3 15%+0.3** 21%+8.5 8%+1.5 6%+1.9 25%+6.4 16%+3 40uM NSC 7%+1.8 24%+9.1 60%+4** 52%+11* 10%+1.3 10%+3.4 39%+11 16%+1.9 80uM NSC 15%+3.5* 73%+14.7** 97%+0.4** 80%+4** 17%+1.2* 46%+10.5** 62%+12.3* 22%+4 (Mean±SEM; n=5; * p<0.05; ** p<=0.01 versus control, bolded columns indicate increased NSC sensitivity) Table 3: % AnnexinV+ cells 7 days after lentiviral transduction of the different cell lines with Rac1 and Rac2-specific shRNA Cell line Jurkat ML-2 Nalm 6 non targeting control 4.3%+0.3 14.2%+8 11.4%+2.2 Rac1 shRNA* 8.0%+3.5 26.3%+7.9 36.8%+8.5 non targeting control 9.6%+4.2 8.1%+4.0 16.2%+3.1 Rac2 shRNA* 18.7%+4.5 35.5%+12.9 43.7%+7.1 (Mean±SEM; n=6; * second set of Rac1 and Rac2 shRNAs gave comparable results) Disclosures: No relevant conflicts of interest to declare.

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Verheul, C., T. V. Kers, A. Van Der Ploeg, M. Van Der Kaaij, M. Aghababazadeh, M. De Wit, Y. Hoogstrate et al. « P11.47 Generation, characterisation and drug screening of patient-derivedIDH1-mutated glioma cell lines ». Neuro-Oncology 21, Supplement_3 (août 2019) : iii54. http://dx.doi.org/10.1093/neuonc/noz126.193.

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Abstract BACKGROUND Despite considerable scientific efforts, endogenous in vitro isocitrate dehydrogenase (IDH)-mutated glioma models remain scarce. Availability of these models is key to understanding underlying molecular mechanisms and vital for the development of new therapeutic interventions. We established and characterized a set of seven cell lines derived from IDH1-mutated gliomas and utilized them to investigate IDH-mutant glioma drug-response in vitro. MATERIAL AND METHODS Fresh tumor material was collected directly from the operating room, mechanically and enzymatically dissociated and cultured under serum-free culture conditions. IDH mutation status was verified at several passages with Sanger sequencing, as well as with whole exome sequencing. D-2-hydroxyglutarate (D2-HG) levels were determined with mass-spectrometry. Genome-wide methylation profiling was performed using the Infinium MethylationEpic BeadChip array. Cell viability was measured using the ATP-based CellTiter-Glo assay. Cell proliferation was determined through cell counting. Drug screens were performed with an FDA-approved anti-cancer drug set from the NIH as well as two IDH-mutant specific inhibitors. RESULTS Over the last decade our lab processed over 800 glioma samples from all WHO grades and IDH mutational status. Optimisation of culture conditions led to the establishment of seven IDH1-mutant glioma cell lines. The IDH1-mutation was present during all tested passages in these cell lines. Whole exome sequencing showed a high concordance between tumor and cell lines with regard to driver gene mutations. Similarly, copy-number changes based on genome-wide methylation data also show a close resemblance between the parental tumor and resulting cell lines. The Heidelberg methylation profiler classified each cell line and its parental tumor as IDH mutant glioma. When exposed to an IDH1-mutant specific inhibitor, all cell lines showed a concentration-dependent decrease of D2-HG levels. The inhibitors showed little to no effect on viability up to 10uM during 8 days, however, long term treatment up to 4 weeks revealed a decrease in cell doubling time in 2 of 6 cultures. Drug screen results either alone or in combination with an IDH1-mutant specific inhibitor identified several interesting candidates that are currently followed up in additional experiments. CONCLUSION We established a unique set of patient-derived IDH1-mutant glioma cell lines that closely resemble their respective tumors and can be used to identify new therapeutic strategies.

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Gozgit, Joseph M., Geraldine A. Bebernitz, Pankaj Patil, Minwei Ye, Jiaquan Wu, Stephanos Ioannidis, Audrey Davies, Tao Wang, Dennis Huszar et Michael Zinda. « Effects of a Novel, Selective Jak2 Inhibitor, AZ60, on STAT5 Signaling and Cellular Growth in Jak2 V617F Cell Lines. » Blood 110, n^o 11 (16 novembre 2007) : 3549. http://dx.doi.org/10.1182/blood.v110.11.3549.3549.

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Abstract A role for Jak2 in the etiology of the myeloproliferative diseases (MPDs) was discovered with the identification of a single activating point mutation, V617F, in the pseudokinase domain of JAK2. We have developed a Jak2 inhibitor, AZ60, which inhibits in vitro JAK2 enzyme activity with a Ki of 0.45 nM. AZ60 demonstrates inhibition of STAT5 phosphorylation and proliferation in a Tel-Jak2 engineered cell line with IC50 values of 18 and 23 nM, respectively. To understand the selectivity versus other Jak kinase family members we engineered three additional cell lines containing Tel fusions with the kinase domains of Jak1, Jak3 and Tyk2. Under these settings, AZ60 demonstrates a 15 to 30-fold selectivity for Tel-Jak2 driven STAT5 phosphorylation when compared to other Jak kinase family members. AZ60 was also tested for its ability to inhibit STAT5 phosphorylation and cellular proliferation in two human hematological cell lines, Set-2 and Hel. Set-2 expresses both wt and V617F Jak2, while Hel is homozygous for the Jak2 V617F mutation. AZ60 decreased phospho-STAT5 levels in a dose-dependent manner in both Set-2 and Hel cells with IC50 values of 15 and 25 nM, respectively. Complete inhibition of proliferation and a marked induction of apoptosis were observed in both cell lines following treatment with AZ60. Induction of apoptosis by AZ60 was characterized by a time- and dose-dependent increase in caspase 3/7 activities and PARP-cleavage. These data demonstrate AZ60 is a potent and selective inhibitor of Jak2 and may help decipher the mechanisms underlying Jak2-driven myeloproliferative disease.

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Sidorchuk, Yu V., A. A. Fomenkov, V. V. Kuznetsov, S. R. Mursalimov, A. A. Zagorskaya, E. A. Uvarova, P. A. Belavin et E. V. Deineko. « Variation in GFP Gene Expression in Arabidopsis thaliana Monoclonal Cell Lines ». Biotekhnologiya 35, n^o 1 (2019) : 58–67. http://dx.doi.org/10.21519/0234-2758-2019-35-1-58-67.

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A set of transgenic Arabidopsis thaliana monoclonal cell lines has been created by selecting individual cells (cell aggregates) with random GFP gene integration events after the Agrobacterium-mediated transformation. The yield of the recombinant GFP ranged from 0.07 to 2.37% of the total soluble protein. Three lines with the highest, about 2% of the total soluble protein, accumulation of the target GFP protein were isolated. Areas of T-DNA insertions into the plant genome for 12 monoclonal cell lines were determined. The variation in this characteristic among 21 examined cell lines can serve as a reference for A. thaliana CRISPR/Cas9 genome editing aimed at the increase in the yield of recombinant proteins in plant expression systems. The lines with the highest level of the recombinant protein accumulation are of interest for the further identification and detailed characteristics of the sites for the transgene integration. These sites can be used as targets for the gene integration during the creation of lines for the production of recombinant proteins. Arabidopsis thaliana, cell suspension culture, Agrobacterium-mediated transformation, gene expression, green fluorescent protein. The work was performed under project no. 17-14-01099 of the Russian Science Foundation.

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Garitano-Trojaola, Andoni, Eva Teufel, Matteo Claudio Da Via', Ana Sancho, Nadine Rodhes, Thorsten Stuehmer, Santiago Barrio Garcia et al. « The Role of NRAS G12D Mutations in the Response to Conventional Chemotherapy and 5-Azacitidine in Secondary AML ». Blood 132, Supplement 1 (29 novembre 2018) : 5148. http://dx.doi.org/10.1182/blood-2018-99-118484.

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Abstract Secondary Acute Myeloid Leukemia (sAML) accounts for 10-30% of all AML. It arises from a preexisting clonal disorder of hematopoiesis, such as myelodysplastic syndromes (MDS) or chronic myeloproliferative neoplasia (cMPN) in most cases (60-70%) or from exposure to a leukemogenic agent e.g. chemotherapy. sAML is generally considered to be of unfavorable prognosis, as treatment sensitivity is reduced, compared to de novo AML (dnAML) and overall survival is shortened. The incidence of AML associated NRAS are similar between sAML and dnAML (10 to 15%, Jelena D. Milosevic et al.). Prognostic impact of such mutations have been controversially discussed, but have been linked to favorable response to high dose cytarabine treatment in dnAML patients (Andreas Neubauer et al.), thus providing the first example of an oncogenic mutation impacting drug response in dnAML. This effect, however, has not yet been shown in sAML, therefore the aim of this work is to study the role of mutated NRAS in the response to chemotherapy and the hypomethylating agent (HMA) 5-azacitidine in sAML. We utilized two sAML cell lines SET-2 and HEL (both NRAS wildtype) in which we stably introduced the NRAS WT and the known activating hotspot mutation NRAS G12D using the sleeping Beauty technology. The dose-response assays of conventional chemotherapy and 5-azacitidine were carried out in the parental cell lines (SET-2/HEL) compared to NRAS WT (SET-2 NRAS WT/HEL NRAS WT) and NRAS G12D (SET-2 NRAS G12D/HEL NRAS G12D). In contrast to our expectations, both NRAS G12D mutation harboring cell lines, SET-2 and HEL developed resistance to cytarabine, idarubicin and 5-azacytidine, whereas the ones with wildtype NRAS remained susceptible to the drugs. To reverse the resistance we tested the MEK inhibitors Binimetinib and Trametinib in our SET-2 NRAS G12D cell line model according to recent reports about preclinical efficacy of MEK inhibition in NRAS mutant dnAML cells (Michael R. Burgess et al.). And in fact, single agent Binetimib and Trametinib treatments reduced cell viability by 20% at 48 hours. Strikingly, in combination with 5-azacitidine, Binimetinib and Trametinib treatments led to a viability reduction by 90%. Next we induced necroptosis in our NRAS mutant cell line models through the combination of Birinapant (SMAC mimetics) and Emricasan (Inhibitor of Caspase 8), as recently described by Brumatti et al. and were, in addition, able to reduce the cell viability by 60 %. In summary, we provide first evidence, that in contrast to dnAML, activating NRAS mutations may promote resistance to conventional chemotherapy and 5-azacitidine in sAML cell lines. Furthermore we were able to demonstrate, that the combination of MEK inhibitors (Binimetinib and Trametinib) and 5-azacitidine as well as the induction of necroptosis such as the combination birinapant and emricasan, may provide a potential strategy to overcome the resistance. Disclosures Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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Hom, J. T., J. M. Stuart et J. M. Chiller. « Murine T cells reactive to type II collagen. I. Isolation of lines and clones and characterization of their antigen-induced proliferative responses. » Journal of Immunology 136, n^o 3 (1 février 1986) : 769–75. http://dx.doi.org/10.4049/jimmunol.136.3.769.

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Abstract Mice of the DBA/1 strain develop arthritis after immunization with native chick type II collagen. Although both a humoral and a cell-mediated response specific to type II collagen are associated with the disease, the underlying immunologic basis remains to be established. As an initial step to analyzing the involvement of cellular immunity in collagen-induced arthritis, we isolated and characterized T cell lines and clones specific to type II collagen. Two sets of T cell lines were obtained by limiting dilution. One set was found to react exclusively with denatured type II collagen, whereas the other set responded to both native and denatured type II collagen. The specificity of such T cells was demonstrated by their inability to respond to other soluble proteins such as type I collagen, HGG, KLH, or OVA. Cells from these lines recognized type II collagen only in an MHC (H-2q)-restricted fashion. Furthermore, the collagen-specific T cells were found to respond to type II collagens obtained from various species, including chick, bovine, and rat. Finally, each set of cells displayed a phenotype characteristic of T helper cells, namely Thy-1+, L3T4+, Lyt-2-.

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Quentmeier, Hilmar, Roderick A. F. MacLeod, Julia Reinhardt, Margarete Zaborski et Hans G. Drexler. « JAK2 V617F Tyrosine Kinase Mutation in Leukemia Cell Lines. » Blood 106, n^o 11 (16 novembre 2005) : 4505. http://dx.doi.org/10.1182/blood.v106.11.4505.4505.

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Abstract It has recently been shown that the majority of patients with polycythemia vera, and substantial numbers of patients with idiopathic myelofibrosis and essential thrombocythemia, carry a single nucleotide mutation in the JAK2 gene. The JAK2 V617F mutation was also found in patients with other myeloproliferative disorders, though at lower percentages. The JAK2 point mutation may contribute to the development of these diseases, and the elucidation of the cellular function of mutated JAK2 might facilitate the development of a suitable drug. In numerous cases, the effects of a mutationally altered protein on cellular functions like proliferation, differentiation or apoptosis, have been revealed using immortalized cell lines that carry the mutation in question. To find such a model system for JAK2 V617F in hematopoietic neoplasia, we tested a panel of 37 cell lines from patients with myeloproliferative disorders, myelodysplastic syndromes (MDS) and the megakaryoblastic variant of acute myeloid leukemia (AML M7) and compared these with cell lines derived from other types of AML (M1-M6), and acute lymphoblastic leukemia (ALL). Cell lines were tested for JAK2 V617F expression applying a DNA tetra-primer amplification refractory mutation system (ARMS), a method that specifically amplifies the normal and mutant sequences plus a positive control band in a single reaction. According to this method, three cell lines carried the JAK2 V167F mutation: cell line MB-02 derived from a patient with a history of myelofibrosis and myeloid metaplasia that developed into AML M7; cell line MUTZ-8 established from a patient with AML M4 25 years after onset of MDS; cell line SET-2 from a patient at the stage of leukemic transformation of essential thrombocythemia. We present data on these cell lines including JAK2 ARMS and sequencing results, cytogenetic analysis focusing on chromosome 9, and JAK-2 protein expression and activation. In summary, we detected three leukemia cell lines that carry the recently discovered JAK2 mutation. These cell lines promise to represent important research tools in the further analysis of this genetic alteration.

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Al-saraireh, Yousef M., Ahmed M. M. Youssef, Fatemah O. F. O. Alshammari, Sameeh A. Al-Sarayreh, Jehad M. Al-Shuneigat, Hamzeh M. Alrawashdeh et Samir S. Mahgoub. « Phytochemical characterization and anti-cancer properties of extract of Ephedra foeminea (Ephedraceae) aerial parts ». Tropical Journal of Pharmaceutical Research 20, n^o 8 (17 février 2022) : 1675–81. http://dx.doi.org/10.4314/tjpr.v20i8.18.

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Purpose: To evaluate the phytochemical profile of methanol extract of Ephedra foeminea and assess its anti-carcer effect on a large set of normal and cancerous cell lines. Methods: Extraction of air-dried powder of aerial parts of E. foeminea was carried out with methanol. The bioactive compounds in the extract were determined using gas chromatography/mass spectrometry (GC-MS). The anti-cancer effect of the extract was determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay against various types of normal and cancer cell lines. Serial concentrations of plant extract were used, ranging from 7.812 to1000 μg/mL. Doxorubicin (DOX) served as standard drug. The half-maximal concentration (IC50) values of the extract and DOX for each cell line were determined, and the selectivity index (SI) was computed. Results: Phytochemical analysis showed that the extract contained several bioactive compounds, including alkaloids, flavonoids, sterols and fatty acids. The hazardous ephedra alkaloids (ephedrine and pseudoephedrine) were absent in the plant extract. The extract showed significant anti-proliferative activity against cancer cell lines, when compared with the positive control, doxorubicin (p < 0.05). Selective and concentration-dependent cytotoxicity was exhibited in cancer cell lines of breast (MCF-7), lung (A549), colon (Caco-2), liver (HepG-2) and prostate (PC-3). Weak selectivity was produced in other cancer cell lines, i.e., human epithelioma (Hep-2) and cervical carcinoma (Hela). Interestingly, non-cancerous cells showed no or weak cytotoxicity. Conclusion: Ephedra foeminea exerts potential selectivity in anti-proliferative effect against some cancer cell lines. Thus, it is a promising drug source for the production of new and selective anti-cancer medicines.

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Bebb, Gwyn, Huong Muzik, Sophia Nguyen, Don Morris et Douglas A. Stewart. « In Vitro and In Vivo Anti Lymphoma Effect of GX15-070 in Mantle Cell Lymphoma. » Blood 108, n^o 11 (16 novembre 2006) : 4756. http://dx.doi.org/10.1182/blood.v108.11.4756.4756.

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Abstract Introduction Mantle cell lymphoma (MCL), an incurable B cell lymphoma, consistently over expresses bcl-2 despite not carrying the t(14;18). The attenuation of apoptosis by bcl-2 is thought to contribute to the malignant process and increase resistance to some cytotoxic agents. We recently demonstrated that GX15-070, a small molecular inhibitor of the BH3 binding groove of bcl-2, has activity against MCL cell lines in vitro. We set out to assess the effect of GX15-070 alone and in combination with Vincristine on the viability of MCL cells in vitro and in vivo. Methods 3 previously characterized bcl-2 over expressing MCL cell lines (JVM-2, Hbl-2, granta) were used. Cells were grown in standard media and exposed to a range of concentrations of GX15-070 with and without Vincristine. Dose-response was assessed by measuring viability at 48 hours using the WST-1 assay. In vivo experiments were conducted on immune deficient mice in which 5×106 cells were injected in the flank then treated IV with GX15-070 (q 2days × 5 doses), Vincristine (q4 days × 3 doses) or both starting 5 days later. Tumours were measured three times weekly. Results All three MCL cell lines over-expressed bcl-2 by western blot. Each MCL cell line showed sensitivity to GX15-070 at a range of concentrations. The addition of GX15-070 to low dose Vincristine (10−6) caused significant growth inhibition of each MCL cell line (see table 1). Discussion Our results demonstrate that using GX15-070 to target bcl-2 is an effective anti neoplastic approach against MCL cell lines in vitro. In addition, our results suggest that combining Vincristine and GX15-070 is a promising strategy in treating MCL. In vivo experiments to confirm this additive activity are still ongoing and will be presented in full. Initial impressions suggest that there is a rationale for the addition of GX15-070 to current cytotoxic regimens used to treat MCL in the setting of clinical trials. Table 1: Effect of Vincristine and GX15-070 on in vitro growth of 3 MCL cell lines Growth as % age of Control Cell Line JVM-2 HBL-2 Granta Vincristine alone (10-6 mg/ml) 92% 48% 89% GX15-070 alone (0.08 uM) 75% 76% 60% Vincristine 10-6 mg/ml and GX15-070 0.08 uM 52% 24% 52%

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Fichtner, A., H. Bohnenberger, O. Elakad, A. Richter, C. Lenz, C. Oing, P. Ströbel, S. Kueffer, D. Nettersheim et F. Bremmer. « Proteomic profiling of cisplatin-resistant and cisplatin-sensitive germ cell tumour cell lines using quantitative mass spectrometry ». World Journal of Urology 40, n^o 2 (27 janvier 2022) : 373–83. http://dx.doi.org/10.1007/s00345-022-03936-1.

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Abstract Purpose Advanced testicular germ cell tumours (GCT) generally have a good prognosis owing to their unique sensitivity towards cisplatin-based chemotherapies. However, cisplatin-resistant GCT have a poor outcome. Further studies are mandatory to better understand resistance mechanisms and develop therapeutic strategies for refractory GCTs. Methods Protein levels in cisplatin-resistant GCT cell lines of NTERA-2, NCCIT and 2102EP were analyzed by quantitative proteomic mass spectrometry (MS) in combination with stable isotope labelling by amino acids in cell culture (SILAC). Differentially abundant protein markers of acquired cisplatin resistance were validated by Western blotting. Comprehensive bioinformatical annotation using gene set enrichment analyses (GSEA) and STRING interaction analysis were performed to identify commonly affected pathways in cisplatin resistance and the data were compared to the GCT cohort of the ‘The Cancer Genome Atlas’. Results A total of 4375 proteins were quantified by MS, 144 of which were found to be differentially abundant between isogenic resistant and sensitive cell line pairs (24 proteins for NTERA-2, 60 proteins for NCCIT, 75 proteins for 2102EP). Western blotting confirmed regulation of key resistance-associated proteins (CBS, ANXA1, LDHA, CTH, FDXR). GSEA revealed a statistically significant enrichment of DNA repair-associated proteins in all three resistant cell lines and specific additional processes for individual cell lines. Conclusion High resolution MS combined with SILAC is a powerful tool and 144 significantly deregulated proteins were found in cisplatin-resistant GCT cell lines. Our study provides the largest proteomic in vitro library for cisplatin resistance in GCT, yet, enabling further studies to develop new treatment options for patients with refractory GCT.

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Abadleh, Mohammed M., Mustafa M. El-Abadelah, Salim S. Sabri, Hanan H. Mohammed, Malek A. Zihlif et Wolfgang Voelter. « Synthesis and Antitumor Activity of Some N2-(Thien-3-yl)amidrazones ». Zeitschrift für Naturforschung B 69, n^o 7 (1 juillet 2014) : 811–16. http://dx.doi.org/10.5560/znb.2014-4062.

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6aA set of new N2-(thien-3-yl)amidrazones (-h) incorporating N-piperazines and related congeners has been synthesized by reacting the hydrazonoyl chloride 4(derived from 3-aminothiophene- 2-carboxylate) with the appropriate sec-cyclic amine. The antitumor activity of these compounds was evaluated on breast cancer (MCF-7) and leukemic (K562) cell lines by a cell viability assay utilizing the tetrazolium dye (MTT). The amidrazone 6d encompassing the N-piperazine moiety, was the most active against MCF-7 and K562 with IC50 of 7.28 and 9:91 μM, respectively.

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Ogato, Denis Mabeya, Eliakim Mbaka Mauti, Godfrey Omare Mauti, Barasa Ambrose et David Keno Kowanga. « Anticancer activity of Eugenia jambolana seeds against Hep2 cell lines ». Journal of Phytopharmacology 4, n^o 6 (2 janvier 2016) : 295–98. http://dx.doi.org/10.31254/phyto.2015.4604.

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Cancer is a life-threatening disease and leads to high rates of mortality worldwide, after cardiovascular disease, is the second leading cause of death. Investigations for finding new plant based anticancer compounds are imperative and interesting. There are many studies on anticancer herb/plant extracts in cell line models. Eugenia jambolana has been reported to contain phytochemicals like coumarin, flavanoids, glycosides, phenols, tannins and steroids. The various part of Eugenia jambolana have therapeutic applications. Plant active components were extracted using the decoction extraction method and the filtrate was obtained by means of filtering through a Whattman no.1 filter paper. The filtrate was evaporated in a weighed flask in a hot air oven set at 50°C. Extracts were reconstituted by re-dissolving in respective solvents. Different concentration i.e. 8, 15.6, 31.25, 62.5, 125, 250, 500 and 1000 µg. of the plant extracts were tested for the anticancer activity. The anticancer assay was performed on Human laryngeal epithiloma cells (Hep 2) obtained from King Institute of Preventive Medicine, Chennai, India. The cell viability was measured using MTT assay. Controls were maintained throughout the experiment (Untreated wells as cell control and diluent treated wells as diluent control). The assay was performed in triplicates for each of the extracts.

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Reinke, Stefan O., Marion Bayer, Markus Berger, Stephan Hinderlich et Véronique Blanchard. « The analysis of N-glycans of cell membrane proteins from human hematopoietic cell lines reveals distinctions in their pattern ». Biological Chemistry 393, n^o 8 (1 août 2012) : 731–47. http://dx.doi.org/10.1515/hsz-2012-0195.

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Abstract Human cell lines are often different in their features and present variations in the glycosylation patterns of cell membrane proteins. Protein glycosylation is the most common posttranslational modification and plays a particular role in functionality and bioactivity. The key approach of this study is the comparative analysis of five hematopoietic cell lines for their N-glycosylation pattern. The N-glycans of membrane proteins were elucidated by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and MALDI-TOF/TOF-MS analyses. Furthermore, the expression of a set of glycosyltransferases was determined via RT-PCR. The B-lymphoma BJA-B and promyelocytic HL-60 cell lines distinguish in levels and linkages of glycan-bound sialic acids. Furthermore, subclones of BJA-B and HL-60 cells, which completely lack UDP-N-acetylglucosamine 2-ēpimerase/N-acetylmannosamine kinase (GNE), the key enzyme of sialic acid biosynthesis, contained almost no sialylated N-glycans. Compared to wild-type cells, the GNE-deficient cells pres\xadented a similar cell surface N-glycosylation pattern in terms of antennarity and fucosylation. The Jurkat T-cell line revealed only partially sialylated N-glycans. Additionally, the different hematopoietic cell lines vary in their level of bisecting GlcNAcylation and antennary fucosylation with the quantities of bisecting N-acetylglucosamine (GlcNAc) and core fucose coinciding with the expression of GnT-III and FucT-VIII. Of note is the occurrence of N-acetyllactosamine (LacNAc) extensions on tetraantennary structures in GNE-deficient cell lines.

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Willis, Clinton, Johanna Nyffeler et Joshua Harrill. « Phenotypic Profiling of Reference Chemicals across Biologically Diverse Cell Types Using the Cell Painting Assay ». SLAS DISCOVERY : Advancing the Science of Drug Discovery 25, n^o 7 (17 juin 2020) : 755–69. http://dx.doi.org/10.1177/2472555220928004.

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Cell Painting is a high-throughput phenotypic profiling assay that uses fluorescent cytochemistry to visualize a variety of organelles and high-content imaging to derive a large number of morphological features at the single-cell level. Most Cell Painting studies have used the U-2 OS cell line for chemical or functional genomics screening. The Cell Painting assay can be used with many other human-derived cell types, given that the assay is based on the use of fluoroprobes that label organelles that are present in most (if not all) human cells. Questions remain, however, regarding the optimization steps required and overall ease of deployment of the Cell Painting assay to novel cell types. Here, we used the Cell Painting assay to characterize the phenotypic effects of 14 phenotypic reference chemicals in concentration–response screening mode across six biologically diverse human-derived cell lines (U-2 OS, MCF7, HepG2, A549, HTB-9 and ARPE-19). All cell lines were labeled using the same cytochemistry protocol, and the same set of phenotypic features was calculated. We found it necessary to optimize image acquisition settings and cell segmentation parameters for each cell type, but did not adjust the cytochemistry protocol. For some reference chemicals, similar subsets of phenotypic features corresponding to a particular organelle were associated with the highest-effect magnitudes in each affected cell type. Overall, for certain chemicals, the Cell Painting assay yielded qualitatively similar biological activity profiles among a group of diverse, morphologically distinct human-derived cell lines without the requirement for cell type–specific optimization of cytochemistry protocols.

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Kozako, Tomohiro, Teruhisa Shoji, Akiyoshi Aikawa, Satoru Hayashida, Yukako Kuramoto, Rie Mononagare, Shinichiro Honda et al. « SIRT1, a Longevity Gene Encoded Protein, Regulates Apoptosis of Adult T-Cell Leukemia Cells and Its Inhibition by Sirtinol Induces Apoptosis. » Blood 114, n^o 22 (20 novembre 2009) : 3684. http://dx.doi.org/10.1182/blood.v114.22.3684.3684.

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Abstract Abstract 3684 Poster Board III-620 Adult T-cell leukemia-lymphoma (ATL) is an aggressive peripheral T-cell neoplasm with a poor prognosis developing after long-term infection with human T-cell leukemia virus-1 (HTLV-1). HTLV-1 Tax is closely related to leukemic cell proliferation through nuclear factor-kappa B (NF-ƒÈB) activation. Recent studies have demonstrated that histone deacetylase class I/II inhibitors induce growth arrest and apoptosis of HTLV-1-infected T-cells via blockade of NF-ƒÈB signaling. SIRT1, an NAD(+)-dependent class III histone deacetylase, is widely recognized for its link to caloric restriction and longevity. SIRT1 plays a crucial role in a variety of physiological processes including metabolism, neurogenesis, cell survival, apoptosis and aging due to its ability to deacetylate numerous substrates such as histone, p53 and NF-ƒÈB. Existing reports on the role of SIRT1 in oncogenesis are controversial, with some evidence of an oncogenic role due to its increased expression in prostate cancer, acute myeloid leukemia and colon cancer, possibly mediated by inactivation of proteins involved in tumor suppression and DNA damage repair. Contrasting evidence of reduced SIRT1 expression in breast and hepatocellular carcinomas may support a tumor suppressor role, especially if the tumor is related to a p53 mutation. Such conflicting reports raise intriguing questions regarding its role in oncogenesis, and even less is known about its role in ATL in particular. We therefore set out to assess the expression of SIRT1 and the effect of its inhibition in HTLV-1 infected cell lines and ATL cells from patients. We observed SIRT1 protein and mRNA expression in ATL patient cells, an HTLV-1-infected cell line (MT-2), an ATL cell line (S1T), as well as HTLV-1 unrelated cell lines, Jurkat and HL60, as controls. SIRT1 expression in ATL patients was significantly higher than asymptomatic HTLV-1-carriers and healthy donors. The SIRT1 inhibitor, sirtinol, inhibited growth of all cell lines tested, with greater selectivity for HTLV-1 related cell lines (Figure 1) and ATL patients. Sirtinol induced apoptosis by activation of caspase-3, 8, 9 (Figure 2) and reducing IkBa phosphorylation, but did not significantly increase p53 acetylation in HTLV-1 infected cell lines. SIRT1 activation by NAD+ augmented apoptosis induction by sirtinol in MT-2 cells. These findings suggest that SIRT1 may be involved in T-cell immortalization by HTLV-1 and may be a crucial anti-apoptotic molecule in ATL cells. SIRT1 inhibition could therefore be useful in treating ATL. Figure 1 Inhibitory effects of sirtinol, SIRT1 inhibitor, on cell viability of leukemic cell lines. Cell lines were treated with sirtinol (0, 0.1, 10, 25 and 50μM) for 24hr. Each bar represents the mean ±S.D. of 3 independent experiments. Figure 1. Inhibitory effects of sirtinol, SIRT1 inhibitor, on cell viability of leukemic cell lines. Cell lines were treated with sirtinol (0, 0.1, 10, 25 and 50μM) for 24hr. Each bar represents the mean ±S.D. of 3 independent experiments. Figure 2 The activities of caspase-3, 8 and 9 in S1T and MT-2. Cell lines were treated with sirtinol (50μM) for 6 hr. Each bar represents the mean ±S.D. of 3 independent experiments. Figure 2. The activities of caspase-3, 8 and 9 in S1T and MT-2. Cell lines were treated with sirtinol (50μM) for 6 hr. Each bar represents the mean ±S.D. of 3 independent experiments. Disclosures: No relevant conflicts of interest to declare.

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Sherman, Emily J., Carmen Mirabelli, Vi T. Tang, Taslima G. Khan, Kyle Leix, Andrew A. Kennedy, Sarah E. Graham et al. « Identification of cell type specific ACE2 modifiers by CRISPR screening ». PLOS Pathogens 18, n^o 3 (1 mars 2022) : e1010377. http://dx.doi.org/10.1371/journal.ppat.1010377.

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SARS-CoV-2 infection is initiated by binding of the viral spike protein to its receptor, ACE2, on the surface of host cells. ACE2 expression is heterogeneous both in vivo and in immortalized cell lines, but the molecular pathways that govern ACE2 expression remain unclear. We now report high-throughput CRISPR screens for functional modifiers of ACE2 surface abundance. In liver-derived HuH7 cells, we identified 35 genes whose disruption was associated with a change in the surface abundance of ACE2. Enriched among these ACE2 regulators were established transcription factors, epigenetic regulators, and functional networks. We further characterized individual HuH7 cell lines with disruption of SMAD4, EP300, PIAS1, or BAMBI and found these genes to regulate ACE2 at the mRNA level and to influence cellular susceptibility to SARS-CoV-2 infection. Orthogonal screening of lung-derived Calu-3 cells revealed a distinct set of ACE2 modifiers comprised of ACE2, KDM6A, MOGS, GPAA1, and UGP2. Collectively, our findings clarify the host factors involved in SARS-CoV-2 entry, highlight the cell type specificity of ACE2 regulatory networks, and suggest potential targets for therapeutic development.

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Hattori, M., T. Sudo, M. Iizuka, S. Kobayashi, S. Nishio, S. Kano et N. Minato. « Generation of continuous large granular lymphocyte lines by interleukin 2 from the spleen cells of mice infected with Moloney leukemia virus. Involvement of interleukin 3. » Journal of Experimental Medicine 166, n^o 4 (1 octobre 1987) : 833–49. http://dx.doi.org/10.1084/jem.166.4.833.

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Continuous cell lines could be reproducibly established by culturing spleen cells from adult mice injected with MLV-producer cells or directly infected with Mo-MLV with rIL-2, whereas the culture of normal splenic cells with rIL-2 induced only transient and limited proliferation resulting in no such lines. All of the lines showed morphological characteristics as LGL with Thy-1+,Lyt-1-,L3T4-,Lyt-2-,AsGM1+,FcR gamma+ phenotype without exception, and most of them exhibited typical NK-patterned cytotoxicity. Analysis of reverse transcriptase activity of the culture supernatants as well as Southern hybridization of the DNA from the lines using an Mo-MLV-specific cDNA probe indicated no evidence of retroviral replication or proviral integration, suggesting that the generation of cell lines reflected a reactive process and viral infection was not directly responsible. It was subsequently revealed that Thy-1+,Lyt-1+,Lyt-2- spleen cells from mice infected with Mo-MLV in vivo spontaneously produced surprising amounts of IL-3 in vitro, leading to the possibility that IL-3 was responsible for the generation of lines. The possibility was directly supported by the observation that continuous lines with identical characteristics could be generated completely in vitro by sequential stimulation with rIL-3 and rIL-2 from normal spleen cells without any involvement of Mo-MLV. The C beta gene of TCR was shown to be rearranged in all the lines examined, indicating the LGL lines were all genetically committed to T cell lineage. Unlike the situation in normal splenic populations expanded by rIL-2, where the expression of IL-2-R was progressively lost, constitutive expression of high-affinity-IL-2-R was observed in all the lines and thus, this was considered to explain the unlimited proliferation of them in response to rIL-2 alone. These results suggested the probable role of IL-3 in the regulation of growth and differentiation of a set of LGL committed to T cell lineage. The possible implications of the phenomenon in the regulation of hematopoiesis as well as in the control of Mo-MLV-induced leukemogenesis were discussed.

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Markovtsov, Vadim, Diane Yu, Marina Gelman, Wayne Lang, Vanessa C. Taylor, Stacey Huynh, Roy Frances et al. « Targeting Myeloproliferative Diseases with JAK2 Inhibitors. » Blood 110, n^o 11 (16 novembre 2007) : 3550. http://dx.doi.org/10.1182/blood.v110.11.3550.3550.

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Abstract Limited options provided by the current standard of care for the patients suffering from myeloproliferative diseases (MPDs) prompted an extensive search for the underlying molecular mechanisms of these disorders. Recent discovery of a single activating mutation (V617F) in JAK2 kinase gene associated with the development of the polycythemia vera (PV), essential thrombocythemia (ET) and chronic idiopathic myelofibrosis (CIMF) opened up a possibility to develop highly targeted therapies against these debilitating ailments. To that end, we engineered cytokine-independent Ba/F3 cell line expressing the V617F mutant of JAK2 to screen a focused small molecule library for potential inhibitors of JAK2 V617F -dependent proliferation. We confirmed the ability of hit compounds to inhibit proliferation of JAK2-dependent tumor cell lines using UKE-1 and SET-2 cells carrying the V617F JAK2 mutation. A FACS-based phosphoSTAT5 assay was then used to demonstrate that the hits directly targeted mutant JAK2. JAK3 activity of each compound was evaluated in IL-2-dependent CTLL-2 cell line using phosphoSTAT5 FACS and proliferation assays. To avoid hits with nonspecific antiproliferative activity, the hits were tested in JAK2-independent MOLT4, A549 and H1299 cell lines. Compound hits with the desirable properties were further evaluated for their ability to inhibit JAK2, JAK3 and other kinases in the context of T cell, B cell, or mast cell activation using a variety of cell-based assays as well as in the in vitro biochemical assays. We identified a number of compounds that potently inhibit growth of the two V617F mutant cell lines with EC50s varying from 20 to 500 nM, but do not affect proliferation of control cell lines MOLT4, A549 and H1299 to the same degree. These compounds induce strong and highly specific suppression of STAT5 phosphorylation with IC50s of 10 to 200 nM in SET-2 and V617F JAK2 expressing Ba/F3 cells. One of the hits with the desirable biological and pharmacokinetic profiles was further evaluated in V617F JAK2 Ba/F3 engraftment mouse model where it demonstrated significant extension of survival at 150 and 200 mg/kg bid. Such potent JAK2 inhibitors could become the basis for the next generation of compounds targeting JAK2-dependent myeloproliferative diseases.

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Li, Liping, Wenyan Lu, Alison R. Moliterno, Lingling Xian, Joseph Kim, Ophelia Rogers, Jerry L. Spivak et Linda Resar. « High Mobility Group A1 Chromatin Regulators : Key Epigenetic Switches and Therapeutic Targets Required for Leukemic Transformation in JAK2 Mutant MPN ». Blood 134, Supplement_1 (13 novembre 2019) : 1680. http://dx.doi.org/10.1182/blood-2019-130262.

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Introduction: Myeloproliferative neoplasms (MPN) are clonal hematopoietic stem cell (HSC) disorders characterized by hyperactive JAK/STAT signaling and increased risk of transformation to myelofibrosis (MF) and acute myeloid leukemia (AML). However, mechanisms driving progression remain elusive and therapies are ineffective after leukemia develops. The High Mobility Group A1/2 (HMGA1/2) genes encode oncogenic chromatin remodeling proteins which are overexpressed in aggressive solid tumors where they portend adverse outcomes. HMGA1/2 genes are also up-regulated in hematologic malignancies and MPN with disease progression. In murine models, Hmga1/2 overexpression drives clonal expansion and deregulated proliferation while Hmga1 overexpression is sufficient for lymphoid leukemic transformation. We therefore sought to: 1) test the hypothesis that HMGA1/2 proteins are rational therapeutic targets required for leukemic transformation in MPN, 2) elucidate mechanisms mediated by HMGA1/2 during disease progression, and, 3) identify therapeutic approaches to disrupt HMGA function and intercept the transition from chronic disease to aggressive leukemia. Methods: We compared HMGA1/2 in JAK2V617F mutant AML cell lines from MPN patients (DAMI, SET-2), CD34+ cells from PV patients during chronic and transformation phases, and JAK2V617F murine models of PV (transgenic JAK2V617F) and PV-AML (transgenic JAK2V617F/MPLSV). To elucidate HMGA1/2 function, we silenced HMGA1 or HMGA2 via short hairpin RNA in human MPN-AML cells and generated murine models of PV-AML with heterozygous Hmga1 or Hmga2 deficiency. To dissect molecular mechanisms underlying HMGA, we compared RNA-Seq from MPN-AML cell lines after gene silencing. Finally, to identify therapies to target HMGA pathways, we integrated the RNA-Seq data with the Broad Connectivity Map (cMAP). Results: There is a marked up-regulation in HMGA1/2 in CD34+ cells from PV patients after transformation to AML and in leukemic blasts from our PV-AML mouse model. Conversely, silencing HMGA1 or HMGA2 in human MPN-AML cell lines (DAMI, SET-2) dramatically halts proliferation, disrupts clonogenicity, and prevents leukemia development in mice. Further, heterozygous Hmga1 deficiency prolongs survival in the transgenic PV-AML murine model with fulminant leukemia and early mortality, although Hmga2 deficiency has no effect. RNA-Seq analyses from human MPN-AML cell lines revealed that HMGA1 up-regulates transcriptional networks involved in cell cycle progressions (E2F targets, mitotic spindle, G2M checkpoint, MYC targets) while repressing immune pathways (inflammation, interferon gamma) and oxidative phosphorylation. HMGA2 up-regulates similar pathways, but represses TNFalpha signaling. cMAP identified inhibitors of histone deacetylation and cell cycle progression as potential agents to target HMGA1 pathways; DNA synthesis inhibitors were predicted to target HMGA2 pathways. Cytotoxicity assays demonstrate that epigenetic therapy with HDAC inhibitors synergizes with Ruxolitinib in JAK2 mutant MPN cells after transformation to leukemia. Conclusions: HMGA1/2 genes are overexpressed in MPN with highest levels after leukemic transformation. Further, silencing HMGA1/2 disrupts leukemogenic phenotypes in vitro and prevents the development of leukemia in mice. In addition, heterozygous deficiency of Hmga1 prolongs survival in a fulminant MPN-AML model. Mechanistically, RNA-Seq analyses revealed that HMGA amplifies transcriptional networks involved cell cycle progression, which can be targeted with epigenetic therapies. Our findings further underscore the key role for HMGA as an epigenetic switch required for leukemic transformation in MPN and opens the door to novel therapeutic approaches to intercept the transition from chronic indolent disease to aggressive leukemia. Disclosures No relevant conflicts of interest to declare.

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Patel, Mrugesh, et Kaushal Patel. « Naphthalene substituted benzo[c]coumarins : Synthesis, characterization and evaluation of antibacterial activity and cytotoxicity ». Heterocyclic Communications 25, n^o 1 (31 décembre 2019) : 146–51. http://dx.doi.org/10.1515/hc-2019-0024.

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AbstractNovel congeners of naphthalene substituted benzo[c]coumarins (2a-f) were synthesized by reaction of various 3-coumarinoyl methyl pyridinium bromide salts (1a-d) with a selected set of acetyl naphthalene in the presence of sodium acetate in refluxing glacial acetic acid. Structures of the synthesized compounds were confirmed by elemental analysis and by various spectroscopic techniques such as 1H-NMR, 13C-NMR, DEPT, and MS spectral data. Synthesised compounds were screened for antibacterial activity and cytotoxicity against different human cancer cell lines including cervix cancer (HeLa), breast cancer (MCF-7) and lung cancer (A549) using tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assay. Although with varying degrees, a significant growth inhibitory and cytotoxic effects were observed on all three cancer cell lines. Compounds 2b and 2e showed significant growth inhibitory and cytotoxicity against aforementioned cancer cell lines.

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Aponte-López, Angélica, Jennifer Enciso, Samira Muñoz-Cruz et Ezequiel M. Fuentes-Pananá. « An In Vitro Model of Mast Cell Recruitment and Activation by Breast Cancer Cells Supports Anti-Tumoral Responses ». International Journal of Molecular Sciences 21, n^o 15 (26 juillet 2020) : 5293. http://dx.doi.org/10.3390/ijms21155293.

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Breast cancer (BrC) affects millions of women yearly. Mast cells (MCs) are common components of breast tumors with documented agonistic and antagonistic roles in tumor progression. Understanding the participation of MCs in BrC may lead to new therapies to control tumor growth. In this study, we looked into mechanistic models of MC responses triggered by BrC cells (BrCC), assessing both early degranulation and late transcriptional activities. We used aggressive and non-aggressive BrCC to model the progressive staging of the disease over HMC1 and LAD-2 human MC lines. We found that both MC lines were chemoattracted by all BrCC, but their activation was preferentially induced by aggressive lines, finding differences in their active transcriptional programs, both at basal level and after stimulation. Among those genes with altered expression were down-regulated SPP1, PDCD1, IL17A and TGFB1 and up-regulated KITLG and IFNG. A low expression of SPP1 and a high expression of KITLG and IFNG were associated with increased overall survival of BrC patients from public databases. The set of altered genes is more often associated with tumor stromas enriched with anti-tumoral signals, suggesting that MCs may participate in tumor control.

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Szafrański, Krzysztof, Jarosław Sławiński, Łukasz Tomorowicz et Anna Kawiak. « Synthesis, Anticancer Evaluation and Structure-Activity Analysis of Novel (E)- 5-(2-Arylvinyl)-1,3,4-oxadiazol-2-yl)benzenesulfonamides ». International Journal of Molecular Sciences 21, n^o 6 (23 mars 2020) : 2235. http://dx.doi.org/10.3390/ijms21062235.

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To learn more about the structure–activity relationships of (E)-3-(5-styryl-1,3,4-oxadiazol-2-yl)benzenesulfonamide derivatives, which in our previous research displayed promising in vitro anticancer activity, we have synthesized a group of novel (E)-5-[(5-(2-arylvinyl)-1,3,4-oxadiazol-2-yl)]-4-chloro-2-R1-benzenesulfonamides 7–36 as well as (E)-4-[5-styryl1,3,4-oxadiazol-2-yl]benzenesulfonamides 47–50 and (E)-2-(2,4-dichlorophenyl)-5-(2-arylvinyl)-1,3,4-oxadiazols 51–55. All target derivatives were evaluated for their anticancer activity on HeLa, HCT-116, and MCF-7 human tumor cell lines. The obtained results were analyzed in order to explain the influence of a structure of the 2-aryl-vinyl substituent and benzenesulfonamide scaffold on the anti-tumor activity. Compound 31, bearing 5-nitrothiophene moiety, exhibited the most potent anticancer activity against the HCT-116, MCF-7, and HeLa cell lines, with IC50 values of 0.5, 4, and 4.5 µM, respectively. Analysis of structure-activity relationship showed significant differences in activity depending on the substituent in position 3 of the benzenesulfonamide ring and indicated as the optimal meta position of the sulfonamide moiety relative to the oxadizole ring. In the next stage, chemometric analysis was performed basing on a set of computed molecular descriptors. Hierarchical cluster analysis was used to examine the internal structure of the obtained data and the quantitative structure–activity relationship (QSAR) analysis with multiple linear regression (MLR) method allowed for finding statistically significant models for predicting activity towards all three cancer cell lines.

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Camacho, Cláudia, Helena Tomás et João Rodrigues. « Use of Half-Generation PAMAM Dendrimers (G0.5–G3.5) with Carboxylate End-Groups to Improve the DACHPtCl2 and 5-FU Efficacy as Anticancer Drugs ». Molecules 26, n^o 10 (14 mai 2021) : 2924. http://dx.doi.org/10.3390/molecules26102924.

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The DACHPtCl2 compound (trans-(R,R)-1,2-diaminocyclohexanedichloroplatinum(II)) is a potent anticancer drug with a broad spectrum of activity and is less toxic than oxaliplatin (trans-l-diaminocyclohexane oxalate platinum II), with which it shares the active metal fragment DACHPt. Nevertheless, due to poor water solubility, its use as a chemotherapeutic drug is limited. Here, DACHPtCl2 was conjugated, in a bidentate form, with half-generation PAMAM dendrimers (G0.5–G3.5) with carboxylate end-groups, and the resulting conjugates were evaluated against various types of cancer cell lines. In this way, we aimed at increasing the solubility and availability at the target site of DACHPt while potentially reducing the adverse side effects. DNA binding assays showed a hyperchromic effect compatible with DNA helix’s disruption upon the interaction of the metallodendrimers and/or the released active metallic fragments with DNA. Furthermore, the prepared DACHPt metallodendrimers presented cytotoxicity in a wide set of cancer cell lines used (the relative potency regarding oxaliplatin was in general high) and were not hemotoxic. Importantly, their selectivity for A2780 and CACO-2 cancer cells with respect to non-cancer cells was particularly high. Subsequently, the anticancer drug 5-FU was loaded in a selected metallodendrimer (the G2.5COO(DACHPt)16) to investigate a possible synergistic effect between the two drugs carried by the same dendrimer scaffold and tested for cytotoxicity in A2780cisR and CACO-2 cancer cell lines. This combination resulted in IC50 values much lower than the IC50 for 5-FU but higher than those found for the metallodendrimers without 5-FU. It seems, thus, that the metallic fragment-induced cytotoxicity dominates over the cytotoxicity of 5-FU in the set of considered cell lines.

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Rødland, Gro Elise, Katrine Melhus, Roman Generalov, Sania Gilani, Francesco Bertoni, Jostein Dahle, Randi Syljuåsen et Sebastian Patzke. « The Dual Cell Cycle Kinase Inhibitor JNJ-7706621 Reverses Resistance to CD37 Targeted Radioimmunotherapy in Activated B Cell like Diffuse Large B Cell Lymphoma Cell Lines ». Blood 134, Supplement_1 (13 novembre 2019) : 2574. http://dx.doi.org/10.1182/blood-2019-123287.

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The CD37 targeting radioimmunoconjugate 177Lu-lilotomab satetraxetan (Betalutin®) is currently being evaluated as monotherapy in a clinical phase 2b trial for patients with follicular lymphoma (FL) and in a phase 1 trial for patients with diffuse large B-cell lymphoma (DLBCL), as well as in a phase 1b trial in combination with rituximab for patients with relapsed/refractory FL. Herein we have investigated the effect of 177Lu-lilotomab satetraxetan in seven activated B-cell like (ABC) DLBCL cell lines. Although the radioimmunoconjugate showed anti-tumor activity, primary resistance was observed in a subset of cell lines: U-2932 and RIVA. Both cell lines are representative for TP53 deficient Double Expressor (DE) DLBCL. Importantly, resistance was not a consequence of reduced binding of the radioimmunoconjugate to cell surface expressed CD37. Thus, we set out to identify drugs able to overcome the resistance to 177Lu-lilotomab satetraxetan in both resistant ABC-DLBCL cell lines. We performed a viability-based screen combining 177Lu-lilotomab satetraxetan with the 384-compound Cambridge Cancer Compound Library. Drug combinations were scored using Bliss and Chou-Talalay algorithms. We identified and characterized the dual-specific CDK1/2 and AURA/B kinase inhibitor JNJ-7706621 as compound able to revert the resistance to radioimmunotherapy (RIT), alongside topoisomerase and histone deacetylases (HDAC) inhibitors. Kinetic studies of the effect of mono- and combination therapy of U-2932 and RIVA cells with JNJ-7706621 and 177Lu-lilotomab satetraxetan are suggestive of a model in which radiation damage induced G2-arrested lymphoma cells eventually enter mitosis (repair or escape) and mitotic entry, progression and exit are impaired by JNJ-7706621 mediated inhibition of CDK1/2 and AURKA/B. Extended residence-time of cells in mitosis due to chromosome condensation and congression defects as well as spindle and mid-spindle assembly failure is likely pivotal for the increased sensitivity to persistent 177Lu-lilotomab satetraxetan deposited DNA damage, ultimately promoting cytokinesis failure (multinucleation, aneuploidy, increased cell size) and cell death. In conclusion, CD37-targeting 177Lu-lilotomab satetraxetan RIT showed activity in several ABC-DLBCL lymphoma cell lines. CD37-independent RIT-resistance was identified in two cell lines representative of aggressive DE ABC-DLBCLs with inactive TP53, and reversed by subsequent inhibition of CDK1/2 and AURKA/B by JNJ-7706621. These findings may be of potential relevance for ongoing clinical trials of 177Lu-lilotomab satetraxetan in relapsed, ASCT-non-eligible DLBCL, and may also be more generally applicable to other 177Lu-based RITs and alternative radionuclide utilizing targeted therapies. Future pre-clinical investigations are required to elucidate the potential application of CDK1/2 and AURKA/B inhibitors as a strategy to revert RIT resistance in TP53 deficient cancers. Disclosures Rødland: Nordic Nanovector ASA: Patents & Royalties, Research Funding. Melhus:Nordic Nanovector ASA: Employment, Equity Ownership, Patents & Royalties. Generalov:Nordic Nanovector ASA: Employment, Equity Ownership, Patents & Royalties. Bertoni:Nordic Nanovector ASA: Research Funding; Oncology Therapeutic Development: Research Funding; PIQUR Therapeutics AG: Other: travel grant, Research Funding; HTG: Other: Expert Statements ; Amgen: Other: travel grants; Astra Zeneca: Other: travel grants; Jazz Pharmaceuticals: Other: travel grants; NEOMED Therapeutics 1: Research Funding; Acerta: Research Funding; ADC Therapeutics: Research Funding; Bayer AG: Research Funding; Cellestia: Research Funding; CTI Life Sciences: Research Funding; EMD Serono: Research Funding; Helsinn: Consultancy, Research Funding; ImmunoGen: Research Funding; Menarini Ricerche: Consultancy, Research Funding. Dahle:Nordic Nanovector ASA: Employment, Equity Ownership, Patents & Royalties. Syljuåsen:Nordic Nanovector ASA: Patents & Royalties, Research Funding. Patzke:Nordic Nanovector ASA: Employment, Patents & Royalties.

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Pietarinen, Paavo, Muntasir Mamun Majumder, Disha Malani, Astrid Murumägi, Tea Pemovska, Perttu Koskenvesa, Olli Kallioniemi et al. « High-Throughput Drug Sensitivity and Resistance Testing (DSRT) Platform Reveals Novel Candidate Drugs For Advanced Phase BCR-ABL1-Positive Leukemia ». Blood 122, n^o 21 (15 novembre 2013) : 2719. http://dx.doi.org/10.1182/blood.v122.21.2719.2719.

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Abstract Background Advanced BCR-ABL1-positive leukemias (chronic myeloid leukemia in blast crisis and Ph+ALL) remain a therapy challenge despite advances in tyrosine kinase inhibitor (TKI) therapy. Emergence of primary and secondary resistance due to gatekeeper and compound mutations within the BCR-ABL1 kinase domain is common even with the novel 2nd and 3rd generation TKIs (dasatinib, nilotinib, ponatinib). We set out to identify novel candidate drugs for advanced BCR-ABL1-positive leukemias by using an unbiased high-throughput drug testing platform and utilizing both primary patient cells and cell lines. Methods As a study material we used 3 CML cell lines representing different types of CML blast phases. In addition to commonly used K562 cells, EM-2 and MOLM-1 cell lines were tested. AML cell lines (AML-193, AP-1060, HL60ATCC, HL60TB, Kasumi-1, KG-1, ME-1, MOLM-13, MONO-MAC-6, MUTZ-2, MV4-11, NOMO-1, SH-2, SHI-1, SIG-M5, SKM-1, THP-1) were used as cell line controls. To verify the results obtained from cell lines, primary bone marrow (BM) cells were derived from 2 TKI-resistant CML BC patients. Patient 1 had developed resistance to imatinib and dasatinib due to a T315I mutation, whereas patient 2 was resistant to nilotinib, dasatinib and ponatinib due to a V299L and a compound mutation. BM cells from 4 healthy individuals were used as controls. The functional profiling of drug responses was performed with a high-throughput drug sensitivity and resistance testing (DSRT) platform comprising of 306 anti-cancer agents (FDA/EMA approved, investigational and experimental compounds). Cells were dispensed to pre-drugged 386-well plates of 5 different concentrations and incubated in a humidified incubator with 5% CO2 at 37 °C for 72 hours. Cell viability was measured by using a luminescent cell viability assay (CellTiter-Glo). From plate reads a Drug Sensitivity Score (DSS) was calculated for each drug as a measure of cytotoxicity. In addition to DSRT, Human Phospho-Kinase Array Kit (R&D systems) was used to analyze the phosphokinase profile in patient samples. Results Based on initial comparisons between CML and AML cells lines, nonspecific cytotoxic drugs, which showed high activity in all cell lines, were omitted from further analysis. The DSS scores from different CML cells lines correlated relatively closely (EM-2 vs. K-562, r=0.89; EM-2 vs. MOLM-1, r=0.82; K-562 vs. MOLM-1, r=0.78; p<0.0001 for all correlations). We next ranked the DSRT data according to the DSS values with most sensitive drugs showing the highest DSS scores. The primary cells from CML BC were further normalized against the median values from healthy controls, resulting in leukemia-specific sensitivity scores (sDSS). Ranked results from the DSRT analysis are shown in the Table. As expected, the cell lines were sensitive to TKIs, with the exception of the MOLM-1, which showed only modest sensitivity. The clinically TKI-resistant patient samples were also TKI-resistant ex vivo, further validating the DSRT assay data. Drugs which showed efficacy in both the cell lines and the TKI-resistant patients included HSP90 inhibitors (NVP-AUY922, BIIB021), a NAMPT inhibitor daporinad and the protein translation inhibitor omacetaxine (homoharringtonine). Phosphokinase antibody array results from the patient samples showed increased expression of the HSP27 protein as a putative biomarker for HSP90 inhibitor response. Conclusions DSRT is a powerful assay for identifying novel candidate molecules for refractory BCR-ABL1-positive leukemias. Our results indicate that HSP90 and NAMPT inhibitors in particular warrant further clinical evaluation both by analyzing a larger set of primary patient samples and by performing proof-of-concept clinical studies. The results also pave way for designing rational combination therapy strategies. Disclosures: Mustjoki: Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau. Porkka:BMS: Consultancy, Research Funding, Speakers Bureau; Novartis: Consultancy, Research Funding, Speakers Bureau.

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Davis, Carter Thomas, Arati V. Rao, Eross Guadalupe, Dale J. Christensen et J. Brice Weinberg. « Fingolimod Is Cytotoxic in Acute Myeloid Leukemia Independent of Additional Chemotherapeutic Agents ». Blood 128, n^o 22 (2 décembre 2016) : 5126. http://dx.doi.org/10.1182/blood.v128.22.5126.5126.

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Abstract INTRODUCTION: Conventional treatment of acute myeloid leukemia (AML) remains largely unchanged for over thirty years. With poor overall survival and disease cure rates, novel therapies are needed. The SET oncoprotein has been implicated in AML as essential for proliferation through inhibition of the tumor suppressor protein phosphatase 2A (PP2A). Interaction between SET and PP2A leads to inactivation of PP2A, leaving cell survival and proliferation signals unchecked. PP2A has been postulated to be an important target in AML. Fingolimod (FTY720), an FDA approved drug for relapsing-remitting multiple sclerosis, is a sphingosine-1 phosphate receptor agonist that has off-target activity to activate PP2A. In this work, we show evidence of FTY720's efficacy in AML cells derived from cell lines and patients, and provide preliminary data regarding SET expression in AML cell lines. METHODS: Cytotoxicity experiments were performed using HL-60, THP-1, MV-4, and Kasumi-3 cell lines, as well as patient-derived samples of AML, obtained through an IRB-approved protocol. Cells were incubated overnight with varied concentrations of FTY720, azacitidine, idarubicin, cytarabine, or drugs in combination. After incubation, cells were analyzed by colorimetric assay. Percent cytotoxicity was estimated as a proportion of light absorbance compared with blank media and untreated control cells. Inhibitory concentration of 50% of cells (IC50) was estimated using GraphPad Prism software, version 6.0. Flow cytometry experiments for confirmation of cytotoxicity were also performed with antibodies against Annexin V and propidium iodide. For estimation of SET expression, we performed ELISA with antibodies against SETα and SETß and quantified measurements by light absorption. RESULTS: FTY720inhibits growth of AML cells independently in both cell lines and patient-derived samples. In the THP-1 cell line, we estimated the IC50 of FTY720 to be 3.4 μM (Figure 1). In the HL-60 cell line, we estimated the IC50 to be 2.5 μM. In patient-derived samples of AML, we had similar findings. The mean IC50 was 3.24 μM (SD = 1.32, n = 8). Flow cytometry of tested samples confirmed induction of both apoptosis and cell death within a 3-hour time frame (Figure 2). Samples were also incubated with combination of FTY720 and conventional cytotoxic chemotherapeutic agents used in AML (Table 1). In the HL-60 cell line, the following IC50s were estimated for these drugs: idarubicin (0.02 μM); cytarabine (0.6 μM); azacitidine (5.7 μM). In combination with FTY720, there was no appreciable change. Results of ELISA showed measurable but low SETα and SETß levels, when compared to a known positive control, the Ramos cell line for Burkitt's lymphoma (Table 2). In the MV-4 AML cell line, the SETα/ß ratio was 0.096. In Kasumi-3 cells, the α/ß ratio was measured at 0.063. DISCUSSION: These data support the assertion that FTY720 is a cytotoxic agent in AML. This effect is independent of other cytotoxic agents, as no additive or synergistic effect was demonstrated when drugs were combined. The micromolar cytotoxicity poses challenges to the adoption of this agent as an active drug in AML, as serum concentrations from currently prescribed doses in multiple sclerosis have been shown to achieve only nanomolar concentrations. It is notable that the volume of distribution of FTY720 is very high and over 90% is concentrated in blood cells, so actual cell concentrations may be substantially higher. Our work has not yielded the same results others have reported with increased SET α/ß ratios in AML cells. In other tumor types, high SET alpha ratios have been associated with higher SET activity; thus, these results would not be suggestive of such a role in AML. Despite our findings, the activity of FTY720 in these cells merits further investigation into SET expression in AML. We have recently a flow cytometric assay for SETα and SETß that can be used to quantify SET levels, and we plan to analyze patient samples used in cytotoxicity experiments to help identify the SET α/ß ratio in AML. We hope that these experiments will establish SET and PP2A as targets for drug development in AML. Figure 1 Cytotoxicity curve of FTY720 in THP-1 cells (n=3) Figure 1. Cytotoxicity curve of FTY720 in THP-1 cells (n=3) Figure 2 Flow cytometric analysis of FTY720 cytotoxicity in HL-60 cells. Figure 2. Flow cytometric analysis of FTY720 cytotoxicity in HL-60 cells. Disclosures Rao: Gilead, Inc.: Employment.

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Cardile, Venera, Rosa Chillemi, Laura Lombardo, Sebastiano Sciuto, Carmela Spatafora et Corrado Tringali. « Antiproliferative Activity of Methylated Analogues of E- and Z-Resveratrol ». Zeitschrift für Naturforschung C 62, n^o 3-4 (1 avril 2007) : 189–95. http://dx.doi.org/10.1515/znc-2007-3-406.

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Abstract The stilbenoids E-resveratrol (E-3,5,4′-trihydroxystilbene, 1), E-3,5,4′-trimethoxystilbene (2), E-3,4,4′-trimethoxystilbene (3) and E-3,4′-dimethoxy-5-hydroxystilbene (4) were converted by photoisomerization to their corresponding Z-isomers 5D8. Compounds 1D8 were subjected to antiproliferative activity bioassays towards a set of four different human cancer cell lines, namely DU-145 (androgen not responsive human prostate tumor), LNCaP (androgen responsive human prostate tumor), M-14 (human melanoma) and KB (human mouth epidermoid carcinoma). The methylated analogues of 1 are more active than the natural lead in the majority of bioassays. The most active compound was Z-3,5,4′-trimethoxystilbene (6), which showed against DU-145 and LNCaP cells GI50 values close to those of the anticancer drug vinorelbine; 6 resulted more active than its E-isomer 2 towards DU-145, LNCaP and especially KB cell lines. A number of methylated Z-isomers displayed a higher activity than their E-isomers, but E-resveratrol (1) was more active than Z-resveratrol (5) towards all the tested cell lines.

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Quentmeier, Hilmar, Sonja Eberth, Roderick AF MacLeod, Stefan Nagel, Julia Romani, Margarete Zaborski et Hans G. Drexler. « CRLF2 and JAK2 Mutations : Occurrence and Function In Pre-B ALL Cell Lines ». Blood 116, n^o 21 (19 novembre 2010) : 5109. http://dx.doi.org/10.1182/blood.v116.21.5109.5109.

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Abstract Abstract 5109 Thymic stromal lymphopoietin (TSLP), a cytokine produced by epithelial cells promotes early B-cell development and activates dendritic cells. It has recently been reported that a subset of B-cell precursor acute lymphoblastic leukemia (pre-B ALL) overexpresses the TSLP receptor CRLF2. CRLF2 overexpression is linked to translocations between sex chromosomes – localizing CRLF2 – and the immunoglobulin heavy chain locus on chromosome 14, or to an interstitial deletion on the gonosomes. Both events, translocation and deletion juxtapose CRLF2 to a different promoter (IgH or P2RY8). Performing quantitative real-time PCR we tested pre-B ALL and acute myeloid leukemia (AML) cell lines for overexpression of CRLF2. AML cell lines were included in the screening because we knew from an earlier TSLP project that the AML cell line MUTZ-3 is TSLP-responsive, and thus positive for the cytokine receptor. Three of 63 (5%) pre-B ALL cell lines tested (INC, MHH-CALL4, MUTZ-5) overexpressed CRLF2 mRNA. CRLF2-high cell lines carry a t(14;Y). With respect to the 58 AML cell lines tested: some expressed CRLF2 mRNA, but none of them rivalled the aforementioned pre-B cell lines. Pre-B ALL cell lines show the association between chromosomal CRLF2 aberrations and JAK2 pseudokinase domain mutations that has been described for primary pre-B ALL cells: cell lines MHH-CALL4 (JAK2I682F) and MUTZ-5 (JAK2R683G) and – newly described - also the CRLF2-high pre-B ALL cell line INC express a mutated version of Janus kinase 2 (JAK2R683G). We established a PCR based assay system that allowed for the rapid detection of the JAK2R683G mutation: none of the CRLF2-low or –negative pre-B ALL cell lines exhibited this mutation. All three CRLF2-high/JAK2mu cell lines showed high phosphorylation levels of the JAK2 downstream target STAT5. Inhibition of the JAK kinase led to dephosphorylation of STAT5. However, repression of 3H-thymidine uptake and induction of apoptosis by inhibition of the JAK2/STAT5 pathway was weaker in the JAK2mu pre-B ALL cell lines than in the JAK2V617F positive essential thrombocythemia-derived cell line SET-2. Provided that these results reflect the situation in primary cells, mutated JAK2 seems to be of lesser importance for growth and survival of pre-B ALL cells than for cells from myeloproliferative neoplasms. The CRLF2-high/JAK2mu cell lines INC, MHH-CALL4 and MUTZ-5 are promising model systems for the study of the roles of high-level CRLF2 expression and of JAK2 mutations in pre-B ALL. Disclosures: No relevant conflicts of interest to declare.

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Luo, Jia, He Xiao, Qian Chen, Yanlan Li, Chuan Chen et Mingying Geng. « Abstract 5720 : Niraparib enhances radiosensitivity of glioblastoma cells with proficient BRCA1/2 through proliferation-related nucleolar protein DDX21 ». Cancer Research 82, n^o 12_Supplement (15 juin 2022) : 5720. http://dx.doi.org/10.1158/1538-7445.am2022-5720.

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Abstract Background Intrinsic resistance to irradiation is one of important factors leading to recurrence of glioblastoma (GBM) patients underwent standard treatment. The radiosensitization by various poly-(ADP-ribose)-polymerase (PARP) inhibitors have been intensively investigated in many solid tumor models especially harboring deficient BRCA1/2. However, the impact of a PARP1/2 inhibitor niraparib on radiosensitivity of glioblastoma cell lines with proficient BRCA1/2 and underling mechanisms has yet not been fully elucidated. Methods GDSC dataset with IC50 of PARP inhibitors and transcriptome profiles as well as NCI-60 cell lines with known survival fraction at 2 Gy (SF2) were used to deduce potential pathways by which PARP inhibitors suppress cell viability and confer radiosensitization through linear regression and reactome gene set enrichment analysis. Two GBM cell lines with proficient BRCA1/2, A172 and U-87-MG were used to evaluate IC50 and radiation dose enhancement factor DEF37 of niraparib by CCK8 experiment and clonogenicity assay. Changes in the distribution of cell cycle and DDX21 protein expression were analyzed with flow cytometry and western blot respectively. The involvement of DDX21 in the mechanism mediating radiosensization of niraparib was performed with small inference RNA experiment. Results Pathways relevant to ribosome biosynthesis and functions such as eukaryotic translation initiation, rRNA processing was found to be responsible for cytotoxicity of niraparib in 519 tumor cell lines. Moreover, mRNA expression of PARP1/2, genes participated in ribosome biosynthesis and homologous recombination (HR) were all significantly negatively associated with SF2 in 44 NCI-60 cell lines. The IC50 of niraparib in A172 and U-87-MG cell lines were 10.77±3.31 and 32.37±2.84 μM respectively. The DEF37 was established as 1.99 at 348 nM, 2.17 at 1044 nM for A172 cell line and 1.10 at 1056 nM, 1.44 at 3169 nM for U-87-MG cell line, respectively. Treatment with niraparib alone and in the combination with radiation (4 Gy) resulted in significant increase in fraction of G2 phase in both cell lines. Treatment with niraparib alone did not change protein expression level of DDX21 in both cell lines. However, knockdown of DDX21 with siRNA significantly reduced proliferation in U-87-MG cells. Niraparib at 1056 nM further decreased clonogenic number in the combination with 4 Gy radiation in U-87-MG cells compared with those treated with radiation alone. Importantly, knockdown of DDX21 resulted in significant increasing in clonogenic number of U-87-MG cells treated with radiation alone or the combination of niraparib and radiation compared with control cells. Conclusions Niraparib has radiosensitization effect for GBM cells with proficient BRCA1/2 at low concentration. DDX21 may be involved in the radiosensitization mechanism of niraparib. Citation Format: Jia Luo, He Xiao, Qian Chen, Yanlan Li, Chuan Chen, Mingying Geng. Niraparib enhances radiosensitivity of glioblastoma cells with proficient BRCA1/2 through proliferation-related nucleolar protein DDX21 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5720.

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Finlay, Jack, Clement Hallou, Pooran Dewari, Steven Pollard, Manav Pathania, Harry Bulstrode et David Rowitch. « Characterising a SOX2+ OLIG2+ glioma stem cell using Crispr-engineered fluorescent reporters in primary patient-derived GBM and DIPG cells. » Neuro-Oncology 21, Supplement_4 (octobre 2019) : iv9. http://dx.doi.org/10.1093/neuonc/noz167.039.

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Abstract Malignant glioma can be understood as a cancer stem cell disease. There is strong evidence that expression of a core set of neural stem cell master regulators is necessary and sufficient for tumour initiation in both adult and childhood disease subtypes, and across a range of underlying mutation signatures and expression profiles. We hypothesise that a double positive SOX2+ OLIG2+ progenitor is the cancer stem cell in H3K27M diffuse intrinsic pontine glioma (DIPG) and adult glioblastoma (GBM) alike. We present expression data in 4 patient-derived cell lines (2 adult GBM, 2 DIPG) in vitro and in a defined mutation mouse model of the H3K27M disease, demonstrating high expression levels of SOX2 and OLIG2 in serum-free culture and especially in the invasive tumour. We then demonstrate derivation of patient-derived human dual reporter lines, using a CRISPR targeting approach to add fluorescent reporters at the endogenous SOX2 and OLIG2 loci. With these tools, we aim to characterise the tumour-initiating potential of the SOX2+ OLIG2+ fraction. We further aim to identify cell surface markers whose expression is driven by this transcription program and which might represent targets for novel molecular therapies.

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Lee, Jinyoung, Sou Hyun Kim, Seung Tae Kim, Heegyu Kim, Huu Long Nguyen, Young-Suk Jung et Hwayoung Yun. « Discovery of 2-Anilinopyrimidine-based Selective Inhibitors against Non-small Cell Lung Cancer Cell Line H1975 ». Yakhak Hoeji 66, n^o 6 (30 décembre 2022) : 345–49. http://dx.doi.org/10.17480/psk.2022.66.6.345.

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Small molecular EGFR-tyrosine kinase inhibitors (EGFR-TKIs) have been proved as a successful and powerful strategy for the treatment of non-small-cell lung cancer (NSCLC). Recent studies have reported that the T790M mutation in EGFR is the most prevalent factor in acquired resistance for NSCLC patients. In an effort to identify small molecules for the inhibition of T790M mutant EGFR, a screening of our in-house chemical library was conducted by an MTT assay. A set of 2-anilinopyrimidine derivatives was rationally selected and then evaluated for their antiproliferative activities against three different type of NSCLC cell lines H358 (EGFR wild-type), HCC827 (EGFR exon 19 deletion) and H1975 (EGFR L858R/T790M double-mutant). Compounds 9, possessing a piperidine in the part A and a 4-methylpiperidine in the part B, selectively showed inhibitory activity in the T790M mutant cell line. In addition, in silico studies of 9 utilizing a SwissADME tool exhibited good drug-like properties.

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Tugcu, Gulcin, Hande Sipahi et Ahmet Aydin. « Application of a Validated QSTR Model for Repurposing COX-2 Inhibitor Coumarin Derivatives as Potential Antitumor Agents ». Current Topics in Medicinal Chemistry 19, n^o 13 (27 août 2019) : 1121–28. http://dx.doi.org/10.2174/1568026619666190618143552.

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Background: The discovery of novel potent molecules for both cancer prevention and treatment has been continuing over the past decade. In recent years, identification of new, potent, and safe anticancer agents through drug repurposing has been regarded as an expeditious alternative to traditional drug development. The cyclooxygenase-2 is known to be over-expressed in several types of human cancer. For this reason cyclooxygenase-2 inhibition may be useful tool for cancer chemotherapy. Objective: The first aim of the study was to develop a validated linear model to predict antitumor activity. Subsequently, applicability of the model for repurposing these cyclooxygenase-2 inhibitors as antitumor compounds to abridge drug development process. Method: We performed a quantitative structure-toxicity relationship (QSTR) study on a set of coumarin derivatives using a large set of molecular descriptors. A linear model predicting growth inhibition on leukemia CCRF cell lines was developed and consequently validated internally and externally. Accordingly, the model was applied on a set of 143 cyclooxygenase-2 inhibitor coumarin derivatives to explore their antitumor activity. Results: The results indicated that the developed QSAR model would be useful for estimating inhibitory activity of coumarin derivatives on leukemia cell lines. Electronegativity was found to be a prominent property of the molecules in describing antitumor activity. The applicability domain of the developed model highlighted the potential antitumor compounds. Conclusion: The promising results revealed that applied integrated in silico approach for repurposing by combining both the biological activity similarity and the molecular similarity via the computational method could be efficiently used to screen potential antitumor compounds among cyclooxygenase-2 inhibitors.

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Kawashima, Nozomu, Yusuke Okuno, Yuko Sekiya, Xinan Wang, Yinyan Xu, Atsushi Narita, Sayoko Doisaki et al. « Generation of Cell Lines Harboring SETBP1 Mutations By the Crispr/Cas9 System ». Blood 124, n^o 21 (6 décembre 2014) : 4622. http://dx.doi.org/10.1182/blood.v124.21.4622.4622.

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Abstract Introduction Recent advances in cancer genetics have led to the identification of somatic mutations in SET-binding protein 1 (SETBP1) in myeloid malignancies categorized as myeloproliferative neoplasm (MPN) and myelodysplastic syndromes (MDS). Heterozygous point mutations in SETBP1 are essentially found at a genomic level in myeloid malignancies, and the frequency of the mutated allele in cDNA suggests somatic heterozygosity without substantial imbalance in allelic expression. Thus, mutant SETBP1 presumably has a dominant altered biological activity. Most mutations in SETBP1 are located in the SKI homologous region. This region is suggested to include regions critical for ubiquitin binding and SETBP1 degradation. SETBP1 binds to SET, which protects against protease cleavage, and thus may result in PP2A inhibition and cell proliferation. Overexpression of SETBP1 resulting from a p.G870S alteration showed higher levels of the protein compared with wild-type (WT), indicating a prolonged halftime of SETBP1, which led to reduced PP2A activity and a higher cell proliferation rate. To date, however, our molecular biological understanding of SETBP1 mutations has been obtained only through observations of exogenous overexpression in cell lines. This may result in bias, considering the predicted dominant-negative function of SETBP1 mutations. Therefore, we used an RNA-guided endonuclease (RGEN), the CRISPR/Cas9 system, to generate a cell line harboring point mutations resulting in only relevant single amino acid substitutions in SETBP1. We analyzed cell signaling using the cell line thus established. Methods pSpCas9(BB) (PX330) was used to express humanized S. pyogenes Cas9 and gRNAs of interest. The gRNAs were designed by searching for NGG protospacer adjacent motif (PAM) sequences near the point mutation target sites. The candidate gRNAs were gRNA#1, 5′-TAGGGAGCCAATCTCGCAC-3′; gRNA#2, 5′-TGTCCCAATGCCGCTGTCGC-3′; gRNA#4, 5′-GTCCCAATGCCGCTGTCGCT-3′; and gRNA#7, 5′-GAGACGATCCCCAGCGACAG-3′. pCAG-EGxxFP harboring the 500 bp target region of WT SETBP1 was constructed for gRNA selection. For homology-dependent repair (HDR), we synthesized 70 mer single-stranded oligonucleotides (ssODN) having both the SETBP1 c.2602G>A, p.D868N mutation and synonymous mutation in the PAM. HEK293T cells were cultured in DMEM with 10% FBS. For cell signaling analysis, the cells were serum-depleted for 16 h prior to western blotting. Anti-SETBP1 antibody (ab98222), anti-phospho-Y307 PP2A antibody (E155), and anti phospho-p44/42 MAPK antibody (CST#4370) were used for cell signaling analysis. Results To validate an efficient sgRNA for DNA scission, we cotransfected pCAG-EGxxFP-SETBP1 and pSpCas9(BB)-SETBP1-gRNA plasmids into HEK293T cells. EGFP fluorescence, whose intensity is correlated with the efficacy of HDR, was observed 48 h later, and we determined that gRNA#2 was the most efficient. Next we cotransfected 293T cells with pCAG-EGxxFP-SETBP1, pSpCas9(BB)-SETBP1-gRNA#2, and ssODN for mutagenesis. Five days after transfection, single EGFP-positive clones were isolated using the FACSAria cell sorting system. Sanger sequencing confirmed that 293T cells harboring the SETBP1 p.D868N homozygous mutation were established. A clone with WT SETBP1 was also maintained as a control. To elucidate the effects of the SETBP1 mutation in 293T cells, we performed cell signaling analysis by western blotting. 293T-SETBP1 p.D868N cells showed higher levels of SETBP1 protein with lower molecular weight compared with WT, indicating a prolonged halftime, possibly due to loss of ubiquitination. In addition, 293T-SETBP1 p.D868N cells showed a higher phosphorylation level of PP2A (Y307, C subunit), a marker of PP2A inactivation. Finally, the phosphorylation level of p44/42 MAPK (ERK1/2) was increased in 293T-SETBP1 p.D868N cells. Conclusions We confirmed that the SETBP1 p.D868N mutation caused a prolonged halftime, resulting in PP2A inactivation and p44/42 MAPK activation in 293T cell lines. Our data suggest a potential therapy target for malignancies harboring SETBP1 mutations. More generally, this work illustrates the utility of RGEN technology for studying hematological malignancies. Disclosures No relevant conflicts of interest to declare.

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Kester, Monique H. A., George G. J. M. Kuiper, Rogier Versteeg et Theo J. Visser. « Regulation of Type III Iodothyronine Deiodinase Expression in Human Cell Lines ». Endocrinology 147, n^o 12 (1 décembre 2006) : 5845–54. http://dx.doi.org/10.1210/en.2006-0590.

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Type I iodothyronine deiodinase (D1) and type II iodothyronine deiodinase (D2) catalyze the activation of the prohormone T4 to the active hormone T3; type III iodothyronine deiodinase (D3) catalyzes the inactivation of T4 and T3. D3 is highly expressed in brain, placenta, pregnant uterus, and fetal tissues and plays an important role in regulating thyroid hormone bioavailability during fetal development. We examined the activity of the different deiodinases in human cell lines and investigated the regulation of D3 activity and mRNA expression in these cell lines, as well as its possible coexpression with neighboring genes Dlk1 and Dio3os, which may also be especially important during development. D1 activity and mRNA were only found in HepG2 hepatocarcinoma cells, and D2 activity was observed in none of the cell lines. D3 activity and mRNA was found in ECC-1 endometrium carcinoma cells, MCF-7 mammacarcinoma cells, WRL-68 embryonic liver cells, and SH-SY5Y neuroblastoma cells, but not in the HepG2 hepatocarcinoma cell line or in any choriocarcinoma or astrocytoma cell line. We demonstrated that the phorbol ester 12-O-tetradecanoylphorbol-13-acetate increased D3 activity 2- to 9-fold in ECC-1, MCF-7, WRL-68, and SH-SY5Y cells. Estradiol increased D3 activity 3-fold in ECC-1, but not in any other cells. Dexamethasone decreased D3 activity in WRL-68 cells only in the absence of fetal calf serum. Incubation with retinoids increased D3 activity 2- to 3-fold in ECC-1, WRL-68, and MCF-7 cells but decreased D3 activity in SH-SY5Y cells. D3 expression in the different cells was not affected by cAMP or thyroid hormone. Interestingly, D3 mRNA expression in the different cell lines strongly correlated with Dio3os mRNA expression and in a large set of neuroblastoma cell lines also with Dlk1 expression. In conclusion, we identified different human D3-expressing cell lines, in which the regulation of D3 expression is cell type-specific. Our data suggest that estradiol may be one of the factors contributing to the induction of D3 activity in the pregnant uterus and that in addition to gene-specific regulatory elements, more distant common regulatory elements also may be involved in the regulation of D3 expression.

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van den Akker, Guus G. H., Lars M. T. Eijssen, Stephen M. Richardson, Lodewijk W. van Rhijn, Judith A. Hoyland, Tim J. M. Welting et Jan Willem Voncken. « A Membranome-Centered Approach Defines Novel Biomarkers for Cellular Subtypes in the Intervertebral Disc ». CARTILAGE 11, n^o 2 (9 avril 2018) : 203–20. http://dx.doi.org/10.1177/1947603518764260.

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Objective Lack of specific marker-sets prohibits definition and functional distinction of cellular subtypes in the intervertebral disc (IVD), such as those from the annulus fibrosus (AF) and the nucleus pulposus (NP). Design We recently generated immortalized cell lines from human NP and AF tissues; these comprise a set of functionally distinct clonal subtypes. Whole transcriptome analyses were performed of 12 phenotypically distinct clonal cell lines (4× NP-Responder, 4× NP-nonResponder, 2× AF-Sheet forming, and 2× AF-nonSheet forming). Data sets were filtered for membrane-associated marker genes and compared to literature. Results Comparison of our immortal cell lines to published primary NP, AF, and articular chondrocytes (AC) transcriptome datasets revealed preservation of AF and NP phenotypes. NP-specific membrane-associated genes were defined by comparison to AF cells in both the primary dataset (46 genes) and immortal cell-lines (161 genes). Definition of AF-specific membrane-associated genes yielded 125 primary AF cell and 92 immortal cell-line markers. Overlap between primary and immortal NP cells yielded high-confidence NP-specific marker genes for NP-R ( CLDN11, TMEFF2, CA12, ANXA2, CD44) and NP-nR (EFNA1, NETO2, SLC2A1). Overlap between AF and immortal AF subtypes yielded specific markers for AF-S ( COLEC12, LPAR1) and AF-nS ( CHIC1). Conclusions The current study provides a reference platform for preclinical evaluation of novel membrane-associated cell type–specific markers in the IVD. Future research will focus on their biological relevance for IVD function in development, homeostasis, and degenerate conditions.

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Gupta, Vikas A., Scott Newman, Nizar J. Bahlis, Jonathan J. Keats, Shannon Matulis, Michael R. Rossi, Daniel Auclair et al. « B-Cell Markers Predict Response to Venetoclax in Multiple Myeloma ». Blood 128, n^o 22 (2 décembre 2016) : 2108. http://dx.doi.org/10.1182/blood.v128.22.2108.2108.

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Abstract BCL2 family members such as MCL1, BCLXL, and BCL2 are critical for cancer cell survival and therefore represent promising therapeutic targets. Both B cells and CLL cells depend primarily on BCL-2 and are thus sensitive to the BCL2 specific inhibitor venetoclax, while plasma cells and multiple myeloma typically depend on Mcl-1 and would therefore be resistant to venetoclax. However, a subset of myeloma is venetoclax sensitive based on recent in vitro and clinical trial data. In preliminary results from a phase I trial of venetoclax in multiple myeloma, 40% of patients positive for t(11;14) had objective responses, while only 6% of t(11;14) negative patients responded. We have made similar observations with in vitro testing of 30 freshly isolated myeloma patient samples, identifying both non-t(11;14) samples sensitive to venetoclax as well as resistant t(11;14) positive samples. Together, these results suggest not only that a subset of multiple myeloma is co-dependent on BCL2 but also that t(11;14) is neither necessary nor sufficient for responding to venetoclax. We therefore set out to identify other factors that may predict BCL2 dependence in multiple myeloma. Previous studies of t(11;14) myeloma have noted increased expression of CD20, CD23, CD79a, and PAX5 which are typically associated with B cells prior to their differentiation into plasma cells. Based on these observations we hypothesized that venetoclax sensitivity in myeloma may be associated with the retention of B cell properties including BCL2 dependence. We probed an online expression database of myeloma cell lines for non-t(11;14) cell lines expressing CD20 and identified two cell lines, OCI-My5 and PCM6, both of which we found to have an IC50 of approximately 50nM when treated with venetoclax. We went on to characterize a panel of 13 cell lines. In addition to OCI-My5 and PCM6, 4 other cell lines were sensitive to venetoclax, all positive for t(11;14). Of the 7 venetoclax resistant cell lines, 2 were t(11;14) positive. Protein levels of MCL1, BCLXL, and BCL2 were comparable among the 13 lines and therefore anti-apoptotic expression is unlikely to be responsible for venetoclax sensitivity. Consistent with our previous co-immunoprecipitation studies, more of the pro-apoptotic BIM was bound to BCL2 in venetoclax sensitive lines compared to resistant lines. In the absence of differences in BCL2 family expression, we next sought to identify other B cell related features correlating with venetoclax sensitivity. We used RNAseq data from our 13 cell lines to compare the expression of 100 genes previously reported to be differentially expressed between normal B cells and plasma cells. Interestingly, unsupervised clustering revealed a group of venetoclax sensitive cells enriched for other B cell associated genes. GSEA revealed enrichment of genes associated with immune system activation at a p < 0.001. We also analyzed the differential expression of genes between our sensitive and resistant lines and again identified overexpression of B cell related genes such as CD20, CD79A, STAT5A, and RASGRP2 in venetoclax sensitive lines, though no single marker was present in all of the venetoclax sensitive lines. We examined the expression of CD20, CD79a, and CD79b in the CoMMpass data set (IA8) as well and found that they were not co-expressed in most patients, again suggesting that no single marker is likely to be predictive. Finally, we created a gene signature from the top differentially expressed genes to predict sensitivity or resistance to venetoclax and used this signature to evaluate a database of 68 myeloma cell lines. One of the top hits predicted to be sensitive by our gene signature is the t(11;14) negative line MOLP2, and indeed this cell line was recently reported to be highly responsive to venetoclax. In conclusion, B cell markers and our gene signature correlate with BCL2 dependence and venetoclax sensitivity independent of t(11;14). Disclosures Bahlis: BMS: Honoraria; Onyx: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Other: Travel Expenses, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Other: Travel Expenses, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria. Nooka:Spectrum, Novartis, Onyx pharmaceuticals: Consultancy. Kaufman:Pharmacyclics: Consultancy; Incyte: Consultancy; Novartis: Consultancy, Research Funding; Celgene: Consultancy, Research Funding. Lonial:Onyx: Consultancy; Onyx: Consultancy; BMS: Consultancy; Janssen: Consultancy; Merck: Consultancy; Celgene: Consultancy; Novartis: Consultancy; Millenium: Consultancy; BMS: Consultancy; Celgene: Consultancy; Novartis: Consultancy; Janssen: Consultancy.

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Johnson, David, Carolyn Conant, Gary Withey, Andrea Löhr, Andro Hsu et Everett Meyer. « Massively parallel phenotyping and clonotyping of single T cells (P3380) ». Journal of Immunology 190, n^o 1_Supplement (1 mai 2013) : 135.16. http://dx.doi.org/10.4049/jimmunol.190.supp.135.16.

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Abstract Flow cytometry captures T cell functionality but not individual T cell receptor clonal sequences. DNA sequencing characterizes the T cell clonotype repertoire, but lacks functional information. We report a method, T cell receptor-Effector Linkage Sequencing (TELS), which uses single-cell microfluidics and next-generation sequencing (NGS) to determine clonotype and phenotype for millions of T cells simultaneously. Single T cells are encapsulated in picoliter droplets. Overlap extension reverse transcriptase PCR amplifies T cell receptor beta (TCRb) and immune effector transcripts separately in early cycles. In later cycles, a linker DNA sequence drives synthesis of fusion products containing both TCRb and effector amplicons. After sequencing the fused products, we process the NGS data to determine clonotype-specific effector expression for each clonotype in the sample. We characterized single-cell expression levels for TCRb, Fas ligand (FasL), and interleukin 2 (IL-2) in four mouse T cell lines using qPCR. Using these cell lines spiked at 0.1% in murine peripheral blood, we produced TELS libraries with a set of multiplexed primers targeting the full TCRb repertoire, FasL, and IL-2. Across >50 experiments, we accurately quantified (p<2.2x10-16) T cell spike-in clonotype and phenotype. TELS could be used routinely for applications where knowing both T cell clonotype and function are helpful for discovery, such as transplantation, vaccine development, and cancer.

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Rahbar, Kambiz, Mark S. Kidd, Ignat A. Drozdov, Alexandra Kitz, Anna Malczewska, Pawel Rajwa, Martin Boegemann, Christof Bernemann, Lisa Bodei et Irvin Mark Modlin. « A blood-based multi-mRNA liquid biopsy with >90% accuracy for diagnosis and assessment of prostate cancers. » Journal of Clinical Oncology 38, n^o 15_suppl (20 mai 2020) : 5574. http://dx.doi.org/10.1200/jco.2020.38.15_suppl.5574.

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5574 Background: There are a paucity of blood-based biomarkers with clinical utility for prostate cancer (PCa). We developed a circulating mRNA (27-gene) prostate cancer signature to diagnose and manage PCa. Methods: Gene identification: Publicly available PCa transcriptome sets ( n= 1,159 samples) were evaluated and compared with normal blood-based transcriptomes using gene co-expression network enrichment, differential expression and functional enrichment analyses to identify candidate markers. Gene expression evaluation: Seven PCA cell lines and two normal prostate epithelial lines were used to assess candidate genes. Marker genes were determined in PCa tumor tissue ( n= 50) and validated in the TCGA-PRAD ( n= 500) dataset. Blood gene expression: Set #I: PCA: n= 132, BPH: n= 44, controls n= 55. Set #II: n= 50 (biochemical recurrence [BCR]). We constructed an artificial intelligence PCa model using classification algorithm analyses. Scoring: normalized algorithmically analyzed gene expression (0 to 100), positive score >20. PSA: BPH ( n= 44) and PCa ( n= 132). Clinical score assessment: Surgical cohort: ( n= 47), samples: pre-surgical and post: 1 week - 14 months. Statistics: Kruskal-Wallis, Pearson-correlation, Fisher’s and AUROC analyses (Mean±SEM). Results: Transcriptomic analysis identified 27 candidates. Cell lines/tissue: Expression levels were significantly elevated ( p< 0.001, 2.1-35.8-fold) in cell lines and PCa surgical samples. All 27 markers were confirmed in TCGA-PRAD samples (average TPM: 58 to 10,366). Blood: In Set#I, levels in PCa were 47±2 ( p< 0.0001) compared to BPH (19±1) and controls (18±0.5); AUROC: 0.92 (BPH) and 0.94 (controls), with an accuracy of 85-88%, a sensitivity of 86% and specificities 82 and 93%. For PSA, the AUROC (PCa vs. BPH) was 0.51 ( p= 0.88). PSA was positive in 86% of BPH and was > 10ng/ml in 30%. PSA was positive in 83% of PCa and > 10ng/ml in 40% (Fisher’s p= 0.28). PSA accuracy ( > 10ng/ml) was 48%. Levels in Set#II (BCR) were 44±3. ProstaTest-was positive in 48 (96%). Surgical cohort ( n= 47): Prostatest accuracy 100% pre-surgery. Resection decreased levels (KW-statistic: 57.4, p< 0.0001) from 52±1 to 23.5±2. Conclusions: A 27-gene blood signature was developed for PCa that exhibited a diagnostic accuracy of 92%; significantly better than PSA (48%, p< 0.0001). Surgical resection significantly ( p< 0.0001) decreased levels. Biochemical recurrence was accurately detected (96%). A multi-gene prostate cancer liquid biopsy is likely to have clinical utility in both diagnosis and monitoring of PCa.

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Stiefelhagen, Marius, Marlon R. Veldwijk, Anna Jauch, Volker Eckstein, Stephanie Laufs, Juergen A. Kleinschmidt, Frederik Wenz, Jens W. Zeller, Anthony D. Ho et Stefan Fruehauf. « Generation and Application of a CML-Specific Recombinant Adeno-Associated Virus (rAAV) Vector. » Blood 106, n^o 11 (16 novembre 2005) : 4417. http://dx.doi.org/10.1182/blood.v106.11.4417.4417.

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Abstract Introduction: Chronic myelogenous leukemia can be controlled but in most patients not be cured by tyrosine kinase inhibition. Direct targeting using gene therapy vectors combined with vaccination strategies may allow to eradicate residual leukemic progenitors. Adeno-associated virus (AAV) vectors are stable DNA vectors which were proven to be effective in the clinical gene therapy for e.g. coagulation disorders. The various AAV serotypes lack specificity for BCR-ABL+ leukemia cells. Recently developed AAV-library techniques allow a retargeting of vectors. We generated a set of rAAV vectors specific for BCR-ABL+ cells. Material and Methods: After four selection rounds on BCR-ABL+ cells, the peptide sequences of the persisting clones were cloned into an AAV helper plasmid. rAAV-GFP stocks (2 K562-specific, 1 random) of each of the mutants were produced. Titers were determined using real time PCR-based titration assay. Both, a panel of leukemic cell lines and CML primary material were transduced with these vectors and gene transfer was determined by FACS analysis. Specificity was tested in a competitive transduction assay using BCR-ABL+ and BCR-ABL− leukemic cell lines. Transduction of primary CML cells was confirmed using FACS-sorted GFP+ cells and subsequent BCR-ABL-FISH. Results: Using the CML-specific rAAV clone on a panel of BCR-ABL+ cell lines, gene transfer rates of >60% could be obtained (random clone: <1%; rAAV-2: <5%), whereas the BCR-ABL− cell lines were not susceptible to these vectors (gene transfer < 1 %). Admixing BCR-ABL− to BCR-ABL+ cells did not result in a significant drop of the gene transfer rates in the BCR-ABL− cell lines, suggesting that vector particles were not blocked by unspecific binding. Using primary material, significant gene transfer was observed (>5%; 6x more efficient than rAAV-2). In those cells, the CML-genotype was confirmed by BCR-ABL-FISH. Conclusion: In this study, we were able to generate and apply a CML-specific rAAV vector on CML cell lines and primary material. Efficient and selective gene transfer in these cells could be obtained compared to standard rAAV-2 vectors and randomly generated clones. These data hold promise for future developments.

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Lahortiga, Idoya, Carlos Graux, Nicole Mentens, Katrien Van Roosbroeck, Kim De Keersmaecker, Frederic Lambert, Peter Vandenberghe et al. « Array-CGH Analysis of T-ALL Patients and Cell Lines. » Blood 108, n^o 11 (16 novembre 2006) : 4469. http://dx.doi.org/10.1182/blood.v108.11.4469.4469.

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Abstract Background Molecular analysis of T-cell acute lymphoblastic leukemia (T-ALL) has provided evidence that a stepwise alteration of at least four specific pathways is required during transformation of thymocytes to leukemic T-cells. Genetic alterations in hematopoietic precursor cells lead to loss of cell cycle control, impaired differentiation, proliferation and survival advantages, and unlimited self-renewal capacity. These defects include inactivation of CDKN2A (P16) present in 96 % of the patients, deregulated expression of transcription factors, and mutation of NOTCH1 in 56% of patients. However, the molecular lesions leading to the proliferative and survival advantages of T-ALL cells are less well characterized, remaining unknown in 80 % of the T-ALLs. Aims Our aim was to set up a genome-wide analysis of T-ALL in order to detect cryptic deletions and amplifications, with a special focus on the 90 protein tyrosine kinase genes present in the human genome. Methods We used the array-CGH (micro-array comparative genomic hybridization) technology with slides containing genomic BAC probes spaced every 1 Mb over the human genome. An additional 480 probes were added covering the genomic locations of each of the 90 protein tyrosine kinases genes. A total of 27 T-ALL cases and 12 cell lines were included in the study. Results An interstitial deletion on chromosome 9p24 directly upstream of JAK2 was identified in one patient. The deletion was confirmed by FISH. Quantitative PCR (qPCR) analyses indicated that the deletion was 700 kb in size including exons 1–4 of JAK2. In two cell lines, deletions affecting ALK and ERBB4 were detected. Molecular analyses to characterize the possible presence of fusion transcripts involving tyrosine kinases are in progress. We did not detected other rearrangements involving tyrosine kinase genes in neither of the 26 other T-ALL cases nor in the 10 cell lines, indicating that cryptic deletions or amplifications involving tyrosine kinase genes are relatively rare in T-ALL. The most frequent aberration was the deletion of CDKN2A (16/27 cases and 9/12 cell lines) ranging from only one clone to almost the complete 9p arm. MYB duplication was found in two cases and 4 cell lines, and was confirmed by qPCR and FISH analysis. Two of the 4 cell lines had overexpression of MYB detected by qPCR, which can interfere with apoptosis enhancing the survival of the cells, as has been previously described. PTEN deletion was present in one case and one cell line. Other unbalanced aberrations of various size were detected: del (2p) in 5/12 cell lines, del (5q) in 1/27 samples and 4/12 cell lines, del(6q) in 3/27 samples and 2/12 cell lines, or del(9p) in 5/27 samples and 4/12 cell lines. Some of these rearrangements were not observed by standard cytogenetics. Strikingly, cell lines had significantly more chromosomal abnormalities than primary T-ALL cases. Conclusions We detected a novel cryptic rearrangement of JAK2 in one T-ALL case, and duplication of MYB in two T-ALL cases (2/27; 7.5%) and four cell lines (4/12; 33%). Our results suggest that cryptic deletions or amplifications involving tyrosine kinase genes are relatively rare in T-ALL.

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Yamada, Yohei, Naotoshi Yamaguchi, Masakazu Ozaki, Yukihiro Shinozaki, Mikako Saito et Hideaki Matsuoka. « An Instant Cell Recognition System Using a Microfabricated Coordinate Standard Chip Useful for Combinable Cell Observation with Multiple Microscopic Apparatuses ». Microscopy and Microanalysis 14, n^o 3 (3 mars 2008) : 236–42. http://dx.doi.org/10.1017/s1431927608080252.

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AbstractDisposable coordinate standard (CS) chips were fabricated by the ejection of melted polystyrene into a metal mold. The CS chip surface was divided into four parts different in height and width. The edge lines of these parts could be recognized as straight lines 2 μm in width in the microscope view and used as the X and Y axes for the culture dish. The CS chip was attached on the bottom of a culture dish outside. Then the dish was set on the microscope stage and moved by means of a motorized automatic stage. The X-Y coordinates of many single-cells in a culture dish were registered, respectively. Once registered, any single-cell could instantly be brought to the center of the microscope view even after displacing the dish from the stage for a while and setting it again on the stage. Therefore, experimenters can easily search any single-cell in any culture dish on any microscope at any time. Such a system is remarkably useful for various modes of single-cell experiment and named “Suguwaculture,” which means “instantly” (“sugu” in Japanese) + “recognizable” (“wakaru” in Japanese) + “culture” (during culture).

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Abdul Razzaq, Eman, Khuloud Bajbouj, Amal Bouzid, Noura Alkhayyal, Rifat Hamoudi et Riyad Bendardaf. « Transcriptomic Changes Associated with ERBB2 Overexpression in Colorectal Cancer Implicate a Potential Role of the Wnt Signaling Pathway in Tumorigenesis ». Cancers 15, n^o 1 (26 décembre 2022) : 130. http://dx.doi.org/10.3390/cancers15010130.

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Colorectal cancer (CRC) remains the third most common cause of cancer mortality worldwide. Precision medicine using OMICs guided by transcriptomic profiling has improved disease diagnosis and prognosis by identifying many CRC targets. One such target that has been actively pursued is an erbb2 receptor tyrosine kinase 2 (ERBB2) (Human Epidermal Growth Factor Receptor 2 (HER2)), which is overexpressed in around 3–5% of patients with CRC worldwide. Despite targeted therapies against HER2 showing significant improvement in disease outcomes in multiple clinical trials, to date, no HER2-based treatment has been clinically approved for CRC. In this study we performed whole transcriptome ribonucleic acid (RNA) sequencing on 11 HER2+ and 3 HER2− CRC patients with advanced stages II, III and IV of the disease. In addition, transcriptomic profiling was carried out on CRC cell lines (HCT116 and HT29) and normal colon cell lines (CCD841 and CCD33), ectopically overexpressing ERBB2. Our analysis revealed transcriptomic changes involving many genes in both CRC cell lines overexpressing ERBB2 and in HER2+ patients, compared to normal colon cell lines and HER2− patients, respectively. Gene Set Enrichment Analysis indicated a role for HER2 in regulating CRC pathogenesis, with Wnt/β-catenin signaling being mediated via a HER2-dependent regulatory pathway impacting expression of the homeobox gene NK2 homeobox 5 (NKX2-5). Results from this study thus identified putative targets that are co-expressed with HER2 in CRC warranting further investigation into their role in CRC pathogenesis.

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Gautier, Emilie-Fleur, Muriel Picard, Camille Laurent, Caroline Marty, Jean-Luc Villeval, Cécile Demur, François Delhommeau et al. « The cell cycle regulator CDC25A is a target for JAK2V617F oncogene ». Blood 119, n^o 5 (2 février 2012) : 1190–99. http://dx.doi.org/10.1182/blood-2011-01-327742.

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Abstract The JAK2V617F mutation is present in the majority of patients with polycythemia vera and one-half of those with essential thrombocythemia and primary myelofibrosis. JAK2V617F is a gain-of-function mutation resulting in constitutive JAK2 signaling involved in the pathogenesis of these diseases. JAK2V617F has been shown to promote S-phase entry. Here, we demonstrate that the CDC25A phosphatase, a key regulator of the G1/S cell-cycle transition, is constitutively overexpressed in JAK2V617F-positive cell lines, JAK2-mutated patient CD36+ progenitors, and in vitro–differentiated proerythroblasts. Accordingly, CDC25A is overexpressed in BM and spleen of Jak2V617F knock-in mice compared with wild-type littermates. By using murine FDC-P1–EPOR and human HEL and SET-2 cell lines, we found that JAK2V617F-induced CDC25A up-regulation was caused neither by increased CDC25A transcription or stability nor by the involvement of its upstream regulators Akt and MAPK. Instead, our results suggest that CDC25A is regulated at the translational level through STAT5 and the translational initiation factor eIF2α. CDC25A inhibition reduces the clonogenic and proliferative potential of JAK2V617F-expressing cell lines and erythroid progenitors while moderately affecting normal erythroid differentiation. These results suggest that CDC25A deregulation may be involved in hematopoietic cells expansion in JAK2V617F patients, making this protein an attracting potential therapeutic target.

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