scholarly journals ATRA Upregulates Cell Surface CD1D on Myeloma Cells and Sensitizes Them to iNKT Cell-Mediated Lysis

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2102-2102
Author(s):  
Xin Li ◽  
Tarun K. Garg ◽  
Sarah K. Johnson ◽  
Susann Szmania ◽  
Justin Stivers ◽  
...  
Keyword(s):  
Gene Expression ◽  
Flow Cytometry ◽  
Retinoic Acid ◽  
High Risk ◽  
Cell Surface ◽  
Cell Lines ◽  
Target Genes ◽  
Inkt Cell ◽  
Inkt Cells

Abstract Invariant natural killer T (iNKT) cells recognize lipid antigens in the context of CD1D and have been shown to effectively kill CD1D-expressing multiple myeloma (MM) cells. It has been reported that iNKT cells in MM patients are deficient and dysfunctional and that CD1D is variably expressed among MM patients and is completely lost in MM cell lines. Recent clinical study suggests that targeting iNKT cells may prevent progression of asymptomatic MM (AMM) to active disease (Richter at al. Blood 2013). All-Trans Retinoic Acid (ATRA) has been used for the treatment of MM but its modes of action have not been completely elucidated. The aims of this study were to shed light on the mechanism of action of ATRA and to identify ATRA target genes in MM. ATRA at clinically relevant concentrations (1-2 μM, 4 days) had minimal growth inhibitory effect on the majority of MM cell lines (10±13% and 14±16%, respectively; n=10). Gene expression profile (GEP) of MM cell lines and primary cases (n=13) treated with ATRA (1 μM, 2 days) revealed upregulation of CD1D (381±204 vs. 5692±1675 mean±SEM GEP signal in CONT and ATRA groups, respectively; p<0.006). Flow cytometry analysis for cell surface CD1D in 21 MM cases treated in vitro with ATRA revealed significant CD1D induction in 11 cases, intermediate induction in 2 cases and no induction in 8 cases. Clinically, MM cells from patients who received ATRA as part of their salvage therapy also had induced CD1D gene expression compared to levels prior to ATRA therapy. Analysis of abnormal plasma cells based on a single 8-color markers flow cytometry showed higher CD1D coefficient of variation (CV) in AMM cases molecularly classified as high-risk (n=5) than low-risk cases (n=9, p<0.003), while mean florescence intensity (MFI) was insignificantly lower in high-risk AMM cases. In vitro ATRA treatment of BM cells from high-risk AMM patient resulted in increased CD1D MFI by 2 folds and reduced CV in abnormal plasma cell restricted population. To test the functional consequences of CD1D upregulation in MM cells, iNKT cells were purified from healthy donors’ peripheral blood mononuclear cells using anti-Vα24Jα18 immunomagnetic beads and expanded in vitro via stimulation with irradiated autologous α-GalCer-loaded mature dendritic cells (DCs) in the presence of IL-2. ATRA treated (1 μM, 2 days) compared to vehicle treated MM cells (n=4) were more sensitive to iNKT cell-mediated lysis in 5 hour 51chromium-release assays or bioluminescence viability assays (e.g. 21% vs. 75% lysis of H929 cells, p<0.0001, effector: target ratio 2.5:1). ATRA-induced MM cell lysis by iNKT cells was markedly blocked by CD1D neutralizing antibody (1-40 μg/ml) indicating that the enhanced cell lysis was mediated through CD1D. Further GEP analysis identified RARRES3 (retinoic acid receptor responder protein 3) as an additional top ATRA target gene in MM cells. RARRES3 gene expression was commonly but variably induced by ATRA whereas MM cells expressing high baseline RARRES3 were resistant to ATRA-induced CD1D gene and protein expression. Expression levels of CD1D and RARRES3 were inversely correlated in MM cells from newly diagnosed patients (n=449, r=-0.4, p<0.0001). Among molecularly classified groups, the MF subtype, known to be associated with poor survival, had the lowest CD1D/highest RARRES3 expression, whereas the favorable CD2 subtype had a reversed pattern. We conclude that CD1D and RARRES3 are ATRA target genes in MM cells and that stimulation of cell surface CD1D expression on MM cells enhances sensitivity to iNKT cell-mediated lysis. Disclosures van Rhee: Senesco: PI Other.

scholarly journals HPVE6-USP46 Mediated Cdt2 Stabilization Reduces Set8 Mediated H4K20-Methylation to Induce Gene Expression Changes

Cancers ◽  
2021 ◽  
Vol 14 (1) ◽  
pp. 30
Author(s):  
Shashi Kiran ◽  
Briana Wilson ◽  
Shekhar Saha ◽  
Julia Ann Graff ◽  
Anindya Dutta
Keyword(s):  
Gene Expression ◽  
Amino Acid ◽  
High Risk ◽  
Cell Lines ◽  
Deletion Mapping ◽  
Helix Loop Helix ◽  
Induced Tumors ◽  
Transformed Cell Lines ◽  
High Risk Hpv

E6 from high-risk strains of HPV is well known to transform cells by deregulating p53. We reported that in HPV transformed cell-lines E6 from high-risk HPV can recruit the USP46 deubiquitinase to substrates such as Cdt2 and stabilize the latter, and that USP46 is important for growth of HPV induced tumors in xenografts. Here we show that in cervical cancer biopsies the stabilization of Cdt2 in the HPV-induced cancers leads to the decrease of a CRL4-Cdt2 substrate, the histone H4K20 mono-methyltransferase Set8, and decrease in H4K20me1 or H4K20me3 that can be detected by immunohistochemistry. In HPV-transformed cancer cell lines in vitro, knockdown of E6 decreases Cdt2 and increases Set8. Co-knockdown of Set8 shows that some of the gene expression changes produced by E6 knockdown is due to the increase of Set8. EGFR and EGFR regulated genes were identified in this set of genes. Turning to the mechanism by which E6 stabilizes Cdt2, we find that a purified E6:USP46 complex has significantly more de-ubiquitinase activity in vitro than USP46 alone, demonstrating that E6 can directly interact with USP46 in the absence of other proteins and that it can substitute for the known activators of USP46, UAF1 and WDR20. Deletion mapping of Cdt2 shows that there are three discrete, but redundant, parts of the substrate that are essential for stabilization by E6: USP46. The helix–loop–helix region or the WD40 repeat driven beta-propeller structure of Cdt2 are dispensable for the stabilization implying that interaction with DDB1 (and the rest of the CRL4 complex) or with the substrate of the CRL4-Cdt2 E3 ligase is not necessary for E6:USP46 to interact with and stabilize Cdt2. The identification of 50 amino acid stretches in the 731 amino acid Cdt2 protein as being important for the stabilization by E6 underlines the specificity of the process. In summary, E6 activates the deubiquitinase activity of USP46, stabilizes Cdt2 utilizing multiple sites on Cdt2, and leads to degradation of Set8 and changes in gene-expression in HPV-transformed cells.

In Vitro Generation of Highly Purified Functional Invariant NKT Cells in Multiple Myeloma: A Strategy for Immunotherapy.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 5104-5104
Author(s):  
Weihua Song ◽  
Hans J.J. van der Vliet ◽  
Yu-Tzu Tai ◽  
Ruojie Wang ◽  
Rao Prabhala ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Immune Responses ◽  
Cell Lines ◽  
Healthy Donor ◽  
Inkt Cell ◽  
Inkt Cells ◽  
Myeloma Cells ◽  
Healthy Donors ◽  
Ifn Γ

Abstract Invariant NKT (iNKT) cells are important immunoregulatory cells that recognize glycolipid antigens with CD1d restriction and contribute to antitumor immune responses through the production of IFN-γ and IL-2. However, in progressive multiple myeloma (MM), the iNKT cell population is decreased along with its capacity to produce IFN-γ. Thus, a novel strategy for the immunotherapy of MM entails the enhancement of iNKT cell functions. In this study, we established iNKT cell lines from MM patients via enrichment with Vα24+ and subsequently with Vβ11+ cells, followed by several rounds of stimulation with α-GalCer-pulsed DCs. These techniques resulted in highly purified iNKT cell lines (&gt;97%). To evaluate potential in vivo interaction between iNKT cells and myeloma cells, we evaluated the CD1d expression on primary myeloma cells as well as MM cell lines. Gene expression profiling revealed compared to normal plasma cells, majority of primary MM cells (11 out of 15) expressed higher levels of CD1d; in contrast, all 6 MM cell lines tested had no expression. Flow cytometric analysis further confirmed the expression of CD1d on primary MM cells and lack of its expression on 12 different MM cell lines. A CD1d-transfected MM1S cell line (MM1S-CD1d) was therefore established for the functional study. To determine whether CD1d-expressing primary MM cells have the antigen presenting capacity, iNKT cell lines from healthy donors (n=2) and MM patients (n=2) were cocultured with 5 cases of CD1d positive primary MM cells with or without α-GalCer. Monitored by the CD25 expression, we demonstrated primary MM cells presented α-GalCer and also endogenous antigen(s) to activate iNKT cells. We have further evaluated the functional profile of expanded iNKT cell lines from MM patients (n=5). Upon stimulation with α-GalCer-pulsed MM.1S-CD1d cells, iNKT cells produced high levels of Th1-type cytokines (IFN-γ and IL-2) compared to low level Th2-type cytokine production (IL-4). Our results thus demonstrate that iNKT cell lines from MM patients were functionally restored by expansion with α-GalCer-pulsed DCs in vitro. To further augment iNKT cells function, we evaluated effects of lenalidomide on iNKT cell lines, an immunomodulatory drug which has been demonstrated to enhance T cell costimulation and NK cell activity. Lenalidomide did not directly stimulate iNKT cells in the presence or absence of α-GalCel. Importantly, upon CD1d-restricted activation by α-GalCer-loaded MM1S-CD1d cells, lenalidomide significantly enhanced the Th1-type immune responses of iNKT cell lines from both healthy donors and MM patients. Compared to those of controls, a significant increase of IFN- γ (healthy donor, p&lt; 0.001, n=7; MM patients, p&lt;0.05, n=3) and IL-2 (MM patients, p&lt;0.0015, n=3) occurred. Meanwhile, lenalidomide had no significant effect on the production of IL-4 by iNKT cell lines (healthy donor, p&gt;0.05, n=7; MM patients, p&gt;0.05, n=3). Taken together, our results provide preclinical feasibility and support a rationale to evaluate efficacy of adoptive transfer of iNKT cells in MM. Moreover, it provides a clinical basis for use of lenalidomide to enhance iNKT cell mediated immunotherapy in myeloma.

Multiple Myeloma Cells Modulate ICAM-3 To Evade Natural Killer Cell-Mediated Lysis

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3105-3105
Author(s):  
Tarun K. Garg ◽  
Ricky D Edmondson ◽  
Shweta S. Chavan ◽  
Junaid Khan ◽  
Susann Szmania ◽  
...  
Keyword(s):  
Amino Acids ◽  
Flow Cytometry ◽  
High Risk ◽  
Cell Surface ◽  
Natural Killer ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Differentially Expressed ◽  
Myeloma Cells ◽  
Chromium Release

Abstract Introduction Ex vivo activated/expanded natural killer (ENK) cells can induce myeloma cell lysis both in vitro and in murine models and are currently being studied clinically in the setting of high-risk relapsing disease and asymptomatic disease at high risk of progression. This prompted us to study, in myeloma cell lines, whether intrinsic resistance to ENK cell lysis exists, whether repeated challenge with ENK leads to increased resistance, and what the underlying mechanisms of resistance are. Of 11 myeloma cell lines tested in standard 4h chromium release assays, 8 were avidly killed (78-89% lysis, E:T Ratio 10:1) whereas 3 lines were less sensitive (41-65% lysis). Repeated exposure to ENK challenge decreased sensitivity in 4 of 11 lines, that was at least in part due to down-regulation of Tumor Necrosis Factor-Related Apoptosis Inducing Ligand-Receptors on the myeloma cell surface (Garg et al, Blood 2012, 120:4020). In this study we investigated the resistance issue further via metabolomics, gene expression profiling (GEP) and flow cytometry analysis of OPM2, which was intrinsically resistant and developed further resistance after challenge with ENK cells. Methods Metabolomics was studied using a quantitative proteomic strategy entailing stable isotope labeling with amino acids in cell culture – mass spectrometry (SILAC-MS). Resistant and parental OPM2 cells were grown either in medium with heavy amino acids (13C6 L-Lysine and 13C6 L-Arginine) or with light amino acids (12C6 L-Lysine and 12C6L-Arginine). Reverse labeling with heavy or light amino acids was also done to confirm the results. Cell lysates from heavy and light amino acid labeled cells were pooled, simultaneously resolved on SDS-PAGE, protein bands were excised and analyzed on a mass spectrometer after trypsin digestion. GEP was performed using the Affymetrix U133 Plus 2.0 microarray platform (Santa Clara, CA). The fold change of signal intensity for genes and proteins in resistant vs. parental OPM2 was calculated. The most differentially expressed genes (top 150-fold up or down) and proteins (up or down by 1.3-fold) were compared for commonality. Cell surface protein expression was determined via flow cytometry. The ability of ENK to lyse myeloma cell targets in the presence of isotype control or ICAM-3 blocking antibody was tested in 4h chromium release assays. Results Metabolomics identified >3800 proteins and revealed that the abundance of 352 proteins was significantly altered in resistant myeloma cells. These altered proteins were mainly associated with cell cycle, morphology, organization, cellular compromise, immune response, and survival. Further, a comparison of these differentially expressed proteins with GEP data revealed 3 commonly up-regulated molecules: TBC1D8B, HSPA1A and IFI16; and 2 down-regulated molecules: intercellular adhesion molecule (ICAM-3) and BAI3. Of these, ICAM-3, a ligand for leukocyte function-associated antigen-1 (LFA-1) and a potent signaling molecule, was selected for further studies. Flow cytometry confirmed that ICAM-3 cell surface expression was > 8-fold lower on resistant versus parental OPM2 cells. Further, blocking of ICAM-3 in cytotoxicity assays resulted in decreased lysis (43% blocked, E:T ratio 5:1), suggesting that this molecule is functionally important and takes part in ENK cell-mediated killing. Conclusion In conclusion, quantitative proteomic analysis demonstrated dynamic changes in the ENK-resistant OPM2 myeloma cells that correlated with GEP and differences in ICAM-3 expression may have functional implications. Studies evaluating the expression of ICAM-3 in myeloma patients at diagnosis and relapse are in progress. Myeloma cells may down-regulate ICAM-3 as a mechanism of escape from immune surveillance and therefore, ICAM-3 may be a useful biomarker to predict sensitivity to ENK cell-mediated killing and aid in the selection of patients most likely to benefit from ENK cell therapy. Disclosures: Barlogie: Celgene: Consultancy, Honoraria, Research Funding; Myeloma Health, LLC: Patents & Royalties.

scholarly journals WT1 and DNMT3A Play an Essential Function and Represent Therapeutic Vulnerabilities in Certain AML Samples, As Shown By CRISPR/Cas9 Mediated Knockout in PDX Models In Vivo

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 377-377
Author(s):  
Maryam Ghalandary ◽  
Yuqiao Gao ◽  
Martin Becker ◽  
Diana Amend ◽  
Klaus H. Metzeler ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Line ◽  
Cell Lines ◽  
Target Gene ◽  
Target Genes ◽  
Nsg Mice ◽  
Essential Function ◽  
Pdx Models

Abstract Background: The prognosis of patients with acute myeloid leukemia (AML) remains poor and novel therapeutic options are intensively needed. Targeted therapies specifically address molecules with essential function for AML and deciphering novel essential target genes is of utmost importance. Functional genomics via CRISPR\Cas9 technology paves the way for the systematic discovery of novel essential genes, but was so far mostly restricted to studying cell lines in vitro, lacking features of, e.g., primary tumor cells and the in vivo tumor microenvironment. To move closer to the clinical situation in patients, we used the CRISPR\Cas9 technology in patient-derived xenograft (PDX) models of AML in vivo. Methods: Primary tumor cells from seven patients with AML were transplanted into immunocompromised NSG mice and serially transplantable PDX models derived thereof. PDX models were selected which carry the AML specific mutations of interest at variant allele frequencies close to 0.5. PDX cells were lentivirally transduced to express the Cas9 protein and a sgRNA; successfully transduced PDX cells were enriched by flow cytometry gating on a recombinant fluorochrome or by puromycin. The customized sgRNA library was designed using the CLUE (www.crispr-clue.de) platform and cloned into a lentiviral vector with five different sgRNAs per target gene, plus positive and negative controls (Becker et al., Nucleic Acids Res. 2020). PDX cells were lentivirally transduced with the CRISPR/Cas9 sgRNA library, transplanted into NSG mice, grown in vivo and cells re-isolated at advanced AML disease. sgRNA distribution was measured by next generation sequencing and compared to input control using the MAGeCK pipeline. Interesting dropout hits from PDX in vivo screens were validated by fluorochrome-guided competitive in vivo experiments in the PDX models, comparing growth of PDX AML cells with knockout of the gene of interest versus control knockout in the same mouse. PDX cells were transduced with lentiviral vectors expressing a single sgRNA, using in parallel three different sgRNAs per target gene. Targeting and control sgRNAs were marked by different fluorochromes; PDX cells expressing targeting or control sgRNA were mixed at a 1:1 ratio, injected into NSG mice and PDX models competitively grown until advanced disease stage, when cell distributions was determined by flow cytometry. Human AML cell lines were studied in vitro for comparison. Results: In search for genes with essential function in AML, we cloned a small customized sgRNA library targeting 34 genes recurrently mutated in AML and tested the library in two PDX AML models in vivo. From the dropouts, we validated most interesting target genes using fluorochrome-guided competitive in vivo assays. Knockout of NPM1 abrogated in vivo growth in all PDX AML models tested, reproducing the known common essential function of NPM1. KRAS proved an essential function in PDX AML models both with and without an oncogenic mutation in KRAS, although with a stronger effect upon KRAS mutation, suggesting that patients with tumors both with and without KRAS mutation might benefit from treatment inhibiting KRAS. Surprising results were obtained for WT1 and DNMT3A. Both genes are frequently mutated in AML, but most AML cell lines tested in vitro do not show an essential function of any of the two genes, in published knockdown or knockout data, including from the Cancer Dependency Map database. On the contrary, knockout of either WT1 or DNMT3A was shown to enhance growth of AML cell lines and increase leukemogenesis in certain models. In PDX models in vivo, we found a clearly essential function for DNMT3A in all AML samples and WT1 in most samples tested and PDX in vivo results were discordant to cell line in vitro data, suggesting that cell line inherent features and/or the in vivo environment influence the function of WT1 and DNMT3A. Conclusion: We conclude that functional genomics in PDX models in vivo allows discovering essentialities hidden for cell line in vitro approaches. WT1 and DNMT3A harbor the potential to represent attractive therapeutic targets in AML under in vivo conditions, warranting further evaluation. Disclosures No relevant conflicts of interest to declare.

All Trans Retinoic Acid (atRA) Differentiation Markers in Normal and Retinoid-Resistant Acute Promyelocytic Leukemia Cells Revealed Induction of atRA Metabolism as Relevant Prognostic of APL Sensitivity to Therapy.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3256-3256 ◽  
Author(s):  
Ronan Quere ◽  
Aurelie Baudet ◽  
Bruno Cassinat ◽  
Gerald Bertrand ◽  
David Piquemal ◽  
...  
Keyword(s):  
Gene Expression ◽  
Retinoic Acid ◽  
Acute Promyelocytic Leukemia ◽  
Cell Lines ◽  
Target Genes ◽  
Expression Profiles ◽  
Promyelocytic Leukemia ◽  
Response Element ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid

Abstract It is now well established that all trans Retinoic Acid (atRA), administered at pharmacological doses to Acute Promyelocytic Leukemia (APL) patients, provides the first example of therapy by differentiation. Clinical remission is often transient as resistance develops. Because mechanisms of installation are still unclear, gene expression changes during APL cells differentiation were identified by Serial Analysis of Gene Expression (SAGE). Construction of proliferative and 48 hours atRA-treated NB4 cells libraries allowed us to identify a set of new transcriptional markers. Expression profiles of atRA response were performed on NB4, two atRA-resistant cell lines (NB4-LR2 and UF1) and APL blasts by real time PCR analyze on Microfluidic Card. We choose a hundred genes from multiple functional classes since there are several potential mechanisms for atRA resistance: target gene expression, transcription regulation, atRA metabolism, proteasome pathways≡ Reliability between SAGE and Microfluidic technologies is high since 95% of significantly modulated SAGE transcripts (p&lt;0.01) show the same modulation by PCR. Analyzes provide valuable markers of the granulocytic phenotype (ICAM3, S100A9, CYP4F3, TMSB10), relevant and new atRA target genes (HIC1, ID2). Moreover, SAGE highlights chromatin remodeling factors, histone deacetylase (HDAC11) or coactivator (NCOA3) which may play a crucial role in the differentiation process. Finally, we insulate markers that correlate with resistance (CEBPA, CRABP2, NDRG1, CYP26). Transcriptome studies were conducted onto blast of patients with distinct long-term sensitivity, established by correlation to the in vitro differentiation rate (Cassinat, B. et al. Blood, 2001). As a result, all patients show transcriptional response to retinoid. However, once blast differentiation reached, induction of atRA-response element genes in high sensitive blasts is reduced. In opposition, transcripts expression of low sensitive patients is still high, revealing a delay in differentiation establishment. Conversely, expression of cytochrome p450 CYP26, involved in the atRA catabolism, is maintained in highly sensitive blast whereas no modulation is observed in low sensitive blast. Because promoter analysis reveals Homeobox response element, involvement of HOX factors found in SAGE librairies was investigated. In cell lines, HOX factors cooperate with retinoid receptors to increase CYP26 transcription. Furthermore, high-pressure chromatography shows a switch between atRA and its metabolites, 6 hours after atRA addition but only in sensitive cell line. To conclude, CYP26 is a relevant marker of resistance prognostic. Since metabolites show similar efficiency for cell growth inhibition and differentiation than atRA, their implication in resistance installation is investigated.

Targeted Knock-Down of CD44 Expression In DLBCL Cells with Retrovirus-Mediated shRNA Interference

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1997-1997
Author(s):  
Ru Feng ◽  
Fangying Zheng ◽  
J. Jessica Yu ◽  
B. Hilda Ye
Keyword(s):  
Gene Expression ◽  
Flow Cytometry ◽  
Cell Surface ◽  
B Cell ◽  
Cell Lines ◽  
Cell Invasion ◽  
Cellular Behavior ◽  
Cd44 Expression ◽  
Knock Down

Abstract Abstract 1997 Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin's lymphoma, characterized by heterogeneity in its clinical, immunophenotypic, and genetic features. Gene-expression profiling studies have distinguished 3 molecular subtypes of DLBCL known as germinal center B-cell-like (GCB) DLBCL, activated B-cell-like (ABC) DLBCL, and type III. When treated with CHOP or other CHOP-like regimens, patients with GCB-DLBCL have a better survival than those with ABC-DLBCL. CD44 is a widely expressed type I transmembrane glycoprotein and functions as the major hyaluronan receptor on many cell types. In a number of solid tumors, CD44 plays an important role in tumor invasion and metastasis. In DLBCL, CD44 is expressed at significantly higher levels in ABC-DLBCL compared to GCB-DLBCL. In addition, co-expression of CD44 splice variants with the hyaluronan receptor predicts a very poor prognosis in patients treated with standard CHOP therapy. Despite these published observations, there is only limited understanding regarding the role of CD44 in the cellular behavior and gene expression control of DLBCL cells. To address this issue, we first profiled cell surface expression of CD44 in 4 DLBCL cell lines by flow cytometry. Two GCB-DLBCL lines (OCI-Ly1 and OCI-Ly8) and two ABC-DLBCL lines (SUDHL-2 and OCI-Ly3) were analyzed. The percentage of CD44 positive cells in SUDHL-2, OCI-Ly3, OCI-Ly1 and OCI-Ly8 cells was 74.57±5.03%, 44.01±3.70%, 0.12±0.05%, 0.14±0.03%, respectively. Next, we evaluated the invasion ability of 3 cell lines by the Matrigel Transwell assay. For the SUDHL-2, OCI-Ly1, and OCI-Ly8 cells, the percentage of invaded cells in 4 hours was 72.7±11.7%, 20.0±8.6%, and 30.3±7.5%, respectively. The ABC-DLBCL line, SUDHL-2, was significantly more invasive compared with OCI-Ly1 and OCI-Ly8 (P&lt;0.01). No significant difference was found between the two GCB-DLBCL lines, OCI-Ly1 and OCI-Ly8 (P=0.238). To study the role of CD44 in cell invasion, we down-regulated the expression of CD44 with retrovirus -mediated short hairpin RNA (shRNA) interference. After stable integration of retroviral vectors, stable subclones were obtained by puromycin selection. Effect of the knock-down on CD44 expression in ABC-DLBCL cells (SUDHL-2) was confirmed by flow cytometry. For SUDHL-2, CD44 level in shRNA expressing cells was reduced to 51.72±0.49% (one week), 38.31±1.24% (two weeks), and 16.29±11.17% (three weeks), respectively. In conclusion, we have confirmed the differential expression pattern of CD44 in DLBCL subtypes using cell line models. Our data also indicates that the level of CD44 expression positively correlates with cell invasion capability, such that ABC-DLBCL cells are more invasive compared with GCB-DLBCL cells. This difference may potentially contribute to the unfavorable treatment outcome of ABC-DLBCL. Lastly, we demonstrate that shRNA-mediated knock-down can stably and substantially reduce the endogenous CD44 expression on the cell surface. This experimental system is expected to provide a useful tool for further investigation of CD44 as a regulator of cellular behavior and chemotherapy response in DLBCL. Disclosures: No relevant conflicts of interest to declare.

Genome-Wide Analysis Reveals Conserved and Divergent Features of Notch1/RBPJ Binding in Human and Murine T Lymphoblastic Leukemia Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 5236-5236
Author(s):  
Jon C Aster ◽  
Hongfang Wang ◽  
James Zou ◽  
Yumi Yashiro-Ohtani ◽  
Bo Zhao ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cell ◽  
Cell Lines ◽  
Binding Sites ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Human T Cell ◽  
Two Factors

Abstract Abstract 5236 Activated Notch1 regulates gene expression by associating with the DNA-binding factor RBPJ and is an important oncoprotein in murine and human T cell acute lymphoblastic leukemia/lymphoma (T-ALL), yet the interplay between Notch1 and other factors that regulate the transcriptional output of T-ALL cells is poorly understood. Using ChIP-Seq and starting with Notch1-dependent human and murine T-ALL cell lines, we find that Notch1 binds preferentially to promoters, to RBPJ binding sites, and near sites for ZNF143, as well as Ets and Runx factors. By ChIP-Seq, ZNF143 binds to ∼40% of Notch1 sites, whereas Ets1 binding is observed within 100 basepairs of ∼70% of genomic Notch1 binding sites. Notch1/ZNF143 “co-sites” have high Notch1 and ZNF143 signals, frequent co-binding of RBPJ to sites embedded within ZNF143 motifs, strong promoter bias, and low mean levels of “activated” chromatin marks. RBPJ and ZNF143 binding to DNA is mutually exclusive in vitro, suggesting RBPJ/Notch1 and ZNF143 complexes exchange on these sites in T-ALL cell lines. In contrast, Ets1 binding sites flank RBPJ/Notch1 binding sites and are associated with high levels of activated chromatin marks, whereas Runx sites are predominantly intergenic. Although Notch1 predominantly binds promoters, ∼75% of direct Notch1 target genes lack promoter binding and appear to be regulated by enhancers, which were identified near MYC, DTX1, IGF1R, IL7R and the GIMAP gene cluster. Both Ets1 and Notch1 binding to an intronic enhancer located in DTX1 were required for expression of this well characterized Notch1 target gene, suggesting that these two factors coordinately regulate DTX1 expression. Although the association of Notch1 binding with ZNF143, Ets, and Runx sites was highly conserved, binding near certain important genes showed substantial divergence. For example, in human T-ALL lines Notch1/RBPJ bind a 3' enhancer near the IL7R gene, whereas in murine T-ALL lines no binding was observed near Il7r. Similarly, in human T-ALL lines Notch1/RBPJ bound an enhancer located ∼565 kb 5' of MYC, whereas in murine T-ALL cells Notch1/RBPJ bound an enhancer located ∼1 Mb 3' of Myc. Human and murine T-ALL genomes also have many sites that bind only RBPJ. Murine RBPJ “only” sites are highly enriched for imputed sites for the corepressor REST, whereas human RPBJ “only” sites lack REST motifs and are more highly enriched for imputed CREB binding sites. Thus, there is a conserved network of cis-regulatory factors that interacts with Notch1 to regulate gene expression in T-ALL cell lines, as well as novel classes of divergent RBPJ “only” sites that also likely regulate transcription. To extend these findings to normal and pathophysiologic tissues, ChIP-Seq was used to identify RBPJ/Notch1 binding sites in primary murine thymocytes and primary murine T-ALL associated with Notch1 gain-of-function mutations. Early findings appear to indicate that primary T-ALLs closely resemble normal DN3a thymocytes in terms of the distribution of Notch1 binding sites and associated chromatin marks. These data suggest that Notch1-driven T-ALLs epigenetically resemble the DN3a stage of T cell development, during which Notch1 signaling is high and cells are rapidly proliferating. Disclosures: No relevant conflicts of interest to declare.

Resveratrol Exerts Antiproliferative Effect and Induces Apoptosis in Waldenstrom’s Macroglobulinemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1383-1383
Author(s):  
Aldo Roccaro ◽  
Xavier Leleu ◽  
Anne-Sophie Moreau ◽  
Antonio Sacco ◽  
Lian Xu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Western Blot ◽  
Cell Lines ◽  
Expression Analysis ◽  
Gene Expression Analysis ◽  
Downstream Target ◽  
Dose Dependent

Abstract Background: Resveratrol is a polyphenolic natural product, synthesized by a wide variety of plant species including grapes. It has gained considerable attention because of its anti-cancer properties, as demonstrated in solid and haematological malignancies. We therefore examined Resveratrol for its anti-tumor activity in Waldenstrom’s Macroglobulinemia (WM). Methods: We examined the effect of increasing concentrations of resveratrol (5–80 μM) on WM cell lines (BCWM.1), IgM secreting low-grade lymphoma cell lines (WM-WSU, MEC-1, RL), peripheral blood mononuclear cells (PBMCs) isolated from healthy donors, primary CD19+ WM cells and bone marrow stromal cells (BMSCs) isolated from bone marrow of patients with WM. [3H]-thymidine uptake and calcein-AM assay were used to evaluate the effect of resveratrol on proliferation and cytotoxicity respectively. Apoptosis and cell cycle analysis were investigated at 24h by flow cytometry using Annexin V-propidium iodide (PI) staining and PI-staining respectively. Apoptotic and cell signaling pathways targeted by resveratrol were investigated by Western Blot at 24 h and 6 h respectively. Since BMCSc confer growth and resistance to conventional treatments, we also tested the effect of resveratrol on WM cells co-cultured with BMSCs. Gene expression analysis has been performed on BCWM.1 cultured in presence or absence of resveratrol. Results: Resveratrol induced significant cytotoxicity and inhibition of DNA synthesis at 24 and 48 h on BCWM.1 with an IC50 of 10–20μM. Similar data was obtained with primary CD19+ WM cells. In contrast, resveratrol did not trigger significant reduction of proliferation of PBMCs. Resveratrol induced apoptosis in BCWM.1, as demonstrated by flow cytometry. Dose-dependent apoptosis at 24h with induction of JNK followed by caspases 3, 8, 9 and PARP cleavage was also observed. Resveratrol induced reduction of Mcl-1 and increase of p53, p63 and p73, as shown by gene expression analysis and western blot, providing an alternative mechanism of cell growth arrest in absence or mutation of p53. In parallel, resveratrol induced down-regulation of cyclin-D1, -D2, -E1, cdk-2, -4, -6 and up-regulation of p21Cip1 and p27Kip1, demonstrated in terms of transcript by gene expression analysis and protein levels by western blotting. We next observed that resveratrol inhibited ERK and Akt phosphorylation in BCWM.1 in a dose-dependent manner, as well as Akt activity, as shown by the in vitro Akt kinase assay. Phosphorylation of GSK3α/β and ribosomal protein-S6, downstream target proteins of Akt, were also markedly inhibited. Resveratrol also down-regulated Wnt signaling pathway with a reduction of nuclear β-catenin levels and a decrease of myc and survivin, both downstream target proteins of β-catenin. Lastly, adherence to BMSCs did not confer protection to WM cells against resveratrol-induced cytotoxicity Furthermore, resveratrol demonstrated synergistic cytotoxicity when combined with dexamethasone, fludarabine and bortezomib. Conclusion: These in vitro data demonstrated that resveratrol has significant antitumor activity in WM, providing the framework for clinical trials in WM patients.

scholarly journals Retinoic Acid Sensitivity of Triple-Negative Breast Cancer Cells Characterized by Constitutive Activation of the notch1 Pathway: The Role of Rarβ

Cancers ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 3027
Author(s):  
Gabriela Paroni ◽  
Adriana Zanetti ◽  
Maria Monica Barzago ◽  
Mami Kurosaki ◽  
Luca Guarrera ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Retinoic Acid ◽  
Cell Lines ◽  
Triple Negative Breast Cancer ◽  
Target Genes ◽  
Triple Negative ◽  
Synergistic Effects ◽  
Acid Sensitivity ◽  
Constitutive Activation

Triple-negative breast cancer (TNBC) is a heterogeneous disease that lacks effective therapeutic options. In this study, we profile eighteen TNBC cell lines for their sensitivity to the anti-proliferative action of all-trans retinoic acid (ATRA). The only three cell lines (HCC-1599, MB-157 and MDA-MB-157) endowed with ATRA-sensitivity are characterized by genetic aberrations of the NOTCH1-gene, causing constitutive activation of the NOTCH1 γ-secretase product, N1ICD. N1ICD renders HCC-1599, MB-157 and MDA-MB-157 cells sensitive not only to ATRA, but also to γ-secretase inhibitors (DAPT; PF-03084014). Combinations of ATRA and γ-secretase inhibitors produce additive/synergistic effects in vitro and in vivo. RNA-sequencing studies of HCC-1599 and MB-157 cells exposed to ATRA and DAPT and ATRA+DAPT demonstrate that the two compounds act on common gene sets, some of which belong to the NOTCH1 pathway. ATRA inhibits the growth of HCC-1599, MB-157 and MDA-MB-157 cells via RARα, which up-regulates several retinoid target-genes, including RARβ. RARβ is a key determinant of ATRA anti-proliferative activity, as its silencing suppresses the effects exerted by the retinoid. In conclusion, we demonstrate that ATRA exerts a significant anti-tumor action only in TNBC cells showing constitutive NOTCH1 activation. Our results support the design of clinical trials involving combinations between ATRA and γ-secretase inhibitors for the treatment of this TNBC subtype.

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