Investigation of the Mechanism of Idelalisib Resistance in the Follicular Lymphoma WSU-Fsccl Cell Line

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2482-2482 ◽  
Author(s):  
Rick Sorensen ◽  
Sarah Meadows ◽  
Anella Yahiaoui ◽  
Li Li ◽  
Peng Yue ◽  
...  
Keyword(s):  
Western Blot ◽  
Growth Inhibition ◽  
Follicular Lymphoma ◽  
Single Cell ◽  
Cell Line ◽  
Wnt Pathway ◽  
Parental Line ◽  
Rna Seq ◽  
Resistance Mutations ◽  
Cell Clones

Abstract Idelalisib (Zydelig®), a first-in-class, selective, oral inhibitor of PI3Kd, is approved in the US and EU for the treatment of patients with follicular lymphoma (FL) who have received at least 2 prior systemic therapies based on the outcome of a phase 2 clinical trial demonstrating an ORR of 54% (Gopal AK, et al. ASH 2014, Abstract 1708; Zydelig® SmPC, Mar 2015). Complete responses are rare (6%) and patients ultimately relapse leading to an urgent need to understand mechanisms of resistance (MOR) to idelalisib. We report here the MOR identified from 3 sets of the transformed FL cell line WSU-FSCCL which were made resistant to idelalisib by continuous in vitro exposure. Methods: Idelalisib resistance was established by continuous passaging of a clonal isolate of WSU-FSCCL in the presence of 1 μM idelalisib. Growth inhibition to idelalisib or other inhibitors was performed using CellTiter Glo viability assay (Promega) at 96 h. Clonal isolates of idelalisib sensitive (FSCCLS) and idelalisib resistant (FSCCLR) cell lines were generated through two rounds of single cell limiting dilution. Characterization of mutations and gene expression in FSCCLS and FSCCLR clones was done by whole exome sequencing (WES, GENEWIZ) and RNA-Seq (Expression Analysis), respectively. Whole cell lysates were analyzed by Simple Western using Peggy Sue (Protein Simple) or SDS-PAGE and Western blot. Results: WSU-FSCCL were highly sensitive to idelalisib and the pan-PI3K inhibitor GDC-0941 with an EC50 of 140 and 180 nM, respectively, indicating that cell viability is driven by PI3Kd, while profiling of BTK inhibitors showed continued lack of activity. FSCCLR showed a loss of sensitivity to idelalisib (1 μM) with a maximal growth inhibition of 16% vs. 85% for parental line (FSCCLS). WES analysisrevealed PIK3CA resistance mutations in 3 independently generated sets of FSCCLR single cell clones, while no PIK3CD, PIK3CB, or PIK3CG resistance mutations were observed. PIK3CA was mutated at N345K (11/11 clones), P539R (3/3 clones) and E970K (3/3 clones). PI3K isoform profiling showed no alterations in expression of any of the four p110 isoforms. Idelalisib activity on PIK3CA mutant clone viability was dramatically shifted (EC50 >5 μM). The sensitivity of PIK3CA mutant FSCCLR to the PI3Kα- specific inhibitor BYL719 was not increased compared to FSCCLS (EC50 > 1.5 μM).Interestingly the combination of idelalisib treatment with BYL719 (0.5 μM) restored the sensitivity of FSCCLR to a concentration in the range of parental cells (EC50 = 0.066 μM, Figure 1). A second set of FSCCLR clones (set 2) were PI3KCA WT and had a 2.7-fold upregulation of PTEN. A survey of compensatory pathway activation revealed upregulation of Src family kinase (SFK) phosphorylation (p-SFK Y416) and specifically of p-Hck Y411 and p-Lyn Y396. SFK phosphorylation was sensitive to treatment with the SFK inhibitor dasatinib. Set 2 FSCCLR were slightly less sensitive to dasatinib (EC50 = 0.058 μM) compared to FSCCLS (EC50 = 0.034 μM). Interestingly, addition of 30 nM dasatinib (EC25 in FSCCLS) to FSCCLR increased the sensitivity of set 2 FSCCLR to idelalisib (EC50 = 0.95 μM) (Figure 2). FSCCLR clones were more resistant to the Syk inhibitor entospletinib (ENTO) (EC50 > 10 μM) as compared to FSCCLS (EC50 = 0.18 μM). Profiling of idelalisib activity on set 2 FSCCLR clones showed resistance to idelalisib as expected (EC50 > 10, μM); the addition of entospletinib at a clinically relevant concentration (0.68 μM), resulted in greater sensitivity than use of either single agent alone (EC50 = 2.27 μM ) (Figure 3). RNA-Seq analysis of the FSCCLR PIK3CA WT single cell clones additionally revealed that a subset of clones (set 3) upregulated a set of WNT pathway genes. Western blot analysis of set 3 FSCCLR showed upregulation of downstream markers of the canonical WNT pathway, including LEF1/TCF, c-Jun, β-catenin, c-Myc and p-GSK3β. Conclusions: Mechanisms of idelalisib resistance in the follicular lymphoma WSU-FSCCL cell line were independent of alterations in p110d but included the likely gain of function mutations in the PIK3CA gene and activation of SFK and WNT pathways. The sensitivity to idelalisib in the resistant clones could be re-established by the combination of idelalisib with inhibitors of compensatory pathways: BYL719 in PIK3CA mutant clones and dasatinib or entospletinib in SFK activated clones. Disclosures Sorensen: Gilead Sciences: Employment, Other: Share holder. Meadows:Gilead Sciences: Employment, Other: Share holder. Yahiaoui:Gilead Sciences: Employment, Other: Share holder. Li:Gilead Sciences: Employment, Other: Share holder. Yue:Gilead Sciences: Employment, Other: Share holder. Kashishian:Gilead Sciences: Employment, Other: Share holder. Queva:Gilead Sciences: Other: Share holder. Tannheimer:Gilead Sciences: Employment, Other: Share holder.

Growth Inhibition of a Myeloid Leukemia Cell Line with 5q Deletion by Beta-Catenin Inhibition

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3608-3608 ◽  
Author(s):  
Amber D. Seba ◽  
Qi Wang ◽  
Ming Ming ◽  
Zhijian Qian
Keyword(s):  
Stem Cells ◽  
Western Blot ◽  
Growth Inhibition ◽  
Hematopoietic Stem Cells ◽  
Cell Line ◽  
Wnt Pathway ◽  
Beta Catenin ◽  
Hematopoietic Stem ◽  
Remission Rates ◽  
5Q Deletion

Abstract Abstract 3608 As the population ages, the incidence of AML with complex cytogenetics continues to increase. These patients have a poor prognosis with 5% percent of patients diagnosed surviving 5 years. New strategies are needed to improve survival in AML patients. 5q deletion is present in 6–10% of cases of AML, and is a common chromosome abnormality in therapy-related AML (t-AML). 5q deletion is associated with haplo-insufficiency of the APC gene in hematopoietic stem cells and progenitors, leading to deregulation of the Wnt pathway and stabilization of beta- catenin (Wang, J Blood 2010). Loss of APC causes rapid exhaustion of hematopoietic stem cells and progenitors in vivo and leads to the accumulation of beta-catenin (Qian, Z et al JEM 2008). Beta-catenin is required for the development of leukemia stem cells in AML, but it is not required for the self-renewal of normal hematopoietic stem cells (Wang, Y et al Science 2010). Development of treatment strategies aimed at blocking the Wnt pathway through suppression of beta-catenin may improve and prolong remission rates. The UoCM1 cell line maintains the unique characteristics of del(5q) leukemia (Qian et al, PNAS 2002). Western Blot shows that the expression of beta-catenin was higher in UoCM1 cells versus REH and MV4–11 cell lines, which do not have del(5q). To block the activation of Wnt signaling, through suppression of beta-catenin, we treated UoCM1, REH and MV4–11 cells with 100uM indomethacin. The UoCM1 cells showed 30% growth inhibition while REH and MV-4 cells displayed 10% growth inhibition. The down-regulation of beta-catenin in UocM1 is confirmed by Western Blot. Curcumin is a dietary polyphenol existing in Indian spice turmeric. We showed suppression of beta-catenin by curcumin in 293T cells by luciferase assay. Next, we treated UoCM1 and MV4–11 cells with 5uM or 10 uM curcumin. The growth of UoCM1 cells was significantly inhibited by curcumin as compared to MV4–11 cells. Treatment of UoCM1 cells with 10 uM curcumin produced 87% growth inhibition, while the growth inhibition was 29% for MV4–11 cells. 5uM concentrations of curcumin caused 48% growth inhibition in UoCM1 cells versus 16% in the MV4–11 cell line. Western Blot was performed to confirm that beta-catenin is down-regulated by curcumin, in the UoCM1 cell line. In summary, blockade of the Wnt pathway through inhibition of beta-catenin suppresses cell growth in UoCM1 leukemia cells, with 5q deletion. Suppression of beta-catenin in combination with cytotoxic therapy may provide a novel means of treating leukemia in patients with a 5q deletion that both improves remission rates and prevents relapse. Disclosures: No relevant conflicts of interest to declare.

scholarly journals ARID1A Controls a Novel Transcriptional Network Regulating FAS in Follicular Lymphoma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3492-3492
Author(s):  
Martina Antoniolli ◽  
Maria Solovey ◽  
Deepak Bararia ◽  
Carolin Dorothea Strobl ◽  
William David Keay ◽  
...  
Keyword(s):  
Western Blot ◽  
Follicular Lymphoma ◽  
Single Cell ◽  
Research Funding ◽  
Chromatin Accessibility ◽  
Mrna Levels ◽  
Rna Seq ◽  
Fas Mrna ◽  
Fas L ◽  
Induced Apoptosis

Abstract Follicular lymphoma (FL) is a clinically and genetically heterogeneous disease. Somatic gene mutations contribute to the heterogeneous clinical course of FL. ARID1A, which encodes for a subunit of the SWI/SNF chromatin remodeling complex, is among the most commonly mutated genes in FL (up to 15% of cases). These mutations are mostly disruptive and are predicted to result in protein haplodeficiency. While we have previously shown that ARID1A mutations are predictive of treatment outcome (Pastore, 2015), the underlying biology of ARID1A loss in FL is unclear. A functional genome-wide in vitro screen showed that ARID1A loss rescued a number of cancer cell lines from FAS-L induced apoptosis (Luo, 2008). FAS-L induced apoptosis plays a critical role in normal B-cell development and homeostasis. Thus, FAS/FAS-L deficiency could contribute to FL development and disease biology. Therefore, we studied the role of ARID1A loss in FAS expression and regulation. We first tested FAS-L induced apoptosis in established lymphoma cell lines that harbor the FL-hallmark translocation t(14;18)[BCL2/IGH] plus ARID1A mutations (Karpas422, WSU-FSCCL) or no ARID1A mutations (OCI-Ly1, OCI-Ly8, SU-DHL16). ARID1A mutant (mut) cells were indeed markedly less sensitive to FAS-L (300 ng/mL/24 hrs) compared to ARID1A wild type (WT) cells (98% vs 52% mean viability by Annexin-V). FAS receptor expression on mutant cells was reduced by almost half compared to WT cells by FACS analysis (N=3, P=0.0004). To test if reduced FAS expression was directly linked to ARID1A loss, we generated single-cell derived clones (from OCI-Ly1 and OCI-Ly8) with either heterozygous (het) loss or complete knock-out (KO) of ARID1A by CRISPR/Cas9. ARID1A loss was validated by Sanger sequencing and Western blot. We consistently observed significantly reduced FAS-L induced apoptosis in het and KO clones (exemplary shown for OCI-Ly8 in Fig A). Remarkably, re-expressed of ARID1A in het cells (het+ARID1A) rescued sensitivity to FAS-L induced apoptosis (Fig A). We confirmed reduced FAS expression on mutant clones by FACS, while re-expression of ARID1A rescued its expression (Fig B). Furthermore, FAS mRNA expression was significantly reduced by qPCR in mut vs WT clones (N=4, P<0.05), while FAS mRNA levels were rescued to WT levels in het+ARID1A cells. To understand the molecular mechanism that links ARID1A loss and reduced FAS expression, we performed ATAC sequencing (Seq) and RNA Seq on 15 single-cell derived clones (9 mut and 6 WT from OCI-Ly1 and OCI-Ly8). RNA Seq confirmed significantly lower ARID1A and FAS mRNA levels (adj p<0.001 each) in the mut clones. We first hypothesized that ARID1A loss could directly affect chromatin accessibility at the FAS promoter. However, we did not observe different chromatin accessibility at the FAS promoter. Next, we searched our data for all known FAS-regulating transcription factors (TFs) (https://dorothea.opentargets.io/#/), but could not identify candidates that were both differentially accessible and differentially expressed. Finally, we searched our data for transcriptional networks, i.e. hubs of all recognized FAS-regulating TFs and their known and predicted interacting partners (https://string-db.org/). Through this, we identified RUNX3, a predicted Co-TF of ETS1, to be both less accessible ("closed chromatin") and less expressed upon ARID1A loss (Fig C), suggesting a novel ARID1A-dependent FAS-regulatory network. To functionally validate our model, we first confirmed reduced RUNX3 expression in ARID1A mutant clones by qPCR and Western blot, and showed that ETS1 levels were unaffected by ARID1A loss. Then, we stably overexpressed RUNX3 in ARID1A mutant clones by lentiviral transduction and could indeed show rescue of FAS surface levels by FACS (Fig D). Lastly, we wanted to validate our findings in primary patients samples. We quantified FAS expression in FL biopsies with known ARID1A mutation status by nCounter gene expression profiling (GEP; N=51, 12 mut vs 39 WT) and quantitative multispectral imaging (QMI; N=44, 10 mut vs 34 WT) (Fig E). Both approaches showed significantly reduced FAS expression in ARID1A mutant FL (P<0.05 for GEP, P<0.0001 for QMI; Fig E). In summary, we show that ARID1A loss is directly linked to reduced FAS expression via a novel RUNX3/ETS1 transcriptional network, potentially opening avenues for therapeutic targeting of this clinically relevant perturbation. Figure 1 Figure 1. Disclosures Subklewe: Pfizer: Consultancy, Speakers Bureau; Takeda: Speakers Bureau; Klinikum der Universität München: Current Employment; Janssen: Consultancy; Seattle Genetics: Consultancy, Research Funding; Roche: Research Funding; Novartis: Consultancy, Research Funding, Speakers Bureau; MorphoSys: Research Funding; Miltenyi: Research Funding; Gilead: Consultancy, Research Funding, Speakers Bureau; Amgen: Consultancy, Research Funding, Speakers Bureau; BMS/Celgene: Consultancy, Research Funding, Speakers Bureau. von Bergwelt: Kite/Gilead: Honoraria, Research Funding, Speakers Bureau; Roche: Honoraria, Research Funding, Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau; Astellas: Honoraria, Research Funding, Speakers Bureau; Miltenyi: Honoraria, Research Funding, Speakers Bureau; BMS: Honoraria, Research Funding, Speakers Bureau; Mologen: Honoraria, Research Funding, Speakers Bureau; MSD Sharpe & Dohme: Honoraria, Research Funding, Speakers Bureau. Weigert: Janssen: Speakers Bureau; Epizyme: Membership on an entity's Board of Directors or advisory committees; Roche: Research Funding.

scholarly journals Characterization of CRISPR/Cas9 RANKL knockout mesenchymal stem cell clones based on single-cell printing technology and emulsion coupling assay as a low-cellularity workflow for single-cell cloning

2020 ◽  
Author(s):  
Tobias Groß ◽  
Csaba Jeney ◽  
Darius Halm ◽  
Günter Finkenzeller ◽  
G. Björn Stark ◽  
...  
Keyword(s):  
Single Cell ◽  
Cell Line ◽  
Large Scale ◽  
Single Cells ◽  
Protein Detection ◽  
Cell Line Development ◽  
Cell Printing ◽  
Cell Clones ◽  
The University

AbstractThe homogeneity of the genetically modified single-cells is a necessity for many applications such as cell line development, gene therapy, and tissue engineering and in particular for regenerative medical applications. The lack of tools to effectively isolate and characterize CRISPR/Cas9 engineered cells is considered as a significant bottleneck in these applications. Especially the incompatibility of protein detection technologies to confirm protein expression changes without a preconditional large-scale clonal expansion, creates a gridlock in many applications. To ameliorate the characterization of engineered cells, we propose an improved workflow, including single-cell printing/isolation technology based on fluorescent properties with high yield, a genomic edit screen (surveyor assay), mRNA rtPCR assessing altered gene expression and a versatile protein detection tool called emulsion-coupling to deliver a high-content, unified single-cell workflow. The workflow was exemplified by engineering and functionally validating RANKL knockout immortalized mesenchymal stem cells showing altered bone formation capacity of these cells. The resulting workflow is economical, without the requirement of large-scale clonal expansions of the cells with overall cloning efficiency above 30% of CRISPR/Cas9 edited cells. Nevertheless, as the single-cell clones are comprehensively characterized at an early, highly parallel phase of the development of cells including DNA, RNA, and protein levels, the workflow delivers a higher number of successfully edited cells for further characterization, lowering the chance of late failures in the development process.Author summaryI completed my undergraduate degree in biochemistry at the University of Ulm and finished my master's degree in pharmaceutical biotechnology at the University of Ulm and University of applied science of Biberach with a focus on biotechnology, toxicology and molecular biology. For my master thesis, I went to the University of Freiburg to the department of microsystems engineering, where I developed a novel workflow for cell line development. I stayed at the institute for my doctorate, but changed my scientific focus to the development of the emulsion coupling technology, which is a powerful tool for the quantitative and highly parallel measurement of protein and protein interactions. I am generally interested in being involved in the development of innovative molecular biological methods that can be used to gain new insights about biological issues. I am particularly curious to unravel the complex and often poorly understood protein interaction pathways that are the cornerstone of understanding cellular functionality and are a fundamental necessity to describe life mechanistically.

scholarly journals SAT-298 Integrative Single-Cell Transcriptomic and Epigenomic Landscape of Mouse Anterior Pituitary Cell Types

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Frederique Murielle Ruf-Zamojski ◽  
Michel A Zamojski ◽  
German Nudelman ◽  
Yongchao Ge ◽  
Natalia Mendelev ◽  
...  
Keyword(s):  
Single Cell ◽  
Cell Line ◽  
Anterior Pituitary ◽  
Cell Types ◽  
Chromatin Accessibility ◽  
Pituitary Cell ◽  
Integrated Analysis ◽  
Pituitary Cells ◽  
Rna Seq ◽  
Cell Type

Abstract The pituitary gland is a critical regulator of the neuroendocrine system. To further our understanding of the classification, cellular heterogeneity, and regulatory landscape of pituitary cell types, we performed and computationally integrated single cell (SC)/single nucleus (SN) resolution experiments capturing RNA expression, chromatin accessibility, and DNA methylation state from mouse dissociated whole pituitaries. Both SC and SN transcriptome analysis and promoter accessibility identified the five classical hormone-producing cell types (somatotropes, gonadotropes (GT), lactotropes, thyrotropes, and corticotropes). GT cells distinctively expressed transcripts for Cga, Fshb, Lhb, Nr5a1, and Gnrhr in SC RNA-seq and SN RNA-seq. This was matched in SN ATAC-seq with GTs specifically showing open chromatin at the promoter regions for the same genes. Similarly, the other classically defined anterior pituitary cells displayed transcript expression and chromatin accessibility patterns characteristic of their own cell type. This integrated analysis identified additional cell-types, such as a stem cell cluster expressing transcripts for Sox2, Sox9, Mia, and Rbpms, and a broadly accessible chromatin state. In addition, we performed bulk ATAC-seq in the LβT2b gonadotrope-like cell line. While the FSHB promoter region was closed in the cell line, we identified a region upstream of Fshb that became accessible by the synergistic actions of GnRH and activin A, and that corresponded to a conserved region identified by a polycystic ovary syndrome (PCOS) single nucleotide polymorphism (SNP). Although this locus appears closed in deep sequencing bulk ATAC-seq of dissociated mouse pituitary cells, SN ATAC-seq of the same preparation showed that this site was specifically open in mouse GT, but closed in 14 other pituitary cell type clusters. This discrepancy highlighted the detection limit of a bulk ATAC-seq experiment in a subpopulation, as GT represented ~5% of this dissociated anterior pituitary sample. These results identified this locus as a candidate for explaining the dual dependence of Fshb expression on GnRH and activin/TGFβ signaling, and potential new evidence for upstream regulation of Fshb. The pituitary epigenetic landscape provides a resource for improved cell type identification and for the investigation of the regulatory mechanisms driving cell-to-cell heterogeneity. Additional authors not listed due to abstract submission restrictions: N. Seenarine, M. Amper, N. Jain (ISMMS).

scholarly journals Single Cell RNA-Seq Characterises Pre-Leukemic Transformation Driven By CEBPA N321D in the Hoxb8-FL Cell Line

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3887-3887
Author(s):  
Moosa Qureshi ◽  
Fernando Calero-Nieto ◽  
Iwo Kucinski ◽  
Sarah Kinston ◽  
George Giotopoulos ◽  
...  
Keyword(s):  
Dendritic Cell ◽  
Single Cell ◽  
Cell Line ◽  
Single Cells ◽  
Mutant Form ◽  
Rna Seq ◽  
Wild Type ◽  
Leukemic Transformation

Abstract The C/EBPα transcription factor plays a pivotal role in myeloid differentiation and E2F-mediated cell cycle regulation. Although CEBPA mutations are common in acute myeloid leukaemia (AML), little is known regarding pre-leukemic alterations caused by mutated CEBPA. Here, we investigated early events involved in pre-leukemic transformation driven by CEBPA N321D in the LMPP-like cell line Hoxb8-FL (Redecke et al., Nat Methods 2013), which can be maintained in vitro as a self-renewing LMPP population using Flt3L and estradiol, as well as differentiated both in vitro and in vivo into myeloid and lymphoid cell types. Hoxb8-FL cells were retrovirally transduced with Empty Vector (EV), wild-type CEBPA (CEBPA WT) or its N321D mutant form (CEBPA N321D). CEBPA WT-transduced cells showed increased expression of cd11b and SIRPα and downregulation of c-kit, suggesting that wild-type CEBPA was sufficient to promote differentiation even under LMPP growth conditions. Interestingly, we did not observe the same phenotype in CEBPA N321D-transduced cells. Upon withdrawal of estradiol, both EV and CEBPA WT-transduced cells differentiated rapidly into a conventional dendritic cell (cDC) phenotype by day 7 and died within 12 days. By contrast, CEBPA N321D-transduced cells continued to grow for in excess of 56 days, with an initial cDC phenotype but by day 30 demonstrating a plasmacytoid dendritic cell precursor phenotype. CEBPA N321D-transduced cells were morphologically distinct from EV-transduced cells. To test leukemogenic potential in vivo, we performed transplantation experiments in lethally irradiated mice. Serial monitoring of peripheral blood demonstrated that Hoxb8-FL derived cells had disappeared by 4 weeks, and did not reappear. However, at 6 months CEBPA N321D-transduced cells could still be detected in bone marrow in contrast to EV-transduced cells but without any leukemic phenotype. To identify early events involved in pre-leukemic transformation, the differentiation profiles of EV, CEBPA WT and CEBPA N321D-transduced cells were examined with single cell RNA-seq (scRNA-seq). 576 single cells were taken from 3 biological replicates at days 0 and 5 post-differentiation, and analysed using the Automated Single-Cell Analysis Pipeline (Gardeux et al., Bioinformatics 2017). Visualisation by t-SNE (Fig 1) demonstrated: (i) CEBPA WT-transduced cells formed a distinct cluster at day 0 before withdrawal of estradiol; (ii) CEBPA N321D-transduced cells separated from EV and CEBPA WT-transduced cells after 5 days of differentiation, (iii) two subpopulations could be identified within the CEBPA N321D-transduced cells at day 5, with a cluster of five CEBPA N321D-transduced single cells distributed amongst or very close to the day 0 non-differentiated cells. Differential expression analysis identified 224 genes upregulated and 633 genes downregulated specifically in the CEBPA N321D-transduced cells when compared to EV cells after 5 days of differentiation. This gene expression signature revealed that CEBPA N321D-transduced cells switched on a HSC/MEP/CMP transcriptional program and switched off a myeloid dendritic cell program. Finally, in order to further dissect the effect of the N321D mutation, the binding profile of endogenous and CEBPA N321D was compared by ChIP-seq before and after 5 days of differentiation. Integration with scRNA-seq data identified 160 genes specifically downregulated in CEBPA N321D-transduced cells which were associated with the binding of the mutant protein. This list of genes included genes previously implicated in dendritic cell differentiation (such as NOTCH2, JAK2), as well as a number of genes not previously implicated in the evolution of AML, representing potentially novel therapeutic targets. Disclosures No relevant conflicts of interest to declare.

scholarly journals DOP23 Single-cell RNA sequencing identifies an important role for class I histone-deacetylase enzymes in intestinal myofibroblasts from patients with Crohn’s Disease strictures

2021 ◽  
Vol 15 (Supplement_1) ◽  
pp. S062-S062
Author(s):  
A Lewis ◽  
B Pan-Castillo ◽  
G Berti ◽  
C Felice ◽  
H Gordon ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Cell Line ◽  
Rna Sequencing ◽  
Histone Deacetylase ◽  
Chromatin Accessibility ◽  
Collagen I ◽  
Class I ◽  
Rna Seq ◽  
Single Cell Rna Sequencing

Abstract Background Histone-deacetylase (HDAC) enzymes are a broad class of ubiquitously expressed enzymes that modulate histone acetylation, chromatin accessibility and gene expression. In models of Inflammatory bowel disease (IBD), HDAC inhibitors, such as Valproic acid (VPA) are proven anti-inflammatory agents and evidence suggests that they also inhibit fibrosis in non-intestinal organs. However, the role of HDAC enzymes in stricturing Crohn’s disease (CD) has not been characterised; this is key to understanding the molecular mechanism and developing novel therapies. Methods To evaluate HDAC expression in the intestine of SCD patients, we performed unbiased single-cell RNA sequencing (sc-RNA-seq) of over 10,000 cells isolated from full-thickness surgical resection specimens of non-SCD (NSCD; n=2) and SCD intestine (n=3). Approximately, 1000 fibroblasts were identified for further analysis, including a distinct cluster of myofibroblasts. Changes in gene expression were compared between myofibroblasts and other resident intestinal fibroblasts using the sc-RNA-seq analysis pipeline in Partek. Changes in HDAC expression and markers of HDAC activity (H3K27ac) were confirmed by immunohistochemistry in FFPE tissue from patient matched NSCD and SCD intestine (n=14 pairs). The function of HDACs in intestinal fibroblasts in the CCD-18co cell line and primary CD myofibroblast cultures (n=16 cultures) was assessed using VPA, a class I HDAC inhibitor. Cells were analysed using a variety of molecular techniques including ATAC-seq, gene expression arrays, qPCR, western blot and immunofluorescent protein analysis. Results Class I HDAC (HDAC1, p= 2.11E-11; HDAC2, p= 4.28E-11; HDAC3, p= 1.60E-07; and HDAC8, p= 2.67E-03) expression was increased in myofibroblasts compared to other intestinal fibroblasts subtypes. IHC also showed an increase in the percentage of stromal HDAC2 positive cells, coupled with a decrease in the percentage of H3K27ac positive cells, in the mucosa overlying SCD intestine relative to matched NSCD areas. In the CCD-18co cell line and primary myofibroblast cultures, VPA reduced chromatin accessibility at Collagen-I gene promoters and suppressed their transcription. VPA also inhibited TGFB-induced up-regulation of Collagen-I, in part by inhibiting TGFB1|1/SMAD4 signalling. TGFB1|1 was identified as a mesenchymal specific target of VPA and siRNA knockdown of TGFB1|1 was sufficient suppress TGFB-induced up-regulation of Collagen-I. Conclusion In SCD patients, class I HDAC expression is increased in myofibroblasts. Class I HDACs inhibitors impair TGFB-signalling and inhibit Collagen-I expression. Selective targeting of TGFB1|1 offers the opportunity to increase treatment specificity by selectively targeting meschenymal cells.

scholarly journals The production of monoclonal antibodies to rubella haemagglutinin and their use in antibody-capture assays for rubella-specific IgM

1982 ◽  
Vol 88 (2) ◽  
pp. 335-350 ◽  
Author(s):  
R. S. Tedder ◽  
J. L. Yao ◽  
M. J. Anderson
Keyword(s):  
Monoclonal Antibodies ◽  
Single Cell ◽  
Cell Line ◽  
Ascitic Fluid ◽  
Solid Phase ◽  
Myeloma Cell ◽  
Myeloma Cell Line ◽  
Cell Clones ◽  
Antibody Capture ◽  
Limiting Dilution

SummaryMice were immunized by three intraperitoneal and one intravenous injection of rubella haemagglutinin. Splenocytes from these mice were fused with the cells of a syngeneic myeloma cell line, and following culture for various periods of time, single-cell clones were derived by the technique of limiting dilution.A total of 139 clones were derived from 13 parent hybrid cultures. To date, four of these cloned cultures have been propagated as ascitic tumours in mice. The preparation of IgG from ascitic fluid and labelling of this antibody with 125I is described. Results indicate that the use of labelled monoclonal antibodies as indicator reagents in solid-phase IgM antibody capture assays for the detection of rubella-specific IgM results in enhanced performance of these tests.

scholarly journals 676 PD-L1 is induced by the periodontal pathogen porphyromonas gingivalis and can be blocked by small molecule gingipain inhibitors, including atuzaginstat

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A715-A715
Author(s):  
Shirin Arastu-Kapur ◽  
Mai Nguyen ◽  
Sean Broce ◽  
Joseph Vacca ◽  
Kirk Ehmsen ◽  
...  
Keyword(s):  
Esophageal Cancer ◽  
Western Blot ◽  
Cell Line ◽  
Cell Lines ◽  
Porphyromonas Gingivalis ◽  
Wnt Pathway ◽  
Host Cells ◽  
Periodontal Pathogen ◽  
Cancer Tissue ◽  
Dependent Manner

BackgroundThe periodontal pathogen Porphyromonas gingivalis (Pg) has been linked to esophageal and other cancers through epidemiology studies. Pg’s protease virulence factors known as gingipains have been identified in esophageal cancer tissue and correlate with worse disease prognosis. Anti-PD-1 antibodies have shown some success in esophageal cancer treatment, but further understanding of the induction of PD-L1 in esophageal cells is needed to identify potential treatment modalities. Pg has been shown to induce PD-L1 on the surface of infected cells, suggesting that the presence of Pg in esophageal cancer cells may contribute to PD-L1 expression and immune escape. One of the pathways known to induce PD-L1 is wnt pathway activation resulting in b-catenin translocation to the nucleus. Prior studies have demonstrated that Pg activates the wnt pathway by a non-canonical mechanism, leading to b-catenin nuclear localization.MethodsAn immortalized non-transformed esophageal cell line, Het-1A, was used to investigate the level of PD-L1 induction by Pg infection using quantitative immunofluorescence. PD-L1 expression was measured using irreversible gingipain inhibitors against lysine-gingipain (Kgp) and arginine-gingipain (Rgp). Pg-induced PD-L1 expression pathways were investigated by Western blot and qPCR. PD-L1 induction by Pg was characterized in cancer cell lines that have an endogenous level of PD-L1 expression, including tongue squamous cell carcinoma (SCC25) and neuroblastoma (SH-SY5Y). PD-L1 induction by Pg was assessed in a murine derived RAW macrophage cell line that is critical for anti-PD-1 responses.ResultsPg infection increased PD-L1 expression on Het-1A cells within 24 hours of infection and increased PD-L1 mRNA within 4 hours of infection. PD-L1 expression level correlated with cellular bacterial burden on the cells in a dose-dependent manner. PD-L1 expression was decreased by the Kgp inhibitor, atuzaginstat, or an Rgp inhibitor, COR613, and PD-L1 expression was completely blocked when both gingipain inhibitors were used together (figure 1). Pg also induced expression of PD-L1 on the surface of infected SCC-25, SH-SY5Y, and RAW cell lines. Western blot analysis and qPCR revealed that Kgp inhibition, but not Rgp inhibition, was able to inhibit the non-canonical activation of b-catenin and down regulation of classical wnt pathway effectors at both the mRNA and protein level.Abstract 676 Figure 1Gingipain inhibitors block PD-L1 induced by PgPg grown with and labeled by red fluorescent membrane-incorporated dye was pre-treated with vehicle or the compounds listed for 30 min. Het-1A cells were infected (MOI = 20) for 24 hours, washed, fixed and stained for visualization of the nuclei (DAPI, blue), PD-L1 protein (anti-PDL1 primary and secondary antibodies, green), and Pg infection (red). Images were captured with immunofluorescent confocal microscopy.ConclusionsIn host cells infected with Pg, gingipains mediate the induction of PD-L1 as a mechanism of immune evasion through the non-canonical activation of the wnt pathway. Further studies to elucidate induction mechanisms are in progress. In esophageal cancer and other cancers infected with Pg, combining gingipain inhibitors with anti-PD-1 therapy may improve treatment outcomes.

The Role of CCAAT Displacement Protein in Neutrophil-Specific Gene Expression.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3558-3558
Author(s):  
Aimee Lee ◽  
Arati Khanna-Gupta ◽  
Terry Zibello ◽  
Caryn St.Clair ◽  
Nancy Berliner
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Cell Line ◽  
Specific Gene ◽  
Gene Promoters ◽  
Nb4 Cells ◽  
Ccaat Displacement Protein ◽  
Cell Clones ◽  
Granule Proteins ◽  
Secondary Granule

Abstract CCAAT displacement protein (CDP) is a highly conserved, ubiquitously expressed homeodomain protein with extensive homology to the Drosophila cut protein. CDP contains three highly conserved DNA-binding repeats called cut repeats, as well as a conserved homeodomain sequence. CDP is a transcriptional repressor of several developmentally regulated genes, including the phagocyte-specific cytochrome heavy chain gene, gp91-phox, and CCAAT enhancer binding protein epsilon (C/EBPε)and its downstream targets the neutrophil secondary granule proteins (SGPs), including lactoferrin (LF). We have previously shown that CDP binds to and represses both the C/EBPε and lactoferrin (LF) gene promoters thereby preventing expression of secondary granule proteins (SGPs) both directly and indirectly. CDP represses expression of SGPs in 32Dcl3 cells, an IL-3 dependent murine myeloid cell line that undergoes differentiation in response to IL-3 withdrawal and G-CSF stimulation. Moreover, LF is not expressed in NB4 cells, a human acute promyelocytic cell line which contains the t(15;17) PML-RARα translocation. CDP has been found to bind persistently to the LF promoter in NB4 cells after morphological differentiation following ATRA induction. Several attempts at generating a CDP knockout mouse have been performed, but all have produced incomplete knockouts. We have generated short hairpin RNA (shRNA) constructs to knock down mouse CDP in myeloid cells by RNA interference. Single cell clones stably expressing the mouse shRNA have been generated in 32Dwt18 cells, and have been shown to decrease CDP expression. Knockdown of CDP does not change the phenotype of the cells, which remain IL3 dependent and undergo phenotypic maturation upon G-CSF induction. However, increased LF gene expression can be seen in uninduced cells expressing CDP shRNA, and the level of LF expression in single cell clones expressing the shRNA for CDP appears to correlate with the level of CDP repression. Control clones do not express LF until induced with G-CSF for several days. The knockdown of CDP does not appear to affect the expression of C/EBPε, suggesting that LF expression reflects direct modulation of CDP binding to its promoter and is not an indirect effect of increased C/EBPε expression. This suggests that CDP can function as the sole negative regulatory element for LF gene expression, and that relief of CDP repression can increase LF expression independent of increased binding of positive regulatory factors.

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