Best Practices for Authentication of Cell Lines to Ensure Data Reproducibility and Integrity

2021 ◽  
Author(s):  
Elisabeth Vicente ◽  
Megan Lesniewski ◽  
Diana Newman ◽  
Zeljko Vujaskovic ◽  
Isabel L. Jackson
Keyword(s):  
Flow Cytometry ◽  
Best Practices ◽  
Cell Line ◽  
Dna Barcoding ◽  
Cell Lines ◽  
Cell Identity ◽  
Purity Analysis ◽  
Primary Rabbit ◽  
Commercial Vendor ◽  
Aortic Endothelial

Cell line misidentification and contamination are major contributors to the reproducibility crisis in academic research. Authentication of cell lines provides assurances of the data generated; however, commercially available cells are often not subjected to rigorous identification testing. In this study, commercially available cell lines underwent testing to confirm cell identity and purity. The methods reported here outline the best practices for cell line authentication. Briefly, a commercially available primary rabbit aortic endothelial cell line was purchased for the intent of producing target proteins necessary for generating species-specific recombinant antibodies. These rabbit-specific antibodies would then be utilized for the development of in-house enzyme-linked immunosorbent assays (ELISA) to evaluate blood-based biomarkers of vascular injury after total-body irradiation. To authenticate the cell line, cell identity and purity were determined by single tandem repeat (STR) testing, flow cytometry, polymerase chain reaction (PCR), and cytochrome c oxidase subunit 1 (CO1) DNA Barcoding in-house and/or through commercial vendors. Fresh cells obtained from a New Zealand White rabbit (Charles River, Wilmington, DE) were used as a positive control. The results of STR and flow cytometry analyses indicated the cells were not contaminated with human or mouse cells, and that the cells were not of endothelial origin. PCR demonstrated that cells were also not of rabbit origin, which was further confirmed by a third-party vendor. An unopened vial of cells was submitted to another vendor for CO1 DNA Barcoding analysis, which identified the cells as being purely of bovine origin. Results revealed that despite purchase through a commercial vendor, the cell line marketed as primary rabbit aortic endothelial cells were of bovine origin. Purity analysis found cells were misidentified rather than contaminated. Further investigation to determine the cell type was not performed. The most cost-effective and efficient methodology for confirming cell line identity was found to be CO1 DNA Barcoding performed by a commercial vendor.

scholarly journals 883 An anti-carcinoma monoclonal antibody (mAb) NEO-201 can also target human acute myeloid leukemia (AML) cell lines in vitro

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A925-A925
Author(s):  
Alessandra Romano ◽  
Nunziatina Parrinello ◽  
Sara Marino ◽  
Enrico La Spina ◽  
Massimo Fantini ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Functional Analysis ◽  
Epithelial Cells ◽  
Cell Line ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Phase 1 ◽  
Adcc Activity ◽  
Phenotypic Analysis

BackgroundNEO-201 is an IgG1 mAb targeting variants of CEACAM5/6 and has demonstrated tumor sensitivity and specificity in epithelial cells. Functional analysis has revealed that NEO-201 can engage innate immune effector mechanisms including ADCC and CDC to directly kill tumor cells expressing its target. A recent Phase 1 clinical trial at the NCI has determined both safety and recommended Phase 2 dosing. We have also seen the expression of the NEO-201 target on hematologic cells, specifically Tregs and neutrophils. Due to epitope being expressed both on malignant epithelial cells as well as several hematologic cells, we designed this study to explore the reactivity of NEO-201 against hematological neoplastic cells in vitro.MethodsPhenotypic analysis was conducted by flow cytometry. Cell lines used were six AML (HL60, U937, MOLM13, AML2, IMS-M2 and OCL-AML3), two multiple myelomas (MM) (OPM2, MM1.S), two acute lymphoblastic leukemia (ALL) (SUP-B15, RPMI8402) and four mantle cell lymphoma (MCL) (Jeko-1, Z138, JVM2 and JVM13). Markers used for flow cytometry analysis were CD15, CD45, CD38, CD138, CD14, CD19 and NEO-201. Functional analysis was performed by evaluating the ability of NEO-201 to mediate ADCC activity against AML cell lines using human NK cells as effector cells.Results5 of 6 AML cell lines tested bind to NEO-201 and the% of positive cells were 47%, 99.5%,100%,100% and 97.8% for HL60, U937, MOLM13, AML3 and IMS-M2, respectively. The% of positive cells in the two MM cell line were 99% and 18% for OPM2 and MM1.S, respectively. NEO-201 binding was not detected in the two ALL and the four MCL cell lines tested. Functional analysis has demonstrated that NEO-201 can mediate ADCC activity against the AML cell line (HL60) tested.ConclusionsThis study demonstrates that NEO-201 mAb’s target is expressed in most of the AML cell lines tested in vitro. In addition, we have shown it can mediate ADCC activity against HL60 cells (AML). Together, these findings provide a rationale for further investigation of the role of NEO-201 in AML as well as MM, further exploring patient PBMCs and bone marrow samples.

Low Expression of hOCT1 May Be a Key Point Mediating Low Intracellular Imatinib Accumulation in Imatinib Mesylate Resistance K562 Cell Line.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4547-4547
Author(s):  
Huanling Zhu ◽  
Ting Liu ◽  
Yongqian Jia
Keyword(s):  
Flow Cytometry ◽  
Cell Line ◽  
Real Time ◽  
Cell Lines ◽  
Real Time Pcr ◽  
K562 Cells ◽  
Pcr Analysis ◽  
Sensitive Cell ◽  
Imatinib Resistance ◽  
Low Expression

Abstract Objective To establish an imatinib resistance cell line and to study its resistant principia. Methods K562 cells were cultured in imatinib at gradually increased concentrations to generate their resistance cell line. Clone imatinib resistance cell lines by limited dilution culture. MTT assay, real time PCR and Semi-quantity PCR, flow cytometry and HPLC were used to clarify the possible mechanisms of the resistance. Results Imatinib resistance cell line K562R was successfully induced by continuous culture in the presence of gradually increasing doses of imatinib up to 5μmol/L. K562R cells were maintained in the media containing 5μmol/L imatinib. Proliferation data showed that cell growth of K562R was not inhibited in 5 μmol/L imatinib, whereas the parental sensitive cell was significantly inhibited by up to 2μM imatinib. The IC50 of K562R was about 7.5μmol/L which was ten times higher than that of the parental cell. HPLC revealed that the intracellular imatinib concentration of K562R was strikingly lower than that of the parental cells (up to 27.8-fold). MDR1 were not detected in mRNA (by RT-PCR)and protein(by flow cytometry) levels on K562R cell, whereas hOCT1 level measured by semi-quantity PCR showed lower expression in K562R cell lines than that of parental sensitive cell, indicating that low intracellular imatinib concentration may be due to lower affluence of imatinib by low level of hOCT1. (5) Real time PCR analysis showed no BCR-ABL/G6PD gene amplification and sequence analysis of the 374bp ABL kinase domain showed no mutation in K562R cell lines. Conclusion An imatinib resistance cell line K562R has been successfully established. Low expression of hOCT1 may be a key point mediating low intracellular imaitnib accumulation in K562R cell lines.

A Small Molecule Stat Inhibitor Blocks Stat3 and Stat5 Phosphorylation and Demonstrates Cytotoxicity In Acute Lymphoblastic Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2904-2904
Author(s):  
Robyn M. Dennis ◽  
Brandon Ballard ◽  
David John Tweardy ◽  
Karen Rabin
Keyword(s):  
Flow Cytometry ◽  
Western Blot ◽  
Cell Line ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Flow Cytometric Analysis ◽  
Positive Control ◽  
Wild Type ◽  
Atp Assay ◽  
Antibody Staining

Abstract Abstract 2904 Survival has improved dramatically in acute lymphoblastic leukemia (ALL), but further gains are unlikely using conventional chemotherapy alone. Several recently discovered, novel cytogenetic lesions with adverse prognostic impact, JAK2 activating mutations and CRLF2 rearrangements, occur in up to 15% of adult and pediatric ALL. These lesions are associated with activation of Jak2 and Stat5, and hold promise as targets for novel therapies affecting these signaling pathways. We performed in vitro testing of a novel small molecule Stat inhibitor, C188-9, in B-lineage ALL cell lines and patient samples with and without JAK2/CRLF2 alterations. C188-9 treatment for one hour at 10 μM inhibited Stat3 and Stat5 phosphorylation in ALL cell lines with JAK2 and CRLF2 alterations, but not in cell lines with wild-type JAK2 and CRLF2, as measured by phospho-flow cytometry (Fig. 1A). Only the cell lines with JAK2 and CRLF2 alterations demonstrated basal Stat5 phosphorylation on Western blot analysis, and this was inhibited by C188-9 treatment (Fig. 1B). C188-9 demonstrated cytotoxicity in ALL cell lines regardless of JAK2/CRLF2 status, with IC50s in the low micromolar concentration range (Fig. 1C). While C188-9 is undergoing investigation currently as a potent inhibitor of Stat3 in acute myeloid leukemia (AML), it also merits further investigation as an agent with Stat5 inhibitory activity and cytotoxicity in ALL. Figure 1. Effects of C188-9 in ALL cell lines. A. Stat3 and Stat5 phosphorylation were determined by flow cytometry in the ALL cell lines MHH-CALL-4 (JAK2/CRLF2 mutated) and Reh (JAK2/CRLF2 wild-type). In each condition, cells were incubated in serum-free media for one hour, followed by incubation with C188-9 or vehicle for one hour, stimulation with vehicle or pervanadate 125 mM for 15 minutes, fixation, permeabilization, phospho-antibody staining for phospho-Stat3 and phospho-Stat5, and flow cytometric analysis. B. Western blot for phospho-Stat5 in K562 cell line (positive control); MHHCALL-4 treated for one hour with C188-9 at 0, 5, or 10 uM; and RS4;11 (JAK2/CRLF2 wild-type ALL cell line). C. IC50 determination by ATP assay for C188-9 in the ALL cell lines MHH-CALL-4 and RS4;11. Each experiment was performed in triplicate. Figure 1. Effects of C188-9 in ALL cell lines. A. Stat3 and Stat5 phosphorylation were determined by flow cytometry in the ALL cell lines MHH-CALL-4 (JAK2/CRLF2 mutated) and Reh (JAK2/CRLF2 wild-type). In each condition, cells were incubated in serum-free media for one hour, followed by incubation with C188-9 or vehicle for one hour, stimulation with vehicle or pervanadate 125 mM for 15 minutes, fixation, permeabilization, phospho-antibody staining for phospho-Stat3 and phospho-Stat5, and flow cytometric analysis. B. Western blot for phospho-Stat5 in K562 cell line (positive control); MHHCALL-4 treated for one hour with C188-9 at 0, 5, or 10 uM; and RS4;11 (JAK2/CRLF2 wild-type ALL cell line). C. IC50 determination by ATP assay for C188-9 in the ALL cell lines MHH-CALL-4 and RS4;11. Each experiment was performed in triplicate. Disclosures: No relevant conflicts of interest to declare.

Identifying Small Molecules That Overcome HoxA9-Mediated Differentiation Arrest in Acute Myeloid Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3513-3513
Author(s):  
David B. Sykes ◽  
Mark K Haynes ◽  
Nicola Tolliday ◽  
Anna Waller ◽  
Julien M Cobert ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Acute Myeloid Leukemia ◽  
Small Molecules ◽  
Cell Line ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Myeloid Differentiation ◽  
Prostacyclin Receptor ◽  
Acute Myeloid

Abstract Abstract 3513 AML in adults is a devastating disease with a 5-year survival rate of 25%. We lack new treatments for AML, and the chemotherapy standard of care remains unchanged in thirty years. One success story in the treatment of AML has been the discovery of drugs that trigger the differentiation of leukemic blasts in the small subset of patients with acute promyelocytic leukemia. However, differentiation therapy is unfortunately not available for the remaining 90% of non-APL acute myeloid leukemia patients. Understanding and targeting the mechanism of differentiation arrest in AML has been under investigation for more than four decades. There is growing evidence to support the role of the homeobox transcription factors in normal hematopoietic differentiation as well as malignant hematopoiesis. The persistent, and inappropriate, expression of the homeobox gene HoxA9 has been described in the majority of acute myeloid leukemias. This implicates HoxA9 dysregulation as a common pathway of differentiation arrest in myeloid leukemias and suggests that by understanding and targeting this pathway, one might be able to overcome differentiation arrest. In cultures of primary murine bone marrow, constitutive expression of HoxA9 blocks myeloid differentiation and results in the outgrowth of immature myeloid cell lines. The mechanism by which HoxA9 causes differentiation arrest is not known and no compounds exist that inhibit HoxA9. We developed a murine cell line model in which the cells were blocked in differentiation by a conditional version of HoxA9. In this system, an estrogen-dependent ER-HoxA9 protein was generated by fusion with the estrogen receptor hormone-binding domain. When expressed in cultures of primary murine bone marrow, immortalized myeloblast cell lines can grow indefinitely in the presence of stem cell factor and beta-estradiol. Upon removal of beta-estradiol, and inactivation of HoxA9, these cell lines undergo synchronous and terminal myeloid differentiation. We took advantage of an available transgenic mouse model in which GFP was expressed downstream of the lysozyme promoter, a promoter expressed only in mature neutrophils and macrophages. Cell lines derived from the bone marrow of this lysozyme-GFP mouse were GFP-negative at baseline and brightly GFP-positive upon differentiation. In this manner, we generated a cell line with a built-in reporter of differentiation. These cells formed the basis of a high-throughput screen in which cells were incubated with small molecules for a period of four days in 384-well plate format. The cells were assayed by multi-parameter flow cytometry to assess for toxicity and differentiation. Compounds that triggered green fluorescence were scored as “HITS” and their pro-differentiation effects confirmed by analysis of morphology and cell surface markers. Given the availability of cells and the simple and reliable assay, we performed both a pilot screen of small molecules at The Broad Institute as well as an extensive screen of the NIH Molecular Libraries Small Molecule Repository. The screen of more than 350,000 small molecules was carried out in collaboration with the University of New Mexico Center for Molecular Discovery. We have identified one lead class of compounds - prostacyclin agonists – capable of promoting myeloid differentiation in this cell line model of AML. Using a parallel cell line derived from a prostacyclin receptor knock-out mouse, we confirmed that activity was due to signaling through the prostacyclin receptor. The role of prostacyclin signaling in myeloid differentiation has not been previously described. Analysis of gene expression demonstrated that the expression of the prostacyclin receptor is seen in ∼60% of in primary human AML samples. This is a potentially exciting finding as prostacyclin agonists (e.g. treprostinil) are clinically relevant as well as FDA-approved. Their potential role in the treatment of acute myeloid leukemia is unknown. Here we present the details of our high-throughput flow cytometry system and preliminary identification of pro-differentiation agents in AML. If successful, we anticipate that one of these small molecules may offer insight into a mechanism for overcoming differentiation arrest, and may also translate into a novel, clinically relevant treatment for acute myeloid leukemia. Disclosures: Sklar: IntelliCyt: Founder of IntelliCyt, the company that sells the HyperCyt high-throughput flow cytometry system. Other. Zon:Fate Therapeutics: Founder Other.

Dramatic Down-Regulation Of MNDA Expression In CP-CML Cells From The LSC Compartment

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2717-2717
Author(s):  
Céline Bourgne ◽  
Alexandre Janel ◽  
Juliette Berger ◽  
Agnès Guerci ◽  
Caroline Jamot ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Differentiation ◽  
Cell Line ◽  
Cell Lines ◽  
Fold Increase ◽  
Positive Control ◽  
Polymorphonuclear Cells ◽  
Primary Cells ◽  
Down Regulation ◽  
Cd34 Cells

Abstract Introduction Chronic Phase - Chronic Myeloid Leukemia (CP-CML) is a myeloproliferative disorder characterized by malignant proliferation of the granulocytic lineage without the arrest of cell differentiation. Tyrosine Kinase Inhibitors (TKI) have revolutionized CML treatment but several studies showed that a combination of TKI and Interferon alpha (IFNα) provides better clinical response. Myeloid Nuclear Differentiation Antigen (MNDA), which belongs to the hematopoietic interferon-inducible nuclear proteins with the 200-amino-acid repeat (HIN200) gene family, encodes a protein expressed in myeloid cells but whose function remains poorly understood. Because of its high expression in polymorphonuclear cells, its involvement in cell differentiation and apoptosis, and its induction by IFNα, we evaluated MNDA expression in CML cells and its modulation after incubation with IFNα. Material and methods We tested MNDA expression in several cell lines (K562, KCL22, LAMA84, TF1 and U937 (positive control)), in polymorphonuclear cells from healthy donors (HD-PMN, n=13) and in primary cells from patients with CP-CML at diagnosis (CP-CML; n=17). The relative expression of the MNDA transcript was analyzed using the 2-ΔΔCt method and was normalized to the endogenous reference gene GAPDH. HD-PMN were used as calibrator. We developed a multiparametric flow cytometry assay (CD45-V500/CD14-APC-H7/CD15-PerCpCy5.5/CD34-PC7/CD38-V450/MNDA-FITC) to detect MNDA protein in the different cell subsets, particularly in CD34+cells. Results As previously described, MNDA was poorly expressed in the K562 cell line. Similarly, mRNA was detected at low levels in two other CML cell lines (KCL22, LAMA84) and in TF1 cells, but at a high level in the U937 cell line, used as a positive control. In each cell line, the transcript expression was correlated to the protein level, as evaluated by flow cytometry (MFI ratio: 2.04±0.21, 2.36±0.24, 1.59±0.14, 1.88±0.11 and 8.77±0.54 for K562, KCL22, LAMA84, TF1 and U937, respectively (n=3)). In CP-CML primary cells, MNDA expression was greatly diminished as compared with HD-PMN in both mRNA (0.20±0.08 (n=17) vs. 1.32±0.21 (n=10); p=1.52x10-6) and protein (MFI ratio: 6.9±0.98 vs. 16.31±1.25, p=0.001). After having verified that IFNα (2000 U/ml, 16 hours) induced MNDA expression in HD mononuclear cells but not in PMN, we observed that induction of MNDA was moderate in CML cell lines K562 and LAMA84 (2-fold increase, n=3) whereas the level of MNDA mRNA was significantly increased in TF1 cells (28-fold increase, n=4). Induction in primary CML cells was variable (3/5 patients). Aiming to evaluate the expression of MNDA in leukemic stem cells (LSC), we first analyzed MNDA expression in CD34+ and CD34+/CD38- cells from HD. We observed that MNDA is down-regulated in healthy CD34+ and CD34+/CD38- cells compared to mature cells (mRNA: about 4 logs, protein: 8-10 fold lower, n=4), but we always detected a significant signal in CD34+cells (MFI ratio: 2.76±0.46, n=3). However, MNDA was not expressed by CML cells from the LSC compartment (n=4). This inhibition does not seem to be antagonized by nilotinib or IFNα (n=2). Discussion/Conclusion MNDA expression appears to be clearly down-regulated in CP-CML cells and dramatically so in the LSC compartment. In some patients, we observed sustained sensitivity to IFNα, but only in the compartment of more mature cells. This suggests early deregulation of MNDA expression which seems to be only partially dependant on differentiation. The mechanisms involved in this down-regulation remain to be elucidated but could be independent to TK activity of BCR-ABL protein and resistant to IFNα in the LSC compartment. This marked deregulation of MNDA in the LSC compartment is an additional argument in favor of intrinsic changes specific to primitive cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Histone Acetyltransferase (HAT) Activator, YF2, Modulates the p53:BCL6 Axis and Antigen Presentation in Diffuse Large B-Cell Lymphomas

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2254-2254
Author(s):  
Brian Estrella ◽  
Yuxuan Liu ◽  
Edd C Ricker ◽  
Manuel A Pazos ◽  
Jole Fiorito ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
B Cell ◽  
Antigen Presentation ◽  
Cell Line ◽  
Cell Lines ◽  
Catalytic Domain ◽  
Dependent Manner ◽  
Rna Seq ◽  
Catalytic Core ◽  
Presentation Pathway

Abstract A hallmark of DLBCL is epigenetic derangements characterized by monoallelic mutations in histone acetyltransferases (HATs); EP300 (p300) and CREBBP (CBP). The intact allele offers the opportunity for targeted therapies designed to overcome mutational dysregulation. We reported the discovery of YF2, a first-in-class HAT activator that demonstrates selective cytotoxicity in HAT-mutated DLBCL and induces HAT-mediated histone acetylation in vitro and in vivo. Here, we detail the mechanisms of action and the downstream effects of YF2 treatment. A unique feature of CBP/p300 is that it harbors a regulatory loop within its catalytic domain that undergoes autoacetylation which is critical for maintaining normal function. In order to determine if YF2 is able to induce the autoacetylation of p300/CBP, thereby increasing its catalytic activity, hypoacetylated CBP/p300 was combined with YF2 and Ac-CoA. YF2 demonstrated significant induction of CBP/p300 autoacetylation. To understand how YF2 interacts with HATs we analyzed the thermal stability, via thermal shift assay, of CBP/p300 subunits in the presence of YF2. We observed a T m shift when utilizing the full p300 (ΔT m = -2.9 oC)/CBP(ΔT m = -3.4 oC) catalytic core, which includes the catalytic, PHD/RING, and bromodomain. YF2 does not interact directly with the catalytic domain as there were no observed T m shift. YF2 significantly interacts with the bromodomain (ΔT m = -5.6 oC). In silico analysis has shown that the bromodomain has 3 TRP domains that are predicted to interact with small molecules. Next, we sought to determine how resistance to HDAC inhibitors (HDACi) and mutations/loss of HATs affects sensitivity to YF2. We first developed cell lines to be 10-fold resistant to HDACi romidepsin. When treated with YF2, resistant-SUDHL-6 was more sensitive to YF2 than the parental cell line (Resistant IC 50 = 2.2µM vs Parental IC 50 = 7.22µM). We found no change in YF2 sensitivity in the HAT wt OCI-Ly1 cell line. We performed CRISPR KO of EP300 in wt OCI-Ly7 cell line. A single cell clone with EP300 mutations was identified (OCI-Ly7-EP300 +/-). ICE analysis revealed that the percentage of indels was 12%. OCI-Ly7-EP300 +/- had lower p300 protein expression and were more sensitive to YF2 (IC 50 = 14.05µM) compared to wt (IC 50 = 23.7µM) when measured by Annexin V and CellTiter Glo assay. CBP/p300 is involved in the transcriptional activation of p53 through direct acetylation. YF2 induced both CBP (EC 50 = 15.47µM) and p300 (EC 50 = 6.05µM) mediated acetylation of p53 in cell free assays. As measured by RNA-Seq, YF2 altered multiple pathways regulated by CBP/p300 such as apoptosis and the p53 pathways. The p53 pathway was significantly upregulated in all cell lines. Validation of this pathway via qPCR, revealed p21, BAI1, ATM, FAS, FOS were upregulated in all cell lines. Additionally, YF2 induced G2/M arrest in a dose dependent manner when assessed via flow cytometry. We also observed modest increases in p21 and decrease CCND1 expression with YF2 treatment. BCL6, a transcriptional repressor linked to B-cell lymphomagenesis, is in part regulated through acetylation by CBP/p300. Mechanistically, CBP and the BLC6/SMRT/HDAC3 repressor complex co-occupy enhancers in the MHC Class II loci. Lack of functional CBP drives BCL6 mediated MHC repression resulting in reduced MHC gene expression and altered antigen presentation. In cell free assays, we YF2 induced p300 mediated BCL6 acetylation (IC 50 = 1.58 µM). We hypothesized HAT activation by YF2 could increase MHC expression in DLBCL. RNA-Seq analysis revealed YF2 led to upregulation of the interferon gamma pathway. Significantly, cell lines treated with YF2 showed increased MHC Class I and II expression when analyzed via flow cytometry. In summary, these findings demonstrate that YF2 interacts with the RING and bromodomains, leading to an allosteric change within the catalytic pocket to facilitate increased acetylation. In addition, YF2 leads to CBP/p300 autoacetylation, further enhancing enzymatic activity. We also demonstrated that YF2 is highly selective to DLBCL harboring HAT mutations and overcomes resistance to HDACi. Additionally, YF2 treatment modulates the p53:BCL6 axis, cell cycle progression, and antigen presentation pathway potentially restoring immune surveillance. These results support future clinical application of YF2 in HAT mutated lymphomas. Figure 1 Figure 1. Disclosures Amengual: Seagen: Consultancy; Daiichi Sankyo, Inc: Consultancy; Epizyme, Inc.: Speakers Bureau; Appia Pharmaceuticals: Research Funding.

scholarly journals Fingolimod Is Cytotoxic in Acute Myeloid Leukemia Independent of Additional Chemotherapeutic Agents

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5126-5126
Author(s):  
Carter Thomas Davis ◽  
Arati V. Rao ◽  
Eross Guadalupe ◽  
Dale J. Christensen ◽  
J. Brice Weinberg
Keyword(s):  
Multiple Sclerosis ◽  
Flow Cytometry ◽  
Acute Myeloid Leukemia ◽  
Cell Line ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Flow Cytometric Analysis ◽  
Chemotherapeutic Agents ◽  
Flow Cytometric ◽  
Acute Myeloid

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.

Understanding the Role of TRKA Receptor in Acute Myeloid Leukemias: From Proliferation and Pro-Survival Signals to a Novel Therapeutic Approach

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3789-3789
Author(s):  
Maurizio L Ghisoli ◽  
Wendy Fang ◽  
Timothy C Graham ◽  
Alejandro G Levy ◽  
Lizhi Zeng ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Line ◽  
Progenitor Cells ◽  
Cell Lines ◽  
Xenograft Model ◽  
Downstream Signaling ◽  
Cell Counts ◽  
Kaplan Meier ◽  
Acute Myeloid

Abstract Background: TRKA (Tropomyosin Receptor Kinase A) is a member of the neurotrophin tyrosine kinase receptor family. Nerve Growth Factor (NGF) binds specifically to TRKA triggering multiple downstream pathways which regulate proliferation and differentiation in a number of neural, epithelial and epidermal cell types. TRKA is also expressed in hematopoietic progenitor cells and NGF has been demonstrated to promote the colony growth and differentiation of myeloid progenitor cells. Interestingly, Pralle et al. found that 44% of patients with acute myeloid leukemia (AML) express TRKA mRNA and up to 30% encode activating TRKA mutations. Furthermore, a constitutively-active TRKA variant has been shown to contribute to myeloid leukemogenesis. Purpose: To evaluate the role of NGF/TRKA signaling on AML proliferation and survival through effects on downstream signaling pathways, and evaluate the effects of TRK inhibition on human AML. Methods: TRKA protein expression was measured in a panel of human AML cell lines (U937, K562, THP1, NB4, HL60, KU812). AlamarBlue (MTT) assays were used to measure proliferation/viability. For proliferation assays, cells were stimulated with NGF (5 ng/ml) for 1–3 days with or without the addition of a TRK inhibitor AZ-23 (25nM). Cell counts, cell cycle and survival were measured using trypan blue cell counts, and propidium iodine-based flow cytometry assays. Downstream signaling events were measured following a 15 minute exposure to recombinant human NGF. Antibodies for phosphorylated and total AKT, ERK, JNK and MEKK were used. In a human AML xenograft model, mice were treated with 100 mg/kg (2.5 mg) of AZ-23 by gavage for 2 weeks without toxicity. Weekly tail vein blood collection and flow cytometry were used to evaluate leukemia tumor burden. Kaplan-Meier curves were used to assess effects on morbidity. Results: Five of six human AML cell lines showed detectable TRKA protein levels by Western. When stimulated with NGF, all five cell lines which expressed TRKA demonstrated enhanced proliferation within 24 hours with mean value of 20% (10 to 45%, p=<0.001), which was abrogated with 25nM AZ-23 (p<0.01). The HL60 cell line showed the most impressive response to NGF (fig 1). The AML cell line which did not express TRKA (U937) and a human pre-B ALL cell line (Nalm-6) did not respond to NGF stimulation, as expected. There were no significant difference between only AZ-23 and the control group (p=>0.5). Following starvation, NGF stimulation induced the phosphorylation of ERK and AKT (2–4 fold), while inhibiting the phosphorylation of MEKK and JNK. All of these signaling effects were blocked with addition of AZ-23. Importantly, human AML-engrafted mice treated with the TRKA inhibitor AZ-23 had a significant decreased AML burden at 3 weeks post-inoculation (70% vs. 18% p=0.004). Kaplan-Meier estimates showed a Hazard Ratio of 10.5 (p=0.01) fig 3. Conclusions: TRKA has been implicated in the proliferation and transformation of myeloid progenitors and may be expressed and activated in a significant subset of AML cases. Our studies provide evidence of an important role for NGF/TRKA signaling in human AML. We show that NGF induces TRK-dependent proliferation while inducing ERK and AKT phosphorylation, consistent with a proliferative and pro-survival effect. Furthermore, we show that NGF stimulation inhibits the phosphorylation of MEKK and JNK, which are associated with NGF withdrawal and promote apoptosis. Finally, we demonstrate in a human AML xenograft model that a selective TRK inhibitor AZ-23 can effectively reduce AML burden and enhance survival. Based on these results, we propose that TRK inhibition may be a therapeutic option for a subgroup of AML, and warrants further pre-clinical development. FIGURE 1 FIGURE 1. FIGURE 2 FIGURE 2.

Extensive Genetic Characterization of MM.1R and MM.1S Identifies Several Events Contributing to Dexamethasone Resistance

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3966-3966
Author(s):  
David K Edwards ◽  
Venkata D Yellapantula ◽  
Kristi Allen ◽  
Wen Yu Wong ◽  
Jessica Albanese ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Exome Sequencing ◽  
Cell Line ◽  
Differential Expression ◽  
Cell Lines ◽  
Model System ◽  
Mrna Sequencing ◽  
Dexamethasone Resistance

Abstract Abstract 3966 The drug treatments currently available for multiple myeloma patients are dramatic improvements over historical regimens, stopping or slowing cancer growth in 80–90% of patients and leading to complete remission in approximately 40% of patients. Many of the new treatment regimens include “novel agents” in combination with dexamethasone, one of the most effective agents used to treat myeloma. The direct mechanism by which dexamethasone works in myeloma is not well characterized but it is assumed that it activates glucocorticoid receptors which results in gene expression changes that promote apoptosis in lymphoid cells. However, often the disease becomes resistant to dexamethasone, and the mechanism for this resistance is not entirely known. To study the mechanism of resistance, two isogenic cell lines, MM.1R and MM.1S, were independently created from the parental cell line MM.1 to represent models of resistance and sensitivity, respectively, to dexamethasone. This model system was created by Steve Rosen and colleagues in the 1990s and was recently deposited in ATCC. Previous studies have demonstrated differential expression of the glucocorticoid receptor NR3C1 but have not precisely identified the genetic difference between MM.1R and MM.1S across the whole genome. To better understand the mechanism behind the differences in drug sensitivity between these isogenic cell lines, we performed extensive characterization of MM.1R and MM.1S. We purchased both lines from ATCC and analyzed each using flow cytometry, CGH, CGH-SNP, mRNA sequencing, and exome sequencing. First, we broadly examined both cell lines, demonstrating a 300,000-fold difference in IC50 of MM.1R to MM.1S after 6 days of dexamethasone treatment. No significant ploidy difference was found between the two lines by flow cytometry analysis. Our CGH results identified 4 copy number differences unique to MM.1R (chr2:p37.1–37.3 deletion, chr4:q32.3–33 deletion, chr5:31.3 deletion, and chr7:q36.3 amplification), the third of which suggested a possible homozygous deletion within NR3C1. To confirm this deletion, we designed primer sets at ∼1kb intervals spanning the entire NR3C1 gene and performed PCR on MM.1R and MM.1S. Our results indicate the presence of a ∼5–8kb deletion of NR3C1 in MM.1R. Additionally, we analyzed our mRNA sequencing data using TopHat-Fusion and identified an inverted fusion between NR3C1 and ARHGAP26, which we confirmed through PCR amplification and Sanger sequencing. From mRNA sequencing, we identified 63 genes with differential expression between MM.1R and MM.1S (FPKM > 5 in either cell line and greater than fourfold change between them). These results demonstrate a reduction in expression of NR3C1 caused by the two independent deletions identified by CGH. The gene with the larges fold change was MGST1, which is associated with drug resistance and thus may be associated with dexamethasone resistance in this model system based on its expression profile. We analyzed our exome sequencing results for high-confidence (called by both SAMtools and GATK) non-synonymous mutations not present in the 1000 Genomes Project and filtered them for expression (FPKM > 5). We identified 218 mutations in MM.1R, 208 mutations which were also expressed in MM.1S and 10 mutations which were not expressed in MM.1S. The 10 genes with these mutations—PDIA5, TCERG1, RANBP9, MMS22L, PHF19, RNMTL1, AURKB, ERN1, GPCPD1, PIGT—present potential additional contributors to dexamethasone resistance. Specifically, for example, overexpression of RANBPM (the protein from RANBP9) results in increased glucocorticoid activity, suggesting that it may work in concert with NR3C1 to mediate the effects of dexamethasone. Ultimately, our results indicate that, unlike previous assumptions, there are several contributors to dexamethasone resistance in this model system and likely even more in the general patient populations, not just differential expression of NR3C1. Furthermore, we have discovered that this differential expression is due to biallelic inactivation of NR3C1 in MM.1R. Future studies will test the relative contribution of each factor to the differential sensitivity to dexamethasone observed in this model system and a broader understanding of this problem in multiple myeloma. Disclosures: No relevant conflicts of interest to declare.

PI3K/Akt/mTOR Pathway Inhibition in Chemotherapy-Sensitive and -Resistant Models of Burkitt Lymphoma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1558-1558 ◽  
Author(s):  
Maria Bhatti ◽  
Thomas Ippolito ◽  
Cory Mavis ◽  
Matthew J. Barth
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Cell Viability ◽  
Cell Line ◽  
Cell Lines ◽  
Propidium Iodide ◽  
Mtor Pathway ◽  
Propidium Iodide Staining ◽  
Induction Of Apoptosis ◽  
Pre Treatment

Abstract Introduction: Burkitt lymphoma (BL) is the most common form of B-cell non-Hodgkin lymphoma (B-NHL) in children. Despite significant improvements in survival with de novo disease, treatment of relapsed or refractory BL remains a significant hurdle with survival in only about 20% of patients. Novel therapeutic approaches are necessary to improve outcomes in this group of childhood B-NHL patients with the worst prognosis. Recent literature has identified a high rate of recurrent mutations that result in activation of the PI3K/Akt pathway in BL and have implicated activation of PI3K/Akt in coordination with Myc in BL lymphomagenesis. Our laboratory has developed rituximab and chemotherapy resistant cell line models and subsequently found that these cell lines exhibit increased activation of Akt. We hypothesized that increased activation of Akt may be contributing to chemoresistance and that targeting the PI3K/Akt/mTOR pathway may increase chemoresponsiveness. To that end, we have investigated the effect of inhibiting the PI3K/Akt/mTOR pathway with either the PI3K-delta inhibitor idelalisib or the pan-PI3K/mTOR inhibitor BEZ-235 in cell line models of BL. Methods: The in vitro effect of idelalisib or BEZ-235 was investigated in BL cell lines including Raji, Raji 2R and Raji 4RH (rituximab-chemotherapy resistant), Raji 7R and Raji 8RH (rituximab resistant), Ramos and Daudi. Cell viability following inhibitor exposure was assessed by Alamar blu and cell-titer glo assays. The effect of inhibitor exposure on cell cycle progression was determined by flow cytometry using propidium iodide staining. Inhibition of Akt activation following inhibitor exposure was determined using phospho-flow cytometry. The activity of cytotoxic chemotherapeutic agents following inhibition by idelalisib or BEZ-235 was assessed using Alamar blu and cell titer glo assays. Results: In vitro exposure of BL cell lines to idelalisib in concentrations from 0.1-100µM for 24, 48 or 72 hours resulted in a dose and time-dependent decrease in viable cells in all cell lines tested with IC50 concentrations of 60-300uM. Pre-treatment with the pan-caspase inhibitor QVD resulted in a small reversal in the decrease in cell viability suggesting only a minimal portion of the activity was caspase dependent. When induction of apoptosis was measured using annexin V-propidium iodide staining, little induction of apoptosis was observed with single agent idelalisib at concentrations up to 100uM. Determination of cell cycle progression following exposure to idelalisib at 1, 10, 50 or 100 uM for 24, 48 or 72 hours indicated a time and dose dependent cell cycle arrest in all cell lines. In chemotherapy-sensitive cell lines the arrest was primarily noted in G1, while the chemotherapy-resistant Raji 2R and Raji 4RH cell lines exhibited arrest primarily in G2/M. A significant reduction in cell viability following chemotherapy exposure for 48 hours was noted in chemotherapy resistant Raji 2R cells following pre-treatment for 48 hours with idelalisib 10uM compared to non-idelalisib exposed cells (doxorubicin 10uM 55% vs 77%, p<0.001; vincristine 0.05uM, 48% vs 61%, P<0.001). At higher idelalisib pre-treatment concentrations (50uM) additional synergistic activity was observed in Raji 2R cells (cisplatin 48% vs 61%, p<0.001; dexamethasone 67% vs 87%, p<0.01). To further assess the effect of dual inhibition of PI3K and mTOR, cell lines were exposed to the dual inhibitor BEZ-235. BEZ-235 exhibited a more potent decrease in cell viability compared to idelalisib with activity at nM concentrations. Unlike idelalisib, exposure to BEZ-235 resulted in significant induction of apoptosis by Annexin V-propidium iodide staining. BEZ-235 also exhibited synergistic activity in combination with chemotherapy in all cell lines. At equivalent dosing, BEZ-235 exposure resulted in a more significant decrease in Akt phosphorylation compared to idelalisib as determined by flow cytometry for p-Akt at Ser and Thr phosphorylation sites. Conclusions: Chemotherapy sensitive and resistant BL cell line models are susceptible to inhibition of the PI3K/Akt/mTOR pathway. Targeted inhibition of this pathway leads to a decrease in AKT activation, decrease in cell viability, cell cycle arrest and an increase in sensitivity to cytotoxic chemotherapeutic agents. Broader inhibition of both PI3K and mTOR is more effective than more targeted inhibition of PI3K-delta alone. Disclosures No relevant conflicts of interest to declare.

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