New Targets in Cytometric Investigation of Acute Leukemia Selected From Gene Profiling Studies

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2536-2536
Author(s):  
Martina Vaskova ◽  
Ester Mejstrikova ◽  
Tomas Brdicka ◽  
Pavla Angelisova ◽  
Tomas Kalina ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Acute Leukemia ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Gene Profiling ◽  
Size Exclusion ◽  
Leukemic Cells ◽  
Grant Support ◽  
Exclusion Chromatography ◽  
Blood Specimens

Abstract Abstract 2536 BACKGROUND. Gene expression profiling (GEP) studies have identified previously unknown molecules that correlate with genotypes of acute leukemia (AL), prognosis and/or the malignant status itself. Although some of these molecules ignited enthusiasm upon their publication, confirmation on independent cohorts and using other reliable methods is needed. Using specific tools on gene or protein level will bring us closer to a conclusion as to what is the biologic importance of the molecules and whether each molecule is worth testing as potential new diagnostic or immunotherapeutic target. METHODS. We have selected genes with probable relevant correlation(s) with conditions listed above. Correlations were then tested using qPCR. Successfully tested genes were selected for the production of new monoclonal antibodies (mAbs), unless satisfactory mAbs were already available. The newly produced mAbs were tested together with those, which were commercially available, using cell lines and diagnostic bone marrow or peripheral blood specimens of children with AL; data were compared also to non-malignant tissues. Flow cytometry was applied both to entire cells and to a novel method of Size Exclusion Chromatography-Microsphere-based Affinity Proteomics (Size-MAP). RESULTS. Eighteen genes (CMTM2, AGPS, EFNB1, DBN1, Clic5a, MYO, LARGE, DDIT4L, TCEAL4, PCLO, ARHGEF4, HYA22, RAG1, Opal 1, Siva, EVI2b, CDC42EP3, and SH3BP5) were selected for testing on qPCR and/or cytometric level. Among them, correlations were confirmed in 9 molecules, excluded in 3 molecules, failed to conclude due to insufficient testing system (cell lines) in 2 molecules and pending in 1 molecule. In 3 molecules, we started mAb production without qPCR testing. The results were combined with those related to previously known mAbs. We have produced 56 novel mAb clones to 9 target molecules. Seventeen mAbs to 9 molecules were further tested using flow cytometry both on cells and by Size-MAP. Of these, mAb to drebrin (DBN1, Figure) was proven to correlate with TEL/AML1 positivity, while its correlation to better prognosis is waiting for a longer follow up.Figure:Two diagnostic specimens of leukemic cells that had negative (left) or high (right) expression of drebrin.Figure:. Two diagnostic specimens of leukemic cells that had negative (left) or high (right) expression of drebrin. Anti-drebrin and anti-OPAL-1 mAbs both showed reproducible signals using Size-MAP. The data were compared with the strong predictors of TEL/AML1 status (CD27 and CD44) that we showed previously. CONCLUSIONS. Some but not all of the molecules that were presented by GEP as promising targets can be detected by flow cytometry and their correlations with molecular genetics was confirmed. Grant support: National Program of Research II (NPVii, project 2B06064), GACR 301/10/1877, MZ CR NS10004-4, NS10480-3 and NS10473-3. Disclosures: No relevant conflicts of interest to declare.

CHILDHOOD Biphenotypic ACUTE LEUKEMIA with High Incidence of Basophilic Differentiation: The CONTRIBUTION of Ultrastructural Methods to Diagnosis.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4698-4698
Author(s):  
Peretz Resnitzky ◽  
Drorit Luria ◽  
Dina Shaft ◽  
Gali Avrahami ◽  
Marta Jeison ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Acute Leukemia ◽  
Nuclear Membrane ◽  
Conflicts Of Interest ◽  
Medical Center ◽  
Leukemic Cells ◽  
Ultrastructural Studies ◽  
High Incidence ◽  
B Lineage ◽  
Biphenotypic Acute Leukemia

Abstract Abstract 4698 The diagnosis of Biphenotypic Acute Leukemia (BAL) is still a challenge in clinical hematology. In spite of the use of the score formulation of the European Group for the Immunological Classification of Leukemias (EGIL) there are still cases difficult to define. We assessed the contribution of ultrastructural morphology and cytochemistry to the diagnosis of BAL in pediatric leukemic patients. Twenty six patients diagnosed at Schneider Children's Medical Center of Israel between the years 1989 to 2004 have been classified as BAL in a retrospective analysis evaluated by various combinations of light-microscopy (LM) morphology, immunophenotype flow-cytometry, cytogenetics and electron-microscopy (EM) including myeloperoxidase (MPO) and platelet peroxidase (PPO) reaction. The identification of myeloid features by EM was based on the presence of 1) MPO positive granules and MPO reactivity in the nuclear membrane, profiles of endoplasmic reticulum (ER) and in the membranes of the Golgi system; 2) PPO reaction in the nuclear membrane and profiles of ER, and 3) the presence of primary basophilic granules in the cytoplasm of the blasts. The EGIL scoring system has been used with the addition of the ultrastructural findings for the definition of BAL. In 24 cases the morphologic appearance and cytochemistry by LM of the leukemic cells were that of undifferentiated blasts. By cytogenetic evaluation abnormal karyotypes were detected in 16 patients, normal in 4 and unknown in 6 patients. Out of 26 patients 15 had the combination of T-ALL with myeloid phenotype and 11 had the B-lineage with myeloid features. Unexpectedly the blasts of 9 of the 26 BAL patients had basophilic differentiation as indicated by the presence of typical primary basophilic granules with MPO reactivity in a scattered pattern as previously reported in basophylic leukemia. Interestingly 7 of these 9 cases had a T lymphoid component. By contrast among 15 patients who had FAB M0, 9 had the B lineage markers. The remaining 2 patients had M1 and M7 features with T lymphoid phenotype. The contribution of EM studies enabled to establish the diagnosis of BAL in 11 out of 26 patients, in other 6 patients it allowed for the refinement of the AML subgroup such as basophilic leukemia and in the remaining 9 patients EM confirmed the diagnosis of BAL as defined by flow cytometry. Conclusions Our study identified a subgroup of children with acute leukemia in whom BAL could be suggested only by the addition of ultrastructural studies. EM could further refine and confirm the diagnosis of BAL in all the other cases. Of interest is the high incidence of basophylic differentiation among BAL pediatric patients, and its association with T lymphoid phenotype. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Study of Diagnosis, Classification and Prognostic Factors Determination of Acute Leukemia By Flow Cytometry Immunophenotyping

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5175-5175
Author(s):  
Alessandra Suelen Jardim Silva ◽  
Juliana Mendonça Freire ◽  
Lenilton Silva DA Silva Júnior ◽  
Gustavo Henrique de Medeiros Oliveira ◽  
Antonia Eduarda Martins Oliveira Elói Silva ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Prognostic Factors ◽  
Acute Leukemia ◽  
Conflicts Of Interest ◽  
Clinical Signs ◽  
Leukemic Cells ◽  
Lymphoid Leukemia ◽  
Acute Leukemias ◽  
Hematological Analysis ◽  
Diagnosis Classification

Introduction: Acute leukemias (AL) are clonal diseases classified into two large groups: acute lymphoid leukemia (ALL), more common in children, and acute myeloid leukemia (AML), more rare in childhood, besides the rare phenotype leukemias such as acute biphenotypic leukemia (ABL) and undifferentiated acute leukemia (UAL). Although the cytomorphology still be relevant in theses leukemias diagnoses, the immunophenotyping by flow cytometry (FC) have become essential in the diagnosis, classification and follow-up of these neoplasms, standing out as a modern and practical methodology, presenting characteristically as a method of multiparametric and quantitative analysis of the leukemic cells. Objective: The objective of this study was realize a retrospective study of immunophenotyping in 371 patients with AL. Methodology: Immunophenotyping was performed biological samples by FC after labeling with a panel of monoclonal antibodies specific for AL directed against lymphoid antigens (B, T and NK cells), myeloid, and markers related to other cell immaturity. At the same time, information was obtained regarding patients such as age, sex, clinical data related to the disease, and previous hematological analysis. Results: From 371 cases, 127 were ALL (71 B lineage and 55 T=ALL), 239 AML, 04 ABL and 2 UAL. In the ALL, it was observed a higher frequency in children, contrasting with the cases of AML, ABL and UAL more prevalent in adults. In ALL, clinical signs and laboratorial data related to disease were more present in the ALL-T and mature-B corroborating with information in the literature. In AML, ABL and UAL the most clinical parameters observed were splenomegaly, hepatomegaly and bleeding. The classification FAB subtypes of AML more predominant were M1, M2 and M4 and lower incidence of the M7 subtype. Conclusion: These data demonstrate the importance of FC technology in the diagnosis, classification and establishment of prognostic factors of these neoplasms. Disclosures No relevant conflicts of interest to declare.

Investigating the Roles of the Yeats Domain in MLL-ENL Mediated Leukemogenesis

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 30-31
Author(s):  
Hsiangyu Hu ◽  
Nirmalya Saha ◽  
Yuting Yang ◽  
Sierrah Marie Grigsby ◽  
Rolf Marschalek ◽  
...  
Keyword(s):  
Cell Growth ◽  
Acute Leukemia ◽  
Transcriptional Activation ◽  
Fusion Proteins ◽  
Conflicts Of Interest ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Fusion Partner ◽  
Wild Type

Approximately 10% of acute leukemia involves rearrangement at chromosome 11q23, giving rise to a relatively aggressive form of acute leukemia characterized by MLL1 (KMT2A) fusion proteins. Despite the identification of >100 MLL1 fusion partners, the majority are members of several similar transcriptional activation complexes including: The Super Elongation Complex (SEC), AEP and EAP (SEC used hereafter). MLL fusion-driven acute leukemia is characterized by deregulated activity of the SEC and the H3K79 methyltransferase DOT1L. This leads to altered epigenetic landscapes at and deregulated transcription of pro-leukemic MLL1-fusion target genes like HoxA9 and Meis1. Thus, targeting these transcriptional and epigenetic complexes has become an attractive therapeutic strategy for treating MLL-fusion leukemia. Eleven-Nineteen-Leukemia (ENL or MLLT1) is the third most common MLL1 fusion partner and a component of the SEC. Recently, wild type ENL was identified as an essential factor for leukemic cell growth. The ENL protein possesses a C-terminal ANC-homology domain (AHD) necessary for SEC recruitment and is essential for MLL-fusion mediated leukemogenesis. In addition, ENL contains a highly conserved N-terminal YEATS domain that functions as an epigenetic reader for acetylated H3K9, H3K18 or H3K27, which is essential for leukemic cell growth. Additionally, the ENL YEATS domain directly interacts with the Polymerase Associated Factor 1 complex (PAF1c), an epigenetic regulator protein complex essential for MLL-fusion mediated leukemogenesis. These studies highlight the importance of the YEATS domain in regulating wild type ENL function in leukemic cells. However, the importance of the YEATS domain in the context of MLL-ENL mediated leukemia remains to be elucidated. In this study, we investigate the clinical relevance and leukemic importance of the ENL YEATS domain in MLL-ENL leukemias. We first analyzed t(11;19) (MLL-ENL) patient data to determine the sites of chromosomal translocation within the ENL gene. We found that the YEATS domain (coded by exons 2 through 4) is retained in 84.1% of MLL-ENL patients (n=302). Specifically, 50.7% (n=153) of these patients possess breakpoints located 5' of the first exon of the ENL gene, while 33.4% (n=101) of the patients display breakpoints within the first intron of ENL gene. These data point towards a tendency for YEATS domain retention in MLL-ENL fusion proteins in t(11;19) patients. We next tested whether the YEATS domain was functional in MLL-ENL mouse leukemia models. Our data shows the YEATS domain is required for MLL-ENL leukemogenesis in vivo, as deletion of the YEATS domain destroys MLL-ENL leukemogenesis and increases apoptosis in cell culture. Transcriptionally, deletion of the YEATS domain decreased expression of pro-leukemic genes such as Meis1 and the anti-apoptotic gene Bclxl. To dissect the contribution of different YEATS domain functions in MLL-ENL leukemogenesis, we engineered YEATS domain mutants defective in interacting with PAF1 or acetylated H3K9/K18/K27. Disrupting the YEATS-PAF1 or YEATS-H3Kac interaction decreased MLL-ENL mediated colony formation exvivo and significantly increased leukemia latency in vivo. The MLL-ENL YEATS domain mutants will be used in future studies to determine how the YEATS domain affects 1) MLL-ENL fusion localization, 2) key protein complexes localization (i.e. SEC and PAF1c) and 3) the epigenetic landscapes (i.e. H3K79me2/3 and H3K4me3) at pro-leukemic targets. To further interrogate the YEATS-PAF1 interaction in MLL-ENL mediated leukemia, we identified the minimal region of the PAF1 protein required for the YEATS-PAF1 interaction. This PAF1 protein fragment will be used to biochemically characterize the structure of the PAF1-YEATS interaction, which might aid in therapeutically targeting specific YEATS interactions in MLL-ENL leukemia. Our results demonstrate for the first time, to our knowledge, an essential role for the YEATS domain in MLL-ENL mediated leukemogenesis. Additionally, our genetic studies elucidate the importance of the YEATS domain interaction with either the PAF1c or H3Kac in MLL-ENL leukemias. Taken together, our study establishes a rationale for exploring the effectiveness of small molecule development aimed at disrupting either the YEATS-H3Kac or the YEATS-PAF1 interaction as a therapeutic intervention for treating MLL-ENL leukemia patients. Disclosures No relevant conflicts of interest to declare.

Expression and Function of the CXCR7 Chemokine Receptor in Leukemias

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2423-2423
Author(s):  
Sergej Konoplev ◽  
Hongbo Lu ◽  
Michael A Fiegl ◽  
Zhihong Zeng ◽  
Wenjing Chen ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Human Leukemia ◽  
Rt Pcr ◽  
Hematopoietic Stem ◽  
Leukemic Cell Lines ◽  
And Function

Abstract Background: Bone marrow produced stromal-derived factor-1a (SDF-1a) is a key chemokine involved in chemotaxis, homing, mobilization, and expansion of hematopoietic stem and progenitor cells. While the majority of well-defined functions of SDF-1a are mediated via its receptor CXCR4, recent studies have characterized CXCR7 as an alternative receptor capable of binding SDF-1a. Although the functions of CXCR7 are still incompletely understood, the receptor was reported to promote migration and adhesion in certain cell types and function as a pro-survival factor in breast cancer cells. CXCR7 expression and function in human leukemia cells has not been characterized. In this study, we examined CXCR7 expression in leukemia cell lines and primary samples from patients with acute lymphoblastic leukemia (ALL) and utilized a small molecule inhibitor of CXCR7 to probe CXCR7’s function. Materials and methods: CXCR4 and CXCR7 expression was determined by flow cytometry, real-time PCR (RT-PCR) and immunocytochemistry (ICC) in leukemic cell lines including AML (OCI-AML2, OCI-AML3, HL60, U937 NB4, Molm13), ALL (REH, Raji, RS4; 11, Nalm6, Molt4) and CML (KBM5, K562) cells. In primary ALL patient samples, CD34+CD19+ gating was applied to detect CXCR7 expression on pre-B leukemic cells by flow cytometry. The migration of leukemic cells towards SDF-1a was studied using a transwell system. CXCR4 inhibitor AMD3100 was purchased from Sigma, and CXCR7 inhibitor CCX-733 was provided by ChemoCentryx Inc., Mountain View, CA. Results: CXCR4 was found to be ubiquitously expressed on the cell surface of all leukemic cell lines tested. CXCR7 mRNA and protein expression was detectable only in Burkitt lymphoma Raji cells, as analyzed by flow cytometry (clone 11G8, R&D systems), RT-PCR and ICC. Curiously, CXCR7 expression was significantly induced in MOLM13 cells under hypoxic (6% O2) conditions (p=0.01). Low levels of surface CXCR7 were found in 8 of the 9 primary ALL samples by flow cytometry. To determine the respective roles of CXCR4 and CXCR7 in migration of leukemic cells, we utilized CXCR4 inhibitor AMD3100 and CXCR7 inhibitor CCR733 in Raji (CXCR7 positive) and RS4;11 (CXCR7 negative) cells. AMD3100 at 25μM significantly inhibited SDF-1a induced migration (from 38.5% to 12%); CCR733 at 10μM also inhibited SDF-1a induced migration (from 38.5% to 24%) and the combination of AMD3100 and CCR733 resulted in 81% inhibition of migration (from 38.5% to 7.2%). AMD3100 blocked SDF-1a induced migration of CXCR4+CXCR7− RS4;11 cells (from 36.5% to 15.8%), while CCR733 had no effect (36.5% and 39.2%). In conclusion, these studies demonstrate functional expression of the SDF-1 receptor CXCR-7 on Raji and primary ALL cells and suggest that CXCR7 plays an active role in the migration of leukemic cells. CXCR-7 may serve as an alternative receptor to CXCR4. Studies addressing the role of CXCR7 in adhesion, SDF-1a-mediated signaling and survival of leukemic cells are in progress.

Superior Cytotoxicity of Clonal Versus Polyclonal Gamma Delta T Cells against Philadelphia Chromosome Positive and B-CLL Derived Leukemic Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3032-3032
Author(s):  
Helena Dhamko ◽  
Gabrielle Melanie Siegers ◽  
Julia Schüler ◽  
Armand Keating
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Conflicts Of Interest ◽  
Philadelphia Chromosome ◽  
Leukemic Cells ◽  
Small Subset ◽  
Target Cells ◽  
Gamma Delta T Cells ◽  
Gamma Delta ◽  
Phenotypic Stability

Abstract Abstract 3032 Poster Board II-1008 Gamma delta T cells (GDTCs), a small subset of T-lymphocytes (<10%) involved in tumor immune surveillance, are promising candidates for adoptive immunotherapy demonstrated by their ability to elicit cytolytic responses against many tumors. We have isolated and expanded GDTCs as a first step in developing a clinical protocol (Siegers, GM et al., ASH 2008). GDTCs exist in subsets whose specificity and function are determined by receptor rearrangement and tissue localization. The Vdelta2 (Vd2) subset in blood recognizes small phosphate containing non-peptide antigens and has been shown to kill myeloma and Burkitt lymphoma cells, whereas Vdelta1 (Vd1) GDTCs are typically found in tissue mucosae and provide defense against epithelial cancers. Although circulating GDTCs are predominantly of the Vdelta2 (Vd2) subset, we found that in 59% of GDTC cultures derived from the peripheral blood of healthy donors (n=17), the Vdelta1 (Vd1) subset was preferentially expanded, comprising 70.5% ± 14.7% (mean ± standard deviation) as determined by flow cytometry. In the remaining cultures, Vd2 GDTCs comprised 75.9 ± 14.2%. Preferential expansion of Vd1 did not correlate with a higher percentage of this subset in donor blood prior to GDTC isolation. In one expanded culture, Vd1 and Vd2 were equally present (40.3% and 41.3% respectively, on day 17). To determine activation status of Vd1 and Vd2 subsets simultaneously when co-incubated for 3 hours at a 1:5 effector:target ratio (E:T) with EM2eGFPluc, Ph(+) leukemic target cells, exposure of the degranulation-induced marker CD107 was determined by flow cytometry. Assays performed on culture days 10 to 17 (n=8) revealed that only 3.4 ± 2.7% Vd1 cells were activated, whereas Vd2 cells exhibited ten-fold activation with 34.1 ± 4.7% expressing CD107. To further investigate the different cytotoxic potential of these GDTC subsets, we generated 3 Vd2 clones from Donor 1 and 7 clones (3 Vd1 and 4 Vd2) from Donor 2. 3 clones were obtained from 200 Vd1-sorted cells, and 4 clones from 600 Vd2-sorted cells, suggesting superior clonogenicity of Vd1. Indeed, Vd1 clones grew faster than Vd2 from this donor. After 40 days in culture, we obtained 57 ± 37 × 106 Vd1 and 37 ± 23 × 106 Vd2 cells from a single cell on day 0. The enhanced growth of Vd1 explains how this subset predominates in most polyclonal GDTC cultures, despite donors having more Vd2 than Vd1 in their blood (Vd2:Vd1 = 5.7±3.2, n=7). Polyclonal expansion of GDTCs from Donor 2 yielded 11.2 × 106 cells on day 20, from 1.7 × 106 on day 0, a 6.7-fold expansion compared to 107-fold achieved with clones from the same donor. Vd2 clones were screened for their ability to lyse EM2eGFPluc in vitro. In a flow-cytometric assay based on propidium iodide staining, Vd2 clones exhibited cytotoxicities ranging 4.5%-10.6% for a 4-hour co-incubation at 2.6:1 E:T. Clones from Donor 1 were tested again and ranking confirmed in a 4-hour cytotoxicity assay at 10:1 E:T, with a range of 23.5%-35.4% for clones A1, B3 and C6, respectively. When C6 was compared to polyclonal GDTCs from the same donor, it was found to be more cytotoxic (9.0% versus 2.0% at 10:1 for 4 hours). Vd2 clones and polyclonal GDTC from Donor 2 were compared; clone E5 exhibited 10-fold (49.2%) and E3 1.4-fold (7.6%) cytotoxicity of polyclonal GDTCs (5.3%). Published reports describe an increase in Vd1 in B-CLL patients, hence we used MEC1, an EBV-positive B-cell line derived from B-CLL, as a target. At a 1.9:1 ratio over 4 hours, % cytotoxicity ranged 7.0% - 13.8% (D3 most cytotoxic). Vd1 clones were compared with polyclonal GDTC cultures derived from Donors 2 and 3, which exhibited 57% and 52% Vd1, respectively. Clone D3 again proved most cytotoxic at 10:1 E:T over 4 hours, with 40.8% compared to 18.6% (Donor 3) and 6.8% (Donor 2). Immunophenotyping indicates phenotypic stability in clones over time that is not evident in polyclonal populations. We conclude that the increased cytotoxicity, superior expansion potential and extended culture duration as well as phenotypic stability of GDTC clones make them a more attractive therapeutic agent than polyclonal cultures for the treatment of hematological malignancies. Our study reveals the potential importance of selecting specific and potent GDT effector cells for treating Ph(+) and B-CLL leukemias with GDTCs. We next plan to test this approach in our established pre-clinical xenogeneic leukemia mouse model. (Dhamko H was the recipient of an ASH Summer Trainee Research Award). Disclosures No relevant conflicts of interest to declare.

All-Trans-Retinoic Acid (ATRA) Causes Extensive Differentiation In the NPM Mutant, Non-APL Leukemic Cell Line OCI-AML3

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3305-3305 ◽  
Author(s):  
Matthew A. Kutny ◽  
Steven J. Collins ◽  
Keith Loeb ◽  
Roland B. Walter ◽  
Soheil Meshinchi
Keyword(s):  
Cell Viability ◽  
Cell Count ◽  
Cell Line ◽  
Cell Lines ◽  
Time Course ◽  
Conflicts Of Interest ◽  
Live Cell ◽  
Leukemic Cells ◽  
Viable Cell Number ◽  
Nuclear Segmentation

Abstract Abstract 3305 The differentiating agent ATRA has been used successfully in the treatment of acute promyelocytic leukemia (APL). By comparison, non-APL AML has not shown similar sensitivity to ATRA induced differentiation. Recent data has suggested that a subset of de novo AML patients with nucleophosmin (NPM1) mutations may benefit from addition of ATRA to conventional therapy. The NPM1 gene has several functions affecting cell cycle proliferation including regulation of ribosome biogenesis and centrosome duplication and it acts as a histone chaperone. Mutation of the NPM1 gene leads to differentiation arrest contributing to AML pathogenesis. We hypothesized that leukemia cells with NPM1 mutations could be induced to undergo differentiation. We tested this hypothesis with the NPM1 mutant AML cell line OCI-AML3 and compared the results to identical assays using the AML cell line HL-60 which has been previously well documented to differentiate in response to ATRA therapy. OCI-AML3 and HL-60 cell lines were treated for 5 days with control media and four ATRA doses including 0.2 μM, 1 μM, 5 μM, and 25 μM. Cell viability was assessed by flow cytometry. Compared to the control condition, OCI-AML3 cells treated with the lowest dose of ATRA (0.2 μM) had a live cell count 21.6% of the control. HL-60 cells treated at even the highest ATRA dose (25 uM) had a live cell count 79.3% of the control. Due to the sensitivity of OCI-AML3 cells to the toxic effects of ATRA, the experiment was repeated with lower doses of ATRA including 0.001 μM, 0.01 μM and 0.1 μM. At the lowest dose of ATRA (0.001 μM), OCI-AML3 cells demonstrated a cell viability of 49% with further decrease to 26% at 0.1 μM dose of ATRA. At similar ATRA doses, cell viability for HL-60 cells was 91% and 85%, respectively (see table 1). Table 1: Cell viability as a percent of control cells after 5 days of treatment at three different doses of ATRA in OCI-AML3 and HL-60 cell lines. Cell Line: ATRA 0.001 μM ATRA 0.01 μM ATRA 0.1 μM OCI-AML3 49% 33% 26% HL-60 91% 91% 85% We subsequently determined the time course of changes in cell growth and the extent of differentiation at each point was determined by morphologic assessment. Both cell lines were treated with ATRA at doses of 0.001 μM, 0.01 μM, 0.1 μM, and 1 μM for a total of 4 days. Each day viable cell number was determined. In contrast to the HL-60 cells which had continued growth in lower ATRA doses, OCI-AML3 cells demonstrated exquisite sensitivity to growth arrest at the lowest doses of ATRA. Cell morphology was assessed daily with modified Wright-Giemsa staining of cells. Cells were examined for signs of myeloid differentiation including decrease in nuclear to cytoplasmic (N/C) ratio, nuclear segmentation, and cytoplasmic granules and vacuoles. At the lowest dose of ATRA (0.001 μM), after 4 days of exposure, significant number of OCI-AML3 cells demonstrated morphologic evidence of differentiation. At this ATRA dose and exposure interval, HL-60 cells showed no evidence of differentiation. At an ATRA dose of 1 μM (considered a standard dose used for differentiation of HL-60 cells), the OCI-AML3 cells showed differentiation changes as early as day 2 with nuclear segmentation and decreased N/C ratio while HL-60 cells did not show any change at this time point. After 4 days of ATRA exposure, most OCI-AML3 cells showed segmented nuclei and vacuolated cytoplasm, whereas HL-60 cells showed less distinct signs of differentiation with some cytoplasm granules and cup shaped nuclei. This data suggests that leukemic cells with NPM mutations may be susceptible to the pro-differentiating properties of ATRA. Further substantiation of this data with primary human specimens may ultimately provide the rationale for a novel therapeutic option using ATRA-based differentiation therapy for subsets of non-APL AML. Disclosures: No relevant conflicts of interest to declare.

Mitochondrial-Mediated Apoptosis by the Sesquiterpene Oil α-Bisabolol and Its Synergism with Imatinib and Nilotinib In BCR-ABL+ Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4456-4456
Author(s):  
Massimiliano Bonifacio ◽  
Antonella Rigo ◽  
Elisabetta Cavalieri ◽  
Emanuele Guardalben ◽  
Christian Bergamini ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Lines ◽  
Mitochondrial Membrane ◽  
Single Agent ◽  
Leukemic Cells ◽  
Parp Cleavage ◽  
Potential Candidate ◽  
Dependent Manner ◽  
Ros Production ◽  
Dna Laddering

Abstract Abstract 4456 Background. The plant-derived agent α-bisabolol is a small oily sesquiterpene alcohol that has been demonstrated to be cytotoxic against human malignant non-hematological and leukemic cells (Bonifacio M et al, Blood, 2009 ASH annual meeting abstracts;114:4800). Here we tested its activity against BCR-ABL+ cell lines and primary cells from patients, alone or in combination with the Tyrosine-Kinase Inhibitors (TKIs) Imatinib and Nilotinib. Also, the mechanism of α-bisabolol cytotoxicity in BCR-ABL+ cells was assessed. Methods. We used the BCR-ABL+ K562, LAMA-84 and CML-T1 cell lines and primary leukemic cells from 14 patients with BCR-ABL+ Acute Lymphoblastic Leukemia at diagnosis. First, the citotoxicity of single-agent α-bisabolol was determined by MTT. Then, mitochondrial membrane potential of treated cells was evaluated by the JC-1 dye in flow cytometry and fluorescence microscopy. Permeabilized leukemic cells were assayed for oxygen consumption by measuring mitochondrial state 3 and uncoupled respiration. Reactive oxygen species (ROS) production in α-bisabolol treated cells were quantified in flow cytometry by oxidation of CM-H2DCFDA, measuring the fluorescence intensity of the DCF products. Apoptosis was studied by the poly(ADP-ribose) polymerase (PARP) cleavage and internucleosomal DNA laddering analysis. Finally, the combination effects between α-bisabolol and Imatinib or Nilotinib (kindly provided by Novartis) were analyzed according to the median-effect method of Chou and Talalay using the CalcuSyn software. Results. α-bisabolol reduced the viability of BCR-ABL+ cells in a dose-dependent manner. The mean IC50 values of α-bisabolol were 46±11 μ M for primary leukemic cells and ranged from 62 to 115 μ M in the cell lines. JC-1 staining of BCR-ABL+ primary leukemic cells treated with 40 μ M α-bisabolol for 3 to 5 hours demonstrated a dissipation of the mitochondrial transmembrane potential (ΔΨm), thus indicating the start of the apoptotic process. Moreover, NADH-supported state 3 respiration in α-bisabolol treated leukemic cells was significantly decreased in comparison with untreated leukemic controls (140.0±70.5 vs 280.7±11.9 pmol O2/min/106 cells; p<.05). Finally, PARP cleavage and DNA laddering followed α-bisabolol exposure of leukemic BCR-ABL+ blasts. The apoptosis induction was accompanied by ROS production. When tested in combination at constant ratio with Imatinib or Nilotinib, α-bisabolol showed overall slight to strong synergistic effects, without evidence for antagonism across a range of doses (Table 1). In 3 patients with mutation of BCR-ABL (T315I, E255V and Y253H, respectively) we observed full activity of α-bisabolol as single agent and confirmed the synergism between α-bisabolol and Imatinib. Conclusion. This study indicates that α-bisabolol is an effective pro-apoptotic agent for human acute BCR-ABL+ leukemia cells via induction of mitochondrial membrane damage. The combination of α-bisabolol with Imatinib or Nilotinib allows a dose reduction up to 90% of each drug to obtain the same cytotoxic effect, so indicating a clear synergism. α-bisabolol may be a potential candidate for the treatment of BCR-ABL+ leukemias and the effective dose of TKIs could be reduced in a combined treatment with α-bisabolol. Disclosures: No relevant conflicts of interest to declare.

Establishment and Characterization of a Novel Human Myeloid Leukemia Cell Line, AMU-AML1, Carrying t(12;22)(p13;q11) with No Fusion of MN1-TEL

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4375-4375
Author(s):  
Mayuko Goto ◽  
Ichiro Hanamura ◽  
Motohiro Wakabayashi ◽  
Hisao Nagoshi ◽  
Tomohiko Taki ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Leukemia Cell ◽  
Leukemic Cells ◽  
Leukemia Cell Line ◽  
Chromosome Band ◽  
Structural Abnormality ◽  
Myeloid Leukemia Cell

Abstract Abstract 4375 Leukemia cell lines are ubiquitous powerful research tools that are available to many investigators. In balanced chromosomal aberration in leukemia, a chimeric fusion gene formed by genes existing on breakpoints is frequently related to leukemogenesis. Cytogenetic abnormalities of chromosome band 12p13 are detected non-randomly in various hematological malignancies and usually involved TEL, which encodes a protein of the ETS transcription factor family. Chromosome band 22q11-12 is one of partners of translocation 12p13 and t(12;22)(p13;q11-12) results in fusion of TEL and MN1 or in just the partial inactivation of TEL. It is important to analyze precisely the breakpoint in a non-random translocation such as t(12;22)(p13;q11-12) and in addition it contributes to the better understanding of the molecular pathogenesis of leukemogenesis. In this study, we established a novel human myeloid leukemia cell line, AMU-AML1, having t(12;22) from a patient with acute myeloid leukemia with multilineage dysplasia and analyzed its characters. Mononuclear cells were isolated by Ficoll-Hypaque sedimentation from patient's bone marrow before initiation of chemotherapy and cultured in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS). After 3 months, cell proliferation became continuous. The cell line, named AMU-AML1, was established. In AMU-AML1, the following pathogens were negative for EBV, CMV, HBV, HCV, HIV-1, HTLV-1 and mycoplasma. A doubling time of AMU-AML1 cells was about 96 hours. Proliferation of the cells was stimulated by rhG-CSF (10 ng/ml), rhGM-CSF (10 ng/ml), M-CSF (50 ng/ml), rhIL-3 (10 ng/ml) and rhSCF (100 ng/ml) but not by IL-5 (10 ng/ml), rhIL-6 (10 ng/ml), and rhEPO (5 U/ml). AMU-AML1 was positive for CD13, CD33, CD117 and HLA-DR, negative for CD3, CD4, CD8 and CD56 by flow cytometry analysis. G-banding combined with SKY analysis of AMU-AML1 cells showed single structural abnormality; 46, XY, t(12;22)(p13;q11.2). Double-color FISH using PAC/BAC clones listed in NCBI website and array CGH analyses indicated that the breakpoint in 12p13 was within TEL or telomeric to TEL and it of 22q11 was centromeric to MN1. A chimeric MN1-TEL transcript and fusion protein of MN1-TEL could not be detected by RT-PCR and western blot analysis. The wild type of MN1 protein was strongly expressed in AMU-AML1 compared with other leukemic cell lines with t(12;22), MUTZ-3 and UCSD/AML1. Our data suggest that AMU-AML1 had a t(12;22)(p13;q11.2) without fusion of MN1-TEL and the expression level of MN1 protein was relatively high, which might have some effects on leukemogenesis. In conclusion, AMU-AML1 is a useful cell line to analyze the biological consequences of the leukemic cells with t(12;22)(p13;q11.2) but no fusion of MN1-TEL. Disclosures: No relevant conflicts of interest to declare.

Aurora Kinases in Childhood Acute Leukemia: The Promise of Aurora Kinase B As Drugable Target

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1476-1476
Author(s):  
Stefanie A. Segers ◽  
C. Michel Zwaan ◽  
Carla Exalto ◽  
Mirjam W.J. Luijendijk ◽  
Valerie S. Calvert ◽  
...  
Keyword(s):  
Acute Leukemia ◽  
Cell Lines ◽  
Aurora Kinase ◽  
Selective Inhibitor ◽  
Leukemic Cells ◽  
Aurora Kinases ◽  
Aurora Kinase B ◽  
Pediatric All ◽  
Childhood Acute Leukemia

Abstract Abstract 1476 AIM: Aurora kinases (AURK) A and B are known regulators of mitosis and are overexpressed in a large number of human cancers, including leukemia. Several AURK-inhibitors have shown anti-tumor activity in vitro and in vivo. However, the efficacy of AURK inhibition in the treatment of childhood acute leukemia is unexplored. We therefore investigated the effect of targeting AURKA and AURKB in leukemic cells of children with newly diagnosed acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). Materials & Methods: Affymetrix gene expression data of 297 ALL, 237 AML and 8 normal bone marrow (nBM) samples were analyzed for AURKA and B mRNA expression levels. Protein expression levels in 172 pediatric ALL and 10 nBM samples were determined with a reverse phase protein array. Functional studies were performed in ALL and AML cell lines, in which AURKA and B were silenced using a short hairpin RNA with a lentiviral delivery system or LNA-containing oligonucleotides. Sensitivity of leukemic cell lines to the AURKB-selective inhibitor Barasertib-hQPA (AZD1152-hQPA) was tested in vitro with an MTS assay. Results: AURKA and B mRNA levels were low in ALL and AML patients. In contrast, Aurora A and B proteins were expressed to a greater extent in patients (p<0.0002), especially in ALL cases with an E2A-PBX1 translocation (p<0.0001) than in nBM mononuclear cells. Silencing of AURKA by shRNA and by LNA-oligonucleotide caused no or only minor growth delay in several cell lines reflecting genetic subtypes typically found in pediatric ALL and AML. In contrast, silencing of AURKB resulted in proliferation arrest and apoptosis in these cells. Furthermore, 18 out of 20 ALL and AML cell lines tested were highly sensitive to the AURKB-selective inhibitor Barasertib-hQPA in the nanomolar range (IC50 = 19–233 nM) whereas less sensitivity was seen for other inhibitors. Conclusion: These data show that inhibition of AURKB but not AURKA has an anti-proliferative and pro-apoptotic effect on acute leukemic cells. Thus, targeting Aurora Kinase B may offer a new strategy to treat pediatric ALL and AML. Disclosures: No relevant conflicts of interest to declare.

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