PDK1 Overexpression In Acute Myeloid Leukemia; Clinical Significance and Potential as a Therapeutic Target

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2162-2162
Author(s):  
Joanna Zabkiewicz ◽  
Lorna Pearn ◽  
Robert Hills ◽  
Gareth Morgan ◽  
Alan Burnett ◽  
...  
Keyword(s):  
Overall Survival ◽  
Clinical Significance ◽  
Cell Lines ◽  
Therapeutic Target ◽  
Conflicts Of Interest ◽  
Ectopic Expression ◽  
Cyclin D3 ◽  
Significant Heterogeneity ◽  
Selective Treatment ◽  
Cd34 Cells

Abstract Abstract 2162 PDK1 is a master kinase responsible for regulating at least six kinase groups including AKT, PKC and S6K. Many of these kinases have been shown to be constitutively active in tumour tissues including leukemia suggesting PDK1 is frequently dysregulated. Here we describe the frequency and significance of PDK1 overexpression in AML and determine the potential of PDK1 as therapeutic target. Analysis of 113 AML patients showed that overexpression of PDK1 compared with normal blast cells was frequently observed in 2 groups of patients defined by FAB M1/M0 (38% PDK1Hi) and M4/M5 (44% PDK1Hi). To establish whether overexpression was a property of the entire leukemic population (including putative leukemic stem cells) we carried out multiparameter flow cytometric intracellular staining. In 7/8 patients, we found PDK1 overexpression to be uniformly expressed in all leukemic sub-populations. Ectopic overexpression of PDK1 in normal CD34+ cells promoted their survival in unsupplemented medium (155% ±38, P<0.01, n=6). Similarly, we found that primary AML blasts overexpressing PDK1 displayed significantly higher survival in vitro when cultured in the absence of growth factors than those with normal PDK1 levels (82.4%±10.1 in PDK1Hi vs 64.4%±12.2 in PDK1Norm; P<0.0001, n=71). To determine whether PDK1Hi AML blasts were more dependent on PDK1 activity for their survival, we assessed their sensitivity to PDK1 inhibition compared with PDK1Norm blasts, using a selective inhibitor, BX-795. We found that PDK1Hi blasts showed significantly increased sensitivity to PDK1 inhibition (PDK1Hi 6.4μM +/−3.07 n=30; PDK1Norm 13.4μM +/−7.4 n=37, P=0.001) indicating that oncogene addiction is a feature of PDK1 overexpression in AML. In contrast, normal bone marrow CD34+ cells were resistant to BX-795 suggesting that PDK1 inhibition selectively targets the survival of AML blasts. Western blotting of BX-795-treated AMLs revealed dose dependent knock down of all detectable PDK1 targets substantiating the specificity of this inhibitor; furthermore BX-795 showed synergistic action when used in combination with AraC (CI= 0.7). We next investigated the clinical significance of PDK1 overexpression (using a statistical model to adjust for known prognostic factors) and discovered significant heterogeneity in overall survival between M1/M0 and M4/M5 AMLs (p=0.001 for interaction) with M1/M0 PDK1Hi patients showing significantly improved overall survival (HR 0.14 (0.003-0.61) p=0.004) whereas M4/M5 PDK1Hi patients showed poorer overall survival (although this did not reach significance: HR 2.08 (0.97-4.46) p=0.06). To examine the basis of this differential survival pattern, we surveyed the targets previously associated with PDK1 hyperactivation. We discovered that PDK1 overexpression in M4/M5 patients exclusively suppressed expression of cyclin D3 (R2 =0.675). Ectopic expression of PDK1 in AML cell lines confirmed that PDK1 suppressed D3 expression in monocytic AML cell lines whereas it induced cyclin D3 in AML lines derived from undifferentiated AML. These data indicate that suppression of cyclin D3 expression by PDK1 may reduce the rate of proliferation in M4/M5 AML, decreasing their sensitivity to standard chemotherapeutic treatments (which are most effective in targeting highly proliferative cells). Taken as a whole these data suggest that therapeutic targeting of PDK1 is an effective and selective treatment for AML, but is likely to be most beneficial for M4/M5 patients. Further, these data demonstrate that developmental context can be a significant factor when establishing the role and significance of abnormalities in AML. Disclosures: No relevant conflicts of interest to declare.

Recurrent Oncogenic Mutations in CCND3 in Aggressive Lymphomas

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 435-435
Author(s):  
Roland Schmitz ◽  
Sameer Jhavar ◽  
Wenming Xiao ◽  
Xuelu Liu ◽  
John Powell ◽  
...  
Keyword(s):  
Rna Interference ◽  
B Cell ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Human Cancer ◽  
Ectopic Expression ◽  
Cyclin D3 ◽  
G1 Arrest ◽  
Sequencing Technologies ◽  
Aggressive Lymphomas

Abstract Abstract 435 The recent development of high throughput mRNA sequencing technologies has allowed major advances in the characterization and quantification of transcriptomes in human cancer. These advances include gene mutation detection, gene fusion discovery, analysis of alternative splicing events, and unbiased gene expression profiling. To comprehensively discover pathogenic sequence variants in lymphomas, we used Illumina technology to sequence mRNA of 206 lymphoma biopsies and cell lines, including 64 diffuse large B-cell lymphomas (DLBCL) of the activated B-cell-like (ABC) subtype, 71 DLBCL of the germinal center (GCB) subtype, and 41 Burkitt's lymphomas (BL). This effort uncovered a large number of mutations that were verified by Sanger sequencing. Using this methodology, we discovered somatic mutations in CCND3, encoding Cyclin D3, in 38 % of BLs, 14 % of ABC DLBCLs, and 10 % of GCB DLBCLs. These mutations stabilized the Cyclin D3 protein by altering a phosphorylation motif at Thr283 that is required for proteasomal degradation. Knockdown of CCND3 by RNA interference confirmed the oncogenic addiction to this gene in the mutated cell lines. In parallel, an RNA interference screen revealed that the cell lines that are dependent on CCND3 are also reliant on cyclin-dependent kinase 6 (CDK6), which together with Cyclin D3 regulates the G1/S transition of the cell cycle. Ectopic expression of the mutant CCND3 isoforms accelerated lymphoma cell line proliferation thereby demonstrating the oncogenic potential of these mutations. Remarkably, treatment of the CCND3/CDK6 addicted cell lines with a small molecule inhibitor of CDK 4/6 caused G1 arrest followed by apoptosis suggesting novel therapeutic strategies for these aggressive lymphomas. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Super-Enhancer Profiling Identifies Novel Oncogenes and Therapeutic Targets in Multiple Myeloma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 362-362
Author(s):  
Jianbiao Zhou ◽  
Yunlu Jia ◽  
Tze King Tan ◽  
Tae-Hoon Chung ◽  
Takaomi Sanda ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Plasma Cell ◽  
Conflicts Of Interest ◽  
Cell Cancer ◽  
Ectopic Expression ◽  
Therapeutic Targets ◽  
Physical Interaction ◽  
Rna Seq ◽  
Normal Plasma

Background: Multiple myeloma (MM) is an aggressive neoplastic plasma cell cancer characterized by diversely cytogenetic abnormalities. MM can be divided into subtypes with immunoglobulin heavy chain (IGH) gene translocations involving CCND1-3, FGFR3/MMSET, MAFs and hyperdiploid myeloma containing trisomies of several odd numbered chromosomes 3, 5, 7, 9, 11, 15, 19, and 21. Although several new drugs have been introduced into clinic, treatment for MM patients remains challenge and refractory/resistant to therapy is often seen. Thus, a better understanding of the molecular pathogenesis of MM can lead to generate new prognostic classification and identify new therapeutic targets. Super-enhancers (SEs) are defined as large clusters of cis-acting enhancers, marked by high level bindings of acetylation of histone H3 lysine 27 (H3K27ac) and mediator complex. SEs have been shown to control genes for maintaining cellular identity and also key tumor drivers in various malignancies. Methods: H3K27Ac ChIP-seq and RNA-seq were performed on primary MM patient samples, MM cell lines. Normal plasma cells and lymphoma cell lines were served as controls. We systematically compared SEs and their associated genes of normal and cancerous tissue. THZ1, a CDK7 inhibitor, was used to efficiently down-regulate SE-associated genes. Combinatory analysis of THZ1-sensitive and SE-associated gene revealed a number of promising MM oncogenes. CRISPR/Cas9 technology and ectopic expression experiments in conjunction with cellular functional assays were performed to determine the effects of candidate SE-genes on MM cells. Circularized chromatin conformation capture followed by sequencing (4C-seq) was applied to explore the direct contact of SE and promoter. Results: SE analysis uncovered some cell lineage-specific transcription factors (TFs) and known oncogenes in MM. Several key TFs, including IRF4, PRDM1, MYC and XBP1, were identified in most MM samples, confirming the origin of MM cells. These data reinforce the concept that SE establishment is a key component of MM biology. The acquisition of SEs around oncogene drivers is widely observed during tumorigenesis. ST3GAL6 and ADM were two known oncogenic drivers in myeloma cells, which were associated with super-enhancers in all MM samples but not in normal plasma cell and lymphoma cells. We also found SE constituents for multiple subtype-specific key oncogenes such as CCND1 in t(11;14) cells, C-MAF in t(14;16) cells, and NSD2 and FGFR3 in t(4;14) cells. Furthermore, THZ1 showed prominent anti-neoplastic effect against MM cells. SE-associated genes were more sensitive to THZ1 compared with those genes associated with typical enhancers (TEs). By overlapping THZ1-sensitve gene with SE-associated genes, we identified a number of novel MM oncogenes, including MAGI2, EDEM3, HJURP, LAMP5, MBD1 and UCK2 as a potential druggable kinase. The expression level of MAGI2 and HJURP confers poor prognosis in several MM datasets. MAGI2 silencing in MM cells decreased cell proliferation and induced apoptosis. qRT-PCR and Western blot analysis confirmed the overexpression of HJURP in t(4;14) cells relative to non-t(4;14) MM cells. Furthermore, 4C-seq analysis revealed the physical interaction between HJURP-SE and promoter and THZ1 treatment diminished this interaction. Motif search at SE constituents revealed a highly significant enrichment of NSD2 recognition. Significant reduction of NSD2 binding at HJURP-SE region was observed in KMS11 infected with NSD2-specific shRNAs. Interestingly, blocking SE sites by CRISPR/Cas9i or silencing HJURP by shRNA led to decreased HJURP expression and cell apoptosis, whereas overexpression of this gene promoted cell growth. Taken together, our data demonstrated that HJURP is a novel SE-associated oncogene in t(4;14) MM. Conclusions: Our integrative approaches by combing H3K27Ac ChIP-seq, RNA-seq and THZ1-sensitive transcript defined the landscape of SE and identified SE-associated novel oncogenes, as well as lineage-specific TFs in MM. Furthermore, we also revealed subtype-specific SE-driving oncogenic program in MM. Taken together, these results not provide novel insight into the MM pathology, but also offer novel, potential therapeutic targets, such as MAGI2, and HJURP for the treatment of MM patients. Disclosures No relevant conflicts of interest to declare.

HOXA9 Is a Novel Therapeutic Target in Multiple Myeloma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 832-832 ◽  
Author(s):  
Michael A Chapman ◽  
Jean-Philippe Brunet ◽  
Jonathan J Keats ◽  
Angela Baker ◽  
Mazhar Adli ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Histone Modification ◽  
Cell Lines ◽  
Therapeutic Target ◽  
Copy Number ◽  
Conflicts Of Interest ◽  
Specific Expression ◽  
Aberrant Expression ◽  
High Level

Abstract Abstract 832 We hypothesized that new therapeutic targets for multiple myeloma (MM) could be discovered through the integrative computational analysis of genomic data. Accordingly, we generated gene expression profiling and copy number data on 250 clinically-annotated MM patient samples. Utilizing an outlier statistical approach, we identified HOXA9 as the top candidate gene for further investigation. HOXA9 expression was particularly high in patients lacking canonical MM chromosomal translocations, and allele-specific expression analysis suggested that this overexpression was mono-allelic. Indeed, focal copy number amplifications at the HOXA locus were observed in some patients. Outlier HOXA9 expression was further validated in both a collection of 52 MM cell lines and 414 primary patient samples previously described. To further verify the aberrant expression of HOXA9 in MM, we performed quantitative RT-PCR, which confirmed expression in all MM patients and cell lines tested, with high-level expression in a subset. To further investigate the mechanism of aberrant HOXA9 expression, we interrogated the pattern of histone modification at the HOXA locus because HOXA gene expression is particularly regulated by such chromatin marks. Accordingly, immunoprecipitation studies showed an aberrantly low level of histone 3 lysine 27 trimethylation marks (H3K27me3) at the HOXA9 locus. H3K27me3 modification is normally associated with silencing of HOXA9 in normal B-cell development. As such, it appears likely that the aberrant expression of HOXA9 in MM is due at least in part to defects in histone modification at this locus. To determine the functional consequences of HOXA9 expression in MM, we performed RNAi-mediated knock-down experiments in MM cell lines. Seven independent HOXA9 shRNAs that diminished HOXA9 expression resulted in growth inhibition of 12/14 MM cell lines tested. Taken together, these experiments indicate that HOXA9 is essential for survival of MM cells, and that the mechanism of HOXA9 expression relates to aberrant histone modification at the HOXA9 locus. The data thus suggest that HOXA9 is an attractive new therapeutic target for MM. Disclosures: No relevant conflicts of interest to declare.

Constitutively Active STAT6 Represses BCL6 in Primary Mediastinal B Cell Lymphoma.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2417-2417
Author(s):  
Olga Ritz ◽  
Jochen K Lennerz ◽  
Karolin Rommel ◽  
Karola Dorsch ◽  
Elena Kelsch ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Cell Lymphoma ◽  
Ectopic Expression ◽  
B Cell Lymphoma ◽  
Proximal Promoter ◽  
Constitutively Active

Abstract Abstract 2417 Primary mediastinal B-cell lymphoma (PMBL) is a subtype of diffuse large B-cell lymphoma (DLBCL) that affects predominantly young women (Swerdlow et al. 2008). Despite improvements due to addition of rituximab, which has become state of the art treatment, 20% of PMBL patients succumb to disease progression or relapse. Notably, here are currently no registered trials that are actively recruiting PMBL-patients and a better understanding of the underlying pathobiology may identify novel therapeutic targets and provide an alternative to dose escalation (Steidl and Gascoyne 2011). BCL6 is a key germinal center B-cell transcription factor that suppresses genes involved in lymphocyte activation, differentiation, cell cycle arrest and DNA damage response gene. BCL6 is aberrantly expressed in certain DLBCL subgroups and BCL6 overexpression is sufficient for lymphomagenesis in mice (Cattoretti et al. 2005). In cellular- and murine DLBCL models, targeting of BCL6 via retroinverted BCL6 peptid inhibitor (RI-BPI) appears effective (Polo et al. 2004; Cerchietti et al. 2010). In conjunction with the relatively restricted expression pattern of BCL6, these data collectively suggest BCL6 as a candidate for targeted therapy in BCL6-positive lymphomas. Despite substantial work on BCL6 in lymphomas, the function of BCL6 in PMBL is unknown. To address the BCL6 function in PMBL, we performed BCL6 depletion by siRNA in all three available PMBL cell lines: K1106, U-2940 and MedB-1. We found that BCL6 acts pro-proliferative and anti-apoptotic; however, PMBL models were only partially dependent on and not addicted to BCL6. Given that BCL6 expression in all PMBL cell lines is variable with a notable fraction of BCL6-negative cells, we argued that increasing the fraction of BCL6-positive cells might increase the level of BCL6-dependence. Since IL-4/STAT6 signaling upregulates BCL6 in mouse lymphocytes (Schroder et al. 2002), we treated PMBL cell lines with IL-4 (or IL-13) and, as expected, observed increased phosphorylated (p)STAT6 levels. Surprisingly, the pSTAT6 increase was not associated with higher – but with drastically lower BCL6 protein levels. Moreover, in untreated cells, co-localization studies for pSTAT6- and BCL6 demonstrated staining in mutually exclusive subsets of cells (Figure 1A), suggesting negative interaction between BCL6 and pSTAT6. Other STAT family members were already shown to participate in the transcriptional regulation of BCL6. Thus, we examined binding of STAT6 to the proximal promoter of BCL6 in all PMBL cell lines using shift assay and chromatin immunoprecipitation. We found that STAT6 can bind all five GAS binding sites within the BCL6 promoter in vitro and in all PMBL cell lines STAT6 was bound to proximal BCL6 promoter in vivo. Furthermore, transient STAT6 depletion by siRNA and/or ectopic expression of constitutively active STAT6 confirms that pSTAT6 is sufficient for transcriptional repression of BCL6. Co-localization studies in primary patient samples demonstrated mutually exclusive BCL6/pSTAT6 distribution as a visual hallmark of the repression mechanism (Figure 1B, C). Thus, our data demonstrate for the first time that constitutively active STAT6 transcriptionally represses BCL6 in PMBL. In conjunction with functional data, the delineated repression mechanism may prevent addiction to one single oncogenic pathway (i.e. BCL6) in PMBL. Figure 1. Mutually exclusive distribution of BCL6 and pSTAT6 in PMBL Figure 1. Mutually exclusive distribution of BCL6 and pSTAT6 in PMBL Disclosures: No relevant conflicts of interest to declare.

Synergism Between PDE4 and PI3Kδ Inhibitors in DLBCL: Improved Clinical Activity with the Potential for Lower Toxicity

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2938-2938 ◽  
Author(s):  
Jeffrey Cooney ◽  
Long Wang ◽  
An-Ping Lin ◽  
Daifeng Jiang ◽  
Avvaru Suhasini ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Ectopic Expression ◽  
Xenograft Model ◽  
B Cell Lymphoma ◽  
Regulatory Subunit ◽  
Pde4 Inhibitor ◽  
Structural Constraints ◽  
Pi3k Activity

Abstract Aberrant activation of the B cell receptor (BCR) is a hallmark of mature B-cell tumors. A better understanding of this process will spearhead effective clinical translation. The initiation and amplification of BCR signaling are well-defined events, and the successful deployment of BTK and PI3Kδ inhibitors in the clinic capitalizes on this knowledge. Conversely, the intricacies of the termination of BCR signals are less well-understood, and to date no rational therapeutic approach has been developed that exploit this aspect of the oncogenic BCR. Cyclic-AMP (cAMP) is a second messenger with marked growth suppression properties towards immune cells, including neoplastic mature B lymphocytes. In earlier work, we showed that inhibition of phosphodiesterase 4 (PDE4), the enzyme that hydrolyzes cAMP, downmodulates the activity of classical effectors of BCR signals, including SYK and PI3K. Herein, we attempted to gain further mechanistic understanding on how cAMP suppresses the proximal BCR activity, and built on this information to pre-clinically test therapeutic strategies that simultaneously attack the BCR at its amplification and termination points. Using diffuse large B cell lymphoma (DLBCL) as a model, we focused our attention on the interplay between cAMP and LCK, as we unexpectedly found that this cAMP-regulated canonical T-cell kinase is also widely expressed in DLBCL. Working with LCK-positive PDE4-low/null DLBCL cell lines, we found a marked increase in the phosphorylation of the inhibitory Y505 of LCK following elevation of intra-cellular cAMP. Next, we showed that ectopic expression of wild-type (WT) PDE4B, but not of a phosphodiesterase-inactive (PI) mutant, abrogated the cAMP-mediated, CSK-dependent, phosphorylation of LCK. Active LCK can phosphorylate PI3K's p85 regulatory subunit, thus freeing the catalytic domain from its structural constraints to promote lipid kinase activity. Thus, we tested whether the cAMP-mediated inhibition of LCK, by suppressing p85 phosphorylation, down-modulated PI3K activity. In LCK-positive PDE4B-null DLBCL, we showed that cAMP readily decreased the phosphorylation of p85 that followed BCR engagement; using the WT and PI PDE4B genetic models, we demonstrated that PDE4B expression abrogated cAMP effects and led to sustained PI3K activity following BCR engagement. These data suggested that inhibition of PDE4, by unleashing the negative effects of cAMP on LCK/p85, could accelerate the termination of PI3K activation that follows BCR engagement. If this hypothesis was correct, then the combination of PI3K and PDE4 inhibitors by attacking the BCR at its amplification and termination points, respectively, may synergistically suppress the growth of DLBCL. In in vitro studies with multiple DLBCL cell lines (WSU-NHL, OCI-Ly7, OCI-Ly18, OCI-Ly3, HBL-1, and OCI-Ly10) we showed that the combination of the FDA-approved PDE4 inhibitor roflumilast with idelalisib synergistically suppresses DLBCL growth (combination index < 1). This synergism was associated with a significant suppression of PI3K and AKT activities (p<0.05, cells treated with the drug combination vs. single agents). We expanded this observation to an in vivo xenograft model of human DLBCL, and showed that mice treated with roflumilast and idelalisib had a significantly smaller tumor burden than those receiving single agents (p< 0.01, two cohorts, n=47 mice). We also found a greater suppression of PI3K activity in the xenografts from mice treated with the combination of PDE4 and PI3Kδ inhibitors (p< 0.0001), as well as increased apoptosis. Together, these data further delineated how cAMP suppresses the BCR and showed that the rational combination PDE4 and PI3Kδ inhibitors synergistically suppresses DLBCL growth. These results are particularly important given the recent evidence of inflammatory/immune toxicity associated with the use of idelalisib, which we propose could be countered by the well-established anti-inflammatory properties of PDE4 inhibitors. Thus, we hypothesize that combining PDE4 and PI3Kδ inhibitors will enhance anti-lymphoma activity while decreasing clinical toxicity. This concept is ripe for clinical testing as we have recently completed a phase Ib trial showing that roflumilast is safe and active in patients with advanced B cell malignancies. Disclosures No relevant conflicts of interest to declare.

Activity and Cytotoxicity of Algae Extracts On Normal and Leukemic Hematopoietic Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4815-4815
Author(s):  
Jeremy Bechelli ◽  
Karen Rosell ◽  
Myra Coppage ◽  
Jane Liesveld
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Gradient Centrifugation ◽  
Leukemia Cell ◽  
Leukemic Cell ◽  
G1 Arrest ◽  
Western Blots ◽  
Prostate Cell ◽  
Cd34 Cells

Abstract Abstract 4815 Algae preparations are commonly used in complementary and alternative medicine for presumed anti-oxidant, anti-inflammatory, and anti-cancer properties. In this study, we have examined the effects of extracts from algae and algae components on the proliferation of normal hematopoietic and leukemia cells. To prepare extracts, 1 gram of Dunaliella salina (Dun), Astaxanthin (Ast), Spirulina (C-phycocyanin) (Spir), or Aphanizomenon flos-aquae (AFA) were added to 10 ml of 70% ethanol and incubated at 4°C on a shaker for 24 hours. The slurry was centrifuged at 400 g for 10 minutes at 4°C, and the supernatant was filtered through 413-grade filter paper. Leukemia cell lines were purchased from ATCC and blood or marrow aspirates from normal subjects or patients with leukemia were subjected to Ficoll-Hypaque density gradient centrifugation. CD34+ cells were isolated using Miltenyi Biotec MiniMACS magnetic bead cell separation columns. To determine effects on cell proliferation, increasing concentrations of algae extracts were added in fresh medium to plated cells, and MTT reagent was added followed by detergent and absorbance readings were recorded at 570 nm. Leukemic cell lines such as HL60 and MV4-11 were significantly inhibited by Ast and AFA at concentration of 0.8 mg/mL of extract (p<0.05 compared with control conditions by two-tailed t-tests). Dun and Spir did not significantly inhibit proliferation of these cell lines. The PC3 prostate cell line and the MCF-7 breast cancer cell line were inhibited only by AFA at 1.5 to 2.0 mg/ml extract at 24 hours as assessed in an MTT assay. Through use of an Annexin V assay to determine effects on apoptosis, enriched primary AML blasts demonstrated increased apoptosis in the presence of AFA at 0.8 and 1.5 mg/ml. Primary CLL cells demonstrated increased apoptosis after 24 hour exposure to 1.5mg/mL Dun, AST, Spir, and AFA (p<0.05) by two-tailed t-tests. Western blots confirmed that apoptosis was mediated in part by increase in cleaved Caspase 3. Only AFA decreased the viability of normal light density marrow cells at 2.0 mg/ml, and normal CD34+ cells were inhibited by 0.8mg/ml Dun and at higher concentrations by Ast, Spir, and AFA. When effect of the extracts on outgrowth of BFU-E and CFU-GM was determined, only Dun and AFA inhibited BFU-E, but all 4 extracts were inhibitory to CFU-GM at 0.8 or 1.5 mg/ml (p<0.05; n=3). AFA was significantly more inhibitory than AST. Cell cycle analysis of AML cell lines exposed to increasing concentrations of all extracts showed G0/G1 arrest. No increase in reactive oxygen species was found using dihydrorhodamine 123. These data suggest that extracts from algae utilized as food supplements have the ability to inhibit AML cell lines and primary leukemia blasts but also demonstrate inhibitory effects on normal hematopoiesis. Each algae extract demonstrated a different pattern of inhibition, and whether these extracts are inhibitory at a stem cell level or would have differential effects on normal or leukemic cell growth in vivo remains to be determined. Disclosures: No relevant conflicts of interest to declare.

Interleukin-16 Is An Important Growth-Promoting Factor and a Novel Diagnostic and Therapeutic Target for Multiple Myeloma

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4056-4056
Author(s):  
Djordje Atanackovic ◽  
York Hildebrandt ◽  
Tim Luetkens ◽  
Axel R. Zander ◽  
Carsten Bokemeyer ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Tumor Cells ◽  
Cell Lines ◽  
Therapeutic Target ◽  
Conflicts Of Interest ◽  
Myeloma Cell ◽  
Antibody Array ◽  
Western Blots ◽  
Myeloma Cells ◽  
Growth Promoting

Abstract Abstract 4056 Background: Multiple myeloma (MM) is a malignancy characterized by the expansion of a plasma cell (PC) clone that localizes to the bone marrow (BM). Myeloma cells and BM stromal cells both produce soluble factors promoting the survival and progression of MM. Interleukin-(IL)-16 is involved in regulating migration and proliferation of normal leukocytes, however, it has been unclear whether IL-16 also plays a role in the pathophysiology of human cancers. Methods: Using an antibody array we screened supernatants of myeloma cell lines for the presence of a variety of cytokines/chemokines. We confirmed IL-16 expression in myeloma cell lines as well as in malignant PC and BM plasma from MM patients applying real-time PCR, western blots, ELISA, and flow cytometry. We applied inhibitory RNA to analyze IL-16 function and we used anti-IL-16 antibodies to evaluate possible therapeutic options for MM. Results: We found IL-16 to be strongly overexpressed in the BM of myeloma patients. Myeloma cell lines as well as primary tumor cells from MM patients constitutively expressed IL-16 RNA and protein and spontaneously secreted soluble IL-16. Functional analyses revealed that IL-16 supports the proliferation of myeloma cells. Accordingly, silencing of IL-16 expression had an anti-proliferative effect on the tumor cells. Most importantly, the application of a monoclonal antibody directed against IL-16had a strong growth-inhibiting influence on myeloma cells. Conclusions: These findings suggest that cytokine IL-16 is an important growth-promoting factor in MM and might represent a novel diagnostic and therapeutic target for this incurable human malignancy. Disclosures: No relevant conflicts of interest to declare.

Overexpression Of Mir-125a In Bone Marrow CD34+ cells Of Patients With Myelodysplastic Syndrome Is Correlated To a Poor Prognosis and May Contribute To The Pathogenesis Of The Disease Through The Modulation Of NF-Kb Activation and Enhancement Of Differentiation Arrest

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5206-5206
Author(s):  
Irene Ganan-Gomez ◽  
Yue Wei ◽  
Hui Yang ◽  
Maria Carmen Boyano-Adánez ◽  
Guillermo Garcia-Manero
Keyword(s):  
Bone Marrow ◽  
Overall Survival ◽  
Conflicts Of Interest ◽  
Fine Tuning ◽  
Luciferase Reporter ◽  
Mirna Cluster ◽  
Luciferase Reporter Gene ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Gene Assay

Abstract Myelodysplastic syndromes (MDS) are a group of clonal malignancies characterized by impaired proliferation and differentiation of hematopoietic stem cells and precursors. The involvement of toll-like receptor (TLR)-mediated signalling in the modulation of myeloid differentiation and its participation in the pathogenesis of MDS are well documented (Wei et al 2013). Increased signaling through this pathway leads to the constitutive activation of NF-kB, which regulates the production of cytokines and mediates cell proliferation and apoptosis (Starczynowski 2010). In addition to the expression of proteins involved in inflammation, the TRL pathway also induces the expression of microRNAs (miRNAs) which participate in the fine-tuning of the inflammatory response (Kawai and Akira 2010). miR-125a and miR-125b are known modulators of hematopoiesis (Gerrits et al. 2012) and have been reported to be involved in several lymphoid and myeloid diseases. Little is known about their role in the pathogenesis of MDS. Interestingly, NF-kB-activating ability has been described for both miR-125a/b (Kim et al. 2012), and miR-125b appears to be upregulated by NF-kB within a positive feedback loop (Zhou et al. 2009; Tan et al. 2012). The aim of this work was to analyze the expression of miR-125a/b in MDS CD34+ cells and to study their relationship with the TLR pathway and differentiation. For this purpose, we analyzed the expression of miR-125a/b by qPCR in bone marrow CD34+ cells of 48 MDS patients, compared it with expression in healthy donors and studied the correlation with overall survival. In our study, we included miR-99b, which is clustered with miR-125a in the genome. Levels of TLR pathway components were detected by qPCR and correlated to those of the miRNAs. Activation of NF-kB was determined in Meg-01 and KG1 cells by the luciferase reporter gene assay, using a vector containing NF-kB responsive elements. Differentiation was studied in K562 and MDS-L cells through colony formation assays combined with analyses of the expression of specific markers by qPCR. For these experiments we used miRNA analogs and a miR-125a anti-sense oligonucleotide. Our results showed that miR-125a, but not miR-125b, is strongly overexpressed in MDS patients (∼15-fold of controls; P<0.01) and that miR-125a levels are significantly and negatively correlated to overall survival of MDS patients (P<0.05). Moreover, expression of miR-99b is also directly connected to the progression of the disease (P<0.05). Both miR-125a and miR-99b cooperate in vitro in the activation of NF-kB (P<0.001); however, we observed a negative correlation between miR-99b/miR-125a expression and levels of TLR2, TLR7 and their downstream proteins MyD88 and JMJD3 (P<0.05), suggesting that NF-kB activation by the miRNA cluster occurs in the absence of TLR signaling. Furthermore, we observed a ∼4-fold increase in NF-kB activity after miR-125a inhibition in the presence of a TLR2 agonist (P<0.001), indicating that miR-125a acts as an NF-kB inhibitor upon TLR stimulation. These results show that miR-125a is involved in the fine-tuning of NF-kB activity and that its effects may depend on the status of the TLR pathway. We then investigated the role of miR-125a in hematopoiesis and found that this miRNA contributes to the blockade of differentiation in the cell lines studied. Therefore, miR-125a could be involved in the pathogenesis or progression of MDS through the modulation of NF-kB activity and differentiation arrest. Thus, this miRNA could be a good prognostic marker and is a potential therapeutic target in MDS. Disclosures: No relevant conflicts of interest to declare.

Gvhd-Associated Early Impairment of Marrow Function Is Frequent after Allogeneic Transplantation and Is a Sensitive Biomarker for Prediction of Treatment Related Mortality and of Overall Survival

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1160-1160
Author(s):  
Giuseppe Milone ◽  
Giuseppe Avola ◽  
Maria Grazia Camuglia ◽  
Salvatore Leotta ◽  
Alessandra Cupri ◽  
...  
Keyword(s):  
Overall Survival ◽  
Cumulative Incidence ◽  
Acute Gvhd ◽  
Conflicts Of Interest ◽  
Univariate Analysis ◽  
Disease Status ◽  
Risk Of Death ◽  
Cd34 Cells ◽  
Treatment Related Mortality ◽  
Related Mortality

Abstract INTRODUCTION: Aim of our study was to determine the role of acute-GVHD in influencing early marrow function and whether assessment of “GVHD-associated marrow impairment” may predict transplant outcome. METHODS: We have prospectively studied 62 patients who received T-replete allogeneic stem cell transplantation because of various malignancies. At day +18/+19, we determined in bone marrow: CD34+ cells, frequency of clonogenic cell (CFU-GM and BFU-E) and, in 20 patients, also CD34+ cells showing apoptosis (AnnexinV/7-AAD). Results were related to acute-GVHD and to clinical outcome in terms of Treatment Related Mortality (TRM), Relapse Rate (RR) and Overall Survival (OS). To distinguish the effect of a-GVHD from that determined by corticosteroid, patients were divided in three groups according to time of presentation of a-GVHD: “Early a-GVHD, “No a-GVHD” and “Impending a-GVHD”. The latter group consisted of patients presenting a-GVHD after engraftment. RESULTS: In univariate analysis “Febrile neutropenia” and” Acute GVHD” were important factors for a reduction of frequency of marrow clonogenic cells assessed on day +18/+19. However, in multivariate analysis only “Acute GVHD” was able to influence frequency of marrow CFU-GM and BFU-E at day +18/+19. Patients not developing “a-GVHD” until day +90, had on day+18 a median growth of CFU-GM of 202/10e5 plated cells while patients suffering “early GVHD” had a marrow CFU-GM growth significantly reduced: 82/10e5 plated cells, (p= 0.0009). Median CFU-GM was found significantly reduced also in patients defined as “impending GVHD” (onset of a-GVHD at day +20-/+90) (p=0.01). Apoptotic CD34+ cells in marrow cells at day +18/+19 were inversely correlated to frequency of marrow BFU-E (r= -0.5, p=0.04). Taking into account competing risks, Cumulative Incidence of TRM at 2 y in the group of patients having a frequency of marrow CFU-GM over median was 5% while it was 30% in the group having CFU-GM below the median (log-rank: p=0.004). Cumulative incidence of Relapse was not significantly different in these two groups (31% versus 34%). OS was significantly better in group having CFU-GM over median: 62% versus 35% (logrank: p=0.02). Frequency of CFU-GM over median at day +18/+19 was a factor important for a reduced risk of death (HR=0.358; p=0.004) also after adjusting, using multivariate Cox analysis, for Disease Status (Early versus Advanced). CONCLUSIONS: acute-GVHD impairs early and significantly marrow function, marrow-GVHD is a sensitive biomarker for prediction of TRM and OS. Disclosures No relevant conflicts of interest to declare.

Protooncogene MYB Activates the Transcription of ROR1 in B Cell Malignancies

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3292-3292
Author(s):  
Ken Watanabe ◽  
Megumi Iida ◽  
Shihoko Suwa ◽  
Miyu Kato ◽  
Osamu Miura ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Ectopic Expression ◽  
Luciferase Reporter ◽  
Regulation Of Transcription ◽  
B Cell Malignancies ◽  
Promoter Assay ◽  
Expression Levels ◽  
Intron 1

Abstract The receptor tyrosine kinase-like orphan receptor 1 (ROR1) is a highly conserved gene that is associated with cell survival. ROR1 is uniquely expressed in some B cell malignancies and epithelial cancer cells although it is barely detected in normal adult tissues. The precise mechanisms of this ectopic expression have remained unknown. We checked ROR1 expression by FACS analysis and by quantitative RT-PCR. ROR1 was highly expressed in the samples from CLL,MCL,SMZL,and BL. Expression levels of ROR1 mRNA and cell surface ROR1 protein showed clear positive correlation (R2=0.55) suggesting that ROR1 expression is regulated mainly transcriptionally. Then, we cloned the 2.5-kb fragment upstream of ROR1 transcriptional start site and performed promoter assay using ROR1 positive and negative cell lines. The promoter activity was observed not only in ROR1 positive lines but also in negative lines, which suggests that this region may not be the only regulatory element. Thus, we hypothesized that conserved regions in intron 1, which is about 230 kb long, may play important roles in regulation of transcription. BLAST search identified 19 regions conserved between human and rat intron 1 of ROR1. Two regions, designated as CRS3 and CRS7, further showed homology with chicken ROR1 gene. Among them, CRS3 region had enhancer activity selectively in ROR1 positive B cell lines. A consensus nucleotide sequence for the binding of protooncogene MYB was found within CRS3 region. Actually, the protein binding to CR3S sequence was verified by EMSA assay using nuclear extract from MYB positive cells. Immunoblot analysis showed that ROR1 positive B cell lines had high expression of MYB. In addition, the expression levels of MYB protein and ROR1 mRNA were positively correlated in 7 CLL samples analyzed (R2=0.49). We introduced shRNA of MYB into ROR1 positive EW36 cell line. MYB shRNA could decrease CRS3 luciferase reporter activity. As a result, MYB shRNA decreased ROR1 expression as well as MYB expression. Collectively, MYB induced ROR1 expression and might contribute to tumorgenesis of B cell malignancies. Disclosures No relevant conflicts of interest to declare.

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