Expression of MicroRNA (miR) miR-15a/miR-16-1 Downregulates Expression of BCL-2 Protein in Chronic Lymphocytic Leukemia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2796-2796 ◽  
Author(s):  
Liguang Chen ◽  
Li Tang ◽  
George Calin ◽  
Carlo M. Croce ◽  
Thomas J. Kipps
Keyword(s):  
Gene Expression ◽  
Chronic Lymphocytic Leukemia ◽  
Human Cancer ◽  
Human Tumor ◽  
Immunoblot Analysis ◽  
Lymphocytic Leukemia ◽  
Control Vector ◽  
Rt Pcr ◽  
Target Mrna

Abstract MicroRNAs (miRNAs) comprise a family of small RNA, each member of which can potentially regulate post-transcription gene expression in a temporal and tissue-specific manner. Each miRNA encodes a transcript of about 22 nucleotides that can modulate expression of specific target mRNA. We identified that the chronic lymphocytic leukemia (CLL) cells of a large proportion of patients have aberrant, low-level expression of two clustered miRNAs located at 13q14, designated miR-15a and miR-16–1, providing the first example of dysregulated miRNA expression in a human cancer (PNAS USA99:15524, 2002). These miRNAs each have sequences that potentially could target mRNA encoding the anti-apoptotic protein bcl-2, which is expressed at high levels in CLL. Transfection of a lymphoma B cell line lacking expression of miR-15a and miR-16–1 with an expression vector encoding miR-15a and miR-16–1, designated pmiR-15/16, attenuated the expression-level of bcl-2 protein and enhanced the susceptibility of such lymphoma B cells to apoptosis in vitro. However, it was not certain whether miR-15a and miR-16–1 could modulate expression of bcl-2 in primary leukemia cells that were not adapted for propagation in vitro. We selected primary CLL cell samples (n = 3) that harbored deletions at 13q14 and that lacked expression of miR-15a and miR-16–1, as assessed via microRNA array and quantitative RT-PCR analyses. We transfected these cells with pmiR-15/16 or a control vector via electroporation and, in parallel studies, also transfected these CLL cells with sense and antisense control oligo-RNAs via transmessenger transfection. Before and after such manipulations we monitored for expression of miR-15a and miR-16–1 by RT-PCR, for relative expression of BCL-2-family member transcripts using a multiplex ligation-dependent probe amplification (MLPA) assay, and for expression of bcl-2 protein using immunoblot analysis and flow cytometry. We found that CLL cells transfected with either pmiR15/16 or with miR-15a and miR-16–1 sense oligo-RNAs had increased expression-levels of miR-15a and miR-16–1 within 24 hours after transfection, whereas CLL transfected with the control vector or antisense oligo-RNA did not. Depite high-level expression of miR-15a and miR-16–1, the relative levels of BCL-2 transcript did not change over the 48 hours after transfection that we examined. However, in this time period we observed that CLL cells made to express miR-15a and miR-16–1, experienced significant reductions in the levels of bcl-2 protein, which were not observed in control transfected CLL cells. This is the first demonstration that miRNAs can effect post-transcriptional regulation of protein expression in a primary human tumor and suggest that miRNAs may have potential therapeutic utility in the modulation of pathogenic gene-expression in CLL and other cancers.

Treatment of Chronic Lymphocytic Leukemia Patients with Lenalidomide Induces Down-Regulation of miR342-3p Associated with Over-Expression of Tumor Suppressor RASSF4,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3885-3885
Author(s):  
Emanuela M. Ghia ◽  
Lillian Werner ◽  
Danelle F. James ◽  
Donna Neuberg ◽  
Laura G Corral ◽  
...  
Keyword(s):  
Gene Expression ◽  
Chronic Lymphocytic Leukemia ◽  
Family Member ◽  
Research Funding ◽  
Human Tumor ◽  
Lymphocytic Leukemia ◽  
Down Regulation ◽  
Antagonistic Effects ◽  
Over Expression ◽  
Pre Treatment

Abstract Abstract 3885 Lenalidomide has promising clinical activity in patients with chronic lymphocytic leukemia (CLL). Unlike other anti-leukemia drugs, lenalidomide is not cytotoxic for CLL cells in vitro. Similar to CD154, lenalidomide can enhance CLL-cell expression of immune co-stimulatory molecules, formation of immunologic synapse, activation of NK-cells, and generation of anti-tumor immunity. Furthermore, lenalidomide repeatedly can enhance expression of CD154, which we had observed was functionally deficient in patients with CLL. However the exact mechanism of action of lenalidomide is still under investigation. Herein, we studied the gene expression profile and microRNA (miR) of CLL cells collected from 20 patients before and at day 8 and day 15 of treatment with 2.5–5 mg of lenalidomide in the CRC014 trial. We observed significant changes in expression level of 54 genes at day 8 versus pre-treatment samples. We identified significant changes in expression level of 189 genes at day 15 versus pre-treatment samples. This included 44 of the 54 (81%) genes noted at day 8. Forty genes were expressed at significantly higher levels at day 8 and day 15 of lenalidomide treatment. We noted that 7 (17%) of these genes were related to Ras pathway and its downstream signaling pathways (i.e. NF-KappaB pathway): Ras association (RalGDS/AF-6) domain family member 4 (RASSF4), a member of RAS oncogene family (RAB13), Ras protein-specific guanine nucleotide releasing factor 1 (RASGRF1), GTPase IMAP family member 6 (GIMAP6), GTP-binding protein ras homolog gene family member S (RND1), kinase suppressor of Ras 2 (KSR2) and toll-like receptor adaptor molecule 2 (TICAM2). Ras signaling affects many cellular functions, which includes cell proliferation, apoptosis, migration, fate specification, and differentiation. In the resting cells, Ras is tightly bound to GDP (Guanosine Diphosphate), which is exchanged for GTP (Guanosine Triphosphate) upon binding to activated cell membrane receptors. In the GTP-bound form, Ras interacts with a broad range of effector proteins to induce a diverse array of biological consequences. Although typically associated with enhanced growth and transformation, activated Ras also may induce growth antagonistic effects such as senescence or apoptosis. Some of the growth-inhibitory properties of Ras are mediated via the RASSF family of Ras effector/tumor suppressors. RASSF4 is the fifth member of this family and it binds directly to activated K-Ras in a GTP-dependent manner via the effector domain, thus exhibiting the basic properties of a Ras effector. Overexpression of RASSF4 induces Ras-dependent apoptosis in 293-T cells and inhibits the growth of human tumor cell lines. Although broadly expressed in normal tissue, RASSF4 is frequently down-regulated by promoter methylation in human tumor cells and primary tumors. However changes in miR expression also may affect the level of gene expression. Therefore we analyzed miRs expression by microarray in pre treatment, day 8, and day 15 CLL samples. We observed significant changes in expression levels of 33 miRs between day 8 and pre treatment samples. We identified significant changes in expression levels of 11 miRs between day 15 and pre treatment samples. Of the 33 miRs differentially expressed at day 8, only 5 were up-regulated whereas the remaining 28 were down-regulated. Interestingly, among these 28 down-regulated miRs, 5 miRs (miR-103, miR-16, miR-30a, miR-30b and miR-342-3p) target RASSF4. Noteworthy, miR-342-3p was one of the 3 miRs (miR-26a, miR-138 and miR-342-3p) down-regulated both at day 8 and at day 15, suggesting that the down-regulation of such miR has a key role in the overexpression of RASSF4 leading to Ras-dependent apoptosis. Further studies are ongoing to elucidate lenalidomide action on CLL cells via RASSF4 overexpression. This study demonstrates that treatment with lenalidomide can induce down-regulation of miRs associated with changes in gene expression by CLL cells, leading to over-expression of RASSF4 and other Ras or GTPase related proteins that can induce growth antagonistic effects and account in part for the activity of this drug in CLL. Disclosures: James: Celgene: Research Funding. Neuberg:Celgene: Research Funding. Corral:Celgene: Employment. Kipps:Igenica: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding; Abbott Industries: Research Funding; Genentech: Research Funding; GSK: Research Funding; Gilead Sciences: Consultancy, Research Funding; Amgen: Research Funding.

Functional Somatic and Germline Variants in the NF-κB Pathway in Chronic Lymphocytic Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 560-560 ◽  
Author(s):  
Ma. Reina Improgo ◽  
Adam Kiezun ◽  
Yaoyu Wang ◽  
Lillian Werner ◽  
Petar Stojanov ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Death ◽  
Chronic Lymphocytic Leukemia ◽  
Transcriptional Activity ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Lymphocytic Leukemia ◽  
Wild Type ◽  
Rt Pcr ◽  
Germline Variants

Abstract Abstract 560 Nuclear factor kappa B (NF-κB) encompasses a family of transcription factors involved in oncogenic processes including cellular proliferation and apoptotic inhibition. Constitutive activation of NF-κB has been observed in hematologic malignancies and is thought to confer resistance to chemotherapeutic agents. Here, we examine the role of the NF-κB pathway in chronic lymphocytic leukemia (CLL). Whole-exome sequencing was performed using tumor and matched germline DNA from 167 CLL patients. We identified 51 patients (30%) carrying 53 non-silent somatic variants in genes of the canonical NF-κB pathway, which consists of 272 genes as defined by the Ingenuity Pathway Analysis tool. Of the 99 patients whose germline sequences have been analyzed to date, 27 patients (27%) carry 34 non-silent germline variants in NF-κB pathway genes. A total of 67 patients (40%) have at least one non-silent somatic or germline variant. Variants in the NFKB1 gene, itself, were also observed: a somatic variant, H66R, found in two patients, and two germline variants, Y89F and R849W, each found in one patient. To evaluate the functional consequences of the NFKB1 variants, we performed site-directed mutagenesis to generate full-length NFKB1 cDNAs encoding these variants. We subsequently measured transcriptional activity of wild-type and mutant NFKB1 via luciferase assays in HEK293T cells using reporter cassettes containing the NFKB1 response element. Transcriptional activity of the three NFKB1 variants was found to be at least 2-fold higher than that of wild-type NFKB1 (p<0.0001). We further hypothesized that this increased transcriptional activity would lead to increased expression of NFKB1 downstream target genes. Analysis of gene expression profiles from Affymetrix HG-U133 Plus 2.0 Arrays of 65 CLL patient samples showed that the NFKB1 downstream targets CCL3, CCL4, and CD69 are upregulated in NFKB1 variants. To validate these results, we performed quantitative RT-PCR in patients with (n=3) or without (n=9) NFKB1 variants and confirmed upregulation of CCL3 (p=0.0286), CCL4 (p=0.0384), and CD69 (p=0.0263). Direct transfection of HEK293T cells with NFKB1 variants also resulted in a 3.3-fold upregulation of CCL3 (p=0.05). To test the hypothesis that deregulation of the NF-κB pathway is a key mechanism in CLL, we compared gene expression profiles of NF-κB pathway genes between CLL patient samples (n=146) and normal B cells (n=16) and found overall upregulation of the NF-κB pathway in CLL (Kolmogorov-Smirnov test, p=2.2e-16). K-means clustering and principal component analysis (PCA) further revealed that CLL patients can be divided into two subgroups exhibiting differential magnitude of NF-κB pathway upregulation. Studies in progress aim to identify the clinical significance of these subgroups. Finally, we assessed the effect of inhibiting the NF-κB pathway using the cell permeant NF-κB inhibitor, SN50. We performed Annexin V/PI staining 24 hours post-treatment in CLL cells with (n=9) or without (n=3) NF-κB pathway variants. SN50 increased cell death 1.8-fold in all cells tested (p<0.0001). Quantitative RT-PCR also showed a 59% decrease in expression of CCL3 one hour post-treatment, confirming inhibition of the NF-κB pathway. In conclusion, our findings demonstrate that a high proportion of CLL patients harbor somatic and germline variants in NF-κB pathway genes, some of which appear to be functional. Furthermore, the NF-κB pathway is upregulated in CLL and pharmacological inhibition of the pathway leads to increased cancer cell death. Functional characterization of NF-κB pathway variants offers mechanistic insight into the disease, providing novel targets for therapy. Disclosures: No relevant conflicts of interest to declare.

Biological and Clinical Relevance of Surrogate Markers of IgVH Mutational Status in B-Cell Chronic Lymphocytic Leukemia.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1062-1062
Author(s):  
Fortunato Morabito ◽  
Marta Lionetti ◽  
Giovanna Cutrona ◽  
Katia Todoerti ◽  
Serena Matis ◽  
...  
Keyword(s):  
Gene Expression ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Candidate Genes ◽  
Lymphocytic Leukemia ◽  
Surrogate Markers ◽  
Rt Pcr ◽  
Mutation Status ◽  
Expression Levels ◽  
Cell Chronic Lymphocytic Leukemia

Abstract B-cell chronic lymphocytic leukemia (B-CLL) is a heterogeneous disease; some patients have a rapidly progressing disease and others exhibit an indolent course and survive for many years without treatment. Mutation status of IgVH genes utilized by CLL cells represents a very reliable predictor of clinical outcome in B-CLL, but its analysis is expensive and beyond the capacities of most diagnostic laboratories. To identify surrogate markers we performed a gene expression profiling analysis of CD19+ purified cells from 80 B-CLL untreated patients in Binet stage A, by means of Affymetrix GeneChip® HGU133A arrays. The comparison of 46 IgVH-unmutated versus 34 mutated samples using the Prediction Analysis of Microarrays software identified 78 differentially expressed probes, specific for 59 well-characterized genes. Specifically, 43 genes had a higher and 16 genes a lower average expression in the IgVH unmutated group. These genes are involved in cellular functions, including cell cycle regulation (SEPT7, SEPT10, CDK2AP1), cell proliferation (SLAMF1, LDOC1), apoptosis (CD63, IFT57, P2RX1, RNF130, TNFRSF1B), cell adhesion (CNTNAP2, C1orf38, PCDH9), immune response (ZAP70, IFI44), signal transduction (AKAP13, RASGRP1, USP6NL, TGFBR3, AKAP12), lipid metabolism and fatty-acid degradation (FADS3, LPL, LASS6), cell-cell signalling (FCRL2), phospholipid biosynthetic process (AYTL2), regulation of circadian rhythm (EGR3, CRY1, OPN3), DNA-dependent regulation of transcription (MYBL1, NR4A2, NRIP1, ZBTB20), muscle development (VAMP5, SRI, DMD). The expression signature identified in the proprietary database was then validated by means of a meta-analysis of a publicly available gene expression dataset of 100 B-CLL (Haslinger et al., 2005), showing classification accuracy measures leading to a global classification rate of 82.93% of the test set and thus suggesting the strength of the identified expression signature. The expression levels of 11 genes (LPL, ZBTB20, ZAP70, CRY1, COBLL1, SEPT10, LDOC1, TNFRSF1B, DMD, SRI, NRIP1) were confirmed by means of quantitative real-time PCR (Q-RT-PCR) in a subset of 40 CLL patients. The prognostic impact for Time To Treatment (TTT) of the 59 candidate genes of our classifier model was investigated in 77 patients. Forty-nine (36.4%) of these received treatment after a median follow up of 4 years. As expected, patients with unmutated IgVH genes had a risk of therapy requirement that was about 3 times higher (HR: 3.1,95% C.I. 1.6–5.8, p&lt;0.0001) than those with mutated IgVH. Based on microarray expression levels, 43/59 genes significantly predicted TTT with a HR ranging from 1.5 (LPL gene) to 4.2 (SRI gene) (value for ZAP-70 = HR: 1.9, 95% C.I. 1.0–3.4, p=0.039). The same analysis performed in the panel of the 11 genes validated by Q-RT-PCR revealed 4 candidate genes which significantly predicted TTT. Specifically, Cox univariate analysis confirmed ZAP-70 as a predictor of disease outcome and underscored the prognostic role of the LPL, TNFRSF1B and CRY1 genes. The predictive power of the novel putative surrogate markers for the IgVH mutation status is now being further validated at protein expression level.

scholarly journals Potent Inhibition of the Growth and Induction of Apoptosis in Lymphoma By the Anthelminthic Drug Niclosamide: In Vitro Data

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5131-5131
Author(s):  
Junaid Ansari ◽  
Paula Polk ◽  
Jeffrey Aufman ◽  
Guillermo A Herrera ◽  
James Cardelli ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Cell Lines ◽  
Mononuclear Cells ◽  
Lymphocytic Leukemia ◽  
Lymphoma Cell ◽  
B Lymphoma

Abstract Background and Purpose: Niclosamide is an anthelminthic drug which has been used for the treatment of human parasitic infections for many years. Niclosamide interacts with lysosomes and induces autophagy. In recent years, it has demonstrated anti-cancer potential in leukemia, breast cancer, colon cancer, myeloma, ovarian, prostate and lung cancer models. Multiple pathways like Wnt/β-catenin, mTORC1, STAT3, NF-κB and notch signaling were reported to be involved. Only limited studies were done in lymphoma models. We hypothesized that niclosamide may also have in vitro and in vivo activities in lymphomas. Non-Hodgkin lymphomas generally respond well to chemotherapy and/ or immunotherapy, however many patients relapse and ultimately become refractory. Relapses are often caused by tumor stem cells not eliminated by cytostatic drugs. Therefore new treatment approaches and new targets are desirable. Materials and Methods: Established B lymphoma cell lines were exposed to different concentrations of niclosamide (0.1-4µM) and IC50 was calculated at 24, 48 and 72 hours. The cell concentration, viability and proliferation were assessed by CellTiter-Blue viability and trypan blue exclusion assays. Apoptosis was assessed by a combined annexin-V/ propidium iodide stain. Gene expression changes were studied using GeneChip Human Transcriptome Array 2.0 (Affymetrix) with 44 699 annotated genes. Colony forming assays were performed in methylcellulose. Ultrastructural changes were studied using a Hitachi electron microscope. As normal controls, peripheral blood mononuclear cells from individuals without active cancer were incubated with niclosamide for up to 72 hours. Samples from patients with chronic lymphocytic leukemia were also treated under the same conditions. Results: Treatment with niclosamide at doses as low as 0.1 μM resulted in time-and dose- dependent apoptosis, cytotoxicity and inhibition of proliferation in aggressive lymphoma cell lines. The 50% inhibitory concentration in a proliferation assay (mean of data at 24, 48 and 72 hours) is shown in the Table below. Niclosamide also inhibited clonal growth in semi-solid media. Electron microscopy showed that filopodia increased and lipid vacuoles developed whereas mitochondria were less numerous and had fewer cristae (when KOPN-8 was treated with 0.5 μM for 48 hours). The viability of mononuclear cells from 8 individuals without lymphoma was unchanged (or minimally decreased) when incubated with niclosamide. As far as cells from two patients with untreated chronic lymphocytic leukemia are concerned, no cytotoxicity was observed at doses between 0.5 and 5 μM. Gene expression changes were studied the cell lines Daudi and KOPN-8 treated with 2.5 μM for 3 and 6 h. 96 genes were consistently overexpressed , 59 down-regulated. Ten out of the 96 overexpressed genes involved the TNF pathway and immunoregulation including CD95. Thirteen out of the 59 down-regulated genes are involved in mitochondrial function. Table.Cell lineDescription of Cell TypeIC 50STDDaudiBurkitt lymphoma cell line0.37 μM± 0.12HBL-2Diffuse large B cell lymphoma cell line0.68 μM± 0.15KOPN-8B precursor ALL cell line0.6 μM± 0.08RamosBurkitt lymphoma cell line0.58 μM± 0.04RajiBurkitt lymphoma cell line0.65 μM± 0.10SU-DHL4-VRVincristine resistant lymphoma cell line0.5 μM± 0.02 Conclusion: Niclosamide effectively inhibits the proliferation of B lymphoma cell lines and induces apoptosis. Preliminary data show that Niclosamide targets genes involved in the TNF pathway and interferes with mitochondrial function. Normal lymphocytes are not sensitive to niclosamide. The in-vitro activity of niclosamide is at least comparable or superior to the activity seen in other malignancies. Niclosamide may target drug-resistant lymphoma stem cells and has clinical potential. We plan to study combination treatments and perform in vivo studies. Acknowledgments: The authors thank Drs. Borje Andersson, Shile Huang, Nakle Saba, Ben Valdez and Ellen Vitetta for their kind gift of cell lines. Disclosures No relevant conflicts of interest to declare.

KCa3.1 Blockers Inhibit Cell Proliferation in Chronic Lymphocytic Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3887-3887
Author(s):  
Eva M Groessinger ◽  
Lukas Weiss ◽  
Elisabeth Hinterseer ◽  
Judith Schmoelzer ◽  
Karin Oberascher ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Peripheral Blood ◽  
Lymphocytic Leukemia ◽  
K Channel ◽  
Relative Reduction ◽  
Rt Pcr ◽  
Ki 67

Abstract Abstract 3887 Potassium (K)-channels play an important role in regulating cell proliferation by maintenance of the membrane potential and subsequent Ca2+ signaling. Out of 80 known human K-channel genes only the voltage gated K-channel Kv1.3 and the calcium-gated K-channel KCa3.1 are expressed in lymphocytes, with expression levels varying greatly depending on lymphocyte maturation and activation status (for review see Cahalan MD, Chandy KG, Immunol Rev. 2009). Accordingly, proliferation of various lymphocyte subtypes can be inhibited by blockade of the respective predominant K-channel. As the modulation of K-channel expression on malignantly transformed lymphocytes and their potential as therapeutic targets has been largely overlooked, we characterized the expression and function of Kv1.3 and KCa3.1 in Chronic Lymphocytic Leukemia (CLL). Primary cells from unselected CLL-patients were isolated from peripheral blood mononuclear cells (PBMCs). Comparison of Kv1.3 and KCa3.1 levels on unstimulated CLL-cells versus PMA/ionomycin-activated CLL-cells revealed a significant reduction in the Kv1.3/KCa3.1 ratio (n=31, 1.526 vs. 0.9054, p=0.0005), as evidenced on mRNA and protein levels by RT-PCR and patch clamp analysis, respectively. Stimulation of CLL-cells with enriched and activated autologous CD4+ T-cells resulted in higher CLL-cell activation as measured by CD80/86 expression, and an even more pronounced reduction of the Kv1.3/KCa3.1 ratio. This stimulation protocol also effectively induced CLL-cell proliferation as verified by Ki-67 expression and CFSE dilution via flow cytometric measurement. Highly activated and/or proliferating CLL-cells consistently up-regulated KCa3.1 (RT-PCR: Ki-67 n=5, p=0.0013; CFSE n=5, p=0.0436; immunofluorescence (IF) staining - n=4, p=0.0121), whereas Kv1.3 was fairly low. Consistent with our in vitro data, CLL-cells in lymph node and bone marrow, believed to be primary sites of CLL-proliferation in vivo, were highly positive for KCa3.1 channels in contrast to CLL-cells from the peripheral blood as revealed by IF staining on paraffin-embedded tissue sections. In light of the significantly increased KCa3.1-expression on activated and proliferating CLL-cells, we investigated whether specific blockade of KCa3.1 could inhibit CLL-cell proliferation. CLL-cells in PBMCs were pre-stimulated by co-culture with CD40L expressing (murine) fibroblasts for 24 hours, and were treated with highly specific blockers for KCa3.1 (TRAM-34 or Clotrimazole) or Kv1.3 (PAP-1 or Psora-4) prior to addition of α-CD3/CD28 beads to activate autologous T-cells. KCa3.1 blockade could effectively diminish CLL-cell cycle entry in all samples investigated (TRAM-34: n=12, p<0.0001; Clotrimazole: n=5, p=0.0625) with a mean relative reduction in Ki-67+ cells of 52% (SD=1.56) for TRAM-34 (see Figure left) and 53% (SD=1.11) for Clotrimazole. This antagonizing effect of TRAM-34 was clearly dose-dependent (n=10) without affecting CLL-cell viability (see Figure right) nor activation. Viability, activation and proliferation of T-cells and fibroblasts present in the co-culture system were not affected by TRAM-34. Blockade of Kv1.3 did not reduce proliferation in CLL-cells. In summary, we showed that CLL-cells exhibit significant changes in their K-channel constitution following activation and proliferation, analogous to healthy B-cells. Notably, in vitro CLL-cell proliferation was effectively inhibited by highly specific KCa3.1 blockers. Thus far in vitro testing of potential CLL-drugs has been primarily performed on G0-arrested CLL-cells, sometimes in co-culture with stromal cells to enhance their viability. Our approach attempts to specifically target proliferating CLL-cells, presumably the most relevant CLL-cell fraction contributing to disease progression. Given their low toxicity profile, KCa3.1 blockers could represent a promising therapeutic option in CLL. Disclosures: No relevant conflicts of interest to declare.

Molecular and Clinical Associations Between Vitamin D and Chronic Lymphocytic Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5282-5282
Author(s):  
Nina Beri ◽  
Daphne R. Friedman ◽  
Tiffany M. Simms ◽  
Maragatha Kuchibhatla ◽  
J. Brice Weinberg ◽  
...  
Keyword(s):  
Gene Expression ◽  
Vitamin D ◽  
Chronic Lymphocytic Leukemia ◽  
Linear Regression ◽  
Vitamin D Insufficiency ◽  
Lymphocytic Leukemia ◽  
Vitamin D Levels ◽  
The Mean ◽  
25 Oh Vitamin D

Abstract Introduction Vitamin D deficiency is common in the general population. Approximately 25-50% of adult patients seen at routine visits in the United States are found to have an insufficient vitamin D level. Vitamin D has been shown to be prognostic in several types of cancers including breast, prostate and colon cancer. Vitamin D activates a nuclear transcription factor that regulates the expression of almost 200 genes which modulate a variety of cellular processes including angiogenesis, differentiation, proliferation, and apoptosis. Recent research has shown that vitamin D levels may have a prognostic effect in patients with chronic lymphocytic leukemia (CLL), where 25-OH vitamin D insufficiency was associated with shortened time to treatment and poorer overall survival. A centrally important unanswered question relates to causation: does vitamin D insufficiency yield more aggressive cancer disease biology, or do intrinsically progressive cases of CLL cause vitamin D insufficiency? We hypothesized that vitamin D insufficiency alters CLL cell biology and favors a more aggressive disease phenotype. Methods Untreated patients within 12 months of initial diagnosis of CLL from Duke University Hospital and the Durham VA were studied. Serum samples from 185 patients were assayed for the 25-OH vitamin D level (immunochemiluminometric assay). A multivariate analysis was performed using: age, race, gender, Rai stage, CD38, Zap70, hierarchical FISH, IGHV, and season of diagnosis to determine whether vitamin D levels are a significant predictor of OS and TTT in this group. Global mRNA expression from 23 patients was analyzed using Affymetrix U133 Plus 2.0 arrays as a function of vitamin D level and gene list generated for those with p values < 10-5. rtPCR was performed on samples from an additional 50 patients to validate the findings from the mRNA expression analysis. Linear regression analysis was conducted to evaluate for significant associations between genes and 25-OH vitamin D levels. An in vitro assessment of 1,25-di-OH vitamin D effects on CLL cell viability in serum free media was evaluated using an MTS assay. Results The mean vitamin D level amongst the group of 185 patients was 25.6± 9.7 ng/mL. Eighty-nine patients had a vitamin D level less than 25 and 96 had a level above 25, which we used as our cutoff, as prior reports have used this level to define insufficiency in CLL. Thirty-one of 95 (33%) of the sufficient vitamin D group were treated versus 39 of 89 (44%) of the insufficient vitamin D group (p=0.12). Among those requiring treatment, the mean TTT was approximately the same between the two groups: 4.7±0.3 yrs for the higher vitamin D group vs. 4.6±0.4 yrs for the insufficient group (p=0.126). OS for the higher vitamin D group was 8.3±0.3 vs. 7.0±0.2 years for the lower vitamin D group (p=0.935). Multivariable analysis showed that IGHV mutation (HR = 0.386; p=0.0159) and Rai stage 0 or 1 (HR = 0.174; p=0.0002) predicted TTT, while age and race influenced OS, with age>62 conferring greater risk of death (p=0.0191) and African Americans having decreased survival (p=0.0110). Preliminary studies of gene expression data identified eight probes that were differentially expressed as a function of vitamin D level. rtPCR was then performed on GPR82, MPZL3, FBXW4, ROR1, and CXCL11 to validate these results. Linear regression confirmed that ROR1 and FBXW4 gene expression correlated with vitamin D level (p=0.0065; r2=0.144 and p=0.0185; r2=0.110, respectively). High levels of ROR1 are observed in B-CLL. FBXW4 has been shown to be mutated or under-expressed in a variety of human cancer cell lines. Early in vitro cytotoxicity of 1,25 di-OH vitamin D in CLL (n = 5 patient derived samples) showed an IC50 = 334 nM. Discussion Our results show that the basal level of vitamin D is not significantly correlated with either OS or TTT in CLL in contrast to previous studies. No interaction between vitamin D levels and race, age, gender, Rai stage, IGHV mutation, season of diagnosis was observed. However, ongoing in vitro experiments show that 1,25 vitamin D is cytotoxic to CLL, raising the intriguing possibility that intermittent bolus dosing could potentially be used therapeutically. Further, we have identified specific genes where quantitative gene expression is correlated with basal vitamin D levels. These findings expand our understanding of the interaction between vitamin D and B cell malignancies. Disclosures: No relevant conflicts of interest to declare.

scholarly journals In vitro enhancement of immunoglobulin gene expression in chronic lymphocytic leukemia.

1984 ◽  
Vol 73 (2) ◽  
pp. 587-592 ◽  
Author(s):  
J Cossman ◽  
L M Neckers ◽  
R M Braziel ◽  
J B Trepel ◽  
S J Korsmeyer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Chronic Lymphocytic Leukemia ◽  
Lymphocytic Leukemia ◽  
Immunoglobulin Gene

Chronic Lymphocytic Leukemia Cells Can Induce Monocytes to Express High Levels of BAFF and Assume Properties of Nurselike Cells In Vitro.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 588-588
Author(s):  
Davorka Messmer ◽  
Tomoyuki Endo ◽  
Bradley T. Messmer ◽  
Thomas J. Kipps
Keyword(s):  
Gene Expression ◽  
Flow Cytometry ◽  
Chronic Lymphocytic Leukemia ◽  
Cell Survival ◽  
Mononuclear Cells ◽  
Leukemia Cell ◽  
Lymphocytic Leukemia ◽  
Soluble Factor ◽  
Blood Mononuclear Cells

Abstract CD14+ blood mononuclear cells co-cultured with chronic lymphocytic leukemia (CLL) B cells differentiate into nurselike cells (NLCs) that in turn can support CLL-cell survival in vitro and possibly in vivo. These cells appear similar to lymphoma-associated macrophages, which were identified in secondary lymphoid tissue of patients with follicular lymphoma and appear more prevalent in patients with therapy-resistant disease. To investigate the relationship between NLC and macrophages, we performed studies on macrophages and NLCs that were induced to differentiate from CD14+ blood mononuclear cells in vitro. Consistent with prior studies, we found that NLCs express significantly higher levels of CD68 than macrophages, as assessed via cytoplasmic flow cytometry. However, Affymatrix U113A microarray analysis of gene expression by NLC, macrophages, and monocytes-derived dendritic cells (DCs) from 3 donors revealed major differences in gene expression between DCs versus macrophages or NLCs, but no major differences in gene expression profiles between NLCs and macrophages. Flow cytometric analyses of NLCs and macrophages revealed that these two cell types also shared similar expression levels of CD16, CD32, CD35, CD86, CD58, MHC-II, CD40, and CD54. However, using flow cytometry we found that NLCs (n=9) expressed significantly higher levels than macrophages of the B-cell activating factor belonging to the tumor necrosis factor family (BAFF). Deconvolution microscopy confirmed the differences in BAFF expression and also revealed that NLCs express higher levels of a proliferation-inducing ligand (APRIL) than macrophages. These are two key factors involved in promoting leukemia/lymphoma B cell survival. Moreover, NLCs maintained high-level expression of BAFF even when cultured apart from CLL cells in fresh medium. We investigated whether co-culture of differentiated macrophages with CLL cells could induce the macrophages to express high-levels of BAFF. Although such co-culture induced progressive increase in macrophages-expression of BAFF, the levels of BAFF induced after even 7 days of co-culture were lower than those noted for NLCs. We cultured CD14+ blood monocytes and CLL cells separated across a transwell membrane to determine whether a soluble factor(s) was responsible for the induction of high BAFF levels noted on NLCs. Following several days in culture, the cultured monocytes acquired expression levels of BAFF similar to those detected for NLCs. These studies indicate that monocytes can respond to a soluble factor(s) elaborated by CLL cells to assume properties similar to those of NLCs. Moreover they suggest that NLCs may be a peculiar type of terminally differentiated macrophages-like cells induced by the leukemia-cell population to have properties that promote CLL cell survival. Agents that can block the maturation of monocytes into NLCs or that inhibit the capacity of NLCs to promote leukemia-cell survival may be effective in the treatment of CLL and related lymphoid malignancies.

The Aberrantly Expressed Long Noncoding RNA, TRERNA1, Predicts for Aggressive Disease in Chronic Lymphocytic Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2911-2911
Author(s):  
Cecelia R Miller ◽  
Amy S. Ruppert ◽  
Kevin Coombes ◽  
Amy M. Lehman ◽  
James S. Blachly ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Damage ◽  
Chronic Lymphocytic Leukemia ◽  
Dna Damage Response ◽  
Lymphocytic Leukemia ◽  
High Expression ◽  
Aggressive Disease ◽  
Damage Response

Abstract Long noncoding RNAs (lncRNAs) have emerged as important regulators of gene expression, and dysregulated expression of lncRNAs has been reported in many cancers. One of the most documented ways by which lncRNAs can contribute to cancer aggressiveness is by altering gene expression through associations with chromatin modifying proteins. However, the vast majority of lncRNAs dysregulated in cancer remain to be functionally characterized. Specifically, there have been few studies investigating the role of lncRNAs in chronic lymphocytic leukemia (CLL). Numerous studies have been published documenting the role of micro RNAs (miRs) in CLL, indicating that non-coding RNAs can play a significant role in this disease. Therefore we hypothesize that dysregulated lncRNA expression in CLL contributes to aggressive disease. We performed microarray analysis using Arraystar Human LncRNA Array v2.0, a platform that analyzes over 30,000 lncRNA transcripts in addition to 30,000 coding transcripts. We found that many lncRNAs are aberrantly expressed in CLL compared to healthy donor B cells. We identified the lncRNA treRNA (TRERNA1) as overexpressed in CLL cells (p = .0014). treRNA has been previously described to have enhancer-like function (Ørom et al., 2010) as well as translational regulatory functions (Gummireddy et al., 2013). It has been reported as overexpressed in breast cancer lymph-node metastases and colon cancer (Gummireddy et al., 2013). In addition to expressing spliced treRNA, CLL cells contain a transcript that retains the intron between the two coding exons due to insufficient splicing. Therefore we investigated the prognostic significance of both spliced and retained intron treRNA (ri-treRNA) in subsequent studies. We obtained 144 well-characterized CLL patient samples from the CLL Research Consortium (CRC) and measured transcript expression by quantitative reverse transcription PCR (qRT-PCR). We separated patients into high or low expressers of treRNA relative to the overall median expression and found that patients with higher expression of treRNA have significantly shorter time to treatment (p = .04). High expression of treRNA also correlates with the poor prognostic indicators unmutated IGHV (p < .0001) and low ZAP70 methylation (p <.001). We validated the correlation with unmutated IGHV in a second cohort of 147 previously untreated CLL samples collected prior to starting therapy in a clinical trial (E2997; Grever et al., 2007) comparing fludarabine to the combination of fludarabine plus cyclophosphamide (FC). We found that high expression (relative to the median) of spliced, but not ri-treRNA, independently predicts for shorter progression free survival in patients receiving FC (HR 3.14, 95% CI 1.61-6.14, p < .001). Since the data from this clinical study suggests a role for treRNA in mediating DNA damage response, we established a stable retroviral system to further study this observation in vitro. We used the CLL cell line established in our lab (OSUCLL; Hertlein et al., 2013) to express empty vector or treRNA. Expression of treRNA does not alter viability, proliferation, or migration. However, OSUCLL expressing treRNA display modest resistance to FC treatment. This correlates with less induction of the DNA damage indicator, γH2AX, as well as the DNA damage response protein, TP53, although these changes were not statistically significant. Consistent with the clinical data, ri-treRNA did not show a differential response to in vitro FC treatment. In summary, we have identified a lncRNA in CLL which may play a role in DNA damage response, and serve as a biomarker predictive of aggressive disease. Disclosures Flinn: Celgene Corporation: Research Funding.

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