Reduced c-FOS Is Associated with Poor Prognosis and Clinical Course in Chronic Lymphocytic Leukemia

Abstract Abstract 2433 We have previously shown that silencing of the tumor suppressor gene, DAPK1, by genetic and epigenetic mechanisms is associated with the pathogenesis of CLL. To elucidate genes and pathways involved in DAPK1 transcriptional regulation, genome-wide siRNA screens were performed using a novel transgene reporter system involving a stably-integrated BAC construct expressing luciferase under the control of the DAPK1 locus. c-FOS and other members of the FOS gene family were identified as positive regulators, implicating the AP-1 pathway in DAPK1 transcription. A subsequent comprehensive examination of the FOS, JUN and ATF gene families in CLL and healthy B cells was performed. As the expression of the AP-1 gene family is highly subject to fluctuations in stress and mitogenic stimuli, freshly isolated cells were cultured using autologous serum conditions, allowing for a uniform establishment of baseline gene expression. This work reveals an alteration of the relative composition of AP-1 transcription factors in CLL, demarked by a substantial reduction in c-FOS gene expression. We find that the inducibility of c-FOS in CLL following MAPK activation by TPA (ERK-MAPK) is impaired 7.0-fold relative to healthy B cells and is completely abrogated following anisomycin (p38-MAPK) stimulation. The level of c-FOS induction following TPA activation can be used to clearly segregate CLL patients into two non-overlapping groups, those with low (mean=4.0-fold reduced expression; range=3.0–7.2; n=9) and very low (mean=21.7-fold reduced expression; range=17.6–36.7; n=8) following one hour induction versus healthy B cells. The very low c-FOS induction cases are characterized by poor prognostic indicators (IGHV unmutated (0/9 in low vs. 5/8 in very low); 17p, 6q deletion (0/9 in low vs. 3/8 in very low); and a substantially shorter time to progression (102.7±40.4 months in low vs. 10.8±8.6 in very low). Patients with very low c-FOS induction also demonstrate elevated c-MYC induction following activation. TPA-dependent ERK phosphorylation and activation of other immediate early genes, such as EGR1 and c-JUN, is intact in both CLL groups, absolving MAPK pathway dysfunction as a relevant c-FOS silencing mechanism. Reduced c-FOS expression can be partially explained, in the majority of very low c-FOS inducible cases, by elevated levels of miR-155 and miR-221 targeting the c-FOS transcript. Reduced expression of c-FOS and the resulting reconfiguration of AP-1 transcription factor composition may be involved in the pathogenesis of CLL and the subsequent silencing of DAPK1. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Relationship Between Expression of TLRs and Disease Activity in CLL.

Blood ◽

10.1182/blood.v114.22.4379.4379 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4379-4379

Author(s):

Wilma Barcellini ◽

Anna Zaninoni ◽

Francesca Guia Imperiali ◽

Gianluigi Reda ◽

Nicola Orofino ◽

...

Keyword(s):

Gene Expression ◽

Immune Response ◽

B Cells ◽

Internal Control ◽

Clinical Course ◽

Conflicts Of Interest ◽

Lymphocytic Leukemia ◽

Mutational Status ◽

Tlr4 Gene ◽

Autoimmune Phenomena

Abstract Abstract 4379 Background Toll-like receptors (TLRs) are major agents of innate immunity and initiators of adaptive immune response by acting as costimulatory signals for B cells and inducing maturation, proliferation and antibody production after pathogen recognition. They are also involved in the self-antigen recognition and could play a role in autoimmune phenomena. It is well known that chronic lymphocytic leukemia (CLL) is characterized by an increased incidence of autoimmune phenomena and immunodeficiency, which can greatly influence the disease outcome leading to a variable clinical course. Aims To evaluate the correlation between the gene expression of TLRs in 41 patients with CLL (mean age±SD 66±18 years, range 42-90, 15 female and 26 male), the clinical course of the disease and the expression of prognostic factors (mutational status of IgVH region, CD38 and ZAP70 expression, and cytogenetic alterations). Methods The gene expression of TLR4, TLR9, and TLR10 was studied. Total RNA was extracted from B cells of patients and controls (pool of 10 healthy donors), cDNA was synthesized, and real-time PCR was performed. For each reaction 50 ng of cDNA were mixed with 1.25μl of TaqMan primer/probe set and 10.25μl of TaqMan Universal Master Mix. The TLR4, TLR9, and TLR10 expression was normalized according to GAPDH as an internal control gene and it was expressed as percentage of control. Results TLR4 gene expression was significantly lower in CLL patients compared to controls (18±3%, mean±SE) while TLR9, and TLR10 gene expression was higher (3457±500% and 2897±440%, respectively). Moreover, considering “progressive” CLL patients (n=20), TLR4 gene expression was significantly lower compared with “indolent” ones (n=21) (10.2±2.2% vs 24.9±4.9%, p=0.01). Patients with unmutated IgVH expression and unfavorable cytogenetic alterations (mostly 11q-) showed even more reduced TLR4 gene expression, but the small numbers did not allow statistical evaluation. TLR4 gene expression was lower in patients with infective diathesis, although not significantly (11.5±2.9% vs 20.0±3.9).The 6 “progressive” CLL patients with autoimmune diseases (hemolytic anemia, Evan's syndrome and phemphigus) showed significantly higher TLR4 gene expression compared with “progressive” patients without autoimmune phenomena (17.7±4.4% vs 6.9±2.1%, p=0.02), possible expression of activated immune system. Conclusion The reduced TLR4 gene expression in CLL patients with “progressive” disease, unmutated IgVH status, and unfavourable cytogenetic alterations is consistent with a reduced ability to a proper immune response to Gram-negative bacterial lipopolysaccarides and consequent increased susceptibility to infections. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Chronic Lymphocytic Leukemia in Venezuela: VH Mutation Status, Expression of ZAP-70, CD38 and Phenotypic Prevalence Among the Venezuelan Population.

Blood ◽

10.1182/blood.v114.22.4395.4395 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4395-4395

Author(s):

Maria Elena Marquez ◽

Pierre-Antoine Deglesne ◽

Osiris Da Costa ◽

Jose Luis Lopez ◽

Mildred Borrego ◽

...

Keyword(s):

B Cells ◽

Gene Family ◽

Prognostic Value ◽

Germ Line ◽

Conflicts Of Interest ◽

Severe Disease ◽

Lymphocytic Leukemia ◽

Clinical Staging ◽

Mutational Status ◽

Venezuelan Population

Abstract Abstract 4395 CLL- B patients have a heterogeneous evolution, while some follow an indolent disease free of complications, others develop a severe disease and die within a few years disregarding of treatment. Recent studies have revealed that the expression of ZAP-70, CD38 high and unmutated immunoglobulin heavy chain variable region genes (U-IgVH) are useful markers for severe disease. Most of these studies have been done in Caucasian and Asian populations. The objective of our study was to find out if such markers are also of prognostic value in Venezuelan CLL patients. We consider this of interest since the Venezuelan population is and admixture of Amerindians, Europeans and Africans. IgVH sequences were done in 82 patients using standard techniques. Sequences with less than 98% homology with the corresponding germ line IgVH were considered mutated. Criteria for ZAP-70 positivity were established by flow cytometry in our laboratory based on the fluorescence intensity values found for B cells in relation with reference values for T cells. Isotypic background was considered. Patients were considered CD38 positive when 20% or more leukemic B cells expressed the marker. We were able to study all the 3 parameters in 55 patients. According to the clinical staging method described by Rai et al. at the time of diagnosis, 53.64% of patients were identified in stage 0; 26.83% in stage I; 7.32% in stage II, 4,88% in stage III and 7.32% in stage IV. Thirty-six (43,9%) patients had U-IgVH, and 46 (56,1%) had mutated M-IgVH. The most frequently expressed VH gene family was found to be VH3 (48,8%) followed by VH1 (24,4%), VH4 (20,7%) and VH5 (6,1%), with no expression of VH2, VH6 or VH7 gene families. VH3-21 were high (9.76%) in our cohort, the VH3-21 gene usage is an unfavorable prognostic marker independent of the IgVH mutational status. Out of the 55 patients studied, 47,3% were ZAP-70 positive and 43,6% patients were CD38 positive. Among ZAP-70 positive patients, 65.4% had U-IgVH; similarly a higher percentage of CD38 positive patients expressed U-IgVH. The majority of patients who required treatment, expressed U-IgVH (62.5%) and ZAP-70 (68,7%) and 43.7% of treated patients expressed both markers. Remarkably, 47.5% of our CLL patients were of European origin which is a clear overrepresentation of Europeans in the Venezuelan population admixture. In conclusion, our results support the prognostic value of ZAP-70 expression and of U-IgVH status for CLL patient evolution and an increased usage of the VH3 gene family, consistent with previous findings elsewhere. Additionally, our study suggest differential susceptibility of CLL in the Venezuelan population being European descendant more prone to suffer the disease. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Overexpression of Rasgrf1, a Guanine Nucleotide Exchange Factor in Chronic Lymphocytic Leukemia.

Blood ◽

10.1182/blood.v114.22.1253.1253 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1253-1253

Author(s):

Sanjai Sharma

Keyword(s):

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Microarray Analysis ◽

Peripheral Blood ◽

Conflicts Of Interest ◽

Mapk Pathway ◽

Lymphocytic Leukemia ◽

Ras Signaling ◽

Guanine Nucleotide ◽

Normal Peripheral Blood

Abstract 1253 Poster Board I-275 We performed microarray expression analysis to identify genes which are differentially expressed in chronic lymphocytic leukemia (CLL) cells. A number of genes from the cohort of maximally upregulated genes were members of the Ras superfamily of proteins function which function as switches between active GTP-complexed and inactive GDP-complexed states. One of the genes upregulated on the microarray analysis in all the CLL cells as compared to normal B cells was RASGRF1. This ras protein specific guanine nucleotide release factor stimulates the dissociation of GDP and activates Ras signaling pathway. To confirm our microarray analysis results, we performed real time PCR analysis on 35 CLL specimens and observed a 10-50 fold upregulation of rasgrf1 RNA as compared to human peripheral blood B cells. This high expression is consistently observed in all CLL specimens regardless of stage indicating that this upregulation is an early important event in the pathogenesis of CLL. Rasgrf1 protein expression was also higher in CLL cells as compared to normal B cells by western blot analysis. Interestingly the rasgrf1 gene is genomically imprinted during development in certain tissues and the expression is downregulated. Demethylation of normal peripheral blood B cells by treatment with azacytidine resulted in increased expression of rasgrf1 with no change in expression of rasgrf1 in CLL cells implying imprinting in normal B cells. Thus malignant transformation of B cells to CLL cells results in a loss of genomic imprinting and an aberrant upregulation of rasgrf1 in CLL cells. One important downstream effect of rasgrf1 upregulation is ERK activation via Ras-MAPK pathway. B cell receptor signaling in CLL results in activation of a number of pro-survival pathways including ERK which is more active in poor prognosis, unmutated immunoglobulin heavy chain CLL. Our results indicate that ERK phosphorylation is also increased in CLL specimens with rasgrf1 overexpression. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Characterization of TET and IDH gene expression in chronic lymphocytic leukemia: comparison with normal B cells and prognostic significance

Clinical Epigenetics ◽

10.1186/s13148-016-0298-y ◽

2016 ◽

Vol 8 (1) ◽

Cited By ~ 14

Author(s):

Michaël Van Damme ◽

Emerence Crompot ◽

Nathalie Meuleman ◽

Marie Maerevoet ◽

Philippe Mineur ◽

...

Keyword(s):

Gene Expression ◽

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Prognostic Significance ◽

Lymphocytic Leukemia

Download Full-text

Dysregulation of TNFα-Induced Necroptotic Signaling In Chronic Lymphocytic Leukemia: Suppression of CYLD Gene by LEF1

Blood ◽

10.1182/blood.v116.21.377.377 ◽

2010 ◽

Vol 116 (21) ◽

pp. 377-377 ◽

Cited By ~ 2

Author(s):

Peng Liu ◽

Bei Xu ◽

Jianyong Li

Keyword(s):

Cell Death ◽

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Molecular Mechanisms ◽

Conflicts Of Interest ◽

Death Receptors ◽

Lymphocytic Leukemia ◽

Ros Production ◽

A Genome ◽

Stimulation Of

Abstract Abstract 377 Impaired cell death program has been noted as one of the hallmarks of Chronic lymphocytic leukemia (CLL) and contributes to its accumulation of malignant monoclonal B cells as well as to chemotherapy resistance. A cell can die through apoptosis or necrosis pathway. While apoptosis is known as a regulated cellular program, necrosis is known as an accidental event caused by overwhelming stress. However, accumulating evidence suggests that necrosis can also be executed by regulated mechanisms, especially in apoptotic-deficient conditions. Recently, the term necroptosis has been used to designate one particular form of programmed necrosis induced by stimulating death receptors with agonists such as TNFα, FasL, and TRAIL. Apoptosis suppression by caspase inhibitors such as zVAD may switch apoptotic response to necroptosis or enhance necroptosis. In contrast to well-characterized apoptotic pathway, the detailed molecular mechanisms underlying necroptosis are still not fully understood. A genome wide siRNA screen revealed two members of the receptor interacting protein (RIP) kinase family, RIP1 and RIP3P, to be essential for necroptosis. Upon stimulation of death receptors, RIP3 is recruited to RIP1 to form a necroptosis-inducing complex which is essential for cell death execution. The deubiquitinase cylindromatosis (CYLD) is recruited to TNFα receptor upon its activation and directly regulates RIP1 ubiquitination. In addition, by activating key enzymes of metabolic pathways, RIP3 regulates TNFα-inducing mitochondrial reactive oxygen species (ROS) production, which partly accounts for its ability to potentiate necroptosis. Until now, much less is known about the significance of necroptosis in malignant disease. Here we demonstrate that primary CLL cells failed to undergo necroptosis upon stimulation of TNFα combined with pan-caspase inhibitor zVAD. Upon TNFα+zVAD stimulation, normal CD19+ B cells increased ROS production > 8 fold, while same treatment only resulted in ∼ 2 fold induction in ROS generation in CLL samples. Two core components of necroptotic machine, RIP3 and CYLD, are markedly down-regulated in CLL compared with normal B cells, at both protein and transcription levels. Moreover, we identified LEF1, a downstream effector of Wnt/β-catenin pathway, as a transcription repressor of CYLD in CLL. LEF1 is highly expressed in CLL cells, whereas normal B cells have very low levels of LEF1 expression. Attenuation of LEF1 expression through RNAi technology resulted in a dramatic increase in CYLD levels in CLL cells, as determined by western blot and real time RT-PCR analysis. Dual-luciferase assays showed that forced expression of LEF1 markedly decreased CYLD promoter activity compared with controls. Mutation of LEF1 responsive elements (LERs) on CYLD promoter significantly abolished transcriptional repression of CYLD by LEF1. Chromatin immunoprecipitation assays showed that LEF1 is recruited to LER region within the CYLD promoter in CLL cells. Additionally, Knocking down LEF1 sensitizes CLL cells to TNFα-induced necroptosis. The present investigation provides the first evidence that CLL cells have defects not only in apoptotic program but also in necroptotic signaling. Targeting the key regulators of necroptotic machine such as LEF1 to restore this pathway may represent a novel approach for CLL treatment. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

B-Cell Chronic Lymphocytic Leukemia (B-CLL) Cells Unresponsive to CD180 Ligation Fail to Respond to Anti-IgM Stimulation as Well

Blood ◽

10.1182/blood.v116.21.3582.3582 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3582-3582

Author(s):

Nino Porakishvili ◽

Peter Lydyard ◽

Anna Bremser ◽

Ketki Vispute ◽

Azka Memon ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Protein Kinases ◽

Cell Activation ◽

Conflicts Of Interest ◽

Lymphocytic Leukemia ◽

Intracellular Signalling ◽

Orphan Receptor ◽

B Cell Activation ◽

Ki 67

Abstract Abstract 3582 Introduction: We have demonstrated that CD180, an orphan receptor of the Toll-like receptor family, is expressed heterogeneously on B-CLL cells, mainly on those with mutated IGVH genes. We further showed that specific ligation of CD180 with mAbs induced activation and cycling of only ~50% CD180+ B-CLL clones (“R”: responders), while CD180+ B-CLL cells unresponsive to CD180 ligation (“NR”: non-responders) or CD180− B-CLL cells could not be activated through either CD40 or IL-4 suggesting anergy. Because CD180 has a short intracellular domain, it presumably, signals through pathways associated with other receptors, such as smIgM. Indeed, engagement of smIgM or CD180 induces Lyn and Syk phosphorylation. Here we compare activation, cycling and phosphorylation of intracellular protein kinases in R and NR and CD180− B-CLL clones and B lymphocytes from healthy subjects upon ligation of smIgM. Methods: B-CLL cells were analyzed for smCD180 and smIgM, and sm CD180+IgM+ B-CLL clones were categorized as R and NR by responsiveness to CD180 ligation. Leukemic clones from 15 smCD180+IgM+R, 14 smCD180+IgM+NR, 12 smCD180−IgM+ untreated B-CLL patients and 14 healthy age-matched individuals were stimulated with goat F(ab’)2 anti-human IgM pAbs for 72h, and stained with PE~anti-CD86 mAbs, or fixed, permeabilized and stained with PE~anti-Ki-67 to assess B-cell activation and cycling, respectively. In order to study early intracellular signalling events, cells were stimulated with the same antibodies for 20 min, fixed, permeabilized and stained with Alexa Fluor~rabbit/mouse antibodies to phospho-Akt, phospho-ERK, phospho-p38MAPK, and phospho-ZAP70/Syk. Unstimulated cells in medium were used as controls. Results were assessed by flow cytometry and analyzed with the Mann-Whitney U test and paired t-test where appropriate. Results: ligation of sIgM on smCD180+IgM+R B-CLL cells resulted in a significant increase in CD86+ cells (66.3±21.7% vs 18.7±12.0%, p=0.00004) and Ki-67+ cells (38.9±10.5% vs 11.1±5.9%, p=0.0001) compared to medium controls; this was not different from the increase in activation and cycling of normal B cells (not shown). In contrast, smCD180+IgM+NR B-CLL cells failed to significantly upregulate CD86 in response to anti-IgM pAbs (20.6±13.8% vs 17.6±13.7%, p=0.334) and Ki-67 (8.4±4.6% vs 5.3±1.4%, p=0.063). Interestingly, smCD180−IgM+ B-CLL cells demonstrated diminished CD86 upregulation following sIgM ligation: 36.9±21.7% vs 11.0±4.7% in medium, p=0.058 (difference with smCD180+IgM+R B-CLL, p=0.0069). Cell cycling was also decreased: 9.7±4.1% vs 5.4±3.6% in medium, p=0.015 (difference with smCD180+IgM+R, p=0.0022). The proximal stages of anti-smIgM responses were further studied by intracellular signalling of protein kinases associated with the IgM-signalling pathway. While ligation of sIgM on control B cells and smCD180+IgM+R B-CLL cells resulted in phosphorylation of all four enzymes studied, smCD180+IgM+NR cells failed to signal downstream from ZAP70/Syk following sIgM ligation (Table 1), although there was a greater heterogeneity in smCD180+IgM+R B-CLL responses, compared to normal B cells. Importantly, smIgM ligation of smCD180−IgM+ B-CLL cells did not increase phosphorylation of Erk or p38MAPK, although some such clones responded to smIgM ligation by phosphorylation of ZAP70/Syk and Akt (data not shown). Conclusions: B-CLL clones that are smCD180+IgM+ but unresponsive to CD180 ligation (~30% of all B-CLL cases) are also unresponsive (anergic) to smIgM ligation measured by intracellular signalling, cell activation and cycling. Meanwhile, smCD180−IgM+ B-CLL clones respond heterogeneously to IgM crosslinking. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

CCR4:CCL17 Interaction Influences TLR-9 Mediated Cell Survival and Proliferation In Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v116.21.3593.3593 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3593-3593

Author(s):

Sonal C. Temburni ◽

Ryon M. Andersen ◽

Luke Janson ◽

Xiao-Jie Yan ◽

Barbara Sherry ◽

...

Keyword(s):

Cell Proliferation ◽

B Cells ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Conflicts Of Interest ◽

Dose Range ◽

Serum Levels ◽

Lymphocytic Leukemia ◽

Novel Therapy

Abstract Abstract 3593 Unlike other hematologic disorders, chronic lymphocytic leukemia(CLL) exhibits remarkable heterogeneity in the rates of disease progression among cases. CLL cells survive by receiving signals from the microenvironment via various receptors: B-cell antigen receptor (BCR), Toll-like receptors (TLRs) and cytokine and chemokine receptors. We previously reported that CLL clones with somatically mutated IGHVs and high (≥30%) percentage of CD38 expressing cells have the highest percentage of CCR4-expressing cells. To further explore the functional contribution of the CCR4:CCL17 axis in CLL, we studied CCL17-induced chemotactic behavior in 16 CLL cases. In transwell cultures we observed a bimodal migratory response to CCL17 at 2 doses in a dose range of 0.78– 25ng/ml, in ~60% of cases; the remaining cases showed maximal migration at a single dose (1.56 or 3.12ng/ml). A comparison of phenotypes of the migrated and non-migrated cell populations was undertaken in 10 cases, analyzing CXCR3, CXCR4, CCR4 and CCR7 that are involved in homing of cells to sites favoring growth, and CD31, CD38 and CD69, activation related molecules. The migrated cells consistently showed significantly higher percentages and densities of CD38 expression than the non-migrated cells suggesting a role for CD38 in the CCR4-mediated downstream pathway. CCR4 ligand, CCL17, is constitutively expressed in the thymus and is produced by dendritic cells, endothelial cells, keratinocytes and fibroblasts, whereas CCL22 is produced by tumor cells and the tumor microenvironment. Serum levels of both these ligands in untreated patients were quantified by ELISA. CCL17 levels ranged between 45-1, 229 pg/ml in U-CLL cases (n=23) and between 43-1, 418 pg/ml in M-CLL cases (n=30). CCL22 levels ranged between 121-5, 497 pg/ml in U-CLL cases (n=23) and 409-5, 502 pg/ml in M-CLL cases (n=30). The percentages of CCR4- expressing B cells directly correlated with percentages of T cells expressing CCR4 in individual cases, whereas they inversely correlated with both, serum levels of CCL17 (p< 0.01) and CCL22 (p< 0.05). CCL17 produced by DCs in peripheral organs may exert an accessory role in BCR- and TLR-9-mediated immune responses in B cells. We therefore tested if CCL17 supported BCR- and TLR-mediated proliferative responses in a cohort of 31 (16 U-CLL and 15M-CLL) CLL cases. CCL17 augmented BCR-mediated B-cell proliferation in 9/16 (56%) U-CLL cases, but only in 3/15 (20%) M-CLL cases. On the other hand, CCL17 showed an additive effect in promoting TLR-9-mediated cell proliferation in 13/15 (87%) M-CLL cases at a dose of 2ng/nl (approximating that detected in serum); it also augmented TLR-9 mediated B cell proliferation in 6/16 U-CLL cases but at a 5-fold or higher dose (10-25 ng/ml). In a subset of this cohort (8 cases) CCL17-induced modulation of molecules involved in the apoptotic process was studied. We found upregulation of anti-apoptotic proteins Mcl-1 and Bcl2 and down-regulation of pro-apoptotic molecules Bim, PUMA, and Bid in 5 of these cases. The pro-survival effects of CCL17 were partially abrogated by the blocking anti-CCR4 mAb (1G1). Taken together, these findings suggest that CCL17 plays a role in modulating TLR-9-mediated signaling and migration in CLL. Therefore, inhibition of CCR4:CCL17 interaction in vivo represents a novel therapy by preventing migration of CLL cells towards an environment that promotes their survival. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Impact of Lenalidomide on Gene Expression Profiles of Malignant and Immune Cells in Patients with Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v118.21.976.976 ◽

2011 ◽

Vol 118 (21) ◽

pp. 976-976 ◽

Cited By ~ 1

Author(s):

John C. Riches ◽

Ajanthah Sangaralingam ◽

Shahryar Kiaii ◽

Tracy Chaplin ◽

Demet Cekdemir ◽

...

Keyword(s):

Gene Expression ◽

T Cells ◽

T Cell ◽

B Cells ◽

Family Member ◽

Cell Function ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Lymphocytic Leukemia ◽

Healthy Donors

Abstract Abstract 976 Lenalidomide has recently been demonstrated to have significant activity in chronic lymphocytic leukemia (CLL). Its mechanism of action in this disease is not well understood, but it is thought to act primarily by enhancing anti-tumor immunity and reducing production of pro-tumoral factors in the CLL microenvironment. We have previously demonstrated alterations in the expression of cytoskeletal genes in T-cells from patients with CLL and have subsequently shown that these changes translate into a deficit in T-cell function, due to impaired actin polymerization resulting in defective immunological synapse formation. Treatment of both autologous T-cells and CLL cells with lenalidomide was necessary to repair this defect, suggesting that this may be a key component of this agent's activity in CLL. Therefore we examined the effect of lenalidomide on the global gene expression profiles of isolated B-cells and T-cell subsets from CLL patients and healthy donors. Peripheral blood mononuclear cells from patients with untreated CLL or healthy donors were cultured in the presence of 1 μM lenalidomide or vehicle control for 48 hours. The lymphocyte subsets were isolated, followed by RNA extraction and gene expression profiling using the Affymetrix HGU133Plus2.0 platform. Lenalidomide treatment had similar effects on gene expression in T-cells from both patients with CLL and healthy donors. The most prominent changes in expression were of genes involved in cytoskeletal signaling including a 20-fold increase in WASF1 (Wiskott Aldrich Syndrome protein family, member 1), and greater than 2-fold increases in the expression of Rac-family member RHOC, (Ras homolog gene family, member C), actin binding proteins CORO1B (Coronin 1B), PARVA (Parvin alpha), and the Rho guanine nucleotide exchange factors (GEFs), ARHGEF5 and ARHGEF7. We also observed changes in genes regulating integrin signaling including PXN (Paxilin) and FAK (Focal adhesion kinase), and a shift towards Th1 differentiation with upregulation of TNF, IL-12R, and IL-18R. In addition, we noted increased expression of the transcription factors IKZF1, IKZF4 and IRF4, genes involved in the Ikaros pathways that are essential for hematopoiesis and control of lymphoid proliferation. These changes in gene expression provide further evidence that an important mechanism of action of lenalidomide is the upregulation of the actin cytoskeletal network including Rho-GTPases and integrin activation signaling, and are consistent with our previous observations concerning the functional repair of T-cells in CLL. Initial analysis of the effect of lenalidomide on the gene expression profiles of the CLL B-cells showed similar changes to those previously described in vivo from CLL patients receiving single agent lenalidomide in a clinical trial (Chen et al. JCO 2010). In our system, lenalidomide treatment resulted in a greater than 2-fold upregulation of 189 genes, and a greater than 2-fold downregulation of 85 genes in CLL B-cells. We observed increased expression of several genes belonging to the TNF superfamily including TNF-α, OX40L, and APRIL, and the receptors DR5, DCR2, and OX40. Many of these are known to mediate apoptosis signaling, and we also observed increased expression of pro-apoptotic genes such as FAS, BID (BH3 interacting domain death agonist), HRK (Harakiri), and CFLAR (CASP8 and FADD-like apoptosis regulator), and cell cycle regulators CDKN1A and CDKN1C (Cyclin-dependent kinase inhibitors 1A and 1C). Lenalidomide also upregulated expression of several genes of known importance in the CLL microenvironment, including the chemokines CCL3 and CCL4, CD40, CD274 (PD-L1), CD279 (PD-1), and adhesion molecules LFA3 and ICAM1. The effect of lenalidomide on the gene expression profiles of normal B-cells was less marked, with greater than 2-fold upregulation of 51 genes and downregulation of 12 genes. However, we did observe that lenalidomide treatment induced upregulation of genes involved in cytoskeletal pathways such as RND1 (Rho family GTPase 1), RHOQ (Ras homolog gene family, member Q), and MYO1B (myosin 1B). In conclusion, investigation of the effect of lenalidomide on gene expression profiling in CLL suggests that the drug acts both to enhance T-cell function, and to render the CLL cells more susceptible to immune cell mediated killing. Disclosures: Gribben: Roche: Honoraria; Celgene: Honoraria; GSK: Honoraria; Mundipharma: Honoraria; Gilead: Honoraria; Pharmacyclics: Honoraria.

Download Full-text

Characterization of lincRNA BALIR-6 in MLL rearranged B-lymphoblastic leukemia

Blood ◽

10.1182/blood.v122.21.3730.3730 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3730-3730

Author(s):

Norma Iris Rodriguez-Malave ◽

Weihong Yan ◽

Giuseppe Basso ◽

Martina Pigazzi ◽

Dinesh S. Rao

Keyword(s):

Gene Expression ◽

B Cells ◽

B Cell ◽

Cell Lines ◽

Microarray Data ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Regulate Gene Expression ◽

In Cis ◽

Regulate Gene

Abstract A new class of non-coding RNA, known as long intergenic non-coding RNAs (lincRNAs), has only recently been described. These lincRNAs have been found to play a role in various molecular processes within the cell including gene regulation, acting as sinks for microRNAs, and regulating splicing, implicating them in development and oncogenic processes. B lymphoblastic leukemia (B acute lymphoblastic leukemia; B-ALL), a malignancy of precursor B-cells, harbors mutations and translocations that result in a dysregulated gene expression. Interestingly, dysregulated expression of lincRNAs has been found in various cancers, but has not yet been described in B-ALL. Recently, we completed a gene expression profiling study in human B-ALL samples, which showed differential lincRNA expression in samples with particular cytogenetic abnormalities. This led us to hypothesize that lincRNAs may be related to disease pathogenesis. Here, we describe a promising lincRNA from our microarray data designated B-ALL associated long intergenic RNA 6 (BALIR-6). Expression of BALIR-6 is highest in patient samples carrying the MLL rearrangement (n=16; when compared to patients with TEL-AML1-translocated, n=39; E2A-PBX1-translocated, n=8; BCR-ABL-translocated, n=3; and cytogenetically normal cases, n=56; 1-way ANOVA p<0.0001) and showed significant variance in the expression level based on the immunophenotype (1-way ANOVA p=0.0004). BALIR-6 is located on chromosome 3p24.3 in humans, and exists in a syntenic gene block in with neighboring genes SATB1 and TBC1D5, and is conserved in mammals. Rapid Amplification of cDNA Ends (RACE) uncovered multiple transcript isoforms; from these, three were cloned out and sequenced, corresponding to the genomic locus as predicted. In B-ALL cell lines, BALIR-6 expression was highest in RS411 cells, which carry the MLL rearrangement, when compared to other B-ALL cell lines. This suggests that the cell lines may show a similar expression pattern to human B-ALL samples. To study the functional role of BALIR-6 we utilized siRNA in a mmu-miR-155 expression cassette to knockdown the transcript. In RS411 cells we observed a reduction in proliferation by MTS assay. Additionally, we observed an increase Sub-G0 cells and a decrease in G2-M phase cells by propidium iodide staining, suggesting an increase in apoptosis. Conversely, overexpression of BALIR-6 in a mouse pre-B cell line (70Z/3), leads to an increase in proliferation. Interestingly, during normal B cell development, BALIR-6 is dynamically expressed, with high expression in pre-B cells and subsequent downregulation, suggesting that a normal role during development is being hijacked in patients with B-ALL. Mechanistically, a few recent studies have described that lincRNAs can regulate gene expression in cis. To explore whether BALIR 6 regulates surrounding genes in cis, we analyzed microarray data of MLL rearranged B-ALL samples, finding that expression of BALIR-6 correlates with expression of surrounding genes SATB1 and TBC1D5. Interestingly for SATB1, this correlation is also seen in human B cell developmental stages. Altering BALIR-6 expression by siRNA mediated knockdown or overexpression causes an effect on the expression of surrounding genes SATB1 and TBC1D5. Previous findings have shown that dysregulated SATB1 has been seen in a variety of malignancies, suggesting a mechanism for how BALIR-6 may produce the changes in cell growth and apoptosis described above. Altogether, these results identify a novel and interesting RNA transcript with the potential to regulate gene expression and pathogenesis in B-ALL with MLL rearrangement, suggesting novel diagnostic, prognostic, and therapeutic implications. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

STAT3-Activated GM-CSFRα Shuttles To The Nucleus and Protects CLL Cells From Apoptosis

Blood ◽

10.1182/blood.v122.21.4118.4118 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4118-4118

Author(s):

Ping Li ◽

Uri Rozovski ◽

David Harris ◽

Lui Zhiming ◽

Alessandra Ferrajoli ◽

...

Keyword(s):

B Cells ◽

Conflicts Of Interest ◽

Lymphocytic Leukemia ◽

Mass Spectrometry Analysis ◽

Luciferase Assay ◽

Cell Fractionation ◽

Mobility Shift ◽

Ligand Interaction ◽

Spectrometry Analysis ◽

Gm Csf

Abstract Granulocyte-macrophage colony stimulating factor (GM-CSF) regulates the survival, proliferation, differentiation, and activation of hematopoietic cells, dendritic cells, and T cells. Upon GM-CSF binding, the α and β subunits of the GM-CSF receptor (GM-CSFR) dimerize and activate signaling. While exploring a possible role for GM-CSF in treating chronic lymphocytic leukemia (CLL), we found that GM-CSF did not enhance the phosphorylation of STAT3, Akt, or ERK, suggesting that GM-CSF does not directly affect CLL cells. Indeed, as in normal B cells, flow cytometry analysis of CLL cells did not detect GM-CSFRβ. However, unlike in normal B cells, GM-CSFRα (CD116) was present in CLL cells. Using confocal microscopy, cell fractionation studies, and GM-CSFRα antibody epitope mapping, we detected GM-CSFRα not only on the surfaces but also in the cytosol and nuclei of CLL cells, suggesting that GM-CSFRα has functions that are unrelated to receptor-ligand interaction. Because STAT3 is constitutively activated in CLL cells and because the promoter of the GM-CSFRα gene harbors putative STAT3 binding sites, we hypothesized that STAT3 activates GM-CSFRα in CLL cells. Both chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSA) analyses revealed that STAT3 binds to the promoter of GM-CSFRα. We therefore cloned the GM-CSFRα promoter in multiple myeloma cell line MM1 and used luciferase assay we identified STAT3 binding activity. To investigate the function of GM-CSFRα, we induced the overexpression of GM-CSFRα in 293FT cells and analyzed the RNA and protein that co-immunoprecipitated with GM-CSFRα. We identified 7200 RNA transcripts that co-immunoprecipitated with GM-CSFRα, including SKI and MAFA oncogenes, two serine/threonine kinase genes, and DEFT1P2 and DEFP1P, which encode proteins that belong to members of the death effector domains family, known to have a role in modulating apoptosis. Overall, the transcripts that co-immunoprecipitated with GM-CSFRα belong to survival pathways, the JAK/STAT pathway, and hematopoietic lineage pathways. Mass spectrometry analysis revealed that housekeeping proteins, chaperon proteins, KAP1, and ISG-15 co-immunoprecipitated with GM-CSFRα. We found that GM-CSFRα–bound KAP1 enhanced the transcriptional activity of STAT3, whereas GM-CSFRα–bound ISG-15 inhibited the nuclear factor-κB pathway. Nevertheless, overexpression of GM-CSFRα protected MM1 cells from dexamethasone-induced apoptosis, and GM-CSFRα-siRNA induced the apoptosis of CLL cells. Taken together, our data suggest that STAT3 induces the transcription of GM-CSFRα, which has distinct, ligand-independent, anti-apoptotic activities. Disclosures: No relevant conflicts of interest to declare.

Download Full-text