High ZAP-70 Expression Research Articles

Identification of hub modules and therapeutic targets associated with CD8+T-cells in HF and their pan-cancer analysis

Heart failure (HF) is a terminal condition of multiple cardiovascular disorders. Cancer is a deadly disease worldwide. The relationship between HF and cancer remains poorly understood. The Gene Expression Omnibus database was used to download the RNA sequencing data of 356 patients with hypertrophic cardiomyopathy-induced HF and non-HF. A co-expression network was established through the weighted correlation network analysis (WGCNA) to identify hub genes of HF and cancer. Cox risk analysis was performed to predict the prognostic risks of HF hub genes in pan-cancer. HF was linked to immune response pathway by the analysis of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). A positive correlation was observed between the expression levels of 4 hub genes and the infiltration of CD8+T-cells in pan-cancer. 4 hub genes were identified as beneficial prognostic factors in several cancers. Western blotting and real-time polymerase chain reaction validated the high expression of GZMM, NKG7, and ZAP70 in both mice and patients with HF compared to control groups. Our study highlights the shared immune pathogenesis of HF and cancer and provides valuable insights for developing novel therapeutic strategies, offering new opportunities for improving the management and treatment outcomes of both HF and cancer.

ZAP-70 expression identifies a chronic lymphocytic leukemia subtype with unmutated immunoglobulin genes, inferior clinical outcome, and distinct gene expression profile

AbstractThe presence or absence of somatic mutations in the expressed immunoglobulin heavy chain variable regions (IgVH) of chronic lymphocytic leukemia (CLL) cells provides prognostic information. Patients whose leukemic cells express unmutated IgVH regions (Ig-unmutated CLL) often have progressive disease, whereas patients whose leukemic cells express mutated IgVH regions (Ig-mutated CLL) more often have an indolent disease. Given the difficulty in performing IgVH sequencing in a routine diagnostic laboratory, this prognostic distinction is currently unavailable to most patients. Pilot gene expression profiling studies in patients with CLL identified genes that were differentially expressed between the Ig-unmutated and Ig-mutated CLL subtypes. Here, we have profiled an expanded cohort of 107 patients and show that ZAP-70 is the gene that best distinguishes the CLL subtypes. Ig-unmutated CLL expressed ZAP-70 5.54-fold more highly than Ig-mutated CLL (P < 10-21). ZAP-70 expression correctly predicted IgVH mutation status in 93% of patients. ZAP-70 expression and IgVH mutation status were comparable in their ability to predict time to treatment requirement following diagnosis. In 7 patients, ZAP-70 expression and IgVH mutation status were discordant: 4 Ig-mutated CLLs had high ZAP-70 expression and 3 Ig-unmutated CLLs had low ZAP-70 expression. Among these ZAP-70 “outliers,” those with Ig-mutated CLL had clinical features that are uncharacteristic of this CLL subtype: 2 required early treatment and 2 used a mutated VH3-21 gene, an IgVH gene that has been associated with progressive disease. We developed reverse transcriptase–polymerase chain reaction and immunohistochemical assays for ZAP-70 expression that can be applied clinically and would yield important prognostic information for patients with CLL.

Flow Cytometric Analysis of ZAP-70 Protein Expression for B-Cell Chronic Lymphocytic Leukemia Prognostication: Usefulness and Limitations.

Objectives: The heterogenous clinical course in B-cell chronic lymphocytic leukemia (B-CLL) can be linked to several genetic and phenotypic characteristics of malignant B-cells. Prognostic analysis in B-CLL is routinely carried out to assist patient management; particularly to predict the time to initiate treatment. Increased ZAP-70 expression is a surrogate marker for unmutated immunoglobulin genes and inferior clinical outcomes which can be quantified to predict future outcomes in B-CLL patients. The study determined the ZAP-70 expression pattern using Z-index in Pakistani patients with B-CLL.Methods: A retrospective analysis of B-CLL cases diagnosed and confirmed on flow cytometry at Aga Khan University Hospital for the last six years which had also undergone ZAP-70 analysis were included. In all these cases, ZAP-70 expression was quantified by measuring mean fluorescence intensities (MFIs) of normal B-cells, T-cells, and CLL-cells (CD19 and CD5 double-positive population). ZAP-70 expression was divided into high, low, and negative categories based on Z-index calculation. Mann-Whitney U test was utilized to determine the significance of ZAP-70 variations in different age groups and genders. P-value <0.05 was considered significant.Results: A total of 120 patients of B-CLL had ZAP-70 analysis during the study period. The median age was 62 with an interquartile range of 35-87 and male to female ratio of 2:1. ZAP-70 expression was high in 18 (15%), low in 52 (43.3%) and negative in 50 (41.7%) cases. No significant difference in ZAP-70 expression with respect to the age or gender of the study population was identified using appropriate statistical calculations.Conclusions: This study showed only 15% of B-CLL cases showing high ZAP-70 expression, a surrogate biomarker for possible aggressive behavior which may necessitate therapeutic intervention and close surveillance.

Mutations in the RAS-BRAF-MAPK-ERK pathway define a specific subgroup of patients with adverse clinical features and provide new therapeutic options in chronic lymphocytic leukemia.

Mutations in genes of the RAS-BRAF-MAPK-ERK pathway have not been fully explored in patients with chronic lymphocytic leukemia. We, therefore, analyzed the clinical and biological characteristics of chronic lymphocytic leukemia patients with mutations in this pathway and investigated the in vitro response of primary cells to BRAF and ERK inhibitors. Putative damaging mutations were found in 25 of 452 patients (5.5%). Among these, BRAF was mutated in nine patients (2.0%), genes upstream of BRAF (KITLG, KIT, PTPN11, GNB1, KRAS and NRAS) were mutated in 12 patients (2.6%), and genes downstream of BRAF (MAPK2K1, MAPK2K2, and MAPK1) were mutated in five patients (1.1%). The most frequent mutations were missense, subclonal and mutually exclusive. Patients with these mutations more frequently had increased lactate dehydrogenase levels, high expression of ZAP-70, CD49d, CD38, trisomy 12 and unmutated immunoglobulin heavy-chain variable region genes and had a worse 5-year time to first treatment (hazard ratio 1.8, P=0.025). Gene expression analysis showed upregulation of genes of the MAPK pathway in the group carrying RAS-BRAF-MAPK-ERK pathway mutations. The BRAF inhibitors vemurafenib and dabrafenib were not able to inhibit phosphorylation of ERK, the downstream effector of the pathway, in primary cells. In contrast, ulixertinib, a pan-ERK inhibitor, decreased phospho-ERK levels. In conclusion, although larger series of patients are needed to corroborate these findings, our results suggest that the RAS-BRAF-MAPK-ERK pathway is one of the core cellular processes affected by novel mutations in chronic lymphocytic leukemia, is associated with adverse clinical features and could be pharmacologically inhibited.

Role of Ts-RNAs in CLL

Chronic lymphocytic leukemia (CLL) is the most common human leukemia. This disease occurs in two forms, aggressive and indolent, both characterized by clonal expansion of CD5+ B-lymphocytes. Hallmarks of aggressive CLL are the high expression level of ZAP-70, the unmutated sequence of IgVH genes, and activation of the TCL1 oncogene. While investigating whether the expression of miR-3676/4521 cluster in CLL is correlated with TCL1 regulation and ZAP-70 methylation in aggressive CLLs, we found that these two microRNAs are associated with tRNA sequences and that this region produces two small RNAs, members of a novel class of small non-coding RNAs (tsRNAs).Ts-3676/4521 cluster is located on the short arm of chromosome 17 and is co-deleted with p53 in 17p deleted CLL patients. We previously demonstrated that ts-3676 and ts-4521 are down-regulated and mutated in all CLL types. Furthermore, ts-3676 targets TCL1 3'UTR, indicating that tsRNAs can interfere with gene expression at a post-transcriptional level in a micoRNA-like manner. However, tsRNAs do not share the biogenesis mechanism of microRNAs but are cleaved from the 3' ends of pre-tRNAs by the endonuclease RNase Z and represent unique sequences starting at the 3' ends of the tRNAs and ending at a sequence of 4 consecutive T nucleotides. Thus tsRNAs are single stranded RNAs, more similar to piRNAs than miRNAs. Since piRNAs interact with Piwi proteins to affect gene methylation, we hypothesized that tsRNAs could also interact with Piwi proteins. By performing a series of RNA immunoprecipitation experiments, we verified the interaction of ts-3676 and ts-4521 with Piwi proteins, suggesting that these molecules could be involved in gene promoter methylation, hence affecting gene expression at a pre-transcriptional level in a piRNA-like manner. Since ts-3676 and ts-4521 are deregulated in CLLs, we studied if other tsRNAs are differentially regulated CLL. We retrieved the tsRNAs sequences from 3' ends of tRNA genes, obtaining a total of 120 tsRNA sequences, and designed a tsRNA microarray chip to investigate signatures of tsRNAs that can distinguish different classes of CLLs. We hybridized total RNAs extracted from 23 CLL samples (11 indolent, 12 aggressive) and 8 CD19+ B cells of healthy donors and found 17 tsRNA differentially expressed in these cohorts of samples. 9 tsRNAs are differentially expressed in aggressive CLL vs. normal B-cells, 10 tsRNAs are differentially expressed in indolent CLL vs. normal B-cells, 15 tsRNAs are differentially expressed in indolent vs. aggressive CLL. In these signatures, we identified ts-46 and ts-47 strongly downregulated in aggressive CLL, suggesting these ts-RNAs as potential tumor-suppressors. Finally, we found that ts-3676 and ts-4521 have a prognostic value for particular subgroups of CLLs. Patients with low ZAP-70 expression and mutated IgVH generally show indolent disease, while patients with high ZAP-70 expression and un-mutated IgVH show aggressive disease. However, a relatively small cohort of patients shows aggressive prognostic markers but clinically indolent disease. Our analysis revealed that expression of ts-3676 is lower in aggressive CLLs vs indolent CLLs, although, we did not test these tsRNAs in cohort of patients with aggressive prognostic markers but indolent disease. Currently there is no good marker to discriminate these patients from aggressive CLLs. Thus, we selected 8 patients with unfavorable prognostic markers but indolent disease and 12 patients with both aggressive clinical course and unfavorable prognosis markers, to test for differences in the expression level of ts-3676 and ts-4521. We found that in patients with unfavorable prognostic markers but indolent disease, expression of ts-3676 and ts-4521 was almost double of that in patients displaying both aggressive clinical course and markers.In conclusion, we provided an evidence that tsRNAs deregulation can affect gene expression by interfering with the epigenetic machinery and/or with the post-transcriptional stability of target genes. We also identified the first tsRNAs important in CLL as new markers and/or targets for therapy: ts-3676, ts-4521, ts-46 and ts-47. DisclosuresKipps:Celgene: Consultancy, Honoraria, Research Funding; Pharmacyclics, LLC, an AbbVie Company: Consultancy, Honoraria; Roche: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Speakers Bureau.

ILK Induction in Lymphoid Organs by a TNFα–NF-κB–Regulated Pathway Promotes the Development of Chronic Lymphocytic Leukemia

The proliferation of chronic lymphocytic leukemia (CLL) cells requires communication with the lymphoid organ microenvironment. Integrin-linked kinase (ILK) is a multifunctional intracellular adaptor protein that transmits extracellular signals to regulate malignant cell motility, metastasis, and cell-cycle progression, but is poorly characterized in hematologic malignancies. In this study, we investigated the role of ILK in the context of CLL and observed high ILK expression in patient samples, particularly in tumor cells harboring prognostic high-risk markers such as unmutated IGHV genes, high Zap70, or CD38 expression, or a signature of recent proliferation. We also found increased numbers of Ki67 (MKI67)-positive cells in regions of enhanced ILK expression in lymph nodes from CLL patients. Using coculture conditions mimicking the proliferative lymph node microenvironment, we detected a parallel induction of ILK and cyclin D1 (CCND1) expression in CLL cells that was dependent on the activation of NF-κB signaling by soluble TNFα. The newly synthesized ILK protein colocalized to centrosomal structures and was required for correct centrosome clustering and mitotic spindle organization. Furthermore, we established a mouse model of CLL in which B-cell-specific genetic ablation of ILK resulted in decelerated leukemia development due to reduced organ infiltration and proliferation of CLL cells. Collectively, our findings describe a TNFα-NF-κB-mediated mechanism by which ILK expression is induced in the lymph node microenvironment and propose that ILK promotes leukemogenesis by enabling CLL cells to cope with centrosomal defects acquired during malignant transformation. Cancer Res; 76(8); 2186-96. ©2016 AACR.

Zeta-Chain-Associated Protein Kinase-70 (Zap-70) Promotes Acute Lymphoblastic Leukemia (ALL) Infiltration into the Central Nervous System By Enhancing Chemokine Receptor 7 (CCR7) Expression

Most children with B-cell precursor ALL (BCP-ALL) have an excellent prognosis but some patients are susceptible to CNS relapse, the mechanisms of which are largely unknown. Zap-70 is a tyrosine kinase mainly expressed in normal T- and NK-cells, but it is also expressed at low levels in normal B-cells. Zap-70 is however overexpressed in B-cell chronic lymphocytic leukemia (B-CLL) and is associated with an aggressive course of the disease. In B-CLL, Zap-70 was shown to up-regulate the expression of CCR7 (Calpe et al., 2011), which in turn allows T-lymphoblasts to enter the CNS (Buonamici et al., 2009). We hypothesized that Zap-70 mediated upregulation of CCR7 may enhance the homing and the survival of CNS-prone BCP-ALL cells in the CNS niche.We first transduced the 697 BCP-ALL cell line with a lentiviral vector carrying a non-targeting GFP (shGFP) or a Zap-70 (shZap-70) specific shRNA. Down-regulation of Zap-70 resulted in a significantly reduced CCR7 mRNA and surface protein expression. 697shGFP and 697shZap-70 cells were then injected intravenously into NSG mice, which were sacrificed when leukemic symptoms developed. There were no statistically significant differences in leukemic bone marrow infiltration and survival between the groups. However, immunohistochemical scoring of CNS infiltration (Krause et al., 2015) revealed that 9/10 animals (90%) in the 697shGFP group were CNS+ and only 2/7 animals (29%) in the 697shZap-70 group showed a CNS+ status (p = 0.0345). This suggests that Zap-70 mediated reduction in CCR7 impairs the CNS-positive phenotype of 697 cells in vivo.We next investigated if the Zap-70/CCR7 axis influences the ability of primary samples from patients to infiltrate the CNS of xenografts. Zap-70 mRNA was measured in pediatric BCP-ALL patients and patients with Zap-70 expression levels higher than the median were considered Zap-70high, and the remaining patients Zap-70low. 1 x 106 cells of 5 Zap-70high and 5 Zap-70low samples were injected intrafemorally into duplicate NSG mice. Interestingly, 7/10 (70%) of the mice injected with Zap-70high cells showed a CNS+ phenotype, whereas only 1/10 (10%) of the mice bearing Zap-70low samples were CNS+ (p = 0.0198). Importantly, leukemic cells of one Zap-70high patient purified from the xenograft CNS showed a marked upregulation of CCR7 expression as compared to cells isolated from bone marrow or spleen. These data suggest that the Zap-70/CCR7 axis is beneficial for patient BCP-ALL cells in the CNS of NSG mice.To test whether Zap-70/CCR7 is associated with CNS involvement in patients, we analyzed Zap-70 and CCR7 mRNA expression in diagnostic primary material of 76 BCP-ALL patients. The cohort contained 21 CNS-positive and 55 CNS-negative patients and was previously published by Cario et al. (2007). Patients were grouped into either Zap-70high/Zap-70low or CCR7high/CCR7low groups according to expression levels higher or lower than the median of the respective gene. Zap-70 expression was highly correlated with CCR7 expression (p = 0.0003; Spearman r = 0.401). There were no statistically significant differences in outcome surrogates such as prednisone response or MRD and there was no correlation between Zap-70/CCR7 expression and outcome (relapse or death). However, a trend towards high CCR7 levels and the occurrence of death could be observed (p = 0.055). Interestingly, high expression of Zap-70 or CCR7 correlated with the presence of blasts in the cytospin of the initial cerebrospinal fluid sample (p = 0.014 and p = 0.045, respectively). Furthermore, there was a significant correlation between high Zap-70 and a higher CNS status and a trend between high CCR7 and a higher CNS status according to AIEOP-BFM 2009 criteria (p = 0.024 and p = 0.098, respectively). We finally performed comparative correlations in 48/76 patients (63.2%) that were Zap-70high/CCR7high (n=24) or Zap-70low/CCR7low (n=24). Most importantly, a significant correlation between Zap-70high/CCR7high expression and a higher CNS status could be observed (p = 0.009). These data suggest that a high expression of Zap-70 or CCR7 is associated with CNS infiltration in BCP-ALL patients and that measurements of Zap-70 and CCR7 expression should be combined.Altogether, we show that Zap-70/CCR7 is important for BCP-ALL cells to enter the CNS and to survive in that niche. We propose Zap-70/CCR7 as a mechanism of CNS leukemia and a diagnostic marker with potential therapeutic implications. DisclosuresNo relevant conflicts of interest to declare.

A CD21 low phenotype, with no evidence of autoantibodies to complement proteins, is consistent with a poor prognosis in CLL.

B-cell chronic lymphocytic leukemia (CLL) is characterized by differential BCR signaling and autoimmune complications. Complement modulates B-cell function via C3d and CD21 cross-linked to the B-cell receptor (BCR). We hypothesized that CD21 contributes to BCR signaling and participates in the autoimmunity associated with CLL. We analyzed CD21 expression on 106 CLL patient samples and matched serum from 50 patients for the presence of soluble CD21 and autoantibodies to CR2, CR1, MCP and FH. CD21 expression on CLL B-cells was significantly lower than that expressed on B-cells from age-matched controls (P < 0.0001) and was inversely correlated with soluble CD21 (r2 = -0.41). We found no evidence of autoantibody to any complement regulator. Low CD21 expression correlated to prognostic subsets of CLL patients, i.e. cases with unmutated IGHV genes (P = 0.0006), high CD38 (P = 0.02) and high ZAP70 expression (P = 0.0017). Low CD21 expression was inversely correlated to the levels of phosphotyrosine induced in CLL cells following BCR ligation with αIgM (r2 = -0.21). Importantly, lower CD21 expression was also predictive for reduced overall survival (P = 0.005; HR = 2.7). In conclusion, we showed that reduced expression of CD21 on CLL B-cells appears functionally relevant and was associated with poor clinical outcomes.

Microrna Mir-21 Is Upregulated after Different Microenvironmental Stimuli and Controls Proliferation, Chemotaxis and Chemoresistance in Chronic Lymphocytic Leukemia

Aberrant expression of diverse microRNAs (miRNAs) has been related to pathogenesis and clinical outcome in patients with CLL. miR-21 is overexpressed in a wide variety of neoplasms where it participates in oncogenic events such as proliferation, resistance to treatment and metastasis. In CLL, miR-21 expression has been associated to refractoriness to fludarabine and to shorter overall survival. In addition, high expression of ZAP-70 protein in CLL is related to shorter overall survival and higher probability of progression. Several biological mechanisms have been described explaining the adverse prognosis associated with high ZAP-70 expression. In this sense, ZAP-70 protein increases the capability of CLL cells to respond to different survival and migration signals provided by the microenvironment. In a previous work, we found that transfected B-cell lines with a ZAP-70 expressing vector (Calpe at al, Blood 2011) had an increased expression of several molecules, including miR-21. Against this background, we studied the relationship between ZAP-70 protein and miR-21. For this, we firstly analyzed the correlation of ZAP-70 with miR-21 in primary CLL cells from 32 patients. In this series we observed that miR-21 expression analyzed by QRT-PCR was significantly higher in patients with high expression of ZAP-70 compared to patients with low expression of ZAP-70 (mean miR-21 in high ZAP-70 (N=17) = 5.781 ± 1.517; mean miR-21 in low ZAP-70 (N=15) = 0.6783 ± 0.2730; p=0.0082). In order to further analyze the molecular mechanisms regulating the induction of miR-21 and the potential role of ZAP-70 protein in this process, we co-cultured primary CLL cells (N=16) in conditions mimicking the microenvironment in the proliferation centers (bone marrow stromal cells with concomitant stimulation of CD40 and TLR9). In these conditions, besides ZAP-70 activation, we observed a 3.6 ± 0.78 mean fold induction in miR-21 expression after 48 hours compared to cells in suspension. Interestingly, this increase was only significant in patients with high expression of ZAP-70 (N=8; p= 0.0379). To define the role of ZAP-70 in miR-21 regulation, we stably transfected Ramos B-cells with ZAP-70 protein and found that both MAPK and STAT3 participate in the induction of miR-21 expression after ZAP-70 activation upon BCR crosslinking. Moreover, using this system we observed downregulation of the tumor suppressors PTEN, PDCD4 and PIAS3, all of which have been found to be targeted by miR-21 in malignant cells. Next, we aimed to study the functional role of miR-21 in primary CLL cells co-cultured in conditions mimicking the microenvironment from the proliferation centers. For this, we analyzed survival, proliferation and response to fludarabine after inhibition of miR-21 expression by transfecting primary CLL cells with antisense miR-21 inhibitor. The co-culture of primary CLL cells significantly increased their survival after 48 hours regardless the inhibition of miR-21. However, the induction of proliferation (measured as percentage of Ki-67 positive cells) was significantly inhibited by the suppression of miR-21 (N=13; p=0.022). Next, the analysis of a small pilot cohort showed a consistent but not yet significant overcoming of the chemoresistance induced by the co-culture after the inhibition of miR-21 Interestingly, the sensitivity to fludarabine of primary CLL cells cultured in suspension was also increased by the inhibition of miR-21. Finally, we examined the ability of primary CLL cells to migrate towards CXCL12 and observed that inhibition of miR-21 resulted in a 2.62-fold reduction of the migration index (ratio of cells migrating towards culture media to cells migrating towards media with 200ng/mL CXCL12). In conclusion, we have showed the correlation and participation of ZAP-70 protein in the regulation of miR-21. We have also observed that miR-21 contributes to CLL proliferation, resistance to fludarabine, and chemotaxis towards CXCL12. Although further experiments are warranted in order to fully elucidate the regulation and functional role of miR-21 in CLL, these results help to enlighten the biology behind the adverse clinical outcome of patients with CLL and high expression of ZAP-70, and could potentially be exploited for the development of new treatments in a near future. DisclosuresBosch:Roche: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding.

Role of Sprouty2 in the Pathogenesis of Chronic Lymphocytic Leukemia

INTRODUCTION: Chronic Lymphocytic Leukemia (CLL) is the most common leukemia in adults representing about 35% of all adult leukemias in the United States. CLL is a B-cell neoplasm which is characterized by accumulation of long lived mature B-cells in peripheral blood (PB), bone marrow (BM) and lymph nodes (LN). We and others have reported that CLL cells in tissue microenvironments have constitutively activated MAPK-Erk and AKT signaling pathways, which promotes their proliferation and survival. However, the molecular mechanisms and the gene(s) responsible for the activation of these pathways in CLL are not clearly understood. We performed gene expression profiling of CLL cells from PB, BM and LN and transcriptome analyses of CLL cells from PB from good and poor prognosis patients. Our studies identified Sprouty 2 (Spry2), a negative feedback regulator as one of the key genes involved in the regulation of MAPK-Erk and Akt pathways in CLL. We observed that Spry2 levels were down-regulated in poor prognosis CLL cells. Interestingly, Spry2 is epigenetically silenced in most human and mouse lymphomas and ectopic expression of Spry2 induces apoptosis in mouse B cells. We categorized CLL patients into good and poor prognosis to further evaluate Spry2 in CLL biology. Poor prognosis patients were defined by unmutated immunoglobulin variable heavy chain (IgVH), 11q22 deletion, 17p deletion, trisomy 12 or high CD38 and Zap70 expression. Good prognosis patients were defined as having mutated IgVH, 13q14 deletions or low CD38 and Zap70 expression.RESULTS: We found that Spry2 expression was significantly decreased by 3.2 log2-fold, (p=0.0001) at transcript and protein levels, respectively, in CLL cells from patients with poor prognosis compared to patients with good prognosis. Spry2 is a negative feedback regulator which attenuates cell proliferation, migration and survival. To determine the effect of Spry2 on CLL cells we transfected Mec-1 cells (stable human CLL cell line) and primary human CLL cells with a Spry2 cDNA containing vector. Ectopic expression of Spry2 induced spontaneous apoptosis in Mec-1 cells and primary CLL cells from poor prognosis patients. These results indicate that Spry2 negatively regulates the survival of human CLL cells. CLL cells have been shown to have active B-cell receptor (BCR) signaling promoting proliferation in vivo. To study the effect of BCR stimulation on Spry2 we stimulated BCR on Mec-1 and primary CLL cells by BCR cross-linking. Unexpectedly, the rate of proliferation of Mec-1 cells decreased upon BCR stimulation. We were able to demonstrate a transient increase in Spry2 levels that peaked at 12 hours post BCR stimulation, which we suspected was mediating the decrease in proliferation observed. We therefore repeated the BCR stimulation experiment using siRNA to inhibit the transient increase in Spry2 in primary CLL cells from seven good prognosis patients and in the Mec-1 cells. As predicted, we observed a significant increase in proliferation in all primary CLL samples and in the Mec-1 cells upon Spry2 knock down following BCR stimulation. Spry2 attenuates ligand induced MAPK-Erk and AKT signaling leading to suppression of proliferation and survival. We also examined the effect of Spry2 on MAPK-Erk and Akt signaling in CLL cells. Upon induction of exogenous Spry2 expression in Mec-1 cells there was a significant decrease in p-Akt and p-Erk levels. Conversely, Spry2 knock down resulted in an increase in p-Erk and p-Akt and enhanced cell proliferation.CONCLUSIONS: Spry2 is significantly down-regulated in the B-cells from patients with poor prognosis CLL. Down-regulation of Spry2 in CLL cells leads to hyperactivation of MAPK-Erk and Akt signaling and exogenous and endogenous expression of Spry2 leads to spontaneous apoptosis in human CLL cells. These results identify Spry2 as a negative regulator of CLL cell survival and proliferation and provide a potential molecular target for therapeutic intervention. DisclosuresBociek:Seattle Genetics, Inc.: Research Funding.

Insulin Growth Factor 1 Receptor Expression Is Associated with NOTCH1 Mutation, Trisomy 12 and Aggressive Clinical Course in Chronic Lymphocytic Leukemia

Insulin growth factor 1 receptor (IGF1R) is emerging as an important gene in many solid and hematological cancers and its over expression has been reported to be associated with aggressive disease and pharmacologic resistance. Specifically, the IGF1R-IGF1-2 interaction was recently described to be involved in the constitutive activation of many important cell signaling such as NOTCH1 and PI3K/Akt pathways that play a key role in many solid and hematological cancers. In this study we performed a clinical and biological investigations about the role of IGF1R expression in a large and representative prospective series of chronic lymphocytic leukemia (CLL) in Binet A clinical stage enrolled in observation O-CLL1 protocol (clinicaltrial.gov identifier NCT00917540). Total RNA extraction, preparation of DNA single-stranded sense target, and hybridization to gene expression profiling arrays were carried out according to manufacturer’s protocols in 217 CLL patients enrolled in the multicentre O-CLL1 protocol. Gene expression data has been deposited in the National Centre for Biotechnology Information’s Gene Expression Omnibus database http://www.ncbi.nlm.mih.gov/geo and are accessible through series accession number GSE51529. High IGF1R expression was significantly associated with IGHV unmutated (IGHV-UM) status (p<0.0001), high ZAP-70 and CD38 expression (p<0.0001) and unfavorable cytogenetic deletion [i.e. del(11)(q23) and del(17)(p13)], particularly with del(11)(q23) (p=0.03). Cases with del(13)(q14) as single lesion were characterized by the lowest IGF1R gene levels. On the contrary, among the most common cytogenetic aberrations, trisomy 12 showed stronger IGF1R expression compared with the other patients (p<0.0001) and this association was independent from IGHV mutational status. Patients with stereotyped HCDR3 sequences showed a greater IGF1R expression compared to not stereotyped HCDR3 (p=0.001) even if this can be related to the high frequency of IGHV-UM among stereotyped HCDR patients. Interestingly, subset #4 patients, who are known to exhibit an indolent clinical course and distinct biological profile, were also characterized by lower IGF1R expression compared to other M-IGHV and UM-IGHV patients. NOTCH1 c.7541_7542delCT mutation was investigated by next generation sequencing Roche 454 technology in 199 (92%) patients (Lionetti et al, BJH 2014). Globally, median depth of coverage was 1510x, ranging from 605 to 2842. Mutant allele frequency estimated by NGS ranged from 0.02% to 75% of total reads per sample. The presence of NOTCH1 mutation was confirmed by ASO-PCR and Sanger sequencing in all patients with allele burden higher > 0.7% (31; 15.5%) and 7% (19; 9.5%) respectively. We considered as mutated only the 31 patients in whom the presence of the dinucleotide deletion was confirmed by ARMS-PCR. Patients carrying NOTCH1 mutation were characterized by a greater IGF1R expression compared with wild type cases (p=0.002). In addition high IGF1R expression was not significantly different comparing patients with low and high NOTCH1 mutation burden. In order to avoid the bias represented by the strong association between NOTCH1 and trisomy 12, we compared the IGF1R expression between NOTCH1 mutated and wild type cases excluding trisomy 12, and confirmed the previous association (p=0.004). IGF1R expression represented a strong clinical prognostic factor in our CLL cohort: by Kaplan-Maier analysis we observed a significant time to first treatment stratification in all CLLs, in all IGHV-UM and in all IGHV-M patients (p<0.0001). Furthermore, IGF1R retained its significance in multivariate analysis with most important clinical and molecular prognostic factors (CD38 expression, unfavorable FISH and IGHV mutational status). Overall, our study shows the importance of IGF1R expression in CLL and its strong association with specific clinical and biological features, confirming the interest for the study of this gene as a potential prognostic factor and its possible role as a therapeutic target in a specific group of CLL patients carrying trisomy 12 and NOTCH1 mutations. DisclosuresNo relevant conflicts of interest to declare.

Detection of p53 gene deletion in Xinjiang patients with chronic lymphocytic leukemia by fluorescence in situ hybridization and its clinical significance

To investigate the presence of p53 gene deletion in Xinjiang patients with chronic lymphocytic leukemia and its clinical significance. Interphase fluorescence in situ hybridization (FISH) was used to detect the p53 gene deletion in 77 patients with CLL. Presence of the deletion and its association with clinical and laboratory features as well as prognostic factors were analyzed. Kaplan-Meier method was used to calculate survivals, and the results were compared using a Log-rank test. p53 gene deletion was found in 10 (12.9%) of the patients but none from the control group (P<0.05). The deletion was found in 12.5% (4/32) of ethnic Hans and 13.3% (6/45) of ethnic Uyghurs (P>0.05). No significant different distribution of p53 gene deletion was found in regard to sex, age, ethnicity, peripheral blood cell count (except for Hb) or the levels of lactate dehydrogenase, β2-micro globulin and CD38 (P>0.05). The deletion rate was higher in the group with high expression of ZAP-70 and patients with advanced stage disease than that in the group of low expression and early-stage CLL (P<0.05). Among 20 patients who received fludarabine therapy, the overall remission rate for those with p53 gene deletion (20%) was lower than those without (75%) (P<0.05). With a median follow-up time of 39.0 (8.0-136.0) months, 11 cases had died (14.3%), among them, 7 cases died from CLL and related complications, and all of them were founded p53 gene deletion. In patients with p53 gene deletion, the progression-free survival (18 months) was shorter than those without the deletion (55 months) (P<0.05). The p53 gene deletion has been found in more than 10% of patients with CLL, and the deletion rate did not significantly differ between ethnic Han and Uyghur patients. The deletion is associated with advanced stage of the disease. High-level ZAP-70 expression and the presence of p53 deletion are associated with shorter survival and poor response to fludarabine containing therapy. Therefore, drugs affecting the p53 signaling pathway should be avoided.

Allele-specific loss and transcription of the miR-15a/16-1 cluster in chronic lymphocytic leukemia.

Deregulation of the miR-15a/16-1 cluster has a key role in the pathogenesis of chronic lymphocytic leukemia (CLL), a clinically heterogeneous disease with indolent and aggressive forms. The miR-15a/16-1 locus is located at 13q14, the most frequently deleted region in CLL. Starting from functional investigations of a rare SNP upstream the miR cluster, we identified a novel allele-specific mechanism that exploits a cryptic activator region to recruit the RNA polymerase III for miR-15a/16-1 transcription. This regulation of the miR-15a/16- locus is independent of the DLEU2 host gene, which is often transcribed monoallellically by RPII. We found that normally one allele of miR-15a/16-1 is transcribed by RNAPII, the other one by RNAPIII. In our subset of CLL patients harboring 13q14 deletions, exclusive RNA polymerase III (RPIII)-driven transcription of the miR-15a/16-1 was the consequence of loss of the RPII-regulated allele and correlated with high expression of the poor prognostic marker ZAP70 (P=0.019). Thus, our findings point to a novel biological process, characterized by double allele-specific transcriptional regulation of the miR-15a/16-1 locus by alternative mechanisms. Differential usage of these mechanisms may distinguish at onset aggressive from indolent forms of CLL. This provides a basis for the clinical heterogeneity of the CLL patients carrying 13q14 deletions.

Serum immunoglobulin levels at diagnosis have no prognostic significance in stage A chronic lymphocytic leukemia: a study of 1113 cases from the Israeli CLL Study Group

Hypogammaglobulinemia, commonly encountered in chronic lymphocytic leukemia (CLL), is one of the main causes of morbidity and mortality; however, its prognostic significance in patients diagnosed in early stages of disease remains uncertain. The aim of this study was to evaluate the predictive power of hypogammaglobulinemia at Bonet stage A. Using the database of the Israeli CLL Study Group, we analyzed the relationship between low serum levels of IgG, IgA, and IgM; the presence of paraproteinemia, as well as other well-recognized prognostic markers in CLL; and time to first treatment (TTT) and overall survival. A total of 1113 patients consecutively diagnosed during the last 25 yrs with Binet stage A CLL were evaluated, and baseline information on serum immunoglobulin levels was found in 857 of the cases. Overall survival times correlated with age >65 yr, male gender, the presence of lymphadenopathy, high serum beta 2-microglobulin (b2m), CD38 and ZAP-70 expression, but not with low levels of immunoglobulin or the presence of paraproteinemia. By univariate analysis, patients with low IgA levels had a shorter TTT; however, on multivariate analysis, the presence of lymphadenopathy (P 0.02), b2m (P 0.04), CD38 (P < 0.001), and ZAP-70 (P < 0.001) was the only laboratory parameters with prognostic significance. In our cohort of patients with early-stage CLL, baseline hypogammaglobulinemia and the presence of paraproteinemia were not found to correlate with prognosis.

Prognostic relevance of angiopoietin-2, fibroblast growth factor-2 and endoglin mRNA expressions in chronic lymphocytic leukemia.

Elevated levels of circulating angiogenic cytokines and increased expression of genes encoding angiogenic factors have been reported in recent years in patients with chronic lymphocytic leukemia (CLL) but data regarding prognostic and predictive significance are still limited. Therefore, in the present study based upon our prior pilot results, we measured mRNA expressions of angiopoietin-2 (Ang-2), fibroblast growth factor-2 (FGF-2) and endoglin (CD105) by reverse transcription quantitative PCR in purified CD19+ cells from 70 untreated CLL patients (median age, 63 years; males, 64%; Rai III/IV stages, 29 %; unmutated IgVH genes, 60 %) and evaluated their possible association with established prognostic factors and clinical course of the disease. Higher expression of Ang-2 was significantly associated with unmutated IgVH genes (n = 55, p = 0.003). Higher CD105 expression was significantly associated with unmutated IgVH genes (n = 55, p < 0.001), high CD38 expression (n = 66, p = 0.022), high ZAP-70 expression (n = 66, p = 0.010), Rai stage I-IV (n = 70, p < 0.001), progressive clinical course of CLL (n = 70, p = 0.001) and shorter time to treatment (n = 70; p < 0.001). Expression of FGF-2 was not significantly associated with any of the prognostic markers. These results indicate that elevated expression of Ang-2 and in particular CD105 by CLL cells is associated with unfavorable prognostic features and clinical outcome; thus, both cytokines appear to play an important role in biology and progression of CLL and warrant further investigation.

ZAP-70 Promotes the Infiltration of Malignant B-Lymphocytes into the Bone Marrow by Enhancing Signaling and Migration after CXCR4 Stimulation

ZAP-70 in chronic lymphocytic leukemia (CLL) is associated with enhanced response to microenvironmental stimuli. We analyzed the functional consequences of ZAP-70 ectopic expression in malignant B-cells in a xenograft mouse model of disseminated B-cell leukemia. Mice injected with B-cells expressing ZAP-70 showed a prominently higher infiltration of the bone marrow. In vitro analysis of the response of malignant B-cells to CXCL12, the main attracting chemokine regulating trafficking of lymphocytes to the bone marrow, or to bone marrow stromal cells, revealed that ZAP-70 induces an increased response in terms of signaling and migration. These effects are probably mediated by direct participation of ZAP-70 in CXCL12-CXCR4 signaling since CXCR4 stimulation led to activation of ZAP-70 and downstream signaling pathways, such as MAPK and Akt, whereas ZAP-70 did not alter the expression of the CXCR4 receptor. In addition, subclones of primary CLL cells with high expression of ZAP-70 also showed increased migrative capacity toward CXCL12. Neutralization of CXCR4 with a monoclonal antibody resulted in impaired in vitro responses to CXCL12 and bone marrow stromal cells. We conclude that ZAP-70 enhances the migration of malignant B-cells into the supportive microenvironment found in the bone marrow mainly by enhancing signaling and migration after CXCR4 stimulation.

Regulation Of Microrna Mir-21 Expression By ZAP-70 Protein In Chronic Lymphocytic Leukemia

Aberrant expression of diverse microRNAs (miRNAs) has been related to pathogenesis and clinical outcome in patients with CLL. In this regard, miR-21 expression has been associated to refractoriness to fludarabine and to shorter overall survival. MicroRNA miR-21 has been found to be overexpressed in a wide variety of neoplasms where it participates in oncogenic events such as proliferation, resistance to treatment, and metastasis. In CLL, high expression of ZAP-70 protein is related to shorter overall survival and higher probability of progression. Moreover, ZAP-70 protein participates in the signaling of the B cell receptor (BCR) and different chemokine receptors, therefore increasing the capability of CLL cells to respond to survival and migration signals provided by the microenvironment. In order to further elucidate the molecular mechanisms defining bad prognosis CLL we studied the relationship between ZAP-70 protein and miR-21. Firstly, we analyzed the correlation of ZAP-70 with miR-21 in primary CLL cells from 32 patients. In this series we observed that miR-21 expression analyzed by QRT-PCR was significantly higher in patients with high expression of ZAP-70 compared to patients with low expression of ZAP-70 (mean miR-21 in high ZAP-70 (N=17) = 5.781 ± 1.517; mean miR-21 in low ZAP-70 (N=15) = 0.6783 ± 0.2730; p=0.0082). In order to further analyze the molecular mechanisms regulating the induction of miR-21 and the potential role of ZAP-70 protein in this process, we co-cultured primary CLL cells (N=16) with bone marrow stromal cells with concomitant stimulation of CD40 and TLR9. In these conditions, besides ZAP-70 activation, we observed a 3.6 ± 0.78 mean fold induction in miR-21 expression after 48 hours of co-culture compared to cells in suspension. Interestingly, when we independently analyzed patients with low or high expression of ZAP-70 (N=8 for each group) we observed that this increase was only significant in patients with high expression of ZAP-70 (p= 0.0379). We therefore decided to specifically study the role of ZAP-70 in the regulation of miR-21. For this, we stably transfected Ramos cells with a vector expressing a ZAP-70-GFP fusion protein or GFP only as a control. The stimulation of the BCR in these cells caused the activation of ZAP-70 protein and the subsequent activation of the MAPK and AKT pathways, as evidenced by western blot analysis. Interestingly, BCR stimulation for 12 hours in Ramos-GFP cells significantly induced the expression miR-21 (19.55 ± 1.49 vs. 65.03 ±12.98; 3.32 fold change) whereas in those cells transfected with ZAP-70 the increase was greater (13.26 ± 2.13 vs. 138.77 ± 11.72; 10.4 fold change), suggesting that ZAP-70 is directly participating in the pathway leading to miR-21 upregulation. In addition, only after 4 hours of BCR stimulation we already observed upregulation of primary-miR21, specially in cells with expression of ZAP-70 (2.1 fold in Ramos GFP vs. 7.3 fold in Ramos GFP-ZAP-70), indicating that the regulation of miR-21 is at the transcriptional level. Furthermore, inhibition of the MAPK pathway, but not of AKT, impaired the upregulation of miR-21 in both cell lines. STAT-3 transcription factor has been shown to participate in the induction of miR-21 expression in different cell types. In this system, we observed that BCR stimulation induced the activation of STAT-3 and that its biochemical inhibition impaired the upregulation of miR-21, indicating that STAT-3 is probably participating in miR-21 induction in malignant B-lymphocytes. Finally, we analyzed the downregulation of potential miR-21 targets. For this, we studied the downregulation of the tumor suppressors PTEN, PDCD4 and PIAS3, all of which have been found to be regulated by miR-21 in malignant cells. We observed that, after 48 hours of BCR stimulation, the protein levels of all three genes were downregulated, indicating that these proteins may be targeted by miR-21 in this cellular system. In conclusion, we have showed the participation of ZAP-70 protein in the regulation of miR-21, an oncomiR that has been related to short survival and resistance to treatment with fludarabine in CLL. Although further experiments are warranted in order to fully elucidate the role of ZAP-70 in miR-21 induction in CLL, these results enlighten the biology behind the adverse clinical outcome of patients with CLL and high expression of ZAP-70, and can potentially be exploited for the development of new treatments. Disclosures:No relevant conflicts of interest to declare.

Allele-Specific Loss Of The Mir-15a/16-1 Cluster Correlates With ZAP70 Expression In CLL Patients With 13q Deletion

Chronic lymphocytic leukemia, the most common form of adult leukemia in the Western world, is characterized by a heterogeneous clinical course, with indolent or progressive forms that entail profoundly different approaches to treatment. The biomarkers useful to predict the clinical course of CLL encompass cytogenetic abnormalities, protein expression (ZAP70, CD38) and IGHV mutation status. Among the many molecular features characterizing biological and clinical aspects of CLL patients, deletion of 13q14 and down-regulation of its related microRNAs (miR-15a and miR-16-1) are the most frequent aberrations in CLL. The involvement of the miR-15a and mir-16-1 in CLL has been extensively described by in vivo and in vitro studies and the genomic region encompassing the miR cluster results frequently deleted, but the mechanisms that regulate the expression of these miRs in CLL are still poorly understood. Here we demonstrate that the G to A single nucleotide polymorphism (SNP) rs115069827 prevents the maturation of miR-15a by reducing the binding of the DROSHA complex to the pri-microRNA 15a/16-1. The study of this functional SNP allowed us to discover a novel mechanism of transcription of the miR-15a/16-1 cluster, independent of DLEU2, its host gene. We demonstrate that the genomic region immediately upstream of the miR-15a/16-1, including the SNP, acts as transcriptional activator. The miR-15a/16-1 cluster is transcribed by allele-specific mechanisms: transcription of one allele is driven by the RNA Polymerase II at the DLEU2 promoter, while the other allele is transcribed by a mechanism that involves occupancy of the newly identified transcription activator region by RNA Polymerase III. Interestingly, the latter mechanism is dominant within CLL cells with mono-allelic 13q14 deletion and high expression of ZAP70 (Fisher's exact test p:0.0128) (Table1). Moreover, in our patient cohorts (training set n=28, validation set n=19) there are CLL samples with cells carrying 13q14 deletions that did not encompass the miR-15a/16-1 locus. Almost all of these show high expression of ZAP70 (Fisher's exact test training set p:0.0055; validation set p:0.0374) and primiR regulation by RPIII, in the presence of both alleles of miR-15a/16-1. By CNV analysis of a more telomeric region (∼30 kb, U59 DLEU2 region), that encompasses the second DLEU2 promoter, we demonstrate that these CLL cells carry a mono-allelic deletion of this region, which entails loss of the primiR regulation by the DLEU2 promoter ( occupied by RPII).Table 1primiR 15a/16-1 transcritpion by RPII independent mechanism correlates with ZAP70 expression in 13q deleted CLL.SampleFISHCopy number variation (CNV)ZAP70IGVHRelative expression after α amanitin treatment (2µg/ml)primiR 15a/16-1RNA PolymeraseU59 DLEU1miR 15a/16-1primiR15a/16-1pretRNAtyrpreACTBpreGAPDHLLC3113qnd-M↓=↓IILLC42NK+/--M=↓↓IIILLC1813q+/--M↓=↓IILLC0713q+/--U↓==IILLC45NK+/--M↓=↓IILLC50NK+/--M==↓IIILLC52A13q+/--M=nd↓IIILLC6113q+/--M↓=↓IILLC8113q+/--M↓=↓IILLC4713q+/--M==↓IIILLC03ANK+/--M↓=ndIICLL4Tw13q+/--M↓=↓IICLL3TwNK+/-+M==↓IIILLC0412tri/13q+/-+/++U=↓↓IIILLC6413q+/-+U=↓↓IIILLC7013q+/++/++U==↓IIILLC3613q+/-+M=↓↓IIILLC80NK+/-+/++M==↓IIILLC17ANK+/-+/++nd=nd↓IIIOverall these data may clarify important genetic features of the major subgroup of CLL patients and highlight the concept that regulation of the expression of the miR-15a/16-1 is the critical event in CLL pathogenesis. Being ZAP70 high expression associated with poor prognosis in CLL, we show that the concomitant presence of different mechanisms of transcriptional regulation of miR-15a and miR-16-1 could have different impacts on the pathogenesis/progression of the CLL.nd: not determined, M: IGVH mutated, U: IGVH unmutated, CNV: Copy number variation, NK, normal karyotype; 13q, deletion of 13q chromosome region; 12tri, 12 trisomy; +/+, presence of both alleles; +/-, monoallelic deletion; -, ZAP70≤20%; +, ZAP70>20%; ↓, downregulation of RNA expression after α amanitin treatment; =, no change in the RNA expression after α amanitin treatment; II, RPII; III, RPIII. Fisher's test: RPIII vs ZAP70, p:0.0128; RPIII vs IGVH, p:0.576 Disclosures:No relevant conflicts of interest to declare.

The Co-Culture Of Primary CLL Cells With Bone Marrow Mesenchymal Cells, CD40 Ligand and CpG ODN Promotes The Proliferation Of Chemoresistant CLL Cells Phenotypically Comparable To Those From The Proliferative Centers

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation and proliferation of monoclonal CD5+ mature B-cells in peripheral blood (PB), lymph nodes (LN), and bone marrow (BM). The microenvironment found in BM and LN, where CLL cells interact with different accessory cells, induces active proliferation of CLL cells and protects them from spontaneous and chemotherapy-induced apoptosis. This may explain why the BM and secondary lymphoid organs are preferential sites for minimal residual disease persistence and for relapses observed in all patients after treatment. Therefore, further characterization of primary CLL cells found in the proliferative niches can facilitate the discovery and study of new targets specifically expressed by this proliferative and resistant subset of primary CLL cells.In order to further characterize the proliferative subset of primary CLL cells we analyzed the characteristics of proliferating subclones of CLL cells found in PB and compared it to the phenotype of primary CLL cells co-cultured ex vivo in conditions mimicking the microenvironment of the proliferative niches. Firstly, we compared actively proliferating CLL cells from 40 patients diagnosed with CLL with their quiescent counterpart. For this, we analyzed by flow cytometry (FC) the differential expression of ZAP-70, CD38, and the chemokine receptors CXCR4, CXCR5 and CCR7 in Ki-67 positive vs. negative CLL cells. Ki-67 positive CLL cells had higher expression levels of ZAP-70 and CD38, while the expression levels of all the chemokine receptors analyzed were significantly lower (Figure 1). This phenotype indicates that these cells may have an increased capability to respond to different survival and migration signals provided by the cellular microenvironment, since high expression of ZAP-70 and CD38 has been related to increased response to this kind of signaling. Moreover, downregulation of chemokine receptors would indicate recent stimulation by chemoatracting cytokines. Furthermore, with the aim of ex vivo mimic the microenvironment found in the proliferative centers, we co-cultured primary CLL cells from 27 patients with the BM-derived stromal cell line UE6E7T-2, 1μg/mL soluble CD40L and 1.5μg/mL CpG ODN, and analyzed the effects in terms of proliferation, modulation of surface molecules and development of chemoresistance. Of note, already 24 hours after co-culture, CLL cells increased their size, resembling stimulated B cells, and tended to be located in sparse clusters that included dividing cells, as observed after immunocytochemistry studies. In this setting, proliferative responses assessed by Ki-67 expression analyzed by FC were already observed after 24 hours (mean % Ki-67 positive cells: 1.48±0.28 in co-culture vs. 0.56±0.11 in suspension, P<0.01) and further increased after 48 hours (mean % Ki-67 positive cells: 2.89±0.51 in co-culture vs. 0.25±0.10 in suspension, P<0.01) and 72 hours (mean % Ki-67 positive cells: 7.68±2.11 in co-culture vs. 0.81±0.24 in suspension, P<0.001). Next, we analyzed the expression levels of ZAP-70, CD38 and the chemokine receptors CXCR4, CXCR5 and CCR7. Interestingly, the modulation of the expression of these molecules in co-cultured CLL cells correlated with the previously mentioned differential expression observed between the Ki-67 positive vs. negative compartments, indicating that the ex vivo co-culture highly resembles the conditions found in proliferative niches (Figure 1). Finally, in order to assess the role of microenvironment in chemoresistance, CLL cells were cultured in suspension and in co-culture for 48 hours and subsequently treated with increasing doses of fludarabine for 24 hours. Interestingly, the co-culture of CLL cells inhibited at such extend the capacity of fludarabine to induce apoptosis that it was not possible to calculate its lethal dose 50, whereas lethal dose 50 for fludarabine in CLL cells in suspension was 416μM (95%CI 125.5-1379).In conclusion, these results suggest that culturing CLL cells in the previously described conditions promotes proliferation and the acquisition of a phenotype which bears many similarities to the one found in actively proliferating CLL cells circulating in PB. This study provides a model for a co-culture system which might serve as a basis for the development and testing of new drugs that target the proliferative and drug resistant CLL cell compartment. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

NFAT2 Is a Critical Regulator Of Anergy Induction In CLL

NFAT is a family of highly phosphorylated proteins residing in the cytoplasm of resting cells. Upon dephosphorylation by calcineurin, NFAT proteins translocate to the nucleus where they orchestrate developmental and activation programs in diverse cell types. CLL is a clonal disorder of mature B cells characterized by the expression of CD19, CD23 and CD5. With respect to prognosis, it constitutes a heterogeneous disease with some patients exhibiting an indolent course for many years and others progressing rapidly and requiring early treatment. Expression of CD38 and ZAP70 define a subgroup of patients with enhanced responsiveness to stimulation of the B cell receptor (BCR) complex and more aggessive disease. In contrast, another subset of CLL patients with more indolent course is characterized by an anergic B cell phenotype refering to B cell unresponsiveness to IgM ligation and essential lack of phosphotyrosine induction and calcium flux. Here, we analyzed the role of NFAT2 in the pathogenesis of B-CLL and in anergy induction in CLL cells.For this purpose, we generated mice with a conditional NFAT2 knock out allele (NFAT2fl/fl). In order to achieve NFAT2 deletion limited to the B cell lineage, we bred NFAT2fl/fl mice to CD19-Cre mice. To investigate the role of NFAT2 in the pathogenesis of CLL we made use of the Eµ-TCL1 transgenic mouse model in which the TCL1 oncogene is expressed under the control of the Eµ enhancer. TCL1 transgenic mice develop a human-like CLL at the age of approximately 14 wks to which the animals eventually succumb at an average age of 10 months. To analyze the role of NFAT2 in CLL, we generated mice (n=10) whose B cells exhibited a specific deletion of this transcription factor in addition to their transgenic expression of the TCL1 oncogene (TCL1 CD19-Cre NFAT2fl/fl). TCL1 transgenic mice without an NFAT2 deletion served as controls (n=10). To identify novel NFAT2 target genes in CLL cells, we performed a comparative gene expression analysis on CLL cells with intact NFAT2 expression and on CLL cells with NFAT2 deletion using affymetrix microarrays.Mice with NFAT2 knock out exhibited a significantly more aggressive disease course with accelerated accumulation of CD5+CD19+ CLL cells and a significantly reduced life expectancy (200 vs. 325 days) as compared to control animals. Flow cytometric analysis at distinct time points showed a pronounced infiltration by CD5+ B cells of the peritoneal cavity, spleen, lymph nodes, liver and bone marrow which was significantly stronger in the NFAT2 ko cohort. Most of the CD5+ B cells in TCL1+NFAT2 ko mice showed high expression of ZAP70 and CD38, whereas TCL1 transgenic mice only demonstrated very few CD5+ B cells with concomitant expression of ZAP70 and CD38. To investigate the effects of an NFAT2 ko on proliferation and apoptosis of CD5+CD19+ CLL cells, we performed in vivo BrdU incorporation assays with subsequent flow cytometric analysis. Interestingly, we could show that CLL cells isolated from spleens, bone marrow and peripheral blood from mice with an NFAT ko exhibited significantly higher rates of proliferation than control animals. To identify NFAT2 target genes resonsible for the observed alterations in the disease phenotype, we subsequently peformed a gene expression analysis with CD5+CD19+ CLL cells from TCL1+NFAT2 ko mice with CLL cells from TCL1+ mice serving as controls. Here, we detected a significantly altered expression of 22 genes associated with B cell anergy in the TCL1+NFAT2 ko cohort. The vast majority of these genes was expressed significantly less in the absence of NFAT2 with Lck, Pacsin1, Hspa14 and CD166 constituting the strongest hits with up to 10fold reduced gene expression. Downregulation of the identified target genes was subsequently confirmed using RT-PCR and Western Blotting.In summary, our data provide strong evidence that NFAT2 is a critical regulator of CD38 and ZAP70 expression and substantially controls cell cycle progression in CLL cells. In addition, we could show that NFAT2 controls the expression of several anergy-associated genes and that its absence prevents the acquisition of an anergic phenotype by the CLL cells correlating with a significantly more aggressive course of the disease. Taken together, our data demonstrate that NFAT2 plays an essential role in the pathogenesis of CLL and implicate this transcription factor as a potential target in its treatment. Disclosures:No relevant conflicts of interest to declare.