CLL Samples Research Articles

Assessment of p53 Functionality in Chronic Lymphocytic Leukemia by Different Assays; An Eric-Wide Approach.

AbstractAbstract 2872▪▪This icon denotes a clinically relevant abstract Introduction:The DNA damage response axis plays a crucial role in chemoresistance in CLL, as indicated by the prognostic impact of deletions of 17p (locus of TP53) and 11q (locus of ATM). These deletions coincide with mutations in the remaining allele, although frequency, especially for ATM varies. Functional read-outs of the p53 axis might add clinical relevant information on the actual DNA damage response. Currently, different p53 function analyses are being developed. These assays are either based on measurement of (i) RNA expression levels of a single gene (RT-PCRp21) or gene sets (RT-MLPA), or protein expression levels (FACSp53-p21) after DNA damage by irradiation or etoposide/nutlin exposition or (ii) gene expression levels at base-line (RT-PCR miR34a). To what extent these different assays correlate is currently unknown. Aim:Detailed side-by-side analysis of available p53 functional assays in well characterized CLL samples Methods:Freshly frozen PBMC’s of 15 different CLL samples (CD19/CD5>90%) were exchanged between 5 research groups that developed the following p53 functional assays: RT-PCRp21 (Ulm), RT-MLPA (bax, puma, p21 and CD95; Amsterdam), FACSp53-p21 (Paris) and RT-PCR miR34a (Salzburg/Brno). FISH-analysis (11q, 17p, 13q and 12) was performed on all samples. In addition mutations in TP53 were determined by FASAY, DHPLC and Sanger sequencing and ATM mutations were assessed by Sanger sequencing. Results:RNA expression levels showed significant correlation (p-value < 0,01) between the different P53 functional assays with high correlation coefficients (range: 0,7–0,94). Based on combination of FISH-analysis, FASAY and Sanger sequencing the investigated CLL samples could be distinguished into 6 different categories; 1. 17p- with TP53 mutation (n=5), 2. Sole TP53 mutation (n=1), 3. 11q- with ATM mutation (n=1), 4. 11q- in absence of TP53/ATM mutation (n=4), 5. 17p- with both TP53 mutation and ATM mutation (n=1) and 6. No 17p- and 11q- in absence of TP53/ATM mutation (n=3). All p53 functional assays showed absent to minimal induction of expression of respective target genes in samples with a cytogenetic abnormality combined with a TP53/ATM mutation (category 1, 3, 5). In contrast, samples without a cytogenetic abnormality in absence of TP53/ATM mutations (category 6) showed a marked increase in expression of respective target genes in all the assays. The patient with a sole TP53 mutation (category 2) showed a normal DNA damage response in all assays except for FACSp53-p21. Samples with an 11q- in absence of TP53/ATM mutation (category 4) showed high inter-assay variation. The two assays with predefined cut-off values (RT-MLPA and FACSp53-p21) assessed 13 samples equally (9 p53 dysfunctional and 4 p53 functional), except for 2 patients (category 2, 3 respectively). Reproducibility of these assays was tested by blinded sending of 3 samples that have been analyzed previously in this study. Except for p21, all assays showed 100% reproducibility. Discussion:For the first time a comparative side-by-side analysis of different available p53 functional assays was performed, and demonstrated strong correlations between the different assays. All assays could detect samples with expected disturbed and normal DNA-damage responses. To what extent these p53 functional assays could be of clinical relevance especially with respect to chemo-responsiveness, should be further studied in prospective studies. Disclosures:No relevant conflicts of interest to declare.

The Kinin-Kallikre System in Chronic Lymphocytic Leukemia - A Potential Target for Therapy?

AbstractAbstract 3904CLL remains an incurable disease with standard therapy regimens. The hyper reactivity of the B cell Receptor (BCR) to unknown antigen ligation plays a pivotal role in B-cell survival. Recent clinical trials of BCR-targeted therapies prompted the need for a better understanding of the biology of BCR signaling. We previously employed proteomics to study protein expression changes associated with BCR ligation. Using 2-dimensional gel electrophoresis (2DE) with MALDI-TOF mass spectrometry (MS) we identified that Kininogen (KNG) was upregulated in 3/3 “poor prognosis” clinical samples upon BCR stimulation. KNG (a critical protein of Kinin-Kallikrein System) is the precursor for Kinins which act via the B1 and B2 receptors (B1R and B2R, respectively) and is known to play a critical role in cell migration, proliferation, vascular permeability, inflammation and intracellular Ca2+ influx. Consequently we hypothesized that there is a functional role for B1R and B2R, Kinins and their precursor KNG in CLL B-cell survival which may offer a potential therapeutic target.Following ethical approval, blood samples were collected from CLL patients. Time to first treatment, clinical stage, IgVh status, CD38 and ZAP-70 data were available. “Poor prognosis” samples were defined as unmutated IgVh status, high WBC and hyper-responsive BCR (p-ERK expression ≥ 2.0- fold increase upon stimulation). Immunoblotting was employed to confirm KNG upregulation and Tissue Kallikrein expression in CLL B-cells. A series of 59 CLL samples was screened for constituent KNG expression using immunoblotting. The presence of mRNA transcripts for KNG was assessed by PCR. An Automated One-stage Factor Assay on the Instrumentation Laboratory ACL-TOP analyser was used to determine the level of Plasma Kallikrein in CLL patients vs healthy controls. Fluorescence Activated Cell Sorting (FACS) was utilized for CLL B-cell enrichment according to CD20 status. Isolated CLL B-cells were subjected to immunodetection and flow cytometry for B1R and B2R cell surface expression identification.Proteomic analysis by 2DE/MALDI-TOF MS previously revealed upregulation of KNG after 5.5 hours of in vitro BCR stimulation in 3/3 clinical samples. Upregulation of KNG after stimulation was confirmed by immunoblotting in 5 samples, including all 3 which were previously analyzed using proteomics. There are 2 forms of KNG: HMWK and LMWK. The analysis of constitutive KNG by immunoblotting revealed positive expression in 72.9% (43/59) and 80% (28/35) for LMWK and HMWK, respectively. No statistical significance was found with preliminary clinical correlations, however, there was a trend towards shorter median survival in LMWK positive cases (152 months vs. 264 months for LMWK negative cases) (p=0.161). PCR confirmed the presence of KNG transcript in CLL samples. An automated one-stage factor assay revealed that 47.2% (26/55) CLL patients expressed elevated Plasma Prekallikrein (the zymogen form of Plasma Kallikrein) level. Tissue Kallikrein overexpression was confirmed in 14 CLL samples, using an anti-KLK 6 antibody. We investigated the presence of inducible B1R and constitutive B2R on CD20+ CLL B-cells and demonstrated that B1R and B2R are expressed by CLL B-cells, however, B1R expression was highly upregulated.We demonstrate for the first time that CLL B-cells express differing levels of KNG protein and that the expression can be significantly increased after BCR ligation. Consequently, high concentration of KNG is achieved in near proximity to B1R and B2R, identified on the surface of CLL B-cells. Therefore, we show that CLL B-cells comprise the complete system for the synthesis and liberation of Kinins which in turn mediate the activation of B1R and B2R via an autocrine loop. We suggest, that simultaneous and sustained activation of B1R and B2R, which is known to be advantageous for immediate triggering of ERK1/2 global phosphorylation and PLCγ2-dependent intracellular Ca2+ mobilization, play a functional role in the induction of pathologic signal transduction leading to B-cells survival and that Ca2+ influx-dependent transcriptional activation and MEK1/ERK1/2 pathway signaling may be dependent upon simultaneous complimentary stimuli input from BCR, B1R and B2R and possibly other disease-associated stimuli. The inhibition of Kinin receptor function may be a potential target for combined therapy in CLL. Disclosures:No relevant conflicts of interest to declare.

Overexpression and Aberrant Nuclear Translocation of NFAT2 in CLL

AbstractAbstract 3909Chronic Lymphocytic Leukemia (CLL) is a hematological malignancy of mature B cells and constitutes the most common leukemia in adults. It is characterized by a progressive accumulation of clonal B cells, which coexpress CD19, CD23 and CD5. The clinical course of CLL can be predicted by serveral prognostic markers like CD38, ZAP70 and cytogenetic abnormalities. While the treatment of CLL has significantly improved during recent years, it remains an essentially uncurable disease and the molecular events that lead to its development are still largely ellusive. NFAT is a family of highly phosphorylated transcription factors residing in the cytoplasm of resting cells. Upon dephosphorylation NFAT proteins translocate to the nucleus where they orchestrate developmental and activation programs in diverse cell types. NFAT is inactivated by a network of several kinases. Several recent studies have demonstrated that Ca2+/NFAT signaling is involved in the pathogenesis of a wide array of different tumor types including pancreatic adenocarcinoma, breast cancer and Non Hodgkin′s lymphoma. In this study we investigated the significance of the Ca2+/NFAT signaling pathway in B-CLL.For this purpose, we analyzed CLL cell lines (MEC-1, JVM-3) as well as primary blood samples from patients with CLL (n=30). The analyzed patient population exhibited a representative distribution of age, sex, Binet stage, WBC count, cytogenetics and IGVH mutational status. PBMC were obtained by ficoll density gradient centrifugation and B cells were subsequently isolated using magnetic bead technology. NFAT2 expression and aberrant nuclear translocation was then assessed by Western Blotting and Immunofluorescence staining. In addition, NFAT2 mRNA levels were measured by qRT-PCR and its DNA binding capacity was assessed using an electrophoretic mobility shift assay (EMSA). Transcriptional activity of NFAT2 proteins in CLL cells was further analyzed by determining the expression of several well characterized NFAT target genes by qRT-PCR and Western Blotting.In our analysis, we detected a profound overexpression of NFAT2 mRNA as well as NFAT2 protein in all CLL samples. Using qRT-PCR we found that CD19+CD5+ CLL cells exhibited an at least three fold overexpression of NFAT2 as compared to CD19+ B cells isolated from healthy donors. In one case, NFAT2 expression in CLL cells was 200 times higher than in the corresponding control cell population. This profound overexpression of NFAT2 in CLL cells could be confirmed on the protein level using Western Blotting and Immunocytochemistry. We could further demonstrate that even under resting conditions significant amounts of NFAT2 protein had translocated to the nucleus in CLL cells, whereas virtually all NFAT2 was in the cytoplasm in healthy B cells. NFAT2 nuclear translocation could be inhibited using pretreatment with Cyclosporin A demonstrating that this process was still calcineurin-dependent in CLL cells. We could further show that nuclear NFAT2 in CLL cells was able to bind DNA but that its transcriptional activity with respect to several apoptosis and cell cycle regulating genes was severely compromised when compared with healthy CD19+CD5+ B cells.In summary, these results provide strong evidence that the Ca2+/NFAT signaling axis is con-stitutively activated in CD19+CD5+ CLL cells. Reduced expression of several apoptosis and cell cycle regulating proteins which are known target genes of NFAT2 potentially links deregulation of this signaling cascade to CLL progression. This is in line with our observation that genetic loss of NFAT2 can induce acceleration of CLL in TCL1 mouse model. Further investigation is warranted to decipher the therapeutic potential of modulating Ca2+/Calcineurin/NFAT signaling in CLL. Disclosures:No relevant conflicts of interest to declare.

Anergic Response to B-Cell Receptor Signaling in Chronic Lymphocytic Leukemia: The Differing Roles of IgD and IgM in the Microenvironment and Peripheral Blood.

AbstractAbstract 2889Chronic Lymphocytic Leukemia (CLL) cells depend on B cell receptor signaling as well as other microenvironmental survival signals (1). Drugs targeting the BCR signaling pathways are showing exciting results in CLL clinical trials. A peculiarity of CLL is that IgD signaling is generally preserved, whilst IgM signaling is decreased and it has been suggested that this pattern mimics anergic B-cells, and might be consistent with chronic autoantigen exposure. We examined the differing roles of IgM and IgD signaling in CLL using a theoretical framework of anergy.Peripheral blood (PB) CLL cells exhibited higher IgD expression, as compared to IgM (n=204, p<0.0001), but this did not have prognostic impact. When we examined IgM and IgD expression in LN biopsies compared to paired PB (n=10) expression, IgM expression was lower in LN (p=0.002) whilst IgD expression was unchanged. Although the number of these paired samples is small, cases with lower LN IgM levels had poorer prognosis, and we are investigating this further with a larger cohort. We hypothesize that reduced LN IgM expression reflects antigen engagement and an anergic response in the microenvironment.We sought to replicate Mockridge et al' s model of reversible anergy (2) by monitoring the dynamic changes in IgM/D expression after in vitro incubation. Most (18/20) PB CLL samples underwent calcium (Ca) flux after IgD crosslinking, whereas only 13/20 cases underwent IgM Ca flux, and the level of Ca flux was less than with IgD, a well recognized anergic pattern. Incubation for 24h in vitro led to partial restoration of IgM Ca flux and some improvement in IgD Ca flux. This was impaired by treatment with anti-IgD or IgM F(ab)2 fragments, mimicking antigen exposure, and in keeping with a model of CLL cells engaging autoantigen in vivo. Further support for the pro-survival role of the BCR in CLL was demonstrated by the finding that both IgD and IgM ligation was associated with reduced apoptosis in vitro, with a significant decrease in apoptosis with IgD ligation as compared to IgM.To examine the mechanistic differences of signaling via IgM and IgD further, we used high-throughput mass-spectrometry based phosphoproteomics. This allows analysis of multiple active signaling pathways without a priori knowledge of which pathways to investigate. 6 CLL samples were compared to 5 tonsil controls. 4,575 unique phosphopeptides were identified using MASCOT proteomics software and quantified using a label-free technique based on extracted ion currents. 174 phosphoproteins (p<0.001, fold change up to >4000-fold) were over-expressed in CLL relative to healthy B-cells. These included components of RNA processing complexes, cytoskeletal regulators and MAPK signaling pathway components. Kinase prediction based on phosphoprotein substrates confirmed activation of kinases known to be active in CLL (such as AKT1, ERK1/2, CK2), but several novel kinases (such as CaMK1, CRIK, ROCK1 and BCKDK) were also active in CLL relative to healthy controls. Evaluation of differentially expressed phosphoproteins after BCR ligation included components of the spliceosome, regulators of the cytoskeleton, as well as known BCR signaling components. BCR-induced kinase activities included mTOR, CDK family members, MAPKs, BCKDK and others. There was much overlap between kinases active after IgM and IgD ligation, but also marked differences in CLL and tonsil BCR signaling.CONCLUSIONSAnergic IgM signaling is contrasted with IgD as a dynamic and plastic process that appears different in the LN and PB compartments in CLL. Mass-spectrometry based phosphoproteomics offers a powerful tool for interrogating intracellular signaling, with networks of phosphorylation characterizing the topology of pathways. BCR signaling in healthy B-cells has not previously been studied using this approach and comparisons with CLL highlight known pathways as well as suggesting novel treatment targets. The ultimate goal is to identify kinases active in CLL that will provide rational and effective drug combinations. Disclosures:Gribben:Celgene: Honoraria; Roche: Honoraria; Pharmacyclics: Honoraria; GSK: Honoraria; Mundipharma: Honoraria; Gilead: Honoraria.

Monoclonal Antibody Against ROR1 in Chronic Lymphocytic Leukemia Cells Induced Apoptosis Via PI3-Kinase/AKT/CREB Pathway

AbstractAbstract 1769 Background:Phosphoinositide 3-kinase (PI3K)/AKT cascade regulates cell survival, proliferation and differentiation in a variety of cells. In CLL cells PI3K pathway is constitutively activated leading to AKT activation and phosphorylation of cAMP response element-binding protein (CREB). CREB is a transcription factor overexpressed and constitutively phosphorylated in a variety of cancers and seems to have a role in tumor pathobiology. There is a great need to develop novel strategies for targeted therapy in CLL. Monoclonal antibodies (mAbs) specifically targeting leukemic cells might be a rewarding approach. ROR1 is a type I transmembrane receptor tyrosine kinase belonging to one of the twenty families of receptor tyrosine kinases (RTKs). ROR1 is overexpressed on CLL cells but not in white blood cells of healthy donors. ROR1 is constitutively phosphorylated in CLL and siRNA transfection induced apoptosis. We have developed a unique anti-ROR1 mAb directed against CRD (cysteine-rich domain) of the extracellular region of ROR1 capable of inducing direct apoptosis of primary CLL cells. Our anti-CRD mAb induced dephosphorylation of the ROR1 molecule. Aims:To study the apoptotic effect of an anti-ROR1 CRD mAb and effects on downstream signaling pathways involved in CLL, specially the PI3-kinase/AKT/CREB pathway using primary CLL cells. Methods:Using a peptide-based mouse mAb generation method we produced several mAbs against the three extracellular domains of ROR1. In the current study we used one of the best anti-ROR1 antibodies, an anti-CRD mAb raised against the CRD region of ROR1 (Daneshmanesh et al., Leukemia. 2012 Jun;26(6):1348-55). Flow cytometry was used for surface staining of ROR1. Primary CLL cells were incubated with the anti-ROR1 CRD mAb and apoptosis was detected by the MTT assay and Annexin V/propidium iodide (flow cytometry) methods in a 24 h assay. Antibody untreated and treated cell lysates were prepared and subjected to Western blot analysis for identification of signaling molecules involved in apoptosis induced by the anti-ROR1 CRD mAb. We analysed total and phosphorylated levels of the following signaling proteins: AKT, p-AKT, PI3K, p-PI3K, CREB, p-CREB, ERK, p-ERK, PKC and p-PKC. Phosphoproteins were measured before incubation with the mAb and after 20 min-2 h. Results:ROR1 surface expression was detected on 80–85% of the CLL cells. The frequency of apoptotic cells induced by the anti-CRD mAb was in the range of 45–50% which is in accordance with our previous reports (see above). Time kinetics experiments using anti-ROR1 CRD mAb incubated with primary CLL cells revealed dephosphorylation of ROR1 downstream signaling molecules. We analysed the following molecules known to be involved in CLL: PKC, PI3-kinase and ERK1/2. After co-culturing CLL cells with the anti-ROR1 CRD mAb, Western blot analysis showed decreased level of phosphorylated AKT in treated compared to untreated samples. No changes in the phosphorylation levels of ERK1/2 and PKC proteins were seen. Furthermore, we analysed the PI3-kinase protein which is upstream of AKT, and noticed that in CLL cells treated with the anti-ROR1 CRD mAb, the phosphorylation intensity of PI3-kinase p85 isoform has decreased but not p55 isoforrn. Moreover, we also studied the CREB phosphorylation in treated and untreated CLL samples and detected dephosphorylation of CREB in treated as compared to untreated samples. Conclusion:Incubation of CLL cells with an anti-ROR1 CRD mAb induced apoptosis of primary CLL cells. Apoptosis was preceded by dephosphorylation within 2 h of PI3-kinase, AKT and CREB proteins indicating deactivation of these signaling proteins by the anti-ROR1 mab. In untreated CLL cells no effect on phosphorylation of these proteins was noted. Furthermore our ROR1 mAb did not dephosphorylate PKC or ERK. Our data may suggest that activation of CREB molecule might occur via the PI3K/AKT pathway and may be a survival signal in CLL cells associated with the aberrant expression of ROR1. The constitutive phosphorylation of PKC and ERK1/2 seen in CLL might not be related to the overexpression of ROR1. Further studies are warranted for a better understanding of signaling pathways associated with ROR1 and the downstream signaling effects of ROR1 targeting drugs. Disclosures:No relevant conflicts of interest to declare.

GSK-3 Spatial Regulation Has an Essential Role in Lenalidomide Activity and Represents a Biomarker of Clinical Response in CLL

AbstractAbstract 1793Thalidomide (THAL) and IMID® immunomodulary drugs lenalidomide (LEN) and (POM) have proven beneficial in the treatment of a variety of hematological malignancies. Pre-clinical studies demonstrate multiple direct and indirect anti-tumor activities including anti-angiogeneic, proapoptotic, anti-proliferative and immunomodulatory effects. Recent studies have identified cerebron (CRBN) as a potential direct physical target of THAL and IMiD compounds and CRBN expression is reportedly required for IMiD compound activity. However, the precise link between CRBN and IMiD compound mechanism of action (MOA) have not been clearly defined. We applied Drosophila as a drug discovery platform to assess the MOA of THAL and IMiD compounds in vivo. THAL or POM fed Drosophila demonstrate morphological phenotypes that replicate wingless (wg) mutants indicating that drugs are inhibitors of Wg/Wnt signaling. In this model system, THAL and IMiD compounds disrupt membrane localization of GSK-3 indicating that the bioactivity of IMiD compounds is achieved through spatial regulation and potentiation of Sgg/GSK-3 in Drosophila. Using epistasis analysis we show that Drosophila expressing genetic mutants lacking GSK-3 activity and myeloma cells in which GSK-3a and GSK-3b have been knocked down by siRNA fail to respond to POM. In both Drosophila and myeloma cells therefore it appears that GSK-3 activity is required for biological responses. To test the clinical validity of GSK-3 as a biomarker, we obtained patient tumor samples from a Phase II clinical trial of single agent LEN for previously untreated CLL (Chen CI et al., J Clin Oncol, 2011; 29:1175). Twenty five patients were enrolled onto this study and received LEN at a starting dose of 2.5 mg days 1–21 of a 28 day cycle with monthly escalation to a target dose of 10 mg. The primary clinical endpoint for the trial was objective response to lenalidomide (complete response (CR) and partial response (PR)) evaluated as defined in the revised 1996 NCI Working Group guidelines. Peripheral blood samples for correlative studies were collected on days 1 (pre-dosing) and 8 of cycles 1 and 2. CRBN expression was evaluated by gene expression profiling and Western blot and found to be uniformly expressed in all 19 evaluable day 1 patient samples regardless of LEN response. Thus CRBN expression does not appear to be a useful predictive biomarker of response in this population of previously untreated patients. However, GSK-3 localization was correlated with response. Paired analysis of CD19 selected CLL cells comparing day 1 vs day 8 revealed focal membrane localization of GSK-3 on day 1 and subcellular redistribution on day 8 in 11 out of 12 evaluable responders (PR or better). By contrast, in the CLL cells from all 6 evaluable non-responders, GSK-3 expression appeared diffusely distributed before and after treatment. In preliminary studies using confocal immunofluorescence microscopy we determined that CRBN and GSK-3 co-localize in day 1 CLL samples of responders but not in those of non-responders. In summary, our results indicate that THAL and the IMiD compounds target GSK-3 function by spatial regulation and identify GSK-3 localization as a potential clinical biomarker of IMiD response. Disclosures:Trudel:Celgene: Honoraria; GlaxoSmithKline: Research Funding; Janssen: Honoraria. Mercurio:Celgene: Equity Ownership, Research Funding. Lopez-Girona:Celgene Corp: Employment, Equity Ownership. Gaidarova:Celgene Corp: Employment, Equity Ownership. Webb:Celgene: Employment, Equity Ownership. Chen:Johnson & Johnson: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; GlaxoSmithKline: Research Funding; Lundbeck: Consultancy. Stewart:Millenium: Consultancy, Honoraria, Research Funding; Onyx: Consultancy; Celgene: Consultancy. Manoukian:Celgene: Research Funding.

The Common Deleted Region in the Short ARM of Chromosome 17 in CLL Includes the MAP2K4 GENE

AbstractAbstract 4562As part of a preliminary study, we show the advantages of whole genome screening of CLL patient samples and highlight several issues. Firstly, 17p rearrangements appear to be far more complex than a simple loss of TP53; secondly, commercially available TP53 FISH probe sets may miss important genetic events for a significant number of CLL patients. Finally we show the possible role of the tumour suppressor MAP2K4 in the pathogenesis of CLL.Specific genome aberrations are recognised prognostic factors in CLL. FISH based genome risk stratifications have been used in clinical decision-making for over a decade. However molecular karyotyping is gaining acceptance as an alternative that not only overcomes the limitations of FISH but also provides a comprehensive whole genome scan. Indeed, several recent studies (Rinaldi et al., BJH 2011, Parker et al., Leukemia 2011, Quilette et al, Blood 2011) using different array platforms have revealed novel clinically relevant cryptic genome aberrations. Here we compare FISH and molecular karyotyping data of 50 diagnostic and 26 follow up samples from CLL patients.Since the loss of the TP53 gene was associated with resistance to chemotherapy, a short survival and a poor prognosis (Dohner et al, NEJM, 2000) CLL patients are routinely screened by FISH to assess its presence. Here we present 6 cases out of the 76 (5 presentation and 1 follow up), that have a deletion of the short arm of chromosome 17 (17p) by whole genome scanning with the 8×60K Agilent array platform. In 4 of the 6 cases the genome loss involved nearly the entire 17p and as expected, they were shown to carry TP53 deletion, when tested by commercial FISH probes. The remaining two cases were found to carry much smaller deletions of 17p. In one of these aCGH found a 5Mb loss within the 17p13.1 cytoband, at address chr17: 10.2–15.2. This region houses many genes, including MAP2K4, but does not include the TP53 (genome address hg19 chr17: 7.57–7.59), although FISH with commercial probes indicated TP53 loss. In the second case, the aCGH analysis identified a cryptic 120Kb loss housing the entire MAP2K4 at 17p12. As expected, commercial FISH probe analysis failed to detect any TP53 changes. However, FISH screening with BACs probes RP11–170H18 (covering the MAP2K4) and RP11–89D11 (TP53) confirmed the array results in both cases.The tumour suppressor gene MAP2K4 (mitogen-activated protein kinase kinase 4) was revealed by our aCGH study to represent the common deleted region in all cases with 17p loss. It was identified in 6 out of 76 CLL samples (8%) by the 60K genomic arrays. MAP2K4 is a putative tumor suppressor gene frequently found to be deleted in various cancer types, including solid tumors, ovarian and breast cancer (Teng DH, et al Cancer Res 1997, Davis SJ, et al BMC Cancer 2011), but to our knowledge has not been described in hematological malignancy. Further work is required to assess the frequency, type and clinical relevance of the genetic aberrations within the short arm of chromosome 17 in CLL patients. Disclosures:No relevant conflicts of interest to declare.

The Gold Compound Auranofin Induces Oxidative Stress and Apoptosis in Primary CLL Cells Independent of Classic Prognostic Markers and the Protective Effect of the Tissue Microenvironment

AbstractAbstract 865Chronic lymphocytic leukemia (CLL), remains incurable, except by allogeneic stem cell transplantation. While multiple chemotherapeutic options are available, relapse and eventually development of chemotherapy resistance are common. Drug resistance of CLL cells is mediated by genetic alterations including deletion of chromosomes 17p (p53 locus) and 11q (ATM locus), and by pro-survival signals emanating from the tissue microenvironment. Thus new therapies are needed that are active against tumor cells in protective tissue sites and those with adverse cytogenetic features. To identify possible new therapies for CLL, we conducted a high throughput screening assay of primary CLL cells exposed to 7 step-wise dilutions of each member of a 2,816 compound library (including FDA-approved drugs and known bio-actives from commercial suppliers). As a control for “unspecific” cytotoxicity, we used PBMCs from normal donors. Of 102 compounds efficacious against CLL cells of all 6 patients tested, 5 were not or minimally toxic in normal lymphocytes. One of these 5 compounds is Auranofin (AF), an FDA-approved oral disease-modifying anti-rheumatic drug.Next, we investigated the efficacy of AF against an expanded cohort of CLL samples (n=50) and investigated its mechanism of action. Samples were characterized for IGHV mutational status, ZAP70 expression, CD38 expression, and FISH cytogenetics. PBMCs were tested against serial dilutions of AF (final concentrations: 0.125–4μM) for 24 hours in flat bottom 96-well plates. MTS assay was performed to quantify cell viability. A dose-dependent response was observed in all samples. The inhibitory concentration at 50% (IC50) was calculated using GraphPad Prism Software. For a majority of samples, the IC50 was <1μM, a concentration easily achieved in vivo. AF cytotoxicity was independent of the IGHV mutational status, ZAP70 and CD38 expression, and cytogenetic subgroups (including 17p and 11q deletion). To assess whether AF is selectively cytotoxic for CLL cells, we determined drug-induced apoptosis of PBMCs of CLL patients for CLL cells (identified as CD19+/CD3-; >95% of those are CLL cells) and T-cells (CD19-/CD3+) separately using Annexin V staining. AF reduced viability of T-cells on average by 10% and of CLL cells by 50% (n=8; P<.05 for comparison between T and CLL cells). To determine the effect of the microenvironment on AF toxicity, we cultured primary CLL cells in the presence or absence of Nurse-Like Cells (NLC), a model of the protective effect of the microenvironment. NCLs enhance CLL cell viability and confer resistance to chemotherapeutic agents such as fludarabine. However, co-culture on NLCs did not protect CLL cells from the cytotoxic effect of AF. To investigate the effect of AF on tumor biology, total RNA (2.5 μg) from CLL PBMCs treated for 4 and 10 hours with AF in vitro was profiled on Human Genome U133 Plus 2.0 arrays (Affymetrix) and compared to untreated controls. There were 81 genes whose expression changed >2-fold at P<.01, most prominently several genes encoding heat-shock proteins and antioxidant enzymes such as heme oxygenase and thiroredoxin reductase. In Ingenuity Pathway analysis, the most significantly upregulated pathway in response to AF was the NRF2-mediated Oxidative Stress Response (P<.001). Combined, this suggests that AF induced oxidative stress resulting in the upregulation of a detoxifying response. To study the effect of AF on redox balance we used dihydroethidium (DHE) and concomitantly measured cell viability using 3,3′-dihexyloxacarbocyanine iodide (DiOC6) by flow cytometry. AF induced a time- and dose-dependent increase in ROS production in CLL cells that correlated with the onset of apoptosis. In conclusion, AF is selectively cytotoxic for CLL cells and its activity is independent of classic mechanisms of chemotherapy resistance. Based on these observations AF is now being studied in a clinical proof of concept phase IIa clinical trial for patients with relapsed/refractory CLL (NCT01419691). Disclosures:No relevant conflicts of interest to declare.

Allosteric Akt Inhibitor MK2206 Synergizes with Bendamustine in Promoting the Apoptosis of Chronic Lymphocytic Leukemia Cells and Selectively Targets B-Cell Receptor Mediated Cytokine Production

AbstractAbstract 3928 Background:Accumulating data support the critical role of PI3K/Akt in CLL B cell receptor (BCR) mediated signal transduction, cell proliferation and survival. In addition recent preclinical and clinical studies indicate that specific PI3K blockade results in robust preclinical and clinical efficacy in CLL. In our model system of CLL B cell-stromal cultures which feature their interaction, platelet derived growth factor (PDGF) present in CLL culture medium drives VEGF production through PI3K/Akt activation in stromal cells (Blood. 2010. 116:2984). Indeed Akt was found to be activated in leukemic cells during the CLL-stroma interaction (Leuk Res. 2008. 32:1565). Therefore, we hypothesized that Akt inhibition should promote CLL B cell apoptosis and abrogate BCR mediated cytokine production. MK2206 is an orally bioavailable highly specific allosteric Akt inhibitor. It has been tested in patients with refractory solid tumors and was demonstrated to be safely administered in a phase I trial. Therefore the goal of this study was to test the preclinical efficacy of MK2206 on both the survival and the BCR mediated cytokine production of CLL leukemic B cells. Methods:Peripheral blood mononuclear cells isolated from CLL patients (n=37) were treated with escalating concentrations of MK2206 (1–16 μM) for 24 hours, 48 hours or 72 hours. The levels of leukemic B cell viability were tested using an (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) assay. The potential impact (antagonistic/additive/synergistic) of Bendamustine in combination with MK2206 was also tested by using the MTT assay. We used the Calcusyn system to calculate the effect of drug interactions. The combination index (CI) as calculated by the program usually indicates synergy when ≤ 0.8 and indicates additive outcomes when between 0.8–1.2. A CI >1.2 indicates antagonism. Downstream signals of Akt activation in CLL B cells were evaluated by testing their expression of Mcl-1, 4EBP1 and p70S6K using immunoblot. The impact of Akt inhibition by MK2206 on cytokine production in response to B-cell receptor ligation with anti-IgM was also tested using a multiplex cytokine analysis (Invitrogen) in a time-course experiment. Results:MK2206 treatment induced concentration- and time-dependent apoptosis in CLL leukemic cells. At 72 hours, the IC50 of MK2206 in the experiments using CLL leukemic cells in vitro is ∼8 mM. MK2206 incubation at 1 or 5 mM cultured with CLL B cells over a 48-hour period abolished of Akt and p70S6K phosphorylation while native PARP was cleaved into the 85 kD polypeptide fragment. However, the expression level of the upstream signal molecule, PI3K, was not changed. Among the CLL patients tested (n = 37), we did not find any difference in sensitivity to MK2206 induced apoptosis based on critical prognostic factors of CD38, ZAP-70, IGHV and del(17p) status. Importantly, we detected synergistic or additive activity between MK2206 and Bendamustine in 11 tested CLL samples when these combinations were used to treat CLL cells in vitro for 72hrs. Thus the median CI value for this group of patients was 0.8 (0.1 – 1.1). Six were found to have CI ≤ 0.8 and five fell within the additive CI values (0.8 – 1.2). Production of immune or chemotactic cytokines (e.g. CCL3, CCL4, MCP-1, IL-1Ra, IL-8 and IL-2R) at 24 hour incubation increased significantly above baseline when CLL cells were stimulated anti-IgM. Akt inhibition with MK2206 selectively abrogated upregulation of CCL3, CCL4, MCP-1 and IL-2R production, but not for IL-8 or IL-1Ra secretion. MK2206 also abolished BCR mediated Akt activation and decreased Erk activation. Conclusion:MK2206, a robust and selective Akt inhibitor, induced significant in vitro apoptosis of CLL B-cells in vitro. Preclinical evidence of a synergistic effect between MK2206 and Bendamustine was also observed independent of prognostic risk. MK2206 abolished BCR mediated Akt activation and selectively abrogates BCR mediated production of cytokines that may promote apoptotic resistance. These findings support the use of MK2206 in treating CLL and indeed we have initiated a phase I/II trial of MK2206 in combination with Bendamustine and Rituximab for relapsed CLL patients(N1087, October 2011). Acknowledgments:This study was funded by the NCI-K23, NCCTG and CLL Global Foundation. Disclosures:Shanafelt:Cephalon: Research Funding; Genentech: Research Funding. Kumar:Genzyme: Research Funding; Novartis: Research Funding; Celgene: Consultancy, Research Funding; Millennium: Research Funding; Merck: Consultancy, Honoraria. Kay:Celgene: Research Funding.

Abstract B10: Genome-wide profiling reveals hyper- and hypomethylation at microRNA promoters in chronic lymphocytic leukemia

Abstract Chronic lymphocytic leukemia (CLL) is the most common leukemia in the Western world. Dysregulation of microRNA (miRNA) expression contributes to CLL pathogenesis. Abnormal promoter methylation is involved in aberrant gene expression in leukemic cells. Here, we investigate the role of epigenetic mechanisms in the global regulation of miRNA expression in CLL. In order to characterize disease-specific aberrant methylation upstream of miRNAs, methylated DNA from 24 CLL patients and 10 healthy age-matched controls was enriched by Methyl-CpG immunoprecipitation. Differentially methylated genomic loci were identified using a custom tiling array covering the genomic sequences 35 kb upstream and 5 kb downstream of 939 annotated miRNAs. We identified putative promoter sequences for 781 miRNAs by determining regions of enriched trimethylated histone 3 lysine 4 (H3K4me3) in CLL samples, healthy B cells or cancer cell lines. The methylation patterns upstream of miRNAs clearly distinguished CLL cells from healthy B cells. Differentially methylated sequences are enriched in H3K4me3, suggesting that miRNA promoters are non-randomly targeted for epigenetic dysregulation. 458 miRNAs display differential methylation in at least 5 out of the 24 CLL samples. Noteworthy, hypomethylation accounted for two thirds of all differentially methylated regions at putative miRNA promoters. Hypomethylated regions are limited to strictly defined sequence stretches, predominantly found outside of CpG islands (89%). Correlating promoter methylation with miRNA expression, we observed loss of methylation and strong transcriptional upregulation of mir-155 in CLL. Similarly, the promoter of mir-1204 showed significantly reduced methylation in CLL associated with increased expression. Mir-129-2, a well known tumor suppressor in solid tumors, could be detected among the epigenetically silenced miRNAs. Furthermore mir-551b, which has not yet been described in the context of CLL, was found to be downregulated and a target of promoter hypermethylation in CLL cells. In conclusion, a combination of DNA methylation profiling and comprehensive miRNA promoter identification has allowed us to identified novel aberrantly regulated miRNAs in CLL. Hypomethylation, which has previously been underestimated, is determined to be a major mechanism for miRNA activation in CLL. Citation Format: Constance Baer, Michael Rehli, John C. Byrd, Clemens-Martin Wendtner, Christoph Plass, Rainer Claus, Lukas Frenzel, Manuela Zucknick, Yoon Jung Park, Lei Gu, Dieter Weichenhan, Martina Fischer, Christian Pallasch. Genome-wide profiling reveals hyper- and hypomethylation at microRNA promoters in chronic lymphocytic leukemia [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer; 2012 Jan 8-11; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(2 Suppl):Abstract nr B10.

C-Mpl Is Not Expressed or Functional At Detectable Levels on Primary Chronic Lymphocytic Leukemia (CLL) Tumor Samples

Abstract 2849The development of thrombocytopenia is associated with progression of CLL to a more advanced stage and is commonly used to classify CLL into the higher disease risk categories. It has been estimated that 5% of CLL patients will present with associated immune thrombocytopenia purpura (ITP) and immune mediated destruction can exacerbate thrombocytopenia related to bone marrow infiltration. Thromopoietin (TPO) mimetics stimulate platelet production though activation of the thrombopoietin receptor (c-Mpl) on megakaryocytes and are currently indicated for chronic ITP. In principle, this approach could be considered for treatment of ITP in CLL patients. In order to rule out the possibility that c-Mpl is functionally expressed in CLL tumor cells; we analyzed expression and TPO-dependent function in primary patient samples.We have developed assays for the analysis of c-Mpl expression on the surface of B-cells in CLL patient samples and to assess functional response to ex-vivo stimulation of theses primary samples with TPO. Peripheral blood was collected from CLL patients and mononuclear cells isolated by Ficoll separation. This cohort included treatment naïve CLL samples (n=57) and CLL samples collected from subjects undergoing active treatment (n=30). CLL cells were analyzed by flow cytometry and identified based upon viability and CD5+/CD19+ expression. c-Mpl expression was measured using a novel c-Mpl-specific monoclonal antibody that was shown to be specific for c-Mpl by flow cytometry. Residual platelets associated with individual PBMC aliquot served as an internal control for c-Mpl expression. To investigate c-Mpl function, CLL cells were stimulated with rhTPO (10 ng/mL for 10 min) and induction of pSTAT5 was measured to assess a functional response to ex-vivo stimulation of TPO. In addition c-Mpl mRNA expression was surveyed in CLL by using mRNA microarray analyses to correlate c-Mpl mRNA expression with protein and functional expression of c-Mpl.Robust c-Mpl protein expression was observed in platelets from normal and CLL patients (fold over control -normal: 31.90 ± 6.39, CLL: 26.76 ± 6.57; mean ± 95%CI), no significant expression of c-Mpl was observed on CD5+/CD19+ CLL cells (fold over control −1.06 ± 0.021; mean ± 95%CI). No induction of STAT5 phosphorylation was detected in B-cell CLL cells in any of the samples stimulated with TPO (fold over control - normal: 0.90 ± 0.02, CLL: 1.04 ± 0.034; mean ± 95%CI). Microarray analysis of the CLL samples demonstrated c-Mpl mRNA expression levels equivalent to background across all CLL samples analyzed (intensity score −10.3 ± 3.0; mean ± 95%CI). In conclusion, we demonstrated a lack of cell surface c-Mpl protein expression in CLL cells. The additional data suggesting the lack of evidence for significant expression of c-Mpl mRNA expression supports the hypothesis that CLL cells do not express c-Mpl and unlikely to be stimulated in patients treated with TPO mimetics. This hypothesis will need to be tested in an appropriate clinical trial to assess the potential benefits of TPO mimetics for the treatment of thrombocytopenia in CLL patients. Disclosures:Loberg:Amgen: Employment, Equity Ownership. Wang:Amgen: Employment, Equity Ownership. Patterson:Amgen: Employment, Equity Ownership. McCaffery:Amgen: Employment, Equity Ownership.

B-Cell Receptor-Mediated Glucosylceramide Synthesis Protects Primary CLL Cells From Ceramide-Dependent Apoptosis

Abstract 1766 Introduction:Survival of CLL cells is triggered by the B-cell receptor (BCR). However, little is known about metabolic processes, which are influenced by the BCR and which are essential for survival of malignant cells such as sphingolipid metabolism. Certain sphingolipids are considered as bioeffector signaling molecules since they regulate several pathways involved in cell metabolism and survival (e.g. mitochondria). For instance ceramide, as the central molecule in sphingolipid metabolism, contributes to apoptosis and growth inhibition. In contrast, glucosylceramide, generated out of ceramide, is responsible for proliferative attributes such as resistance to apoptosis and to several chemotherapeutics. We therefore investigated the role of sphingolipid metabolism in survival and apoptosis-resistance of CLL cells. Methods and Results: We performed liquid chromatography electrospray ionization tandem mass spectrometry of 8 CLL samples in order to determine sphingolipid levels. Prior analysis, cells were either incubated with anti-IgM immunobeads for 24h or were left native. IgM stimulation significantly increased survival of primary CLL cells (n=9; p=0.0246) shown by flow cytometry. Our mass spectrometric analysis revealed a significant decrease of apoptosis-inducing ceramide in BCR-stimulated CLL cells compared to native controls (16:0 p<0.0001, 22:0 p=0.0325, 24:0 p<0.0001, 24:1 p=0.0010). Simultaneously, glucosylceramide synthesis was significantly increased after BCR engagement pointing out its pro-survival effect (16:0 p=0.0004, 18:0 p=0.0343, 24:1 p=0.0012, 26:1 p=0.0027). The total amount of ceramide and glucosylceramide did not change after IgM stimulation. Most importantly, the ratio between pro-apoptotic ceramide and pro-survival glucosylceramide became almost completely reverted towards glucosylceramide after IgM stimulation. Via PCR, we could identify the enzyme UDP-glucose ceramide glucosyltransferase (UGCG) to catalyze the synthesis of glucosylceramide out of ceramide after BCR engagement (p=0.0126). In order to investigate the functional impact of this observation, we tested whether inhibition of UGCG (UGCGi) in combination with a ceramide-inducing drug might lead to increased apoptosis during IgM stimulation. Thereby, we identified ABT-737 as agent that induces apoptosis through up-regulation of ceramide. As UGCG enzyme inhibitor, we used N-(n-Butyl)deoxygalactonojirimycin (OGB-1) and N-(n-Nonyl)deoxygalactonojirimycin (OGB-2). While IgM stimulation protected CLL cells partly from ABT-737-induced apoptosis as determined by AnnexinV-7AAD and JC-1 staining (mitochondrial outer membrane permeabilization) and subsequent flow cytometry, UGCGi reverted this effect leading to a significantly higher amount of apoptotic cells (n=9; p=0.0021). In order to prove that ABT-737-induced apoptosis influenced the ratio of ceramide:glucosylceramide in primary CLL cells, we performed additional mass spectrometric analyses. Most importantly, we could show that UGCGi reverted the ratio between ceramide:glucosylceramide towards ceramide after IgM stimulation. Protection from ABT-737 by IgM stimulation was also measurable by glucosylceramide-dominated ratio. Finally, inhibition of UGCG during IgM stimulation and ABT-737 treatment resulted in higher apoptosis accompanied by ceramide-dominated ratio. Conclusion: Here we identified how BCR engagement controls lipid metabolism and thereby survival and apoptosis-resistance of primary CLL cells. Our findings suggest that ceramide and glucosylceramide may be mediators of survival of CLL cells upon BCR stimulation. The ratio between ceramide and glucosylceramide seems to be crucial to induce resistance to apoptosis. This study provides potential targets for treatment of CLL beyond current concepts.C.M.W. and L.P.F contributed equally to this work. Disclosures:No relevant conflicts of interest to declare.

P53 Homolog TAp63 elicits apoptosis in Chronic Lymphocytic Leukemia (CLL) B-Cells,

Abstract 3894p63 is a p53 homolog whose function depends on the cellular context. The full-length TAp63 variant may carry an anti-oncogenic potential in solid tumor models, where it mediates Ras-induced cellular senescence, antagonizes tumorigenesis and suppresses the development of metastases. In hepatoma cells TAp63 is involved in activation of both extrinsic and intrinsic apoptosis pathways. By contrast, ΔNp63, an amino-terminally truncated p63 variant, is oncogenic in tumors of squamous cell origin. TAp63 is the predominantly expressed p63 isoform in lymphoid malignancies. Increased expression of TAp63 in diffuse large B-cell lymphoma confers an unfavorable prognosis. In CLL TAp63 was shown to mediate B-cell homing to the bone marrow, thus possibly contributing to apoptosis evasion. We studied expression of p63 in CLL and determined whether p63 plays a role in CLL B-cell survival and sensitivity to chemotherapy.We enrolled 25 previously untreated subjects with B-CLL at the Norris Cotton Cancer Center (Lebanon, NH). CLL B-cells were isolated from peripheral blood with standard Ficoll-Hypaque technique and purified using a B-cell (CLL) Isolation Kit. Small interfering RNA against p63 were delivered using Lonza Nucleofector with transfection efficiency of 20–50% and viability of 60–80% at 24 h. Viability was enhanced when cells were cocultured with “feeder cells”. To test sensitivity to chemotherapy, cells were treated with 10 μM chlorambucil and 5 μM fludarabine for 48–72 h. For apoptosis analysis cells were stained with Annexin V and 7-AAD and assayed by flow cytometry. Five lymph node tissues were analyzed for p63 expression by immunohistochemistry using a mouse monoclonal p63 antibody (clone 4A4).Of 25 CLL patients 15 were males (60%). Median age was 61 years. Median follow up was 3 years. Most patients presented in Rai stage 0–1 (92%). TAp63 mRNA transcripts were expressed in all CLL samples, while ΔNp63 variant was not detected. TAp63α was the predominantly expressed splicing variant. A C-terminally truncated TAp63γ variant was detected at a low level. TAp63 mRNA transcript levels were higher in CLL B-cells than in normal B-cells, but 10- to 100-fold lower than in high-grade lymphoma cell lines. Of five lymph nodes analyzed, weak nuclear p63 staining was detected in one. In this small cohort, p63 expression in the peripheral blood CLL B-cells did not correlate with ZAP-70 expression. CD38-positive (>30%) CLL samples expressed higher TAp63 mRNA transcript levels (p<0.05). siRNA knockdown of p63 in CLL cells resulted in protection from spontaneous apoptosis at 24–48 h, which persisted when cells were cultured on a “feeder layer”. Furthermore, p63 knockdown conferred protection against chlorambucil and fludarabine. B-cell receptor crosslinking with IgM resulted in a 2.5-fold repression of TAp63 transcription, consistent with the pro-survival role of a B-cell receptor in CLL.Thus, TAp63α is the predominantly expressed p63 variant in the peripheral blood CLL cells. TAp63a contributes to the intrinsic apoptosis program in CLL and may play a role in sensitizing CLL B-cells to standard chemotherapy drugs. Disclosures:No relevant conflicts of interest to declare.

Pro-Apoptotic Effect of An Anti-CD37 Scfv-Fc Fusion Protein, in Combination with the Anti-CD20 Antibody, Ofatumumab, on Tumor Cells From B-Cell Malignancies

Abstract 1662 Background:SMIP-016, a mouse/human chimeric SMIP™ (mono-specific protein therapeutic) targeting human CD37, was shown to have preclinical antitumor activity against B-cell lymphoid malignancies. SMIP-016 demonstrated synergistic or additive activity in non-Hodgkin’s lymphoma cells in combination with rituximab, rapamycin, doxorubicin and bendamustine. TRU-016, a humanized form of SMIP-016, is currently in phase 1 clinical trial in refractory or relapsed patients with CLL or small lymphocytic lymphoma. The aim: The goal of this study was to evaluate the antitumor activity of SMIP-016 against tumor cells from CLL and other B-cell lymphoid malignancies when combined with monoclonal anti-CD20 antibody, ofatumumab (OFA) or routine cytostatics. Material and Methods: Cytotoxic effects of SMIP-016 alone and in combinations with OFA or purine analogues, fludarabine (FA) or cladribine (2-chlodeoxyadenozine; 2-CDA), were assessed ex vivo in peripheral blood samples from 21 untreated CLL patients. Moreover, activity of SMIP-016 alone and combined with OFA was assessed in vitro on cell lines derived from B-cell lymphoblastic leukemia (NALM-6), Burkitt lymphoma (Raji) and diffuse large B-cell lymphoma (Toledo). Effects of that treatment on multiple myeloma (MM) cells, both in vitro (RPMI 8226 line) and ex vivo (bone marrow), were also studied. Cells were cultured with drugs for 24–72 hrs. SMIP-016 was used together with a secondary cross-linking Fc-specific IgG antibody (a-Fc). Antitumor effect was evaluated by the Annexin-V(Ann-V) flow cytometric assay. Apoptotic index (AI) was calculated as a percentage of Ann-V-positive cells. For assessing potential synergistic/additive effects of combinations the combination index (CI) was calculated. Additionally, mitochondrial potential and activation of caspases-8, -9 and -3 were assessed. Expression of several apoptosis-regulating proteins, including Bcl-2 protein and inhibitor of apoptosis protein (IAP) family, were investigated by flow cytometry and Western blot. Results: Both SMIP-016 and OFA were analyzed at the doses ranging from 0.1 to 100 mg/ml for in vitro studies. For ex vivo studies, 10mg/ml of SMIP-016 and 5mg/ml of OFA were chosen, as the lowest doses inducing significant pro-apoptotic effect compared to the control, whereas for in vitro studies, the dose of 1mg/ml of both SMIP-016 and OFA were used. 2-CdA and FA were used at concentrations 50ng/ml and 1mg/ml, respectively. After 48 hrs of incubation of ex vivo CLL cells with study drugs, SMIP-016 treated cells had median AI of 30.5% (vs. control – p<0.01) and OFA treated cells had median AI of 32.0%. Interestingly, in our experiments both SMIP-016 and OFA induced significant loss of mitochondrial potential, activation of caspases-9, -3, and overexpression of Bax protein. 2-CdA and FA induced median AI of 41.0% and 36.3%, respectively. Combination of SMIP-016 and OFA exerted additive effect (CI=0.95), whereas SMIP-016 combined with FA or 2-CdA showed synergistic interactions (CIs <0.8). In the in vitro models, SMIP-016 was significantly active against Raji (median AI 36.1%; vs. control p=0.007) and RPMI 8226 (median AI 40.5%; vs. control p<0.001), but did not affect NALM-6 or Toledo cell lines. In combination studies, SMIP-016 and OFA exerted a synergistic effect (median AI for Raji and RPMI 8226 cells - 58.0% and 70.2%, respectively; CI<0.8). Importantly, both agents induced cytotoxicity at very low concentrations which may decrease their potential side effects in clinical practice. Similar strong activity of these drug combinations were confirmed also in ex vivo cultures of MM patients cells. The mechanism of action for SMIP-016 cytotoxicity in all the examined models was likely apoptosis, triggered evidently by activation of both mitochondrial and external caspase pathways. Additionally, SMIP-016 induced drop of Bcl-2/Bax ratio (p<0.01), downregulation of cIAP1 (p<0.03) and overexpression of Smac/DIABLO (p<0.003) apoptosis-regulation proteins. Conclusion: Our study demonstrated strong pro-apoptotic activity of SMIP-016, especially in combination with CD20-targeting treatment in B cell tumor lines, CLL and MM patient samples. The data presented provides supporting evidence for further combination studies in preclinical models in vivo and may be a promising therapeutic concept for treatment of B-cell lymphoid malignancies, especially CLL, Burkitt lymphoma or MM. Disclosures:Stromatt:Emergent Product Development Seattle, LLC, Seattle, WA: Employment.

Heat-Shock Protein Signature Is Associated with Refractory Chronic Lymphocytic Leukemia Cells From Different In Vivo Microenvironments,

Abstract 3866Chronic Lymphocytic Leukemia (CLL) is a monoclonal B-cell disorder with accumulation of leukemic cells in peripheral blood, bone marrow and lymphoid organs. It presents with a heterogeneous clinical course. Many patients survive long periods of time without any need for treatment, whereas other patients show resistance to treatment or relapse soon after administration of therapy. Although some prognostic markers such as mutational status of immunoglobulin variable heavy chain, chromosomal abnormalities, CD38 levels, or ZAP-70 expression may help predict at initial diagnosis which patients will have more aggressive disease, the exact factors that can determine chances of remission in CLL are still not clear, making treatment challenging. Furthermore, CLL remains an incurable disease, necessitating a way for controlling its progression. Identifying novel molecular signatures associated with refractory CLL disease may help devise targeted treatment strategies and thus may prolong survival times and prevent the progression of CLL in relapsed patients. Considering this, we performed gene expression profiling (GEP) on peripheral blood (PB), bone marrow (BM) and lymph node (LN) samples collected at the time of diagnosis. We divided CLL samples into 3 groups based on their response to treatment; i) Stable CLL group: asymptomatic patients requiring no treatment, ii) Treated but stable CLL group: patients required treatment but had stable disease for at least one year after the end of the treatment cycle, and iii) Relapsed CLL: patients who relapsed within a year of end of the treatment cycle. Significance analysis of microarray (SAM) revealed that the heat-shock protein (HSP) signature (HSJ2, HSP70, HSP90, HSP60, HSP10, HSP 105, HSP40, HSP27, HSPA2, HSJ1, HSF4, HSPCA), BCR signaling pathway (JUN, NFATC4, NFKBIE, PPP3CB, TRAF3, CD81, CCT4), activation markers (CD81, CD83) and MMPs (MMP3, MMP9) were overexpressed in relapsed PB-CLL (n=3) compared to stable PB-CLL (n=6) and treated but stable PB-CLL (n=10). Overexpression of heat-shock protein signature genes were further observed in additional relapsed PB-CLL (n=6) group compared to other two PB-CLL (n=22) group. Interestingly, the HSP signature was consistently overexpressed in relapsed BM-CLL (n=6) and LN-CLL (n=12) compared to stable and treated but stable BM-CLL (n=11) and LN-CLL (n=3) groups. HSPs are considered chaperones of tumorigenesis and known to enhance survival, migration, and proliferation of tumor cells which may contribute to relapse in patients. Furthermore, the HSPs genes (HSP90 and HSP70) were significantly overexpressed in LN-CLL as compared to PB-CLL which implies important role of the microenviroment in rendering CLL refractory. To investigate the link between the expression of the individual genes with the aggressiveness of the disease, Kaplan-Meier log-rank tests were performed. We found that the higher expression of HSP90A, HSP90B, HSJ, and MMP9 were significantly (p<0.05) associated with shorter time to treatment. In summary, our study suggests that HSP genes are overexpressed in refractory CLL patients and thus are promising targets to improve clinical outcome. Disclosures:No relevant conflicts of interest to declare.

Th17/IL-17 Might Play a Protective Role in CLL Immunity

Abstract 2843 Background:Immune system disorders play an important role in the development and progression of CLL. Many deficiencies, contributing to disease progression, were found in T cell populations. Th17 cells represent a recently-discovered distinct lineage of helper T-cells, characterized by IL-17 secretion. IL-17 play an active role in inflammation and autoimmune diseases, however the role of IL-17 and Th17 cells in CLL immunopathogenesis remains undefined. We investigated IL-17A mRNA and IL-17A protein expression in peripheral blood (PB) CD4+ T cells and the plasma levels of IL-17 in 70 untreated patients with CLL in the context of clinical and immunological parameters. Th17 cell percentage was measured also in the bone marrow of CLL patients. The control group consisted of 20 healthy age-matched subjects. Materials and methods:Quantitative ‘real-time' reverse transcription-polymerase chain reaction (qRT-PCR) was used for the analysis of IL-17A mRNA in PB CD4+ T-cells. Intracellular IL-17A expression in CD3+/CD4+cells was analyzed using a three-color flow cytometry technique. Plasma levels of IL-17A, IL-4 and IL-10 were measured by ELISA. Results:None of the CD4+ T cells in the samples obtained from healthy volunteers (HV) contained detectable amounts of IL-17 mRNA, whereas it could be found in 5 out of 70 CLL samples. All CLL patients with detectable IL-17 mRNA in T-cells were in 0 Rai stage and negative both for ZAP-70 and CD38 expression. It is possible that IL-17 mRNA in these cells is expressed only for a short time thus evading detection by PCR, or that CLL cells contain low amounts of the IL-17 transcript and the RT-PCR method was not sufficiently sensitive to detect these small amounts. Frequently, in HV as well as in CLL patients, the percentage of CD4+ T cells with intracellular IL-17 expression in non-activation assays was lower than 1%, comparable to the level of autofluorescence. Despite the fact that IL-17 mRNA was not detected in the T cells of the majority of CLL patients, IL-17 protein was present in T cells after PMA and ionomycin stimulation. We found a significantly higher median percentage of CD4+/CD3+/IL-17+ cells in CLL patients (18.2%) than in HV (2.9%) (p<0.01). Median percentage of Th17 cells was significantly higher in CLL patients in stages 0–1 according to Rai, as compared with stages 2–4 (36.2% vs 4.5%, p<0.05). There were no differences in Th17 percentages between the PB and BM of CLL patients. Accordingly, the IL-17 plasma level was significantly higher in CLL patients than in HV (101.9 pg/ml vs 75.6 pg/ml; p<0.05) and there was a significant correlation between Th17 cell percentage and IL-17 plasma level. There was also significant correlation between the percentages of Th17 and iNKT cells (R=0.56; p<0.05), and inverse correlations between the percentage of Th17 and: Treg cells (R=−0.28; p<0.05), CD4+IL-4+ (R=−0.55; p<0.05) and CD4+TNF+ T cells (R=−0.59; p<0.01). The percentage of Th17 inversely correlated with β2-microglobulin serum level (R=−0.29; p<0.05) and the expression of ZAP-70 (R=−0.244; p<0.05) and CD38 (R=−0.33; p<0.01). The plasma level of IL-17 inversely correlated with the stage of disease (R=−0.23; p<0.05), IL-10 (R=−0.56; p<0.01) and TNF (R=−0.66; p<0.01) plasma levels, CD38 antigen (R=−0.25; p<0.05) and ZAP-70 expression (R=−0.28; p<0.05). In patients requiring therapy during the observation period, the median percentage of Th17 cells and median plasma IL-17 level were significantly lower comparing to untreated ones (5.7% vs 23.1%, p<0.05; 53.8 vs 95.6 pg/ml, p<0.05; respectively). Moreover, median plasma IL-17A level correlated with the time from CLL diagnosis to the start of therapy (R=0.49; p<0.01). Conclusions: both the Th17 cell percentage, and the IL-17 plasma level are significantly higher in the PB of untreated CLL patients compared with the HV, and IL-17 mRNA expression was found only in the T cell of CLL patients, but not in the control group. Correlations found with disease activity parameters, the subsets of T cells involved in tumor surveillance, and also with the length of time from diagnosis to the start of therapy, suggest an important protective role for Th17 cells in CLL immunity. Disclosures:No relevant conflicts of interest to declare.

Overexpression of the Autoimmunity-Associated Tyrosine Phosphatase PTPN22 in CLL Cells Enhances Antiapoptotic B-Cell Receptor Signals Through Selective Activation of the AKT Pathway

Abstract 619The phosphatase PTPN22 is an important negative regulator of T cell receptor (TCR) signaling. A gain of function polymorphism in this phosphatase is strongly linked with increased risk for the development of several common autoimmune diseases, including rheumatoid arthritis, insulin-dependent diabetes mellitus and systemic lupus erythematosus. The exact mechanism how the more active PTPN22 variant contributes to the development of these autoimmune diseases is still unclear, but it is has been postulated that increased activity of this enzyme may prevent negative selection of autoreactive T cells by blocking proapoptotic TCR signals induced by autoantigen.We now report that PTPN22 is significantly overexpressed in CLL B cells. Of the 91 investigated CLL samples, in 66 (73%) the levels of PTPN22 were at least 2 fold higher and often exceeded by 10 fold the levels in normal tonsilar B cells (CLL, mean PTPN22 levels 5.7, SD +/−4.3; normal tonsilar B cells, mean PTPN22 levels 0.9, SD +/−0.1). PTPN22 levels were somewhat higher in CD38−positive than CD38−negative cases (P=0.031), whereas no association was observed with IGHV mutation status, ZAP-70 expression and time to treatment.To determine the functional consequences of PTPN22 overexpression, we performed RNA interference experiments in primary CLL B cells with high PTPN22. We evaluated the effects of PTPN22 silencing in unstimulated CLL cells as well as CLL cells stimulated through the B cell receptor (BCR), given the important role of the BCR signaling pathway in the pathophysiology of CLL. Both soluble and immobilized anti-IgM antibodies were used for BCR stimulation, as these two BCR crosslinking agents have opposing effects on leukemic cell survival.Silencing of PTPN22 had no effect on the viability of unstimulated CLL cells (n=11, viable CLL cells: siControl 49%, siPTPN22 48%, P=n.s.), but significantly inhibited the antiapoptotic effect of immobilized anti-IgM (viable CLL cells: siControl 55%, siPTPN22 48%, P=0.001) and enhanced the proapoptotic effect of soluble anti-IgM (viable CLL cells: siControl 44%, siPTPN22 31%, P<0.001). Consistent with these data, overexpression of PTPN22 in the lymphoma B cell line B104 significantly blocked apoptosis induced by soluble anti-IgM (control 22%, PTPN22 35%) without affecting the survival of unstimulated cells (control 52%, PTPN22 51%).The previous experiments suggested that PTPN22 functions as a molecular switch that enhances antiapoptotic and inhibits proapoptotic BCR signals. To establish the molecular mechanisms underlying these different activities, we studied BCR signal transduction in primary CLL cells transfected with control or PTPN22-specific siRNA. Downregulation of PTPN22 increased anti-IgM-induced activation of several BCR signaling molecules, including LYN, SYK, ERK, JNK and the proapoptotic p38MAPK, confirming that PTPN22 is primarily a negative regulator of BCR signaling. However, activation of the antiapoptotic AKT kinase was substantially reduced by silencing of PTPN22, as evidenced by decreased phosphorylation of AKT and its direct targets GSK3b and FOXO3a. Lack of PTPN22 expression was further associated with increased activity of the phosphatase SHIP, a key negative regulator of the AKT kinase. Opposite effects on the activity of these signaling molecules were observed when PTPN22 was overexpressed in B104 cells, thus validating the data obtained by PTPN22 knockdown in primary CLL cells.In summary, this study shows that the phosphatase PTPN22, which has been implicated in the pathogenesis of several common autoimmune diseases, is significantly overexpressed in CLL B-cells. Overexpression of PTPN22 inhibits activation of molecules that propagate the proapoptotic BCR signal, such as p38MAPK, but at the same time enhances the antiapoptotic signal delivered through the AKT kinase. The mechanism how PTPN22 enhances activation of AKT in response to BCR engagement is by preventing Lyn-mediated activation of the negative regulator SHIP. Collectively, these data show that PTPN22 is an important regulatory molecule in CLL and a potential therapeutic target. Inhibitors of this phosphatase, which are currently being developed for the treatment of autoimmune diseases associated with the gain of function PTPN22 variant, would be expected to convert the BCR survival signal into a death signal and may thus provide means for selective targeting of the malignant clone. Disclosures:No relevant conflicts of interest to declare.

Association of reactive oxygen species-mediated signal transduction with in vitro apoptosis sensitivity in chronic lymphocytic leukemia B cells.

BackgroundChronic lymphocytic leukemia (CLL) is a B cell malignancy with a variable clinical course and unpredictable response to therapeutic agents. Single cell network profiling (SCNP) utilizing flow cytometry measures alterations in signaling biology in the context of molecular changes occurring in malignancies. In this study SCNP was used to identify proteomic profiles associated with in vitro apoptotic responsiveness of CLL B cells to fludarabine, as a basis for ultimately linking these with clinical outcome.Methodology/Principal FindingSCNP was used to quantify modulated-signaling of B cell receptor (BCR) network proteins and in vitro F-ara-A mediated apoptosis in 23 CLL samples. Of the modulators studied the reactive oxygen species, hydrogen peroxide (H2O2), a known intracellular second messenger and a general tyrosine phosphatase inhibitor stratified CLL samples into two sub-groups based on the percentage of B cells in a CLL sample with increased phosphorylation of BCR network proteins. Separately, in the same patient samples, in vitro exposure to F-ara-A also identified two sub-groups with B cells showing competence or refractoriness to apoptotic induction. Statistical analysis showed that in vitro F-ara-A apoptotic proficiency was highly associated with the proficiency of CLL B cells to undergo H2O2-augmented signaling.Conclusions/SignificanceThis linkage in CLL B cells among the mechanisms governing chemotherapy-induced apoptosis increased signaling of BCR network proteins and a likely role of phosphatase activity suggests a means of stratifying patients for their response to F-ara-A based regimens. Future studies will examine the clinical applicability of these findings and also the utility of this approach in relating mechanism to function of therapeutic agents.

2.14 The Kinetics of Phosphorylation of the CD5 Regulatory Domain in Chronic Lymphocytic Leukemia B Cells Reflects Ongoing Activation of the B-Cell Receptor

gene transcription. Similarly, the ataxia telangiectasia mutated kinase (ATM), which senses double strand breaks, is required for DNA damage–induced activation of NFB in breast cancer cells. Furthermore, other reports showed that the DNA damage–signalling enzyme, DNA-dependent protein kinase (DNA-PK) activates NFB by regulating MEK/ERK signalling, the IKK complex, and the p50 subunit. We previously demonstrated that inhibition of DNA-PK chemosensitises CLL cells and that DNA-PK is highly expressed in CLL cells from patients with a poor prognosis. These data collectively led us to hypothesise that, like ATM, part of the DNA-PK–mediated DNA damage response involves regulation of NFB activity. First, we quantified p50, p65, p52, c-rel, and Rel-B subunits in 68 CLL samples (standardised enzyme-linked immunosorbent assay) to determine their role in disease progression and correlation with prognostic markers and resistance. High levels of p50 and p65 subunits correlated with shorter overall survival (hazard ratio 5.8 and 5.7, respectively) and identified patients harbouring del(17p) or del(11q). High levels of p52 and c-Rel had no significant effect on outcome, but in 17 cases of known ATM mutation status, Rel-B levels were significantly higher (p 0.005) in patients with ATM mutations. Increased NFB activity correlated with ex vivo chemoresistance (XTT assay) to fludarabine (p 0.001), chlorambucil (p 0.0001), and mitoxantrone (p 0.03). Of note, in 4 cases, drug-induced activity of the p50 and p65 NFB subunits in fresh CLL cells was abrogated by at least 50% by DNA-PK inhibition (using 1 M of NU7441), indicating that catalytic activity of DNAPKcs is required for drug-induced NFB signalling. In support of this concept, human glioblastoma cells deficient in DNA-PKcs (M059J) were 3-fold more sensitive to the NFB antagonist, BAY 11-7082, than the DNA-PKcs–proficient counterpart (M059J Fus1). These data indicate that in addition to its role in double strand break repair, DNA-PKcs can mediate chemosensitisation by decreasing NFB activity. Pilot data using chromatin immunoprecipitation indicate that NFB subunits are capable of binding to the promoter region of DNA-PKcs, and future studies using knockdown of DNA-PKcs will elucidate the role of DNA-PK on NFB– dependent gene transcription. Collectively, these data suggest that targeting DNA damage activated NFB activity represents a novel approach to overcoming chemoresistance in patients with CLL who have increased levels of the p65 and p50 NFB subunits.

2.16 Overexpression of the Autoimmunity-Associated Tyrosine Phosphatase PTPN22 in CLL Cells Enhances Antiapoptotic B-Cell Receptor Signals Through Selective Activation of the AKT Pathway

Abstract Abstract 619 The phosphatase PTPN22 is an important negative regulator of T cell receptor (TCR) signaling. A gain of function polymorphism in this phosphatase is strongly linked with increased risk for the development of several common autoimmune diseases, including rheumatoid arthritis, insulin-dependent diabetes mellitus and systemic lupus erythematosus. The exact mechanism how the more active PTPN22 variant contributes to the development of these autoimmune diseases is still unclear, but it is has been postulated that increased activity of this enzyme may prevent negative selection of autoreactive T cells by blocking proapoptotic TCR signals induced by autoantigen. We now report that PTPN22 is significantly overexpressed in CLL B cells. Of the 91 investigated CLL samples, in 66 (73%) the levels of PTPN22 were at least 2 fold higher and often exceeded by 10 fold the levels in normal tonsilar B cells (CLL, mean PTPN22 levels 5.7, SD +/−4.3; normal tonsilar B cells, mean PTPN22 levels 0.9, SD +/−0.1). PTPN22 levels were somewhat higher in CD38−positive than CD38−negative cases (P=0.031), whereas no association was observed with IGHV mutation status, ZAP-70 expression and time to treatment. To determine the functional consequences of PTPN22 overexpression, we performed RNA interference experiments in primary CLL B cells with high PTPN22. We evaluated the effects of PTPN22 silencing in unstimulated CLL cells as well as CLL cells stimulated through the B cell receptor (BCR), given the important role of the BCR signaling pathway in the pathophysiology of CLL. Both soluble and immobilized anti-IgM antibodies were used for BCR stimulation, as these two BCR crosslinking agents have opposing effects on leukemic cell survival. Silencing of PTPN22 had no effect on the viability of unstimulated CLL cells (n=11, viable CLL cells: siControl 49%, siPTPN22 48%, P=n.s.), but significantly inhibited the antiapoptotic effect of immobilized anti-IgM (viable CLL cells: siControl 55%, siPTPN22 48%, P=0.001) and enhanced the proapoptotic effect of soluble anti-IgM (viable CLL cells: siControl 44%, siPTPN22 31%, P The previous experiments suggested that PTPN22 functions as a molecular switch that enhances antiapoptotic and inhibits proapoptotic BCR signals. To establish the molecular mechanisms underlying these different activities, we studied BCR signal transduction in primary CLL cells transfected with control or PTPN22-specific siRNA. Downregulation of PTPN22 increased anti-IgM-induced activation of several BCR signaling molecules, including LYN, SYK, ERK, JNK and the proapoptotic p38MAPK, confirming that PTPN22 is primarily a negative regulator of BCR signaling. However, activation of the antiapoptotic AKT kinase was substantially reduced by silencing of PTPN22, as evidenced by decreased phosphorylation of AKT and its direct targets GSK3b and FOXO3a. Lack of PTPN22 expression was further associated with increased activity of the phosphatase SHIP, a key negative regulator of the AKT kinase. Opposite effects on the activity of these signaling molecules were observed when PTPN22 was overexpressed in B104 cells, thus validating the data obtained by PTPN22 knockdown in primary CLL cells. In summary, this study shows that the phosphatase PTPN22, which has been implicated in the pathogenesis of several common autoimmune diseases, is significantly overexpressed in CLL B-cells. Overexpression of PTPN22 inhibits activation of molecules that propagate the proapoptotic BCR signal, such as p38MAPK, but at the same time enhances the antiapoptotic signal delivered through the AKT kinase. The mechanism how PTPN22 enhances activation of AKT in response to BCR engagement is by preventing Lyn-mediated activation of the negative regulator SHIP. Collectively, these data show that PTPN22 is an important regulatory molecule in CLL and a potential therapeutic target. Inhibitors of this phosphatase, which are currently being developed for the treatment of autoimmune diseases associated with the gain of function PTPN22 variant, would be expected to convert the BCR survival signal into a death signal and may thus provide means for selective targeting of the malignant clone. Disclosures: No relevant conflicts of interest to declare.