CLL Cell Lines Research Articles

Human ROR1 Activates AKT and Accelerates Leukemia Cell Proliferation

AbstractAbstract 3872ROR1 is an orphan-receptor-tyrosine-kinase-like developmental surface-antigen that is expressed by embryonic cells, chronic lymphocytic leukemia (CLL) cells, and neoplastic cells of many other types of cancer, but not by virtually all normal adult tissues. ROR1 can serve as a receptor for Wnt5a and potentially complex with other surface receptors for growth/survival factors elaborated by the tumor microenvironment or expressed by the tumor itself. In prior studies, we and others found that silencing ROR1 could impair tumor-cell growth and/or survival in vitro and in vivo. Although the CLL cells of ≥96% of all examined patients (N= 800) expressed ROR1, we found that a B-cell line derived from CLL cells, namely MEC1 (Leuk Res 23:127, 1999), lacked expression of this protein. This provided us with an opportunity to assess how introduction of ROR1 could affect the biology of this CLL-cell line, which has relatively slow doubling-time of ≈40 hours. We transfected MEC1 cells with either an expression vector encoding human ROR1 or a control vector, and then selected for stable transfectants in selection medium. MEC1 cells transfected with the ROR1 vector expressed high-levels of ROR1, but not MEC1 cells transfected with the control vector. MEC1 cells made to express ROR1 had higher levels of phosphorylated and activated-AKT and activated cAMP response-element-binding (CREB) protein than non-transfected MEC1 cells or cells transfected with the control vector. Furthermore, MEC1 made to express ROR1 had significantly greater proportions of cells in S/G2/M phase than did control-transfected cells 16 hours after transfer from serum-free medium to complete growth medium, indicating that MEC1 cells made to express ROR1 had increased relative proportions of cells undergoing cell division. Consistent with this, we found that ROR1+ MEC1 cells had significantly shorter doubling times in culture than did comparably cultured parental MEC1 cells or MEC1 cells transfected with the control vector. This study indicates that expression of ROR1 in the MEC1 CLL cell line can activate AKT/CREB and accelerate leukemia cell growth, providing us with a model system with which to interrogate the function of ROR1 in vitro. Disclosures:No relevant conflicts of interest to declare.

Lymphocyte Cytosolic Protein 1 (LCP1) Is a Membrane Associated Molecular Target in Chronic Lymphocytic Leukemia and Is Activated in Microenvironment Signaling

Abstract 3866Chronic lymphocytic leukemia (CLL) is an incurable accumulation of malignant B-lymphocytes in peripheral circulation, which home to supportive leukemic microenvironments within the bone marrow, lymph nodes, and spleen. Membrane associated antigens are critical to the pathogenesis of CLL since they facilitate leukemic cell signaling, microenvironment homing, proliferation, and survival. Targeting CLL surface molecules and associated signaling patterns is a current focus of CLL therapeutic development with antibodies and vaccine-based approaches. Rituximab, ofatumumab, and alemtuzumab are widely used antibody-based therapeutics that are proven to be effective at targeting CLL membrane associated antigens. However, despite enhanced patient survival, targeting of non-obligate antigens such as CD20 ultimately leads to antibody resistance. In addition, the expression of CD52 across multiple healthy immune cells often produces unacceptable off-target toxicity. Targeting immunosurveilance antigens may provide a key to overcome these obstacles. A number of relevant tumor antigens such as p53, EGFR, WT1, BCR-Abl and CLL-specific ROR1 elicit robust autoimmune responses in cancer patients due to high levels of ectopic expression, high relative immunogenicity and obligate oncogenic signaling that forces expression despite autoimmune recognition. In many cases, autoantibody signatures were realized long after identification of the oncologic target antigens; however, the convergence of these two datasets implies that such signatures could be utilized to identify novel targets. To further explore this possibility, we interrogated anti-tumor humoral reactivity specifically directed against autologous CLL membrane antigens. Immunoreactive leads were identified by mass spectrometry. Potential membrane antigens were further confirmed using immunoblot and ELISA based techniques. Our analysis revealed Lymphocyte Cytosolic Protein 1 (LCP1), a membrane associated lymphocyte-specific target that is constitutively expressed on CLL and ectopically expressed in cancers of various histological subtypes. Subsequent confirmatory assays unveiled high frequency, robust, LCP1-specific IgG autoimmune responses in CLL patients despite a profound absence of reactivity to common viral and vaccine antigens, implying continued antigenic stimulation in an immunosuppressed host. LCP1 plays a critical role in B-cell biology by cross linking f-actin filaments, thereby solidifying cytoskeletal structures and providing a scaffold for CLL-critical signaling pathways such as PKC. In healthy B-cells phosphorylation of LCP1 at serine 5 is a key step in responding to CXCL12, a powerful lymph node and stromal cell chemokine. To further explore the role of LCP1 in CLL microenvironment recall signaling, we developed stable LCP1 knockdown and Ser-5 phosphomimetic CLL cell lines. Transwell assays confirmed that LCP1 activity contributed to the migration of CLL cells towards CXCL12. Our data reveals that LCP1 is a novel membrane associated target antigen in CLL with evidence of differential immune response as compared to common vaccine antigens. We also demonstrate that LCP1 plays a role in CLL microenvironment signaling. Future studies will focus on targeting specific moieties within LCP1 which are critical for its oncogenic signature. Disclosures:No relevant conflicts of interest to declare.

Vascular Endothelial Growth Factor (VEGF) Increases B-CLL CELL Migration Through Regulation of CXCR4/SDF1 AXIS

Abstract 3917 INTRODUCTIONVEGF is an important pro-angiogenic factor involved in survival and dissemination of chronic lymphocytic leukemia cells. At high concentrations such as those present in bone marrow niches, VEGF reduces MMP9 production and favours bone marrow retention. But on the other hand, it is well known that autologous VEGF and CD49d are required for B-CLL cell migration. In other cancer cell lines, this effect seems to be mediated by CXCR4/SDF-1 axis. In this work we studied the role of exogenous VEGF on B-CLL migration as well its relationship with the G-coupled proteins CXCR4, CXCR7 and CD49d. MATERIALS AND METHODSWe obtained peripheral blood mononuclear cells from 33 patients diagnosed of CLL according to established clinical and laboratory protocols at our institution after informed consent. We also used the CLL cell line Mec-1. B-lymphocytes were purified by Ficoll-Hypaque density gradient centrifugation anti-CD19 conjugated Dynabeads and stored in liquid nitrogen. Then we analysed by flow cytometry the expression of several chemokines and adhesion molecules, CXCR4, CXCR7 and CD49d before and after exogenous VEGF exposure (50 ng/mL), VEGF-R2 inhibitor (70 nM) or both. In order to evaluate the role of VEGF in the motility of these cells, we performed an in vitro 24-hours transmigration assay towards a media containing (or not) SDF-1. B-CLL (5 × 105 cells) cells were incubated on the upper chamber of transwell filters coated with human umbilical vein endothelial cells in the presence or absence (control) of vascular endothelial growth factor (VEGF) ([50 ng/ml], 24 hours), VEGFR2/KDR inhibitor [70nM] or both simultaneously. We quantified the migration ratio between those stimulated with VEGF compared with non-stimulated controls, inhibitor-treated cells versus control and both (R2-inh plus VEGF) versus inhibitor-treated cells. Migration ratio was given as mean ± SD. Statistical analysis was performed by non-parametric Wilcoxon test using SPSS statistical software (version 19.0). RESULTSBasal CXCR4, CXCR7 and CD49d expression levels of B-CLL cells were highly variable among the 33 patients analyzed. Mean fluorescent intensity (MFI) of CXCR4 expression was significantly higher on cells treated with VEGF versus untreated cells (average increase, 9.64 ± 95, p=0.028). However, we did not detect a significant difference in the percentage of cells expressing this receptor. On the other hand, VEGF treatment did not influence either the mean fluorescent intensity or the number of CXCR7 and CD49d expressing cells. Exposure to a VEGFR2 inhibitor reduced the percentage of cells expressing CXCR4 and CXCR7, suggesting a potential regulatory role of this receptor in the expression of these chemokines. Concerning B-CLL migration, we observed a significant increase in cell migration of cells treated with exogenous VEGF versus the control ones in both the Mec-1 cell line and the primary cells (27.66 ± 69.97 p= 0.03). Furthermore, the treatment with VEGFR2 inhibitor reduced significantly the migration index (−23.18 ± 33.5, p= 0.001) and the motility was restored by the addition of VEGF to the R2-inhibitor treated cells (36.04 ± 39.33, p=0.001). DISCUSSIONThis preliminary data suggest that VEGF seems to be involved in B-CLL migration through the regulation of CXCR4 expression levels. Detailed molecular mechanisms implicated in this process should be further studied. New therapeutic strategies focussed in blocking both the SDF1-CXCR4 axis and/or VEGF pathway could have a potential therapeutic implication by decreasing B-CLL cell migration into lymph nodes and bone marrow microenvironment. [Display omitted] 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.

Abstract 3027: Dishevelled proteins are highly upregulated in chronic lymphocytic leukemia

Abstract Chronic Lymphocytic Leukemia (CLL) is the most common adult leukemia and is characterized by the progressive accumulation of CD5+ B cells. An important signaling pathway in CLL cells might be Wnt signaling pathways. Wnt signaling pathways include the Wnt/ β-catenin pathway and the non-canonical Wnt pathways. Dishevelled proteins (DVL1, DVL2, and DVL3) are important mediators in Wnt signaling and have been shown in to be expressed many cancer types but their precise role is not clear. The aim of the study was to characterize the expression of DVL1, DVL2 and DVL3 proteins in indolent and progressive CLL and compare to normal white blood cells (PBMC) as well as to assess the expression of other important proteins of the canonical pathway as β-catenin and the non-canonical pathways as GSK-3β, PKC, ERK and AKT respectively. Leukemic cells from indolent (n=9) and progressive (n=9) CLL patients were isolated by Ficoll gradient centrifugation as well as PBMC from healthy donors (n=9). Six cell lines including four CLL cell lines EHEH, CII, I83, 232 B4, and the Jurkat and Lucas cell lines were also used. DVL 1, DVL 2, DVL 3 were analyzed by Western blot and densitometric calculations as well as PKC, GSK-3β, AKT and ERK using antibodies against the total protein and the phosphorylated protein respectively. Overexpression of DVL1, DVL 2, and DVL3 was seen in all indolent and progressive CLL patients and the six cell lines. There was a significantly higher expression of DVL 1 and DVL 3 in indolent compared to progressive CLL patients (p<0.01 and p<0.01 respectively). In contrast to DVL 1 and DVL 3, DVL 2 was highly expressed in indolent as compared to progressive CLL patients. ≤ -catenin followed the same expression pattern as DVL2 being highly expressed in indolent CLL as compared to progressive CLL patients (p<0.001). There was no significant difference in the degree phosphorylation of PKC and GSK-3β between indolent CLL, progressive CLL and healthy donors.The degree of AKT and ERK phosphorylation was significantly higher in indolent than in progressive CLL patients (p<0.001 and p<0.01 respectively). In summary DVL1, DVL2 and DVL3 are highly expressed in CLL patients and CLL cell lines but their expression in normal PBMC is almost negligible but expression of other canonical and non-canonical pathways proteins are seen both in CLL and healthy donors. DVL1, DVL2 and DVL3 may have a key role in the transduction of Wnt mediated signals in CLL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3027. doi:1538-7445.AM2012-3027

New Therapeutic Strategy for Sensitisation of CLL Cells with Inactivation of the DNA Damage Response by Targeting the Deubiquitylating Enzyme USP7-Dependent Pathways,

Abstract 3865Chronic Lymphocytic Leukaemia (CLL) is characterised by marked clinical heterogeneity and tumours with deletion or mutation of the TP53 and ATM genes on chromosomes 17p and 11q are associated with poor outcome. ATM is a protein kinase that, following induction of DNA double strand breaks (DSBs), phosphorylates a number of downstream targets and synchronises a network of cellular responses including p53 activation that leads to induction of pro-apoptotic genes and activation of apoptosis. Consequently, loss of integrity of ATM/p53 pathway results in apoptotic resistance, retention of cells with genomic damage, and tumour progression.Cellular p53 levels are regulated through a p53-Mdm2 regulatory loop whereby Mdm2, a ubiquitin ligase, facilitates p53 polyubiquitination and targeting of p53 for proteasome degradation. It was recently shown that stabilisation of p53 can be achieved by manipulation of this regulatory loop through use of small molecule inhibitors of this p53 degradation pathway, termed nutlins, which prevent p53 ubiquitination. More recently, a class of deubiquitinating enzymes (DUBs) highlighted an additional level of p53-Mdm2 regulation. In particular, a specific DUB, (USP7/HAUSP), has a high affinity for Mdm2 and functions by antagonizing Mdm2 ubiquitination. Unlike nutlins, USP7 also has been implicated in the regulation of cell cycle, mitosis and DNA damage response and we reasoned that USP7 inhibition may sensitise CLL tumours with ATM and TP53 defects.Our analysis of 25 primary CLL tumours with different ATM and TP53 status indicated that USP7 was robustly expressed in all CLL tumour cells tested. Through collaboration with Hybrigenics we obtained a specific USP7 inhibitor, HBX19818. To determine if cell killing could be induced in ATM or TP53 deficient tumours by inhibiting the USP7-Mdm2 pathway, we analysed the induction of cell death over a range of HBX19818 concentrations using both isogenic CLL cell lines with and without ATM and/or p53, as well as 18 primary CLL tumours. We observed a significant cytotoxic effect of HBX19818 in isogenic CLL lines, at concentrations between 1–10μM, irrespective of their ATM and TP53 status. Strikingly, the majority of primary CLL tumours were sensitive to HBX19818 concentrations between 8μM and 16μM to which non-tumour PBMCs were resistant.Western blotting was used to monitor the induction of p53, apoptosis (associated with caspase 7 and PARP1 cleavage), and also whether DNA damage was induced (as measured by H2AX phosphorylation) in response to HBX19818. Our analysis revealed that pharmacological inhibition of USP7 led to p53 upregulation in the p53 proficient CLL cells associated with a robust induction of p21 indicating that the stabilised p53 was active. This response was absent in Mec-1 cell line with non-functional p53. Interestingly, caspase 7 and PARP1 were only cleaved in the ATM wild type CLL cell lines suggesting that activated p53 was capable of inducing cell death. In contrast, the ATM and p53 deficient CLL cell lines did not exhibit any markers suggesting an apoptotic mode of cell death. Rather, these cell lines displayed elevated levels of phospho-H2AX, suggesting the induction of DNA damage, possibly caused by an underlying DNA repair defect. Consistent with our hypothesis, both p53 proficient and p53 non-functional CLL cell lines failed to induce the recruitment of the HR protein Rad51 to sites of IR-induced DNA double strand breaks.Taken together, our data implies that in addition to p53 activation, pharmacological inhibition of USP7 can exert a cytotoxic effect by further mechanisms, possibly by modulating DNA double strand break repair. This is consistent with previous reports suggesting that USP7 regulates monoubiquitination of transcription factor FOXO4 and is involved in the regulation of DNA repair and mitotic progression via its interactions with Claspin and Chfr respectively. We suggest that pharmacological inhibition of USP7 represents a promising target for the treatment of tumours with defective ATM and p53 signalling. Disclosures:No relevant conflicts of interest to declare.

In vitro and in vivo model of a novel immunotherapy approach for chronic lymphocytic leukemia by anti-CD23 chimeric antigen receptor

AbstractChronic lymphocytic leukemia (CLL) is characterized by an accumulation of mature CD19+CD5+CD20dim B lymphocytes that typically express the B-cell activation marker CD23. In the present study, we cloned and expressed in T lymphocytes a novel chimeric antigen receptor (CAR) targeting the CD23 antigen (CD23.CAR). CD23.CAR+ T cells showed specific cytotoxic activity against CD23+ tumor cell lines (average lysis 42%) and primary CD23+ CLL cells (average lysis 58%). This effect was obtained without significant toxicity against normal B lymphocytes, in contrast to CARs targeting CD19 or CD20 antigens, which are also expressed physiologically by normal B lymphocytes. Moreover, CLL-derived CD23.CAR+ T cells released inflammatory cytokines (1445-fold more TNF-β, 20-fold more TNF-α, and 4-fold more IFN-γ). IL-2 was also produced (average release 2681 pg/mL) and sustained the antigen-dependent proliferation of CD23.CAR+ T cells. Redirected T cells were also effective in vivo in a CLL Rag2−/−γc−/− xenograft mouse model. Compared with mice treated with control T cells, the infusion of CD23.CAR+ T cells resulted in a significant delay in the growth of the MEC-1 CLL cell line. These data suggest that CD23.CAR+ T cells represent a selective immunotherapy for the elimination of CD23+ leukemic cells in patients with CLL.

Abstract 4581: IMGN529 is an anti-CD37-maytansinoid conjugate with potent in vitro and in vivo activity against NHL and CLL cell lines

Abstract IMGN529 is an antibody-drug conjugate consisting of the functional anti-CD37 antibody K7153A conjugated by a non-cleavable SMCC linker to the highly cytotoxic maytansinoid, DM1. Expression of CD37 in normal tissues is mainly limited to B-cells in blood and lymphoid tissues such as spleen, tonsil and lymph nodes. To evaluate the potential for targeting CD37 in B-cell malignancies by IMGN529, CD37 expression was compared to that of CD20, a well-validated target for antibody-directed therapy in lymphoma. CD37 exhibited a similar prevalence as CD20 in subtypes of NHL such as follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), Burkitt's lymphoma (BL) and mantle cell lymphoma (MCL) as assessed by immunohistochemistry using tumor microarrays. In addition, CD37 and CD20 antigen levels were measured by quantitative flow cytometry in NHL and B-CLL cell lines. All of the CD20+ B-cell lines examined also expressed CD37 at antigen levels ranging from 17,000 to 360,000 ABC (antibodies bound per cell). The activity of IMGN529 and its antibody component (K7153A) were evaluated against NHL and CLL cell lines in vitro. K7153A was active against an array of NHL-derived lymphoma cell lines including the DLBCL cell line SU-DHL-4, the FL cell line DOHH-2, the MCL cell line Granta-519, as well as B-CLL cell lines such as JVM-2 and JVM-3. The antibody showed only minimal activity against the BL cell lines Daudi and BJAB. Importantly, IMGN529 demonstrated enhanced and specific cytotoxicity against these cell lines when compared to either unconjugated K7153A antibody or a non-targeted control conjugate (EC50 10 – 400 pM). In contrast, the anti-CD37 compound, TRU-016, had no direct effect on any of these cell lines. The anti-CD20 antibody rituximab showed less direct activity than K7153A in most of these cell lines despite the often higher CD20 expression levels. In vivo, while the K7153A antibody showed anti-tumor efficacy in established tumor xenograft models of DLBCL (SU-DHL-4), FL (DOHH-2) and B-CLL (JVM-3), IMGN529 was significantly more active in these models. In addition, IMGN529 was active against established BL-derived BJAB tumors at doses (5 or 10 mg/kg single dose) where the unconjugated antibody was not active. In the FL-derived DOHH-2 tumor model, a single treatment of IMGN529 at 10 mg/kg resulted in tumor growth delay that was similar to rituximab given at 5 mg/kg twice weekly for 3 weeks and was better than treatment with CVP (cyclophosphamide/vincristine/prednisone). A single 5 mg/kg dose of IMGN529 demonstrated better activity than the anti-CD20 antibody ofatumumab (5 mg/kg twice weekly × 3) or bendamustine (50 mg/kg x1) in the B-CLL-derived JVM-3 xenograft model. In conclusion, IMGN529 is highly active in vitro and in vivo against representative B-cell lines from NHL and CLL suggesting the utility of this functional antibody-maytansinoid conjugate as a therapeutic in these disease indications. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4581. doi:10.1158/1538-7445.AM2011-4581

Milatuzumab Improves Efficacy of Current Treatments for B-Cell Malignancies.

AbstractAbstract 2837 Introduction:Milatuzumab (Immunomedics, Inc.) is a humanized anti-CD74 monoclonal antibody in clinical evaluation for therapy of multiple myeloma, CLL, and NHL. CD74, the MHC class-II chaperone molecule, also functions as the cellular receptor for the proinflammatory cytokine, macrophage migration-inhibitory factor, and initiates a signaling cascade resulting in proliferation and survival. Preclinically, milatuzumab demonstrates therapeutic activity against various B-cell malignancies when used alone, and the therapeutic efficacies of bortezomib, doxorubicin, and dexamethasone are enhanced in multiple myeloma cell lines when given combined with milatuzumab. In addition, milatuzumab acts through distinct mechanisms from rituximab, and exhibits different expression and sensitivity profiles. Here we examine milatuzumab given in combination with rituximab or fludarabine in human NHL, CLL, and ALL cell lines. Methods:Three human NHL (WSU-FSCCL, Raji, and RL); two ALL (MN60 and REH), and two CLL (MEC-1 and WAC) cell lines were tested, with evaluation of therapeutic efficacies of milatuzumab and fludarabine performed in NHL and CLL cell lines. Results:Anti-proliferative activity was augmented in vitro when milatuzumab and rituximab were combined. For example in WSU-FSCCL cells, which are relatively insensitive to rituximab, inhibition of proliferation in the presence of 33.3 nM rituximab increased from 12.6±3.7% in the absence of milatuzumab to 85.5±0.0% (P=.023) in the presence of 33.3 nM milatuzumab. In Raji, a more sensitive cell line, inhibition of proliferation in the presence of 22.2 nM rituximab increased from 64.8±1.3% without milatuzumab to 86.6±0.9% (P=.018) with 22.2 nM milatuzumab. Significant increases in the anti-proliferative activity of rituximab were similarly observed in all but one of the tested NHL, CCL, and ALL cell lines, REH, which was not sensitive to killing by either milatuzumab or rituximab. Unlike rituximab, milatuzumab induces little or no ADCC or CDC. However, in vitro exposure of cells to milatuzumab does not affect rituximab mediated ADCC or CDC. Moreover, the combination of milatuzumab and rituximab was shown to result in a more potent decrease in the mitochondrial potential in rituximab-sensitive cell lines.In the 3 NHL and 2 CLL cell lines, it was found that milatuzumab increased the efficacy of fludarabine. For example, in Raji cells, which are relatively insensitive to fludarabine, inhibition of proliferation in the presence of 4 nM fludarabine increased from no inhibition in the absence of milatuzumab to 76.9±0.7% (P=.009) in the presence of 33.3 nM milatuzumab. In WSU-FSCCL cells, a more fludarabine-sensitive cell line, inhibition of proliferation in the presence of 0.8 nM fludarabine increased from 41.3±0.3% in the absence of milatuzumab to 79.7±0.1% (P<.0001) with 33.3 nM milatuzumab. Conclusions:Milatuzumab, a promising new therapeutic for B-cell malignancies as a naked antibody, can significantly add to the efficacy of currently approved therapies for these diseases, including fludarabine and rituximab. (Supported in part by USPHS grants P01-CA103985 and R01-CA109474 from the NIH and NJDHSS grant 07-1824-FS-N-0.) Disclosures:Goldenberg:Immunomedics, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

CLL Xenograft Models to Address the Impact of Single Gene Loss (ATM) on Proliferation and Cytotoxic Response

AbstractAbstract 2439Chronic lymphocytic leukaemia (CLL) is a malignancy characterised by gradual accumulation of mature B cells in peripheral lymphoid organs. This accumulation is a consequence of both prolonged survival but also active proliferation restricted to a small population of tumour cells. Access to the proliferating tumour population in CLL patients is limited and there is a need for establishment of human CLL xenograft models that can recapitulate human disease. Several xenograft models of CLL have been already established, both with primary tumours and CLL cell lines, and they exhibit either a limited length of engraftment or tissue distribution that only partially recapitulates CLL disease. Furthermore, there is an absence of CLL xenograft models that address the impact of a single gene loss that confers a poor clinical outcome. The purpose of this study was to a) establish a range of CLL xenografts that exhibit different kinetics and distribution of CLL engraftment b) develop individual xenograft models with isogenic CLL cell lines, with and without the principal DNA damage response gene ATM whose loss confers chemoresistance, c) obtain simultaneous engraftment of differentially labelled ATM null and ATM wild type isogenic CLL cells and d) demonstrate that such CLL xenograft models can be used to measure the cytotoxic impact of new treatments. Initially, we addressed the kinetics of engraftment and tissue distribution of the following CLL cell lines: MEC-1 (Stacchini A at al. Leuk Res. 1999; 23:127–36), HG3 (Klein and Rosen, in prep), CII (Najfeld V et al.Int J Cancer. 1980;26:543–549) and PGA (Lewin N et al. Int J Cancer. 1988;41:892–895) administered intravenously into NOD/SCID mice. Engraftment and tissue distribution varied considerably between the different cell lines, with CII recapitulating CLL most closely. Detectable levels of peripheral blood engraftment of all cell lines occurred within 2–4 weeks. Mice engrafted with HG3 cells rapidly succumbed to CLL engraftment, surviving for a maximum of 6 weeks, whilst engraftment of other CLL cell lines was more prolonged (PGA up to 13 weeks; CII up to 16 weeks; MEC-1 up to 21 weeks). MEC-1 and CII cells were found to reside mainly in haemopoietic organs such as spleen, bone marrow and blood of injected animals, whereas HG3 and PGA showed a wider distribution that included kidneys, liver, uterus, gastrointestinal tract, bladder and neck. We subsequently established a subcutaneous model of the CLL cell line CII that both recapitulated CLL disease by dissemination into lymphoid organs but also allowed direct measurement of tumour size. Solid subcutaneous tumours were evident within 2 weeks and engraftment in the peripheral blood, spleen and bone marrow was demonstratable within 5 days of subcutaneous cell injection. In order to recapitulate the effect of ATM loss on engraftment and proliferation we simultaneously injected isogenic cell line pairs (MEC-1, HG3, CII), with and without stable ATM knock down. Isogenic cell line pairs were differentially labelled with either SNARF (ATM wild-type) or CFSE (ATM null), mixed in equal numbers (3×106) and intravenously injected into multiple NOD/SCID mice. The relative proportion of each cell type in the peripheral blood was assessed weekly by FACS analysis and using an IVIS whole body imager. We observed a predominance of ATM null CLL cells (MEC-1, HG3, CII) in the peripheral blood and spleen within three weeks of engraftment, whereas equivalent levels of ATM null and control isogenic cells were found in the bone marrow. Finally, to determine whether the CLL xenografts could serve as a valid models for testing new CLL treatments, the effect of a PARP inhibitor (Olaparib) on tumour load and animal survival was examined in a MEC-1 xenograft with stable ATM knockdown. Daily oral administration of olaparib (100mg/kg/mouse) to mice engrafted with ATM null MEC-1 cells compared to the vehicle-treated animals resulted in a reduction in the tumour load both in the bone marrow and to a lesser extent the spleen with concurrent lengthening of overall survival. We conclude that xenograft models with isogenic CLL cell lines provide a robust system where the impact of a single gene loss on engraftment, proliferation and treatment response can be addressed. Disclosures:No relevant conflicts of interest to declare.

Proteasome Inhibition Leads to Dephosphorylation and Downregulation of Protein Expression of Members of the Akt/mTOR Pathway In MCL

AbstractAbstract 4449 Background:Mantle cell Lymphoma (MCL) is a distinct B-cell subtype characterized by the chromosomal translocation t(11;14)(q13;q32), an especially poor clinical outcome and low response to chemotherapy. The proteasome inhibitor bortezomib is approved for treatment of relapsed and refractory MCL and achieves a response rate of 30–40%. However, little is known which molecules represent the critical targets of proteasome inhibition and how different regulators of cell cycle and apoptosis are affected. Bortezomib has been shown to inactivate the NFkB pathway in MCL, but recent findings indicate Bortezomib is also active in a proteasome –independent manner suggesting Bortezomib targets multiple pathways. Method:Four MCL cell lines (HBL2, Granta 519, Jeko-1, NCEB-1), two CLL cell lines (Mec1, Mec2) and two hematological control cell lines (Jurkat, Karpas 422) were exposed to Bortezomib at a previously defined cytotoxic concentration (25nmol). Western blot and mRNA analysis were performed for various members of the PI3K/Akt/mTOR and the MEK/ERK pathway after 24h Bortezomib exposure. Results were compared to cell proliferation (WST1, trypan blue staining), induction of apoptosis (Annexin V PE/7-AAD staining) and cell cycle data (FACS). Result:Western blot analysis revealed reduced phosphorylation of Akt at Ser473 in all cell lines while autophosphorylation of mTOR at Ser2481 was completely downregulated only in the susceptible cell lines. In addition further members of the mTOR pathway were affected by bortezomib treatment. Dephosphorylation of the 4EBP1, downregulation of p70S6 protein expression was detected in all cell lines whereas EIF4E dephosphorylation was only observed in the sensitive MCL cell lines. In contrast Bortezomib did not affect members of the MEK/ERK pathway (MEK1/2, p42/44MAPK). Interestingly the mRNA and protein expression profile of CCND1 were differently altered suggesting an involvement of bortezomib in the regulation of translation initiation. This data were also confirmed by microarray analysis. Conclusion:In this study Bortezomib treatment was shown to target the Akt/mTOR pathway especially by dephosphorylation of the translation initiation factor EIF4E and other molecules of this signal pathway. This knowledge will play a crucial role in the development of future combinations of biologicals to target the molecular pathogenesis of MCL. Disclosures:Dreyling:Johnson & Johnson: support of investigators initial trials.

CD74 Expression by AML Cell Lines and Bone Marrow Specimens, and Augmented In Vitro Cytotoxicity of Anti-CD74 Antibody After Interferon-Gamma (IFN-γ) Treatment

AbstractAbstract 2888 Background:CD74 (HLA-DR-associated invariant chain) is expressed alone or along with DR in a wide range of hematologic cancers and solid tumors. Humanized anti-CD74 mAb, milatuzumab (Immunomedics, Morris Plains, NJ), exhibits direct cytotoxicity for NHL, CLL and MM cell lines, and is undergoing clinical evaluation for treatment of these malignancies. CD74 is upregulated by interferons in hematologic and epithelial cancer cell lines. Here we present the results of our analysis of CD74 expression and function in AML, and the effect of CD74 upregulation by treatment with IFN-γ on the cytotoxicity of milatuzumab for AML cell lines. Methods:CD74 expression in bone marrow biopsy (BMB) specimens from non-M3 AML patients was evaluated by immunohistochemistry and, for the 3 human AML cell lines, by flow cytometry, with/without permeabilization and with/without IFN-γ (40 and 200 U/mL). These cell lines were also tested in proliferation assays for responses to milatuzumab, with/without IFN-γ. Also, assessment of apoptosis and cellular signaling was performed. Results:In the initial group of AML cases, 13/14 BMB specimens showed moderate to strong CD74 expression by leukemic blasts, which was mostly intracellular, usually with a perinuclear distribution. Three AML cell lines also showed moderate to strong expression of CD74, which was mostly intracellular. Without IFN-γ, surface expression of CD74 was present, but IFN-γ treatment of these 3 lines resulted in upregulation of surface CD74 by 69–117%. Much higher levels of intracellular CD74 were observed in all 3 lines (with and without IFN-γ), with IFN-γ-induced upregulation of intracellular CD74 in all 3 lines (from 85%-868%; P<0.001). In 2/3 lines, IFN-γ increased milatuzumab-mediated growth inhibition (23.7 to 44.8% and -3.9 to 30.9%, P=0.01 and P<0.05, respectively). Cytotoxicity was in part due to apoptosis, as significant increases in Annexin V binding (P=0.01) were observed after treatment with IFN-γ plus milatuzumab. Initial experiments addressing cellular signaling suggest a role for AKT, because phosphorylated AKT levels increased (P=0.06) in response to IFN-γ + milatuzumab. Conclusions:CD74 is expressed in AML patient specimens and in AML cell lines, with the majority of CD74 expression found intracellularly. Cell surface and cytoplasmic expression of CD74 were upregulated in AML lines after IFN-γ exposure. This increased expression resulted in increased cytotoxicity of the anti-CD74 mAb, milatuzumab, in 2/3 AML lines. This effect was through apoptosis and involved the AKT pathway. Thus, AML is another cancer type where combined IFN-γ and milatuzumab treatment may be useful.Supported in part by NIH grant PO1-CA103985 (DMG). Disclosures:No relevant conflicts of interest to declare.

Expression of CD74 by AML blasts and cell lines, and enhanced in vitro cytotoxicity of anti-CD74 antibody after interferon-gamma (IFN-γ) treatment.

6576 Background: CD74 (DR-associated invariant chain) is expressed alone or with DR in a wide range of hematologic cancers and solid tumors. Humanized anti-CD74 mAb, milatuzumab (Immunomedics, Morris Plains, NJ), exhibits direct cytotoxicity for NHL, CLL and MM cell lines, and is in clinical evaluation for therapy of NHL, MM and CLL. Also, CD74 is induced by interferons in multiple cancer cell lines. Here we report an evaluation of CD74 expression and function in AML, and the effect of CD74 upregulation by treatment with IFN-γ on the cytotoxicity of milatuzumab for AML cell lines. Methods: CD74 expression in bone marrow biopsy (BMB) specimens from non-M3 AML patients was evaluated by immunohistochemistry and, for the 3 human AML cell lines, by flow cytometry, with/without permeabilization and with/without IFN-γ (40 and 200 U/mL). These cell lines were also tested in proliferation assays for responses to milatuzumab, with/without IFN-γ. Results: In 13/14 BMB specimens, there was moderate to strong CD74 expression by leukemic blasts, which was mostly intracellular, usually with a perinuclear distribution. Three AML cell lines also showed moderate to strong expression of CD74, which was mostly intracellular. WithoutIFN-g, surface expression of CD74 was present, but IFN-γ treatment of these 3 lines resulted in upregulation of surface CD74 by 69-117%. Much higher levels of intracellular CD74 were observed in all 3 lines, with and without IFN-γ. IFN- γ induced intracellular CD74 in all 3 lines (from 85%-868%). In 2/3 lines, IFN-γ increased milatuzumab-mediated growth inhibition (23.7 to 44.8% and -3.9 to 30.9%, respectively). Conclusions: CD74 is expressed in AML patient specimens and in AML cell lines, with the majority of CD74 expression found intracellularly. Cell surface and cytoplasmic expression of CD74 were upregulated in AML lines after IFN-γ exposure. This increased expression resulted in increased cytotoxicity of the anti-CD74 mAb, milatuzumab, in 2/3 AML lines. Thus, AML is another cancer type where combined IFN-γ and milatuzumab treatment may be useful. Supported in part by NIH grant PO1- CA103985. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Immunomedics, Inc. Immunomedics, Inc.

Immune recovery after fludarabine–cyclophosphamide–rituximab treatment in B-chronic lymphocytic leukemia: implication for maintenance immunotherapy

Thirty B-cell chronic lymphocytic leukemia patients were treated with fludarabine-cyclophosphamide-rituximab (FCR) and immune cell counts (natural killer (NK) cells, CD4, CD8, Tgammadelta and monocytes) were monitored from the end of treatment (EOT) up to 36 months (M36). Moreover, nonleukemic peripheral blood lymphocyte cytotoxicity (PBL/CTC) as well as rituximab (RTX)-dependent PBL/CTC was also measured at the initiation of therapy, EOT and M12. These parameters were correlated with post-FCR monitoring of the minimal residual disease (MRD) level in blood using a four-color flow cytometry technique. FCR induced a profound and sustained depletion of all T-cell populations, Tgammadelta being the most affected, whereas NK cells were relatively preserved. Both basal and interleukin-2-stimulated nonleukemic PBL/CTC against MEC-2, a CLL cell line, increased during the post-FCR period. There was no correlation between immune recovery parameters and MRD progression profile, except that patients with high post-FCR CD4(+) counts experienced rapid MRD progression. MRD at M12 predicts clinical relapse. The limited data show RTX-mediated LBL/CTC activity against autologous B-cell cells in individuals with <1% residual disease at M12, opening avenues for immunomodulation post-FCR with anti-CD20 antibodies. To conclude, our study suggests that MRD increase at M12 precedes disease evolution post-FCR, and should be assessed as a surrogate marker for proactive management of CLL relapse.

Therapy of B-Cell Malignancies by Anti-HLA-DR Humanized Monoclonal Antibody, IMMU-114, Is Mediated through Hyperactivation of ERK and JNK MAP Kinase Signaling Pathways.

Abstract 3738Poster Board III-674 IntroductionHLA-DR is currently under investigation as a target for antibody (mAb) therapy of B-cell malignancies. IMMU-114, a humanized IgG4 form of the murine anti-HLA-DR mAb, L243, recognizes a conformational epitope in the a-chain of HLA-DR. We have previously reported that IMMU-114 lacks effector-cell functions while displaying potent antitumor activity against B-cell lymphomas in vitro and in vivo. Here, we further investigated the cytotoxic effects of IMMU-114 on leukemia, lymphoma, and multiple myeloma (MM) cell lines, as well as clinical specimens of CLL, and explored the signaling pathways involved. MethodsBinding and cytotoxicity of IMMU-114 was examined on a panel of NHL, AML, MCL, ALL, and CLL cell lines. Induction of apoptosis (Annexin V binding), ROS generation (dihydoehidium staining), changes in mitochondrial membrane potential (ψM, TMRE staining) and effects on the ERK1/2 and JNK pathways (immunoblot analyses) were evaluated on selected cell lines. The effects of various inhibitors on IMMU-114-mediated apoptosis also were determined. Studies were performed in tumor-bearing SCID mice. ResultsHigh expression of HLA-DR was detected on the cell surface of all MCL, ALL, HC, CLL, and NHL cell lines tested, as well as 2/3 AML and 5/6 MM cell lines. IMMU-114 was cytotoxic to 2/2 MCL, 2/2 CLL, 2/4 ALL, 1/1 HC, 2/2 NHL and 2/2 MM cell lines without any added anti-Fc second crosslinking antibody. IMMU-114 was consistently more cytotoxic than anti-CD20 mAbs on these cell lines, as well as on rituximab-resistant variants of Raji and SU-DHL-4. Despite positive staining, AML cell lines, Kasumi-3 and GDM-1, were not killed by IMMU-114. Four of 6 CLL patient samples expressed HLA-DR at markedly higher levels than CD20, with the remaining 2 showing similar HLA-DR and CD20 expression. Approximately 60% cytotoxicity was obtained after incubation with IMMU-114 in CLL patient cells. Induction of apoptosis as well as changes in ψM and ROS correlated with sensitivity of cell lines to IMMU-114-mediated cytotoxicity. Time-course analyses demonstrated that IMMU-114 induced approximately 46% change of ψM and 24% enhancement of ROS in as little as 30 min in Raji cells. These changes were abrogated in the presence of ROS inhibitor, N-acetyl cysteine. Immunoblot analyses revealed that IMMU-114 induces phosphorylation of ERK and JNK mitogen-activated protein (MAP) kinases in all cells defined as IMMU-114-sensitive by the cytotoxicity assays, but not IMMU-114-resistant cell lines. The p38 pathway was found to be constitutively active in these cell lines. Inhibition of ERK, JNK, or ROS by their respective inhibitors decreased apoptosis, although the inhibition was not complete when any single inhibitor was used. This could be due to activation of multiple pathways, since inhibition of 2 or more pathways by specific inhibitors abolishes the apoptosis induced by IMMU-114. Activation (5-fold increases of phosphorylation) of ERK1/2 and JNK signaling pathways also was observed in CLL patient samples following IMMU-114 incubation. Further studies revealed caspase-independent apoptosis and activation of apoptosis-inducing factor (AIF) mediated by IMMU-114. The therapeutic activity of IMMU-114 was evaluated in vivo in 3 NHL models, WSU-FSCCL, Namalwa, and Raji. IMMU-114 was more effective than anti-B-cell mAbs such as rituximab, in all models. For example, in WSU-FSCCL, IMMU-114 treatment yielded 100% long-term survival. Although rituximab also induced significant responses compared to untreated mice (median survival time of 87.5 vs. 36 days for untreated controls, P<0.0001), no long-term survivors were seen. ConclusionIMMU-114 is cytotoxic to a variety of hematological malignancies, and its potent activity can be related to high antigen expression of target cells and hyperactivation of ERK and JNK pathways upon binding to HLA-DR. Clinical evaluation of IMMU-114 is planned. Disclosures:Gupta:Immunomedics Inc.: Employment. Stein:Garden State Cancer Center: Employment. Cardillo:Immunomedics Inc.: Employment. Chang:Immunomedics Inc.: Employment. Goldenberg:Immunomedics Inc.: Consultancy, Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Autocrine Function of Cysteinyl-Leukotrienes and Their Receptor CysLT1 in Chronic Lymphocytic Leukemia (B-CLL).

Abstract 2325Poster Board II-302Leukocytes, including B cells, represent the physiological source of lipid mediators of the cysteinyl-leukotriene family. In malignant B cells, particularly in B-CLL, even higher levels of enzymes involved in leukotriene biosynthesis have been detected. The major receptor for cysteinyl-leukotrienes, CysLT1, mediates pleiotropic effects on cell migration, proliferation and survival. In the present study, we found that CysLT1 was consistently overepressed in B-CLL with mRNA levels 2-4-fold greater than in normal B cells, as detected by real-time PCR. The CysLT1 receptor was highly active in CLL cell lines (MEC-1, EHEB) and primary B-CLL cells, as demonstrated by ligand-induced intracellular calcium fluxes and actin polymerization (measured by flow cytometry), chemotaxis (Boyden chamber assay), and MAP-kinase/Erk1/2 phosphorylation (analyzed with Western blot). The effects were completely blocked by the CysLT1 antagonist MK-571. Upregulation of both the CysLT1 receptor and enzymes of leukotriene biosynthesis, in conjunction with the CysLT1-mediated activation of the MAP-kinase/Erk signaling pathway, suggest an autocrine loop contributing to cell survival. Indeed, incubation of CLL cells in serum-free medium with the CysLT1 antagonist reduced their viability in a dose-dependent manner by up to 70% (24 h) and 80% (48 h) compared to cells which were cultured without the antagonist. Our results provide evidence that cysteinyl-leukotrienes and their receptor regulate survival of CLL cells in an autocrine fashion, and imply a therapeutic potential of CysLT1 receptor antagonists, currently used in asthma therapy, also in the treatment of B-CLL. Disclosures:No relevant conflicts of interest to declare.

A Novel Paradigm to Trigger Apoptosis in Chronic Lymphocytic Leukemia.

Abstract 731Chronic lymphocytic leukemia (CLL) is characterized by the abnormal accumulation of malignant monoclonal B cells, which has been largely attributed to defective apoptosis rather than aberrant proliferation. This calls for new strategies to re-activate apoptosis programs in CLL in order to develop new therapeutic strategies. “Inhibitor of Apoptosis” (IAP) proteins such as XIAP are aberrantly expressed in many human cancers and block apoptosis at a key node by inhibiting activation of caspases. In the present study, we therefore explored whether targeting XIAP is a suitable strategy to overcome apoptosis resistance of CLL.Here, we provide first evidence that small molecule XIAP inhibitors in combination with the death receptor ligand TRAIL present a novel approach to trigger apoptosis in CLL even in subgroups with resistant disease. Analysis of apoptosis regulatory proteins reveals that XIAP, cIAP1 and cIAP2 are expressed at high levels in primary CLL samples. Proofs of concept studies in CLL cell lines demonstrate that subtoxic concentrations of several distinct XIAP inhibitors significantly enhance TRAIL-induced apoptosis. In addition, XIAP inhibitors sensitize CLL cells for CD95-mediated apoptosis, whereas they have no effect on fludarabine- or chlorambucil-induced apoptosis. This indicates that XIAP inhibitors in particular enhance death receptor-triggered apoptosis in CLL cells. By comparison, no sensitization for death receptor-induced apoptosis is observed in the presence of a structurally related control compound that only weakly binds to XIAP, demonstrating the specificity of the sensitization effect of XIAP inhibitors. Importantly also in primary CLL samples, XIAP inhibitors act in concert with TRAIL to trigger apoptosis in 18 of 27 cases (67%). Analysis of combination index reveals that this interaction of XIAP inhibitor and TRAIL is highly synergistic. Mechanistic studies in primary CLL cells show that the addition of XIAP inhibitor profoundly enhances TRAIL-induced cleavage of caspase-3 into active fragments and significantly increases caspase-3 enzymatic activity upon treatment with TRAIL. The broad range caspase inhibitor zVAD.fmk completely blocks apoptosis in response to combination treatment with XIAP inhibitor and TRAIL, demonstrating that apoptosis occurs in a caspase-dependent manner. Importantly, the cooperative interaction of XIAP inhibitor and TRAIL is even evident in distinct subgroups of patients with poor prognostic features, including patients with 17p deletion, TP53 mutation, chemotherapy-refractory disease or unmutated VH genes. This suggests that the combination treatment with XIAP inhibitor and TRAIL may present a novel approach to trigger apoptosis in CLL patients that are resistant to other treatment options. Interestingly, we found that cases with unmutated VH genes are significantly more sensitive to XIAP inhibitor- and TRAIL-induced apoptosis compared to VH gene mutated samples. This points to a role of B-cell receptor signaling in the regulation of apoptosis in CLL cells. In conclusion, we demonstrate for the first time that XIAP inhibitors in combination with TRAIL present a new strategy to trigger apoptosis even in resistant forms and poor prognostic subgroups of CLL. These findings have important implications for the development of novel strategies to overcome the intrinsic resistance to apoptosis in CLL. Since IAP inhibitors as well as TRAIL receptor agonists as single agents are currently already under evaluation in early clinical trials, it is feasible that such combination protocols of XIAP inhibitors and TRAIL could be translated into clinical application in CLL. Disclosures:No relevant conflicts of interest to declare.

Restoring the Functional Immunogenicity of Chronic Lymphocytic Leukemia Using Epigenetic Modifiers.

Abstract 5116 BackgroundChronic lymphocytic leukemia (CLL) is a malignancy arising from immune cells (B-lymphocytes) endowed with intrinsic antigen-presenting capabilities. Such a function however is lost during malignant transformation and CLL cells are well known for their inability to process and present antigens to the T-cell arm of the immune system. Instead, malignant CLL cells elicit a vast array of immune regulatory mechanisms conducive to T-cell dysfunction and immunosupression. Recent evidence suggests that DNA methylation inhibitors (DNMTi) and histone deacetylase inhibitors (HDACi) can have lasting cancer-specific effects on the expression of highly immunogenic cancer-testis antigens (CTAs) as well as modulating costimulatory and major histocompatability molecule (MHC) surface expression. MethodsTo investigate the potential efficacy of a combined therapy of DNMTi (5-aza-2'-deoxycytidine) and HDACi (LAQ) in human CLL we performed a gene expression microarray profile and further confirmed the expression of CTAs. Furthermore we investigated phenotypic alterations due to changes in costimulatory molecules (CD40, 80, and 86), and MHC molecules (HLA-A, B, C, and HLA-DR) in both cell lines and primary cells from patients with CLL. To confirm that alterations in molecule surface expression correlated to more potent immune stimulation we characterized the formation of the immune-synapse between the CLL antigen presenting cell and healthy T-lymphocytes in treated and untreated samples using confocal microscopy and flow conjugation assay. To demonstrate functional significance we subjected treated and untreated CLL cell lines to mixed lymphocyte proliferation assays with CD4, CD8, and NK cell subsets. ResultsAs expected we demonstrate by gene expression profile and RT-PCR that many highly immunogenic CTAs (SSX family, MAGE family, NY-ESO-1, GAGE-2 and NXF2) are significantly upregulated after treatment with an HDACi and DNMTi in combination. Interestingly, preliminary data showed interferon gamma mRNA upregulation. Consistent with this information, our data also indicates that a more proinflammatory signaling pathway is developed between the CLL B-cell and the T-lymphocyte after combination treatment demonstrated by upregulated surface expression of CD86, 80, 40, HLA-A,B,C, and HLA-DR as well as the formation of significantly more, robust, numerous, and organized actin-mediated immune-synapses, changes which were not duplicated using either drug alone. Functionally, only the combined HDACi/DNMTi treatment of CLL cells led to increased allogenic T and NK cell proliferation and cytotoxicity. ConclusionsTaken together these data indicate that combined HDACi/DNMTi may benefit current antigen specific vaccine strategies for CLL by inducing the expression of a highly antigenic CTAs, increasing CLL costimulatory capacity, repairing immunological synapse, and functionally increasing the proinflammatory status of the CLL APC. DisclosuresPinilla:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Research Funding; exelixis: Research Funding.

Protein kinase A and Epac (Exchange Protein Activated by cAMP) are Pro‐and Anti‐Apoptotic Mediators, respectively, in Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL), which can be classified as "indolent" or "aggressive", is generally characterized by the accumulation of B‐cells. Increasing the level of cAMP can promote apoptosis of CLL cells but mechanisms for this pro‐apoptotic response are not known. The actions of cAMP are largely mediated by protein kinase A (PKA) and exchange protein activated by cAMP (Epac). We sought to investigate the role of these two downstream mediators in cAMP‐induced apoptosis using CLL cells and EHEB cells, a CLL‐cell line. Activation of PKA by N6‐ Phenyladenosine‐cAMP (50 μM, 48 hr) and 8‐Chloro‐cAMP (50 μM, 48 hr) both induced apoptosis of CLL cells and decreased the proliferation of EHEB cells. In contrast, Epac activation by 8‐pCPT‐2Me‐cAMP (50 μM, 48 hr), which stimulated Rap‐1 activation, protected the CLL from spontaneous apoptosis and increased EHEB proliferation. Further analysis revealed that CLL cells (isolated from both indolent and aggressive patients) have increased Epac‐1 mRNA and protein expression compared to normal PBMC and normal B‐cells. These data suggest that increased expression of Epac‐1 contributes to anti‐apoptosis in CLL. Efforts to decrease the expression and function of Epac‐1 may thus be beneficial for the treatment of CLL by increasing the pro‐apoptotic effects of drugs that act through raising cAMP and activating PKA. Supported by the Lymphoma and Leukemia Society and NIH.

Raf-1 Is Over Expressed in Chronic Lymphocytic Leukemia and Promotes Cell Survival by Phosphorylation of ERK and BAD

Introduction: The serine threonine kinase Raf-1 plays a protective role in many cell types, but its expression and function in CLL cells has not been studied in detail. In the present study, we analyzed Raf-1 expression and tested the hypothesis that Raf-1 is critical for CLL cell survival.Materials and Methods: By using qRT-PCR and western blot, we compared the expression of Raf-1 of mRNA and protein levels in purified B cells from 45 CLL cases and CD38 negative B cells from 5 reactive tonsils. By western blot, we analyzed the activity of phospho-Raf-1 (ser338) and its downstream targets (phospho-ERK1/2, phospho-BAD) in 23 CLL cases and 4 CLL cell lines (JVM-3, MEC-1, MEC-2 and MO1043) before and after IgM stimulation. By immunoprecipitation, we analyzed if Raf-1 co-localizes with Bcl-2. We correlated the change in phosphorylation status (Raf-1, ERK and BAD) in response to IgM stimulation with the ZAP-70/SYK mRNA ratio, which was detected by qRT-PCR in purified B cells from CLL cases. After using specific inhibitors, including the Raf-1 inhibitor GW5074, the Raf-1 destabilizer Geldanamycin and Bcl-2 inhibitor YC137, we investigated apoptosis by Annexin V flowcytometry in CLL cases and CLL cell lines as well as cell cycle changes in the 4 cell lines by flowcytometry.Results: In comparison to normal resting (CD38 negative) B cells, there was a strongoverexpression of Raf-1 in CLL cells, both at at the mRNA and protein level. Using qRT-PCR there was an almost linear correlation between Raf-1 and Bcl-2 expression. Moreover, the phosphorylation status of Raf-1 and ERK in response to IgM stimulation strongly correlated with the ZAP-70/SYK mRNA ratio. Using immunoprecipitation and confocal miscroscopy we found colocalization of Raf-1 with Bcl-2, which might account for the observed constitutive activation of BAD in CLL cells. The Raf-1 inhibitor GW5074, Raf-1 destabilizer Geldanamycin and Bcl-2 inhibitor YC137 all led to p-Raf-1 inhibition as well as downregulation of p-ERK and p-BAD. Additionally, all three inhibitors downregulated cyclin D3 and cyclin E, which are important for G0/G1 transition. We also found that GW5074 induced apoptosis in CLL cell lines and primary cells of CLL cases.Conclusion/Discussion: In conclusion, our study identifies Raf-1 as a critical anti-apoptotic and cell cycle regulating kinase in CLL cells.