ALBERT

Description: Abstract 4609 Active NOTCH signaling is observed in an increasing number of human neoplasias including chronic lymphocytic leukemia (CLL) and represents a potential therapeutic target. We have recently reported that the γ-secretase inhibitor DAPT may not be effective in all cases of CLL (Hubmann et al; BJH 2010). The aim of this study was to evaluate the therapeutic value of a newly identified NOTCH transactivation inhibitor (gliotoxin) and to compare its efficiency with the highly selective γ-secretase inhibitor (GSI) DAPT in CLL cells. Electrophoretic mobility shift assays (EMSA) showed that gliotoxin completely blocked the formation of DNA-bound NOTCH2 in CLL cells independent of their sensitivity to DAPT. The inhibition of NOTCH2 signaling by gliotoxin was associated with down regulation of its target gene CD23 and induction of apoptosis. The IC50 of Gliotoxin was variable between patients (n=20) and ranged between 50–100 nM irrespective to their sensitivity to GSI. Short term (4 hours) exposure of CLL cells to gliotoxin revealed that gliotoxin modulates the mRNA expression of several NOTCH-related genes including JAG1, PRKCD (PKC-δ), and NR4A1. We (Shehata et al; BLOOD 2010) and others have reported on the supportive effect of primary bone marrow stromal cells (BMSC) for CLL cells. Therefore, we tested whether gliotoxin may overcome this supportive effect. FACS analysis and MTT assays showed that gliotoxin abolished the supportive effect of BMSC under co-culture conditions. The effect of gliotoxin was dose dependent and selectively induced apoptosis in CLL cells accompanied by down regulation of NOTCH2 and CD23 transcription. Western blotting analysis demonstrated that gliotoxin also decreased the phosphorylation of Akt-pSer473 suggesting a link to PI3-K signaling. NOTCH1 has been recently shown to be affected by “gain of function” mutations in a subset of CLL patients. We observed that co-culture of CLL cells with BMSC was associated with increased NOTCH1 mRNA expression which could be decreased upon exposure to gliotoxin. In addition, gliotoxin inhibited DNA-bound NOTCH1 complexes in the T-ALL cell line SupT1 which is known to express high NOTCH1 activity. This indicates that gliotoxin may target both NOTCH1 and NOTCH2 isoforms. In conclusion, the data show that gliotoxin effectively targets NOTCH activity in CLL cells in a mechanism which is independent of γ-secretase. Thus, gliotoxin might have a beneficial effect in a wider range of CLL patients and warrants further evaluation. Disclosures: No relevant conflicts of interest to declare.

Description: Members of the Notch family encode transmembrane receptors that modulate differentiation, proliferation, and apoptotic programs of many precursor cells, including hematopoietic progenitors. Stimulation of Notch causes cleavage followed by translocation of the intracellular domain (NotchIC) to the nucleus, where it activates transcription of CBF1 responsive genes. The aim of this study was to elucidate the mechanisms leading to the overexpression of CD23, a striking feature of B-cell chronic lymphocytic leukemia (B-CLL) cells. By electrophoretic mobility shift assays, we identified a transcription factor complex (C1) that binds sequence specific to one known and 4 newly identified putative CBF1 recognition sites in the CD23a core promoter region. With the use of Epstein-Barr virus (EBV)–infected B cells as a model for CBF1 mediated CD23a expression, C1 was found to be EBV inducible. Supershift assays revealed that the nuclear form of Notch2 is a component of C1 in B-CLL cells, supporting a model in which NotchIC activates transcription by binding to CBF1 tethered to DNA. Transient transfection of REH pre–B cells with an activated form of Notch2 induced endogenous CD23a, confirming thatCD23a is a target gene of Notch2 signaling. Finally, reverse transcription-polymerase chain reaction and kinetic analysis demonstrated that the Notch2 oncogene is not only overexpressed in B-CLL cells but might also be related to the failure of apoptosis characteristic for this disease. In conclusion, these data suggest that deregulation of Notch2 signaling is involved in the aberrant expression of CD23 in B-CLL.

Description: B-cell chronic lymphocytic leukemia (B-CLL) represents a clonal expansion of self-reactive CD5+ B-lymphocytes which seems to be resistant to apoptosis in vivo. One of the characteristics of B-CLL lymphocytes is the high expression of the B-cell differentiation/activation marker CD23 which we recently identified as a target gene for NOTCH2 signaling. NOTCH2 is implicated in the development/homeostasis of murine CD5+ (B-1a) B-cells, suggesting a function for human NOTCH2 in B-CLL leukemogenesis. Here we show that peripheral B-CLL lymphocytes overexpress a transcriptionally active form of NOTCH2 (N2IC) irrespective of their prognostic marker profile (ie. IgVH mutational status, CD38 expression, and cytogenetics). Although the majority of unstimulated B-CLL samples downregulate their N2IC activity in vitro, DNA-bound N2IC complexes could be maintained by the protein kinase C (PKC) activator TPA (12-O-tetradecanoylphorbol 13-acetate) accompanied by an upregulation of the NOTCH2 target gene CD23 and increased B-CLL cell viability. These effects are sensitive to the PKC-δ selective inhibitor Rottlerin. In 80% of B-CLL cases, NOTCH2 signaling was found to be resistant to the γ-secretase inhibitors (GSI’s) Dapt and compound E, indicative for the expression of truncated forms of NOTCH2 which do not require γ-secretase for processing and function. Inhibition of NOTCH2 either by Dapt in GSI sensitive B-CLL cases or, more specifically, by RNA interference downregulates CD23 expression on the mRNA and protein level and sensitizes B-CLL cells for apoptosis. Since self-reactive B-cells are normally eliminated either by chronic (anergy) or apoptotic (negative selection) B-cell receptor (BCR) signaling, we asked whether NOTCH2 modulates B-cell fate decisions triggered by the BCR. For this reason, we stably transduced the human B-cell line BL41 with constitutive active forms of NOTCH2 and found that NOTCH2 inhibits BCR mediated apoptosis induced by surface-IgM cross-linking. In summary, the data demonstrate that NOTCH2 signaling is deregulated in B-CLL cells and might be critically involved in the PKC-dependent maintenance of their malignant phenotype.

Description: The prognosis of patients with B-CLL is largely determined by the karyotype of the malignant clone. Microarray technology has facilitated linkage between chromosomal aberrations and gene expression signatures. We have investigated the gene expression profile associated with trisomy 12 (+12). Expression data were obtained by microarray analysis of mRNA from unselected PBMNC of 4 patients with +12 and compared with 16 B-CLL controls. 146 genes were at least 2-fold over- or underexpressed in samples with +12. Five of the 16 genes showing the strongest correlation with +12 were selected for further analysis (HIP1R FC=3,43; MYF6 FC=3,92; P2RY14 FC=−9,59; RASGRP3 FC=−3,85; SLC2A6 FC=2,13) and validation by real time PCR: HIP1R located on chromosome 12q24, with a fold change (FC) of 3,43, MYF6 (chromosome 12q21, FC=3,92), P2RY14 (chromosome 3q21–q25, FC-9,59) RASGRP3 (chromosome 2p25.1–p24.1, FC=−3,85). SLC2A6 (chromosome 9q34, FC=2,13). Quantitative PCR was performed with mRNA from 61 patients (29 with +12, 32 B-CLL controls) and 2 healthy donors. Only 3 genes were significantly associated with +12 compared to the B-CLL-controls in this evaluation: HIP1R (3,486; p

Description: Abstract 2433 Chronic lymphocytic leukemia (CLL) is characterized by the clonal expansion of B lymphocytes which typically express CD19 and CD5. The disease remains incurable and recurrence often occurs after current standard therapies due to residual disease or probably due to the presence of therapy-resistant CLL precursors. Based on the growing evidence for the existence of leukemia stem cells, this study was designed to search for putative CLL precursors/stem cells based on the co-expression of CLL cell markers (CD19/CD5) with the hematopoietic stem cell marker (CD34). Forty seven CLL patients and 17 healthy persons were enrolled in the study. Twenty four patients had no previous treatment and 23 had pre-therapy. Twenty two patients were in Binet stage C and 25 patients in B. Twenty two patients had unmutated and 18 mutated IgVH gene (7: ND). Cytogenetic analysis by FISH showed that 14 patients had del 13q, 8 had del 11q, 4 had del 17p and 9 had trisomy 12. Peripheral blood and bone marrow mononuclear cells were subjected to multi-colour FACS analysis using anti-human antibodies against CD34, CD19 and CD5 surface antigens. The results revealed the presence of triple positive CD34+/CD19+/CD5+ cells in CLL samples (mean 0.13%; range 0.01–0.41) and in healthy donors (0.31%; range 0.02–0.6) within the CD19+ B cells. However, due to the high leukocyte count in CLL patients, the absolute number of these cells was significantly higher in CLL samples (mean: 78.7; range 2.5–295 cells /μL blood) compared to healthy persons (mean: 0.45: range 0.04–2.5 cells/μl)(p

Description: The activation of anti-apoptotic mechanisms in the leukemic B cells in chronic lymphocytic leukemia (B-CLL) through the interaction with their microenvironment may lead to prolonged survival and the accumulation of the malignant clone. The aim of this study is to elucidate the influence of the lymphoid microenvironment in the activation of the potent anti-apoptotic PI3-K/Akt pathway and to investigate the pattern of expression of the tumor suppressor PTEN in B-CLL. Stromal fibroblasts of bone marrow (BMF), spleen (SF) and lymph gland (LGF) were used as an in vitro model for lymphoid microenvironment. Pharmacological inhibitors and siRNAs against PI3-K and Akt were applied to explore the anti-apoptotic effect of this pathway in B-CLL. The results showed that co-cultivation of B-CLL cells with human BMF, LGF, and SF significantly inhibited apoptosis and prolonged survival of the leukemic cells in comparison to suspension cultures. To explore the involvement of PI3-K/Akt pathway in the anti-apoptotic effect of stromal fibroblasts, co-cultures were performed in presence of PI3-K inhibitors (wortmannin or LY294002) or siRNAs against PI3-K and Akt1. These inhibitors significantly reduced the supportive effect of stromal fibroblasts and induced apoptosis in B-CLL cells. The leukemic cells were more sensitive to PI3-K inhibition than T cells, monocytes and stromal fibroblasts. Induction of apoptosis was associated with a significant decrease in the intracellular levels of PIP3, PI3-K, PDK1, Akt1, NF-kappa B, IKK, and dephosphorylation (activation) of PTEN. Since PTEN activity, as a negative regulator for PI3-K signalling, is controlled by its phosphorylation at the tail domain, we studied the pattern of PTEN protein expression in B-CLL. Western blotting demonstrated that the total PTEN in PBMC of B-CLL patients (n=40) is comparable to healthy individuals (n=8). However, using phosphospecific anti-PTEN antibody demonstrated that samples of B-CLL patients highly express phosphorylated (inactive) forms of PTEN in comparison to healthy persons. In conclusion, the results demonstrate that PI3-K/Akt pathway is involved in inhibition of apoptosis of B-CLL cells and suggest that interaction of the leukemic cells with lymphoid microenvironment may lead to the activation of this pathway. The data also suggest that targeting this pathway represents a feasible therapeutic approach in B-CLL.

Description: We have previously shown that NOTCH2 signaling is involved in the overexpression of CD23 in B-cell chronic lymphocytic leukemia (B-CLL) cells (Hubmann et al., BLOOD 2002 May 15;99(10):3742–7). NOTCH2 plays a determining role in the development/homeostasis of CD5+ B1 B-cells and of the related marginal zone (MZ) B2 B-cells, suggesting a potential role for NOTCH2 in B-CLL leukemogenesis. Using electrophoretic mobility shift assays (EMSA) we demonstrate that freshly isolated B-CLL patient samples (n=30) express an activated form of nuclear NOTCH2 (N2IC) irrespective of their prognostic marker profile (ie. IgVH mutational status, CD38 surface expression, cytogenetics). Although the majority of cultured B-CLL samples lose their N2IC activity within one day, DNA-bound N2IC complexes could be maintained by low concentrations of the PKC-stimulating phorbol esther PMA (1ng/ml). This was accompanied by the upregulation of CD23. The effect of PMA on N2IC activation and CD23 expression was abrogated by the PKC-δ inhibitor Rottlerin. Since wild type NOTCH2 signaling is regulated through binding to its ligand followed by γ-secretase mediated cleavage and release of the intracellular domain (N2IC), we induced NOTCH2 signaling by PMA in the presence or absence of the γ-secretase inhibitors (GSI) DAPT and compound E. Results demonstrated that the PMA-induced NOTCH2 activity is resistant to GSI treatment in 24 out of 30 B-CLL cases (80%). This suggests that the leukemic cells from the majority of B-CLL patients express an activated form of N2IC which is independent from γ-secretase cleavage and, thus, do not reqire NOTCH2 ligands for signaling. In conclusion, the results suggest that PKC-δ is involved in the activation/nuclear translocation of N2IC in B-CLL cells. The data may also suggest that deregulated NOTCH2 signaling is an early step in the development of B-CLL and might be critically involved in the aberrant response of the malignant clone to cell fate modulating stimuli acting through PKC.

Description: Evidence suggests that tumor microenvironment is critically involved in supporting survival of chronic lymphocytic leukemia (CLL) cells. However, the molecular mechanisms of this effect and the clinical significance are not fully understood. We applied a microenvironment model to explore the interaction between CLL cells and stromal cells and to elucidate the role of phosphatidylinositol 3 kinase (PI3-K)/Akt/phosphatase and tensin homolog detected on chromosome 10 (PTEN) cascade in this process and its in vivo relevance. Primary human stromal cells from bone marrow, lymph nodes, and spleen significantly inhibited spontaneous apoptosis of CLL cells. Pan–PI3-K inhibitors (LY294002, wortmannin, PI-103), isotype-specific inhibitors of p110α, p110β, p110γ, and small interfering RNA against PI3-K and Akt1 counteracted the antiapoptotic effect of the stromal cells. Induction of apoptosis was associated with a decrease in phosphatidylinositol-3,4,5-triphosphate, PI3-K–p85, and dephosphorylation of phosphatidylinositol-dependent kinase-1 (PDK-1), Akt1, and PTEN. Freshly isolated peripheral blood mononuclear cells from patients with CLL (n = 44) showed significantly higher levels of phosphorylated Akt1, PDK-1, PTEN, and CK2 than healthy persons (n = 8). CK2 inhibitors (4,5,6,7-tetrabromo-1H-benzotriazole, apigenin, and 5,6-dichloro-1-β-D-ribofuranosylbenzimidazol) decreased phosphorylation of PTEN and Akt, induced apoptosis in CLL cells, and enhanced the response to fludarabine. In conclusion, bone marrow microenvironment modulates the PI3-K/Akt/PTEN cascade and prevents apoptosis of CLL cells. Combined inhibition of PI3-K/Akt and recovery of PTEN activity may represent a novel therapeutic concept for CLL.

Description: Abstract 3916 There is accumulating evidence that green tea extract EGCG [(-)-epigallocatechin-3-gallate] may exert a preventive or a direct anti-tumor effect in several tumor types including chronic lymphocytic leukemia (CLL) and clinical trials with EGCG are already on-going. However, EGCG has a broad spectrum of activities and downstream targets. Therefore, it would be necessary to precisely characterize the key targets of this compound and identify the CLL patients who would most likely profit from EGCG. Therefore, the aim of this study was to evaluate the effect of EGCG on the viability of CLL cells in a well characterized cohort of patients and to get insight into its mechanism of action in CLL. Peripheral blood mononuclear cells (PBMC) of 27 CLL patients were included in this study. Patients were characterized according to the Rai/Binet stage, IgVH mutation status and cytogenetics (13q-del, 11q-del, 17p-del, trisomy-12). The percentage of the leukemic cells (CD19+/CD5+) ranged between 60–98%. CLL cells were exposed to a wide range of concentrations of EGCG (0.1 – 200μM) and cell viability was evaluated by cell titer blue (CTB) assays and FACS analysis after 4 hours, 1, 2 and 3 days. Treatment with EGCG was performed in suspension cultures and under co-culture with primary human bone marrow stromal cells (BMSC). Cell viability assays demonstrated a dose and time dependent decrease in the cell viability after the exposure to EGCG with an IC50 ranging between 50–80μM (25–50μg/ml). A moderate variation in the response to EGCG was observed between patients demonstrating the heterogeneity of the disease. No clear correlation between the in vitro response to EGCG and the clinical background and prognostic markers could be observed in this cohort of patients. Annexin V/propidium iodide (Anx/PI) staining showed that EGCG increased the percentage of early apoptotic (Anx+/PI-) and late apoptotic/necrotic cells (Anx+/PI+). These data suggest that EGCG exerts a pro-apoptotic effect and activates other cell killing mechanisms in CLL cells. The leukemic cells (CD19/CD5) were relatively more sensitive to the compound compared to T cells and monocytes. Co-culture experiments showed that EGCG effectively overcomes the supportive effect of BMSC and induces apoptosis/cell killing in CLL cells. BMSC were less sensitive to the compound and a toxic effect was observed at a concentration of 200 μM or higher. RT-PCR showed a downregulation of the catalytic domain p110a and the regulatory domain p85 of phosphoinositide 3-kinases (PI3K) as well as Bcl-2 and Mcl-1 mRNA expression after exposure to EGCG. Western blotting analysis demonstrated a decrease in the phosphorylation of Akt particularly at pThr308 residue and de-phosphorylation of the tumor suppressor PTEN at pSer380 residue in parallel to the induction of PARP cleavage. In addition, EGCG induced a decrease in the protein expression of the activation marker CD23 and the adhesion molecule CD44. Furthermore, proteasome assays showed that EGCG inhibits the chymotrypsin-like activity within 4 hours of incubation in parallel to induction of early apoptosis. This effect was more remarkable after 24 hours. However, EGCG was less effective in proteasome inhibition compared to Bortezomib. In conclusion, these data demonstrate that EGCG induces cell death in CLL cells through a complex mechanism which may involve the inactivation of PI3K/Akt signaling cascade and inhibition of proteasome activity. The results also point to a potential therapeutic effect of EGCG in CLL which warrants further evaluation. Disclosures: No relevant conflicts of interest to declare.