Triggering of CD40 Antigen Inhibits Fludarabine-Induced Apoptosis in B Chronic Lymphocytic Leukemia Cells

Abstract We analyzed the effect of CD40 triggering on the fludarabine-induced apoptosis of B chronic lymphocytic leukemia (B-CLL) cells. Peripheral blood samples obtained from 15 patients were incubated with fludarabine in the absence or the presence of the anti-CD40 monoclonal antibody (MoAb) G28-5. In 12 patients a significant proportion of apoptotic cells, ranging from 22% to 38% (mean ± SE: 28.5 ± 1.6), were detected after 3 days of culture. In 9 of these samples, the addition of G28-5 reduced apoptosis by at least 30.1% and by 57.1% ± 7.8% on average (P = .0077). Because the CD40 antigen activates NF-κB/Rel transcription factors in B cells, and NF-κB/Rel complexes can inhibit cell apoptosis, we investigated whether the antiapoptotic effect of G28-5, in our system, could be related to modulation of NF-κB/Rel activity. As expected, B-CLL cells displayed significant levels of nuclear NF-κB/Rel activity; p50, RelA, and c-Rel components of the NF-κB/Rel protein family were identified in these complexes. After exposure to fludarabine, NF-κB/Rel complexes were decreased in the nuclei. The addition of G28-5 upregulated the NF-κB/Rel levels. To determine the involvement of NF-κB/Rel activity in the G28-5–mediated inhibition of apoptosis, we blocked the transcription factor with a decoy oligonucleotide, corresponding to the NF-κB/Rel consensus sequence. Cells incubated with the anti-CD40 MoAb in the presence of the decoy oligonucleotide but not a control oligonucleotide displayed a complete impairment of the G28-5 antiapoptotic effect, indicating that NF-κB/Rel activity was required for the inhibition of apoptosis. These results suggest that CD40 triggering in vivo could counteract the apoptotic effect of fludarabine on B-CLL cells and that its neutralization, or the use of NF-κB/Rel inhibitors, could improve the therapeutic effect of fludarabine. © 1998 by The American Society of Hematology.

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Triggering of CD40 Antigen Inhibits Fludarabine-Induced Apoptosis in B Chronic Lymphocytic Leukemia Cells

Blood ◽

10.1182/blood.v92.3.990.415a08_990_995 ◽

1998 ◽

Vol 92 (3) ◽

pp. 990-995 ◽

Cited By ~ 5

Author(s):

Maria Fiammetta Romano ◽

Annalisa Lamberti ◽

Pierfrancesco Tassone ◽

Fiorella Alfinito ◽

Silvia Costantini ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Consensus Sequence ◽

Significant Proportion ◽

Lymphocytic Leukemia ◽

Antiapoptotic Effect ◽

Apoptotic Effect ◽

American Society ◽

Induced Apoptosis ◽

Decoy Oligonucleotide

We analyzed the effect of CD40 triggering on the fludarabine-induced apoptosis of B chronic lymphocytic leukemia (B-CLL) cells. Peripheral blood samples obtained from 15 patients were incubated with fludarabine in the absence or the presence of the anti-CD40 monoclonal antibody (MoAb) G28-5. In 12 patients a significant proportion of apoptotic cells, ranging from 22% to 38% (mean ± SE: 28.5 ± 1.6), were detected after 3 days of culture. In 9 of these samples, the addition of G28-5 reduced apoptosis by at least 30.1% and by 57.1% ± 7.8% on average (P = .0077). Because the CD40 antigen activates NF-κB/Rel transcription factors in B cells, and NF-κB/Rel complexes can inhibit cell apoptosis, we investigated whether the antiapoptotic effect of G28-5, in our system, could be related to modulation of NF-κB/Rel activity. As expected, B-CLL cells displayed significant levels of nuclear NF-κB/Rel activity; p50, RelA, and c-Rel components of the NF-κB/Rel protein family were identified in these complexes. After exposure to fludarabine, NF-κB/Rel complexes were decreased in the nuclei. The addition of G28-5 upregulated the NF-κB/Rel levels. To determine the involvement of NF-κB/Rel activity in the G28-5–mediated inhibition of apoptosis, we blocked the transcription factor with a decoy oligonucleotide, corresponding to the NF-κB/Rel consensus sequence. Cells incubated with the anti-CD40 MoAb in the presence of the decoy oligonucleotide but not a control oligonucleotide displayed a complete impairment of the G28-5 antiapoptotic effect, indicating that NF-κB/Rel activity was required for the inhibition of apoptosis. These results suggest that CD40 triggering in vivo could counteract the apoptotic effect of fludarabine on B-CLL cells and that its neutralization, or the use of NF-κB/Rel inhibitors, could improve the therapeutic effect of fludarabine. © 1998 by The American Society of Hematology.

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Inhibition of Bortezomib-Induced Apoptosis in Chronic Lymphocytic Leukemia by Red Blood Cell Uptake.

Blood ◽

10.1182/blood.v106.11.5027.5027 ◽

2005 ◽

Vol 106 (11) ◽

pp. 5027-5027

Author(s):

Luise M.C. Wheat ◽

Susan L. Kohlhaas ◽

Johan Monbaliu ◽

Roland De Coster ◽

Aneela Majid ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Whole Blood ◽

Mononuclear Cells ◽

Lymphocytic Leukemia ◽

Cell Uptake ◽

Reversible Inhibitor ◽

Apoptotic Pathway ◽

Mutational Status ◽

Induced Apoptosis

Abstract Bortezomib (PS-341/Velcade™) is a reversible inhibitor of the proteasome that has shown promising activity in clinical trials in several malignancies including multiple myeloma, mantle cell lymphoma and follicular lymphoma, including those with refractory disease. However, results have been less encouraging in chronic lymphocytic leukemia (CLL) and we have, therefore, sought to determine the barriers to effective therapy with bortezomib in this disease. Patients with CLL were eligible but were required to have received no therapy in the six months prior to the study. In a panel of 26 patients with CLL, both purified mononuclear cells and whole blood were tested for their apoptotic response to bortezomib (1–100 nM) up to 24 h by flow cytometry and western blotting. In all cases, purified CLL cells were sensitive to bortezomib-induced apoptosis in a concentration and time-dependent fashion, irrespective of stage of disease, resistance to prior therapy, IGHV mutational status or the presence of TP53 mutations. Apoptosis was induced at low (>10 nM) nanomolar concentrations of bortezomib by activation of the intrinsic apoptotic pathway. Bortezomib-induced apoptosis correlated with levels of ubiquitination, Bax activation, and caspase cleavage. Apoptosis of CLL cells was obtained at drug levels readily obtained in vivo using currently-used dosing protocols. However, in vitro, it was necessary to maintain these concentrations for 16–24 hours to obtain maximal apoptosis. Apoptosis measured in a whole blood apoptosis assay was markedly less than in isolated lymphocytes at comparable time points and concentrations. Activity of bortezomib in purified cells was not diminished by addition of exogenous plasma but was abrogated by addition of autologous red blood cells (RBC), suggesting preferential active uptake of the drug by these cells. These data were confirmed in animal models showing preferential distribution of bortezomib to the RBC fraction. RBC uptake may therefore account for the low serum levels of bortezomib attained in vivo during terminal half-life and thus the lack of activity against cells in the peripheral blood. Together with pharmacokinetic and in vivo data, these studies suggest that different dosing schedules of bortezomib other than bolus injections may be more effective in patients with CLL.

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IGF1R Inhibition Induces Apoptosis in Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v120.21.3864.3864 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3864-3864

Author(s):

Niuscha Yaktapour ◽

Rudolf Uebelhart ◽

Christine Dierks ◽

Meike Burger ◽

Dietmar Pfeifer ◽

...

Keyword(s):

Flow Cytometry ◽

Growth Factor ◽

Chronic Lymphocytic Leukemia ◽

Growth Factor Receptor ◽

Lymphocytic Leukemia ◽

Multikinase Inhibitor ◽

Sorafenib Treatment ◽

Igf1r Inhibitor ◽

Induced Apoptosis

Abstract Abstract 3864 Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of monoclonal B lymphocytes. For decades, nucleoside analogs, alkylating agents, and immunotherapeutics have remained the mainstay in treating this disease. Despite major advances in this field, CLL remains incurable with standard therapy. In recent years, preclinical and early clinical data on the use of kinase inhibitors have sparked new hope in the treatment of CLL. The multikinase inhibitor sorafenib, targeting RAF, platelet-derived growth factor receptor (PDGFR), KIT, FMS-like tyrosine kinase 3 (FLT3), and vascular endothelial growth factor receptor (VEGFR), has been approved for the treatment of renal cell carcinoma and hepatocellular carcinoma. Recent studies suggested that CLL cells might be also susceptible to this compound, however the precise mode of action in CLL cells remains elusive. In this study, we identified the Insulin-like growth factor receptor-I (IGF1R) pathway as novel target of sorafenib inducing cell death in CLL cells. Treatment with 10 μM of sorafenib significantly increased apoptosis in primary CLL cells as determined by AnnexinV/PI staining via flow cytometry. Commensurate with its RAF inhibiting properties, the apoptotic effect of sorafenib was accompanied with ERK pathway inhibition. Moreover, sorafenib treatment decreased phosphorylation of SRC and AKT, molecules implicated with IGF1R and insulin receptor (IR) signaling. Interestingly, the latter were strongly expressed in primary CLL cells compared with healthy B cells. Similar to sorafenib, 24 hour treatment of CLL cells with the three structurally distinct IGF1R inhibitors Picropodophyllin, AG1024, and Linsitinib significantly increased apoptosis compared with vehicle control resulting in decreased phophorylation of MEK, ERK, SRC, and AKT. Sorafenib and the IGF1R inhibitor AG1024 also downregulated the expression of IGF1R on CLL cells but not on healthy B cells. To test whether sorafenib modulates IGF-1 binding and thereby influences the IGF1R activation, we biotinylated recombinant IGF-1 and tested its binding to the IGF1R via flow cytometry. We observed a reduced IGF-1 binding after sorafenib treatment. IGF-1 binding after treatment with different IGF1R inhibitors was performed as an internal control. In order to further establish the functional relevance of IGF1R expression in CLL, we performed IGF1R specific and non-silencing siRNA experiments in primary CLL cells. In line with our previous results, IGF1R knockdown resulted in a significant decrease of cell viability and in downregulation of RAF-1 expression, and MEK, ERK, SRC, and AKT phosphorylation. The stromal microenvironment protects CLL cells from spontaneous and drug-induced apoptosis. Sorafenib, AG1024, and Picropodophyllin counteracted the protective effect of microenvironmental factors simulated by the presence of the murine stromal cell line M210B4, the chemokine CXCL12, and the integrin CD49d. Finally, we used the Eμ-Tcl1 transgenic mouse model to validate these results in vivo. Male and female mice (n=8) were treated with 25 mg/kg of the IGF1R inhibitor Linsitinib per oral gavage for 7 days and the amount of CD5/CD19 positive cells was determined flow cytometrically at different time points. We observed a reduction of CD5/CD19-positive cells by 26,1% and 23,2% after 4 and 8 days of treatment, respectively. Our results provide a novel mechanism of action of the multikinase inhibitor sofarenib in CLL cells by blocking IGF1R mediated signaling. IGF1R inhibition by itself induced apoptosis in CLL cells in vitro and in vivo, thus identifying IGF1R as promising target for therapeutic approaches and proposing IGF1R inhibitors for clinical assessment in the therapy of CLL. Disclosures: No relevant conflicts of interest to declare.

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IL-21 mediates apoptosis through up-regulation of the BH3 family member BIM and enhances both direct and antibody-dependent cellular cytotoxicity in primary chronic lymphocytic leukemia cells in vitro

Blood ◽

10.1182/blood-2007-07-099531 ◽

2008 ◽

Vol 111 (9) ◽

pp. 4723-4730 ◽

Cited By ~ 74

Author(s):

Aruna Gowda ◽

Julie Roda ◽

Syed-Rehan A. Hussain ◽

Asha Ramanunni ◽

Trupti Joshi ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Family Member ◽

Lymphocytic Leukemia ◽

Cellular Cytotoxicity ◽

Antibody Dependent Cellular Cytotoxicity ◽

Bh3 Domain ◽

Induced Apoptosis ◽

Primary Chronic Lymphocytic Leukemia

Abstract Interleukin-21 (IL-21) is a recently identified γ-chain receptor cytokine family member that promotes B-cell apoptosis as well as activation of innate immune system. Based on this, we hypothesized that IL-21 might enhance the apoptosis induced by fludarabine and rituximab and also play a role in augmenting immune-mediated clearance of the chronic lymphocytic leukemia (CLL) cells. Our studies demonstrate that the majority of CLL patients have surface IL-21 receptor-α, and its expression correlates with apoptosis, tyrosine phosphorylation of STAT1, and up-regulation of the proapoptotic BH3 domain protein BIM. IL-21–induced BIM up-regulation is critical for apoptosis because inhibition of BIM expression using small interfering RNA prevented IL-21–induced apoptosis. IL-21 treatment of CLL cells but not normal T cells with fludarabine or rituximab additively enhanced the direct cytotoxic effect of these therapies. In addition to its proapoptotic effect, IL-21 promoted STAT1 and STAT5 phosphorylation in natural killer cells with concurrent enhanced antibody-dependent cellular cytotoxicity against rituximab-coated CLL cells in vitro. These data provide justification for combination studies of IL-21 with fludarabine and rituximab in CLL and suggest that BIM up-regulation might serve as relevant pharmacodynamic end point to measure biologic effect of this cytokine in vivo.

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Sildenafil and vardenafil, types 5 and 6 phosphodiesterase inhibitors, induce caspase-dependent apoptosis of B-chronic lymphocytic leukemia cells

Blood ◽

10.1182/blood-2002-01-0075 ◽

2003 ◽

Vol 101 (1) ◽

pp. 265-269 ◽

Cited By ~ 80

Author(s):

Marika Sarfati ◽

Véronique Mateo ◽

Sylvie Baudet ◽

Manuel Rubio ◽

Christine Fernandez ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Phosphodiesterase Inhibitors ◽

Inhibitory Effect ◽

Lymphocytic Leukemia ◽

Interleukin 4 ◽

Drug Induced ◽

Pde Inhibitors ◽

Induced Apoptosis

Abstract Type 4 phosphodiesterase (PDE4) inhibitors reportedly induce apoptosis in chronic lymphocytic leukemia (CLL) cells. Following clinical improvement of one previously untreated CLL patient with sildenafil therapy, we evaluated the in vitro induction of apoptosis in CLL cells by 4 PDE5/6 inhibitors, including sildenafil, vardenafil, zaprinast, and methoxyquinazoline (MQZ). After 24 hours of culture, the various PDE inhibitors differed in their ability to induce apoptosis, with zaprinast displaying no killing effect. Normal B cells isolated from control donors were totally resistant to PDE-induced apoptosis. Vardenafil was 3 and 30 times more potent an inducer of apoptosis than sildenafil and MQZ, respectively. Both vardenafil and sildenafil failed to elevate adenosine 3′5′ cyclic monophosphate (cAMP) levels, largely excluding an inhibitory effect on cAMP-PDE3, -PDE4, and -PDE7. Vardenafil- or sildenafil-treated B-CLL cells displayed up to 30% intracellular active caspase 3. Drug-induced apoptosis was inhibited by the caspase inhibitor z-VAD.fmk, prevented by interleukin-4 (IL-4), and significantly reduced by stromal-derived factor1-α (SDF-1α). We conclude that vardenafil and sildenafil induce caspase-dependent apoptosis of B-CLL cells in vitro and thus might be considered in the treatment of CLL patients. However, further in vivo investigations should be warranted.

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Mediation of apoptosis by and antitumor activity of lumiliximab in chronic lymphocytic leukemia cells and CD23+ lymphoma cell lines

Blood ◽

10.1182/blood-2007-03-082024 ◽

2008 ◽

Vol 111 (3) ◽

pp. 1594-1602 ◽

Cited By ~ 57

Author(s):

Nuzhat I. Pathan ◽

Peter Chu ◽

Kandasamy Hariharan ◽

Carolyn Cheney ◽

Arturo Molina ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Antitumor Activity ◽

B Cell ◽

Cell Lines ◽

Human Monoclonal Antibody ◽

B Cell Lymphoma ◽

Lymphocytic Leukemia ◽

Synergistic Cytotoxicity ◽

Induced Apoptosis

AbstractLumiliximab is a chimeric macaque-human monoclonal antibody to CD23, a protein expressed on virtually all chronic lymphocytic leukemia (CLL) cells. We examined the ability of lumiliximab to mediate apoptosis, antibody-dependent cellular cytotoxicity, and complement-dependent cytotoxicity against primary CLL cells and CD23-expressing B-cell lines. Our data suggest that lumiliximab kills CLL cells and CD23-expressing B cells predominantly by apoptosis, which occurs through the intrinsic pathway. Lumiliximab-induced apoptosis was accompanied by the down-regulation of antiapoptotic proteins Bcl-2, Bcl-XL, and XIAP, activation of Bax, and release of cytochrome c from the mitochondria. We also found that the addition of lumiliximab to rituximab or fludarabine results in synergistic cytotoxicity of primary CLL cells and CD23-expressing B-cell lines. We investigated the in vivo activity of lumiliximab in a human disseminated CD23+ B-cell lymphoma SCID mouse model and found greater antitumor activity with it than with control antibody. We also found that paralysis-free survival was greater with lumiliximab plus rituximab or fludarabine than with any of those agents alone. These results suggest that lumiliximab may be an effective treatment alone or in combination with rituximab or chemotherapy agents in CLL or other CD23-overexpressing B-cell malignancies.

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Regulation of Berg-36 Gene Expression by Survival and Apoptotic Stimuli in B-Chronic Lymphocytic Leukaemia Cells.

Blood ◽

10.1182/blood.v104.11.4767.4767 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4767-4767

Author(s):

Andrew P. Jewell ◽

Maria Baou ◽

Kwee L. Yong ◽

Robert Carr ◽

John Murphy

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Calcium Ionophore ◽

Early Response ◽

Lymphocytic Leukemia ◽

Apoptotic Cells ◽

Cd20 Antibody ◽

Induced Apoptosis ◽

Anti Cd20

Abstract Chronic lymphocytic leukemia is characterized by the accumulation of monoclonal malignant CD5+ B cells that show resistance to apoptosis in vivo. Berg 36 (ZFP36L1, TIS11b, BRF1, cMG1, ERF1) is a zinc finger containing early response gene that was cloned from PMA-stimulated B chronic lymphocytic leukemia cells. Induced expression of this gene has been linked to calcium ionophore and anti-CD20-induced apoptosis of human B lymphoma cells. Berg36 protein has, in common with two other members of the small gene family (TIS11, TIS11d), been reported to function as an mRNA-binding protein that may promote instability of cytokine mRNAs. We have therefore studied the regulation of Berg36 expression in B-CLL cells in vitro, in response to cytokines and other signals that regulate apoptosis. B-CLL cells from 12 patients were purified and incubated with the following agents either alone or in combinations for a number of hours; IL-4, CD40 ligand, PMA or anti-CD20 antibody (rituximab). Apoptosis was measured after 24 hours by Annexin/PI staining and Berg-36 expression by Northern blot analysis. Spontaneous apoptosis in unstimulated B-CLL cells was 20.9±5.1%. Co-incubation of B-CLL cells with IL-4 reduced the percentage of apoptotic cells to 6.2±1.2%, but had no effect on Berg-36 expression. In contrast, both PMA and CD40 stimulation reduced the percentage of apoptotic cells (to 14.8±3.4% and 10.2±4.2% respectively), and markedly induced Berg36 expression. On the other hand, anti-CD20 antibody induced apoptosis (36.2±6.6%), but also induced Berg36 expression. Specific inhibitors of various intracellular signalling molecules confirmed that induction of Berg36 by different agents was mediated through different signalling pathways. In conclusion, expression of Berg36 can be induced in B-CLL cells by stimuli that induce either survival or apoptosis in these cells.

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Interleukin-6 dimers produced by endothelial cells inhibit apoptosis of B-chronic lymphocytic leukemia cells

Blood ◽

10.1182/blood.v97.1.242 ◽

2001 ◽

Vol 97 (1) ◽

pp. 242-249 ◽

Cited By ~ 38

Author(s):

Ana Moreno ◽

Marı́a Luisa Villar ◽

Carmen Cámara ◽

Rosario Luque ◽

Constantino Cespón ◽

...

Keyword(s):

Endothelial Cells ◽

Chronic Lymphocytic Leukemia ◽

Gel Filtration ◽

Lymphocytic Leukemia ◽

Cell Contact ◽

Phagocytic Capacity ◽

Antiapoptotic Effect ◽

Dimeric Form

Abstract Tumoral lymphocytes from patients with B-chronic lymphocytic leukemia (B-CLL) are long-lived cells in vivo, but they die rapidly by apoptosis in vitro. Here, it is reported that endothelial cells (ECs) inhibit the apoptosis of B-CLL cells, as determined by 4 different flow cytometric methods, and that this antiapoptotic effect is mediated mainly by soluble factor(s), as can be deduced from the following findings. First, EC-conditioned medium (ECCM) inhibited the apoptotic rate in B-CLL to approximately 50% of control. Second, the antiapoptotic effect mediated by EC/B-CLL cell contact was more apparent than real; using a fluorescence-based phagocytosis assay, it was demonstrated that this effect was due to the phagocytic capacity of ECs, which internalized apoptotic cells. Third, the protective effect of ECCM was associated neither with proliferation nor differentiation signals. Fourth, the survival factor was a dimeric form of IL-6 because anti–IL-6 antibodies completely neutralized the antiapoptotic effect mediated not only by the crude ECCM but also by the 45- to 55-kd active fractions obtained after gel filtration, which contained high levels of IL-6. These IL-6 dimers (IL-6D) were noncovalently associated. Sixth, human recombinant IL-6D(hrIL-6D) inhibited B-CLL apoptosis, whereas hrIL-6 monomers (hrIL-6M) did not. Binding and functional competition experiments showed not only that monomers and dimers had similar affinity for the IL-6R, but also that hrIL-6Minhibited the antiapoptotic activity of hrIL-6D. These data suggest that IL-6D derived from ECs promote the survival of B-CLL cells.

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