B-cell Chronic Lymphocytic Leukemia Cells Research Articles

PO-032 Displacement of hexokinase 2 from mitochondria induces mitochondrial Ca2 +overload and caspase-independent cell death in cancer cells

IntroductionHexokinase 2 (HK2) phosphorylates glucose for starting its utilisation in glycolysis and pentose phosphate pathway. In many cancer cell types these processes are enhanced and HK2 expression is strongly induced and mainly localised to the outer mitochondrial membrane, where it also exerts an anti-apoptotic activity. Genetic ablation in mouse highlights HK2 importance in tumour formation. Therefore, HK2 is a good target for antineoplastic strategies, but HK2 inhibitors can have important side effects as they affect glucose metabolism. Here we have developed an antineoplastic strategy based on HK2 detachment from mitochondria in order to induce tumour cell death without inhibiting hexokinase enzymatic activity.Material and methodsPeptide design and synthesis; hexokinase enzymatic activity assays. Measurements of mitochondrial membrane potential, intracellular Ca2+ levels, cell death and in vitro and in vivo tumorigenic assays on human and mouse cancer cell models (CT26 colon cancer cells, 4 T1 breast cancer cells, HeLa cervix carcinoma cells and primary human B-CLL cells).Results and discussionsWe have observed that in cancer cells HK2 locates at contact sites between mitochondria and endoplasmic reticulum called MAMs (mitochondria-associated membranes). We could selectively detach HK2 from MAMs by using a peptide that does not perturb hexokinase enzymatic activity. This treatment rapidly induces opening of the Inositol-3-Phospate-Receptor and the ensuing Ca2+ transfer from endoplasmic reticulum to mitochondria. As a consequence, a Ca2+ overload occurs in mitochondria, leading to permeability transition pore opening, mitochondrial membrane depolarization and apoptosis in a caspase-independent way. Peptide administration reduces allografic growth of breast and colon cancer cells without any noxious effect on healthy tissues, and elicits death of B-cell chronic lymphocytic leukaemia (B-CLL) cells freshly obtained by patients and in vivo.ConclusionWe have reported that HK2 locates in MAMs of cancer cells, where it acts as an important player in the control of their survival. Targeting HK2 with a peptide-based strategy constitutes a novel and promising anti-neoplastic approach.

SPOT-008 Neurotensin receptor type 2 protects B-cell chronic lymphocytic leukaemia cells from apoptosis

Introduction B-cell chronic lymphocytic leukaemia (B-CLL) cells are resistant to apoptosis, and consequently accumulate to the detriment of normal B cells and patient immunity. Because current therapies fail to eradicate these apoptosis-resistant cells, it is essential to identify alternative survival pathways as novel targets for anticancer therapies. Material and methods B-cells were isolated from the peripheral blood of healthy donors and CLL patients (n=34) using the MACSxpress human B-CLL Cell Isolation Kit. Gene expression was assessed by RT-qPCR, protein expression and signalling pathway activation was assessed by Western Blot. Protein-protein interaction was confirmed by both Indirect Immuno-Fluorescence and co-immunoprecipitations. Cell death was assessed by Elisa Cell Death as well as Flow Cytometry. Studied receptors were inhibited either by RNA interference or pharmacological inhibitors. Results and discussions In this study, we identified neurotensin receptor 2 (NTSR2) as an essential driver of apoptosis resistance in B-CLL. NTSR2 was highly expressed in B-CLL cells, whereas expression of its natural ligand, neurotensin (NTS), was minimal in both B-CLL cells and patient plasma. Surprisingly, NTSR2 remained in a constitutively active phosphorylated state, caused not by a mutation-induced gain-of-function but rather by an interaction with the oncogenic tyrosine kinase receptor TrkB. Functional and biochemical characterisation revealed that the NTSR2–TrkB interaction acts as a conditional oncogenic driver requiring the TrkB ligand BDNF, which unlike NTS is highly expressed in B-CLL cells. Together, NTSR2, TrkB and BDNF induce autocrine and/or paracrine survival pathways that are independent of mutation status and indolent or progressive disease course. The NTSR2–TrkB interaction activates survival signalling pathways, including the Src and AKT kinase pathways, as well as expression of the anti-apoptotic proteins Bcl-2 and Bcl-xL. When NTSR2 was downregulated, TrkB failed to protect B-CLL cells from a drastic decrease in viability via typical apoptotic cell death. Conclusion Together, our findings demonstrate that the NTSR2–TrkB interaction and the sustained activation of the signalling pathways under the control of these two actors plays a crucial role in B-CLL cell survival, suggesting that inhibition of NTSR2 represents a promising targeted strategy for treating B-CLL malignancy.

Proteomics and metabolomics identify molecular mechanisms of aging potentially predisposing for chronic lymphocytic leukemia

B cell chronic lymphocytic leukemia (B-CLL), the most common type of leukemia in adults, is still essentially incurable despite the development of novel therapeutic strategies. This reflects the incomplete understanding of the pathophysiology of this disease. A comprehensive proteome analysis of primary human B-CLL cells and B cells from younger as well as elderly healthy donors was performed. For comparison, the chronic B cell leukemia cell line JVM-13 was also included. A principal component analysis comprising 6,945 proteins separated these four groups, placing B cells of aged-matched controls between those of young donors and B-CLL patients, while identifying JVM-13 as poorly related cells. Mass spectrometric proteomics data have been made fully accessible via ProteomeXchange with identifier PXD006570-PXD006572, PXD006576, PXD006578, and PXD006589-PXD006591. Remarkably, B cells from aged controls displayed significant regulation of proteins related to stress management in mitochondria and ROS stress such as DLAT, FIS1, and NDUFAB1, and DNA repair, including RAD9A, MGMT, and XPA. ROS levels were indeed found significantly increased in B cells but not in T cells or monocytes from aged individuals. These alterations may be relevant for tumorigenesis and were observed similarly in B-CLL cells. In B-CLL cells, some remarkable unique features like the loss of tumor suppressor molecules PNN and JARID2, the stress-related serotonin transporter SLC6A4, and high expression of ZNF207, CCDC88A, PIGR and ID3, otherwise associated with stem cell phenotype, were determined. Alterations of metabolic enzymes were another outstanding feature in comparison to normal B cells, indicating increased beta-oxidation of fatty acids and increased consumption of glutamine. Targeted metabolomics assays corroborated these results. The present findings identify a potential proteome signature for immune senescence in addition to previously unrecognized features of B-CLL cells and suggest that aging may be accompanied by cellular reprogramming functionally relevant for predisposing B cells to transform to B-CLL cells.

CD150 AND CD180 ARE INVOLVED IN REGULATION OF TRANSCRIPTION FACTORS EXPRESSION IN CHRONIC LYMPHOCYTIC LEUKEMIA CELLS

Sequential stages of B-cell development is stringently coordinated by transcription factors (TFs) network that include B-lineage commitment TFs (Ikaros, Runx1/Cbfb, E2A, and FOXO1), B-lineage maintenance TFs (EBF1and PAX5) and stage specific set of TFs (IRF4, IRF8, BCL6, BLIMP1). Deregulation of TFs expression and activity is often occurs in malignant B cells. The aim of this study was to evaluate TFs expression in chronic lymphocytic leukemia cells taking into consideration CD150cell surface expression. From other side we attempted to regulate TFs expression via CD150and CD180cell surface receptors. Studies were performed on normal peripheral blood B-cell subpopulations and chronic lymphocytic leukemia(CLL) cells isolated from peripheral blood of 67primary untreated patients with CLL. Evaluation of TFs expression was performed on mRNA level using qRT-PCR and on protein level by western blot analysis. Median of PAX5and EBF1mRNA expression was higher in cell surface CD150positive (csCD150+) compared to csCD150- CLL cases or normal CD19+ and CD19+CD5+ B-cell subsets. Differences in mRNA expression of IRF8, IRF4and BLIMP1between studied groups of CLL and normal B cells were not revealed. All CLL cases were characterized by downregulated expression of PU.1and BCL6mRNAs in comparison to normal B cells. At the same time elevated SPIB mRNA expression level was restricted to CLL cells. Protein expression of IRF4, IRF8 and BCL6was uniformly distributed between csCD150- and csCD150+ CLL cases. PU.1protein and CD20that is direct PU.1target gene positively correlated with CD150cell surface expression on CLL cells. Ligation of CD150and CD180alone or in combination upregulated IRF8and PU.1while downregulated the IRF4mRNA expression. Signaling via CD150or CD180alone elevated the level of BCL6mRNA. Strong downregulation of IRF4mRNA was observed after CD150, CD180or CD150 andCD180 coligation on CLL cells. We found that in CLL cells CD150is a negative regulator of SPIB while CD180is involved in upregulation of EBF1expression level. Moreover, CD180ligation on CLL cells caused increase of CD150mRNA level that is a one of the EBF1 target genes. Analysis of TFs expression profile revealed upregulated SPIB mRNA level and downregulated PU.1 inCLL cells. CD150and CD180receptors may modulate transcriptional program in CLL cells by regulating the TFs expression levels.

Neurotensin receptor type 2 protects B-cell chronic lymphocytic leukemia cells from apoptosis

B-cell chronic lymphocytic leukemia (B-CLL) cells are resistant to apoptosis, and consequently accumulate to the detriment of normal B cells and patient immunity. Because current therapies fail to eradicate these apoptosis-resistant cells, it is essential to identify alternative survival pathways as novel targets for anticancer therapies. Overexpression of cell-surface G protein-coupled receptors drives cell transformation, and thus plays a critical role in malignancies. In this study, we identified neurotensin receptor 2 (NTSR2) as an essential driver of apoptosis resistance in B-CLL. NTSR2 was highly expressed in B-CLL cells, whereas expression of its natural ligand, neurotensin (NTS), was minimal in both B-CLL cells and patient plasma. Surprisingly, NTSR2 remained in a constitutively active phosphorylated state, caused not by a mutation-induced gain-of-function but rather by an interaction with the oncogenic tyrosine kinase receptor TrkB. Functional and biochemical characterization revealed that the NTSR2–TrkB interaction acts as a conditional oncogenic driver requiring the TrkB ligand brain-derived neurotrophic factor (BDNF), which unlike NTS is highly expressed in B-CLL cells. Together, NTSR2, TrkB and BDNF induce autocrine and/or paracrine survival pathways that are independent of mutation status and indolent or progressive disease course. The NTSR2–TrkB interaction activates survival signaling pathways, including the Src and AKT kinase pathways, as well as expression of the anti-apoptotic proteins Bcl-2 and Bcl-xL. When NTSR2 was downregulated, TrkB failed to protect B-CLL cells from a drastic decrease in viability via typical apoptotic cell death, reflected by DNA fragmentation and Annexin V presentation. Together, our findings demonstrate that the NTSR2–TrkB interaction plays a crucial role in B-CLL cell survival, suggesting that inhibition of NTSR2 represents a promising targeted strategy for treating B-CLL malignancy.

Role for ZAP-70 Signaling in the Differential Effector Functions of Rituximab and Obinutuzumab (GA101) in Chronic Lymphocytic Leukemia B Cells.

Rituximab (RTX) has been the hallmark anti-CD20 mAb for the treatment of B cell neoplasms, including B cell chronic lymphocytic leukemia (B-CLL). Recently, a novel humanized anti-CD20 mAb obinutuzumab (GA101) has been implemented as first-line CLL therapy. Treatment of CLL patients with RTX is associated with CD20 loss via an FcγR-mediated process, trogocytosis. RTX-induced trogocytosis has been characterized as both the means of resistance to therapy, via loss of cell surface target proteins (antigenic modulation), as well as a process that alters B cell phenotype and function. This study investigates the nature and clinical relevance of GA101-mediated trogocytosis. In this study, we demonstrate that GA101 is a more potent mediator of trogocytosis than RTX in vitro in both normal B cells and B-CLL cells. Qualitative differences in the effector function of these anti-CD20 Abs appear specific to B-CLL cells. GA101-mediated CD19 and CD20 trogocytosis from B-CLL cells is associated with its ability to induce homotypic adhesion (HA). The degree of HA varies between CLL patients and positively correlates with the expression of ZAP-70, a BCR-associated kinase. Deregulation of ZAP-70 using tyrosine kinase inhibitors, gefitinib or ibrutinib, diminishes HA formation and trogocytosis by GA101. Taken together, these findings elucidate the differences in trogocytosis and HA formation mediated by anti-CD20 mAbs RTX and GA101, as well as provide a novel link between ZAP-70 expression and these effector functions.

Enhanced CDC of B cell chronic lymphocytic leukemia cells mediated by rituximab combined with a novel anti-complement factor H antibody.

Rituximab therapy for B cell chronic lymphocytic leukemia (B-CLL) has met with mixed success. Among several factors to which resistance can be attributed is failure to activate complement dependent cytotoxicity (CDC) due to protective complement regulatory proteins, including the soluble regulator complement factor H (CFH). We hypothesized that rituximab killing of non-responsive B-CLL cells could be augmented by a novel human monoclonal antibody against CFH. The B cells from 11 patients with B-CLL were tested ex vivo in CDC assays with combinations of CFH monoclonal antibody, rituximab, and a negative control antibody. CDC of rituximab non-responsive malignant B cells from CLL patients could in some cases be augmented by the CFH monoclonal antibody. Antibody-mediated cytotoxicity of cells was dependent upon functional complement. In one case where B-CLL cells were refractory to CDC by the combination of rituximab plus CFH monoclonal antibody, additionally neutralizing the membrane complement regulatory protein CD59 allowed CDC to occur. Inhibiting CDC regulatory proteins such as CFH holds promise for overcoming resistance to rituximab therapy in B-CLL.

Activated Allogeneic NK Cells Preferentially Kill Poor Prognosis B-Cell Chronic Lymphocytic Leukemia Cells.

Mutational status of TP53 together with expression of wild-type (wt) IGHV represents the most widely accepted biomarkers, establishing a very poor prognosis in B-cell chronic lymphocytic leukemia (B-CLL) patients. Adoptive cell therapy using allogeneic HLA-mismatched Natural killer (NK) cells has emerged as an effective and safe alternative in the treatment of acute myeloid and lymphoid leukemias that do not respond to traditional therapies. We have described that allogeneic activated NK cells eliminate hematological cancer cell lines with multidrug resistance acquired by mutations in the apoptotic machinery. This effect depends on the activation protocol, being B-lymphoblastoid cell lines (LCLs) the most effective stimulus to activate NK cells. Here, we have further analyzed the molecular determinants involved in allogeneic NK cell recognition and elimination of B-CLL cells, including the expression of ligands of the main NK cell-activating receptors (NKG2D and NCRs) and HLA mismatch. We present preliminary data suggesting that B-CLL susceptibility significantly correlates with HLA mismatch between NK cell donor and B-CLL patient. Moreover, we show that the sensitivity of B-CLL cells to NK cells depends on the prognosis based on TP53 and IGHV mutational status. Cells from patients with worse prognosis (mutated TP53 and wt IGHV) are the most susceptible to activated NK cells. Hence, B-CLL prognosis may predict the efficacy of allogenic activated NK cells, and, thus, NK cell transfer represents a good alternative to treat poor prognosis B-CLL patients who present a very short life expectancy due to lack of effective treatments.

Fluorescence lifetime imaging of DAPI-stained nuclei as a novel diagnostic tool for the detection and classification of B-cell chronic lymphocytic leukemia.

B-cell chronic lymphocytic leukaemia (B-CLL) and B-cell precursor acute lymphoblastic leukaemia (B-ALL) are the most common type of leukaemia in adults and children, respectively. Today, fluorescence in situ hybridization (FISH) is the standard for detecting chromosomal aberrations that reflect adverse and favorable outcome. This study revealed a new, simple, and fast diagnostic tool to detect pathological cells by measuring and imaging the fluorescence lifetime (FLT) using FLT imaging microscopy (FLIM) of the peripheral blood (PB) cells of B-CLL samples that were labeled with the DNA binder, DAPI. The FLT of DAPI in healthy individuals was found to be 2.66 ± 0.12 ns. In contrast, PB cells of B-CLL and BM cells of B-ALL patients were characterized by a specific group distribution of the FLT values. The FLT of DAPI was divided into four subgroups, relative to 2.66 ns: short+, normal, prolonged, and prolonged+. These alterations could be related to different chromatin arrangements of B-CLL and B-ALL interphase nuclei. Notably, extremely long FLT of nuclear DAPI correlate with the presence of extra chromosome 12, while moderate increases compared to normal characterize the deletion of p53. Such correlations potentially enable a FLT-based rapid automatic diagnosis and classification of B-CLL even when the frequency of genetic and chromosomal abnormalities is low. © 2016 International Society for Advancement of Cytometry.

The prognostic significance of surface dipeptidylpeptidase IV (CD26) expression in B-cell chronic lymphocytic leukemia

A number of factors related to B-cell chronic lymphocytic leukemia (B-CLL) patients' prognosis have been identified. However, still some factors better reflecting disease activity in individual cases are explored. The study aimed to evaluate prognostic significance of dipeptidylpeptidase IV/CD26 expression on B-CLL cells and its relationship with other well established prognostic factors. The study included 94 patients with newly diagnosed B-CLL and involved analysis of clinical, laboratory, flow-cytometry and cytogenetic data. Detailed analysis showed that CD26 expression on B-CLL cells correlates with Rai’s clinical stage of the disease at diagnosis (p=0.034), β2-microglobulin concentration (p=0.012), lactic acid dehydrogenase activity (p=0.045) and absolute lymphocytes’ count (p=0.027) in the blood. The multivariate analysis revealed that time to treatment (TTT) was significantly influenced by Rai clinical stage, LDH activity in blood and CD26 expression on B-CLL cell’s. Moreover, in the multivariate analysis restricted to the group of patients with documented cytogenetic risk (n=36) CD26 expression, Rai clinical stage and cytogenetic profile remained their independent impact on TTT. The results of our study indicate that the CD26 expression should be incorporated in B-CLL patients risk assessment along with well known prognostic factors, since it seems to have a relationship with the tumor mass and influences TTT.

The role of IL-10 in B-cell chronic lymphocytic leukemia cell survival

Abstract B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the progressive accumulation of abnormal lymphocytes in the periphery. Typically the disease progression is slow, but as the number of leukemic cells increases, they interfere with the production of other important blood cells, causing the patients to be in an immunosuppressive state. To study B-CLL we use cells from the transgenic Eμ-Tcl1 mouse, which spontaneously develop B-CLL. Previously we showed that Eμ-Tcl1 CLL cells produce an anti-inflammatory cytokine, IL-10, constitutively. Here we studied the role of IL-10 in CLL cell survival in vitro and the development of CLL in vivo. We found that neutralization of IL-I0 using anti-IL-10 antibodies or blocking the IL-10 receptor (IL-10R) using anti-IL-10R antibodies did not effect the survival of CLL cells in vitro. When we adoptively transferred Eμ-Tcl1 cells into IL-10R KO mice, we found that cells grew at a slower rate in IL-10R KO mice vs. wild type mice. We also found that there was a significant reduction in CLL cell engraftment in the spleen, bone marrow, peritoneal cavity and liver of the IL-10R KO mice. Further studies revealed that IL-10 could be playing a role in the tumor microenvironment possibly by affecting anti-tumor immunity. This was seen by a reduction in the activation of CD8+ T cells as well as a significantly lower production of IFN-γ by CD4+ T cells purified from CLL-injected WT mice in compare to the IL-10R KO injected mice. In current in vivo studies, CLL growth will be monitored in WT mice after IL-10 neutralization using anti-IL-10 monoclonal antibody to further elucidate the role of IL-10 in CLL development. These studies demonstrate that CLL cells suppress host anti-tumor immunity via IL-10 production.

Synergistic induction of apoptosis in B-cell chronic lymphocytic leukemia cells after treatment with all-trans retinoic acid in combination with interleukin-21 and rituximab.

B-cell chronic lymphocytic leukemia (B-CLL) is the most common adult leukemia in the Western world and characterized by the progressive expansion of malignant B lymphocytes in peripheral blood. In spite of advances in sciences to recognize the number of effective agents for the treatment of chronic lymphocytic leukemia (CLL), this leukemia is thought as incurable one. Introducing a new therapy that has a direct effect on B-CLL lymphocytes and no cytotoxic effects on the other cells is a great wish. Twenty-one patients with B-CLL were enrolled in the study. Peripheral blood mononuclear cells (PBMCs) were isolated from patients' blood samples and were further treated with all-tans retinoic acid (ATRA), interleukin (IL-21), and rituximab at concentrations of 30 ng/ml, 25 ng/ml, and 4 μg/ml, respectively. ATRA, IL21, and rituximab were used alone or in various combinations and their effects on apoptosis were measured using annexin V-fluorescein isothiocyanate apoptosis detection kit. Treatment of the patients' cells with IL21 and rituximab showed a synergistic effect on the induction of apoptosis, in comparison with untreated CLL cells (P < 0.05). The induction of apoptosis by ATRA in combination with IL21 and rituximab were significantly increased compared to untreated CLL cells as a negative control (P < 0.01). Treatment of patients' PBMCs by ATRA in combination with IL21 and rituximab and also IL21 in combination with rituximab showed synergistic induction of apoptosis compared to untreated CLL cells as a negative control (P < 0.01). It seems that ATRA in combination with IL21 and rituximab activate different pathways of apoptosis (extrinsic pathway, intrinsic pathway, and granzyme B pathway).

Quantitative and Qualitative Analysis of Regulatory T Cells (Tregs) in B Cell Chronic Lymphocytic Leukemia (B-CLL)

Introduction: T regulatory cells are immunosuppressive cells considered to play an important role in cancer biology and autoimmunity by suppressing host immune response and autoreactive lymphocytes respectively. Several studies reveal that Treg cells act by suppressing anti-tumor immune response, through the targeting of other immune cells, such as T cells, B cells and dendritic cells. Accumulated data indicate a significant role of T cell dysfunction in the pathogenesis of CLL.Aims: The scope of this study is the analysis of numerical and functional abnormalities of Tregs in B-CLL with the view to elucidate their role in the pathogenesis of the disease.Methods: Treg cells derived from 44 untreated B-CLL patients with a median age 62 and 17 healthy donors were analyzed by Flow cytometry, using the following antibodies: CD45Ro-FITC/CD45RA-PE/CD4-ECD/CD25-PC5/CD127-PC7, CD1a-FITC/CD137-PE/CD4-ECD/CD25-PC5/CD127-PC7, CD95-FITC/cyCD152-PE/CD4-ECD/CD25-PC5/CD127-PC7, beads/FoxP3-PE/CD4-ECD/CD25-PC5/CD127-PC7, Annexin V-FITC/CD4-ECD/CD25-PC5/CD127-PC7. For the functional analysis, peripheral blood was obtained from 20 patients with B-CLL. Mononuclear cells were isolated using Ficoll-Paque gradient centrifugation. CD4+ CD25+ (Treg cells), CD4+ CD25- (T effectοr cells, Teff), CD5+ CD19+ (B-CLL) and CD5- CD19+ (Normal B, NB) cells were separated using magnetic antibody cell sorting. To test the functionality of the assayed Tregs, the isolated cell populations were cultured in a 96-well plate (Tregs, Teff, B-CLL cells, NB cells, B-CLL cells: Tregs in 1:20 ratio, B-CLL cells: Teff in 1:20 ratio, NB cells: Tregs in 1:20 ratio, NB cells: Teff in 1:20 ratio) and their proliferative capacity was measured using the BrdU assay. To further analyze the functional role of Tregs, peripheral blood was obtained from 22 patients with CLL and 22 healthy donors. Mononuclear cells were isolated using Ficoll-Paque gradient centrifugation. CD4+ CD25+ CD127dim/- (Treg cells), CD5+ CD19+ (B-CLL) and CD5- CD19+ (Normal B, NB) cells were separated using magnetic antibody cell sorting and were co-cultured in a 96-well plate in a 1:10 ratio. The apoptosis of B cells was determined by the Annexin V/PI method.Results: FACS analysis of the Treg cells resulted at the following observations: The Treg absolute cell number (cells/μL), estimated either as the number of CD4+ CD25+ CD127- cells or as the number of CD4+ CD25+ FoxP3+ cells, was statistically significantly higher in patients' samples than in controls (CD127- 21.65 vs 7.35, p=0.001; FoxP3+ 20.42 vs 6.5, p= 0.001). Annexin V expression in Treg cells from BCLL patients was significantly lower compared to controls (3.626 vs 38.615, p=0.003).The functional analysis of Treg cells through BrdU assay indicated that CLL Tregs were able to suppress the proliferation of Teff cells (p=0.002) and that Teff cells were in turn able to significantly suppress the proliferation of B-CLL cells (p=0.05).Moreover, FACS analysis through Annexin V/PI method indicated that Treg CLL cells significantly decrease the apoptosis rate of NB cells after their co-culturing, compared to NB cells (p<0,02). On the contrary, healthy donors derived Treg cells significantly increase the apoptosis of B-CLL cells after their co-culturing, compared to B-CLL cells (p<0.025). Interestingly, no significant alterations were observed after culturing NB cells with Tregs from healthy donors and B-CLL cells with Treg CLL cells.Conclusions: In CLL patients, Treg cells are significantly higher and present with lower apoptotic levels compared to healthy donors. The functional analysis indicates that T effector cells suppress the proliferation of B-CLL cells and T effector cells are suppressed by Tregs indicating that the increased number of Tregs observed in CLL contributes indirectly to the proliferation of the CLL clone. These data are further supported by our observations that CLL derived Treg cells appear rather incapable of inducing apoptosis of both NB cells and B-CLL cells, in contrast to normal Tregs, suggesting an immunoediting effect of B-CLL cells on Tregs which negatively affects the functionality of the latter. Therefore, Treg cells in CLL do not efficiently eliminate the abnormal clone and play an important role in the pathogenesis of the disease. The molecular underlying mechanisms need to be further elucidated. DisclosuresNo relevant conflicts of interest to declare.

TLR-9 and IL-15 Synergy Promotes the In Vitro Clonal Expansion of Chronic Lymphocytic Leukemia B Cells.

Clinical progression of B cell chronic lymphocytic leukemia (B-CLL) reflects the clone's Ag receptor (BCR) and involves stroma-dependent B-CLL growth within lymphoid tissue. Uniformly elevated expression of TLR-9, occasional MYD88 mutations, and BCR specificity for DNA or Ags physically linked to DNA together suggest that TLR-9 signaling is important in driving B-CLL growth in patients. Nevertheless, reports of apoptosis after B-CLL exposure to CpG oligodeoxynucleotide (ODN) raised questions about a central role for TLR-9. Because normal memory B cells proliferate vigorously to ODN+IL-15, a cytokine found in stromal cells of bone marrow, lymph nodes, and spleen, we examined whether this was true for B-CLL cells. Through a CFSE-based assay for quantitatively monitoring in vitro clonal proliferation/survival, we show that IL-15 precludes TLR-9-induced apoptosis and permits significant B-CLL clonal expansion regardless of the clone's BCR mutation status. A robust response to ODN+IL-15 was positively linked to presence of chromosomal anomalies (trisomy-12 or ataxia telangiectasia mutated anomaly + del13q14) and negatively linked to a very high proportion of CD38(+) cells within the blood-derived B-CLL population. Furthermore, a clone's intrinsic potential for in vitro growth correlated directly with doubling time in blood, in the case of B-CLL with Ig H chain V region-unmutated BCR and <30% CD38(+) cells in blood. Finally, in vitro high-proliferator status was statistically linked to diminished patient survival. These findings, together with immunohistochemical evidence of apoptotic cells and IL-15-producing cells proximal to B-CLL pseudofollicles in patient spleens, suggest that collaborative ODN and IL-15 signaling may promote in vivo B-CLL growth.

Metformin inhibits cell cycle progression of B-cell chronic lymphocytic leukemia cells.

B-cell chronic lymphocytic leukemia (CLL) was believed to result from clonal accumulation of resting apoptosis-resistant malignant B lymphocytes. However, it became increasingly clear that CLL cells undergo, during their life, iterative cycles of re-activation and subsequent clonal expansion. Drugs interfering with CLL cell cycle entry would be greatly beneficial in the treatment of this disease. 1, 1-Dimethylbiguanide hydrochloride (metformin), the most widely prescribed oral hypoglycemic agent, inexpensive and well tolerated, has recently received increased attention for its potential antitumor activity. We wondered whether metformin has apoptotic and anti-proliferative activity on leukemic cells derived from CLL patients. Metformin was administered in vitro either to quiescent cells or during CLL cell activation stimuli, provided by classical co-culturing with CD40L-expressing fibroblasts. At doses that were totally ineffective on normal lymphocytes, metformin induced apoptosis of quiescent CLL cells and inhibition of cell cycle entry when CLL were stimulated by CD40-CD40L ligation. This cytostatic effect was accompanied by decreased expression of survival- and proliferation-associated proteins, inhibition of signaling pathways involved in CLL disease progression and decreased intracellular glucose available for glycolysis. In drug combination experiments, metformin lowered the apoptotic threshold and potentiated the cytotoxic effects of classical and novel antitumor molecules. Our results indicate that, while CLL cells after stimulation are in the process of building their full survival and cycling armamentarium, the presence of metformin affects this process.

Constitutive IL-10 production by normal and malignant B-1 cells is dependent on B cell receptor signaling (IRM10P.620)

Abstract The most common human leukemia is B-cell chronic lymphocytic leukemia (B-CLL), which can have immunosuppressive effects. CLL cells share multiple phenotypic markers with B-1 B cells such as their surface expression of CD5. This study identifies some functional similarities between B-1 and CLL cells. B-1 B cells have been shown to produce a high level of interleukin-10 (IL-10) constitutively and upon toll-like receptor stimulation, a characteristic property of regulatory B cells. Eμ-Tcl1 mice, which express the Tcl1 oncogene in a B cell specific manner, spontaneously develop B-CLL. We show that Eμ-Tcl1 mouse B-CLL cells also produce IL-10 constitutively. The constitutively produced IL-10 was not required for CLL survival in vitro. This study describes a novel role of B cell receptor (BCR) signaling pathway in constitutive IL-10 secretion by normal and malignant B-1 cells. Thus inhibition of Src family kinases, spleen tyrosine kinase (Syk) or Bruton’s tyrosine kinase (Btk), all of which are downstream of BCR, reduces constitutive IL-10 production by both normal and malignant B-1 cells. Analysis of a published microarray study showed that IL-10 levels were higher in peripheral blood of CLL patients than normal human blood samples, which correlated positively with STAT3 levels. Interestingly, we found that BCR signaling induced IL-10 production is independent of STAT3. Current studies are directed at understanding the mechanisms by which BCR signaling affects IL-10 production.

ID helix-loop-helix proteins as determinants of cell survival in B-cell chronic lymphocytic leukemia cells in vitro.

BackgroundMembers of the inhibitor of DNA-binding (ID) family of helix-loop-helix proteins have been causally implicated in the pathogenesis of several types of B-cell lineage malignancy, either on the basis of mutation or by altered expression. B-cell chronic lymphocytic leukemia encompasses a heterogeneous group of disorders and is the commonest leukaemia type in the Western world. In this study, we have investigated the pathobiological functions of the ID2 and ID3 proteins in this disease with an emphasis on their role in regulating leukemic cell death/survival.MethodsBioinformatics analysis of microarray gene expression data was used to investigate expression of ID2/ID3 in leukemic versus normal B cells, their association with clinical course of disease and molecular sub-type and to reconstruct a gene regulatory network using the ‘maximum information coefficient’ (MIC) for target gene inference. In vitro cultured primary leukemia cells, either in isolation or co-cultured with accessory vascular endothelial cells, were used to investigate ID2/ID3 protein expression by western blotting and to assess the cytotoxic response of different drugs (fludarabine, chlorambucil, ethacrynic acid) by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. ID2/ID3 protein levels in primary leukemia cells and in MEC1 cells were manipulated by transduction with siRNA reagents.ResultsDatamining showed that the expression profiles of ID2 and ID3 are associated with distinct pathobiological features of disease and implicated both genes in regulating cell death/survival by targeting multiple non-overlapping sets of apoptosis effecter genes. Consistent with microarray data, the overall pattern of ID2/ID3 protein expression in relation to cell death/survival responses of primary leukemia cells was suggestive of a pro-survival function for both ID proteins. This was confirmed by siRNA knock-down experiments in MEC1 cells and in primary leukemia cells, but with variability in the dependence of leukemic cells from different patients on ID protein expression for cell survival. Vascular endothelial cells rescued leukemia cells from spontaneous and cytotoxic drug-induced cell death at least in part, via an ID protein-coupled redox-dependent mechanism.ConclusionsOur study provides evidence for a pro-survival function of the ID2/ID3 proteins in chronic lymphocytic leukemia cells and also highlights these proteins as potential determinants of the pathobiology of this disorder.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-014-0286-9) contains supplementary material, which is available to authorized users.

Human NK cells activated by EBV+ lymphoblastoid cells overcome anti-apoptotic mechanisms of drug resistance in haematological cancer cells

Natural killer (NK) cells recognize and eliminate transformed or infected cells that have downregulated MHC class-I and express specific activating ligands. Recent evidence indicates that allogeneic NK cells are useful to eliminate haematological cancer cells independently of MHC-I expression. However, it is unclear if transformed cells expressing mutations that confer anti-apoptotic properties and chemoresistance will be susceptible to NK cells. Allogeneic primary human NK cells were activated using different protocols and prospectively tested for their ability to eliminate diverse mutant haematological and apoptotic-resistant cancer cell lines as well as patient-derived B-cell chronic lymphocytic leukemia cells with chemotherapy multiresistance. Here, we show that human NK cells from healthy donors activated in vitro with Epstein Barr virus positive (EBV+)-lymphoblastoid cells display an enhanced cytotoxic and proliferative potential in comparison to other protocols of activation such a K562 cells plus interleukin (IL)2. This enhancement enables them to kill more efficiently a variety of haematological cancer cell lines, including a panel of transfectants that mimic natural mutations leading to oncogenic transformation and chemoresistance (e.g., overexpression of Bcl-2, Bcl-XL and Mcl-1 or downregulation of p53, Bak/Bax or caspase activity). The effect was also observed against blasts from B-cell chronic lymphocytic leukemia patients showing multi-resistance to chemotherapy. Our findings demonstrate that particular in vitro activated NK cells may overcome anti-apoptotic mechanisms and oncogenic alterations frequently occurring in transformed cells, pointing toward the use of EBV+-lymphoblastoid cells as a desirable strategy to activate NK cells in vitro for the purpose of treating haematological neoplasia with poor prognosis.

HA14-1 potentiates apoptosis in B-cell cancer cells sensitive to a peptide disrupting IP 3 receptor / Bcl-2 complexes.

Anti-apoptotic B-cell lymphoma 2 (Bcl-2) is commonly upregulated in hematological cancers, including B-cell chronic lymphocytic leukemia (B-CLL) and diffuse large B-cell lymphoma (DLBCL), thereby protecting neoplastic cells from oncogenic-stress-induced apoptosis. Bcl-2 executes its anti-apoptotic function at two different sites in the cell. At the mitochondria, Bcl-2 via its hydrophobic cleft interacts with pro-apoptotic Bcl-2 family members to inhibit apoptosis. At the endoplasmic reticulum (ER), Bcl-2 via its Bcl-2 homology (BH)4 domain, prevents excessive Ca(2+) signals by interacting with the inositol 1,4,5-trisphosphate receptor (IP3R), an intracellular Ca(2+)-release channel. A peptide tool (BIRD-2) that targets the BH4 domain of Bcl-2 reverses Bcl-2's inhibitory action on IP3Rs and can trigger pro-apoptotic Ca(2+)signals in B-cell cancer cells. Here, we explored whether HA14-1, a Bcl-2 inhibitor that also inhibits sarco/endoplasmic reticulum Ca(2+)-ATPases (SERCA), could potentiate BIRD-2-induced cell death. We measured apoptosis in Annexin V/7-AAD stained cells using flow cytometry and intracellular Ca(2+) signals in Fura2-AM-loaded cells using an automated fluorescent plate reader. HA14-1 potentiated BIRD-2-induced Ca(2+) release from the ER and apoptosis in both BIRD-2-sensitive DLBCL cell lines (SU-DHL-4) and in primary B-CLL cells. BIRD-2-resistant DLBCL cells (OCI-LY-1) were already very sensitive to HA14-1. Yet, although BIRD-2 moderately increased Ca(2+) levels in HA14-1-treated cells, apoptosis was not potentiated by BIRD-2 in these cells. These results further underpin the relevance of IP3R-mediated Ca(2+) signaling as a therapeutic target in the treatment of Bcl-2-dependent B-cell malignancies and the advantage of combination regimens with HA14-1 to enhance BIRD-2-induced cell death.

Effects of TNF secreting HEK cells on B lymphocytes' apoptosis in human chronic lymphocytic leukemias.

Tumor necrosis factor (TNF) related apoptosis-inducing ligand (TRAIL) is an antitumor candidate in cancer therapy. This study focused on effects of TRAIL, as a proapototic ligand that causes apoptosis, in B-CELL chronic lymphocytic leukemia cells (B-CLL) . A population of HEK 293 cells was transducted by lentivirus that these achieved ability for producing the TRAIL protein and then HEK 293 cells transducted were placed in the vicinity of CLL cells. After 24 hours of co-culture, apoptosis of CLL cells was assessed by annexin V staining. The amount of Apoptosis was examined separately in four groups: 293 HEK TRAIL (16.17±1.04%); 293 HEK GFP (2.7±0.57%); WT 293 HEK (2±2.6%); and CLL cells (0.01±0.01%). Among the groups studied, the maximum amount of apoptosis was in the group that the vector encoding TRAIL was transducted. In this group, the mean level of soluble TRAIL in the culture medium was 253 pg/ml; also flow cytometry analyzes showed that proapotosis in this group was 32.8±1.6%, which was higher than the other groups. In this study, we have demonstrated that TNF secreted from HEK 293 cells are effective in death of CLL cells.