Poly [ADP-Ribose] Polymerase 1 (PARP-1) Expression Before and After Immunochemotherapy In Patients With B Chronic Lymphocytic Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5293-5293
Author(s):  
Panagiotis Theodorou Diamantopoulos ◽  
Maria Sofotasiou ◽  
Vasiliki Papadopoulou ◽  
Katerina Polonyfi ◽  
Theodoros Iliakis ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Healthy Volunteers ◽  
Conflicts Of Interest ◽  
Lymphocytic Leukemia ◽  
Response To Treatment ◽  
Mrna Levels ◽  
Protein Levels ◽  
Significant Difference ◽  
Before And After ◽  
Parp 1

Abstract Introduction Poly [ADP-ribose] polymerase 1 (PARP-1) has a central role in the repair of single-stranded DNA (ssDNA) breaks, thus protecting the cell from genomic instability. This enzyme is a promising target in anticancer treatment. The aim of the present study is to investigate the role of PARP1 in B chronic lymphocytic leukemia (B-CLL) patients pre- and post- treatment. Patients and Methods Blood samples were collected from B-CLL patients before treatment and following 3 cycles of immunochemotherapy (chosen by the treating physician according to common clinical practice). Quantification of PARP1-mRNA levels was performed by a SYBR-green based PCR performed on BIORADCFX96 (BIORAD). The results were expressed in correlation to the mRNA levels of the endogenous housekeeping gene of beta actin (ACTB). The detection of the gene product was performed by Western Blot using anti-PARP1 antibody (clone 47-258; Calbiochem). We detected the 70 KD protein segment that results from the in vivo fractionation of PARP1 by caspase-3. The protein bands were visualized using an enhanced chemiluminescence detection system (Amersham Biosciences) and quantitated by densitometry using the gel analysis software ImageJ. We correlated the PARP1 protein levels to those of a group of healthy volunteers. We also studied the differences between PARP1-mRNA and protein levels before and after treatment and correlated them to clinical response. The related samples Wilcoxon Signed Rank test (IBM SPSS statistics, version 19.0) was used for the statistical analysis of the results. Results Samples were obtained from 24 B-CLL patients before treatment and from 15/24 following 3 cycles of immunochemotherapy. The patients' characteristics are shown in Table 1. Pre-treatment PARP1 protein levels were similar to those of 15 healthy volunteers (p=0.426). No statistically significant difference was noted between the PARP1-mRNA and protein levels following treatment (p=0.507 and 0.650 respectively) (Table 1). Multivariate analysis did not reveal statistically significant differences in the mRNA and protein levels in correlation to the stage of disease, the peripheral blood lymphocyte count, the LDH levels, the response to treatment and the overall prognosis of the patients. Conclusions The expression of PARP1 in patients with B-CLL seems to be comparable to that of healthy persons. We could not distinguish patients displaying reduced levels of PARP1, as previously reported by others. Moreover, PARP1 expression is not affected by immunochemotherapy, irrespective of stage and response to treatment. Thus, this molecule seems not to participate in the mechanisms involved in DNA repair and apoptosis that are impaired in B-CLL. The study is still ongoing with the addition of more subjects. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Akt Inhibitors Induce Apoptosis in Chronic Lymphocytic Leukemia Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1731-1731
Author(s):  
Mercè de Frias ◽  
Daniel Iglesias-Serret ◽  
Ana M Cosialls ◽  
Llorenç Coll-Mulet ◽  
Antonio F Santidrián ◽  
...  
Keyword(s):  
T Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Conflicts Of Interest ◽  
Therapeutic Option ◽  
Mrna Level ◽  
Lymphocytic Leukemia ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Healthy Donors ◽  
Induced Apoptosis

Abstract Abstract 1731 Poster Board I-757 Phosphatidylinositol-3-kinase (PI3K)/Akt pathway has been described to be critical in the survival of chronic lymphocytic leukemia (CLL) cells. Here, we have analyzed the effect of two selective chemical inhibitors of Akt (Akti-1/2 and A-443654) in the survival of CLL cells. We studied by cytometric analysis the cytotoxic effects of Akt inhibitors on peripheral B and T lymphocytes from patients with CLL and from healthy donors. Both inhibitors induced apoptosis in CLL cells in a dose-dependent manner. Moreover, B cells from CLL samples were more sensitive to Akt inhibitors than T cells from CLL samples, and B or T cells from healthy donors. Survival factors for CLL cells, such as IL-4 and SDF-1a, were not able to block the apoptosis induced by both Akt inhibitors. We studied the changes induced by Akti-1/2 and A-443654 at mRNA level by performing reverse transcriptase multiplex ligation–dependent probe amplification (RT-MLPA). Akti-1/2 did not induce any change in the mRNA expression profile of genes involved in apoptosis, while A-443654 induced some changes, including an increase in NOXA and PUMA mRNA levels, suggesting the existence of additional targets for A-443654. We also studied the changes induced by both Akt inhibitors in some BCL-2 protein family members on CLL cells by Western blot. Both inhibitors induced an increase in PUMA and NOXA protein levels, and a decrease in MCL-1 protein level. Moreover, Akti-1/2 and A-443654 induced apoptosis irrespective of TP53 status. These results demonstrate that Akt inhibitors induce apoptosis of CLL cells and might be a new therapeutic option for the treatment of CLL. Disclosures No relevant conflicts of interest to declare.

Comparative Efficacy of First-Line Chemotherapy-Free Combinations in Chronic Lymphocytic Leukemia (CLL): A Network Meta-Analysis

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 25-26
Author(s):  
Philip A Haddad ◽  
Nowell Ganey ◽  
Kevin M. Gallagher
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
English Language ◽  
Conflicts Of Interest ◽  
Residual Disease ◽  
Meta Analysis ◽  
The Elderly ◽  
Lymphocytic Leukemia ◽  
First Line ◽  
Significant Difference ◽  
Anti Cd20

Introduction: Chronic Lymphocytic Leukemia (CLL) is an incurable B-cell malignancy which disproportionately affects the elderly. Although first-line chemoimmunotherapy (CIT) improved CLL clinical outcomes, recent randomized trials revealed superior outcomes with novel chemotherapy-free combinations (CFC) incorporating anti-CD20 monoclonal antibodies and inhibitors of BTK or Bcl-2. So far, these CFC have not been compared head-to-head. We conducted this network meta-analysis to evaluate their relative efficacy to each other. Methods: A review of the medical literature was conducted using online databases. Inclusion criteria consisted of English language; diagnosis of CLL; trials that explored the efficacy of first-line CFC with Obinutuzumab (O), Rituximab (R), Ibrutinib (IB), Acalabrutinib (ACAL), Venetoclax (VEN) compared to standard CIT that included Chlorambucil (CHLOR) with either R or O, Bendamustine+Rituximab, or Fludarabine+ Cyclophosphamide+R; and phase 3 randomized studies reporting responses, progression, death, and adverse (AE) events. A frequentists network meta-analysis was conducted using netmeta package and random-effects model. Results: Five studies comprising a total of 2,272 participants were included. When O-based CFC data was analyzed, only ACAL-O had a significant lower relative risk (RR) of progression and death (P&D). There were no significant differences with respect to overall response rates (ORR), complete remission (CR), minimal residual disease (MRD), or grade >3 adverse events (Grd3+) among O-based CFC. When R-based CFC data was analyzed, IB and IB-R were not different with respect to RR of P&D, ORR, CR, MRD, or Grd3+. When the data was analyzed as CFC versus combined CIT, only ACAL-O was found to be significantly superior to other O- and R-based CFC with respect to RR of P&D. ORR and Grad3+ rates of O- and R-based CFC were not significantly different. While ACAL-O, IB-O, and VEN-O had superior CR and MRD rates compared to other CFC, there were no significant differences among each other. Conclusions: This network meta-analysis is the first to compare and rank first-line CFC therapies in CLL. It indicates that ACAL-O has a superior profile having the lowest RR of P&D without significant difference in Grd3+ among CFC. Disclosures No relevant conflicts of interest to declare.

Monocytes/Macrophages Are the Major Targets of the CCL3 Chemokine Produced by CD38+CD49d+ Chronic Lymphocytic Leukemia Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2350-2350
Author(s):  
Antonella Zucchetto ◽  
Dania Benedetti ◽  
Claudio Tripodo ◽  
Riccardo Bomben ◽  
Fleur Bossi ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Chronic Lymphocytic Leukemia ◽  
T Lymphocytes ◽  
Conflicts Of Interest ◽  
Cell Types ◽  
Lymphocytic Leukemia ◽  
Bone Marrow Biopsies ◽  
Tnf Α ◽  
Significant Difference

Abstract Abstract 2350 Poster Board II-327 Introduction: CD38 and CD49d are associated negative prognosticators in chronic lymphocytic leukemia (CLL). Recent gene expression profiling studies comparing CLL cases expressing low versus high levels of CD38 and CD49d, identified CCL3 as a gene upregulated by CD38+CD49d+ CLL. The release of CCL3 by cultured CLL cells was also demonstrated upon CD38 triggering, and CCL3 protein was found in CLL cells from bone marrow biopsies (BMB) of CD38+ cases (Zucchetto et al., Cancer Res, 2009; 69:4001-9). Given the role of CCL3 as potent chemoattractant for different cell types, we aimed at identifying the major targets of CCL3, as produced by CD38+CD49d+ CLL cells. Methods: CLL infiltrates of BMB were characterized by immunohistochemistry (IHC). Expression of the CCL3 receptors CCR1 and CCR5 by PB CLL subpopulations was evaluated by flow cytometry. T lymphocyte and monocyte migrations were performed by in-vitro transwell chemotaxis assays. Results: IHC analysis of BMB from 16 CLL cases revealed a higher number of infiltrating CD68+ cells in the context of CLL-involved areas of BMB from CD38+CD49d+CCL3+, compared to CD38−CD49d−CCL3− cases (p=0.01). CD3+ lymphocytes were interspersed in the CLL aggregates, but with no significant difference between the two subgroups. Evaluation of CCR1 and CCR5 in PB cell subpopulations from 40 CLL cases expressing or not surface CD38 and CD49d, showed the highest mean fluorescence intensity (MFI) levels for both CCR1 (624±60) and CCR5 (64±9) in the monocytic component, irrespective of CD38 and CD49d expression by CLL cells. Conversely, both CLL cells and residual T lymphocytes showed low MFI levels for CCR1 (19±4 and 14±3) and CCR5 (21±2 and 20±2). High CCR1 and CCR5 expression levels were detected in in-vitro differentiated monocytes from purified PB cells of four CD38+CD49d+ CLL. Accordingly, CCR1 expression was documented in macrophage-like cells in BMB from CD38+CD49d+ CLL. Next, we evaluated the capability of purified monocytes and T lymphocytes from 10 CLL cases to migrate in response to CCL3. In keeping with the strong expression of CCR1, monocytes migrated toward CCL3 at a concentration of 3 ng/mL (migration index, MI= 8.8±0.9, p=0.03), whereas T lymphocytes required a higher CCL3 concentration (100 ng/mL) to display slight migration capability (MI= 1.6±0.2, p=ns). The increased infiltration of macrophages in BMB from CCL3-producing CD38+CD49d+ CLL, prompted us to verify the capability of CCL3-stimulated macrophages to induce the expression by endothelial cells (EC) of the CD49d specific ligand VCAM-1. By using two different EC models (HUVEC and ADMEC), we documented a significant up-regulation of VCAM-1 by EC exposed to conditioned media (CM) collected from cultures of macrophages challenged in-vitro with CCL3 (p=0.002). Notably, increased levels of the pro-inflammatory cytokine TNF-α were detected in CCL3-CM (p=0.006), and neutralization of TNF-α by specific antibodies reverted the capability of CCL3-CM to induce VCAM-1 by EC models. In agreement with these in-vitro data, we found a more prominent meshwork of VCAM-1+ stromal/endothelial cells in lymphoid infiltrates from CD38+CD49d+ CLL compared to CD38−CD49d− cases (p=0.002), and engagement of CD49d by VCAM-1 was able to significantly delay the spontaneous apoptosis observed in cultured CLL cells. Conclusions: CD68+ monocytes/macrophages are likely the main targets for the CLL3 chemokine produced by CD38+CD49d+ CLL cells, and are active in determining, through the release of TNF-α and other yet unidentified cytokines, the overexpression of VCAM-1 by endothelial cells. Experiments aimed at investigating further roles of CD68+ monocytes/macrophage in CLL are currently matter of study. Disclosures: No relevant conflicts of interest to declare.

Bendamustine in the Treatment of Chronic Lymphocytic Leukemia -Consistent Superiority Over Chlorambucil in Elderly Patients and Across Clinically Defined Risk Groups.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2367-2367 ◽  
Author(s):  
Wolfgang Ulrich Knauf ◽  
Toshko Lissitchkov ◽  
Ali Aldaoud ◽  
Anna Liberati ◽  
Javier Loscertales ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Remission Rate ◽  
Risk Groups ◽  
The Elderly ◽  
Lymphocytic Leukemia ◽  
Phase Iii ◽  
Response To Treatment ◽  
Open Label ◽  
Significant Difference ◽  
Binet Stage

Abstract Abstract 2367 Poster Board II-344 Introduction: Bendamustine is a purine analog/alkylator hybrid agent with a unique mechanism of action, which has shown good clinical efficacy and acceptable tolerability in various hematological malignancies. Chronic lymphocytic leukemia (CLL) is a disease of the elderly, and presents with a variety of clinical characteristics which influence the prognosis. We analyzed tolerability and efficacy of bendamustine (BEN) in comparison to chlorambucil (CLB) in clinical risk groups defined by age and specific indicators of disease activity. Patients and Methods: The efficacy and safety of BEN and CLB have been compared in a randomized, open-label, multicenter, phase III trial in patients with previously untreated advanced (Binet stage B/C) CLL. Patients were randomized to receive BEN (100 mg/m2 on days 1 + 2) or CLB (0.8 mg/kg on days 1 and 15) for up to 6 treatment cycles. The primary endpoints were overall remission rate (ORR), which was defined as complete or partial response, and progression-free survival (PFS). Secondary endpoints included overall survival (OS) and safety. The response to treatment was evaluated by a blinded Independent Response Assessment Committee. Results: A total of 319 patients were randomized (162 bendamustine, 157 chlorambucil), of whom all were included in the efficacy analysis, while 312 were evaluable for safety. Median age was 64 years (35 to 78). The median number of treatment cycles was 6 in both study arms, regardless of an age above or below 65 years. The median observation time was 35 months. ORR was significantly higher with BEN than with CLB (68% versus 31%, P<0.0001). The median PFS was 21.6 months with BEN and 8.3 months with CLB (P<0.0001). So far, there is no difference in OS (median not reached with BEN versus 65.4 months with CLB; p = 0.16). No significant difference in the remission rates became apparent when comparing patients below and above the age of 65 years (ORR 71.6 % versus 63.5 % with BEN, p>0.3; and 28.4 % versus 32.5 % with CLB, p>0.6). PFS was not influenced by age above 65 years, stage of disease (Binet stage B versus C), or elevated LDH. However, patients without B symptoms had a longer median PFS with BEN than those patients with B symptoms (30.4 months versus 17.7 months; p<0.0001), whereas median PFS was not affected by the presence of B symptoms in patients with CLB (8.9 months in both patient groups). Conclusion: This study has shown that bendamustine offers significantly greater efficacy than chlorambucil in the elderly and across clinically defined major risk groups, even in the presence of B symptoms. BEN should be considered as first-line chemotherapy for patients with advanced CLL. Disclosures: Knauf: mundipharma: Consultancy, Honoraria; cephalon: Consultancy, Honoraria. Klein:mundipharma: Consultancy, Honoraria. Merkle:mundipharma: Consultancy, Honoraria. Montillo:mundipharma Italy: Consultancy, Research Funding.

Aspirin Induces Apoptosis in Human Leukemia Cells Independently of NF-κB and MAPKs through Alteration of the Mcl-1/Noxa Balance.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4825-4825
Author(s):  
Ana M Cosialls ◽  
Daniel Iglesias-Serret ◽  
Maria Piqué ◽  
Montserrat Barragán ◽  
Antonio F Santidrián ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Leukemia Cell ◽  
Cell Types ◽  
Lymphocytic Leukemia ◽  
Leukemia Cells ◽  
Human Leukemia ◽  
Mrna Levels ◽  
Protein Levels ◽  
Human Leukemia Cells ◽  
Induced Apoptosis

Abstract Abstract 4825 Aspirin and other non-steroidal anti-inflammatory drugs (NSAIDs) induce apoptosis in most cell types. We examined the mechanism of aspirin-induced apoptosis in human leukemia cells. Our results show that aspirin induced apoptosis in leukemia Jurkat T cells independently of NF-κB. Although aspirin induced p38 MAPK and c-Jun N-terminal kinase (JNK) activation, selective inhibitors of these kinases did not inhibit aspirin-induced apoptosis. We studied the regulation of Bcl-2 family members in aspirin-induced apoptosis. The mRNA levels of some pro-apoptotic members, such as BIM, NOXA, BMF or PUMA, were induced by aspirin. However, none of these pro-apoptotic proteins increased and the levels of Mcl-1 protein were reduced. Interestingly, in the presence of aspirin the protein levels of Noxa remained high. This alteration of the Mcl-1/Noxa balance was also found in other leukemia cell lines and primary chronic lymphocytic leukemia cells (CLL). Furthermore, in CLL cells aspirin induced an increase in the protein levels of Noxa. Knockdown of Noxa or Puma significantly attenuated aspirin-induced apoptosis. These results indicate that aspirin induces apoptosis through alteration of the Mcl-1/Noxa balance. Disclosures No relevant conflicts of interest to declare.

scholarly journals 98% IGHV Gene Identity Is Still the Optimal Cutoff to Predict the Prognosis of Chronic Lymphocytic Leukemia Patients in China

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5545-5545
Author(s):  
Yi Xia ◽  
Ke Shi ◽  
Qian Sun ◽  
Chun Qiao ◽  
Huayuan Zhu ◽  
...  
Keyword(s):  
Prognostic Factor ◽  
Chronic Lymphocytic Leukemia ◽  
Conflicts Of Interest ◽  
Somatic Hypermutation ◽  
Lymphocytic Leukemia ◽  
Study Cohort ◽  
Optimal Cutoff ◽  
Entire Cohort ◽  
Mutational Status ◽  
Significant Difference

Abstract Background: Immunoglobulin heavy chain variable region (IGHV) has been an important prognostic factor for chronic lymphocytic leukemia (CLL) for decades. 98% being a cut-off value for IGHV is a mathematical choice and researches on the best cut-off value have never been stopped. Chinese CLL patients are known to differ from Caucasian CLL patients on both clinical and genetical features. However, the optimal cutoff for IGHV mutational status has not yet been studied in this particular ethnic group. Method: We carried out a study on 595 Chinese CLL patients in order to find out whether 98% is the best cut-off value for IGHV in Chinese CLL patients. Genomic DNA from peripheral blood or bone marrow was subjected to PCR amplification following the IGH Somatic Hypermutation Assay v2.0 protocol (InVivoScribe). Sequences were aligned to ImMunoGeneTics/V-QUEry and Standardization (IMGT/-VQUEST) database. Result: 600 sequences were received after IGHV rearrangement sequencing. IGHV3-23, IGHV4-34, IGHV3-7, IGHV4-39 and IGHV1-69 were the most frequently used IGHV genes. 352 (58.7%) cases were IGHV-mutated while 248 (41.3%) cases were IGHV-unmutated if the classical 98% classification by ERIC was used. In order to determine the optimal cut-off value, we used 1% as the interval to divide the entire cohort into 7 groups according to the mutational rate, which were <95%, 95%-95.99%, 96%-96.99%, 97%-97.99%, 98%-98.99%, 99%-99.99% and 100% respectively. Binet A patients had a relatively indolent course of disease and cases with different IGHV mutational rates had no significant differences in time to first treatment (TTFT) apart from truly unmutated (100%) cases. For the whole study cohort, significant difference appeared at 98% interval (P<0.001 and P=0.005 for TTFT and OS respectively) while intervals less than 98% had no significant difference compared with the <95% group. Similarly, there was no clear dissimilarities among 98%, 99% and 100% intervals (Table 1a and b). All the other prognostic factors including del(17p), del(11q), TP53 mutation, MYD88 mutation, NOTCH1 mutation, SF3B1 mutation, CD38, ZAP-70, Binet staging, gender, β2-microglobulin and EBV-DNA were differently distributed between group <98% and group ³98%, but not among subgroups in ³98%. In multivariate analysis, the 98% IGHV was also an independent prognostic factor for TTFT and OS. Conclusion: 98% is the optimal cutoff value for IGHV mutational status to predict the prognosis of CLL patients in China. Table 1. Table 1. Disclosures No relevant conflicts of interest to declare.

Reducing Mcl-1 by Homoharringtonine Induces Apoptosis In Chronic Lymphocytic Leukemia Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2896-2896
Author(s):  
Rong Chen ◽  
Lei Guo ◽  
Yuling Chen ◽  
Yingjun Jiang ◽  
William G. Wierda ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Stromal Cell ◽  
Conflicts Of Interest ◽  
Lymphocytic Leukemia ◽  
Myelogenous Leukemia ◽  
Leucine Incorporation ◽  
Immunoblotting Analysis ◽  
Protein Levels ◽  
Mitochondrial Membrane Depolarization ◽  
Apoptotic Proteins

Abstract Abstract 2896 Homoharringtonine (HHT, omacetaxine) is an inhibitor of translation elongation that is currently in clinical evaluation in chronic myelogenous leukemia. We hypothesized that this compound would be active in inducing apoptosis in chronic lymphocytic leukemia (CLL) based on the following rationale: 1): CLL cells are characterized by their dependence on the overexpression of anti-apoptotic proteins to maintain their survival; 2): one of the key anti-apoptotic proteins, Mcl-1, turns over rapidly. Thus, upon transient exposure to a translation inhibitor, Mcl-1 expression would decrease rapidly and allow the pro-apoptotic proteins to activate the mitochondria-dependent apoptosis. Using tritiated leucine incorporation as an indicator of new protein synthesis, our results in primary CLL cells demonstrated that HHT was more potent at inhibiting translation than cycloheximide. A polysome profiling analysis confirmed that Mcl-1 translation was blocked by HHT. Mcl-1 protein levels were reduced significantly after HHT exposure, measured by an immunoblotting analysis. This led to mitochondrial membrane depolarization and initiation of apoptosis, analyzed by flow cytometry. Sensitivity to HHT varied in different CLL samples. Further studies showed that this heterogeneous response was independent of the CLL prognostic factors such as the mutation status of the IgVH or ZAP70 expression. Rather, it correlated significantly to the baseline Mcl-1 expression in each CLL sample and the extent of reduction of Mcl-1 protein. A detailed time course study showed that Mcl-1 reduction was evident as early as 2 h and continue to decrease in the next 6 to 8 h; cell death started in 2 h and continued to increase for 24-h. The reduction of Mcl-1 was not affected by the pan caspase inhibitor zVAD, indicating that reduction of Mcl-1 was the initiation event of apoptosis, rather than the results of caspase cleavage. On the contrary, the proteosome inhibitor MG-132 caused retention of the Mcl-1 level in the presence of HHT, suggesting that when Mcl-1 synthesis was blocked by HHT, the remaining Mcl-1 in the cells was degraded rapidly in a proteosome-dependent mechanism. As the action of HHT relied largely on the rapid turn-over of Mcl-1 protein, blocking Mcl-1 degradation by the proteosome inhibitor would counteract the effect of HHT. This explained the observed antagonistic reaction of HHT and MG-132 when they were used together. Further, interaction of CLL cells with the microenvironment has been shown to protect CLL cells from chemotherapy. Incubating CLL cells on either a murine or human stromal cell layer in vitro clearly protected CLL cells from the toxicity of fludarabine. There was an induction of both Mcl-1 transcript and protein levels, which could explain this protection. However, this induction was reversed by HHT, overcoming stromal cell-mediated protection. Finally, because of the important role of Mcl-1 in the pathogenesis CLL, we hypothesized a sequential blockade strategy that inhibits both Mcl-1 transcription and translation to completely block Mcl-1 synthesis. Median effect analysis showed that combining the transcription inhibitor SNS-032 together with HHT killed the CLL cells synergistically. An immunoblotting analysis demonstrated that the combination of the two drugs depleted Mcl-1 more completely than either single drug at the same concentration, which correlated to a greater induction of apoptosis than either drug alone. Taken together, our data demonstrated that transiently inhibiting translation and depleting Mcl-1 was the major mechanism of HHT-induced apoptosis in CLL cells. This action overcame stromal cell-mediated protection and was synergistic with inhibitors of transcription. Thus, these data provided rationale for the clinical development of HHT in CLL, and for the design of combination strategies. Disclosures: No relevant conflicts of interest to declare.

Stroma-Induced TCL1 Expression In Chronic Lymphocytic Leukemia Cells Is Associated with Down Regulation of TCL1A-Targeting miRNAs

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 52-52
Author(s):  
Mariela Sivina ◽  
Elena Hartmann ◽  
Jorge Boucas ◽  
Michael J Keating ◽  
William G. Wierda ◽  
...  
Keyword(s):  
T Cell ◽  
Chronic Lymphocytic Leukemia ◽  
Conflicts Of Interest ◽  
Independent Set ◽  
Inverse Correlation ◽  
Lymphocytic Leukemia ◽  
Mrna Levels ◽  
Cell Leukemia ◽  
Prolymphocytic Leukemia ◽  
Qrt Pcr

Abstract Abstract 52 The T cell leukemia/lymphoma 1 (TCL1) oncogene was initially identified as a target of chromosomal translocations and inversions at the 14q32.1 chromosome breakpoint region in T-cell prolymphocytic leukemia (T-PLL). Deregulation of TCL1 in mouse B cells results in a CD5+ B cell leukemia that mimics aggressive human chronic lymphocytic leukemia (CLL), indicating that TCL1 plays an important role in the pathogenesis of CLL. However, chromosomal aberrations that constitutively activate TCL1 have not been identified in CLL, and the mechanism(s) of TCL1 activation in CLL remain unclear. We co-cultured CLL cells with two marrow stromal cell (MSC) lines (KUSA-H1, NK-Tert) and found TCL1 to be among the top genes up regulated after co-cultures with MSC. This was based on at least 3-fold up-regulation in paired samples, using Affymetrix HG U133 plus 2.0 oligonucleotide arrays. We also found an up-regulation of TCL1 mRNA by qRT-PCR and TCL1 protein by immunoblotting and flow cytometry. qRT-PCR revealed significant increases in TCL1A mRNA levels with up to 100 fold increases after 2 and 7 days of co-culture in an independent set of 8 CLL samples (Fig. a). Because TCL1 is a co-activator of the AKT oncoprotein and its downstream target MCL-1, we next explored AKT and MCL-1 activation after MSC co-culture by immunoblotting. We found increases in TCL1A protein to be associated with robust increases of pAKT, AKT isoforms 1 and 2, and MCL-1 after MSC co-culture. In order to investigate the possible mechanism of regulation of TCL1 expression by MSC, we next examined the levels of TCL1 negative regulators miR-29b, miR-181b and miR34b targeting the 3’ UTR of TCL1 (Pekarsky et al., Cancer Res. 66:11590-3, 2006). qRT-PCR revealed decreased levels of miR-29b, miR-181b, and miR-34b after 7 days of co-culture with MSC (Fig. b), which paralleled the increased levels of TCL1. This inverse correlation was more marked in some cases than in others, and this heterogeneity did not correlate with the initial miRNA or TCL1A levels. The timing of these events suggests, that miRNA based modulation of TCL1 levels is only in part responsible for TCL1 up regulation in MSC co-cultures. It rather implicates multiple mechanisms to be responsible for milieu mediated TCL1 regulation, i.e. transcriptional and/or post-transcriptional. The fact that all analyzed miRNAs are simultaneously down regulated in our system also suggests a common superimposed regulatory mechanism in miRNA processing/regulation that might contribute to both direct and miRNA mediated TCL1 up regulation in MSC co-cultures. In summary, our results indicate that MSC induce TCL1 expression uniformly in primary CLL; the induction of TCL1 is associated with downregulation of miR-29b, miR-181b, and miR34b, along with induction of pAKT, AKT 1/2, and MCL-1. Collectively, these findings suggest that the microenvironment plays a proactive role in regulation of TCL1 expression. Figure Figure. Disclosures: No relevant conflicts of interest to declare.

Chronic Lymphocytic Leukemia (CLL)/Small Lymphocytic Leukemia (SLL) Exhibit Altered Cytokine Protein Levels in Peripheral Blood Serum

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2646-2646
Author(s):  
Laura A Paganessi ◽  
Robin R Frank ◽  
Sucheta Jagan ◽  
Reem Karmali ◽  
Yan Li ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Peripheral Blood ◽  
Conflicts Of Interest ◽  
Fold Change ◽  
Lymphocytic Leukemia ◽  
P Value ◽  
Protein Levels ◽  
Healthy Donors ◽  
Cytokine Levels

Abstract Abstract 2646 Introduction: Chronic lymphocytic leukemia (CLL)/Small lymphocytic leukemia (SLL) is one of the most common forms of indolent lymphomas in elderly adults. Currently, CLL is not treated until it develops into later stage disease. An increase in the knowledge of the biology of CLL could aid in the development of new treatment strategies for early stage CLL, thus positively impacting disease progression. We therefore investigated the cytokine levels present in peripheral blood (PB) serum of CLL patients and compared them to levels in healthy donors. Methods: PB was obtained from 25 untreated CLL and 3 untreated SLL patients and 29 normal healthy donors under an IRB approved protocol. Serum was stored at −80°C until analyzed. 86% of the patients were Rai 0/1, 3.5% Rai stage 2, and 10.5% unknown at diagnosis. Risk stratification based on cytogenetics and FISH found of the enrolled patients, 47% good, 25% intermediate, and 14% poor risk. The risk factor was unknown in 14%. β-2 microglobulin and 54 cytokine protein levels in 2 different serum samples per patient (collected at least one year post diagnosis) were measured in duplicate using MILLIPLEX™, a multiplex Luminex based technology. Genespring 11.5.1 software was used to convert the data into base-2 logarithmic values and then apply a median baseline transformation across all samples. Data is grouped according to the source of the sample (CLL or normal PB serum). A Filter on Volcano Plot analysis is then done. This filter analysis performs an unpaired t-test, which yields a p-value as well as computes the fold change of each cytokine. Results: As expected, β-2 microglobulin was significantly higher in CLL patients compared to normal samples (p=5.17×10−7, 1.79 fold). Using a minimum of a 1.5 fold change and p-value≤0.05, 16 cytokines had significantly higher expression and 9 cytokines had significantly lower expression in CLL samples compared to normal samples (see Table 1). Conclusion: These changes in cytokine levels help provide insight to the peripheral blood microenvironment, in which circulating CLL cells reside. Some cytokines were substantially higher in CLL patients; soluble IL-2 receptor alpha (sIL-2Rα) and stem cell factor (SCF). The substantially higher levels of sIL-2Rα have also been observed in follicular lymphoma (FL) and appear to predict which FL patients will have a reduced survival (Yang et al., Blood 2011). Levels of sIL-2Rα correlating to survival may be a phenomenon seen in all B-cell lymphomas. SCF is thought to be important in early B-cell development but its receptor c-kit (CD117) has not been reported to be expressed on B-CLL cells. The chemokine receptors CXCR4, CXCR5 and CCR7 are expressed on B-CLL cells. Interestingly, their corresponding ligands, CXCL12, CXCL13, and CCL21 are significantly higher (Table 1) in the PB serum of CLL patients. It is likely that existing immunomodulatory therapies, such as IMiDs, lenalidomide or thalidomide alter the PB levels of the cytokine milieau. Other successful hematological malignancy therapies involve targeted monoclonal antibodies. Therefore, a deeper understanding of the cytokine microenvironment will provide guidance in the development of future targeted therapies or highlight current therapies for other malignancies that could be promising for CLL. Disclosures: No relevant conflicts of interest to declare.

Dishevelled Proteins of Wnt Signaling Pathways Are Highly Expressed in Chronic Lymphocytic Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4583-4583
Author(s):  
Abdul Salam Khan ◽  
Åsa Sandin ◽  
Anders Österborg ◽  
Mohammad Hojjat-Farsangi ◽  
Håkan Mellstedt ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Wnt Signaling ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Lymphocytic Leukemia ◽  
Healthy Donors ◽  
Significant Difference ◽  
Gsk 3Β ◽  
Progressive Accumulation

Abstract Abstract 4583 Chronic Lymphocytic Leukemia (CLL) is the most common leukemia in the Western world and is characterized by progressive accumulation of CD5+, CD19+, and CD23+ B cells in the blood, bone marrow, and secondary lymphoid organs. The progressive accumulation of malignant B cells is partly due to defective apoptosis. An important constitutively activated signaling pathway in CLL cells might be the Wnt signaling cascades that include the Wnt/b-catenin pathway and the non-canonical Wnt pathway. These pathways might be activated downstream of the constitutively phosphorylated ROR1. Dishevelled family proteins (Dvl1, Dvl2, and Dvl3) are important cytoplasmic mediators of Wnt signaling and have recently been shown to be expressed in many cancer types. The expression and precise roles of individual Dvl proteins are however not clear. The aim of the study was to characterize the expression of Dvl1, Dvl2 and Dvl3 proteins in progressive and non-progressive CLL and compare to normal white blood cells (PBMC) and to assess the expression of other important proteins of the canonical pathway as b-Catenin and GSK-3β as well as the non-canonical pathways as GSK-3α, PKC, ERK and AKT respectively. Leukemic cells from progressive (n=9) and non-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 proteins were analyzed by Western blot and densitometric calculations as well as PKC, GSK-3α, AKT and ERK using antibodies against the total protein and the phosphorylated protein respectively. Immunoprecipitation was performed to check the phosphorylation status of Dvl proteins. Overexpression of Dvl1, Dvl2, and Dvl3 was detected in all progressive and non-progressive CLL patients and the six cell lines. There was a significantly higher expression of Dvl1 and Dvl3 in progressive compared to non-progressive CLL patients (p<0.01 and p<0.01 respectively). In contrast to Dvl1 and Dvl3, Dvl2 was highly expressed in non-progressive as compared to progressive CLL patients. Dvl1 was phosphorylated at serine residues while Dvl2 was phosphorylated at tyrosine residues. b-Catenin followed the same expression pattern as Dvl2 being highly expressed in non-progressive CLL as compared to progressive CLL patients (p<0.001) whereas GSK-3β phosphorylation was significantly higher in progressive than non-progressive CLL patients (p<0.01). There was no significant difference in the degree phosphorylation of PKC and GSK-3α comparing non-progressive CLL, progressive CLL and healthy donors. In summary Dvl1, Dvl2 and Dvl3 are highly upregulated in CLL patients and CLL cell lines but the expression in normal PBMC is almost negligible. Dvl1, Dvl2 and Dvl3 may have a key role in the transduction of Wnt mediated signals in CLL. Disclosures: No relevant conflicts of interest to declare.

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