Mode of Action of the SDF-1/CXCL12 Inhibiting Spiegelmer® Nox-A12 and Its Impact On Chronic Lymphocytic Leukemia (CLL) Cell Motility and Chemosensitization

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 318-318
Author(s):  
Dirk Zboralski ◽  
Julia Hoellenriegel ◽  
Christian Maasch ◽  
Anna Kruschinski ◽  
Jan A. Burger
Keyword(s):  
Bone Marrow ◽  
Chronic Lymphocytic Leukemia ◽  
Stromal Cells ◽  
Mode Of Action ◽  
Leukemia Cell ◽  
Lymphocytic Leukemia ◽  
Lymphoid Tissues ◽  
Cell Trafficking ◽  
Dependent Manner

Abstract Abstract 318 NOX-A12 is a novel Spiegelmer®-based antagonist of SDF-1/CXCL12, a chemokine involved in the regulation of chronic lymphocytic leukemia (CLL) cell trafficking. Spiegelmers® are mirror-image oligonucleotides that are identified to specifically bind to proteins in a manner conceptually similar to antibodies. Unlike aptamers, however, Spiegelmers® are built from the non-natural L-isomer form of nucleotides which confers resistance to the action of nucleases and avoids potential immunogenicity. CXCL12 is constitutively secreted and presented by bone marrow stromal cells (BMSC) via glycosaminoglycans (GAG) and acts as a homing factor for normal and malignant hematopoietic cells to the bone marrow (BM) and secondary lymphoid tissues via CXCR4 receptors that are expressed at high levels on circulating CLL cells. The microenvironment in the BM and secondary lymphoid tissues, in particular the CXCL12-CXCR4 axis, favors survival and chemotherapy-resistance of leukemic cells. We therefore investigated the effects of NOX-A12 in an in vitro co-culture system to model the interaction of CLL cells with their microenvironment. Surprisingly we observed that NOX-A12 increased pseudoemperipolesis in vitro, i.e. spontaneous leukemia cell migration beneath BMSC. Interestingly, this NOX-A12 induced trans-migration of CLL cells was completely inhibited by the CXCR4 antagonist AMD3100, suggesting a CXCL12/CXCR4 dependent mechanism. We postulated that this observation might result from a direct effect of NOX-A12 on CXCL12 release by the stromal cells. Therefore, we investigated this hypothesis in different BMSC lines (MS-5, R15C, and TSt-4) and we found that NOX-A12 induced a significant CXCL12 release in all three tested cell lines. We asked whether this NOX-A12 dependent increase of CXCL12 of BMSCs is due to release from either intracellular or extracellular storages. Intracellular staining of CXCL12 using flow cytometry did not reveal significant changes when BMSCs were incubated with NOX-A12. Furthermore, the transcription of CXCL12 was not found to be altered after NOX-A12 incubation over a period of three days as shown by quantitative RT-PCR. Rather, CXCL12 is released from extracellular storages of BMSCs. First hints were obtained through a rapid CXCL12 release within five minutes of incubation with NOX-A12. To confirm that CXCL12 is bound to the extracellular surface (by GAGs like heparin) and is being detached by NOX-A12 we first incubated BMSCs with NOX-A12, followed by a wash step and the addition of recombinant CXCL12. Recombinant CXCL12 was bound by BMSCs that were pre-incubated with NOX-A12 but not with a non-functional control (revNOX-A12), indicating that NOX-A12 strips off CXCL12. To corroborate the findings we incubated the BMSCs with heparin which also led to the release of CXCL12 in a dose dependent manner. Of note, the EC50 of heparin regarding CXCL12 release was much higher compared to the EC50 of NOX-A12 (≈ 12 μM vs. 5 nM) revealing the high affinity of NOX-A12 to CXCL12. The competition of NOX-A12 with heparin regarding CXCL12 binding was confirmed by Biacore experiments. Based on these findings, we developed a novel adapted co-culture approach to examine the ability of NOX-A12 to chemosensitize CLL cells. In this setting, we first strip off CXCL12 from BMSCs by NOX-A12 and subsequently add CLL cells which will be either non-treated or treated with chemotherapy (fludarabine combined with bendamustine). We found that NOX-A12 slightly decreased CLL cell viability. As expected, a strong viability decrease was observed with chemotherapy, which could be even further decreased by the combination with NOX-A12, suggesting synergistic effects. In conclusion, we propose that NOX-A12's mode of action is the release of extracellular bound CXCL12 and its subsequent inhibition. Since CXCL12 induces leukemia cell trafficking and homing to tissue microenvironment and also favors leukemia cell survival, we believe that targeting CXCL12 is an attractive approach to remove the protective effects of CXCL12-secreting BMSCs in order to sensitize CLL cells for subsequent chemotherapy. Thus, NOX-A12 represents a very promising agent to significantly improve the treatment of CLL. The compound is currently being tested in a Phase IIa study in relapsed CLL patients. Disclosures: Zboralski: NOXXON Pharma AG, Berlin, Germany: Employment. Maasch:NOXXON Pharma AG: Employment. Kruschinski:NOXXON Pharma AG: Employment. Burger:NOXXON Pharma AG: Consultancy, Research Funding.

Standardizing Co-Culture Conditions Between Chronic Lymphocytic Leukemia Cells and Marrow Stromal Cells: Towards a Reliable and Reproducible System to Assess Cell Adhesion-Mediated Drug Resistance

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3149-3149
Author(s):  
Antonina Kurtova ◽  
Maite P. Quiroga ◽  
William G. Wierda ◽  
Michael Keating ◽  
Jan A. Burger
Keyword(s):  
Bone Marrow ◽  
Drug Resistance ◽  
Cell Adhesion ◽  
Chronic Lymphocytic Leukemia ◽  
Stromal Cells ◽  
Lymphocytic Leukemia ◽  
Marrow Stromal Cells ◽  
Hpv E6 ◽  
Induced Apoptosis

Abstract Contact between chronic lymphocytic leukemia (CLL) cells and accessory stromal cells in tissue microenvironments is considered to play a major role in regulating CLL cell survival and disease progression. Stromal cells of various origins and species, and variable stromal-CLL cell ratios have been used in the past to study CLL-stromal cell interactions and to assess cell-adhesion mediated drug resistance (CAM-DR). Because of the heterogeneity of the currently used in vitro systems to study CLL-MSC interactions, and the importance of these co-culture systems for development and testing of novel agents, we tested a panel of murine and human MSC lines for their capacities to support CLL cell survival and CAM-DR, using various CLL-MSC ratios and fludarabine (F-ara-A) to induce CLL cell apoptosis. We tested four murine, non-transformed MSC lines derived from bone marrow: M210B4, KUM4, ST-2 and KUSA-H1. Also, we tested three human transformed cell lines: Stroma-NKtert, derived from bone marrow and immortalized by human telomerase reverse transcriptase (hTERT), UE6E7-T2 derived from bone marrow and transformed with human papilloma viruses (HPV) E6, E7 and hTERT, and UCB408E6E7Tert33 derived from umbilical cord blood and transformed with hTERT and HPV E6, E7. CLL cells were isolated from peripheral blood of untreated patients and each cell line was tested with at least three different patients according to the following protocol: viability of CLL was tested after 24, 48 and 72 hours by flow cytometry after staining with DiOC6 and propidium iodide. The following conditions were assayed on each of the MSC lines: CLL cells in suspension culture, CLL cells in suspension culture with 10 mM F-ara-A, CLL cells in co-culture with MSC, and CLL cells in co-culture with MSC and with 10 mM F-ara-A. Firstly, we performed titration experiments in order to identify the most appropriate ratio between stromal and CLL cells, using CLL-MSC ratios of 5:1, 10:1, 20:1, 50:1 and 100:1. We found a decline in MSC-derived CLL cell protection at the highest ratio of 100:1, suggesting that ratios of 50:1 or lower provide optimal conditions for in vitro assays. Results shown in Table 1 were assayed using a 20:1 ratio and represented relative viabilities when compared to untreated controls (mean±SEM). Regarding the protective effect of different MSC, we found that all MSC lines demonstrated remarkable protection of CLL cells from spontaneous and F-ara-A-induced apoptosis. We also found that stromal cells that had round shape morphology and easily formed confluent monolayer (M210B4, KUSA-H1, Stroma-NKTert) showed more prolonged protective effect in comparison to cell lines with more spindle shaped morphology (ST-2, KUM4, UE6E7-T2). The failure of UE6E7-T2 and UCB408E6E7Tert33 to demonstrate long-term protection of CLL cells could be related to their own sensitivity to F-ara-A. In this comparative study we demonstrated that both murine and human MSC provide substantial and comparable levels of protection from spontaneous and drug-induced apoptosis. CLL:MSC ratios of 50:1 or lower can be considered ideal for co-culture experiments. Further experiments have to be done to determine the levels of MSC-derived protection in a larger series of CLL samples and in different laboratories for validation. Collectively, in these co-culture assays we can study CLL-MSC interactions and CLL drugs under more standardized conditions that may allow us to evaluate the efficacy of new treatments that target the CLL microenvironment. Time points 24 hours 48 hours 72 hours +Flu + MSC + MSC +Flu +Flu + MSC + MSC +Flu +Flu +MSC + MSC +Flu M210B4 85.2±2.4 117.2±5.0 110.5±4.9 30.8±12.6 138.1±9.5 113.0±2.2 5.2±3.1 138.1±5.1 120.4±3.4 ST-2 93.6±3.0 99.9±2.6 103.1±0.5 51.6±9.4 111.9±2.6 89.8±8.7 13.9±6.3 112.6±5.7 87.0±16.4 KUM-4 93.6±3.0 106.4±1.8 104.2±1.9 51.6±9.4 112.4±2.6 100.8±2.8 13.9±6.3 111.8±6.7 88.5±11.4 KUSA-H1 79.4±7.4 125.1±3.7 118.2±2.0 33.9±10.9 136.0±3.6 107.2±7.0 11.3±6.1 133.6±5.4 84.9±7.6 Stroma-NKTert 79.3±7.0 118.6±7.0 111.0±7.0 30.5±9.5 130.7±9.5 115.6±8.0 7.1±4.3 133.0±11.5 122.7±9.0 UE6E7-T2 79.3±7.0 113.4±3.9 109.3±3.0 30.5±9.5 118.4±4.8 85.0±7.1 7.1±4.3 119.2±6.9 51.0±10.1 UCB408 E6E7Tert33 81.5±7.2 120.2±5.4 111.8±2.7 36.7±9.4 123.7±6.3 86.7±7.7 8.5±6.7 119.7±6.1 50.8±13.0 Table 1. Flu: fludarabine (10mM/ml), MSC: marrow stromal cells

Chronic Lymphocytic Leukemia Cells With Trisomy 12 Home To The Bone Marrow In a CXCR4-Independent Manner and Are Prone To Proliferate In Vitro

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 870-870
Author(s):  
Evelyn Hutterer ◽  
Elisabeth Hinterseer ◽  
Sylvia Ganghammer ◽  
Gabriele Brachtl ◽  
Daniela Asslaber ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Chronic Lymphocytic Leukemia ◽  
Research Funding ◽  
Lymphocytic Leukemia ◽  
Dependent Manner ◽  
Independent Manner ◽  
Atypical Cell ◽  
Trisomy 12 ◽  
Inside Out

Abstract Trisomy 12 (tri12) is a frequent chromosomal aberration in chronic lymphocytic leukemia (CLL) associated with atypical cell morphology, high in vivo tumor proliferation activity and a predisposition to Richter’s transformation. Tri12 harboring CLL cells express increased levels of the negative prognostic marker CD49d, the α4 subunit of the integrin very late antigen 4 (VLA-4), which we previously identified as a key regulator of CLL cell homing to bone marrow (BM). During this process, inside-out activation of VLA-4 upon CXCR4 binding to endothelially displayed CXCL12 is thought to upregulate the adhesive properties of VLA-4 and augment the arrest of CLL cells on the VCAM-1 presenting vessels. Here, we investigated the functional interplay of VLA-4 and CXCR4 in CLL carrying tri12. We first found that the upregulation of CD49d expression in this subset (MFIR CD49d 9.8±5.3 (n=22) vs. 2.7±3.9 (n=126), p<0.0001) was paralleled by their reduced CXCR4 expression (MFIR CXCR4 11.8±7.2 (n=22) vs. 22.7±14.2 (n=126), p=0.0003). Using short term adoptive transfers, we compared the ability of tri12 and no tri12 CLL cells to home to the BM of NOD/SCID mice. 5-10x106 CLL cells were injected into tail vein and homing was evaluated after 3 hours. Based on their more frequent CD49d high phenotype, we observed increased homing rates (homed human CLL cells per 106 injected cells per 106 acquired murine cells) of tri12 compared to no tri12 CLL (225±160 (n=7) vs. 90±117 (n=20), p=0.025). However, when comparing CD49d+ tri12 and CD49d+ no tri12 subsets, we did not observe any significant differences in their homing capacity. To further study CXCL12/CXCR4 function in BM homing, we pretreated mice with either the novel CXCL12 antagonist NOX-A12 or the CXCR4 inhibitor AMD3100 prior to CLL cell injection. While homing of no tri12 CLL cells (n=3, in duplicates) was reduced by both pretreatments (homing rates 137 vs 38 vs 30), the homing capacity of tri12 CLL cells (n=3, in duplicates) was not affected. We next tested whether VLA-4 expressed on these cells was able to undergo CXCL12-induced activation and support cell arrest under shear conditions. To this end, we perfused CLL cells over VCAM-1 or VCAM-1/CXCL12 substrates and analyzed rates and categories of cell tethering at a single cell level by videomicroscopy. CXCL12 induced the arrests of no tri12 CLL cells (n=3) on VCAM-1 under shear flow in a CXCR4 and VLA-4 dependent manner. In contrast, tri12 CLL cells (n=3) robustly tethered to VCAM-1 in the absence of the chemokine, and interactions could not be further enhanced by additional CXCL12 nor could they be abrogated by use of AMD3100. This failure of CXCR4-induced adhesion was not based on a general defect in CXCR4 functionality as in vitro chemotaxis of tri12 CLL cells (n=5) towards CXCL12 was fully maintained. To detect potential differences in VLA-4 affinity regulation, we used a conformationally sensitive antibody that recognizes epitopes induced by VLA-4 ligation, and an LDV-containing VLA-4 specific ligand to probe resting integrin affinity. Also, we used a small fluorescent ligand to study rapid VLA-4 affinity changes during inside-out chemokine induced activation. On resting tri12 CLL, VLA-4 exhibited an affinity state similar to that observed on circulating lymphocytes, and tri12 CLL cells failed to undergo the rapid affinity up-regulation triggered by CXCL12 pretreatment, in keeping with tethering experiments. Next, we investigated whether the tumor microenvironment has a different influence on the behavior of the tri12 subset. Therefore we subjected the cells to in vitro co-cultures mimicking the lymphoid proliferation centers. Basal levels of the early activation marker CD69 were similar in tri12 CLL compared to no tri12 cases. Tri12 CLL, however, underwent stronger activation when cultured in presence of accessory cells (%CD69+ cells 60.0±18.5 (n=4) vs. 17.7±20.1 (n=19), p=0.008). Moreover, in several setups, proliferation rates of these cells were increased, irrespective of the proliferative stimulus and detection method used. In summary, our results provide a mechanistical basis at least in part explaining the peculiar and clinical features of the tri12 CLL subset. In light of the specific migratory and proliferative properties of tri12 cells and novel agents targeting particularly these functions, our findings may also imply therapeutical consequences. Disclosures: Greil: NOXXON Pharma AG: Research Funding. Hartmann:NOXXON Pharma AG: Research Funding.

BMS-936564 (MDX1338): A Fully Human Anti-CXCR4 Antibody Induces Apoptosis in an in Vitro Model of Stromal – Leukemia Cell Interaction for Chronic Lymphocytic Leukemia.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2887-2887
Author(s):  
Manoj Kumar Kashyap ◽  
Deepak Kumar ◽  
Harrison Jones Jones ◽  
Michael Y. Choi ◽  
Johanna Melo-Cardenas ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Stromal Cells ◽  
Leukemia Cell ◽  
Cell Interaction ◽  
Lymphocytic Leukemia ◽  
Leukemia Cells ◽  
Drug Induced ◽  
Induced Apoptosis ◽  
Cells Cultured

Abstract Abstract 2887 Chronic lymphocytic leukemia (CLL) remains incurable despite advances in the biology and treatment of this disease. Current data support the notion that resistance to therapy is promoted by a “protective” tumor microenvironment in which non-leukemia cells produce factors that enhance the resistance of CLL cells to spontaneous or drug-induced apoptosis. One such factor is the chemokine CXCL12, which interacts with its receptor CXCR4 on CLL cells to promote cancer cell survival. To examine the therapeutic potential of blocking CXCL12-CXCR4 interactions, we studied the effect of BMS-936564, a fully human IgG4 anti-CXCR4 antibody, using an in vitro co-culture model of human bone marrow derived stomal-NKter cells – leukemia cell interaction. Such stromal-NKter cells secrete CXCL12 and enhance the resistance of CLL cells to apoptosis in vitro. We observed that primary CLL cells co-cultured with stromal-NKter cells had significantly greater viability than CLL cells cultured alone (20–60% above baseline at 48 hours). Moreover, CLL cells co-cultured with stromal cells had enhanced resistance to drug-induced apoptosis. We found that BMS-936564 antibody at concentrations of 2–200nM could enhance the rate of apoptosis of CLL cells cultured alone or in the presence of stromal cells. CLL cells that expressed unmutated IgVH genes or ZAP-70 appeared equally susceptible to treatment with BMS-936564 as did CLL cells that lack these adverse prognostic markers, as did CLL cells that harbored deletions in 17p13.2 and that were resistant to chemotherapeutic agents, such a fludarabine monophosphate. BMS-936564 antibody inhibited CXCL12 mediated F-Actin polymerization in CLL cells at lower concentrations (20–200nM) compared to AMD-3100 (Mozobil), a small molecule CXCR4 inhibitor (50–150μM). In addition, AMD-3100 did not induce apoptosis in CLL cells (10–300μM). In summary, we observed that the anti-CXCR4 antibody BMS-936564 inhibited CXCL12 mediated activation of the CXCR4 receptor in CLL cells and induced apoptosis in leukemia cells. The pro-apoptotic activity of BMS-936564 was observed in cells cultured alone or together with stromal cells suggesting that this antibody had direct cytotoxic effect on leukemia cells and that it can overcome the protective tumor microenvironment. More over, the activity of BMS-936564 was independent of the presence of poor prognostic factors such as del(17p) suggesting that its mechanism of action is P53 independent. These findings show evidence that the CXCR4-CXCL12 pathway is a valid therapeutic target in CLL and provide additional biological rationale for ongoing clinical trials in CLL and other hematological malignancies using BMS-936564. Disclosures: Kuhne: Bristol-Myers Squibb: Employment. Sabbatini:Bristol-Myers Squibb: Employment. Cohen:Bristol-Myers Squibb: Employment. Shelat:Bristol-Myers Squibb: Employment. Cardarelli:Bristol-Myers Squibb: Employment. Kipps:Abbott: Consultancy, Research Funding.

Targeting CK2 Overexpression as a Novel Therapeutic Tool in Chronic Lymphocytic Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 803-803
Author(s):  
Leila R. Martins ◽  
Paulo Lúcio ◽  
Paula Gameiro ◽  
Maria Gomes Silva ◽  
Joao T Barata
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Cell Survival ◽  
Peripheral Blood ◽  
Leukemia Cell ◽  
Lymphocytic Leukemia ◽  
Great Promise ◽  
Dependent Manner ◽  
Healthy Controls ◽  
Primary Leukemia

Abstract Abstract 803 Characterization of the molecular mechanisms that regulate viability of B cell chronic lymphocytic leukemia (CLL) cells may provide novel insights into the biology of this incurable malignancy and reveal prognostic markers and therapeutic targets. In particular, specific inhibition of signaling elements essential for leukemia cell survival offers great promise for the design of more efficient and selective therapies. The protein serine/threonine kinase CK2 is frequently upregulated in different cancers and mounting evidence implicates CK2 in tumorigenesis. In the present study, we evaluated whether CK2 may play a significant role in CLL. Peripheral blood mononuclear cells (PBMCs) from CLL patients (n=15) and healthy controls (n=7 ) were initially compared by Western blot densitometry analysis. CK2α, but not CK2β, was significantly upregulated in primary CLL patient samples (P=0.0003; Unpaired t-test), and CK2α expression clearly correlated with CK2 in vitro kinase activity (P value=0.0165). To test the functional significance of CK2 overexpression in CLL, we cultured PBMCs collected from leukemia patients (n=53) or purified CLL cells (n=7) in the presence of the CK2-specific small molecule inhibitors TBB and DRB. As determined by Annexin V-APC/7AAD staining and analysis of procaspase 3 cleavage, both TBB and DRB clearly promoted apoptosis of CLL cells (CD19+CD5+) in a time- and dose-dependent manner. Importantly, neither CK2 antagonist induced significant cell death in the normal T-cell population (CD19-CD3+) present in each PBMC patient sample. Consequently, the percentage of normal T-cells dramatically increased upon TBB/DRB treatment. Further, B-cells from healthy controls (n=7) were not affected by TBB or DRB, confirming the selectivity of the CK2 inhibitors towards leukemia cells. Notably, although co-culture with OP9 stromal cells promoted primary leukemia cell survival in vitro, it did not prevent apoptosis of CLL cells mediated by CK2 inhibition. The pro-apoptotic effect of TBB/DRB did not correlate with clinical parameters such as lymphocyte doubling time, ZAP-70 expression or IGHV mutational status. Interestingly, there was a significant association between the percentage of CLL cells in the peripheral blood and sensitivity to CK2 inhibition (e.g. 25μM TBB, P=0.0043), and treatment with 12.5μM TBB correlated with Binet stage (P=0.0043). We previously showed that CK2 phosphorylates and thereby inactivates PTEN in primary T-ALL cells leading to the hyperactivation of PI3K signaling pathway (Silva et al, JCI 2008). Here, we found that primary CLL samples displayed higher P-PTEN/PTEN ratios than healthy controls (P=0.0034), which correlated with CK2 expression (P=0.0064). Furthermore, treatment with TBB/DRB abrogated PTEN phosphorylation in 2 primary leukemia samples analyzed, suggesting that CK2 negatively regulates the activity of the tumor suppressor PTEN in CLL and raising the possibility that the pro-survival effect of CK2 may be dependent, at least in part, on the activation of PI3K pathway. Overall, our study suggests that CK2 plays an important role in the biology of CLL, and lays the groundwork for the inclusion of CK2 antagonists into future therapeutic options. Disclosures: No relevant conflicts of interest to declare.

The Immunophenotype Signature CD49d+CD38+ Identifies Chronic Lymphocytic Leukemia Cases with a Higher Potential for Migration Beneath Marrow Stromal Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 356-356 ◽  
Author(s):  
Antonina Kurtova ◽  
Mariela Sivina ◽  
Maite P. Quiroga ◽  
William G. Wierda ◽  
Michael J. Keating ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Chronic Lymphocytic Leukemia ◽  
Stromal Cells ◽  
Conflicts Of Interest ◽  
Residual Disease ◽  
Lymphocytic Leukemia ◽  
Marrow Stromal Cells ◽  
Lymphoid Tissues ◽  
Mutational Status

Abstract Abstract 356 Adhesion of chronic lymphocytic leukemia (CLL) cells to stromal cells in the marrow and secondary lymphoid tissues confers drug resistance and may account for survival and maintenance of residual CLL cells after conventional treatments, paving the way to relapses. Therefore, targeting the cross talk between CLL cells and stromal cells represents an attractive and necessary approach to overcome minimal residual disease in CLL. We previously reported that fractions of CLL cells and other neoplastic B cell spontaneously migrate beneath marrow stromal cells (MSC) in a CXCR4- and CD49d-(VLA-4) dependent fashion, an in vitro phenomenon termed pseudoemperipolesis (PEP). Also, we reported that cells that migrated beneath and underneath MSC were largely protected from cytotoxic drugs, in contrast to cells that remained in the supernatant (Blood 113:4604-13, 2009). The aim of this study was to identify correlations between CLL surface markers and their ability to migrate beneath MSC. We tested samples from 116 different CLL patients in co-culture assays for their ability to migrate beneath MSC (KUSA H1 cells). After 6 hours of incubation, CLL cells were vigorously washed off the MSC layers, and PEP+ cases were identified by phase contrast microscopy as cases, in which CLL cells were visualized within the stromal layers, characterized by the dark appearance of CLL cells that had migrated into the same focal plane as the MSC. We characterized 45 PEP+ and 71 PEP- CLL cases. Next, we profiled the expression of adhesion molecules and chemokine receptors, including CD44, CD49d, CD62L, CXCR3, CXCR4 and CXCR5 by flow cytometry in the PEP+ and PEP- subgroups. Based on the mean fluorescence intensity ratios (MFIR), we found that both groups displayed comparable levels of CD44 (58.8±4.6 in PEP+ vs. 58.7±3.5 in PEP-), CD62L (7.8±1.3 vs. 9.3±1.3), CXCR3 (7.9±1.0 vs. 8.0±0.8), CXCR4 (87.6±9.3 vs. 81.4±4.6), and CXCR5 (47.4±2.9 vs. 49.3±2.7). In contrast, the levels of CD49d were significantly different between these two groups; the MFIR for CD49d was 14.0±2.4 in PEP+ cases and 4.0±0.7 in PEP- cases (p<0.0001, see Fig. 1). Interestingly, in PEP+ cases, higher levels of CD49d correlated with lower levels of CXCR4 (r=-0.43; p<0.003), which could be due to higher affinity to a CXCL12-rich microenvironment, with respective CXCR4 down regulation. Analysis of prognostic factors (CD38, ZAP-70 and mutational status) revealed that PEP+ cases displayed higher level of CD38 expression: 29.8±5.8% vs. 15.6±2.7% (MFIR, p<0.05), whereas there was no difference in ZAP-70 expression or mutational status between PEP+ or PEP- cases. When comparing cytogenetic profiles, favorable risk (del13q14.3 and normal karyotype) cases represented 43.6% in the PEP+ vs. 71.2% in PEP- groups; trisomy12 cases 23.1% vs. 4.5%, and poor risk cytogenetics (del11q, del17p, complex) represented 33.3% in the PEP+ vs. 24.3% in PEP- group. Collectively, the distinct immunophenotype of CD49d+CD38+ identifies CLL cases with a higher affinity for migration beneath MSC. Consequently, CLL cells in such cases are expected to be more difficult to eradicate by conventional treatments because of higher levels of stroma-mediated drug resistance. Drugs, such as Natalizumab or Plerixafor, that target CLL-stroma interactions, could be of particular benefit for such patients by disrupting CLL-stroma crosstalk and mobilization of CLL cells from protective marrow niches. Disclosures: No relevant conflicts of interest to declare.

scholarly journals T cell chronic lymphocytic leukemia: presence in bone marrow and peripheral blood of cells that suppress erythropoiesis in vitro

Blood ◽  
1978 ◽  
Vol 52 (1) ◽  
pp. 255-260 ◽  
Author(s):  
R Hoffman ◽  
S Kopel ◽  
SD Hsu ◽  
N Dainiak ◽  
ED Zanjani
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
Chronic Lymphocytic Leukemia ◽  
T Lymphocytes ◽  
Peripheral Blood ◽  
Lymphocytic Leukemia ◽  
Transfusion Therapy ◽  
Cell Chronic Lymphocytic Leukemia ◽  
Marrow Cells

Abstract The pathogenesis of the anemia associated with malignancy was investigated in a patient with T cell chronic lymphocytic leukemia. The plasma clot culture system was used as a measure in vitro of erythropoiesis. The patient's peripheral blood and marrow T lymphocytes obtained both before and after transfusion therapy suppressed erythroid colony formation by normal human bone marrow cells. Pretreatment of the patient's bone marrow T cells by antithymocyte globulin (ATG) and complement reversed this suppression. In addition, pretreatment of the patient's marrow cells with ATG and complement markedly augmented erythropoiesis in vitro. The expression of erythroid activity caused by the selective destruction of the suppressor T lymphocytes in the patient's bone marrow with ATG and the suppression of normal erythropoiesis by the patient's bone marrow and peripheral blood lymphocytes suggest that interaction between the malignant T cell and the erythropoietin-responsive stem cell is important in production of anemia in this patient.

Reconstitution of PTEN activity by CK2 inhibitors and interference with the PI3-K/Akt cascade counteract the antiapoptotic effect of human stromal cells in chronic lymphocytic leukemia

Blood ◽  
2010 ◽  
Vol 116 (14) ◽  
pp. 2513-2521 ◽  
Author(s):  
Medhat Shehata ◽  
Susanne Schnabl ◽  
Dita Demirtas ◽  
Martin Hilgarth ◽  
Rainer Hubmann ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Chronic Lymphocytic Leukemia ◽  
Stromal Cells ◽  
Molecular Mechanisms ◽  
Mononuclear Cells ◽  
Lymphocytic Leukemia ◽  
Therapeutic Concept ◽  
Antiapoptotic Effect ◽  
Human Stromal Cells ◽  
Ck2 Inhibitors

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

scholarly journals Involvement of CED-3/ICE Proteases in the Apoptosis of B-Chronic Lymphocytic Leukemia Cells

Blood ◽  
1997 ◽  
Vol 89 (9) ◽  
pp. 3378-3384 ◽  
Author(s):  
Beatriz Bellosillo ◽  
Mireia Dalmau ◽  
Dolors Colomer ◽  
Joan Gil
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Lymphocytic Leukemia ◽  
Interleukin 4 ◽  
Leukemia Cells ◽  
Parp Cleavage ◽  
Dependent Manner ◽  
Kinase C ◽  
Dose Dependent ◽  
Pkc Activation

Abstract B-chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of long-lived B lymphocytes that express high levels of Bcl-2. We examined the involvement of CED-3/ICE-like proteases in the apoptosis of B-CLL cells. One of the substrates of these proteases is poly(ADP [adenosine 5′-diphosphate]-ribose) polymerase (PARP). The effect of different factors that induce the apoptosis of B-CLL cells on the proteolytic cleavage of PARP has been studied. Treatment of B-CLL cells with different concentrations of dexamethasone (1 to 1,000 μmol/L) induced in a dose-dependent manner the cleavage of PARP. Dexamethasone induced PARP cleavage after 12 hours of incubation, which was almost complete at 48 hours. PARP cleavage during apoptosis of B-CLL cells was studied in cells from eight patients and a correlation was found between cell viability and the degree of PARP cleavage. Incubation in vitro of B-CLL cells with fludarabine for 48 hours induced PARP cleavage in all the cases studied. Protein kinase C (PKC) activation with 100 nmol/L TPA (12-O-tetradecanoylphorbol 13-acetate) or incubation with interleukin-4 (10 ng/mL) prevented either dexamethasone- or fludarabine-induced proteolysis of PARP. Incubation of B-CLL cells with the CED-3/ICE–like protease inhibitor Z-VAD.fmk inhibited spontaneous and dexamethasone-induced PARP cleavage and DNA fragmentation in a dose-dependent manner. Furthermore, Z-VAD.fmk prevented the cytotoxic effect of dexamethasone. These results indicate that CED-3/ICE–like proteases play an important role in the apoptosis of B-CLL cells.

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