Chronic Lymphocytic Leukemia-Exosomes Switch Endothelial and Mesenchymal Stromal Cells into Cancer-Associated Fibroblasts to Sustain Leukemic Cell Survival

Abstract Chronic lymphocytic leukemia (CLL), the most common hematologic malignancy in Western countries, is mostly affecting the elderly over 65 year-old. CLL is characterized by the accumulation of mature but non-functional B lymphocytes of clonal origin in the blood and the primary lymphoid organs. CLL was previously considered as a relatively static disease resulting from the accumulation of apoptosis-resistant but quiescent B lymphocytes. However, recent studies using heavy water labeling indicated that CLL is in fact a very dynamic disease with alternation of proliferation phases and peripheral circulation. A focus on the trafficking of CLL cells in vivo has shown that leukemic cells circulate between the blood and the lymphoid organs but have a preference for the bone marrow. Recent next-generation sequencing of CLL cells indicated the presence of different genetic subclones. This intraclonal heterogeneity observed in CLL subpopulations may be in part determined by the interactions that leukemic cells entertain with their microenvironment when B cells migrate into the lymph nodes and the bone marrow. Indeed, tumor-stroma interactions are not only providing signals necessary for leukemic cells survival but may also influence the clonal architecture and evolution. One of these interactions involves CLL-derived exosomes. Here, we show that CLL-exosomes efficiently transfer nucleic acids, including functional microRNAs, and proteins, including MHC-Class II molecules and B-cell specific proteins, to bone marrow mesenchymal stem cells and endothelial cells. CLL-exosomes also activate signaling pathways, including PI3K and NF-κB pathways, in these stromal cells. As a consequence, gene expression is strongly modified indicating a switch towards a cancer-associated fibroblast phenotype. Functionally, exosome-stimulated stromal cells show a striking actin cytoskeleton remodeling characterized by the formation of stress fibers, and enhanced proliferation, motility and angiogenic properties. We also identified several proteins synthesized and secreted by stromal cells that promote leukemic cell adhesion and survival ex vivo. To confirm the involvement of CLL-exosomes in CLL pathology in vivo, MEC-1-eGFP cells were subcutaneously injected into immunocompromised NSG mice together with CLL-exosomes. We observed a significant increase in tumor size and a reduction in survival of exosome-treated animals. Flow cytometry analysis of selected organs indicated an enrichment in leukemic cells in the kidney, providing a potential explanation to the renal failures observed in CLL patients. In conclusion, the communication between CLL cells and stromal cells may be a critical factor influencing CLL progression by promoting leukemic cell survival. This study demonstrates the crucial role of exosomes as mediators of the communication between leukemic cells and their microenvironment. Exosomes could thus represent a suitable target for therapeutic intervention in CLL. Disclosures No relevant conflicts of interest to declare.

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Expression of ZAP-70 Does Not Accelerate Leukemia Development and Progression in the Eμ-TCL1 Transgenic Mouse Model of Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v120.21.925.925 ◽

2012 ◽

Vol 120 (21) ◽

pp. 925-925

Author(s):

Stefania Gobessi ◽

Francesca Belfiore ◽

Sara Bennardo ◽

Brendan Doe ◽

Luca Laurenti ◽

...

Keyword(s):

T Cells ◽

Chronic Lymphocytic Leukemia ◽

Mouse Model ◽

Leukemic Cell ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Leukemia Development ◽

Low Levels ◽

And Behavior

Abstract Abstract 925 One of the most relevant prognostic factors in chronic lymphocytic leukemia (CLL) is expression of the protein tyrosine kinase ZAP-70. Typically, patients whose leukemic cells express ZAP-70 at 30–100% of the levels in normal T cells have aggressive disease, whereas patients whose leukemic cells do not express ZAP-70 or express only low levels of this protein have indolent disease. Previously, we and others demonstrated that ZAP-70 modulates B-cell receptor signaling and thus affects the capacity of the leukemic cells to respond to antigen stimulation. However, a direct link between an altered antigen response and CLL pathogenesis has still not been established and, more importantly, the question whether ZAP-70 directly contributes to the aggressiveness of the disease or is just a marker of aggressive CLL still remains to be answered. We have now addressed these issues by analyzing in vivo the impact of forced expression of ZAP-70 on the development and behavior of leukemias that arise in the Eμ-TCL1 transgenic (tg) mouse model of CLL. This model is characterized by the development of antigen-driven leukemias that resemble human CLL in many aspects but are always ZAP-70-negative. To force the expression of ZAP-70 in TCL1 leukemias, we generated two tg lines with targeted expression of ZAP-70 in the B cell compartment (ZAP70high and ZAP70low) and crossed them with Eμ-TCL1 tg mice. B cells in ZAP70high tg mice express similar levels of ZAP-70 as normal mouse T cells, whereas the levels of ZAP-70 in B cells of ZAP70lowtg mice are approximately 10 times lower. Both cohorts of Eμ-TCL1/ZAP70 double tg mice developed characteristic TCL1 leukemias. Eμ-TCL1/ZAP70low tg mice developed leukemias with onset and rate of progression similar to their ZAP-70-negative littermates, indicating that low levels of ZAP-70 do not alter the development and behavior of the disease. Surprisingly, Eμ-TCL1/ZAP70high tg mice developed leukemias with an approximately 2 month delay compared to their ZAP-70-negative Eμ-TCL1 tg littermates, which was contrary to the expectation that high ZAP-70 expression will accelerate leukemia development. The delay in leukemia development was especially evident at 6 months of age, when leukemic cells could be detected in the PB of 77% (10/13) of Eμ-TCL1 tg mice and only 24% (4/17) of Eμ-TCL1/ZAP70hightg mice (P=0.011). Since ZAP-70 expression can affect the migratory and adhesion capacity of human CLL cells in vitro, we first investigated if the delayed appearance of leukemic cells in the PB of Eμ-TCL1/ZAP70high tg mice could be due to increased retention of the leukemic cells in the lymphoid tissues. Assessment of tumor burden in the spleen, peritoneal cavity (PC), bone marrow and PB of 7 months old mice showed that the number of tumor cells in each compartment was significantly lower in Eμ-TCL1/ZAP70hightg mice than their Eμ-TCL1 littermates, suggesting that the delay in leukemia appearance is not caused by increased tissue retention but rather by reduced tumor growth. To investigate if ZAP-70 impairs tumor growth by affecting proliferation, we performed in vivo BrdU incorporation analysis of leukemic cells from spleen and PC of Eμ-TCL1 and Eμ-TCL1/ZAP70high tg mice. Spleen and PC samples were analyzed because they are the major sites of leukemia proliferation in Eμ-TCL1 tg mice. Interestingly, while the percentage of proliferating leukemic cells in the spleens of Eμ-TCL1 and Eμ-TCL1/ZAP70high tg mice was similar (mean % of BrdU+ cells ±SD: 6.81 ±1.67 and 6.15 ±2.92, respectively; P=n.s.), the percentage of proliferating leukemic cells in the PC of Eμ-TCL1/ZAP70high tg mice was significantly lower (mean % of BrdU+cells ±SD: 1.74 ±1.05 and 0.56 ±0.39, respectively; P=0.024). In summary, this study shows that ZAP-70 expression, per se, is unable to accelerate leukemia development and progression in an established in vivo model of CLL and suggests that ZAP-70 is not directly responsible for the greater disease severity in the poor prognosis subset of CLL. In addition, this study reveals that ZAP-70 in certain tissue environments can function as a negative regulator of leukemic cell proliferation, contrary to the widespread perception of ZAP-70 as a positive regulator of leukemic cell responses. Disclosures: No relevant conflicts of interest to declare.

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In vitro and in vivo model of a novel immunotherapy approach for chronic lymphocytic leukemia by anti-CD23 chimeric antigen receptor

Blood ◽

10.1182/blood-2010-10-311845 ◽

2011 ◽

Vol 117 (18) ◽

pp. 4736-4745 ◽

Cited By ~ 51

Author(s):

Greta Maria Paola Giordano Attianese ◽

Virna Marin ◽

Valentina Hoyos ◽

Barbara Savoldo ◽

Irene Pizzitola ◽

...

Keyword(s):

T Cells ◽

Chronic Lymphocytic Leukemia ◽

B Lymphocytes ◽

Chimeric Antigen Receptor ◽

Antigen Receptor ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Car T Cells ◽

Car T

Abstract Chronic lymphocytic leukemia (CLL) is characterized by an accumulation of mature CD19+CD5+CD20dim B lymphocytes that typically express the B-cell activation marker CD23. In the present study, we cloned and expressed in T lymphocytes a novel chimeric antigen receptor (CAR) targeting the CD23 antigen (CD23.CAR). CD23.CAR+ T cells showed specific cytotoxic activity against CD23+ tumor cell lines (average lysis 42%) and primary CD23+ CLL cells (average lysis 58%). This effect was obtained without significant toxicity against normal B lymphocytes, in contrast to CARs targeting CD19 or CD20 antigens, which are also expressed physiologically by normal B lymphocytes. Moreover, CLL-derived CD23.CAR+ T cells released inflammatory cytokines (1445-fold more TNF-β, 20-fold more TNF-α, and 4-fold more IFN-γ). IL-2 was also produced (average release 2681 pg/mL) and sustained the antigen-dependent proliferation of CD23.CAR+ T cells. Redirected T cells were also effective in vivo in a CLL Rag2−/−γc−/− xenograft mouse model. Compared with mice treated with control T cells, the infusion of CD23.CAR+ T cells resulted in a significant delay in the growth of the MEC-1 CLL cell line. These data suggest that CD23.CAR+ T cells represent a selective immunotherapy for the elimination of CD23+ leukemic cells in patients with CLL.

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The PI3 Kinase δ Inhibitor, CAL-101 (GS-1101), Inhibits Chronic Lymphocytic Leukemia (CLL) Cell Survival in Endothelial and Marrow Stromal Cell Co-Cultures.

Blood ◽

10.1182/blood.v118.21.1769.1769 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1769-1769 ◽

Cited By ~ 1

Author(s):

Stefania Fiorcari ◽

Wells S Brown ◽

Bradley W McIntyre ◽

Susan O'Brien ◽

Mariela Sivina ◽

...

Keyword(s):

Bone Marrow ◽

Endothelial Cells ◽

Chronic Lymphocytic Leukemia ◽

Cell Survival ◽

Stromal Cells ◽

Lymphocytic Leukemia ◽

Clinical Activity ◽

Spontaneous Apoptosis ◽

Cell Protection ◽

Survival Signals

Abstract Abstract 1769 CLL cells are characterized by their ability to resist apoptosis in vivo, but in vitro they undergo spontaneous apoptosis. This suggests that interactions between CLL cells and accessory cells in the tissue microenvironments, such as mesenchymal stromal cells (MSC), nurselike cells (NLC), T-cells, and endothelial cells are critical for maintaining CLL cell survival. CLL cells display constitutive PI3K pathway activation, presumable due to CLL interactions with the microenvironment. CAL-101 is a potent and selective inhibitor of the p110d PI3K isoform and has shown promising clinical activity in chronic lymphocytic leukemia (CLL) in early stage clinical trials. Here, we investigated the ability of CAL-101 to disrupt interactions between CLL and endothelial cells (EC) or bone marrow stromal cells (BMSC). We tested two EC lines human umbilical vein endothelial cells (HUVEC) and UV-2 mouse vascular endothelial cells, and two BMSC lines, stroma-NKtert derived from human bone marrow, and KUSA-H1, a murine BMSC line. CLL cells were cultured for 72h in presence or absence of EC or BMSC. Fig A displays mean (±SEM) CLL cell viabilities of cells from 7 different patients. We found that both, EC and BMSC rescue CLL cells from spontaneous apoptosis with significantly higher CLL cell viabilities in the presence of EC and BMSC (*P< 0.05; **P< 0.01). For example, after 48h significantly higher CLL cell viabilities were noticed with HUVEC (53.2%±4.3%, p<0.05,), UV2 (61.8%±5.3%, p<0.01), stroma-NKtert (96.7%±5.3%, p<0.01) and KUSA-H1 (93.7%±0.95%, p<0.01), when compared to CLL cultured in medium alone (37.5%±4.1%). To test the effects of CAL-101 on EC- and BMSC-mediated CLL cell protection, CLL cells were cultured on ECs or BMSCs in presence or absence of CAL-101 (0.5μM and 5μM), and CLL cell viabilities were assessed at 24h, 48h and 72h. Viabilities of CAL-101 treated samples were normalized to the viabilities of control samples at the respective timepoints (100%). Fig B depicts the mean relative viabilities of CLL cells co-culture with ECs or BMCSs in presence of 5μM CAL-101, compared to CLL cells in the absence of CAL-101. We found a significant reduction of the viability of CLL cells in co-culture with EC and BMSC with both concentrations of CAL-101 (*P< 0.05; **P< 0.01; n=7). These data demonstrate that marrow stromal and endothelial cells both support the viability and protect CLL cells from apoptosis. When comparing BMSC with EC, we noticed that BMSC were more effective than EC in protecting CLL cells, which may explain why the marrow is a preferred site for residual disease and relapses in patients with CLL. CAL-101 can overcome both, BMSC- and EC-mediated CLL cell protection, indicating that CAL-101 inhibits BMSC- and EC-derived pro-survival signals. Ongoing experiments investigate the role of adhesion molecules on BMSC- and EC-derived survival signals and CLL cell adhesion to BMSC versus EC, and how adhesion molecule function is affected by CAL-101. These studies will give us better insight into the mechanism of action of this interesting new drug. Disclosures: O'Brien: Gilead: Consultancy, Research Support. Lannutti:Gilead Sciences: Employment.

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The Valproic Acid-Fludarabine Combination Induces a Synergistic Response in Chronic Lymphocytic Leukemia Via a Mechanism Involving the Lysosomal Protease Cathepsin B.

Blood ◽

10.1182/blood.v120.21.2892.2892 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2892-2892

Author(s):

Ju-Yoon Yoon ◽

David Szwajcer ◽

Ganchimeg Ishdorj ◽

Pat Benjaminson ◽

James B Johnston ◽

...

Keyword(s):

Valproic Acid ◽

Cell Death ◽

Chronic Lymphocytic Leukemia ◽

Cathepsin B ◽

Leukemic Cell ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Lysosomal Protease

Abstract Abstract 2892 Chronic Lymphocytic Leukemia (CLL) is the most common haematological malignancy in the western world. Fludarabine, a nucleoside analogue, is commonly used to treat Chronic Lymphocytic Leukemia (CLL) in untreated and relapsed CLL. However, patients commonly develop resistance to fludarabine. We hypothesize that the addition of Valproic Acid (VPA), an inhibitor of histone deacetylases (HDACs), can improve fludarabine-based therapy. The VPA-Fludarabine combination induced a synergistic response in human leukemic cells and primary CLL cells. Fludarabine also interacted synergistically with three other HDAC inhibitors, suberoylanilide hydroxamic acid (SAHA), Trichostatin A, and sodium butyrate, while the synergy was not observed with valpromide, the VPA analogue that does not inhibit HDACs. We confirmed that fludarabine treatment activates caspases-8, -9 and caspase-3, and we also show that fludarabine treatment activates caspase-2, an upstream caspase that has been implicated in cell death associated with lysosome membrane permeabilization (LMP). Activation of all four caspases was enhanced by the addition of VPA. Enhanced activation of caspases was associated with down-regulation of two prominent anti-apoptotic proteins, Mcl-1 and XIAP. The down-regulation of Mcl-1 and XIAP was dependent on the lysosomes, as their alkalinization using either chloroquine or NH4Cl partially stabilized both proteins, leading to reduced apoptosis. Chemical inhibition of a specific lysosomal protease, cathepsin B, using CA074-Me, was sufficient to stabilize Mcl-1 and XIAP, reduce caspase activation and apoptosis. Treatment with fludarabine or the VPA-fludarabine combination led to the loss of lysosome integrity, as visualized by fluorescent staining, thus suggesting a leakage of the lysosomal content into the cytosol in response to the drugs. Addition of purified cathepsin B to leukemic cell lysates led to the reduction in protein levels of Mcl-1, XIAP and pro-caspase-2, thus suggesting that the re-localization of cathepsin B into the cytosol is sufficient to drive cell death. VPA treatment enhanced cathepsin B levels in both leukemic cell lines and primary CLL cells. When cathepsin B activity was examined using zRR-AMC, a fluorogenic substrate of cathepsin B, VPA also increased cathepsin B activity, and this activity was abolished by the addition of CA074-Me. In parallel with the in vitro/ex vivo experiments, we had launched a phase II clinical trial at CancerCare Manitoba. Six relapsed CLL patients who had received at least one prior therapy with fludarabine were examined. No responses were seen after 28 days using VPA alone, in line with the in vitro observation of minimal cytotoxicity of VPA at low doses. However, in five patients who continued on VPA with fludarabine, three patients showed a >50% fall in lymphocyte/lymph node size after receiving five cycles of the combination. When the leukemic cells from VPA-treated CLL patients were examined, VPA administration induced increased levels of histone-3 acetylation and cathepsin B in vivo. In summary, a novel mechanism for fludarabine cytotoxicity has been elucidated, where fludarabine induces a loss of lysosomal integrity, leading to cathepsin B-dependent cell death. VPA interacted with fludarabine synergistically, and this synergy was associated with the VPA-induced increase in VPA level and activity. VPA induced increase in histone-3 acetylation and cathepsin B in vivo, and this induction of cathepsin B is likely to be contributing to the clinical response observed in fludarabine-relapsed/refractory CLL patients. Disclosures: Off Label Use: Valproic acid as adjunct therapy in Chronic Lymphocytic Leukemia. Johnston:Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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In Chronic Lymphocytic Leukemia the JAK2/STAT3 Pathway Is Constitutively Activated and Its Inhibition Leads to CLL Cell Death Unaffected by the Protective Bone Marrow Microenvironment

Cancers ◽

10.3390/cancers11121939 ◽

2019 ◽

Vol 11 (12) ◽

pp. 1939 ◽

Cited By ~ 6

Author(s):

Severin ◽

Frezzato ◽

Visentin ◽

Martini ◽

Trimarco ◽

...

Keyword(s):

Bone Marrow ◽

Cell Death ◽

Chronic Lymphocytic Leukemia ◽

Leukemic Cell ◽

Bone Marrow Microenvironment ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Bone Marrow Niche ◽

Stat3 Pathway ◽

Jak2 Inhibitor

The bone marrow microenvironment promotes proliferation and drug resistance in chronic lymphocytic leukemia (CLL). Although ibrutinib is active in CLL, it is rarely able to clear leukemic cells protected by bone marrow mesenchymal stromal cells (BMSCs) within the marrow niche. We investigated the modulation of JAK2/STAT3 pathway in CLL by BMSCs and its targeting with AG490 (JAK2 inhibitor) or Stattic (STAT3 inhibitor). B cells collected from controls and CLL patients, were treated with medium alone, ibrutinib, JAK/Signal Transducer and Activator of Transcription (STAT) inhibitors, or both drugs, in the presence of absence of BMSCs. JAK2/STAT3 axis was evaluated by western blotting, flow cytometry, and confocal microscopy. We demonstrated that STAT3 was phosphorylated in Tyr705 in the majority of CLL patients at basal condition, and increased following co-cultures with BMSCs or IL-6. Treatment with AG490, but not Stattic, caused STAT3 and Lyn dephosphorylation, through re-activation of SHP-1, and triggered CLL apoptosis even when leukemic cells were cultured on BMSC layers. Moreover, while BMSCs hamper ibrutinib activity, the combination of ibrutinib+JAK/STAT inhibitors increase ibrutinib-mediated leukemic cell death, bypassing the pro-survival stimuli derived from BMSCs. We herein provide evidence that JAK2/STAT3 signaling might play a key role in the regulation of CLL-BMSC interactions and its inhibition enhances ibrutinib, counteracting the bone marrow niche.

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Soluble CD14 is a novel monocyte-derived survival factor for chronic lymphocytic leukemia cells, which is induced by CLL cells in vitro and present at abnormally high levels in vivo

Blood ◽

10.1182/blood-2010-05-284505 ◽

2010 ◽

Vol 116 (20) ◽

pp. 4223-4230 ◽

Cited By ~ 52

Author(s):

Martina Seiffert ◽

Angela Schulz ◽

Sibylle Ohl ◽

Hartmut Döhner ◽

Stephan Stilgenbauer ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Cell Survival ◽

Nuclear Factor Κb ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Enzyme Linked Immunosorbent Assay ◽

Nuclear Factor ◽

Soluble Cd14

Abstract Accumulation of leukemic cells in patients with chronic lymphocytic leukemia (CLL) is due to prolonged cell survival rather than increased proliferation. Survival of CLL cells depends on microenvironmental factors. Even though long-lived in vivo, CLL cells rapidly die by spontaneous apoptosis in vitro unless cocultured with stromal cells or their conditioned medium. In the present study, we show that survival of CLL cells is maintained in high cell density cultures, where the main prosurvival activity is delivered by monocytes. Cytokine array and enzyme-linked immunosorbent assay studies revealed increased expression of soluble CD14 by monocytes in the presence of CLL cells. The addition of recombinant soluble CD14 to primary CLL cells resulted in significantly increased cell survival rates, which were associated with higher activity nuclear factor κB. Quantification of serum levels of soluble CD14 revealed abnormally high levels of this protein in CLL patients, indicating a potential role of soluble CD14 in vivo. In summary, the presented data show that monocytes help in the survival of CLL cells by secreting soluble CD14, which induces nuclear factor κB activation in these cells, and that CLL cells actively shape their microenvironment by inducing CD14 secretion in accessory monocytes.

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Role of Hematopoietic-Specific Protein 1 (HS1) in Apoptosis in B-Chronic Lymphocytic Leukemia.

Blood ◽

10.1182/blood.v108.11.2809.2809 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2809-2809

Author(s):

Livio Trentin ◽

Antonella Contri ◽

Anna Maria Brunati ◽

Federica Frezzato ◽

Martina Frasson ◽

...

Keyword(s):

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Cell Survival ◽

B Lymphocytes ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Cell Fractionation ◽

Normal Peripheral Blood ◽

Protein Levels ◽

The Mean

Abstract B-cell chronic lymphocytic leukemia (B-CLL) is the most common leukemia in adults and is characterized by the accumulation of clonal CD5+ B lymphocytes. Several protein kinase pathways have been claimed to be involved in the regulation of apoptosis and cell survival. We previously demonstrated that Src kinase Lyn is overexpressed at the protein level in leukemic cells as compared to normal B lymphocytes with substantial amount of the kinase anomalously present in the cytosol. Moreover, most of Lyn is constitutively active in resting leukemic cells and is poorly responsive to BCR engagement. The finding that B CLL cells contained cytosolic Lyn fraction and are defective in programmed cell death suggest that the tyrosine phosphorlation of specific cytosolic targets might account, at least in part, for cell resistance to apoptosis. The 75 KDa HS1 protein is one of the major substrate of Lyn kinase upon BCR cross-linking that plays a crucial role in BCR- induced apoptosis in the mouse B lymphoma cell line WEHI-231. A recent study demonstrates that most HS1 protein was constitutively phosphorylated in B CLL patients with poor prognosis whereas only a fraction was phosphorylated in patients with good prognoses. In the present study, the relative HS1 protein levels were measured by Western blot analysis in 50 CLL patients belonging to different clinical stages. The relative HS1 protein levels were compared with corresponding levels in normal peripheral blood and with Jurkat cells. For normal B cells, the mean ± SD for HS1: actin ratio was 0,88 ± 0,10. There was considerable variation in the levels of HS1/actin ratio in CLL cells, which ranged from 0,49 to 2,50. Thus, compared to normal B cells, 15 CLL patients had a HS1 level which fell within the mean ± 1SD HS1 levels for normal B cells, while 9 patients had lower levels and 26 patients had higher levels. When assessed by flow cytometry, HS1 expression was normally distributed among CLL cells in individual patients and the mean levels correlated with those obtained by Western blotting. A difference in the levels of HS1 was also observed between mutated and unmutated patients. Using confocal microscopy and subcellular cell fractionation, we observed that HS1 protein was abnormally distributed in malignant cells as compare with normal B cells: a 4–7% aliquot of HS1 was anomalously present in the nucleus of leukemic cells. When primary CLL cells were in vitro treated whith dexamethazone, cyclosporin A, chlorambucil, or fludarabine the HS1 levels decreased correlating with the sensitivity of these cells to undergo apoptosis. Using a polyclonal antiserum against HS1 a major cleavage product of the apparent molecular weight of 64 KDa and one minor product of approximately 46 Kda was detected in B CLL cells cultured for 24 hours whith drugs. These findings suggest that HS1 plays a pivotal role in the regulation of cell survival of leukemic B cells and suggest that HS1 might represent a target for the development of new drugs to be used in vivo in these patients.

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Chronic Lymphocytic Leukemic B Cells But Not Normal B Cells Are Rescued From Apoptosis by Contact With Normal Bone Marrow Stromal Cells

Blood ◽

10.1182/blood.v91.7.2387.2387_2387_2396 ◽

1998 ◽

Vol 91 (7) ◽

pp. 2387-2396 ◽

Cited By ~ 9

Author(s):

L. Lagneaux ◽

A. Delforge ◽

D. Bron ◽

C. De Bruyn ◽

P. Stryckmans

Keyword(s):

Bone Marrow ◽

B Cells ◽

B Lymphocytes ◽

Stromal Cells ◽

Cell Apoptosis ◽

Bone Marrow Stromal Cells ◽

Ex Vivo ◽

Marrow Stromal Cells ◽

Leukemic Cells

The leukemic B lymphocytes from chronic lymphocytic leukemic (CLL) patients have a long survival in vivo, although ex vivo they rapidly die by apoptosis. To further investigate the mechanism of this, we have studied the influence of bone marrow stromal cells from normal subjects on apoptosis of B-CLL cells and normal umbilical cord blood (UCB) B lymphocytes. After 48 hours of incubation in medium alone, leukemic and normal B cells showed, respectively, 22 ± 3% and 31 ± 5% of apoptosis. Cocultures with stromal cells reduced the percentage of leukemic cells undergoing apoptosis (8 ± 2%, P< .0005) and prevented the loss of bcl-2 protein expression. In contrast, stromal cells slightly increased normal B-cell apoptosis (37 ± 6%). Direct contact between leukemic cells and stromal cells was found to be essential for inhibition of leukemic cell apoptosis; indeed, separation of leukemic cells from stromal cells by microporous membrane increased spontaneous apoptosis, and comparable results were obtained with stromal cell conditioned medium. The difference in behavior observed between normal and leukemic B cells plated on stromal cells can be explained by the fact that only a few normal B cells adhere to stromal cells in comparison with B-CLL cells. B-CLL cell adhesion to stromal cells is mediated by β1 and β2 integrins acting simultaneously. Contact between B-CLL cells and bone marrow stromal cells seems to play a major role in the accumulation and survival of B-CLL cells in the bone marrow.

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Chronic Lymphocytic Leukemic B Cells But Not Normal B Cells Are Rescued From Apoptosis by Contact With Normal Bone Marrow Stromal Cells

Blood ◽

10.1182/blood.v91.7.2387 ◽

1998 ◽

Vol 91 (7) ◽

pp. 2387-2396 ◽

Cited By ~ 274

Author(s):

L. Lagneaux ◽

A. Delforge ◽

D. Bron ◽

C. De Bruyn ◽

P. Stryckmans

Keyword(s):

Bone Marrow ◽

B Cells ◽

B Lymphocytes ◽

Stromal Cells ◽

Cell Apoptosis ◽

Bone Marrow Stromal Cells ◽

Ex Vivo ◽

Marrow Stromal Cells ◽

Leukemic Cells

Abstract The leukemic B lymphocytes from chronic lymphocytic leukemic (CLL) patients have a long survival in vivo, although ex vivo they rapidly die by apoptosis. To further investigate the mechanism of this, we have studied the influence of bone marrow stromal cells from normal subjects on apoptosis of B-CLL cells and normal umbilical cord blood (UCB) B lymphocytes. After 48 hours of incubation in medium alone, leukemic and normal B cells showed, respectively, 22 ± 3% and 31 ± 5% of apoptosis. Cocultures with stromal cells reduced the percentage of leukemic cells undergoing apoptosis (8 ± 2%, P< .0005) and prevented the loss of bcl-2 protein expression. In contrast, stromal cells slightly increased normal B-cell apoptosis (37 ± 6%). Direct contact between leukemic cells and stromal cells was found to be essential for inhibition of leukemic cell apoptosis; indeed, separation of leukemic cells from stromal cells by microporous membrane increased spontaneous apoptosis, and comparable results were obtained with stromal cell conditioned medium. The difference in behavior observed between normal and leukemic B cells plated on stromal cells can be explained by the fact that only a few normal B cells adhere to stromal cells in comparison with B-CLL cells. B-CLL cell adhesion to stromal cells is mediated by β1 and β2 integrins acting simultaneously. Contact between B-CLL cells and bone marrow stromal cells seems to play a major role in the accumulation and survival of B-CLL cells in the bone marrow.

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PKC Inhibitor AEB071 Demonstrates Pre-Clinical Activity In Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v122.21.4187.4187 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4187-4187

Author(s):

Dalia ElGamal ◽

Yiming Zhong ◽

Katie Williams ◽

Chia-Hsien Wu ◽

Ching-Shih Chen ◽

...

Keyword(s):

T Cells ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Cell Survival ◽

Stromal Cells ◽

Lymphocytic Leukemia ◽

Clinical Activity ◽

Pkc Inhibitor ◽

Pkc Β

Abstract Targeting B-cell receptor (BCR) downstream pathways is of therapeutic importance in eradicating chronic lymphocytic leukemia (CLL) cells. Members of the protein kinase C (PKC) family play an important role in B-cell activation. PKC-β has recently been shown to be over-expressed in CLL and essential to CLL development in the TCL1 mouse model. Mice deficient in PKC-β exhibit a survival defect in response to BCR stimulation, correlating with an inability to induce the NF-κB-dependent anti-apoptotic proteins as Bcl-xL and A1. Moreover, PKC-β-dependent activation of NF-κB in stromal cells is pivotal for the survival of B-CLL cells in vivo; wherein PKC-β inhibition was shown to prevent microenvironment protection of CLL. Additionally, PKC-β lies downstream of PLC-γ2 where activating mutations have been noted in BTK (ibrutinib) resistant patients, which conveys a potential mechanism to target resistance related to mutations in this target protein. Therefore exploration of a PCK-β inhibitor in CLL is highly justified and innovative. Sotrastaurin (AEB071) is an orally administered potent inhibitor of classical and novel PKC isotypes; with strong and specific activity on PKC-α, PKC-β and PKC-θ and lesser activity on PKC-δ, PKC-ε, and PKC-η. Pre-clinically, AEB071 has demonstrated in vivo pre-clinical activity in activated B-cell diffuse large B-cell lymphoma (DLBCL) models and is currently being tested for efficacy in CD79b mutated DLBCL. Since PKC-β is indispensable for BCR-induced NF-κB activation and B-cell survival, herein we evaluate the impact of AEB071 on CLL cell survival as a promising therapeutic to target this pathway. Our preliminary work demonstrated that AEB071 was markedly cytotoxic to CLL cells in a dose-dependent (≤6.25uM, p<0.001) and time-dependent manner (p=0.011) as measured by MTS analysis. In a whole blood assay, AEB071 exhibits a retained selective cytotoxicity against tumor cells with a modest reduction in B-CLL cells whereas no effect on T-cells or natural killer cells was detected in CLL patient samples. Notably, upon treatment of blood from healthy subjects, AEB071 showed no toxic effects on normal B-cells, T-cells and natural killer cells. AEB071 inhibits CPG-induced survival of CLL cells in vitro (p<0.01), and effectively blocks the protection induced by soluble factors such as CD40L, IL-4, and TNF (p<0.01), which are known to reduce the spontaneous apoptosis associated with CLL cells. Similar effects were observed with stromal cell contact; wherein AEB071 showed enhanced cytotoxic potency on CLL cells under co-culture conditions with stromal cells compared to CLL alone (p<0.05). Additionally, AEB071 attenuated anti-IgM-induced survival of CLL cells with a modest induction of apoptosis (p<0.001). Furthermore, treatment of PMA- or BCR-activated CLL cells with AEB071 could effectively abrogate downstream survival pathways including ERK1/2, p38MAPK, AKT, GSK3β, and NF-κB as revealed by immunoblot analysis. Collectively, this data indicate that therapeutic strategies to inhibit PKC-β have the potential to disrupt signaling from the microenvironment that lead to in vivo CLL cell survival and potentially drug resistance. Current studies are ongoing to evaluate the in vivo tolerability and therapeutic efficacy of AEB071 in the Eμ-TCL1 transgenic mouse model of CLL. In conclusion, PKC-β represents an innovative target for CLL and therefore, future efforts targeting PKC with the PKC inhibitor AEB071 as monotherapy in clinical trials of relapsed and refractory CLL patients may be warranted. Disclosures: No relevant conflicts of interest to declare.

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