Supplementary Figure 3 from Cell Motility in Chronic Lymphocytic Leukemia: Defective Rap1 and αLβ2 Activation by Chemokine

Supplementary Plot 1 from c-Abl Expression in Chronic Lymphocytic Leukemia Cells: Clinical and Therapeutic Implications

Abstract Abstract 1763 Chronic Lymphocytic Leukaemia (CLL) is characterised by a clonal expansion of activated CD5+ mature B-lymphocytes. On antigen recognition by the B-cell receptor (BCR), a normal B cell may undergo apoptosis, progress to late B-cell differentiation, or become anergic. While many stimuli have been implicated in their survival and proliferation, CLL cells are selected and likely continually stimulated by antigen. Antigen recognition does not appear to preferentially induce either plasma-cell (PC) differentiation or apoptosis, but instead CLL lymphocytes remain activated and proliferate in an anergy-like state. The inability of CLL cells to differentiate into PC's, produce antibody and potentially eliminate the stimulating antigen is not well understood. PRDM1, a tumour suppressor gene, is essential for PC differentiation and antibody production. Its loss of expression, and function, has recently been shown to cause the manifestation of an activated B cell (ABC)-like Diffuse Large B cell Lymphoma (DLBCL). Similar to ABC-DLBCL, CLL cells also exhibit constitutive activation of the canonical NF-κB pathway. Whilst it is known that CLL cells express little or no PRDM1, the effect of PC inducing stimuli on PRDM1 expression in CLL cells has not been reported. The aim of this study was therefore to establish whether functional PRDM1 could be induced in CLL. We first studied the effects of IL-21 and CpG-ODN on CLL cells because of their potent effect on inducing PRDM1 in normal B cells. We found that after in vitro stimulation with either IL-21 or CpG-ODN, PRDM1 could be readily induced in the CLL cells from ∼50% of patients. Induction of PRDM1 was associated with PC differentiation and antibody secretion from these responsive clones (R-CLL). In contrast, a failure to induce PRDM1 from non-responsive CLL lymphocytes (NR-CLL) following stimulation was associated with a lack of PC differentiation and antibody production. Failure to induce PRDM1 expression in NR-CLL was due to a block in transcription despite efficient and preserved signal transduction from the IL-21 receptor or toll-like receptor 9 (TLR9; CpG-ODN receptor) as evidenced by induction of phosphorylated Tyr705 in STAT3 and activation of the p65 subunit of NF-κB respectively. Furthermore, there was no difference in induction of Mcl-1 (IL-21R target) or IκBα and IκBζ (TLR9 targets) in R- and NR-CLL clones after treatment with IL-21 or CpG-ODN. Importantly, additional co-stimuli such as CD40L and BCR crosslinking failed to overcome the block in PRDM1 transcription in NR-CLL. The failure to induce PRDM1 in NR-CLL was associated, significantly, with IGVH1-69 gene usage (n=8/8; P=0.03), while the IGVH3 gene-family expressing clones showed a tendency to induce PRDM1 (18/29; P=0.065). There was no significant association of PRDM1 induction with other well-characterised prognostic variables in CLL. We conclude and hypothesise that a failure to induce PRDM1 expression may be of pathobiological and therapeutic significance in some CLL patients (NR-CLL). A correlation with particular IGVH gene usage suggests a link to the antigen that might have selected and is driving the disease in vivo. Furthermore, this work illustrates that strategies that promote a forced induction of PRDM1 (e.g. with either IL-21 or CpG-ODN), may be exploited for therapeutic benefit in a subset of CLL patients, to induce terminal differentiation and cell death. Disclosures: No relevant conflicts of interest to declare.

Two-way interactions between HCs and the microenvironment are especially prominent in HCL. Many of these interactions are now reasonably well understood and are summarised in this article.

PURPOSE OF REVIEW:The biology of hairy-cell leukaemia is reviewed, focussing first on the hairy cell itself and then on its interactions with the microenvironment.RECENT FINDINGS:Hairy cells are highly activated clonal B cells related to memory cells, normally resident in the marginal zone of the spleen. Their activation results from multiple stimuli arising from the microenvironment, autocrine cytokines and the still unknown transforming oncogenic event(s) responsible for the disease. Protein kinase Cepsilon is a central player in the activation process.SUMMARY:The activation of hairy cells makes them unusually sensitive to interferon and nucleosides. Future important research topics include characterization of the oncogenic events responsible for the disease and for its associated differentiation block.

Background The aims of the present study were to ascertain the activation status of Akt in the primary cells of chronic lymphocytic leukemia and to investigate the effects of specific Akt inhibition on chronic lymphocytic leukemia-cell survival. Design and Methods Anti-phospho-Akt (Ser473 or Thr308) antibodies and western blotting were used to establish the activation status of Akt. The effects of two different, specific small-molecule inhibitors (A-443654 or Akti-1/2) or small interfering RNA on cell survival and downstream targets of Akt were assessed. Apoptosis was determined by fluorescence-activated cell sorting analysis of phosphatidylserine exposure and by measurement of PARP cleavage. The phosphorylation status of GSK-3 and MDM2, two immediate downstream substrates of Akt, levels of the anti-apoptotic proteins BCL2 and MCL1, and expression of p53 and p21 were all measured by western blotting. Results Fully activated Akt was demonstrable in all chronic lymphocytic leukemia clones examined (n=26). These results were validated with extensive controls and it was shown that a harsh method of cell extraction is needed for detection of the active enzyme. Specific inhibition of Akt induced extensive apoptosis of chronic lymphocytic leukemia cells, which was associated with both a rapid loss of MCL1 through proteasomal degradation and increased expression of p53. Moreover, the Akt inhibitors, at concentrations that induced extensive apoptosis in chronic lymphocytic leukemia cells, had little or no effect on normal peripheral blood mononuclear cells. Conclusions Chronic lymphocytic leukemia clones consistently contain activated Akt which plays a pivotal role in maintaining cell survival. Inhibition of the Akt pathway may be of potential value as a novel therapeutic strategy in chronic lymphocytic leukemia.

As in all malignancies, the biology of HCL reflects both the behaviour of the malignant cells (hairy cells) themselves and their two-way interaction with the microenvironment. However, the tissue interactions of HCs are particularly striking and involve extensive remodelling of bone marrow, spleen and liver, with relative sparing of lymph nodes. The mechanistic basis of this remodelling is now largely understood and is described herein. Regarding HCs themselves, they are late clonal B cells which have often undergone heavy-chain-isotype class switching and whose VH genes are usually mutated. HCs are highly activated cells in which a number of signalling pathways are constitutively active. This activation determines many of the specific features of HCs, but its cause remains unknown.

AbstractChemokine-induced activation of α4β1 and αLβ2 integrins (by conformational change and clustering) is required for lymphocyte transendothelial migration (TEM) and entry into lymph nodes. We have previously reported that chemokine-induced TEM is defective in chronic lymphocytic leukemia (CLL) and that this defect is a result of failure of the chemokine to induce polar clustering of αLβ2; engagement of α4β1 and autocrine vascular endothelial growth factor (VEGF) restore clustering and TEM. The aim of the present study was to characterize the nature of this defect in αLβ2 activation and determine how it is corrected. We show here that the αLβ2 of CLL cells is already in variably activated conformations, which are not further altered by chemokine treatment. Importantly, such treatment usually does not cause an increase in the GTP-loading of Rap1, a GTPase central to chemokine-induced activation of integrins. Furthermore, we show that this defect in Rap1 GTP-loading is at the level of the GTPase and is corrected in CLL cells cultured in the absence of exogenous stimuli, suggesting that the defect is the result of in vivo stimulation. Finally, we show that, because Rap1-induced activation of both α4β1 and αLβ2 is defective, autocrine VEGF and chemokine are necessary to activate α4β1 for ligand binding. Subsequently, this binding and both VEGF and chemokine stimulation are all needed for αLβ2 activation for motility and TEM. The present study not only clarifies the nature of the αLβ2 defect of CLL cells but is the first to implicate activation of Rap1 in the pathophysiology of CLL. [Cancer Res 2008;68(20):8429–36]

Hairy-cell leukemia is characterised by a marked sensitivity of the malignant cells to the cytotoxic effects of therapeutically administered interferon-alpha. The aim of this study was to assess the role of protein tyrosine phosphatases in the maintenance of hairy-cell (HC) viability and their sensitivity to interferon-alpha. The selective tyrosine phosphatase inhibitor mpV(pic) killed HCs, but not normal B lymphocytes or chronic lymphotic leukemia (CLL) cells. HCs displayed increased expression of the phosphatases SHP-1 and SHP-2 when compared with normal B lymphocytes. Phosphatase inhibition also enhanced the cytotoxic effect of interferon-alpha against HCs in four of the five cases tested. Therefore, HCs, but not normal B cells or CLL-cells, require tyrosine phosphatase activity for preservation of their viability. In addition, HC sensitivity to interferon is down-regulated by this activity.

We have previously identified the presence of Ras/Raf-independent constitutive activation of extracellular signal-regulated kinase (ERK) in the hairy cells (HCs) of hairy cell leukemia. The aim of the present study was to characterize the signaling components involved in this activation and their relationship to the reported activation of Rac1. We found that both Rac1 and ERK activation in HCs are downstream of active Src and protein kinase C (PKC). Inhibition with toxin B showed that Rac1 plays no role in ERK activation in HCs. However, toxin B inhibited p60src and the Rac1-GEF Vav, demonstrating a positive feedback/activation of p60src by Rac1. Treatment with specific small interfering RNA for various PKC isoforms, or with PKC isoform-specific inhibitors, demonstrated a central role for PKCepsilon in the constitutive activation of Rac1 and ERK in HCs. PKCepsilon and active ERK were mutually associated and co-localized with mitochondria in HCs. Furthermore, active PKCepsilon was nitrated on tyrosine, pointing to a reactive oxygen species-dependent mechanism of activation. By being involved in activation of ERK and Rac1, PKCepsilon plays roles in both the survival of HCs and in the cytoskeletal dynamics responsible for the distinctive morphology and tissue homing of these cells. Our study therefore describes novel aspects of signaling important for the pathogenesis of hairy cell leukemia.

Abstract c-Abl is important for normal B-cell development, but little is known about the function of this nonreceptor tyrosine kinase in chronic lymphocytic leukemia (CLL). Therefore, the aim of the present study was to examine the clinical, therapeutic, and pathogenetic importance of c-Abl in this disease. We show that the malignant cells of CLL predominantly express the type 1b splice variant of c-Abl and that the expression of c-Abl protein is higher in CLL cells than in normal peripheral blood B cells. Moreover, we show that the levels of c-Abl protein expression correlate positively with tumor burden and disease stage, and negatively with IgVH mutation. We also show that STI-571, an inhibitor of c-Abl kinase activity, induces apoptosis of CLL cells with high c-Abl expression levels through a mechanism involving inhibition of nuclear factor κB. We conclude that overexpression of c-Abl is likely to play a pathogenetic role in CLL and that STI-571 may be of potential use in the treatment of this disease. (Cancer Res 2006; 66(15): 7801-9)

HCs are clonal late B cells that are related to memory cells and display specific features of activation. Many of the distinctive features of HCs (eg, morphology, TRAP) are related to this specific activation. Many of the distinctive histologic features of HCL can be related to constitutive production of cytokines (eg, FGF, fibrosis) and to the expression/activation of adhesion receptors (eg, alpha(4)beta(1), alpha(5)beta(1) and alpha(v)beta(3) integrins, CD44v3). HCs usually have mutated IGVH genes and have no consistent or specific chromosome abnormalities (5q additions and 7q deletions in a minority). The signals that are responsible for several of the phenotypic features of HCs have been identified, but the nature of the underlying oncogenic events remains unknown.

Vascular endothelial cell growth factor (VEGF) is a multifunctional cytokine involved in tumor formation. In chronic lymphocytic leukemia (CLL), it is known that the malignant cells secrete VEGF and possess VEGF receptors. This suggests that an autocrine loop might be important in the pathogenesis of CLL. Here we show that, in patients with lymphadenopathy, autocrine VEGF and alpha(4)beta(1) integrin are involved in the chemokine-dependent motility of CLL cells on and through endothelium-processes important for the invasion of lymphoreticular tissues, a major determinant of disease outcome. In contrast, normal lymphocytes were not dependent on autocrine VEGF or alpha(4)beta(1) for either type of cell movement. Moreover, in contrast to normal B lymphocytes, CLL cells failed to cluster and activate alpha(L)beta(2) in response to chemokines, unless VEGF receptor(s) and alpha(4)beta(1) were also engaged by their respective ligands. This is the first demonstration that autocrine VEGF is involved in CLL-cell motility, and that the alpha(L)beta(2) on the malignant cells is functionally altered compared with that of normal B cells in not undergoing activation in response to chemokine alone. Given the importance of cell motility for tissue invasion, the present results provide a rationale for a trial of VEGF and alpha(4) blockade in patients with CLL who have tissue disease.

Hairy cells (HCs) are mature malignant B cells that contain a number of constitutively active signaling molecules including GTP-bound Rac1, protein kinase C, and Src family kinases. Because Rac1 is a component of the reactive oxidant species (ROS)-generating NADPH oxidase system, we investigated the role of this GTPase in ROS production in HCs. In this study, we show that ROS production in HCs involves a flavin-containing oxidase dependent on Ca2+, but not on GTP-Rac1 or protein kinase C. This suggests the involvement of the nonphagocytic NADPH oxidase NOX5, an enzyme found in lymphoid tissues, but not in circulating lymphocytes. By using RT-PCR and Southern and Western blotting and by measuring superoxide anion production in membrane fractions in the absence of cytosolic components, we demonstrate for the first time that HCs (but not circulating normal B cells or some other lymphoid cell types) express NOX5. We also demonstrate that inhibition of NADPH oxidase in HCs results in a selective increase in the activity of Src homology region 2 domain-containing phosphatase 1 (SHP-1). Furthermore, SHP-1 in HCs coimmunoprecipitates with tyrosine phosphorylated CD22 and localizes in the same cellular compartment as NOX5. This allows the inactivation of SHP-1 by NOX5-generated ROS and contributes to the maintenance of the constitutive activation of HCs.

CLL is a heterogeneous disease, but the mechanisms responsible for this heterogeneity are not fully understood. Previous studies have shown that global tyrosine phosphorylation in response to B-cell receptor (BCR) engagement is more pronounced in cases with low IgVH somatic hypermutation and worse prognosis, indicating an involvement of protein tyrosine kinases in progression of the disease. c-Abl, a non-receptor protein tyrosine kinase, plays an important role in B cell development, differentiation and BCR-mediated signalling. Inhibition of c-Abl with a specific reagent STI-571 induces CLL- cell death in vitro, and the sensitivity of CLL clones to this inhibitor seems to be variable. Since expression of c-Abl in CLL cells also varies, we hypothesized that response to STI-571 may be related to the level of c-Abl expression which, in turn, may be critical for protection of some CLL cells from apoptosis and therefore be associated with the disease progression. Western blot analysis confirmed that expression of c-Abl in malignant cells varies among CLL cases with a 13-fold difference between the minimum and the maximum. On average, the expression in CLL cells was 7.3-flold higher than in normal B-cells (n=7). Analysis of c-abl mRNA by quantitative RT-PCR confirmed this over-expression, and also showed that CLL cells exclusively express the anti-apoptotic 1b isoform of this protein. c-Abl expression in CLL cells showed a negative correlation with the extent of IgVH mutation (n=41, r=−0.55, P=0.003), but no correlation with either p53 functional status or CD38 positivity. Furthermore, c-Abl levels in CLL cells showed a positive correlation with peripheral blood WBC counts at sampling (n=43, r=0.570, P<0.001), and cases with a high c-Abl level (above the mean) were more likely to be at Binet stage B or C compared to the group with lower c-Abl (18/24 cases vs 3/18 cases, P<0.001). Functional studies showed that CLL clones with high c-Abl were more sensitive to STI-571-induced killing than CLL clones with low c-Abl expression (n=26, r=0.571, P=0.002). STI-571-induced killing of CLL-cells was associated with induction of apoptosis as indicated by mitochondrial depolarization (n=8) and by PARP cleavage (n=7). Further analysis revealed that STI-571 had no effect on Erk1/2 phosphorylation, despite a positive correlation between Erk1/2 tyrosine phosphorylation and c-Abl expression (n=31, r=0.606, P<0.001). However, c-Abl appears to play a role in NF-kB pathway signalling in CLL cells. Thus treatment of these cells either with STI-571 or with c-abl siRNA inhibited this pathway, particularly in those CLL cases with high c-Abl expression. In conclusion, this study has identified a group of prognostically unfavourable CLL cases in which malignant cells selectively overexpress the myristoylated 1b isoform of c-Abl. Treatment of these cells with STI-571 led to the inhibition of apparently constitutive activation of NF-kB and accelerated in vitro cell death by apoptosis. This indicates that, in CLL cases with overexpressed c-Abl, pharmacological inhibition of this enzyme may be of therapeutic benefit.

CD154 is an important regulator of chronic lymphocytic leukaemia (CLL)-cell survival. In CLL, high serum levels of VEGF are a feature of advanced disease, and we and others have previously shown that CLL cells produce and secrete this growth factor. Since CD154 stimulates VEGF production in other cell types, and VEGF is known to promote cell survival, we examined whether the cytoprotection of CLL cells by CD154 involves VEGF. We report for the first time that treatment of CLL cells with CD154 results in increased VEGF production and demonstrate involvement of NF- κ B in this process. Moreover, we show that CD154-induced CLL-cell survival is reduced by anti-VEGF-neutralising antibody and by inhibiting VEGF receptor (VEGFR) signalling with SU5416. However, addition of exogenous VEGF alone or blocking secreted autocrine VEGF had little or no effect on CLL-cell survival. We therefore conclude that CLL-cell cytoprotection in the presence of CD154 requires combined signalling by both CD40 and VEGFR. This combined signalling and resulting cytoprotection were shown to involve NF- κ B activation and increased survivin production. In conclusion, our findings identify autocrine VEGF as an important mediator of the antiapoptotic effect of CD40 ligation, and thus provide new insights into CLL-cell rescue by CD154 in lymphoreticular tissues.