Hallmark Of Chronic Lymphocytic Leukemia Research Articles

Protein Kinase C-β Dependent Activation of NF-κB in Stromal Cells Is Indispensable for the Survival of Chronic Lymphocytic Leukemia B-Cells in Vivo

AbstractAbstract 314Defects in the apoptosis program are a hallmark of chronic lymphocytic leukemia (CLL), characterized by high expression levels of bcl2 and Mcl1. Notably, this de-regulation of anti-apoptotic proteins is not sufficient to maintain long-term survival of CLL cells, which remain highly dependent on pro-survival factors provided by the leukemia-microenvironment. Bone marrow stromal cells (BMSCs) play an important role for microenvironment mediated survival of CLL cells, based on the provision of soluble and membrane-bound factors. The stroma-CLL interactions not only protect CLL cells from spontaneous, but also from drug-induced apoptosis, clinically recognized as minimal-residual disease. Therefore, understanding the molecular mechanisms of CLL-stroma interactions may offer new therapeutic options and help in eradicating CLL cells from the bone-marrow niche.Here we describe that monoclonal B-cells from CLL patients impose morphological and genetic changes in stromal cells, which become reminiscent of cancer-associated fibroblast (CAF). Comparative gene expression profiles indicate that contact with primary CLL cells induce the expression of pro-inflammatory genes in stromal cells. Further characterization of the underlying signaling pathways activated in stromal cells revealed that CLL cells induce the expression of protein-kinase C-β in BMSCs. Blocking the up-regulation of PKC-β by siRNA abrogated the pro-survival effects of stromal cells on CLL cells. Furthermore, following induction of PKC-β, BMSCs activate NF-kappaB through a Bcl10-independent, but NEMO/IKKgamma-dependent pathway. Gene expression profiling of NEMO-proficient and deficient BMSCs indicated that NF-kappaB regulates the expression of pro-inflammatory cytokines and adhesion molecules by stromal cells, required to promote survival of CLL. Interference with the NF-kappaB activation in BMSCs abrogated the pro-survival effects of stromal cells on CLL, similar to PKC-β deficient stromal cells.To demonstrate that this pathway is also important in vivo, Tcl1-CLL was transplanted into syngeneic PKC-β knock-out and wild-type mice. Notably, all PKC-β wild-type mice died of a CLL-like disease, whereas PKC-β kock-out animals were entirely resistant to CLL transplants. Immunofluorescence staining of PKC-β in bone marrow trephine biopsies indicated that this pathway is also activated in mesenchymal stromal cells of CLL patients. Importantly, our data provide further evidence that the PKC-β – NF-kappaB pathway is also activated in stomal cells by monoclonal B-cells from ALL and mantle-cell lymphoma (MCL) patients.Conclusively, we describe a novel survival signaling pathway activated by monoclonal B-cells in BMSCs. Interference with the PKC-β-NF-kappaB pathway activated in the leukemia/lymphoma microenvironment may offer new therapeutic options to fully eradicate malignant B-cells from bone-marrow niches. Disclosures:No relevant conflicts of interest to declare.

Identification of a novel recurrent gain on 20q13 in chronic lymphocytic leukemia by array CGH and gene expression profiling

The presence of genetic changes is a hallmark of chronic lymphocytic leukemia (CLL). The most common cytogenetic abnormalities with independent prognostic significance in CLL are 13q14, ATM and TP53 deletions and trisomy 12. However, CLL displays a great genetic and biological heterogeneity. The aim of this study was to analyze the genomic imbalances in CLL cytogenetic subsets from both genomic and gene expression perspectives to identify new recurrent alterations. The genomic imbalances and expression levels of 67 patients were analyzed. The novel recurrent abnormalities detected with bacterial artificial chromosome array were confirmed by FISH and oligonucleotide microarrays. In all cases, gene expression profiling was assessed. Copy number alterations were identified in 75% of cases. Overall, the results confirmed FISH studies for the regions frequently involved in CLL and also defined a new recurrent gain on chromosome 20q13.12, in 19% (13/67) of the CLL patients. Oligonucleotide expression correlated with the regions of loss or gain of genomic material, suggesting that the changes in gene expression are related to alterations in copy number. Our study demonstrates the presence of a recurrent gain in 20q13.12 associated with overexpression of the genes located in this region, in CLL cytogenetic subgroups.

Substantial Susceptibility of Chronic Lymphocytic Leukemia to BCL2 Inhibition: Results of a Phase I Study of Navitoclax in Patients With Relapsed or Refractory Disease

BCL2 overexpression is a hallmark of chronic lymphocytic leukemia (CLL). The novel BH3 mimetic navitoclax (ABT-263) specifically inhibits BCL2 and related proteins BCL-x(l) and BCL-w, potently inducing apoptosis of CLL cells in vitro. A phase I trial in patients with CLL was conducted to evaluate the safety, pharmacokinetics, and biologic activity of oral navitoclax. Twenty-nine patients with relapsed or refractory CLL received daily navitoclax for 14 days (10, 110, 200, or 250 mg/d; n = 15) or 21 days (125, 200, 250, or 300 mg/d; n = 14) of each 21-day cycle. Dose escalation decisions were informed by continual reassessment methodology. Lymphocytosis was reduced by more than 50% in 19 of 21 patients with baseline lymphocytosis. Among 26 patients treated with navitoclax ≥ 110 mg/d, nine (35%) achieved a partial response and seven maintained stable disease for more than 6 months. Median treatment duration was 7 months (range, 1 to ≥ 29 months). Median progression-free survival was 25 months. Activity was observed in patients with fludarabine-refractory disease, bulky adenopathy, and del(17p) CLL. Thrombocytopenia due to BCL-x(l) inhibition was the major dose-limiting toxicity and was dose-related. Low MCL1 expression and high BIM:MCL1 or BIM:BCL2 ratios in leukemic cells correlated with response. We determined that the navitoclax dose of 250 mg/d in a continuous dosing schedule was optimal for phase II studies. BCL2 is a valid therapeutic target in CLL, and its inhibition by navitoclax warrants further evaluation as monotherapy and in combination in this disease.

Dysregulation of TNFα-induced necroptotic signaling in chronic lymphocytic leukemia: suppression of CYLD gene by LEF1

Impaired cell death program has been noted as one of the hallmarks of chronic lymphocytic leukemia (CLL) and contributes to its accumulation of malignant monoclonal B cells as well as to chemotherapy resistance. A cell can die through the apoptosis or necrosis pathway. Recent investigations suggest that in apoptotic-deficient conditions, such as most types of cancer, a process of programmed necrosis, called necroptosis, prevails. However, the detailed molecular mechanisms underlying this alternative cell death pathway are still not fully understood. Here we demonstrate that CLL cells failed to undergo necroptosis upon stimulation of TNFα combined with pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD). Two core components of necroptotic machine, RIP3 and deubiquitinase cylindromatosis (CYLD), are markedly downregulated in CLL. Moreover, we identified lymphoid enhancer-binding factor 1 (LEF1), a downstream effector of the Wnt/β-catenin pathway, as a transcription repressor of CYLD in CLL. Knocking down LEF1 sensitizes CLL cells to TNFα/zVAD-induced necroptosis. The present investigation provides the first evidence that CLL cells have defects not only in apoptotic program but also in necroptotic signaling. Targeting the key regulators of necroptotic machine, such as LEF1, to restore this pathway may represent a novel approach for CLL treatment.

Lenalidomide Treatment Enhances Immunological Synapse Formation of Cord Blood Natural Killer Cells with B Cells Derived From Chronic Lymphocytic Leukemia

Abstract 1794Immune dysfunction is a hallmark of chronic lymphocytic leukemia (CLL) including suppressed humoral and cell-mediated immune responses. The immunomodulatory agent lenalidomide has shown effective clinical activity against CLL, but its mechanism of action is poorly understood. Previous work has demonstrated that the T cell immunological synapse and functional defects in CLL can be reversed following lenalidomide treatment (J Clin Invest. 2008; 118). Polymerization of F-actin at the NK cell immunological synapse with tumor cells is required for signaling molecules to assemble and regulate NK cell activation and effector function. Confocal microscopy was used to visualize and analyze F-actin polymerization at the immune synapse between NK cells and CLL cells. The impaired immune synapse defect identified in CLL could result from not only the defects of CLL B cells but also defects in the CLL NK cells or a combination of both factors. To investigate the contribution of each factor, we examined synapse formation in experiments using CLL B cells with autologous CLL NK cells or healthy allogeneic NK cells. Conjugates formed with healthy NK cells and CLL B cells exhibited a strong band of F-actin at the immune synapse. In contrast, significantly less actin polymerization at the synapse was observed in autologous CLL NK cells and CLL B cells (P < 0.01). These results indicate CLL B cells, together with CLL NK cells contributed to the immune dysfunction in CLL. As autologous NK cell function in CLL is suppressed, we investigated the utility of CB as a potential functional source of NK cells for CLL immunotherapy. We examined the effect of lenalidomide on NK cell immune synapse function with CLL B cells acting as APCs. We demonstrated that ex vivo treatment of CLL cells with lenalidomide (500 ng/ml) for 48 hours caused a significant increase in the ability of autologous CLL NK cells to form F-actin immune synapses with CLL B cells. The same treatment of CLL B cells also significantly increased the ability of CB-NK cells to form F-actin immunological synapses with these treated CLL B cells compared to untreated CLL B cells (33.6% to 67.3%, P < 0.01, n=6).Our results also show that lenalidomide treatment of autologous NK cells from CLL patients enhanced synapse formation with treated CLL cells compared to experiments using untreated NK cells, but with reduced function compared to CB NK cells. Of note, lenalidomide treatment was shown to increase the recruitment of the signaling molecule Lck to NK cell:CLL cell synapse site, that is known to regulate lytic synapse function. Importantly, lenalidomide treatment significantly increased CB-NK killing of CLL B cells compared to untreated CLL B cells (20.5% versus 48.2%, E:T ratio of 10:1, n = 6, p < 0.001). These results provide insight into the potential mechanism of action of lenalidomide’s anti-leukemic function – priming CLL tumor cells for enhanced NK cell lytic synapse formation and effector function. In addition, the data suggests that immunotherapeutic strategies utilizing a combination of CB-NK cells and lenalidomide has an enhanced clinical efficacy in CLL. Disclosures:Gribben:Roche: Honoraria; Celgene: Honoraria; GSK: Honoraria; Mundipharma: Honoraria; Gilead: Honoraria; Pharmacyclics: Honoraria.

Proliferation centers in chronic lymphocytic leukemia: correlation with cytogenetic and clinicobiological features in consecutive patients analyzed on tissue microarrays

To better define the significance of proliferation centers (PCs), the morphological hallmark of chronic lymphocytic leukemia (CLL), lymph node biopsies taken from 183 patients were submitted to histopathologic and fluorescence in situ hybridization (FISH) studies using a 5-probe panel on tissue microarrays. Seventy-five cases (40.9%) with confluent PCs were classified as 'PCs-rich' and 108 cases (59.1%) with scattered PCs were classified as 'typical'. Complete FISH data were obtained in 101 cases (55.1%), 79 of which (78.2%) displayed at least one chromosomal aberration. The incidence of each aberration was: 13q- 36,7%, 14q32 translocations 30.8%, 11q- 24.7%, trisomy 12 19.5% and 17p- 15.6%. Five cases showed extra copies of the 14q32 region. The 'PCs-rich' group was associated with 17p-, 14q32/IgH translocation, +12, Ki-67>30%. The median survival from the time of tissue biopsy for PCs-rich and typical groups was 11 and 64 months, respectively (P=0.00001). The PCs-rich pattern was the only predictive factor of an inferior survival at multivariate analysis (P=0.022). These findings establish an association between cytogenetic profile and the amount of PC in CLL, and show that this histopathologic characteristic is of value for risk assessment in patients with clinically significant adenopathy.

The BH3-only protein Noxa is stimulated during apoptosis of chronic lymphocytic leukemia cells triggered by M2YN, a new plant-derived extract.

Deficiency of apoptosis is a hallmark of chronic lymphocytic leukemia (CLL) cells. M2Yn is a natural extract from plants of central Asia, identified for its antiangiogenic properties and its ability to block the migration of malignant cells. Here, we report that in vitro treatment of cells derived from CLL patients with M2Yn results in internucleosomal DNA fragmentation, phosphatidylserine externalization, mitochondrial membrane depolarization, caspase-3 activation and cleavage of the caspase substrate PARP-1. The extents of these effects depend on the patients and are mostly comparable to those of flavopiridol or hyperforin, two known plant-derived apoptosis inducers of CLL cells. M2Yn does not modulate Mcl-1 expression, while downregulation of this antiapoptotic protein is involved in the action of flavopiridol. By contrast, M2Yn, like hyperforin, upregulates the Noxa protein, possibly by inhibiting proteasomal activity. This BH3-only protein is known to trigger the activation of the pro-apoptotic protein Bak through displacement of the Mcl-1/Bak complex at the mitochondrial membrane, as actually observed here in M2Yn-treated cells. Our data, therefore, show that M2Yn can induce the caspase-dependent mitochondrial pathway of apoptosis in CLL cells via a mechanism resembling that of hyperforin. Our data also confirm that the BH3-only protein Noxa is a relevant target for CLL therapy.

Dysregulation of TNFα-Induced Necroptotic Signaling In Chronic Lymphocytic Leukemia: Suppression of CYLD Gene by LEF1

AbstractAbstract 377Impaired cell death program has been noted as one of the hallmarks of Chronic lymphocytic leukemia (CLL) and contributes to its accumulation of malignant monoclonal B cells as well as to chemotherapy resistance. A cell can die through apoptosis or necrosis pathway. While apoptosis is known as a regulated cellular program, necrosis is known as an accidental event caused by overwhelming stress. However, accumulating evidence suggests that necrosis can also be executed by regulated mechanisms, especially in apoptotic-deficient conditions. Recently, the term necroptosis has been used to designate one particular form of programmed necrosis induced by stimulating death receptors with agonists such as TNFα, FasL, and TRAIL. Apoptosis suppression by caspase inhibitors such as zVAD may switch apoptotic response to necroptosis or enhance necroptosis. In contrast to well-characterized apoptotic pathway, the detailed molecular mechanisms underlying necroptosis are still not fully understood. A genome wide siRNA screen revealed two members of the receptor interacting protein (RIP) kinase family, RIP1 and RIP3P, to be essential for necroptosis. Upon stimulation of death receptors, RIP3 is recruited to RIP1 to form a necroptosis-inducing complex which is essential for cell death execution. The deubiquitinase cylindromatosis (CYLD) is recruited to TNFα receptor upon its activation and directly regulates RIP1 ubiquitination. In addition, by activating key enzymes of metabolic pathways, RIP3 regulates TNFα-inducing mitochondrial reactive oxygen species (ROS) production, which partly accounts for its ability to potentiate necroptosis. Until now, much less is known about the significance of necroptosis in malignant disease.Here we demonstrate that primary CLL cells failed to undergo necroptosis upon stimulation of TNFα combined with pan-caspase inhibitor zVAD. Upon TNFα+zVAD stimulation, normal CD19+ B cells increased ROS production > 8 fold, while same treatment only resulted in ∼ 2 fold induction in ROS generation in CLL samples. Two core components of necroptotic machine, RIP3 and CYLD, are markedly down-regulated in CLL compared with normal B cells, at both protein and transcription levels.Moreover, we identified LEF1, a downstream effector of Wnt/β-catenin pathway, as a transcription repressor of CYLD in CLL. LEF1 is highly expressed in CLL cells, whereas normal B cells have very low levels of LEF1 expression. Attenuation of LEF1 expression through RNAi technology resulted in a dramatic increase in CYLD levels in CLL cells, as determined by western blot and real time RT-PCR analysis. Dual-luciferase assays showed that forced expression of LEF1 markedly decreased CYLD promoter activity compared with controls. Mutation of LEF1 responsive elements (LERs) on CYLD promoter significantly abolished transcriptional repression of CYLD by LEF1. Chromatin immunoprecipitation assays showed that LEF1 is recruited to LER region within the CYLD promoter in CLL cells. Additionally, Knocking down LEF1 sensitizes CLL cells to TNFα-induced necroptosis.The present investigation provides the first evidence that CLL cells have defects not only in apoptotic program but also in necroptotic signaling. Targeting the key regulators of necroptotic machine such as LEF1 to restore this pathway may represent a novel approach for CLL treatment. Disclosures:No relevant conflicts of interest to declare.

TAp63 Regulates VLA-4 Expression and Chronic Lymphocytic Leukemia Cell Migration to the Bone Marrow in a CD74-Dependent Manner

The hallmark of chronic lymphocytic leukemia (CLL) is the relentless accumulation of mature lymphocytes, mostly due to their decreased apoptosis. CD74 was recently shown to serve as a survival receptor on CLL cells. In this study, we show that stimulation of CD74 with its natural ligand, migration inhibitory factor, initiates a signaling cascade that results in upregulation of TAp63, which directly regulates CLL survival. In addition, TAp63 expression elevates the expression of the integrin VLA-4, particularly during the advanced stage of the disease. Blocking of CD74, TAp63, or VLA-4 inhibits the in vivo homing of CLL cells to the bone marrow (BM). Thus, CD74 and its target genes TAp63 and VLA-4 facilitate migration of CLL cells back to the BM, where they interact with the supportive BM environment that rescues them from apoptosis. These results could form the basis of novel therapeutic strategies aimed at blocking homing of CLL cells in their return to the BM and attenuating their survival.

Abstract 4508: Targeting XIAP as a novel paradigm to induce apoptosis in chronic lymphocytic leukemia

Abstract Evasion of apoptosis is a hallmark of chronic lymphocytic leukemia (CLL), calling for new concepts to overcome apoptosis resistance. We therefore investigated whether targeting the antiapoptotic protein XIAP by small molecule inhibitors would sensitize for apoptosis. Here, we provide first evidence that XIAP inhibitors in combination with the death receptor ligand TRAIL present a new strategy to synergistically trigger apoptosis in CLL even in subgroups with resistant disease. Analysis of apoptosis regulatory proteins reveals that XIAP, cIAP1 and cIAP2 are expressed at high levels in primary CLL samples. Proofs of concept studies in CLL cell lines demonstrate that subtoxic concentrations of several distinct XIAP inhibitors significantly enhance TRAIL-induced apoptosis. By comparison, no sensitization for death receptor-induced apoptosis is observed in the presence of a structurally related control compound that only weakly binds to XIAP, demonstrating the specificity of the sensitization effect of XIAP inhibitors. Importantly also in primary CLL samples, subtoxic concentrations of XIAP inhibitors act in concert with TRAIL to trigger apoptosis in 18 of 27 cases (67%), whereas primary CLL cells are resistant to treatment with TRAIL alone. Analysis of combination index reveals that this interaction of XIAP inhibitor and TRAIL is highly synergistic. Mechanistic studies in primary CLL cells show that the addition of XIAP inhibitor profoundly enhances TRAIL-induced cleavage of caspase-3 into active fragments and significantly increases caspase-3 enzymatic activity. The broad range caspase inhibitor zVAD. fmk completely blocks apoptosis in response to combination treatment with XIAP inhibitor and TRAIL, pointing to caspase-dependent apoptosis. Intriguingly, the cooperative interaction of XIAP inhibitor and TRAIL is even evident in several distinct subgroups of CLL patients with poor prognostic features, including patients with 17p deletion, TP53 mutation, chemotherapy-refractory disease or unmutated VH genes. Interestingly, we found that cases with unmutated VH genes are significantly more sensitive to XIAP inhibitor- and TRAIL-induced apoptosis compared to VH gene mutated samples, pointing to a role of B-cell receptor signaling in the regulation of apoptosis in CLL cells. In conclusion, we provide first evidence that XIAP inhibitors in combination with TRAIL present a novel strategy to trigger apoptosis even in resistant forms and poor prognostic subgroups of CLL. These findings have important implications for the development of innovative approaches to overcome the intrinsic resistance to apoptosis in CLL. Since IAP inhibitors as well as TRAIL receptor agonists as single agents are currently already under evaluation in early clinical trials, it is feasible that such combination protocols of XIAP inhibitors and TRAIL could be translated into clinical application in CLL. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4508.

A Novel Paradigm to Trigger Apoptosis in Chronic Lymphocytic Leukemia

Evasion of apoptosis is a hallmark of chronic lymphocytic leukemia (CLL), calling for new strategies to bypass resistance. Here, we provide first evidence that small-molecule X-linked inhibitor of apoptosis (XIAP) inhibitors in combination with the death receptor ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) present a novel approach to trigger apoptosis in CLL, including subgroups with resistant disease or unfavorable prognosis. XIAP, cellular IAP (cIAP) 1, and cIAP2 are expressed at high levels in primary CLL samples. Proof-of-concept studies in CLL cell lines show that subtoxic concentrations of XIAP inhibitors significantly enhance TRAIL-induced apoptosis and also sensitize for CD95-mediated apoptosis. Importantly also in primary CLL samples, XIAP inhibitor acts in concert with TRAIL to trigger apoptosis in 18 of 27 (67%) cases. This XIAP inhibitor-induced and TRAIL-induced apoptosis involves caspase-3 activation and is blocked by the caspase inhibitor zVAD.fmk. The cooperative interaction of XIAP inhibitor and TRAIL is even evident in distinct subgroups of patients with poor prognostic features (i.e., with 17p deletion, TP53 mutation, chemotherapy-refractory disease, or unmutated V(H) genes). Interestingly, cases with unmutated V(H) genes were significantly more sensitive to XIAP inhibitor-induced and TRAIL-induced apoptosis compared with V(H) gene-mutated samples, pointing to a role of B-cell receptor signaling in apoptosis regulation. By showing that XIAP inhibitors in combination with TRAIL present a new strategy to trigger apoptosis even in resistant forms and poor prognostic subgroups of CLL, our findings have important implications for the development of apoptosis-based therapies in CLL.

STAT3 Is Constitutively Phosphorylated on Serine 727 Residues, Activates Transcription, and Plays a Major Role in the Pathogenesis of B-Cell Chronic Lymphocytic Leukemia.

Signal transducer and activator of transcription 3 (STAT3) plays a pivotal role in cellular physiology and organ development. Tyrosine phosphorylation of STAT3 provides cells with survival advantage and induces proliferation, whereas deletion of STAT3 results in embryonic lethality. As might be expected, STAT3 is commonly activated in neoplastic cells. Constitutive phosphorylation of STAT3 on tyrosine 705 residues has been found in a number of solid tumors and hematologic malignancies. A decade ago, Frank et al. (JCI 100:3149, 1997) reported that unlike other neoplasms in B-cell chronic lymphocytic leukemia (CLL), STAT3 is constitutively phosphorylated (p) on serine 727 but not on tyrosine residues. This observation prompted us to investigate the role of STAT3 in CLL. We studied peripheral blood (PB) cells from ~100 patients with CLL using Western immunoblotting and found that in all patients, regardless of PB count, disease stage, or treatment status, STAT3 is constitutively phosphorylated on serine 727 but not on tyrosine residues. Serine pSTAT3 was detected in CD19+ but not in CD19− CLL PB-derived cells or in normal PB-derived B-lymphocytes isolated by immunomagnetic beads. Unlike constitutive serine pSTAT3, tyrosine pSTAT3 could be induced by incubating CLL cells with cytokines, such as interleukin (IL)-6, or anti-IgM antibodies. However, expression of cytokine-induced tyrosine pSTAT3 was transient and was no longer detected shortly (2 hours) after removal of the stimulating agent. In contrast, serine pSTAT3 levels remained stable in vitro for up to 72 hours, confirming that serine phosphorylation of STAT3 is constitutive in CLL cells. Because CLL is the only disease in which STAT3 is constitutively phosphorylated exclusively on serine residues and since tyrosine phosphorylation is thought to be required for STAT3 activation, we sought to determine whether serine pSTAST3 is biologically active. First, we isolated cytoplasmic and nuclear extracts and used Western immunoblotting to demonstrate that pSTAT3 is present both in the cytoplasm and the nucleus. Then, we confirmed these findings using confocal microscopy. After confirming that serine pSTAT3 translocates to the nucleus, we asked whether serine pSTAT3 binds to DNA. Using the electrophoretic mobility shift assay we demonstrated that nuclear STAT3 binds to a STAT3-specific DNA probe and that STAT3-DNA binding was eliminated by both anti-STAT3 and anti-serine pSTAT3 antibodies. Furthermore, although induction of tyrosine pSTAT3 by exogenous IL-6 slightly increased STAT3-DNA binding, complete dephosphorylation of tyrosine pSTAT3 by T-cell tyrosine phosphatase did not affect STAT3-DNA binding, suggesting that STAT3-DNA binding does not require tyrosine phosphorylation. Finally, pull-down of STAT3 with biotin-labeled DNA further confirmed that serine pSTAT3 binds to DNA in CLL cells. We then asked whether serine pSTAT3 initiates transcription. To answer this question we infected CLL cells with a lentiviral GFP-STAT3-shRNA and, as control, with GFP-empty vector and calculated changes in RNA levels by relative real-time PCR. In several CLL samples, infection with GFP-STAT3-shRNA induced a ~4-fold time-dependent reduction in the STAT3-regulated genes Bcl2, Pim1, Bcl-xL, Cyclin D1, p21 Waf1, and c-Myc, implying that serine pSTAT3 initiates transcription. Taken together, our data suggest that constitutive phosphorylation of STAT3 on serine 727 residues is a hallmark of CLL and induces activation of proliferation and survival genes. Therefore, STAT3 should be considered a therapeutic target in this disease.

Sphingosine 1-Phosphate (S1P) Induces Migration and ERK/MAP-Kinase-Dependent Proliferation in Chronic Lymphocytic Leukemia (B-CLL) Due to Expression of the G Protein-Coupled Receptors S1P1/4.

Egress of lymphocytes from lymphoid organs into the circulation has been shown to depend on the presence of the lipid mediator sphingosine 1-phosphate (S1P) in the peripheral blood, and expression of corresponding S1P receptors (i.e., S1P1), that belong to the family of 7-transmembrane G protein-coupled receptors (GPCR). As circulating lymphocytic lymphoma cells are a hallmark of chronic lymphocytic leukemia, we analyzed expression of different S1P receptors and the effects of S1P on B-CLL cells. By qualitative and quantitative (TaqMan) RT-PCR, significant mRNA expression of S1P1 and S1P4 was found in CLL cell lines (EHEB, MEC-1) and in most samples (S1P1 in 88%, S1P4 in 100%) of primary CD19+ cells isolated from the peripheral blood of untreated B-CLL patients. mRNA of other S1P receptors (S1P2, S1P3, S1P5) was less consistently detected. Normal, nonmalignant B cells were strongly positive for S1P1, while other S1P receptors were weakly expressed or negative. S1P induced typical effects of chemotactic GPCR, such as actin polymerization (analyzed by flow cytometry) and chemotaxis (measured in a modified Boyden chamber assay) in CLL cell lines and primary B-CLL cells. After serum deprivation in vitro, S1P induced phosphorylation of ERK/MAP-kinase as analyzed by Western blot, demonstrating that S1P receptors expressed in CLL were able to activate signaling pathways of GPCR not only related to cell migration and chemotaxis, but also to cell proliferation. Of note, the S1P1 ligand FTY720, which induces receptor internalization after prolonged exposure and acts as an antagonist, resulted in apoptosis in CLL cell lines and primary CLL cells in vitro, as measured by MTT-test and staining with Annexin-FITC, respectively. We conclude that sphingosine 1-phosphate, which is present in the peripheral blood in considerable amounts, contributes to the trafficking of B-CLL cells expressing the GPCRs S1P1/4, and to their prolonged survival.

Angiogenic growth factors: autocrine and paracrine regulation of survival in hematologic malignancies.

Recent research has focused on the role of angiogenic growth factors and their ability to mediate tumor growth and metastases, both in solid tumors and in hematologic malignancies. The bone marrow microenvironment is the setting for a wealth of complex interactions that include cell-to-cell contacts as well as secretion of and response to soluble factors. Abundant evidence supports the role of basic fibroblast growth factor (bFGF) in contributing to the dysregulation of apoptosis that is the hallmark of chronic lymphocytic leukemia (CLL). In fact, CLL cells themselves express bFGF; intracellular levels of this cytokine correlate with clinical CLL stage. Other stromal factors mediate the inhibition of apoptosis in CLL as well, suggesting that strategies to block the responses of CLL cells to these factors may represent effective therapies. More broadly, the class of agents known as angiogenesis inhibitors may offer important advantages with respect to the treatment of numerous types of malignancies. Currently, a number of clinical trials are under way to evaluate the clinical potential of several different angiogenesis inhibitors in several hematologic neoplasms.