Inbal Hazan-Halevy

STAT3 is constitutively phosphorylated on serine 727 residues, binds DNA, and activates transcription in CLL cells

Chronic lymphocytic leukemia (CLL) is the most common leukemia in the Western hemisphere, but its pathogenesis is still poorly understood. Constitutive tyrosine phosphorylation (p) of signal transducer and activator of transcription (STAT) 3 occurs in several solid tumors and hematologic malignancies. In CLL, however, STAT3 is constitutively phosphorylated on serine 727, not tyrosine 705, residues. Because the biologic significance of serine pSTAT3 in CLL is not known, we studied peripheral blood cells of 106 patients with CLL and found that, although tyrosine pSTAT3 was inducible, serine pSTAT3 was constitutive in all patients studied, regardless of blood count, disease stage, or treatment status. In addition, we demonstrated that constitutive serine pSTAT3 translocates to the nucleus by the karyopherin-beta nucleocytoplasmic system and binds DNA. Dephosphorylation of inducible tyrosine pSTAT3 did not affect STAT3-DNA binding, suggesting that constitutive serine pSTAT3 binds DNA. Furthermore, infection of CLL cells with lentiviral STAT3-small hairpin RNA reduced the expression of several STAT3-regulated survival and proliferation genes and induced apoptosis, suggesting that constitutive serine pSTAT3 initiates transcription in CLL cells. Taken together, our data suggest that constitutive phosphorylation of STAT3 on serine 727 residues is a hallmark of CLL and that STAT3 be considered a therapeutic target in this disease.

WP1066 Disrupts Janus Kinase-2 and Induces Caspase-Dependent Apoptosis in Acute Myelogenous Leukemia Cells

Several cytokines and growth factors that stimulate the proliferation of acute myelogenous leukemia (AML) cells transduce their signals by activating the transcription factor Janus-activated kinase 2 (JAK2). Accordingly, the inhibition of JAK2 or of its downstream signaling pathways suppresses the proliferation of AML cells. Because (E)-3(6-bromopyridin-2-yl)-2-cyano-N-((S0-1-phenylethyl)acrylamide) (WP1066) is a novel analogue of the JAK2 inhibitor AG490, we tested its activity in AML cells and investigated its mechanism of action. Using clonogenic assays, we found that although WP1066 had a marginal effect on normal marrow progenitors, it inhibited the proliferation of AML colony-forming cells obtained from patients with newly diagnosed AML and that of the AML cell lines OCIM2 and K562. WP1066 inhibited OCIM2 cell multiplication by inducing accumulation of cells at the G(0)-G(1) phase of the cell cycle. Similar to its parent compound AG490, WP1066 inhibited the phosphorylation of JAK2, but unlike AG490, WP1066 also degraded JAK2 protein, thereby blocking its downstream signal transducer and activator of transcription (STAT) and phosphoinositide-3-kinase pathways. These effects resulted in the activation of the caspase pathway. Incubation of both OCIM2 and K562 cells with WP1066 activated caspase-3, induced cleavage of poly(ADP-ribose) polymerase, and caused caspase-dependent apoptotic cell death. Thus, WP1066 is a potent JAK2 inhibitor whose effects in AML and other hematologic malignancies merit further investigation.

Activation of CD44, a receptor for extracellular matrix components, protects chronic lymphocytic leukemia cells from spontaneous and drug induced apoptosis through MCL-1

Survival of chronic lymphocytic leukemia (CLL) cells in vivo is supported by the tissue microenvironment, which includes components of the extracellular matrix. Interactions between tumor cells and the extracellular matrix are in part mediated by CD44, whose principal ligand is hyaluronic acid. Here, we show that CD44 is more highly expressed on CLL cells of the clinically more progressive immunglobulin heavy chain variable gene (IGHV)-unmutated subtype than on cells of the IGHV-mutated type. Engagement of CD44 activated the phosphatidylinositol 3-kinase (PI3K)/AKT and mitogen activated protein kinase (MAPK)/ERK pathways and increased myeloid cell leukemia sequence 1 (MCL-1) protein expression. Consistent with the induction of these anti-apoptotic mechanisms, CD44 protected CLL cells from spontaneous and fludarabine-induced apoptosis. Obatoclax, an antagonist of MCL-1, blocked the pro-survival effect of CD44. In addition, obatoclax synergized with fludarabine to induce apoptosis of CLL cells. In conclusion, components of the extracellular matrix may provide survival signals to CLL cells through engagement of CD44. Inhibition of MCL-1 is a promising strategy to reduce the anti-apoptotic effect of the microenvironment on CLL cells.

Stimulation of NADPH oxidase by angiotensin II in human neutrophils is mediated by ERK, p38 MAP-kinase and cytosolic phospholipase A2.

Objective The present research was designed to study the involvement of ERK and p38 MAP-kinase in cytosolic phospholipase A2 (cPLA2) and NADPH-oxidase activation by angiotensin II (Ang II) in human neutrophils. Methods NADPH-oxidase activity was measured by reduction of cytochrome C. cPLA2 activity was measured in cell lysate using sonicated dispersions of 1-stearoyl-2-[14C]arachidonyl phosphatidylcholine. Cells were incubated with MEK inhibitor UO126 or with p38 MAP-kinase inhibitor SB202190 prior to stimulation with Ang II. Translocation of p47phox, p67phox and cPLA2 and phosphorylation of ERK and p38 MAP-kinase were measured by immunoblot analysis. Results Ang II induced a dose-dependent activation of NADPH oxidase in neutrophils and monocytes as well as in differentiated PLB-985 cells towards neutrophil or monocyte lineages, but not in cPLA2-deficient differentiated PLB-985 cells. An immediate activation of both ERK and p38 MAP-kinase and of cPLA2 was induced by Ang II in human neutrophils. In addition, Ang II induced translocation of the cytosolic oxidase components, detected by translocation of p47phox, which preceded the translocation of cPLA2 induced by this agonist. The p38 MAP-kinase inhibitor SB202190 or the MEK–ERK pathway inhibitor UO126 totally inhibited the activation of both NADPH oxidase and cPLA2 as well as the translocation of cytosolic oxidase components and of cPLA2 to the membrane fractions. Conclusions These results suggest that either ERK or p38 MAP-kinase are involved in the activation of both cPLA2 and NADPH oxidase, and that cPLA2 is required for activation of the NADPH oxidase by Ang II in human neutrophils.

The Cytokine Midkine and Its Receptor RPTPζ Regulate B Cell Survival in a Pathway Induced by CD74

Lasting B cell persistence depends on survival signals that are transduced by cell surface receptors. In this study, we describe a novel biological mechanism essential for survival and homeostasis of normal peripheral mature B cells and chronic lymphocytic leukemia cells, regulated by the heparin-binding cytokine, midkine (MK), and its proteoglycan receptor, the receptor-type tyrosine phosphatase ζ (RPTPζ). We demonstrate that MK initiates a signaling cascade leading to B cell survival by binding to RPTPζ. In mice lacking PTPRZ, the proportion and number of the mature B cell population are reduced. Our results emphasize a unique and critical function for MK signaling in the previously described MIF/CD74-induced survival pathway. Stimulation of CD74 with MIF leads to c-Met activation, resulting in elevation of MK expression in both normal mouse splenic B and chronic lymphocytic leukemia cells. Our results indicate that MK and RPTPζ are important regulators of the B cell repertoire. These findings could pave the way toward understanding the mechanisms shaping B cell survival and suggest novel therapeutic strategies based on the blockade of the MK/RPTPζ-dependent survival pathway.

Stimulation of the B-cell receptor activates the JAK2/STAT3 signaling pathway in chronic lymphocytic leukemia cells

In chronic lymphocytic leukemia (CLL), stimulation of the B-cell receptor (BCR) triggers survival signals. Because in various cells activation of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway provides cells with survival advantage, we wondered whether BCR stimulation activates the JAK/STAT pathway in CLL cells. To stimulate the BCR we incubated CLL cells with anti-IgM antibodies. Anti-IgM antibodies induced transient tyrosine phosphorylation and nuclear localization of phosphorylated (p) STAT3. Immunoprecipitation studies revealed that anti-JAK2 antibodies coimmunoprecipitated pSTAT3 and pJAK2 in IgM-stimulated but not unstimulated CLL cells, suggesting that activation of the BCR induces activation of JAK2, which phosphorylates STAT3. Incubation of CLL cells with the JAK1/2 inhibitor ruxolitinib inhibited IgM-induced STAT3 phosphorylation and induced apoptosis of IgM-stimulated but not unstimulated CLL cells in a dose- and time-dependent manner. Whether ruxolitinib treatment would benefit patients with CLL remains to be determined.

STAT-3 ACTIVATES NF-KAPPAB IN CHRONIC LYMPHOCYTIC LEUKEMIA CELLS

NF-κB plays a major role in the pathogenesis of B-cell neoplasms. A broad array of mostly extracellular stimuli has been reported to activate NF-κB, to various degrees, in chronic lymphocytic leukemia (CLL) cells. Because CLL cells harbor high levels of unphosphorylated STAT-3 (USTAT-3) and USTAT-3 was reported to activate NF-κB, we sought to determine whether USTAT-3 activates NF-κB in CLL. Using the electrophoretic mobility shift assay (EMSA), we studied peripheral blood low-density cells from 15 patients with CLL and found that CLL cell nuclear extracts from all the samples bound to an NF-κB DNA probe, suggesting that NF-κB is constitutively activated in CLL. Immunoprecipitation studies showed that STAT-3 bound NF-κB p65, and confocal microscopy studies detected USTAT-3/NF-κB complexes in the nuclei of CLL cells, thereby confirming these findings. Furthermore, infection of CLL cells with retroviral STAT-3-short hairpin RNA attenuated the binding of NF-κB to DNA, as assessed by EMSA, and downregulated mRNA levels of NF-κB–regulated genes, as assessed by quantitative PCR. Taken together, our data suggest that USTAT-3 binds to the NF-κB p50/p65 dimers and that the USTAT-3/NF-κB complexes bind to DNA and activate NF-κB–regulated genes in CLL cells. Mol Cancer Res; 9(4); 507–15. ©2011 AACR.Nuclear factor (NF)-κB plays a major role in the pathogenesis of B-cell neoplasms. A broad array of mostly extracellular stimuli has been reported to activate NF-κB, to various degrees, in chronic lymphocytic leukemia (CLL) cells. Because CLL cells harbor high levels of unphosphorylated (U) signal transducer and activator of transcription (STAT)-3 protein and U-STAT3 was reported to activate NF-κB, we sought to determine whether U-STAT3 activates NF-κB in CLL. Using the electrophoretic mobility shift assay (EMSA) we studied peripheral blood low-density cells from 15 patients with CLL and found that CLL cell nuclear extracts from all the samples bound to an NF-κB DNA probe, suggesting that NF-κB is constitutively activated in CLL. Immunoprecipitation studies showed that STAT3 bound NF-κB p65, and confocal microscopy studies detected U-STAT3/NF-κB complexes in the nuclei of CLL cells, thereby confirming these findings. Furthermore, infection of CLL cells with retroviral STAT3-shRNA attenuated the binding of NF-κB to DNA, as assessed by EMSA, and downregulated mRNA levels of NF-κB-regulated genes, as assessed by quantitative polymerase chain reaction. Taken together, our data suggest that U-STAT3 binds to the NF-κB p50/p65 dimers and that the U-STAT3/NF-κB complexes bind to DNA and activate NF-κB-regulated genes in CLL cells.

Signal Transducer and Activator of Transcription (STAT)-3 Activates Nuclear Factor (NF)-κB in Chronic Lymphocytic Leukemia Cells

NF-κB plays a major role in the pathogenesis of B-cell neoplasms. A broad array of mostly extracellular stimuli has been reported to activate NF-κB, to various degrees, in chronic lymphocytic leukemia (CLL) cells. Because CLL cells harbor high levels of unphosphorylated STAT-3 (USTAT-3) and USTAT-3 was reported to activate NF-κB, we sought to determine whether USTAT-3 activates NF-κB in CLL. Using the electrophoretic mobility shift assay (EMSA), we studied peripheral blood low-density cells from 15 patients with CLL and found that CLL cell nuclear extracts from all the samples bound to an NF-κB DNA probe, suggesting that NF-κB is constitutively activated in CLL. Immunoprecipitation studies showed that STAT-3 bound NF-κB p65, and confocal microscopy studies detected USTAT-3/NF-κB complexes in the nuclei of CLL cells, thereby confirming these findings. Furthermore, infection of CLL cells with retroviral STAT-3-short hairpin RNA attenuated the binding of NF-κB to DNA, as assessed by EMSA, and downregulated mRNA levels of NF-κB–regulated genes, as assessed by quantitative PCR. Taken together, our data suggest that USTAT-3 binds to the NF-κB p50/p65 dimers and that the USTAT-3/NF-κB complexes bind to DNA and activate NF-κB–regulated genes in CLL cells. Mol Cancer Res; 9(4); 507–15. ©2011 AACR.Nuclear factor (NF)-κB plays a major role in the pathogenesis of B-cell neoplasms. A broad array of mostly extracellular stimuli has been reported to activate NF-κB, to various degrees, in chronic lymphocytic leukemia (CLL) cells. Because CLL cells harbor high levels of unphosphorylated (U) signal transducer and activator of transcription (STAT)-3 protein and U-STAT3 was reported to activate NF-κB, we sought to determine whether U-STAT3 activates NF-κB in CLL. Using the electrophoretic mobility shift assay (EMSA) we studied peripheral blood low-density cells from 15 patients with CLL and found that CLL cell nuclear extracts from all the samples bound to an NF-κB DNA probe, suggesting that NF-κB is constitutively activated in CLL. Immunoprecipitation studies showed that STAT3 bound NF-κB p65, and confocal microscopy studies detected U-STAT3/NF-κB complexes in the nuclei of CLL cells, thereby confirming these findings. Furthermore, infection of CLL cells with retroviral STAT3-shRNA attenuated the binding of NF-κB to DNA, as assessed by EMSA, and downregulated mRNA levels of NF-κB-regulated genes, as assessed by quantitative polymerase chain reaction. Taken together, our data suggest that U-STAT3 binds to the NF-κB p50/p65 dimers and that the U-STAT3/NF-κB complexes bind to DNA and activate NF-κB-regulated genes in CLL cells.

Harnessing RNAi-based nanomedicines for therapeutic gene silencing in B-cell malignancies

Significance RNA interference (RNAi) holds great promise as a novel therapeutic approach. Small interfering RNAs (siRNAs) that manipulate gene expression in leukocytes could be used to treat blood cancers. However, the lack of strategies for delivering siRNAs to leukocytes systemically has hampered the development of RNAi-based therapeutics. Here, we show that lipid-based nanoparticles coated with anti-CD38 monoclonal antibodies specifically target mantle cell lymphoma (MCL) cells and induce cell-specific therapeutic gene silencing in vivo. CD38-targeted nanoparticles that contain cyclin D1 siRNAs prolong survival of mice bearing MCL lymphomas in the bone marrow. This strategy opens a new avenue for treating MCL that might be applied to other hematological malignancies. Despite progress in systemic small interfering RNA (siRNA) delivery to the liver and to solid tumors, systemic siRNA delivery to leukocytes remains challenging. The ability to silence gene expression in leukocytes has great potential for identifying drug targets and for RNAi-based therapy for leukocyte diseases. However, both normal and malignant leukocytes are among the most difficult targets for siRNA delivery as they are resistant to conventional transfection reagents and are dispersed in the body. We used mantle cell lymphoma (MCL) as a prototypic blood cancer for validating a novel siRNA delivery strategy. MCL is an aggressive B-cell lymphoma that overexpresses cyclin D1 with relatively poor prognosis. Down-regulation of cyclin D1 using RNA interference (RNAi) is a potential therapeutic approach to this malignancy. Here, we designed lipid-based nanoparticles (LNPs) coated with anti-CD38 monoclonal antibodies that are specifically taken up by human MCL cells in the bone marrow of xenografted mice. When loaded with siRNAs against cyclin D1, CD38-targeted LNPs induced gene silencing in MCL cells and prolonged survival of tumor-bearing mice with no observed adverse effects. These results highlight the therapeutic potential of cyclin D1 therapy in MCL and present a novel RNAi delivery system that opens new therapeutic opportunities for treating MCL and other B-cell malignancies.

Kit inhibitor APcK110 induces apoptosis and inhibits proliferation of acute myeloid leukemia cells.

Kit is a membrane-bound tyrosine kinase and receptor for stem cell factor (SCF) with a crucial role in hematopoiesis. Mutations of KIT occur in almost half of patients with core-binding factor leukemias, in which they have been associated with worse outcome. Development of new compounds targeting Kit may therefore hold promise for therapy. We investigated the activity and mechanism of action of APcK110, a novel Kit inhibitor, in the mastocytosis cell line HMC1.2 (KITV560G and KITD816V), acute myeloid leukemia (AML) lines OCIM2 and OCI/AML3 (both wild-type), and primary samples from patients with AML. We show that (a) APcK110 inhibits proliferation of the mastocytosis cell line HMC1.2 and the SCF-responsive cell line OCI/AML3 in a dose-dependent manner; (b) APcK110 is a more potent inhibitor of OCI/AML3 proliferation than the clinically used Kit inhibitors imatinib and dasatinib and at least as potent as cytarabine; (c) APcK110 inhibits the phosphorylation of Kit, Stat3, Stat5, and Akt in a dose-dependent fashion, showing activity of APcK110 on Kit and its downstream signaling pathways; (d) APcK110 induces apoptosis by cleavage of caspase-3 and poly(ADP-ribose) polymerase; and (e) APcK110 inhibits proliferation of primary AML blasts in a clonogenic assay but does not affect proliferation of normal colony-forming cells. Although APcK110 activity may partly depend on cytokine responsiveness (e.g., SCF) and not exclusively KIT mutation status, it remains a potent inhibitor of AML and mastocytosis cell lines and primary AML samples. APcK110 and similar compounds should be evaluated in clinical trials of patients with AML.