julilili42/papers huggingface dataset:
5-Azacytosine (Azacytidine) and 2′-Deoxy-5-Azacytidine (Decitabine) Mediate Opposite Effects on NK Cell Anti-Tumor Reactivity
2008-11-16

Abstract The cytosine analogues 5-azacytosine (azacytidine) and 2′-deoxy-5-azacytidine (decitabine) display substantial therapeutic potential in patients with AML and MDS. Besides causing DNA demethylation, azanucleosides also mediate cytotoxic effects, and it appears that clinical responses are influenced by both epigenetic alterations and by apoptosis induction. However, the molecular changes induced by these drugs are still poorly understood. NK cells play an important role in tumor-immunosurveillance by confining development and progression of hematopoietic malignancies and are also important after therapeutic intervention like e.g. haploidentical stem cell transplantation. Thus it is important to define how a given therapeutic agent influences NK cell reactivity. Here we studied the effect of pharmacological concentrations of azacytidine and decitabine on NK cell effector functions. After preincubation with the azanucleosides (12h or more), NK cell cytotoxicity was found to be significantly enhanced by decitabine while, in contrast, azacytidine nearly completely abolished NK cell lysis of K562 and Raji target cells (up to 50% increase and 88% reduction, respectively; E:T ratio 10:1). Of note, modulation of NK cell cytotoxicity was also observed when the compounds were, after NK cell pretreatment, absent during the cytotoxicity assays. In contrast, neither the presence of either agent in cytotoxicity assays without preincubation nor pretreatment of target cells with either agent for 4h (corresponding to the time of the cytotoxicity assay) altered NK cell reactivity. These results indicate that azanucleosides are capable to modulate NK cell responsiveness to activating stimuli. In line, after pretreatment with the compounds NK cell IFN-γ production upon stimulation with IL-2 and IL-15 or in cocultures with target cells was found to be enhanced by decitabine but inhibited by azacytidine (up to 46% increase and 85% reduction, respectively). NK cell effector functions were not affected by deoxycytidine and cytidine, the physiological counterparts of the azanucleosides. While azacytidine treatment substantially induced NK cell apoptosis (about 30% after 24h) which may explain its inhibitory effect, no induction of apoptosis by decitabine was observed. Our data demonstrate that azacytidine and decitabine differentially affect NK cell anti-tumor reactivity and suggest that, while azacytidine causes NK cell apoptosis, decitabine enhances NK cell responsiveness via a yet unknown mechanism which is presently under study.
Radioimmunotherapy with Yttrium-90-Ibritumomab Tiuxetan as Part of a Reduced Intensity Conditioning Regimen for Allogeneic Hematopoietic Cell Transplantation in Patients with Advanced Indolent Non-Hodgkin Lymphoma: Interim Analysis of a Phase II Study.
2008-11-16

Abstract Allogeneic hematopoietic cell transplantation (HCT) using reduced intensity conditioning (RIC) regimens offers a potential curative therapy to patients with advanced indolent NHL. RIC HCT induces potent graft-versus-lymphoma effects, with best results in patients with low tumor burden at time of HCT. Combined use of radioimmunotherapy (RIT) with RIC may increase anti-lymphoma activity of RIC. A multicenter phase II study of allogeneic HCT combining RIT using yttrium-90-ibritumomab tiuxetan (Y90-CD20, Zevalin®) with 0.4 mCi (15 MBq)/kg on day -14 combined with RIC using fludarabine (30 mg/m2 day -4 to -2) and 2 Gy TBI (day 0) followed by allogeneic HCT from matched related or unrelated donors was initiated. Postgrafting immunosuppression consists of cyclosporine and mycophenolate mofetil. Targeted enrolment is 40 patients with dosimetry on day -21 in the first five patients. Primary objective of this study was the evaluation of treatment related mortality and engraftment after RIT-RIC HCT. Secondary objectives were disease response, relapse rate, disease free and overall survival, GVHD and immune reconstitution. To date, 36 patients are evaluable. Diagnoses were follicular lymphoma (n=15), mantle cell lymphoma (n=8), chronic lymphocytic leukemia (n=11), marginal zone lymphoma (n=1) and immunocytoma (n=1). Median age was 56 (range, 34–68) years. PBSC grafts were either from matched related (n=11) or matched unrelated donors (n=25). All patients were “high risk” with refractory disease or relapse after preceding autologous HCT. Disease stage at time of HCT was CR=7, PR=24, SD=5. No additional toxicity due to RIT in comparison to our previous experience with the same RIC was observed. Engraftment was rapid and sustained with no graft rejections. Median time to &amp;gt;500 granulocytes/μL was 14 (range, 0–69) days, and to &amp;gt;20000 platelets/μL 8 (range, 0–69) days. In 14 patients platelets never dropped below 20000/μL and in 8 patients granulocytes never below 500/μL, illustrating the non-myeloablative intensity of the conditioning regimen. TRM in the first 100 days was 8% (n=3) and overall 25% (n=9). Incidence of grade II-IV GVHD was 44% (II=4, III=10, IV=2). To date, extensive chronic GVHD occurred in 6 patients. Deaths occurred due to infections=5, GVHD=2, relapse=2 and multi organ failure=2. 25/36 (69%) of all patients are alive with a median follow up of 453 (range, 39–713) days, resulting in a Kaplan-Meier estimate 1 year survival of 67%. Disease status of patients alive is CR=18 and PR=7. In conclusion, a combination of RIT with RIC is feasible with no additional toxicity due to RIT and with stable engraftment in all patients. Disease response and treatment related mortality seems promising even in this elderly and heavily pretreated cohort but requires further follow-up.
Differential Effects of the BCR-ABL-Inhibitors Imatinib, Nilotinib and Dasatinib on NK Cell Reactivity against Chronic Myeloid Leukemia (CML)
2008-11-16

Abstract CML is characterized by the BCR-ABL fusion protein, which mediates the oncogenic signaling. This led to the development of BCR-ABL inhibitors revolutionizing therapy of CML. However, as recently reported for Dasatinib (Schade et al., Blood 111:1366 (2008); Blake et al., Blood 111:4415 (2008)), these agents may impair the activity of immune effector cells like NK cells and T cells. After initiating oncogenic events, development and progression of clinically apparent malignancy is dependent on the evasion of the tumor cells from immunosurveillance. In light of the important role of NK cell reactivity against leukemia we compared the influence of Imatinib, Nilotinib and Dasatinib on the reactivity of both resting and IL-2 activated NK cells against CML cells to identify the compound with the least immuno-compromising side effects. First, the effects of the compounds on NK cell reactivity in concentrations corresponding to plasma peak levels were studied. Dasatinib (200nM) completely abolished NK cell granule mobilization, cytotoxicity and IFN-γ production, while no substantial inhibition was observed with Imatinib (5μM) and Nilotinib (3.6μM) mediated a minor but significant inhibition (p&amp;lt;0.05, Student’s T-test). Presence of the compounds in concentrations corresponding to IC50 levels (Imatinib 600nM, Nilotinib 30nM, Dasatinib 10nM) revealed no influence of Imatinib and Nilotinib, while Dasatinib still significantly reduced NK cell cytotoxicity and IFN-γ production up to 60%. Since Dasatinib, in addition to BCR-ABL, potently inhibits SRC kinases, which are involved in the activation of MAPK pathways and thus crucial for NK cell cytotoxicity, we determined the influence of the compounds on ERK phosphorylation. While no inhibitory effect was observed using Imatinib and Nilotinib, Dasatinib markedly reduced ERK phosphorylation in NK cells. Our data demonstrate that NK cell anti-tumor reactivity is not inhibited by clinically relevant concentrations of Imatinib. While Nilotinib may mediate a minor effect, Dasatinib substantially impairs NK cell reactivity by inhibition of signaling pathways crucial for NK cell effector functions. For a given patient, the choice and dosing of the most suitable BCR-ABL inhibitor may thus require careful consideration of its influence on the immune system, especially in view of the important role of NK cells in the immunesurveillance of residual leukemia.
Mechanisms of Synergy Between Toll-Like Receptor 4 and Triggering Receptor Expressed on Myeloid Cells-1 in Human Neutrophils
2008-11-16

Abstract The triggering receptor expressed on myeloid cells 1 (TREM-1) is an important player in the innate inflammatory response to microbial infections. Activation and expression of TREM-1 by polymorphonuclear neutrophils (PMN) occurs in concert with Toll-like receptors (TLR) such as TLR4 for bacterial lipopolysaccharide. However, it is currently unclear how this is mediated on a molecular level. Using pharmacologic inhibitors and western blot analysis we demonstrate that phosphatidyl inositide 3-kinase, phospholipase C and the mitogen activated kinase p38 are essential for the TREM-1 and TLR4 mediated respiratory burst of human PMN. The down stream phosphorylation of protein kinase B and extracellular signal related kinase show characteristic phosphorylation patterns upon single or co-ligation indicating individual activation pathways of both receptors. TREM-1, but not TLR4 mediated respiratory burst depended on calcium flow via store operated calcium entry channels, while transient receptor potential channels were important for TLR and TREM-1. Taken together, we provide new insights on the mechanisms how TREM-1 and TLR interact creating synergistic activation in PMN. These results shed a new light on our understanding how the innate inflammatory responses are regulated and might contribute to the development of future concepts for the treatment of severe inflammatory conditions such as sepsis.
The Leukemogenic Potential of Cdx2 Is Dependent on Its N-Terminal Serine Sites and Can Be Counteracted by MAPK Inhibitors
2008-11-16

Abstract The homeobox transcription factor Cdx2 is one of the most frequent ectopically expressed proto-oncogenes in human AML and when retrovirally expressed causes AML in mice (Rawat et. al. PNAS 2004 and Blood 2008). As the leukemogenic potential of Cdx2 was dependent on its N-terminal transactivation domain, we now extended structure-function analyses by inactivating the evolutionarily conserved MAPK phosphorylation sites (S60, S99, S108, S156, S60-S99-108 (3S), and S60-S99-108-156 (4S)) in Cdx2. Peripheral blood analysis of the mice, transplanted with bone marrow cells retrovirally transduced with different Cdx2 serine mutants (n=5) after six weeks, revealed that all the Cdx2 serine mutants showed significant growth advantage over non-transduced carrier cells. However, assessment of engraftment after 16 weeks showed that mice transplanted with BM expressing the S99, the S108 mutants, the 3S and 4S failed to develop leukemic engraftment in contrast to wild type Cdx2, indicating that the S99 and S108 serine sites are critical for leukemic transformation. Furthermore, mice transplanted with BM expressing Cdx2 wild type (n=24), the S60 (n=3) or S156 mutant (n=5) developed AML after median latency of 120, 90 and 167 days post transplantation, respectively. In contrast S99, S108 and 3S mice developed AML after very long latency of 328 (5/5 mice), 341(3/5 mice) and 336 (3/5 mice) days post transplantation, respectively. Interestingly, 4S mice (n=5), did not develop any disease up to an observation time of 400 days, indicating that the transforming potential of Cdx2 was dependent on multiple N-terminal serines. As it was shown that the transcriptional activity of Cdx2 is dependent on the phosphorylation status of N-terminal serines in non-hematopoietic tissue we tested the phosphorylation status in hematopoietic cells: murine cells retrovirally transduced with Cdx2 and human AML cell lines positive for CDX2 expression showed a phosphorylated form of CDX2 and an activated Erk1/2 pathway, in contrast to AML cell lines negative for CDX2 expression. Based on this we tested whether inhibition of the MAPK pathway would impair the transforming potential of Cdx2. When Cdx2 transduced BM cells were incubated with MEK1/2 inhibitors (PD98059, U0126) for 14 days in liquid culture, viability of the cells was reduced by 78% (n=3, p&lt;0.03). Incubation with the inhibitors decreased the activity of Cdx2 at the level of the short-term repopulating stem cell 8-fold as assessed in the rCFU-S after 7 days in vitro culture (n=7, p&lt;0.001). In contrast, Mek1/2 inhibitors did not change the yield of day 12 CFU-S for AML1-ETO expressing progenitor cells in this assay. These data demonstrate that the leukemogenic potential of the homeobox gene Cdx2 depends on N-terminal serines. Furthermore, our data link the oncogenic capacity of the transcription factor Cdx2 to the MAPK signaling, opening the possibility to counteract homeobox-associated leukemogenesis by kinase inhibitors.
PI3 Kinase Inhibition Induces Apoptotic Cell Death in Acute Lymphoblastic Leukemia Via the Mitochondrial Pathway.
2008-11-16

Abstract The phosphatidylinositol 3-kinase (PI3K) pathway regulates many cellular processes that are involved in tumor progression. Aberrant activation of the PI3K pathway due to an alteration of its elements like PTEN or Akt occurs quite frequently in malignant cells. Thus, inhibition of this pathway represents a promising option for the treatment of cancer patients. The best characterized PI3K inhibitors are LY294002 and wortmannin that were shown to disrupt downstream signaling and induce apoptotic cell death in tumor cells. Acute lymphoblastic leukemia (ALL) is a malignancy mainly found in young children and elderly with constitutive activation of PI3K pathway. In our study we analyzed the effect of PI3K inhibition in cell lines deduced from ALL (Jurkat, BV173, SD1, T-2) and primary leukemic cells by incubating them with increasing concentrations of inhibitors of the PI3K signaling. We found that treatment of ALL cells with LY294002, the mTOR inhibitor rapamycin or Akt inhibitor SH5 induced apoptotic cell death that was accompanied by caspase-3 activation and PARP-cleavage and interfered with intracellular PI3K/Akt signaling as analyzed by phosphorylation and expression of mTOR or P70S6K. In line with these results apoptotic cell death could be inhibited by the pan-caspase inhibitor zVAD. In order to determine the pathway of apoptosis induction we took advantage of Jurkat cells (T-ALL) overexpressing or lacking molecules involved in apoptotic pathways such as FADD, an adaptor molecule recruited to the death receptor upon ligand binding, Caspase-8, Caspase-9 or Bcl-2, an anti-apoptotic protein that prevents the release of cytochrom c from mitochondria. PI3K inhibition by LY294002 induced apoptotic cell death in cells deficient of FADD or caspase-8 with no difference to wild type cells. In contrast, cells overexpressing Bcl2 or lacking caspase-9 were resistant to apoptotic death indicating that PI3-kinase inhibition is independent of the external death receptor signaling and is mediated via the mitochondrial pathway. These results were confirmed by analyzing PARP cleavage and caspase-3 activation in utilized leukemic cell lines. Furthermore, we found that the PI3K inhibitor LY294002 induced apoptosis in ALL cells that could be increased by the etoposide, a topoisomerase inhibitor, or TRAIL. In addition, in contrast to etoposide, treatment of ALL cells with TRAIL could overcome the resistance of ALL cells to PI3K inhibition even in caspase-9 deficient Jurkat cells. Our results provide an interesting approach in designing novel therapeutic strategies to target the PI3K pathway in ALL to overcome the resistance to cytotoxic agents.
Gene Expression Profiling Reveals a Crucial Role for C/EBPbeta in Proliferation Pathways of ALK+ ALCL Cell Lines
2008-11-16

Abstract Introduction: ALK+ anaplastic large cell lymphomas (ALCL) overexpress C/EBPβ, as a consequence of NPM-ALK kinase activity. C/EBPβ is a leucine zipper transcription factor, which plays a major role in cellular differentiation, inflammation, proliferation and metabolism control. To determine the role of C/EBPβ in ALK+ ALCL transformation, and to identify its downstream targets, a highly specific C/EBPβ-shRNA was used to knockdown C/EBPβ. The consequences of C/EBPβ gene-silencing were analyzed by gene expression profiling. Materials and Methods: Four ALK+ ALCL cell lines, SUDHL-1, Kijk, Karpas 299 and SUP-M2 were transfected with lentivirus containing the C/EBPβ shRNA or the vector without shRNA in triplicates. Western Blot analysis and qRT-PCR were performed to quantify the knockdown effect. At day three after infection, RNA was extracted and used for Gene Chip expression analysis (Affymetrix). Using Anova software for statistical analysis, we identified genes, which were regulated in all four cell lines. The effect of C/EBPβ knockdown on proliferation, cell cycle, and viability was analyzed by MTT assay and FACS analysis. Results: In all four ALK+ ALCL, efficient C/EBPβ knockdown resulted in profound growth retardation (up to 84%) compared to control cells after 6 days of infection, and a clear shift from the S phase to the G1 phase in the cell cycle was observed. To identify genes regulated by C/EBPβ in all four cell lines, we performed statistical analysis applying a false discovery rate of 20%, and accepted only genes with a &amp;gt;1,1 and &amp;lt;0,9 fold ratio. We identfied 435 genes regulated after C/EBPβ knockdown (117 upregulated, 318 downregulated). Classification of the differentially expressed genes into biological categories revealed overrepresentation of genes involved in the regulation of kinase activity, cell cycle and proliferation, lymphocyte differentiation, and metabolic processes. In particular, kinases involved in the regulation of JNK activity, which have been shown previously to be involved in proliferation of ALCL, were highly affected by C/EBPβ knockdown. Genomatix Bibliosphere Pathway Analysis revealed C/EBPβ to be connected to pathways involving cell cycle (RUNX3, CCNG1, CDKN2A), apoptosis (FAS, PTPRC, BCL2A1, BIRC3) and MAPK cascades (TRIB1 and several MAP3Ks). Several of the genes identified contain known C/EBPβ binding sites. Conclusions: C/EBPβ silencing induces growth arrest in ALK+ALCL, which correlates with differential expression of genes involved in cell cycle, apoptosis and differentiation. This study reveals C/EBPβ as a master transcription regulator of NPM-ALK induced cellular proliferation, and therefore, an ideal candidate for targeted therapeutic intervention.
The G Protein-Coupled Receptor cysLT1 Mediates Gi Protein-Dependent Chemotaxis and Adhesion of CD34+ Cells in Vitro Similar to CXCR4, but Not Progenitor Mobilization in Vivo.
2008-11-16

Abstract CysLT1, which is expressed in several cell types during inflammation and allergy, and the homing related chemokine receptor CXCR4 both belong to the family of G protein-coupled receptors (GPCR). Similar to CXCR4, the cysteinyl-leukotriene receptor cysLT1 is strongly expressed in CD34+ cell lines and CD34+ hematopoietic progenitor cells (HPC). We therefore compared the effects mediated by cysLT1 on HPC to those observed after activation of CXCR4. The most potent cysLT1 ligand LTD4 induced chemotaxis and adhesion of CD34+ HPC to endothelial cells, immobilized VCAM-1 and fibronectin, which was comparable to effects induced by the CXCR4 ligand SDF-1. CysLT1- and CXCR4-mediated effects were inhibited by pertussis toxin (PTX), suggesting that both GPCRs employ the same Gi-protein-dependent signaling pathways in CD34+ HPC. This is supported by identical time courses of intracellular calcium fluxes and actin polymerization induced by LTD4 and SDF-1, as measured by time-dependent flow cytometry. Given the striking similarities of cysLT1- and CXCR4-mediated effects in vitro, one might expect also overlapping functions in vivo. We therefore investigated whether blocking of cysLT1 is associated with HPC mobilization. As cysLT1 antagonists are established for therapy and prophylaxis in patients with allergic and exercise-induced asthma, circulating CD34+ progenitors were enumerated after initiation of a treatment with the cysLT1 antagonist montelukast, used as asthma prophylaxis in otherwise healthy subjects. The number of CD34+ cells or white blood counts did not differ significantly from the baseline value 2, 4, 8, 12, and 24 hours after administration of 10 mg montelukast, in contrast to previous studies analyzing stem cell mobilization induced by CXCR4 antagonists. This corresponds with our in vitro findings that LTD4 is produced by bone marrow endothelium and stromal cells only when deprived of hematopoietic cells, in contrast to the constitutive production of SDF-1. We conclude that cysLT1 is not involved in bone marrow retention of HPC during steady-state hematopoiesis, but may modulate HPC homing when its ligands are produced either locally (i.e. bone marrow aplasia) or systemically (i.e. inflammation).
NK Cell Anti-Tumor Immune Responses Are Impaired by Sorafenib but Not by Sunitinib
2008-11-16

Abstract Natural killer (NK) cells play an important role in the surveillance of tumors by exerting cellular cytotoxicity and also due to their ability to secrete immunoregulatory cytokines. A variety of intracellular kinases like PI3K, MAPKs or STAT molecules are involved in the regulation of NK cell functions. Therefore, treatment of tumor patients with small molecule inhibitors targeting various protein kinases may also largely affect NK cell anti-tumor reactivity. The multikinase inhibitors Sunitinib (SUTENT®) and Sorafenib (NEXAVAR®) have been approved for the treatment of patients with metastatic Renal Cell Carcinoma (RCC), and are now also being evaluated in various other tumor entities including AML. Both compounds target, among others, PDGF receptors, VEGF receptors, Flt3 and c-kit. Sorafenib additionally inhibits the MAPK pathway members B-RAF and c-RAF. Here we report that Sorafenib but not Sunitinib substantially reduces NK cell cytotoxicity as revealed by analysis of cell surface CD107a expression as a surrogate marker for mobilization of cytolytic granules on NK cells from ten healthy donors. In line, pharmacological levels of Sorafenib but not Sunitinib largely reduced lysis of several solid tumor and leukemia cell lines by primary NK cells, and this inhibitory effect of Sorafenib occurred concentration dependently. Furthermore, Sorafenib but not Sunitinib concentration dependently impaired NK cell IFN-γ production in coculture experiments with different tumor cell lines. Since Sorafenib, in contrast to Sunitinib, also potently inhibits RAF which is involved in MAPK activation we determined the influence of the two compounds on ERK phosphorylation which is important for the activation of NK cell anti-tumor reactivity. While Sunitinib had no effect, Sorafenib, alike the specific MEK inhibitor PD98059, markedly reduced ERK phosphorylation in NK cells. Taken together, out results indicate that NK cell-mediated anti-tumor immune responses are impaired by Sorafenib but not by Sunitinib via inhibition of signaling pathways crucial for NK cell effector functions. Thus, Sunitinib is exquisitely suitable in diseases and for therapeutic approaches which rely on a sufficient anti-tumor immune response like e.g. allogenic stem cell transplantation in AML or immunotherapeutic approaches in RCC patients.