技术资料

Multiple myeloma is characterized by the proliferation of clonal plasma cells that have a heterogeneous expression of various cell surface markers,precluding successful use of monoclonal antibodies for therapeutic targeting of the tumor cell. Thymoglobulin (rabbit-derived polyclonal anti-thymocyte globulin),by virtue of its method of preparation,contains antibodies against several B-cell and plasma cell antigens and offers an attractive option for immunotherapy of myeloma. Here,we demonstrate potent anti-myeloma activity of the rabbit anti-thymocyte globulin preparation Thymoglobulin in vitro and in vivo in an animal model of myeloma. Thymoglobulin was able to induce dose- and time-dependent apoptosis of several myeloma cell lines,including those resistant to conventional anti-myeloma agents. Importantly,the anti-myeloma activity was preserved even when myeloma cells were grown with different cytokines demonstrating the ability to overcome microenvironment-mediated resistance. Thymoglobulin induced apoptosis of freshly isolated primary myeloma cells from patients. Using a competitive flow cytometric analysis,we were able to identify the potential antigen targets for Thymoglobulin preparation. Finally,in a plasmacytoma mouse model of myeloma,Thymoglobulin delayed the tumor growth in a dose-dependent manner providing convincing evidence for continued evaluation of this agent in the clinic in patients with myeloma,either alone or in combination with other agents. View Publication

Although toll-like receptor (TLR) agonists,such as CpG,are used as immunotherapeutic agents in clinical trials for cancer and infectious diseases,their effects are limited and the underlying mechanism(s) that restrains CpG efficacy remains obscure. Here,we show that signal transducer and activator of transcription 3 (Stat3) plays a key role in down-modulating immunostimulatory effects of CpG. In the absence of interleukin-6 (IL-6) and IL-10 induction,CpG directly activates Stat3 within minutes through TLR9. Ablating Stat3 in hematopoietic cells results in rapid activation of innate immunity by CpG,with enhanced production of IFN-gamma,tumor necrosis factor-alpha,IL-12,and activation of macrophages,neutrophils,and natural killer cells marked with Stat1 activation. Innate immune responses induced by CpG in mice with a Stat3-ablated hematopoietic system cause potent antitumor effects,leading to eradication of large (textgreater1 cm) B16 melanoma tumors within 72 h. Moreover,ablating Stat3 in myeloid cells increases CpG-induced dendritic cell maturation,T-cell activation,generation of tumor antigen-specific T cells,and long-lasting antitumor immunity. A critical role of Stat3 in mediating immunosuppression by certain cytokines and growth factors in the tumor microenvironment has been recently documented. By demonstrating direct and rapid activation of Stat3 by TLR agonists,we identify a second level of Stat3-mediated immunosuppression. Our results further suggest that targeting Stat3 can drastically improve CpG-based immunotherapeutic approaches. View Publication

Acute lymphoblastic leukemia (ALL) still frequently recurs after hematopoietic stem cell transplantation (HSCT),underscoring the need to improve the graft-versus-leukemia (GvL) effect. Natural killer (NK) cells reconstitute in the first months following HSCT when leukemia burden is at its lowest,but ALL cells have been shown to be resistant to NK cell-mediated killing. We show here that this resistance is overcome by NK cell stimulation with TLR-9-activated plasmacytoid dendritic cells (pDCs). NK cell priming with activated pDCs resulted in TRAIL and CD69 up-regulation on NK cells and IFN-γ production. NK cell activation was dependent on IFN-α produced by pDCs,but was not reproduced by IFN-α alone. ALL killing was further enhanced by inhibition of KIR engagement. We showed that ALL lysis was mainly mediated by TRAIL engagement,while the release of cytolytic granules was involved when ALL expressed NK cell activating receptor ligands. Finally,adoptive transfers of activated-pDCs in ALL-bearing humanized mice delayed the leukemia onset and cure 30% of mice. Our data therefore demonstrate that TLR-9 activated pDCs are a powerful tool to overcome ALL resistance to NK cell-mediated killing and to reinforce the GvL effect of HSCT. These results open new therapeutic avenues to prevent relapse in children with ALL. View Publication

The dose response curve is the gold standard for measuring the effect of a drug treatment,but is rarely used in genomic scale transcriptional profiling due to perceived obstacles of cost and analysis. One barrier to examining transcriptional dose responses is that existing methods for microarray data analysis can identify patterns,but provide no quantitative pharmacological information. We developed analytical methods that identify transcripts responsive to dose,calculate classical pharmacological parameters such as the EC50,and enable an in-depth analysis of coordinated dose-dependent treatment effects. The approach was applied to a transcriptional profiling study that evaluated four kinase inhibitors (imatinib,nilotinib,dasatinib and PD0325901) across a six-logarithm dose range,using 12 arrays per compound. The transcript responses proved a powerful means to characterize and compare the compounds: the distribution of EC50 values for the transcriptome was linked to specific targets,dose-dependent effects on cellular processes were identified using automated pathway analysis,and a connection was seen between EC50s in standard cellular assays and transcriptional EC50s. Our approach greatly enriches the information that can be obtained from standard transcriptional profiling technology. Moreover,these methods are automated,robust to non-optimized assays,and could be applied to other sources of quantitative data. View Publication

Ectopic C/EBPalpha expression in p210(BCR/ABL)-expressing hematopoietic cells induces granulocytic differentiation,inhibits proliferation,and suppresses leukemogenesis. To assess the underlying mechanisms,C/EBPalpha targets were identified by microarray analyses. Upon C/EBPalpha activation,expression of c-Myb and GATA-2 was repressed in 32D-BCR/ABL,K562,and chronic myelogenous leukemia (CML) blast crisis (BC) primary cells but only c-Myb levels decreased slightly in CD34(+) normal progenitors. The role of these 2 genes for the effects of C/EBPalpha was assessed by perturbing their expression in K562 cells. Ectopic c-Myb expression blocked the proliferation inhibition- and differentiation-inducing effects of C/EBPalpha,whereas c-Myb siRNA treatment enhanced C/EBPalpha-mediated proliferation inhibition and induced changes in gene expression indicative of monocytic differentiation. Ectopic GATA-2 expression suppressed the proliferation inhibitory effect of C/EBPalpha but blocked in part the effect on differentiation; GATA-2 siRNA treatment had no effects on C/EBPalpha induction of differentiation but inhibited proliferation of K562 cells,alone or upon C/EBPalpha activation. In summary,the effects of C/EBPalpha in p210(BCR/ABL)-expressing cells depend,in part,on transcriptional repression of c-Myb and GATA-2. Since perturbation of c-Myb and GATA-2 expression has nonidentical consequences for proliferation and differentiation of K562 cells,the effects of C/EBPalpha appear to involve dif-ferent transcription-regulated targets. View Publication

Mature mammary epithelial cells are generated from undifferentiated precursors through a hierarchical process,but the molecular mechanisms involved,particularly in the human mammary gland,are poorly understood. To address this issue,we isolated highly purified subpopulations of primitive bipotent and committed luminal progenitor cells as well as mature luminal and myoepithelial cells from normal human mammary tissue and compared their transcriptomes obtained using three different methods. Elements unique to each subset of mammary cells were identified,and changes that accompany their differentiation in vivo were shown to be recapitulated in vitro. These include a stage-specific change in NOTCH pathway gene expression during the commitment of bipotent progenitors to the luminal lineage. Functional studies further showed NOTCH3 signaling to be critical for this differentiation event to occur in vitro. Taken together,these findings provide an initial foundation for future delineation of mechanisms that perturb primitive human mammary cell growth and differentiation. View Publication

The identification of succinate dehydrogenase (SDH),fumarate hydratase (FH) and isocitrate dehydrogenase (IDH) mutations in human cancers has rekindled the idea that altered cellular metabolism can transform cells. Inactivating SDH and FH mutations cause the accumulation of succinate and fumarate,respectively,which can inhibit 2-oxoglutarate (2-OG)-dependent enzymes,including the EGLN prolyl 4-hydroxylases that mark the hypoxia inducible factor (HIF) transcription factor for polyubiquitylation and proteasomal degradation. Inappropriate HIF activation is suspected of contributing to the pathogenesis of SDH-defective and FH-defective tumours but can suppress tumour growth in some other contexts. IDH1 and IDH2,which catalyse the interconversion of isocitrate and 2-OG,are frequently mutated in human brain tumours and leukaemias. The resulting mutants have the neomorphic ability to convert 2-OG to the (R)-enantiomer of 2-hydroxyglutarate ((R)-2HG). Here we show that (R)-2HG,but not (S)-2HG,stimulates EGLN activity,leading to diminished HIF levels,which enhances the proliferation and soft agar growth of human astrocytes. These findings define an enantiomer-specific mechanism by which the (R)-2HG that accumulates in IDH mutant brain tumours promotes transformation and provide a justification for exploring EGLN inhibition as a potential treatment strategy. View Publication

The chimeric transcription factor E2a-Hlf is an oncoprotein associated with a subset of acute lymphoblastic leukemias of early B-lineage derivation. We employed a retroviral transduction-transplantation approach to evaluate the oncogenic effects of E2a-Hlf on murine B-cell progenitors harvested from adult bone marrow. Expression of E2a-Hlf induced short-lived clusters of primary hematopoietic cells but no long-term growth on preformed bone marrow stromal cell layers comprised of the AC6.21 cell line. Coexpression with Bcl-2,however,resulted in the sustained self-renewal of early preB-I cells that required stromal and interleukin-7 (IL-7) support for growth in vitro. Immortalized cells were unable to induce leukemias after transplantation into nonirradiated syngeneic hosts,unlike the leukemic properties and cytokine independence of preB-I cells transformed by p190(Bcr-Abl) under identical in vitro conditions. However,bone marrow cells expressing E2a-Hlf in combination with Bcl-2,but not E2a-Hlf alone,induced leukemias in irradiated recipients with long latencies,demonstrating both a requirement for suppression of apoptosis and the need for further secondary mutations in leukemia pathogenesis. Coexpression of IL-7 substituted for Bcl-2 to induce the in vitro growth of pre-B cells expressing E2a-Hlf,but leukemic conversion required additional abrogation of undefined stromal requirements and was associated with alterations in the Arf/Mdm2/p53 pathway. Thus,E2a-Hlf enhances the self-renewal of bone marrow B-cell progenitors without inciting a p53 tumor surveillance response or abrogating stromal and cytokine requirements for growth,which are nevertheless abrogated during progression to a leukemogenic phenotype. View Publication

The JAK2(V617F) mutation was found in most patients with myeloproliferative disorders (MPDs),including polycythemia vera,essential thrombocythemia,and primary myelofibrosis. We have generated transgenic mice expressing the mutated enzyme in the hematopoietic system driven by a vav gene promoter. The mice are viable and fertile. One line of the transgenic mice,which expressed a lower level of JAK2(V617F),showed moderate elevations of blood cell counts,whereas another line with a higher level of JAK2(V617F) expression displayed marked increases in blood counts and developed phenotypes that closely resembled human essential thrombocythemia and polycythemia vera. The latter line of mice also developed primary myelofibrosis-like symptoms as they aged. The transgenic mice showed erythroid,megakaryocytic,and granulocytic hyperplasia in the bone marrow and spleen,displayed splenomegaly,and had reduced levels of plasma erythropoietin and thrombopoietin. They possessed an increased number of hematopoietic progenitor cells in peripheral blood,spleen,and bone marrow,and these cells formed autonomous colonies in the absence of growth factors and cytokines. The data show that JAK2(V617F) can cause MPDs in mice. Our study thus provides a mouse model to study the pathologic role of JAK2(V617F) and to develop treatment for MPDs. View Publication

Reversal of promoter DNA hypermethylation and associated gene silencing is an attractive cancer therapy approach. The DNA methylation inhibitors decitabine and azacitidine are efficacious for hematological neoplasms at lower,less toxic,doses. Experimentally,high doses induce rapid DNA damage and cytotoxicity,which do not explain the prolonged time to response observed in patients. We show that transient exposure of cultured and primary leukemic and epithelial tumor cells to clinically relevant nanomolar doses,without causing immediate cytotoxicity,produce an antitumor memory" response� View Publication

Throughout its history,chronic myeloid leukemia (CML) has set precedents for cancer research and therapy. These range from the identification of the first specific chromosomal abnormality associated with cancer to the development of imatinib as a specific,targeted therapy for the disease. The successful development of imatinib as a therapeutic agent for CML can be attributed directly to decades of scientific discoveries. These discoveries determined that the BCR-ABL tyrosine kinase is the critical pathogenetic event in CML and an ideal target for therapy. This was confirmed in clinical trials of imatinib,with imatinib significantly improving the long-term survival of patients with CML. Continuing in this tradition of scientific discoveries leading to improved therapies,the understanding of resistance to imatinib has rapidly led to strategies to circumvent resistance. Continued studies of hematologic malignancies will allow this paradigm of targeting molecular pathogenetic events to be applied to many additional hematologic cancers. View Publication