Latest Articles Include:
- A Syk inhibitor for sick platelets?
Woulfe DS - Blood 113(14):3133-3134 (2009)
- The VWD2B saga continues to Montreal
Nurden P - Blood 113(14):3134-3135 (2009)
- Reduced-intensity allogeneic transplantation for myeloma: reality bites
Stewart AK - Blood 113(14):3135-3136 (2009)
- Backache and breast cancer
- Blood 113(14):3137 (2009)
- Regulation of macrophage function in tumors: the multifaceted role of NF-{kappa}B
Hagemann T Biswas SK Lawrence T Sica A Lewis CE - Blood 113(14):3139-3146 (2009)
The pivotal role of tumor-associated macrophages (TAMs) in tumor progression is now well established. TAMs have been shown to influence multiple steps in tumor development including the growth, survival, invasion, and metastasis of tumor cells as well as angiogenesis and lymphangiogenesis in tumors. The molecular circuits that polarize TAMs toward such a protumoral phenotype are now the focus of intense investigation. The transcription factor, nuclear factor-{kappa}B (NF-{kappa}B), is a master regulator of many cellular processes and been shown to regulate various pathways that impact on the function of TAMs. Much evidence for this has come from the use of elegant transgenic murine tumor models in which modification of single components of the NF-{kappa}B signaling pathway has been shown to regulate the pro-tumor repertoire of TAMs. Here, we outline this evidence and attempt to reconcile the various views that have emerged recently over the exact role of NF-{kappa}B in this phenomenon. - How I treat and monitor viral hepatitis B infection in patients receiving intensive immunosuppressive therapies or undergoing hematopoietic stem cell transplantation
Liang R - Blood 113(14):3147-3153 (2009)
Hepatitis B virus (HBV) reactivation is a serious but preventable complication of immunosuppression. Full HBV serologic profile must be obtained from all patients receiving intensive immunosuppressive therapy. In general, preemptive anti-HBV therapy is more effective than giving treatment after development of reactivation. Prompt lamivudine therapy should be given to at-risk patients who are hepatitis B surface antigen (HBsAg)-positive. It is recommended that lamivudine be continued until at least 6 months after the cessation of immunosuppression. Some patients requiring a longer duration of lamivudine therapy are at risk of developing drug resistance. The newer anti-HBV agents are effective in overcoming lamivudine resistance. Early use of these agents may be considered. HBV reactivation was observed in HBsAg-negative patients with occult HBV infection (HBV DNA-positive) who are on heavy immunosuppression. The optimal management of this group of patients is unclear. Fo r patients receiving allogeneic HSC transplants, the HBV status of the donors requires special attention. To minimize the risk of transmission of infection to recipients, HBsAg-positive donors should receive adequate anti-HBV therapy before HSC donation. As the result of adoptive immune transfer, clearance of HBsAg is observed in HBsAg-positive patients receiving HSC transplants from donors who are positive for hepatitis B surface and core antibodies. - Of mice and men: an open-label pilot study for treatment of immune thrombocytopenic purpura by an inhibitor of Syk
Podolanczuk A Lazarus AH Crow AR Grossbard E Bussel JB - Blood 113(14):3154-3160 (2009)
To determine whether inhibition of Syk would be useful in Fc{gamma}R-dependent cytopenias such as immune thrombocytopenic purpura (ITP) or autoimmune hemolytic anemia, mouse models were used to evaluate efficacy of R406, an inhibitor of Syk function, in treating cytopenia. Both disease models responded favorably to treatment, with amelioration of ITP being more dramatic. Thus, phase 2 clinical trial was initiated to study the effects of Syk inhibition in humans with ITP. Sixteen adults with chronic ITP were entered into an open-label, single-arm cohort dose-escalation trial beginning with 75 mg and escalating as high as 175 mg twice daily. Doses were increased until a persistent response was seen, toxicity occurred, or 175 mg twice daily was reached. Eight patients achieved persistent responses with platelet counts greater than 50 x 109/L (50 000 mm3) on more than 67% (actually 95%) of their study visits, including 3 who had not persistently responded to thrombopoietic agents. Four others had nonsustained responses. Mean peak platelet count exceeded 100 x 109/L (100 000 mm3) in these 12 patients. Toxicity was primarily GI-related with diarrhea (urgency) and vomiting; 2 patients had transaminitis. In conclusion, inhibition of Syk was an efficacious means of increasing and maintaining the platelet count in half the patients with chronic refractory ITP. (ClinicalTrials.gov, no. NCT00706342 ). - Bone marrow angiogenesis magnetic resonance imaging in patients with acute myeloid leukemia: peak enhancement ratio is an independent predictor for overall survival
Shih TT Hou HA Liu CY Chen BB Tang JL Chen HY Wei SY Yao M Huang SY Chou WC Hsu SC Tsay W Yu CW Hsu CY Tien HF Yang PC - Blood 113(14):3161-3167 (2009)
Emerging evidence suggests that progression of hematologic malignancies is associated with angiogenesis. Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) can provide global and functional imaging of tumor angiogenesis. In this study, we performed bone marrow DCE-MRI prospectively at diagnosis and after induction chemotherapy in 78 de novo acute myeloid leukemia (AML) patients and correlated it with treatment outcome. An algorithm to assess bone marrow angiogenesis by measuring the DCE-MRI time-intensity curve pixel by pixel was developed using 3 distinct parameters: peak enhancement ratio (Peak) to indicate tissue blood perfusion; amplitude (Amp) to reflect vascularity; and volume transfer constant (K trans) to indicate vascular permeability. The Peak and Amp decreased significantly at remission status after induction chemotherapy. Patients with higher Peak or Amp at diagnosis had shorter overall survival and disease-free survival than others. Cox multivar iate analysis identified higher Peak value (hazard ratio, 9.181; 95% confidence interval, 1.740-48.437; P = .009) as an independent predictor for overall survival in addition to unfavorable karyotype and old age. Our findings provide evidence that increased bone marrow angiogenesis measured by DCE-MRI can predict adverse clinical outcome in AML patients. DCE-MRI may help to select high-risk phenotype AML patients for tailored antiangiogenic therapy and to monitor treatment response. - Relevance of the immunoglobulin VH somatic mutation status in patients with chronic lymphocytic leukemia treated with fludarabine, cyclophosphamide, and rituximab (FCR) or related chemoimmunotherapy regimens
Lin KI Tam CS Keating MJ Wierda WG O'Brien S Lerner S Coombes KR Schlette E Ferrajoli A Barron LL Kipps TJ Rassenti L Faderl S Kantarjian H Abruzzo LV - Blood 113(14):3168-3171 (2009)
Although immunoglobulin VH mutation status (IgVH MS) is prognostic in patients with chronic lymphocytic leukemia (CLL) who are treated with alkylating agents or single-agent fludarabine, its significance in the era of chemoimmunotherapy is not known. We determined the IgVH somatic mutation status (MS) in 177 patients enrolled in a phase 2 study of fludarabine, cyclophosphamide, and rituximab (FCR) and in 127 patients treated with subsequent chemoimmunotherapy protocols. IgVH MS did not impact significantly on the complete remission (CR) rate of patients receiving FCR or related regimens. However, CR duration was significantly shorter in patients with CLL that used unmutated IgVH than those whose CLL used mutated IgVH (TTP 47% vs 82% at 6 years, P < .001). In a multivariate model considering all baseline characteristics, IgVH MS emerged as the only determinant of remission duration (hazard ratio 3.8, P < .001). Our results suggest that postremission interventions should be targeted toward patients with unmutated IgVH status. - Functional involvement of RINF, retinoid-inducible nuclear factor (CXXC5), in normal and tumoral human myelopoiesis
Pendino F Nguyen E Jonassen I Dysvik B Azouz A Lanotte M Segal-Bendirdjian E Lillehaug JR - Blood 113(14):3172-3181 (2009)
Retinoids triggers differentiation of acute promyelocytic leukemia (APL) blasts by transcriptional regulation of myeloid regulatory genes. Using a microarray approach, we have identified a novel retinoid-responsive gene (CXXC5) encoding a nuclear factor, retinoid-inducible nuclear factor (RINF), that contains a CXXC-type zinc-finger motif. RINF expression correlates with retinoid-induced differentiation of leukemic cells and with cytokine-induced myelopoiesis of normal CD34+ progenitors. Furthermore, short hairpin RNA (shRNA) interference suggests for this gene a regulatory function in both normal and tumoral myelopoiesis. Interestingly, RINF localizes to 5q31.3, a small region often deleted in myeloid leukemia (acute myeloid leukemia [AML]/myelodysplasia [MDS]) and suspected to harbor one or several tumor suppressor gene. - Abnormal megakaryocyte morphology and proplatelet formation in mice with megakaryocyte-restricted MYH9 inactivation
Eckly A Strassel C Freund M Cazenave JP Lanza F Gachet C Léon C - Blood 113(14):3182-3189 (2009)
Mutations in the MYH9 gene encoding nonmuscle myosin IIA lead to macrothrombocytopenia as observed in MYH9-related disorders. We used mice with megakaryocyte-restricted MYH9 inactivation to explore the role of myosin in thrombopoiesis. In situ, bone marrow MYH9{Delta} megakaryocytes were irregularly shaped, appearing leaky with poorly defined limits. The demarcation membranes were abnormally organized and poorly developed, pointing to an insufficient reservoir for the future formation of platelets. The cytoskeletal-rich peripheral zone was lacking due to the absence of the myosin filament network that normally surrounds the granular zone in wild-type cells. In vitro studies of cultured cells showed that MYH9{Delta} megakaryocytes were unable to form stress fibers upon adhesion to collagen, suggesting that the leaky shape results from defects in internal tension and anchorage to the extracellular environment. Surprisingly, the proportion of cells extending proplatelets w as increased in MYH9{Delta} megakaryocytes and the proplatelet buds were larger. Overall, this study provides evidence for a role of myosin in different steps of megakaryocyte development through its participation in the maintenance of cell shape, formation and organization of the demarcation membranes and the peripheral zone, anchorage to the extracellular matrix, and proplatelet formation. - Circulating Ly-6C+ myeloid precursors migrate to the CNS and play a pathogenic role during autoimmune demyelinating disease
King IL Dickendesher TL Segal BM - Blood 113(14):3190-3197 (2009)
Mature myeloid cells (macrophages and CD11b+ dendritic cells) form a prominent component of neuroinflammatory infiltrates in multiple sclerosis and experimental autoimmune encephalomyelitis (EAE). The mechanism by which these cells are replenished during relapsing and chronic neuroinflammation is poorly understood. Here we demonstrate that CD11b+CD62L+Ly6Chi monocytes with colony-forming potential are mobilized into the bloodstream by a granulocyte-macrophage colony-stimulating factor-dependent pathway immediately before EAE relapses. Circulating Ly6Chi monocytes traffic across the blood-brain barrier, up-regulate proinflammatory molecules, and differentiate into central nervous system dendritic cells and macrophages. Enrichment of Ly6Chi monocytes in the circulating pool is associated with an earlier onset and increased severity of clinical EAE. Our studies indicate that granulocyte-macrophage colony-stimulating factor-driven release of Ly6Chi precursors from the bone marrow prevents exhaustion of central nervous system myeloid populations during relapsing or chronic autoimmune demyelination, suggesting a novel pathway for therapeutic targeting. - p85{beta} phosphoinositide 3-kinase regulates CD28 coreceptor function
Alcazar I Cortes I Zaballos A Hernandez C Fruman DA Barber DF Carrera AC - Blood 113(14):3198-3208 (2009)
CD28 is a receptor expressed on T cells that regulates their differentiation after antigen stimulation to long-term-survival memory T cells. CD28 enhances T-cell receptor signals and reduces expression of CBL ubiquitin ligases, which negatively control T-cell activation. In the absence of CD28 ligation during the primary stimulation, CBL levels remain high and T cells fail to mount an efficient secondary response. CD28 associates with p85{alpha}, one of the regulatory subunits of phosphoinositide-3-kinase (PI3K), but the relevance of this interaction is debated. We examined here the contribution of the other ubiquitous PI3K regulatory subunit, p85{beta}, in CD28 function. We describe that p85{beta} bound to CD28 and to CBL with greater affinity than p85{alpha}. Moreover, deletion of p85{beta} impaired CD28-induced intracellular events, including c-CBL and CBL-b down-regulation as well as PI3K pathway activation. This resulted in defective differentiation of activated T cells, which failed to exhibit an efficient secondary immune response. Considering that p85{beta}-deficient T cells fail in recall responses and that p85{beta} binds to and regulates CD28 signals, the presented observations suggest the involvement of p85{beta} in CD28-mediated activation and differentiation of antigen-stimulated T cells. - Identification of a particular HIV-specific CD8+ T-cell subset with a CD27+ CD45RO-/RA+ phenotype and memory characteristics after initiation of HAART during acute primary HIV infection
Lécuroux C Girault I Urrutia A Doisne JM Deveau C Goujard C Meyer L Sinet M Venet A - Blood 113(14):3209-3217 (2009)
CD8+ T cells play an important role in controlling viral infections. Defective CD8+ T-cell responses during HIV infection could contribute to viral persistence. Early initiation of highly active antiretroviral therapy during acute primary HIV infection helps to preserve HIV-specific immune responses. Here, we describe a particular CD27+ CD45RO-/RA+ HIV-specific CD8+ T cell in participants treated early during the primary infection. These cells, which were present at a very low frequency during primary HIV infection, increased markedly after early treatment, whereas their frequency remained unchanged in untreated participants and in participants treated later. These nonnaive antigen-experienced cells are in a resting state and have characteristics of long-lived memory cells. They also possess direct effector capabilities, such as cytokine production, and are able to proliferate and to acquire cytotoxic functions on reactivation. Our results suggest that these HIV-specifi c CD27+ CD45RO-/RA+ CD8+ T cells, observed when early viral replication is inhibited, form a pool of resting cells with memory characteristics. - Activin-A attenuates several human natural killer cell functions
Robson NC Wei H McAlpine T Kirkpatrick N Cebon J Maraskovsky E - Blood 113(14):3218-3225 (2009)
Dendritic-cell (DC) and natural killer (NK)-cell interactions are critical in sculpting the adaptive immune response. However, the mechanisms by which DCs down-regulate NK-cell functions are not well understood. NK-cell function is inhibited by transforming growth factor beta (TGF-{beta}), but DCs do not appear to produce TGF-{beta}. We have previously shown that activated human DCs produce large amounts of activin-A, a TGF-{beta} superfamily member, which autoregulates DC function. The present report shows that NKcells express type I and II activin receptors and that activin-A triggers NK-cell Smad 2/3 signaling. Furthermore, activin-A directly regulates NK cell functions by (1) down-regulating the T-box transcription factor T-bet and interferon gamma (IFN-{gamma}) but not perforin or granzyme mRNA; (2) suppressing NK-cell IFN-{gamma} production as potently as TGF-{beta}; and (3) suppressing NK-cell CD25 expression and proliferation and sculpting NK-cell cytokine and c hemokine profiles. Interestingly, unlike TGF-{beta}, activin-A weakly down-regulates the NK-cell natural cytotoxicity receptors (NCRs) NKp30 and NKG2D but does not attenuate their cytotoxic function. These findings provide the first evidence for a novel immune regulatory role of activin-A during DC-mediated NK-cell regulation, highlighting the potential of antagonizing activin-A signaling in vivo to enhance NK cell-mediated immune functions and adaptive immunity. - A critical role for DAP10 and DAP12 in CD8+ T cell-mediated tissue damage in large granular lymphocyte leukemia
Chen X Bai F Sokol L Zhou J Ren A Painter JS Liu J Sallman DA Chen YA Yoder JA Djeu JY Loughran TP Epling-Burnette PK Wei S - Blood 113(14):3226-3234 (2009)
Large granular lymphocyte (LGL) leukemia, or LGLL, is characterized by increased numbers of circulating clonal LGL cells in association with neutropenia, anemia, rheumatoid arthritis, and pulmonary artery hypertension (PAH). Emerging evidence suggests that LGLL cells with a CD8+CD28null phenotype induce these clinical manifestations through direct destruction of normal tissue. Compared with CD8+CD28null T cells from healthy controls, CD8+CD28null T cells from LGLL patients have acquired the ability to directly lyse pulmonary artery endothelial cells and human synovial cells. Here, we show that LGLL cells from patients possess enhanced cytotoxic characteristics and express elevated levels of activating natural killer receptors as well as their signaling partners, DAP10 and DAP12. Moreover, downstream targets of DAP10 and DAP12 are constitutively activated in LGLL cells, and expression of dominant-negative DAP10 and DAP12 dramatically reduces their lytic capacity. These a re the first results to show that activating NKR-ligand interactions play a critical role in initiating the DAP10 and DAP12 signaling events that lead to enhanced lytic potential of LGLL cells. Results shown suggest that inhibitors of DAP10 and DAP12 or other proteins involved in this signaling pathway will be attractive therapeutic targets for the treatment of LGLL and other autoimmune diseases and syndromes. - TLR2-dependent eosinophil interactions with mycobacteria: role of {alpha}-defensins
Driss V Legrand F Hermann E Loiseau S Guerardel Y Kremer L Adam E Woerly G Dombrowicz D Capron M - Blood 113(14):3235-3244 (2009)
Peripheral blood and tissue eosinophilia are a prominent feature in allergic diseases and during helminth infections. Eosinophil recruitment also frequently occurs upon mycobacterial infections, particularly in lung granuloma. However, the mechanism by which eosinophils interact with mycobacteria remains largely unknown. Because eosinophils recently have been shown to be involved in innate immune responses, we investigated the direct interactions of eosinophils with Mycobacterium bovis BCG as a study model. We show that live BCG attracts human eosinophils and induces reactive oxygen species (ROS) synthesis, granule protein release, and tumor necrosis factor (TNF)-{alpha} secretion. Using anti-TLR2 neutralizing antibodies before exposure of eosinophils to BCG, we showed a critical role of TLR2 signaling in ROS and eosinophil peroxidase release. BCG-induced eosinophil activation is mediated through the p38 mitogen-activated protein (MAP) kinase and nuclear factor (NF)-{ka ppa}B pathways. In addition, a mycobacterial wall component, lipomannan, induced a TLR2-dependent eosinophil activation. In addition, we showed that eosinophils express and produce {alpha}-defensins upon stimulation with BCG and lipomannan and that {alpha}-defensins could inhibit mycobacterial growth in synergy with eosinophil cationic protein. These results suggest a role for human eosinophils as direct effectors in TLR2-mediated innate immunity against mycobacteria and confer to these cells potent cytotoxic functions through defensin and eosinophil cationic protein production. - The transcription factor c-Myc enhances KIR gene transcription through direct binding to an upstream distal promoter element
Cichocki F Hanson RJ Lenvik T Pitt M McCullar V Li H Anderson SK Miller JS - Blood 113(14):3245-3253 (2009)
The killer cell immunoglobulin-like receptor (KIR) repertoire of natural killer (NK) cells determines their ability to detect infected or transformed target cells. Although epigenetic mechanisms play a role in KIR gene expression, work in the mouse suggests that other regulatory elements may be involved at specific stages of NK-cell development. Here we report the effects of the transcription factor c-Myc on KIR expression. c-Myc directly binds to, and promotes transcription from, a distal element identified upstream of most KIR genes. Binding of endogenous c-Myc to the distal promoter element is significantly enhanced upon interleukin-15 (IL-15) stimulation in peripheral blood NK cells and correlates with an increase in KIR transcription. In addition, the overexpression of c-Myc during NK-cell development promotes transcription from the distal promoter element and contributes to the overall transcription of multiple KIR genes. Our data demonstrate the significance of t he 5' promoter element upstream of the conventional KIR promoter region and support a model whereby IL-15 stimulates c-Myc binding at the distal KIR promoter during NK-cell development to promote KIR transcription. This finding provides a direct link between NK-cell activation signals and KIR expression required for acquisition of effector function during NK-cell education. - Notch signaling is required for proliferation but not for differentiation at a well-defined {beta}-selection checkpoint during human T-cell development
Taghon T Van de Walle I De Smet G De Smedt M Leclercq G Vandekerckhove B Plum J - Blood 113(14):3254-3263 (2009)
Notch signaling is absolutely required for {beta}-selection during mouse T-cell development, both for differentiation and proliferation. In this report, we investigated whether Notch has an equally important role during human T-cell development. We show that human CD34+ thymocytes can differentiate into CD4+CD8{beta}+ double positive (DP) thymocytes in the absence of Notch signaling. While these DP cells phenotypically resemble human {beta}-selected cells, they lack a T-cell receptor (TCR)-{beta} chain. Therefore, we characterized the {beta}-selection checkpoint in human T-cell development, using CD28 as a differential marker at the immature single positive CD4+CD3-CD8{alpha}- stage. Through intracellular TCR-{beta} staining and gene expression analysis, we show that CD4+CD3-CD8{alpha}-CD28+ thymocytes have passed the {beta}-selection checkpoint, in contrast to CD4+CD3-CD8{alpha}-CD28- cells. These CD4+CD3-CD8{alpha}-CD28+ thymocytes can efficiently differentiate into C D3+TCR{alpha}{beta}+ human T cells in the absence of Notch signaling. Importantly, preselection CD4+CD3-CD8{alpha}-CD28- thymocytes can also differentiate into CD3+TCR{alpha}{beta}+ human T cells without Notch activation when provided with a rearranged TCR-{beta} chain. Proliferation of human thymocytes, however, is clearly Notch-dependent. Thus, we have characterized the {beta}-selection checkpoint during human T-cell development and show that human thymocytes require Notch signaling for proliferation but not for differentiation at this stage of development. - HSV ICP0 recruits USP7 to modulate TLR-mediated innate response
Daubeuf S Singh D Tan Y Liu H Federoff HJ Bowers WJ Tolba K - Blood 113(14):3264-3275 (2009)
Pattern recognition receptors represent the first line of defense against invading pathogens. Herpes simplex virus (HSV) encodes multiple ligands detected by these receptors, yet persists in the majority of infected individuals indicating a breakdown in host defense against the virus. Here we identify a novel mechanism through which HSV immediate-early protein ICP0 inhibits TLR-dependent inflammatory response by blocking NF-{kappa}B and JNK activation downstream of TLR signal activation. This process depends on ICP0-mediated translocation of USP7 (HAUSP) from the nucleus to cytoplasm. We show that nuclear USP7 migrates to the cytoplasm in response to TLR engagement, a process that contributes to termination of TLR response. Cytoplasmic USP7 binds to and deubiquitinates TRAF6 and IKK{gamma}, thus terminating TLR-mediated NF-{kappa}B and JNK activation. These findings suggest that USP7 is part of a negative feedback loop regulating TLR signaling and that ICP0 exploits thi s physiologic process to attenuate innate response to HSV. ICP0 inhibition of the TLR response serves to uncouple the innate and adaptive immune response, thereby playing a key role in HSV pathogenesis and persistence. - The NAD biosynthesis inhibitor APO866 has potent antitumor activity against hematologic malignancies
Nahimana A Attinger A Aubry D Greaney P Ireson C Thougaard AV Tjornelund J Dawson KM Dupuis M Duchosal MA - Blood 113(14):3276-3286 (2009)
APO866 inhibits nicotinamide phosphoribosyltransferase (NMPRTase), a key enzyme involved in nicotinamide adenine dinucleotide (NAD) biosynthesis from the natural precursor nicotinamide. Intracellular NAD is essential for cell survival, and NAD depletion resulting from APO866 treatment elicits tumor cell death. Here, we determine the in vitro and in vivo sensitivities of hematologic cancer cells to APO866 using a panel of cell lines (n = 45) and primary cells (n = 32). Most cancer cells (acute myeloid leukemia [AML], acute lymphoblastic leukemia [ALL], mantle cell lymphoma [MCL], chronic lymphocytic leukemia [CLL], and T-cell lymphoma), but not normal hematopoietic progenitor cells, were sensitive to low concentrations of APO866 as measured in cytotoxicity and clonogenic assays. Treatment with APO866 decreased intracellular NAD and adenosine triphosphate (ATP) at 24 hours and 48 to72 hours, respectively. The NAD depletion led to cell death. At 96 hours, APO866-mediated c ell death occurred in a caspase-independent mode, and was associated with mitochondrial dysfunction and autophagy. Further, in vivo administration of APO866 as a single agent prevented and abrogated tumor growth in animal models of human AML, lymphoblastic lymphoma, and leukemia without significant toxicity to the animals. The results support the potential of APO866 for treating hematologic malignancies. - Expression of CD133 on leukemia-initiating cells in childhood ALL
Cox CV Diamanti P Evely RS Kearns PR Blair A - Blood 113(14):3287-3296 (2009)
Optimization of therapy for childhood acute lymphoblastic leukemia (ALL) requires a greater understanding of the cells that proliferate to maintain this malignancy because a significant number of cases relapse, resulting from failure to eradicate the disease. Putative ALL stem cells may be resistant to therapy and subsequent relapses may arise from these cells. We investigated expression of CD133, CD19, and CD38 in pediatric B-ALL. Cytogenetic and molecular analyses demonstrated that karyotypically aberrant cells were present in both CD133+/CD19+ and CD133+/CD19- subfractions, as were most of the antigen receptor gene rearrangements. However, ALL cells capable of long-term proliferation in vitro and in vivo were derived from the CD133+/CD19- subfraction. Moreover, these CD133+/CD19- cells could self-renew to engraft serial nonobese diabetic-severe combined immunodeficient recipients and differentiate in vivo to produce leukemias with similar immunophenotypes and karyoty pes to the diagnostic samples. Furthermore, these CD133+/CD19- ALL cells were more resistant to treatment with dexamethasone and vincristine, key components in childhood ALL therapy, than the bulk leukemia population. Similar results were obtained using cells sorted for CD133 and CD38, with only the CD133+/CD38- subfraction demonstrating xenograft repopulating capacity. These data suggest that leukemia-initiating cells in childhood B-ALL have a primitive CD133+/CD19- and CD38- phenotype. - Potentiating effects of RAD001 (Everolimus) on vincristine therapy in childhood acute lymphoblastic leukemia
Crazzolara R Cisterne A Thien M Hewson J Baraz R Bradstock KF Bendall LJ - Blood 113(14):3297-3306 (2009)
Despite advances in the treatment of acute lymphoblastic leukemia (ALL), the majority of children who relapse still die of ALL. Therefore, the development of more potent but less toxic drugs for the treatment of ALL is imperative. We investigated the effects of the mammalian target of rapamycin inhibitor, RAD001 (Everolimus), in a nonobese diabetic/severe combined immunodeficiency model of human childhood B-cell progenitor ALL. RAD001 treatment of established disease increased the median survival of mice from 21.3 days to 42.3 days (P < .02). RAD001 together with vincristine significantly increased survival compared with either treatment alone (P < .02). RAD001 induced a cell-cycle arrest in the G0/1 phase with associated dephosphorylation of the retinoblastoma protein, and reduced levels of cyclin-dependent kinases 4 and 6. Ultrastructure analysis demonstrated the presence of autophagy and limited apoptosis in cells of RAD001-treated animals. In contrast, cleaved poly( ADP-ribose) polymerase suggested apoptosis in cells from animals treated with vincristine or the combination of RAD001 and vincristine, but not in those receiving RAD001 alone. In conclusion, we have demonstrated activity of RAD001 in an in vivo leukemia model supporting further clinical development of target of rapamycin inhibitors for the treatment of patients with ALL. - Pharmacogenetic study in Hodgkin lymphomas reveals the impact of UGT1A1 polymorphisms on patient prognosis
Ribrag Vincent - Blood 113(14):3307-3313 (2009)
Hodgkin lymphoma is a highly curable malignancy, but treatment outcome might be influenced by inherited gene polymorphisms determining anticancer agent metabolism. We prospectively collected peripheral blood lymphocytes from 313 patients with Hodgkin lymphomas to analyze GSTP1, GSTM1, GSTT1, UGT1A1, and CYP3A4 enzyme gene polymorphisms. All patients were treated with chemotherapy, associated with radiotherapy when they had localized disease. There was no difference for GSTP1, GSTM1, and GSTT1 as well as for UGT1A1 and CYP3A4 polymorphism distributions between Hodgkin lymphoma patients and healthy controls. Patients carrying 1 or 2 UGT1A1*28 allele had a significantly (P < .05) better freedom from progression and time to treatment failure than those homozygous for the UGT1A1 TA6/TA6 allele. Multivariate prognostic analyses showed that the UGT1A1 polymorphism was as an independent prognostic parameter for all the studied endpoints, the wild-type homozygous UGT1A1 TA6/TA6 genotype being associated with a significantly worse prognosis than genotypes with at least one UGT1A1*28 allele (overall survival; relative risk [RR] = 2.54, 95% confidence interval [CI], 1.05-6.14; P = .04; freedom from progression, RR = 2.70, 95% CI, 1.37-5.31; P = .004; time to treatment failure, RR = 2.37, 95% CI, 1.28-4.40, P = .006). UGT1A1 polymorphism on TA repeats, which are thought to determine several anticancer drugs metabolism, influence Hodgkin lymphoma patient outcome. - Regulation of mir-196b by MLL and its overexpression by MLL fusions contributes to immortalization
Popovic R Riesbeck LE Velu CS Chaubey A Zhang J Achille NJ Erfurth FE Eaton K Lu J Grimes HL Chen J Rowley JD Zeleznik-Le NJ - Blood 113(14):3314-3322 (2009)
Chromosomal translocations involving the Mixed Lineage Leukemia (MLL) gene produce chimeric proteins that cause abnormal expression of a subset of HOX genes and leukemia development. Here, we show that MLL normally regulates expression of mir-196b, a hematopoietic microRNA located within the HoxA cluster, in a pattern similar to that of the surrounding 5' Hox genes, Hoxa9 and Hoxa10, during embryonic stem (ES) cell differentiation. Within the hematopoietic lineage, mir-196b is most abundant in short-term hematopoietic stem cells and is down-regulated in more differentiated hematopoietic cells. Leukemogenic MLL fusion proteins cause overexpression of mir-196b, while treatment of MLL-AF9 transformed bone marrow cells with mir-196-specific antagomir abrogates their replating potential in methylcellulose. This demonstrates that mir-196b function is necessary for MLL fusion-mediated immortalization. Furthermore, overexpression of mir-196b was found specifically in patients w ith MLL associated leukemias as determined from analysis of 55 primary leukemia samples. Overexpression of mir-196b in bone marrow progenitor cells leads to increased proliferative capacity and survival, as well as a partial block in differentiation. Our results suggest a mechanism whereby increased expression of mir-196b by MLL fusion proteins significantly contributes to leukemia development. - Runx2 induces acute myeloid leukemia in cooperation with Cbf{beta}-SMMHC in mice
Kuo YH Zaidi SK Gornostaeva S Komori T Stein GS Castilla LH - Blood 113(14):3323-3332 (2009)
The core-binding factor (CBF) is a master regulator of developmental and differentiation programs, and CBF alterations are frequently associated with acute leukemia. The role of the CBF member RUNX2 in hematopoiesis is poorly understood. Genetic evidence suggests that deregulation of Runx2 may cause myeloid leukemia in mice expressing the fusion oncogene Cbfb-MYH11. In this study, we show that sustained expression of Runx2 modulates Cbf{beta}-smooth muscle myosin heavy chain (SMMHC)-mediated myeloid leukemia development. Expression of Runx2 is high in the hematopoietic stem cell compartment and decreases during myeloid differentiation. Sustained Runx2 expression hinders myeloid progenitor differentiation capacity and represses expression of CBF targets Csf1R, Mpo, Cebpd, the cell cycle inhibitor Cdkn1a, and myeloid markers Cebpa and Gfi1. In addition, full-length Runx2 cooperates with Cbf{beta}-SMMHC in leukemia development in transplantation assays. Furthermore, we sho w that the nuclear matrix-targeting signal and DNA-binding runt-homology domain of Runx2 are essential for its leukemogenic activity. Conversely, Runx2 haplo-insufficiency delays the onset and reduces the incidence of acute myeloid leukemia. Together, these results indicate that Runx2 is expressed in the stem cell compartment, interferes with differentiation and represses CBF targets in the myeloid compartment, and modulates the leukemogenic function of Cbf{beta}-SMMHC in mouse leukemia. - Molecular mimicry of host sialylated glycans allows a bacterial pathogen to engage neutrophil Siglec-9 and dampen the innate immune response
Carlin AF Uchiyama S Chang YC Lewis AL Nizet V Varki A - Blood 113(14):3333-3336 (2009)
Human neutrophil Siglec-9 is a lectin that recognizes sialic acids (Sias) via an amino-terminal V-set Ig domain and possesses tyrosine-based inhibitory motifs in its cytoplasmic tail. We hypothesized that Siglec-9 recognizes host Sias as "self," including in cis interactions with Sias on the neutrophil's own surface, thereby dampening unwanted neutrophil reactivity. Here we show that neutrophils presented with immobilized multimerized Sia{alpha}2-3Gal{beta}1-4GlcNAc units engage them in trans via Siglec-9. The sialylated capsular polysaccharide of group B Streptococcus (GBS) also presents terminal Sia{alpha}2-3Gal{beta}1-4GlcNAc units, and similarly engages neutrophil Siglec-9, dampening neutrophil responses in a Sia- and Siglec-9-dependent manner. Reduction in the neutrophil oxidative burst, diminished formation of neutrophil extracellular DNA traps, and increased bacterial survival are also facilitated by GBS sialylated capsular polysaccharide interactions with Siglec -9. Thus, GBS can impair neutrophil defense functions by coopting a host inhibitory receptor via sialoglycan molecular mimicry, a novel mechanism of bacterial immune evasion. - ETS2 and ERG promote megakaryopoiesis and synergize with alterations in GATA-1 to immortalize hematopoietic progenitor cells
Stankiewicz MJ Crispino JD - Blood 113(14):3337-3347 (2009)
ETS2 and ERG are transcription factors, encoded on human chromosome 21 (Hsa21), that have been implicated in human cancer. People with Down syndrome (DS), who are trisomic for Hsa21, are predisposed to acute megakaryoblastic leukemia (AMKL). DS-AMKL blasts harbor a mutation in GATA1, which leads to loss of full-length protein but expression of the GATA-1s isoform. To assess the consequences of ETS protein misexpression on megakaryopoiesis, we expressed ETS2, ERG, and the related protein FLI-1 in wild-type and Gata1 mutant murine fetal liver progenitors. These studies revealed that ETS2, ERG, and FLI-1 facilitated the expansion of megakaryocytes from wild-type, Gata1-knockdown, and Gata1s knockin progenitors, but none of the genes could overcome the differentiation block characteristic of the Gata1-knockdown megakaryocytes. Although overexpression of ETS proteins increased the proportion of CD41+ cells generated from Gata1s-knockin progenitors, their expression led to a significant reduction in the more mature CD42 fraction. Serial replating assays revealed that overexpression of ERG or FLI-1 immortalized Gata1-knockdown and Gata1s knockin, but not wild-type, fetal liver progenitors. Immortalization was accompanied by activation of the JAK/STAT pathway, commonly seen in megakaryocytic malignancies. These findings provide evidence for synergy between alterations in GATA-1 and overexpression of ETS proteins in aberrant megakaryopoiesis. - The Montreal platelet syndrome kindred has type 2B von Willebrand disease with the VWF V1316M mutation
Jackson SC Sinclair GD Cloutier S Duan Z Rand ML Poon MC - Blood 113(14):3348-3351 (2009)
Montreal platelet syndrome (MPS), hitherto described in only one kindred, is a hereditary thrombocytopenia associated with mucocutaneous bleeding, giant platelets, and spontaneous platelet aggregation in vitro. These are features shared with some forms of type 2B von Willebrand disease (VWD); however, the MPS kindred had not been investigated for VWD. We found that all affected MPS family members had borderline to normal von Willebrand factor antigen (VWF:Ag; 0.43-0.75 U/mL), discrepantly low ristocetin cofactor activity (VWF:RCo; 0.16-0.29 U/mL), and normal factor VIII coagulant activity (FVIII:C; 0.57-1.04 U/mL). Unaffected family members all had normal VWF:Ag, VWF:RCo, and FVIII:C levels. In addition, persons with MPS, but not unaffected family members, had loss of plasma (but not platelet) high molecular weight VWF multimers, and were heterozygous for the previously reported V1316M type 2B VWD mutation. Thus, in reevaluating this kindred, we determined that patients with MPS have type 2B VWD with the V1316M VWF mutation. - Novel roles for erythroid Ankyrin-1 revealed through an ENU-induced null mouse mutant
Rank G Sutton R Marshall V Lundie RJ Caddy J Romeo T Fernandez K McCormack MP Cooke BM Foote SJ Crabb BS Curtis DJ Hilton DJ Kile BT Jane SM - Blood 113(14):3352-3362 (2009)
Insights into the role of ankyrin-1 (ANK-1) in the formation and stabilization of the red cell cytoskeleton have come from studies on the nb/nb mice, which carry hypomorphic alleles of Ank-1. Here, we revise several paradigms established in the nb/nb mice through analysis of an N-ethyl-N-nitrosourea (ENU)-induced Ank-1-null mouse. Mice homozygous for the Ank-1 mutation are profoundly anemic in utero and most die perinatally, indicating that Ank-1 plays a nonredundant role in erythroid development. The surviving pups exhibit features of severe hereditary spherocytosis (HS), with marked hemolysis, jaundice, compensatory extramedullary erythropoiesis, and tissue iron overload. Red cell membrane analysis reveals a complete loss of ANK-1 protein and a marked reduction in {beta}-spectrin. As a consequence, the red cells exhibit total disruption of cytoskeletal architecture and severely altered hemorheologic properties. Heterozygous mutant mice, which have wild-type levels of ANK-1 and spectrin in their RBC membranes and normal red cell survival and ultrastructure, exhibit profound resistance to malaria, which is not due to impaired parasite entry into RBC. These findings provide novel insights into the role of Ank-1, and define an ideal model for the study of HS and malarial resistance. - Alternative pre-mRNA splicing switches modulate gene expression in late erythropoiesis
Yamamoto ML Clark TA Gee SL Kang JA Schweitzer AC Wickrema A Conboy JG - Blood 113(14):3363-3370 (2009)
Differentiating erythroid cells execute a unique gene expression program that insures synthesis of the appropriate proteome at each stage of maturation. Standard expression microarrays provide important insight into erythroid gene expression but cannot detect qualitative changes in transcript structure, mediated by RNA processing, that alter structure and function of encoded proteins. We analyzed stage-specific changes in the late erythroid transcriptome via use of high-resolution microarrays that detect altered expression of individual exons. Ten differentiation-associated changes in erythroblast splicing patterns were identified, including the previously known activation of protein 4.1R exon 16 splicing. Six new alternative splicing switches involving enhanced inclusion of internal cassette exons were discovered, as well as 3 changes in use of alternative first exons. All of these erythroid stage-specific splicing events represent activated inclusion of authentic anno tated exons, suggesting they represent an active regulatory process rather than a general loss of splicing fidelity. The observation that 3 of the regulated transcripts encode RNA binding proteins (SNRP70, HNRPLL, MBNL2) may indicate significant changes in the RNA processing machinery of late erythroblasts. Together, these results support the existence of a regulated alternative pre-mRNA splicing program that is critical for late erythroid differentiation. - Role of activated protein C and its receptor in inhibition of tumor metastasis
Bezuhly M Cullen R Esmon CT Morris SF West KA Johnston B Liwski RS - Blood 113(14):3371-3374 (2009)
Engagement of endothelial protein C receptor (EPCR) by activated protein C (aPC) decreases expression of endothelial adhesion molecules implicated in tumor-endothelium interactions. We examined the role of the aPC/EPCR pathway on tumor migration and metastasis. In vitro, B16-F10 melanoma cells showed decreased adhesion to and transmigration through endothelium treated with recombinant human aPC (rhaPC). In murine B16-F10 metastasis models, transgenic EPCR overexpressing (Tie2-EPCR) mice exhibited marked reductions in liver (50%) and lung (92%) metastases compared with wild-type (WT) animals. Intravital imaging showed reduced B16-F10 entrapment within livers of Tie2-EPCR compared with WT mice. A similar reduction was observed in WT mice treated with rhaPC. Strikingly, rhaPC treatment resulted in a 44% reduction in lung metastases. This was associated with decreased lung P-selectin and TNF-{alpha} mRNA levels. These findings support an important role for the aPC/EPCR path way in reducing metastasis via inhibition of tumor cell adhesion and transmigration. - Nonmyeloablative allografting for newly diagnosed multiple myeloma: the experience of the Gruppo Italiano Trapianti di Midollo
Bruno B Rotta M Patriarca F Mattei D Allione B Carnevale-Schianca F Sorasio R Rambaldi A Casini M Parma M Bavaro P Onida F Busca A Castagna L Benedetti E Iori AP Giaccone L Palumbo A Corradini P Fanin R Maloney D Storb R Baldi I Ricardi U Boccadoro M - Blood 113(14):3375-3382 (2009)
Despite recent advances, allografting remains the only potential cure for myeloma. From July 1999 to June 2005, 100 newly diagnosed patients younger than 65 years were enrolled in a prospective multicenter study. First-line treatment included vincristin, adriamycin, and dexamethasone (VAD)-based induction chemotherapy, a cytoreductive autograft (melphalan 200 mg/m2) followed by a single dose of nonmyeloablative total body irradiation and allografting from an human leukocyte antigen (HLA)-identical sibling. Primary end points were the overall survival (OS) and event-free survival (EFS) from diagnosis. After a median follow-up of 5 years, OS was not reached, and EFS was 37 months. Incidences of acute and chronic graft-versus-host disease (GVHD) were 38% and 50%, respectively. Complete remission (CR) was achieved in 53% of patients. Profound cytoreduction (CR or very good partial remission) before allografting was associated with achievement of posttransplantation CR (haza rd ratio [HR] 2.20, P = .03) and longer EFS (HR 0.33, P < .01). Conversely, development of chronic GVHD was not correlated with CR or response duration. This tandem transplantation approach allows prolonged survival and long-term disease control in patients with reduced tumor burden at the time of allografting. We are currently investigating the role of "new drugs" in intensifying pretransplantation cytoreduction and posttransplantation graft-versus-myeloma effects to further improve clinical outcomes. (http://ClinicalTrials.gov; NCT-00702247.) - Long-term outcome of patients with multiple myeloma after autologous hematopoietic cell transplantation and nonmyeloablative allografting
Rotta M Storer BE Sahebi F Shizuru JA Bruno B Lange T Agura ED McSweeney PA Pulsipher MA Hari P Maziarz RT Chauncey TR Appelbaum FR Sorror ML Bensinger W Sandmaier BM Storb RF Maloney DG - Blood 113(14):3383-3391 (2009)
Autologous hematopoietic cell transplantation (HCT) followed by nonmyeloablative allogeneic HCT (auto/alloHCT) provides cytoreduction and graft-versus-myeloma effects. We report on long-term outcomes of 102 patients with multiple myeloma who received auto/alloHCT with a median follow-up of 6.3 years. Treatment consisted of high-dose melphalan and autograft followed by 2-Gy total body irradiation, with or without fludarabine, and alloHCT from human leukocyte antigen-identical siblings. Postgrafting immunosuppressive agent was cyclosporine or tacrolimus and mycophenolate mofetil. Forty-two percent of patients developed grade 2 to 4 acute graft-versus-host disease (GVHD) and 74% extensive chronic GVHD. Five-year nonrelapse mortality after allografting was 18%, 95% related to GVHD or infections. Among 95 patients with detectable disease, 59 achieved complete remissions. Median time to progression was 5 years. Median overall survival (OS) was not reached. Median progression- free survival (PFS) was 3 years. Five-year OS and PFS were 64% and 36%, respectively. Seventy-three patients receiving autoHCT within 10 months from treatment initiation had 5-year OS of 69% and PFS of 37%. In multivariate analysis, {beta}-2-microglobulin of more than 3.5 {micro}g/mL at diagnosis and auto/alloHCT more than 10 months after treatment initiation correlated with shorter OS (P = .03 and P = .02) and PFS (P = .04 and P = .03), whereas Karnofsky scores less than 90% at allotransplantation correlated with shorter PFS only (P = .005). Long-term disease control and GVHD remain key issues.
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