ALBERT

Description: Thrombopoietin (TPO) and TPO receptor agonists have substantially broadened the therapeutic options for patients with immune thrombocytopenia (ITP). Platelet count response of ITP is usually maintained during the medication; however, once it is stopped, platelet counts commonly drop to pretreatment levels. We report three corticosteroid-resistant ITP patients achieving sustained complete remission with one short-term application of recombinant human thrombopoietin (rhTPO). Thirty-two adult ITP patients (18 females and 14 males; age range 18-72 years, median 45 years) were enrolled between December, 2011 and July, 2013 at the Department of Hematology, Qilu Hospital, Shandong University. Patients were diagnosed according to the recently published criteria (Rodeghiero F et al, Blood 2009). All patients had a baseline platelet count (PC) of 〈 30 x 109/L and bleeding symptoms, and were resistant to prior corticosteroid therapy or splenectomy. Recombinant human thrombopoietin (rhTPO, a truncated and non-glycosylated TPO developed by 3SBIO Pharmaceutical Co., LTD, Shenyang, China, approved by China State Food and Drug Administration) was given subcutaneously at a daily dose of 1.0 µg/kg for 14 days. Withdrawal of rhTPO could be performed while platelet count rose to above 100 x 109/L in less than 14 days. Responses were required to be independent of supportive medications. The criteria for response were defined as follows: (1) complete response (CR): platelet count 〉 100 x 109/L; (2) response (R): platelet count 〉 30 x 109/L and at least 2-fold increase of the baseline platelet count and absence of bleeding; (3) no response (NR): platelet count 〈 30 x 109/L or less than 2-fold increase of the baseline platelet count or bleeding. Of the 32 ITP patients treated with rhTPO, the complete response (CR), response (R), overall response (OR) and no response (NR) rates were 37.50% (12/32), 28.12% (9/32), 65.62% (21/32) and 34.38% (11/32), respectively. Among the 32 patients, three (9.38%) acquired sustained complete response (i.e., platelet count 〉 100 x 109/L and no clinical symptoms for at least six months after the medication). Of these three patients, the time to peak response was 28, 20 and 28 days, and the peak values of platelet counts were 215 x 109/L, 245 x 109/L and 296 x 109/L, respectively. These three patients’ information was shown in Table 1. Their platelet count responses sustained for 56, 40 and 28 weeks without any ITP-specific treatments after the cessation of rhTPO (Figure 1). In addition, only mild adverse events (WHO grades 1-2) were observed, including fever, fatigue and insomnia.Table 1Characteristics of the three ITP patients who achieved a durable complete response associated with the use of rhTPO.PatientAge(years)SexBaseline Platelet Count (x109/L)rhTPO Daily Dose(µg/kg)rhTPO Duration(days)Time to Peak Response(days)Peak Value of Platelet Count(x109/L)Complete Remission off rhTPO(Weeks)172Female21122821556234Female51102024540354Male81142829628Figure 1Serial platelet counts of three ITP patients who achieved a definite rhTPO-induced sustained remission.Figure 1. Serial platelet counts of three ITP patients who achieved a definite rhTPO-induced sustained remission. In conclusion, some ITP patients can achieve sustained complete remission after a short-course of rhTPO. However, the mechanism behind this is unclear. Disclosures: No relevant conflicts of interest to declare.

Description: The NFAT (nuclear factor of activated T-cells) family of transcription factors functions as integrators of multiple signaling pathways by binding to chromatin in combination with other transcription factors and coactivators to regulate genes central for cell growth and survival in hematopoietic cells. Recent experimental evidence has implicated the calcineurin/NFAT signaling pathway for involvement in the pathogenesis of various malignancies, including large B-cell lymphoma (LBCL), a non-Hodgkin’s lymphoma subgroup that is generally responsive to conventional cancer therapies (R-CHOP), but relapse is common that subsequently leads to therapeutic resistance. Although we have shown previously that NFAT family member NFATc1 is constitutively activated and has the ability to maintain cell growth and survival in LBCL cell lines and primary cells, the molecular mechanism(s) underlying how NFATc1 regulates cell growth and survival in LBCL is still unclear. In this study, we demonstrate that the well-known oncogene c-myc is transcriptionally regulated by the transcription factor NFATc1 in LBCL, through a chromatin remodeling mechanism that involves the recruitment of the SWI/SNF chromatin-remodeling complex. In aggressive B-cell lymphoma cell lines, c-myc oncogene protein expression was shown to correlate with NFATc1 protein expression. We further showed that NFATc1 binds to a specific DNA binding element within the proximal c-myc promoter and up-regulates c-myc transcription. The SWI/SNF proteins Brg-1 and Brm, chromatin-remodeling proteins that utilize ATP hydrolysis for energy to modify chromatin structure in order to regulate gene expression, also were shown to bind to the NFAT binding site on the c-myc promoter. Confocal microscopic analysis showed that NFATc1 colocalizes with Brg-1, and co-immunoprecipitation assays showed that Brg-1 interacts with NFATc1. Both proteins interact with the c-myc promoter within the NFAT binding site, as demonstrated by chromatin-immunoprecipitation (ChIP) analysis. Induction of a constitutively active mutant of NFATc1 (caNFATc1) in an NFATc1 negative lymphoma cell line induces c-myc protein expression. Constitutively active NFATc1 also enhances Brg-1 binding to the c-myc promoter when analyzed by ChIP-qPCR assays, suggesting that NFATc1 recruits Brg-1 to the NFAT binding site in the c-myc promoter. Down-regulation of NFATc1 by chemical inhibitors (FK-506) or by validated shRNA of NFATc1, inhibited c-myc protein expression and in-vitro lymphoma cell growth. Our data indicates a novel control mechanism for the transcriptional regulation of c-myc in the pathophysiology of aggressive lymphoma B cells and suggests that targeting NFATc1 could have therapeutic value.

Description: Key Points The impaired function of MSCs in proliferation ability and in inducing tolerogenic DCs may play a role in the pathogenesis of ITP. The effect of THD in correcting dysfunctions of MSCs may suggests a therapeutic potential of THD in ITP patients.

Description: The tumor necrosis factor (TNF) family (TNF-R; CD40; BAFF-R) plays a key role in neoplastic as well as normal B cell growth and survival mechanisms. TNF receptor-associated factor-6 (TRAF-6) is an adapter molecule that regulates several important signaling pathways critical for cell growth and cell survival. It is a member of seven closely related TRAF proteins that serve as signaling molecules, coupling to TNF-receptor superfamily to intracellular signaling, particularly in the CD40 Signalosome. TRAF6 has shown to be over-expressed and play an important role in cell growth and cell survival through the activation of the key transcription factor NF-kB in aggressive non-Hodgkin’s lymphoma B cells (NHL-B), common B cell neoplasm that have been increasing in recent years. Although much of TRAF-6 functions have focused primarily as an adaptor molecule in signaling pathways in the cytoplasm, the role of TRAF-6 in other cellular compartments has not been investigated. Here, we demonstrate, by confocal microscopy as well as cellular fractionation studies that TRAF-6 resides not only in the cytoplasm but also in the nucleus of lymphoma B cells. Immunoprecipitation studies show that TRAF6 is auto-ubiquitinated in the cytoplasm but not in the nucleus, suggesting that nuclear TRAF6 functions differently than cytoplasmic TRAF6. Chromatin immunoprecipitation (ChiP) cloning assays using anti-TRAF6 polyclonal antibody reveal over 200 clones, one of which contains a 130 bp fragment belonging to the proximal 5′ end of the c-myb oncogene promoter. Further experiments demonstrate that nuclear TRAF6 co-localized with SUMO1 and c-myb, suggesting that TRAF-6 may enter the nucleus through SUMO1 interaction and serve as an E3 sumo ligase, in addition to its known adapter role in cytoplasmic signaling. Over-expression studies show that TRAF6 enhances c-myb sumoylation in lymphoma B cells, where this oncogene is over-expressed. C-myb correlates with TRAF6 protein and mRNA expressions in NHL-B cells, suggesting that TRAF6 may be involved in the modulation of c-myb expression through sumoylation, regulating key genes that are regulated by c-myb. Small interfering RNA (siRNA) targeting c-myb results in inhibition of lymphoma cell survival, suggesting that SUMO1/TRAF6/c-myb interactions are important in cell survival pathways in aggressive NHL-B. Such pathways could represent novel targets for the development of therapeutic agents for aggressive B cell lymphomas.

Description: CD40 is a member of TNF receptor family, which plays important roles in the proliferation, survival and differentiation of lymphocytes. Constitutively active CD40 signaling is one of the hallmarks of aggressive B cell lymphoma development and mediates pivotal functions through its downstream pathways, involving NF-kBs. Recently, our group has reported the nuclear localization of CD40, opening a new paradigm in the functional role of CD40 in non-Hodgkin lymphomas of B cell origin (NHL-B). In this study, we show that CD40 co-localizes and immuno-precipitates with nuclear pore complex (NPC) proteins, which suggests NPC proteins are involved in the classic karyopherin pathway that facilitates the nuclear translocation of CD40 protein. Importantly, our co-immunoprecipitation assays and confocal microscopy have demonstrated that, in NHL-B cells, nuclear localized CD40 interacts with the NF-kB protein-c-Rel, but not p65. Chromotin-IP assay further indicates that CD40 forms complexes with c-Rel on the promoters of several proliferation and survival genes-CD154, Blys/BAFF and Bfl-1/A1 in various NHL-B cell lines. Co-transfection of CD40 and c-Rel in NHL-B cells synergistically enhance Blys promoter-driven luciferase activity while CD40 and p65 do not show such synergy. In normal B cells, transfection of c-Rel has synergistically enhanced the proliferation of B lymphocyte after CD40 ligand stimulation, which results in accumulation of CD40 in the nucleus. Furthermore, co-transfection of c-Rel and wild type CD40 also synergizes the proliferation of NHL-B cells, while co-transfection of c-Rel and nuclear localization signal (NLS) mutated CD40 does not. This study suggests that the nuclear CD40 plays an important role in lymphoma growth and survival mechanisms through its interaction with c-Rel, an oncogene that is amplified in many cases of NHL-B that may contribute to the pathophysiology of the disease process. Modulating the nuclear function of CD40 could provide a new targeted therapeutic approach for lymphoma therapy.

Description: Constitutive NF-kB activation is considered a “hallmark” in B-cell malignancies, especially in large B-cell lymphoma (LBCL), a common but heterogenous Non-Hodgkin’s lymphoma type. However, the mechanism(s) of activation and interactions of NF-kB components in the nuclear compartment of B-cell lymphomas are still poorly defined. Our findings demonstrate that both the canonical and the alternative NF-kB pathways are constitutively activated in both LBCL cell lines and primary lymphoma cells. NF-kB DNA binding ELISA analysis showed that the canonical NF-kB components p65 and c-rel as well as the non-canonical NF-kB components p52 and relB are constitutively activated in 14 LBCL cell lines and in 14 primary lymphoma patients’ cells (both ABC-and GCB-like subtypes). Based on the ELISA data, we found that the p65/c-rel ratio is relatively higher in ABC-like LBCL subtype when compared to GCB-like LBCL subtype, but the alternative NF-kB members’ p52 and rel-B are differentially activated in both GCB- and ABC-like LBCL subtypes. Micro-tissue array (MTA) studies confirmed that both NF-kB pathways are constitutively active in LBCL biopsy-cores. The components of both NF-kB pathways were also found to merge and form functional “hybrid” NF-kB complexes in the nuclear compartment. Besides the common hetero-dimeric complexes occur between p50, p65, and c-rel of the canonical NF-kB pathway and between p52 and relB of the alternative NF-kB pathway, components of both pathways also merged to form “hybrid” dimeric complexes, such as p52-relB, p52-c-rel, relB-p65, and relB-c-rel in LBCL cells. Interestingly, p65 and c-rel binds to relB and p52, respectively, with higher affinity in GCB- than in ABC-like LBCL subtype. Our data also showed that activation of both NF-kB pathways and their cross-interaction are not restricted to only neoplastic B cells but that they are also activated and utilized in highly proliferative activated normal B-lymphocyte. Specific siRNA that target individual NF-kB members p65, c-rel, and rel-B have inhibitory activity in lymphoma cell growth, but similar p52 siRNAs do not have inhibitory activity. These studies indicated that in both LBCL cell lines and patient samples (GCB-and ABC-like subtypes), both the canonical and the alternative NF-kB pathways are not only constitutively activated but also interact to form multimeric NF-kB complexes in the nuclear compartment, giving rise to an NF-kB signaling module that not only controls lymphoma cell growth and survival but likely contributes to the heterogeneity of the disease processes. These findings reveal additional aspects of molecular heterogeneity and complexity in NF-kB signaling mechanisms and interacting transcriptional modules, contributing to the pathophysiology of LBCL. These results also highlight the critical importance of multiple constitutively active NF-kB signaling pathways in LBCL and may also contribute to therapeutic refractoriness, particularly in relapsed/refractory LBCLs.

Description: Key Points The impaired suppressive function of myeloid-derived suppressor cells plays a role in the pathogenesis of immune thrombocytopenia. The effect of dexamethasone in correcting dysfunction of myeloid-derived suppressor cells suggests a new therapeutic mechanism of high-dose dexamethasone in patients with immune thrombocytopenia.

Description: Increased macrophage phagocytosis of antibody-coated platelets, as well as decreased numbers and/or impaired function of CD4+CD25+Foxp3+ regulatory T (Treg) cells, has been shown to participate in the pathogenesis of immune thrombocytopenia (ITP). Low-dose histone deacetylase inhibitors (HDACi’s) are anti-inflammatory and immunomodulatory agents that can enhance immunosuppression in graft-versus-host disease by increasing the number and function of Foxp3+ Treg cells, but it is unclear whether they have the potential to promote immune tolerance and platelet release in ITP. In this study, we performed in vitro and in vivo experiments and found that a low-dose HDACi (chidamide) alleviated thrombocytopenia in passive and active murine models of ITP. Further, low-dose HDACi’s attenuated macrophage phagocytosis of antibody-coated platelets, stimulated the production of natural Foxp3+ Treg cells, promoted the peripheral conversion of T cells into Treg cells, and restored Treg cell suppression in vivo and in vitro. Finally, we confirmed that low-dose HDACi’s could regulate CTLA4 expression in peripheral blood mononuclear cells through modulation of histone H3K27 acetylation. Low-dose HDACi treatment in ITP could be offset by blocking the effect of CTLA4. Therefore, we propose that low-dose chidamide administration has potential as a novel treatment for ITP in the clinic.

Description: Introduction: ITP is an autoimmune hemorrhagic disorder characterized by increased platelet destruction and insufficient platelet production. The classical mechanisms of platelet destruction include premature platelet clearance mediated by antiplatelet autoantibodies in the reticuloendothelial system and cytotoxic T lymphocyte (CTL)-mediated platelet lysis. Platelet desialylation, the process of removing sialic acid residues from glycoproteins, which results in the exposure of beta-galactose on platelet surface, can initiate platelet recognition by asialoglycoprotein receptors (ASGPRs)-expressing liver macrophages and hepatocytes and then lead to platelet clearance in the liver. The status and role of platelet desialylation in ITP have not been studied yet. In this study, we detected the level of platelet desialylation and analyzed its association with antiplatelet autoantibody- and CTL- mediated platelet destruction in ITP. Methods: Ethylenediaminetetraacetic acid anti-coagulated whole blood was obtained from 74 active ITP patients and 34 matched controls. Platelets, CD8+ T lymphocytes and plasma were isolated. The levels of platelet desialylation in ITP and controls were evaluated by the binding level of FITC conjugated Ricinus communis agglutinin I (RCA-I) on platelets by flow cytometry. In addition, the modified antibody-specific immobilization of platelet antigens (MAIPA) and assay of CTL-mediated cytotoxicity towards platelets were performed to evaluate antiplatelet autoantibody- and CTL-mediated platelet destruction in ITP patients. Results: Our results showed that the binding level of RCA-I on platelets was significantly elevated in ITP patients compared with controls (Figure 1). Among these 74 ITP patients, 10 were single anti-GPIIb/IIIa positive, 13 were single anti-GPIb positive, 16 were positive for both, and 35 were negative for autoantibodies. There was neither significant difference in the binding level of RCA-I on platelets between patients with anti-GPIIb/IIIa and those without anti-GPIIb/IIIa antibodies, nor between patients with anti-GPIb and those without anti-GPIb antibodies (data not shown). Moreover, we conducted the assay of CTL-mediated cytotoxicity towards platelets in 40 ITP patients and 13 controls. Positively induced platelet apoptosis (greater than mean plus 2 SD for the controls) was detected in 22 of 40 ITP patients, which was regarded as group A. The other 18 patients was referred to as group B. Interestingly, the binding level of RCA-I was significantly higher in group A than that in group B (Figure 2). These findings indicated that the level of platelet desialylation was markedly elevated in ITP patients than controls, and significantly higher in ITP patients with CTL-mediated platelet destruction. We supposed that neuraminidase-1, which could translocate to platelet surface after platelet lesion and cause platelet desialylation, was elevated from an internal platelet store after CTL-mediated platelet destruction, and then caused increased beta-galactose exposure and led to further platelet clearance in the liver. The specific mechanism still needs further study. Conclusion: In conclusion, our results suggest that platelet desialylation may be involved in the pathogenesis of ITP and closely associated with CTL-mediated platelet destruction. Inhibiting platelet desialylation may be a novel therapeutic strategy for ITP patients with CTL-mediated platelet destruction. Figure 1 The level of platelet desialylation in ITP patients and controls. A. The representative flow cytometry results. B. The binding level of RCA-I was higher in ITP patients than controls (***P 〈 0.001). Figure 1. The level of platelet desialylation in ITP patients and controls. A. The representative flow cytometry results. B. The binding level of RCA-I was higher in ITP patients than controls (***P 〈 0.001). Figure 2 Figure 2. The level of platelet desialylation was higher in ITP patients with CTL-mediated platelet destruction. A. Induced platelet apoptosis by CTLs in controls, group A and group B. B. The representative scattergrams of platelet apoptosis. Dots in the lower right corner represented apoptotic platelets. C. The binding level of RCA-I was significantly higher in group A or B than controls (***P 〈 0.001; *P = 0.042). Moreover, it was higher in group A than group B (*P = 0.015). Disclosures No relevant conflicts of interest to declare.

Description: CD40 plays important roles in the proliferation, survival and differentiation of lymphocytes. Constitutively active CD40 recruits TRAFs and IKKs within the lipid rafts to form a signalosome that mediates pivotal downstream proliferation and survival mechanisms involving NF-kB. Recently, we have reported that nuclear localization of CD40, through its interaction with c-Rel, promotes growth, cell cycle progression and survival in large B cell lymphoma. Our studies have opened a new paradigm in the functional role of CD40 in non-Hodgkin lymphomas of B cell origin (NHL-B). However, the mechanism about how CD40 enters nuclear still remains elusive. In this study, we show that CD40 ligation enhances its nuclear accumulation with activation of c-Rel in both normal B-lymphocytes and B cell lymphoma cells with cell fractionation assay and con-focal microscopy. Over-expression of c-Rel in B cell lymphoma cells drives CD40 into cell nucleus. We hypothesize that the route CD40 enters nucleus may involve endosome-endoplasmic reticulum-nuclear pore complex. Indeed, further studies show CD40 co-localizes with endosome marker-EEA1 and endoplasmic reticulum marker-Sec61. Furthermore, our co-immunoprecipitation assay has demonstrated CD40 interacts with Sec61. CD40 also co-localizes and immuno-precipitates with nuclear pore complex (NPC) proteins-NUP62 in normal B-lymphocytes and B lymphoma cells, which suggests NPC proteins may facilitate the nuclear translocation of CD40 protein. Overall, our study suggests that translocation of CD40 into cell nucleus involves multiple pathways. Blocking nuclear localization may modulate the function of CD40 in lymphoma cells; which could provide a new-targeted therapeutic approach for lymphoma therapy.