ALBERT

Description: Abstract 3559 Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL) is triggered by constitutively activated BCR-ABL and SRC family tyrosine kinases. They interact each other, then activate downstream growth-signaling pathways including Raf/MEK/ERK,Akt/mTOR and STAT5 pathways. The BCR-ABL tyrosine kinase inhibitor imatinib is the standard treatment for Ph+ leukemia. However, response rate of Ph+ ALL to imatinib is low, relapse is frequent and quick. Studies have documented the potential anti-tumor activities of curcumin, a yellow colored polyphenol from the perennial herb Curcuma longa. However, whether curcumin can be used in the therapy for Ph+ALL remains obscure. Here, we reported that curcumin induced autophagic cell death by activating RAF/MEK/ERK pathway in early stage of the 24-hour exposure course, later induced apoptosis by inhibiting AKT/mTOR, ABL/STAT5 signalings, down-regulating expression of bcr/abl gene and Bcl2 anti-apoptosis protein, and up-regulating the expression of pro-apoptosis protein BAX in Ph+ALL cell line SUP-B15. Furthermore, we found curcumin exerted synergetic anti-leukemia effect with imatinib by inhibiting imatinib-mediated up-regulation of the activation of AKT/mTOR signaling and down-regulating expression of bcr/abl gene. It is worth noting that curcumin provide advantages over dexmethasone as to synergetic anti-leukemia effect with imatinib because dexmethasone improved the imatinib-mediated up-regulation of the activation of AKT/mTOR/P70S6 signaling. In primary samples from Ph+ALL patients, curcumin inhibit growth signaling not only in newly-diagnosised patient but also in imatinib-resistant patient. Moreover, curcumin effectively exhibited anti-leukemia efficacy and synergetic anti-leukemia effect with imatinib in Ph+ALL mouse models. These results demonstrate that curcumin may be a promising agent for the treatment of patients with Ph+ ALL, and curcumin might be particularly effective when used with current induction regimens consisting of imatinib with or without chemotherapy for treating Ph+ ALL. [Grant Support:National Natural Science Foundation of China (No.30770912), Foundation of the Science & Technology Department of Sichuan Province (No.2008SZ0017)]. Disclosures: No relevant conflicts of interest to declare.

Description: Recent studies have shown that anemia is commonly observed after exposure to pathogens or pathogen-derived products, which are recognized via Toll-like receptor 9 (TLR9). In the current study, we demonstrate that CpG oligodeoxynucleotide-2006, a TLR9 ligand with phosphodiester (PO; 2006-PO) but not with the phosphorothioate backbone, selectively inhibits the erythroid growth derived from human CD34+ cells. The 2006-PO was internalized by the erythroid progenitors within 30 minutes; however, expression of TLR9 mRNA was not detected in these cells. The 2006-PO directly inhibited burst-forming unit-erythroid growth, resulted in the accumulation of cells in S and G2/M phases, and increased cell size and frequency of apoptotic cells. These features were similar to those observed in erythroid progenitors infected with human parvovirus B19 that causes pure red cell aplasia. The consensus sequence of 2006-PO was defined as 5′-GTTTTGT-3′, which was located in the P6-promoter region of B19 and inhibited erythroid growth in a sequence-specific manner and down-regulated expression of erythropoietin receptor (EPOR) mRNA and EPOR. B19 genome extracted from serum also inhibited erythroid growth and down-regulated expression of EPOR on glycophorin A+ cells. These results provide a possible insight into our understanding of the mechanisms of human parvovirus B19-mediated inhibition of erythropoiesis.

Description: Mammalian erythroblasts undergo enucleation, a process thought to be similar to cytokinesis. Although an assemblage of actin, non-muscle myosin II, and several other proteins is crucial for proper cytokinesis, the role of non-muscle myosin II in enucleation remains unclear. In this study, we investigated the effect of various cell-division inhibitors on cytokinesis and enucleation. For this purpose, we used human colony-forming unit-erythroid (CFU-E) and mature erythroblasts generated from purified CD34+ cells as target cells for cytokinesis and enucleation assay, respectively. Here we show that the inhibition of myosin by blebbistatin, an inhibitor of non-muscle myosin II ATPase, blocks both cell division and enucleation, which suggests that non-muscle myosin II plays an essential role not only in cytokinesis but also in enucleation. When the function of non-muscle myosin heavy chain (NMHC) IIA or IIB was inhibited by an exogenous expression of myosin rod fragment, myosin IIA or IIB, each rod fragment blocked the proliferation of CFU-E but only the rod fragment for IIB inhibited the enucleation of mature erythroblasts. These data indicate that NMHC IIB among the isoforms is involved in the enucleation of human erythroblasts.

Description: Abstract 3429 Background: Idiopathic PRCA and secondary PRCA associated with thymoma and large granular lymphocyte leukemia are major subtypes of adult-onset chronic PRCA. We have previously shown that these types of PRCA are responsive to immunosuppressive therapy but most patients require long-term maintenance immunosuppressive treatment. These results suggest that acquired chronic PRCA is an autoimmune disorder mediated by T lymphocytes and pathogenic T cell clones may be persistently present during remission. We have previously made an interesting observation that a thymoma-associated PRCA patient had an increase of Vd1 gd T cells in blood. We have also reported that recipients of allogeneic hematopoietic stem cell grafts had an oligoclonal expansion of Vd1 gd T cells and that Vd1 gd T clones had cytotoxicity against autologous EBV-transformed B cell line. Thus, gd T cell repertoires may be altered in PRCA patients in response to certain antigens. Objective: In order to clarify the role for gd T cells in the pathogenesis of chronic acquired PRCA, we have examined the gd T cell receptor repertoire in acquired chronic PRCA patients. Materials and Methods: Nineteen PRCA (8 idiopathic, 6 thymoma, 3 LGL-leukemia and 2 SLE) and 107 healthy volunteer donors were included in the study. This study was approved by the Institutional Review Board at Akita University and conducted in accordance with the Declaration of Helsinki. Blood lymphocyte subsets were analyzed by flow cytometry. Clonality of T cells was determined by complementarity-determining region 3 (CDR3) size distribution analysis and junctional sequence was determined by subcloning of PCR products and DNA sequencing. In some experiments, purified gd T cells from PRCA patients were co-cultured with allogeneic erythroid progenitor cells derived from CD34-positive cells in vitro in order to learn whether patient's gd T cells would exert cytotoxic or growth-inhibitory effect on erythroid progenitor cells. Results: The absolute numbers of ab T cells and gd T cells were normal in patients with PRCA, but there were an increase of Vd1 gd T cells and a decrease of Vd2 T cells (Table 1). More than 50% of Vd1 T cells from PRCA patients expressed HLA-DR, while 20 to 30% of those from healthy individuals expressed HLA-DR (Fig. 1). CDR3 size spectratyping revealed that CDR3 size distribution patterns were skewed in 9 out of 13 PRCA patients examined, although skewed CDR3 size distribution patterns were also observed in 7 out of 10 healthy individuals. In order to determine whether a particular Vd1-Jd rearrangement size was selected in PRCA patients, we performed statistical analysis comparing the CDR3 size distribution of 115 Vd1 TCR clones obtained by subcloning of PCR products in 7 PRCA patients versus 7 controls. No significant difference was found between the two groups (p=0.795 by Mann-Whitney test). Moreover, no apparent consensus amino acid motifs were identified in PRCA patients. Although the T cell clone carrying the -YWGIR- sequence in the CDR3d region was detected in 3 PRCA patients, the T cell clone carrying the -YWGIR- sequence was also detected in one healthy donor. Purified gdT lymphocytes from idiopathic PRCA neither showed an inhibitory effect on proliferation nor cytotoxicity against erythroid progenitor cells in vitro. Adjusted p value was calculated by Kruskal-Wallis ANOVA test. Conclusions: Expansion of Vd1 T cells and depletion of Vd2 T cells are unique features for chronic acquired PRCA. Expansion of Vd1 T cells does not seem to be the consequence of CDR3-dependent selection. Depletion of Vd2 T cells may be the result of chronic stimulation, because our previous study has revealed that the numbers of Vd2 T cells show an age-dependent decrease and Vd2 T cells are susceptible to activation-induced cell death (Int J Hematol, in press). Failure to demonstrate the cytotoxicity of gd T cells from a PRCA patient against erythroid progenitor cells suggests that expanded gd T cells are not effector T cells. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4992 Hydroa vacciniform-like lymphoma is an unusual pediatric cutaneous T-cell lymphoma, which rarely involved with adults. Chemotherapy and/or radiotherapy had little or no benefit. Patients may have a response to α-interferon. We report here an adult case with interferon resistant hydroa vacciniform-like lymphoma that has been successfully treated with sirolimus, an mTOR pathway inhibitor. The patient is a thirty-five year old women,she begun with recurrent cutanous rash and small vesicule around her mouth and nose 3 years ago. One year before, the symptoms were exacerbated with obvious vesiculopapular eruption, edematous, blisters, ulcers, scarring and crusts in her face. Scattered vesiculopapular could be seen in her upper chest and extremities accompanied with fever and submandibular lymphadenopathy. Facial skin biopsy showed angiocentric infiltrates from the epidermis to the subjacent dermis. The infiltrate cells showed a cytotoxic T-cell phenotype, with positive TCR gene rearrangement and EBER expression in situ hybridization. EBV serum test showed EBV-IgA and IgG positive, but serum EBV-DNA was negative. Patient was given α-interferon 300 million units, intramuscularlly, two times a week, plus prednisone 30mg per day, orally. Patients' skin lesions were improved, but present recurrent episodes of papulovesicular eruptions. For the past 3 month patient had a recurrent high fever and exacerbated vesiculopapular eruptions and blisters. Laboratory test showed anemia and neutropenia, serum biochemistry and marrow examination confirmed that a lymphoma associated hematophagocytosis was complicated. Sirolimus was started at an initial dose of 1.5mg, orally, every 12 hours, combined with dexamethasone 10mg/day, intravenously. Three days later the fever begun to resolved and the skin lesion gradually subsided. Two weeks later the skin lesions disappeared and the dose of sirolinmus begun to tapered to 1.0mg, orally, every 12 hours, with prednisone 30mg/day, orally. By three month of follow-up the patient still on stable and be observed. Disclosures: No relevant conflicts of interest to declare.