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Background：Downregulation of CD20, a molecular target for monoclonal antibodies (mAbs), is a clinical problem leading to decreased efficacy of anti-CD20-based therapeutic regimens.Up to one-third of diffuse large B cell lymphoma (DLBCL) patients eventually develop resistance to R-CHOP regimen, since the remaining therapeutic options are limited. LW-213, a derivative of wogonin, is reported to possess antineoplastic properties in a variety of cancers, but whether it has effects on DLBCL is n-ot known. Studies have reported that upregulation of CD20 expression b-y either HDACi or silenced SOX2 expression showed sensitizing potential in Rituximab-induced cell death in malignant B cells. Our study was to explore whether LW-213 could sensitize DLBCL to Rixutimab thus improve therapeutic efficacy. Methods： Two DLBCL cell lines, RI-1 (ABC subtype) and Su-DHL -8 (GCB subtype), were used in our study. RI-1 and Su-DHL-8 cells were treated with LW-213 at different doses and for different times, and their proliferation and viability were detected by Cell counting kit-8 (CCK8).Flow cytometry was used to determine surface CD20 expression. Western blotting and q-PCR were applied to examine the protein and mRNA levels of CD20, SOX2, Ace-H3 and Ace-H3K27. CDC assay was used to evaluate the synergistic effects of LW-213 and Rixutimab. Results：We showed that LW-213 inhibited the proliferation of human DLBCL cell lines (Su-DHL-8、RI-1 ) in dose-and time-dependent manners with IC 50 values at the low μmol/L levels, meanwhile it potently inhibited primary lymphoma cells derived from peripheral blood of B-cel-l lymphoma patients. Furthermore, LW-213 significantly increased CD20 surface expression and the acetylation level of histone in DLBCL cell li-nes. Inversely，the SOX2 expression level remarkably decreased. Finally,Combination with LW-213 significantly synergized Rituximab-induced cell death in vitro. Conclusion： The results demonstrate that LW-213 sensitizes DLBCL cells to Rituximab in vitro by upregulating CD20 expression and the SOX2/ace-H3K27/ace-H3 axis may plays a critical role in CD20 upregulation processing. Even though this strategy is important in vitro models,the upregulating CD20 expression therapy against DLBCL proposed in this study warrants further study in vivo and clinical trials . Keywords：CD20 DLBCL Rituximab SOX2 Histone Deacetylation Disclosures No relevant conflicts of interest to declare.

Background:T-cell malignancies are characterized by the excessive proliferation of hematopoietic precursor cells of T-cell lineage lymphocytes in bone marrow. T-cell malignancies consist of T-cell lymphoblastic leukemia (T-ALL) and T-cell non-Hodgkin lymphoma (T-NHL). Effective therapeutic options are limited for T-cell malignancies patients. Apoptosis is one of the most important means to cure cancer. Autophagy, a conserved process that sequesters and targets cellular components for lysosomal degradation, also plays highly context-specific roles in mediating cell death. Our research found that LW-218, a new synthetic flavonoid compound, exerted anti-T-cell malignancies cell lines effects by autophagy-mediated apoptosis induction. Methods: In vitrostudy, T-ALL cell lines (Jurkat, Molt4) and T-NHL cell lines (Hut102, Hut78) were used to investigated LW-218-induced apoptosis effects by survival assays (Flow cytometry, Annexin V/PI). A series of experiment were used to assess the autophagy flux triggered by LW-218, by using mCherry-GFP-LC3 construct, Lysotracker Red staining, and determining the expression ratio of LC3-II/LC3-I. Western blot assay, RT-qPCR, transfection with siRNA, and immunofluorescence were used to investigate the underlying mechanism. In vivostudies, the anti-tumor effects of LW-218 were evaluate in T-ALL xenografts which established by subcutaneous injection of Jurkat cells. Results:Mechanistic studies demonstrated that LW-218-induced cell apoptosis in T-cell malignancies cell lines was correlated with activation of caspase 8, caspase 3 and caspases 9, and the substrate PARP1. In addition, LW-218 induced autophagy flux in T-cell malignancies, as increased ratios of LC3B-II/LC3B-I and reductions of P62 expression. The increased numbers of GFP-mCherry puncta and fluorescent intensity also indicated the increase in autolysosomes. The autophagy-mediated cell death must strictly follow the criteria that the inhibition of autophagy, through either genetic or chemical means, prevents cell death. LW-218-induced cell apoptosis and the activation of caspases3/8/9 could be prevented when the autophagy flux was blocked by autophagy inhibitors. mTOR is a master regulator of cellular metabolism and governs the programmed cell death pathways of apoptosis and autophagy. The results showed that LW-218 inhibited the expression of p-mTOR (Ser2448). And the restored activation of mTOR correlated with the inhibition of autophagy flux, which indicated mTOR might be involved in LW-218-induced autophagy. LW-218 also induced cleavage of Bid, the substrate of caspases 8, which indicated that the apoptosis mediated by autophagy might via caspases 8 and Bid activation. Thein vivostudies also verified that LW-218 inhibited the growth of tumors. Conclusions:Our study provides a new insight into the mechanism of LW-218-induced autophagy-mediated apoptosis, suggesting the potency of LW-218 to be a promising agent against T-cell malignancies. Disclosures No relevant conflicts of interest to declare.

Wogonoside is the main flavonoid component derived from the root of Scutellaria baicalensis Georgi. It is a popular Chinese herbal medicine with the potential to treat hematologic malignancies. In this study, we investigated the anticancer effects of wogonoside in acute myeloid leukemia (AML) cell lines and primary patient-derived AML cells. Wogonoside exerted antiproliferative properties both in vitro and in vivo. Furthermore, it efficiently inhibited the proliferation of U937 and HL-60 cells through the induction of G1 phase arrest and the promotion of differentiation. We also demonstrated that wogonoside significantly increased the transcription of phospholipid scramblase 1 (PLSCR1) due to its influence on the expression of cell cycle- and differentiation-related genes, including the upregulation of p21waf1/cip1 and downregulation of the oncogenic protein c-Myc. Wogonoside also promoted PLSCR1 trafficking into the nucleus and facilitated its binding to the inositol 1,4,5-trisphosphate receptor 1 (IP3R1) promoter, thus increasing the expression of IP3R1. Finally, inhibition of PLSCR1 expression with small interfering RNA partially blocked wogonoside-induced cell cycle arrest and differentiation and disturbed the wogonoside-associated molecular events. The results of this study therefore suggest that wogonoside may represent a therapeutic candidate for the treatment of AML.

The LMAN1-MCFD2 (lectin, mannose binding 1/multiple coagulation factor deficiency protein 2) cargo receptor complex transports coagulation factors V (FV) and VIII (FVIII) from the endoplasmic reticulum (ER) to the ER-Golgi intermediate compartment (ERGIC). LMAN1 (ERGIC-53) is a hexameric transmembrane protein with a carbohydrate recognition domain (CRD) on the ER luminal side. Here, we show that mutations in the first beta sheet of the CRD abolish MCFD2 binding without affecting the mannose binding, suggesting that LMAN1 interacts with MCFD2 through its N-terminal beta sheet, consistent with recently reported crystal structures of the CRD-MCFD2 complex. Mutations in the Ca2+- and sugar-binding sites of the CRD disrupt FV and FVIII interactions, without affecting MCFD2 binding. This interaction is independent of MCFD2, as LMAN1 mutants defective in MCFD2 binding can still interact with FVIII. Thus, the CRD of LMAN1 contains distinct, separable binding sites for both its partner protein (MCFD2) and the cargo proteins (FV/FVIII). Monomeric LMAN1 mutants are defective in ER exit and unable to interact with MCFD2, suggesting that the oligomerization of LMAN1 is necessary for its cargo receptor function. These results point to a central role of LMAN1 in regulating the binding in the ER and the subsequent release in the ERGIC of FV and FVIII.

Background: Depression is more common in patients with multiple myeloma (30%) than in the general population (7%), similar to other patients with advanced cancer (PMID 27141015, 20187072). It may be more difficult to detect depression in cancer patients as psychiatric symptoms commonly overlap with medical symptoms from their disease or treatment. Additionally, it is difficult to discern differences between normal and pathologic reactions to such a serious diagnosis. Depressive symptoms, even mild presentations, have been shown to lower quality of life, affect medication compliance, and increase suicide risk, which increase all-cause mortality in the general population, and this is no different in cancer patients, including those with multiple myeloma (PMID 26029972, 23677523). Although depression has a significant impact on cancer patients, there have not been large studies to evaluate antidepressant use, including the frequency of its initiation after diagnosis. Methods: Patients included in this study were participants in the Ohio Myeloma Initiative (NCT01408225), an observational registry collected from patients in Ohio State University's myeloma clinic. Medications were collected and patients who took antidepressants were filtered into a list. The order date of the medications was compared to the patient's diagnosis date. Patients with order dates occurring after date of diagnosis were collected. Chart review was then performed to determine when the antidepressant was started and its indication. Results: Fourteen hundred eight-nine multiple myeloma patients were enrolled in the registry and their medications (35,390) compiled. Of those 1,489 patients, 207 (14%) had an antidepressant on their medication list. The most commonly prescribed antidepressants were duloxetine (14%), sertraline (13%), and citalopram (9%). There were 133 male patients and 74 female patients and the average age of diagnosis was 60.2 years. Although 148 patients (148/207, 71.4%) had documentation of antidepressant use after diagnosis. Of these patients, 106 patients (106/148) had the indication of depression with or without anxiety. Other common indications included neuropathy (24/148, 16.2%) and insomnia (13/148, 8.8%). Fibromyalgia, smoking cessation, anorgasmia, and dizziness were rare but documented indications. Only 32 patients (32/207, 22%) were initiated on an antidepressant with the indication of depression after their diagnosis. There were 25 male patients and 7 female patients. Sertraline (22%), citalopram (22%), and escitalopram (18.6%) were the most common antidepressants started for depression. Conclusions: Only 14% of our patients were on antidepressants despite an average 30% incidence of depressive symptoms reported in prior studies. Of those on antidepressant medications, it would appear that younger patients tended to initiate treatment for their depression, which is congruent with a study by Lamers et al. 2013, which found that younger patients showed the greatest interest in treating their depression. Multiple myeloma is a unique malignancy in that its treatments have side effects, most notably polyneuropathy, which benefit from administration of a SSRI or TCA. Therefore, physicians treating these patients may unconsciously prescribe these antidepressants for a "two birds with one stone" approach. Because documentation of anti-depressant use is often lacking, prospective registries will need to separately document the indications for psychotropic medications. Disclosures Hofmeister: Janssen: Pharmaceutical Companies of Johnson & Johnson: Research Funding; Incyte, Corp: Membership on an entity's Board of Directors or advisory committees; Signal Genetics, Inc.: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Karyopharm Therapeutics: Research Funding; Arno Therapeutics, Inc.: Research Funding; Takeda Pharmaceutical Company: Research Funding; Teva: Membership on an entity's Board of Directors or advisory committees.

GL-V9, a newly synthetic flavonoid derivative, is an active cytotoxic component and exhibits anticancer activities. Here, we demonstrated the sequence of death reponses to different concentration of GL-V9 in human T-cell malignancies cell Jurkat and HuT78. Responses included transient accumulation of cells at the G2/M border followed by also transient rise in several mitotic markers and mitotic attempts were largely abnormal, resulting in numerous micronucleated and multinucleated cells in low drug concentration (2 μM), as shown by flow cytometre analyses and Giemsa staining. These events, indicative of mitotic catastrophe, were not associated with immediate cell death. However, MTT assay showed that high concentration of GL-V9 (4, 8, 16 μM) causes near complete cell growth inhibition at 12 h. Also, the classical manifestations of apoptosis (including phosphatidylserine externalization, mitochondrial dysfunction, and caspase activation) were marginal at 24 h, as shown by Annexin V-PI staining, JC-1, and western blot assay. The mechanism research reveals that low dose of GL-V9 induced mitotic catastrophe via decreasing EEA1, which is required for cytokinesis, and increasing the expression of PTEN. Besides, high concentration of GL-V9 induced cell apoptosis by up-regulating the expression of apoptosis-related proteins caspase 3 and Bim in lymphoid tumor cell lines and primary ALL cells. Collectively, these findings show that different concentrations of GL-V9 can induce mitotic catastrophe and ultimate cell death event of human T-cell malignancies respectively, which would be a potential therapeutical compound for treating human lymphoid tumor. Disclosures No relevant conflicts of interest to declare.

Combined deficiency of factor V and factor VIII (F5F8D) is a bleeding disorder caused by mutations in either LMAN1 or MCFD2. LMAN1 (ERGIC-53) and MCFD2 form a Ca2+-dependent cargo receptor that cycles between the endoplasmic reticulum (ER) and the ER-Golgi intermediate compartment for efficient transport of FV/FVIII from the ER to the Golgi. Here we show that the C-terminal EF-hand domains are both necessary and sufficient for MCFD2 to interact with LMAN1. MCFD2 with a deletion of the entire N-terminal non-EF hand region still retains the LMAN1-binding function. Deletions that disrupt core structure of the EF-hand domains abolish LMAN1 binding. Circular dichroism spectroscopy studies on missense mutations localized to different structural elements of the EF-hand domains suggest that Ca2+-induced folding is important for LMAN1 interaction. The EF-hand domains also mediate the interaction with FV and FVIII. However, mutations in MCFD2 that disrupt the tertiary structure and abolish LMAN1 binding still retain the FV/FVIII binding activities, suggesting that this interaction is independent of Ca2+-induced folding of the protein. Our results suggest that the EF-hand domains of MCFD2 contain separate binding sites for LMAN1 and FV/FVIII that are essential for cargo receptor formation and cargo loading in the ER.

Tumor necrosis factor alpha (TNFα) is a complicated cytokine which could induce differentiation of acute myeloid leukemia (AML) cells. We have found that Oroxylin A, a natural compound isolated from Scutellariae radix, markedly enhanced TNFα-induced NBT reduction and CD11b/CD14 expression of AML cells. Besides, Giemsa staining also displayed that the combination group induced U937-MDR cells to differentiate into monocyte-like cells yet NB4 and HL-60-resistant cells to granulocytic-like cells. Further study showed that TNFα induced PI3K subunit p85α binding to N-terminal truncated nuclear receptor RXRα (tRXRα) proteins and activating AKT. On the contrary, these could be inhibited by Oroxylin A through inhibiting interaction between tRXRα and p85α in NB4 and HL-60-resistant cells. Moreover, Oroxylin A inhibited the activation of NF-κB signaling and the DNA binding activity by TNFα proved by EMSA in these two AML cells. All these suggested that Oroxylin A was able to inhibit NF-κB signaling and RXRα-dependent AKT signaling, the negative effects of TNFα for AML therapy, suggesting that combination of Oroxylin A and TNFα might be a novel candidate for differentiation therapy for AML cells and required further investigation. Disclosures No relevant conflicts of interest to declare.

Wogonin, a plant-derived flavone, has been shown to inhibit tumor angiogenesis. Angiogenesis is associated with multiple myeloma (MM) progression. However, the mechanisms that Wogonin inhibits angiogenesis in MM microenvironment are not well known. In this study, we investigated Wogonin inhibited tumor angiogenesis that correlated with the inhibition the secretion of vascular endothelial growth factor (VEGF) in MM cells. The c-Myc/Hif-1α–dependent pathway was essential to regulate MM cell-mediated angiogenesis and contributed to tumor growth. Wogonin inhibited c-Myc activation and down-regulated the expression of c-Myc and Hif-1α. Functionally, based on transient knockdowns and overexpression, our data delineate Wogonin inhibited VEGF production and secretion mediating tumor angiogenesis via c-Myc/Hif-1α–dependent pathway. Our data, therefore, identify Wogonin as a novel anti-angiogenesis compound to inhibit tumor promotion in MM. Disclosures No relevant conflicts of interest to declare.

Drug resistance is a major obstacle in cancer chemotherapy, especially in the treatment of leukemia. Hypoxia is an inseparable component of the solid tumor microenvironment as well as the bone marrow (BM) microenvironment. In this study, we investigated the effect of the new nanoparticles polyethylene glycol (PEG)-poly L-lysine (PLL)-poly lactic-co-glycolic acid (PLGA) (PEG-PLL-PLGA) modified by transferrin (Tf) combined with daunorubicin (DNR) on K562 cells under hypoxia. We found the IC50 of DNR and DNR-PEG-PLL-PLGA in K562 cells increased under hypoxia, while the IC50 of DNR-Tf-PEG-PLL-PLGA in K562 cells decreased under hypoxia according to the results of MTT assay. The apoptosis of cells and the intracellular concentration of DNR were detected by flow cytometry (FCM). From the datas, we found that the new targeted nanoparticles can overcome hypoxia induced drug resistance of K562 cells. By targeting the transferrin receptor (TfR) on the surface of K562 cells, the new targeted drug led to an increase in intracellular accumulation of DNR. Transcription and Protein of P-glycoprotein (P-gp) and TfR increased in K562 cells under hypoxia, which probably had a relationship with the decreased degradation of the Hypoxia-Inducible Factor-1α (HIF-1α). We further detected the levels of HIF-1α, Bcl-2, Bax and Caspase-3 of K562 cells in transcription and protein under hypoxia regulated by DNR, DNR-PEG-PLL-PLGA and DNR-Tf-PEG-PLL-PLGA, respectively. DNR-Tf-PEG-PLL-PLGA had the best effect when compared with other groups. Furthermore, intravenous injection of DNR-Tf-PEG-PLL-PLGA expressed the best effect of suppressing proliferation of K562 cells injected to nude mice among the three drugs. Therefore, the new drug DNR-Tf-PEG-PLL-PLGA may be a potential cancer therapy for human leukemia. Disclosures No relevant conflicts of interest to declare.

Abstract 3970 The objective of this paper is to study the biocompatibility and security of self-prepared magnetic nanoparticles Fe3O4 (Fe3O4-MNPs) loaded with daunorubicin (DNR), which has the potential application in hematologic malignancies therapy. Hemolysis test was carried out to estimate it s blood toxicity; Fe3O4-MNPs loaded with DNR were intra peritoneally injected into mouse to calculate the LD50; micronucleus (MN) assay was reckoned to identify its genotoxicity; acute toxicity testing was evaluated its influence to mouse hepatic and renal functions. The result of hemolysis rate (HR) of Fe3O4-MNPs loaded with DNR was 2.908%, far less than 5%. Therefore, we concluded that self-prepated Fe3O4-MNPs loaded with DNR nanoparticles had no hemolytic reaction, and they consistent with the requirement of hemolytic test of biomaterials. The LD50 of Fe3O4-MNPs loaded with DNR nanoparticles to the mice was 1009.71mg/kg (relative content of DNR was 10mg/kg) and the 95% confidence interval was 769.11∼1262.40mg/kg, it had no significant difference compared with LD50 of using DNR only, which data was 8.51mg/kg and the 95% confidence interval was 6.48∼10.37mg/kg, and it had wide safety value circumscription. In micronucleus assay, compared Fe3O4-MNPs loaded with DNR nanoparticles experimental groups with negative control group, we found that the result had no significant difference (P > 0. 05) in micronucleus formation rate, while compared experimental groups with positive control group, the result had significant difference (P < 0. 05). The result indicated that Fe3O4 magnetic nanoparticles had no cacogenesis and mutagenesis. Acute toxicity testing showed that mice body weigh of control group, Fe3O4-MNPs loaded with DNR nanoparticles experimental group and isodose DNR group had no significant difference in 24h, 48h, and 72h after intra peritoneally injection; they had normal activity, eating and evacuation; toxic reactions such as instability of gait, convulsion, paralysis and respiratory depression were not been found; the alanine transarninase (ALT), blood urea nitrogen (BUN), and creatinine clearance rate (CCr) of Fe3O4-MNPs loaded with DNR nanoparticles experimental group was 66.0±28.55u/L, 9.06±1.05mmol/L, and 18.03±1.84umol/L, respectively, which had no significant difference compared with control group and isodose DNR group. From the results of our experiment, we could consider that self-prepared Fe3O4-MNPs loaded with DNR nanoparticles is a kind of high biocompatibility and security materials and perhaps is suitable for further application in hematologic malignancies therapy. Disclosures: No relevant conflicts of interest to declare.

Abstract 4947 Objective: The aim of this study was to investigate the expression of survivin and the apoptosis induced by DNR and BrTet in the leukemic cells K562/A02. Methods: In a typical experiment, the K562/AO2 cells were treated with daunorubicin (DNR), 5-bromotetrandrine (BrTet), or DNR and BrTet for 48 hours, and the cells treated without any drugs were used as control group. Cell proliferation was analyzed by MTT assay. Cells apoptosis and the concentration of DNR within the cells were measured by Flow cytometry (FCM). The expressions of mRNA and protein of survivin were determined by semi-quantitative reverse transcription PCR (RT-PCR) and Western blot, respectively. Results: The results of MTT assay indicated that DNR and BrTet were both able to inhibit the proliferation of K562/AO2 cells in dose-dependent manner. The fresh evidence from flow cytometry showed that a higher apoptosis rate could be induced and a higer concentration of DNR could be detected in K562/AO2 cells by DNR and BrTet as compared with those by DNR or BrTet in the same concentrations(P0.05). Western bolt demonstrated that the expression of survivin protein was much lower in the DNR and BrTet group(P Conclusion: BrTet could increase the concentration of DNR and reverse the multidrug resistance(MDR) in the K562/AO2 cells. Survivin may play an important role in apoptosis induced by DNR. Survivin could be a target for the treatment of MDR in haematopoietic malignancies. Disclosures: No relevant conflicts of interest to declare.

Abstract 4856 Objective: The multidrug resistance (MDR) in leukemia is a major chemotherapy obstacle, rendering many currently available chemotherapeutic drugs ineffective. The aim of this study was to explore the new strategy to early diagnose the MDR by electrochemical biosensor based on carbon nanotubes–drug supramolecular interaction. Methods: The carbon nanotubes modified glassy carbon electrodes (CNTs/GCE) as a sensor were directly immersed into the cells suspension of the sensitive leukemia cells K562 and/or its MDR cells K562/A02 to detect the response of the electrochemical probe of daunorubicin (DNR) residues after incubated with cells. Results: The fresh evidence from the electrochemical studies based on CNTs/GCE demonstrated that the homogeneous, label-free strategy could directly measure the function of cell membrane transporters in MDR cancer cells, identify the cancer cell phenotype (sensitive or MDR). The cathodic peak current showed good linear response to the changes of the fraction of MDR with a correlation coefficient of 0.995 when we further took the different ratios of the sensitive leukemia cells K562 and its MDR ones K562/A02 as the model of MDR levels to simulate the MDR occurrence in leukemia. Then, we can easily predict the MDR fraction based on the calibration curve of the cathodic peak current versus the fraction of MDR according to the obtained peak current. Conclusion: These results indicated that the biosensing assay could provide a powerful tool for assessment of MDR in leukemia. The new electrochemical biosensor based on carbon nanotubes–drug supramolecular interaction could represent promising approach in the rapid diagnosis of MDR in leukemia. Disclosures: No relevant conflicts of interest to declare.

Abstract 4432 Object This study was purposed to investigate the expression of HIF-1α and VEGF in leukemia cell lines and the effect of YC-1 on the regulation of HIF-1α and VEGF and the induction of apoptosis in U937 cell, exploring the mechanism of apoptosis after treatment of YC-1. Methods RT-PCR was used to determine the levels of HIF-1α mRNA and VEGF mRNA in K562,U937 and Jurkat cells. After treatment of U937 cell with 4μmol/L YC-1 for 0 hour, 8 hours,16 hours and 24 hours respectively, the changes of morphologic features were observed by DAPI staining under fluorescent microscope and the apoptotic rates were assayed by flow cytometry with Annexin V-FITC/PI staining; the levels of HIF-1α mRNA and VEGF mRNA were measured with RT-PCR ; the protein expression of HIF-1α, VEGF, Bax,Bcl-2 and Caspase-3 were measured by Western blot. Results HIF-1α mRNA and VEGF mRNA were expressed in all three leukemia cell lines. After treatment of U937 cell with 4μmol/L YC-1 for 0 hour,8 hours,16hours and 24hours respectively, the typical apoptotic morphologic features of U937 cells were observed under fluorescent microscope and the apoptotic rates were significantly increased in a time-dependent manner, they were (4.87±0.70)%, (27.27±2.00)%, (51.53±2.81) and (60.5±3.20)% respectively, the level of VEGF mRNA reduced, while the level of HIF-1α mRNA had no obviously changes. Furthermore, the expression of HIF-1α, VEGF and Bcl-2 decreased, while the expression of Bax and Caspase-3 increased in a time-dependent manner. Conclusion HIF-1α mRNA and VEGF mRNA are expressed in leukemia, YC-1 has significant effect of down-regulation the protein expression of HIF-1α and VEGF and induction of apoptosis in U937, its mechanisms may involve in up-regulating Bax/Bcl-2 ratio and expression of Caspase-3. Disclosures: No relevant conflicts of interest to declare.

Abstract 2140 Poster Board II-117 Coagulation factor V (FV) and factor VIII (FVIII) play key roles in hemostasis and thrombosis. The LMAN1 (ERGIC-53)-MCFD2 complex is a mammalian cargo receptor for efficient transport of FV and FVIII from the endoplasmic reticulum (ER) to the Golgi. Mutations in either LMAN1 or MCFD2 cause a bleeding disorder, combined deficiency of factor V and factor VIII. LMAN1 is a type-1 transmembrane protein with a Ca2+-dependent carbohydrate recognition domain homologous to leguminous lectins. MCFD2 is a small soluble protein with an N-terminal sequence of unknown structure and two Ca2+-binding EF-hand domains at the C terminus. LMAN1 and MCFD2 form a Ca2+-dependent protein complex in the ER-Golgi intermediate compartment (ERGIC), an organelle between the ER and Golgi that is unique to higher eukaryotic cells. FV and FVIII interact with the LMAN1-MCFD2 complex in a Ca2+ -dependent manner. To elucidate the role of Ca2+ in regulating the ER-to-Golgi transport of FV and FVIII, we determined the structural features important for the organization of the receptor complex and the interaction of this complex with its client cargo FV and FVIII. We show that the C-terminal Ca2+-binding EF hand domains of MCFD2 are both necessary and sufficient for interaction with LMAN1. The EF hand domains also mediate the interaction with FV and FVIII. All MCFD2 missense mutants identified in F5F8D patients are localized to the EF hand domains and fail to bind LMAN1. However, these mutants still retain the FV and FVIII binding activities. Circular dichroism spectroscopy studies on missense mutations localized to different structural elements of the EF hand domains suggest that Ca2+-induced folding of MCFD2 is important for LMAN1 interaction, but not essential for FV and FVIII binding. We also demonstrate that the carbohydrate recognition domain (CRD) of LMAN1 contains separate binding sites for MCFD2 and FV/FVIII. Mutations in the Ca2+ and sugar binding sites of CRD disrupt FV and FVIII interaction, without affecting MCFD2 binding, suggesting that the Ca2+ binding sites in LMAN1 are primarily required for the recognition of sugar residues in FV and FVIII. These results support a model in which Ca2+ plays a critical role in regulating the binding in the ER and the subsequent release in the ERGIC of FV and FVIII. Ca2+ concentration is higher in the ER than in the ERGIC and the Golgi. In the ER lumen, FV and FVIII loading is initiated by a flexible interaction with MCFD2 and stabilized by the follow-up interaction of sugar side chains of FV and FVIII with the carbohydrate binding site of LMAN1. The LMAN1-FV/FVIII interaction is more sensitive to Ca2+ concentration than the LMAN1-MCFD2 interaction, so that the lower Ca2+/pH in the ERGIC triggers the release of FV and FVIII but not the dissociation of the LMAN1-MCFD2 receptor complex. The empty receptor complex is subsequently recycled back to the ER for the next round of cargo loading. Disclosures: No relevant conflicts of interest to declare.

Objective Aberrant DNA methylation of CpG site is among the earliest and most frequent alterations in cancer. The aim of this study was to study whether phenylhexyl isothiocyanate can reduce the methylation level of P16 gene. This study used a microarray-based method for quantificationally detecting changes of P16 gene methylation in leukemia patient and U266 cell line. And to simply discuss the effect of phenylhexyl isothiocyanate on tumor methylation. Methods This method used bisulfite-modified DNA as a template for PCR amplification, resulting in conversion of unmethylated cytosine, but not methylated cytosine, into thymine within CpG islands of interest. One set of oligonucleotide probes were designed to fabricate a DNA microarray to detect the methylation changes of P16 gene CpG islands. Each set contained a pair of methylated and unmethylated oligonucleotides for interrogating 3 CpG sites in close proximity. By TA cloning, PCR, sequencing, positive and negative DNA targets were required. Next drawing a standard curve by fluorescence intensity. Leukemia samples DNA were abstracted and bisulfite-modified. Sample DNA targets were required by PCR amplification and were hybridized with the microarry. Finally the microarry was scanned with ScanArray Lite microarray analysis systems. Results The linear relationship (R2=0.9660) was established and it could be used to eliminate background noise. The methylation level of U266 is hypermethylation, after cultured with PHI, the level reduce. Eleven patients have P16 gene hypermethylation in thrity. After culture with PHI, seven patients showed level reduce, one patient showed raise, three patients showed remaining. Conclusion PHI can reduce the methylation level of P16 gene in U266 cell line, and also can reduce the methylation level of P16 gene of leukemia patient samples in vitro culture.

Objective: To establish the xenograft leukemia model with stable multiple drug resistance in nude mice; to investigate the reversal effect of 5-Bromotetrandrine and Magnetic nanoparticle of Fe3O4 combined with DNR in vivo and to search for the possible reversal mechanisms. Methods: K562 and K562/A02 cells were respectively inoculated subcutaneously into back of athymic nude mice (1×107 cells/each) to establish the xenograft models. The tumor formation was evaluated by animal ultrasonic inspection. Tumors-bearing nude mice were assigned randomly to five groups which were treated with NS (A group); DNR 1mg/kg (B group); nanoparticle of Fe3O4 combined with DNR 0.63mg/kg(C group): 5-BrTet 2.5mg/kg combined with DNR(D group); 5-Bromotetrandrine 2.5mg/kg and Magnetic nanoparticle of Fe3O4 combined with DNR 0.63mg/kg(E group) respectively. The incidence of tumor formation, growth characteristics, weight and volume of tumor were observed. The histopathologic examination of tumors and organs were detected. For resistant tumors, the protein levels of P-glycoprotein (P-gp) were detected by Western blot. Results: The tumor incidence was 100% in the nude mice inoculated with either K562 or K562/A02 cells. In 6 to 9 days,the tumors reached a volume of more than 1 00 mm3. In vivo, MTT assay showed K562/A02 tumor maintained the drug resistance. For K562 cells xenograft tumors, there were no apparent differences in tumor suppression effect between the B AC AD AE group. For K562/A02 cells xenograft tumors, 5-BrTet and Magnetic nanoparticle of Fe3O4 combined with DNR significantly suppressed growth of tumor: the inhibition rate was 62.76% while DNR alone be used, the inhibition rate was 3.68%. Pathologic examination of resistant tumors showed the tumors necrosis obviously in E group. Application of 5-BrTet and Magnetic nanoparticle of Fe3O4 inhibited the overexpression of P-gp. Conclusion: The xenograft leukemia nude mice model was maintain the multiple drug resistance. 5-Bromotetrandrine and Magnetic nanoparticle of Fe3O4 combined with DNR had a significant tumor-suppressing effect on MDR leukemia cells xenograft model.