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This study investigated the cytotoxic effect of uvangoletin on HL-60 cells, and the effects of uvangoletin on myelosuppression, leucopenia, gastrointestinal tract disturbances and the possible cytotoxic mechanisms by using CCK-8, flow cytometry, western blot, xenograft, cyclophosphamide-induced leucopenia, copper sulfate-induced emesis and ethanol-induced gastric mucosal lesions assays. The results of CCK-8, flow cytometry and western blot assays indicated that uvangoletin showed the cytotoxic effect on HL-60 cells and induced the apoptosis of HL-60 cells by downregulating the expression levels of anti-apoptotic proteins (Survivin, Bcl-xl and Bcl-2), upregulating the expression levels of pro-apoptotic proteins (Smac, Bax, Bad, c-caspase-3 and c-caspase-9), and promoting the release of cytochrome c from mitochondria to cytoplasm. Further, the results of xenograft assay suggested that uvangoletin inhibited the HL-60-induced tumor growth without adverse effect on body weight of nude mice in vivo by regulating the expression levels of above apoptotic proteins. The results indicated that the reductions of WBCs count and thighbone marrow granulocytes percentage in cyclophosphamide-induced leucopenia assay, the incubation period and number of emesis in copper sulfate-induced emesis assay and the gastric mucosal lesions in ethanol-induced gastric mucosal lesions assay were not exacerbated or reversed by uvangoletin. In conclusion, the research preliminarily indicated that uvangoletin induced apoptosis of HL-60 cells in vitro and in vivo without adverse reactions of myelosuppression, leucopenia and gastrointestinal tract disturbances, and the pro-apoptotic mechanisms may be related to mitochondria-mediated apoptotic pathway.

Multiple myeloma (MM) is one of the most common causes of mortality from hematological malignancy in China. Recent studies have demonstrated that cancerous inhibitor of protein phosphatase 2A (CIP2A) may exhibit a role in promoting the growth of cancer; however, the function of CIP2A in MM remains unknown. In the present study, the expression and molecular mechanism underlying the effects of CIP2A in patients with MM and in MM cell lines were elucidated. Firstly, the expression of CIP2A was detected in patients with MM and in MM cell lines by reverse transcription‑quantitative polymerase chain reaction. Furthermore, silencing of CIP2A with short hairpin RNA was performed in MM cells, and the impact on the proliferation and apoptosis of RPMI‑8226 cells was analyzed (as endogenous CIP2A is highly expressed in RPMI‑8226 cell lines compared with other cells). CIP2A was significantly elevated in patients with MM and in MM cell lines, and silencing of CIP2A could inhibit the proliferation ability of RPMI‑8226 cells in vitro. In addition, CIP2A knockdown induced apoptosis and led to substantial reduction of c‑Myc protein levels in MM cell lines. This study suggested that CIP2A inhibition may provide a promising therapeutic strategy for patients with MM.

Objective To investigate the safety and the efficacy of Umibilical cord mesenchymal stem cells(UCMSC) on severe aplastic anemia(SAA). Methods 30 cases who diagnosed SAA in our department from October 2013to January 2016 and treated with UCMSC on the basis of conventional treatment,were enrolled in this study prospectively.The cultured UCMSC using Serum-free culture system in vitro were given to patients with AA, interval of 15 days, 3 times for a course.the conventional treatments include CsA,male hormone and Chinese herb.Observed the changes of Th / Ts before and after treatmen ,Clinical efficacy and UCMSC infusion adverse reactions. Compared the efficacy of UCMSC therapy with conventional therapy of 30 cases of SAA at the same time. Results:Among 30 cases using umbilical cord mesenchymal stem cell infusion with UCMSC each infusion volume 3.2 × 106 / kg ~ 4.2 × 106 / kg, the average of 2.9 × 106 / kg, the average influsinon three times , 8 cases acquired basic cure, 8 cases acquired remission , 6 cases acquired significant progress in 6 cases, 8 cases were ineffective,with an effective rate of 73.3%.Before UCMSC treatment Th / Ts 0.67 ± 0.41, after1.33 ± 0.45, there was significant difference (P Conclusions:In vitro serum-free culture system can be used to culture and expand clinical scale UCMSC.UCMSC infusion can improve the SAA patient's Th / Ts proportion of T lymphocyte subpopulation , can improve the efficacy of new SAA, also more secure, no serious adverse reactions. Disclosures No relevant conflicts of interest to declare.

Background: Acute myeloid leukemia (AML) is characterized by the clonal expansion of immature myeloid cells. AML is treated primarily with chemotherapy, but the 5-year survival rate has only marginally increased over the past few decades, highlighting the need for novel therapies to achieve higher cure rates with more acceptable toxicities. Bcl-2 family proteins, together with TP53, are the central regulators of apoptosis. Overexpression of Bcl-2 protein is associated with leukemia progression and chemoresistance. We have observed elevated expression of Bcl-2 in AML and recently demonstrated that Bcl-2 inhibition by ABT-199 effectively induced apoptosis in AML (Pan, et.al., Cancer Discovery, 2014). However, resistance to ABT-199 was observed in cells expressing high levels of Mcl-1 or Bcl-xL. Moreover, a recent study showed heterogeneous but overlapping expression of Bcl-2, Mcl-1, and Bcl-xL proteins in 577 AML patient samples (Bogenberger, et. al., Leukemia, 2014). Although common in solid tumors, p53 mutations are relatively rare in AML. However, p53 functions are diminished by overexpression of MDM2 protein, an E3 ubiquitin ligase of p53 and an inhibitor of p53 transactivation. We previously reported MDM2 overexpression in 53% of primary AMLs (Kojima et al., Blood, 2005). Our group also demonstrated that p53 activation by Nutlins, the prototypical MDM2 inhibitors, induced apoptosis and growth inhibition in AML. Rationale: Since p53 activation by MDM2 inhibitors upregulates pro-apoptotic Bcl-2 proteins like NOXA, PUMA, and Bax, which counteract Mcl-1 and Bcl-xL, we hypothesized that the second-generation MDM2 inhibitor RG7388 could overcome AML resistance to Bcl-2-specific ABT-199, and that the combination could synergistically enhance apoptosis in AML. Results: We first demonstrated that RG7388 induced apoptosis exclusively in p53 wild type (wt) cells. RG7388 was essentially ineffective in p53 mutant or null AML cell lines such as HL-60, KG1 and THP1 (48h IC50s > 5 μM). Nonetheless, it showed high potency against p53 wt cell lines (48h IC50s: MOLM13 = 21.7 nM, MV-4-11 = 29.2 nM). Furthermore, stable knockdown of TP53 rendered the wt cell lines completely resistant to RG7388 (IC50s > 5 μM), confirming TP53-specificity. To study if RG7388 was able to overcome inherent resistance to ABT-199, we tested its efficacy on OCI-AML3 cells, which are inherently resistant to ABT-199, AraC and Idarubicin. As a single agent, RG7388 potently killed OCI-AML3 cells (48h IC50 = 148 nM). Importantly, RG7388 was ~20-fold more effective in OCI-AML3 cells than its predecessors Nutlin-3a and RG7112. We also examined the time- and dose-response of RG7388 in several genetically diverse AML cell lines (p53 wt) and found that 100 nM RG7388 was able to induce apoptosis and inhibit cell growth within 12 h. Next we studied whether RG7388 synergizes with ABT-199 to kill the refractory OCI-AML3 cells. A combination index of 0.35 (Chou-Talalay method) indicated a strong synergy between the two compounds. The combination exhibited higher activity in killing OCI-AML3 cells than either agent alone (48h IC50s: ABT-199 = 1680 nM, RG7388 = 148 nM, ABT+RG = 28 nM). Similar synergy was observed in additional AML cell lines and in primary samples. Next, we generated ABT-199 resistant cells by continuous exposure of initially sensitive AML cells to escalating concentrations of ABT-199. While 1000 nM ABT-199 had no effects on the viability of these cells, additional treatment with 30 nM RG7388 effectively killed them. This finding suggested that RG7388 was able to overcome acquired resistance to ABT-199. The mechanisms underlying this resensitization and its synergism with ABT-199 are under investigation using in vitro and in vivo model systems. Conclusions: The novel MDM2 inhibitor RG7388 induces growth arrest and apoptosis selectively in p53 wt AML cells. Importantly, the combination of RG7388 with ABT-199 synergistically induced apoptosis in AML cell lines and primary patient cells, and RG7388 was able to overcome inherent or acquired resistance to ABT-199. Since both Bcl-2 and MDM2 overexpression are associated with poor prognosis in AML, the proposed combination of the two clinical-stage compounds could have considerable clinical potential. We will report on ongoing experiments with primary AML cells in NSG mice to determine the potential of this combinatorial approach to eliminate AML stem cells. Disclosures Nichols: Roche: Employment, Equity Ownership. Leverson:abbvie: Employment, Equity Ownership. Dangl:Roche: Employment, Patents & Royalties. Konopleva:Abbvie: Research Funding. Andreeff:Roche: Research Funding; Abbvie: Research Funding.

Objective To evaluate the clinical efficacy and safety of allogenic dendritic cells (DCs) and cytokine-induced killer (CIK) cells in the eliminating minimal residual leukemia (MRL). Methods 48 acute leukemia patients with hematological complete remission (CR) but without molecular biological remission (CRM), or patients with minimal residual Leukemia (MRL) were selected from Ping’an Hospital of Shijiazhuang during Jan. 2009 to Jun. 2011. According to the patients’ will, 48 patients divided into combined treatment group and chemotherapy group 24 each. All the patients were in the same general information and disease level. The combined treatment group was treated with DC-CIK and consolidation chemotherapy, and the chemotherapy group was treated with consolidation chemotherapy. PBMCs were collected from healthy donors (the patient's parents or children) to prepare DC-CIK cells. DC-CIK cells were intravenous injected into patients once every 15 days, a total of 4-6 times infusion. The blood routine, bone marrow cells, leukemia related genes, urine and stool routine, liver and kidney biochemistry function, and ECG were observed. Changes of peripheral lymphocyte subsets in patients were detected by flow cytometry. Adverse reactions were examined. Results (1)Eleven cases in the combined treatment group achieved CRM, and the CRM rate was 45.8%; whereas only 2 cases in the chemotherapy group achieved CRM and the CRM rate was 8.3%,the difference was statistically significant(χ2=8.55, P Conclusion DC-CIK combined with chemotherapy can inhibit leukemia gene, promote the negative conversion rate of CFIM, facilitate the clear of MRL, improve immune function and prolong remission of the patients.No serious adverse reactions were found in patients with DC-CIK infusion. Disclosures: No relevant conflicts of interest to declare.

Abstract 4923 Objective To explore the safety and clinic therapeutic effects of using tumor activated dendritic cells (DC) accompanied cytokine induced killer (CIK) in order to feinforce opposing anti-tumor function and to eliminate minimal residual disease. Methods 59 patients entered into DCIK plus chemotherapy consolidation teams as achieved the 1st complete remission and after 1 or 2 cycle themotherapy. Collect 56 case's bone marrow monocyte cells at diagnosising to prepare tumor antigen, Collect bone marrow monocyte cells at complete remission to culture DC and CIK, using tumor antigen to activate DCs. After 7 days culturing separately then co-culture together for 1 day, collect these cells as DCs activated CIK, after scrubbing, infusing back to patients. Then these patients accept 10–15 day's immuno-regulation treatment, such as interleukine-2(IL-2) 500000u/d for one week or interferon -α(IFN-α)3,000,000u/d three times one week or thymopentin 1mg/d,2 times every week for two weeks. Those cases (one case AML-M3a,one case ALL-B,one case AML-M4a)only have routine culturing and infusion of DCs and CIK and the DCs are not activated by tumor antigen because of having no conservation leukemic cells. Results 59 cases accept DCIK immuno-therapy for 6 to 16 times respectively, in the 24 to 78 months observation stage, 42 cases OS(overall survival)and 33 cases DFS(disease free survival). No one developed therapeutic-dependent auto-allergic disease, such as fever, swollen, diarrhea, rash et al. Those three cases who accepted routine culturing and infusion of CIK and DCs that had not activated by tumor antigen didn't occure any treatment-dependency side-effects and keep hematologic complete remission, minimal residual disease test outcome persist lower under 103. Conclusion Tumor antigen activated DCs combined with CIK can reinforce anti-tumor immune function and have no obviously side-effects,accompanying with chemotherapy,those patients maybe benefit of this kind consolidation treatment and avoid routine chemo-therapeutic side reaction, temporary curative effect are satisfaction and worth popularized. Disclosures: No relevant conflicts of interest to declare.

Abstract 3226 Objective To evaluate mesenchymal stem cells(MSCs) combination with routine therapy to treat refractory aplastic anemia and bone marrow fibrosis, observe the course of the hematopoitic reconstitution and evaluate the security and curative effect. Methods 12 primary myelofibrosis and 19 chronic aplastic anemia patients who had all accepted routine therapy more than 6 months,because of pathogenetic condition, they needed infusing constituent blood such as at least condensed red blood cells 6 units and platelet 4 units per months, and despite this, whole blood cells counts were very lower. Those patients all had intermittent dizzy, fatigue, bad appetite and purpura. Because of age, complication, and psychological reasons, they refused Allogenic stem cell transplantation. So besides routine therapy, they received umbilical MSCs 1×107, every two weeks one time, those cells were injected directly into medullary cavity of ilium. Results 28 recipients showed hematopoietic reconstitution when PLT was up to 20×109/L and granulocytes was up to 2.0×109/L on day 28 post MSCs infusion. Bone marrow biopsy showed hyperplasia from hypoplasia to evident hyperplasia. On day 28, Th cells ascended from 23.0 to 36.2, Ts cells descended from 24.2 to 14.6, Th.. MTs from 0.95 to 2.47:1, IgG from 6.6 to 13.3, NK cells from 2.40 to 13.5, indicating earlier immune recorrect. At the last follow-up of 12 months, 17 patients showed normal hemoglobin and platelet counts, immune globulin was in the normal level, indicating the hematopoietisis and immune persistent reconstitution. 5 patients still needed infusing constituent blood cells, although, the interval of platelet and red blood cells infusion obviously prolonged, and the times to see doctor decreased and life quality elevated greatly. Conclusion This is a novel clinical research of successful treatment of refractory CAA and MF, it tell us infusion HLA-mismatched allogenic MSCs directly into medullary cavity of ilium is efficiency and security. Disclosures: No relevant conflicts of interest to declare.

Purpose: To explore the efficacy and toxicity of adoptive immunotherapy with donor lymphocyte infusion(DLI) after allogeneic nonmyeloablative stem cell transplantation (allo-NST). Methods: We retrospectively sum up the clinical data of 28 patients who had been performed with allo-NST among 41 patients and had been treated with DLI for 72 times between October 1999 and October 2004. Among these patients, there are 20 chronic myeloid leukemia (CML), 16 acute leukemia (AL), 2 multiple myeloma (MM) and 3 solid tumor. We used two different preparative regimens with or without Fludarabin. Cyclosporin A and MMF were used in the prevention of graft versus host disease (GVHD). The median dose of DLI was positive CD3 cells 2.7×107/kg (1.0×107/kg–2.0×108/kg). According to the patients’ situation and chimerism, we divided these patients into two groups, one group included 15 patients whose chimerism decreased for the reason of disease relapsed or advanced, the other included 13 patients whose chimerism decreased because of disease remission or stable. We analysis the change of chimerism and patients’ situation after DLI and the side effects of DLI. Results: Among 28 patients, there are 16 CML, 6 acute myeloid leukemia (AML), 3 acute lymphocytic leukemia (ALL), 1 MM and 2 solid tumor. 13 of them were with increased donor chimerism and 11 of them were with full chimerism. In 11 of the 16 CML patients, whose chimerism decreased because of relapsed or advanced, only one patient who achieved partial remission (PR) and other 10 died for the sake of no effect after DLI. With DLI, 5 patients whose chimerism decreased during the course of disease remission got increasing donor chimerism, 3 got complete remission (CR), 2 got PR, 4 survived and 1 PR patient died of GVHD. Among 6 AML patients with DLI, 1 died due to disease relapsed or advanced and decreased chimerism, other 5 who were with disease remission or stable situation and decreased chimerism obtained CR and survival according to the increased donor chimerism after DLI. Among 3 B-ALL patients post DLI, one alive and the other two died. One MM patient died of no effect. Two late phrase lung cancer patients got PR. One has survived for 14 months and another died of lung virus infection. Their rates of donor chimerism were higher than 80 percent and 96 percent. Among all these 28 patients, their average following up time was 30 months (10–66 months). 12 of them survived (42.6 percent) and total efficient rate was 57.14 percent. 3 developed grade II aGVHD that was DLI-associated. 1 died and 5 was complicated with grade II to IV cGVHD (3 extensive and 2 local). Given active treatment, they were alive. 2 patients had ever been with transient complete blood cell decreasing and had recovered after active surppoting treatment. Conclusion: DLI is a potential therapy that can make chimerism increase and prevent disease relapse post allo-NST. Its toxicity and side effects can be accepted.

Objective In this article, we explore the feasibility of dendritic cells (DCs) induced from chronic myeloid leukemia (CML) with different cytokine cocktails and the mechanisms of immunological responses by specific T lymphocytes primed by this kind of DC vaccine. Methods Monocytes (MNCs) for the generation of DC were collected from patients with high numbers of circulating peripheral blood CML cells, then cultured with different cytokine cocktails in different cultural medium (Fetal calf serum or Human serum albumin medium) to induce DCs. DCs were studied for morphology and immunofluorescent staining. RT-PCR was used to analyze the specific fusion genes and chromosome banding technique were used to detect Philadelphia chromosome (ph’). In vitro, DCs were used to induce auto-lymphocytes to be activated CTL, killing rates and T subgroups were assayed, IL-12 contents in the supernents were assayed also. Results After induction, all these CML-derived MNCs developed typical DC morphology and the DC-associated surface molecules upregulated. The contents of IL-12 in the co-culture system of CML-DC and lymphocytes increased and the activated lymphocyte skewed from Th1 to Th2. At and effector: target ratio of 20:1, 80.67±6.43%, 62.33±12.01%, 23.92±5.62% cytotoxicity was noted with CML-DC/AL against autologous leukemia cells, K562, HL60, exhibiting more killing activities to auto-CML cells than K562 and HL60 cell. Conclusion The results of this research will supply new approach for specific immunotherapy of CML and supply the application value for the CML derived DC vaccine against CML.