ALBERT

Description: Acute myeloid leukemia (AML) is a malignant heterogeneous disease characterized by rapid clonal growth of myeloid lineage blood cells. This year there will be an estimated 20,830 new AML cases and an estimated 10,400 deaths from this deadly disease in the United States. Overall survival rates remain low despite advances in treatment with overall survival rates of 25% for adults and 65% for children. Resistance to frontline chemotherapy remains a major cause of treatment failure, highlighting the need for new therapies. Overexpression of the anti-apoptotic Bcl-2 family members is associated with chemoresistance in leukemic cell line models and with poor clinical outcome. Anti-apoptotic Bcl-2 family members, such as Bcl-2, Bcl-xL, and Mcl-1, sequester pro-apoptotic BH3-only proteins, such as Bim, which activate pro-apoptotic proteins Bax and Bak causing mitochondrial outer membrane permeabilization resulting in cytochrome c release and apoptosis. Thus, inhibition of anti-apoptotic Bcl-2 family members represents a promising approach for the treatment of AML. We previously demonstrated preclinical efficacy of a pan-Bcl-2 inhibitor, GX15-070, in combination with cytarabine in AML cell lines and primary patient samples. Another promising inhibitor, ABT-263, has shown preclinical efficacy, but has been associated with thrombocytopenia due to inhibition of Bcl-xL, thus much attention has been focused on inhibition of Bcl-2. ABT-199, a Bcl-2 selective inhibitor, has demonstrated encouraging results in AML, acute lymphoblastic leukemia, chronic lymphocytic leukemia, mantle cell lymphoma, multiple myeloma, and breast cancer. We previously demonstrated that ABT-199 has a wide range of activity in AML cells (Niu X, et al. Leukemia. 2014; 28: 1557-1560.) However, it has limited efficacy in Bcl-xL and Mcl-1 dependent malignancies. Thus, intrinsic drug resistance remains a concern. Understanding the molecular mechanisms of resistance to ABT-199 will allow for rationally designed combination regimens to increase its antileukemic efficacy. In this study, we investigated the molecular mechanism underlying intrinsic resistance to ABT-199 in AML cells. Immunoprecipitation of Bim from ABT-199 treated cells demonstrated decreased association with Bcl-2, but increased association with Mcl-1, without corresponding change in mitochondrial outer membrane potential. ABT-199 treatment resulted in increased levels of Mcl-1 protein and unchanged or decreased Mcl-1 transcript levels. shRNA knockdown of Bim almost completely abolished ABT-199 treatment-induced increase of Mcl-1 protein levels, suggesting that the association with Bim plays an important role in stabilizing Mcl-1 protein. AML cells treated with ABT-199 in the presence of the protein translation inhibitor cycloheximide resulted in significantly longer Mcl-1 half-life and treatment with the proteasome inhibitor MG-132 resulted in increased Mcl-1 protein level and no further enhancement was detected when treated with combined MG-132 and ABT-199, suggesting that ABT-199 affects Mcl-1 protein stability. Combining conventional chemotherapeutic agent cytarabine or daunorubicin with ABT-199 resulted in increased DNA damage, decreased Mcl-1 protein levels, decreased association of Mcl-1 with Bim, and synergistic induction of cell death compared to ABT-199 alone, in both AML cell lines and primary patient samples obtained from AML patients at diagnosis independent of their sensitivities to ABT-199, thus providing evidence that screening for ABT-199 resistance is not necessary. Our results demonstrate that sequestration of Bim by Mcl-1 is a mechanism of intrinsic ABT-199 resistance, and this mechanism of resistance can be overcome by combining ABT-199 with daunorubicin or cytarabine in AML cells. Our findings, though in a limited number of primary patient samples, provide new insights into the mechanism of ABT-199 resistance in AML cells and support the clinical development of the combination of daunorubicin or cytarabine and ABT-199 in the treatment of AML. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 2302 Although allogeneic peripheral blood stem cell transplantation from a matched related donor (RD-PBSCT) presents the best curable opportunity for many patients with hematologic malignancies, only about one third of individuals have HLA matched sibling donors. Fortunately, from unrelated donor peripheral blood stem cell transplantation has been acceptable and expanded recently. In order to evaluate the effect of allogeneic peripheral blood stem cell transplantation from unrelated donor (URD-PBSCT), we compare results of URD-PBSCT and RD-PBSCT that were done for 172 consecutive adult patients with hematologic maligancies from Jan 2002 to Dec 2008 at a single-center. Among these patients, 62 cases underwent URD-PBSCT and 110 cases underwent RD-PBSCT. Myeloablative conditioning was adopted for all patients. In graft versus host disease (GVHD) prophylaxis, 49 URD-PBSCT recipients received CSA, MTX, MMF and ATG (URD-ATG group), 13 recipients were given simulect more in base of URD-ATG group (URD-ATG+CD25 group) while RD-PBSCT recipients (RD group) received CSA, MTX and MMF. Engraftment was achieved in 98.4% of URD-PBSCT patients and 98.2% of RD-PBSCT patients (100% for patients surviving beyond 28 days after transplant). The cumulative incidence of acute GVHD (grade II-IV) was 15.4%, 36.7% and 29.1% respectively in the URD-ATG+CD25, URD-ATG and RD groups (P = 0.275). The cumulative incidence of chronic GVHD was 0%, 45.6%, 39.6% respectively and significant lower in URD-ATG+CD25 group than the other two groups (P = 0.0134). Relapse incidence was 53.8%, 12.2%, 14.5% respectively and significant higher in URD-ATG+CD25 group than the other s (P = 0.0059), while there was no different between the URD-ATG and RD groups (P = 0.610). Estimated overall survival (OS) at 5 years was 30.8%, 69.4% and 67.3% respectively and no significant difference between URD-ATG group and RD group (p=0.898). Adverse prognosis factors for relapse and OS included transplant not in remission and GVHD prophylaxis with simulect. Our results indicate PBSCT from unrelated donor may be considered comparable to those from related donor. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4582 Objectives Bone marrow mesenchymal stem cells (BMSCs) have multilineage differentiation potential and highly proliferative potential in vitro. Cell therapy with BMSCs has great promise in regenerating the damaged tissues, restoring their function and treating ischemic related failures, but the chief limitation may be the poor viability of BMSCs transplanted into the pre-existing ischemic environment, where massive death of transplanted cells has been detected. The serum deprivation (SD) is a component of ischemia. Lidocaine can be used for relief of pain and has been one of the most widely used local anesthetics. Intravenously, it has been used successfully for emergency treatment of ventricular arrhythmias. It reportedly also showed anti- proliferative, anti-inflammatory and protective effects when administered before or after the ischemic insult. Which effects has it on BMSCs during the BMSCs transplanted in the patient with lidocaine treatment? The experimental objective is to investigate the effects of lidocaine on the proliferation of BMSCs cultured in high glucose plus serum deprivation DMEM. Methods BMSCs were separated from adult male SD rat bone marrow. The BMSCs were cultured in the high glucose (4.5g/L) DMEM containing 10% fetal bovine serum (FBS). All experiments were performed using BMSCs from the 3rd passage adherent cell fraction. The BMSCs were divided into 10 groups according to the concentrations of lidocaine and FBS: 0μg/ml, 2μg/ml, 20μg/ml, 200μg/ml, 2000μg/ml lidocaine, containing 10% FBS; 0μg/ml, 2μg/ml, 20μg/ml, 200μg/ml, 2000μg/ml lidocaine, containing 0% FBS. Cell proliferation of the BMSCs was assessed by MTT assay at the time points of 24h, 48h and 72h respectively. Results As the MTT colorimetric assay revealed, lidocaine with 2μg/ml, 20μg/ml, 200μg/ml, 2000μg/ml all inhibited the proliferation of the BMSCs at under the condition of 10% FBS and under the condition of 0% FBS. The cells cultured in 0μg/ml lidocaine, 10% FBS displayed a significant proliferation compared to those cultured in 0μg/ml lidocaine,0% FBS (serum deprivation) (P