Transfer of Cellular Content from the Allogeneic Cell-Based Cancer Vaccine DCP-001 to Host Dendritic Cells Hinges on Phosphatidylserine and Is Enhanced by CD47 Blockade

DCP-001 is a cell-based cancer vaccine generated by differentiation and maturation of cells from the human DCOne myeloid leukemic cell line. This results in a vaccine comprising a broad array of endogenous tumor antigens combined with a mature dendritic cell (mDC) costimulatory profile, functioning as a local inflammatory adjuvant when injected into an allogeneic recipient. Intradermal DCP-001 vaccination has been shown to be safe and feasible as a post-remission therapy in acute myeloid leukemia. In the current study, the mode of action of DCP-001 was further characterized by static and dynamic analysis of the interaction between labelled DCP-001 and host antigen-presenting cells (APCs). Direct cell–cell interactions and uptake of DCP-001 cellular content by APCs were shown to depend on DCP-001 cell surface expression of calreticulin and phosphatidylserine, while blockade of CD47 enhanced the process. Injection of DCP-001 in an ex vivo human skin model led to its uptake by activated skin-emigrating DCs. These data suggest that, following intradermal DCP-001 vaccination, local and recruited host APCs capture tumor-associated antigens from the vaccine, become activated and migrate to the draining lymph nodes to subsequently (re)activate tumor-reactive T-cells. The improved uptake of DCP-001 by blocking CD47 rationalizes the possible combination of DCP-001 vaccination with CD47 blocking therapies.

Synergistic Depression of Wnt Pathway by Sequential Combination of Decitabine and Idarubicin in Human Myeloid Leukemic Cell Lines,

Abstract 3628 PURPOSE: In the preliminary experiment, we investigated the effect of anti-leukemia drugs (idarubicin, daunorubicin cytarabine, thalidomide, homoharringtonine) in combination with decitabine of inhibiting growth of myeloid leukemic cell lines. The result showed notable synergistic effect of sequential combination of decitabine and idarubicin. In searching the associated mechanism, we assessed the effect of sequential combination of decitabine and idarubicin in apoptosis, cell cycle arrest, and depression of wnt pathway. Methods: Myeloid leukemia cells (U937, HEL and SKM-1) were treated with decitabine alone, idarubicin alone, and decitabine sequential with idarubicin to determine their impact on cellular proliferation, cell cycle regulation, apoptosis and the regulation of wnt pathway by detecting DNA methylation status, mRNA level and protein expression. Results: The sequential combination of decitabine and idarubicin produced synergistic inhibition of myeloid leukemia cells growth by apoptosis and cell cycle arrest. Sequential treatment with both agents induced synergistic effect on decreasing methylation status of wnt antagonists (SFRP1, HDPR1 and DKK3) which result in re-expression or up-expression of these genes. Furthermore, we found that the down-steam genes of wnt pathway including c-myc and β-catenin were down-regulated. Conclusion: Taken together, these findings showed that decitabine sequentially combined with idarubicin has synergistic anti-leukemia effect, which suggest clinical potential in the treatment of myeloid leukemias. Disclosures: No relevant conflicts of interest to declare.

High Expression of Decoy Receptor TRAIL-R3 on AML Blasts Is Associated to Shortened Overall Survival. TRAIL-R3 Induced Apoptosis Resistance Can Be Overcome by Specifically Targeting TRAIL-R2 in Vitro

In vivo, effector T cells and NK cells do express membrane-bound TRAIL (TNF-related apoptosis-inducing ligand). T cells also secrete a soluble form of TRAIL (sTRAIL). Binding of TRAIL to one of the receptors on target cells can induce apoptosis. In human, 4 membranebound receptors for TRAIL have been identified: two of them (TRAIL-R1 and TRAIL-R2) contain a functional death domain, while the two other (TRAIL-R3 and TRAIL-R4) lack a functional death domain and function as decoy receptors. Most normal cells express TRAIL-R3 and TRAIL-R4, where many tumor cells express TRAIL-R1 and TRAIL-R2, making activation of the TRAIL mediated apoptotic pathway a possible new model for targeted therapy. We investigated bone-marrow samples from 119 patients with de novo AML and from 11 healthy donors for TRAIL receptor expression on CD45dim/SSClow blasts by flow-cytometry. Expression profiles of patients were correlated to survival data. Functional data were obtained by incubation of 6 myeloid leukemic cell lines and fresh AML samples with different concentrations of sTRAIL/Apo2L (n=10) and specific antibodies directly targeting R1 and R2 (HGS-ETR1 and HGS-ETR2) (n=18). Apoptosis was assessed by flow–cytometry after staining with 7AAD and annexinV. R3 expression on the cell surface of one cell line (MM6) was down-modulated with PI-PLC (Phosphatidyl-inositol phospholipase C ).When comparing AML and normal blasts, significantly more pro-apoptotic TRAIL-R1 and TRAIL-R2 receptor expression on myeloid leukemic blasts (p<0.0005) was measured however without association with outcome. About 30% of AML patients had a high anti-apoptotic TRAIL-R3 expression, which was strongly correlated to a shortened overall survival (log rank, p=0.0051). In multivariate analysis, R3 expression remained a significant prognostic factor next to cytogenetics (p=0.03 and p= 0.015 respectively). In vitro, studies on 6 myeloid leukemic cell lines confirmed sTRAIL sensitivity in cell lines that expressed presumably TRAIL-R1 and TRAIL-R2. Moreover, down-modulation of TRAIL-R3 by treatment with PI-PLC (TRAIL-R3 is the only GPI-linked TRAIL receptor) on cell lines improved sTRAIL sensitivity. Fresh myeloid leukemic samples were treated with sTRAIL. Mean apoptosis induction was 14% (0–54%). sTRAIL can bind to both the pro-apoptotic as the decoy receptors. Bypassing of the decoy R3 expression by treatment with the antibody directly targeting TRAIL-R2 (anti-TRAIL-R2) resulted in much higher rates of apoptosis (mean 30%, 0–80%). Apoptosis induction via direct targeting of TRAIL-R2 was, as expected, independent of TRAIL-R3 expression. All leukemic blasts that showed apoptosis with anti-TRAIL-R2 expressed the R2 receptor, but not all blasts that expressed TRAIL-R2 were sensitive for anti-TRAIL-R2, which could be due to inhibitory factors downstream the apoptotic cascade. In conclusion, AML blasts do express pro-apoptotic TRAIL receptors. However, co-expression with the decoy receptor TRAIL-R3 results in significant shortened overall survival. AML blasts are sensitive to targeting the pro-apoptotic TRAIL-R2 receptor, yielding a new therapeutic option for AML patients.