trichostatin-a and bryostatin-1

Natural killer (NK) cells are potent effectors of innate antitumor defense and are currently exploited for immune-based therapy of human leukemia. However, malignant blood cells in acute myeloid leukemia (AML) display low levels of ligands for the activating immunoreceptor NKG2D and can thus evade NK immunosurveillance. We examined the possibility of up-regulating NKG2D-specific UL16-binding protein (ULBP) ligands using anti-neoplastic compounds with myeloid differentiation potential. Combinations of 5-aza-2'-deoxycytidine, trichostatin A, vitamin D3, bryostatin-1, and all-trans-retinoic acid, used together with myeloid growth factors and interferon-gamma, increased cell surface ULBP expression up to 10-fold in the AML cell line HL60 and in primary AML blasts. Up-regulation of ULBP ligands was associated with induction of myelomonocytic differentiation of AML cells. Higher ULBP expression increased NKG2D-dependent sensitivity of HL60 cells to NK-mediated killing. These findings identify NKG2D ligands as targets of leukemia differentiation therapy and suggest a clinical benefit in combining a pharmacological approach with NK cell-based immunotherapy in AML.

Topics: Acute Disease; Antineoplastic Combined Chemotherapy Protocols; Azacitidine; Bryostatins; Cell Differentiation; Cell Line, Tumor; Cholecalciferol; Cytotoxicity, Immunologic; Decitabine; Flow Cytometry; GPI-Linked Proteins; Humans; Hydroxamic Acids; Intercellular Signaling Peptides and Proteins; Killer Cells, Natural; Leukemia, Myeloid; Reverse Transcriptase Polymerase Chain Reaction; Tretinoin; Up-Regulation

Primary acute myeloid leukaemia (AML) blasts can be induced to differentiate into dendritic-like leukaemia cells (DLLC) by culture with certain cytokine combinations. DLLC offer potential for use as autologous vaccines based on their ability to present putative leukaemia-specific antigens to T cells. It has been reported, however, that in around 30-50% of AML cases the leukaemia cells are not capable of undergoing DLLC differentiation. The purpose of this study was to identify the features that represent successful DLLC differentiation and, for those cases shown to be resistant to cytokine-induced differentiation, to use differentiating agents in an attempt to overcome the differentiation block. Leukaemia cells derived from 42 patients with AML were cultured in vitro with cytokines GM-CSF, IL-4 and TNFalpha/CD40L. In 22 cases the leukaemic cells underwent DLLC differentiation based on characteristic morphological changes and expression of costimulatory and dendritic cell-associated molecules. Four cases were not evaluable because of poor viability over the culture period. The remaining 16 cases failed to show evidence of DLLC differentiation. Many of these differentiation resistant cases were associated with poor risk karyotypic features. Nine of the resistant cases were selected for further study. Differentiating agents trichostatin (TSA), azacytidine (AZA) and bryostatin (BRYO) were used in combination with cytokines for the first 96 h of the culture period. Bryostatin (BRYO) alone was shown to be capable of overcoming differentiation resistance and allowing DLLC differentiation to proceed.

Topics: Acute Disease; Adolescent; Adult; Aged; Aged, 80 and over; Antigens, Neoplasm; Azacitidine; Bryostatins; CD40 Ligand; Cell Differentiation; Child; Cytokines; Dendritic Cells; Drug Resistance; Drug Synergism; Female; Granulocyte-Macrophage Colony-Stimulating Factor; HLA-D Antigens; Humans; Hydroxamic Acids; Immunophenotyping; In Situ Hybridization, Fluorescence; Interleukin-12; Interleukin-4; Karyotyping; Lactones; Leukemia, Myeloid; Lymphocyte Culture Test, Mixed; Macrolides; Male; Middle Aged; Neoplasm Proteins; Neoplastic Stem Cells; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha