Abstract 4713: Detrimental effects of a histone deacetylase inhibitor on human lymphocytes

Abstract

Abstract Background:Histone deacetylase inhibitors (HDACi) have been shown to increase cancer-testing and melanomasomal tumor antigen expression, which may allow their use as adjuvants to immunotherapy for melanoma. For this use, a key question is to test the effects of HDACi on lymphocytes compared to melanoma cells. Methods: We tested the effects on cell viability, cell cycle, apoptosis and DNA damage of the pan-HDACi LBH589 on peripheral blood mononuclear cells (PBMC) from a healthy donor (HD), four patients with metastatic melanoma (MM), two previously established human melanoma cell lines (M229 and M370), and two bone marrow samples of patients with multiple myeloma previously treated with GM-CSF. In addition, phospho-flow cytometry was used to study the effects of the HDACi on intracellular signalling in lymphocytes with or without pulsing with IL2 (400IU) or IFNα (10,000). For some experiments, HD PBMC were genetically modified to express the T cell receptor (TCR) for the melanoma antigen MART-1 using retroviral transduction for testing in in vitro cytotoxicity assays. Results. In replicate experiments the 50% inhibition concentration (IC50) of LBH589 for PBMC was low (< 20 nM) in comparison to the melanoma cells (> 600 nM). LBH589 induced > 20% (10 nM) and > 40% (1-10 mM) apoptotic cell death demonstrated by double presence of a sub-G0/G1 peak and cleaved poly (ADP-ribose) polymerase (PARP) in PBMC samples, while it was < 10% in melanoma cell lines at these same concentrations. In a DNA damage assay, there was around 2-fold increase in the phosphorylation of the histone variant H2A.X in HD PBMC at 1 nM, while it required 10 nM for the similar effect in the melanoma cell line M229. However, the phosphorylation of H2A.X in PBMCs of three patients with MM (1 nM LBH589) was slightly inhibited. The pH2A.X of the other cell line and one of the MM patients was a 1.5- fold increase. The maximal effects on signaling pathways were seen after 30 minutes of treatment. LBH589 slightly inhibited phosphorylation of STATs 1, 3, 5 and 6 and MAPK proteins (p38, ERK), p53, cyclin D3 and histone H3 in flow gated B and T cells from the HD. On the contrary, the same phosphoproteins were activated up to six times higher in the MM patient samples and in a bone marrow sample. Conclusions. The HDACi LBH589 induced cytotoxic effects at nanomolar concentrations on human lymphocytes and altered key signaling pathways involved in lymphocyte activation. These effects are at lower concentrations than the antitumor activity in melanoma in vitro, resulting in an adverse therapeutic window. Therefore, LBH589 should be used with caution if intended to sensitize melanoma to immunotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4713. doi:1538-7445.AM2012-4713