5-Azacytidine and Decitabine Induce FAS Re-Expression, Exert Major Proapoptotic Effects, and Cooperate with the FAS Ligand in Producing Apoptosis in Neoplastic Human Mast Cells,

Abstract 3457

Aggressive systemic mastocytosis (ASM) and mast cell leukemia (MCL) are advanced myeloid neoplasms with a poor prognosis. In these patients, neoplastic mast cells (MC) are resistant against most conventional drugs. Demethylating agents reportedly exert beneficial effects in several advanced myelogenous neoplasms, including myelodysplastic syndromes. We examined the effects of two demethylating agents, 5-Azacytidine and 5-Aza-2`Deoxycytidine (Decitabine) on growth and survival (apoptosis) of neoplastic MC and the human MC line HMC-1. Two HMC-1 subclones were used, HMC-1.1 lacking KIT D816V and HMC-1.2 exhibiting KIT D816V. Both demethylating agents were found to induce apoptosis and growth inhibition in HMC-1.1 cells and HMC-1.2 cells in a dose-dependent manner (IC₅₀: 5-Azacytidine: 5–10 μM, Decitabine: 1–5 μM). Interestingly, only Decitabine but not 5-Azacytidine induced a major G2/M cell cycle arrest in HMC-1 cells. Drug-induced apoptosis in HMC-1 cells was accompanied by cleavage and activation of Caspase-8 and Caspase-3 as well as an increased expression of proapoptotic FAS/CD95, whereas no major effects on expression of other surface antigens were seen. We also found that both demethylating agents synergize with the FAS-ligand in inducing apoptosis in neoplastic MC. Methylation-specific PCR and bisulfite genomic sequencing revealed that the FAS-promoter is hypermethylated in HMC-1 cells. In addition, qPCR demonstrated that exposure to 5-Azacytidine or Decitabine leads to re-expression of FAS in neoplastic MC, which was confirmed by flow cytometry. Correspondingly, a FAS-specific siRNA was found to block drug-induced expression of FAS and drug-induced apoptosis in HMC-1 cells. Although other key regulators and tumor suppressor molecules such as p16 were also found to be hypermethylated in HMC-1 cells, no major demethylating effects of 5-Azacytidine or Decitabine were seen. Neither 5-Azacytidine nor Decitabine induced substantial apoptosis or growth arrest in normal human cord blood progenitor-derived MC or in control bone marrow cells. Together, our data show that 5-Azacytidine and Decitabine exert growth-inhibitory and pro-apoptotic effects in neoplastic MC. These effects are mediated through FAS re-expression and are augmented by the FAS ligand. Whether epigenetic drugs produce anti-neoplastic effects in vivo in patients with advanced SM including MCL, remains to be determined in clinical trials.