5-Azacytidine and Decitabine Induce Demethylation and Re-Expression of FAS (CD95) and Promote Apoptosis in Neoplastic Cells in Acute Myeloid Leukemia (AML),

Abstract 3463

Epigenetic and apoptosis-regulating mechanisms have been implicated as critical factors contributing to the progression from myelodysplastic syndromes (MDS) to secondary acute myeloid leukemia (AML). However, the exact molecular mechanisms and genes involved in disease evolution have not been identified yet. We screened for epigenetically regulated pro-apoptotic effector molecules in neoplastic cells in patients with MDS (n=50) and AML (n=30). Among a series of potential regulators, we identified FAS (CD95) as an epigenetically regulated critical death regulator in neoplastic cells. As assessed by qPCR, bone marrow cells obtained from patients with low risk MDS were found to display high levels of FAS, whereas FAS mRNA levels were lower or undetectable in patients with advanced MDS (with excess of blasts) or secondary AML. Moreover, we were able to show by multicolor flow cytometry that CD34+/CD38+ progenitor cells and CD34+/CD38- stem cells in MDS and AML display measurable FAS (CD95) on their surface, with slightly higher levels detectable in progenitor cells in low risk MDS compared to high risk MDS and secondary AML. Methylation-specific PCR and qPCR revealed that the FAS-promoter is hypermethylated in primary AML cells as well as in various AML cell lines including KG1 and HL60 thus repressing mRNA-synthesis. In addition, we found that exposure to 5-Azacytidine or Decitabine leads to demethylation of CpG-rich regions closest to the transcription starting sites, and thus to re-expression of FAS in AML cells. In vitro-targeting of AML cells by demethylating drugs was also found to revert epigenetic inactivation of other tumor suppressor genes such as CDKN2B (P15), CDKN2A (P16), ESR1 (estrogen-receptor alpha), with subsequent normalization of mRNA expression levels. Next, we asked whether CD95 acts as a critical death regulator involved in drug-induced apoptosis in neoplastic cells. In these experiments, both demethylating agents, 5-Azacytidine or Decitabine, were found to induce dose-dependent apoptosis and growth inhibition in primary AML cells, primary MDS cells, and in all AML cell lines examined. Drug-induced apoptosis in AML cells was accompanied by activation of caspase 8 and caspase 3 as well as increased expression of proapoptotic FAS/CD95 as determined by qPCR, Western blotting, and flow cytometry. Moreover, both drugs were found to induce expression of the FAS-ligand and DAPK1 in neoplastic cells. We then applied a siRNA against FAS. The siRNA-induced knock-down of FAS was found to block drug-induced FAS expression and FAS-induced apoptosis in KG1 cells and HL60 cells. In conclusion, our data show that FAS is hypermethylated in neoplastic cells in patients with advanced MDS and AML, that demethylating agents lead to re-expression of FAS, and that drug-induced FAS expression mediates apoptosis in leukemic cells. As FAS is also expressed on neoplastic stem cells, these observations may have clinical implications and may explain beneficial effects seen with 5-Azacytidine or Decitabine in patients with advanced MDS.