5-azacytidine and decitabine exert proapoptotic effects on neoplastic mast cells: role of FAS-demethylation and FAS re-expression, and synergism with FAS-ligand

Abstract

IntroductionSystemic mastocytosis (SM) is a myeloid neoplasm characterized by pathologic accumulation of mast cells (MCs) in one or more extracutaneous organs. 1,2 Indolent and aggressive variants of SM have been described. [1][2][3][4][5] In most patients, the transforming KIT mutation D816V is detectable independent of the category of SM. [6][7][8] Patients with indolent SM (ISM) have an excellent prognosis with normal or near-normal life expectancy. [1][2][3][4][5]9,10 In patients with aggressive SM (ASM) and mast cell leukemia (MCL), the prognosis is grave, [1][2][3][4][5]9,10 and responses to conventional drugs and most targeted drugs are poor. [1][2][3][4]9,[11][12][13][14][15] During the past few years, several attempts have been made to define new molecular targets in neoplastic MCs and to establish new treatment concepts for these patients. 16,17 Abnormal DNA methylation and histone-acetylation are frequently observed in various neoplasms and supposedly contribute to disease evolution and drug resistance. [18][19][20] In hematologic malignancies, epigenetic abnormalities have been reported in acute and chronic leukemias as well as in myelodysplastic syndromes (MDS). [21][22][23] Especially in patients with MDS, abnormal methylation of the genome has been described. 21,22 Correspondingly, epigenetically active drugs such as 5-azacytidine and 5-aza2Јdeoxycytidine (decitabine) reportedly act as antineoplastic agents in these patients. [23][24][25] More recent data suggest that demethylating agents also exert beneficial effects in patients in whom neoplastic cells exhibit myeloproliferative and myelodysplastic features, such as chronic myelomonocytic leukemia (CMML), and sometimes even in patients with advanced myeloproliferative neoplasms (MPN). 26,27 Advanced SM is a myeloid neoplasm that often presents with myeloproliferative features and sometimes with bone marrow (BM) dysplasia. [1][2][3][4][5] In addition, advanced SM may coexist with MDS (SM-MDS), CMML, or a JAK2-mutated MPN. 28 However, so far, no data on the effects of demethylating agents on neoplastic cells in advanced SM or SM-AHNMD are available. In addition, little is known about methylation patterns in neoplastic MCs. We explored the effects of 2 widely used demethylating agents, 5-azacytidine and decitabine, on growth and survival of neoplastic MCs, and examined the mechanism(s) of action of these drugs. MethodsDrugs, monoclonal antibodies, and other reagents PKC412 (midostaurin) was kindly provided by Dr J. Roesel and Dr P. W. Manley (Novartis). 5-azacytidine, decitabine, recombinant human FASligand, and propidium iodide (PI) were purchased from Sigma-Aldrich, Submitted September 27, 2011; accepted February 29, 2012. Prepublished online as Blood First Edition paper, March 21, 2012; DOI 10.1182 DOI 10. /blood-2011 The online version of this article contains a data supplement.The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''advertis...

Fas expression at diagnosis as a biomarker of azacitidine activity in high-risk MDS and secondary AML

“…Indeed, azacitidine favors the re-expression of FAS in neoplastic mast cells or PI-PLCbeta1 genes in MDS or sAML. 7,12 Here we show that the in vivo use of the drug also favors the re-appearance of Fas receptor in sAML-derived CD45 lo /CD34 þ cells, together with FAS promoter demethylation (Supplementary Figure S1).…”

“…7,11 The mechanism by which FAS promoter is overmethylated in sAML is still unclear. However, the 5 0 -CpG1-8 are located immediately downstream of the docking site of Dnmt1, which promotes FAS promoter methylation in KRAS-transformed NIH3T3 cells.…”

“…4 FAS/TNFRSF6/APO1 that belongs to a set of genes involved in the control of apoptosis is hypermethylated in tumors. [5][6][7] We have previously shown that Fas is overexpressed in low/int-1-risk (LR)-MDS. 8 Conversely, decreased expression of pro-apoptotic Fas, or Fas-associated via death domain (FADD) or increased anti-apoptotic Bcl2 protein in int-2 or HR-MDS could participate in resistance to apoptosis and contribute to disease progression.…”

“…Variations of the methylation level at FAS promoter have already been described in one CpG island located over the transcription start site ( þ 1) and in a 5 0 -regulatory region located upstream the CpG island in the NIH3T3 cell line transformed by KRAS or in primary tumor cells. [5][6][7]9 Thus, we extensively studied DNA methylation of the promoter region spanning between À 899 and þ 231, which contains 35 CpG dinucleotides, of which 26 belong to a CpG island. The FAS promoter was amplified from bisulfite-treated genomic DNA extracted from CD34 þ cells in 18 LR-MDS, 8 HR-MDS, 11 sAML and 8 controls (Supplementary Table S2 for primers).…”

Epigenetic Modifications: Therapeutic Potential in Cancer

“…It has been reported that 5-azacytidine and decitabine can induce apoptosis in the neoplastic mast cells through re-expression of Fas receptor (Ghoshal et al 2005). It has also been shown that 5-azacytidine treatment leads to apoptosis of both myeloid precursor PC-MDS cells and leukemia K562 cells, mainly through the induction of Bak mRNA expression (Ghanim et al 2012). Dihydro-5-azacytidine (DHAC) is hydrolytically more stable (Beisler et al 1977) and less cytotoxic (Presant et al 1981) than 5-Aza-CR, and has been proved to be an inhibitor of DNA methylation in human lymphoid and leukaemia cell lines, as well as in tumour-bearing mice (Antonsson et al 1987;Powell and Avramis 1988;Kees and Avramis 1995).…”