Promoter Methylation of RASSFIA, SHP1 and SOCS1 Genes in Acute Myeloid Leukaemia (AML) and Myelodysplastic Syndromes (MDS).

Mutations in the receptor tyrosine kinase (RTK)/RAS signalling pathway frequently provide a proliferative signal in AML, especially those with inv(16) and t(15;17). We have reported, for example, that approximately 70% of AML and inv(16) have mutations in FLT3, c-KIT and RAS. However, the role of RASSFIA, SHP1 and SOCS1, negative regulators of RTK/RAS signalling, has not been extensively investigated in AML. RASSFIA is a tumour suppressor gene that serves as a RAS effector that inhibits cell cycle progression and mediates apoptotic cell death, while SHP1 and SOCS1 are negative regulators of the Jak/STAT pathway. In many cancers, the CpG islands of RASSFIA, SHP1 and SOCS1 are aberrantly hypermethylated, resulting in gene inactivation due to repression of transcription. In this study, methylation-specific polymerase chain reaction (MS-PCR) was employed to determine if aberrant promoter methylation of these negative regulators are involved in the pathogenesis of AML and MDS. Patients with MDS (n=107), AML (n=154), JMML (n=5) were studied, together with 15 normal controls. Primers were located in the promoter region of each gene as well as within exon 2 of SOCS1 as previously reported. Methylation of RASSFIA was found in five of 55 (11%) MDS patients, but in none of 57 AML cases. RASSFIA methylation was present in one case (20%) of JMML, which lacked a RAS, NF1 or PTPN11 mutation, a finding that strengthens the concept of mutual exclusivity of genetic and epigenetic changes in this disorder. SHP1 methylation was identified in 13 of 121 (11%) AML cases, including 3 AML with inv(16) and c-KIT 419 mutations. In contrast, none of the 65 MDS or 5 JMML cases showed SHP1 methylation. MS-PCR for SOCS1 promoter and exon 2 gave discrepant results. Promoter analysis was negative in the 15 controls whereas 2 controls showed exon 2 positivity. SOCS1 promoter methylation was seen in eight of 74 (11%) MDS patients, but not in the five JMML cases. Methylation of RASSFIA and SOCS1 promoters in MDS were mutually exclusive. No case of AML showed SOCS1 promoter methylation whereas 19 of 47 (40%) cases exhibited exon 2 methylation. In conclusion, promoter methylation may lead to epigenetic silencing of RASSFIA and SOCS1 in MDS but not in AML, whereas SHP1 gene silencing may be important in the pathogenesis of AML. SHP1 methylation studies suggest that some patients with inv(16) AML and RTK mutations possess additional epigenetic events. Finally, SOCS1 exon 2 methylation does not appear to be pathogenetically important, since methylation may occur in normal samples and results do not correlate with promoter methylation.