Characterization of N-RAS promoter mutation in leukemia

Many hematological malignancies are characterized by molecular abnormalities which impact upon RAS protein-mediated signal transduction pathways. In particular, mutations of N-RAS at codon 12, 13 or 61 are common in AML. Since overexpression of the normal N-RAS gene can also transform cells in vitro, we postulated the existence of mutations in the promoter region which had the potential to deregulate N-RAS expression, thus representing an alternative mechanism of N-RAS activation in leukemia. Single-stranded conformational polymorphism, combined with DNA sequencing and/or restriction mapping, was used to screen the N-RAS promoter in cells from 26 AML, 19 CML, 1 CLL and 13 non-leukemic samples. One AML patient had an A-to-G mutation at position 409 which abolished protein binding in the region of a c-MYB binding site. The mutation persisted during remission and relapse, implicating it as an early event in leukemogenesis. A T-to-A mutation at position 520 in a second AML patient altered DNA-protein interactions in the region of the AP-1 and c-MYC binding sites. Another T-to-A mutation lying within a protein binding site at position 378 was found in a patient with CLL. In addition, we have identified a novel CA polymorphism at position 390; loss of heterozygosity studies utilising this polymorphism have proved helpful in studying clonal evolution in a patient with CML. In conclusion, the N-RAS promoter appears to be a frequent site of mutagenic activity in human leukemia, and these mutations extend the spectrum of abnormalities which have the potential to disrupt RAS-mediated signal transduction in hematological malignancies.