Overexpression of Rasgrf1, a Guanine Nucleotide Exchange Factor in Chronic Lymphocytic Leukemia.

Abstract 1253

Poster Board I-275

We performed microarray expression analysis to identify genes which are differentially expressed in chronic lymphocytic leukemia (CLL) cells. A number of genes from the cohort of maximally upregulated genes were members of the Ras superfamily of proteins function which function as switches between active GTP-complexed and inactive GDP-complexed states. One of the genes upregulated on the microarray analysis in all the CLL cells as compared to normal B cells was RASGRF1. This ras protein specific guanine nucleotide release factor stimulates the dissociation of GDP and activates Ras signaling pathway. To confirm our microarray analysis results, we performed real time PCR analysis on 35 CLL specimens and observed a 10-50 fold upregulation of rasgrf1 RNA as compared to human peripheral blood B cells. This high expression is consistently observed in all CLL specimens regardless of stage indicating that this upregulation is an early important event in the pathogenesis of CLL. Rasgrf1 protein expression was also higher in CLL cells as compared to normal B cells by western blot analysis. Interestingly the rasgrf1 gene is genomically imprinted during development in certain tissues and the expression is downregulated. Demethylation of normal peripheral blood B cells by treatment with azacytidine resulted in increased expression of rasgrf1 with no change in expression of rasgrf1 in CLL cells implying imprinting in normal B cells. Thus malignant transformation of B cells to CLL cells results in a loss of genomic imprinting and an aberrant upregulation of rasgrf1 in CLL cells. One important downstream effect of rasgrf1 upregulation is ERK activation via Ras-MAPK pathway. B cell receptor signaling in CLL results in activation of a number of pro-survival pathways including ERK which is more active in poor prognosis, unmutated immunoglobulin heavy chain CLL. Our results indicate that ERK phosphorylation is also increased in CLL specimens with rasgrf1 overexpression.