Rasgrf1 Overexpression Amplifies B Cell Receptor Signaling in Chronic Lymphocytic Leukemia Specimens

Abstract 926

The B-cell receptor (BCR) signaling is a hallmark of chronic lymphocytic leukemia cells and results in cell proliferation, survival and resistance to apoptosis. B cell receptor (BCR) signaling in CLL specimens activates the Ras/ERK/MAPK pathway and is critical for survival and growth of CLL cells. Rasgrf1 is a guanine exchange factor that accelerates the removal of inactive GDP from Ras so that active GTP can bind Ras that results in Ras activation. In an Affymetrix microarray expression analysis screen of CLL specimens (n=5) and peripheral blood normal B cells (n=2), Rasgrf1 was found to be over-expressed (ranked within top 100 overexpressed genes) in CLL. This finding was further confirmed by quantitative RT-PCR as a 5–300 fold overexpression in CLL specimens (n=20) was noted as compared to normal B cells. Western blot analysis data also showed over-expression of Rasgrf1 protein in CLL specimen lysates but interestingly the expressed protein is of a smaller size in CLL specimens (70-80 kDa) as compared to the Rasgrf1 in normal peripheral blood mononuclear cells (140 kDa) This truncated protein retains the important c-terminal GEF (guanine exchange factor) domain but lacks the regulatory domains and is known to function as a hyperactive GEF in other tumor model systems. Knockdown of Rasgrf1 in CLL specimens (n=4) resulted in inhibition of the BCR-Ras/ERK/MAPK pathway as determined by a decrease in ERK phosphorylation. Similar Rasgrf1 knockdown experiments with BCR crosslinking also showed identical results in additional CLL specimens. Transient SiRNA mediated Rasgrf1 knockdown also resulted in higher percentage of annexin positive or apoptotic CLL cells as compared to the scrambled SiRNA control transfection. Lysates from the Rasgrf1 knockdown cells showed an increase in PARP cleavage by western blot analysis as well indicating that continued expression of this protein was necessary for survival of the leukemic cells. To confirm that the Rasgrf1 knockdown resulted in a decrease in the GEF function, the active Ras (Ras-GTP) in the CLL specimens was evaluated by an ELISA assay. In two CLL specimens the assay confirmed that the active Ras in the CLL cells was decreased with Rasgrf1 knockdown. Overall the findings implicate a novel GEF protein, Rasgrf1 as an amplifier of BCR signaling in CLL cells.