Influence of Mir-155 On B-Cell Receptor Signaling in Chronic Lymphocytic Leukemia.

Abstract 2343

Poster Board II-320

B-cell receptor (BCR) signaling arguably plays an important role in the pathogenesis and/or progression of chronic lymphocytic leukemia. Ligation of the BCR by F(ab)2 anti-μ can induce phosphorylation of p72^Syk, BLNK, phospholipase C-gamma (PLCγ) and other downstream adapter/signaling molecules, inducing intracellular calcium flux and cellular activation. Prior studies found that CLL cells that expressed unmutated Ig heavy-chain variable region genes (IGHV) and the zeta-associated protein of 70 kD (ZAP-70) generally experienced greater levels of activation following treatment with anti-μ than did CLL cells that lacked expression of ZAP-70. However, we found unusual cases that lacked expression of ZAP-70 that also responded vigorously to treatment with anti-μ, suggesting that other factors contribute to the noted differences in BCR-signaling. Analyses for expression of microRNAs by microarray revealed that CLL cells that used unmutated IGHV and that expressed ZAP-70 expressed higher levels of certain microRNAs than did cases that used mutated IGHV and that lacked expression of ZAP-70. One of such microRNA, miR-155, was found to target mRNA encoding SHIP-1, a phosphatase that plays a critical role in modulating the level of BCR signaling in normal B cells. Using quantitative assays for miR-155 we found high-level expression of this microRNA was associated with proficient BCR signaling in CLL. To examine whether miR-155 could modulate the levels of SHIP-1 and/or BCR signaling in CLL cells we transfected primary leukemia cells from each of multiple patients with control oligo-RNAs, miR-155, or a specific inhibitor of miR-155 (miR-155 inhibitor). Twenty-four hours later the cells were stimulated with anti-μ or control antibody and then examined 10 minutes later for expression of SHIP-1, induced calcium influx, or phosphorylation of kinases and adapter proteins that are involved in BCR signaling. CLL cells that had low expression levels of miR-155 and that were poorly responsive BCR had significantly higher levels of calcium influx and phosphorylated p72^Syk, BLNK, and PLCγ in response to anti-μ following transfection with miR-155 than following mock transfection or transfection with control oligo-RNA. Conversely, CLL cells that had high expression levels of miR-155 and highly responsive BCR were made to have significantly higher amounts of SHIP-1 protein and to have significantly lower relative levels of phosphorylated protein and calcium influx in response to anti-μ following transfection with the miR-155 inhibitor than did mock transfected CLL cells. These results identify miR-155 as a factor that can modulate BCR signaling in CLL in part by regulating the relative expression level of SHIP-1. These results demonstrate that differential expression of microRNAs in CLL can influence physiologic features that potentially contribute to disease progression.