Diagnostic and Therapeutic Implications of Expression and Phosphorylation of the Translation Regulatory Protein 4E-Binding Protein (BP)-1 in B-Cell Lymphomas and Reactive Lymphoid Tissues.

Abstract 3919

Poster Board III-855

Identifying pathogenic mechanisms that contribute to the development of lymphomas and influence clinical behavior is critical for developing targeted therapies, and selecting patients who may benefit from such drugs. An important level of control of gene expression occurs during initiation of cap-mediated mRNA translation by the eukaryotic initiation factor-4F (eIF-4F) trimolecular complex (eIF-4E, eIF-4G and eIF-4A), in which eIF-4E is rate limiting and oncogenic. eIF-4F hyperactivity plays a key role in human cancers by mediating expression of proteins critical for cell growth, transformation and tumorigenesis. eIF-4F activity is controlled by repressor eIF-4 binding proteins (BPs). 4E-BP1 activity is regulated by phosphorylation. Hypo/non-phosphorylated 4E-BP1 is active, binds eIF-4E and impedes eIF-4F formation, blocking translation and inducing apoptosis. Phosphorylation of 4E-BP1 (p4E-BP1) releases bound eIF-4E, which initiates cap-dependent translation. Because only limited information is available on the expression and phosphorylation of 4E-BP1 in lymphomas, and since agents (e.g., antisense oligonucleotides and small molecules) that target eIF-4E have been developed, we examined the frequency and level of expression of 4E-BP1 and its phosphorylation in various subtypes of mature B cell non-Hodgkin's lymphomas (BCL). Forty-six BCLs (12 follicular [FL], 13 diffuse large B-cell [DLBCL], 7 mantle cell, 5 extranodal marginal zone, and 9 small lymphocytic [SLL] lymphomas), 4 FL with incipient/partial lymph node involvement, and 11 reactive lymphoid tissues were examined using immunohistochemistry for total and phosphorylated 4E-BP1. Staining intensity was graded as from 0 to 3+. Western immunoblotting (WB) was performed on lysates of 5 mature BCLs (2 FL, 3 DLBCL) and 2 reactive lymph nodal tissues for eIF-4G (total), eIF-4E and 4E-BP1 (total and phosphorylated) expression. In reactive lymphoid tissues, there was regional and cellular specificity of expression of 4E-BP1, with either lack of, or minimal (0 to 1+) cytoplasmic expression in follicular center cells and paracortical T-cells, 2+ expression in follicular dendritic cells and paracortical zone Langerhan cells, and 3+ expression in mantle and marginal zones. p4E-BP1 expression was inverted, with 3+ cytoplasmic immunoreactivity in reactive follicular center cells and no expression in the mantle and marginal zone cells or T-cells, and 2+ or 3+ immunoreactivity in follicular dendritic cells and paracortical zone Langerhan cells. In BCLs, a consistently high level (2+ or 3+) of cytoplasmic 4E-BP1 expression was seen in neoplastic lymphocytes in 45/46 (98%) cases. In contrast, p4E-BP1 was moderately or strongly expressed in 19/46 (41%) cases of BCL, being negative in 17 (37%) cases, and only dimly expressed in the remaining 10 (22%) cases. Three of 4 cases with incipient/partial involvement by FL were easily distinguishable from reactive germinal centers by strong, diffuse staining with 4E-BP1 (and 1+ staining in the 4^th case) in neoplastic follicles, distinct from negative/weak staining of adjacent reactive germinal centers. In SLL, slightly higher 4E-BP1 expression was noted in proliferation centers in comparison to surrounding small mature lymphocytes. WB confirmed that non-phosphorylated and p4E-BP1 were expressed in reactive nodes, FL and DLBCL. Other components of the eIF-4F complex including eIF-4G, total and p-eIF-4E and total 4E-BP1 were detectable in whole tissue lysates from BCL samples. We conclude that (a) while 4E-BP1 is almost uniformly expressed in various subtypes of BCL, its level of phosphorylation (indicative of activity) varies widely and has regional and cellular specificity, and (b) 4E-BP1 expression may identify minimal/early lymphomatous involvement in tissues. We speculate that 4E-BP1 phosphorylation may influence the biological behavior of BCLs, since in other investigations we found that the level of phosphorylation of 4E-BP1 correlates with survival after CHOP-based chemotherapy in DLBCL. Our findings support therapeutic trials targeting the eIF-4E pathway in many BCL subtypes, particularly in patients where immunostaining identifies high levels of 4E-BP1 phosphorylation.