Non-Hodgkin's Lymphomas (NHL) are common tumors of the human immune system, primarily of B cell lineage (NHL-B) that have been showing significant unexplained increases in incidence for the last three decades. Unlike normal B-lymphocytes aggressive forms of NHL-B show rapid, dysregulated B lymphocyte growth characteristics, while retaining typical B cell immuno-phenotypes, including expression of characteristic CD40 and SIg. cell surface receptors. Normal B cells, involved in inflammatory, or other immune functions, transduce signals to activate and release, the key transcription factor, NF-kappaB from its cytoplasmic inhibitor; but aggressive NHL-B cells, such as Large B cell Lymphomas (LBCL), show constitutive expression of nuclear NF-kappaB. Our studies have shown that this is accomplished by continually maintaining an assembled, scaffold-like, signaling platform as a concatenate molecular aggregate, called a Signalosome within a lipid raft microdomains, contained within or subjacent to the lymphoma cell membrane. We have developed a hypothetical model of aggressive NHL-B cell pathophysiology that envisions dysregulation of the CD40 mediated signaling pathways as the major mechanism controlling tumor cell growth and other parameters of malignancy. The CD40 Signalosome appears to be initiated through autochthonous production and cognate ligand binding of CD154 (CD40L, gp39) to the CD40 receptor on the lymphoma cell surface. Our studies have indicated that the necessary and sufficient conditions for CD154 expression seem to differ in NHL-B cells from normal activated T lymphocytes, suggesting that the neoplastic lymphocytes show dysregulated gene expression and signaling pathways to mediate autonomous tumor cell growth. Constitutive expression of NF-kappaB in NHL-B can be down-regulated by treatment with specific antibodies or antisense oligos to CD40 or CD154, that disrupt the integrity of the CD40 Signalosomes, resulting in the inhibition of lymphoma cell growth, and the induction of lymphoma cell death in vitro. In this proposal, we will study the molecular characteristics of the CD40 Signalosome, by isolating and sequencing the component proteins of the canonical CD40/NFkB pathway contained within the signalosome, and demonstrate the signaling capabilities of this macromolecular structure. We will also study the signaling pathways controlling gene expression of the CD40 ligand, CD154, that we believe is intimately involved with driving the signalosome pathway, to ascertain the mechanism of its "ectopic" expression, as well as its possible role the aggressive growth pattern and resistance to cell death shown by aggressive NHL-B. In addition to these abnormalities involving lymphoma cell proliferation and cell viability, NHL-B cells also show aberrant expression of the TGF-beta/SMAD-SNO/SKI system, that is responsible for negative growth regulation in normal B lymphocytes (as well as many other functions in most cell types). Our preliminary studies have shown that abnormalities in SNO/SKI gene expression are involved in abrogating the negative regulatory influence of this signaling system in NHL-B that also appears to be linked to the CD40/NFkB system in NHL-B. Our studies will further examine the role of this system in potentiating the biologic, and possibly the clinical aggressiveness of these lymphoid tumors, and explore whether inhibition of the dysregulatory elements in this pathway can have therapeutic potential. We believe that our CD40 Signalosome model will provide an important "roadmap" into the important control mechanisms involved in NHL-B, that can be used for developing new therapeutic approaches for this very important group of human lymphoid neoplasms.