Although the requirement of the NF-kB pathway in B-cell lymphomas is clear, the role of the individual REL (c-rel, relA, and relB) proteins is not understood. This is particularly interesting for several reasons. The first is the observation that c-rel is often amplified in several lymphomas: 50% of Hodgkin's (HL) and 20-30% of diffuse large B-cell lymphomas (DLBCL). However, the specific role of c-rel amplification, as opposed to relA and rel B is not known. Secondly, the lack of similarities in the primary sequences of the three RELs is considerable. This disparity does not lie within the DNA binding and dimerization Rel Homology Domain (RHD), but rather in the C-terminal transcriptional transactivation domains (TAD) of the three. There is only 10% similarity between c-rel and relA, for example. We have therefore constructed the hypothesis that the three REL proteins are functionally distinct and unique. The differences in the TADs suggest that each REL protein has different promoter targets and unique transcriptional biochemistry. Furthermore, the specificity of the c-rel amplification implies that the REL proteins each will have distinct, nonoverlapping functions in lymphoma tumorigenesis. We are therefore addressing the functions of the REL family in B-cell lymphomas in vivo and in vitro. We are addressing REL family functions in several ways. We have begun an in vitro analysis of the protein factors that are necessary for the transcriptional activity of each REL family member. As stated above, we expect these factors to be different for each REL protein. Secondly, we have embarked on an extensive analysis of the genomic distribution of the REL proteins and the promoters they regulate. We are analyzing several different human B-cell lines that represent both Hodgkin's and non-Hodgkin's lymphomas. Finally, we are using cell viability assays and shRNAs that target each REL member to assess their relative importance in the maintenance of lymphoma cell viability.We are in the process of publishing our first paper on the results of our NF-kB project. We unexpectedly found that although all germinal center lymphomas we tested required relA and c-rel, only Hodgkin's lymphoma (HL) was additionally dependent on relB. This underscores the unique functions of relB in HL and argues that the defining characteristic of HL is relB expression. We have found this requirement in several HL cell lines, all showing the constitutive activity of nuclear relB. Furthermore, we have shown that the noncanonical pathway (required to activate relB) is active in HL cell lines and in 49/50 primary HL tumors. This is an important contribution to understanding the etiology of HL: however, and HL is made, the activation of the noncanonical pathway is an essential requirement. Lastly, we have identifed a small molecule that inhibits NIK which is an enzyme required for the activation of the noncanonical pathway. This results in a reduction in cell proliferation in the tested HL cell lines as well as in several multiple myeloma cell lines that are also dependent on NIK expression. These experiments suggest that NIK-dependent tumors are targetable with small molecules that inhibit NIK phosphorylation activity. In collaboration with Dr. Waldmann we are attempting to expand these observations to other lymphomas.The genomic distribution of the REL factors appears to be unique for each type of lymphoma indicating that each REL regulates a unique transcriptional regulatory network and which probably serve as the defining feature of these lymphoma classes. We have found that the REL distributions are different both within a particular lymphoma, suggesting that each REL regulates its own network of genes, and between lymphomas, suggesting that each REL network and function is different in different lymphomas. That is, different REL regulatory networks establish different lymphomas. We are now putting these data together in a manuscript.Our future work will focus on more the functional biochemistry of the REL proteins themselves to answer the question of how each REL activates transcription. They clearly have different transactivation domains and therefore will require different coactivators for gene activation. The isolation and identification of these coactivators will undoubtedly contribute to our understanding of REL function of gene networks in lymphomas.
