Distinct Interaction Networks of the MUM1/IRF4 in B- and T-Cell Lymphomas Identified by Tandem Mass Spectrometry.

Multiple myeloma oncogene 1/interferon regulatory factor 4 (MUM1/IRF4) is a 52-kDa transcriptional activator protein which plays an important role in interferon-stimulated response element (ISRE)-regulated signal transduction mechanisms important in lymphoid cell development and differentiation. MUM1/IRF4 is also critical for the generation of immunoglobulin-secreting plasma cells. MUM1/IRF4 is expressed in the activated B-cell (ABC)-like subset of diffuse large B-cell lymphomas (DLBCL) and is targeted by chromosomal translocations in a subset of multiple myelomas and peripheral T-cell lymphomas. Despite its importance in lymphocyte and lymphoma biology, and its addiction demonstrated in multiple myelomas, the proteomic network of MUM1/IRF4 interacting proteins has not been determined.

We determined the proteomic interaction networks of MUM1/IRF4 in B-cell and T-cell lymphoma contexts, by using a functional proteomic approach. Total cell lysates were prepared from MUM1/IRF4 expressing cell lines including OCI-LY10 ((ABC)-like DLBCL) and HH (mycosis fungoides derived-T-cell) and, as a negative control, from non-MUM1/IRF4 expressing SUDHL4 cell line. Immunoprecipitates of the MUM1/IRF4 expressing B- and T-cell line were compared to that of hyperimmune mouse immunoglobulin by 1-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis. Coomassie-stained protein bands from both immunocomplexes were excised and analyzed by electrospray liquid chromatography tandem mass spectrometry (ESI-LC/MS/MS). Peptide sequences were identified by searching the MS/MS data against human IPI protein using database X!Tandem with k-score plug-in. Proteins found in the two control sets (SUDHL4 and IgG isotype) were manually subtracted from the proteins found in the experimental set. MS/MS results were validated using immunoprecipitation and Western blotting and some of the interacting proteins were further confirmed by reciprocal immunoprecipitation.

A total of 163 and 94 proteins were identified from 12 Coomassie-stained bands which were unique to the MUM1/IRF4 immunocomplex in OCI-Ly10 and HH cell lines, respectively. Previously reported proteins in the MUM1/IRF4 signal pathway were identified including FK506 and TRAF family protein. More importantly, many proteins previously not associated with MUM1/IRF4 were identified. In common to both subsets were proteins such as ARHGDIA-involved in RhoA-mediated signaling. In the ABC-like B-cell (OCI-LY10) specific context, these included proteins known to be critical regulators of lymphocyte proliferation (RAB2A), motility, trafficking and cell adhesion. In the T-cell specific context, proteins known to play important roles in endocytosis (Reggies) and T-cell activation (GDI2, Guanine nucleotide exchange factors) were identified in the MUM1/IRF4 interactome. A subset of the proteins identified by MS were confirmed by western blotting and reciprocal immunoprecipitation.

Our studies reveal that although a minor subset of MUM1/IRF4 interacting proteins are common in B- and T-cells, MUM1/IRF4 exhibits distinct interaction partners dependent of specific cellular contexts. The diverse interaction networks implicate MUM1/IRF4 in previously undescribed functional roles in lymphocyte-specific subsets. Comprehensive elucidation of the protein-protein interaction networks in the specific cellular contexts will provide opportunities for exploitation of the knowledge for design of rational interventions targeting the critical nodes and modules in MUM1/IRF4-deregulation in B- and T-cell malignancies.

No relevant conflicts of interest to declare.