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

Abstract

Abstract 3925Poster Board III-861 IntroductionMultiple 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. MethodsWe 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. ResultsA 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. ConclusionsOur 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. Disclosures:No relevant conflicts of interest to declare.