PROJECT SUMMARY Diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL) arise from germinal center (GC) B cells; a stage at which precursor B cells undergo rounds of proliferation, edit their immunoglobulins by somatic hypermutation and class-switch recombination, followed by affinity selection via interactions with T follicular helper (Tfh) cells and follicular dendritic cells (FDCs). The entry to, and exit from, the GC reaction requires extensive changes in gene expression that are controlled by transcription factors that recruit co-activators or co- repressors that drive epigenetic and transcriptional changes. The dysregulation of these molecular programs perturbs the normal B cell differentiation and contributes to FL/DLBCLs. Prior studies indicate that (a) EZH2 regulates the hyperproliferative nature of GC B cells and (b) gain-of-function mutations of the EZH2 Y641 residue “initiate” lymphomagenesis by attenuating GC B cell requirement for Tfh cell help and shifting the dependency to FDCs through increased expression of receptors through which B-cells interface with these stromal cells. Ezh2Y641F GC B cells evade Tfh-directed clonal selection and affinity maturation to escape Tfh-mediated elimination, leading to lymphomagenesis. Notably, compared to EZH2WT, GC B cells carrying Ezh2Y641F show increased and decreased BAFFR and CD40 expressions, respectively, and escape CD40L blockade-mediated impairment of GC reaction. It is unclear whether the impairment of Tfh help manifests as dysregulation of the mechanical control of Tfh–B immunological synapse (IS) and alteration of their intercellular receptor-ligand interactions and signaling in FL/DLBCLs. The overarching hypothesis is that in contrast to GC B cells with EZH2WT, the EZH2Y641 mutation in GCB DLBCL/FL results in dysregulation of key immunoreceptors mediating the interactions of GC B cells with Tfh cells and FDCs manifesting as altered receptor forces, ligand binding, and mechanotransduction. The dysregulated receptor mechanobiology impacts downstream B cell signaling, epigenetics, and sensing of lymphoid microenvironmental cues responsible for extinguishing the “pseudo- malignant” phenotype of GC B cells, thereby driving the cancerous transformation of B cells. Our transcriptomic analysis suggests changes in the levels of proteins involved in B–FDC and B–Tfh interactions, which should affect cellular function. The goal is to understand how the dynamic epigenetic states of DLBCL/FL-driving EZH2Y641 mutations dysregulate B cell mechanobiology, leading to malignancy.

PROJECT NARRATIVE The proposal will determine the previously biophysical signals that control histone-modifying complexes during the normal immune response and how somatic mutations of histone-modifying enzymes reprograms B cells to undergo malignant transformation by disrupting these signals. Based on functional studies, he will identify and test the efficacy of epigenetic- targeted drugs that counteract the effect of these mutations on the epigenome and sensitize lymphomas to other targeted agents and immunotherapies.