IRF2BP2 mutations activate IL-1β-mediated NF-κB signaling and confer actionable anti-IL-1β sensitivity in DLBCL

Abstract Diffuse large B cell lymphoma (DLBCL) is the most prevalent lymphoid malignancy in adults and exhibits significant heterogeneity. Historically classified into activated B cell-like (ABC) and germinal center B cell-like (GCB) subtypes based on cell-of-origin, DLBCL has been further stratified into distinct clusters (A53, ST2, N1, BN2, EZB, and MCD) according to tumor mutational signatures. Although frontline chemo-immunotherapy achieves cure rates of approximately 65%, management of relapsed or refractory disease presents significant challenges, especially in ABC-DLBCL and MCD subtypes. The transcriptional repressor interferon regulatory factor 2 binding protein 2 (IRF2BP2) undergoes frequent mutation in MCD patients, with most mutations anticipated to confer loss-of-function. Consistent with findings in human DLBCL, Irf2bp2 emerges as one of the most commonly co-mutated genes in established MCD mouse models featuring B cell-specific Prdm1 loss, mutant Myd88p.L252P expression, BCL2 overexpression, and Cd79b ITAM mutations. Despite this prevalence, the functional significance of IRF2BP2 in lymphoma pathogenesis and its potential tumor suppressor role in DLBCL remain unexplored. To investigate the landscape of IRF2BP2 DNA binding sites in DLBCL, we performed a series of IRF2BP2 and H3K27Ac ChIP-seq and ATAC-seq experiments in human ABC/MCD-DLBCL cell lines. We found a strong overrepresentation of interferon regulatory factor (IRF) motifs in IRF2BP2 binding peaks with differential signals in the H3K27Ac and ATAC-seq datasets. IRF motifs are often found upstream of cytokine genes that are crucial for regulating inflammation and immune response. Further multiplex cytokine arrays revealed that secretion of several inflammatory cytokines, including IL-1β, were significantly increased in the IRF2BP2 knockout setting. This increase of IL-1β secretion was further validated via ELISA. At the RNA level, differences in IL-1β expression were unchanged in the IRF2BP2-proficient vs. IRF2BP2-deficient setting, suggesting that the differences we see at the protein-level are a result of differential cleavage of pro-IL-1β. To validate this hypothesis, we found that IRF2BP2-deficient cells exhibited enhanced activation of caspase-1, the IL-1 converting enzyme responsible for processing pro-IL-1β into its secreted bioactive form. Additionally, the genetic loci of several regulators of inflammasome activation are bound by IRF2BP2 and change in H3K27Ac after IRF2BP2 knockout, e.g. ZBP1, TET2 and STK24. Furthermore, we observed that CRISPR/Cas9-mediated IRF2BP2 knockout ABC/MCD-DLBCL cells with increasedIL-1β secretion, also displayed enhanced proliferation and elevated NF-κB signaling compared to IRF2BP2-intact cells. This active IL-1β-NF-κB autocrine signaling pathway in IRF2BP2-deficient cells can be therapeutically exploited, and IRF2BP2-deficient cells demonstrated sensitivity to IL-1β inhibition both in vitro and in vivo, while IRF2BP2-proficient cells remained refractory. Moreover, murine lymphoma cell lines derived from MCD mouse models bearing spontaneous Irf2bp2 mutations also displayed anti-IL1β sensitivity in vitro, with sustained responsiveness over extended culture periods. Overall, these results implicate IL-1β as the principal driver of enhanced NF-κB signaling following IRF2BP2 disruption and suggest anti-IL-1β therapy as a promising therapeutic approach for IRF2BP2-mutant patients.