P-216: IRF2 Inhibition Impairs Cell Migration and Triggers Necroptosis in Multiple Myeloma

Introduction: Multiple Myeloma (MM) is the second most common hematologic neoplasia, but it remains incurable. A recent study of the epigenome of MM cells described de novo chromatin activation as a unifying factor underlying the disease. These de novo active regions were enriched in specific Transcription Factor Binding Sites (TFBS) suggesting that TFs may exert a significant role in the development and progression of MM. Here we identify and characterize the function of essential TFs involved in the pathogenesis of MM.

Methods: na

Results: CRISPR/CAS9 library screening was conducted against 54 TFs that presented TFBS at de novo active chromatin regions revealing that 22 TFs were essential for MM. Strikingly, there were three members of the IRF family: IRF1, IRF2, and IRF5, along with the positive control IRF4. Single knockdowns identified IRF2 and IRF4 as the most essential TFs. Besides, using the CoMMpass database we detected that MM patients with higher expression of IRF2 had worse progression-free and overall survival, both in univariate and multivariate analyses considering high-risk genetic factors. Then, to characterize the biological function of IRF2 in MM, its chromatin localization was determined by CUT&RUN. IRF1 (non-essential) and IRF4 were also assessed to compare the role of the three IRFs. We identified 16300 chromatin regions bound by IRF2, significantly enriched in active promoters compared to IRF4 (7512 peaks) and IRF1 (5891 peaks). The expression of the IRF2 target genes was analyzed in plasma cells from healthy donors, MGUS, SMM and MM patients, observing a dysregulation from MGUS state that suggests the role of IRF2 in MM development. Moreover, IRF2 co-localized with IRF1/IRF4 in 40% of the chromatin regions and regulated the immune system and GTPases. Whereas most of the regions were regulated exclusively by IRF2 and regulated cell cycle and GTPases. Intriguingly, IRF2 was present in 32% (498/1556) of de novo active chromatin regions, more than IRF1 or IRF4. 201 peaks were unique to IRF2 and regulate genes involved in cell migration and osteoblast differentiation, while 297 peaks co-localized with IRF1/IRF4 and were related to oxidative stress genes. Finally, transcriptome analysis revealed that IRF2 inhibition downregulated the adaptive immune system, cell proliferation, and GTPases were also dysregulated leading to a decrease in cell-cell adhesion and cell migration. Then, we observed an upregulation of the innate immune system, dsRNA responses, membrane biogenesis and necroptosis, which was confirmed by the increase of MLKL phosphorylation.

Conclusions: IRF2 plays a significant role as a prognostic biomarker and as an essential gene in the pathogenesis of MM by participating in the regulation of relevant signaling pathways such as cell cycle, migration and adhesion. Moreover, the inhibition of IRF2 promotes necroptosis paving the way to the development of new therapeutic strategies.