Loss of IRF4 Results in Multiple Myeloma Cell Apoptosis through the Transcriptional up-Regulation of the BH3-Only Proteins Bmf and BIM

The transcription factor IRF4 is essential for the survival of both normal plasma cells and the plasma cell malignancy multiple myeloma (MM). However the basis for this dependency remains uncertain, with the prevailing view that IRF4 loss in MM induces "death by a thousand cuts." Here we have explored the genetic basis for IRF4 addiction through CRISPR-Cas9 mediated deletion of IRF4 in three lenalidomide sensitive MM cells lines (MM1S, OPM2 and H929) and one resistant cell line (RPMI-8226). Loss of IRF4 resulted in marked loss of viability, irrespective of lenalidomide sensitivity, which was driven by two distinct mechanisms: 1) induction of apoptosis, which was rescued through prior deletion of apoptotic mediators BAX and BAK, and 2) proliferative arrest, which persisted despite rescue from apoptosis. RNA-sequencing following IRF4 inactivation identified a core group of 86 genes whose dysregulation was shared across all 4 MM cell lines. We identified 31 common down-regulated genes (IRF4 activated), including several with previously described roles in survival (such as KLF2, TNFRSF17/BCMA and MYB). Furthermore, MYC, a previously identified target of IRF4 known to play an important role in MM pathogenesis and survival, was down-regulated in 3 of the 4 cell lines. Most surprisingly, IRF4 inactivation resulted in 55 common up-regulated genes that included two BH3-only pro-apoptotic proteins, BMF and BCL2L11 (BIM). Up-regulation of BMF appeared more marked than BIM with an average fold change of 6.5x (range 2.7 - 10.3) vs 1.9x (range: 1.8 - 2.2) respectively. Remarkably, genetic ablation of BMF in the OPM2 cell line and BMF/BIM in the MM1S largely protected from the cell death observed following IRF4 inactivation, suggesting that IRF4 maintains MM survival through the transcriptional repression of BMF and BIM. Our results confirm the vital role of IRF4 for MM proliferation and survival, and shed further light into its critical downstream transcriptional targets. Most interestingly, we have demonstrated that the 'killer blow' appears to be the concerted effect of the BH3-only pro-apoptotic proteins BMF and BIM. Disclosures Huang: Genentech: Patents & Royalties: DCSH is an employee of the Walter and Eliza Hall Institute which receives milestone and royalty payments related to venetoclax.