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K. Ponnusamy et al.
ZBP1-IRF3 interaction requires the RHIM domain-con- taining C-terminus of ZBP1 (Online Supplementary Figure S5B, C). Furthermore, while total IRF3 and TBK1 levels were not appreciably altered in ZBP1-depleted cells, pIRF3 and pTBK1 levels markedly decreased in both constitutive shRNA-transduced (Figure 4F) and doxycycline-induced shRNA targeting ZBP1 (Online Supplementary Figure S5D) in MM.1S cells. These findings suggest a post-translational dependency of IRF3 and TBK1 constitutive phosphoryla- tion on ZBP1. Finally, shRNA-mediated depletion of TBK1 resulted in a decrease of IRF3 phosphorylation (Figure 4G). Together these data support a model whereby ZBP1 serves as a scaffold for TBK1-dependent constitutive phos- phorylation of IRF3 in MMCL.
IRF3 regulates the cell cycle in myeloma cells
As observed for ZBP1, IRF3 depletion also induces cell cycle arrest and apoptosis and thereby inhibits myeloma cell growth (Figure 4H, I and Online Supplementary Figure S6A-C). Of note, a similar effect was observed after deple- tion of TBK1 (Online Supplementary Figure S6D-G). In addi- tion, transcriptome analysis of IRF3-depleted MM.1S cells revealed that among 185 genes downregulated by both anti-IRF3 shRNA1 and shRNA2, 109 genes were also com- monly downregulated upon ZBP1 depletion (Figure 5A, B and Online Supplementary Table S4) and these are also enriched for cell cycle regulation (Figure 5C and Online Supplementary Table S5). However, only 23 genes were shared among those upregulated upon depletion of ZBP1 or IRF3.
To identify candidate transcriptional targets of IRF3 in myeloma cells we generated and mapped its genome- wide binding by IRF3 ChIP-sequencing in MM.1S cells. IRF3-bound regions (promoter, intergenic and intronic) (Online Supplementary Figure S7A) were highly enriched for IRF3-binding motifs (Figure 5D). In the same cells, we cor- related genome-wide IRF3 binding with chromatin acces- sibility as assessed by an assay for transposase-accessible chromatin (ATAC)-sequencing, RNA polymerase II bind- ing, and activating (H3K27ac and H3K4me1/2/3) and repressive (H3K27me3) histone marks (Figure 5E). This showed that IRF3 binding occurs in nearly 28,000 highly accessible chromatin regions with activating transcription- al potential as revealed by Pol II binding and the presence of activating histone marks. Thus, constitutively phospho- rylated IRF3 in myeloma cells is highly transcriptionally active in the nucleus.
Next, to obtain the compendium of genes directly regu- lated by IRF3 in MM.1S cells, we integrated the IRF3- depleted transcriptome for each anti-IRF3 shRNA with the IRF3 cistrome using BETA-plus software (Figure 5F and Online Supplementary Figure S7D). After intersection of anti-IRF3 shRNA1 and shRNA2 data, we found that the 770 genes predicted to be directly activated by IRF3, included the key cell cycle regulators E2F1, E2F2, AURKB, CCNE1, MKI67 and MCM2–7 complex (Figure 5G, Online Supplementary Figure S7E, F and Online Supplementary Table S6), and were significantly enriched for cell cycle regula- tion pathways (Online Supplementary Figure S7B and Online Supplementary Table S7). Notably, the 339 genes predicted to be repressed by IRF3 were not enriched for IFN type I response genes (Figure 5F, Online Supplementary Figure S7C and Online Supplementary Tables S6 and S7). Consistent with this, while IRF3 binds in the regulatory regions of IFNA1 (but not of IFNB1) and ISG15, which are hallmark
type I IFN response genes, expression of these genes was not altered upon IRF3 depletion (Online Supplementary Figure S7G, H).
IRF3 regulates transcription of and co-operates with IRF4 to regulate the cell cycle in myeloma cells. IRF4 is a transcription factor critical for normal PC development34 while in myeloma PC it co-operates with MYC to estab- lish a transcriptional circuitry to which myeloma cells are highly addicted.41 Since the IRF4 motif was among the top-most enriched regions bound by IRF3 (Figure 5D), we explored potential synergy between IRF3 and IRF4 by overlaying our in-house-generated genome-wide binding profile of IRF3 with that of previously published IRF4 ChIP-sequencing in MM.1S cells.42 First, we observed co- binding of the two transcription factors at the promoter and the previously established super-enhancer of IRF443 (Figure 6A). This observation and the fact that IRF4 expression is significantly downregulated following IRF3 as well as ZBP1 depletion (Figure 6B) suggested that IRF4 transcriptional regulation is, at least in part, under control of the ZBP1-IRF3 axis.
At a genome-wide level, 21,614 IRF3-bound chromatin regions were co-bound by IRF4 (Figure 6C, D). Correlating these with transcriptome changes following IRF3 deple- tion, we identified 612 and 267 genes predicted to be co- activated or co-repressed, respectively, by both IRF3 and IRF4 (Figure 6D, E) with the former highly enriched in cell cycle regulators (Figure 6F and Online Supplementary Tables S8 and S9). To validate this on-chromatin association of IRF3 with IRF4, we performed ChIP-re-ChIP assays at IRF3-IRF4 co-binding regions using a region upstream of IRF4 in which no binding of either transcription factor was observed as a negative control. In all tested regions we found specific co-occupancy of IRF3 and IRF4 including in the IRF4 promoter and super-enhancer regions (Figure 6G, H) and also at genes regulating the cell cycle including E2F1, E2F2, MCM2 and AURKB (Online Supplementary Figure S8A, B).
Discussion
Here we demonstrate that the Z-nucleic acid sensor ZBP1 is an important and novel determinant of MM biolo- gy. We link the myeloma-selective constitutive expression of ZBP1 to constitutive IRF3 activation and regulation of IRF3-dependent IRF4 expression and myeloma cell prolif- eration.
While other nucleic acid sensors, e.g., cGAS-STING, are expressed constitutively and are activated upon nucleic acid binding,14,16 ZBP1 differs in that its expression is only detected in response to nucleic acids, viral pathogens or inflammatory stimuli including interferons.17,8,20,44 Our extensive analysis confirmed that ZBP1 expression is low or not detected in all human normal and cancer cells tested with the striking exception of cells in the late B-cell devel- opment trajectory and in particular PC. Reflecting their cell of origin, we found constitutive ZBP1 expression also in MMCL and primary myeloma PC.
While our data do not address the molecular role of Zbp1 in late B-cell development, at a cellular level, they identify suboptimal humoral immune response to a T-cell- dependent antigen in Zbp1-/- mice. Of note, in contrast to our results, antibody levels in Zbp1-/- mice were previously found to be intact in response to DNA vaccination;18 the
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haematologica | 2022; 107(3)