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ZBP1 regulates myeloma cell proliferation via IRF3/IRF4
different routes and process of immunization might account for these differences.
We found that ZBP1 depletion had a profound and selec- tive effect on MMCL proliferation and survival in vitro and in vivo. Similarly, in primary myeloma PC which are less proliferative than MMCL, depletion of ZBP1 also induced cell cycle arrest. Based on appropriate design of ZBP1-tar- geting shRNA we could determine that myeloma cell pro- liferation is sustained by the isoform 1, which retains both Zα1 and Zα2 domains, but not by the isoform 2, which retains only the Zα2 domain. Future research will explore the nature and origin of nucleic acids that are bound by the Zα domain and their impact on the pro-proliferative func- tion of ZBP1 in myeloma cells.
Transcriptomes of ZBP1-depleted myeloma cells, which are driven by distinct primary oncogenes, i.e., MAF (MM.1S cells) and MMSET (H929 cells), highlighted cell cycle regulation as one of the main pathways regulated by ZBP1. This novel pro-proliferative function of ZBP1 con- trasts with the anti-proliferative potential of IFNβ which can induce and sustain expression of ZBP1.45,20 However, although GSEA suggested that ZBP1 mediates repression of IFN type I response in ZBP1-depleted MM.1S cells, study of a large number of primary myeloma PC transcrip- tomes revealed a strong IFN type I response transcriptional signature as well as enrichment for cell cycle pathways among upregulated genes in ZBP1hi myeloma PC. Together, these findings are consistent with a model whereby an active IFN type I response restrains myeloma PC proliferation in the low proliferative early phase myelo- ma PC but sustains ZBP1 expression, which exerts a limit- ed pro-proliferative function. In contrast, while the IFN type I transcriptional program is attenuated or even repressed in the highly proliferative MMCL such as MM.1S cells, which are representative of advanced MM,21,46 persistent ZBP1 expression regulates proliferation which is not constrained by the IFN type I response. In line with this model, a recent comparison of primary myeloma PC and MMCL transcriptomes demonstrated enrichment for the IFN type I response gene signature in myeloma PC in more than 700 MM patients at diagnosis while prolifer- ative but not IFN type I gene signatures were dominant in relapsed disease myeloma PC and in MMCL.21
The physiological role of ZBP1 is to promote necroptosis and inflammation through interaction with RIPK3 in response to pathogens or cellular dsRNA.47,10,12 While inter- action of ZBP1 with IRF3 and TBK1 has been shown pre- viously in an ectopic expression system,17,40 whether it reg- ulates the type I IFN response has been disputed.18 Here we confirmed direct and functional interactions of endoge- nous ZBP1-IRF3-TBK1 in myeloma cells which highlights ZBP1 as a physical platform that directs activation of TBK1 and IRF3.
While transcriptional activation by phosphorylation of IRF3 in response to inflammatory stimuli is expected to be transient, we found that IRF3 is constitutively phosphory- lated in both primary myeloma cells and cell lines. This is not unique to MM since constitutively phosphorylated IRF3 has been reported in several ZBP1-negative cancer lines39 but not functionally investigated although a pro- proliferative effect of IRF3 has been reported in acute myeloid leukemia cells at a cellular level.48 Importantly, we
demonstrate that IRF3 binds to transcriptionally active regions of the genome and it directly regulates genes that promote cell cycle progression in myeloma cells. Accordingly, IRF3 depletion in myeloma cells leads to cell cycle arrest and apoptosis.
We also show that TBK1 depletion results in cell cycle arrest and apoptosis in myeloma cells. Although this cellu- lar effect is likely linked to downstream regulation of cell cycle genes by pIRF3, other mechanisms are also possible since TBK1 is a pleiotropic kinase.49
IRF4, the lineage-defining transcription factor in PC development,34 establishes an aberrant transcriptional cir- cuity with MYC that renders myeloma PC highly dependent on an oncogenic program that includes activa- tion of the cell cycle among other pathways.41 Here, based on IRF3 binding to the super-enhancer and promot- er of IRF4 and the fact that depletion of IRF3 (also ZBP1) results in significant IRF4 downregulation, we demon- strate direct transcriptional activation of IRF4 by IRF3. Indeed, since as little as 50% reduction in IRF4 expression levels is toxic to myeloma cells,41 the greater than 50% reduction in IRF4 mRNA induced by IRF3 depletion would be expected to contribute significantly to myelo- ma cell death. Our genome-wide and sequential ChIP assays demonstrated and validated extensive co-occupan- cy of IRF3 and IRF4 in the myeloma regulatory genome including at the super-enhancer of IRF4, with genes involved in cell cycle control being among the targets of the IRF3-IRF4 synergy.
In summary, our data show that like other nucleic acid sensors, ZBP1 can regulate cellular pathways critical for cancer biology. We show a constitutively active ZBP1-IRF3 axis that is co-opted into promoting proliferative pathways in myeloma PC and regulating expression of the critical myeloma oncogene IRF4. Guided by our initial delineation of the structural requirements of the ZBP1-IRF3 interaction in myeloma cells, disruption of the ZBP1-IRF3 axis will offer an opportunity for targeted and relatively selective therapeutic intervention in MM.
Disclosures
No conflicts of interest to disclose.
Contributions
KP and AK conceived and designed the study; KP and MMT performed the co-immunoprecipitation and doxycycline-inducible study in vitro; KP and MB performed the shTBK1 study. PT and KN performed immunohistochemistry; AC processed RNA- sequencing data for ZBP1; MER processed RNA-sequencing and ChIP-sequencing data for ZBP1 and IRF3; KP performed all other experiments in vitro, in vivo and integrated all the ChIP-sequencing and RNA-sequencing data and performed all other bioinformatics analysis and created all the figures; DI provided erythroblast cells; VSC, AK, NT, XX, IVK, IR, and HWA provided reagents; AK supervised the study. KP and AK wrote the manuscript.
Funding
We acknowledge funding from Blood Cancer UK (to KP, NT, XX, and VC), KKLF (to AK), and the Imperial NIHR Biomedical Research Centre, LMS/NIHR Imperial Biomedical Research Centre Flow Cytometry Facility, Imperial BRC Genomics Facility and the MRC/LMS Sequencing Facility for support.
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