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K. Ponnusamy et al.
In addition, like other nucleic acid sensors, such as cGas/STING, ZBP1 has been shown to associate with ser- ine/threonine kinase TBK1 with subsequent phosphoryla- tion of the transcription factor IRF3 (pIRF3). Activation of STING and other nucleic acid sensors triggers transloca- tion of pIRF3 to the nucleus where it directly activates transcription of interferon (IFN) type I response genes;14-16 however, this has been disputed in the case of ZBP1.17,18 Therefore, the role of ZBP1-TBK1-IRF3 in innate sensing or in the context of cancer remains unclear. Nevertheless, it is clear that ZBP1 itself is an IFN-inducible gene as its expression is induced by type I and II IFN which leads to RIPK3-dependent necroptosis7,13,19 and IFNAR1-/- cells fail to upregulate virus-induced ZBP1 expression.20 Since recent work demonstrated an active type I IFN versus pro- liferative transcriptional signature prevailing in early diag- nosis versus relapsed patients’ myeloma PC or myeloma cell lines, respectively,21 we hypothesized that ZBP1 might regulate myeloma PC biology.
Methods
Cell culture, primary samples
U266 and NCI-H929 (Deutsche Sammlung von Mikroorganismen und Zellkulturen, Germany), MM.1R and MM.1S (American Type Culture Collection, Manassas, VA, USA), and HeLa, HEK293T, DU145, MCF7, HCC95, SF295, LNCAP, K562, Jurkat and C1R cells were cultured in either 10% fetal bovine serum, RPMI-1640 or Dulbecco modified Eagle medium. Primary myeloma cells were collected under ethical committee approval (REC n. 11/H0308/9) and cultured with IL-6 (10 ng/mL).
Cell cycle, proliferation and apoptosis
Cell cycle status was assessed using flow cytometry after stain- ing cells with 10 mM Hoechst-33342 in RPMI-1640 medium for 1 h. The cytostatic effects of constitutive or inducible shRNA-trans- duced cells were measured as shown in Online Supplementary Figure S3C. Annexin V (BioLegend) staining was performed according to the manufacturer’s instructions.
Subcutaneous tumor model, immunization
The subcutaneous tumor study was performed in NOD/SCIDγ mice (license PPL70/8586), by subcutaneously injecting 107 doxy- cycline-inducible shRNA- transduced cells with Matrigel (Corning). Tumor volume was calculated by the formula (1/2[length×width2]). Animals were administered 100 mg/mL doxycycline in drinking water and 0.2 mg/kg intraperitoneally.
Zbp1-/- animals18 were obtained from Manolis Pasparakis, Institute of Genetics (Cologne, Germany). Ten- to 12-week-old lit- termates were immunized by intraperitoneal injection of 4 mg/kg NP-KLH (Santacruz Biotech) with Alum Adjuvant (Thermoscientific) at a 3:1 ratio followed by a booster dose of NP- KLH on day 4. Serum NP-KLH-specific antibodies were measured.
Quantitative polymerase chain reaction analysis, RNA-sequencing
Total RNA was isolated using a Nucleospin RNA kit (Macherey- Nagel) followed by cDNA synthesis using a RevertAid cDNA syn- thesis kit (Thermoscientific). Indicated cDNA were quantified using the primers shown in the Online Supplementary Methods. Poly(A)-tail mRNA was isolated, from total RNA of fluorescence- activated cell sorting (FACS)-purified green fluorescent protein (GFP)+ cells with either scrambled or shRNA targeting ZBP1 or IRF3 on day 4 after transduction when >80% knockdown was
achieved, using the NEBNext poly(A) mRNA Magnetic Isolation Module, and libraries were prepared using NEBNext Ultra II RNA library prep kits (New England Biolabs). A 2 nM DNA library (350- 400 bp) was sequenced using Illumina HiSeq 2500 (for shZBP1) or NextSeq500 (for shIRF3) for paired-end 150 bp reads.
Chromatin immunoprecipitation (ChIP)-sequencing, ChIP-re-ChIP
MM.1S cells were cross-linked with 1% formaldehyde and lysed with hypotonic lysis buffer followed by nuclear lysis buffer. The lysate was sonicated to shear the chromatin up to 500 bp fol- lowed by pre-clear with protein A/G magnetic beads and immunoprecipitation with IRF3 antibody or equivalent isotype control. ChIP DNA was collected with AMPure XP-beads after reverse cross-linking. ChIP DNA (1 ng) was used for library prepa- ration with an NEBNext kit. The 2 nM DNA library (400-500 bp) was sequenced using Illumina NextSeq500. For ChIP-re-ChIP, pulled chromatin was eluted in 1% sodium dodecylsulfate (SDS) and diluted 10 times with elution buffer and repeated re-ChIP with the appropriate antibody. The detailed protocols are provid- ed in the Online Supplementary Methods.
Co-immunoprecipitation, immunoblotting
Co-immunoprecipitation was performed as described previous- ly12 with 5% anti-strep-tagII-magnetic bead slurry (IBA) or 2 mg anti-ZBP1 or anti-V5-Tag or anti-IRF3 or their equivalent isotype control antibodies conjugated with protein A/G magnetic beads. For immunoblotting, total cell proteins denatured in 1x LDS Sample Buffer (ThermoFisher Scientific), were resolved in 10% SDS polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes followed by probing with appropriate antibodies shown in the Online Supplementary Methods.
Data analysis
The details of the data analysis are provided in the Online Supplementary Methods. In brief, RNA-sequencing and ChIP- sequencing reads were aligned using STAR or Salmon and BWA MEM,22,23 respectively, to the GRCh38 genome. Differential expression analysis was performed using DESeq2 or limma- voom.22,24,25 ChIP-sequencing peaks were called with MACS226 and tracks with Deeptools27 and visualized in IGV.28 Homer was used for Motif analysis29 and the BETA-plus package30 to integrate RNA-sequencing and ChIP-sequencing data. Gene set enrichment analysis (GSEA)31 and the Enrichr web tool32 were used for path- wayanalysis.
Data availability
All next-generation sequencing data for RNA-sequencing and ChIP-sequencing experiments can be accessed via the Gene Expression Omnibus (GSE163497).
Additional methods
The details of shRNA sequences, cloning strategies, reagents, antibodies and additional methodologies are provided in the Online Supplementary Methods.
Results
Restricted and constitutive expression of ZBP1 in normal and myeloma plasma cells
By searching for genes selectively expressed in MM we identified ZBP1 as highly and selectively expressed in MM cell lines (MMCL) but not in other cancer cells (CCLE
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haematologica | 2022; 107(3)