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K. Ponnusamy et al.
weeks in control or dox-treated animals (D, E) (n=4-5 mice/group). (F) RNA-sequencing was performed with poly(A) tail-enriched RNA from FACS-purified GFP+ live cells of scr or shRNA-transduced cells. Venn diagram showing the number of commonly and differentially (up- and down-regulated) expressed genes based on log2 fold-change to scr control with a cut-off adjusted P-value (Padj) <0.05 among the two ZBP1-depleted transcriptomes of the multiple myeloma cell lines (MMCL) H929 and MM.1S and both anti-ZBP1 sh1 or sh2 (n=2). (G) Heatmap showing the expression patterns of the top 289 commonly and differentially expressed genes, based on log2 fold-change to scr control with a cut off Padj <0.05, shared by the two ZBP1-depleted MMCL H929 and MM.1S and both anti-ZBP1 sh1 or sh2. (H) Enrichr pathway enrichment analysis of the shared 270 genes that are commonly downregulated between two MMCL treated with anti-ZBP1 sh1 or sh2 as compared to the scr control. (I) A representative histogram shows depletion of ZBP1 by anti-ZBP1 sh1 or sh2 induces cell cycle arrest in MMCL H929 as compared to the scr control, and its cumulative data shown for MMCL H929 and MM.1S cells. The analysis was performed on GFP+ cells on day 4 after transduction (n=3). (J) A representative histogram showing that anti-ZBP1 sh1- or sh2-mediated ZBP1 depletion induces cell cycle arrest as compared to the scr control in multiple myeloma (MM) patient- derived bone marrow myeloma cells purified for the CD138+ marker, and the quantitative data. The flow cytometry plot at the top shows the gating strategy for the GFP+ population in transduced cells. The analysis was performed on GFP+ cells on day 4 after transduction (n=3 MM bone marrow samples). The error bars of all the cumulative data indicate the mean ± standard error of mean. A two-tailed unpaired t-test was applied to determine the P values. *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001, ns: not significant (P>0.05). The number of experiments performed is indicated separately in each panel legend.
the role of ZBP1 in GCB and PC development, we assessed the humoral immune responses against the T-cell-dependent antigen NP-KLH using alum as an adju- vant. We found that while Zbp1 mRNA is undetectable in FACS-purified GCB cells and PC from Zbp1-/- mice in response to either NP-KLH-alum or alum-only control, it increased from GCB cells to PC in wild-type (WT) litter- mates and this increase was more pronounced upon immunization with NP-KLH-alum (Figure 2A).
Splenic frequencies of B220+CD19+GL7+CD95+ GCB cells (Figure 2B, C) and B220loCD138+ PC (Figure 2D, E) were not different at baseline between Zbp1-/- and their WT littermates. Similarly GCB-cell frequencies were not significantly different between WT and Zbp1-/- animals after NP-KLH-alum immunization; however, the increase in PC frequency and NP-KLH-specific IgG (but not IgM) serum levels in immunized Zbp1-/- animals was signifi- cantly lower compared to that in immunized WT litter- mates (Figure 2D-G). These findings suggest that although Zbp1 is not required for GCB cell and PC development under steady-state, it is required for optimal T-cell-depen- dent humoral immune responses. Whether cellular nucleic acids in complex with Zbp1 play a role in this process remains to be addressed.
ZBP1 is required for myeloma cell proliferation and survival
To investigate the functional role of ZBP1 in MM, we depleted ZBP1 expression in MMCL by targeting both of its main isoforms, i.e., isoform 1 comprising Zα1 and Zα2 domains and isoform 2 which lacks Zα1. Depletion of either isoform 1 or both isoforms 1 and 2 by shRNA1- or shRNA2-mediated knockdown, respectively, was toxic to H929 and U266 cells while shRNA3 did not deplete ZBP1 and behaved like the scrambled control without affecting cell viability (Figure 3A and Online Supplementary Figure S3A-C). Depletion of ZBP1 by shRNA1/2 was also toxic to MMCL MM.1S and its dexamethasone-resistant deriva- tive MM.1R (Figure 3B and Online Supplementary Figure S3D). These findings suggest that the observed effect is mediatedbydepletionofisoform1andcannotberescued by isoform 2. This effect was specific because the anti- proliferative function was not observed in the shRNA- transduced erythromyeloid K562 or epithelial HeLa cells (Figure 3C) which lack ZBP1 expression (Online Supplementary Figures S1F and 3E). Furthermure, depletion of shRNA1-transduced myeloma cells was at least in part rescued by overexpression of ZBP1 cDNA with appropri- ate silent mutations (Online Supplementary Figure S3F). Mutating the seed region of shRNA1, aimed at eliminating off-target effects,35 did not alter either the expression of ZBP1 or the cytotoxic effects in MM.1S cells (Online Supplementary Figure S3G,H). Using a doxycycline-
inducible shRNA,36 we found that ZBP1 depletion inhibit- ed myeloma cell growth in vitro (Online Supplementary Figure S3I,J) and also subcutaneous myeloma tumor growth in vivo (Figure 3D, E and Online Supplementary Figure S3K,L). Together, these findings suggest an impor- tant role of ZBP1 in myeloma cell biology.
In line with these observations, transcriptome analysis of two ZBP1-depleted MMCL, in which oncogenic tran- scriptomes are driven by MAF (MM.1S) or MMSET (H929) oncogenes, revealed 270 genes that are significant- ly downregulated in both cells and by both shRNA (Figure 3F, G and Online Supplementary Table S1). These genes were highly enriched for cell cycle control pathways (Figure 3H and Online Supplementary Table S2).
We also validated reduction of the mRNA expression levels of the cell cycle regulators Ki-67, FOXM1 and E2F1 upon ZBP1-depletion by quantitative polymerase chain reaction analysis (Online Supplementary Figure S4A, B) and confirmed the decrease in proteins by immunoblotting (FOXM1 and E2F1) and flow-cytometry (Ki-67) (Online Supplementary Figure S4C-E). Flow-cytometric analysis of the cell cycle in ZBP1-depleted cells revealed arrest at the G0/G1 phase (Figure 3I) in conjunction with increased apoptosis as assessed by annexin V staining in MM.1S and H929 cells (Online Supplementary Figure S4F). Notably, we also confirmed that both anti-ZBP1 shRNA induced cell cycle arrest in MM patient-derived, bone marrow myelo- ma CD138+ PC (Figure 3J and Online Supplementary Figure S4G), thus confirming the role of ZBP1 in cell cycle regu- lation in primary myeloma PC as well as MMCL.
In addition to downregulation of cell cycle pathways, GSEA also showed significant enrichment for the IFN type I pathway in upregulated genes induced by both shRNA1 and shRNA2 in MM.1S cells but in downregulat- ed genes by only shRNA1 in H929 cells (Online Supplementary Figure S4H). This disparate effect of ZBP1 depletion on IFN type I response genes might reflect the distinct transcriptomes of MM.1S and H929 MMCL imposed by their primary driver oncogenes. Previous work demonstrated that a transcriptional proliferative signature identifies a minority of MM patients with adverse prognosis.37,38 Accordingly, GSEA of myeloma PC transcriptomes with the top 5% highest versus 90% low- est ZBP1 expression revealed significant enrichment in the former for cell cycle regulation pathways among over- expressed genes (Online Supplementary Figure S4I, J and Online Supplementary Table S3). Interestingly, among these overexpressed genes in the subgroup of ZBP1hi patients, we also observed significant enrichment for IFN type I signaling consistent with the role of ZBP1 as an IFN- response gene (Online Supplementary Figure S4I, J and Online Supplementary Table S3).
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haematologica | 2022; 107(3)