Induction of viral mimicry upon loss of DHX9 and ADAR1 in breast cancer cells

UNLABELLED: Detection of viral double-stranded RNA (dsRNA) is an important component of innate immunity. However, many endogenous RNAs containing double-stranded regions can be misrecognized and activate innate immunity. The IFN-inducible ADAR1-p150 suppresses dsRNA sensing, an essential function for adenosine deaminase acting on RNA 1 (ADAR1) in many cancers, including breast. Although ADAR1-p150 has been well established in this role, the functions of the constitutively expressed ADAR1-p110 isoform are less understood. We used proximity labeling to identify putative ADAR1-p110-interacting proteins in breast cancer cell lines. Of the proteins identified, the RNA helicase DHX9 was of particular interest. Knockdown of DHX9 in ADAR1-dependent cell lines caused cell death and activation of the dsRNA sensor PKR. In ADAR1-independent cell lines, combined knockdown of DHX9 and ADAR1, but neither alone, caused activation of multiple dsRNA sensing pathways leading to a viral mimicry phenotype. Together, these results reveal an important role for DHX9 in suppressing dsRNA sensing by multiple pathways. SIGNIFICANCE: These findings implicate DHX9 as a suppressor of dsRNA sensing. In some cell lines, loss of DHX9 alone is sufficient to cause activation of dsRNA sensing pathways, while in other cell lines DHX9 functions redundantly with ADAR1 to suppress pathway activation

Identifying Molecular Targets of ADAR1 Dependency in Breast Cancer

Innate immunity is a fundamental defense mechanism against non-self. Similar to its role in pathogenic infections, innate immunity is capable of eliminating cancerous cells by activating cytotoxic inflammatory signaling. Adenosine deaminase acting on RNA 1 (ADAR1) is a key regulator of cell-intrinsic innate immunity. ADAR1 converts adenosines to inosines on double-stranded RNAs (dsRNAs), altering dsRNA structure and its interactive properties. This suppresses cytoplasmic sensing of immunogenic dsRNA and activation of the type I interferon (IFN) signaling pathway. Frequently, suppressing endogenous dsRNA from triggering cytotoxic inflammatory signaling is essential for cancer cell survival. This is known as ADAR1 dependency, which has been proposed as a potential therapeutic target in cancer. In this dissertation, I aimed to analyze cellular mechanisms mediating ADAR1 dependency in breast cancer to identify novel molecular targets.As ADAR1 dependency is associated with elevated type I IFN signature, I investigated a potential interplay between ADAR1 and hypoxia-inducible factor 1 (HIF1) in the context of cell-intrinsic type I IFN signaling activated by co-depletion of tumor suppressors p53 and ARF. In the mouse embryonic fibroblast model and human breast cancer cell lines, knockdown of ADAR1 decreased the normoxic expression of HIF1 α subunit (HIF1α). Knockdown of HIF1α in breast cancer cell lines phenocopied ADAR1 knockdown, characterized by reduced proliferation and translational shutdown. However, unlike the ADAR1 dependency mechanism, the translational shutdown upon HIF1α knockdown was not mediated by protein kinase R (PKR). This suggests that HIF1α is not a downstream mediator of ADAR1 dependency. Therefore, in tumors with ADAR1-driven expression of HIF1α, targeting ADAR1 can potentially activate multiple stress response kinases to trigger translational shutdown. Additionally, tumor suppressor von Hippel-Lindau (VHL) was downregulated by ADAR1 post-translationally although it was not coupled with HIF1α expression. Next, I sought to determine the factors that establish ADAR1 dependency or ADAR1 independency. Using proximity labeling, putative ADAR1-interacting proteins were identified in breast cancer cell lines. Of the proteins identified, DExH box helicase 9 (DHX9) was of particular interest. Knockdown of DHX9 in ADAR1-dependent cell lines caused cell death and activation of PKR. In ADAR1-independent cell lines, combined knockdown of DHX9 and ADAR1, but neither alone, caused activation of multiple dsRNA sensing pathways leading to a viral mimicry phenotype. Furthermore, the dsRNA-binding domain of DHX9 was sufficient to prevent PKR activation upon combined knockdown. Taken together, DHX9 plays an important role in suppression of dsRNA sensing, and its functional coordination with ADAR1 distinguishes ADAR1-dependent from -independent cells. Another RNA helicase identified by proximity labeling, DDX54, was examined for its potential role in ADAR1 dependency. However, DDX54 did not suppress ADAR1 dependency. Unexpectedly, a short hairpin RNA targeting DDX54 was shown to directly activate PKR. Other dsRNA-sensing pathways were not activated, suggesting that the shRNA acts as a specific agonist of PKR rather than a broad immunostimulant. Collectively, this dissertation highlights pathway analyses surrounding ADAR1 function and identifies molecular determinants of ADAR1 dependency. It excludes HIF1α as a mechanistic component of ADAR1-driven cancer cells and proposes DHX9 as a novel target in ADAR1-associated viral mimicry. The findings establish dysregulation of RNA metabolism as a major driver of cancer cell-intrinsic inflammatory signaling. In the broader context of molecular cell biology, this work demonstrates the use of genetic and interactome-based approaches to uncover functional interplays between cellular pathways

Supplementary Figure 12 from Induction of Viral Mimicry Upon Loss of DHX9 and ADAR1 in Breast Cancer Cells

Supplementary Figure 12</p

Supplementary Figure 7 from Induction of Viral Mimicry Upon Loss of DHX9 and ADAR1 in Breast Cancer Cells

Supplementary Figure 7</p

FIGURE 5 from Induction of Viral Mimicry Upon Loss of DHX9 and ADAR1 in Breast Cancer Cells

Induction of a viral mimicry phenotype upon knockdown of DHX9 and ADAR1 in MCF-7. A, Volcano plot showing changes in RNA expression upon knockdown of DHX9 and ADAR1 in MCF-7, a volcano plot for SK-BR-3 is in Supplementary Fig. S6F. RNA was isolated from cells 5 days after transduction with the shRNAs listed. Fold change of RNA expression shown in A was determined using an interaction term between ADAR1 and DHX9 knockdown, volcano plots for fold change of RNA expression for single knockdown of ADAR1 or DHX9 is in Supplementary Fig. S6A–S6B and S6D–S6E. B, GO terms identified by gene set enrichment analysis of the RNA-seq data in A. C–E, Heat maps and summary box plots showing RNA expression changes in MCF-7 and SK-BR-3 upon knockdown of ADAR1 and/or DHX9. C shows RNA expression for core ISGs (22, 35), D shows ATF4 targets, and E shows NFκB targets with ISGs removed. Genes are clustered by RNA expression across all four samples. Clustering was performed for each gene set (ISGs, ATF4 targets, or NFκB targets) independently. For more information, see GitHub repository link in Data and Code Availability.</p

FIGURE 8 from Induction of Viral Mimicry Upon Loss of DHX9 and ADAR1 in Breast Cancer Cells

Rescue of PKR activation by ADAR1-p110 and ADAR-p150. A, Representative immunoblot showing the phenotype of ADAR1 and DHX9 knockdown with ADAR1-p110 or ADAR1-p150 overexpression in SK-BR-3. Immunoblots for other replicates and uncropped blots can be found in Source Data Figures. B, Fold change of PKR phosphorylation at Thr-446 upon ADAR1 and DHX9 knockdown with ADAR1 isoform overexpression, quantified from immunoblots in A. C, Representative foci formation phenotype of ADAR1 and DHX9 knockdown ADAR1 isoform overexpression. Quantification of relative foci area is shown in D. Quantification of protein expression for other proteins of interest can be found in Supplementary Fig. S11A–S11F. Timepoints for collecting protein lysates and foci formation are the same as described in Fig. 4 for SK-BR-3. Bars represent the average of at least three replicates, error bars are ± SD. ***, P P values determined by Dunnett test.</p

FIGURE 3 from Induction of Viral Mimicry Upon Loss of DHX9 and ADAR1 in Breast Cancer Cells

DHX9 is overexpressed in breast cancer and suppresses PKR activation. A, Schematic showing the domain structure of PKR, ADAR1, DHX9 and other helicases identified by proximity labeling in Fig. 1, dsRBD refers to the dsRNA binding domain. B, Pearson and Spearman correlation coefficients for the correlation between ADAR1 expression at the RNA level and the expression of each indicated helicase at the RNA level, data from breast tumors within TCGA. Scatterplots showing the correlation between ADAR1-p110 (C), or ADAR1-p150 (D), and DHX9 expression in normal breast or breast tumors. E, Expression of DHX9 at the RNA level in normal breast, non-TNBC or TNBC tumors. F, Waterfall plot showing DHX9 dependency of breast cancer cell lines using data from DepMap, ER = estrogen receptor–positive cell lines, ERRB2 = HER2-positive cell lines. G, Foci formation assay following knockdown of DHX9 with two different shRNAs in four TNBC cell lines. Cells were plated for foci formation 2 days after transduction and foci were stained after 10 days. G, Representative immunoblot following knockdown of DHX9 with two different shRNAs in four TNBC cell lines, same cells as used in G. Protein lysates were collected from cells 4 days after transduction with lentivirus encoding the shRNAs listed. Immunoblots for other replicates and uncropped blots can be found in Source Data Figures. I, Quantification of PKR phosphorylation as determined by the immunoblot in H. Quantification of protein expression for other proteins of interest can be found in Supplementary Fig. S4A–S4E. Bars represent the average of at least three replicates, error bars are ± SD. *, P P P P values determined by Dunnett test.</p

Supplementary Figure 9 from Induction of Viral Mimicry Upon Loss of DHX9 and ADAR1 in Breast Cancer Cells

Supplementary Figure 9</p

Source Data Figures from Induction of Viral Mimicry Upon Loss of DHX9 and ADAR1 in Breast Cancer Cells

Source Data Figures</p

FIGURE 7 from Induction of Viral Mimicry Upon Loss of DHX9 and ADAR1 in Breast Cancer Cells

Rescue of PKR activation by DHX9 mutants in MCF-7. A, Representative immunoblot showing the phenotype of ADAR1 and DHX9 knockdown with DHX9, DHX9K417R or dsRBD-EGFP overexpression in MCF-7. Immunoblots for other replicates and uncropped blots can be found in Source Data Figures. B, Fold change of PKR phosphorylation at Thr-446 upon ADAR1 and DHX9 knockdown with DHX9, DHX9K417R or dsRBD-EGFP overexpression in MCF-7, quantified from immunoblots in A. Quantification of protein expression for other proteins of interest can be found in Supplementary Fig. S9B–S9G. C, Representative foci formation phenotype of ADAR1 and DHX9 knockdown with DHX9, DHX9K417R or dsRBD-EGFP overexpression in MCF-7. Quantification of relative foci area is shown in D. E, RNA expression of representative genes from upregulated pathways in Fig. 5 as determined by qRT-PCR for the MCF-7 rescue experiment. Statistical analysis for each pathway is shown in Supplementary Fig. S9H–S9J. F, Analysis of rRNA integrity upon knockdown of ADAR1 and DHX9 in MCF-7 with overexpression of EGFP or dsRBD-EGFP. Arrows indicate canonical RNase L cleavage products (64). Timepoints for collecting protein lysates, isolating RNA, and foci formation are the same as described in Fig. 4 for MCF-7. Bars represent the average of at least three replicates, error bars are ± SD. *, P P P P values determined by Dunnett test.</p