Triple-negative breast cancer (TNBC) is the deadliest form of breast cancer. Unlike other types of breast cancer that can be effectively treated by targeted therapies, no such targeted therapy exists for all TNBC patients. The ADAR1 enzyme carries out A-to-I editing of RNA to prevent sensing of endogenous double-stranded RNAs. ADAR1 is highly expressed in breast cancer including TNBC. Here, we demonstrate that expression of ADAR1, specifically its p150 isoform, is required for the survival of TNBC cell lines. In TNBC cells, knockdown of ADAR1 attenuates proliferation and tumorigenesis. Moreover, ADAR1-knockdown leads to robust translational repression. ADAR1-dependent TNBC cell lines also exhibit elevated IFN stimulated gene expression. IFNAR1 reduction significantly rescued the proliferative defects of ADAR1 loss. These findings establish ADAR1 as a novel therapeutic target for TNBC tumors.

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 interferon inducible ADAR1-p150 suppresses dsRNA sensing, an essential function for 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.

14Triple-negative breast cancer (TNBC) is the deadliest form of breast cancer. Unlike other 15 types of breast cancer that can be effectively treated by targeted therapies, no such targeted 16 therapy exists for all TNBC patients. The ADAR1 enzyme carries out A-to-I editing of RNA to 17 prevent sensing of cellular double-stranded RNAs (dsRNA). ADAR1 is highly expressed in 18 breast cancer including TNBC. Here, we demonstrate that ADAR1 expression and editing 19 activity is required in TNBC cell lines but not in ER+ and/or Her2+ cells. In TNBC cells, 20 knockdown of ADAR1 attenuates proliferation and tumorigenesis. PKR expression is elevated in 21 TNBC and its activity is induced upon ADAR1-knockdown, which correlates with a decrease in 22 translation. ADAR1-dependent TNBC cell lines also exhibit elevated IFN stimulated gene 23 expression. IFNAR1 reduction significantly rescues the proliferative defects of ADAR1 loss. 24These findings establish ADAR1 as a novel therapeutic target for TNBC tumors. 25 26 Keywords: ADAR1, triple-negative breast cancer, RNA editing, Type I IFN, PKR 27 28 65highlighting the therapeutic potential of ADAR1 inhibitors for the treatment of TNBC. 66 Results 67ADAR1 is highly expressed in all breast cancer subtypes 68 Using publicly available data from TCGA (The Cancer Genome Atlas) (Han et al., 2015, 69 Fumagalli et al., 2015, we found that high expression of ADAR1 correlated with poor prognosis 70 of breast cancers ( Figure 1A). Recent studies indicated that ADAR1 promotes tumorigenesis of 71 metaplastic breast cancers, and that high expression of ADAR1 correlates with poor prognosis in 72 basal-like breast cancers (Sagredo et al., 2018, Dave et al., 2017. Since both basal-like and 73 5 metaplastic breast cancers share similar characteristics with TNBC, we sought to determine the 74 importance of ADAR1 in the tumorigenesis of TNBC. By evaluating the TCGA database, we 75 found that while mRNA expression of ADAR1 was higher in TNBC compared to normal, it was 76 not significantly different between TNBC and non-TNBC tumors ( Figure 1B). Additionally, 77 ADAR1 expression was not significantly higher in any one subtype of breast cancer based on 78 PAM50 classification(Lehmann et al., 2016) (Supplemental Figure 1A). This observation is 79 consistent with data from the Cancer Cell Line Encyclopedia (CCLE), which uses both RNA-seq 80 and Reverse Phase Protein Array (RPPA) to determine RNA and protein expression levels in 81 numerous cancer cell lines (Supplemental Figure 1B-C). Data from both the TCGA and CCLE 82 datasets also revealed that both p150 and p110 isoforms of ADAR1 were expressed at similar 83 levels between TNBC and non-TNBC specimen (Supplemental Figure 1D-H), with p110 84 expression being consistently higher than p150 in all samples. Additionally, we assessed p150 85 isoform expression by immunohistochemistry in TNBC and non-TNBC patient tumors, Figure 86 1D. We sought to determine the protein expression level of the ADAR1-p150 isoform in a panel 87 of established breast ca...