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.

ObjectiveThe diversity of the tumour microenvironment (TME) of intrahepatic cholangiocarcinoma (iCCA) has not been comprehensively assessed. We aimed to generate a novel molecular iCCA classifier that incorporates elements of the stroma, tumour and immune microenvironment (‘STIM’ classification).DesignWe applied virtual deconvolution to transcriptomic data from ~900 iCCAs, enabling us to devise a novel classification by selecting for the most relevant TME components. Murine models were generated through hydrodynamic tail vein injection and compared with the human disease.ResultsiCCA is composed of five robust STIM classes encompassing both inflamed (35%) and non-inflamed profiles (65%). The inflamed classes, named immune classical (~10%) and inflammatory stroma (~25%), differ in oncogenic pathways and extent of desmoplasia, with the inflammatory stroma showing T cell exhaustion, abundant stroma and KRAS mutations (p<0.001). Analysis of cell–cell interactions highlights cancer-associated fibroblast subtypes as potential mediators of immune evasion. Among the non-inflamed classes, the desert-like class (~20%) harbours the lowest immune infiltration with abundant regulatory T cells (p<0.001), whereas the hepatic stem-like class (~35%) is enriched in ‘M2-like’ macrophages, mutations in IDH1/2 and BAP1, and FGFR2 fusions. The remaining class (tumour classical: ~10%) is defined by cell cycle pathways and poor prognosis. Comparative analysis unveils high similarity between a KRAS/p19 murine model and the inflammatory stroma class (p=0.02). The KRAS-SOS inhibitor, BI3406, sensitises a KRAS-mutant iCCA murine model to anti-PD1 therapy.ConclusionsWe describe a comprehensive TME-based stratification of iCCA. Cross-species analysis establishes murine models that align closely to human iCCA for the preclinical testing of combination strategies.

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...

OBJECTIVES/SPECIFIC AIMS: Triple-negative breast cancer (TNBC) accounts for one-fifth of the breast cancer patient population. The heterogeneous nature of TNBC and lack of options for targeted therapy make its treatment a constant challenge. The co-deficiency of tumor suppressors p53 and ARF is a significant genetic signature enriched in TNBC, but it is not yet clear how TNBC is regulated by this genetic alteration. METHODS/STUDY POPULATION: To answer this question, we established p53/ARF-defective murine embryonic fibroblast (MEF) to study the molecular and phenotypic consequences in vitro. Moreover, transgenic mice were generated to investigate the effect of p53/ARF deficiency on mammary tumor development in vivo. RESULTS/ANTICIPATED RESULTS: Increased transformation capability was observed in p53/ARF-defective cells, and formation of aggressive mammary tumors was also seen in p53-/-ARF-/- mice. RNA-editing enzyme ADAR1 was identified as a potential mediator for the elevated oncogenic potential. Interestingly, we found that the overexpression of ADAR1 is also prevalent in human TNBC cell lines and patient specimen. Using short hairpin RNA (shRNA) to reduce ADAR1 expression abrogated the oncogenic potential of human TNBC cell lines, while non-TNBC cells are less susceptible. Different levels of RNA editing of known ADAR1 targets were detected in shRNA-treated human TNBC cell lines, suggesting that ADAR1-mediated RNA editing contributes to TNBC pathogenesis. DISCUSSION/SIGNIFICANCE OF IMPACT: These results indicate critical roles played by the tumor suppressors p53 and ARF in the pathogenesis of TNBC, partially through affecting ADAR1-mediated RNA editing. Further understanding of this pathway could shed light on potential vulnerabilities of TNBC and inform the development of personalized therapies based on patients’ genetic signiatures.

Research Overview and Objective Triple-negative breast cancer (TNBC) accounts for one-fifth of the breast cancer patient population. The heterogeneous nature of TNBC and lack of options for targeted therapy make its treatment a constant challenge. The co-deficiency of tumor suppressors p53 and ARF is a significant genetic signature enriched in TNBC, but it is not yet clear how TNBC is regulated by this genetic alteration. Methods To answer this question, we established p53/ARF-defective murine embryonic fibroblast (MEF) to study the molecular and phenotypic consequences in vitro. Moreover, transgenic mice were generated to investigate the effect of p53/ARF deficiency on mammary tumor development in vivo. Results Increased transformation capability was observed in p53/ARF-defective cells, and formation of aggressive mammary tumors was also seen in p53−/−ARF−/− mice. RNA-editing enzyme ADAR1 was identified as a potential mediator for the elevated oncogenic potential. Interestingly, we found that the overexpression of ADAR1 is also prevalent in human TNBC cell lines and patient specimen. Using short hairpin RNA (shRNA) to reduce ADAR1 expression abrogated the oncogenic potential of human TNBC cell lines, while non-TNBC cells are less susceptible. Different levels of RNA editing of known ADAR1 targets were detected in shRNA-treated human TNBC cell lines, suggesting that ADAR1-mediated RNA editing contributes to TNBC pathogenesis. Implication/Discussion These results indicate critical roles played by the tumor suppressors p53 and ARF in the pathogenesis of TNBC, partially through affecting ADAR1-mediated RNA editing. Further understanding of this pathway could shed light on potential vulnerabilities of TNBC and inform the development of personalized therapies based on patients’ genetic signiatures. Citation Format: Che-Pei Kung, Catherine E Kuzmicki, Emily A Bross, Sua Ryu, Eric Freeman, Thwisha Sabloak, Emily R Bramel, Leonard B Maggi Jr, Jason D Weber. Tumor suppressors p53 and ARF control ADAR1-driven tumorigenicity in triple negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-08-03.

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