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Despite efforts in understanding its underlying mechanisms, the etiology of chromosomal instability (CIN) remains unclear for many tumor types. Here, we identify CIN initiation as a previously undescribed function for APOBEC3A (A3A), a cytidine deaminase upregulated across cancer types. Using genetic mouse models of pancreatic ductal adenocarcinoma (PDA) and genomics analyses in human tumor cells we show that A3A-induced CIN leads to aggressive tumors characterized by enhanced early dissemination and metastasis in a STING-dependent manner and independently of the canonical deaminase functions of A3A. We show that A3A upregulation recapitulates numerous copy number alterations commonly observed in patients with PDA, including co-deletions in DNA repair pathway genes, which in turn render these tumors susceptible to poly (ADP-ribose) polymerase inhibition. Overall, our results demonstrate that A3A plays an unexpected role in PDA as a specific driver of CIN, with significant effects on disease progression and treatment. Woermann and colleagues describe a deaminase-independent function for APOBEC3A in initiating chromosomal instability and STING-dependent metastasis in human and mouse pancreatic cancer.

Adenosine deaminase acting on RNA 1 (ADAR1) is an RNA-binding protein that deaminates adenosine(A) to inosine(I). A-to-I editing alters post-transcriptional RNA processing making ADAR1 a critical regulator of gene expression. Consequently, Adar1 has been implicated in organogenesis. To determine the role of Adar1 in pancreatic development and homeostasis, we specifically deleted Adar1 from the murine pancreas (Ptf1aCre/+; Adar1Fl/Fl). The resulting mice had stunted growth likely due to malabsorption associated with exocrine pancreas insufficiency. Analyses of pancreases revealed ductal expansion, heightened interferon-stimulated gene expression and an increased influx of immune cells. In addition, we observed an increased prevalence of CD4+ T and natural killer cells in their splenic tissue. These results indicate an association between loss of pancreatic Adar1 with dysregulation of systemic immunity. Concurrent deletion of Adar1 and Mavs, a signaling protein implicated in the innate immune pathway rescued the degenerative phenotype and resulted in normal pancreatic development. Taken together, our work suggests that the primary function of Adar1 in the pancreas is to prevent aberrant activation of the Mavs-mediated innate immune pathway, thereby maintaining pancreatic homeostasis.Summary statementThis work defines the role of Adar1 in pancreatic development and homeostasis.

While genetic mutations have been shown to drive pancreatic ductal adenocarcinoma (PDAC), epigenomic alterations in cancer cells may also contribute to tumor initiation and progression. Adenosine-to-inosine (A-to-I) editing is the most common type of RNA editing in the cell and is mediated by the adenosine deaminase acting on RNA (ADAR) enzyme. ADAR has been shown to be upregulated in various tumor types, it improves tumor cell viability and loss of Adar1 in melanoma cells increases efficacy of checkpoint blockade therapy in vivo. However, its function in PDAC is currently not well understood. In order to investigate the role of Adar1 in PDAC we developed a mouse model with a conditional knockout of Adar1 in the pancreas using the pancreas specific ptf1a Cre. However, the CYA (ptf1a [cre/wt], Rosa26 [LSL-YFP/LSL-YFP], Adar [F/F]) mice are born runted compared to littermate controls and die preweaning. We find that a conditional loss of Adar1 in the pancreas induces acinar cell death and the pancreatic tissue degenerates over a period of fifteen days. Adar1 inhibits interferon (IFN) responses that are activated by cellular double-stranded RNA (dsRNA) sensors through mitochondrial antiviral signaling protein (Mavs). To rescue the apoptotic and inflammatory phenotype of the CYA mice, we developed a CYAM mouse (ptf1a [cre/wt], Rosa26 [LSL-YFP/LSL-YFP], Adar [F/F], Mavs [-/-]). We find that these mice develop normally and their pancreas is devoid of apoptotic cells. Furthermore, to understand the function of Adar1 in pancreas homeostasis of adult mice, we developed an inducible model wherein in adult CiYA (ptf1aCre-ERTM [cre/wt], Rosa26 [LSL-YFP/LSL-YFP], Adar [F/F]) Adar1 loss was induced with intraperitoneal tamoxifen injections. We find that loss of Adar1 in the CiYA adult pancreas induces cell death and inflammation and the mice decline rapidly. We tested expression of a panel of 5 ISGs (Mx1, Isg15, IfnB1, Ifit1, Ifit2) by RT-qPCR and show that there is an elevated expression of ISGs in the pancreas of CiYA mice compared to WT controls. In conclusion, we show that pancreatic acinar cells are extremely sensitive to Adar1 loss both during developmental stages and in adult mice. Current in vivo models to study the function of Adar1 in development and cancer are limited by the lethal phenotype caused by upregulation of interferon stimulated genes (ISGs) and cell death in tissues where Adar1 is knocked out. Our work establishes a mouse model wherein pancreas-specific role of Adar1 in development and cancer can be studied without activation of Mavs-mediated inflammation. We are currently exploring how the CYAM mouse model can be utilized to advance our understanding of Adar1’s role in pancreas development and cancer. Citation Format: Dhwani N. Rupani, Robert W. Cowan, Andrew D. Rhim. Loss of Adar1 in pancreatic acinar cells leads to cell apoptosis and inflammation [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr A45.

The transferrin receptor is the primary means of iron importation for most mammalian cells and understanding its regulatory mechanisms is relevant to hematologic, oncologic, and other disorders in which iron homeostasis is perturbed. The 3′ UTR of the transferrin receptor mRNA contains an instability element that is protected from degradation during iron depletion through interactions of iron regulatory proteins (IRPs) with five iron-responsive elements (IREs). The structural features required for degradation and the site of IRP binding required for in situ protection remain unclear. An RNA-CLIP strategy is described here that identifies the predominant site of IRP-1 interaction within a nontransformed primary cell line. This approach avoided complications associated with the use of elevated concentrations of protein and/or mRNA and detected interactions within the native environment of the mRNA. A compensatory mutagenesis strategy indicates that the instability element at minimum consists of three non-IRE stem–loops that can function additively, suggesting that they are not forming one highly interdependent structure. Although the IREs are not essential for instability, they enhance instability when IRP interactions are inhibited. These results are supportive of a mechanism for a graded response to the intracellular iron resulting from a progressive loss of IRP protection.