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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.

PurposeThe clinical utility of circulating tumor DNA (ctDNA) has been shown in advanced pancreatic ductal adenocarcinoma (PDA). However, diagnostic sensitivity of many ctDNA assays is low in resectable and locally advanced disease, where tumor burden is substantially lower. We have previously described Multiplex Enrichment using Droplet Pre-Amplification (MED-Amp), a multiplexed panel for the detection of the most common oncogenic KRAS mutations in PDA. In this study, we aimed to assess the diagnostic sensitivity of MED-Amp for detection of rare mutant alleles present in the plasma of patients with localized PDA.Experimental DesignWe retrospectively analyzed ninety-eight plasma samples from 51 patients with various stages of localized disease. For comparison, we measured ctDNA levels in 20 additional patients with metastatic PDA. The MED-Amp assay was used to measure the abundance of the four most common KRAS codon 12 mutations (G12C/D/R/V). We correlated the presence and quantity of ctDNA with overall survival (OS) as well as progression-free survival (PFS). Using serial plasma draws, we also assessed the relationship between changes in ctDNA allelic frequency and progression.ResultsKRAS-positive ctDNA was detected in 52.9% of localized PDA and 75% of metastatic samples tested using DNA inputs as low as 2 ng. As previously reported, the presence of KRAS mutant ctDNA was correlated with worse OS for all disease stages (p = 0.02). In patients with localized PDA high ctDNA levels also correlated with significantly worse median OS (533 days vs 1090 days) and PFS (192 days vs 787 days). We also studied a small cohort of serial plasma draws to observe the relationship between ctDNA fold change and PFS. We found 83% of patients with increased fold change in mutant KRAS experienced disease progression (n=6). In contrast, 75% (n=4) of patients with decreased fold change remained disease-free (p=0.03).ConclusionsMED-Amp is a flexible and cost-effective approach for measurement of ctDNA in patients with localized cancer. Though this study focused on KRAS mutation detection, this assay could be adapted for a number of common oncogenic alterations.Statement of translational relevanceOnly 25% of pancreatic ductal adenocarcinoma (PDA) patients with localized disease survive five years post-resection. It is hypothesized PDA undergoes dissemination at the earliest stages of tumor formation, driving formation of occult metastases which go undetected using conventional screening methods. Development of high specificity, high sensitivity biomarkers is critical to improving patient mortality. Circulating tumor DNA (ctDNA) has gained increasing acceptance as a non-invasive prognostic in metastatic disease. However, the sensitivity of most targeted ctDNA assays precludes reliable detection of localized and resected disease. Here, we present a digital droplet PCR assay for multiplexed enrichment and detection of KRAS mutations, the most commonly mutated oncogene in PDA. This assay preserves ctDNA allelic frequency in the original sample, while increasing the molecular signal over 50-fold. This study shows the ctDNA has potential diagnostic value in early-stage PDA, and that digital preenrichment of cell-free DNA increases overall assay sensitivity without sacrificing specificity.

INTRODUCTION: Chronic pancreatitis is associated with an increased risk of developing pancreatic cancer, and patients with inherited forms of pancreatitis are at greatest risk. We investigated whether clinical severity of pancreatitis could also be an indicator of cancer risk independent of etiology by performing targeted DNA sequencing to assess the mutational burden in 55 cancer-associated genes. METHODS: Using picodroplet digital polymerase chain reaction and next-generation sequencing, we reported the genomic profiles of pancreases from severe clinical cases of chronic pancreatitis that necessitated palliative total pancreatectomy with islet autotransplantation. RESULTS: We assessed 57 tissue samples from 39 patients with genetic and idiopathic etiologies and found that despite the clinical severity of disease, there was no corresponding increase in mutational burden. The average allele frequency of somatic variants was 1.19% (range 1.00%–5.97%), and distinct regions from the same patient displayed genomic heterogeneity, suggesting that these variants are subclonal. Few oncogenic KRAS mutations were discovered (7% of all samples), although we detected evidence of frequent cancer-related variants in other genes such as TP53, CDKN2A, and SMAD4. Of note, tissue samples with oncogenic KRAS mutations and samples from patients with PRSS1 mutations harbored an increased total number of somatic variants, suggesting that these patients may have increased genomic instability and could be at an increased risk of developing pancreatic cancer. DISCUSSION: Overall, we showed that even in those patients with chronic pancreatitis severe enough to warrant total pancreatectomy with islet autotransplantation, pancreatic cancer–related mutational burden is not appreciably increased.

Introduction: Apolipoprotein B mRNA editing enzyme, catalytic polypeptide 3A (A3A), is an endogenous deaminase acting upon gDNA at specific sequence motifs to create C>T edits and is upregulated in a number of cancers. Large-scale sequencing studies have demonstrated an enriched APOBEC mutational signature in a variety of solid tumors, indicating a strong mutagenic role of APOBEC in cancer. Although A3A has been found to be overexpressed and associated with poor overall survival in hPDACs (TCGA), a rather weak APOBEC mutational signature is present in hPDACs, leading us to the hypothesis that A3A may drive independent of its ability to create point mutations, an aggressive tumor burden in hPDAC. Here, we established a genetically engineered mouse model (GEMM) to unravel the novel function of A3A beyond its deaminase activity in PDAC. Methods: Unlike humans, mice have only one APOBEC3 isoform, lacking genomic DNA deaminase activity. Thus, to model A3A independent of its deaminase function in PDAC we generated a A3A (Exon1/2) transgenic mouse that we bred to a well-established GEMM of PDAC, yielding LSL-KrasG12D; p53fl/+; Pdx1-Cre; RosaLSL-YFP; hAPOBEC3A+/- (hA3A; PKCY) mice. hA3A;PKCY tumors were genotyped with Ilumina exome capture sequencing, whole-genome sequencing, and comparative RNAseq. In addition, PKCY tumors cells expressing deaminase deficient and competent A3A were analyzed for chromosomal instability (CIN) and metastatic capacity. Results: Although high expression of A3A was associated with significantly shorter survival in hPDACs (p=0.04; n=178 (TCGA)), levels of A3A did not correlate with the overall mutational rate. Interestingly, deaminase-deficient A3A mice (hA3A;PKCY) (n=29) expired significantly faster than PKCY (n=40) controls (p Conclusions: Our study suggests that A3A beyond deaminase function and creating point mutations may drive CIN and a more aggressive and metastatic phenotype in PDAC. As opposed to mutagenesis, we found that the more relevant and novel deaminase independent function of A3A is initiation of widespread CIN, SVs and copy number alterations. We unraveled an A3A-mediated BRCAness signature of deep deletion that could be used in PDAC patients to predict tumor biology, outcome, and therapeutic response. This abstract is also being presented as Poster A56. Citation Format: Sonja Maria Woermann, Robert Cowan, Susan M. Ross, Andrew D. Rhim. A novel deaminase independent function of APOBEC3A catalyzes widespread chromosomal instability to drive an aggressive metastatic phenotype in pancreatic cancer [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 PR03.

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 progression from normal cells to invasive pancreatic ductal adenocarcinoma (PDAC) requires the accumulation of multiple inherited or acquired mutations. Activating point mutations in the KRAS oncogene are prevalent in pancreatic cancer and result in the stimulation of several pathways including the RAF-mitogen-activated protein kinase pathway and the phosphoinositide 3-kinase pathway. Other genetic alterations, including telomere shortening and the inactivation of tumor suppressor genes such as CDKN2A, TP53, and SMAD4, which encode p16, p53, and SMAD4, respectively, also contribute to the progression of pancreatic cancer. These, and other genetic events, can present at different stages in the development of PDAC at histologically defined precursor lesions known as pancreatic intraepithelial neoplasia, intraductal papillary mucinous neoplasms, or mucinous cystic neoplasms. Each precursor lesion represents alternate routes to PDAC formation and has a unique presentation and somewhat distinct genetic events controlling its development. Despite the advances in the understanding of the genetics of PDAC, the prognosis for this cancer remains poor, and several important aspects of its pathogenesis must be clarified to improve therapeutics, including the timing and method of metastases, as well as the relationship of the tumor cells with the desmoplastic stroma, which is a characteristic feature of the cancer. This review discusses the principal genetic alterations in PDAC and its precursor lesions, including their effects on promoting carcinogenesis.

Introduction: Apolipoprotein B mRNA Editing enzyme, Catalytic polypeptide 3A (A3A) is a C to U cytidine deaminase, preferentially deaminating the lagging strand of double-stranded DNA at specific sequence motifs. A3A is overexpressed across multiple tumor types, and in pancreatic ductal adenocarcinoma (PDA), high expression is associated with significantly decreased survival in early stage patients. Large scale sequencing studies of various human tumors (TCGA and ICGC), including PDA, have implicated A3A as a potential driver of tumor mutagenesis. However, the biological relevance of the deamination function of A3A has not yet been shown. Here, we utilize a novel genetically engineered mouse model to show that A3A supports the development of aggressive PDA independent of mutagenic capabilities. Methods: As opposed to humans, mice only contain one APOBEC3 isoform which has limited to no deaminating activity on genomic DNA. Thus, to dissect the function of A3A on PDA initiation and progression, we made mice with a germline knockin for the coding sequence for human A3A and bred to a well-established GEMM of PDA to yield LSL-KrasG12D; p53fl/+; Pdx1-Cre; RosaLSL-YFP; A3A+/- (KPCY;A3A) mice and compared to KPCY mice. We genotyped tumors from both cohorts using VarScan after exome capture sequencing. Tumor and immune cells were FACS sorted comparative RNAseq performed. The immune contexture of murine and 155 untreated and treated human PDAs was analyzed by performing 7-color multiplex staining and immune cell spatial interaction analysis. Results: All KPCY;A3A (n=11) mice developed tumors and expired significantly faster compared to KPCY (n=18) controls (3.9 v. 7.0 mo; p Conclusions: Counter to our hypothesis, A3A supports the initiation and growth of PDA in vivo through effects independent of deamination or mutagenesis of cancer cell genomes but rather indirectly through suppression of the tumor immune response. Future studies will address the precise mechanisms underlying A3A-mediated tumor immune evasion. Citation Format: Sonja Woermann, Robert Cowan, Susan M. Ross, Andrew D. Rhim. APOBEC3A inhibits tumor immune response independent of deamination in a novel genetic model of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4751.

Background & Aims Intraductal papillary mucinous neoplasias (IPMNs) are precancerous cystic lesions that can develop into pancreatic ductal adenocarcinomas (PDACs). These large macroscopic lesions are frequently detected during medical imaging, but it is unclear how they form or progress to PDAC. We aimed to identify cells that form IPMNs and mutations that promote IPMN development and progression. Methods We generated mice with disruption of Pten specifically in ductal cells ( Sox9CreER T2 ; Pten flox/flox ; R26R YFP or Pten ΔDuct/ΔDuct mice) and used Pten ΔDuct/+ and Pten +/+ mice as controls. We also generated Kras G12D ; Pten ΔDuct/ΔDuct and Kras G12D ; Pten ΔDuct/+ mice. Pancreata were collected when mice were 28 weeks to 14.5 months old and analyzed by histology, immunohistochemistry, and electron microscopy. We performed multiplexed droplet digital polymerase chain reaction to detect spontaneous Kras mutations in Pten ΔDuct/ΔDuct mice and study the effects of Ras pathway activation on initiation and progression of IPMNs. We obtained 2 pancreatic sections from a patient with an invasive pancreatobiliary IPMN and analyzed the regions with and without the invasive IPMN (control tissue) by immunohistochemistry. Results Mice with ductal cell–specific disruption of Pten but not control mice developed sporadic, macroscopic, intraductal papillary lesions with histologic and molecular features of human IPMNs. Pten ΔDuct/ΔDuct mice developed IPMNs of several subtypes. In Pten ΔDuct/ΔDuct mice, 31.5% of IPMNs became invasive; invasion was associated with spontaneous mutations in Kras . Kras G12D ; Pten ΔDuct/ΔDuct mice all developed invasive IPMNs within 1 month. In Kras G12D ; Pten ΔDuct/+ mice, 70% developed IPMN, predominately of the pancreatobiliary subtype, and 63.3% developed PDAC. In all models, IPMNs and PDAC expressed the duct-specific lineage tracing marker yellow fluorescent protein. In immunohistochemical analyses, we found that the invasive human pancreatobiliary IPMN tissue had lower levels of PTEN and increased levels of phosphorylated (activated) ERK compared with healthy pancreatic tissue. Conclusions In analyses of mice with ductal cell–specific disruption of Pten , with or without activated Kras, we found evidence for a ductal cell origin of IPMNs. We also showed that PTEN loss and activated Kras have synergistic effects in promoting development of IPMN and progression to PDAC.

Introduction: The apolipoprotein B editing complex 3 (APOBEC3) family of enzymes are possible candidates for inducing mutations across a number of tumors, including pancreatic ductal adenocarcinoma (PDA; Alexandrov et al, 2013; Roberts et al, 2013). APOBEC3A (hA3A) is one of eight identified isoforms in humans and known to deaminate cytidines in genomic ssDNA at a specific sequence motif. Large scale sequencing studies demonstrate significant enrichment of this signature in a number of human cancers. Furthermore, hA3A is overexpressed in a variety of solid tumors, including PDA. Taken together, these data suggest that hA3A may catalyze point mutations in cancer and drive cancer initiation and progression. However, direct evidence to support this hypothesis in vivo are lacking. Here, we utilize a novel genetically engineered mouse model to determine the precise effects of hA3A on PDA initiation and progression. Methods: As opposed to humans, mice only contain one APOBEC3 isoform which has limited to no deaminating activity on genomic DNA. Thus, to delineate the function of hA3A on PDA initiation and progression, we exchanged the murine APOBEC3 protein coding sequence for hA3A, leaving the murine 5’ and 3’ endogenous regulatory sequences intact to ensure physiologic expression (murine APOBEC3 and hA3A reflect similar expression kinetics during PDA development). We then bred these mice to a well-established GEMM of PC (Rhim et al., Cell 2012) to yield LSL-KrasG12D; p53fl/+; Pdx1-Cre; RosaLSL-YFP; hA3A+/- (KPCY;hA3A) mice. We compared these mice to KPCY mice. We genotyped tumors from both cohorts with Ilumina exome capture sequencing with variant calling by a custom caller based on VarScan. Results: All KPCY; hA3A (n=11) mice developed tumors and expired significantly faster compared to KPCY (n=18) controls (3.9 v. 7.0 mo; p Conclusions: These data show for the first time that physiologic expression of hA3A in vivo leads to increased mutations, altered immune response and more aggressive cancer. Future studies will address the precise mechanisms by which hA3A catalyzes tumorigenesis using our novel gain of function model. Furthermore, our data suggest that incorporating our humanized A3A germline allele may provide a more genomically recapitulative model of cancer. Citation Format: Sonja Woermann, Robert Cowan, Susan M. Ross, Andrew D. Rhim. Direct evidence for a pro-tumor role of APOBEC3A in cancer initiation and progression in vivo: enhanced mutagenesis and immune suppression in a novel humanized autochthonous model of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1035. doi:10.1158/1538-7445.AM2017-1035