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During B cell activation, the DNA lesions that initiate somatic hypermutation and class switch recombination are introduced by activation-induced cytidine deaminase (AID). AID is a highly mutagenic protein that is maintained in the cytoplasm at steady state, however AID is shuttled across the nuclear membrane and the protein transiently present in the nucleus appears sufficient for targeted alteration of immunoglobulin loci. AID has been implicated in epigenetic reprogramming in primordial germ cells and cell fusions and in induced pluripotent stem cells (iPS cells), however AID expression in non-B cells is very low. We hypothesised that epigenetic reprogramming would require a pathway that instigates prolonged nuclear residence of AID. Here we show that AID is completely re-localised to the nucleus during drug withdrawal following etoposide treatment, in the period in which double strand breaks (DSBs) are repaired. Re-localisation occurs 2-6 hours after etoposide treatment, and AID remains in the nucleus for 10 or more hours, during which time cells remain live and motile. Re-localisation is cell-cycle dependent and is only observed in G2. Analysis of DSB dynamics shows that AID is re-localised in response to etoposide treatment, however re-localisation occurs substantially after DSB formation and the levels of re-localisation do not correlate with γH2AX levels. We conclude that DSB formation initiates a slow-acting pathway which allows stable long-term nuclear localisation of AID, and that such a pathway may enable AID-induced DNA demethylation during epigenetic reprogramming.

The CD155/TIGIT axis can be co-opted during immune evasion in chronic viral infections and cancer. While insights regarding mechanisms of immune escape have fueled tremendous clinical successes in a broad range of tumor types, microsatellite-stable pancreatic adenocarcinoma (PDAC), which represents greater than 98% of patients, is largely refractory to available immune checkpoint blockade. The recent recognition that a subset of pancreas cancer harbors potential neoantigens has intensified interest in defining the molecular and cellular mechanisms of immune evasion in PDAC to guide effective therapeutic strategies that leverage the adaptive immune system in this disease. However, difficulty in precisely defining the tumor-reactive T cell compartment has hampered prior efforts to delineate the full spectrum of mechanisms by which PDAC evades immune eradication. We recently developed multiple organoid-based and autochthonous preclinical models to delineate the molecular and cellular mechanisms of immune evasion in this subset of patients. Specifically, we utilized a high affinity MHC class I-restricted antigen (OVA257–264) or recently described missense-derived MHC class I-restricted neoantigens (mutations in the laminin 4 alpha subunit or in alpha-1,3-glucosyltransferase). While a subset of animals either successfully clear these tumors, or are arrested in a state of immune-mediated tumor dormancy, all three neoantigen models lead to a significant subset of tumors that acquire the ability to evade immune clearance. In these preclinical models, we demonstrate that intratumoral neoantigen-specific CD8+ T cells adopt multiple states of dysfunction, typified by T cell exhaustion (best marked by TIGIT+PD1+ co-positivity). Global profiling of the tumor-immune microenvironment in neoantigen-expressing murine PDAC compared to non-neoantigen-expressing PDAC identified an expected increase in CD8+ T cells, and specifically in neoantigen-specific CD8+ T cells, but also identified additional changes within the broader leukocyte contexture. Using flow cytometric and single-cell transcriptomic analyses, we demonstrate that human PDAC TILs express similar dysfunctional programs and are enriched in TIGIT+PD1+ TILs. Furthermore, we show that CD155, the high affinity TIGIT ligand, is highly expressed on the surface of murine and human PDAC tumor cells. Using genetic (CRISPR-activation) and/or pharmacologic modulation, we functionally interrogate the CD155/TIGIT axis and demonstrate that increased signaling through CD155 and/or TIGIT is sufficient to promote immune evasion in neoantigen-expressing PDAC. Lastly, we identified a combination immunotherapy (TIGIT/PD-1 co-blockade plus CD40 agonism) that elicits profound anti-tumor responses in these preclinical models. Using a suite of high-resolution analyses, we are dissecting the mechanisms of effectiveness and resistance to this combination immunotherapy, which is set to enter clinical trials in pancreatic cancer later this year. Citation Format: William Freed-Pastor, Laurens Lambert, Zackery Ely, Nimisha Pattada, Arjun Bhutkar, Alex Jaeger, George Eng, Kim Mercer, William Hwang, Tyler Jacks. Functional interrogation of immune escape in neoantigen-expressing pancreatic cancer identifies a critical role for the CD155/TIGIT axis [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-063.

Pancreatic adenocarcinoma (PDAC) carries a dismal prognosis and efforts to harness immune-based therapies are in high demand. Using retrograde pancreatic duct instillation of lentiviral particles, we have established and characterized a preclinical autochthonous mouse model for immunogenic PDAC. Specifically, pancreatic tumors are initiated in KRASLSL-G12D/+; p53flox/flox (KP) mice using lentiviral particles expressing Cre recombinase and a tumor-specific antigen (TSA) to track the endogenous antigen-specific T cell response in both precursor lesions (PanIN) and adenocarcinoma (PDAC). Using this model system, we demonstrate profound immunoediting in the autochthonous pancreas, which occurs primarily at the preinvasive (PanIN) stage. In parallel, we derived a biologic library of immunogenic or nonimmunogenic pancreatic organoids and developed an ex vivo co-culture system of pancreatic organoids with antigen-specific CD8 T cells that recapitulates the phenomena of immunoediting. Additionally, we examined the effect of TSA expression in established adenocarcinomas and performed preclinical trials in which the CD8 T-cell compartment was transiently depleted and then restored, to evaluate at what point during tumor progression pancreatic tumors gain the ability to evade immune clearance. Intriguingly, we identified both “regressor” tumors as well as “progressor” tumors in response to CD8 restoration. Longitudinal imaging was paired with flow cytometric analysis to track both clinical and antigen-specific T-cell responses. Excitingly, we found that a subset of “regressor” tumors undergo a complete pathologic response following CD8 T-cell restoration, highlighting the translational potential for novel insights regarding immune escape in PDAC. In addition, we demonstrate that both the magnitude and kinetics of the antigen-specific T-cell response tracks with the clinical response of these tumors, with “progressor” tumors eliciting both delayed kinetics and a blunted response compared with “regressor” tumors. Additionally, we developed an immunogenic genetically defined organoid system in which KP organoids were engineered to express a defined neoantigen from a safe harbor locus. In line with prior studies, a subset of immunogenic organoids was completely rejected upon orthotopic transplantation and this clearance was dependent upon CD8 T cells. However, in contrast to observations using two-dimensional monolayer cell lines, we also identified a significant subset of orthotopically transplanted immunogenic pancreatic organoids that successfully evade immune clearance, despite eliciting an antigen-specific immune response. Immunophenotyping the tumor microenvironment revealed that antigen-specific CD8 T cells within these “escaper” tumors display hallmarks of T-cell exhaustion. These complementary systems now provide a robust preclinical platform to rapidly interrogate potential therapeutic interventions. This abstract is also being presented as Poster C17. Citation Format: William A. Freed-Pastor, Laurens Lambert, Ana P. Garcia, George Eng, Omer Yilmaz, Tyler Jacks. Preclinical models to dissect immune escape 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 PR14.

Objective: Pancreatic ductal adenocarcinoma (PDAC) remains a treatment-refractory disease as existing molecular subtypes are insufficient and do not currently inform clinical decisions. Rare cell types, including those responsible for resistance, are difficult to detect with bulk transcriptomic profiling. Indeed, several previously identified transcriptomic subtypes of PDAC are unintentionally driven by “contaminating” stromal components. Single-cell transcriptomics provides an unprecedented degree of resolution into the properties of individual cells. However, RNA extraction from RNase- and stroma-rich pancreatic tissue is difficult and prior single-cell efforts have been limited by suboptimal dissociation/RNA quality. We developed a robust single-nucleus RNA-seq (sNuc-seq) technique compatible with frozen archival PDAC specimens and computational techniques to identify the transcriptomic programs driving tumor subtypes and therapeutic resistance. Methods: Patients with localized PDAC undergoing surgical resection with or without neoadjuvant chemoradiotherapy were consented for this IRB-approved study. Specimens were screened for RNA Integrity Number >6. Single nuclei suspensions were extracted from flash-frozen primary PDAC specimens and organoids. Approximately 8,000 nuclei were loaded on the 10x Genomics Chromium platform per sample to generate and sequence 3’ gene expression libraries (Illumina HiSeq 2500, 125 bp paired-end reads). sNuc-seq derived reads were processed using the 10X CellRanger v3.0.2 pipeline. Unsupervised clustering was utilized to identify different cell populations and known marker genes from literature were used to label cell types. Results: Both treatment-naïve (n=12) and treatment-resistant (n=11) specimens yielded high-quality sNuc-seq data (>1,000 nuclei per sample, >1,000 median genes per nucleus). In each tumor, distinct clusters with gene expression profiles consistent with ductal, fibroblast, endothelial, endocrine, lymphocyte, and myeloid cell populations were identified. Malignant cells were confirmed by inferred copy number variation analysis (InferCNV v3.9) and segregated into several distinct clusters for each individual patient highlighting intratumoral heterogeneity. While some malignant clusters corresponded to previously identified basal-squamous and classical-progenitor bulk subtypes, others featured expression profiles distinct from known subtypes, including cells with upregulation of hypoxia-associated or cytoskeletal genes. Conclusions: Applying sNuc-seq to treatment-naïve and pretreated PDAC specimens, we uncovered significant intratumoral heterogeneity in the malignant and stromal compartments and identified malignant cells featuring transcriptomic programs that do not fit previously identified bulk subtypes. Characterization of therapeutic resistance programs, spatial relationships among cell types, and association with clinical outcomes is ongoing. Citation Format: William L. Hwang, Karthik A. Jagadeesh, Orr Ashenberg, Eugene Drokhlyansky, George Eng, Nicholas Van Wittenberghe, William Freed-Pastor, Clifton Rodriguez, Danielle Dionne, Julia Waldman, Michael Cuoco, Alexander Tsankov, Connor Lambden, Caroline Porter, Jason Schenkel, Laurens Lambert, Debora Ciprani, Andrew J. Aguirre, Mari Mino-Kenudson, Theodore S. Hong, Orit Rozenblatt-Rosen, Carlos Fernandez-del Castillo, Andrew S. Liss, Aviv Regev, Tyler E. Jacks. Molecular subtypes and resistance programs in pancreatic ductal adenocarcinoma elucidated with single-nucleus RNA-seq [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 A22.