Transition from pluripotency to differentiation is a pivotal yet poorly understood developmental step. Here, we show that the tumour suppressor RASSF1A is a key player driving the early specification of cell fate. RASSF1A acts as a natural barrier to stem cell self-renewal and iPS cell generation, by switching YAP from an integral component in the β-catenin-TCF pluripotency network to a key factor that promotes differentiation. We demonstrate that epigenetic regulation of the Rassf1A promoter maintains stemness by allowing a quaternary association of YAP–TEAD and β-catenin–TCF3 complexes on the Oct4 distal enhancer. However, during differentiation, promoter demethylation allows GATA1-mediated RASSF1A expression which prevents YAP from contributing to the TEAD/β-catenin–TCF3 complex. Simultaneously, we find that RASSF1A promotes a YAP–p73 transcriptional programme that enables differentiation. Together, our findings demonstrate that RASSF1A mediates transcription factor selection of YAP in stem cells, thereby acting as a functional “switch” between pluripotency and initiation of differentiation.

The pancreas is a gland composed mainly by endocrine and exocrine cells, giving rise to three main tumour types. Pancreatic neuroendocrine tumour or PNET arise from the endocrine portion of the pancreas. On the contrary, pancreatic exocrine neoplasms include pancreatic ductal adenocarcinoma (PDAC) and acinar cell carcinoma. PDAC is the most common type of pancreatic cancer and one of the leading causes of cancer-related death. It has been shown that less than 3% of PDAC patients have an overall survival of up to 5 years in the U.K. This mainly arises since the majority of patients diagnosed with PDAC present with advanced unresectable disease, which is highly resistant to all forms of chemotherapy and radiotherapy. Activating mutations of an isoform of the RAS protein, KRAS, are found in almost all PDAC cases and occur during early stages of malignant transformation. KRAS mutations play a critical role as they are involved in both initiating and maintaining PDAC development. The interaction of RAS with GDP/GTP along with its recruitment to the membrane affects transduction of its activating signals to downstream effectors. In this review, we aim to summarise different mutations of RAS and their prevalence in pancreatic cancer along with other RAS-induced tumours. In addition, we briefly discuss the genetically engineered mouse models that have been developed to study KRAS-mutated adenocarcinomas in the pancreas. These provide an opportunity to also address the importance of targeting RAS for better treatment response in PDAC patients along with the challenges incurred herein.

BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDAC) is characterized by advanced disease stage at presentation, aggressive disease biology, and resistance to therapy, resulting in an extremely poor 5-year survival rate of <10%. PDAC is classified into transcriptional subtypes with distinct survival characteristics, although how these arise is not known. Epigenetic deregulation, rather than genetics, has been proposed to underpin progression, but exactly why is unclear and is hindered by the technical limitations of analyzing clinical samples. METHODS: We performed genome-wide epigenetic mapping of DNA modifications 5-methylcytosine and 5-hydroxymethylcytosine (5hmc) using oxidative bisulfite sequencing from formalinembedded sections. We identified overlap with transcriptional signatures in formalin-fixed, paraffin-embedded tissue from resected patients, via bioinformatics using iCluster and mutational profiling and confirmed them in vivo. RESULTS: We found that aggressive squamous-like PDAC subtypes result from epigenetic inactivation of loci, including GATA6, which promote differentiated classical pancreatic subtypes. We showed that squamous-like PDAC transcriptional subtypes are associated with greater loss of 5hmc due to reduced expression of the 5-methylcytosine hydroxylase TET2. Furthermore, we found that SMAD4 directly supports TET2 levels in classical pancreatic tumors, and loss of SMAD4 expression was associated with reduced 5hmc, GATA6, and squamous-like tumors. Importantly, enhancing TET2 stability using metformin and vitamin C/ascorbic acid restores 5hmc and GATA6 levels, reverting squamous-like tumor phenotypes and WNTdependence in vitro and in vivo. CONCLUSIONS: We identified epigenetic deregulation of pancreatic differentiation as an underpinning event behind the emergence of transcriptomic subtypes in PDAC. Our data showed that restoring epigenetic control increases biomarkers of classical pancreatic tumors that are associated with improved therapeutic responses and survival.

Background and Aims: Pancreatic ductal adenocarcinoma (PDAC) is characterised by advanced disease stage at presentation, aggressive disease biology and resistance to therapy resulting in extremely poor five-year survival <10%. PDAC is classified into transcriptional subtypes with distinct survival characteristics, although how these arise is not known. Epigenetic deregulation, rather than genetics, has been proposed to underpin progression but exactly why is unclear and hindered by analysis of clinical samples. Methods: Genome-wide epigenetic mapping of DNA modifications 5-hydroxymethylcytosine (5mc) and 5-hydroxymethylcytosine (5hmc) using oxidative bisulphite sequencing (oxBS). Bioinformatics using iCluster and mutational profiling to identify overlap with transcriptional signatures in FFPE from resected patients and confirmation in vivo. Results: We find that more aggressive squamous-like PDAC subtypes result from epigenetic inactivation of loci including GATA6 that promote differentiated classical-pancreatic subtypes. We show that squamous-like PDAC transcriptional subtypes are associated with greater loss of 5hmc due to reduced expression of the 5mc-hydroxylase TET2. Furthermore, we find that SMAD4 directly supports TET2 levels in the pancreas and classical-pancreatic tumors and loss of SMAD4 expression is associated reduced 5hmc, GATA6 and squamous-like tumors. Importantly, enhancing TET2 stability using Metformin and VitaminC/ascorbic acid (AA) restores 5hmc and GATA6 levels, reverting squamous-like tumor phenotypes and WNT-dependence in vitro and in vivo. Conclusions: We identify epigenetic deregulation of pancreatic differentiation as an underpinning event behind the emergence of transcriptomic subtypes in PDAC. Our data shows that restoring epigenetic control increases biomarkers of classical-pancreatic tumors and raises the possibility that combination of Vitamin C and Metformin may prolong survival in patients with squamous-like pancreatic cancer.

Purpose Multi-slice scanning in the abdomen and thorax of small animals is compromised by the effects of respiration unless imaging and respiration are synchronised. To avoid the signal modulations that result from respiration motion and a variable TR, blocks of fully relaxed slices are typically acquired during inter-breath periods, at the cost of scan efficiency. This paper reports a conceptually simple yet effective prospective gating acquisition mode for multi-slice scanning in free breathing small animals at any fixed TR of choice with reduced sensitivity to respiratory motion. Methods Multi-slice scan modes have been implemented in which each slice has its own specific projection or phase encode loop index counter. When a breath is registered RF pulses continue to be applied but data are not acquired, and the corresponding counters remain fixed so that the data are acquired one TR later, providing it coincides with an inter-breath period. The approach is refined to reacquire the slice data that are acquired immediately before each breath is detected. Only the data with reduced motion artefact are used in image reconstruction. The efficacy of the method is demonstrated in the RARE scan mode which is well known to be particularly useful for tumour visualization. Results Validation in mice with RARE demonstrates improved stability with respect to ungated scanning where signal averaging is often used to reduce artefacts. SNR enhancement maps demonstrate the improved efficiency of the proposed method that is equivalent to at least a 2.5 fold reduction in scan time with respect to ungated signal averaging. A steady-state magnetisation transfer contrast prepared gradient echo implementation is observed to highlight tumour structure. Supplementary simulations demonstrate that only small variations in respiration rate are required to enable efficient sampling with the proposed method. Conclusions The proposed prospective gating acquisition scheme enables efficient multi-slice scanning in small animals at the optimum TR with reduced sensitivity to respiratory motion. The method is compatible with a wide range of complementary methods including non-Cartesian scan modes, partially parallel imaging, and compressed sensing. In particular, the proposed scheme reduces the need for continual close monitoring to effect operator intervention in response to respiratory rate changes, which is both difficult to maintain and precludes high throughput.

740 Background: SCALOP was a multi-centre phase II RCT where 114 patients with LAPC were received 3 cycles of Gemcitabine and Capecitabine (GEMCAP) and those with stable/responding disease (n = 74) were randomised to Gem-RT or Cap-RT. The trial showed superiority of Cap-RT. Baseline blood samples of randomised patients were analysed for 35 circulating biomarkers. In vivo study was undertaken with candidate biomarker (CCL5) to test actionability. Methods: Patient bloods were tested using R&D multiplexed magnetic Luminex assays and IGF-1, TGF-b1 and b-NGF DuoSet ELISA. Orthotropic KrasG12D;P53R172H;PDXcre (KPC) tumors were implanted in Bl6-mice and treated with Gem, CCR5-inhibitor (CCR5i) maraviroc (MV), PD1 inhibitor (PD1i), PD1i+MV alone and in combination with MRI guided small animal Radiotherapy (RT). Immunophenotyping was performed by IHC and Aurora Cytek spectral flow cytometry. Results: Baseline biomarker data was available on 63/74 randomised patients. Of the 35 biomarkers tested, only CCL5 was found to be significantly associated with OS with a median OS of 18.5 (95% CI: 11.76-21.32) vs 11.3 (9.86-15.51) months (low vs high), and HR 1.37 (95% CI:1.04-3.65; p = 0.037) in the Cox multivariable model. Treatment of orthotopic KPC tumors revealed that combination of MV+PD1i+RT resulted in tumour growth inhibition and a switch of tumour macrophages from M2 to M1 accompanied by increase in infiltration of cytotoxic CD8+ Tcells and NK cells. Conclusions: Previous pre-clinical studies reported CCL5-CCR5 axis as a poor prognostic marker and a possible cause of immune-resistance in pancreatic cancer. Herein we have demonstrated in prospectively collected clinical trial blood samples that high circulating CCL5 is associated with poor prognosis in LAPC. CCR5 inhibitor in combination with RT+PD1i may overcome immune-resistance, and should be tested in clinical trials. Clinical trial information: 96169987 .

Background: Pancreatic ductal adenocarcinoma (PDAC) is the most prevalent form of pancreatic cancer with poor survival outcomes. Results from the clinic have demonstrated the lack of efficacy when either radiotherapy (RT) or immunotherapy are used as a monotherapy. Recent publications revealed a synergistic effect on RT-induced immune modulation and reduced immune suppression when the immunotherapy was administrated concurrently with RT in mouse models. Other publications demonstrate that immune evasion in PDAC depends on the CCL5/CCR5 axis to recruit immunosuppressive T-regulatory cells (Tregs) into the tumor microenvironment. Therefore, targeting the migration of Tregs through modulation of CCL5/CCR5 axis can potentially inhibit tumor growth in pancreatic cancer. Aim: This preclinical study is evaluating the impact of fractionated MR-Image Guided Radiotherapy (MR-IGRT) in combination CCR5 inhibitor and simultaneous inhibition of the immune checkpoint axis PD1/PD-L1 on pancreatic cancer. Methods: For the purpose of this study we generated a syngeneic orthotopic pancreatic mouse model. Tumor cells derived from the Lox-Stop-Lox (LSL)-KrasG12D; LSL-Trp53R172H; Pdx1-cre (KPC) mouse model are injected in the tail of the pancreas. We are able to monitor tumor growth using a respiratory motion desensitized T2-weighted MRI imager, allowing the generation of high-resolution and high-contrast MRI data. Taking advantage of in-house developed technology, the MR-IGRT was delivered using the Small Animal Radiation Research Platform (SARRP) in combination with MRI imaging to deliver MR-guided fractionated radiotherapy. Concurrently with the radiotherapy, CCR5 inhibitor (maraviroc) and PD1 inhibitor were administered at specific time points. To investigate the immunologic microenvironment, we developed a 17-color flow cytometry (FC) panel to immune-phenotype cytotoxic T, T regulatory, NK, NK/T and B cells, M-MDSC, PMN-MDSC, M1 and M2 macrophages in the peripheral blood and tumor infiltrate. Results/Conclusions: Using the established orthotopic mouse model, our aim is to investigate how the combination of MRI-guided radiotherapy and immune therapies can modulate the tumor microenvironment and the immune response in pancreatic cancer. This project is part of an ongoing preclinical study, and preliminary results will be presented at the meeting. Citation Format: Simone Lanfredini, Sophie Hughes, Asmita Thapa, Fiona Bangs, Jennifer Morton, Danny Allen, Veerle Kersemans, Paul Kinchesh, Sean Smart, Amy Elliot, James Thompson, Mark Hill, Somnath Mukherjee, Eric O'Neill. Assessment of CCR5i/maraviroc immunotherapy in combination with PD1 and MR-guided radiotherapy for treatment of 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 B30.

Background: mRNA datasets have defined two molecular subtypes of pancreatic ductal adenocarcinoma (PDAC), classical and squamous, with distinct clinical characteristics and raising the possibility of subtype specific therapies (Collisson et al., 2019). The aggressive squamous subtype is characterized by poorer patient survival and loss of endodermal differentiation markers GATA6 and PDX1, but neither PDAC subtypes nor metastasis can be explained by genetic mutations alone (Reiter et al., 2018). Conversely, widespread epigenetic reprogramming is associated with progression to more aggressive phenotypes (McDonald et al., 2017), suggesting that aggressive molecular subtype is likely to be epigenetically driven. The role of DNA 5’methylcytosine (5’mc) in PDAC has been previously unclear due to an inability to distinguish this mark from the reciprocal activation mark DNA 5’hydroxymethylcytosine (5’hmC), mediated by Ten-eleven-translocation (TET) enzymes. Moreover, 5’hmc is dynamically regulated during pancreatic differentiation, but a role during PDAC initiation or progression has not been previously addressed. Methods: PDAC patients who underwent surgical resection at the Churchill Hospital, Oxford (n=146) had FFPE tumor tissue mutation comprehensively profiled by illumina hotspot array (300X), mRNA microarray, and the first epigenetic separation of 5’mc and 5’hmc on FFPE via oxidative bisulphite sequencing (oxBS) and illumina EPIC arrays. iCluster was used to highlight epigenetic PDAC subtypes and compare to existing TCGA datasets. Orthotopic models were employed to test whether the Squamous subtype is defined by 5’hmc loss and if this can be converted to the classical subtype in vivo. Results: Compared to healthy tissue, PDAC tumors demonstrate loss of 5’hmC at genes critical for pancreatic development and associated with PDAC progression, including MAPK signaling and TP53 targets. 5’hmC was preferentially lost in aggressive molecular subtypes (squamous) compared to the classical subtypes and was associated with SMAD4 mutations and reduced TET2. Overexpression of TET2 in squamous tumor cells restored 5’hmc and the expression of classical associated genes (e.g., GATA6 and PDX1), suggesting that 5’hmC is a master epigenetic regulator of PDAC molecular subtypes. Notably, we find TET2 stability is regulated by glucose levels, implying the widespread hypoglycemia seen in PDAC patients may contribute to progression of aggressive subtypes through loss of 5’hmC. Concomitantly, as metformin and vitamin C independently enhance endogenous TET2 activity, we find that this combination acts synergistically on squamous tumors to increase 5’hmC and expression of a classical-subtype phenotype, suggesting subtype switching is achievable in vitro and in vivo. Conclusion: These results identify 5’hmC as a regulator of molecular subtype that can be targeted in vivo using well-tolerated drugs. This abstract is also being presented as Poster A39. Citation Format: Michael Eyres, Simone Lanfredini, Frances Willenbrock, Asmita Thapa, Andrew Blake, Adam Burns, Ahmad Sabbagh, Aswin Abraham, Timothy Maughan, Zahir Soonawalla, Anna Schuh, Somnath Mukhergee, Eric O'Neill. Loss of TET2 activity results in epigenetic instability and drives PDAC molecular subtypes [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 PR02.