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8-Nitro-2′-deoxyguanosine (8-nitrodG) is a relatively unstable, mutagenic lesion of DNA that is increasingly believed to be associated with tissue inflammation. Due to the lability of the glycosidic bond, 8-nitrodG cannot be incorporated into oligodeoxynucleotides (ODNs) by chemical DNA synthesis and thus very little is known about its physicochemical properties and base-pairing preferences. Here we describe the synthesis of 8-nitro-2′-O-methylguanosine, a ribonucleoside analogue of this lesion, which is sufficiently stable to be incorporated into ODNs. Physicochemical studies demonstrated that 8-nitro-2′-O-methylguanosine adopts a syn conformation about the glycosidic bond; thermal melting studies and molecular modelling suggest a relatively stable syn-8-nitroG·anti-G base pair. Interestingly, when this lesion analogue was placed in a primer-template system, extension of the primer by either avian myeloblastosis virus reverse transcriptase (AMV-RT) or human DNA polymerase β (pol β), was significantly impaired, but where incorporation opposite 8-nitroguanine did occur, pol β showed a 2:1 preference to insert dA over dC, while AMV-RT incorporated predominantly dC. The fact that no 8-nitroG·G base pairing is seen in the primer extension products suggests that the polymerases may discriminate against this pairing system on the basis of its poor geometric match to a Watson–Crick pair.

e14094 Background: Wnt signalling initiates oncogenic pathways involved in tumour initiation, growth, differentiation and metastasis.1 Targeting the Wnt pathway is an attractive approach to cancer treatment. Porcupine (PORCN) is a membrane-bound O-acyltransferase dedicated to the palmitoylation of Wnt ligands, an essential step in the processing of Wnt into active ligands.2 A PORCN inhibitor may benefit patients with cancers in which Wnt signalling is implicated. It has been shown that PORCN inhibition is efficacious in cell lines with upstream mutations in this pathway, eg RNF43 loss of function.3 Methods: Analysis of TCGA data was carried out to identify cancer types harbouring RNF43 mutations.4 Redx PORCN inhibitor RXC004 was evaluated in vitro for Wnt pathway inhibition and anti-proliferative effects using assays previously described for early lead compounds.5Anti-tumour effects of RXC004 were evaluated using a pancreatic CAPAN-2 xenograft in SCID Bg. mice and a gastric PDX model in nu/nu mice. Both models have RNF43 loss of function mutations. Results: TCGA data revealed that both gastric cancer and pancreatic cancer exhibit mutations in the RNF43 gene (18% and 4% respectively),4expected to result in loss of function. RXC004 showed activity in preclinical models of cancers with this genetic background. RXC004 significantly inhibited tumour growth in a CAPAN-2 pancreatic cancer xenograft and in a gastric cancer PDX model when dosed orally once or twice daily. Conclusions: Taken together, these data indicate that RXC004 is a potent inhibitor of tumour growth in RNF43 loss of function preclinical cancer models. RXC004 will be evaluated in a first in human Phase 1 study in 2017. Phase 1a aims are to characterise the safety profile and PK/PD relationship in cancer patients and establish a Phase 1b dose. The efficacy of RXC004 in gastric and pancreatic cancer patients prospectively selected for RNF43 loss of function mutations will be the focus of Phase 1b expansion groups. 1 . Nat. Rev. Cancer, 2013, 13 (1): 11-26.2. J. Biol. Chem., 2012, 287 (27): 23246-23254. 3. PNAS, 2013, 110 (31): 12649-12654 4. Sci. Signal, 2013, 269 (6): 1-34; Cancer Discov., 2012, 2 (5): 401-404 5. AACR Annual Meeting, 2015, 75 (15): abs. 5071.

Background: RXC004, a potent and selective porcupine (PORCN) inhibitor, is being investigated in a safety and tolerability study in cancer patients with solid tumours (CT 2017-000720-98). In addition to the tumour targeting role of RXC004 and other Wnt pathway inhibitors, we present pre-clinical data which suggests further potential for RXC004 in modulating the immune system of the tumour microenvironment. Materials and Methods: To evaluate the potential of a novel porcupine inhibitor, RXC004, as an immunomodulatory anti-cancer agent, sub cutaneous B16F10 melanoma (C57BL/6 mice) and CT26 colorectal (BALB/c mice) murine tumour models were utilised. Mice in both models were treated with RXC004 alone or in combination with mouse anti-PD-1 antibody. Flow cytometry analysis was utilised to measure key immune cell populations in the tumour microenvironment. To probe the underlying mechanism of immune modulation in these models and to provide a link to the emerging clinical data suggesting a role for Wnt pathway activation in immune escape, human monocytic cells were isolated from PBMCs and human dendritic cells were derived in vitro. The Wnt pathway was induced in derived Dendritic cells and expression of IDO was measured. Results: In the murine CT26 model, RXC004 treatment reduced tumour size when dosed in combination with anti-PD-1 antibody, causing regression and cures in some animals. Furthermore, flow cytometry showed RXC004 in combination with anti-PD-1 antibody increased the proportion of CD8+ cytotoxic T cells as well as decreasing FoxP3+ regulatory T cells when compared to the monotherapy anti-PD-1 arm. In a syngeneic murine melanoma B16F10 model, RXC004 monotherapy at a dose of 5mg/kg QD orally significantly inhibited tumour growth, as did RXC004 combined with anti-PD-1. RXC004 had no effect on the proliferation of B16F10 cells in vitro, suggesting this was not caused by the compound directly affecting B16 cell proliferation. Flow cytometry analysis of the B16 tumours showed significant immune modulatory effects in the tumour microenvironment. In addition to mouse model data, Wnt pathway activation in human dendritic cells was shown to increase IDO expression. Conclusion: Taken together, data from murine syngeneic mouse models corroborate literature data suggesting that inhibiting the Wnt pathway may promote the immune response against human cancers. Citation Format: Inder Bhamra, Richard Armer, Matilda Bingham, Catherine Eagle, Alicia Edmenson Cook, Caroline Phillips, Simon Woodcock. Porcupine inhibitor RXC004 enhances immune response in pre-clinical models of 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 3764.