Abstract Background: Gastrointestinal (GI) cancers cause more cancer deaths than any other body system, with 3.4 million related deaths in 2018. Wnt pathway activation is common in GI cancers, with the pathogenic upstream Wnt pathway variant sub-population (RNF43 loss of function/RSPO gain of function) showing a combined prevalence of 4.7% in GI cancer patients; pre-clinically this sub-population shows exquisite sensitivity to RXC004, a small molecule Porcupine inhibitor. A striking co-occurrence of upstream Wnt pathway variants with MAPK pathway driver mutations (KRAS, BRAF, NRAS) was detected in 77% of Microsatellite stable (MSS) GI cancers, suggesting co-inhibiting these pathways could enhance clinical activity in these patients. Methods: In vitro proliferation assays were performed in pre-clinical models of upstream Wnt pathway mutated MSS GI cancer (SNU-1411, HPAF-II and AsPC-1) using Wnt (RXC004) or MAPK (Trametinib) pathway inhibitors at multiple concentrations as single agents or in combination. Control cells with downstream Wnt pathway mutations (WiDr and HCT116) were also tested. Synergistic combination effects were detected by BLISS scores. RXC004 (1.5mg/kg QD) and Trametinib (0.3mg/kg QD) were evaluated in an RSPO-fusion xenograft model (SNU-1411) as single agents or in combination. Efficacy was measured by tumour volume and weight; PD markers were assessed in end of study tumour samples. Relative AXIN2 and DUSP6 gene expression changes were determined upon inhibitor treatment by quantitative Polymerase Chain Reaction (qPCR). Results: RXC004 in combination with Trametinib demonstrated strong anti-proliferative synergy in SNU-1411 cells and moderate synergy/additivity in HPAF-II and AsPC-1 cells; no synergy was observed in control cells. In vivo, RXC004 and Trametinib monotherapy led to significant moderate tumour growth control (p<0.05) at the doses tested, whereas their combination led to significantly enhanced efficacy (p<0.05). Gene expression analysis demonstrated a reciprocal Wnt/MAPK signaling mechanism; RXC004 strongly suppressed Wnt signaling (AXIN2) but increased MAPK signaling (DUSP6), whilst Trametinib strongly suppressed MAPK signaling (DUSP6) but increased Wnt signaling (AXIN2). Combination of RXC004 and Trametinib sustained inhibition of both Wnt and MAPK signalling. Similar effects were observed with inhibitors of other MAPK pathway components. Conclusion: Wnt and MAPK pathways are frequently co-activated in GI cancers, particularly in the upstream Wnt pathway mutated sub-population. Pre-clinically we show that Wnt and MAPK pathways act as potential reciprocal resistance mechanisms following single agent inhibition of either pathway; co-inhibition of these pathways leads to synergistic effects in vitro and enhanced efficacy in vivo. This provides a clear rationale to assess this combination approach in the clinic. Citation Format: Simon A. Woodcock, Dorottya Keppel, Catherine Eagle, James Kelly, Eimear Flanagan, Emma Bishop, Inder Bhamra, Clifford D. Jones, Richard Armer, Jane Robertson, Caroline Phillips. Pre-clinical activity of the Wnt pathway inhibitor RXC004 in combination with MAPK pathway inhibitors in GI cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 944.

Supplementary Figure 1: Effect of RXC004 on gene expression, caspase activity and cell growth in colorectal and pancreatic cancer cell lines in vitro. Supplementary Figure 2: RXC004 effects on gene expression in in vivo xenograft tumor models. Supplementary Figure 3: Pharmacokinetic/pharmacodynamic (PK/PD) analysis of RXC004 in SNU-1411/NOD-SCID mice. Supplementary Figure 4: Dose-dependent effects of RXC004 on tumor markers, intestinal Ki67 and bodyweight. Supplementary Figure 5: Effect of RXC004 with or without anti-PD-1 on body weight and survival in in vivo models. Supplementary Figure 6: Dose-dependent effects of RXC004 alone or in combination with anti-CTLA-4 in B16F10/C57BL/6 syngeneic model. Supplementary Figure 7: In vitro PBMC co-culture additional cytokines and monocultures. Supplementary Table 1: In vivo exposure data for RXC004.

Background: RXC004 is a potent and selective inhibitor of the Wnt pathway regulator Porcupine, and is currently being investigated in phase 2 studies in patients with advanced cancers, including Biliary Tract Cancers (BTCs) +/- Pembrolizumab (NCT04907851 and NCT04907539). Whilst genetically-defined ‘upstream’ Wnt pathway alterations, including loss-of-function RNF43 mutations and RSPO gene fusions, sensitise Gastrointestinal (GI) cancers to RXC004, other non-genetic factors remain to be determined. Wnt pathway mutations are rare in BTCs, however these tumors have been shown to overexpress various Wnt ligands, with evidence that this high expression is associated with worse prognosis. Methods: RXC004 was evaluated as a monotherapy against a panel of patient-derived xenograft (PDX) models of Biliary Tract Cancer for efficacy and pharmacodynamic (PD) activity. RXC004 was dosed at 5mg/kg QD and efficacy was measured by tumor volume, tumor weight and survival endpoint, whilst tumor pharmacodynamic (PD) effects were measured by quantitative Real-Time PCR (qPCR) for gene expression and histological staining methods. Baseline transcriptome characteristics of the PDX models were determined against the PanCancer Pathways and PanCancer IO360 codesets using Nanostring nCounter analysis. Results: RXC004 monotherapy was efficacious in multiple PDX models of Biliary Tract Cancer, significantly reducing tumor volume and tumor weight (range p<0.05 to p<0.0001). Tumor PD effects of RXC004 treatment on gene expression by qPCR included Wnt pathway genes (AXIN2, cMYC), differentiation genes (MUC2, MUC4, MUC5AC) and secreted Wnt pathway regulators (sFRPs, DKKs), and on histology included Ki67 and Mucin (periodic acid-Schiff’s (PAS)/Alcian blue staining). Hierarchical clustering of the PDX models was performed based on the expression of Wnt signalling genes in the Nanostring analysis. Furthermore, the gene expression profiles were used to predict whether models were likely to be RXC004 sensitive using Random Forest machine learning algorithms. Conclusion: These data demonstrate that (1) RXC004 is efficacious in pre-clinical PDX models of BTC, (2) RXC004 induces multiple PD effects in BTC models at the level of gene expression, cell proliferation and cell differentiation, and (3) PDX models of BTC can be clustered based on baseline transcriptomic profiles of Wnt signalling genes and predict RXC004 sensitivity. Citation Format: Simon A. Woodcock, Dorottya Keppel, Catherine Eagle, Inder Bhamra, Eleanor Platt, Lucy Frost, Kevin Randall, Richard Armer, Jane Robertson, Gayle Marshall, Caroline Phillips. Pre-clinical activity of the Wnt/Beta-catenin pathway inhibitor RXC004 in models of biliary tract cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1654.

Background Tumour-derived Wnt-ligand signalling leads to a reprogramming of the immune microenvironment and is implicated in intrinsic and adaptive resistance to Immune Checkpoint Inhibitor (ICI) therapy. Specifically, Wnt-ligand signaling is correlated with reduced CD8+ve T-cell infiltration,1 and ICI resistance2 in multiple cancers.3 Inhibition of Wnt-ligand signalling can enhance ICI efficacy by (i) reversing dendritic cell tolerization, (ii) decreasing generation of Treg cells, and (iii) reducing the recruitment of myeloid-derived suppressor cells in tumor models.4 RXC004 is a novel small molecule inhibitor of PORCN, a protein-serine-O-palmitoyltransferase [5]. PORCN is essential for post-translational modification of Wnt ligands which is required for downstream Wnt signaling. RXC004 thus has potential for monotherapy efficacy in Wnt-ligand driven tumors i.e. cancers with RNF43 mutations or R-Spondin fusions, or with high Wnt-ligand activity. Furthermore, RXC004 can reverse immune evasion in mouse models,5 and may therefore restore ICI sensitivity in ICI resistant tumors when co-administered. This abstract reports the second module of a multi-modular adaptive design protocol (NCT03447470). The first module was previously reported6 and the recommended Phase 2 dose (RP2D) for RXC004 monotherapy was 2mg QD. Methods This was an open label, 3+3 dose escalation study. Following a single dose with a 7-day washout, patients received RXC004 QD in 28-day cycles, and nivolumab 480mg i.v every 4 weeks. The primary objectives were to assess safety and tolerability and define a RP2D of RXC004 to combine with ICIs. Secondary objectives were Pharmacokinetics (PK) and RECIST response. Exploratory objectives included changes in circulating immune subsets by flow cytometry, and cytokines by a bead-based multiplexed immunoassay. Results Between 24/03/2021 and 30/06/2022, 14 patients with unselected advanced solid tumors received RXC004 at doses of 1mg and 1.5mg QD, in combination with nivolumab. The AE profile for the combination was broadly similar to RXC004 monotherapy. The most common treatment-related AEs were nausea, dysgeusia, fatigue, anorexia and weight loss. No grade 4/5 AEs, bone events or immune-related AEs were reported. RXC004 PK exposure in the combination did not exceed the 2mg QD monotherapy exposure. Disease control was observed in some patients in the 1.5mg QD combination cohort. Conclusions In patients with unselected cancers, RXC004 was safe and tolerated at doses up to 1.5mg QD in combination with standard dose nivolumab. The RP2D was 1.5mg QD. RXC004+ICI combinations will now be investigated in selected patients with Wnt-ligand driven tumors. Trial Registration EudraCT No: 2017-000720-98 References Spranger S, Bao R, Gajewski TF. Melanoma-intrinsic β-catenin signalling prevents anti-tumour immunity. Nature. 2015;523(7559):231–5. doi: 10.1038/nature14404. Epub 2015 May 11. PMID: 25970248. Abril-Rodriguez et al. PAK4 inhibition reverses immune cell exclusion and overcomes resistance to checkpoint blockade therapy. Journal for ImmunoTherapy of Cancer. 2018;6(suppl 1):O39 Luke JJ, Bao R, Sweis RF, Spranger S, Gajewski TF. WNT/β-catenin Pathway Activation Correlates with Immune Exclusion across Human Cancers. Clin Cancer Res. 2019;25(10):3074–3083. doi: 10.1158/1078-0432.CCR-18-1942. Epub 2019 Jan 11. PMID: 30635339 DeVito NC, Sturdivant M, Thievanthiran B, Xiao C, Plebanek MP, Salama AKS, Beasley GM, Holtzhausen A, Novotny-Diermayr V, Strickler JH, Hanks BA. Pharmacological Wnt ligand inhibition overcomes key tumor-mediated resistance pathways to anti-PD-1 immunotherapy. Cell Rep. 2021;35(5):109071. doi: 10.1016/j.celrep.2021.109071. PMID: 33951424; Phillips C. et al. Wnt/â-Catenin pathway inhibitor RXC004 enhances the immunity of pre-clinical models of cancer. Cancer Res. 2019;79(13_Supplement):506 Cook N. et al Phase I study of the porcupine (PORCN) inhibitor RXC004 in patients with advanced solid tumours Annals of Oncology. 2021;32(suppl_5):S583-S620. 10.1016/annonc/annonc699 Ethics Approval This study was approved by West Midlands-Edgbaston Research Ethics Committee and Health Research Authority in UK; approval number 222362 (IRAS ID).

Gastrointestinal cancers are responsible for more cancer deaths than any other system of the body. This review summarises how Wnt pathway dysregulation contributes to the development of the most common gastrointestinal cancers, with a particular focus on the nature and frequency of upstream pathway aberrations. Tumors with upstream aberrations maintain a dependency on the presence of functional Wnt ligand, and are predicted to be tractable to inhibitors of Porcupine, an enzyme that plays a key role in Wnt secretion. We summarise available pre-clinical efficacy data from Porcupine inhibitors in vitro and in vivo, as well as potential toxicities and the data from early phase clinical trials. We appraise the rationale for biomarker-defined targeted approaches, as well as outlining future opportunities for combination with other therapeutics.

Wnt signaling is implicated in the etiology of gastrointestinal tract cancers. Targeting Wnt signaling is challenging due to on-target toxicity concerns and lack of druggable pathway components. We describe the discovery and characterization of RXC004, a potent and selective inhibitor of the membrane-bound o-acyl transferase Porcupine, essential for Wnt ligand secretion. Absorption, distribution, metabolism, and excretion and safety pharmacology studies were conducted with RXC004 in vitro, and pharmacokinetic exposure assessed in vivo. RXC004 effects on proliferation and tumor metabolism were explored in genetically defined colorectal and pancreatic cancer models in vitro and in vivo. RXC004 effects on immune evasion were assessed in B16F10 immune “cold” and CT26 immune “hot” murine syngeneic models, and in human cell cocultures. RXC004 showed a promising pharmacokinetic profile, inhibited Wnt ligand palmitoylation, secretion, and pathway activation, and demonstrated potent antiproliferative effects in Wnt ligand–dependent (RNF43-mutant or RSPO3-fusion) colorectal and pancreatic cell lines. Reduced tumor growth and increased cancer cell differentiation were observed in SNU-1411 (RSPO3-fusion), AsPC1 and HPAF-II (both RNF43-mutant) xenograft models, with a therapeutic window versus Wnt homeostatic functions. Additional effects of RXC004 on tumor cell metabolism were confirmed in vitro and in vivo by glucose uptake and 18fluorodeoxyglucose-PET, respectively. RXC004 stimulated host tumor immunity; reducing resident myeloid-derived suppressor cells within B16F10 tumors and synergizing with anti-programmed cell death protein-1 (PD-1) to increase CD8+/regulatory T cell ratios within CT26 tumors. Moreover, RXC004 reversed the immunosuppressive effects of HPAF-II cells cocultured with human peripheral blood mononuclear cells, confirming the multiple anticancer mechanisms of this compound, which has progressed into phase II clinical trials. Significance: Wnt pathway dysregulation drives many gastrointestinal cancers; however, there are no approved therapies that target the pathway. RXC004 has demonstrated the potential to block both tumor growth and tumor immune evasion in a genetically defined, clinically actionable subpopulation of Wnt ligand–dependent gastrointestinal cancers. The clinical utility of RXC004, and other Porcupine inhibitors, in such Wnt ligand–dependent cancers is currently being assessed in patient trials.

Abstract Background: RXC004 is a porcupine inhibitor, and thus a potent and selective inhibitor of Wnt ligand-dependent signalling. RXC004 is currently being investigated in a safety and tolerability study in cancer patients with solid tumours (NCT03447470). Certain Wnt pathway alterations, including loss-of-function RNF43 mutations and RSPO gene fusions, result in higher levels of the Wnt receptor Fzd on the cell surface increasing Wnt-ligand dependent signalling. These alterations are implicated in colorectal and pancreatic cancer. We present pre-clinical data on the effects of RXC004 in genetically defined models of cancer. Methods: RXC004 in vitro effects on the metabolic activity of both sensitive (genetically selected RNF43 mutant or RSPO fusion models) and insensitive (beta-catenin mutant model) colorectal and pancreatic tumour cells lines were assessed. To test if these effects translated in vivo, RXC004 was also evaluated in an in vivo FDG-PET study in an RSPO fusion tumour cell line. In addition, as we had previously noted that RXC004 caused cell differentiation in an RSPO fusion model, we studied if pre-treatment with RXC004 would affect subsequent tumour growth post RXC004 induced cell differentiation in vivo. Results: In tumour cell lines in vitro, RXC004 significantly reduced glucose uptake only in genetically selected tumour cell lines. In vivo FDG-PET studies also showed a reduction in FDG-PET signals post 7 days treatment of 5mg/kg QD RXC004. Imaging demonstrated that MaxSUV (maximum standardised uptake value) decreased by 31% in RXC004 treated versus vehicle treated tumours, furthermore biodistribution of PET ligand was significantly inhibited in tumours post 7 days RXC004 treatment. Pre-treatment of RXC004 in an in vivo mouse xenograft model for 7 days resulted in a significant reduction in tumour growth when tumour fragments from these pre-treated mice were re-implanted into naïve mice (P<0.05). Conclusion: These data demonstrate that RXC004 monotherapy results in tumour cell differentiation accompanied by reduced metabolic activity in genetically selected tumours. RXC004-induced cell differentiation may therefore continue to provide patient benefit even post dosing. Moreover, FDG-PET imaging in clinical studies may allow early detection of the positive effects of RXC004 in a non-invasive manner, and prior to any changes in tumour volume by conventional RECIST measurements. Citation Format: Caroline Phillips, Simon Woodcock, Catherine Eagle, Andrew Saunders, Craig Tilston, Inder Bhamra, Richard Armer. Mechanism of action of RXC004, a Wnt pathway inhibitor, in genetically-defined models of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 998.

Background: RXC004, a potent and selective inhibitor of the Wnt/β-Catenin pathway regulator porcupine, is being investigated in a safety and tolerability study in cancer patients with solid tumors (NCT03447470). We present pre-clinical data confirming the potential for RXC004 in modulating the immune system of the tumor microenvironment. Materials and Methods: To evaluate RXC004 as an immunomodulatory anti-cancer agent we took two complementary approaches; first utilizing a syngeneic B16F10 melanoma model in C57BL/6 mice. A range of doses and schedules of RXC004 were tested for 28 days and efficacy was measured by tumor volume. To further probe the mechanism of immune modulation by RXC004 in syngeneic models, global gene expression in the tumor microenvironment (TME) was analyzed using the Nanostring IO 360 gene set, and immune cell populations in the blood and spleen were analyzed by flow cytometry. In the second approach, modulation of a range of immune-relevant biomarkers by RXC004 was assessed in a panel of Wnt ligand-dependent human cancer cell lines using flow cytometry and gene expression. Results: In the syngeneic B16F10 melanoma model, RXC004 treatment significantly reduced tumor volume in a dose-dependent manner at 0.5, 1.5 and 5 mg/kg QD. In addition, 5mg/kg QD of RXC004 scheduled 5 days on, 2 days off also gave significant efficacy. RXC004 treatment in this model is known to decrease myeloid-derived-suppressor-cells (MDSCs) within the TME. Consistent with this, RXC004 treatment (5mg/kg QD) increased the proportion of MDSCs circulating in blood. MDSCs are therefore either retained in the TME through Wnt pathway signaling, or Wnt pathway signaling promotes chemotaxis of these cells to the TME. Analysis of a panel of immune genes using Nanostring IO 360 demonstrated RXC004 treatment (5mg/kg QD) increased expression of chemokine and cytokine signaling components in the TME, such as the T cell chemoattractant CXCL9. CXCL9 is reported to be produced by CD103+ve dendritic cells in the mouse TME and absence of these dendritic cells is linked to a lack of T cell infiltrate (Spranger et al., 2017). In a panel of Wnt ligand-dependent human cancer cells, RXC004 treatment regulated levels of markers associated with tumors evading the immune system e.g. MYC expression levels were reduced significantly. Modulation of such markers in paired clinical sample may indicate an immune-modulatory response to RXC004. Conclusion: Taken together, Wnt pathway suppression by RXC004 treatment can enhance the immune response against tumors by 1) directly regulating levels of immune-relevant markers on cancer cells, and 2) enhancing the immunity of the TME by decreasing MDSCs and increasing chemokine and cytokine signaling. Spranger et al Cancer Cell 2017 8 31(5):711-723 Citation Format: Caroline Phillips, Inder Bhamra, Catherine Eagle, Alicia Edmenson Cook, Cliff Jones, Simon Woodcock. Wnt/â-Catenin pathway inhibitor RXC004 enhances the immunity of pre-clinical models of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 506.

Background: RXC004, a potent and selective inhibitor of the Wnt/β-Catenin pathway regulator porcupine, is being investigated in a safety and tolerability study in cancer patients with solid tumours (NCT03447470). Wnt pathway alterations, including loss-of-function RNF43 mutations and RSPO gene fusions, result in higher levels of the Wnt receptor Fzd on the cell surface, increasing Wnt-ligand dependent signalling. These alterations are implicated in colorectal, gastric, pancreatic and biliary cancer. We present pre-clinical data on the direct tumour-targeting effects of RXC004 in genetically-defined models of cancer. Methods: RXC004 in vitro effects on sensitive (RNF43 mutant or RSPO fusion) or insensitive (APC/β-Catenin mutant) colorectal and pancreatic tumour cells lines were assessed against a panel of potential Wnt target genes measured using qPCR. To test if these effects translated in vivo, RXC004 was evaluated in several RNF43 mutant or RSPO fusion tumour line efficacy, PK and PD studies. A range of oral doses and schedules of RXC004 were tested for either 7 or 26 days, for PK/PD or efficacy measurements respectively. Efficacy was measured by tumour volume/weight, RXC004 PK was measured in plasma and tumour, and PD effects were measured by tumour qPCR and histological methods. Results: In sensitive cell lines, RXC004 inhibited expression of the Wnt pathway negative feedback genes Axin2 and RNF43. Decreases in c-Myc correlated with the anti-proliferative effects of RXC004. Additionally, MMP7 and CD44 were downregulated, whilst Mucin gene expression increased. In contrast, RXC004 had no anti-proliferative effects on APC mutant colorectal cancer cells in vitro, and no modulation of these Wnt target genes. In vivo, RXC004 demonstrated significant efficacy and PD effects in multiple RNF43 mutant and RSPO fusion xenograft models. Focusing on an RSPO fusion model, RXC004 reduced tumour volume in a dose-dependent manner at 1.5 and 5 mg/kg QD, and 1.5mg/kg BID. In addition, 1.5mg/kg BID of RXC004 scheduled 5 days on, 2 days off also gave significant efficacy. PK analysis was consistent with the predicted dose response, showing excellent absorption and tumour distribution. PD analysis confirmed the RXC004-induced Wnt target gene changes identified in vitro translated in vivo, whilst histology showed a clear reduction in the proliferation marker Ki67 and concomitant increase in the staining of Mucins, indicative of mucinous differentiation of the tumour cells. Conclusion: Taken together, these data demonstrate that RXC004 monotherapy has the potential to benefit patients with tumours bearing RNF43 mutations or RSPO fusions, supporting a genetically-defined patient selection strategy for ongoing RXC004 clinical studies. Citation Format: Simon Woodcock, Inder Bhamra, Cliff Jones, Alicia Edmenson Cook, Catherine Eagle, Caroline Phillips. Efficacy of the Wnt/Beta-Catenin pathway inhibitor RXC004 in genetically-defined models of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3874.

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.

Abstract Harnessing the immune system via immune checkpoint blockade (e.g. anti PD-1, anti PD-L1, anti CTLA4) has led to fast and long lived responses in cancer patients. Response rates however are low and new treatments that enhance these rates are needed. Recent studies have shown that the administration of immune checkpoint blockers is associated with the overexpression of indoleamine 2,3-dioxygenase 1 (IDO1). The resulting immunoregulatory phenotype counteracts immune checkpoint blockade and allows for cancer progression. The discovery of IDO1 inhibitors, and the potential to combine them with immune checkpoint blockers, therefore represents an attractive strategy to fight cancer. We carried out a ligand-based virtual screen with > 1,000,000 commercially available small molecules. In vitro screening of the resulting 610 virtual hits provided us with 2 IDO1-selective, 2 TDO2-selective and 2 IDO1/TDO2-dual confirmed hits. (TDO2 is a protein with similar biochemical activity to IDO1 that is essential to tryptophan homeostasis.) A subsequent Hit to Lead campaign led to the identification of novel chemotypes that display potency similar or superior to IDO1 inhibitors currently under clinical investigation in IFN-γ stimulated (i.e. IDO1+) HeLa cells, with no sign of cytotoxicity. We have demonstrated that these compounds are > 1000-fold selective for IDO1 over TDO2 using cellular assays. IDO1 upregulation by cancer cells is known to be one of the mechanisms by which cancer cells evade the immune system. In an in vitro co-culture assay of cancer cells and T cells we have demonstrated our compounds can rescue T cell proliferation with EC50 values between 10 and 50 nM. We have also demonstrated that our compounds inhibit IDO1 in monocyte derived human dendritic cells. Interestingly, despite this potent cellular activity demonstrated in multiple disease relevant cellular assays, this chemotype failed to inhibit recombinant IDO1 in an isolated biochemical assay performed under reducing conditions, whereas the reference compound epacadostat provided activity comparable to literature values. In order to confirm our cellular effects were due to direct inhibition of IDO1 we set up thermal shift assays. Thermal shift assays using purified IDO1 protein have demonstrated that our compounds directly bind IDO1, and cellular thermal shift assays have confirmed direct target engagement in intact cells (stimulated for IDO1 expression). These compounds have physicochemical properties that would support oral dosing and display low in vitro CYP450 and hERG inhibition, thus reducing the risk of toxicity in the clinic. Our IDO1 inhibitors show a novel differentiated mode of action at the cellular level, and the consequences of this profile in terms of in vivo characterisation is ongoing. Citation Format: Thomas Pesnot, Sachin Mahale, Philip MacFaul, John Maclean, Caroline Phillips, Matilda Bingham, Catherine Eagle, James Kelly, Abhijith Thippeswamy, Simon Armitage, Aleksandr Grisin, Sheenagh Aiken, Lucy Cartwright, Richard Armer. Development of 2nd generation indoleamine 2,3-dioxygenase 1 (IDO1) selective inhibitors [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 5581. doi:10.1158/1538-7445.AM2017-5581