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Acetylation of histones and additional nuclear proteins is a key mechanism in the regulation of gene expression. Aberrant acetylation has been linked to a wide range of diseases including cancer, inflammation, and neurodevelopmental disorders. Histone acetylation is introduced by histone acetyltransferases complexes (HATs), where substrate specificity is dramatically enhanced by scaffolding proteins that activate and target them to specific chromatin sites. A protein of interest with both epigenetic acetyl reader and scaffolding function is the protein Bromodomain and PHD finger-containing protein 1 (BRPF1). The Protein contains domains of two plant homeodomain (PHD) fingers separated by a zinc knuckle (PZP domain), a bromodomain, and a proline-tryptophan-tryptophan-proline (PWWP) Tudor domain. Native BRPF1 complexes can contain either MOZ/MORF or HBO1 as the catalytic MYST-family acetyltransferase subunit and the stable complex with Moz-Tif2 is known to lead to the development of human acute myeloid leukemia (AML). Given this, we chose to pursue the development of BRPF1 degraders to probe cancer disease biology especially in AML, where new therapies are required to overcome several unmet needs such as less-toxic treatments and relapsed/refractory disease paradigms. Here we present the design and synthesis of a range of BRPF1 degraders in highly desirable physicochemical space utilizing in-silico modeling. The prepared degraders which utilize multiple E3 ligases were then screened against cell lines harboring Mixed-lineage leukemia (MLL) translocations specifically the THP-1 cell line. In addition to this, we investigated the ability of the compounds to effectively degrade the target and suitability of our degraders for potential in-vivo exposure through a panel of routine ADMET assays. Citation Format: Daniel Joseph Glynn, Rosie Crampton, Thomas Pesnot, Andrew Scott, Anne-Chloe Nassoy, Ralph Kirk, Daniele Narducci, Gary Nelson, Lynette Ongiri, Habiba Begum, Vincent Rao, Matilda Bingham, Rhoanne McPherson, Darryl Turner. The development of BRPF1 degraders as a potential treatment for acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 419.

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.

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

REDX05358 has been identified as a novel, highly selective and potent next generation pan RAF inhibitor with improved therapeutic potential and predicted safety profile. Aberrant signalling via the MAPK pathway is commonly seen in cancer. RAF inhibitors such as Dabrafenib and Vemurafenib have been approved for the treatment of unresectable and metastatic BRAF mutant melanoma, but these agents lack efficacy in BRAF mutant colorectal cancer (CRC), partly because of EGFR-mediated feedback reactivation of the MAPK pathway via CRAF. Furthermore, RAF inhibitor treatment of RAS-mutant, BRAF wildtype melanomas has been associated with other skin cancers, such as cutaneous squamous cell carcinoma due to MAPK pathway paradoxical activation mediated by CRAF. There is therefore a clinical need for novel agents targeting the MAPK pathway that do not have these undesirable properties. Here we present, REDX05358 that demonstrates subnanomolar binding affinity for BRAF and CRAF with high selectivity profile against a panel of 468 kinases that exhibits negligible paradoxical activation due to inhibition of both RAF monomers and dimers. As a result, REDX05358 not only inhibits MAPK signalling in BRAF V600E mutant tumor cells, but also in those harbouring NRAS and KRAS mutations. Furthermore, REDX05358 does not induce feedback reactivation of the pathway through its ability to sustain inhibition of MAPK signalling in CRC cell lines. Correspondingly, profiling of REDX05358 in a panel of CRC, melanoma and NSCLC cell lines shows it has potent anti-proliferative activity in cell lines harbouring BRAF or RAS mutations. REDX05358 is an orally bioavailable, well tolerated small molecule that has demonstrated in vivo efficacy in BRAF V600E CRC xenograft model. In contrast, first generation inhibitors such as Vemarafenib and Dabrafanib have been reported to be ineffective in this genetic background in CRC cell lines and patients. Thus, we have developed a pan RAF inhibitor with unique pharmacological properties enabling it to have utility in treating BRAF and RAS mutant cancers Citation Format: Helen Mason, Simon Scrace, Richard Testar, Julie Rainard, Fatima Talab, Ryaka Poonawala, Philippa Smith, Harriet Brooke, Sarah Frith, Jonathan Ahmet, Jonathan Hall, David Sorrell, Jon Moore, Caroline Phillips, Dennis France, Matilda Bingham, Richard Armer. Development of REDX05358, a novel highly selective and potent pan RAF inhibitor and a potential therapeutic for BRAF and RAS mutant tumors [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 5160. doi:10.1158/1538-7445.AM2017-5160

Redx Oncology has developed novel, differentiated, reversible small molecule inhibitors of BTK, combining current best-in-class potency with improved selectivity profiles, which are suitable for oral, once daily dosing, designed to be equipotent against wild-type and C481S BTK. BTK is a member of the src-related Tec family of cytoplasmic tyrosine kinases. BTK plays a key role in the BCR signaling pathway, which is required for the development, activation and survival of B-cells. BTK inhibitors have therefore been developed with the aim of treating B-cell malignancies that are dependent on BCR signaling, such as CLL and NHL. Ibrutinib is an irreversible BTK inhibitor that has been approved for the treatment of CLL, MCL and WM. Irreversible and covalent reversible BTK inhibitors specifically target a cysteine residue C481 within BTK. Following treatment with ibrutinib, cases of secondary resistance have emerged in both CLL and MCL patients. Acquired mutations within BTK such as C481S, C481Y, C481R, C481F have been reported in the literature and clearly interfere with covalent drug binding. It has been predicted that the incidence of observed resistance will increase as clinical use outside clinical trials expands over time. Here, we present our reversible BTK inhibitor series demonstrating subnanomolar binding affinity for WT and C481S forms of BTK, that inhibits formation of pBTK in both WT and C481S BTK expressing cells. In addition, the compound demonstrated significant in vitro potency against a number of lymphoma cell lines including inhibition of BCR signaling and proliferation in OCI-Ly10 cells at nanomolar concentrations. To further investigate the binding nature of these compounds, PBMC wash out studies, measuring CD69 as a marker of B-cell activation, were used to highlight the reversible activity of these compounds. Some BTK inhibitors also inhibit ITK, which plays a critical role in FcR-stimulated NK cell function that is required for ADCC. ADCC is the mechanism that anti-CD20 antibodies, such as rituximab, are believed to activate, and ibrutinib has been shown to antagonize this mechanism in vitro. As rituximab-combination chemotherapy is today's standard of care in B-cell malignancies, it would be desirable to have a BTK inhibitor with high selectivity for BTK over ITK. At 1 μM, our lead compound is highly selective when tested against 469 kinase and did not show significant inhibition against other kinases involved in BCR signaling (e.g. Syk, Lyn). Furthermore, the compound has high selectivity versus structurally related cysteine-containing kinases (including EGFR and ITK) both in enzyme and cellular assays. Our lead compound has a favorable in vitro safety profile and drug-like properties, displaying an improved CYP profile to competitor compounds. In vivo PK demonstrated good oral bioavailability and in vivo efficacy has been demonstrated. Citation Format: Nicolas E S Guisot, Victoria Walker, Stuart A. Best, Valentina Abet, Rose Chappell, Juliette Emmerich, Kelvin Ho, James R. Kelly, Kristina Lyons, Melanie Müller, Julienne Refuerzo, Louise Sargent, Fatima Talab, Madelene Waldron, Matilda Bingham, Mary-Ann Campbell, Caroline Phillips, Richard Armer. Development of novel, selective, reversible inhibitors equipotent against wild-type and C481S Bruton's tyrosine kinase (BTK). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4795.