G-protein-coupled receptors (GPCRs) are the largest and most versatile cell surface receptor family with a broad repertoire of ligands and functions. We've learned an enormous amount about discovering drugs of this receptor class since the first GPCR was cloned and expressed in 1986, such that it's now well-recognized that GPCRs are the most successful target class for approved drugs. Here we take the reader through a GPCR drug discovery journey from target to the clinic, highlighting the key learnings, best practices, challenges, trends and insights on discovering drugs that ultimately modulate GPCR function therapeutically in patients. The future of GPCR drug discovery is inspiring, with more desirable drug mechanisms and new technologies enabling the delivery of better and more successful drugs.

Abstract Resistance to androgen receptor (AR) blockade in castration-resistant prostate cancer (CRPC) is associated with sustained AR signaling, including through alternative splicing of AR (AR-SV). Inhibitors of transcriptional coactivators that regulate AR activity, including the paralog histone acetyltransferase proteins p300 and CBP, are attractive therapeutic targets for lethal prostate cancer. Herein, we validate targeting p300/CBP as a therapeutic strategy for lethal prostate cancer and describe CCS1477, a novel small-molecule inhibitor of the p300/CBP conserved bromodomain. We show that CCS1477 inhibits cell proliferation in prostate cancer cell lines and decreases AR- and C-MYC–regulated gene expression. In AR-SV–driven models, CCS1477 has antitumor activity, regulating AR and C-MYC signaling. Early clinical studies suggest that CCS1477 modulates KLK3 blood levels and regulates CRPC biopsy biomarker expression. Overall, CCS1477 shows promise for the treatment of patients with advanced prostate cancer. Significance: Treating CRPC remains challenging due to persistent AR signaling. Inhibiting transcriptional AR coactivators is an attractive therapeutic strategy. CCS1477, an inhibitor of p300/CBP, inhibits growth and AR activity in CRPC models, and can affect metastatic CRPC target expression in serial clinical biopsies. See related commentary by Rasool et al., p. 1011. This article is highlighted in the In This Issue feature, p. 995

Whilst the advent of Immune Checkpoint Blockade has revolutionized the management of cancer, a significant proportion of patients have limited or absent response to these therapies. A key cause of this immune insensitivity is the hostile solid tumor microenvironment (TME) dominated by immunosuppressive myeloid cells. We previously identified the acid sensing G protein coupled receptor (GPCR), GPR65, as a primary determinant of these suppressive cells. In mice, genetic deletion of Gpr65 or oral administration of small molecule GPR65 inhibitors in vivo causes a profound repolarization of immunosuppressive tumor associated macrophages, an increase in infiltrating effector cells and potent anti tumor effects in syngeneic models. In TCGA data, across all tumors, patients homozygous for a hypomorphic coding variant in GPR65 (I231L) show increased overall survival, providing compelling genetic evidence of the clinical potential of GPR65 inhibition. To further explore the translational potential of GPR65 we employed a range of techniques to define the human biology of this receptor in different contexts. At the mechanistic level, single cell RNA sequencing (scRNAseq) of human PBMCs obtained from healthy donors demonstrated a pronounced effect of low pH on the myeloid compartment, with a clear polarization of these cells toward an immunosuppressive character and modulation of GPR65 expression. In parallel, pharmacological inhibition of GPR65 in human monocyte derived macrophages exposed to low pH demonstrated that equivalent gene expression changes are primarily due to GPR65 activation. To examine the relevance of these findings to the intact acidic human TME, we performed studies in fresh primary human tumor histocultures from clear cell renal cell carcinoma (ccRCC) patients with immunohistochemically confirmed high macrophage infiltration and carbonic anhydrase 9 (CA9) expression. In these cultures, GPR65 inhibition caused a dose dependent suppression of a geneset closely overlapping with that modulated by GPR65 in primary macrophages. Furthermore, we observed a marked decrease in immune suppressive IL10 secretion with coincident elevation of specific proinflammatory chemokines. Consistent with these findings, in vivo administration of a small molecule GPR65 inhibitor elicited similar changes in human CA9 expressing RCC PDX tumors implanted in myeloid boosted CD34+ stem cell engrafted NCG mice. In summary, inhibition of GPR65 provides a unique and genetically validated approach to favorably modify the immunosupressive TME with features highly conserved between mouse and human contexts. We propose that GPR65 inhibition holds significant clinical promise, with specific evidence around ccRCC as a potential standout indication. Citation Format: Barbara Cipriani, Alastair Corbin, David Miller, Alan Naylor, Faraz Khan, Gavin Milne, Barbara Young, Rupert Satchell, Sourav Sarkar, Mussa Quareshy, Anastasia Nika, Preeti Singh, Gavin Knox, Darryl Turner, Satish Sankaran, Nandini Pal Basak, Toszka Bohn, Tobia Bopp, Surya Koturan, Bo Sun, Benjamin Fairfax, Tom McCarthy, Stuart Hughes. The translational biology of small molecule GPR65 inhibitors: shared effects between mouse models and human primary tumors highlight the unique transformative potential of targeting a genetically validated innate immune checkpoint [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 668.

Background An important facet of the hostile tumor microenvironment (TME) and a recognized driver of cancer immunosuppression is extracellular acidification which results from the glycolytic nature of rapidly proliferating cancer cells. Pathios identified GPR65, a pH-sensing G protein-coupled receptor (GPCR), as the primary sensor of this acidity in human immune cells. Activation of GPR65 is particularly pronounced in macrophages where it leads to the downregulation of key chemokines and anti-tumorigenic cytokines, and the upregulation of pro-tumorigenic wound repair and remodeling factors. In dendritic cells GPR65 signaling suppresses antigen presentation pathways, whereas in T cells and natural killer cells it leads to the suppression of chemokine receptors required for effective tumor homing. Consequently, acid sensing by GPR65 can explain tumor resistance to current immunotherapies. Validating the relevance of this target in immuno-oncology, cancer patients homozygous for the hypomorphic GPR65 variant (I231L) exhibit profoundly improved survival compared to patients with other genotypes. Methods Pathios has developed PTT-4256, a first-in-class small molecule inhibitor of GPR65. The in vitro efficacy of PTT-4256 under low pH conditions was determined by cAMP screening, gene expression and cytokine release profiling of primary immune populations. Anti-tumor activity was assessed using the MC38 and B16.F10 syngeneic mouse models, whilst immunomodulatory effects in MC38 tumor-infiltrating leukocytes were profiled by targeted RNAseq. Notably, PTT-4256 is markedly more potent at human GPR65 versus the mouse receptor. To determine its full potential in patients and to develop an accurate prediction of efficacious human exposure, we employed a range of approaches including molecular pharmacology, site-directed mutagenesis, PK/PD modelling, and genetically engineered mice. Results PTT-4256 fully counteracts the low pH-driven immunosuppressive transcriptional program in human and murine immune cells and restores the secretion of key pro-inflammatory cytokines and chemokines in macrophages. PTT-4256 displays an excellent oral PK profile across species and elicits significant monotherapy efficacy in MC38 and B16.F10 mouse models. Through molecular modelling and site-directed mutagenesis we identified three key residues that explained the difference in potency of PTT-4256 between mouse and human GPR65. Incorporating these residues in a transgenic knock-in mouse fully restored human pharmacology in vitro and provided an important translational platform. Conclusions GPR65 is a key checkpoint on tumor-infiltrating immune cells that links the chronically acidic TME to tumor-promoting immunosuppression. The GPR65 inhibitor PTT-4256 restores anti-tumor immunity and demonstrates pronounced single-agent efficacy in mice, and is therefore being advanced towards clinical development. Ethics Approval Protocols or procedures involving the care and use of animals in studies in China were reviewed and approved by the Institutional Animal Care and Use Committee of Crown Bioscience. During studies, the care and use of animals was conducted in accordance with the regulation of the Association for Assessment and Accreditation of Laboratory Animal Care. Studies involving the welfare and use of animals within the UK complied with the UK Animals Scientific Procedures Act 1986 (ASPA) in line with Directive 2010/63/EU of the European Parliament and Council of 22/September/2010 on the protection of animals used for scientific purposes and UK Home Office guidance on the implementation of the Act and applicable codes of practice for the care and housing of laboratory animals.

Background High frequencies of Tumor Associated Macrophages (TAMs) are related to poor patient prognosis. The Tumor Microenvironment (TME) is characterised by resource scarcity, toxic metabolic by-products, and low pH, together creating an immunosuppressive environment which polarises TAMs towards a pro-tumorigenic state. Methods We identified the proton-sensing G-Protein-Coupled Receptor 65 (GPR65) as a key determinant of low-pH-induced immunosuppression in human cancers, specifically via modulating TAM phenotype in response to the acidic TME. The importance of GPR65 in human cancers is highlighted by three key findings: (1) cancer patients homozygous for the hypomorphic I231L variant exhibit a pronounced survival benefit, (2) GPR65 and downstream pathway genes are highly expressed in innate immune cells from all human solid tumors when assessed by single cell RNA sequencing, and (3) low pH treatment of macrophages in vitro leads to a marked suppression of inflammatory genes and an upregulation of a tissue repair signature. Results We have identified potent and selective small-molecule antagonists of human GPR65 that inhibit the low pH-induced accumulation of cAMP in recombinant cell systems and primary human macrophages with single-digit nanomolar potencies. These compounds dose-dependently prevent the low pH-driven suppression of inflammatory cytokine and chemokine genes and counteract the upregulation of pro-tumorigenic and tissue repair genes in both human and mouse macrophages. Oral administration of our exemplar compound PTT-3213 in subcutaneous MC38 tumor-bearing mice caused gene expression changes consistent with those observed in primary macrophages in vitro, indicative of a dramatic impact on the TME. Weekly dosing of PTT-3213 significantly reduced MC38 Tumor Volume (TV) compared to vehicle (46%). This monotherapy activity was comparable to bi-weekly administration of anti-PD1, whilst combination of PTT-3213 and anti-PD-1 led to a more pronounced curtailment of TV vs vehicle-treated animals (61%). In accordance with the increased expression of chemokine genes, PTT-3213 monotherapy in MC38-bearing mice markedly elevated the frequency of tumor-infiltrating NK cells (up to 22-fold). There was also an increase in the CD8+/CD4+ T cell ratio which attained statistical significance in combination with anti-PD-1. Conclusions Taken together, we have identified GPR65 as a key innate immune checkpoint and therapeutic target in solid tumors and propose that macrophage conditioning via GPR65 inhibition may provide an efficacious strategy to counteract the immunosuppressive action of the acidic TME on TAMs in patients. Ethics Approval Protocols or procedures involving the care and use of animals in studies in China were reviewed and approved by the Institutional Animal Care and Use Committee of Crown Bioscience. During studies, the care and use of animals was conducted in accordance with the regulation of the Association for Assessment and Accreditation of Laboratory Animal Care Studies involving the welfare and use of animals within the UK complied with the UK Animals Scientific Procedures Act 1986 (ASPA) in line with Directive 2010/63/EU of the European Parliament and Council of 22/September/2010 on the protection of animals used for scientific purposes and UK Home Office guidance on the implementation of the Act and applicable codes of practice for the care and housing of laboratory animals.

The acidic tumour microenvironment (TME) and the abundance of tumour associated macrophages (TAMs) are key features of solid tumours that drive immune suppression, support tumour growth and limit the efficacy of approved therapies. We identified the pH sensing GPCR, GPR65, as a key determinant of low pH induced immune suppression in human cancers, particularly in TAMs, based on the following observations: 1) cancer patients who are homozygous for a hypomorphic coding variant in GPR65 (I231L) show a statistically significant increase in survival and altered expression of key immune system genes compared to other genotypes; 2) single cell RNA sequencing (scRNAseq) data from multiple solid tumors show that GPR65 and downstream pathway genes are ubiquitously expressed in myeloid and other innate immune cells in human cancers; and 3) low pH acting via GPR65 profoundly alters gene expression in human macrophages in vitro, bringing about a pronounced suppression of proinflammatory cytokines and a marked upregulation of tissue repair genes. These findings identify GPR65 as a novel innate immune check point, and suggest that GPR65 inhibition could be highly beneficial in cancer. Indeed, previous work has shown that genetic ablation of the GPR65 signaling pathway in B16.F10 tumour bearing mice upregulates immunostimulatory genes in TAMs and significantly reduces tumor growth1. Pathios has identified potent and selective antagonists of human GPR65 with excellent oral bioavailability and pharmacokinetics. In line with their potencies in recombinant cell systems, lead molecules are able to inhibit low pH induced cAMP elevations in primary human macrophages with IC50 values in the single digit nM range. In macrophages subjected to acidic conditions, the inhibitory effects on cAMP are accompanied by a reduction in the expression of anti inflammatory and tissue repair genes, and the enhancement of immunostimulatory genes. In particular, GPR65 inhibition counteracts the pronounced low pH induced suppression of key Type I/II interferon (IFN) response genes and chemokines such as CXCL9, CXCL10 and CXCL11. Following oral administration in tumour bearing mice, lead compounds up-regulate the expression of anti-tumor immune response genes at systemic exposures that significantly suppress GPR65 signalling in vitro. Results from tumour growth inhibition studies in mice with our GPR65 inhibitors will be also be presented. In summary, ‘Macrophage Conditioning’ via GPR65 inhibition holds substantial promise as a novel immunoncology strategy to counteract the immunosuppressive effects of the acidic TME on TAMs, and could be deployed either as monotherapy or in combination with T cell checkpoint inhibitors or other standard of care treatments. 1Nat. Immunol. 19:1319 Citation Format: Barbara Cipriani, David Miller, Alan Naylor, Gavin Milne, Barbara Young, Rupert Satchell, Suorav Sarkar, Zoe Smith, Rhoanne McPherson, Anastasia Nika, Preeti Singh, Toszka Bohn, Tobias Bopp, Tom McCarthy, Stuart Hughes. Inhibition of GPR65 counteracts low pH induced immunosuppressive polarization of macrophages: In vitro and in vivo characterization of potent, selective and orally bioavailable small molecule GPR65 antagonists [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 2162.

Resistance to androgen receptor (AR) blockade in castration-resistant prostate cancer (CRPC) is associated with sustained AR signaling, including through alternative splicing of AR (AR-SV). Inhibitors of transcriptional coactivators that regulate AR activity, including the paralog histone acetyltransferase proteins p300 and CBP, are attractive therapeutic targets for lethal prostate cancer. Herein, we validate targeting p300/CBP as a therapeutic strategy for lethal prostate cancer and describe CCS1477, a novel small-molecule inhibitor of the p300/CBP conserved bromodomain. We show that CCS1477 inhibits cell proliferation in prostate cancer cell lines and decreases AR- and C-MYC-regulated gene expression. In AR-SV-driven models, CCS1477 has antitumor activity, regulating AR and C-MYC signaling. Early clinical studies suggest that CCS1477 modulates KLK3 blood levels and regulates CRPC biopsy biomarker expression. Overall, CCS1477 shows promise for the treatment of patients with advanced prostate cancer. SIGNIFICANCE: Treating CRPC remains challenging due to persistent AR signaling. Inhibiting transcriptional AR coactivators is an attractive therapeutic strategy. CCS1477, an inhibitor of p300/CBP, inhibits growth and AR activity in CRPC models, and can affect metastatic CRPC target expression in serial clinical biopsies.See related commentary by Rasool et al., p. 1011.This article is highlighted in the In This Issue feature, p. 995.

Tumor-associated macrophages (TAMs) are the major innate immune component in the microenvironment of solid tumors. These cells are highly heterogeneous and plastic but often display a pronounced immunosuppressive phenotype that supports primary tumor growth and metastasis. A recently identified determinant of the immunosuppressive properties of TAMs is the activation of the pH-sensing G protein-coupled receptor, GPR65, on these cells by the acidic microenvironment that is inherent to many advanced solid tumours1. Previous work in mouse macrophages has shown that GPR65 activation leads to an elevation of inducible cAMP early repressor (ICER), an isoform of the CREM gene, which in turn suppresses the expression of a host of proinflammatory mediators1. Consistent with a high expression of GPR65 and CREM in human myeloid cells, and particularly in human TAMs, we now show that equivalent immunosuppressive signaling is also present in human macrophages in response to low pH. Further substantiating GPR65 as an innate immune checkpoint in human cancers, we also show that subjects that are homozygous for a hypomorphic coding variant in GPR65 (I231L) have a significantly improved survival across a range of cancers compared to other genotypes. This survival advantage is maintained in patients with highly glycolytic tumors that would otherwise be predicted to respond poorly to immunotherapy, and is consistent with studies showing that genetic deletion of ICER attenuates the growth of anti-PD-1-resistant B16.F10 melanoma tumors in mice1. With a view to developing a therapeutic agent able to reverse low pH-dependent immunosuppression in TAMs, we identified potent and selective small molecule inhibitors of GPR65 from a screening campaign. These molecules were able to fully ablate the acidic pH-induced expression of ICER in human macrophages with downstream implications for pro-inflammatory cytokines known to support T cell anti-tumor responses. Additional medicinal chemistry optimization led to molecules exhibiting excellent oral bioavailability in preclinical species and systemic exposure that completely suppressed GPR65 signaling following oral dosing in mice. Profiling of these inhibitors in relevant mouse tumor models is currently ongoing. In conclusion, we have (i) demonstrated that GPR65 is a key determinant of low pH-induced immunosuppression in human macrophages and thus an important innate immune checkpoint in cancer, and (ii) identified selective small molecule inhibitors of GPR65 with potent in vivo activity. This work provides the basis for developing novel “macrophage conditioning” therapeutic agents that may have utility across a range of cancers, either as single agents or in combination with other approved immunoncology drugs. 1Nat Immunol 19:1319. Citation Format: Barbara Cipriani, Alan Naylor, Gavin Milne, Barbara Young, Rupert Satchell, Sourav Sarkar, Zoe Smith, Louise Healy, John Unitt, Jessica Holien, Rhoanne McPherson, Anastasia Nika, Jessy Cartier, Tozska Bohn, Tobias Bopp, Tom McCarthy, Stuart Hughes. GPR65 is a critical mediator of low pH induced immunosuppressive signalling in tumor associated macrophages: Human target validation of GPR65 as a novel innate immune checkpoint and discovery of potent, selective GPR65 antagonists [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 1631.

Background: Histone acetyl transferases E1A binding protein (p300) and CREB binding protein (CBP) are known co-activators of several key transcription factors that contribute to tumor progression including HIF1a, BRCA-1, p53, c-myc and androgen receptor (AR). A large proportion of AR regulated gene expression has been shown to be dependent on p300 either through direct regulation of AR interaction with promoters of AR regulated genes or subsequent histone modification events. Both p300 and CBP are highly expressed in advanced prostate cancer and androgen deprivation leads to upregulation of both proteins. CCS1477 is a potent, selective inhibitor of the bromodomain in CBP/p300 that has been shown to inhibit prostate tumor cell proliferation in vitro and tumor growth in vivo. Methods: 22Rv1 prostate tumor cells that express both AR and AR variants were transplanted in nude mice. Established tumors were treated with CCS1477, 20mg per kg, once daily p.o. for 28 days. CCS1477 treatment resulted in virtually complete inhibition of tumor growth that was maintained up to 24 days after cessation of treatment by which time tumor recurrence was evident. Tumors were excised from vehicle and CCS1477 treated animals at day 7, day 28, and day 52; mRNA was isolated and gene expression analysis was carried using Affymetrix Clarion D microarrays. Differential gene expression based on fold change (FC) >1.5 and FDR-adjusted p-value <0.05 identified a number of genes with significant FC in CCS1477 vs vehicle treated control tumors. Results: Although ~1.5 fold downregulation of AR was maintained from day 7 to day 52, downregulation of AR target genes ETS2, TMPRSS2 and NKX3.1 recovered after treatment cessation. Similarly, expression of c-myc was significantly reduced at day 7 (-2.7 FC) and recovered by day 28. Of note, among the top downregulated genes were CIART and BHLHE40 circadian clock regulated genes that provide negative feedback loops that in conjunction with downregulation of AR and c-myc would disrupt circadian gene regulation. VEGFA mRNA that was downregulated >1.5 fold at all time points, together with c-myc and p300 are all under circadian regulation. Expression of the histone demethylase KDM3A was reduced >1.5 fold at all time points. KDM3A is known to function as an AR coactivator of key AR target genes including NKX3.1 and c-myc. Conclusions: Gene expression analysis of CCS1477 treated prostate tumors suggests an underlying mechanism involving the inhibition of key drivers of prostate cancer progression including AR and c-myc and a network of interacting pathways. Citation Format: Paul Elvin, Neil Pegg, Simone Daminelli, Izabela Eden, Barbara Young, Amy Prosser, Jenny Worthington, Nigel Brooks. P300/CBP inhibitor CCS1477 targets 22Rv1 prostate tumor AR and c-myc gene expression in vivo [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 1019.

E1A binding protein (p300) and CREB binding protein (CBP) are two closely related histone acetyl transferases with oncogenic roles in acute myeloid leukaemia (AML) and multiple myeloma (MM). Here we describe the pre-clinical characterization of CCS1477, an orally bioavailable, potent and selective inhibitor of the bromodomain of p300/CBP and its therapeutic application in AML and MM. CCS1477 binds to p300 and CBP with high affinity (KD=1.3/1.7nM), and selectivity (e.g. KD=222nM; BRD4) in a surface plasmon resonance assay. It is a potent inhibitor of cell proliferation in a panel of 16 AML and 9 MM human cell lines. MM cells were particularly sensitive to CCS1477 with the majority of lines having a GI50 below 100nM. In stromal co-culture assays CCS1477 also inhibited proliferation of primary patient AML blast cells from a range of patients with a variety of molecular subtypes. These anti-proliferative effects were due to a combination of cell cycle arrest (with a decrease of cells in S-phase and an increase in cells in G1/G0) and induction of differentiation, as confirmed by up regulation of selected differentiation markers (e.g. CD11b & CD86) flow cytometric analyses. In xenograft models of AML (MOLM-16) and MM (OPM-2), daily oral dosing with CCS1477 as monotherapy, caused a dose-dependent reduction in tumour growth with regressions observed at the highest dose of 20mg/kg. After cessation of treatment with 20mg/kg CCS1477, there was sustained inhibition of tumour growth for approximately 11 days before re-growth began. The inhibition of tumour growth during drug treatment, was accompanied by significant reduction in tumour expression of MYC and IRF4 by qPCR in the OPM-2 model and in MYC expression in MOLM-16. The effects of CCS1477 were further evaluated in murine models by comparison with candidate standard of care regimens for AML & MM. In the MOLM-16 (AML) model, CCS1477 administered daily by oral gavage (10mg/kg) demonstrated superior tumour growth inhibition by comparison with azacitidine or cytarabine. There was also a significant combination benefit of CCS1477 when administered with these two agents in this model. In OPM-2 MM cells that are sensitive to lenalidomide, CCS1477 was a significantly more potent inhibitor of cell proliferation (GI50 = 5nM; CCS1477 vs. 100nM; lenalidomide). CCS1477 also retains exquisite anti-proliferative potency in MM cells that are either intrinsically resistant to lenalidomide (KMS-11 and RPMI 8226) or in OPM-2 cells that have developed resistance after long-term culture in lenalidomide. CCS1477 shows significant synergy when combined with lenalidomide in OPM-2 cells in vitro and also in an OPM-2 xenograft model in vivo. Significant combination benefit of CCS1477 with vorinostat or velcade is also observed in this xenograft model. These data support the clinical testing of CCS1477 in haematological malignancies, including MM and AML, with a strong pre-clinical rationale for use as monotherapy or in combination with standard of care agents, such as lenalidomide. CCS1477 is currently in Phase I/II clinical trials. Disclosures Brooks: CellCentric Ltd: Employment, Equity Ownership. Somervaille:Novartis: Consultancy. Pegg:CellCentric Ltd: Employment, Equity Ownership.

Background: CCS1477 is a potent and selective p300/CBP bromodomain inhibitor, currently in a Ph1 trial for patients with metastatic castration resistant prostate cancer (mCRPC). CCS1477 works by inhibiting the expression and function of the androgen receptor (AR), as well as inhibiting c-Myc. Bromodomain and extraterminal domain (BET) protein inhibitors are also being developed in mCRPC. We have established a BET inhibitor (BETi) resistant 22Rv1 prostate cancer cell line and used this, alongside parental 22Rv1 cells, to characterise the differential effects of p300/CBP vs BET bromodomain inhibition. Methods: 22Rv1 cells which express both the wild-type and splice variant forms of AR were cultured in the presence of increasing concentrations (30-500nM) of JQ1 for several months. A parallel set of 22Rv1 cells were cultured in the presence of vehicle (0.1% DMSO). The anti-proliferative effects of JQ1, iBet762, OTX-015 and CCS1477 (10 nm-10 µM dose range) was determined in resistant and parental 22Rv1 cells in a 5d CellTitre Glo assay. The effects of combining CCS1477 with JQ1 was measured in parental 22Rv1 cells. Protein biomarker (AR, AR-splice variant, c-Myc) responses were measured by Western blot and qPCR was used to determine changes in the expression of selected genes (AR, AR-V7, c-Myc, KLK3, TMPRSS2). Gene expression microarrays (Clariom D) were used to assess global gene expression changes in cells treated for 24h with 500nM CCS1477 or JQ1. Results: JQ1 resistant 22Rv1 cells were significantly less sensitive to JQ1 compared with parental cells. (IC50; Res, 7.3 µM vs parental, 0.06 µM). There was also cross-resistance to other chemically distinct BET inhibitors, iBET762 and OTX-015. JQ1 potently inhibited c-Myc protein and gene expression in parental cells, a response that was abrogated in the JQ1 resistant line. The inhibitory effects of JQ1 on AR gene and protein expression were reduced in the resistant line. In contrast potent anti-proliferative effects of CCS1477 were retained in JQ1 resistant cells, as was the inhibitory effect on c-Myc and AR. Combination of CCS1477 & JQ1 resulted in a highly synergistic inhibitory effect on proliferation in normal 22Rv1 cells. Global gene expression analysis revealed significantly fewer altered genes after CCS1477 (27 up, 119 down) compared to JQ1 (196 up, 655 down). Conclusions: These studies provide three lines of evidence for a differentiated mode of action of CCS1477 vs BETi. First, CCS1477 continues to inhibit proliferation and relevant response biomarkers in a cell line that is resistant to BETi. Second, there is a synergistic, rather than additive effect of combining CCS1477 with JQ1. Third, there are significantly fewer genes and a distinct pattern of gene change after CCS1477 vs. JQ1. Collectively, these data point to a differentiated and more selective profile after p300/CBP inhibition with CCS1477. Citation Format: Nigel Brooks, Amy Prosser, Barbara Young, Luke Gaughan, Paul Elvin, Neil Pegg. CCS1477, a potent and selective p300/CBP bromodomain inhibitor, is targeted & differentiated from BET inhibitors in prostate cancer cell lines in vitro [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 3826.

Background: Sustained androgen receptor pathway activation is the hallmark of castration-resistant prostate cancer (CRPC). Therapeutic strategies for CRPC include non-ligand-binding domain targeted degradation of androgen receptor (AR) and AR variants (ARV). E1A binding protein (p300) and CREB binding protein (CBP) are two closely related histone acetyl transferase proteins that are critical transcriptional regulators of the androgen receptor. We have developed CCS1477, which is a potent, selective, and orally active small-molecule inhibitor of the bromodomain of p300/CBP, and investigated its role in regulating androgen receptor expression and function. Methods: Binding affinity of CCS1477 to p300, CBP and BRD4 was measured in a surface plasmon resonance (SPR) assay. Potency and functional activity (proliferation, cell cycle analysis, and biomarker knockdown) was demonstrated in a panel of prostate cells lines representing hormone-responsive (LNCaP), hormone-independent (DU145, PC3), and castration-resistant disease (22Rv1, VCaP, LNCaP-AR). A JQ1 resistant cell line was generated by culturing cells in increasing doses of JQ1 over a period of approximately 9 months and used to differentiate potency and functional activity of BET vs p300/CBP inhibition in vitro. In vivo efficacy, linked to inhibition of biomarkers, was determined in 22Rv1 and LNCaP xenograft models. Results: CCS1477 binds to p300 and CBP with high affinity (KD=1.3/1.7nM) and selectivity (KD=222nM; BRD4). It is a potent inhibitor of cell proliferation in castration-resistant cell lines (IC50= 96nM,22Rv1; 49nM,VCaP; 150nM,LNCaP-AR) with minimal effect in AR-ve lines (PC3, DU145). These inhibitory effects on cell proliferation were accompanied by a decrease in the number of cells in S phase and an increase in the number of cells in the G1/G0 phase of the cell cycle. We have developed a JQ1 resistant 22Rv1 cell line and observed a marked reduction in sensitivity (proliferation end point) to JQ1 as expected, which is accompanied by cross-resistance to a variety of other BET inhibitors, including iBET-151 and OTX-015. In contrast, and as evidence for a differentiated mechanism, sensitivity to CCS1477 is retained in this JQ1 resistant line. Inhibition of the bromodomain of p300/CBP downregulates AR-FL, AR-V7 and c-Myc protein in 22Rv1 cells by Western blot and this is accompanied by profound inhibition of c-Myc, KLK3 and TMPRSS2 genes measured by qPCR. The in vivo PK properties of CCS1477 are consistent with qd or qod oral dosing in mouse. CCS1477 dosed at 10mg/kg qd, 20mg/kg qd, or 30mg/kg qod caused complete tumor growth inhibition over 28 days in a 22Rv1 xenograft model, including extended duration in the absence of the drug for a further 24 days. This was accompanied by complete inhibition of plasma PSA and significant knockdown of tumor AR-FL, AR-V7, and c-Myc protein as well as c-Myc and TMPRSS2 mRNA expression. At 100mg/kg, a single dose of CCS1477 induces expression of cleaved PARP and when dosed orally every three days, causes tumor regression in the 22Rv1 model. Furthermore, CCS1477 results in almost complete tumor growth inhibition in a bicalutamide-resistant LNCaP xenograft model, when given alone or in combination with enzalutamide. Conclusions: Taken together, these data support the concept that therapeutic targeting of the p300/CBP bromodomain results in a novel and differentiated approach to targeting androgen receptor pathway activation in castration-resistant prostate cancer. Citation Format: Nigel Brooks, Neil Pegg, Jenny Worthington, Barbara Young, Amy Prosser. Therapeutic targeting of the p300/CBP bromodomain for the treatment of castration-resistant prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A091.

Background: E1A binding protein (p300) and CREB binding protein (CBP), two paralogue histone acetyl transferase proteins, act as transcriptional co-activators of a variety of cancer related genes. We have developed CCS1477, a potent, selective and orally active small molecule inhibitor of the bromodomain of p300/CBP and investigated its role in regulating AR and c-Myc expression and function, for the treatment of prostate cancer and haematological malignancies. We have also examined the role of p300/CBP in driving synthetic lethality in tumours with loss of function mutations (eg. bladder cancer). Methods: Potency and functional activity was evaluated in a panel of prostate cells lines representing hormone responsive, hormone independent and castration resistant disease. Effects of CCS1477 on AR, AR-V7 splice variant and c-Myc protein, as well as KLK3, c-Myc, UBE2C, CCNA2 and TMPRSS2 gene expression, were assessed. Inhibition of proliferation and function by CCS1477 was also examined in acute myeloid leukaemia cell lines and patient derived primary AML cells. In addition, potency was determined in bladder cell lines possessing a loss of function mutation in p300/CBP and compared to wild type. Results: CCS1477 is a potent inhibitor of cell proliferation in castration resistant prostate cell lines (IC50 = 96nM 22Rv1; 49nM VCaP) with minimal effect in AR-ve lines (PC3 and DU145). Treatment of 22Rv1 and VCaP cells with CCS1477 significantly reduced expression of KLK3, UBE2C and CCNA2 in the presence and absence of enzalutamide indicating compromised signalling via AR and AR-SV. Furthermore, AR and AR-SV protein levels were inhibited in response to CCS1477 treatment. Utilising an enzalutamide-resistant cell line (LNCaP-ARF876L), CCS1477 treatment down-regulated both androgen and enzalutamide-stimulated KLK3 and TMPRSS2 gene expression. CCS1477 dosed at 20mg/kg qd caused complete tumour growth inhibition in a 22Rv1 xenograft model. CCS1477 is also a potent inhibitor of proliferation in AML cell lines (IC50 ~ 100nM; THP-1; MV4-11), with effects mediated by G1 cell cycle arrest and accompanied by myeloid differentiation. Comparable results are observed on patient derived primary AML cells. In bladder cancer cell lines, we observed differential sensitivity to CCS1477 with p300/CBP deficient lines (IC50 = 300nM VM-CUB-2 and 647V) compared with wild type (no activity at 30uM, RT112). Conclusions: Taken together these data support the clinical testing of p300/CBP inhibition in patients in three settings; (i) castration resistant prostate cancer through down-regulating AR, AR-SV and c-Myc expression and function; (ii) haematological cancers by effects on cell cycle arrest and myeloid differentiation, and (iii) patients with loss of function mutations in p300 or CBP by driving synthetic lethality. Citation Format: Neil Pegg, Jenny Worthington, Barbara Young, Amy Prosser, Luke Gaughan, Gary Spencer, Tim Somervaille, Julie Burns, Margaret Knowles, Nigel Brooks. Novel small molecule inhibitors of p300/CBP down-regulate androgen receptor (AR) and c-Myc for the treatment of prostate cancer and beyond [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 3991.

11590 Background: Targeted degradation of androgen receptor (AR) and AR variants (ARV) remains an attractive therapeutic opportunity for patients with castrate resistant prostate cancer (CRPC). E1A binding protein (p300) and CREB binding protein (CBP) are two closely related transcriptional activators of AR. We have developed CCS1477 which is a potent, selective and orally active small molecule inhibitor of the bromodomain of p300/CBP and investigated its role in regulating androgen receptor expression and function. Methods: Binding of CCS1477 to p300, CBP and BRD4, was measured in a surface plasmon resonance (SPR) assay. Potency and functional activity (proliferation and biomarker knockdown) was demonstrated in prostate cell lines in vitro (22Rv1, VCaP). Cross species in vivo pharmacokinetic (PK) properties were assessed, and in vivo efficacy, linked to inhibition of biomarkers, was determined in 22Rv1 and LNCaP xenograft models. Results: CCS1477 binds to p300 and CBP with high affinity (Kd = 1.3/1.7nM) and selectivity (Kd = 222nM; BRD4). It is a potent inhibitor of cell proliferation in prostate cell lines (IC50 = 96nM,22Rv1; 49nM,VCaP) with minimal effect in AR-ve lines. In 22Rv1 cells, p300/CBP inhibition down-regulates AR-FL, AR-V7 and c-Myc protein by Western, an effect not seen with the BET inhibitor, JQ1 at equivalent proliferation IC50s. Inhibition of p300/CBP also reduces c-Myc, KLK3 and TMPRSS2 gene expression (qPCR) in 22Rv1 cells in vitro. The in vivo PK properties of CCS1477 are consistent with qd or qod oral dosing in mouse. CCS1477 dosed at 10mg, 20mg/kg qd or 30mg/kg qod, caused complete tumour growth inhibition over 28 days in a 22RV1 xenograft model, including extended duration in the absence of the drug for a further 24 days. This was accompanied by complete inhibition of plasma PSA and significant knockdown of tumour AR-FL, AR-V7, and C-Myc protein as well as C-Myc and TMPRSS2 mRNA expression. Conclusions: Taken together these data support the clinical testing of CCS1477 in castrate resistant prostate cancer by down-regulation of AR, AR-SV and c-MYC expression and function.

168Background: Targeted degradation of androgen receptor (AR) and androgen receptor variants (ARV) remains an attractive therapeutic opportunity for patients with castrate resistant prostate cancer (CRPC). E1A binding protein (p300) and CREB binding protein (CBP) are two closely related histone acetyl transferase proteins that act as transcriptional activators of AR. We have developed potent, selective and orally active small molecule inhibitors of the bromodomain of p300/CBP and investigated their role in regulating the expression and function of AR and ARV. Methods: Binding affinity to p300, CBP and BRD4 was measured in a surface plasmon resonance (SPR) assay and potency and functional activity was demonstrated in a panel of prostate cells lines representing hormone responsive (LNCaP), hormone independent (DU145, PC3) and castrate resistant disease (22Rv1, C4-2, VCaP, LNCaP-AR). Effects of p300/CBP inhibitors (and the BET inhibitor, JQ1), on AR, AR-V7 splice variant and c-Myc protein, as well as c-Myc, ...

Abstract Background: E1A binding protein (p300) and CREB binding protein (CBP) are two closely related, paralogue histone acetyl transferase proteins that act as transcriptional co-activators of a variety of cancer related genes. We have developed potent, selective and orally active small molecule inhibitors of the bromodomain of p300/CBP and investigated their role in regulating androgen receptor expression and function. We have also examined their role in driving synthetic lethality in tumours. Loss of function mutations in either p300 or CBP (including in significant proportions of lung and bladder tumours), can lead to a dependency on the corresponding paralogue protein. Methods: Binding affinity to p300, CBP and BRD4 was measured in a surface plasmon resonance (SPR) assay. Potency and functional activity was demonstrated in a panel of prostate cells lines representing hormone responsive (LNCaP), hormone independent (DU145, PC3) and castrate resistant disease (22Rv1, C4-2, VCaP, LNCaP-AR) as well as wildtype (A549) and CBP deficient (H520, H1703, LK2) lung cancer cells. Combination effects of p300/CBP inhibitors with a PARP or CDK4/6 inhibitor were determined in LNCaP and C4-2 cells. Effects of p300/CBP inhibitors (and by comparison, the BET inhibitor, JQ1), on AR, AR-V7 splice variant and c-Myc protein, as well as c-Myc, KLK3 and TMPRSS2 gene expression, were assessed in 22Rv1 cells in vitro. In vivo effects on biomarkers were measured in a 22Rv1 xenograft model. Results: CCS1357, an in vitro probe compound, binds to p300 and CBP with high affinity (Kd=4nM) and selectivity (Kd=245nM; BRD4). It is a potent inhibitor of cell proliferation in castrate resistant cell lines (IC50=100nM in LnCaP-AR; 350nM in 22Rv1) with minimal effects in hormone independent lines. CCS1357 combined with palbociclib (CDK4/6) or olaparib (PARP) in LNCaP or C4-2 cells, showed reduced cell viability compared with any of these drugs given alone. In 22RV1 cells, CCS1357 significantly down-regulated AR-FL, AR-V7 and c-Myc protein by Western, an effect not seen with JQ1 at equivalent proliferation IC50s. CCS1357 effects were reversed by the proteasome inhibitor, MG132. CCS1357 also caused a profound inhibition of c-Myc, KLK3 and TMPRSS2 genes measured by qPCR in 22Rv1 cells in vitro. A preclinical candidate (CCS1477) given as a single oral dose (30mg/kg) inhibited plasma PSA and tumour AR, AR-V7 and c-Myc in a 22Rv1 xenograft model. In the lung cancer cell lines, we observed differential sensitivity to CCS1357; CBP deficient lines were more sensitive (cell proliferation) compared with normal. Conclusions: Taken together these data support the clinical testing of p300/CBP inhibition in patients in two settings; firstly, castrate resistant prostate cancer by down-regulating of AR, AR-SV and c-MYC expression and function; and secondly in patients with loss of function mutations in p300 or CBP by driving synthetic lethality. Citation Format: Nigel Brooks, Neil Pegg, Jenny Worthington, Barbara Young, Amy Prosser, Jordan Lane, David Taddei, Matthew Schiewer, Renee deLeeuw, Jennifer McCann, Karen Knusden. Novel small molecule inhibitors of p300/CBP down-regulate AR and c-Myc for the treatment of castrate resistant prostate cancer [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 1575. doi:10.1158/1538-7445.AM2017-1575