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PDF - 56KB, Supplementary methods detailing Relative peptide quantification by selected reaction monitoring (SRM)using mass spectrometry

PDF - 464KB, Supplementary Figure 1: Inhibition of AR transcripts with AZD3514 treated LNCaPs in steroid free media and the timecourse of AZD3514 effects in the presence and absence of DHT. Supplementary Figure 2: Effect of AZD3514 on growth of AR negative cells. Supplementary Figure 3: AR and PSA expression in LAPC4 cells treated with AZD3514. Supplementary Figure 4: AR degradation and synthesis rates of a C-terminal AR peptide, as measured by Mass Spectrometry. Supplementary Figure 5: Comparison of ARD1 & AZD3514 effects on seminal vesicle weight in rats, and pharmacokinetic effects in mice and rats Supplementary Figure 6: Effect of ARD1 on growth and AR in the HID28 model. Supplementary Figure 7: Dose response effects of AZD3514 on AR nuclear translocation and foci formation.

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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

PDF - 329KB, Supplementary Table 1: TSQ Vantage mass spectrometry instrument settings. Supplementary Table 2: 4000 QTRAP mass spectrometry instrument settings. Supplementary Table 3: The half-life of AR for the first 6 hours following treatment calculated for both the N-terminal (i) and C-terminal (ii) AR peptides assuming exponential decay. Supplementary Table 4: Summary table of pharmacokinetics of ARD1 and AZD3514 in the mouse.

Supplementary figures

Figure S1: Evidence of FGFR1 heterogeneity by FISH in samples from the same patient. FGFR1 FISH images from Patient 4 (A) and patient untreated on the study (B, Patient X) are shown. Patient 4's samples were both taken from the lung, with one showing amplification and the other showing polysomy. Patient X's samples were taken from a soft tissue lesion and the lung, with the former showing gene amplification and the latter, 2-3 copies per cell without amplification. Figure S2: C2D1 and C3D1 PK data with reference to response

PDF file - 106K, Detection of FGFR1 gene amplification in DMS114 cells using FISH analysis

Supplementary Figure Legend

PDF file - 94K, Supplementary Table 1. AZD4547 GI50 comparisons between FGFR1-amplified DMS114 and EGFR-mutant NCI-H1975 cell lines. Supplementary Table 2. Dynamic modulation of pERK, pS6, Ki67 and CC3 markers in model L121 tumor sections treated with a single dose of 12.5mg/kg AZD4547. Supplementary Table 3. Statistical association analysis of tumor histology and patient smoking status. Supplementary Table 4. FGFR1 amplification shows high mutual exclusivity with EGFR, Kras and EML4-ALK status in Chinese NSCLC.

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Purpose: Squamous cell lung cancers (SQCLC) account for 25% of all NSCLCs, yet the prognosis of these patients is poor and treatment options are limited. Amplified FGFR1 is one of the most common oncogenic events in SQCLCs, occurring in approximately 20% of cases. AZD4547 is a potent and selective FGFR1-3 inhibitor with antitumor activity in FGFR1-amplified SQCLC cell lines and patient-derived xenografts.Experimental Design: On the basis of these data, we performed a phase I study of AZD4547 in patients with previously treated stage IV FGFR1-amplified SQCLCs (NCT00979134). FGFR1 amplification (FGFR1:CEP8 ≥ 2) was determined by FISH. The primary endpoint was safety/tolerability. Secondary endpoints included antitumor activity, pharmacokinetics, pharmacodynamics, and molecular analyses.Results: Fifteen FGFR1-amplified patients were treated. The most common related adverse events (AE) were gastrointestinal and dermatologic. Grade ≥3–related AEs occurred in 3 patients (23%). Thirteen patients were evaluable for radiographic response assessment. The overall response rate was 8% (1 PR). Two of 15 patients (13.3%) were progression-free at 12 weeks, and the median overall survival was 4.9 months. Molecular tests, including next-generation sequencing, gene expression analysis, and FGFR1 immunohistochemistry, showed poor correlation between gene amplification and expression, potential genomic modifiers of efficacy, and heterogeneity in 8p11 amplicon.Conclusions: AZD4547 was tolerable at a dosage of 80 mg oral twice a day, with modest antitumor activity. Detailed molecular studies show that these tumors are heterogeneous, with a range of mutational covariates and stark differences in gene expression of the 8p11 amplicon that likely explain the modest efficacy of FGFR inhibition in this disease. Clin Cancer Res; 23(18); 5366–73. ©2017 AACR.

MSK-IMPACT gene list

AZD4547 dose intensity

PDF file - 219K, AZD4547 demonstrates potent anti-tumor efficacy in the standard FGFR1-amplified xenograft lung model, NCI-H1581

Next-generation sequencing data by patient

Percent tumor cell content in H&E slides from patients where available

nanoString codeset list

Treatment history for each patient where available

Purpose: To investigate the incidence of FGFR1 amplification in Chinese non–small cell lung cancer (NSCLC) and to preclinically test the hypothesis that the novel, potent, and selective fibroblast growth factor receptor (FGFR) small-molecule inhibitor AZD4547 will deliver potent antitumor activity in NSCLC FGFR1–amplified patient-derived tumor xenograft (PDTX) models.Experimental Design: A range of assays was used to assess the translational relevance of FGFR1 amplification and AZD4547 treatment including in vitro lung cell line panel screening and pharmacodynamic (PD) analysis, FGFR1 FISH tissue microarray (TMA) analysis of Chinese NSCLC (n = 127), and, importantly, antitumor efficacy testing and PD analysis of lung PDTX models using AZD4547.Results: The incidence of FGFR1 amplification within Chinese patient NSCLC tumors was 12.5% of squamous origin (6 of 48) and 7% of adenocarcinoma (5 of 76). AZD4547 displayed a highly selective profile across a lung cell line panel, potently inhibiting cell growth only in those lines harboring amplified FGFR1 (GI50 = 0.003–0.111 μmol/L). AZD4547 induced potent tumor stasis or regressive effects in four of five FGFR1-amplified squamous NSCLC PDTX models. Pharmacodynamic modulation was observed in vivo, and antitumor efficacy correlated well with FGFR1 FISH score and protein expression level.Conclusions: This study provides novel epidemiologic data through identification of FGFR1 gene amplification in Chinese NSCLC specimens (particularly squamous) and, importantly, extends the clinical significance of this finding by using multiple FGFR1-amplified squamous lung cancer PDTX models to show tumor stasis or regression effects using a specific FGFR inhibitor (AZD4547). Thus, the translational science presented here provides a strong rationale for investigation of AZD4547 as a therapeutic option for patients with squamous NSCLC tumors harboring amplification of FGFR1. Clin Cancer Res; 18(24); 6658–67. ©2012 AACR.

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BackgroundThe sustainability of crop production is impacted by climate change and land degradation, and the advanced application of nanotechnology is of paramount importance to overcome this challenge. The development of nanomaterials based on essential nutrients like zinc could serve as a basis for nanofertilizers and nanocomposite synthesis for broader agricultural applications and quality human nutrition. Therefore, this study aimed to synthesize zinc oxide nanoparticles (ZnO NPs) using pecan (Carya illinoinensis) leaf extract and investigate their effect on the growth, physiology, nutrient content, and antioxidant properties of mustard (Brassica juncea).MethodsThe ZnO NPs were characterized by UV-Vis spectrophotometry, Dynamic Light Scattering (DLS), X-ray diffractometer (XRD), Scanning Electron Microscopy (SEM), and Fourier Transform Infra-Red Spectroscopy (FTIR). Mustard plants were subjected to different concentrations of ZnONPs (0, 20, 40, 60, 80, 100 and 200 mg L-1) during the vegetative growth stage.ResultsThe UV-Vis spectra of ZnO NPs revealed the absorption maxima at 362 nm and FTIR identified numerous functional groups that are responsible for capping and stabilizing ZnO NPs. DLS analysis presented monodispersed ZnO NPs of 84.5 nm size and highly negative zeta potential (-22.4 mV). Overall, the application of ZnO NPs enhanced the growth, chlorophyll content (by 53 %), relative water content (by 46 %), shoot biomass, membrane stability (by 54 %) and net photosynthesis significantly in a dose-dependent manner. In addition, the supplement of the ZnO NPs augmented K, Fe, Zn and flavonoid contents as well as overcome the effect of reactive oxygen species by increasing antioxidant capacity in mustard leaves up to 97 %.ConclusionsIn conclusion, ZnO NPs can be potentially used as a plant growth stimulant and as a novel soil amendment for enhancing crop yields. Besides, the biofortification of B. juncea plants with ZnO NPs helps to improve the nutritional quality of the crop and perhaps potentiates its pharmaceutical effects.

Prostate cancer (PCa) is the second leading cause of cancer-related deaths in men in the US. There is a largely unmet clinical need to identify and develop novel strategies, that work either alone or in concert with AR-directed therapeutics, to combat CRPC. The highly conserved histone acetyltransferases CBP/p300 are potent co-activators for AR, and high p300 expression is associated with locally advanced disease and castration-resistant AR function. This study shows that CBP and p300 are highly expressed and correlate closely with AR gene expression and AR activity score in primary PCa and CRPC. By employing clinically relevant PCa models, the clinical significance of CBP/p300 expression in PCa patients as well as mechanistic evaluation of CBP/p300 transcriptional reprogramming and DNA damage response pathways have been undertaken. The molecular response to CBP/p300 inhibition will be assessed to discern novel metrics for precision medicine for PCa patients to improve therapeutic efficacy. Previous studies have relied on non-specific compounds and genetic silencing to target CBP/p300. CCS1477 (inobrodib) is a first-in-class bromodomain inhibitor developed by Cell Centric and targeted to inhibit CBP/p300 mediated bromodomain activity, and thus regulate cell survival. Inhibition of the CBP/p300 bromodomain resulted in significant downregulation of AR-FL, AR-V7, and its targets’ mRNA expression, as well as inhibition of associated factors such c-MYC and its downstream targets, in multiple PCa models. Transcriptomic analysis indicated that both CBP and p300 expression correlate with expression of genes involved in double strand break (DSB) DNA repair process including homologous recombination (HR) and non-homologous end joining (NHEJ) in both primary PCa and CRPC models. CCS1477 directly impacted DNA damage response and repair dynamics, as shown via delay in time to resolution of DNA damage foci formation, including RAD51 and γH2AX foci. Inhibition of CBP/p300 activity decreased tumor cell proliferation, blocked CRPC xenograft growth in vivo, and decreased proliferation ex vivo in patient-derived prostate tumor explants. Importantly, CBP/p300 expression correlated with HR genes in human prostate tissue samples in different cohorts. Lastly, inhibition of CBP/p300 activity also decreased HR gene expression in patients further supporting the essential role CBP/p300 plays in DNA repair. In sum, CBP/p300 inhibition mediates HR repair and impacts patient outcome. In conclusion, these studies identify CBP/p300 as a driver of PCa tumorigenesis through coordinated control of critical transcriptional events and lay the groundwork to optimize therapeutic strategies for advanced PCa via CBP/p300 inhibition, potentially in combination with AR-directed therapies. Combined, these studies have the capacity for significant near-term impact in the prevention and/or management of metastatic disease. Citation Format: Sumaira Sardar, Lakshmi Ravindranath, Christopher McNair, Saswati Chand, Wei Yuan, Denisa Bogdan, Jon Welti, Adam Sharp, Matthew Schiewer, Lisa Butler, Johann de Bono, Kris Frese, Nigel Brooks, Neil Pegg, Karen Knudsen, Ayesha Shafi. Targeting CBP/p300 and its downstream transcriptional machinery in advanced prostate 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 1449.

Background: Blockade of programmed death-ligand 1 (PD-L1) by therapeutic antibodies has shown to be a promising strategy in cancer therapy, yet clinical response in many types of cancer is limited. Tumor cells secrete PD-L1 through exosomes or splice variants, which has been described as a new mechanism for the resistance to PD-L1 blockade therapy in multiple cancers. Our previous findings showed that the histone acetyltransferases p300/CBP could be recruited to the promoter of CD274 (encoding PD-L1) to regulate the expression of PD-L1. P300/CBP inhibition abrogated this process and reduced the secretion of exosomal PD-L1 by blocking the transcription of CD274, which combined with the anti-PD-L1 antibody to reactivate T cells function for tumor attack. In this study we have tested the outcome of combined immune checkpoint inhibitors with CCS1477, a novel p300/CBP bromodomain inhibitor, in pre-clinical models of prostate cancer and melanoma. Results: CCS1477 significantly decreased human prostate (DU145/PC-3) and melanoma (A375/SK-MEL-28) cancer cell line PD-L1 expression, as well as exosome secreted PD-L1 after 24 hours treatment. Moreover, consistent with these findings in vitro, CCS1477 (2.5mg//kg) also reduced PD-L1 expression in a syngeneic mouse prostate model (TRAMP-C2) and syngeneic mouse melanoma model (B16-F10). Meanwhile, CCS1477 treatment significantly increased tumor-infiltrating CD8+ and CD4+ T cells in syngeneic mouse models. Monotherapy treatment with CCS1477 or immune checkpoint inhibitors (anti-PD-L1 antibody for prostate cancer or anti-PD-L1 and anti-CTLA4 antibody for melanoma) resulted in significant but modest efficacy. Strikingly, the combination of immune checkpoint inhibitors with CCS1477 showed robust synergistic responses resulting in tumor regression. Mechanistically, CCS1477 treatment induced significant loss of H3K27 acetylation from super-enhancers of CD274, which subsequently induced a significant reduction of PD-L1 expression and exosome secreted-PD-L1. Also, we found that CCS1477 treatment significantly decreased the abundance of circulating and tumor-infiltrating myeloid-derived suppressor cells (MDSCs), which are known to play important roles in tumor immune evasion by inhibiting T cell’s activation. Reduction of MDSCs and exosome secreted-PD-L1 preserved T-cell function and elicited robust immunotherapy response combined with immune checkpoint blockade agents. Conclusions: These observations illuminate a clinically relevant hypothesis for combining CCS1477 with immune checkpoint blockade in the treatment of cancer. Citation Format: Jinghui Liu, Xinyi Wang, Katelyn Jones, Nigel Brooks, Neil Pegg, Xiaoqi Liu. p300/CBP bromodomain inhibitor CCS1477 enhances the efficacy of immune checkpoint blockade therapy in cancer treatment [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 1345.

The nucleotide analogue azacitidine (AZA) is currently the best treatment option for patients with high-risk myelodysplastic syndromes (MDS). However, only half of treated patients respond and of these almost all eventually relapse. New treatment options are urgently needed to improve the clinical management of these patients. Here, we perform a loss-of-function shRNA screen and identify the histone acetyl transferase and transcriptional co-activator, CREB binding protein (CBP), as a major regulator of AZA sensitivity. Compounds inhibiting the activity of CBP and the closely related p300 synergistically reduce viability of MDS-derived AML cell lines when combined with AZA. Importantly, this effect is specific for the RNA-dependent functions of AZA and not observed with the related compound decitabine that is only incorporated into DNA. The identification of immediate target genes leads us to the unexpected finding that the effect of CBP/p300 inhibition is mediated by globally down regulating protein synthesis., Azacitidine (AZA) treatment is used for patients with myelodysplasias that cannot undergo bone marrow transplantation; however, AZA treatment is only partially effective. Here the authors show synergy of AZA with compounds inhibiting the chromatin regulators CBP and p300, which is mediated by the RNA-dependent functions of AZA affecting protein translation.

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

E1A binding protein (p300) and CREB binding protein (CBP) are two closely related histone acetyl transferases that co-activate key oncogenes such as MYC and IRF4 which are relevant in a number of haematological malignancies. Here we describe the effects of CCS1477, a new orally available inhibitor of the p300/CBP bromodomains, given alone or in combination with azactidine or venetoclax in pre-clinical models of AML and B-cell lymphoma. As a monotherapy, daily oral dosing with CCS1477 (5, 10 and 20mg/kg) caused a significant dose-dependent reduction in tumour growth in a MOLM-16 xenograft model of AML, with regressions observed at the highest and well tolerated dose of 20mg/kg/qd. The inhibition of tumour growth during drug treatment was accompanied by a significant reduction in tumour expression of MYC by qPCR. In murine retroviral transduction and transplantation models of human AML initiated by either MLL-AF10 or MLL-AF9, daily monotherapy by oral gavage with CCS1477 at 30mg/kg for 42 days, caused a highly significant prolongation of survival. All seven control treated mice died of AML within 160 days whereas only one of seven CCS1477 treated mice had died in each model by 300 days. In the MOLM-16 xenograft model, a sub-maximal dose of CCS1477 (10mg/kg) demonstrated superior tumour growth inhibition by comparison with azacitidine (0.5mg/kg i.p) and with significant combination benefit when these two agents were combined. In this same model venetoclax (50 or 100 mg/kg/qd oral) had no effect on tumour growth, indicating that MOLM-16 tumours are intrinsically resistant. CCS1477 dosed daily at 10mg/kg restored sensitivity to venetoclax as evidenced by significantly greater tumour growth inhibition when compared with CCS1477 given alone. In a venetoclax-sensitive xenograft model of AML (MV-4-11), CCS1477 (10mg/kg) and venetoclax (100mg/kg) resulted in a similar and partial reduction in tumour growth over a 21 day dosing period. When venetoclax and CCS1477 were combined there was a significant combination benefit compared to either drug given alone. The DOHH-2 xenograft model of B-cell lymphoma was also sensitive to venetoclax (50mg/kg) and with a similar impact on tumour growth compared with CCS1477 (10mg/kg). In this model the combination of CCS1477 and venetoclax was more profound and caused complete tumour stasis during the dosing period. These data support the clinical testing of CCS1477 in combination with azacitidine and/or venetoclax in AML and B-cell lymphomas. CCS1477 is currently in Phase I/II clinical trials in AML, Non-Hodgkin lymphoma (including B-cell lymphoma) and multiple myeloma. (NCT04068597). Disclosures Brooks: CellCentric Ltd: Current Employment, Current holder of individual stocks in a privately-held company. Knurowski: CellCentric Ltd: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Hughes: CellCentric Ltd: Consultancy, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Clegg: CellCentric Ltd: Current Employment, Current holder of individual stocks in a privately-held company. West: CellCentric Ltd: Current Employment, Current holder of individual stocks in a privately-held company. Pegg: CellCentric Ltd: Current Employment, Current holder of individual stocks in a privately-held company. Somervaille: Novartis: Consultancy, Honoraria.

Purpose: Squamous cell lung cancers (SQCLC) account for 25% of all NSCLCs, yet the prognosis of these patients is poor and treatment options are limited. Amplified FGFR1 is one of the most common oncogenic events in SQCLCs, occurring in approximately 20% of cases. AZD4547 is a potent and selective FGFR1-3 inhibitor with antitumor activity in FGFR1-amplified SQCLC cell lines and patient-derived xenografts. Experimental Design: On the basis of these data, we performed a phase I study of AZD4547 in patients with previously treated stage IV FGFR1-amplified SQCLCs (NCT00979134). FGFR1 amplification (FGFR1:CEP8 ≥ 2) was determined by FISH. The primary endpoint was safety/tolerability. Secondary endpoints included antitumor activity, pharmacokinetics, pharmacodynamics, and molecular analyses. Results: Fifteen FGFR1-amplified patients were treated. The most common related adverse events (AE) were gastrointestinal and dermatologic. Grade ≥3–related AEs occurred in 3 patients (23%). Thirteen patients were evaluable for radiographic response assessment. The overall response rate was 8% (1 PR). Two of 15 patients (13.3%) were progression-free at 12 weeks, and the median overall survival was 4.9 months. Molecular tests, including next-generation sequencing, gene expression analysis, and FGFR1 immunohistochemistry, showed poor correlation between gene amplification and expression, potential genomic modifiers of efficacy, and heterogeneity in 8p11 amplicon. Conclusions: AZD4547 was tolerable at a dosage of 80 mg oral twice a day, with modest antitumor activity. Detailed molecular studies show that these tumors are heterogeneous, with a range of mutational covariates and stark differences in gene expression of the 8p11 amplicon that likely explain the modest efficacy of FGFR inhibition in this disease. Clin Cancer Res; 23(18); 5366–73. ©2017 AACR.

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 first in class potent, selective and orally bioavailable inhibitor of the bromodomains of p300 and CBP, two closely related histone acetyl transferases with oncogenic roles in haematological malignancies. In pre-clinical studies, CCS1477 was found to be a potent inhibitor of cell proliferation in acute myeloid leukaemia (AML) multiple myeloma (MM) and non-Hodgkin lymphoma (NHL) cell lines. In primary patient AML blast cells CCS1477 inhibited proliferation through a combination of cell cycle arrest at the G1/S transition and an induction of differentiation (up-regulation of CD11b and CD86). CCS1477 has significant anti-tumour activity, inducing tumour regressions in xenograft models of AML and MM. These effects were accompanied by significant reductions in tumour MYC and IRF4 expression. Additionally, there are molecular features of certain haematological malignancies that are likely to increase the sensitivity to p300/CBP inhibition with CCS1477. For example, in B-cell lymphomas there are frequent loss of function mutations in CBP that are associated with heightened sensitivity to pre-clinical inhibition of corresponding non-mutated p300. CCS1477 represents a novel and differentiated approach to inhibiting cell proliferation and survival and offers a potential new therapeutic option for patients who have relapsed or are refractory to current standard of care therapies in AML, MM or NHL. Study Design and Methods This study is the first time that CCS1477 is being dosed in patients with haematological malignancies. The Phase I/IIa study aims to determine the maximum tolerated dose (MTD) and/or recommended Phase II dose (RP2D) and schedule(s) of CCS1477 and investigate clinical activity of CCS1477 monotherapy in patients with haematological malignancies. This study will also characterise the pharmacokinetics (PK) of CCS1477 and explore potential biological activity by assessing pharmacodynamic and exploratory biomarkers. The trial aims to enrol approximately 90 patients and is currently recruiting in the UK with plans to open additional sites in the USA. Key inclusion criteria include patients with confirmed (per standard disease specific diagnostic criteria), relapsed or refractory haematological malignancies (AML, MM and NHL). Patients must have received standard therapy which for the majority of therapeutic indications is at least 2 prior lines of therapy. Single dose and steady state pharmacokinetics will be determined in all patients. AML response will be measured in bone marrow samples. Myeloma response will be evaluated according to the 'International Myeloma Working Group Response Criteria' based on changes in M protein in blood and/or urine, changes in serum free light chains if measurable, and changes on imaging and/or bone marrow if applicable and according to the guidelines. In NHL patients, tumour assessments will be done for measurable disease, non-measurable disease, and new lesions on CT (or magnetic resonance imaging [MRI]) and/or combined with visual assessment of [18F]2-fluoro-2-deoxy-D-glucose-positron emission tomography (FDG-PET) for response assessment per recent International Working Group consensus criteria (RECIL 2017), until progression The study will begin with two parallel monotherapy dose-escalation arms; Arm 1: Relapsed or refractory NHL and MM; Arm2: Relapsed or refractory AML/high-risk MDS. Once a recommended phase 2 dose/schedule is reached, three monotherapy expansion arms will be opened in AML/high-risk MDS (15 patients), MM (15 patients) and NHL (30 patients). Blood samples along with bone marrow biopsies and aspirates will be collected for exploratory biomarker analysis to understand mechanisms of response to treatment or disease progression. This will include the analysis of tumour-specific and circulating biomarkers, such as tumour DNA, mRNA, proteins or metabolites. In NHL patients, analysis of CBP (and p300) mutations will be undertaken to allow retrospective correlation with tumour response and to determine if loss of function mutations in the genes for either proteins can be utilised as response predictive biomarkers in future studies. Disclosures Clegg: CellCentric Ltd: Employment, Equity Ownership. Brooks:CellCentric Ltd: Employment, Equity Ownership. Ashby:CellCentric Ltd: Employment, Equity Ownership. Pegg:CellCentric Ltd: Employment, Equity Ownership. West:CellCentric Ltd: Employment, Equity Ownership. Somervaille:Novartis: Consultancy. Knapper:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Daiichi Sankyo: Honoraria; Jazz: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Tolero: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees. Davies:ADCT Therapeutics: Honoraria, Research Funding; MorphoSys AG: Honoraria, Membership on an entity's Board of Directors or advisory committees; BioInvent: Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Kite Pharma: Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Research Funding; Karyopharma: Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta Pharma: Honoraria, Research Funding; GSK: Research Funding; Pfizer: Honoraria, Research Funding; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Research Funding; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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.

e16534 Background: Growth and survival of prostate cancer cells are initially dependent upon androgens, and androgen deprivation therapy (ADT) is used to control tumor growth. Unfortunately, resistance to ADT inevitably occurs, and patients relapse with lethal castrate-resistant prostate cancer (CRPC). Increased expression of the androgen receptor (AR) and constitutively active AR variants are hallmarks of CRPC, and treatments targeting aberrant AR signaling are urgently required. CCS1477 is an inhibitor of p300/CBP currently in a Phase I/IIa study for CRPC. CCS1477 enhances degradation of numerous cellular proteins including the AR and AR variants in prostate cancer cells. Our preclinical studies with this compound demonstrated potent single-agent efficacy of CCS1477 using in vitro and in vivo models of prostate cancer and, when used in combination, CCS1477 enhances the efficacy of enzalutamide, a clinical AR antagonist. Understanding the response of clinical tumors to CCS1477, and their potential adaptive evolution, is essential to personalize treatment and predict potential resistance mechanisms. Methods: To assess CCS1477 in human disease, we used a unique model in which clinical prostate tumors from radical prostatectomy are cultured as explants with maintenance of tissue integrity, cell proliferation and androgen signaling. Tumors from 13 patients were cultured in the absence or presence of CCS1477 (10µM) or enzalutamide (10µM) for 48 or 72 hours; micromolar doses were selected to account for altered small molecule uptake and penetration into tissues compared to cell lines, as previously reported. Proliferation, apoptosis and androgen signaling were all analyzed post-culture. Results: Whereas the tumor explants exhibited highly heterogenous proliferative responses to enzalutamide, tumors from all patients exhibited a marked antiproliferative response to CCS1477 (mean reduction in Ki67 immunoreactivity of > 90% compared to vehicle control; p < 0.0005). Culture with CCS1477 was associated with repression of androgen signaling in the prostate tissues, measured by expression and secretion of the clinical biomarker prostate specific antigen (PSA). Conclusions: The consistent and pronounced efficacy of CCS1477 in this patient-derived model would support further investigation of this class of epigenetic agents in the castrate-sensitive prostate cancer setting.

TPS5089 Background: CCS1477 is a potent, selective and orally bioavailable inhibitor of the bromodomain of p300 and CBP, two homologous and critical co-activators of the androgen receptor (AR) and its variant forms, including mutated, amplified and spliced AR, as well as c-Myc. CCS1477 represents a new therapeutic option for prostate cancer patients who have progressed after failure of anti-androgen therapy and in combination with anti-androgens such as enzalutamide or abiraterone. Methods: This is a Ph I/IIa study to determine the maximum tolerated dose (MTD) and/or recommended Phase II dose and schedule(s) of CCS1477 and investigate clinical activity of CCS1477 monotherapy and CCS1477 in combination with abiraterone or enzalutamide in patients with metastatic castration resistant prostate cancer (mCRPC). The trial aims to enrol approximately 150 patients and is currently recruiting in the UK with plans to open additional sites in the USA (NCT03568656). Key inclusion criteria (for the mCRPC) require previous treatment with abiraterone and/or enzalutamide, taxane as well as evidence of disease progression (PCWG-3 guidelines). Single dose and steady state pharmacokinetics will be determined along with changes in plasma PSA, LDH and ALKP and in circulating tumour cell number. Anti-tumour activity will be determined by standard imaging according to PCWG-3 guidelines. Paired tumour biopsies for biomarker assessment are being collected. Cohort 1 of the monotherapy dose-escalation (rolling 6 design; 3-6 patients/cohort) has completed. Enrolment to cohort 2 began in January 2019. Dose finding in combination (CCS1477 + abiraterone; CCS1477 + enzalutamide) will be open once monotherapy dose escalation completes. Following definition of a recommended phase 2 dose and schedule for monotherapy and in combination, three expansion arms in patients with mCRPC will be opened in parallel (25 patients/arm); CCS1477 monotherapy; CCS1477 + abiraterone; CCS1477 + enzalutamide. A further expansion in patients with advanced solid tumours with a mutation in p300 or CBP will also be opened. Clinical trial information: NCT03568656.

Continued androgen receptor (AR) expression and signaling is a key driver in castration-resistant prostate cancer (CRPC) after classical androgen ablation therapies have failed, and therefore remains a target for the treatment of progressive disease. Here, we describe the biological characterization of AZD3514, an orally bioavailable drug that inhibits androgen-dependent and -independent AR signaling. AZD3514 modulates AR signaling through two distinct mechanisms, an inhibition of ligand-driven nuclear translocation of AR and a downregulation of receptor levels, both of which were observed in vitro and in vivo. AZD3514 inhibited testosterone-driven seminal vesicle development in juvenile male rats and the growth of androgen-dependent Dunning R3327H prostate tumors in adult rats. Furthermore, this class of compound showed antitumor activity in the HID28 mouse model of CRPC in vivo. AZD3514 is currently in phase I clinical evaluation. Mol Cancer Ther; 12(9); 1715–27. ©2013 AACR.

Chemical starting points were investigated for downregulation of the androgen receptor as an approach to treatment of advanced prostate cancer. Although prototypic steroidal downregulators such as 6a designed for intramuscular administration showed insufficient cellular potency, a medicinal chemistry program derived from a novel androgen receptor ligand 8a led to 6-[4-(4-cyanobenzyl)piperazin-1-yl]-3-(trifluoromethyl)[1,2,4]triazolo[4,3-b]pyridazine (10b), for which high plasma levels following oral administration in a preclinical model compensate for moderate cellular potency.

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.

Secretion of gonadotropin-releasing hormone (GnRH) at the median eminence is the essential activator of the reproductive axis. The mechanisms by which embryonic GnRH neurons migrate from the olfactory placode to the preoptic area and then elaborate neurites that course through the hypothalamus to terminate at the median eminence are largely unknown. We investigated the hypothesis that GnRH neurite outgrowth is promoted by brain-derived neurotrophic factor (BDNF) because GnRH neurites course through BDNF-rich areas of the forebrain during their development. Confocal microscopy revealed that most (86%) cultured embryonic GnRH cells tagged with a green fluorescent protein reporter were immunoreactive for TrkB. In primary cultures of E12.5 olfactory tissue, treatment with BDNF induced a dose-dependent increase in neurite outgrowth, but had no discernible effect on branching. BDNF induced phosphorylation of Ca(2+)/cAMP response element-binding protein (pCREB) in both GnRH and non-GnRH cells in these cultures. This was not associated with phosphorylation of ERK in GnRH-immunoreactive cells, though BDNF treatment did stimulate pERK in neighbouring non-GnRH cells. Promotion of neurite outgrowth is unlikely therefore to result from activation of the Ras-MAPK/ERK pathway. We conclude that the developing GnRH secretory system is directly sensitive to BDNF and that this polypeptide functions as a neurotrophic factor for GnRH neurons.

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

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.

168 Background: 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, KLK3 and TMPRSS2 gene expression, were assessed in 22Rv1 cells in vitro. In vivoeffects 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) and 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 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 and AR-V7 in a 22Rv1 xenograft model. Conclusions: Small molecule inhibition of the bromodomain of p300/CBP, leads to down-regulation of AR, ARV and c-Myc as well as inhibition of key downstream PD biomarkers including PSA and TMPRSS2 and represents a promising new approach for the treatment of CRPC.

Several studies have demonstrated that antagonists of the corticotrophin releasing factor (CRF) receptor markedly inhibit experimentally induced excitotoxic, ischaemic and traumatic brain injury in the rat, and that CRF expression is elevated in response to experimentally induced stroke or traumatic brain injury. CRF is also induced by the pro-inflammatory cytokine interleukin 1 (IL-1), which participates in various forms of neurodegeneration. The aim of this study was to test the hypothesis that CRF is toxic directly in vivo or in vitro. In primary cultures of rat cortical neurons, exposure to CRF (10 pM-100 nM) for 24 h failed to cause cell death directly, or to modify the neurotoxic effects of N-methyl-D-aspartate (NMDA). Similarly, infusion of CRF (0.3-5 microg) into specific brain regions of the rat did not induce cell death and did not significantly alter the neuronal damage produced by infusion of excitatory amino acids. These data demonstrate that CRF is not directly neurotoxic, and suggest that either CRF mediates neuronal damage by indirect actions (e.g. on the vasculature) and/or that CRF is not the endogenous ligand which contributes to neurodegeneration through activation of CRF receptors.

Hepatotoxicants can elicit liver damage by various mechanisms that can result in cell necrosis and death. The changes induced by these compounds can vary from gross alterations in DNA repair mechanisms, protein synthesis, and apoptosis, to more discrete changes in oxidative damage and lipid peroxidation. However, little is known of the changes in gene expression that are fundamental to the mechanisms of hepatotoxicity. We have used DNA microarray technology to identify gene transcription associated with the toxicity caused by the hepatotoxicant carbon tetrachloride. Labeled poly A+ RNA from cultured human hepatoma cells (HepG2) exposed to carbon tetrachloride for 8 hours was hybridized to a human microarray filter. We found that 47 different genes were either upregulated or downregulated more than 2-fold by the hepatotoxicant compared with dimethyl formamide, a chemical that does not cause liver cell damage. The proinflammatory cytokine interleukin-8 (IL-8) was upregulated over 7-fold compared with control on the array, and this was subsequently confirmed at 1 hour and 8 hours by Northern blot analyses. We also found that carbon tetrachloride caused a time-dependent increase in interleukin-8 protein release in HepG2 cells, which was paralleled by a decrease in cell viability. These data demonstrate that carbon tetrachloride causes a rapid increase in IL-8 mRNA expression in HepG2 cells and that this increase correlates with a later and significant increase in the levels of interleukin-8 protein. These results illustrate the potential of microarray technology in the identification of novel gene changes associated with toxic processes.

Removal of the basic piperazine nitrogen atom, introduction of a solubilising end group and partial reduction of the triazolopyridazine moiety in the previously-described lead androgen receptor downregulator 6-[4-(4-cyanobenzyl)piperazin-1-yl]-3-(trifluoromethyl)[1,2,4]triazolo[4,3-b]pyridazine (1) addressed hERG and physical property issues, and led to clinical candidate 6-(4-{4-[2-(4-acetylpiperazin-1-yl)ethoxy]phenyl}piperidin-1-yl)-3-(trifluoromethyl)-7,8-dihydro[1,2,4]triazolo[4,3-b]pyridazine (12), designated AZD3514, that is being evaluated in a Phase I clinical trial in patients with castrate-resistant prostate cancer.