Binding Protein P300 Research Articles

Abstract 12567: Cardiomyocyte Hypertrophy is Regulated by P300 Binding Protein 1, a Novel Binding Protein

Background: Hypertrophic stimuli such as hemodynamic overload activate the cardiac signaling pathway that terminates in cardiomyocyte nuclei, thus changing the pattern of gene expression associated with cardiomyocyte hypertrophy. This hypertrophy eventually leads to systolic dysfunction and decompensated heart failure. Nuclear acetylation is a critical event in the activation of this hypertrophic gene expression and is mediated in part by intrinsic histone acetyltransferases (HAT) such as coactivator p300. p300-HAT activity is a potential pharmacological target for the prevention of heart failure. However, the regulation mechanism of p300-HAT activity is still unclear. In this study, we identified novel p300 binding partners by proteomic analysis and investigated the role of one of these proteins, p300 binding protein 1 (p300BP1), in the hypertrophic responses of cardiomyocytes. Methods and Results: To identify novel p300 binding proteins, cardiac-specific FLAG-p300 overexpressing mice were generated. Protein fractions from heart tissues of these mice and wild-type mice were purified with anti-FLAG-M2 agarose. Mass spectrometry analysis identified 553 novel p300 binding proteins, including kinases, phosphatases, methyltransferases, and transcriptional regulators. p300BP1 was selected for analysis. Binding was found to occur between baculovirus-expressed the recombinant genes FLAG-p300 full length and HA-p300BP1. The aa762-aa1098 fragment of p300 was required for this binding. In cultured cardiomyocytes, knockdown of p300BP1 prevented phenylephrine-induced increases in cardiomyocyte surface area and hypertrophic gene transcription factors such as ANF and BNP. Conversely, the overexpression of p300BP1 increased the surface area of cardiomyocytes and hypertrophic gene transcriptions. Furthermore, overexpression of p300BP1 significantly increased H3K9 acetylation in cultured cardiomyocytes. A reporter gene assay showed that p300BP1 enhanced p300/GATA4-mediated ANF and BNP promoter activation in HEK293T cells. Conclusions: We have identified multiple novel p300 binding proteins using a proteomics approach. p300BP1 may play an important role in p300/GATA4-dependent hypertrophic responses in cardiomyocytes.

Abstract 4114: Evaluation of AU-18069, a novel small molecule CBP/p300 bromodomain inhibitor for the treatment of cancers

Abstract Background: E1A binding protein (p300) and its paralog CREB binding protein (CBP or CREBBP) are ubiquitously expressed acetyl transferases (HAT) that also act as co-activators for number of transcription factors including HIF1a, BRCA-1, p53, c-Myc and androgen receptor (AR). Both CBP and p300 possess bromodomain (BRD) and a lysine acetyltransferase (KAT) domain. These two closely related epigenetic modulators are known to play an oncogenic role in a variety of cancers. Functional synthetic lethal screens have identified preferential killing in CBP-deficient and/or MYC-dependent hematological cancer cells by suppression of the paralogue p300. CBP/p300 BRD inhibitor could also prevent AR and ER signaling, thereby potentially inhibiting growth of AR and ER -dependent cancer cells. Thus, targeting CBP/p300 represents an attractive approach for developing personalized cancer therapies. Experimental Results: Multiple potent and selective CBP/p300 BRD inhibitors that are structurally unrelated to known inhibitors were identified by iterative medicinal chemistry and SAR based approaches. The lead compound AU-18069 was optimized towards attaining good potency, selectivity, physicochemical properties and DMPK profile. AU-18069 potently inhibited proliferation of a wide range of cell lines derived from prostate cancer, breast cancer and CBP mutant/MYC-dependent hematological cancers. The lead compound demonstrated good PK profile in rodents as well as in dogs. Excellent anti-tumor efficacy was achieved in leukemia and lymphoma xenograft models at well-tolerated doses. Significant downregulation of c-Myc was observed in a single dose PK-PD study. Conclusion: Lead candidate AU-18069 demonstrated that selective CBP/p300 bromodomain inhibitors are efficacious in models of hematologic malignancies and solid cancers in vitro and in vivo. Further evaluation of efficacy in other xenograft models, as a single agent as well as in combination with other agents is planned. Long term toxicological and safety pharmacology evaluation in different species and other IND enabling studies are in progress to support progression of this compound to clinical trials. Citation Format: Chandrasekhar Abbineni, Saravanan Thiyagarajan, Aravind A B, Amit A Dhudashiya, Sivapriya Marappan, Naveen Kumar R, Raghavendra N R, Mamon Dey, Avinash Kumar, Uma Bharathi B V, Girish Aggunda Renukappa, N Venkata Siva Reddy, Asha Babu, Aakanksha J Shetty, Amith A, Narasimha Rao K, Suraj T Gore, Mahaboobi Jaleel, Ramesh S Senaiar, Vijaya Shankar Nataraj, Subhendu Mukherjee, Samiulla D S, Thomas Antony, Girish Daginakatte, Rajesh Eswarappa, Kavitha Nellore, Shekar Chelur, Murali Ramachandra, Susanta Samajdar. Evaluation of AU-18069, a novel small molecule CBP/p300 bromodomain inhibitor for the treatment of cancers [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 4114.

A novel high throughput screen to identify candidate molecular networks that regulate spermatogenic stem cell functions†.

Spermatogenic regeneration is key for male fertility and relies on activities of an undifferentiated spermatogonial population. Here, a high-throughput approach with primary cultures of mouse spermatogonia was devised to rapidly predict alterations in functional capacity. Combining the platform with a large-scale RNAi screen of transcription factors, we generated a repository of new information from which pathway analysis was able to predict candidate molecular networks regulating regenerative functions. Extending from this database, the SRCAP-CREBBP/EP300 (Snf2-related CREBBP activator protein-CREB binding protein/E1A binding protein P300) complex was found to mediate differential levels of histone acetylation between stem cell and progenitor spermatogonia to influence expression of key self-renewal genes including the previously undescribed testis-specific transcription factor ZSCAN2 (zinc finger and SCAN domain containing 2). Single cell RNA sequencing analysis revealed that ZSCAN2 deficiency alters key cellular processes in undifferentiated spermatogonia such as translation, chromatin modification, and ubiquitination. In Zscan2 knockout mice, while spermatogenesis was moderately impacted during steady state, regeneration after cytotoxic insult was significantly impaired. Altogether, these findings have validated the utility of our high-throughput screening approach and have generated a transcription factor database that can be utilized for uncovering novel mechanisms governing spermatogonial functions.

Abstract 1143: Evaluation of AU-18069, a novel small molecule CBP/p300 bromodomain inhibitor for the treatment of cancers

Abstract Background: E1A binding protein (p300) and its paralog CREB binding protein (CBP or CREBBP) are ubiquitously expressed acetyl transferases (HAT) that also act as co-activators for number of transcription factors including HIF1a, BRCA-1, p53, c-Myc and androgen receptor (AR). Both CBP and p300 possess bromodomain (BRD) and a lysine acetyltransferase (KAT) domain. These two closely related epigenetic modulators are known to play oncogenic roles in a variety of cancers. Functional synthetic lethal screens have identified preferential killing in CBP-deficient and MYC-dependent hematological cancer cells by suppression of the paralogue p300. CBP/p300 BRD inhibitor could also prevent its coactivator function at AR, thereby potentially inhibit growth of AR-dependent prostate cancer cells. Thus, targeting CBP/p300 represents an attractive approach for developing personalized therapies. Experimental procedures and Results: Multiple potent and selective CBP/p300 BRD inhibitors that are structurally unrelated to known inhibitors have been identified by iterative medicinal chemistry and SAR based approaches. The lead compound, AU-18069 was optimized towards attaining good potency, physicochemical properties and DMPK profile. AU-18069 potently inhibited viability and proliferation of a wide range of cell lines derived from prostate cancer, CBP mutant and MYC-dependent hematological cancers and demonstrated improved PK profile in rodent models in comparison with a compound, currently in clinical trials. Excellent efficacy with significant tumor growth inhibition (TGI) was observed in MV4-11 xenograft model at well-tolerated doses along with downregulation of cMYC in a single dose PK-PD study. AU-18069 also showed modulation of different immune phenotypes including CD4+ T cell subsets. In summary, our lead candidate AU-18069 demonstrated that selective CBP/p300 bromodomain inhibitors are potent in models of hematologic malignancies and solid tumors in vitro and in vivo. Further evaluation of immune activation potential, efficacy studies in various xenograft models, long term toxicological evaluation in different species and other IND enabling studies are in progress. Citation Format: Chandrasekhar Abbineni, Saravanan Thiyagarajan, Ramesh S Senaiar, Subhendu Mukherjee, Mahaboobi Jaleel, Sivapriya Marappan, Raghavendra N R, Girish Aggunda Renukappa, Aravind A B, Naveen Kumar R, Venkata Siva Reddy, Asha Babu, Akhila Srinivas, Prasad Yadlapalli, Suraj T Gore, Pathange Hemasankar, Mamon Dey, Samiulla D S, Chandranath D Naik, Thomas Antony, Kavitha Nellore, Shekar Chelur, Girish Daginakatte, Mikko Myllymäki, Gerd Wohlfahrt, Murali Ramachandra, Susanta Samajdar. Evaluation of AU-18069, a novel small molecule CBP/p300 bromodomain inhibitor for the treatment of cancers [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 1143.

Insights into interaction mechanism of inhibitors E3T, E3H and E3B with CREB binding protein by using molecular dynamics simulations and MM-GBSA calculations

ABSTRACT CREB binding protein (CBP) and its paralog E1A binding protein (p300) are related to the development of inflammatory diseases, cancers and other diseases, and have been potential targets for the treatment of human diseases. In this work, interaction mechanism of three small molecules E3T, E3H, and E3B with CBP was investigated by employing molecular dynamics (MD) simulations, principal component analysis (PCA), and molecular mechanics/generalized born surface area (MM-GBSA) method. The results indicate that inhibitor bindings cause the changes of movement modes and structural flexibility of CBP, and van der Waals interactions mostly drive associations of inhibitors with CBP. In the meantime, the results based on inhibitor-residue interactions not only show that eight residues of CBP can strongly interact with E3T, E3H and E3B but also verify that the CH-CH, CH-π, and π-π interactions are responsible for vital contributions in associations of E3T, E3H and E3B with CBP. In addition, the H-O radial distribution functions (RDFs) were computed to assess the stability of hydrogen bonding interactions between inhibitors and CBP, and the obtained information identifies several key hydrogen bonds playing key roles in bindings of E3T, E3H and E3B to CBP. Potential new inhibitors have been proposed.

Abstract P3-06-06: The importance of p300/CBP (CREBBP)-survivin (BIRC5) for cell cycle and apoptosis in triple negative breast cancer

Abstract In breast cancer, both incidence and mortality have been increasing. Triple negative breast cancer (TNBC), one of breast cancer subtypes, is a heterogeneous nature that is negative for estrogen receptor (ER), progesterone receptor (PR) and human epithelial growth factor receptor type 2 (HER2). Due to the lack of expression of therapeutic targets, new therapeutic strategy and elucidation the mechanism of action in TNBC are strongly desired. Histone acetyltransferases E1A binding protein (p300) and CREB binding protein (CBP) are coactivators of a large number of essential transcription factors that contribute to cell survival and proliferation. p300/CBP (CREBBP) interacts with transcription factors and other proteins through histone acetyltransferase (HAT) and regulates several gene expression. Dysregulation of CREBBP HAT activity could contribute to various diseases, including cancer. It is reported that the high expression of p300 correlates with poor prognosis in breast cancers. However, the mechanism of CREBBP HAT activity remains unclear. We, therefore, focused on CREBBP and clarified its important mechanism in TNBC. In breast cancer cell lines, p300 expression was higher than the normal mammary epithelial cell (MCF10A) in mRNA and protein levels and positively correlated with that of CBP. To evaluate the roles of p300 and CBP in cell survival and proliferation, the loss of function studies was conducted. In TNBC cell line (MDA-MB-231), cell survival and proliferation significantly reduced in p300 knockdown cells. The reduction in cell survival and proliferation was also observed using p300 inhibitor (L002). To examine the mechanism of action for the reduction of cell survival and proliferation in p300 inhibition, cell cycle assay and apoptosis assay were performed. We found that the cell-cycle progression was arrested at the cell cycle G2/M phase and apoptosis induction in p300 knockdown cells. These results suggest that cell cycle G2/M phase arrest and the late stage of apoptosis are caused by p300 inhibition and p300 inhibition enhances the anti-tumor effect. To identify a mechanism responsible for the reduction of cell survival and proliferation in p300 inhibition, antibody array was performed. As a result of this assay and TCGA patients cohorts analysis, we identified survivin (BIRC5), a member of the inhibitor of apoptosis (IAP) family, as a functional novel biomarker of TNBC with significantly higher expression. These results suggest that suppression of BIRC5 through CREBBP inhibitor result in G2/M phase cell cycle arrest and apoptotic cell death in vitro and in vivo. In conclusion, CREBBP-BIRC5 transcriptional complex could be a novel therapeutic target for TNBC. Citation Format: Sunao Tanaka, Natsuki Wariishi, Nobuko Kawaguchi-Sakita, Souichi Adachi, Masakazu Toi, Yasuhiko Kamikubo. The importance of p300/CBP (CREBBP)-survivin (BIRC5) for cell cycle and apoptosis in triple negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-06-06.

Abstract 2694: A first-in-class small-molecule modulator of the p300/CBP interactome exhibits broad efficacy in xenograft models of solid tumors and hematologic malignancies

Abstract Background: Dysregulation of the cellular transcription machinery is a fundamental feature of cancer. E1A binding protein (p300) and CREB binding protein (CBP) are two closely related paralog transcription co-activators involved in the expression of oncogenic drivers in cancer cells. Methods: Small molecule ligands of the CH1/TAZ1 domain of p300/CBP were rationally designed and optimized for their anti-proliferative activity in vitro. Subsequent characterization focused on their ability to modulate downstream biological processes controlled by p300/CBP, including hormone-dependent androgen receptor signalling, the HIF-1 alpha/VEGF pathway and reactivation of p53 in HPV-positive carcinomas. The in vivo efficacy of this novel compound class was assessed in a variety of patient and cell line derived xenograft models, and pharmacodynamic effects were investigated. Results: Several modulators of p300/CBP exhibited anti-proliferative effects at low nanomolar concentrations in more than 60 cancer cell lines inducing both senescence and apoptosis in a highly context dependent manner. Transcriptome analyses revealed the downregulation of various drivers of cell cycle progression. Once daily oral administration triggered strong growth-inhibitory effects in several xenograft models up to complete tumor eradication at well tolerated doses. p300/CBP dependent biomarkers like VEGF (colon cancer) and PSA (prostate cancer) were significantly downregulated in the tumors of treated animals. In an orthotopic model of acute myeloid leukemia, the growth of primary tumors and metastasis was almost completely inhibited. Conclusions: Reprogramming the transcriptional profile of cancer cells by modulation of p300/CBP activity by targeting the CH1/TAZ1 domain represents a novel and broadly applicable approach for the treatment of cancer. The presented data illustrate the clinical potential of this new class of therapeutics against a broad variety of tumor types, including prostate and colon cancer as well as hematological malignancies. Citation Format: Bernd Hentsch, Valentino Cattori, Marc Labelle, Ulrich Kessler. A first-in-class small-molecule modulator of the p300/CBP interactome exhibits broad efficacy in xenograft models of solid tumors and hematologic malignancies [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 2694.

Abstract 1019: P300/CBP inhibitor CCS1477 targets 22Rv1 prostate tumor AR and c-myc gene expression in vivo

Abstract 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.

Abstract A091: Therapeutic targeting of the p300/CBP bromodomain for the treatment of castration-resistant prostate cancer

Abstract 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.

Oral small molecule modulators of p300/CBP to reprogram cancer cells and reactivate the p53 pathway in HPV-associated carcinomas.

e14581Background: Dysregulation of the basal cellular transcription machinery is a fundamental feature of cancer. E1A binding protein (p300) and CREB binding protein (CBP) are two closely related p...

Abstract 1575: Novel small molecule inhibitors of p300/CBP down-regulate AR and c-Myc for the treatment of castrate resistant prostate cancer

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

A novel small molecule inhibitor of p300/CBP for the treatment of castration-resistant prostate cancer: Preclinical evaluation.

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.

JMY protein, a regulator of P53 and cytoplasmic actin filaments, is expressed in normal and neoplastic tissues

JMY is a p300-binding protein with dual action: by enhancing P53 transcription in the nucleus, it plays an important role in the cellular response to DNA damage, while by promoting actin filament assembly in the cytoplasm; it induces cell motility in vitro. Therefore, it has been argued that, depending of the cellular setting, it might act either as tumor suppressor or as oncogene. In order to further determine its relevance to human cancer, we produced the monoclonal antibody HMY 117 against a synthetic peptide from the N-terminus region and characterized it on two JMY positive cell lines, MCF7 and HeLa, wild type and after transfection with siRNA to switch off JMY expression. JMY was expressed in normal tissues and heterogeneously in different tumor types, with close correlation between cytoplasmic and nuclear expression. Most noticeable was the loss of expression in some infiltrating carcinomas compared to normal tissue and in in situ carcinomas of the breast, which is consistent with a putative suppressor role. However, as in lymph node metastases, expression of JMY was higher than in primary colorectal and head and neck carcinomas, it might also have oncogenic properties depending on the cellular context by increasing motility and metastatic potential.

Cited2 modulates hypoxia‐inducible factor–dependent expression of vascular endothelial growth factor in nucleus pulposus cells of the rat intervertebral disc

To determine whether nucleus pulposus cells of the intervertebral disc express hypoxia-inducible factor 2alpha (HIF-2alpha), and to assess the role of HIF-1 and HIF-2 in controlling cited2 and vascular endothelial growth factor (VEGF) expression. Rat cells were cultured under normoxic (21% O2) or hypoxic (2% O2) conditions, and expression and promoter activity of HIF-2 target genes were evaluated. Gain- or loss-of-function experiments were performed to investigate the contribution of HIF isoforms to cited2 activity as well as the role of cited2 in regulating VEGF expression. We found that HIF-2alpha protein was expressed in vivo and that protein and messenger RNA expression were similar under both normoxic and hypoxic conditions. However, there was a significant increase in HIF-2alpha transactivation under hypoxic conditions. With respect to functional activity, unlike the case in most other tissues, HIF-2 failed to increase the transcriptional activities of superoxide dismutase 2 and frataxin, 2 common target genes involved in radical dismutation. However, under hypoxic conditions, HIF-2 preferentially regulated the expression and promoter activity of cited2, a p300 binding protein. When HIF-2alpha or HIF-1alpha was suppressed, cited2 promoter activity was inhibited. Finally, we showed that forced expression or suppression of cited2 resulted in corresponding changes in expression of VEGF, a common target gene for HIF-1 and HIF-2 in the nucleus pulposus cells. Results of this study indicate that in nucleus pulposus cells, HIF-2 and HIF-1 modulate their own transcriptional activity through cited2. We suggest that the 2 arms of the regulatory circuit serve to maintain survival activities and inhibit angiogenesis in the healthy disc.

Calcium-dependent enhancement of transcription of p300 by human T-lymphotropic type 1 p12 I

Human T-lymphotropic virus type 1 (HTLV-1) p12 I localizes to the endoplasmic reticulum and Golgi causing sustained release of calcium, T cell activation, and enhanced expression of several calcium-regulated genes. In recent microarray studies, p300 mRNA was increased in T cells expressing p12 I. The co-activator p300 is a key regulator of cellular and viral transcription; however, factors that influence its transcriptional regulation are less well studied. We hypothesized that the transcription of p300 is calcium dependent and that sustained low magnitude increases in intracellular calcium may enhance the transcription of p300. Herein, we report enhanced expression of p300 in T cells by p12 I in a calcium-dependent, but calcineurin-independent manner. Sustained low magnitude calcium release induced by ionomycin in T cells was sufficient to increased mRNA and protein levels of p300 resulting in enhanced transcription from a p300-dependent promoter. Promoter analysis of the p300 gene was used to predict calcium-responsive transcription factor binding sites. Using mutant forms of p12 I, we demonstrate that ER localization of the viral protein is required to increase p300. In addition, p12 I reversed the repression of HTLV-1 LTR-driven transcription by HTLV-1 p30 II, a p300-binding protein. HTLV-1 p12 I-mediated enhancement of p300 expression represents a novel mechanism of regulation of cellular gene expression by viral proteins. By targeting a ubiquitous second messenger such as calcium, HTLV-1 p12 I may regulate the expression of the cellular transcriptional co-activator p300 to modulate viral gene expression and promote lymphocyte survival.

Human T-lymphotropic virus type 1 p30(II) regulates gene transcription by binding CREB binding protein/p300.

The highly conserved coadapters CREB binding protein (CBP) and p300 form complexes with CREB as well as other DNA binding transcription factors to modulate chromatin remodeling and thus transcription. Human T-lymphotropic virus type 1 (HTLV-1) transcription is controlled, in part, by the CREB/ATF family of transcription factors which bind promoter sequences and function as complexes with the viral oncogenic protein Tax. We have reported that the nuclear localizing protein p30(II) of HTLV-1 functions as a transcription factor, differentially modulates CREB-responsive promoters, and is critical for maintenance of proviral loads in rabbits. In this study, we tested whether p30(II) directly interacts with CBP/p300 to modulate gene transcription. Gal4(BD)-p30(II)-mediated transactivation was enhanced following exogenous expression of p300 and was competitively repressed by the p300 binding protein, adenovirus E1A, and E1ACR2 (mutated for retinoblastoma binding but retaining p300 binding). In contrast, E1ACR1 (mutated for p300 binding) failed to alter Gal4(BD)-p30(II)-mediated transactivation. In addition, Gal4(BD)-p30(II)-mediated transactivation was competitively inhibited by the cotransfection of CMV-p30(II)-HA and CMV-Tax but could be rescued by exogenous p300. Importantly, we demonstrate that p30(II) colocalizes with p300 in cell nuclei and directly binds to CBP/p300 in cells. Deletion mutants of CBP/p300 were used to localize the site critical for binding p30(II) to a highly conserved KIX region. DNA binding assays confirmed the interference of p30(II) with the assembly of CREB-Tax-p300/CBP multiprotein complexes on 21-bp repeat oligonucleotides in vitro. Collectively, our results demonstrate that CBP/p300 is a cellular protein target for HTLV-1 p30(II) and mediates its transcriptional effects in vivo.

Inhibition of p53-dependent transcription by BOX-I phospho-peptide mimetics that bind to p300.

The N-terminal BOX-I domain of p53 containing a docking site for the negative regulator MDM2 and the positive effector p300, harbours two recently identified phosphorylation sites at Thr18 or Ser20O whose affect on p300 is undefined. Biochemical assays demonstrate that although MDM2 binding is inhibited by these phosphorylations, p300 binding is strikingly stabilized by Thr18 or Ser20 phosphorylation. Introducing EGFP-BOX-I domain peptides with an aspartate substitution at Thr18 or Ser20 induced a significant inhibition of endogenous p53-dependent transcription in cycling cells, in irradiated cells, as well as in cells transiently co-transfected with p300 and p53. In contrast an EGFP-wild-type BOX-I domain peptide stimulated p53 activity via inhibition of MDM2 protein binding. These results suggest that phosphorylation of p53 at Thr18 or Ser20 can activate p53 by stabilizing the p300-p53 complex and also identify a class of small molecular weight ligands capable of selective discrimination between MDM2- and p300-dependent activities.

A novel Rb- and p300-binding protein inhibits transactivation by MyoD.

The retinoblastoma protein (Rb) regulates both the cell cycle and tissue-specific transcription, by modulating the activity of factors that associate with its A-B and C pockets. In skeletal muscle, Rb has been reported to regulate irreversible cell cycle exit and muscle-specific transcription. To identify factors interacting with Rb in muscle cells, we utilized the yeast two-hybrid system, using the A-B and C pockets of Rb as bait. A novel protein we have designated E1A-like inhibitor of differentiation 1 (EID-1), was the predominant Rb-binding clone isolated. It is preferentially expressed in adult cardiac and skeletal muscle and encodes a 187-amino-acid protein, with a classic Rb-binding motif (LXCXE) in its C terminus. Overexpression of EID-1 in skeletal muscle inhibited tissue-specific transcription. Repression of skeletal muscle-restricted genes was mediated by a block to transactivation by MyoD independent of G(1) exit and, surprisingly, was potentiated by a mutation that prevents EID-1 binding to Rb. Inhibition of MyoD may be explained by EID-1's ability to bind and inhibit p300's histone acetylase activity, an essential MyoD coactivator. Thus, EID-1 binds both Rb and p300 and is a novel repressor of MyoD function.