MICA: Characterisation of a novel bromodomain inhibitor AZD5153 in castrate resistant prostate cancer

Lead Research Organisation: Newcastle University
Department Name: Translational and Clinical Res Institute

Abstract

Context of Research:

Prostate cancer kills approximately 10,000 men in the UK each year. A protein found in prostate cells called the androgen receptor (AR) is an important driver of prostate cancer development and is the main target for therapeutic intervention. Drugs that reduce circulating androgen levels or directly antagonise the AR (called anti-androgens) block AR activity and initially cause the primary cancer to regress/shrink. Unfortunately, after approximately 2 years, patients usually relapse and the tumour re-appears in a more aggressive form called castrate-resistant prostate cancer (CRPC) that is no longer responsive to the initial treatments. This type of disease is very difficult to treat and is usually fatal. Recent advances have seen the introduction of new AR-targeted therapies, such abiraterone and enzalutamide, that have shown some promise in the clinic. However, even these treatments are not completely effective in all patients and resistance occurs to these agents rendering them useless. At this advanced stage, all treatment options have been exhausted and thus the development of new strategies to target the AR and treat CRPC patients is vital.

Aims and Objectives:

The research teams within the Northern Institute for Cancer Research, Newcastle University, and AstraZeneca are aiming to validate a newly developed drug as an AR-targeting therapy for the treatment of CRPC. This drug is aimed at inactivating the AR by blocking an important group of proteins in the cell called BET proteins. These are important for enabling the AR to become hyperactive in prostate cancer and thus by inactivating them, we hypothesise that we can disrupt AR activity and cause tumours to disappear. Importantly, during CRPC development, the AR undergoes several distinct changes that make it function in the presence of the current therapeutic agents; enabling progression of CRPC. It is important, therefore, that any new therapy targeting the AR has to be additionally effective against these altered receptors that are present in CRPC patients. Consistent with this, the main objective of this study is to address whether the new AstraZeneca drug works on normal, as well as altered AR proteins present in therapy-resistant CRPC. Key to this project is the identification of 'biomarkers' that will indicate whether the drugs are effective at inactivating the normal and altered forms of the AR and causing tumour cells to die.

Application and Benefits:

The study will use two main model systems to assess the effect of these new drugs on normal and altered AR activity: (i) cell line models of CRPC that reflect tumour cells growing in patients with different forms of the AR within them; (ii) biopsied primary prostate cancer specimens that are grown in the laboratory containing normal and altered AR proteins. Together, these strategies have the potential to indicate the effectiveness of the new drug in CRPC patients. Using 'Biomarkers' identified in the study, we can assess whether normal and altered AR proteins found in CRPC are affected by the new drug. This is an important aspect of the study as we would be only aiming to treat patients in the clinic with this new drug if it proved effective at inactivating the forms of AR present in their tumour, a process termed 'patient stratification'. By doing this, we will be treating a specific cohort of patients who will receive benefit from the new therapy and, importantly, not treating patients who would receive no benefit from drug treatment. Ultimately, the overarching goal of the study is to provide new treatment options in CRPC patients that are effective against disease that have failed conventional therapies.

Technical Summary

Prostate cancer (PC) is the most prevalent UK male cancer, accounting for 10,000 deaths per year. The androgen receptor (AR) is a transcription factor that transmits androgenic signals to drive prostate growth and cellular transformation. Inactivation of the AR by anti-androgens is initially effective causing tumour regression. Unfortunately, most patients relapse with a more aggressive form of the disease termed castrate-resistant PC (CRPC) that is unresponsive to treatment, but is AR-dependent. The development of next-generation AR-targeted therapies, including enzalutamide, have shown promise in the treatment of CRPC, but response rates of just 50% and the development of resistance have limited their success in the clinic. Importantly, several aberrations to the AR signalling cascade have been identified in CRPC, including AR mutation and the formation of receptor splice variants, that facilitate AR activity in castrate conditions and contribute to conventional and next-generation anti-androgen treatment failure. A key challenge is to develop novel treatments for CRPC that antagonise all ARs and repress tumour progression.

Consistent with a role for Bromodomain and extraterminal motif (BET) proteins enhancing activity of CRPC-relevant forms of the androgen receptor, pre-clinical testing of first-generation BET inhibitors have been shown to compromise PC growth in vitro and in vivo. This study will assess a new and more efficacious BET inhibitor developed by AstraZeneca to attenuate receptor signalling and prevent tumour growth using CRPC cell line models and human PC biopsy samples ex vivo. The ability of this agent to inactivate all AR isoforms has major clinical implications and is the main focus of the project. By determining the efficacy of the BET inhibitor against clinically-relevant AR mutants and splice variants, we will advance our understanding of the receptor in CRPC, and comprehensively validate the compound for patient-tailored treatment.

Planned Impact

Who will benefit from the research?

The study has several different categories of beneficiaries outside of the immediate academic professional circle, including
the large cohort of prostate cancer (PC) patients in the UK who would benefit directly from the research, and distinct private
and public sector workforces who would benefit from the commercialisation and educational impact of the work (as
discussed below).

PC patients:

The ultimate research objective of the study is to improve strategic healthcare for advanced PC in the medium to long term by provision of patient-tailored treatments that are beneficial to health and wellbeing. PC is a disease of ageing and hence poses a major clinical challenge in the developed world where life-expectancy is increasing year on year. In the UK alone, 10,000 deaths result from PC per year and this figure is likely to rise over the next 10 years. The development of new therapies that are efficacious in patients who have failed current therapies is key for the health and wellbeing of a large population of men in the UK. Therefore, our research has the capacity to benefit this expanding group of individuals by indicating novel therapeutic regimens in patients who have exhausted all treatment options.

Industrial Beneficiaries:

The research will be generating a number of specialist reagents that can be commercialised through our links with Cancer Research Technology (CRT) who have a repository of reagents for use in research. The CAS9/CRISPR-modified LNCaP and CWR22Rv1 cell lines will be marketed by CRT to provide researchers avenues for exploiting enzalutamide- and abiraterone-resistance mechanisms for new CRPC treatments. Additionally, we have forged strong links with Gillette/Proctor and Gamble through our Movember alliance with Prostate Cancer UK and hence we can facilitate knowledge transfer and impact on this funding stream by engagement with P&G representatives.

Public Sector:

We have engagement with local schools where our research is explained as part of distinct modules in lower school and sixth form. Students and teachers will directly benefit from this research as they will be introduced to the concepts of disease modelling in laboratories and technical advancements in cancer research.

How will they benefit?

PC patients

Specific cohorts of PC patients will benefit from this research by the provision of more effective therapies that could increase life-expectancy and improve quality of life. Ultimately, many thousands of males in the UK have the potential to be impacted by this study and outputs from our research could see a change in clinical practise within the next 5 years.

Public sector:

Provision of information from this study as teaching aids for students interested in cancer biology, biotechnology and other relevant modules in years 12 and 13 would indicate real-world relevance of scientific endeavours and how research can benefit general healthcare of the UK population. This is a key time to introduce these concepts as students may be attracted to pursuing a scientific career from being exposed to real-world examples of research in practise. Importantly, this project has very focussed objectives and deliverables that are relevant in today's society.

MICA-funded research associate:

The research associate who will undertake the day to day experimental work primarily at Newcastle, but will also be trained in bioinformatics and in vivo experimentation at Newcastle and AstraZeneca, respectively. This dual-site work schedule is one that the collaborative team have previously used and provides added value to the learning and technical outcomes of the study that will facilitate the career development of the researcher. Additionally, the project will also improve transferable skills of the researcher, including communication, data analysis and interpretation, that are key attributes for many employment sector
 
Description BBSRC CASE IP
Amount £130,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 09/2019 
End 09/2023
 
Description Filling a critical knowledge gap in androgen receptor variant splicing to enable development of new prostate cancer therapies
Amount £440,243 (GBP)
Organisation Prostate Cancer Research Centre (PCRC) 
Sector Private
Country United Kingdom
Start 01/2020 
End 01/2024
 
Title Developing Splicing Factor Detection Approaches 
Description We are currently optimising novel tools to facilitate the detection of factors involved in the generation of androgen receptor splicing variants. By combining the RNA-targeting capability of Cas13b (CasRX) and the biotinylation-catalysing APEX2 protein, we have developed a system to detect proteins involved in splicing of pre-mRNA. We are currently validating this approach to enable us to provide much needed information on which splicing factors are required for androgen receptor splice variant production. 
Type Of Material Technology assay or reagent 
Year Produced 2019 
Provided To Others? No  
Impact There is currently no discernible impact resulting from this work. 
 
Title Epigenetic screening assay 
Description We have developed a novel method called SCRIPTURE which is currently being optimised to examine epigenetic and transcriptional regulators (transcription factors, co-regulators and histone post-translational modifications) at distinct genetic loci using a combination of CRISPR-based chromatin cleavage, capture and mass spectrometry. Where this excels over other methods (ChIP, ChIP-Seq) is that it is not reliant on antibodies and is adaptable to any chromatin region within the genome. 
Type Of Material Technology assay or reagent 
Year Produced 2019 
Provided To Others? No  
Impact We have only recently embarked on optimising this approach, but envisage disseminating the work later this year as part of a study we are conducting. 
 
Title Generation of transcriptomics data 
Description We have data from advanced prostate cancer cell lines of the effect of the bromodomain inhibitor AZD5153 on global transcriptomics. This is very important data as it will allow us to utilise gene targets of the compound as key in vivo biomarkers when the animal study is undertaken. Moreover, given that gene expression profiling of other bromodomain inhibitors is available in the public domain, we can compare directly the functionality of AZD5153 against other drugs and make our findings available to other researchers in the field. 
Type Of Material Physiological assessment or outcome measure 
Year Produced 2018 
Provided To Others? No  
Impact We are currently analysing the data, but we envisage to identify distinct functionality compared to earlier bromodomain inhibitors. Although we are not making the data public until it has been fully annotated and validated, it will be of use to other researchers in this field when disseminated over the next year or so. 
 
Title Novel bromodomain inhibitor-resistant cell line 
Description In an effort to understand the process of bromodomain inhibitor resistance in prostate cancer, we chronically applied AZD5153 to the prostate cancer cell line CWR22Rv1 for 6 months to develop a derivative that no longer responds to drug treatment. As a consequence, we now have a CWR22Rv1 cell line that is resistant to AZD5153; and of interest is also cross-resistant to other clinically-relevant BET inhibitors. 
Type Of Material Cell line 
Year Produced 2018 
Provided To Others? No  
Impact No impact as of yet. 
 
Title AZD5153-treated VCaP cell line RNA-sequencing 
Description We have transcriptomics data from VCaP prostate cancer cells treated with AZD5153 for 8 and 24 hours and downstream bioinformatics analyses. . 
Type Of Material Data analysis technique 
Year Produced 2020 
Provided To Others? No  
Impact We have been able to define potential biomarkers of AZD5153 treatment from these experiments which may be of value for clinical application of the drug. 
 
Title RNA-sequencing data-set comparing parental CWR22Rv1 cells to AZD5153-resistant CWR22Rv1 cells 
Description As reported previously, we have generated an AZD5153-resistant CWR22Rv1 derivative cell line that has been grown chronically in the presence of 1 uM AZD5153 now for upwards of two years. We embarked on transcriptomic profiling of this derivative versus the parental CWR22Rv1 cell line to identify mechanism/s of resistance to this agent. We have analysed the data and identified a number of altered pathways that may be important for enabling resistance to the BET inhibitor. These are currently being validated in the laboratory. The data is raw RNA-sequencing data which has been successfully aligned and subsequent differential gene expression in RStudio using DSeq2 has been performed. We anticipate the work to be published in the next 6 months which would provide public access to the data. 
Type Of Material Database/Collection of data 
Year Produced 2021 
Provided To Others? No  
Impact The findings from this RNA-Seq work may highlight translationally-relevant mechanisms of AZD5153 resistance that could be important if the compound is continued for use in patients. 
 
Title Transcriptomics data from in vivo study (conducted by AstraZeneca) 
Description Xenograft studies were conducted by AstraZeneca to assess the efficacy of the novel bromodomain inhibitor in models of prostate cancer. Resultant tumour samples have been harvested and RNA sequencing on these samples will be conducted within the next two months to provide biomarkers for treatment efficacy and comparative data for our human cell line data generated last year. 
Type Of Material Database/Collection of data 
Year Produced 2019 
Provided To Others? No  
Impact There is no discernible impact from this data yet. 
 
Description AZ and LMB collaboration 
Organisation Medical Research Council (MRC)
Department MRC Laboratory of Molecular Biology (LMB)
Country United Kingdom 
Sector Academic/University 
PI Contribution This is an exciting interaction between the already collaborating Newcastle and AstraZeneca teams and the group headed by Prof Madan Babu at the LMB to study protein complexes involved in androgen receptor regulation.
Collaborator Contribution The Newcastle team have developed a process to identify factors involved in androgen receptor splicing and Prof Babu's group are facilitating the identification and characterisation of protein complexes involved in this important cancer-related process. Currently, Prof Babu's group are examining some of our previous data-sets and we will be visiting the team in March 2019 to detail further experimental developments. To help the running of the collaboration, we have set up a data repository between the three groups which allows big data to be transferred between the groups.
Impact This interaction is very much multi-disciplinary as we have molecular, cellular and translational biology expertise within the Newcastle and AZ teams which is complemented by systems biology and bioinformatics expertise within the lab of Prof Babu. This collaboration was initiated in December 2018 and therefore we do not have any robust outcomes or outputs as of yet.
Start Year 2018
 
Description Newcastle Structural Biology Collaboration 
Organisation Newcastle University
Country United Kingdom 
Sector Academic/University 
PI Contribution Over the course of the first 12 months of the project, the Gaughan/Robson/McCracken team have identified some key issues regarding androgen receptor-BRD4 interaction that will be answered appropriately using in vitro biophysical techniques. By providing in-cell data that we have generated, the creation and utility of informative biophysical approaches can be applied to help decipher the dynamics of AR-BRD4 interaction.
Collaborator Contribution The Structural Biology Team have a wealth of experience creating and using biophysical approaches, such isothermal calorimetry (ITC), to study protein-protein interactions. They will help us decipher how BRD4 and the AR interact and if post-translational modifications of the receptor impact on this association in vitro. Based on thee read-outs, we can then model the interaction in cell line experiments which will be of value to the mechanism of action studies described in the application.
Impact This is a very new collaboration and therefore we have no experimental outputs to reports currently.
Start Year 2018
 
Description University of Oslo 
Organisation University of Oslo
Country Norway 
Sector Academic/University 
PI Contribution Shared resources (cell lines and transcriptomics data) to study bromodomain inhibitor compounds in prostate cancer.
Collaborator Contribution Plan to perform in vivo studies to complement in vitro work conducted at Newcastle and Oslo.
Impact No outputs (research papers, posters etc) have arisen yet as the collaboration is only 2 months old.
Start Year 2019
 
Description Hosted prostate cancer patient and family for interactive lab tour 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Patients, carers and/or patient groups
Results and Impact I was made aware of a prostate cancer patient who was a good friend of a colleague. I contacted the patient and invited him and his family to visit the NICR and experience a lab tour and presentation on our work. This was a great event and we now interact on a regular basis to undertake other engagement activities in his local town (one of which is planned for April 2019). He has a very interesting background of music production and has a lot of contacts within this field where he is able to disseminate his own experiences on social media which is raising awareness to all of his followers.
Year(s) Of Engagement Activity 2018
 
Description Movember talk at Proctor and Gamble, Newcastle 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Industry/Business
Results and Impact I was involved in presenting a prostate cancer awareness and treatment talk to supporters of the Movember charity at Proctor and Gamble, Newcastle. Here, I was joined by a prostate cancer patient who I have known for a number of years and we discussed symptoms, GP visits, PSA testing and treatments in an interactive workshop. I received excellent feedback from participants and have made additional external links with a neighbouring school to disseminate information to year 10 students.
Year(s) Of Engagement Activity 2018
 
Description Open discussion forum 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact I presented a talk on prostate cancer alongside one of my colleagues Prof Jim Allan and a prostate cancer survivor Mr Mark Harborough at a book store in Alnwick, Northumberland. Here we provided an insight into cancer biology and genetics of cancer, and then the real-life journey from diagnosis to treatment. Principally, this was to raise awareness of prostate cancer and get people to talk about their own personal experiences, but was also to say a big thank you to the fundraisers who have supported cancer research in the local area.
Year(s) Of Engagement Activity 2020
 
Description Patient group workshop 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Patients, carers and/or patient groups
Results and Impact We hosted two prostate cancer patients who had recently been diagnosed with locally-advanced prostate cancer. These patients were personally known to the one of the principle investigators on the grant and it was agreed to host them for a half-day workshop. Here, we provided a lab tour of the Northern Institute for Cancer Research and then a prostate cancer-focused interactive talk which aligned their disease sub-type to our own research. This was extremely valuable to the group as we were able to disseminate some of our recent findings. Importantly, the two prostate cancer patients have put us in contact with a larger cohort of middle-aged/elderly gentlemen (local sport club) where we plan to go out and raise awareness of prostate cancer and symptoms, and how GP visits are likely to have a considerable benefit for treating disease if detected earlier.
Year(s) Of Engagement Activity 2019
 
Description Patient worshop and lab tour 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Patients, carers and/or patient groups
Results and Impact The Gaughan/Robson/McCracken Group at the Northern Institute for Cancer Research hosted a lab tour/workshop for a prostate cancer patient (and family) to learn more about the research that we conduct in Newcastle and how we are helping develop less invasive techniques for disease detection and improved therapies for treatment. The patient in question is currently working in the music industry and is assembling a host of professional musicians to compose and produce a song to boost awareness of prostate cancer in the UK. We talked at length about how the prostate group at Newcastle could help with this process and we envisage close contact with the patient over the next few years to help him achieve his goal. If successful, this will have great significance for the UK as many of the artists to be included in the work will be relevant to the age of males who would be considering PSA testing/experiencing prostate symptoms.
Year(s) Of Engagement Activity 2018
 
Description Primary school visit during Science Week 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact I attended a local primary school during Science Week to give an insight into the biology of cells and DNA. This was quite challenging as the age ranged from 5-11 (Year groups 6 down to reception) and some of the information was possibly lost on the very young members of the audience. However, the children seemed to enjoy it and liked the forensics section where we had to identify the individual who had eaten all of the biscuits in the staff room based on personal characteristics controlled by genes (hair colour etc).

I am going to do this at a number of other schools this year during Science Week 2020.
Year(s) Of Engagement Activity 2019