CDK-containing macromolecular assemblies

Lead Research Organisation: Newcastle University
Department Name: Northern Institute for Cancer Research

Abstract

The behaviour of a cell is defined by the set of genes it expresses and by its commitment to either a quiescent or a proliferating state. The cyclin-dependent kinases (CDKs) bind to non-catalytic cyclin subunits to form CDK-cyclin complexes that play central roles in regulating both gene-expression and cell-proliferation. Consequently, CDK-cyclins are important in normal cells and in a variety of disease conditions. We are proposing a programme of research to understand how CDK-cyclins work, how they in turn are regulated, and how their inappropriate activity can contribute to the development of disease.

Because certain CDK-cyclin complexes can function interchangeably, expression of all but one cell-cycle regulatory CDK can be inhibited without compromising the cells ability to divide. The exception is CDK1, which must possess unique properties that make it indispensable. We have solved the structure of CDK1-containing complexes, and found that it has a small number of unique kinetic and structural properties. We propose to use mutagenesis to engineer these properties into CDK2, the closest homologue of CDK1, and see if the resulting CDK2-variant can functionally complement CDK1. In this way, we will identify what is the property of CDK1 that explains its uniqueness, resolving a key conundrum at the heart of CDK-biology.

CDK1 and CDK2 play important roles in meiosis, the process by which sperm and egg cells are generated. These CDKs can be regulated by binding of non-cyclin partners and by covalent modifications other than phosphorylation. We will explore how these regulatory mechanisms work, and how conventional and unconventional regulatory mechanisms operate in meiosis.

Different CDK-cyclin complexes are characterised as regulating either transcription (e.g. CDK9-cyclin T) or proliferation (e.g. CDK4-cyclin D). A growing body of evidence, however, suggests that this classification is over simplified. In particular cyclin D appears to work together with nuclear hormone receptors (such as the androgen and oestrogen receptors) to regulate transcription, and we will investigate the mechanisms that underlie this. Because inhibition of androgen receptor signalling is a well validated way to combat prostate cancer, a better understanding of how cyclin D contributes to androgen receptor function may suggest ways to tackle forms of prostate cancer that have become resistant to current therapies.

Several transcriptional and cell-cycle regulatory CDKs depend on a chaperone system to shepherd them into the correct shape and/or correct set of complexes to carry out their cellular roles. The chaperoning of CDKs is mediated by HSP90, a promiscuous chaperone, and by Cdc37, a more specific adaptor. We have reconstituted the process of "hand-off" from chaperone to functional CDK-cyclin complexes in cell-free experiments. We now propose to characterise the structural mechanism of hand off, using a combination of biophysical and structural techniques.

X-ray crystallography has provided a structural explanation of how cyclins both activate CDKs and, to some extent, direct them towards appropriate substrates. For CDK9-cyclin T, a further level of regulation is provided by interchange between two settings: an inhibitory particle termed the 7SK ribonucleoprotein (7SKsnRNP) and an activated setting, where CDK9-cyclin T is recruited to genes that are being expressed. Recent technical developments make large complexes such as the 7SKsnRNP accessible to high resolution structural study by cryoelectron microscopy, and we will use this technique to image the 7SKsnRNP to understand the mechanism by which it holds CDK9-cyclin T inactive, and how it subsequently switches into the activated setting.

Technical Summary

This programme will exploit our collaborations and strengths in CDK reagent generation, biochemical/biophysical assay, target structural biology to illuminate the mechanisms that underlie essential roles of CDKs in regulating the cell cycle, meiosis, and transcription.

Through collaborations with Professor Mary Herbert and Professor Angel Nebrada, and building on structural studies of CDK1- and CDK2-containing complexes, we will investigate the roles of CDKs 1 and 2, as regulated by either canonical (cyclin A, cyclin B, cyclin E) or non-canonical (SPDY/RINGO) cyclin regulators, in meiosis and mitosis.

We have dissected a site of direct contact between CDK2-cyclin A and Skp1-Skp2. We will complete the characterization of this site and implement cellular studies to explore its role in the regulation of the localization and stability of p27, and of SKP2 itself (via interactions with CDH1).

Cyclin E can play a driver role in cancer. Through collaboration with the target validation group at the NICR, we will identify the properties of cyclin E that enable this role, suggesting mechanisms to target in anti-cancer drug discovery.

We hypothesize that cyclin D forms complexes that regulate transcription by a mosaic of interactions involving multiple peptide motifs binding to corresponding docking sites. Some of these sites are established but we have observed and/or inferred others in structural studies. We will characterize these interactions in cell-free systems, and test their relevance to (e.g.) AR-dependent transcription and proliferation in cellular systems through collaboration with Dr Luke Gaughan.

We will use biochemical and biophysical tools to study the interactions of P-TEFb, a key regulator of transcription, with its positive and negative regulators. In collaboration with Professor Hans Elmlund, we will solve high resolution cryoelectron microscopy structures for various states of the 7SK snRNP, a complex that sequesters and inhibits P-TEFb.

Planned Impact

The major impact of our research will come from subsequent translational research informed by better understanding of CDK function. In addition, we expect to generate impact by ways that include:

i) Training a cohort of skilled researchers in interdisciplinary structural biology
Macromolecular crystallography (MX) is an essential enabling technology for the cellular and molecular biosciences, and consequently for UK pharmaceutical and biotechnological industries. The biotechnological and pharmaceutical industry that is dependent on this discipline contributes hugely to the UK economy: in 2010 the pharmaceutical sector provided 67,000 jobs, each contributing £195,000 of GVA, with 25,000 of these positions being in high skill R&D activities (source: http://www.abpi.org.uk). Use of MX in UK pharma/biotech requires a cohort of skilled practitioners, trained in interdisciplinary structural biology and with an awareness of the potential for contribution to medical and biotechnological practice. Support of this proposal will allow the Noble and Endicott groups to continue their contribution in this area.

ii) Building capacity in a cryoelectron microscopy
RCUK has recognized cryo EM as an important area for development, following recent exciting technical advances in the field. The proposed collaborative EM research will build UK presence and international networks in this area and will train a named researcher in the relevant experimental and computational techniques.

iii) Identifying important interactions to be targeted in the development of personalized medicine by commercial and/or academic drug discoverers
CDKs are the end effectors of pathways that function aberrantly in diseases ranging from HIV-AIDS (CDK9), through cardiac hypertrophy (CDK9) to cancer (CDK9, CDK4/6, CDK2). Accordingly, there are CDK inhibitors in preclinical development and/or clinical trials in each of these indications. These inhibitors all target the ATP-binding site. While thi strategy has demonstrated utility, it does have drawbacks relating to selectivity and the potential for resistant mutations. This proposal will characterize the complexes in which CDKs participate, identifying protein:protein interactions that are essential to CDK function. Such interactions are increasingly being targeted for drug discovery, being often more amenable to selective inhibition. Particularly promising in this respect are cyclin-transcription factor interactions, such as cyclin D binding to the androgen receptor. Understanding these interactions in AR-driven proliferation may suggest new strategies to overcome castrate-resistant prostate cancer.

iv) Generating assays and reagents to enable the study of CDKs in diverse biological contexts
While study of the CDKs in regulating cell-cycle, transcription, cancer and meiosis is the focus of this programme, CDKs are implicated in other biological processes and diseases. The phosph-CDK2-cyclin A generating reagents and protocols that we developed have been widely distributed by JE to academic and commercial groups and commercialized by New England Biolabs. Similar impact can be expected from the proposed programme.

v) Participating in national and international networks
Networks provide a vehicle for disseminating results, know-how and practice that contribute broadly to the community. MN and JE will share outputs of the proposed programme through key networks that promote anticancer drug discovery, and structural biology software development.

vi) Inspiring and educating biomedical undergraduates in basic and applied research
The research will be conducted in the NICR, which contributes to Newcastle's undergraduate lecture courses, and hosts undergraduate and Masters students through 2-3 month projects. In both of these undertakings, the students will be exposed to the cutting-edge research supported by this grant, to the mutual benefit of researchers and students.
 
Description BBSRC-DTP
Amount £100,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2017 
End 03/2021
 
Description MRC Confidence in Concept
Amount £60,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 01/2019 
End 12/2019
 
Description MRC DTP (DiMeN) studentship
Amount £100,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 10/2018 
End 03/2022
 
Description MRC Doctoral Training Programme
Amount £100,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 10/2016 
End 03/2020
 
Description MRC-DTP
Amount £100,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 10/2017 
End 03/2021
 
Title CDK1-cyclin B crystals 
Description CDK1-cyclin B suitable for assay and crystallisation. 
Type Of Material Technology assay or reagent 
Year Produced 2016 
Provided To Others? Yes  
Impact None to date. 
 
Title CDK9/cyclin T crystals 
Description Crystals of CDK9/cyclin T suitable for use in determining binding modes of ATP-competitive inhibitors. Information is useful for the structure-aided design of selective CDK9 inhibitors in academia / pharmaceutical industry. 
Type Of Material Technology assay or reagent 
Year Produced 2010 
Provided To Others? Yes  
Impact Structure determination of CDK9/cyclin T/inhibitor complex structures. 
 
Description CDC37/HSP90/CDK complexes 
Organisation University of Sussex
Department Genome Damage and Stability Centre
Country United Kingdom 
Sector Academic/University 
PI Contribution Provision of CDK4/6 complexes, biochemical, biophysical and structural characterisation of CDK/CDC37 and CDK/CDC37/HSP90 complexes
Collaborator Contribution Provision of CDC37 and HSP90 proteins, biochemical, biophysical and structural characterisation of CDK/CDC37 and CDK/CDC37/HSP90 complexes
Impact PMID: 29091774
Start Year 2013
 
Description CDK PTMs 
Organisation Leiden University Medical Center
Country Netherlands 
Sector Multiple 
PI Contribution Generation of CDK1- and CDK2-cyclin complexes.
Collaborator Contribution Identification of novel post-translational modifications on CDKs. Characterisation by mass spectrometry
Impact Exchange of reagents.
Start Year 2015
 
Description CDK complexes controlling meiosis 
Organisation Newcastle University
Department School of Civil Engineering and Geosciences
Country United Kingdom 
Sector Academic/University 
PI Contribution Expertise in the determination and analysis of CDK and other protein kinase-containing macromolecular complex structures. Biochemical and biophysical assays. Provision of ATP-competitive inhibitors.
Collaborator Contribution Expertise in model mouse oocyte systems, imaging and analysis.
Impact Award of an MRC DTP studentship entitled: "How do meiotic cells navigate the precarious transition between two M-Phases?" Start date 17/10/2016.
Start Year 2016
 
Description CDK1 and CDK2 complexes with RINGO/Speedy regulators 
Organisation Institute for Research in Biomedicine (IRB)
Country Spain 
Sector Academic/University 
PI Contribution Preparation of CDK1 and CDK2 proteins together with various regulators. Biochemical and structural characterisation of CDK-containing complexes.
Collaborator Contribution Biochemical characterisation of RINGO/Speedy proteins. Provision of unpublished results relating to RINGO/Speedy protein expression and characterisation. Provision of cDNAs, mutants and other RINGO/Speedy reagents. Mouse models to interrogate the functions of RINGO/Speedy in vivo
Impact Collaborator provided data that assisted in the preparation of an application to the MRC for programme grant support.
Start Year 2015
 
Description CDK1/cyclin B/inhibitor complexes 
Organisation Astex Pharmaceuticals
Department Astex Therapeutics Ltd
Country United Kingdom 
Sector Private 
PI Contribution Provision of CDK1/cyclin B protein for structure determination.
Collaborator Contribution Provision of proprietary CDK1/cyclin B-selective inhibitors
Impact None as of 11/2015
Start Year 2013
 
Description CDK2 inhibitor studies 
Organisation Astex Pharmaceuticals
Country United States 
Sector Private 
PI Contribution Provision of CDK/cyclin complexes for structure determination by X-ray crystallography
Collaborator Contribution Provision of proprietary CDK inhibitors and cell-based assay protocols and reagents
Impact PMID 29063678, PMID 29118092, PMID 28005359 Multidisciplinary: small molecule chemical synthesis, biochemical and cellular assays, X-ray crystallography, drug design
Start Year 2013
 
Description CDK2 inhibitor studies 
Organisation Cancer Research UK
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution Provision of CDK/cyclin complexes for structure determination by X-ray crystallography
Collaborator Contribution Provision of proprietary CDK inhibitors and cell-based assay protocols and reagents
Impact PMID 29063678, PMID 29118092, PMID 28005359 Multidisciplinary: small molecule chemical synthesis, biochemical and cellular assays, X-ray crystallography, drug design
Start Year 2013
 
Description CDK2 inhibitor studies 
Organisation Newcastle University
Department School of Chemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution Provision of CDK/cyclin complexes for structure determination by X-ray crystallography
Collaborator Contribution Provision of proprietary CDK inhibitors and cell-based assay protocols and reagents
Impact PMID 29063678, PMID 29118092, PMID 28005359 Multidisciplinary: small molecule chemical synthesis, biochemical and cellular assays, X-ray crystallography, drug design
Start Year 2013
 
Description CDK4/6-Androgen Receptor 
Organisation Newcastle University
Department Northern Institute for Cancer Research Newcastle
Country United Kingdom 
Sector Academic/University 
PI Contribution Provision of CDK and cyclin constructs and protein for in vitro and cell-based studies to elucidate CDK-androgen receptor interactions
Collaborator Contribution Provision of appropriate cell lines and expertise for cell-based studies of androgen receptor function (transfection, si RNA, ChIP and lenti-viral-mediated knockdown),
Impact No publications as of 11/2015. Results of collaborative experiments were included in a subsequent application to the MRC for programme grant funding.
Start Year 2013
 
Description CDKs and the DNA damage response 
Organisation University of Liverpool
Department Institute of Integrative Biology
Country United Kingdom 
Sector Academic/University 
PI Contribution Provision of CDK containing macromolecular complexes for structural, biophysical and biochemical characterisation. Expertise in the determination of structures by X-ray crystallography.
Collaborator Contribution Provision of CDK complexes and cell-based systems.
Impact BBSRC DTP studentship between University of Liverpool and Newcastle University, entitled: "CDK18-dependent phosphorylation and the DNA damage response". Start date 01/10/2017.
Start Year 2017
 
Description Cellular studies of CDK4 
Organisation Newcastle University
Department Northern Institute for Cancer Research Newcastle
Country United Kingdom 
Sector Academic/University 
PI Contribution Generation of constructs for cellular studies and supporting biochemical, biophysical and structural characterisation
Collaborator Contribution Provision of expertise in cellular studies and cell imaging
Impact Generation of stable transfected cell lines for further functional characterisation. Collaboration is multidisciplinary: cell culture, FACS and imaging at NICR, biochemical, biophysical and structural characterisation of CDK4-containing complexes at Newcastle.
Start Year 2010
 
Description Characterisation of the interaction between CDK1 and RGC32 
Organisation University of Sussex
Country United Kingdom 
Sector Academic/University 
PI Contribution Provision of CDK1-containing complexes, expertise in biophysical assays and associated reagents
Collaborator Contribution Knowledge of RGC32 biology. Provision of RGC32 for assay.
Impact No outputs. Research is multidisciplinary: cellular studies carried out at Sussex, biophysical and biochemical analyses at Newcastle.
Start Year 2016
 
Description Determination of CDK9/cyclin T/ATP-competitive inhibitor structures 
Organisation University of Nottingham
Department School of Pharmacy
Country United Kingdom 
Sector Academic/University 
PI Contribution Provision of CDK9/cyclin T for structure determination of inhibitor complexes by X-ray crystallography
Collaborator Contribution Provision of CDK9-specific inhibitors for co-crystallisation studies for structure lead inhibitor design. Hold a CRUK DDERP award (01/01/14-31/12/15) on which we are a collaborator and receive £5000/annum towards consumables.
Impact Multidisciplinary: inhibitor synthesis and in vitro assays at Nottingham, structural characterisation started at Oxford. Activity now transferred to NICR, Newcastle University. Two papers published: 1. Shao,H., Shi,S., Huang, S., Hole, A.J., Abbas, A.Y., Baumli, S., Liu,X., Lam, F., Foely, D., Fischer, P.M., Noble, M.E.M., Endicott, J.A., Pepper, C. and Wang, S. (2013) Substituted 4-(thiazol-5-yl)-2-(phenylamino)pyrimidines are highly active CDK9 inhibitors: synthesis, x-ray crystal structure, SAR and anti-cancer activities. J.Med. Chem. 56:640-59. 2. Hole, A.J., Baumli, S., Shao, H., Shi, S., Huang, S., Abbas, A.Y., Liu, X., Lam, F., Pepper, C., Fischer, P.F., Wang, S., Endicott, J.A. and Noble, M.E.M. (2013) Comparative binding of 2-amino-4-heteroaryl-pyrimidine inhibitors to CDK2 and CDK9. J.Med. Chem. 56:660-70. 3. Successful application for further funding: CRUK Science Committee - Drug Discovery Project Award (PI P. Fischer, Nottingham University, Award ref: C18648/A17648)
Start Year 2009
 
Description EM structure determination of P-TEFb complexes 
Organisation Monash University
Country Australia 
Sector Academic/University 
PI Contribution Preparation of samples for EM analysis.
Collaborator Contribution Expertise in EM data collection and analysis of protein-RNA complexes. Access to a Titan Krios microscope.
Impact With advice from partners, successful generation of samples suitable for data collection. Structure determination in progress.
Start Year 2015
 
Description Identification of CDK1 substrates 
Organisation University of Edinburgh
Country United Kingdom 
Sector Academic/University 
PI Contribution CDK1 kinase assays
Collaborator Contribution Identification of CDK1 substrates
Impact No outputs. Multidisciplinary: Biochemical and biophysical assays of CDK1 activity at Newcastle University; Mass spectrometry at University of Edinburgh; cell-based functional assays at University of Sussex.
Start Year 2017
 
Description Identification of CDK1 substrates 
Organisation University of Sussex
Country United Kingdom 
Sector Academic/University 
PI Contribution CDK1 assays
Collaborator Contribution Cell-based CDK1 functional studies.
Impact No outputs. Multidisciplinary: Biochemical and biophysical assays of CDK1 activity at Newcastle University; Mass spectrometry at University of Edinburgh; cell-based functional assays at University of Sussex.
Start Year 2017
 
Description Myc-P-TEFb interactions 
Organisation University of Leeds
Department Faculty of Medicine and Health
Country United Kingdom 
Sector Academic/University 
PI Contribution Provision of P-TEFb protein complexes and structural biology expertise. Complementary expertise in biochemical and biophysical assays and cellular models.
Collaborator Contribution Provision of Myc family proteins and mutants. Complementary expertise in structural biology, biophysical and biochemical and structural methods.
Impact Joint MRC-DTP studentship awarded to Leeds and Newcastle. Start date 01/10/2017. Title: "Connecting Myc to transcriptional regulation through cyclin-dependent kinases"
Start Year 2017
 
Description Structure/function studies of CDK and CDK regulatory protein complexes 
Organisation Ludwig Institute for Cancer Research
Department Oxford Branch
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution Generation of constructs for cellular studies, proteins and supporting biochemical, biophysical and structural characterization
Collaborator Contribution Provision of expertise in cellular studies and cell imaging for functional studies.
Impact Two manuscripts published: (1) Lu, M., Breyssens, H., Salter, V., Zhong, S., Hu, Y., Baer, C., Sullivan, A., Brown, N.R., Endicott, J.A., Knapp, S., Kessler, B., Middleton, M.R., Siebold, C., Jones, Y., Sviderskaya, E.V., Cebon, J., John, T., Goding, C.T. and Lu, X. (2013) Restoring p53 function in human melanoma cells by inhibiting mdm2 and cyclinB1/cdk1 phosphorylated nuclear iASPP. Cancer Cell, 23:618-33. (2) Lu, M., Zak, J., Chen, S., Sanchez-Pulido, L., Severson, D.T., Endicott, J., Ponting, C.P., Schofield, C.J., and Lu, X. (2014) A code for RanGDP binding in ankyrin repeats defines a nuclear import pathway. Cell 157:1130-450. Collaboration is multidisciplinary: cell culture, FACS and imaging at Oxford, biochemical, biophysical and structural characterisation of CDK-containing complexes at Newcastle.
Start Year 2009
 
Description CRUK Race for Life (Newcastle) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Public engagement in cancer research. Short talk and thank you to women running a "Race for Life".

Highlights the work of the Institute - interested groups ask for building tours and to meet scientists.
Year(s) Of Engagement Activity 2012,2014,2016,2017
 
Description Inspiring the Future L'Oréal Women in Science Event 
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 50-60 school children attended an in-school science presentation with an emphasis on talking to girls about possible careers in science.
Year(s) Of Engagement Activity 2017
 
Description Institute tour (Newcastle) 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Tours of the Institute given to groups of cancer patients and their families and various supporter groups. Aim is to showcase the research going on in the institute, to inform and to say thank you for their fund-raising.
Year(s) Of Engagement Activity 2016,2017
 
Description Pint of Science 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact General Science outreach in local pubs. Three different topics ("Atoms and Galaxies", "Our Body" and "Beautiful Mind" (as examples, topics selected in 2016)).
Year(s) Of Engagement Activity 2016,2017,2018
 
Description School visit (Newcastle) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Visit to a class of 20 year 1 children to introduce them to doing experiments and analysing the results- chromatography of inks and discussion of life being a scientist as examples.
Year(s) Of Engagement Activity 2014,2015,2016,2017