Molecular mechanisms of eukaryotic ribosome assembly

Lead Research Organisation: University of Cambridge
Department Name: Cambridge Institute for Medical Research

Abstract

Understanding how the acquisition of a genetic mutation by a single cell drives a cancer by outcompeting its normal healthy cell counterparts is a key question in cancer biology. The genetic disorder Shwachman-Diamond syndrome (SDS) is an excellent model with which to address this question as individuals affected by the disorder have an increased propensity to develop blood cancers and the initiating genetic lesions are known. MRC-supported work in my lab defined the primary defect in SDS as impaired assembly of ribosomes, molecular nanomachines that make all the proteins in our cells. However, the basic mechanisms of ribosome assembly and how defects in this process promote cancer remain poorly understood. These are important questions, as mutations in ribosomal protein genes are now recognised as a common vulnerability in human cancers. However, there are two major barriers to progress in the field. The first is the lack of high-resolution structures of native human 60S ribosome assembly intermediates, which are highly dynamic and often transient. The second barrier is the inherent difficulty in tracking blood stem cell clones in vivo in human disease states.

To overcome these obstacles, we will systematically determine the structures of key native 60S ribosomal subunit maturation intermediates using the latest imaging technologies. Furthermore, we will use acquired mutations in blood stem cells as an endogenous barcoding tool to determine how defects in ribosome assembly promote the development of cancer by outcompeting their normal counterparts.

Technical Summary

Understanding how the acquisition of a genetic mutation by a single cell drives a cancer by outcompeting its normal healthy cell counterparts is a key question in cancer biology. The autosomal recessive disorder Shwachman-Diamond syndrome (SDS) is an excellent model with which to address this question as affected individuals have an increased propensity to develop acute myeloid leukaemia (AML) and the initiating genetic lesions (germline mutations in the SBDS, EFL1 or DNAJC21 genes) are known. MRC-supported work in my lab defined SDS as a ribosomopathy that is caused by defective maturation of the large ribosomal subunit. However, the basic mechanisms of ribosome assembly and how defects in this process promote cancer remain poorly understood. These are important questions, as haploinsufficiency of ribosomal protein genes is now recognised as a common vulnerability in human cancers and haematological malignancies. However, there are two major barriers to progress in the field. The first is the lack of high-resolution structures of native human pre-60S ribosome assembly intermediates, which are highly dynamic and often transient. The second barrier is the inherent difficulty in tracking haematopoietic stem cell clones in vivo in disease states such as human ribosomopathies.

To overcome these obstacles, we will systematically determine the structures of key native pre-60S ribosomal subunit maturation intermediates using the latest advances in single-particle cryo-electron microscopy (cryo-EM). Furthermore, we will use somatic mutations as an endogenous stem cell barcoding tool to determine how ribosomal stress promotes the development of malignant clones in vivo that outcompete their normal counterparts to drive leukaemia. The feasibility and timeliness of this programme is supported by our published work and strong preliminary data that demonstrate our expertise in applying latest advances in cryo-EM to visualise 60S ribosomal subunit maturation states.

Planned Impact

Our research will have significant impact for a wide range of beneficiaries.

Biomedical research community
Our research brings innovative approaches to key questions which are of critical concern to researchers across the sector. The basic mechanisms of ribosome assembly are a fundamental, universally conserved process with wide ranging relevance but they remain poorly understood. Our research will make a key contribution to their study. Our data and in particular the high-resolution structures of native human pre-60S ribosome assembly intermediates we will produce will be vital information for ribosome researchers. We will however also be tackling the effects of defects in ribosome assembly. These are implicated in a wide range of conditions giving our research far reaching relevance. In particular, by addressing in depth their promotion of cancer, we will ensure significant impact. We will use the autosomal recessive disorder Shwachman-Diamond syndrome (SDS) as a model condition and so our research will have significant impact for researchers studying it and other ribosomopathies.

Patients and their families
Our work will benefit many stakeholders in the rare disease community including patients, patient's representatives, health care professionals, industry, policymakers and representatives of Members States of the European Organisation for Rare Diseases (EURORDIS). There are 30 million people affected by rare disorders in the EU, and all of these patients face the same problems of lack of access to information, to diagnosis, to care, to drugs and to appropriate support. By increasing awareness of the pathophysiology of rare diseases and drawing molecular links between them, our work will help promote the extension of new policy on rare disease internationally to promote the development of new drugs and optimise access to health care.

Cryo-electron microscopy community
Cryo-EM is an evolving discipline which faces several challenges. The great insights it offers are attracting more researchers to its use but there are insufficient skilled researchers and much beamtime is wasted. We will continue to operate at the leading edge of single particle cryo-EM research, further improving our techniques and developing new methods. These will serve as guides to less experienced users. We will also train a number of new users both within our own group and across the University of Cambridge.

Healthcare Community
Advances in our basic understanding of bone marrow failure will benefit patients, their families, their physicians, nurses, dieticians and clinical psychologists. Greater understanding of the consequences of disease mutations in SDS helps patients and their families come to terms with and better understand their disease. Patients will also benefit by reconfiguration of the commissioning of health care provision in the UK to National Centres of Excellence, thereby increasing the referral base, and enhancing physician expertise in managing the multidisciplinary problems with which these patients present. This will benefit Addenbrookes NHS Trust by raising its clinical profile and bringing in more resource to treat the increased number of specialist referrals.

Industry
Feedback on our use of cutting edge cryo-EM equipment and bespoke software will be of great benefit to its manufacturers as they continue to develop their products. Cryo-EM is an expanding international multi-million pound industry employing many people in the UK. We have also received interest in commercialising our research. The insights produced by our research will be potentially exploitable to design novel therapeutics targeting the aberrant translational programme in cancer cells. The interest by industry partners in our research shows its economic potential.

General public
Significant public interest in our research is shown by a recent BBC Television News item on the translational impact of our use of new technology.

Publications

10 25 50
 
Description Congenital and Acquired Chronic Neutropenias: Challenges, Perspectives and Implementation of the EuNet-INNOCHRON Action
Geographic Reach Europe 
Policy Influence Type Membership of a guideline committee
Impact The EuNet-INNOCHRON is an open network focused on the investigation, characterization, harmonization of diagnosis and treatment of chronic neutropenias (CNP), promotion of research, technology and entrepreneurial innovation, consisting so far of more than 30 countries. The Action was built on a network of experts in the field of CNP with long-standing collaborations and further enriched with scientists of diverse expertise to achieve an interdisciplinary and multidisciplinary approach to study the pathogenesis of CNP and is open to researchers, particularly at early career stages, related scientific societies, patient organizations, biotechnology and pharmaceutical companies. A key intention of EuNet-INNOCHRON is to foster broad collaborations and open the way for cutting-edge research and transfer of knowledge to the clinic. The consortium is anticipated to strengthen the interactions among experts in the field of CNP at the European level and beyond, to facilitate and extend the collaboration between existing outstanding networks such as EHA-Scientific Working Groups, SCNIR and national Registries for the introduction of common policies in CNP Registries and Biobanks, and, most importantly, to contribute to the generation of the future leading physicians and researchers in the field of CNPs with appropriate knowledge and skills to promote further research, innovation and personalized patient treatment.
 
Description Leukaemia Research Programme Grant
Amount £1,400,000 (GBP)
Organisation Leukaemia and Lymphoma Research 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2008 
End 04/2013
 
Description Programme Grant
Amount £2,000,000 (GBP)
Organisation Leukaemia and Lymphoma Research 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2013 
End 03/2019
 
Description Targeting the ribosome to treat blood cancer
Amount £299,968 (GBP)
Funding ID 1302 
Organisation Blood Cancer UK 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2021 
End 09/2024
 
Description Dictyostelium ribosomes by cryo-EM 
Organisation Medical Research Council (MRC)
Department MRC Laboratory of Molecular Biology (LMB)
Country United Kingdom 
Sector Academic/University 
PI Contribution Resulted in submission of a scientific manuscript
Collaborator Contribution Culture of Dictyostelium discoideum cells
Impact Weis F, Giudice E, Churcher M, Jin L, Li Jin, Hilcenko C, Wong CC, Traynor D, Kay RR, Warren AJ. Preparing the 60S ribosomal subunit for translation: structural basis of eIF6 release (revision submitted).
Start Year 2008
 
Description Quality control of human 60S ribosomal subunit assembly 
Organisation Children's Hospital of Philadelphia
Country United States 
Sector Hospitals 
PI Contribution Cryo-EM analysis of human ribosome assembly intermediates
Collaborator Contribution Functional and genetci studies in mice
Impact Manuscript Lv K, Gong C, Aruljothi C, Han X, Ren J-G, Donaghy R, Cheng Y, Pellegrino S, Warren, AJ, Paralkar VR and Tong, W. HectD1 Ubiquitin Ligase Dependent Control of Ribosome Biogenesis Regulates Hematopoietic Stem Cell Regeneration. Cell Stem Cell (2021). Mar 4; S1934-5909(21)00058-8. doi: 10.1016/j.stem.2021.02.008. PMID 33711283
Start Year 2021
 
Description Shwachman-Diamond syndrome pathogenesis 
Organisation National Institute of Health and Medical Research (INSERM)
Department UMR 1163
Country France 
Sector Public 
PI Contribution Primary research
Collaborator Contribution Assessed function of acquired eIF6 mutations in bone marrow cells from patients with inherited bone marrow failure
Impact Research publication
Start Year 2018
 
Description mitochondrial ribosome assembly 
Organisation Medical Research Council (MRC)
Department MRC Mitochondrial Biology Unit
Country United Kingdom 
Sector Academic/University 
PI Contribution Collection and processing of cryo-EM data
Collaborator Contribution Functional studies of mitochondriall ribosome assembly
Impact Rebelo-Guiomar P, Pellegrino S, Dent KC, Sas-Chen A, Miller-Fleming L, Garone C, Van Haute L, Rogan JF, Dinan A, Firth AE, Andrews B, Whitworth AJ, Schwartz S, Warren AJ, Minczuk M. A late-stage assembly checkpoint of the human mitochondrial ribosome large subunit. Nature Communications (2022) Feb 17;13(1):929. doi: 10.1038/s41467-022-28503-5. PMID: 35177605
Start Year 2020
 
Company Name SDS THERAPEUTICS LIMITED 
Description Company aims to develop targeted therapeutics for the treatment of the bone marrow failure disorder Shwachman-Diamond syndrome 
Year Established 2020 
Impact Only running since 1st Jan 2021
 
Description COST action CA18233 Management Committee Meeting 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact 84 professional attended a European working group on neutropenia, which involved data presentation by myself and others, panel discussion concerning future training goals and drafting of international guidelines to improve patient treatment.
Year(s) Of Engagement Activity 2021
 
Description International scientific meeting 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Hosted an international working group (EuNET INNOCHRON) for 84 professional practioners in March 2020 (remotely, due to COVID19) to present scientific data, draw up draft international clinical management guidelines and broaden the scope of international collaboration.
Year(s) Of Engagement Activity 2020
 
Description Workshop 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact China-UK Precision Medicine in Haematology Workshop, Thursday 3rd - Friday 4th June 2021, Plenary talk
Year(s) Of Engagement Activity 2021
 
Description web based seminar to patient group 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Patients, carers and/or patient groups
Results and Impact Family group in the USA broadcast an educational seminar on Facebook which sparked questions and discussions afterwards, driving selection of topic for further discussion at forthcoming international research meeting.
Year(s) Of Engagement Activity 2021