Molecular mechanisms of eukaryotic ribosome assembly

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.

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.

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.