Unravelling signatures of clonal response, resistance and evolution of high-risk essential thrombocythaemia at single-cell resolution

In recent years, substantial advances in genetic testing have transformed our ability to carry out analysis of the entire human DNA code (human genome) and its mirroring RNA sequence (transcriptome), a surrogate for gene expression, from minute amounts of genetic material. This has propelled our ability to analyse single-cells at a genome/transcriptome wide level, which is now possible with unprecedented scale and resolution. These techniques are now poised for clinical use in precision medicine to improve diagnosis, risk stratification, disease monitoring and drug discovery. However, translating this powerful new technology through to direct clinical application requires integration of clinical and scientific expertise as well as evidence of how such an approach might benefit patients in a specific disease area. Ideally, this should be assessed within the context of a prospective clinical trial. This is the overarching problem/challenge that we aim to address in this research i.e. to provide proof of principle of the clinical utility of single-cell genomic analysis in a specific disease with an unmet need: high-risk essential thrombocythaemia (ET).

ET is a form of chronic myeloproliferative neoplasm (MPN) characterised by a high platelet count, increased risk of thrombosis, bleeding and progression to aggressive blood cancers such as myelofibrosis (MF) and acute myeloid leukaemia (AML). MPNs arise from the earliest form of a blood cell, a stem cell, in the bone marrow which acquires a genetic change, leading to changes in gene expression which promotes uninhibited cell growth. Patients with ET have a variable disease course, with approximately 20% of high-risk patients developing resistance to standard first line therapy with hydroxycarbamide (HC). HC-resistant ET patients have an increased risk of disease progression to MF or AML and significantly reduced overall survival (26% at 10 years). Current therapies prevent bleeding or thrombosis through reduction of blood cells but none have shown ability to prevent disease progression. Ruxolitinib, a JAK inhibitor, is the first targeted treatment approved in MPNs in MF and polycythaemia vera (PV). It has been evaluated in high-risk HC-resistant or intolerant ET in the MAJIC study with variable responses observed and in a proportion, disease progression occurred on this treatment. This project aims to apply single-cell technology to understand the cellular and molecular differences in patients with HC-resistant/intolerant ET on the MAJIC study and in doing so, determine mechanisms for ruxolitinib response (or lack of response) and disease progression.

Firstly, I will compare clinical information of these patients with mutation testing of over 30 genes associated with MPNs to understand if these influence ruxolitinib response and disease progression. Guided by these results, I will then carry out extensive single-cell genomic analysis of mutated bone marrow stem and progenitor cells of subgroups of patients to understand whether gene expression differences between mutated and non-mutated stem cells at a single-cell level identifies mechanisms influencing responses and disease progression. There is increasing evidence to support that the bone marrow environment is abnormal in blood cancers and may influence the disease course. Therefore, I will also study non-mutated stem cells in HC-resistant/intolerant ET patients using single-cell analysis to determine if increased number of these cells or their altered gene expression may be associated with response to treatment, disease progression or side effects of the treatment.

This clinical research training fellowship will provide a state-of-the-art training in the application of single-cell genomics in clinical medicine. We anticipate a direct, tangible impact on patient outcomes through better approaches to diagnose, monitor (identify biomarkers of early relapse) and treat high-risk ET.

Single-cell genomics techniques have enormous potential to resolve intratumoural heterogeneity, of importance for precision medicine in cancer. However, translating this powerful new technology through to direct clinical application requires evidence that such an approach might benefit patients in specific disease area. The overarching aim of this research is to provide this proof of principle of the clinical utility of single-cell genomic analysis in a disease with an unmet need: high-risk essential thrombocythaemia (ET). We hypothesise that single-cell analysis of haematopoietic stem and progenitor cells (HSPCs) in hydroxycarbamide-resistant/intolerant ET patients in the MAJIC study will reveal considerable heterogeneity reflecting distinct molecular signatures, not detected by mutation screening of bulk cell populations, and will therefore facilitate the identification of novel biomarkers and therapeutic targets. We will use a novel technique developed in the Mead laboratory (TARGET-seq) to carry out this analysis. This technique allows ultrasensitive mutation analysis (with allelic dropout rates <3%) combined with unbiased whole transcriptome analysis of the same cell. No currently published method allows this combined analysis and we believe that this will prove to be an extremely powerful method for precision medicine. The technique also allows identification on non-clonal HSPCs to provide insights into cell-extrinsic disruption in the bone marrow microenvironment in high-risk ET.

I am committed to a career as an academic haematologist and am excited about the potential application of single-cell genomics technologies in clinical medicine. This innovative technology is likely to move towards wider clinical application in the coming years and I anticipate that the state-of-the-art training in this field that I will receive, as described in this proposal, will set the foundation for a career as a clinician scientist working in this exciting area of research.

Advances in next generation sequencing (NGS) in recent years have rapidly transformed our ability to carry out genetic analysis from minute amounts of genomic material. This includes analysis of single-cells, which is now possible with unprecedented scale and resolution, leading to a large number of recent key scientific discoveries. Single-cell genomics techniques are now poised for clinical application in diagnostics, risk stratification, disease monitoring and therapeutic discovery. A number of different parties stand to benefit from the outcomes of this research, both in the short-term and long-term. In the short-term, this work would interest clinicians and researchers in haematology and oncology and researchers in the field of single-cell genomics.

A next essential group to consider are patients with myeloproliferative neoplasms (MPNs). These disorders significant impact patients' health and quality of life during chronic phase and at disease transformation. No adequate methods exist to accurately predict progression to myelofibrosis or leukaemia. Furthermore, current therapies mainly prevent development of bleeding or thrombosis, key MPN symptoms, through reduction of blood cells but none have shown ability to prevent disease progression to an aggressive stage. As such, there is an important unmet need in management of these patients and research to address this is essential. This project will help to tackle this by seeking to identify the genetic alterations at a single-cell level associated with poor response to current targeted therapy (JAK inhibitors) and disease progression.

Through this work we aim to enhance the quality of life of those living with MPN. Firstly, improving the ability to identify patients suitable for ruxolitinib treatment will mean that those who will not respond are not subjected to an ineffective treatment, with potential harmful side effects. More resources can then be allocated into recruiting these patients to future trials of new drugs. For those who are deemed suitable for ruxolitinib therapy, they can immediately benefit from this treatment. We predict that if we identify a useful biomarker for patient stratification, this can be implemented in clinical practice within a few years following validation.

Secondly, by identifying other biological mechanisms involved in driving treatment progression, we hope to identify new therapeutic targets to inform the development of new treatments for MPN. Not only do patients, their families and clinicians stand to benefit from this, but the commercial sector as well, through new drug discovery pipelines. This is a longer-term impact from this research.

More broadly, this project will provide a proof of principle for the application of single cell genomics techniques towards direct clinical application, an area of key strategic importance. Although this may appear to be "science fiction", not so long ago the possibility of routine clinical use of whole genome sequencing would have seemed far-fetched, yet this is now being integrated into diagnostic algorithms on a large scale. Whilst considerable hurdles remain before new single cell genomics techniques can be applied directly in the clinic for patient benefit, the next few years are likely to see extensive efforts towards translation of this new technology towards personalised medicine. This includes biomarker identification to predict response to specific therapies, prognostic risk stratification and therapeutic target discovery. This is an area where the MRC has made a major investment in the UK in recent years, including at the MRC WIMM in Oxford, which hosts the Oxford Single Cell Biology Consortium.

This Fellowship will also have an impact on my professional development, by allowing me to develop important laboratory and data analysis skills. These will be crucial for a career in academic haematology, with a direct beneficial impact for patients, colleagues and students.