Unravelling biological heterogeneity in neoplastic myeloproliferative stem cells

One of the key challenges in cancer biology is to better understand why patients with the same subtype of cancer show remarkably different responses to treatment. One emerging explanation for this observation is that not all cells within a specific cancer behave in the same way. This partly reflects variability in the types of DNA damage or mutation(s) carried within each cell but might also be related to the type of cell which carries the mutation(s) as only some cancer cells, known as cancer stem cells (CSCs), are able to propagate disease relapse in patients. Our ability to understand the biology underlieing this variability is limited as most methods of analysing cancer cells involve pooling large populations of cancer cells, often many millions of cells, and thus the variability within individual cells is lost. This is important as the cancer cell which ultimately cause relapse following treatment may in some cases only account for a small proportion of cancer cells at diagnosis; akin to "finding a needle in a haystack". One approach that can be used to overcome this is to analyse single cancer cells. Whilst single cell analysis is not a new approach, previous methods have been limited by issues of sensitivity and technical variation making it difficult to understand which of the findings are "real" and which simply reflect "noise" in the testing process. An exciting new technology, introduced to the WIMM by AJM, allows analysis of single cancer cells, thus overcoming the above limitations. This technology will allow both mutation testing and analysis of the expression of genes all from the same cell, thus allowing presence of specific mutations to be correlated with the biology of single cells and also, most importantly, outcome of the patient.

During this fellowship, AJM will develop and apply single cell analysis to a particular form of blood cancer known as myeloproliferative neoplams (MPN). Chronic myeloid leukaemia (CML) is a type of MPN which can now be highly effectively treated with a drug called imatinib. Whilst imatinib induces remissions in almost all patients with CML, the disease remains detectable in the large majority of patients due to the presence of imatinib resistant CML-CSCs. As these CML-CSCs are "hidden" amongst the normal bone marrow stem cells, single cell analysis is required to distinguish CML-CSCs form their normal counterparts in order to understand how these CSCs might be more effectively treated. A similar strategy will be used to investigate high-risk MPN patients receiving novel JAK2 inhibitor treatment, an exciting new treatment for patients with certain types of MPN. Whilst JAK2 inhibitor treatment induces symptomatic improvement in patients, it is also clear that MPN-CSCs are largely unaffected by the treatment and single cell analysis of MPN-CSCs will help to understand the reasons for this. Importantly, whilst these studies are specific to MPNs, as this is the area of clinical medicine in which AJM specialises, the findings of these studies will also be more broadly applicable across cancer biology.

In order to understand how different types of cells respond to the acquisition of specific cancer causing mutations, a complementary approach is required. AJM will develop a number of model systems allowing the selective targeting of combinations of different MPN causing mutations to different types of bone marrow stem cells. It is possible, for example, that the same mutation may behave very differently depending on the cell type which originally acquires the mutation, thus influencing influence the resulting disease type, even though the mutation might remain the same.

Through application of these state of the art cell and molecular biology techniques in humans and model systems, these studies will provide a substantial contribution to the understanding of MPN-CSCs and the findings will highlight the clinical applicability of single cell technology across cancer biology.

In haematologic malignancies, and cancers in general, there is increasing appreciation of complex subclonal genetic structures in cancer stem cell populations which may intersect with the differentiation and quiescence state of the cells and dictate variability in self-renewal and chemoresistance. To unravel this complexity requires robust single cell analysis in a high throughput manner. The Fluidigm C1 mircofluidics Platform allows capture of single primary cells and the interrogation of RNA and DNA content by multiplex PCR or through whole transcriptome/genome analysis, all at the single cell level. The technology will be used to define single cell heterogeneity in human and mouse malignant stem and progenitor populations to allow us to better define, purify and therapeuticlaly target these critical populations of cells. Furthermore, the novel mouse models generated as part of the fellowship, allowing specific targeting of different stem cell subsets with oncogenic mutations, is a further major technical advance in the field.

Both these techniques are central to the aspiration to develop stratified medicine in the UK, using state of the art genomic approaches to dissect heterogeneity in patients and to understand this heterogeneity in model systems.

This research will have a clear beneficial impact well beyond the immediate professional circle carrying out similar research. For example:

1. Economic impact: It is estimated that tyrosine kinase inhibitor (TKI) therapy for patients with chronic myeloid leukaemia (CML) will cost the health service between £2-3 billion over the coming 10 years. Thus, it is of major economic importance to be able to identify which patients might be able to safely stop TKI treatment. In those patients with TKI resistant CML-SCs, it is important to identify the pathways propagating these cells in order that they might be eradicated allowing therapy discontinuation. This is a key part of the "pathway to cure" developed by Novartis, the pharmaceutical company which manufactures imatinib and nilotinib, two NICE approved TKIs.

2. People: As outlined in the communication plan, AJM has considerable experience of disseminating scientific research findings more broadly with the public including schools and also for inspiring young potential scientists.

3. New processes: Single cell technology is likely to develop rapidly over the coming years. This will potentially have a key role in stratified medicine particularly in cancer biology and this fellowship will place AJM and the MRC at the forefront of these developments. As an example, AJM introduced the Fluidigm BioMark system to the WIMM and HSCB laboratory in 2011. We were the first to use this technology in Europe for low cell number analysis. We are now the reference center for BioMark in Europe and AJM regularly receives requests for information on our experience of using this platform and requests for collaboration, as illustrated by the research outputs listed on AJM's CV. As a direct result of these interactions, a number of other research groups (at least 4) have introduced the Fluidigm BioMark system in their center. The Fluidigm C1 microfluidics Platform allows capture of single primary cells and the interrogation of RNA and DNA content by multiplex PCR or through whole transcriptome/genome analysis, all at the single cell level. The technology will be used to define single cell heterogeneity in human and mouse malignant stem and progenitor populations to allow us to better define, purify and therapeutically target these critical populations of cells. It is anticipated that this will generate much more interest even than the BioMark system, with benefits for many other groups interested in working with genomics at the single cell level in the UK and further afield. This fruitful interaction with Fluidigm (the manufacturer of the BioMark and C1-prep systems) has clear and mutual beneficial impact.

4. New products: The access to serial samples from patients treated on innovate clinical trials, for example the MAJIC study assessing the role of ruxolitinib therapy (made by Novartis) in high-risk patients with myeloproliferative neoplasms will help identify specific patients/groups of patients who are likely to benefit from these new treatments, accelrating introduction of these new treatments with benefits for patients, the health service and industry alike. A number of other pharmaceutical companies have JAK2 inhibitor treatments in development (Sanofi Aventis, Cellular Therapeutics, Gilead) and AJM is already in the process of developing fruitful collaborations with these industry partners which will be further facilitated by the pioneering work outlined in this fellowship proposal.

5. Inward investment: AJM has developed a number of collaborations with industry on related but distinct projects. Industry stakeholders are extremely interested in single cell technology but do not have the skills or expertise to analyse, interpret or apply this emerging new technology. Thus, it is anticipated that a number of collaborations with industry will arise as a result of developing single cell expertise in the WIMM led by AJM