Single cell analysis of malignant stem cell clones in myelofibrosis

Aims

To develop a new method to allow parallel mutational analysis, RNA sequencing and lineage tracing of single stem cell clones.

To apply this technology to explore biological heterogeneity of single stem/progenitor populations in patients with myelofibrosis.

Background

Haematopoiesis is an ideal tractable model for the application of pioneering techniques in single cell genomics. Over the last few years, the Haematopoietic Stem Cell (HSC) Biology Laboratory at the WIMM has established state of the art techniques to study gene expression in single haematopoietic stem and progenitor cells. However, major challenges for the application of single cell transcriptome analysis in malignant stem cell biology remain. One of these relates to a lack of sensitivity for mutation detection using standard RNA-sequencing approaches due to a combination of technical "drop outs" in the data as well as true biological heterogeneity of expression of mutations at the single cell level. A second limitation of single-cell RNA-sequencing of stem cells is that this only provides a “snapshot” of the cells at a certain point in time, with no indication of their output in terms of clonal progeny.

The purpose of this studentship is to develop and optimise a new technique for mutation detection with parallel analysis of the whole transcriptome of single stem cells. This will involve targeted gDNA and global RNA analysis from the same single cells through refined procedures for generating single cell RNA-seq libraries in order to minimise technical drop outs in the data. This technique will also encompass lineage tracing of stem cell clones using either the specific mutations detected or through lentiviral based cellular barcoding. This will provide excellent experience in state of the art molecular and stem cell biology techniques as well as training in the use of high throughput robotics platforms and bioinformatics.

This technique will then be used for the analysis of stem cells from patients with myelofibrosis, a poor prognosis form of blood cancer which is propagated by rare malignant stem cell populations. Myelofibrosis is an ideal tractable model system to apply the above single cell approach but with more broad applicability for cancer biology. Normal and myelofibrosis HSCs will be analysed using a combination of in vitro and in vivo stem cell assays combined with the above single cell analysis technique in order to characterise driver mutations in single malignant stem cells and to correlate this mutational information with whole transcriptome analysis of stem cells and readout of their clonal progeny. The analysis will focus on disruption of megakaryocytic differentiation pathways during the development of myelofibrosis. This information will be correlated with clinical outcome and response to targeted therapies in patients, with potential to provide unique insights into the biology of myelofibrosis, with potential for clinical benefit in patients through biomarker and therapeutic target discovery.