Dominique Bonnet: Projects

Our group is interested in studying normal human haematopoietic stem cells (HSCs) and leukaemic stem cells (LSCs). At present we are investigating the relationship between normal HSCs, LSCs and their microenvironment. For this we have developed in vivo imaging techniques allowing us to visualise and define normal and leukaemic stem cells niche in vivo. We hope to dissect the role of different components of the stem cell niche and see whether we can intervene in this niche to expand normal stem cells and eradicate LSCs. All these projects should shed light into pathways or interactions that are more specifically used by LSCs and where therapeutic intervention might be developed.

CD34- HSC at the apex of human HSC

In addition to well-characterised CD34+ Haematopoietic Stem and Progenitor Cells (HSPCs), the human HSC hierarchy contains a rare CD34− population with a severely combined immunodeficiency-repopulating capacity. However, little is known about the molecular characteristics of these CD34− cells or their relationship to the CD34+ populations.

We have recently shown that the self-renewing Lin−CD34−CD38−CD93hi population contains cells that not only function as HSCs, but can also be placed above the CD34+ populations in the haematopoietic hierarchy. These cells have an active Notch pathway, in which signalling through Delta4 is crucial for maintenance of the primitive state, and the combined signals from Jagged1 and TGF-β are important in controlling its quiescence. They are also refractory to proliferative signals, and show a repressed canonical Wnt pathway, in part regulated by Notch.

Overall, therefore, CD34− cells represent an immature and quiescent human HSC population maintained through a distinctive network of cellular signalling interactions. Future work will aim to define the ontogeny of these cells, the role of this fraction under stress conditions, and ways to expand these cells (Anjos-Afonso et al., 2013; Cell Stem Cell. 13(2): 161-74).

 

Role of HIF-2α in the regulation of both normal HSC and LSC

HSPCs are exposed to low levels of oxygen in the bone marrow niche, and Hypoxia-Inducible Factors (HIFs) are the main regulators of cellular responses to oxygen variation.

Recent studies using conditional knockout mouse models have unveiled a major role for HIF-1α in the maintenance of murine HSCs; however, the role of HIF-2α is still unclear.

Figure 1. HIF–2α: as the safeguard of Human HSC against ROS generation.

Figure 1. HIF–2α: as the safeguard of Human HSC against ROS generation. (Click to view larger image)

We recently demonstrated that knockdown of HIF-2α, and to a much lesser extent HIF-1α, impedes the long-term repopulating ability of human CD34+ umbilical cord blood cells. HIF-2α-deficient HSPCs display increased production of Reactive Oxygen Species (ROS), which subsequently stimulate Endoplasmic Reticulum (ER) stress and triggers apoptosis by activation of the Unfolded-Protein-Response (UPR) pathway (Figure 1). HIF-2α deregulation also significantly decreased engraftment ability of human acute myeloid leukaemia (AML) cells.

Overall, our data demonstrates a key role for HIF-2α in the maintenance of human HSPCs and in the survival of primary AML cells. Whether inhibition of this pathway could be used to selectively target AML has still to be determined (Rouault-Pierre et al., 2013; Cell Stem Cell. 13(5): 549-63).

Cross-talk between LSCs and their microenvironment

It has become clear both in solid tumours and in leukaemia that cancer stem cells depend on their microenvironment to grow and expand.

In AML, we know that LSCs cannot be maintained ex vivo without the addition of a stroma support, indicating that LSCs are dependent on their microenvironment for their survival/maintenance. Understanding the crosstalk between LSCs and their microenvironment is thus crucial to better understand this dependency and potentially use this to target LSC in vivo.

We thus started a project trying to better define the factors involved in this crosstalk. AML samples were co-cultured ex vivo with Mesenchymal Stroma Cells (MSC), and after one week micro-array analysis was performed on sorted stroma cells. Using pathway analysis and the Gene Go program, we built a network of the combined datasets.

Further studies will be undertaken to evaluate the effect of the different keys factors. If any of these factors affect the growth/differentiation/apoptosis of AML, we will further investigate one/more pathway(s) in more detail in vitro but also in vivo using knock-down approaches.

Seeing is believing: the stem cell niche

In order to better characterise the bone marrow microenvironment, stem cells and their niches, we have developed different technologies for in vivo contrasting procedures as well as for tracking normal and leukaemic cells in vivo, combining whole body near infrared fluorescence, bioluminescence imaging, intravital microscopy of intact live bone marrow as well as histology and flow cytometry.

Using improved contrasting procedures for the bone marrow endothelium we demonstrated that osteoblastic niches are in close proximity to vascular niches in flat but also in long bones. We believe that the combined use of advanced multimodal and multiscale analysis of the bone marrow will very likely shed new light on our understanding of haematopoietic stem cells and their niches in health and disease (Lassailly et al., 2010; Blood. 115(26): 5347-54; Lassailly et al., 2013; Blood. 122(10): 1730-40).

By using this technique on live animals, adding time-lapse imaging, we can visualise the arrival of human HSCs in the bone marrow and their behaviour over time. This should allow us to better define the niche and see whether LSCs use the same niche as normal HSCs (Foster et al., In Revision).

 

Dominique Bonnet

Dominique Bonnet

dominique.bonnet@crick.ac.uk
+44 (0)20 379 61198

  • Qualifications and history
  • 1993 PhD, University of Paris VII, France
  • 1993-1997 Postdoctoral Research Fellow, Hospital for Sick Children, Canada
  • 1998 Assistant Professor, Coriell Institute for Medical Research, USA
  • 2001 Adjunct Assistant Professor, University of Pennsylvania, USA
  • 2001 Established lab at the Imperial Cancer Research Fund, UK (in 2002 the Imperial Cancer Research Fund became Cancer Research UK)
  • 2015 Group Leader, the Francis Crick Institute, London, UK