Leuko/Stromal interactions in normal and pathological hematopoiesis

 

Hematopoiesis is the process by which Hematopoietic Stem Cells (HSCs) replenish platelets, red blood cells and immune cells over lifetime. It occurs in the bone marrow (BM) of adult mammals and requires the retention of HSCs in specialized “niches” that control HSCs quiescence, proliferation and differentiation. Over the last decade, numerous studies have tried to characterize stromal cells forming the bone marrow microenvironment that supports hematopoiesis. In most of them, if not all, the working hypothesis was that different stromal cells of different ”niches” must provide specific signals to hematopoietic cells at each stage of differentiation in order to maintain homeostasis. Such mechanisms are thought to be responsible for leukemic cell drug resistance. 

Our research aims at the characterization of molecular mechanisms involved in leuko/stromal interactions in order to define new adjuvant therapeutic strategies to reduce leukemic cell drug resistance in leukemia. This research program started through the study of our molecules of interest in normal hematopoiesis: JAM-B/JAM-C and Pre-BCR. In vivo studies are implemented with cutting edge imaging technologies aiming at the characterization of dynamic interactions occuring between leukemic and stromal cells. 

Project 1: Junctional Adhesion Molecules in Leuko/Stromal interactions

We have recently found that the Junctional Adhesion Molecule-C (JAM-C) is expressed by HSC and interacts with JAM-B expressed by bone marrow stromal cells (Arcangeli et al, Stem Cells, 2014; Blood, 2011). The interaction occurs across species (i.e. human JAM-C interacts with mouse JAM-B) and contributes to maintenance of HSC quiescence. In the follow-up study, we will translate our results to pathological situation such as AML and better characterize the molecular mechanisms regulating JAM-B and JAM-C expression. Indeed, our preliminary results indicate that JAM-C is regulated by transcriptional and post-transcriptional mechanisms, some of which involving protein trafficking. We actually use these changes in JAM-C expression to dissect HSC heterogeneity at the single cell level (Figure) and to generate new knock-out mice strains that will be relevant to the regulation of JAM-B and JAM-C expression.

In addition, we currently develop several approaches to inhibit the JAM-B/JAM-C adhesion axis:

  • blocking monoclonal antibodies
  • small compounds inhibiting protein/protein interaction

Principal Investigator: Michel Aurrand-Lions

Project 2: Regulation of normal and pathological B cell development/survival by stromal cell niches

The differentiation of normal B cells depends on specific cues transmitted by specialized stromal cell niches of the bone marrow. Indeed, while pro-B cells are found in the vicinity of cells secreting IL-7, we have demonstrated that pre-B cells are in contact with galectin-1 expressing stromal cells, which are distinct from the IL-7+ pro-B cell niche (Mourcin et al, Blood 2011). The project we currently develop aims to decipher the molecular mechanisms allowing specific migration and adhesion of the different B cell subsets to their respective niches.

In addition, some surface receptors involved in interactions between B cell progenitor acute lymphoblastic leukemia (BCP-ALL, malignant counterparts of developing B cells), and bone marrow stromal cells have been identified in functional screenings. Their role in leukemic progression is under investigation. The generation of antagonists of BCP-ALL / stromal cells interactions is a promising new adjuvant therapeutic strategy which should allow circumventing resistance to chemotherapies. Characterization of such inhibitors and defining indications of their use in pre-clinical models is the main aim of this project.

 

Principal investigator: Stéphane Mancini

 

 

Project 3: Videonanoscopy of adhesion molecules

Interactions between hematopoietic and stromal cells are profoundly remodeled by leukemia and myeloablative treatments. Advances in optical microscopy now allow detecting individual fluorescent molecules with nearly nanometric precision. Thus, tracking JAM dynamics at the membrane and into the cell by videonanoscopy allows studying the development and evolution of cell junctions. 

The analysis of molecular trajectories using a dedicated homemade algorithm, Multi-Target Tracing (Sergé et al. Nature Methods 2008Rouger et al. J Vis Exp 2012), unravels the signature of interaction, stabilization or intracellular trafficking events. Our objective is to characterize the mechanisms of tumor cell interaction and migration as well as stem cell retention in bone marrow niches.

We have also developed an automated method named For3D (Sergé et al. J Immunol Methods 2015) for 3D rendering and quantification of organs of interest.

Principal investigator: Arnauld Sergé

 

 

OP9 stromal cells stained with an anti-JAM-B antibody (green) co-cultured with KG1 leukemia cells stained with an anti-JAM-C antibody (red). Left: video projection of 500 images (100 ms/frame). Right: JAM-B (green) and JAM-C (red) trajectories superimposed on the phase image. Colocalisation events are labeled by a yellow circle.
3D rendering of a mouse thymic lobe (cortex in green and medulla in red) starting from images of serial sections labeled by immunohistochemistry