Temporal Single Cell Analysis of Myeloid Leukemia Microenvironment

Signals from the bone marrow microenvironment are known to be critical for the support and development of adult hematopoietic stem cells (HSCs) and for leukemic initiation and progression. While niche-driven signals are known to promote cancer progression, less is known about temporal changes that occur in signal producing stromal populations during leukemia initiation and progression. To comprehensively map changes in the bone marrow microenvironmental populations, we carried out single cell RNA-sequencing in a blast crisis chronic myeloid leukemia murine model. Our temporal analysis identified 7 major stromal populations including arteriolar and sinusoidal endothelial cells, chondrocytes, fibroblasts, pericytes, mesenchymal stromal cells (MSCs), and osteo-associated cells. In particular, we note an expansion in arteriolar endothelial cells, chondrocytes and MSCs, and a loss in sinusoidal endothelial cells and osteo-associated cells. We identify changes in gene expression that may support leukemia progression, including downregulation of bone mineralization and osteoblast differentiation in MSCs, and reduced endothelial focal adhesion. This indicates that leukemic progression may be associated with an arrest in osteogenic differentiation and expansion of MSCs as well as a loss of endothelial cell integrity and function. We integrate our microenvironmental scRNA-seq data with our RNA-seq analysis of human LSCs from distinct aggressive myeloid cancer subtypes and our CRISPR based in vivo LSC dependency map to develop a temporal receptor-ligand interactome essential for disease progression. In addition to identifying key ligand-receptor interactions, our analyses identified distinct patterns of LSC-specific microenvironmental ligand expression. While the expression of some genes remained steady in the same population at all time points (e.g., Jam2 and Ihh), expression of some were lost over time (e.g. Pcdh7 and Il7), especially during disease end point. Expression of genes like Anxa1 first increased during disease progression but declined towards the end. While a fourth category of genes were expressed at all time points, the microenvironmental populations expressing them changed over time. These included Cdo1, Apoe and Agt. Importantly, we find that inhibiting expression of candidate ligands in niche cells strongly impairs the growth and survival of co-cultured leukemia cells, confirming a functional role of niche-driven ligands in disease progression. Our temporal analysis thus identifies signals that are likely necessary not only at different stages of disease progression, but also those that are critical during the entire course of the disease.

Liesveld:Syros: Membership on an entity's Board of Directors or advisory committees; Servier: Membership on an entity's Board of Directors or advisory committees; Dalichi Sankyo: Membership on an entity's Board of Directors or advisory committees.