Identifying the hematopoietic and leukemic stem cell niches

Acute myeloid leukemia (AML) is a devastating disease originating from immature hematopoietic cells in the bone marrow (BM) of patients. Leukemic stem cells (LSC) maintain the disease and likely cause relapse after treatment. Current treatment strategies in AML are mainly targeting actively dividing leukemic cells, but do not tackle quiescent and self-renewing LSCs, which likely explains its limited effectiveness. Therefore, to eradicate the disease, treatment must eliminate the LSC fraction. However, approaches to eradicate LSCs remain undeveloped. LSCs are sustained by unique interactions with the BM microenvironment (aka niche) defined by specific molecular/cellular determinants. Here, we propose a strategy to identify these determinants using a novel synthetic Notch reporter system (SynNotch). A synthetic Notch receptor contains the core of the Notch receptor but can be customized for their sensing and response behaviors by swapping the extracellular recognition domain and intracellular signaling domain to create a reporter of the receptor activity. Thereby one can monitor user-specified environmental clues such as direct cellular contact or specific extracellular ligand binding in cells expressing the SynNotch receptor. We will engineer our SynNotch system to identify BM niche cells that were in direct cellular contact with leukemic cells by the activation fluorescent reporter expression. The fluorescent cells will be isolated analyzed by single cells RNA sequencing to identify the molecular determinants specific to LSC niche interactions. Concluding, our proposal addresses the principal question whether it is possible to eradicate the LSC fraction of AML cells by targeting the LSC-niche.

This project set out to answer a simple but fundamental question: Which cells in the bone marrow physically "host" healthy blood stem cells and leukemic stem cells? Knowing who those "host" cells are is crucial for understanding how leukemia starts, persists, and relapses, and for designing therapies that target the cancer's home rather than only the cancer cells themselves. To do this, we adapted a cutting edge technology called synthetic Notch receptor (SynNotch). In simple terms, bone marrow stromal cells (the "host cells") are genetically equipped with an artificial receptor that recognizes a marker on neighboring blood or leukemia cells. When physical contact occurs, the bone marrow stromal cell switches on a bright fluorescent signal. This makes the interacting stromal cells visible and ammenable for enrichment and more further detailed analysis. We successfully used CRISPR-Cas9 and viral tools to insert SynNotch receptors into bone marrow stromal cell genomes and fine tuned the system to reduce unwanted background activation to ensure that only true cell-cell contact turns the signal on. We therefore extensively tested different designs (original vs. improved versions that reduce background noise), different cell types and various ways to present the activating signal. Although several challenges had to be overcome to finally demonstrate that engineered primary stromal cells can indeed switch on the fluorescent reporter when co cultured with model leukemia cells. Our experiments clarified an important trade off: Versions of the system that are very "clean" (little background) are also harder to be activated. This knowledge did eventually guide the cruical experiments for the characterization of the very stromal cells that physically interact with human blood and leukemic stem cells.