Clonal evolution of acute myeloid leukemia genomes

Human acute myeloid leukemia (AML) is a very heterogeneous and aggressive hematopoietic malignancy with a poor prognosis. In AML development, sequential acquisition of genetic mutations within the hematopoietic stem and progenitor cell (HSPC) compartment results in subclones of pre-leukemic hematopoietic stem cells (HSCs). These pre-leukemic HSCs undergo further evolution to produce heterogeneous leukemia stem cells (LSCs), again composed of distinct subclones that can replenish the disease or contribute to relapse. According to this hypothesis, human AML exhibits clonal heterogeneity, which has significant implications for pathogenesis and treatment. Pre- leukemic HSCs and/or LSCs may persist following therapy and can eventually expand and give rise to refractory or relapsed disease. This project aims to identify clonal heterogeneity among pre-leukemic HSCs and LSCs through its functional and genomic characterization. We have previously shown that subcutaneous implanted human mesenchymal stromal cells (MSCs) are capable of forming a highly susceptible and easily accessible humanized hematopoietic niche that provides an ideal microenvironment for allowing the preferential engraftment of human hematopoietic cells. Based on sophisticated polychromatic flow cytometry protocols, single pre-leukemic HSCs and LSCs can be isolated from primary AML samples. Our novel xeno-transplantation mouse model allowing for optimal human engraftment will be used for intra-bone single cell transplantation. Functional analysis will be carried out to evaluate either normal hematopoietic reconstitution (in the case of pre-leukemic HSCs) or leukemia initiation (in the case of LSCs). As a second step, engrafted single cell-derived progeny will undergo next generation exome-sequencing to analyze its mutation status. Comparative analysis of functional and genomic data will help to identify novel mutations present either in single pre-leukemic HSCs or LSCs that are involved in driving malignant transformation and leukemia development. These results should provide new insights into the genetic signatures and molecular events necessary and responsible for the evolution of AML.

Both hematopoietic stem cell (HSC) and leukemia cell survival and propagation require complex interactions between stroma cells, extracellular matrix, growth factors, and cell adhesion molecules in the bone marrow compartment. Human leukemia cells, however, require human-specific factors for optimal expansion, and their absence in mice likely explains why many types of human primary myeloid leukemias are difficult to propagate in mice. We could describe a new engraftment model in which a human ossicle a bone marrow-like organoid - placed into the flank of mice acts as a stem cell niche. The human ossicle enables faster and superior engraftment of normal human hematopoietic stem and progenitor cels, as well as both patient-derived myeloid and lymphoid leukemias. Human leukemias preferentiallly colonize the human organoid ossicles over the mouse hosts endogenous bone marrow, supporting the conclusion that the human ossicles contain a niche that provides key components for the preferential engraftment, growth, and expansion of human leukemias that is not provided by mouse BM. Successful engraftment of acute promyelocytic leukemia (APL) and myelofibrosis (MF), both previously shown little to no engraftment in NSG mice, further supports this notion. All together, our model represents a significant step forward in the development of preclinical tools to improve characterization and treatment of human leukemias. This work was published in Nature Medicine (2016) and Nature Protocols (2017).