Characterization of functional heterogeneity in a mouse model of HoxA9/Meis1 dependent acute myeloid leukemia through fluorescent genetic barcoding.

• Applicant: Dr. Tobias Maetzig
• Subject Area: Hematology, Oncology
• Term: from 2016 to 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 328854319

Acute myeloid leukemia (AML) is a severe hematopoietic disease with low long-term survival rates. The reason for this lies in the failure of standard therapies to fully eradicate the leukemic stem cell (LSC) population. For studying the disease, mouse models mimicking human AML are of utmost importance. One of these models relies on the transplantation of murine bone marrow cells overexpressing HoxA9 and Meis1 (H9M) transcription factors. According to current literature, the H9M LSC pool contains at least three phenotypically defined subpopulations that clonally reconstitute each other and that give rise to the whole leukemic hierarchy upon serial transplantation. However, our own data indicate that these phenotypically defined LSC may be hierarchically organized. Given the reported differences in drug sensitivities of the LSC subpopulations, understanding the molecular pathways that govern LSC properties, and their characterization in healthy hematopoiesis may pave the way for better treatment options. Therefore, this proposal aims at 1. Characterizing transcription programs and signaling pathways that regulate the phenotypic identity as well as the phenotypic plasticity of H9M LSC; 2. Identifying H9M collaboration partners that increase the aggressiveness of the disease and may be suitable as prognostic markers; and 3. Investigating the functional implications of leukemia associated genes from Aims 1 and 2 for healthy hematopoiesis. Key to these studies will be the utilization of a newly developed lentiviral gene marking system for uniquely labeling of up to 12 cell populations. This approach thus offers the opportunity to directly monitor and compare the fate of multiple H9M populations in the same mouse, while concomitantly reducing required mouse numbers. Based on this strategy, we will first employ multiplex transplantation assays to gain deeper insights into the hierarchical organization of the H9M LSC pool. We will then utilize comparative molecular studies and functional studies for characterizing gene expression pathways that control the phenotype of individual LSC populations and potentially govern their hierarchical organization. We furthermore hypothesize that lentiviral gene marking may influence LSC features by triggering secondary genetic lesions that collaborate with H9M signaling. These rare mutants will be identified through multiplex H9M transplantation assays and subsequent integration site analyses. The prognostic value of experimentally verified H9M collaboration partners on patient survival will then be assess by interrogating publicly available data sets (TCGA), before investigating the function of leukemia associated genes in healthy hematopoiesis, e.g. transformation potential, self-renewal capacity and lineage skewing. This work will thus lead to the identification and characterization of mechanisms controlling LSC properties, which may aid the development of LSC directed therapies for improving patient survival.

• DFG Programme: Research Grants