Molecular and Functional Dynamics of Hematopoietic Progenitor Cell Fate During Ontogeny

Hematopoietic stem and progenitor cells (HSPCs) in the fetus and the adult possess distinct molecular signatures that regulate cell fate and change their susceptibility to initiation and progression of hematological malignancies, such as leukemia. It is well established that fetal and adult hematopoiesis are functionally distinct processes; however, the underlying molecular mechanisms that govern ontogenic differences in normal and malignant hematopoiesis have thus far been poorly explored on the proteomic level. We hypothesize that an intrinsically programmed proteomic switch in HSPCs during ontogeny controls both the outcome of normal hematopoiesis and the susceptibility to initiation of leukemia, and that the proteomic make–up of the leukemia-initiating cell (LIC) has an instructive role in determining the pathogenic outcome of the resulting cancer. Here, we have addressed our hypothesis by implementing mass spectrometry (MS)-based quantitative proteomics in combination with advanced functional assays to comprehensively characterize factors that are responsible for the differences in fetal and adult hematopoiesis in both a normal and a malignant setting. Our initial work resulted in the identification of numerous proteins and biological processes that distinguish fetal and adult LinSca-1+ cKit+ (LSK) HSPCs, with particularly strong differences between the two cell types in processes related to immune response, protection against reactive oxygen species (ROS) and proteolysis. We showed that fetal and adult HSPCs exhibit distinct responses to modulation of Type I interferon (IFN) signaling and neutrophil serine protease (NSP) activity. We subsequently investigated redox homeostasis in fetal and adult HSPCs, and showed that in line with the lower expression of proteins involved in antioxidant defense, the fetal HSPC proteome has a significantly higher oxidation level than the adult, and undergoes even further oxidation upon leukemic initiation. Our subsequent work focused on characterization of ontogenic proteomic and functional changes that occur in lineage-biased hematopoietic progenitor cells (HPCs). We revealed an intrinsically programmed difference in the lineage bias of fetal and adult lymphomyeloid multipotent progenitors (LMPPs), common lymphoid progenitors (CLPs) and granulocyte monocyte progenitors (GMPs), with the adult cells showing a considerably stronger myeloid potential compared to the fetal. The difference in myeloid potential was in part due to a significantly lower expression of Irf8 and a concomitant defect in production of mature monocytes in fetal relative to adult GMPs, which could be partially rescued by increasing Irf8 expression. Finally, we focused our investigations on leukemia driven by the MLL-ENL fusion oncogene, a translocation which represents a highly prevalent mutational event in in utero-derived infant acute lymphoblastic leukemia (ALL). We showed that fetal MLL-ENL-expressing LMPPs possess an intrinsic bias towards generation of immature lymphoid cells ex vivo. In a transplantation model, however, expression of MLL-ENL in fetal as well as adult LMPPs drives acute myeloid leukemia (AML) in adult recipients, suggesting that an interplay between intraand extracellular factors determines disease progression and outcome in MLLr leukemia. Molecularly, we found that MLL-ENL-mediated leukemia initiation is hallmarked by a differentiation arrest and metabolic reprogramming in fetal as well as adult cells. Remarkably, while expression of proteins associated with inflammation is increased in adult leukemic relative to wild-type (WT) LMPPs, such signatures instead appear to be suppressed in fetal-origin leukemia. Thus, we identified shared as well as ontogeny-specific features of MLL-ENL-mediated leukemogenesis which could potentially be exploited in the development of age-tailored anti-cancer therapies. Collectively, this thesis work has uncovered numerous novel ontogeny-specific molecular and functional features of fetal and adult HSPCs, both in normal development and in leukemia. The results from the included studies will aid in guiding future efforts aimed at developing novel strategies for prevention and treatment of pediatric and adult acute leukemia.