Inflammatory responses in human hematopoietic stem cells: the effect of aging and clonal hematopoiesis

PROJECT SUMMARY Hematopoietic stem cells (HSC) in the bone marrow (BM) are responsible for the production of mature blood cells throughout life. These HSCs maintain a highly regulated balance between self-renewal and differentiation, allowing them to preserve the HSC pool while simultaneously responding to inflammatory stimuli. However, with both age and the acquisition of mutations in genes such as TET2, the ability of HSCs to regulate their response to inflammation is fundamentally altered, resulting in susceptibility to blood disorders such as acute myeloid leukemia. In murine models, the pro-inflammatory cytokine IL-1β has been linked to the progression of both age- related blood dysfunction and aberrant clonal expansion of TET2-mutated HSCs. However, our understanding of how age and TET2 mutations affect human HSC responses to pro-inflammatory cytokines such as IL-1β is limited, representing a major roadblock to identifying translatable targets for the prevention of associated blood disorders. This proposal addresses this key knowledge gap by examining the hypothesis that aging and TET2- mutations alter the engagement of inflammatory response pathways in human HSCs, resulting in lineage-biased differentiation and diminished clonal diversity. In Specific Aim 1, I will utilize human BM samples in both ex vivo models and in vivo humanized mouse models to investigate age-dependent functional responses to IL-1β. I will also examine the molecular underpinnings of age-dependent inflammatory responses by assessing engagement of NF-κB-dependent pathways using confocal microscopy, chromatin immunoprecipitation sequencing, and unbiased RNA- and ATAC-sequencing. In Specific Aim 2, I will use an innovative BM-on-a-chip, complemented by humanized mouse models, to determine how IL-1β promotes aberrant expansion of aged TET2-mutated HSCs. I will also examine how transcriptional regulatory networks in TET2-mutated HSCs are perturbed in response to inflammation using RNA-sequencing, and then validate the functional importance of these pathways using CRISPR-Cas9. Collectively, these aims will provide a comprehensive understanding of the functional and molecular consequences of aging and TET2-mutation on responses to IL-1β-induced inflammation, with the goal of revealing therapeutic targets to prevent associated blood disorders. Additionally, this proposal outlines a five- year career development plan that will enable my transition to a position as an independent physician-scientist. The research studies outlined in this proposal build upon my background in molecular and cell biology, as well as my clinical expertise in hematology, to provide the basis for independent investigations on how alterations in human HSCs and their microenvironment affect the progression of malignant hematopoiesis. I have assembled a world-renowned mentorship team with expertise in each of my training objectives and an intense commitment to my career development. The proposed studies, in combination with the strong mentorship and institutional support described in this proposal, provide the ideal foundation to launch an independent scientific career.