PROJECT SUMMARY Clonal hematopoiesis (CH) is a pre-malignant condition characterized by the clonal expansion of the mutated hematopoietic stem and progenitor cells (HSPCs). CH represents a significant public health concern, over 10% of individuals aged 70 and above carry CH mutations. This blood disorder predominantly affects older adults and elevates the risk of hematologic malignancies nearly 13 folds. As the US population projected to be markedly aged in the next three decades, the implications of CH will become increasingly significant. First being recognized as a distinct category of precursor myeloid disease state by WHO in 2022, there is not yet any FDA approved treatment toward CH, highlighting an urgent medical need. This project aims to investigate how inflammation, especially that originating form the non-hematopoietic cells within the bone marrow microenvironment, contributes to the CH progression. We have found Gasdermin D (GSDMD) loss-of-function in the non-hematopoietic stromal cells significantly mitigated the CH progression in mice. To further translate our findings from mice to humans, we have developed a novel human bone marrow organoid model. This model autonomously generates multi-lineage hematopoietic cells and non-hematopoietic stromal cells within a 3D vascular network, closely resembling the human bone marrow microenvironment. Notably, we generated GSDMD-/- organoids and engrafted them with patient-derived HSPCs carrying TET2 mutations, we observed a significant reduction in myeloid-biased hematopoiesis of the engrafted HSPCs with in GSDMD-/- organoids compared to WT organoids. Moving forward, we will employ spatial transcriptomic analysis coupled with single-cell RNA sequencing to uncover the mechanisms by which GSDMD influences cell-cell interactions in both mouse models and human organoid models. Ultimately, we plan to create a novel patient-derived xenograft mouse model by implanting the organoid into NSG immunodeficient mice, further advancing our ability to model human CH in vivo. The successful completion of this project will provide critical insights into the cellular and molecular mechanisms of the protective effect of GSDMD deficiency in the bone marrow microenvironment. It will lay the groundwork for potential therapeutic strategies targeting dysregulated inflammation in non- hematopoietic cells, serving as a stepping stone for my transition to independent investigations as a PI.

PROJECT NARRATIVE Clonal hematopoiesis (CH) is a pre-malignant condition that arises from accumulated mutations in hematopoietic stem cells, leading to increased risk of hematologic malignancies and posing a significant public health concern for aging populations. The role of GSDMD-induced inflammation in non- hematopoietic bone marrow stromal cells during CH progression remains to be studied. By utilizing state-of-the- art technologies, this research seeks to elucidate the mechanisms and develop therapeutic strategies targeting dysregulated inflammation, potentially paving the way for novel interventions to halt the progression of CH to myeloid malignancies.