Role of inflammation in TET2 mediated clonal hematopoiesis in the context of bone marrow niche

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Clonal hematopoiesis (CH) occurs due to the accumulation of somatic mutations in hematopoietic stem and progenitor cells (HSPCs). The presence of CH increases the risk for the development of hematological malignancies by 12-fold and heart disease by more than 2-fold. TET2, a gene involved in regulation of cytosine methylation, is one of the most frequently mutated drivers of CH. Mounting evidence and our preliminary data shows that expansion of HSPCs with Tet2 loss-of-function (Tet2-KO) is strongly promoted by inflammation. Unfortunately, the underlying mechanisms driving expansion of Tet2-KO HSPCs, as well as the relative contribution of the niche, is not defined. My long-term goal is to understand which targetable pathways are regulated by inflammatory stress to promote CH. Identified factors could serve as early intervention and disease monitoring strategies. We have found in vivo that interleukin 1 beta (IL1β) promotes preleukemic myeloproliferation and expansion of Tet2-KO HSPCs over healthy cells. Furthermore, in vitro assessment shows that self-renewal ability of Tet2-KO HSPCs is significantly increased upon IL1β exposure. Interestingly, IL1β- mediated myeloid expansion of Tet2-KO HSPCs is also supported by non-hematopoietic bone marrow niche cells. Further IL1β alters the composition of the bone marrow stromal niche when Tet2-KO hematopoietic cells are present. To identify transcriptional changes impacting self-renewal and myeloid expansion of Tet2-KO HSPCs, single cell RNA sequencing of bone marrow derived Tet2-KO and healthy HSPCs treated with or without IL1β was performed. These data revealed upregulation of pathways and genes related to self-renewal, cytokine signaling, and myeloid differentiation in the IL1β treated Tet2-KO HSPCs relative to healthy HSPCs. Cumulatively these data led me to my overarching hypothesis that IL1β drives expansion of Tet2-KO HSPCs through the alteration of transcriptional and epigenetic signaling while impacting niche interactions with HSPCs. I will address my hypothesis by following two aims. Aim 1 will identify the mechanism driving expansion of Tet2-KO HSPCs in the context of IL1β induced chronic inflammation. I will utilize genetic and pharmacological inhibition approaches to identify the role of candidate genes impacting fitness of Tet2-KO HSPCs relative to healthy cells. Further, I will use rapid immunoprecipitation mass spectrometry of endogenous proteins (RIME) and chromatin immunoprecipitation (ChIP-seq) to identify how IL1β and TET2 alter methylation to promote expansion of Tet2-KO HSPCs. In Aim 2, I will determine how reprogramming of the bone marrow niche by IL1β impacts fitness of Tet2-KO HSPCs relative to healthy cells. Specifically, I will use cytometry by time of flight (CyTOF) and differentiation assays to characterize how IL1β alters the stromal component of the bone marrow niche. Finally, I will examine how niche cells in response to IL1β impacts Tet2-KO HSPCs expansion using in vitro coculture assays. Cumulatively, these studies will define the inflammation-mediated mechanisms driving TET2 CH, providing targets for early intervention and detection by reducing the fitness of TET2 HSPCs.