Investigating Innate Immune Signaling in Models of TET2 Deficient Hematopoiesis

Abstract

Loss of function mutations in the gene Tet methylcytosine dioxygenase 2 (TET2) are common in clonal hematopoiesis (CH) and myeloid malignancies such as myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Loss of TET2 function leads to hypermethylation of DNA and biases hematopoietic stem and progenitor cells (HSPCs) towards the myeloid lineage and enhances stem cell self-renewal. TET2 deficiency also exacerbates the response of hematopoietic cells to inflammatory stimulation in a cell-type specific manner, with LPS driving expansion of Tet2KO HSPCs and causing more differentiated macrophages to produce higher levels of inflammatory cytokines such as IL-1and IL-6. While the effects of TET2 loss on stem cell self-renewal and inflammation are well characterized, data on the role of TET2 in driving cell-type specific cytokine responses across the hematopoietic hierarchy are lacking. Here, I investigate cell-type specific effects of Tet2KO on inflammatory cytokine exposure using a wide range of modalities. In Chapter 2, I show with mass cytometry and flow cytometry that Tet2KO myeloid progenitors and monocytes have enhanced STAT1 phosphorylation in response to IFN and elevated STAT1 pathway output that can be targeted with the JAK/STAT inhibitor, ruxolitinib. In Chapter 3, RNA-seq showed that Tet2KO myeloid progenitor cells respond to IL-1 by upregulating transcription factors involved in proliferation/differentiation as well as inflammatory cytokines, while Mature Tet2KO cells produce copious amounts of cytokine. Both effects were ablated by inhibiting NFB signaling. In Chapter 4, I used PROseq to profile nascent transcription in response to IFNin Tet2KO HSPCs. Here, Tet2KO led to upregulation of several enhancers involved in inflammation and differentiation. Analysis of gene body transcription revealed Tet2KO HSPCs express lower levels of inflammation linked molecules and myeloid driving transcription factors, identifying a potential mechanism for the enhanced self-renewal capacity of Tet2KO HSPCs. Future work will focus on probing both the perturbed enhancer expression of dysregulated gene body transcription to better understand the effects of Tet2KO on HSPC self-renewal.