Multi-Omics Analysis Reveals That TET2 Loss Epigenetically Primes Monocytes for Inflammatory Responses Via AP-1 Signaling

Abstract

Loss of function mutations in TET2 are common in clonal hematopoiesis (CH) and myelodysplastic syndromes (MDS). Increased inflammation and stem cell capacity have been reported in Tet2 KO mouse models, but molecular mechanisms and cell type-specific contributions to explain this phenotype are lacking. To create models of CH-like TET2 deficiency, we transplanted Mx1-CRE Tet2 fl/fl (Tet2 KO) or Tet2 fl/fl (both CD45.1 + ) in CD45.2 + recipientsfollowing radiation-free conditioning with ACK2 (anti-cKit) antibody injection. Six months post-transplant, we flow sorted CD45.1 + Tet2 KO and Tet2 fl/fl Lin -Sca-1 +cKit + (LSK) cells, granulocyte-monocyte progenitors (GMP), and monocytes for RNA and assay for transposase-accessible chromatin (ATAC) sequencing. Differential gene expression analysis identified 902 (468 up, 434 down) differentially expressed genes (DEGs) in LSK, 134 (95 up, 39 down) in GMP, and 474 (125 up, 349 down) in monocytes (p adj<0.05). Gene set enrichment analysis (GSEA) identified that the top Hallmark gene sets in Tet2 KOLSK cells were related to MYC targets, oxidative phosphorylation, and E2F targets. In contrast, GMP and monocytes were most enriched for IFN-alpha and gamma gene sets along with IL6/JAK/STAT3 signaling. ATACseq revealed increased accessibility of myeloid-related transcription factor (TF) binding motifs in Tet2 KOLSK cells (CEBPA, PU.1-IRF(ETS:IRF), and AP-1 TF FOSL2(bZIP)), while prediction of cis-regulating regions via GREAT identified downregulated peaks associated with stem cell maintenance and upregulated peaks associated with myeloid mediated immunity. Peaks upregulated in Tet2 KOGMP cells were enriched for the motifs recognized by CEBPE, ISRE(IRF)) and the AP-1 TF JUND, while upregulated peaks in monocytes were enriched for RUNX1 and CEBPA motifs, as well as AP-1 TF FOS::JUNB. GREAT analysis in GMPs identified cis-regulatory regions related to myeloid mediated immunity, while monocytes possessed those associated with biotic stimuli. To better dissect heterogeneity across primitive and mature cell types driven by TET2 loss, we performed scRNAseq on Tet2 KO and Tet2 WT wholebone marrow (WBM) and sorted LSK cells. We identified 18 transcriptionally distinct cell subsets in a combined analysis of WBM. Tet2 KOprogenitor and myeloid subsets were all enriched for gene sets related to IFN-alpha and gamma and immune signaling. Interestingly, primitive and cycling HSPCs were also enriched for MYC target and oxidative phosphorylation gene sets, while these were negatively enriched for GMP, monocyte progenitor, and monocyte Tet2 KO subsets. Further, the IL-6/JAK/STAT3 signaling gene set was enriched in later myeloid cell types, but not in early or cycling HSPCs. These observations led us to hypothesize that Tet2 KO BM cell populations have differentially enhanced phosphoprotein signaling responses to key cytokines. We used a 36-antibody mass cytometry panel to perform deep immunophenotyping and signaling response studies in Tet2 KO BM. FlowSOM analysis identified clusters corresponding to cKIT +CD34 + myeloid progenitors, a Ly6C +CD34 +cKIT + progenitor, and Ly6Chi monocyte clusters were enriched in Tet2 KO BM, while manual gating revealed expansion of LSK, MPP3, and monocytes. We found that activation of pSTAT3 and pSTAT1, in response to either IL-6 or IFNγ, respectively, were significantly higher in Tet2 KO Ly6Chi monocytes, while the more primitive Ly6C +CD34 +cKIT + progenitors demonstrated only an increased response to IFNγ at pSTAT1. Further, prolonged exposure to IL-6 in immortalized Tet2 KO Hoxb8-ER GMP-like cells led to significantly higher levels of pSTAT3 over 4 hours. Since AP-1 TFs are drivers of inflammation and were enriched in our ATACseq dataset, we tested the effects of an AP-1 inhibitor on the Tet2 KO Hoxb8-ER cells. We found that the AP-1 inhibitor T5224 dose dependently reduced the expansion of Tet2 KO Hoxb8 progenitor cells in base media and inhibited the IL-6 mediated expansion of Tet2 KO Hoxb8 cells. Our data show that TET2 loss rewires BM HSPCs towards an inflammatory, myeloid phenotype. Starting at the GMP stage, we see increases in phosphoprotein responses to IFNγ, and then the addition of hypersensitivity to IL-6 in monocytes. TET2 loss leads to an increase in accessible AP-1 motifs in Tet2 KO cells, while inhibition of AP-1 reduces fitness of cells, suggesting a possible therapeutic strategy for TET2-mutated myeloid malignancy.