Session: 503. Clonal Hematopoiesis, Aging and Inflammation: Poster III
Hematology Disease Topics & Pathways:
Research, Fundamental Science, CHIP, hematopoiesis, Diseases, Biological Processes
To better dissect heterogeneity across primitive and mature cell types driven by TET2 loss, we performed scRNAseq on Tet2KO and Tet2WT whole bone marrow (WBM) and sorted LSK cells. We identified 18 transcriptionally distinct cell subsets in a combined analysis of WBM. Tet2KO progenitor 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 Tet2KO 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 Tet2KO 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 Tet2KO BM. FlowSOM analysis identified clusters corresponding to cKIT+CD34+ myeloid progenitors, a Ly6C+CD34+cKIT+ progenitor, and Ly6Chi monocyte clusters were enriched in Tet2KO 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 Tet2KO 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 Tet2KO 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 Tet2KO Hoxb8-ER cells. We found that the AP-1 inhibitor T5224 dose dependently reduced the expansion of Tet2KO Hoxb8 progenitor cells in base media and inhibited the IL-6 mediated expansion of Tet2KO 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 Tet2KO cells, while inhibition of AP-1 reduces fitness of cells, suggesting a possible therapeutic strategy for TET2-mutated myeloid malignancy.
Disclosures: Ferrell: Novartis: Research Funding.
See more of: Oral and Poster Abstracts