Chronic IL1β Exposure Enhances Self-Renewal Ability and Myeloid Priming in TET2 Deficient Stem and Progenitor Cells

Background: Clonal hematopoiesis (CH) increases risk for the development of hematological malignancy (HM). Patients with CH have high levels of circulatory cytokines (such as IL1b, IL6, TNFa) leading to increased inflammatory stress. Inflammation is associated with increased risk for the development of HM. However, how inflammation alters population dynamics of hematopoietic stem and progenitor cells (HSPCs) in CH, is not well understood. Here we modelled inflammation using the proinflammatory cytokine IL1β in TET2 mediated CH.

Methods and Results: To identify the impact of chronic IL1β exposure on HSPCs in CH, we used pan-hematopoietic Tet2 loss-of-function mice (VavCre Tet2^fl/fl, referred as Tet2 KO). We treated WT and Tet2 KO mice with IL1β for five weeks. Tet2 KO mice treated with IL1β exhibited splenomegaly and elevated neutrophils in the spleen and bone marrow (BM) compared to vehicle treated or WT mice. In a parallel study, we used a murine competitive transplantation model with a doxycycline inducible shRNA targeting Tet2 (shTet2) BM cells competing against WT. Similar to Tet2 KO, shTet2 cells were expended in the upon IL1β stimulation for six weeks compared to vehicle (85.6% vs 61.8%, p <0.05) with significant myeloid bias in PB, BM and spleen. Specifically, IL1β stimulation led to the significant expansion (3-4 fold) of ly6c^hi monocyte/macrophages in the BM and spleen of Tet2 KO mice relative to WT and with no change in anti-inflammatory ly6c^lo monocyte/macrophages in the spleen and 13-fold (p <0.001) reduction in the BM. Further, Myeloid biased MPP2/3 were elevated upon IL1β treatment relative to lymphoid biased MPP4s in shTet2 and Tet2 KO compared to WT mice. The ratio of myeloid biased GMPs to megakaryocyte/erythroid biased MEPs was increased within Tet2 KO mice in all conditions. Notably, LT-HSCs were elevated in frequency in the BM of both shTet2 (9.5-fold, p <0.01) and Tet2 KO (1.9-fold, p <0.05) relative to WT upon IL1β treatment. Colony formation assays with serial replating demonstrated IL1β further enhanced the self-renewal ability of Tet2 KO progenitor cells (LSK, Lin^-Sca1⁺ckit⁺) 10.6-fold by the quaternary plating (p <0.0001) over that of untreated Tet2 KO LSKs.

To identify the mechanisms by which IL1β promotes myeloid bias and self-renewal potential of Tet2 KO HSCs, we used scRNAseq and whole genome bisulfite sequencing (WGBS) analyses. scRNAseq identified that the signatures for inflammatory pathways and chronically stressed old HSCs were upregulated in Tet2 KO HSCs relative to WT at steady state and upon IL1β stimulation for 5 weeks in vivo. Additionally, seven gene sets and genes (Erg, Meis1, and Egr1) associated with enhanced self-renewal ability were upregulated in Tet2 KO HSCs relative to WT upon IL1β stimulation, whereas only a single gene set was significant at steady state. WGBS identified that IL1β stimulation for 5 weeks significantly reduced global methylation in WT LSKs, CMPs, and GMPs, but not in Tet2 KO progenitors. Using published ChIPseq data for 35 proteins, we identified 4 transcription factors known to drive terminal hematopoietic differentiation (ELF4, PU.1, CEBPB, JUN) among the top hypermethylated factors specifically in Tet2 KO LSKs relative to WT upon IL1β stimulation, thus reducing terminal differentiation.

Finally, to understand the therapeutic potential of IL1 signaling in CH, we examined the PB of aged Tet2 KO, WT, interleukin 1 receptor 1 knockout (Il1r1^-/-), and Il1r1^-/- Tet2 KO (double knockout, DKO) mice. We found DKO was protected from the myeloid expansion present in Tet2 KO mice. Accordingly, IL1-driven myeloid expansion is blocked by anakinra, a clinically approved IL1 antagonist, in a murine Tet2 KO model in vivo or TET2 edited human progenitors in a colony formation assay.

Conclusions: IL1β mediated chronic inflammatory stress promotes expansion of Tet2 KO HSCs, myeloid biased progenitors, pro-inflammatory monocytes and neutrophils. IL1β drastically increased self-renewal capacity of Tet2 KO HSPCs with potent upregulation for transcriptional signatures driving self-renewal and hypermethylation of transcription factor binding sites driving terminal differentiation to preserve the stem cell pool. Finally, genetic or pharmacological mitigation of IL1 signaling reduced myeloid expansion, providing strong evidence for therapeutic targeting of IL1 signaling to prevent or delay premalignant myeloid expansion.

No relevant conflicts of interest to declare.