Tet2-associated inflammatory signaling alters healthy hematopoiesis with clonal expansion Free

Age-related clonal hematopoiesis (CH) is often driven by mutations in epigenetic regulators such as TET2. These mutations in epigenetic regulators predispose individuals to systemic inflammation, immune dysfunction, and the progression of malignant disease. Importantly, CH driven by TET2 mutations is associated with an increased risk of cardiovascular events and leukemia, underscoring its clinical significance. Statistically, individuals with TET2-driven CH have a higher incidence of these severe conditions. This highlights the urgent need to understand mechanisms involved. While much research has focused on how TET2-loss of function cells gain a competitive advantage, it has yet to be appreciated how healthy, non-mutated (mutant-exposed) hematopoietic stem and progenitor cells (HSPCs) are affected by the perturbed environment.

Prior work from our group has established that mutant-exposed HSPCs are altered upon exposure to leukemic cells. To address this, we used a competitive transplant model in which TET2 knockout (Tet2^KO) cells were intravenously injected into non-irradiated congenic hosts, allowing us to assess the impact of mutant cells on otherwise healthy, non-mutated (mutant-exposed) HSPCs. Unlike irradiated hosts, non-irradiated hosts preserve native niche signals, which are central to our investigation of microenvironmental effects. This approach ensures that the observed influences are a direct result of mutant cellular interactions, rather than artifacts from disrupted environments. Peripheral blood was analyzed monthly using complete blood counts, and flow cytometry to test for engraftment of chimeric mutant cells as well as the percentages of T cells, B cells, and myeloid cells. Mice were sacrificed, bone marrow was analyzed, and further assays were conducted up to 12-months post-transplant. Consistent with prior studies, Tet2^KO progenitors were myeloid biased, characterized by increased frequencies of MPP3 (myeloid-biased) cells and reduced MPP4 (lymphoid-biased) populations. Interestingly, frequencies of mutant-exposed MPP3s were also increased compared to controls, while frequencies of mutant-exposed MPP4s were decreased with frequencies being comparable to Tet2^KO cells. This signifies that environmental inflammation alone can replicate key phenotypes of Tet2 loss. However, downstream progenitors (GMPs) and mature myeloid cells (Ly6G⁺ neutrophils, CD11b⁺Ly6G⁻ monocytes) were most significantly expanded in Tet2^KO-derived cells only. Suggesting that TET2 loss confers maximal advantage at the point of myeloid commitment, not in upstream HSPCs.

To determine potential mechanisms contributing to these effects, we performed single-cell transcriptomics on Lineage⁻Sca-1⁺c-Kit⁺ (LSK, HSPCs) cells from chimeric control and Tet2^KO mice. We defined six HSPC clusters. Mutant-exposed MPP3s upregulate genes involved in NFĸB, STAT, and MAPK signaling, highlighting their heightened responsiveness to Tet2^KO inflammatory cues. The activation of the MAPK/NFĸB pathways in these MPP3s likely contributes to their reduced fitness, aligning both our biological and transcriptional data. In contrast, Tet2^KO MPP3s did not activate these regulatory pathways, implying an inflammatory ignorance that may drive their selective expansion. Functionally, we cultured MPP3 populations from each cohort with Tet2^KO serum or cytokines. While all cells exhibited altered differentiation, only Tet2^KO MPP3s showed increased myeloid output, reinforcing their unique response to this inflammatory signaling.

Collectively, we observed mutant-exposed and Tet2^KO cells share similar trends at the primitive progenitor level indicating they're responding similarly to the altered inflammatory environment. However, upon commitment to the myeloid lineage, mutant-exposed healthy cells are lost in favor of Tet2^KO cell expansion. Transcriptomic analysis indicates mutant-exposed cells are activating regulatory signaling genes. Therefore, Tet2^KO progenitors thrive by ignoring inflammatory constraints, whereas healthy cells are altered by them. These data suggest that the selective advantage of mutant clones may derive not only from cell-intrinsic mutations, but also from an environment that obstructs normal hematopoiesis.