Compartmentalized Niche and Spatially Heterogenous Inflammatory Landscape Regulate Tet2+/-- Mediated Clonal Hematopoiesis

Background: Tet2 loss-of-function in hematopoietic stem cells (HSCs) is prevalent in clonal hematopoiesis (CH) and widely affects myeloid neoplasms. It is understood that both the mutation-associated mechanisms and cell-extrinsic factors, such as inflammation, lead to selective expansion of these mutant cells within the bone marrow (J. Exp Med. 218:e20201544 2021; Cell Stem Cell 28:1428, 2021). Despite the presence of systemic factors, our prior work showed that clonal expansion of activated HSCs is compartmentalized by bone marrow cavities with bone resorptive (remodeling) activities, whereas these cells remain solitary at sites predominated by bone formation (Nature 578:278, 2020). These results raised questions of whether the resident microenvironment cells within individual marrow cavities are heterogeneous, and whether anti-resorptive osteoporosis management impacts progression of pre-malignant clonal disorders. Microenvironment that regulates CH clonal development has not been studied in a spatially resolved manner. Elucidating this has promise for devising therapeutic targets leveraging the bone marrow microenvironment to intervene clonal disorders.

Methods: We recently reported that ultralow-dose irradiation enables engraftment and intravital tracking of disease initiating niches in clonal hematopoiesis (pre-print). Leveraging this approach, we transplanted 2x10⁶ Tet2^+/-/GFP⁺ whole bone marrow cells into 0.5 Gy-irradiated C57/BL6 adult mice and performed intravital imaging to determine the spatial landscape of CH clones in the calvarial bone marrow. To further assess niche factors from specific marrow cavities, we harvested the marrow cavities of growth hot spots (dense Tet2^+/- cells), cold spots (solitary Tet2^+/- cells) under image guidance. Subsequently, niche cells were profiled with single cell transcriptomics (10x Genomics). The effects of anti-resorptive bisphosphonates on the engraftment of Tet2^+/- cells and the resident niches were further evaluated.

Results: Consistent with prior work, intravital imaging showed that Tet2^+/-/CD34⁺ cell expansion was restricted within the marrow cavities undergoing active bone remodeling. Notably, modulating the compartment using anti-resorptive Zoledronic Acid (ZOL) led to a marked reduction of Tet2^+/- cells in the marrow. This is accompanied by a reduction of Tet2^+/- KLS (Kit⁺Lin^-Sca1⁺) stem and progenitor cells while the healthy counterpart remained unchanged. Further longitudinal tracking of resident niche cells revealed host CD206⁺ tissue resident macrophages (TRMs) proximal to Tet2^+/- throughout disease progression, suggesting participation of healthy microenvironment cells from early stage of clonal development. ZOL; however, disrupted this spatial association with CD206⁺ TRMs. Via spatial single cell transcriptomics, we showed that TRMs are transcriptionally distinct between cold and hot spot cavities. Gene ontology (GO) analysis revealed that macrophages exhibited immunosuppressive characteristics and significantly downregulated antigen presentation within the cold spots, even when compared to niche cells from a healthy bone marrow. In accordance, the CD34⁺ healthy progenitor populations in the cold spots are enriched with quiescent signatures, with significantly downregulated oxidative phosphorylation (FDR = 9.47E-11) and mitochondrial ATP synthesis (FDR = 9.63E-8).

Conclusion: This work identified compartmentalized niche defined by bone turnover and revealed a previously unrecognized heterogeneity in inflammatory landscape associated with Tet2^+/- clonal hematopoiesis and the potency of healthy progenitor cells. Validation via expanding the size or enhancing the niche factors within the cold spots may provide a therapeutic insight for intervening clonal disorders.