Variation in Mesenchymal KITL/SCF and IGF1 Expression at Middle Age Underlies Steady-State Hematopoietic Stem Cell Aging.

Intrinsic molecular programs and extrinsic factors including pro-inflammatory molecules are understood to regulate hematopoietic aging. This is based on foundational studies using genetic perturbation to evaluate causality. However, individual organisms exhibit natural variation in hematopoietic aging phenotypes and the molecular basis of this heterogeneity is poorly understood. Here, we generated individual single cell transcriptomic profiles of hematopoietic and non-hematopoietic cell types in five young adult and nine middle-aged C57BL/6J female mice, providing a web-accessible transcriptomic resource for the field. Among all assessed cell types, hematopoietic stem cells (HSCs) exhibited the greatest phenotypic variation in expansion among individual middle-aged mice. We computationally pooled samples to define modules representing the molecular signatures of middle-aged HSCs and interrogated which extrinsic regulatory cell types and factors would predict variance in these signatures between individual middle-aged mice. Decline in signaling mediated by ADIPOQ, KITL and IGF1 from mesenchymal stromal cells (MSCs) was predicted to have the greatest transcriptional impact on middle-aged HSCs, as opposed to signaling mediated by endothelial cells or mature hematopoietic cell types. In individual middle-aged mice, lower expression of Kitl and Igf1 in MSCs highly correlated with reduced lymphoid lineage commitment of HSCs and increased signatures of differentiation-inactive HSCs. These signatures were independent of expression of aging-associated pro-inflammatory cytokines including IL1, IL6, TNF and RANTES. In sum, we find that Kitl and Igf1 expression are co-regulated and variable between individual mice at middle age and expression of these factors is predictive of HSC activation and lymphoid commitment independently of inflammation.

Mesenchymal Stromal Cell Senescence Induced by Dnmt3a -Mutant Hematopoietic Cells is a Targetable Mechanism Driving Clonal Hematopoiesis and Initiation of Hematologic Malignancy.

Clonal hematopoiesis (CH) can predispose to blood cancers due to enhanced fitness of mutant hematopoietic stem and progenitor cells (HSPCs), but the mechanisms driving this progression are not understood. We hypothesized that malignant progression is related to microenvironment-remodelling properties of CH-mutant HSPCs. Single-cell transcriptomic profiling of the bone marrow microenvironment in Dnmt3a R878H/+ mice revealed signatures of cellular senescence in mesenchymal stromal cells (MSCs). Dnmt3a R878H/+ HSPCs caused MSCs to upregulate the senescence markers SA-ß-gal, BCL-2, BCL-xL, Cdkn1a (p21) and Cdkn2a (p16), ex vivo and in vivo . This effect was cell contact-independent and can be replicated by IL-6 or TNFα, which are produced by Dnmt3a R878H/+ HSPCs. Depletion of senescent MSCs in vivo reduced the fitness of Dnmt3a R878H/+ hematopoietic cells and the progression of CH to myeloid neoplasms using a sequentially inducible Dnmt3a ; Npm1 -mutant model. Thus, Dnmt3a -mutant HSPCs reprogram their microenvironment via senescence induction, creating a self-reinforcing niche favoring fitness and malignant progression. Statement of Significance: Mesenchymal stromal cell senescence induced by Dnmt3a -mutant hematopoietic stem and progenitor cells drives clonal hematopoiesis and initiation of hematologic malignancy.

Transcriptional and functional consequences of Oncostatin M signaling on young Dnmt3a-mutant hematopoietic stem cells.

Age-associated clonal hematopoiesis (CH) occurs due to somatic mutations accrued in hematopoietic stem cells (HSCs) that confer a selective growth advantage in the context of aging. The mechanisms by which CH-mutant HSCs gain this advantage with aging are not comprehensively understood. Using unbiased transcriptomic approaches, we identified Oncostatin M (OSM) signaling as a candidate contributor to age-related Dnmt3a-mutant CH. We found that Dnmt3a-mutant HSCs from young adult mice (3-6 months old) subjected to acute OSM stimulation do not demonstrate altered proliferation, apoptosis, hematopoietic engraftment, or myeloid differentiation. Dnmt3a-mutant HSCs from young mice do transcriptionally upregulate an inflammatory cytokine network in response to acute in vitro OSM stimulation as evidenced by significant upregulation of the genes encoding IL-6, IL-1ß, and TNFα. OSM-stimulated Dnmt3a-mutant HSCs also demonstrate upregulation of the anti-inflammatory genes Socs3, Atf3, and Nr4a1. In the context of an aged bone marrow (BM) microenvironment, Dnmt3a-mutant HSCs upregulate proinflammatory genes but not the anti-inflammatory genes Socs3, Atf3, and Nr4a1. The results from our studies suggest that aging may exhaust the regulatory mechanisms that HSCs employ to resolve inflammatory states in response to factors such as OSM.

Oncostatin M is a Master Regulator of an Inflammatory Network in Dnmt3a -Mutant Hematopoietic Stem Cells.

Age-associated clonal hematopoiesis (CH) occurs due to somatic mutations accrued in hematopoietic stem cells (HSCs) that confer a selective advantage in the context of aging. The mechanisms by which CH-mutant HSCs gain this advantage with aging are not comprehensively understood. Using unbiased transcriptomic approaches, we identify Oncostatin M (OSM) signaling as a candidate contributor to aging-driven Dnmt3a -mutant CH. We find that Dnmt3a -mutant HSCs from young mice do not functionally respond to acute OSM stimulation with respect to proliferation, apoptosis, hematopoietic engraftment, or myeloid differentiation. However, young Dnmt3a -mutant HSCs transcriptionally upregulate an inflammatory cytokine network in response to acute OSM stimulation including genes encoding IL-6, IL-1ß and TNFα. In addition, OSM-stimulated Dnmt3a -mutant HSCs upregulate the anti-inflammatory genes Socs3, Atf3 and Nr4a1 , creating a negative feedback loop limiting sustained activation of the inflammatory network. In the context of an aged bone marrow (BM) microenvironment with chronically elevated levels of OSM, Dnmt3a -mutant HSCs upregulate pro-inflammatory genes but do not upregulate Socs3, Atf3 and Nr4a1 . Together, our work suggests that chronic inflammation with aging exhausts the regulatory mechanisms in young CH-mutant HSCs that resolve inflammatory states, and that OSM is a master regulator of an inflammatory network that contributes to age-associated CH.

Distinct Tumor Necrosis Factor Alpha Receptors Dictate Stem Cell Fitness versus Lineage Output in Dnmt3a-Mutant Clonal Hematopoiesis.

Clonal hematopoiesis resulting from the enhanced fitness of mutant hematopoietic stem cells (HSC) associates with both favorable and unfavorable health outcomes related to the types of mature mutant blood cells produced, but how this lineage output is regulated is unclear. Using a mouse model of a clonal hematopoiesis-associated mutation, DNMT3AR882/+ (Dnmt3aR878H/+), we found that aging-induced TNFα signaling promoted the selective advantage of mutant HSCs and stimulated the production of mutant B lymphoid cells. The genetic loss of the TNFα receptor TNFR1 ablated the selective advantage of mutant HSCs without altering their lineage output, whereas the loss of TNFR2 resulted in the overproduction of mutant myeloid cells without altering HSC fitness. These results nominate TNFR1 as a target to reduce clonal hematopoiesis and the risk of associated diseases and support a model in which clone size and mature blood lineage production can be independently controlled to modulate favorable and unfavorable clonal hematopoiesis outcomes. SIGNIFICANCE: Through the identification and dissection of TNFα signaling as a key driver of murine Dnmt3a-mutant hematopoiesis, we report the discovery that clone size and production of specific mature blood cell types can be independently regulated. See related commentary by Niño and Pietras, p. 2724. This article is highlighted in the In This Issue feature, p. 2711.