Cell-Extrinsic Stressors from the Aging Bone Marrow (BM) Microenvironment Promote Dnmt3a-Mutant Clonal Hematopoiesis

During aging, intrinsic and extrinsic stressors are thought to contribute to clonal expansion of HSCs carrying somatic mutations, leading to clonal hematopoiesis (CH). While CH is benign in the majority of individuals, the presence of CH is associated with an increased risk of developing hematological malignancies. Therefore, it is critical to understand the mechanisms by which, and extent to which, aging-associated stressors cause CH to distinguish benign from high-risk CH and to develop strategies to prevent progression of CH to hematological malignancy.

We recently reported an inducible mouse model of a common hotspot mutation in DNA methyltransferase 3A found in CH and AML (Dnmt3a^R878H). This mutation increases long-term HSC (LT-HSC) frequency and competitive advantage upon transplant that accumulates with aging (Loberg et al. Leukemia 2019). These findings are consistent with previous work showing that Dnmt3aknockoutdrives LT-HSC expansion and can immortalize HSCs by blocking their differentiation(Challen et al. Nat Genet 2012; Jeong et al. Cell Reports 2018). Current definitions of CH in humans, including CH of indeterminate potential (CHIP), age-related clonal hematopoiesis (ARCH), and idiopathic cytopenias of undetermined significance (ICUS), typically define variant allele frequency (VAF) in unfractionated peripheral blood (PB) or bone marrow (BM) samples. This leads to a paradox; if CH-driving somatic mutations cause impaired or blocked differentiation of LT-HSCs, would they not be expected to be observed at high VAF in LT-HSCs but at low VAF in mature hematopoietic cells? Here, we propose a model that CH detected in unfractionated PB or BM increases with aging due to HSC-extrinsic stressors that overcome impaired differentiation of Dnmt3a-mutant HSCs.

To test this concept, we transplanted BM from young Dnmt3a^R878H/+ mice into young and aged congenic recipient mice. We observed that transplant into aged recipient mice accelerated the expansion of phenotypically-defined Dnmt3a^R878H/+ short-term (ST)-HSCs as well as their progeny including multipotent progenitor MPP3 cells. To determine the mechanisms by which these populations were expanded in aged recipients, we performed global transcriptome analysis of Dnmt3a^R878H/+ LT-HSCs re-isolated from aged and young recipient mice. Dnmt3a^R878H/+ LT-HSCs re-isolated from an aged BM microenvironment were found to upregulate gene signatures associated with pro-myeloid differentiation, immune response and immune system processes while downregulating gene signatures associated with chromatin organization and cell cycle regulation. Together, these data indicate that HSC-extrinsic alterations in the aged BM microenvironment can overcome impaired differentiation and promote myeloid differentiation of Dnmt3a-mutant LT-HSCs.

To identify candidate factors in the aged BM microenvironment, we analyzed our RNA-seq data using Ingenuity Pathway Analysis (IPA) for upstream regulators predicted to elicit the observed transcriptional alterations. This analysis identified increased TNF-α and M-CSF-mediated signaling caused by the aged BM microenvironment. We confirmed that TNF-α and M-CSF are increased in the local BM microenvironment of aged animals using ELISA analysis of BM fluid. To directly test the effect of TNF-α and M-CSF on Dnmt3a^R878H/+ LT-HSCs, we utilized ex vivo culture systems. We found that both TNF-α and M-CSF overcome the Dnmt3a^R878H/+ LT-HSC differentiation block and the resultant LT-HSC expansion phenotype in favor of pro-myeloid differentiation, compared to WT LT-HSCs.

Together, these data support that HSC-extrinsic alterations in the aged BM microenvironment provide altered selection pressure and stressors that differentially impact wild-type and CH-mutant HSC clones, driving differentiation and detectable clonal expansion within mature hematopoietic cell compartments. These mechanisms represent attractive targets to prevent clonal hematopoietic expansion and progression of CH to hematological malignancy.