Bone Marrow Stromal Cell Senescence Induced By Dnmt3a-Mutant Hematopoietic Stem and Progenitor Cells Accelerates Clonal Hematopoiesis and Progression to Leukemia

Abstract

Clonal hematopoiesis (CH) is commonly driven by mutation in the DNA methyltransferase DNMT3A. While DNMT3A catalyzes DNA methylation, the mechanism(s) by which DNMT3A mutations confer a clonal selective advantage to hematopoietic stem cells (HSCs), and how this can lead to development of acute myeloid leukemia (AML), is not completely understood. Non-hematopoietic cell types in the bone marrow (BM) microenvironment have recently emerged as key contributors in AML by creating a favorable environment for cell growth, including pro-inflammatory changes, accumulation of senescent cells, and the senescence-associated secretory phenotype (SASP). As these alterations have also been found in the context of normal aging, we hypothesized that they contribute to CH-mutant HSC expansion and initiation of AML. To investigate alterations in the BM microenvironment induced by Dnmt3a-mutant hematopoiesis, we transplanted Dnmt3a-mutant or control BM cells into wild-type recipient mice and performed single cell RNA-seq on hematopoietic and non-hematopoietic BM cell fractions. We identified a cellular senescence signature as being enriched in the BM stromal cell (BMSC) subsets Osteo-CAR and Adipo-CAR. We hypothesized that Dnmt3a-mutant hematopoietic stem and progenitor cells (HSPC) remodel the BM microenvironment by induction of senescence to favor their selective advantage. To test this, we developed an ex vivo co-culture system using primary murine BMSC and Dnmt3a-mutant HSPC. Primary BMSCs cultured with Dnmt3a-mutant HSPCs were found to have increased expression of senescence markers (P16, P21, b-galactosidase) and SASP (IL-6, IL-1a) compared to BMSC cultured with control wild-type (WT) HSPCs. This phenotype was similarly observed in BMSCs in vivo after Dnmt3a-mutant cells were transplanted into WT recipient mice, in addition to increased expression of the anti-apoptotic proteins BCL-2 and BCL-xL in BMSCs. We then pursued mechanistic experiments to reveal how Dnmt3a-mutant HSPCs induce senescence of BMSCs. Transwell assays supported that senescence induction was cell contact independent. Cytokine profiling of conditioned media from Dnmt3a-mutant HSPCs, and well as BM fluid of transplanted recipient mice, revealed elevated levels of the inflammatory cytokines IL-6, S100A8/9 and TNFα. We directly tested the effect of IL-6 on primary BMSC by culture with recombinant IL-6 alone and found that this was sufficient to increase b-galactosidase staining in BMSCs. To determine the extent to which deletion of senescent cells can abrogate Dnmt3a-mutant selective advantage and progression to hematologic malignancy, we used our inducible mouse model of Dnmt3a;Npm1-mutant AML (Loberg et al., Leukemia 2019). We transplanted cells carrying the Dnmt3a mutation into WT recipient mice, administered the senolytic Navitoclax or vehicle control, and then induced Npm1 mutation in vivo. We found that Navitoclax-treated mice had reduced peripheral blood myeloid cell expansion, a biomarker of Dnmt3a;Npm1-mutant transformation. Furthermore, Navitoclax-treated mice had reduced WBC and monocyte counts, reduced bone marrow engraftment, reduced myeloid colony-forming units (CFU), and maintained lower BMSC senescence. Together, we report that Dnmt3a-mutant HSPCs induce senescence of BMSCs through production of the pro-inflammatory cytokine IL-6. Depletion of these senescent cells using Navitoclax reduces myeloproliferation and transformation of Dnmt3a-mutant HSPCs to AML. We suggest that SASP may be used as a biomarker of transformation risk in individuals with CH, and that targeting the induced senescent BM microenvironment may be used prophylactically to prevent transformation to AML.