Session: 503. Clonal Hematopoiesis, Aging and Inflammation: Insights from Animal Models
Hematology Disease Topics & Pathways:
Research, Fundamental Science, Translational Research
If the apoptotic resistance was merely due to delayed cell cycle activation from quiescence, proliferative activation should normalize the apoptotic levels of IFNg-exposed Dnmt3a-mutant HSCs. To test this, we induced HSCs into cell cycle by injecting 5-fluorouracil (5FU) into control (Vav-cre-; Dnmt3af/f) and mice lacking Dnmt3a in hematopoietic cells (3aKO, Vav-cre+; Dnmt3af/f). Kinetic analysis by BrdU labeling revealed that HSCs reached proliferation peak 8-days post-5FU treatment (>90% BrdU+). To capture the maximal number of proliferative HSCs, we injected PBS or recombinant mouse IFNg into 5FU-treated control and 3aKO mice on day 7 and 8. Phenotypic HSCs (Lin-cKit+EPCR+CD150+CD48-) were purified 2h after the second injection and analyzed for apoptosis. Cycling control HSCs were significantly more apoptotic upon IFNg exposure, while cell-cycle activated 3aKO HSCs remained insensitive to IFNg-primed apoptosis. Functional validation by colony formation assay showed IFNg-exposed cycling control HSCs formed significantly fewer colonies, while 3aKO HSCs gave rise to significantly more colonies regardless of exposure. These data suggest that Dnmt3a mutation may render HSCs resistant to IFNg-primed apoptosis via cell-cycle independent mechanisms. It has been documented that inflammation causes HSC apoptosis by inducing DNA damage through various mechanisms. Thus, the apoptotic resistance observed in Dnmt3a mutant HSCs could be due to 1) resistance to inflammation-induced DNA damage or 2) enhanced resolution of DNA damage.
To test if Dnmt3a mutation renders HSCs resistant to IFNg-primed DNA damage, neutral comet assay was performed using PBS or IFNg-exposed quiescent HSCs. Interestingly, both genotypes showed increased DNA double-strand breaks (DSBs) upon IFNg exposure, suggesting that quiescent Dnmt3a-mutant HSCs are susceptible to IFNg-primed DSBs. DNA repair is one mechanism enabling DSB resolution, and is functionally active in proliferative HSCs. Thus, we performed neutral comet assay on cycling HSCs to quantify DSBs following PBS/IFNg exposure. Expectedly, IFNg significantly increased DSBs in cycling control but not in 3aKO HSCs, suggesting Dnmt3a loss may endow HSCs with augmented DNA repair. Similarly, when DNA damage was determined in control and Dnmt3a-knockout (3aKO) murine myeloblast cells (32D) 4h and 24h post-IFNg exposure, a comparable increase in DNA damage was observed 4h post-exposure in both genotypes. Interestingly, DNA damage levels were significantly reduced 24h later only in the 3aKO cells. This data paralleled HSC data, implying that Dnmt3a ablation facilitates augmented DNA repair. DSBs are repaired primarily via three universal yet distinct pathways, homologous recombination (HR), non-homologous end joining (NHEJ), and alternative NHEJ. To investigate how Dnmt3a loss facilitates enhanced DNA repair in response to IFNg exposure, we performed a DNA repair reporter assay in 32D genotypes 24h after IFNg exposure. The plasmid repair reporter assay was built on the detection of a green fluorescent protein (GFP) expression, which is otherwise disrupted by the insertion of I-SceI restriction enzyme recognition sites. After cutting with I-SceI, GFP can only be expressed after specific repair by one of the three pathways. Results showed Dnmt3a loss specifically enhanced the NHEJ pathways upon IFNg exposure, while other pathways were comparable between genotypes and treatments.
In summary, our results demonstrated that Dnmt3a loss protects cell-cycle activated HSCs from inflammation-primed DSBs and apoptosis via augmented NHEJ as one mechanism. Future studies will investigate the molecular mechanisms underlying the enhanced NHEJ in Dnmt3a-mutant HSCs in response to chronic inflammation.
Disclosures: Challen: Incyte: Consultancy, Other: Sponsored Research agreements.
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