The role of the Dnmt3aR882H mutation in the evolution of clonal haematopoiesis.

Abstract

DNMT3A mutations are very frequent events in clonal haematopoiesis of indeter- minate potential (CHIP), and considered one of the earliest genetic events during the development of haematopoietic malignancies. Individuals with CHIP, in spite of having a normal full blood count are at increased risk of cardiovascular and pulmonary diseases as well as haematological neoplasms. Somatic mutations in DNMT3A are drivers of CHIP, but decades may elapse between the acquisition of a mutation and CHIP. Besides, only a minority of these individuals will go onto developing a haematological cancer, this wide interindividual heterogeneity suggests that environmental factors influence clonal expansion. It is in particular emerging, that individuals with CHIP have a proinflamma- tory phenotype, additionally, chronic inflammation is typically considered deleterious to normal haematopoiesis, as it leads to haematopoietic stem cells (HSCs) exhaustion. R882H Using a conditional knock-in mouse model heterozygous for Dnmt3a mon DNMT3A mutation, we have assessed the responses of Long-Term Haematopoietic Stem Cells (LT-HSCs) to inflammatory "stress" in vitro and in vivo. We have also em- ployed bulk LT-HSCs genomics studies to interrogate differences in the transcriptional landscape in response to inflammation. We have demonstrated that Dnmt3a-mutant LT- HSCs have a globally abnormal response to inflammation, characterised by faster exit from quiescence and subsequent hyper-proliferation. Interestingly, as well as a larger differentiated output, Dnmt3aR882H HSCs were also better preserving their progenitor compartment and their self-renewal ability compared to wild-type HSCs exposed to the same stimulus, giving some insight into potential mechanisms that lead to clonal expan- sion within an inflammatory microenvironment. We linked this phenotype to the reduced expression of two TP53 target genes: Niam/Tbrg1, involved in the TP53-mediated activation of the p21 pathway that leads to G1 growth arrest upon exposure to "stress", and Necdin/Ndn a regulator of LT-HSCs quiescence. We then went on to demonstrate that TP53 and DNMT3A might function redundantly within the same functional pathway, and that Dnmt3a loss-of-function can lead to re- duced stem cell quiescence in vivo, and increased genomic instability when Dnmt3aR882H progenitors are challenged with genotoxic “stressors”. Interestingly, in spite of accumu- lating more DNA damage, Dnmt3aR882H cells did not lose clonogenic ability and showed similar viability to wild-type cells, suggesting that these cells are more tolerant to the accumulation of DNA damage. Subtle changes of this nature are likely to play an im- portant role in a phenomenon that develops over the lifetime of an individual, and could create a permissive state for the retention and accumulation of subsequent mutations, eventually leading to transformation.