3010 – THE IMPACT OF AGING AND INFLAMMATORY STRESS ON GENOME STABILITY IN HEMATOPOIETIC STEM CELLS

The acquisition of mutations within the genome of hematopoietic stem cells (HSCs) is of particular importance as this is a likely driver of malignant transformation for many leukemias, as well as a hallmark of aging. Importantly, the study of normal physiologic mediators of HSC mutation acquisition is an underdeveloped area. We have developed a methodology that allows the assessment of clonal mutations occurring within single HSCs in vivo, which we then validated by sequencing a clone to >90X coverage followed by down-sampling. Importantly, from the perspective of establishing the absolute mutation frequency of an individual HSC, we established that the number of mutations called per cell increases in an almost linear fashion with increasing coverage, until a plateau is reached around 30X coverage. We also developed an optimal bioinformatics pipeline for data analysis, which demonstrated a much-improved capacity for discerning true positive and false positive mutations at low coverage, when compared to previously published methodologies. Using this optimized sequencing pipeline, we collected and sequenced HSC clones from young and aged mice, as well as those exposed to stress agonists. We additionally employed a genetic label-retention system to segregate dormant and actively cycling HSCs in order to assess whether mutations are predominantly acquired during replication. Genomic coverage ranged from 30-40X. As seen in humans, we found a progressive increase in mutation burden with age within the murine HSC compartment, corresponding to a rate of ∼38 SNVs per month. These HSCs had a mutational signature corresponding to that observed in aged human tissues. In contrast to previous reports, data from the label-retention shows that this increase correlated with HSC replication, as dormant aged HSCs had similar mutation burdens to young HSCs. We envisage that these findings will be an important step towards interrogating whether replication stress is a biologically relevant driver of genome instability in HSCs.