Replication Stress and Ribosome Biogenesis in Hematopoietic Stem Cell Aging

PROJECT SUMMARY/ABSTRACT Diseases of the elderly are an increasingly urgent societal problem due to the worldwide increases in lifespan. The aging of the hematopoietic system is caused by dysfunction in hematopoietic stem cells (HSC) and is characterized by anemia, thrombocytosis, and overproduction of myeloid cells at the expense of lymphopoiesis. Together these defects play a key role in the development of cardiovascular diseases, loss of adaptive immunity that impedes vaccination, and establishment of chronic systemic inflammation that damages tissue and contributes to frailty. HSC aging is conserved in mammals, with human and murine old HSCs (oHSC) both exhibiting reduced regenerative potential, genomic instability, epigenetic drift, metabolic rewiring, and altered cell-cell communication. Although these overt phenotypic features are widely understood to be significant characteristics of oHSCs, we still know little about their underlying molecular mechanisms and functional consequences. This gap in knowledge has hindered efforts to delay or reverse HSC aging at its root. Our lab identified replication stress as a potent driver of oHSC dysfunction and impaired regenerative potential. This is especially severe at fragile ribosomal DNA loci, leading to loss of ribosome biogenesis. This project aims to determine the functional consequences of reduced ribosome biogenesis for oHSCs, and to identify the programs that underly replication stress initiation with a goal to target them to restore oHSC function. Our preliminary data suggest that oHSCs are defective in their capacity for protein translation, even though their mitogenic signaling pathways are overactive. They also suggest chronic activation of the Nucleolar Stress Response (NSR) as a consequence of replication stress in oHSCs. Furthermore, we have evidence for epigenetic alterations and cell cycle transcriptional repression consistent with Mcm downregulation and replication stress initiation. In Aim 1, we will determine the extent of defective protein translation in quiescent and activated oHSCs using in vitro and in vivo approaches. We will also interrogate the signaling pathways driving defective protein translation in oHSCs focusing in particular on NSR activation using a mouse genetic approach. These experiments will establish how defective proteostasis contribute to HSC aging, and the connection between replication and nucleolar stress in driving oHSC impaired regeneration potential. In Aim 2, we will identify the transcription factors or cell cycle regulators responsible for replication stress initiation, and also uncover the epigenetic basis for this defect. We will then assess whether pharmocological tuning of specific epigenetic modifiers can restore oHSC function. These experiments will dissect the molecular underpinnings of replication stress and determine whether correcting this cell-intrinsic hallmark of HSC aging will improve oHSC regenerative potential. Altogether, our proposed investigations are promising avenues to better understand and treat HSC aging. They have exciting implications for identifying actionable targets for promoting HSC functional longevity, a logical strategy towards restoring blood and immune function in the elderly.