Dissecting and engineering reversible cell cycle states

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7. PROJECT SUMMARY/ABSTRACT Numerous diseases across organ systems as well as the deleterious effects of aging are attributable to the loss of terminally differentiated cells that facilitate organ function. In these cases, there is no active stem cell population capable of generating new functional cells. Moreover, any functional cells that remain cannot replace lost cells as the former have permanently exited the cell cycle as part of their differentiation process. A means of making terminally differentiated cells re-enter the cell cycle would dramatically alleviate and potentially cure these disease states. In principle, cellular differentiation does not necessitate cell cycle exit. Indeed some differentiated cell types, such as hepatocytes and lymphocytes, retain the ability to re-enter the cell cycle. These cell types are considered to be quiescent rather than terminally differentiated. Some of the extracellular signals that drive quiescent cells back into the cell cycle as well as the pathway for cell cycle entry, the Cyclin D-Cdk4,6 pathway, have been defined. However, it is unclear what allows a quiescent cell to receive these signals and reactivate the Cyclin D-Cdk4,6 pathway while a terminally differentiated cell cannot do so. As such, the difference between terminally differentiated cells and quiescent cells is a functional classification lacking a molecular explanation. Although mammalian cell culture models have provided tractable systems for investigating quiescence, they have failed to recapitulate the complexity of cell cycle regulation in tissues. There is therefore a need to study quiescence in an organismal context in order to provide a comprehensive understanding of this state and facilitate its translation to regenerative medicine. This project will establish the mouse liver as a tractable physiologic system to understand the reversibility of the quiescent state ultimately in order to confer this ability to terminally differentiated cells and enable their proliferation in the setting of disease. The approach will consider two non-mutually exclusive means of poising the Cyclin D- Cdk4,6 pathway for reactivation: direct modulation of the pathway itself or involvement of a factor extrinsic to this cascade. Aim 1 will determine whether there are functionally relevant differences in the activity of Cyclin D- Cdk4,6 pathway components in quiescent hepatocytes compared to other cell cycle states in tissues. Aim 2 will establish the first genome-wide, loss-of-function screen in the liver to identify any genes outside of this pathway that are required for the reversibility of quiescence. Finally, aim 3 will define conserved molecular features of quiescence by determining which features identified in aims 1 and 2 are also required for the reversibility of this state in lymphocytes. Together, these experiments will elucidate the core molecular signature of quiescence and thus the distinction between quiescence and terminal differentiation in tissues. Core factors would then be introduced into terminally differentiated cells and evaluated for their ability to confer proliferative capacity. These findings will not only fill a critical gap in our understanding of the mammalian cell cycle but also unlock a novel means of treating myriad diseases currently lacking effective therapy.