Dissecting cell death, cellular senescence, and aging with genetic screens
Keywords: Genetics.

Forward genetic screens identifying long-lived mutants catalyzed a revolution in aging research. A decade later a second wave of research, spurred by the methodological development of unbiased genome-scale RNAi screens, pushed beyond the identification of individual longevity genes and began to describe genetic and molecular pathways that regulate aging. The discovery of CRISPR-Cas9 technology and its application in genetic screening has just begun to further advance the aging field. An evolving therapeutic strategy for age-related disease involves the selective ablation, using small molecule "senolytics", of senescent cells, which accumulate both with age and in certain pathologies. While a promising therapeutic approach, current senolytic agents are limited to local administration due to their side effects and potency. Genetic CRISPR screens could be applied to senescent cells and help identify improved senolytics, however current screening methodology is not optimized for studying genes which regulate cell death or for non-dividing cell types. Forward genetic screens have not been widely implemented for non-dividing, primary, post-mitotic, or quiescent cell types as many forward genetic screens often require cell sorting or growth-based phenotypes, both of which could be impossible in many cell types. Furthermore, millions of cells are required for sufficient coverage in traditional screens and this scale of experiment is prohibitively expensive and challenging with many specialized cell types. Here, I present new technology to improve the ability of genetic screening to study cell death and bring the power of CRISPR screening to disease-relevant cell types. This technique enables discovery of the molecular mechanisms of cell death subroutines with enhanced signal-to-noise ratios and could help identify drug targets for varied pathological states, such as cancer and neurodegeneration. I have applied this technology in the context of cellular senescence to discover genetic modifiers of senolytics, demonstrate a combination of senolytic drug targets which act synergistically, and provide proof-of-concept that this synergistic senolytic drug combination clears senescent cells in vivo to ameliorate age-related pathology. Chapter 2 introduces this optimized genetic screening methodology, termed "Death-seq", for modifiers of cell death and applies it in a genome-wide screen studying the senolytic agent ABT-263. Chapter 3 demonstrates the ability of Death-seq to find senolytic modifiers with other drugs beyond ABT-263, including drugs which induce negligible cell death on their own, to demonstrate the importance of the drug target SMAC. Chapter 4 details the fact that the BH3 mimetics ABT-263 and ABT-199 act synergistically with SMAC mimetics to selectively ablate senescent cells. Chapter 5 demonstrates that the combination of ABT-199 and the SMAC mimetic birinapant enables the clearance of senescent cells in vivo in mice systemically and ameliorates aspects of certain models of age-related pathology. Overall, these chapters demonstrate the ability of the new genetic screening methodology, Death-seq, to identify modifiers of cell death and show proof-of-concept for this method's ability to discover novel approaches in the context of cellular senescence with therapeutic potential for age-related disease