Cellular senescence is believed to be a key driver of several age-related diseases, including COPD and IPF. Therefore, senothereapeutics represent an attractive area of research with potential for future therapies against these debilitating diseases. To identify novel targets of senescence in an unbiased manner, we set up an arrayed whole genome CRISPR knock-out screen in primary human lung fibroblasts (NHLFs) assessing etoposide-induced nuclear p21 expression as a marker of senescence using confocal microscopy. The NHLF screen achieved a hit rate of 1.6%. To identify genes that also modulate senescence in a cell model relevant to COPD, we followed-up the top 250 hits from the NHLF screen with a validation screen in small airway epithelial cells (SAECs). Again, the assay used etoposide-induced nuclear p21 expression as well as modulated morphology endpoints such as nuclear- and cellular area, all of which are characteristics of senescent cells.

The SAEC screen identified 38 targets that significantly reduced nuclear p21. Of those, we triaged 12 hits that were also effective in reducing cell and nuclear area. In silico pathway analysis showed involvement of these targets in exocytosis, DNA damage response, splicing, protein synthesis and growth hormone signaling; the role of these processes in driving or maintaining cellular senescence is largely unknown.

In conclusion, we have successfully identified novel targets involved in senescence. These targets will be further investigated to understand their role in disease and pursued for the development of new therapeutic approaches for targeting senescence.