ROCK2 Knockout Improves Proliferation Rate in a Cellular Model of Down Syndrome

Abstract

In a cellular model of Down Syndrome, hTERT immortalized RPE-1 (human retinal pigment epithelial-1) cells carrying an extra copy of chromosome 21 exhibit reduced fitness, in part, as an increase in doubling time (or a reduction in cell proliferation rate) in culture. ROCK2 (Rho associated coiled-coil containing kinase 2) was identified in a whole genome CRISPR knockout (KO) screen designed to identify genes and pathways that could be therapeutically targeted to improve cell proliferation (Replogle JM, et al. manuscript in preparation). ROCK2 KO cell lines of both RPE-1 euploid and trisomy 21 aneuploid cells were created using CRISPR. Trisomy 21 ROCK2 KO cell lines showed a modest increase in cell proliferation rate compared to the parental aneuploid cells, similar to the relative effect that ROCK2 knockout had in the whole genome CRISPR screen. Euploid ROCK2 KO cell lines showed no difference in growth rate vs their ROCK2 expressing counterparts. Changes in doubling time in response to two pharmaceutical ROCK inhibitors, Fasudil and Y27632, also showed the same modest increase in cell proliferation rate in the trisomy 21 cells. The actin cytoskeleton, a target of ROCK2 regulation, exhibited long stress fibers that aligned across multiple contiguous cells in confluent trisomy 21 ROCK2 KO cells compared to the disorganized stress fibers of the parental trisomy 21 cells with normal ROCK2 expression.