CRISPR/Cas9-Mediated Point Mutation in Prolongs Protein Half-Life and Reverses Effects Allelic Loss.

Bowen C, Shibata M, Zhang H, Bergren SK, Shen MM, Gelmann EP. 2020. CRISPR/Cas9-Mediated Point Mutation in Prolongs Protein Half-Life and Reverses Effects Allelic Loss. Cancer Res. 80:4805–4814. doi:10.1158/0008-5472.CAN-20-1742.

is the most commonly deleted gene in prostate cancer and is a gatekeeper suppressor. is haploinsufficient, and pathogenic reduction in protein levels may result from genetic loss, decreased transcription, and increased protein degradation caused by inflammation or PTEN loss. NKX3.1 acts by retarding proliferation, activating antioxidants, and enhancing DNA repair. DYRK1B-mediated phosphorylation at serine 185 of NKX3.1 leads to its polyubiquitination and proteasomal degradation. Because NKX3.1 protein levels are reduced, but never entirely lost, in prostate adenocarcinoma, enhancement of NKX3.1 protein levels represents a potential therapeutic strategy. As a proof of principle, we used CRISPR/Cas9-mediated editing to engineer a point mutation in murine to code for a serine to alanine missense at amino acid 186, the target for Dyrk1b phosphorylation. , and mice were analyzed over one year to determine the levels of Nkx3.1 expression and effects of the mutant protein on the prostate. Allelic loss of caused reduced levels of Nkx3.1 protein, increased proliferation, and prostate hyperplasia and dysplasia, whereas mouse prostates had increased levels of Nkx3.1 protein, reduced prostate size, normal histology, reduced proliferation, and increased DNA end labeling. At 2 months of age, when all mice had normal prostate histology, mice demonstrated indices of metabolic activation, DNA damage response, and stress response. These data suggest that modulation of Nkx3.1 levels alone can exert long-term control over premalignant changes and susceptibility to DNA damage in the prostate. SIGNIFICANCE: These findings show that prolonging the half-life of Nkx3.1 reduces proliferation, enhances DNA end-labeling, and protects from DNA damage, ultimately blocking the proneoplastic effects of allelic loss.