Generation of CRISPR engineered prostate cancer cell line models to study androgen receptor signalling in advanced prostate cancer

Abstract

Prostate cancer resistance to AR targeted therapies due to the emergence of AR point mutations and AR splice variants that cannot be targeted by the currently available agents comprise a major clinical challenge. There is paucity of models that accurately reflect the mechanisms of AR regulation in advanced disease. This highlights the high demand of generating novel disease relevant models. A CRISPR pipeline was developed to generate cell line models which harbour specific point mutations in the LBD of AR as well as stop codons in AR exon 5 which resulted in AR-FL knock-out so that the remaining endogenous AR-Vs could be studied discriminately of interfering AR-FL. Using a streptavidin-tagged Cas9 in conjugation with a biotinylated donor template resulted in high donor template knock-in efficiencies and yielded (i) an ARW741L CWR22Rv1 cell line derivative and (ii) an AR-FL knock-out cell line derivative called CWR22Rv1-AR-EK (Exon Knock-out). CWR22Rv1-AR-EK cells retained all endogenous AR-Vs following AR gene editing. AR-Vs acted unhindered following AR-FL deletion to drive cell growth and expression of androgenic genes. Global transcriptomics demonstrated that AR-Vs drive expression of a cohort of cell cycle and DNA damage response genes and depletion of AR-Vs sensitised cells to ionising radiation. To date, elimination of AR-Vs by pharmacological inhibition remains challenging. However, disruption of AR pre-mRNA splicing is nowadays a highly attractive option. A CRISPR-based approach, called CRIME (Cas9-directed Rapid Immunoprecipitation Mass Spectrometry of Endogenous proteins) was developed to isolate and identify the AR-V7-specific spliceosome, in association with a nuclease deficient Cas9 at AR cryptic exon 3. Mass spec-derived hits were screened for their ability to alter AR-V7 mRNA levels in CWR22Rv1 cells. SRSF3 was identified as a potential AR-V7 splicer which promotes cryptic exon 3 skipping in normal prostate. In CRPC, SRSF3 is significantly downregulated and hence cryptic exon 3 inclusion in the mature AR-V7 mRNA transcript is permitted leading to generation of AR-V7.