Colorectal cancer (CRC) remains a critical challenge due to limited therapeutic options. CDK5 has emerged as a potential target; however, its structural similarity to other CDKs complicates direct inhibition. Targeted protein degradation (TPD), which utilizes the ubiquitin-proteasome system, offers a novel approach. The dTAG system, based on PROTACs, enables specific protein degradation for functional studies in vitro and in vivo. Our aim was to lay the foundation to generate a PROTAC drug against CDK5 as a novel treatment for CRC.

HT-29 and LoVo CRC cell lines were knocked out of endogenous CDK5 using CRISPR-cas9. FKBP12F36V-tagged CDK5 was reintroduced using lentiviral vectors. Degradation via dTAGv-1 was validated by western blot (WB) (D1F7M, Cell Signaling). Degradation of CDK5 in vivo was measured in subcutaneous (SC) HT-29_Nluc_CDK5dTAG and WT mock cells in BALB/c (nu/nu) (n=3/group). Tumours were treated with vehicle (5% DMSO in 20% solutol/saline) or dTAGv-1 via intraperitoneal (IP) (40 or 80 mg/kg), SC (40 mg/kg), or intratumoral (IT) (40 mg/kg) administration for 3 days. Tumours were harvested on day 4, snap-frozen, and analysed for protein degradation via WB. To assess liver metastasis, HT-29_Nluc_CDK5dTAG cells were injected into the spleen of BALB/c (nu/nu) (n=10/group), and metastases were tracked via bioluminescence imaging over 4 weeks (IVIS Illumina II). Mice were treated daily with 40 mg/kg dTAGv-1 or vehicle intraperitoneally. At endpoint, ex vivo fluorescence was measured, and liver samples were paraffin-embedded. Statistical analysis used was one-way ANOVA with Tukey’s multiple comparison for SC and bioluminescence data, and Mann-Whitney U test for fluorescence.

CRISPR/Cas9 efficiently knocked out CDK5, and FKBP12F36V-tagged CDK5 was successfully reintroduced in both cell lines. From 500nM down to 10nM of dTAGv-1 induced a dose-dependent degradation of CDK5, confirmed by WB. In vivo, IT administration achieved the highest degradation (5.27% ±2,58%, p<0.01), outperforming IP and SC routes. Degradation rates for IP (40 mg/kg: 59.46% ±10,36%; 80 mg/kg: 40.23% ±8,4%) and SC (48.15% ±20,37%) routes showed no significant differences. All treatment groups exhibited significant protein degradation compared to vehicle (p<0.001) (mean ±SD). Metastases were significantly reduced in dTAGv-1-treated mice vs. vehicle controls as measured by bioluminescence (3.73×10⁷ ±1,13 x107 vs. 3.65×10⁸ ±1,15x108; p/s/cm²/sr, mean ±SEM; p<0.05) and fluorescence [4.45×10⁸ ±1,59x108 vs. 3.68×109 ± 2,67x109; (p/s/cm²/sr)/(µW/cm²) mean ±SEM; p<0.01], respectively.

The dTAG system effectively degraded CDK5 in vitro and in vivo, reducing tumour burden and metastases without observed toxicities. These findings highlight CDK5 TPD’s potential as a therapeutic strategy in CRC.