Targeted Degradation of Oncogenic KRASG12C by VHL-Recruiting PROTACs

Abstract

KRAS is mutated in ∼20% of human cancers and is one of the most sought-after targets for pharmacological modulation, despite having historically been considered "undruggable." The discovery of potent covalent inhibitors of the KRAS^G12C mutant in recent years has sparked a new wave of interest in small molecules targeting KRAS. While these inhibitors have shown promise in the clinic, we wanted to explore PROTAC-mediated degradation as a complementary strategy to modulate mutant KRAS. Herein, we report the development of LC-2, the first PROTAC capable of degrading endogenous KRAS^G12C. LC-2 covalently binds KRAS^G12C with a MRTX849 warhead and recruits the E3 ligase VHL, inducing rapid and sustained KRAS^G12C degradation leading to suppression of MAPK signaling in both homozygous and heterozygous KRAS^G12C cell lines. LC-2 demonstrates that PROTAC-mediated degradation is a viable option for attenuating oncogenic KRAS levels and downstream signaling in cancer cells.

Figure 1

MRTX849-VHL PROTACs engage and degrade endogenous KRAS^G12C in NCI-H2030 cells. (A) Chemical structures of MRTX849, LC-1 (inactive PROTAC), LC-2 (active PROTAC), and LC-2 Epimer. (B) LC-1 engages KRAS^G12C in a dose-dependent manner. Quantitation on the right. (C) LC-2 degrades KRAS^G12C in a dose-dependent manner. Cells were treated for 24 h. Immunoblots show lysates from independent wells harvested side-by-side on the same day. Quantitation on the right. Quantified data represents mean ± SD from two independent biological replicates. Not Significant (N.S.); * p < 0.05; ** p < 0.01; **** p < 0.001.

Figure 2

Degradation of endogenous KRAS^G12C is via a PROTAC mechanism. (A) LC-2 Epimer does not induce KRAS^G12C degradation at 2.5 μM and LC-2 induced degradation is rescued by VHL ligand competition, proteasome inhibition with epoxomicin (Epox) and neddylation inhibition with MLN4924 (MLN) in NCI-H2030 cells. Immunoblots show lysates from independent wells harvested side-by-side on the same day. Quantitation is below. (B) Inhibition of neddylation, but not inhibition of lysosomal acidification, rescues LC-2 induced KRAS^G12C degradation in NCI-H23 cells. Quantitation is below. Quantified data represents mean ± SD for two biological replicates. Not Significant (N.S.); *** p < 0.005

Figure 3

KRAS^G12C degradation is rapid, with maximal degradation induced as early as 4 h. (A) Time course in NCI-H2030 cells. LC-2 and LC-2 Epimer engage within 1 h with maximal degradation observed by 8 h and maintained up to 24 h. Quantitation on the right. (B) Time course in SW1573 cells. LC-2 and LC-2 epimer engage KRAS within 1 h and maximal degradation is observed at 12 h and maintained up to 24 h. Quantitation on the right. LC-2 Epimer is a quantification of the higher molecular weight, PROTAC Epimer modified band to monitor engagement of KRAS^G12C overtime rather than total KRAS levels. Quantified data represents mean ± SD for two biological replicates. Not Significant (N.S.); * p < 0.05; ** p < 0.01; *** p < 0.005; **** p < 0.001.

Figure 4

Degradation of endogenous KRAS^G12C is sustained over 72 h in multiple cancer cell lines. (A) 72 h time course in MIA PaCa-2 cells. Degradation occurs at 6 h and is maintained up to 72 h. Quantitation on the right. (B) 72 h time course in NCI-H23 cells. Degradation occurs within 6 h, reaches a maximum at 24 h, and begins to rebound by 72 h. Immunoblots show lysates from independent wells harvested side-by-side on the same day. Quantitation on the right. Quantified data represents mean ± SD for two biological replicates. Not Significant (N.S.); ** p < 0.01; *** p < 0.005; **** p < 0.001.

Figure 5

Degradation of endogenous KRAS^G12C modulates Erk signaling in homozygous and heterozygous KRAS^G12C cell lines. (A) Degradation of KRAS^G12C in homozygous NCI-H2030 cells attenuates pErk in a dose-dependent manner. Quantitation on the right. Immunoblots show lysates from independent wells harvested side-by-side on the same day. (B) Degradation of KRAS^G12C in heterozygous NCI-H23 cells decreases pErk in a dose-dependent manner. Quantitation on the right. For statistical analysis, see Supplemental Tables 2 and 3. Quantified data represents mean ± SD for two biological replicates.

Figure 6

Effect of KRAS^G12C degradation and inhibition on Erk signaling over time. (A) Inhibition and degradation of KRAS^G12C decreases pErk signaling at 6 and 24 h in homozygous MIA PaCa-2 cells. Quantitation on the right. (B) Inhibition and degradation of KRAS^G12C decreases pErk signaling at 6 and 24 h in heterozygous NCI-H23. Immunoblots show lysates from independent wells harvested side-by-side on the same day. Quantitation on the right. For statistical analysis, see Supplemental Tables 4 and 5. Quantified data represents mean ± SD for two biological replicates.