Characterisation of a novel KRAS G12C inhibitor ASP2453 that shows potent anti-tumour activity in KRAS G12C-mutated preclinical models

Abstract

Background: KRAS is one of the most frequently mutated oncogenes in various cancers, and several novel KRAS G12C direct inhibitors are now in clinical trials. Here, we characterised the anti-tumour efficacy of ASP2453, a novel KRAS G12C inhibitor, in preclinical models of KRAS G12C-mutated cancer.

Methods: We evaluated the in vitro and in vivo activity of ASP2453, alone or in combination with targeted agents and immune checkpoint inhibitors, in KRAS G12C-mutated cancer cells and xenograft models. We also assessed pharmacological differences between ASP2453 and AMG 510, another KRAS G12C inhibitor, using an SPR assay, washout experiments and an AMG 510-resistant xenograft model.

Results: ASP2453 potently and selectively inhibited KRAS G12C-mediated growth, KRAS activation and downstream signalling in vitro and in vivo, and improved the anti-tumour effects of targeted agents and immune checkpoint inhibitors. Further, ASP2453 had more rapid binding kinetics to KRAS G12C protein and showed more potent inhibitory effects on KRAS activation and cell proliferation after washout than AMG 510. ASP2453 also induced tumour regression in an AMG 510-resistant xenograft model.

Conclusions: ASP2453 is a potential therapeutic agent for KRAS G12C-mutated cancer. ASP2453 showed efficacy in AMG 510-resistant tumours, even among compounds with the same mode of action.

Fig. 1. ASP2453 is a potent, selective and covalent KRAS G12C inhibitor.

a Chemical structure of ASP2453. b Interaction between ASP2453 and KRAS G12C (mean ± SD). c ASP2453, among the proteome in NCI-H1373 cells, selectively binds to KRAS G12C after treatment for 2 h at 30 nmol/L. d Inhibitory activity of ASP2453 on SOS-mediated KRAS G12C-Raf or KRAS WT-Raf interactions (mean ± SEM, three independent experiments).

Fig. 2. ASP2453 selectively inhibits KRAS signalling and proliferation of KRAS G12C-mutated in cancer cells in vitro.

a Inhibitory activity of ASP2453 on p-ERK 1/2 after treatment for 2 h (mean ± SEM, three independent experiments). b Inhibitory activity of ASP2453 on the proliferation of human cancer cells harbouring KRAS G12C mutations in 3D spheroid culture (mean ± SEM, three independent experiments). c Effect of ASP2453 on KRAS activation, KRAS mobility shift and downstream signals in NCI-H1373 cells after treatment for 2 h. d Effect of ASP2453 on KRAS activation and KRAS mobility shift in KRAS-mutated cell lines after treatment for 2 h.

Fig. 3. Oral administration of ASP2453 induces anti-tumour activity in an NCI-H1373 xenograft model.

a Anti-tumour activity of ASP2453 in a subcutaneous KRAS G12C-mutated NCI-H1373 xenograft model (n = 5 mice per group, mean ± SEM). ****P < 0.0001, Dunnett’s multiple comparison test compared with vehicle. b Effect of ASP2453 on KRAS activation, KRAS mobility shift, p-ERK 1/2 and p-S6 in NCI-H1373 tumours at 6 h after administration of a single dose. c Pharmacodynamics of ASP2453 on KRAS-GTP, p-ERK 1/2 and p-S6 in NCI-H1373 tumours. Tumour samples were collected at 1, 2, 4, 6, 24 and 48 h after administration of a single dose (n = 3 mice per group, mean ± SEM). d Plasma and tumour concentrations of ASP2453. Plasma and tumour samples were collected at 1, 2, 4, 6, 24 and 48 h after administration of a single dose (n = 3 mice per group, mean ± SD).

Fig. 4. ASP2453 shows potent anti-tumour activity in multiple human cancer xenograft models.

a Anti-tumour activity of ASP2453 in a subcutaneous KRAS G12C-mutated MIA PaCa-2 xenograft model (n = 5 mice per group, mean ± SEM). *P < 0.05, ****P < 0.0001, Dunnett’s multiple comparison test compared with vehicle. b Anti-tumour activity of ASP2453 in a subcutaneous KRAS G12C-mutated LXFA592 PDX xenograft model (vehicle or 30 mg/kg-treated group: n = 8 mice per group, 10 mg/kg-treated group: n = 7 mice per group, mean ± SEM). ***P < 0.001, Dunnett’s multiple comparison test compared with vehicle. c Anti-tumour activity of ASP2453 in a subcutaneous KRAS WT A375 xenograft model (n = 5 mice per group, mean ± SEM). d Bioluminescence imaging of mice orthotopically inoculated with NCI-H1373-Luc cells administered ASP2453. Day 0: day of randomisation. Representative images of 10 mice from each group are shown. e Survival of mice orthotopically inoculated with NCI-H1373-Luc cells administered ASP2453 (n = 10 mice per group). **P < 0.01, ****P < 0.0001, two-sided Mantel–Cox test compared with vehicle.

Fig. 5. Combination ASP2453 with erlotinib or anti-PD-1 antibody inhibits cell proliferation in vitro and tumour growth in vivo.

a Combination ASP2453 (1 nmol/L) with erlotinib (500 nmol/L) under EGF stimulation (0.1 ng/mL) inhibits the proliferation of KRAS G12C-mutated NCI-H358 cells in 3D spheroid culture (mean ± SD). b Anti-tumour activity of ASP2453 (10 mg/kg) in combination with erlotinib (100 mg/kg) in a subcutaneous NCI-H358 xenograft model (n = 5 mice per group, mean ± SEM). ***P < 0.01, ****P < 0.001, Dunnett’s multiple comparison test compared with vehicle. ^††P < 0.01, two-tailed Student’s t-test compared with erlotinib-treated group. c Anti-tumour activity of ASP2453 in combination with anti-PD-1 antibody in a subcutaneous CT26.WT_mKRAS (p.G12C) (KI) syngeneic model (n = 5 mice per group, mean ± SEM).

Fig. 6. Comparison of ASP2453 and AMG 510 in vitro and in vivo.

a Chemical structures and binding modes of ASP2453 (left) and AMG 510 (right). Chemical structures are shown in the insets. ASP2453, AMG 510, GDP, Mg²⁺ ion, and water molecules bound to the Mg²⁺ ion are shown as ball-and-stick figures and other atoms are shown as sticks. Each element is represented by a different colour (white: hydrogen, cyan or grey: carbon, blue: nitrogen, red: oxygen, yellow: sulfur). The colours of the protein surfaces are based on electrostatic potential (blue: positive, red: negative, white: neutral). For clarity, non-polar hydrogen atoms have been omitted and the protein surfaces at the front of ASP2453 and AMG 510 are hidden. b SPR sensorgram results for the association of ASP2453 and AMG 510 to KRAS G12C-GDP protein. c Time course of KRAS activation and KRAS mobility shift in MIA PaCa-2 cells. MIA PaCa-2 cells were treated with ASP2453 or AMG 510 for the indicated period of time. KRAS activation and KRAS mobility shift were detected using a RAF-RBD pulldown assay and immunoblotting. d Time course of KRAS activation, KRAS mobility shift and downstream signals in MIA PaCa-2 cells after removal of ASP2453 and AMG 510. MIA PaCa-2 cells were treated with ASP2453 or AMG 510 for 2 h. After treatment, each well was washed three times with medium and incubated for 0, 2, 6 or 24 h. KRAS activation, KRAS mobility shift and downstream signals were detected using a RAF-RBD pulldown assay and immunoblotting. e Inhibitory activity of ASP2453 and AMG 510 on the proliferation of MIA PaCa-2 cells after washout (mean ± SD). f Anti-tumour activity of ASP2453 and AMG 510 in a subcutaneous KRAS G12C-mutated MIA PaCa-2 xenograft model (n = 5 mice per group, mean ± SEM). *P < 0.05, Dunnett’s multiple comparison test compared with AMG 510 (30 mg/kg)-treated group. g Anti-tumour activity of ASP2453 and AMG 510 in an AMG 510-resistant MIA PaCa-2 xenograft model (mean ± SEM). AMG 510 (30 mg/kg) was orally administered once a day for 28 days. Mice were randomised into four groups (n = 5) based on tumour volume and ASP2453 or AMG 510 was administered orally once a day. *P < 0.05, ****P < 0.0001, Dunnett’s multiple comparison test compared with AMG 510 (30 mg/kg)-treated group.