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. 2022 May 17;12(1):8121.
doi: 10.1038/s41598-022-12401-3.

The K-Ras(G12D)-inhibitory peptide KS-58 suppresses growth of murine CT26 colorectal cancer cell-derived tumors

Affiliations

The K-Ras(G12D)-inhibitory peptide KS-58 suppresses growth of murine CT26 colorectal cancer cell-derived tumors

Kotaro Sakamoto et al. Sci Rep. .

Abstract

Mutations in the cell proliferation regulator K-Ras are found with a variety of cancer types, so drugs targeting these mutant proteins could hold great clinical potential. Very recently, a drug targeting the K-Ras(G12C) mutant observed in lung cancer gained regulatory approval and several clinical trials are currently underway to examine the efficacy of this agent when combined with other drugs such as a monoclonal antibody inhibitor of programmed cell death 1 receptor (anti-PD-1). Alternatively, there are currently no approved drugs targeting K-Ras(G12D), the most common cancer-associated K-Ras mutant. In 2020, we described the development of the K-Ras(G12D) inhibitory bicyclic peptide KS-58 and presented evidence for anticancer activity against mouse xenografts derived from the human pancreatic cancer cell line PANC-1 stably expressing K-Ras(G12D). Here, we show that KS-58 also possess anticancer activity against mouse tumors derived from the colorectal cancer cell line CT26 stably expressing K-Ras(G12D). Further, KS-58 treatment reduced phosphorylation of ERK, a major downstream signaling factor in the Ras pathway, confirming that KS-58 inhibits K-Ras(G12D) function. Unexpectedly; however, KS-58 did not show additive or synergistic anticancer activity with mouse anti-PD-1. Morphological analysis and immunostaining demonstrated no obvious differences in CD8+ cells infiltration or PD-L1 expression levels in CT26-derived tumors exposed to monotherapy or combination treatment. Nonetheless, KS-58 demonstrated reasonable stability in blood (t1/2 ≈ 30 min) and no obvious systemic adverse effects, suggesting clinical potential as a lead molecule against colorectal cancer.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
KS-58 suppressed in vitro proliferation of the murine colorectal cancer cell line CT26. (A) Chemical structure of KS-58. (B) Dose-dependent suppression of CT26 cell proliferation by KS-58. Results in (B) are expressed as mean ± S.E.M of n = 4 independent experiments using separately treated cultures (*p < 0.05 and **p < 0.01 vs. DMSO by Dunnett’s test). (C) Dose-dependent suppression of phosphorylated ERK (pERK) in CT26 cells by KS-58 (n = 6, mean ± S.E.M, **p < 0.01 vs. DMSO control by Dunnett’s test). The phosphorylation levels were shown as % value compared to DMSO set as 100% and no-serum stimulation set as 0%. (D) Colony growth suppression activity of KS-58 (30 μM) (yellow bar: 50 μm, n = 6, mean ± S.E.M, **p < 0.01 vs. DMSO control by Student’s t-test). The colony growth levels were shown as % value compared to DMSO at day 3 set as 100% and initial at day 0 set as 0%. (E) Apoptosis inducing effect of KS-58 (30 μM and 100 μM) (n = 8, mean ± S.E.M, *p < 0.05 and **p < 0.01 vs. DMSO by Dunnett’s test). The value on vertical axis means actual values minus average value of DMSO control.
Figure 2
Figure 2
Anticancer activities of KS-58 alone and KS-58 plus anti-PD-1 antibody against CT26 allografts. (A) Schematic of experimental procedures. Major groups (n = 8 mice per group) were tested to evaluate anti-cancer activity. Tumors, livers and kidneys of satellite groups (n = 3 mice per group) were collected for morphological analysis of Fig. 4. (B) Changes in tumor volume and body weight for each major treatment group (n = 8 mice per group, expressed as mean ± SD, *p < 0.05 and **p < 0.01 vs. Vehicle control by Dunnett’s test). (C) Weights of tumor, liver, and kidney (right) collected from major groups on day 17 (n = 8 mice per group, mean ± SD, *p < 0.05 and **p < 0.01 vs. Vehicle control by Dunnett’s test). (D) Appearance of tumors collected from major groups. KS-58 doses were 40 mg/kg (high) and 10 mg/kg (low) in all experiments.
Figure 3
Figure 3
Inhibition of K-Ras(G12D) in CT26 cell-derived tumors by KS-58 measured using phosphorylated ERK as a biomarker. Tumors collected from major groups within 3 h after peptide or vehicle injection were homogenized, and phosphorylated ERK (pERK), total ERK and GAPDH were quantified by Western blotting. Expression levels of pERK were normalized by total ERK and GAPDH, respectively. Expression levels of total ERK was normalized by GAPDH. These expression levels are presented as the fold change relative to the vehicle group (n = 3 tumors per group, mean ± S.E.M, *p < 0.05 and **p < 0.01 vs. Vehicle by Dunnett’s test).
Figure 4
Figure 4
Pharmacokinetics and stability of KS-58. (A) KS-58 was intravenously injected at 10 mg/kg (n = 3 mice) and blood samples were collected at the indicated times. The concentration of KS-58 (mean ± SD) in plasma was determined by LC–MS/MS and the pharmacokinetics parameters t1/2, AUCinf, and Vdss were calculated as described in “Materials and methods” section. (B) KS-58 remaining in human and mouse liver microsomes (Ms) following 10 and 60 min of incubation. (C) Residual KS-58 in mouse whole blood after 60 and 120 min of incubation. (D) Translocation of KS-58 to blood cells. (E) Binding (%) of KS-58 to human and mouse plasma proteins.
Figure 5
Figure 5
Effects of KS-58 alone and combined with anti-PD-1 on tumor and non-target organ histopathology. Tumors, livers, and kidneys collected from satellite mouse groups were fixed in 10% neutral-buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. (A) CD8+ cells infiltration (black bar: 200 μm) and PD-L1 expression (black bar: 50 μm) were evaluated by immunostaining. (B) Cell degeneration and necrosis were evaluated by morphological analysis using light microscopy (black bar: 100 μm in kidney and 20 μm in liver). CV central vein, P portal area in liver.

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