VS-5584, a novel and highly selective PI3K/mTOR kinase inhibitor for the treatment of cancer

Abstract

Dysregulation of the PI3K/mTOR pathway, either through amplifications, deletions, or as a direct result of mutations, has been closely linked to the development and progression of a wide range of cancers. Moreover, this pathway activation is a poor prognostic marker for many tumor types and confers resistance to various cancer therapies. Here, we describe VS-5584, a novel, low-molecular weight compound with equivalent potent activity against mTOR (IC(50) = 37 nmol/L) and all class I phosphoinositide 3-kinase (PI3K) isoforms IC(50): PI3Kα = 16 nmol/L; PI3Kβ = 68 nmol/L; PI3Kγ = 25 nmol/L; PI3Kδ = 42 nmol/L, without relevant activity on 400 lipid and protein kinases. VS-5584 shows robust modulation of cellular PI3K/mTOR pathways, inhibiting phosphorylation of substrates downstream of PI3K and mTORC1/2. A large human cancer cell line panel screen (436 lines) revealed broad antiproliferative sensitivity and that cells harboring mutations in PI3KCA are generally more sensitive toward VS-5584 treatment. VS-5584 exhibits favorable pharmacokinetic properties after oral dosing in mice and is well tolerated. VS-5584 induces long-lasting and dose-dependent inhibition of PI3K/mTOR signaling in tumor tissue, leading to tumor growth inhibition in various rapalog-sensitive and -resistant human xenograft models. Furthermore, VS-5584 is synergistic with an EGF receptor inhibitor in a gastric tumor model. The unique selectivity profile and favorable pharmacologic and pharmaceutical properties of VS-5584 and its efficacy in a wide range of human tumor models supports further investigations of VS-5584 in clinical trials.

Figure 1

VS-5584 effectively blocks PI3K/mTOR signaling in cancer cells with different genetic background. A, PC3 cells were treated with VS-5584 for 3 hours as indicated. After lysis, phosphorylation status of pS6 and pAkt were detected by immunoblotting. B, structure of everolimus. MV4-11 (C), NCI-N87 (D), COLO-205 (E), MDA-MB-231 (F), and HUH-7 (G) cells were treated with VS-5584 for 3 hours as indicated or everolimus (100 nmol/L for 3 hours). After lysis, phosphorylation status of pS6, pAkt, pmTOR, and pERK1/2 was detected by immunoblotting.

Figure 2

Pharmacokinetic (PK)/pharmacodynamic properties of VS-5584. A, PC3 tumor-bearing mice received various single doses of VS-5584 as indicated. Mice were sacrificed 6 hours postdosing and the concentration of VS-5584 was determined in blood plasma and tumor tissue. B, the phosphorylation status of pAKT(S473) and pS6(S240/244) in tumor lysates 6 hours postdosing was determined by immunoblotting. C, PC3 tumor-bearing mice received a single dose of 33 mg/kg VS-5584 as indicated. Mice were sacrificed 1, 6, and 24 hours postdosing and the concentration of VS-5584 was determined in blood plasma and tumor tissue. D, 1, 6, and 24 hours postdosing the phosphorylation status of pAKT(S473) and pS6(S240/244) in tumor lysates was determined by immunoblotting.

Figure 3

VS-5584 is efficacious in a PTEN^null human prostate PC3 xenograft model and in a rapamycin-resistant human colorectal COLO-205 xenograft model. A, PC3 tumor-bearing mice (n = 7/group) were treated daily for 28 days as indicated and the TGI determined. ANOVA with Dunnett posttest was conducted; ***, P < 0.001. B and C, 6 hours after the last treatment on day 27 the phosphorylation status of pS6, pAkt(S473) in tumor tissue was analyzed. ANOVA with Dunnett posttest was conducted; **, P < 0.01. D, Colo-205 tumor-bearing nude mice (n = 13/group) were treated daily for 18 days and the tumor volume monitored. E, tumor weight for each group is shown. ANOVA with Dunnett posttest was conducted; ***, P < 0.001. F, 6 hours after the last treatment on day 17 the phosphorylation status of S6, Akt(S473) in tumor tissue were determined. G, on day 17, active vessels in the tumors were stained with FITC-conjugated Ricinus communis agglutinin I and the vessel score determined. t test was conducted; *, P < 0.05.

Figure 4

High dose of VS-5584 or a low dose in combination with an EGFRi is efficacious in a gastric xenograft model. A, NCI-N87 tumor-bearing mice (n = 12/group) were treated for 17 days with 25 mg/kg orally daily. VS-5584 or with 25 mg/kg 5-FU intraperitoneally every Tuesday, Thursday, and Saturday for 2 weeks, followed by 1 week break. TGI on volume is indicated. ANOVA with Dunnett posttest was conducted, ***, P < 0.001. B, structure of 5-FU. C and D, 6 hours after dosing on day 16 phosphorylation status of mTOR, S6, Akt, and ERK1/2 and total actin was determined in tumor tissue. E, NCI-N87 tumor-bearing mice (n = 10/group) were treated for 26 days with 11 mg/kg orally daily. VS-5584, 150 mg/kg gefitinib orally dosed for 5 day-on and 2 day-off in cycles and combined treatment of 11 mg/kg VS-5584 with 150 mg/kg gefitinib (dosing schedule was the same as monotherapy). ANOVA with Dunnett posttest was conducted, ***, P < 0.001. F, structure of gefitinib.

Figure 5

Cell line profiling identifies an association between VS-5584 sensitivity and PIK3CA mutational status. A, a volcano plot representation from a multivariate ANOVA for cancer gene mutations associated with sensitivity and resistance to VS-5584. Each circle represents the correlation between VS-5584 and a single cancer gene. The effect on drug response (x-axis) and significance of the association (y-axis; inverted scale) is shown, and circle size is proportional to the number of cell lines screened with the given mutation (range, 1–291 depending on the gene). B, a scatterplot of IC₅₀ values plotted on a log scale for WT and PIK3CA-mutated cell lines. Each circle represents the IC₅₀ from a single cell line and the gray bar indicates the geometric mean. C, VS-5584 IC₅₀ values in PIK3CA-mutated cell lines categorized by tissue type. PIK3CA WT cell lines are shown for comparison. D, a comparison of IC₅₀ values in WT and PIK3CA-mutated breast cancer cell lines. E, sensitivity to VS-5584 is dependent on both PIK3CA and ERBB2 mutational status. The + and − symbols indicate the presence or absence of the indicated mutation. Kruskal–Wallis nonparametric analysis of variance was used comparing WT cells to the indicated mutant cell line populations. Only significant associations are indicated.