Identification of a promising PI3K inhibitor for the treatment of multiple myeloma through the structural optimization

Abstract

Background: We previously reported a PI3K inhibitor S14161 which displays a promising preclinical activity against multiple myeloma (MM) and leukemia, but the chiral structure and poor solubility prevent its further application.

Methods: Six S14161 analogs were designed based on the structure-activity relationship; activity of the compounds in terms of cell death and inhibition of PI3K were analyzed by flow cytometry and Western blotting, respectively; anti-myeloma activity in vivo was performed on two independent xenograft models.

Results: Among the six analogs, BENC-511 was one of the most potent compounds which significantly inhibited PI3K activity and induced MM cell apoptosis. BENC-511 was able to inactivate PI3K and its downstream signals AKT, mTOR, p70S6K, and 4E-BP1 at 1 μM but had no effects on their total protein expression. Consistent with its effects on PI3K activity, BENC-511 induced MM cell apoptosis which was evidenced by the cleavage of Caspase-3 and PARP. Notably, addition of insulin-like growth factor 1 and interleukin-6, two important triggers for PI3K activation in MM cells, partly blocked BENC-511-induced MM cell death, which further demonstrated that PI3K signaling pathway was critical for the anti-myeloma activity of BENC-511. Moreover, BENC-511 also showed potent oral activity against myeloma in vivo. Oral administration of BENC-511 decreased tumor growth up to 80% within 3 weeks in two independent MM xenograft models at a dose of 50 mg/kg body weight, but presented minimal toxicity. Suppression of BENC-511 on MM tumor growth was associated with decreased PI3K/AKT activity and increased cell apoptosis.

Conclusions: Because of its potent anti-MM activity, low toxicity (LD50 oral >1.5 g/kg), and easy synthesis, BENC-511 could be developed as a promising agent for the treatment of MM via suppressing the PI3K/AKT signaling pathway.

Figure 1

BENC-511 displays potent inhibitory effects on AKT activation. (A) The structures of the analogs of S14161, including DQJ-610, DJY-611, WQD-612, QDF-510, BENC-511. (B) OPM2 cells were treated with 4 μM of S14161, BENC (BENC-511), QDF (QDF-510), DQJ (DQJ-610), DJY (DJY-611), or WQD (WQD-612) for 24 hours. After incubation, cells were harvested and total proteins were isolated. Expression of p-AKT (S473) and total AKT were measured by immunoblotting. (C) RPMI-8226 (8226), JJN3, OCI-MY5 (MY5), U266, LP1, OPM2 cells were treated with 4 μM of BENC-511 or DMSO for 24 hours followed by the analysis of the expression of p-AKT (S473) and total AKT. (D) RPMI-8226, LP1 and OPM2 cells were treated with increasing concentration of BENC-511 for 24 hours. Expression of p-AKT (S473 and T308), and total AKT were measured by immunoblotting. (E) RPMI-8226 and OPM2 cells were treated with increasing concentration of BENC-511 for 12 hours followed by the analysis of AKT activation. GAPDH was used as a loading control. T-AKT: total AKT.

Figure 2

BENC-511 suppresses AKT activation triggered by IGF-1. (A) Myeloma (RPMI-8226, JJN3, LP1 and OPM2) cells were starved overnight and then treated with 100 μM of S14161, 50 μM of BENC-511, or DMSO for 2 hours, followed by 100 ng/mL of IGF-1 for 15 minutes. After incubation, cells were harvested and total proteins were isolated. Expression of AKT, p-AKT (T308), p-AKT (S473), and GAPDH was measured by immunoblotting. (B) RPMI-8226 and OPM2 cells were treated with increasing concentration of BENC-511 for 0.5 or 1 hour, S14161 for 2 hours, followed by IGF-1 stimulation. Cells were then harvested and total proteins isolated. Expression of AKT, p-AKT (S473), and GAPDH was measured by immunoblotting. (C) OPM2 cells were starved overnight and then treated with BENC-511 (8 μM), or DMSO for the indicated time followed by 100 ng/mL IGF-1 or 50 ng/mL IL-6 for 15 minutes. After incubation, cells were harvested and total proteins isolated. Expressions of total AKT, p-AKT (S473), PARP, and GAPDH were measured by immunoblotting. T-AKT: total AKT.

Figure 3

BENC-511 downregulates PI3K/AKT downstream signals. OPM2, RPMI-8226 and LP1 cells were treated with increasing concentrations of BENC-511 for 24 hours. Whole lysates were subjected to Western blot analysis. (A) p-mTOR (Ser2448), T-mTOR, Raptor; (B) p-p70S6K, p70S6K, p-4E-BP1, and 4E-BP1; (C) p-GSK-3β (Ser9). β-actin was used as an internal control.

Figure 4

BENC-511 activates apoptotic signaling in MM cells. (A) Myeloma cells (RPMI-8226, JJN3, OCI-MY5, U266, LP1) were treated with 4 μM BENC-511 or DMSO for 24 hours followed by the analysis of the expression of PARP, Caspase-3, and GAPDH. (B) OPM2, RPMI-8226 and LP1 cells were treated with increasing concentrations of BENC-511 for 24 hours. (C) OPM2 and RPMI-8226 were incubated with 4 μM of BENC-511 for the indicated time. After incubation, cells were harvested and total proteins were isolated. Cell lysates were applied for PARP and GAPDH analyses.

Figure 5

BENC-511 induces MM cell apoptosis. LP1, OCI-MY5, OPM2 and RPMI-8226 were treated with 1 μM of S14161, 0.5 or 1 μM of BENC-511 (BENC) and DMSO for 24 hours. Cells were then stained with Annexin V-FITC and PI and analyzed on a BD FACSCalibur™ flow cytometer.

Figure 6

BENC-511 induces MM cell apoptosis in the presence of IL-6 or IGF-1. (A) OPM2 cells were co-treated with IL-6, IGF-1 and BENC-511 with the increasing concentration for 24 hours. After incubation, cells were harvested and cell lysates were measured for the expression of PARP and GAPDH by Western blotting. (B) MM cell lines OPM2 was co-cultured overnight with human bone marrow stromal cell HS-5, followed by treatment with BENC-511 overnight. Cells were then collected for PARP, p-AKT (S473) and total AKT (T-AKT) analysis.

Figure 7

BENC-511 induces MM cell death in vivo and delays tumor growth in myeloma xenograft models. Human multiple myeloma cells (RPMI-8226 and OPM2) were injected subcutaneously into nude mice with a density of 30 million cells/site. When tumors were palpable, mice (n = 10/group) were orally given BENC-511 (50 mg/Kg body weight) in PBS containing 10% Tween 80 and 10% DMSO daily for continuous 20 days. Tumor volumes (A) were monitored every other day. *, p < 0.05; #, p < 0.01. Tumor samples from each group were subject to immunoblotting analysis for PARP, Caspase-3, phospho-AKT, T-AKT, p-mTOR, T-mTOR, p-p70S6K, and p70S6K with specific antibodies (B, C, and D).