Preclinical evaluation of a novel triple-acting PIM/PI3K/mTOR inhibitor, IBL-302, in breast cancer

Abstract

The proviral integration of Moloney virus (PIM) family of protein kinases are overexpressed in many haematological and solid tumours. PIM kinase expression is elevated in PI3K inhibitor-treated breast cancer samples, suggesting a major resistance pathway for PI3K inhibitors in breast cancer, potentially limiting their clinical utility. IBL-302 is a novel molecule that inhibits both PIM and PI3K/AKT/mTOR signalling. We thus evaluated the preclinical activity of IBL-302, in a range of breast cancer models. Our results demonstrate in vitro efficacy of IBL-302 in a range of breast cancer cell lines, including lines with acquired resistance to trastuzumab and lapatinib. IBL-302 demonstrated single-agent, anti-tumour efficacy in suppression of pAKT, pmTOR and pBAD in the SKBR-3, BT-474 and HCC-1954 HER2+/PIK3CA-mutated cell lines. We have also shown the in vivo single-agent efficacy of IBL-302 in the subcutaneous BT-474 and HCC-1954 xenograft model in BALB/c nude mice. The combination of trastuzumab and IBL-302 significantly increased the anti-proliferative effect in HER2+ breast cancer cell line, and matched trastuzumab-resistant line, relative to testing either drug alone. We thus believe that the novel PIM and PI3K/mTOR inhibitor, IBL-302, represents an exciting new potential treatment option for breast cancer, and that it should be considered for clinical investigation.

Fig. 1. IBL-302 was tested in a panel of 40 breast cancer cell lines using CellTiter-Glo anti-proliferation assay (72 h of incubation).

The panel of 40 breast cancer cell lines are subdivided into their clinical and molecular subtypes: HER+ (Green), ER+ (Orange) and TNBC (Blue). Each cell line’s corresponding PIM 1, PIM 2, PIM 3 and MYC expression levels, P53, PIK3CA, BRCA1 and BRCA2 mutation status, MYC amplification status and ER and PR status are displayed in Table 1.

Fig. 2. Using the clinical subtype, molecular subtype and PIM 1/3 expression values of 40 breast cancer cell lines, IBL-302 sensitivity (based on IC₅₀) was examined.

(a) Compares triple-negative versus HER2-amplified/ER+ breast cancer cell lines in terms of IBL-302 IC₅₀ values. (b) Compares Basal A triple-negative with basal B triple-negative breast cancer cell lines in terms of IBL-302 IC₅₀ values. (c) Compares PIM 1 expression across the 40 breast cancer cell lines with IBL-302 IC₅₀ values and (d) compares PIM 3 expression across the 40 breast cancer cell lines with IBL-302 IC₅₀ values.

Fig. 3. Impact of IBL-302 on PIM/AKT/PI3K signalling components and induction of apoptosis in breast cancer cells.

a SKBR-3 cells were treated with DMSO and IBL-302 (1 µM) for 3 h. The resulting total AKT, pAKT-(S473), pAKT-(T308), total mTOR, pmTOR-(S2448), pmTOR-(2481) and pBAD-(S138). b–g BT-474 and HCC-1954 cells were treated with ascending doses of BKM120 and IBL-302 (0.1, 0.3 and 1 µM) for 3, 8 and 24 h. The resulting pAKT-(S473) and pBAD-(S112) were analysed via western blot analysis. h, i BT-474 and HCC-1954 cells were treated with increasing doses of BKM120 and IBL-302 (0–10 µM) for 3 h. The resulting caspase 3/7 induction was analysed via caspase assay.

Fig. 4. In vivo efficacy of IBL-302 was determined in BT-474 and HCC-1954 subcutaneous xenograft models in BALB/c nude mice.

a BT-474 xenograft mice were treated with vehicle control and IBL-302 (50 mg/kg). BT-474 tumour (mean ± SE) volumes were measure and plotted as a function of time. P values were determined via Student t test and (p < 0.05) was considered to be statistically significant. b HCC-1954 xenograft mice were treated with vehicle control, IBL-302 (50 mg/kg) and dose adjustment IBL-302 (as described in ‘Materials and methods’ section). HCC-1954 tumour (mean ± SE) volumes were measured and plotted as a function of time. P values were determined via Student t test and (p < 0.05) was considered to be statistically significant.

Fig. 5. Acid phosphatase assay showing the efficacy of IBL-302 on viability in trastuzumab and lapatinib resistant breast cancer cells.

a SKBR-3 trastuzumab-resistant (SKBR-3-T) and (b) SKBR-3 lapatinib-resistant (SKBR-3-L) cells. c BT-474 parental (BT-474-P) and (d) BT-474 trastuzumab resistant (BT-474-T). e HCC-1954 parental (HCC-1954-P) and (f) HCC-1954 lapatinib-resistant (HCC-1954-L) (g) cells after the combination of ascending amounts of IBL-302 (nM) with a static amount of trastuzumab (10 µg/ml). Significance was determined via Kruskal–Wallis, non-parametric test through utilisation of Prism software (* > 0.05) (** > 0.01) (*** > 0.001).