Ridaforolimus (MK-8669) synergizes with Dalotuzumab (MK-0646) in hormone-sensitive breast cancer

Abstract

Background: Mammalian target of rapamycin (mTOR) represents a key downstream intermediate for a myriad of oncogenic receptor tyrosine kinases. In the case of the insulin-like growth factor (IGF) pathway, the mTOR complex (mTORC1) mediates IGF-1 receptor (IGF-1R)-induced estrogen receptor alpha (ERα) phosphorylation/activation and leads to increased proliferation and growth in breast cancer cells. As a result, the prevalence of mTOR inhibitors combined with hormonal therapy has increased in recent years. Conversely, activated mTORC1 provides negative feedback regulation of IGF signaling via insulin receptor substrate (IRS)-1/2 serine phosphorylation and subsequent proteasomal degradation. Thus, the IGF pathway may provide escape (e.g. de novo or acquired resistance) from mTORC1 inhibitors. It is therefore plausible that combined inhibition of mTORC1 and IGF-1R for select subsets of ER-positive breast cancer patients presents as a viable therapeutic option.

Methods: Using hormone-sensitive breast cancer cells stably transfected with the aromatase gene (MCF-7/AC-1), works presented herein describe the in vitro and in vivo antitumor efficacy of the following compounds: dalotuzumab (DALO; "MK-0646"; anti-IGF-1R antibody), ridaforolimus (RIDA; "MK-8669"; mTORC1 small molecule inhibitor) and letrozole ("LET", aromatase inhibitor).

Results: With the exception of MK-0646, all single agent and combination treatment arms effectively inhibited xenograft tumor growth, albeit to varying degrees. Correlative tissue analyses revealed MK-0646 alone and in combination with LET induced insulin receptor alpha A (InsR-A) isoform upregulation (both mRNA and protein expression), thereby further supporting a triple therapy approach.

Conclusion: These data provide preclinical rationalization towards the combined triple therapy of LET plus MK-0646 plus MK-8669 as an efficacious anti-tumor strategy for ER-positive breast tumors.

Keywords: Aromatase inhibitors/therapeutic use; Breast neoplasms/drug therapy; Disease models, Animal; Drug resistance, Neoplasm; Receptor, IGF type 1; Receptor, Insulin; mTOR inhibitor.

Fig. 1

IGF-1R inhibition (MK-0646) does not enhance hormonal therapy (letrozole or tamoxifen) in MCF-7/AC-1 xenografts. Ovariectomized female nu/nu mice between the ages of 7–8 weeks old were inoculated with MCF-7/AC-1 tumor cells in each flank and immediately supplemented with androstenedione (100 μg/day). Once bilateral flank tumors (both left and right) reached the appropriate size (250–300 mm³), mice were randomized (n ≥ 8 mice/cohort) and the appropriate treatments initiated (control, MK-0646, letrozole (LET), LET + MK-0646, tamoxifen (TAM), TAM + MK-0646) for a total of 28 days. Tumor volumes were measured twice weekly and depicted as percent change relative to day 0. Error bars represent SEM and results are representative of three independent experiments

Fig. 2

MK-0646 increases Insulin Receptor A Isoform expression in MCF-7/AC-1 xenografts. MCF-7/AC-1 xenograft tumors were harvested and immediately flash frozen following 28 days of treatment (Fig. 1). RNA and protein were extracted for Insulin Receptor and IGF-1R quantification by qPCR (a) and western blot analysis (b) as described in the methods. a Absolute IGF1R (Top) and IRA/B isoform (Bottom) copy number normalized to RPL19 housekeeper. b Protein from pooled group replicates isolated at indicated time points were subject to western blotting with indicated antibodies, as described in Materials and Methods section. Error bars represent SEM. *, P < 0.05; **, P < 0.01; ***, P <0.001; **** P < 0.0001

Fig. 3

Persistent mTOR signaling in response to IGF-1R inhibition is overcome by MK-8669. Western Blot analysis of MCF-7/AC-1 (a) and MCF-7/AC-1/IGF-2 (b) cells treated with one or more of the following agents for 24 h at the indicated dose(s): LET (1uM), MK-0646 (1.25, 2.5, 5 ng/uL) and/or MK-8669 (100, 500, 1000 nM). IGF-1 was added for 10 min prior to harvest for the indicated samples

Fig. 4

MK-0646 and MK-8669 synergistically inhibit clonogenesis. a MCF-7/AC-1 (Top) and MCF-7/AC-1/IGF-2 (Bottom) cells were exposed to increasing concentrations of MK-8669 (closed squares), MK-0646 (closed triangles) or the combination MK-8669 + MK-0646 (closed diamonds) and assessed for colony outgrowth (normalized to control). Synergy is indicated by combination index (CI) values, where < 1 is synergistic. Error bars represent SEM and results representative of three independent experiments. b Surface response modeling was employed to demonstrate the multiplicative benefit of adding MK-8669 to existing MK-0646 or vice versa. MCF-7/AC-1 cells were treated with MK-0646 (x-axis), MK-8669 (y-axis) or the combination (z-axis) at varying doses. Percent inhibition is depicted as the fraction affected (Fa)

Fig. 5

In vivo activity of LET +/− MK-8669 +/− MK-0646 in MCF-7/AC-1 xenografts. a As previously described, ovariectomized female nu/nu mice between the ages of 7–8 weeks old were inoculated with MCF-7/AC-1 tumor cells in each flank and immediately supplemented with androstenedione (100 μg/day). Once bilateral flank tumors (both left and right) reached the appropriate size (250–300 mm³), mice were randomized (n ≥ 9 mice/cohort) and the appropriate treatments initiated (Control, LET, MK-8669, MK-0646, MK-8669 + MK-0646, LET + MK-0646, LET + MK-8669 or LET + MK-0646 + MK-8669) for a total of 28 days. Tumor volumes were measured weekly and depicted as percent change relative to day 0. Error bars represent SEM and results are representative of two independent experiments. b Tumors from treatment groups were collected 28 days post treatment initiation, immediately snap frozen and lysates pooled (n ≥ 3 samples/treatment cohort) for Western Blot analysis

Fig. 6

RNAseq analysis reveals both overlapping and non-overlapping gene expression patterns in MCF-7/AC-1 xenografts. a Venn Diagram of the overlapping and unique number of genes according to treatment cohort(s). b Heatmap depicting the most significantly up (red) and down (green) regulated genes in LET + MK-8669 + MK-0646-treated tumors compared to the remaining treatment cohorts. Color bar indicates log-fold expression change compared to control tumors