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. 2013 Oct 1;73(19):6013-23.
doi: 10.1158/0008-5472.CAN-13-1191. Epub 2013 Aug 5.

Combination of antibody that inhibits ligand-independent HER3 dimerization and a p110α inhibitor potently blocks PI3K signaling and growth of HER2+ breast cancers

Joan T Garrett  1 , Cammie R Sutton, Richard Kurupi, Carl Uli Bialucha, Seth A Ettenberg, Scott D Collins, Qing Sheng, Jerry Wallweber, Lisa Defazio-Eli, Carlos L Arteaga
Affiliations

Combination of antibody that inhibits ligand-independent HER3 dimerization and a p110α inhibitor potently blocks PI3K signaling and growth of HER2+ breast cancers

Joan T Garrett et al. Cancer Res. .

Abstract

We examined the effects of LJM716, an HER3 (ERBB3) neutralizing antibody that inhibits ligand-induced and ligand-independent HER3 dimerization, as a single agent and in combination with BYL719, an ATP competitive p110α-specific inhibitor, against HER2-overexpressing breast and gastric cancers. Treatment with LJM716 reduced HER2-HER3 and HER3-p85 dimers, P-HER3 and P-AKT, both in vitro and in vivo. Treatment with LJM716 alone markedly reduced growth of BT474 xenografts. The combination of LJM716/lapatinib/trastuzumab significantly improved survival of mice with BT474 xenografts compared with lapatinib/trastuzumab (P = 0.0012). LJM716 and BYL719 synergistically inhibited growth in a panel of HER2+ and PIK3CA mutant cell lines. The combination also inhibited P-AKT in HER2-overexpressing breast cancer cells and growth of HER2+ NCI-N87 gastric cancer xenografts more potently than LJM716 or BYL719 alone. Trastuzumab-resistant HER2+/PIK3CA mutant MDA453 xenografts regressed completely after 3 weeks of therapy with LJM716 and BYL719, whereas either single agent inhibited growth only partially. Finally, mice with BT474 xenografts treated with trastuzumab/LJM716, trastuzumab/BYL719, LJM716/BYL719, or trastuzumab/LJM716/BYL719 exhibited similar rates of tumor regression after 3 weeks of treatment. Thirty weeks after treatment discontinuation, 14% of mice were treated with trastuzumab/LJM716/BYL719, whereas >80% in all other treatment groups were sacrificed due to a recurrent large tumor burden (P = 0.0066). These data suggest that dual blockade of the HER2 signaling network with an HER3 antibody that inhibits HER2-HER3 dimers in combination with a p110α-specific inhibitor in the absence of a direct HER2 antagonist is an effective treatment approach against HER2-overexpressing cancers.

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Figures

Figure 1
Figure 1. LJM716 inhibits HER3-PI3K signaling
Breast cancer cell lines were treated with 10 μg/ml of LJM716 over a 24-h time course. Whole cell lysates were prepared and separated in a 7% SDS gel followed by immunoblot analysis with indicated antibodies.
Figure 2
Figure 2. HER3 antibody disrupts HER2/HER3 interactions
A. Mice bearing BT474 or MDA453 xenografts were treated with two doses of 20 mg/kg LJM716 delivered i.p. over a period of 72 h and sacrificed 4 h after the last dose. Formalin fixed paraffin embedded tumor sections were subjected to VeraTag analysis as indicated in Methods. B. BT474 and SKBR3 cells treated with 10 μg/ml LJM716 for 0–4 h. Cell lysates were prepared and were precipitated with a C-terminus HER3 antibody. Antibody pulldowns were next subjected to immunoblot analysis with HER2 and HER3 antibodies.
Figure 3
Figure 3. HER3 antibody in combination with HER2 inhibitors improves survival
A. Female athymic mice were injected with BT474 cells and randomized to vehicle or the indicated combinations of 20 mg/kg LJM716, 20 mg/kg trastuzumab and 100 mg/kg lapatinib. Treatment was administered for 3 weeks. Tumors were measured two to three times a week with calipers. Number of mice per treatment group is indicated in Figure. Each data point represents the mean tumor volume in mm3 + SEM. B. Therapy was stopped at 3 weeks; mice in the lapatinib/trastuzumab, lapatinib/trastuzumab/LJM716 and trastuzumab/LJM716 treatment groups were monitored for tumor re-growth. Plot of overall mouse survival is shown. The x-axis indicates the weeks after discontinuation of treatment. Per institutional guidelines, mice were sacrificed once tumor burden was ≥2000 mm3.
Figure 4
Figure 4. LJM716 and p110α inhibitor synergistically inhibit tumor cell growth and PI3K
A. Heatmap representing percent growth inhibition for the listed cell lines 5 days after treatment with 33 nM (5 μg/ml) of LJM716, 330 nM BYL719 or the combination, relative to untreated cells as assessed by the CellTiterGlo Assay. Values for LJM716 were the average of two independent dose-titration curves. Synergistic inhibition (synergy score ≥2.0) was observed for the following cell lines: EFM192A, AU565, SKBR-3, BT474, MDA361 and MDA453 (all with HER2 gene amplification). Percent inhibition relative to IgG-treated (control) cells is visualized in the form of a heat map colored from blue (0% inhibition) to red (100% inhibition). Cell lines harboring PIK3CA hotspot mutations are highlighted in red. B. Cells were plated (1–5×104 cells/well) in 6-well plates and treated in triplicate with DMSO, 10 μg/ml LJM716 ± 1 μM BYL719. Media and drugs were replenished every 3–4 days. Cells were stained with crystal violet when the DMSO-treated (control) monolayers became confluent, ranging from 14–21 days. Quantification of integrated intensity (% control) is shown (*, p<0.05, t test). C. Cells were seeded in Matrigel and allowed to grow in the absence or presence of 10 μg/ml LJM716 and/or 1 μM BYL719 as indicated. Cell media and drugs were replenished every 3 days. Images shown were recorded 15–19 days after seeding. D. Cells were treated with 10 μg/ml LJM716 ± 1 μM BYL719 for 1 or 24 h. Whole cell lysates were prepared and separated in a 7% SDS gel followed by immunoblot analysis with the indicated antibodies.
Figure 5
Figure 5. Combination of LJM716 and BYL719 inhibits growth of HER2+ xenografts
A. Mice with established HCC1954 or MDA453 xenografts were treated over a 72-h period with two doses of 20 mg/kg LJM716 and three daily doses of 30 mg/kg BYL719. Mice received BYL719 and LJM716 one and 24 h before sacrifice, respectively. Tumor cell lysates were prepared and separated in a 7% SDS gel followed by immunoblot analysis with the indicated antibodies. B. NCI-N87 tumor xenografts were grown in nude mice and treated with IgG (20 mg/kg q 2 days), LJM716 (20 mg/kg q 2 days), BYL719 (12.5 mg/kg daily) or LJM716/BYL719. Treatment of mice in the control and LJM716-treated groups was terminated at 34 days. The remaining groups were kept on treatment for an additional 14 days before the study was terminated. C. Female athymic mice were injected with MDA453 cells and randomized to vehicle or 20 mg/kg LJM716 three times per week and/or 30 mg/kg BYL719 p.o. daily. Treatment was administered for 21 days. Tumors were measured two to three times a week with calipers. Each data point represents the mean tumor volume in mm3 + SEM.
Figure 6
Figure 6. Combination of p110α inhibitor, HER3 antibody and trastuzumab improves survival
A. Female athymic mice were injected with BT474 cells as indicated in Methods. Once tumors reached a volume of ≥400 mm3, there were randomized to treatment with vehicle (controls) or the indicated combinations of 20 mg/kg LJM716, 20 mg/kg trastuzumab and 30 mg/kg BYL719. Treatment was administered for 24 days. Tumor diameters were measured two to three times a week with calipers and volume in mm3 was calculated. Each data point represents the mean tumor volume in mm3 + SEM. B, C. After 24 days, treatment was discontinued and mice were monitored for tumor recurrence. The x-axis indicates the number of weeks after treatment discontinuation. B. Plot of tumor-free mice over time after termination of therapy; tumors ≥200 mm3 in volume were scored as recurrences. C. Plot of overall mouse survival. Per institutional guidelines, mice were sacrificed once tumor burden was ≥2000 mm3.
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