Combination inhibition of PI3K and mTORC1 yields durable remissions in mice bearing orthotopic patient-derived xenografts of HER2-positive breast cancer brain metastases

Abstract

Brain metastases represent the greatest clinical challenge in treating HER2-positive breast cancer. We report the development of orthotopic patient-derived xenografts (PDXs) of HER2-expressing breast cancer brain metastases (BCBM), and their use for the identification of targeted combination therapies. Combined inhibition of PI3K and mTOR resulted in durable tumor regressions in three of five PDXs, and therapeutic response was correlated with a reduction in the phosphorylation of 4EBP1, an mTORC1 effector. The two nonresponding PDXs showed hypermutated genomes with enrichment of mutations in DNA-repair genes, which suggests an association of genomic instability with therapeutic resistance. These findings suggest that a biomarker-driven clinical trial of PI3K inhibitor in combination with an mTOR inhibitor should be conducted for patients with HER2-positive BCBM.

Conflict of interest statement

Competing Financial Interests

T.M.R. is a consultant of Novartis and has received a research grant from Novartis. E.P.W. has received research grants from Genentech and Roche. I.E.K. is a consultant of Amgen and has received research funding from Genentech. N.U.L. has received research grants from Genentech, Array Biopharma, GlaxoSmithKline, Kadmon, and Novartis. The remaining authors declare no competing financial interests.

Figures

Figure 1. Establishment of orthotopic HER2-positive BCBM PDXs

(a) Schematic depicting the process of generating orthotopic PDX BCBM models for use in preclinical studies. Fresh brain metastatic tissues from patients with BCBM were grafted directly into the brains of female SCID mice. The xenografts in the brain were explanted, dissociated and transduced with a luciferase gene, and then re-injected into new cohorts of mice. P0, primary graft; P1–P5, passage number in mice. DF-BM: Dana-Farber Brain Metastases samples. (b) Representative histologic and immunophenotypic analyses of two patient surgical biopsies and corresponding PDXs. (Scale bars = 25 μm). (c) Compiled result of PTEN immunohistochemistry performed on 27 human HER2-positive BCBM samples. 0, no staining in > 90% of tumor cells; 1+, weak staining in > 75% of tumor cells; 2+, strong staining in > 75% of tumor cells.

Figure 2. Differential responses of HER2-positive BCBM PDXs to the combination of BKM120/RAD001

(a) Representative bioluminescence imaging analysis of mice bearing DF-BM355 tumor before and after treatment with combined BKM120 (30 mg/kg) and RAD001 (7.5 mg/kg), n = 5. (b) Representative MRI of DF-BM355-bearing mice treated with vehicle control or combined BKM120 with RAD001, n = 3. (c) Quantification of the regions of interest (ROI) determined at each imaging time point, n = 2. (d) Kaplan–Meier survival of DF-BM355-bearing mice treated with vehicle control or BKM120 + RAD001, n = 6. (e) IHC analyses of p-4EBP1, p-S6RP, Ki67 and cleaved caspase-3 on DF-BM355 tumors treated for 4 days with indicated treatments (Scale bars = 25 μm). Graphs represent mean ± s.d. (n = 6–10 images per group, *P < 0.05, **P < 0.01, one-way ANOVA followed by Dunnett’s test). (f) Kaplan–Meier survival of mice bearing DF-BM354, DF-BM463, DF-BM507, and DF-BM590 with vehicle control or compound as indicated. n = 5–9. (g) Transcriptome analysis of AKT/mTOR-dependent signature genes from brain xenograft tumor tissues from untreated mice. Boxplots correspond to the first and third quartiles with upper and lower whisker extending to the highest value that is within 1.5 times the interquartile range (n = 4–6 per group, P = 0.0004, Student’s t-test). (h) Number of somatic mutations in HER2-positive BCBM PDXs identified by WES. (i) Mutational profiling of a panel of DNA repair genes.