Combined PI3K/mTOR and MEK inhibition provides broad antitumor activity in faithful murine cancer models

Abstract

Purpose: Anticancer drug development is inefficient, but genetically engineered murine models (GEMM) and orthotopic, syngeneic transplants (OST) of cancer may offer advantages to in vitro and xenograft systems.

Experimental design: We assessed the activity of 16 treatment regimens in a RAS-driven, Ink4a/Arf-deficient melanoma GEMM. In addition, we tested a subset of treatment regimens in three breast cancer models representing distinct breast cancer subtypes: claudin-low (T11 OST), basal-like (C3-TAg GEMM), and luminal B (MMTV-Neu GEMM).

Results: Like human RAS-mutant melanoma, the melanoma GEMM was refractory to chemotherapy and single-agent small molecule therapies. Combined treatment with AZD6244 [mitogen-activated protein-extracellular signal-regulated kinase kinase (MEK) inhibitor] and BEZ235 [dual phosphoinositide-3 kinase (PI3K)/mammalian target of rapamycin (mTOR) inhibitor] was the only treatment regimen to exhibit significant antitumor activity, showed by marked tumor regression and improved survival. Given the surprising activity of the "AZD/BEZ" combination in the melanoma GEMM, we next tested this regimen in the "claudin-low" breast cancer model that shares gene expression features with melanoma. The AZD/BEZ regimen also exhibited significant activity in this model, leading us to testing in even more diverse GEMMs of basal-like and luminal breast cancer. The AZD/BEZ combination was highly active in these distinct breast cancer models, showing equal or greater efficacy compared with any other regimen tested in studies of over 700 tumor-bearing mice. This regimen even exhibited activity in lapatinib-resistant HER2(+) tumors.

Conclusion: These results show the use of credentialed murine models for large-scale efficacy testing of diverse anticancer regimens and predict that combinations of PI3K/mTOR and MEK inhibitors will show antitumor activity in a wide range of human malignancies.

Figure 1

RAS-driven melanoma is refractory to standard chemotherapy but is sensitive to combined PI3K/mTOR/MEK inhibition. TRIA mice were stratified by tumor size and randomly assigned to a treatment cohort. A, of the 15 treatment regimens tested, combined AZD/ BEZ treatment was the only regimen that produced tumor regression as measured by percent change in tumor volume at day 21.B,waterfall plot distribution of tumor response from combined AZD/BEZ compared with the average tumor size of untreated animals on day 21. AZD/BEZ response represents the best response seen on day 21 or beyond. Negative values indicate tumor shrinkage. C, combined AZD/BEZ treatment prolonged median survival from 21 to 61 days; MS, median survival.

Figure 2

Transcriptional analysis of murine models of mammary carcinoma and melanoma. A, an unsupervised cluster of all probes with a log₂ absolute expression value greater than 2 on at least 3 microarrays (2,584 probes). The colored bars correspond to the region of the cluster for the gene lists in parts C to E. B, highlighted is the location in the dendrogram of the C3-TAg, MMTV-c-neu, claudin-low (including T11 OST tumors), and melanoma tumors (including TRIA tumors). Below are markers of the claudin-low subtype as defined by Prat and colleagues (25). C, a cluster of genes that are downregulated in both claudin-low and melanoma tumors. D, a cluster of genes that are upregulated in tumors from 6 different claudin-low and 4 different melanoma models. E, a melanoma specific gene cluster. F, a rank of median differentiation scores for tumors from the indicated breast and melanoma models.

Figure 3

Combined PI3K/mTOR/ MEK inhibition is the most effective treatment regimen in claudin-low breast cancer model. T11 mice were stratified by tumor size and randomly assigned to a treatment cohort. A, combined AZD/BEZ was the most effective treatment regimen as measured by percent change in tumor volume at day 14. B, waterfall plot distribution of tumor response from combined AZD/BEZ compared to the average tumor size of untreated animals on day 14. AZD/ BEZ response represents the best response seen on day 14 or beyond. Negative values indicate tumor shrinkage. C, combined AZD/BEZ treatment prolonged median survival from 15 to 32 days; MS, median survival.

Figure 4

Basal-like breast cancer model is exquisitely sensitive to combined PI3K/mTOR/MEK inhibition. C3-TAg mice were stratified by tumor size and randomly assigned to a treatment cohort. A, combined AZD/BEZ was the most effective treatment regimen, resulting in a median percent change in tumor volume at day 21 of —84%. B, waterfall plot distribution of tumor response from combined AZD/BEZ compared with the average tumor size of untreated animals on day 21. AZD/BEZ response represents the best response seen on day 21 or beyond. Negative values indicate tumor shrinkage. C, combined AZD/BEZ treatment prolonged median survival from 33 to 71 days; MS, median survival.

Figure 5

A GEM model of luminal breast cancer is exceptionally sensitive to combined PI3K/mTOR/ MEK inhibition. MMTV-c-neu mice were stratified by tumor size and randomly assigned to a treatment cohort. A, lapatinib and combined AZD/BEZ treatment regimens provide nearly complete tumor regression as measured by percent change in tumor volume at day 21. B, waterfall plot distribution of tumor response from combined AZD/BEZ compared with the average tumor size of untreated animals on day 21. AZD/BEZ response represents the best response seen on day 21 or beyond. Negative values indicate tumor shrinkage. C, combined AZD/ BEZ treatment prolonged median survival from 29 to 173 days, whereas lapatinib prolonged median survival to only 112 days; MS, median survival.

Figure 6

Inhibition of AZD6244 and BEZ235 targets in tumor-derived cell lines. To evaluate the molecular response to AZD6244, BEZ235, or combined AZD/ BEZ treatment, tumor-derived cell lines from TRIA, T11, and C3Tag tumors were treated for 24 hours with AZD6244 (1 µmol/L) and/or BEZ235 (250 nmol/L) and evaluated by Western blot analysis for target inhibition. Single-agent AZD led to PI3K activation (as evidenced by increased S473 phosphorylation of AKT), whereas single-agent BEZ led to ERK activation (as evidenced by increased phosphorylation of ERK1/2). Combined treatment with AZD/BEZ produced combined inhibition of the PI3K/mTOR and MEK/ERK pathways.

Figure 7

Combined PI3K/mTOR/MEK inhibition is an effective treatment regimen in lapatinib resistant HER2⁺ breast cancer. A, MMTV-c-neu mice stratified by tumor size and assigned to continuous treatment with lapatinib were followed until tumors progressed through treatment. Resistant tumor were harvested and passaged into the mammary fat pad of 6-to 8-week-old female FVB mice. Following injection, tumors were allowed to reach minimum size of at least 5 mm in any one-dimension before initiating second line therapy (either rechallenge of lapatinib or dual AZD/BEZ).B, retreatment with lapatinib has no effect on tumor growth, whereas the combined AZD/BEZ treatment results in median tumor regression of −57%, as measured by percent change in tumor volume at day 21. C, combined AZD/BEZ treatment prolonged median survival from 29 to 113 days; MS, median survival. D, lapatinib-resistant MMTV-c-neu tumors display upregulated phosphorylation of AKT, MEK1/2, and ERK1/2. Treatment of mice bearing lapatinib resistant tumors with AZB/BEZ returns phosphorylation of AKT, MEK1/2, and ERK1/2 to preresistant levels.