Cancer Therapy: Preclinical In vivo Antitumor Activity of MEK and Phosphatidylinositol 3-Kinase Inhibitors in Basal-Like Breast Cancer Models Klaus P. Hoeflich,2 Carol O’Brien,1Zachary Boyd,1Guy Cavet,3 Steve Guerrero,3 Kenneth Jung,3 To m Ja n u a r i o , 1 Heidi Savage,1Elizabeth Punnoose,1Tom Truong,2 Wei Zhou,2 Leanne Berry,2 Lesley Murray,2 Lukas Amler,1Marcia Belvin,4 Lori S. Friedman,4 and Mark R. Lackner1 Abstract Purpose: The pathways underlying basal-like breast cancer are poorly understood, and as yet, there is no approved targeted therapy for this disease.We investigated the role of mitogen- activated protein kinase kinase (MEK) and phosphatidylinositol 3-kinase (PI3K) inhibitors as targeted therapies for basal-like breast cancer. Experimental Design: We used pharmacogenomic analysis of a large panel of breast cancer cell lines with detailed accompanying molecular information to identify molecular predictors of response to a potent and selective inhibitor of MEK and also to define molecular mechanisms underlying combined MEK and PI3K targeting in basal-like breast cancer. Hypotheses were confirmed by testing in multiple tumor xenograft models. Results:We found that basal-like breast cancer models have an activated RAS-like transcriptional program and show greater sensitivity to a selective inhibitor of MEK compared with models rep- resentative of other breast cancer subtypes.We also showed that loss of PTEN is a negative pre- dictor of response to MEK inhibition, that treatment with a selective MEK inhibitor caused up-regulation of PI3K pathway signaling, and that dual blockade of both PI3K and MEK/extracel- lular signal ^ regulated kinase signaling synergized to potently impair the growth of basal-like breast cancer models in vitro and in vivo. Conclusions: Our studies suggest that single-agent MEK inhibition is a promising therapeutic modality for basal-like breast cancers with intact PTEN, and also provide a basis for rational combination of MEK and PI3K inhibitors in basal-like cancers with both intact and deleted PTEN. Recent molecular profiling studies have suggested that breast poor prognosis if untreated, and significant clinical benefit cancers represent at least three distinct diseases with a common from the HER2 targeting monoclonal antibody trastuzumab tissue of origin but different underlying biological aberrations. (Herceptin, Genentech; ref. 3). Basal-like breast cancers Gene expression studies have shown that primary breast tumors typically lack expression of HER2, ER, and progesterone may be classified into at least three major subtypes by receptor and hence overlap with tumors immunohistochemi- unsupervised hierarchical clustering analysis and that the cally defined as ‘‘triple-negative’’ tumors (4). Basal-like breast subtypes have different prognostic outcomes in terms of patient cancers have a poor prognosis and currently have not been survival (1). Luminal breast cancers are typically estrogen shown to respond to any targeted therapy and thus represent a receptor (ER) positive and characterized by coordinate expres- particularly pressing unmet medical need (5). Interestingly, the sion of a number of epithelial specific genes, a relatively good majority of basal-like breast cancers express high levels of the prognosis, and good response rates to targeted hormonal epidermal growth factor receptor (EGFR) and have an EGFR- therapies (2). HER2-positive breast cancers are characterized by associated gene expression signature (6). Because growth HER2 high-level gene amplification of the oncogene, relatively factor stimulation of EGFR results in potent activation of the signaling pathway composed of RAS, RAF, mitogen-activated protein kinase (MAPK) kinase (MEK), and extracellular Authors’ Affiliations: 1Development Oncology Diagnostics, 2Translational 3 4 signal–regulated kinase (ERK; ref. 7), we sought to determine Oncology, Bioinformatics, and Cancer Signaling Pathways, Genentech, Inc., South San Francisco, California whether the downstream arm of this pathway might be a target Received2/9/09;revised3/19/09;accepted3/24/09;publishedOnlineFirst6/30/09. of therapeutic intervention in breast cancer. The costs of publication of this article were defrayed in part by the payment of page The protein kinase MEK occupies a crucial signaling node advertisement charges. This article must therefore be hereby marked in accordance downstream of RAS and RAF and directly upstream of ERK and with 18 U.S.C. Section 1734 solely to indicate this fact. Note: Supplementary data for this article are available at Clinical Cancer Research as such has been the subject of intensive drug discovery Online (http://clincancerres.aacrjournals.org/). activities (8). Genetic and biochemical analyses of MEK K. Hoeflich, C. O’Brien, and Z. Boyd contributed equally to this work. function have suggested that MEK activity is necessary for the Requests for reprints: Mark R. Lackner, Genentech, Inc., 1DNA Way, South San transforming and proliferative effects of this pathway, suggest- Francisco, CA 94080. Phone: 650-467-1846; Fax: 650-225-7571; E-mail: [email protected]. ing that therapeutics that completely inhibit MEK function may F 2009 American Association for Cancer Research. have utility in the treatment of cancers driven by activation of doi:10.1158/1078-0432.CCR-09-0317 the RAS/RAF axis. Such efforts have led to the development of www.aacrjournals.org 4649 Clin Cancer Res 2009;15(14) July 15, 2009 Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2009 American Association for Cancer Research. Cancer Therapy: Preclinical cancer based on in vitro analyses (16). Our studies are consistent Translational Relevance with those findings and, moreover, offer extensive in vivo confirmation of the important role MEK signaling plays in We find that basal-like breast cancer models are sensitive basal-like breast cancer. In addition, we also show that activation in vitro in vivo and to the inhibitory effects of mitogen- of the PI3K/Akt pathway by loss of the tumor suppressor PTEN activated protein kinase kinase (MEK) inhibition relative to occurs in a subset of basal-like breast cancer cell line models and luminal and HER2-amplified tumors and identify the tumor is associated with diminished response to MEK inhibition, and suppressor PTEN as a negative predictor factor for that siRNA ablation of PTEN in responsive models blocks cell response to MEK inhibition.We also find that a combination cycle and proliferative changes triggered by MEK inhibition. of MEK and phosphatidylinositol 3-kinase inhibitors Based on the apparent role of PTEN and activation of the PI3K/ results in enhanced efficacy in both PTEN wild-type and Akt pathway in mediating resistance to MEK inhibition, we use PTEN-null basal-like tumors and provide a mechanistic both in vitro and in vivo combination studies with potent and explanation for this observation. Thus, our studies provide selective MEK and PI3K inhibitors to show that simultaneous a rational hypothesis for biomarker evaluation and patient treatment with a MEK inhibitor and a potent and selective stratification in clinical trials seeking to evaluate the efficacy inhibitor of PI3K results in synergistic decreases in cell viability, of MEK and phosphatidylinositol 3-kinase inhibitor enhanced down-regulation of cyclin D1, and increased apoptosis treatment in this poor-prognosis tumor type. specifically in basal-like breast cancer models. molecules such as PD0325901 and AZD6244 that act through a Materials and Methods novel noncompetitive allosteric mechanism of inhibition by binding to a site adjacent to the ATP-binding site and locking Cell lines and cell viability experiments. Details and sources of all the kinase in a closed and catalytically inactive conformation cell lines used in this study have been described previously (11, 17) and (9). This pathway is frequently activated by mutations in RAS are also detailed in Supplementary Materials and Methods. All studies family members or BRAF in a number of solid tumor types, and in this report describing MEK inhibition have used the potent and elegant studies in cell lines and mouse tumor models have selective MEK1/2 inhibitor PD0325901 (Pfizer) to evaluate the effects suggested that mutations in BRAF and, to a lesser extent, KRAS of MEK inhibition in breast cancer preclinical models, with the exception of a small number of confirmatory studies described in may be useful to stratify patients enrolled in clinical trials of Supplementary materials that use a second potent and selective MEK MEK inhibitors (10). We sought to address whether MEK inhibitor (9), purchased from Synthesis MedChem.5 For cell viability inhibition might have therapeutic value in breast cancer studies, cells were plated in quadruplicate at a density of 3,000 per well through evaluation of a large panel of breast cancer cell lines in 384-well plates in normal growth medium and allowed to adhere and tumor xenograft models that have been characterized in overnight. Compounds were added in 10 concentrations based on a detail at the molecular level and shown to reflect many of the 3-fold dilution series. Cell viability was measured 72 h later using the genomic alterations commonly found in breast tumors (11). CellTiter-Glo Luminescent Cell Viability Assay (Promega). The concen- We also set out to examine the relationship between RAS/ tration of drug resulting in 50% of maximal inhibition (EC50) was RAF/MEK/ERK pathway activity and phosphatidylinositol calculated from a four-parameter