Network quantification of EGFR signaling unveils potential for targeted combination therapy.

The epidermal growth factor receptor (EGFR) signaling network is activated in most solid tumors, and small-molecule drugs targeting this network are increasingly available. However, often only specific combinations of inhibitors are effective. Therefore, the prediction of potent combinatorial treatments is a major challenge in targeted cancer therapy. In this study, we demonstrate how a model-based evaluation of signaling data can assist in finding the most suitable treatment combination. We generated a perturbation data set by monitoring the response of RAS/PI3K signaling to combined stimulations and inhibitions in a panel of colorectal cancer cell lines, which we analyzed using mathematical models. We detected that a negative feedback involving EGFR mediates strong cross talk from ERK to AKT. Consequently, when inhibiting MAPK, AKT activity is increased in an EGFR-dependent manner. Using the model, we predict that in contrast to single inhibition, combined inactivation of MEK and EGFR could inactivate both endpoints of RAS, ERK and AKT. We further could demonstrate that this combination blocked cell growth in BRAF- as well as KRAS-mutated tumor cells, which we confirmed using a xenograft model.

PK-PD modeling of combination efficacy effect from administration of the MEK inhibitor GDC-0973 and PI3K inhibitor GDC-0941 in A2058 xenografts.

PURPOSE: Mutations and activations of the MEK and PI3K pathways are associated with the development of many cancers. GDC-0973 and GDC-0941 are inhibitors of MEK and PI3K, respectively, currently being evaluated clinically in combination as anti-cancer treatment. The objective of these studies was to characterize the relationship between the plasma concentrations of GDC-0973 and GDC-0941 administered in combination and efficacy in A2058 melanoma xenograft. METHODS: GDC-0973 and GDC-0941 were administered to A2058 tumor-bearing mice daily (QD) or every third day (Q3D) either as single agents or in combination. A semi-mechanistic population anti-cancer model was developed to simultaneously describe the tumor growth following QD/Q3D single-agent and QD combination treatments. The interaction terms ψ included in the model were used to assess whether the combination was additive. Using this model, data from the Q3D combination regimen were simulated and compared with the observed tumor volumes. RESULTS: The model consisting of saturable tumor growth provided the best fit of the data. The estimates for ψ were not significantly different from 1, suggesting an additive effect of GDC-0973 and GDC-0941 on tumor growth inhibition. The population rate constants associated with tumor growth inhibition for GDC-0973 and GDC-0941 were 0.00102 and 0000651 µM(-1) h(-1), respectively. Using the model based on single-agent and QD combination efficacy data, simulations adequately described the tumor growth from the Q3D combination regimen. CONCLUSIONS: These findings suggest that, based on minimal data, it is possible to predict the effects of various combinations preclinically and also assess the potential clinical efficacy of combinations using human pharmacokinetic inputs.

GDC-0941, a novel class I selective PI3K inhibitor, enhances the efficacy of docetaxel in human breast cancer models by increasing cell death in vitro and in vivo.

PURPOSE: Docetaxel is a front-line standard-of-care chemotherapeutic drug for the treatment of breast cancer. Phosphoinositide 3-kinases (PI3K) are lipid kinases that regulate breast tumor cell growth, migration, and survival. The current study was intended to determine whether GDC-0941, an orally bioavailable class I selective PI3K inhibitor, enhances the antitumor activity of docetaxel in human breast cancer models in vitro and in vivo. EXPERIMENTAL DESIGN: A panel of 25 breast tumor cell lines representing HER2+, luminal, and basal subtypes were treated with GDC-0941, docetaxel, or the combination of both drugs and assayed for cellular viability, modulation of PI3K pathway markers, and apoptosis induction. Drug combination effects on cellular viability were also assessed in nontransformed MCF10A human mammary epithelial cells. Human xenografts of breast cancer cell lines and patient-derived tumors were used to assess efficacy of GDC-0941 and docetaxel in vivo. RESULTS: Combination of GDC-0941 and docetaxel decreased the cellular viability of breast tumor cell lines in vitro but to variable degrees of drug synergy. Compared with nontransformed MCF10A cells, the addition of both drugs resulted in stronger synergistic effects in a subset of tumor cell lines that were not predicted by breast cancer subtype. In xenograft models, GDC-0941 enhanced the antitumor activity of docetaxel with maximum combination efficacy observed within 1 hour of administering both drugs. GDC-0941 increased the rate of apoptosis in cells arrested in mitosis upon cotreatment with docetaxel. CONCLUSION: GDC-0941 augments the efficacy of docetaxel by increasing drug-induced apoptosis in breast cancer models.

Intermittent administration of MEK inhibitor GDC-0973 plus PI3K inhibitor GDC-0941 triggers robust apoptosis and tumor growth inhibition.

Combinations of MAP/ERK kinase (MEK) and phosphoinositide 3-kinase (PI3K) inhibitors have shown promise in preclinical cancer models, leading to the initiation of clinical trials cotargeting these two key cancer signaling pathways. GDC-0973, a novel selective MEK inhibitor, and GDC-0941, a class I PI3K inhibitor, are in early stage clinical trials as both single agents and in combination. The discovery of these selective inhibitors has allowed investigation into the precise effects of combining inhibitors of two major signaling branches downstream of RAS. Here, we investigated multiple biomarkers in the mitogen-activated protein kinase (MAPK) and PI3K pathway to search for points of convergence that explain the increased apoptosis seen in combination. Using washout studies in vitro and alternate dosing schedules in mice, we showed that intermittent inhibition of the PI3K and MAPK pathway is sufficient for efficacy in BRAF and KRAS mutant cancer cells. The combination of GDC-0973 with the PI3K inhibitor GDC-0941 resulted in combination efficacy in vitro and in vivo via induction of biomarkers associated with apoptosis, including Bcl-2 family proapoptotic regulators. Therefore, these data suggest that continuous exposure of MEK and PI3K inhibitors in combination is not required for efficacy in preclinical cancer models and that sustained effects on downstream apoptosis biomarkers can be observed in response to intermittent dosing.

Neuregulin-1-mediated autocrine signaling underlies sensitivity to HER2 kinase inhibitors in a subset of human cancers.

HER2 kinase inhibitors, such as lapatinib, have demonstrated clinical efficacy in HER2-amplified breast cancers. By profiling nearly 700 human cancer cell lines, we identified a subset of non-HER2 amplified cancer cells with striking sensitivity to HER2 kinase inhibition-particularly from head and neck tumors. These cells were found to depend on a neuregulin-1 (NRG1)-mediated autocrine loop driving HER3 activation, which can be disrupted by lapatinib. Elevated NRG1 expression and activated HER3 are strongly associated with lapatinib sensitivity in vitro, and these biomarkers were enriched in a subset of primary head and neck cancer samples. The findings suggest that patients with NRG1-driven tumors lacking HER2 amplification may derive significant clinical benefit from HER2:HER3-directed therapies.

Navitoclax enhances the efficacy of taxanes in non-small cell lung cancer models.

PURPOSE: To explore the potential of navitoclax in combination with taxane-based chemotherapy in the treatment of non-small cell lung cancer (NSCLC) by defining mechanism of synergy and identifying correlative biomarkers. EXPERIMENTAL DESIGN: We treated a panel of NSCLC lines with a dose matrix of paclitaxel and navitoclax (formerly ABT-263), an inhibitor of Bcl-2, Bcl-x(L), and Bcl-w (1), and evaluated synergy. We next used time-lapse microscopy to explore mechanism of synergy. Finally, we developed an immunohistochemical assay and assessed prevalence of Bcl-x(L) in NSCLC tumor tissues. RESULTS: All cell lines exhibit greater than additive response to the combination of navitoclax and a taxane. These results were extended to mouse xenograft tumor models, in which the combination is more efficacious than either single-agent docetaxel or navitoclax. Addition of navitoclax to paclitaxel decreases the time from mitotic entry to cell death and changes cell fate from mitotic slippage to death during mitotic arrest. The relative levels of Bcl-x(L) and Mcl-1 correlate with the extent of synergy, suggesting that cancers with elevated levels of Bcl-x(L) will be relatively resistant to taxane-based therapy but could benefit from the addition of navitoclax to taxane treatment. Finally, a significant percentage of NSCLC patient samples exhibit relatively high Bcl-x(L) levels. CONCLUSIONS: The addition of navitoclax to taxane-based chemotherapy in NSCLC has the potential to increase efficacy, particularly in patients whose tumors express high levels of Bcl-x(L).

Effects of anti-VEGF treatment duration on tumor growth, tumor regrowth, and treatment efficacy.

PURPOSE: Inhibition of the vascular endothelial growth factor (VEGF) axis is the basis of all currently approved antiangiogenic therapies. In preclinical models, anti-VEGF blocking antibodies have shown broad efficacy that is dependent on both tumor context and treatment duration. We aimed to characterize this activity and to evaluate the effects of discontinuation of treatment on the dynamics of tumor regrowth. EXPERIMENTAL DESIGN: We evaluated the effects of anti-VEGF treatment on tumor growth and survival in 30 xenograft models and in genetic mouse models of cancer. Histologic analysis was used to evaluate the effects of treatment on tumor vasculature. We used a variety of treatment regimens to allow analysis of the effects of treatment duration and cessation on growth rate, survival, and vascular density. RESULTS: Preclinical tumor models were characterized for their varied dependence on VEGF, thereby defining models for testing other agents that may complement or augment anti-VEGF therapy. We also found that longer exposure to anti-VEGF monoclonal antibodies delayed tumor growth and extended survival in established tumors from both cell transplants and genetic tumor models and prevented regrowth of a subset of residual tumors following cytoablative therapy. Discontinuation of anti-VEGF in established tumors resulted in regrowth at a rate slower than that in control-treated animals, with no evidence of accelerated tumor growth or rebound. However, more rapid regrowth was observed following discontinuation of certain chemotherapies. Concurrent administration of anti-VEGF seemed to normalize these accelerated growth rates. CONCLUSIONS: In diverse preclinical models, continuous VEGF suppression provides maximal benefit as a single agent, combined with chemotherapy, or as maintenance therapy once chemotherapy has been stopped.

Predictive biomarkers of sensitivity to the phosphatidylinositol 3' kinase inhibitor GDC-0941 in breast cancer preclinical models.

PURPOSE: The class I phosphatidylinositol 3' kinase (PI3K) plays a major role in proliferation and survival in a wide variety of human cancers. A key factor in successful development of drugs targeting this pathway is likely to be the identification of responsive patient populations with predictive diagnostic biomarkers. This study sought to identify candidate biomarkers of response to the selective PI3K inhibitor GDC-0941. EXPERIMENTAL DESIGN: We used a large panel of breast cancer cell lines and in vivo xenograft models to identify candidate predictive biomarkers for a selective inhibitor of class I PI3K that is currently in clinical development. The approach involved pharmacogenomic profiling as well as analysis of gene expression data sets from cells profiled at baseline or after GDC-0941 treatment. RESULTS: We found that models harboring mutations in PIK3CA, amplification of human epidermal growth factor receptor 2, or dual alterations in two pathway components were exquisitely sensitive to the antitumor effects of GDC-0941. We found that several models that do not harbor these alterations also showed sensitivity, suggesting a need for additional diagnostic markers. Gene expression studies identified a collection of genes whose expression was associated with in vitro sensitivity to GDC-0941, and expression of a subset of these genes was found to be intimately linked to signaling through the pathway. CONCLUSION: Pathway focused biomarkers and the gene expression signature described in this study may have utility in the identification of patients likely to benefit from therapy with a selective PI3K inhibitor.

Molecular predictors of response to a humanized anti-insulin-like growth factor-I receptor monoclonal antibody in breast and colorectal cancer.

The insulin-like growth factor-I receptor (IGF-IR) pathway is required for the maintenance of the transformed phenotype in neoplastic cells and hence has been the subject of intensive drug discovery efforts. A key aspect of successful clinical development of targeted therapies directed against IGF-IR will be identification of responsive patient populations. Toward that end, we have endeavored to identify predictive biomarkers of response to an anti-IGF-IR-targeting monoclonal antibody in preclinical models of breast and colorectal cancer. We find that levels of the IGF-IR itself may have predictive value in these tumor types and identify other gene expression predictors of in vitro response. Studies in breast cancer models suggest that IGF-IR expression is both correlated and functionally linked with estrogen receptor signaling and provide a basis for both patient stratification and rational combination therapy with antiestrogen-targeting agents. In addition, we find that levels of other components of the signaling pathway such as the adaptor proteins IRS1 and IRS2, as well as the ligand IGF-II, have predictive value and report on the development of a pathway-focused panel of diagnostic biomarkers that could be used to test these hypotheses during clinical development of IGF-IR-targeting therapies.

In vivo antitumor activity of MEK and phosphatidylinositol 3-kinase inhibitors in basal-like breast cancer models.

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 representative of other breast cancer subtypes. We also showed that loss of PTEN is a negative predictor 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/extracellular 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.

Antitumor efficacy of the novel RAF inhibitor GDC-0879 is predicted by BRAFV600E mutational status and sustained extracellular signal-regulated kinase/mitogen-activated protein kinase pathway suppression.

Oncogenic activation of the BRAF serine/threonine kinase has been associated with initiation and maintenance of melanoma tumors. As such, development of pharmacologic agents to target RAF proteins or their effector kinases is an area of intense investigation. Here we report the biological properties of GDC-0879, a highly selective, potent, and orally bioavailable RAF small-molecule inhibitor. We used extracellular signal-regulated kinase (ERK)-1/2 and mitogen-activated protein kinase/ERK kinase (MEK)-1/2 phosphorylation as biomarkers to explore the relationship between tumor outcome and pharmacodynamic inhibition of the RAF-MEK-ERK pathway. In GDC-0879-treated mice, both cell line- and patient-derived BRAF(V600E) tumors exhibited stronger and more sustained pharmacodynamic inhibition (>90% for 8 hours) and improved survival compared with mutant KRAS-expressing tumors. Despite the involvement of activated RAF signaling in RAS-induced tumorigenesis, decreased time to progression was observed for some KRAS-mutant tumors following GDC-0879 administration. Moreover, striking differences were noted for RAF and MEK inhibition across a panel of 130 tumor cell lines. Whereas GDC-0879-mediated efficacy was associated strictly with BRAF(V600E) status, MEK inhibition also attenuated proliferation and tumor growth of cell lines expressing wild-type BRAF (81% KRAS mutant, 38% KRAS wild type). The responsiveness of BRAF(V600E) melanoma cells to GDC-0879 could be dramatically altered by pharmacologic and genetic modulation of phosphatidylinositol 3-kinase pathway activity. These data suggest that GDC-0879-induced signaling changes are dependent on the point of oncogenic activation within the RAS network. Taken together, these studies increase our understanding of the molecular determinants for antitumor efficacy resulting from RAF pathway inhibition and have implications for therapeutic intervention in the clinic.

Blocking neuropilin-2 function inhibits tumor cell metastasis.

Metastasis, which commonly uses lymphatics, accounts for much of the mortality associated with cancer. The vascular endothelial growth factor (VEGF)-C coreceptor, neuropilin-2 (Nrp2), modulates but is not necessary for developmental lymphangiogenesis, and its significance for metastasis is unknown. An antibody to Nrp2 that blocks VEGFC binding disrupts VEGFC-induced lymphatic endothelial cell migration, but not proliferation, in part independently of VEGF receptor activation. It does not affect established lymphatics in normal adult mice but reduces tumoral lymphangiogenesis and, importantly, functional lymphatics associated with tumors. It also reduces metastasis to sentinel lymph nodes and distant organs, apparently by delaying the departure of tumor cells from the primary tumor. Our results demonstrate that Nrp2, which was originally identified as an axon-guidance receptor, is an attractive target for modulating metastasis.

Quantification of viable tumor microvascular characteristics by multispectral analysis.

Tumor heterogeneity complicates the quantification of tumor microvascular characteristics assessed by dynamic contrast-enhanced MRI (DCE-MRI). To address this issue a novel approach was developed that combines DCE-MRI with diffusion-based multispectral (MS) analysis to quantify the microvascular characteristics of specific tumor tissue populations. Diffusion-based MS segmentation (feature space: apparent diffusion coefficient, T(2) and proton density) was performed to identify tumor tissue populations and the DCE-MRI characteristics were determined for each tissue class. The ability of this MS DCE-MRI technique to detect microvascular changes due to treatment with an antibody (G6-31) to vascular endothelial growth factor-A (VEGF) was evaluated in a tumor xenograft mouse model. Anti-VEGF treatment resulted in a significant reduction in K(trans) for the MS viable tumor tissue class (-0.0034 +/- 0.0022 min(-1), P < 0.01) at 24 hr posttreatment that differ significantly from the change observed in the control group (0.0002 +/- 0.0025 min(-1)). Viable tumor K(trans) for the anti-VEGF group was also reduced 62% relative to the pretreatment values (P < 0.01). Necrotic tissue classes were found to add only noise to DCE-MRI estimates. This approach provides a means to measure physiological parameters within the viable tumor and address the issue of tumor heterogeneity that complicates DCE-MRI analysis.