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Supplementary Figures 1-4 from Inhibition of Tumor Growth Progression by Antiandrogens and mTOR Inhibitor in a Pten-Deficient Mouse Model of Prostate Cancer

Supplementary Figures 1-3, Tables 1-2 from Downregulation of Notch Pathway by a γ-Secretase Inhibitor Attenuates AKT/Mammalian Target of Rapamycin Signaling and Glucose Uptake in an ERBB2 Transgenic Breast Cancer Model

Activation of the phosphoinositide 3-kinase pathway is commonly observed in human prostate cancer. Loss of function of phosphatase and tensin homolog (PTEN) is associated with the activation of AKT and mammalian target of rapamycin (mTOR) in many cancer cell lines as well as in other model systems. However, activation of mTOR is also dependent of kinases other than AKT. Here, we show that activation of mTOR is not dependent on AKT in a prostate-specific PTEN-deficient mouse model of prostate cancer. Pathway bifurcation of AKT and mTOR was noted in both mouse and human prostate tumors. We demonstrated for the first time that cotargeting mTOR and AKT with ridaforolimus/MK-8669 and M1K-2206, respectively, delivers additive antitumor effects in vivo when compared to single agents. Our preclinical data suggest that the combination of AKT and mTOR inhibitors might be more effective in treating prostate cancer patients than current treatment regimens or either treatment alone.

ERBB2/neu and Notch signaling are known to be deregulated in many human cancers. However, pathway cross-talk and dependencies are not well understood. In this study, we use an ERBB2-transgenic mouse model of breast cancer (neuT) to show that Notch signaling plays a critical role in tumor maintenance. Inhibition of the Notch pathway with a γ-secretase inhibitor (GSI) decreased both the Notch and the mammalian target of rapamycin/AKT pathways. Antitumor activity resulting from GSI treatment was associated with decreased cell proliferation as measured by Ki67 and decreased expression of glucose transporter Glut1. Positron emission tomography (PET) imaging showed that the functional consequences of decreased Glut1 translated to reduced glucose uptake and correlated with antitumor effects as measured by micro-computed tomography imaging. The decrease of Glut1 in neuT tumors was also observed in several human breast cancer cell lines following GSI treatment. We provide evidence that ∼27% of ERBB2-positive human breast cancer specimens display high expression of HES1, phospho-S6RP, and GLUT1. Together, these results suggest that pathways downstream of Notch signaling are, at least in part, responsible for promoting tumor growth in neuT and also active in both neuT and a subset of human breast cancers. These findings suggest that GSI may provide therapeutic benefit to a subset of ERBB2-positive breast cancers and that [18F]FDG-PET imaging may be useful in monitoring clinical response. Cancer Res; 70(6); 2476–84

Background and purpose: γ-Secretase inhibitors (GSIs) block NOTCH receptor cleavage and pathway activation and have been under clinical evaluation for the treatment of malignancies such as T-cell acute lymphoblastic leukaemia (T-ALL). The ability of GSIs to decrease T-ALL cell viability in vitro is a slow process requiring >8 days, however, such treatment durations are not well tolerated in vivo. Here we study GSI's effect on tumour and normal cellular processes to optimize dosing regimens for anti-tumour efficacy. Experimental approach: Inhibition of the Notch pathway in mouse intestinal epithelium was used to evaluate the effect of GSIs and guide the design of dosing regimens for xenograft models. Serum Aβ40 and Notch target gene modulation in tumours were used to evaluate the degree and duration of target inhibition. Pharmacokinetic and pharmacodynamic correlations with biochemical, immunohistochemical and profiling data were used to demonstrate GSI mechanism of action in xenograft tumours. Key results: Three days of >70% Notch pathway inhibition was sufficient to provide an anti-tumour effect and was well tolerated. GSI-induced conversion of mouse epithelial cells to a secretory lineage was time- and dose-dependent. Anti-tumour efficacy was associated with cell cycle arrest and apoptosis that was in part due to Notch-dependent regulation of mitochondrial homeostasis. Conclusions and implications: Intermittent but potent inhibition of Notch signalling is sufficient for anti-tumour efficacy in these T-ALL models. These findings provide support for the use of GSI in Notch-dependent malignancies and that clinical benefits may be derived from transient but potent inhibition of Notch.

Androgen receptors have been shown to play a critical role in prostate cancer. We used ultrasound imaging techniques to track tumor response to antiandrogen and rapamycin treatment in a prostate-specific Pten-deleted mouse model of cancer. Depletion of androgens by either surgical or chemical castration significantly inhibited tumor growth progression without altering the activation of Akt and mammalian target of rapamycin (mTOR). We also showed for the first time that targeting mTOR along with antiandrogen treatment exhibited additive antitumor effects in vivo when compared with single agents. Our preclinical data suggest that combination of antiandrogens with mTOR inhibitors might be more effective in treating androgen-dependent prostate cancer patients. [Cancer Res 2009;69(18):7466–72]

Mutations in NOTCH1 are frequently detected in patients with T-cell acute lymphoblastic leukemia (T-ALL) and in mouse T-ALL models. Treatment of mouse or human T-ALL cell lines in vitro with γ-secretase inhibitors (GSIs) results in growth arrest and/or apoptosis. These studies suggest GSIs as potential therapeutic agents in the treatment of T-ALL. To determine whether GSIs have antileukemic activity in vivo, we treated near-end-stage Tal1/Ink4a/Arf+/− leukemic mice with vehicle or with a GSI developed by Merck (MRK-003). We found that GSI treatment significantly extended the survival of leukemic mice compared with vehicle-treated mice. Notch1 target gene expression was repressed and increased numbers of apoptotic cells were observed in the GSI-treated mice, demonstrating that Notch1 inhibition in vivo induces apoptosis. T-ALL cell lines also exhibit PI3K/mTOR pathway activation, indicating that rapamycin may also have therapeutic benefit. When GSIs are administered in combination with rapamycin, mTOR kinase activity is ablated and apoptosis induced. Moreover, GSI and rapamycin treatment inhibits human T-ALL growth and extends survival in a mouse xenograft model. This work supports the idea of targeting NOTCH1 in T-ALL and suggests that inhibition of the mTOR and NOTCH1 pathways may have added efficacy.

Microbeam radiation therapy is an experimental modality using parallel arrays of thin (100 micro m) slices of synchrotron-generated X rays (microplanar beams, microbeams). We used EMT-6 murine mammary carcinoma subcutaneously inoculated in the hind legs of mice to compare the therapeutic efficacies of single-fraction, unidirectional (1) "co-planar" microbeams (an array of vertically oriented microplanar beams), (2) "cross-planar" microbeams (two arrays of parallel microbeams propagated in the same direction, one with vertically and the other with horizontally oriented microplanar beams), and (3) seamless (broad) beams from the same synchrotron source. The microbeams were 90 micro m wide and were spaced 300 micro m on center; the median energy in all beams was 100 or 118 keV. Tumor ablation rates were 4/8, 4/8 and 6/7 for a 410-, 520- and 650-Gy in-slice cross-planar microbeam dose, respectively, and 1/8, 3/8, 3/7 and 6/8 for a 23-, 30-, 38- and 45-Gy broad-beam dose, respectively. When the data were pooled from the three highest doses (same average tumor ablations of 50-60%), the incidences of normal-tissue acute toxicity (moist desquamation and epilation) and delayed toxicity (failure of hair regrowth) were significantly lower for cross-planar microbeams than broad beams (P0.025). Furthermore, for the highest doses in these two groups, which also had the same tumor ablation rate (75%), not only were the above toxicities lower for the cross-planar microbeams than for the broad beams (P0.02), but severe leg dysfunction was also lower (P0.003). These findings suggest that single-fraction microbeams can ablate tumors at high rates with relatively little normal-tissue toxicity.

The Notch pathway is an evolutionarily conserved regulator of cellular differentiation in many tissues during development and later through adult life. It has been reported that particular cancer t...

Radiotherapeutic doses for malignant gliomas are generally palliative because greater, supposedly curative doses would impart clinically unacceptable damage to nearby vital CNS tissues. To improve radiation treatment for human gliomas, we evaluated microbeam radiation therapy, which utilizes an array of parallel, microscopically thin (

Ovarian cancer, the sixth most frequent cause of cancer death among women in the developed world, is a heterogeneous disease characterized by a diverse set of genetic alterations. These factors underscore the need for more effective treatment options accompanied by biomarker strategies to identify patients who will have a greater likelihood to respond to novel therapy. A combination strategy to target the PI3K pathway with the mTOR inhibitor ridaforolimus and anti-IGF1R antibody dalotuzumab is currently undergoing clinical development. Previous translational work has suggested that low RAS activity, as determined by a RAS gene expression signature score, and high levels of IGF1R pathway activation may enrich for response to this combination therapy. Ranking of tumors in the Moffitt tumor database with a low RAS and high IGF profile suggested that ER+ breast and ovarian cancers are enriched for these putative response biomarkers. Consistent with these observations, clinical responses were noted for several ER+ breast or ovarian cancer patients in a Phase I trial for ridaforolimus and dalotuzumab combination therapy. To provide further support for low RAS and high IGF as response biomarkers, the anti-tumor activity of ridaforolimus and dalotuzumab was assessed in 12 patient derived primary ovarian cancer xenograft models developed at START. These models have been extensively characterized by the South Texas Accelerated Research and Therapeutics (START) group for response to standard of care drugs, and the status of many commonly mutated genes in ovarian cancer. Molecular analyses of these tumors suggest that they represent a diverse cross section of ovarian cancer. Similarly, responses to ridaforolimus and dalotuzumab combination therapy ranged from minimal to significant regression. Importantly, the responsive tumor models were associated with a low RAS gene signature and a moderate to high IGF expression level. Tumors with KRAS mutations or a high RAS gene score were generally resistant to therapy. These results support the further development of low RAS and high IGF as enrichment biomarkers for ridaforolimus and dalotuzumab combination therapy in ovarian carcinoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1868. doi:1538-7445.AM2012-1868

Prostate cancer is the third most common cause of cancer-related deaths in men and is most prevalent in developed countries. The androgen signaling pathway plays a critical role in the development and maintenance of the prostate gland and is hyper-activated in prostate cancer. Additionally, gain of function mutations in the PI3 kinase pathway is associated with progression of this disease. Using the prostate-specific Pten-deleted mouse model of cancer, we show that these two pathways are distinct for tumor growth and disease progression. Depletion of androgens by either surgical or chemical castration significantly inhibits tumor growth without altering the activation of Akt and mTOR. We demonstrate that inhibition of mTOR with ridaforolimus or inhibition of Akt with MK-2206, combined with androgen deprivation (bicalutamide), results in additive anti-tumor effects in vivo when compared to single agent treatment. In this model, we also demonstrate that Akt inhibition does not block mTOR kinase activation, and that inhibiting both Akt and mTOR kinases together significantly enhances anti-tumor efficacy. Our data suggest that the combination of an anti-androgen with an mTOR inhibitor and/or Akt inhibitor may be more effective than standard therapies in treating androgen-dependent prostate cancer patients with an activated PI3 kinase pathway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-284.

c-Met and ErbB family of receptors including EGFR, ErbB-2, ErbB-3 and ErbB-4 are co-expressed in many human tumors including Non-Small Cell Lung Cancers (NSCLC). Several reports suggest that the activation of c-Met and ErbB family receptors is associated with poor prognosis in multiple tumors types. Recently it has been shown that activation of c-Met is common in Erlotinib and Gefitinib-resistant NSCLC patients. Combining c-Met and EGFR inhibitors has been proposed as a strategy for treatment in EGFR inhibitor-refractory lung cancers. To understand the cooperation of these pathways, we used a c-Met activated human NSCLC cell line (EBC-1), where EGFR signaling network is also active. In vitro treatment of EBC-1 cells with a combination of Erlotinib and MK-8033 (an inhibitor of c-Met and RON kinases) resulted in greater growth inhibition relative to treatment with either agent alone. In order to validate these in vitro data, we have tested MK-8033 and EGFR inhibitors (Erlotinib and Gefitinib) in vivo. The data suggests the effect of MK-8033 with Gefitinib or Erlotinib on tumor growth is greater when combined as compared to monotherapy in this preclinical xenograft model. PK data suggests no drug-drug interaction of MK-8033 and EGFR inhibitor when administered together. Exposure of these agents was sufficient to inhibit both kinases in tumors. Our studies provide a rationale for the combination of MK-8033 and Erlotinib or Gefitinib in EGFRi refractory patients with NSCLC. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C209.

Gastric cancer is currently the second most frequent cancer-related cause of death with an incidence rate of approximately one million cases annually. The present worldwide standard of care treatment regimen for gastric cancer is the combination of 5-fluorouracil (5-FU) and platinum analog (cisplatin). Many receptor tyrosine kinases are deregulated in gastric cancer including erbB2, c-Met, and EGFR. The purpose of our study was to dissect the role of c-Met signaling in gastric cancer and test the effects of our c-Met inhibitor (MK-8033) alone and in combination with the standard of care agents 5-FU and cisplatin. As a monotherapy, MK-8033 potently inhibited proliferation in a panel of gastric cancer cell lines in vitro. When administered simultaneously with cisplatin and 5-FU, the effects of the combination were diverse, ranging from additive to antagonistic. Altering the treatment schedule by the addition of 5-FU and cisplatin prior to MK-8033 resulted in an overall combination benefit across the panel. In a gastric cancer xenograft model, while MK-8033 alone significantly inhibited tumor growth progression, the co-administration of 5-FU and cisplatin with MK-8033 showed greater benefit than either the monotherapy or the standard regimen. In vitro cell cycle and survival analysis support the observed in vivo data, and provides a mechanistic rationale for the observed combination benefit. Our data suggest the combination of c-Met inhibitors with standard of care agents 5-FU and cisplatin may be beneficial in gastric cancer patients with activated c-Met signaling. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C204.