Supplementary Figure 8 from Constitutive Activation of Signal Transducers and Activators of Transcription Predicts Vorinostat Resistance in Cutaneous T-Cell Lymphoma

Supplementary Figure 7 from Constitutive Activation of Signal Transducers and Activators of Transcription Predicts Vorinostat Resistance in Cutaneous T-Cell Lymphoma

Supplementary Figure 5 from Constitutive Activation of Signal Transducers and Activators of Transcription Predicts Vorinostat Resistance in Cutaneous T-Cell Lymphoma

Supplementary Figure 2 from Constitutive Activation of Signal Transducers and Activators of Transcription Predicts Vorinostat Resistance in Cutaneous T-Cell Lymphoma

Supplementary Figure 4 from Constitutive Activation of Signal Transducers and Activators of Transcription Predicts Vorinostat Resistance in Cutaneous T-Cell Lymphoma

Supplementary Figure 1 from Constitutive Activation of Signal Transducers and Activators of Transcription Predicts Vorinostat Resistance in Cutaneous T-Cell Lymphoma

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

Supplementary Figure 3 from Constitutive Activation of Signal Transducers and Activators of Transcription Predicts Vorinostat Resistance in Cutaneous T-Cell Lymphoma

Supplementary Figure 6 from Constitutive Activation of Signal Transducers and Activators of Transcription Predicts Vorinostat Resistance in Cutaneous T-Cell Lymphoma

Aberrant kinase activation resulting from mutation, amplification, or translocation can drive growth and survival in a subset of human cancer. FGFR2 is amplified in breast and gastric cancer, and we report here the first characterization of FGFR2 gene amplification in colorectal cancer in the NCI-H716 colorectal cancer cell line. FGFR2 is highly expressed and activated in NCI-H716 cells, and FGFR selective small molecule inhibitors or FGFR2 shRNA strongly inhibited cell viability in vitro, indicating "addiction'' of NCI-H716 cells to FGFR2. NCI-H716 growth in a xenograft model was also inhibited by an FGFR small molecule inhibitor. FGFR2 was required for activation of multiple downstream signaling proteins including AKT, ERK, S6RP and NFKB. Inhibition of downstream kinases such as AKT or ERK alone had modest effects on proliferation, whereas combined inhibition of AKT and ERK signaling resulted in a loss of viability similar to FGFR2 inhibition. We identified elevated FGFR2 expression in a small subset of primary colorectal cancer, however FGFR2 amplification was not observed. Although FGFR2 amplification is not common in primary colon cancer or lymph node and liver metastases, other subsets of colorectal cancer such as ascites, from which the NCI-H716 cell line was derived, have yet to be tested. These results suggest that emerging FGFR inhibitor therapeutics may have efficacy in a subset of colon cancer driven by FGFR2 amplification.

Abstract Vintafolide (also known as EC145 or MK-8109), is a small molecule drug conjugate for the treatment of cancers expressing high affinity folate receptor (FR). Vintafolide consists of the anti-mitotic vinca alkaloid desacetylvinblastine monohydrazide (DAVLBH) chemically linked to folic acid. Binding of vintafolide to FR located on the cell surface delivers the chemotherapy payload directly to the tumor cell. Clinical investigations of vintafolide are underway in ovarian and lung cancer, indications with a high prevalence of FR expression. Triple negative breast cancer (TNBC) may represent an additional indication for vintafolide due to the prevalence of FR expression (∼30%), sensitivity to vinca alkaloids, and unmet medical need. Vintafolide and/or DAVLBH were evaluated in preclinical models of TNBC as monotherapies and in combination with taxanes (paclitaxel or docetaxel). Taxanes represent a commonly used standard of care therapy for TNBC. A panel of TNBC cell lines was generally sensitive to DAVLBH, with IC50s ranging from 4-67 nM. In the majority of cell lines tested, DAVLBH in combination with paclitaxel provided combination benefit and induced more cell death than either single agent. Vintafolide and DAVLBH were further evaluated in vivo in the FR-high MDA-MB-231 and FR-low CAL51 TNBC xenograft models. Vintafolide was dosed at its MTD of 9.6 mg/kg three times per week (TIW), and at 1.5 mg/kg TIW. DAVLBH was dosed at its MTD of 0.77 mg/kg TIW. Mice were dosed for 3 weeks followed by a 6 week follow-up. Both vintafolide doses resulted in marked MDA-MB-231 tumor regressions of 56-78% at the end of therapy (Day 21) and 75% cures (6 of 8 mice) over the 6 week follow-up period. In the CAL51 model, vintafolide produced 8% and 76% tumor growth inhibition (TGI), respectively, at the 1.5 and 9.6 mg/kg doses, and no cures. DAVLBH was less efficacious compared to vintafolide, giving 96% TGI in the MDA-MB-231 model and 46% TGI in the CAL51 model. In mechanism of action studies in the MDA-MB-231 xenograft model, a dose of vintafolide that gave tumor regressions/cures was associated with increased phospho-histone H3 staining, indicative of a mitotic block of tumor cells, and consistent with the mechanism of action of vinca alkaloids. In combination therapy experiments in vivo, docetaxel (20 mg/kg weekly) monotherapy achieved near tumor stasis (98% TGI) of MDA-MB-231 tumors over 3 weeks of therapy, but all tumors re-grew upon cessation of treatment. Since vintafolide monotherapy at 1.5 mg/kg TIW gave significant regressions and cures, a combination benefit with docetaxel could not be determined. However, DAVLBH and docetaxel combination therapy delayed tumor re-growth over single agents upon follow-up, suggesting benefit of a vinca alkaloid / taxane combination. Taken together, these preclinical data support further investigation of vintafolide monotherapy and in combination with taxane therapy in TNBC. Citation Format: Brian B. Haines, Jennifer O'Neil, Marlene C. Hinton, Christopher Ware, Tammie C. Yeh, Tianxiao Sun, Kristen L. Picard, Theresa Zhang, Emmett V. Schmidt, Isabelle Dussault. Preclinical activity of Vintafolide/MK-8109 monotherapy and in combination with standard of care therapy in triple-negative breast cancer models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1677. doi:10.1158/1538-7445.AM2014-1677

Most non-small cell lung cancer (NSCLC) patients harboring activating epidermal growth factor receptor (EGFR) mutations respond to tyrosine kinase inhibitor (TKI) therapy. However, about 30% exhibit primary resistance to EGFR TKI therapy. Here we report that Met protein expression and phosphorylation were associated with primary resistance to EGFR TKI therapy in NSCLC patients harboring EGFR mutations, implicating Met as a de novo mechanism of resistance. In a separate patient cohort, Met expression and phosphorylation were also associated with development of NSCLC brain metastasis and were selectively enriched in brain metastases relative to paired primary lung tumors. A similar metastasis-specific activation of Met occurred in vitro in the isogenous cell lines H2073 and H1993, which are derived from the primary lung tumor and a metastasis, respectively, from the same patient. We conclude that Met activation is found in NSCLC before EGFR-targeted therapy and is associated with both primary resistance to EGFR inhibitor therapy and with the development of metastases. If confirmed in larger cohorts, our analysis suggests that patient tumors harboring both Met activation and EGFR mutation could potentially benefit from early intervention with a combination of EGFR and Met inhibitors.

Abstract 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

Aberrant activation of the serine/threonine kinase Akt, a central node of the PI3K pathway, has been found in a significant proportion of human solid tumors, making Akt an attractive target for therapeutic intervention. MK-2206 is a potent inhibitor of Akt isozymes 1, 2, and 3 with in vitro IC50 values of 8, 12, and 65 nM, respectively. The compound is an allosteric inhibitor of Akt, requiring the presence of the Pleckstrin homology domain for activity. As a consequence, MK-2206 is highly selective against Akt, exhibiting no inhibitory activities against over 250 protein kinases when tested at 1\#956;M. In several cancer cell lines, MK-2206 potently inhibited Akt1 kinase activity (IC50 \#8776; 20 nM), and blocked Akt2 and Akt3 activities 2- to 6-fold less potently. MK-2206 potently inhibited phosphorylation of T308 and S473 of Akt in these cell lines and prevented Akt-mediated phosphorylation of down-stream signaling molecules, including TSC2, PRAS40 and ribosomal S6 proteins. MK-2206 exhibited potent anti-proliferative activity against a number of cancer cell lines harboring one or more of the following genetic defects: 1) constitutive activation of receptor tyrosine kinases such as HER2; 2) PTEN mutation, 3) PI3KCA mutation, and 4) Akt2 amplification. In nude mice bearing A2780 ovarian cancer xenografts, a single oral dose of MK-2206 at 240 mg/kg caused sustained inhibition (>70 %) of phospho-Akt1/2 (T308 and S473) in the tumors. In the same tumor model, MK-2206 inhibited tumor growth by \#8776; 60% when administrated orally at 240 mg/kg per day three times a week. These preclinical results support further clinical development of MK-2206. Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 3714.

Two genetically engineered, conditional mouse models of lung tumor formation, K-ras(LSL-G12D) and K-ras(LSL-G12D)/p53(LSL-R270H), are commonly used to model human lung cancer. Developed by Tyler Jacks and colleagues, these models have been invaluable to study in vivo lung cancer initiation and progression in a genetically and physiologically relevant context. However, heterogeneity, multiplicity and complexity of tumor formation in these models make it challenging to monitor tumor growth in vivo and have limited the application of these models in oncology drug discovery. Here, we describe a novel analytical method to quantitatively measure total lung tumor burden in live animals using micro-computed tomography imaging. Applying this methodology, we studied the kinetics of tumor development and response to targeted therapy in vivo in K-ras and K-ras/p53 mice. Consistent with previous reports, lung tumors in both models developed in a time- and dose (Cre recombinase)-dependent manner. Furthermore, the compound K-ras(LSL-G12D)/p53(LSL-R270H) mice developed tumors faster and more robustly than mice harboring a single K-ras(LSL-G12D) oncogene, as expected. Erlotinib, a small molecule inhibitor of the epidermal growth factor receptor, significantly inhibited tumor growth in K-ras(LSL-G12D)/p53(LSL-R270H) mice. These results demonstrate that this novel imaging technique can be used to monitor both tumor progression and response to treatment and therefore supports a broader application of these genetically engineered mouse models in oncology drug discovery and development.

Aberrant c-Met activity has been implicated in the pathogenesis of a variety of human tumors and is therefore an attractive target for therapeutic intervention. We have identified MK-8033, a highly selective inhibitor of c-Met and Ron kinases. MK-8033 potently inhibits the kinase activity of c-Met (IC50 1.3 nM) and Ron (IC50 14 nM) while exhibiting no inhibitory activities against over 220 protein kinases when tested at 1\#956;M. The compound is a novel ATP-competitive inhibitor that is equally potent against wild type c-Met and naturally occurring oncogenic c-Met mutants, including activation loop mutants. MK-8033 inhibits both ligand stimulated and constitutive c-Met phosphosphorylation. As a result c-Met driven in vitro cell proliferation and scattering are inhibited by MK-8033. Genomic amplification and constitutive activation of c-Met are predictors of tumor cell line response to treatment in vitro. In a gastric cancer xenograft model driven by MET amplification, MK-8033 inhibits autophosphorylation of c-Met docking site tyrosine residues and suppresses downstream signaling events such as ERK1/2 and Akt phosphorylation. Tumor growth is inhibited at well tolerated oral doses of the compound in MET amplified gastric and NSCLC xenograft models. These preclinical data provide support for ongoing clinical evaluation of MK-8033 as a potential targeted therapy for patients with c-Met driven tumors. Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 1751.

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 gamma-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.