Thorsten Füreder

The PI3-Kinase/mTOR-Targeting Drug NVP-BEZ235 Inhibits Growth and IgE-Dependent Activation of Human Mast Cells and Basophils

The phosphoinositide 3-kinase (PI3-kinase) and the mammalian target of rapamycin (mTOR) are two major signaling molecules involved in growth and activation of mast cells (MC) and basophils (BA). We examined the effects of the dual PI3-kinase/mTOR blocker NVP-BEZ235 on growth of normal and neoplastic BA and MC as well as immunoglobulin E (IgE)-dependent cell activation. Growth of MC and BA were determined by measuring 3H-thymidine uptake and apoptosis. Cell activation was determined in histamine release experiments and by measuring upregulation of CD63 and CD203c after challenging with IgE plus anti-IgE or allergen. We found that NVP-BEZ235 exerts profound inhibitory effects on growth of primary and cloned neoplastic MC. In the MC leukemia cell line HMC-1, NVP-BEZ235 showed similar IC50 values in the HMC-1.1 subclone lacking KIT D816V (0.025 µM) and the HMC-1.2 subclone expressing KIT D816V (0.005 µM). Moreover, NVP-BEZ235 was found to exert strong growth-inhibitory effects on neoplastic MC in a xenotransplant-mouse model employing NMR1-Foxn1nu mice. NVP-BEZ235 also exerted inhibitory effects on cytokine-dependent differentiation of normal BA and MC, but did not induce growth inhibition or apoptosis in mature MC or normal bone marrow cells. Finally, NVP-BEZ235 was found to inhibit IgE-dependent histamine release in BA and MC (IC50 0.5–1 µM) as well as anti-IgE-induced upregulation of CD203c in BA and IgE-dependent upregulation of CD63 in MC. In summary, NVP-BEZ235 produces growth-inhibitory effects in immature neoplastic MC and inhibits IgE-dependent activation of mature BA and MC. Whether these potentially beneficial drug effects have clinical implications is currently under investigation.

Safety and feasibility of every-other-week maintenance cetuximab after first-line chemotherapy in patients with recurrent or metastatic head and neck squamous cell cancer

In patients with recurrent and/or metastatic head and neck squamous cell cancer (HNSCC), there are no data about an every‐other‐week cetuximab maintenance schedule after chemotherapy plus cetuximab as first‐line treatment.

BEZ235 impairs gastric cancer growth by inhibition of PI3K/mTOR in vitro and in vivo

Background Gastric cancer at advanced stage of disease is a major health problem and the prognosis with the current therapeutic treatment strategies remains poor. PI3K/mTOR pathway mutations, especially PTEN, PI3K3C and AKT mutations and pS6 overexpression, are found frequently in gastric cancer patients and are linked with poor outcome. Thus, we evaluated the dual PI3K and mTOR inhibitor BEZ235 against gastric cancer in vitro and in vivo.

Dose study of the multikinase inhibitor, LY2457546, in patients with relapsed acute myeloid leukemia to assess safety, pharmacokinetics, and pharmacodynamics

Background: Acute myeloid leukemia (AML) is a life-threatening malignancy with limited treatment options in chemotherapy-refractory patients. A first-in-human dose study was designed to investigate a safe and biologically effective dose range for LY2457546, a novel multikinase inhibitor, in patients with relapsed AML. Methods: In this nonrandomized, open-label, dose escalation Phase I study, LY2457546 was administered orally once a day. Safety, pharmacokinetics, changes in phosphorylation of target kinases in AML blasts, and risk of drug–drug interactions (DDI) were assessed. Results: Five patients were treated at the starting and predicted minimal biologically effective dose of 50 mg/day. The most commonly observed adverse events were febrile neutropenia, epistaxis, petechiae, and headache. The majority of adverse events (81%) were Grade 1 or 2. One patient had generalized muscle weakness (Grade 3), which was deemed to be a dose-limiting toxicity. Notably, the pharmacokinetic profile of LY2457546 showed virtually no elimination of LY2457546 within 24 hours, and thus prevented further dose escalation. No significant DDI were observed. Ex vivo flow cytometry studies showed downregulation of the phosphoproteins, pcKIT, pFLT3, and pS6, in AML blasts after LY2457546 administration. No medically relevant responses were observed in the five treated patients. Conclusion: No biologically effective dose could be established for LY2457546 in chemotherapy-resistant AML patients. Lack of drug clearance prevented safe dose escalation, and the study was terminated early. Future efforts should be made to develop derivatives with a more favorable pharmacokinetic profile.

PD-1 and PD-L1 expression in HNSCC primary cancer and related lymph node metastasis - impact on clinical outcome

Expression profiles and clinical impact of programmed cell death ligand 1 (PD‐L1) and programmed cell death 1 (PD‐1) expressing tumour infiltrating lymphocytes (TILs) in head and neck squamous cell carcinoma (HNSCC) are not elucidated fully. This study evaluates expression patterns in primary HNSCC and related lymph node metastasis and the impact on patients’ clinical outcome.

Abstract 4081: Sensitivity of gastric cancer cell lines against 2-deoxy-D-glucose is independent of PI3K/mTOR inhibition

2-deoxy-D-glucose (2-DG) is currently studied in Phase I clinical trials as a novel therapeutic strategy for anti-cancer therapy. Several preclinical studies have suggested that the anti-proliferative activity of 2-DG might be linked, amongst others, to the inhibition of the PI3K/mTOR signalling pathway. Blocking the PI3K/mTOR pathway has become a major focus for molecular targeted drug development. In the current study we assessed the biological relevance of PI3K/mTOR signalling for the anti-proliferative activity of 2-DG in gastric cancer cells. The gastric cancer cell lines (N87, MKN-28, MKN-45) were treated with 2-DG and evaluated for cell proliferation, apoptosis induction, cell cycle arrest, and PI3K/mTOR signalling. Everolimus, a specific mTORC1 inhibitor, was employed as positive control. Treatment with 2-DG potently suppressed cell proliferation resulting in an up to 300% reduced IC50 in the gastric cancer cell lines. Cell cycle inhibition and apoptosis induction varied substantially among cell lines. However, the distinct 2-DG sensitivity of gastric cancer cell lines was not reflected by any alteration in the PI3K/mTOR signalling pathway. In all cancer cell lines, no major inhibition of pERK and pAKT was observed, whereas both, 2-DG as well as everolimus strongly inhibited phosphorylation of S6. In contrast to 2-DG everolimus induced also a clear inhibition of 4EBP1 phosphorylation. In conclusion, inhibition of PI3K/mTOR signalling pathway by 2-DG differs from inhibition by everolimus and is not linked with 2-DG anti-proliferative sensitivity in gastric cancer cell lines. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4081. doi:10.1158/1538-7445.AM2011-4081

Abstract 4470: The dual PI3K/mTOR inhibitor BEZ235 impairs gastric cancer growth in vitro and in vivo

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Background Gastric cancer is still a major health problem and the prognosis at advanced stage of disease with the current chemotherapeutic treatment strategies remains poor. Therefore novel treatment strategies and molecular targets for gastric cancer therapy are desperately needed. PI3K/mTOR pathway mutations, especially PTEN, PI3K3C and AKT mutations and pS6 overexpression, frequently occur in gastric cancer. Thus, we tested the activity of the dual PI3K and mTOR BEZ235 against gastric cancer in vitro and in vivo. Materials and Methods Three gastric cancer cell lines (N87, Mkn28 and MKN45) were treated with BEZ235 (20-80nM) and assessed for cell viability, cell death and cell cycle via celltiter blue and FACS analysis, respectively. PI3K/mTOR protein target modulation was measured by Western blotting. For in vivo studies athymic nude mice were inoculated with N87 or Mkn45 cells bilaterally and treated daily with 20 or 40mg/kg BEZ235. Results In vitro, treatment of gastric cancer cells with 20-80nM BEZ235 decreased cell growth in a dose dependent manner in all cell lines tested (up to −70%). This anti-proliferative activity was accompanied with a G1 cell cycle arrest of gastric cancer cells (up to 75%), whereas no significant levels of cell death were detected. On the molecular level, BEZ235 therapy resulted in a decrease of phosphorylation of AKT and S6 protein at 80nM. Notably, lower concentrations abolished mTOR downstream signalling but had no effect on AKT phosphorylation. In vivo, treatment with 20 and 40mg/kg BEZ235 resulted in a significant anti-tumor effect in a N87 gastric cancer xenograft mouse model. Interestingly, BEZ235 therapy displayed no anti-tumor activity in the MKN45 gastric cancer xenograft mouse model. In both models, we observed similar results in terms of PI3K/mTOR pathway downregulation in xenografts. Conclusion BEZ235 has pronounced anti-tumor activity in gastric cancer cells at low nanomolar range, which is linked with PI3K/mTOR downregulation and G1 cell cycle arrest. In vivo, N87 gastric cancer cells responded to BEZ235 treatment, whereas MKN45 cells were resistant. Thus, in our models in vitro sensitivity of BEZ235 did not predict for in vivo activity. We are currently conducting a microPET mouse study in order to evaluate whether PET is a suitable tool to predict BEZ235 sensitivity in vivo and the results will be presented at the meeting. 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 4470.

Vertical inhibition of the mTORC1/mTORC2/PI3K pathway shows synergistic effects against melanoma in vitro and in vivo

The phosphatidyl inositol 3-kinase/mammalian target of rapamycin (PI3K/mTOR) pathway has been shown to be involved in the development of melanoma. PI-103 is a kinase inhibitor blocking PI3K class IA and mTOR complex 1 and 2. Here, we studied the effect of targeting the PI3K/mTORC1/mTORC2 pathway by PI-103 and rapamycin in melanoma cells and in a melanoma mouse model. Dual targeting of PI3K and mTOR by PI-103 induced apoptosis and cell-cycle arrest, and inhibited viability of melanoma cells in vitro. Combined treatment with PI-103 and the prototypic mTORC1 inhibitor rapamycin led to the synergistic suppression of AKT and ribosomal S6 protein phosphorylation and to the induction of apoptosis. In vivo, PI-103 and rapamycin displayed only modest singleagent activity, but the combination significantly reduced the tumor growth compared with both single agents. These data show that blocking the PI3K/mTORC1/mTORC2 pathway using the combination of two distinct smallmolecule inhibitors (‘‘vertical inhibition’’) leads to superior efficacy against malignant melanoma in vitro and in vivo.

Abstract A124: ERCC1 expression correlates with EGFR expression and is regulated by cisplatin, cetuximab, and dasatinib in head and neck cancer cells

Background: ERCC1 has been linked to resistance against platin‐based chemotherapy. Cetuximab sensitizes colorectal cancer cells to oxaliplatin. This sensitization coincides with ERCC1 downregulation, but the link between EGFR and ERCC1 is not fully understood. It is known that treatment with cetuximab inhibits nuclear transport of EGFR and DNA repair in response to irradiation. Moreover, cisplatin treatment results in phosphorylation at Y845 and nuclear transport of EGFR. We intended to investigate the regulation of ERCC1 by cisplatin, cetuximab and dasatinib, a src inhibitor stabilizing EGFR in the membrane, its relevance for chemosensitivity and its correlation with EGFR and its phosphorylation at Y845 in head and neck cancer cells. Material and Methods: In UM‐SCC 14c head and neck cancer cell line, ERCC1, EGFR and EGFR (pY845) expression was determined by western blot and FACS analysis. Cell viability, cell cycle and apoptosis induction was assessed by cell titer blue assay, propidium iodide and caspase 3 staining, respectively. Results: In cell viability assay, combination of cisplatin with cetuximab led to a significant (p Conclusion: 14c cells are sensitized to cisplatin treatment by adding cetuximab. ERCC1 expression correlated with phosphorylation status of EGFR (pY845) following treatment with cisplatin and cetuximab. However, treatment with the src inhibitor dasatinib before adding cisplatin and cetuximab reduced phosphorylation of EGFR (Y845), but increased ERCC1 and EGFR expression. Whether the increase of ERCC1 is a late effect due to transcriptional upregulation of EGFR after dasatinib treatment remains to be determined. However, the dose‐effect relationship and the lack of regulation of ERCC1 expression in peripheral blood leukocytes after treatment with cisplatin and cetuximab support the notion that ERCC1 expression is regulated by EGFR in 14c cells. Further studies elucidating the subcellular transport of EGFR and its transcriptional regulation after dasatinib treatment are ongoing and will be presented. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A124.

Targeting sphingosine kinase 1 with LNA oligonucleotides in gastric cancer.

Background Gastric cancer is the fourth most common cancer worldwide. Despite advances in diagnosis of gastric cancer, the prognosis at advanced stage of disease with the current chemotherapeutic treatment strategies remains poor. Hence, new agents and molecular targets for gastric cancer therapy are desperately needed. Sphingosine kinase 1 represents a promising novel target for anti-cancer therapy. However, the most common used small molecule inhibitors of SphK are unspecific inhibitors of SphK1. Here we investigated the effect of targeted downregulation of SphK1 by locked nucleic acid antisense oligonucleotides (LNA-ASO) in gastric cancer cell lines.