mrk-003 and Precursor-T-Cell-Lymphoblastic-Leukemia-Lymphoma

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.

Topics: Amyloid Precursor Protein Secretases; Animals; Apoptosis; Basic Helix-Loop-Helix Transcription Factors; Blotting, Western; Carrier Proteins; Cell Proliferation; Cyclic S-Oxides; Cyclin-Dependent Kinase Inhibitor p16; Disease Models, Animal; Flow Cytometry; Humans; Immunoenzyme Techniques; Mice; Mice, Transgenic; Phosphotransferases (Alcohol Group Acceptor); Precursor T-Cell Lymphoblastic Leukemia-Lymphoma; Proto-Oncogene Proteins; Receptor, Notch1; Signal Transduction; T-Cell Acute Lymphocytic Leukemia Protein 1; Thiadiazoles; TOR Serine-Threonine Kinases; Tumor Cells, Cultured

NOTCH signaling is deregulated in the majority of T-cell acute lymphoblastic leukemias (T-ALL) as a result of activating mutations in NOTCH1. Gamma secretase inhibitors (GSI) block proteolytic activation of NOTCH receptors and may provide a targeted therapy for T-ALL. We have investigated the mechanisms of GSI sensitivity across a panel of T-ALL cell lines, yielding an approach for patient stratification based on pathway activity and also providing a rational combination strategy for enhanced response to GSI. Whereas the NOTCH1 mutation status does not serve as a predictor of GSI sensitivity, a gene expression signature of NOTCH pathway activity does correlate with response, and may be useful in the selection of patients more likely to respond to GSI. Furthermore, inhibition of the NOTCH pathway activity signature correlates with the induction of the cyclin-dependent kinase inhibitors CDKN2D (p19(INK4d)) and CDKN1B (p27(Kip1)), leading to derepression of RB and subsequent exit from the cell cycle. Consistent with this evidence of cell cycle exit, short-term exposure of GSI resulted in sustained molecular and phenotypic effects after withdrawal of the compound. Combination treatment with GSI and a small molecule inhibitor of CDK4 produced synergistic growth inhibition, providing evidence that GSI engagement of the CDK4/RB pathway is an important mechanism of GSI action and supports further investigation of this combination for improved efficacy in treating T-ALL.

Topics: Amyloid Precursor Protein Secretases; Cell Line, Tumor; Cyclic S-Oxides; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p19; Cyclin-Dependent Kinase Inhibitor p27; G1 Phase; Gene Expression Profiling; Humans; Intracellular Signaling Peptides and Proteins; Phosphorylation; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma; Protease Inhibitors; Receptor, Notch1; Retinoblastoma Protein; S Phase; Signal Transduction; Thiadiazoles; Transcription, Genetic; Transfection

gamma-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.. 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 Abeta(40) 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.. 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.. 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.

Topics: Amyloid beta-Peptides; Amyloid Precursor Protein Secretases; Animals; Antineoplastic Agents; Apoptosis; Cell Differentiation; Cell Line, Tumor; Colon; Cyclic S-Oxides; Down-Regulation; Drug Administration Schedule; Humans; Intestinal Mucosa; Membrane Potential, Mitochondrial; Mice; Mice, Nude; Mitochondrial Proteins; Neoplasm Transplantation; Peptide Fragments; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma; Receptor, Notch1; Signal Transduction; Thiadiazoles; Transplantation, Heterologous