mrk-003 and Disease-Models--Animal

Next-generation sequencing was used to identify Notch mutations in a large collection of diverse solid tumors. NOTCH1 and NOTCH2 rearrangements leading to constitutive receptor activation were confined to triple-negative breast cancers (TNBC; 6 of 66 tumors). TNBC cell lines with NOTCH1 rearrangements associated with high levels of activated NOTCH1 (N1-ICD) were sensitive to the gamma-secretase inhibitor (GSI) MRK-003, both alone and in combination with paclitaxel, in vitro and in vivo, whereas cell lines with NOTCH2 rearrangements were resistant to GSI. Immunohistochemical staining of N1-ICD in TNBC xenografts correlated with responsiveness, and expression levels of the direct Notch target gene HES4 correlated with outcome in patients with TNBC. Activating NOTCH1 point mutations were also identified in other solid tumors, including adenoid cystic carcinoma (ACC). Notably, ACC primary tumor xenografts with activating NOTCH1 mutations and high N1-ICD levels were sensitive to GSI, whereas N1-ICD-low tumors without NOTCH1 mutations were resistant.. NOTCH1 mutations, immunohistochemical staining for activated NOTCH1, and HES4 expression are biomarkers that can be used to identify solid tumors that are likely to respond to GSI-based therapies.

Topics: Amyloid Precursor Protein Secretases; Animals; Antineoplastic Agents; Apoptosis; Biomarkers; Carcinoma, Adenoid Cystic; Cell Line, Tumor; Cellular Senescence; Cyclic S-Oxides; Disease Models, Animal; Drug Resistance, Neoplasm; Exome; Female; Gene Expression Regulation, Neoplastic; Gene Rearrangement; Genes, myc; High-Throughput Nucleotide Sequencing; Humans; Models, Molecular; Mutation; Prognosis; Protease Inhibitors; Protein Conformation; Protein Interaction Domains and Motifs; Receptors, Notch; Signal Transduction; Thiadiazoles; Treatment Outcome; Triple Negative Breast Neoplasms; Xenograft Model Antitumor Assays

Human breast tumors comprise a minor sub-population of tumor-initiating cells (TICs), commonly termed cancer stem cells. TICs are thought to sustain tumor growth and to confer resistance to current anticancer therapies. Hence, targeting TIC may be essential to achieving durable cancer cures. To identify molecular targets in breast TIC, we employed a transgenic mouse model of ERBB2 breast cancer; tumors arising in this model comprise a very high frequency of TIC, which is maintained in tumor cell populations propagated in vitro as non-adherent tumorspheres. The Notch pathway is dysregulated in human breast tumors and overexpression of constitutively active Notch proteins induces mammary tumors in mice. The Notch pathway has also been implicated in stem cell processes including those of mammary epithelial stem cells. Hence, we investigated the potential that the Notch pathway is required for TIC activity. We found that an antagonist of Notch signaling, a gamma (γ)-secretase inhibitor termed MRK-003, inhibited the survival of tumorsphere-derived cells in vitro and eliminated TIC as assessed by cell transplantation into syngeneic mice. Whereas MRK-003 also inhibited the self-renewal and/or proliferation of mammosphere-resident cells, this effect of the inhibitor was reversible thus suggesting that it did not compromise the survival of these cells. MRK-003 administration to tumor-bearing mice eliminated tumor-resident TIC and resulted in rapid and durable tumor regression. MRK-003 inhibited the proliferation of tumor cells, and induced their apoptosis and differentiation. These findings suggest that MRK-003 targets breast TIC and illustrate that eradicating these cells in breast tumors ensures long-term, recurrence-free survival.

Topics: Amyloid Precursor Protein Secretases; Animals; Cyclic S-Oxides; Disease Models, Animal; Enzyme Inhibitors; Female; Gene Expression Profiling; Genes, erbB-2; Mammary Neoplasms, Experimental; Mice; Mice, Transgenic; Neoplastic Stem Cells; Receptors, Notch; Thiadiazoles

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