mrk-003 and Brain-Neoplasms

Notch signaling can promote tumorigenesis in the nervous system and plays important roles in stem-like cancer cells. However, little is known about how Notch inhibition might alter tumor metabolism, particularly in lesions arising in the brain. The gamma-secretase inhibitor MRK003 was used to treat glioblastoma neurospheres, and they were subdivided into sensitive and insensitive groups in terms of canonical Notch target response. Global metabolomes were then examined using proton magnetic resonance spectroscopy, and changes in intracellular concentration of various metabolites identified which correlate with Notch inhibition. Reductions in glutamate were verified by oxidation-based colorimetric assays. Interestingly, the alkylating chemotherapeutic agent temozolomide, the mTOR-inhibitor MLN0128, and the WNT inhibitor LGK974 did not reduce glutamate levels, suggesting that changes to this metabolite might reflect specific downstream effects of Notch blockade in gliomas rather than general sequelae of tumor growth inhibition. Global and targeted expression analyses revealed that multiple genes important in glutamate homeostasis, including glutaminase, are dysregulated after Notch inhibition. Treatment with an allosteric inhibitor of glutaminase, compound 968, could slow glioblastoma growth, and Notch inhibition may act at least in part by regulating glutaminase and glutamate.

Topics: Brain Neoplasms; Cell Line, Tumor; Cyclic S-Oxides; Glioblastoma; Glutamic Acid; Glutaminase; Homeostasis; Humans; Metabolome; Receptors, Notch; Thiadiazoles

Pilocytic astrocytoma (PA) is the most common primary brain tumor in children; various signaling pathways have been implicated in its biology. The Notch signaling pathway has been found to play a role in the development, stem cell biology, and pathogenesis of several cancers, but its role in PA has not been investigated. We studied alterations in Notch signaling components in tumor tissue from 18 patients with PA and 4 with other low-grade astrocytomas to identify much needed therapeutic targets. We found that Notch pathway members were overexpressed at the mRNA (NOTCH1, NOTCH2, HEY1, HEY2) and protein (HES1) levels in PAs at various anatomic sites compared with non-neoplastic brain samples. These changes were not associated with specific BRAF alterations. Inhibiting the Notch pathway in the pediatric low-grade astrocytoma cell lines Res186 and Res259 using either RNA interference or a γ-secretase inhibitor resulted in variable, but significant, reduction in cell growth and migration. This study suggests a potential role for Notch signaling in pediatric low-grade astrocytoma tumorigenesis and that Notch signaling may be a viable pathway therapeutic target.

Topics: Adolescent; Antineoplastic Agents; Astrocytoma; Basic Helix-Loop-Helix Transcription Factors; Brain Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Child; Child, Preschool; Core Binding Factors; Cyclic S-Oxides; Enzyme Inhibitors; Female; Gene Expression Regulation, Neoplastic; Homeodomain Proteins; Humans; Immunoglobulin J Recombination Signal Sequence-Binding Protein; Male; Receptors, Notch; Signal Transduction; Thiadiazoles; Transcription Factor HES-1; Young Adult

Molecular targeted therapy can potentially provide more effective treatment for patients with high-grade gliomas. Notch and Akt are notable target molecules as they play important roles in a variety of cellular processes, such as regeneration, differentiation, proliferation, migration, and invasion. Here, we assessed the therapeutic possibility of inhibiting Notch and Akt in gliomas using the clinically available, selective small molecule inhibitors MRK003 and MK-2206. We evaluated their efficacy individually and as a combination therapy in U251 and U87 glioma cell lines. We confirmed that MK-2206 effectively inhibits Akt phosphorylation in a dose-dependent manner, whereas MRK003 inhibits Notch signaling and Akt phosphorylation. Both MRK003 and MK-2206 significantly inhibited cell growth, migration, and invasion in a dose-dependent manner. Akt dephosphorylation was enhanced by combination therapy with MRK003 and MK-2206. However, the effect of combination treatment did not exceed that of MK-2206 monotherapy in proliferation assay. Inhibition of invasion, further enhanced by combination therapy, correlated with increased Akt inactivation. In summary, combination therapy with MRK003 and MK-2206 may be effective for inhibiting invasion but not proliferation.

Topics: Antineoplastic Agents; Brain Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclic S-Oxides; Drug Interactions; Glioma; Heterocyclic Compounds, 3-Ring; Humans; Molecular Targeted Therapy; Neoplasm Invasiveness; Phosphorylation; Proto-Oncogene Proteins c-akt; Receptors, Notch; Signal Transduction; Thiadiazoles

Multiple developmental pathways including Notch, Hedgehog, and Wnt are active in malignant brain tumors such as medulloblastoma and glioblastoma (GBM). This raises the possibility that tumors might compensate for therapy directed against one pathway by upregulating a different one. We investigated whether brain tumors show resistance to therapies against Notch, and whether targeting multiple pathways simultaneously would kill brain tumor cells more effectively than monotherapy.. We used GBM neurosphere lines to investigate the effects of a gamma-secretase inhibitor (MRK-003) on tumor growth, and chromatin immunoprecipitation to study the regulation of other genes by Notch targets. We also evaluated the effect of combined therapy with a Hedgehog inhibitor (cyclopamine) in GBM and medulloblastoma lines, and in primary human GBM cultures.. GBM cells are at least partially resistant to long-term MRK-003 treatment, despite ongoing Notch pathway suppression, and show concomitant upregulation of Wnt and Hedgehog activity. The Notch target Hes1, a repressive transcription factor, bound the Gli1 first intron, and may inhibit its expression. Similar results were observed in a melanoma-derived cell line. Targeting Notch and Hedgehog simultaneously induced apoptosis, decreased cell growth, and inhibited colony-forming ability more dramatically than monotherapy. Low-passage neurospheres isolated from freshly resected human GBMs were also highly susceptible to coinhibition of the two pathways, indicating that targeting multiple developmental pathways can be more effective than monotherapy at eliminating GBM-derived cells.. Notch may directly suppress Hedgehog via Hes1 mediated inhibition of Gli1 transcription, and targeting both pathways simultaneously may be more effective at eliminating GBMs cells.

Topics: Antineoplastic Combined Chemotherapy Protocols; Basic Helix-Loop-Helix Transcription Factors; Brain Neoplasms; Cell Line, Tumor; Cyclic S-Oxides; Drug Evaluation, Preclinical; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Glioblastoma; Hedgehog Proteins; Homeodomain Proteins; Humans; Receptors, Notch; Signal Transduction; Thiadiazoles; Transcription Factor HES-1; Transcription Factors; U937 Cells; Veratrum Alkaloids; Zinc Finger Protein GLI1