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Concurrent MEK targeted therapy prevents MAPK pathway reactivation during BRAFV600E targeted inhibition in a novel syngeneic murine glioma model.

ABSTRACT: Inhibitors of BRAFV600E kinase are currently under investigations in preclinical and clinical studies involving BRAFV600E glioma. Studies demonstrated clinical response to such individualized therapy in the majority of patients whereas in some patients tumors continue to grow despite treatment. To study resistance mechanisms, which include feedback activation of mitogen-activated protein kinase (MAPK) signaling in melanoma, we developed a luciferase-modified cell line (2341luc) from a BrafV600E mutant and Cdkn2a- deficient murine high-grade glioma, and analyzed its molecular responses to BRAFV600E- and MAPK kinase (MEK)-targeted inhibition. Immunocompetent, syngeneic FVB/N mice with intracranial grafts of 2341luc were tested for effects of BRAFV600E and MEK inhibitor treatments, with bioluminescence imaging up to 14-days after start of treatment and survival analysis as primary indicators of inhibitor activity. Intracranial injected tumor cells consistently generated high-grade glioma-like tumors in syngeneic mice. Intraperitoneal daily delivery of BRAFV600E inhibitor dabrafenib only transiently suppressed MAPK signaling, and rather increased Akt signaling and failed to extend survival for mice with intracranial 2341luc tumor. MEK inhibitor trametinib delivered by oral gavage daily suppressed MAPK pathway more effectively and had a more durable anti-growth effect than dabrafenib as well as a significant survival benefit. Compared with either agent alone, combined BRAFV600E and MEK inhibitor treatment was more effective in reducing tumor growth and extending animal subject survival, as corresponding to sustained MAPK pathway inhibition. Results derived from the 2341luc engraftment model application have clinical implications for the management of BRAFV600E glioma.

SUBMITTER: Grossauer S

PROVIDER: S-EPMC5342782 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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Concurrent MEK targeted therapy prevents MAPK pathway reactivation during BRAFV600E targeted inhibition in a novel syngeneic murine glioma model.

Grossauer Stefan S Koeck Katharina K Murphy Nicole E NE Meyers Ian D ID Daynac Mathieu M Truffaux Nathalene N Truong Albert Y AY Nicolaides Theodore P TP McMahon Martin M Berger Mitchel S MS Phillips Joanna J JJ James C David CD Petritsch Claudia K CK

Oncotarget 20161101 46

Inhibitors of BRAFV600E kinase are currently under investigations in preclinical and clinical studies involving BRAFV600E glioma. Studies demonstrated clinical response to such individualized therapy in the majority of patients whereas in some patients tumors continue to grow despite treatment. To study resistance mechanisms, which include feedback activation of mitogen-activated protein kinase (MAPK) signaling in melanoma, we developed a luciferase-modified cell line (2341luc) from a BrafV600E ...[more]

PMID: 27713119

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Project description:BACKGROUND:Germinal Vesicle (GV) stage mouse oocytes in first meiotic prophase exhibit highly active HCO(3)(-)/Cl(-) exchange--a class of transport nearly ubiquitously involved in regulation of intracellular pH and cell volume. During meiosis, however, oocyte HCO(3)(-)/Cl(-) exchange becomes inactivated during first metaphase (MI), remains inactive in second metaphase (MII), and is reactivated only after egg activation. Previous work using pharmacological manipulations had indicated that activity of the MEK/MAPK signaling pathway was negatively correlated with HCO(3)(-)/Cl(-) exchange activity during meiosis. However, the mechanism by which the exchanger is inactivated during meiotic progression had not been determined, nor had the role of MEK/MAPK been directly established. METHODOLOGY/PRINCIPAL FINDINGS:Expression of a constitutively active form of MEK (MAP kinase kinase), which prevented the normal downregulation of MAPK after egg activation, also prevented reactivation of HCO(3)(-)/Cl(-) exchange. Conversely, suppression of endogenous MAPK activity with dominant negative MEK activated the normally quiescent HCO(3)(-)/Cl(-) exchange in mature MII eggs. A GFP-tagged form of the HCO(3)(-)/Cl(-) exchanger isoform Ae2 (Slc4a2) was strongly expressed at the GV oocyte plasma membrane, but membrane localization decreased markedly during meiotic progression. A similar pattern for endogenous Ae2 was confirmed by immunocytochemistry. The loss of membrane-localized Ae2 appeared selective, since membrane localization of a GFP-tagged human dopamine D1 receptor did not change during meiotic maturation. CONCLUSIONS:Direct manipulation of MAPK activity indicated that GFP-tagged Ae2 localization depended upon MAPK activity. Inactivation of HCO(3)(-)/Cl(-) exchange during the meiotic cell cycle may therefore reflect the loss of Ae2 from the oocyte plasma membrane, downstream of MEK/MAPK signaling. This identifies a novel role for MEK/MAPK-mediated cytostatic factor (CSF) activity during meiosis in membrane protein trafficking in mouse oocytes, and shows for the first time that selective retrieval of membrane proteins is a feature of meiosis in mammalian oocytes.

Project description:There is a clinically unmet need for effective treatments for triple-negative breast cancer (TNBC), as it remains the most aggressive subtype of breast cancer. Herein, we demonstrate a promising strategy using a tumor-targeting alkylphosphocholine (NM600) for targeted radionuclide therapy of TNBC. Methods: NM600 was radiolabeled with 86Y for PET imaging and 177Lu for targeted radionuclide therapy. 86Y-NM600 PET imaging was performed on female BALB/C mice bearing syngeneic 4T07 (nonmetastatic) and 4T1 (metastatic) TNBC tumor grafts (n = 3-5). Quantitative data derived from a PET-image region-of-interest analysis, which was corroborated by ex vivo biodistribution, were used to estimate the dosimetry of 177Lu-NM600 treatments. Weight measurement, complete blood counts, and histopathology analysis were performed to determine 177Lu-NM600 toxicity in naïve BALB/C mice administered 9.25 or 18.5 MBq. Groups of mice bearing 4T07 or 4T1 grafts (n = 5-6) received excipient or 9.25 or 18.5 MBq of 177Lu-NM600 as a single or fractionated schedule, and tumor growth and overall survival were monitored. Results: Excellent tumor targeting and rapid normal-tissue clearance of 86Y-NM600 were noted in both 4T07 and 4T1 murine models. Ex vivo biodistribution corroborated the accuracy of the PET data and validated 86Y-NM600 as a surrogate for 177Lu-NM600. 177Lu-NM600 dosimetry showed absorbed doses of 2.04 ± 0.32 and 1.68 ± 0.06 Gy/MBq to 4T07 and 4T1 tumors, respectively, which were larger than those delivered to liver (1.28 ± 0.09 Gy/MBq) and to bone marrow (0.31 ± 0.05 Gy/MBq). The 177Lu-NM600 injected activities used for treatment were well tolerated and resulted in significant tumor growth inhibition and prolonged overall survival in both tested TNBC models. A complete response was attained in 60% of treated mice bearing 4T07 grafts. Conclusion: Overall, our results suggest that 177Lu-NM600 targeted radionuclide therapy has potential for TNBC and merits further exploration in a clinical setting.

Dataset Information

Concurrent MEK targeted therapy prevents MAPK pathway reactivation during BRAFV600E targeted inhibition in a novel syngeneic murine glioma model.

Publications

Concurrent MEK targeted therapy prevents MAPK pathway reactivation during BRAFV600E targeted inhibition in a novel syngeneic murine glioma model.

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