Gerald Moncayo

MAP Kinase-Interacting Kinase 1 Regulates SMAD2-Dependent TGF-β Signaling Pathway in Human Glioblastoma

Glioblastoma multiforme (GBM) is the most common aggressive brain cancer with a median survival of approximately 1 year. In a search for novel molecular targets that could be therapeutically developed, our kinome-focused microarray analysis identified the MAP (mitogen-activated protein) kinase-interacting kinase 1 (MNK1) as an attractive theranostic candidate. MNK1 overexpression was confirmed in both primary GBMs and glioma cell lines. Inhibition of MNK1 activity in GBM cells by the small molecule CGP57380 suppressed eIF4E phosphorylation, proliferation, and colony formation whereas concomitant treatment with CGP57380 and the mTOR inhibitor rapamycin accentuated growth inhibition and cell-cycle arrest. siRNA-mediated knockdown of MNK1 expression reduced proliferation of cells incubated with rapamycin. Conversely, overexpression of full-length MNK1 reduced rapamycin-induced growth inhibition. Analysis of polysomal profiles revealed inhibition of translation in CGP57380 and rapamycin-treated cells. Microarray analysis of total and polysomal RNA from MNK1-depleted GBM cells identified mRNAs involved in regulation of TGF-β pathway. Translation of SMAD2 mRNA as well as TGF-β-induced cell motility and vimentin expression was regulated by MNK1 signaling. Tissue microarray analysis revealed a positive correlation between the immunohistochemical staining of MNK1 and SMAD2. Taken together, our findings offer insights into how MNK1 pathways control translation of cancer-related mRNAs including SMAD2, a key component of the TGF-β signaling pathway. Furthermore, they suggest MNK1-controlled translational pathways in targeted strategies to more effectively treat GBM.

Mer receptor tyrosine kinase promotes invasion and survival in glioblastoma multiforme

The infiltration of glioma cells into adjacent tissue is one of the major obstacles in the therapeutic management of malignant brain tumours, in most cases precluding complete surgical resection. Consequently, malignant glioma patients almost invariably experience tumour recurrences. Within the brain, glioma cells migrate rapidly either amoeboidly or mesenchymally to invade surrounding structures, in dependence on the extracellular environment. In addition, radiotherapy, frequently applied as adjuvant therapeutic modality, may enhance tumour cell mobility. Here, we show that the receptor tyrosine kinase Mer (MerTK) is overexpressed in glioblastoma multiforme (GBM) and that this is accompanied with increased invasive potential. MerTK expression is maintained in primary GBM-derived tumour spheres under stem cell culture conditions but diminishes significantly in serum-containing cultures with concomitant downregulation of Nestin and Sox2. Depletion of MerTK disrupts the rounded morphology of glioma cells and decreases their invasive capacity. Furthermore, the expression and phosphorylation of myosin light chain 2 are strongly associated with MerTK activity, indicating that the effect of MerTK on glioma cell invasion is mediated by actomyosin contractility. Finally, DNA damage robustly triggers the upregulation and phosphorylation of MerTK, which protects cells from apoptosis. This effect is strongly impaired upon MerTK depletion or overexpression of an inactive MerTK mutant. Collectively, our data suggests that MerTK is a novel therapeutic target in the treatment of the malignant gliomas.

MNK1 pathway activity maintains protein synthesis in rapalog-treated gliomas

High levels of mammalian target of rapamycin complex 1 (mTORC1) activity in malignant gliomas promote tumor progression, suggesting that targeting mTORC1 has potential as a therapeutic strategy. Remarkably, clinical trials in patients with glioma revealed that rapamycin analogs (rapalogs) have limited efficacy, indicating activation of resistance mechanisms. Targeted depletion of MAPK-interacting Ser/Thr kinase 1 (MNK1) sensitizes glioma cells to the mTORC1 inhibitor rapamycin through an indistinct mechanism. Here, we analyzed how MNK1 and mTORC1 signaling pathways regulate the assembly of translation initiation complexes, using the cap analog m7GTP to enrich for initiation complexes in glioma cells followed by mass spectrometry-based quantitative proteomics. Association of eukaryotic translation initiation factor 4E (eIF4E) with eIF4E-binding protein 1 (4EBP1) was regulated by the mTORC1 pathway, whereas pharmacological blocking of MNK activity by CGP57380 or MNK1 knockdown, along with mTORC1 inhibition by RAD001, increased 4EBP1 binding to eIF4E. Furthermore, combined MNK1 and mTORC1 inhibition profoundly inhibited 4EBP1 phosphorylation at Ser65, protein synthesis and proliferation in glioma cells, and reduced tumor growth in an orthotopic glioblastoma (GBM) mouse model. Immunohistochemical analysis of GBM samples revealed increased 4EBP1 phosphorylation. Taken together, our data indicate that rapalog-activated MNK1 signaling promotes glioma growth through regulation of 4EBP1 and indicate a molecular cross-talk between the mTORC1 and MNK1 pathways that has potential to be exploited therapeutically.

Phosphatidylinositol 3-Kinase Signaling in Thymocytes: The Need for Stringent Control

PI3K signaling is important in the development of normal and malignant thymocytes. The thymus serves as the primary site for the lifelong formation of new T lymphocytes; hence, it is essential for the maintenance of an effective immune system. Although thymocyte development has been widely studied, the mechanisms involved are incompletely defined. A comprehensive understanding of the molecular events that control regular thymocyte development will not only shed light on the physiological control of T cell differentiation but also probably provide insight into the pathophysiology of T cell immunodeficiencies, the molecular basis that underpins autoimmunity, and the mechanisms that instigate the formation of T cell lymphomas. Phosphatidylinositol 3-kinases (PI3Ks) play a critical role in thymocyte development, although not all of their downstream mediators have yet been identified. Here, we discuss experimental evidence that argues for a critical role of the PI3K-phosphoinositide–dependent protein kinase (PDK1)–protein kinase B (PKB) signaling pathway in the development of both normal and malignant thymocytes, and we highlight molecules that can potentially be targeted therapeutically. The immune system is composed of the closely interacting innate and adaptive effector systems, and it serves to eliminate almost all types of pathogens as well as some malignancies. Whereas the innate arm of the immune system provides an immediate, but nonspecific, form of defense against pathogens, it is the system’s adaptive response, which is mediated by B and T lymphocytes, that assures antigen specificity and immunological memory, albeit after a delay of several days. The vast majority of T cells are formed in the thymus, which provides cell-surface and soluble ligands that are essential for T lineage commitment and differentiation. Among the pathways that are important for the de novo formation of T cells, signals that engage phosphatidylinositol 3-kinases (PI3Ks) are of particular importance, which is a finding consistent with a physiological role of PI3K signaling in cell differentiation in many different tissues. Moreover, uncontrolled activation of PI3K in thymocytes is associated with the formation of tumors, which highlights the requirement for a precise control of PI3K activity. In this Review, including two figures and 174 references, we discuss the importance of the PI3K-phosphoinositide–dependent protein kinase (PDK1)–protein kinase B (PKB) signaling pathway for the development of both normal and malignant thymocytes, and we outline the potential to target this pathway for therapeutic interventions.

Deltex-1 Activates Mitotic Signaling and Proliferation and Increases the Clonogenic and Invasive Potential of U373 and LN18 Glioblastoma Cells and Correlates with Patient Survival

Glioblastoma (GBM) is a highly malignant primary tumor of the central nervous system originating in glial cells. GBM results in more years of life lost than any other cancer type. Low levels of Notch receptor expression correlates with prolonged survival in various high grade gliomas independent of other markers. Different downstream pathways of Notch receptors have been identified. We tested if the Notch/Deltex pathway, which is distinct from the canonical, CSL-mediated pathway, has a role in GBM. We show that the alternative or non-canonical Notch pathway functioning through Deltex1 (DTX1) mediates key features of glioblastoma cell aggressiveness. For example, DTX1 activates the RTK/PI3K/PKB and the MAPK/ERK mitotic pathways and induces anti-apoptotic Mcl-1. The clonogenic and growth potential of established glioma cells correlated with DTX1 levels. Microarray gene expression analysis further identified a DTX1-specific, MAML1-independent transcriptional program - including microRNA-21- which is functionally linked to the changes in tumor cell aggressiveness. Over-expression of DTX1 increased cell migration and invasion correlating to ERK activation, miR-21 levels and endogenous Notch levels. In contrast to high and intermediate expressors, patients with low DTX1 levels have a more favorable prognosis. The alternative Notch pathway via DTX1 appears to be an oncogenic factor in glioblastoma and these findings offer new potential therapeutic targets.

Inhibition of MNK pathways enhances cancer cell response to chemotherapy with temozolomide and targeted radionuclide therapy

Current standard-of-care treatment for malignant cancers includes radiotherapy and adjuvant chemotherapy. Here, we report increased MAP kinase-interacting kinase (MNK)-regulated phosphorylation of translation initiation factor 4E (eIF4E) in glioma cells upon temozolomide (TMZ) treatment and in medullary thyroid carcinoma (MTC) cells in response to targeted radionuclide therapy. Depletion of MNK activity by using two MNK inhibitors, CGP57380 or cercosporamide, as well as by MNK1-specific knockdown sensitized glioblastoma (GBM) cells and GBM-derived spheres to TMZ. Furthermore, CGP57380 treatment enhanced response of MTC cells to (177)Lu-labeled gastrin analogue. In order to understand how MNK signaling pathways support glioma survival we analyzed putative MNK substrates by quantitative phosphoproteomics in normal condition and in the presence of TMZ. We identified MNK inhibitor-sensitive phosphorylation sites on eIF4G1, mutations of which either influenced eIF4E phosphorylation or glioma cell response to TMZ, pointing to altered regulation of translation initiation as a resistance mechanism. Pharmacological inhibition of overexpressed MNK1 by CGP57380 reduced eIF4E phosphorylation and induced association of inactive MNK1 with eIF4G1. Taken together, our data show an activation of MNK-mediated survival mechanisms in response to either glioma chemotherapy or MTC targeted radiation and suggest that inhibition of MNK activity represents an attractive sensitizing strategy for cancer treatments.

SYK inhibition blocks proliferation and migration of glioma cells and modifies the tumor microenvironment

Background Glioblastoma (GBM) is one of the most aggressive human brain tumors, with a median survival of 15-18 months. There is a desperate need to find novel therapeutic targets. Various receptor protein kinases have been identified as potential targets; however, response rates in clinical studies have been somewhat disappointing. Targeting the spleen tyrosine kinase (SYK), which acts downstream of a range of oncogenic receptors, may therefore show more promising results. Methods Kinase expression of brain tumor samples including GBM and low-grade tumors were compared with normal brain and normal human astrocytes by microarray analysis. Furthermore, SYK, LYN, SLP76, and PLCG2 protein expressions were analyzed by immunohistochemistry, western blot, and immunofluorescence of additional GBM patient samples, murine glioma samples, and cell lines. SYK was then blocked chemically and genetically in vitro and in vivo in 2 different mouse models. Multiphoton intravital imaging and multicolor flow cytometry were performed in a syngeneic immunocompetent C57BL/6J mouse GL261 glioma model to study the effect of these inhibitors on the tumor microenvironment. Results SYK, LYN, SLP76, and PLCG2 were found expressed in human and murine glioma samples and cell lines. SYK inhibition blocked proliferation, migration, and colony formation. Flow cytometric and multiphoton imaging imply that targeting SYK in vivo attenuated GBM tumor growth and invasiveness and reduced B and CD11b+ cell mobility and infiltration. Conclusions Our data suggest that gliomas express a SYK signaling network important in glioma progression, inhibition of which results in reduced invasion with slower tumor progression.

Abstract 4041: Glioma cells display a prognostically significant hematopoietic phenotype

Glioblastoma multiforme (GBM) is one of the most aggressive human cancers. Glioma cells expressed immunoreceptor tyrosine-based activation motif (ITAM) bearing molecules, such as Fc receptors, T cell receptor components and C-type lectins and expression was up regulated in two distinct tumor-derived prognostic signatures identified in GBM and low-grade gliomas. Targeting SYK, downstream of ITAM molecules, attenuated GBM tumor growth and invasiveness, and reduced B and CD11b+ cell mobility and infiltration resulting in improved survival. In addition, GBM tumor cells were found to display phagocytic characteristics in vitro and in vivo by flow cytometry and two photon imaging. Our data demonstrate that gliomas are hematopoietic-like tumors that express an immune signaling network important in glioma progression, and identify targets for therapeutic intervention. Citation Format: Gerald Moncayo. Glioma cells display a prognostically significant hematopoietic phenotype. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4041.