14 research outputs found
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Targeting neuronal activity-regulated neuroligin-3 dependency in high-grade glioma
Summary High-grade gliomas (HGG) are a devastating group of cancers, representing the leading cause of brain tumor-related death in both children and adults. Therapies aimed at mechanisms intrinsic to the glioma cell have translated to only limited success; effective therapeutic strategies will need to also target elements of the tumor microenvironment that promote glioma progression. We recently demonstrated that neuronal activity robustly promotes the growth of a range of molecularly and clinically distinct HGG types, including adult glioblastoma (GBM), anaplastic oligodendroglioma, pediatric GBM, and diffuse intrinsic pontine glioma (DIPG)1. An important mechanism mediating this neural regulation of brain cancer is activity-dependent cleavage and secretion of the synaptic molecule neuroligin-3 (NLGN3), which promotes glioma proliferation through the PI3K-mTOR pathway1. However, neuroligin-3 necessity to glioma growth, proteolytic mechanism of secretion and further molecular consequences in glioma remain to be clarified. Here, we demonstrate a striking dependence of HGG growth on microenvironmental neuroligin-3, elucidate signaling cascades downstream of neuroligin-3 binding in glioma and determine a therapeutically targetable mechanism of secretion. Patient-derived orthotopic xenografts of pediatric GBM, DIPG and adult GBM fail to grow in Nlgn3 knockout mice. Neuroligin-3 stimulates numerous oncogenic pathways, including early focal adhesion kinase activation upstream of PI3K-mTOR, and induces transcriptional changes including upregulation of numerous synapse-related genes in glioma cells. Neuroligin-3 is cleaved from both neurons and oligodendrocyte precursor cells via the ADAM10 sheddase. ADAM10 inhibitors prevent release of neuroligin-3 into the tumor microenvironment and robustly block HGG xenograft growth. This work defines a promising strategy for targeting neuroligin-3 secretion, which could prove transformative for HGG therapy
Inhibition of O-GlcNAc transferase activity reprograms prostate cancer cell metabolism
Metabolic networks are highly connected and complex, but a single enzyme, O-GlcNAc transferase (OGT) can sense the availability of metabolites and also modify target proteins. We show that inhibition of OGT activity inhibits the proliferation of prostate cancer cells, leads to sustained loss of c-MYC and suppresses the expression of CDK1, elevated expression of which predicts prostate cancer recurrence (p=0.00179). Metabolic profiling revealed decreased glucose consumption and lactate production after OGT inhibition. This decreased glycolytic activity specifically sensitized prostate cancer cells, but not cells representing normal prostate epithelium, to inhibitors of oxidative phosphorylation (rotenone and metformin). Intra-cellular alanine was depleted upon OGT inhibitor treatment. OGT inhibitor increased the expression and activity of alanine aminotransferase (GPT2), an enzyme that can be targeted with a clinically approved drug, cycloserine. Simultaneous inhibition of OGT and GPT2 inhibited cell viability and growth rate, and additionally activated a cell death response. These combinatorial effects were predominantly seen in prostate cancer cells, but not in a cell-line derived from normal prostate epithelium. Combinatorial treatments were confirmed with two inhibitors against both OGT and GPT2. Taken together, here we report the reprogramming of energy metabolism upon inhibition of OGT activity, and identify synergistically lethal combinations that are prostate cancer cell specific
A homogeneous SIRPα-CD47 cell-based, ligand-binding assay: Utility for small molecule drug development in immuno-oncology.
CD47 is an immune checkpoint protein that downregulates both the innate and adaptive anti-tumor immune response via its counter receptor SIRPα. Biologics, including humanized CD47 monoclonal antibodies and decoy SIRPα receptors, that block the SIRPα-CD47 interaction, are currently being developed as cancer immunotherapy agents. However, adverse side effects and limited penetration of tumor tissue associated with their structure and large size may impede their clinical application. We recently developed a quantitative high throughput screening assay platform to identify small molecules that disrupt the binding of SIRPα and CD47 as an alternative approach to these protein-based therapeutics. Here, we report on the development and optimization of a cell-based binding assay to validate active small molecules from our biochemical screening effort. This assay has a low volume, high capacity homogenous format that relies on laser scanning cytometry (LSC) and associated techniques to enhance signal to noise measurement of cell surface binding. The LSC assay is specific, concentration dependent, and validated for the two major human SIRPα variants (V1 and V2), with results that parallel those of our biochemical data as well as published studies. We also utilized the LSC assay to confirm published studies showing that the inhibition of amino-terminal pyroglutamate formation on CD47 using the glutaminyl cyclase inhibitor SEN177 disrupts SIRPα binding. The SIRPα-CD47 interaction could be quantitatively measured in live and fixed tumor cells. Use of fixed cells reduces the burden of cell maintenance and provides stable cell standards to control for inter- and intra-assay variations. We also demonstrate the utility of the assay to characterize the activity of the first reported small molecule antagonists of the SIRPα-CD47 interaction. This assay will support the screening of thousands of compounds to identify or validate active small molecules as hits, develop structure activity relationships and assist in the optimization of hits to leads by a typical iterative medicinal chemistry campaign
Inhibition of O-GlcNAc transferase renders prostate cancer cells dependent on CDK9
O-GlcNAc transferase (OGT) is a nutrient-sensitive glycosyltransferase that is overexpressed in prostate cancer, the most common cancer in males. We recently developed a specific and potent inhibitor targeting this enzyme, and here, we report a synthetic lethality screen using this compound. Our screen identified pan-cyclin-dependent kinase (CDK) inhibitor AT7519 as lethal in combination with OGT inhibition. Follow-up chemical and genetic approaches identified CDK9 as the major target for synthetic lethality with OGT inhibition in prostate cancer cells. OGT expression is regulated through retention of the fourth intron in the gene and CDK9 inhibition blunted this regulatory mechanism. CDK9 phosphorylates carboxy-terminal domain (CTD) of RNA Polymerase II to promote transcription elongation. We show that OGT inhibition augments effects of CDK9 inhibitors on CTD phosphorylation and general transcription. Finally, the combined inhibition of both OGT and CDK9 blocked growth of organoids derived from patients with metastatic prostate cancer, but had minimal effects on normal prostate spheroids. We report a novel synthetic lethal interaction between inhibitors of OGT and CDK9 that specifically kills prostate cancer cells, but not normal cells. Our study highlights the potential of combining OGT inhibitors with other treatments to exploit cancer-specific vulnerabilities
A Small Molecule That Inhibits OGT Activity in Cells
<i>O</i>-GlcNAc transferase (OGT) is an essential mammalian
enzyme that regulates numerous cellular processes through the attachment
of <i>O</i>-linked <i>N</i>-acetylglucosamine
(<i>O</i>-GlcNAc) residues to nuclear and cytoplasmic proteins.
Its targets include kinases, phosphatases, transcription factors,
histones, and many other intracellular proteins. The biology of <i>O</i>-GlcNAc modification is still not well understood, and
cell-permeable inhibitors of OGT are needed both as research tools
and for validating OGT as a therapeutic target. Here, we report a
small molecule OGT inhibitor, OSMI-1, developed from a high-throughput
screening hit. It is cell-permeable and inhibits protein <i>O</i>-GlcNAcylation in several mammalian cell lines without qualitatively
altering cell surface <i>N</i>- or <i>O</i>-linked
glycans. The development of this molecule validates high-throughput
screening approaches for the discovery of glycosyltransferase inhibitors,
and further optimization of this scaffold may lead to yet more potent
OGT inhibitors useful for studying OGT in animal models