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Therapeutic strategies for diffuse midline glioma from high-throughput combination drug screening.Lin GL, et al.Sci Transl Med , (11), 2019. Article Pubmed Diffuse midline gliomas (DMGs) are universally lethal malignancies occurring chiefly during childhood and involving midline structures of the central nervous system, including thalamus, pons, and spinal cord. These molecularly related cancers are characterized by high prevalence of the histone H3K27M mutation. In search of effective therapeutic options, we examined multiple DMG cultures in sequential quantitative high-throughput screens (HTS) of 2706 approved and investigational drugs. This effort generated 19,936 single-agent dose responses that inspired a series of HTS-enabled drug combination assessments encompassing 9195 drug-drug examinations. Top combinations were validated across patient-derived cell cultures representing the major DMG genotypes. In vivo testing in patient-derived xenograft models validated the combination of the multi-histone deacetylase (HDAC) inhibitor panobinostat and the proteasome inhibitor marizomib as a promising therapeutic approach. Transcriptional and metabolomic surveys revealed substantial alterations to key metabolic processes and the cellular unfolded protein response after treatment with panobinostat and marizomib. Mitigation of drug-induced cytotoxicity and basal mitochondrial respiration with exogenous application of nicotinamide mononucleotide (NMN) or exacerbation of these phenotypes when blocking nicotinamide adenine dinucleotide (NAD+) production via nicotinamide phosphoribosyltransferase (NAMPT) inhibition demonstrated that metabolic catastrophe drives the combination-induced cytotoxicity. This study provides a comprehensive single-agent and combinatorial drug screen for DMG and identifies concomitant HDAC and proteasome inhibition as a promising therapeutic strategy that underscores underrecognized metabolic vulnerabilities in DMG.
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Structure-Based Evolution of Low Nanomolar O-GlcNAc Transferase Inhibitors.Martin SES, Tan ZW, Itkonen HM, Duveau D, Paulo JA, Janetzko J, Boutz PL, Törk L, Moss FA, Thomas C, Gygi SP, Lazarus MB, Walker SJ. Am. Chem. Soc. , (140), 13542-13545, 2018. Article Pubmed Reversible glycosylation of nuclear and cytoplasmic proteins is an important regulatory mechanism across metazoans. One enzyme, O-linked N-acetylglucosamine transferase (OGT), is responsible for all nucleocytoplasmic glycosylation and there is a well-known need for potent, cell-permeable inhibitors to interrogate OGT function. Here we report the structure-based evolution of OGT inhibitors culminating in compounds with low nanomolar inhibitory potency and on-target cellular activity. In addition to disclosing useful OGT inhibitors, the structures we report provide insight into how to inhibit glycosyltransferases, a family of enzymes that has been notoriously refractory to inhibitor development.
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High-grade gliomas (HGG) are a devastating group of cancers, and represent the leading cause of brain tumour-related death in both children and adults. Therapies aimed at mechanisms intrinsic to glioma cells have translated to only limited success; effective therapeutic strategies will need also to target elements of the tumour microenvironment that promote glioma progression. Neuronal activity promotes the growth of a range of molecularly and clinically distinct HGG types, including adult and paediatric glioblastoma (GBM), anaplastic oligodendroglioma, and diffuse intrinsic pontine glioma (DIPG). An important mechanism that mediates this neural regulation of brain cancer is activity-dependent cleavage and secretion of the synaptic adhesion molecule neuroligin-3 (NLGN3), which promotes glioma proliferation through the PI3K-mTOR pathway. However, the necessity of NLGN3 for glioma growth, the proteolytic mechanism of NLGN3 secretion, and the further molecular consequences of NLGN3 secretion in glioma cells remain unknown. Here we show that HGG growth depends on microenvironmental NLGN3, identify signalling cascades downstream of NLGN3 binding in glioma, and determine a therapeutically targetable mechanism of secretion. Patient-derived orthotopic xenografts of paediatric GBM, DIPG and adult GBM fail to grow in Nlgn3 knockout mice. NLGN3 stimulates several oncogenic pathways, such as early focal adhesion kinase activation upstream of PI3K-mTOR, and induces transcriptional changes that include upregulation of several synapse-related genes in glioma cells. NLGN3 is cleaved from both neurons and oligodendrocyte precursor cells via the ADAM10 sheddase. ADAM10 inhibitors prevent the release of NLGN3 into the tumour microenvironment and robustly block HGG xenograft growth. This work defines a promising strategy for targeting NLGN3 secretion, which could prove transformative for HGG therapy.
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Matrix Screen Identifies Synergistic Combination of PARP Inhibitors and Nicotinamide Phosphoribosyltransferase (NAMPT) Inhibitors in Ewing Sarcoma.Heske CM, Davis MI, Baumgart JT, Wilson K, Gormally MV, Chen L, Zhang X, Ceribelli M, Duveau D, Guha R, Ferrer-Alegre M, Arnaldez FI, Ji J, Tran HL, Zhang Y, Mendoza A, Helman LJ, Thomas CClin. Cancer Res. , 2017. Article Pubmed Purpose: Although many cancers are showing remarkable responses to targeted therapies, pediatric sarcomas, including Ewing sarcoma, remain recalcitrant. To broaden the therapeutic landscape, we explored the in vitro response of Ewing sarcoma cell lines against a large collection of investigational and approved drugs to identify candidate combinations.Experimental Design: Drugs displaying activity as single agents were evaluated in combinatorial (matrix) format to identify highly active, synergistic drug combinations, and combinations were subsequently validated in multiple cell lines using various agents from each class. Comprehensive metabolomic and proteomic profiling was performed to better understand the mechanism underlying the synergy. Xenograft experiments were performed to determine efficacy and in vivo mechanism.Results: Several promising candidates emerged, including the combination of small-molecule PARP and nicotinamide phosphoribosyltransferase (NAMPT) inhibitors, a rational combination as NAMPTis block the rate-limiting enzyme in the production of nicotinamide adenine dinucleotide (NAD(+)), a necessary substrate of PARP. Mechanistic drivers of the synergistic cell killing phenotype of these combined drugs included depletion of NMN and NAD(+), diminished PAR activity, increased DNA damage, and apoptosis. Combination PARPis and NAMPTis in vivo resulted in tumor regression, delayed disease progression, and increased survival.Conclusions: These studies highlight the potential of these drugs as a possible therapeutic option in treating patients with Ewing sarcoma. Clin Cancer Res; 23(23); 1-11. ©2017 AACR.
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Selective targeting of JAK/STAT signaling is potentiated by Bcl-xL blockade in IL-2-dependent adult T-cell leukemia.Zhang M, Mathews Griner LA, Ju W, Duveau D, Guha R, Petrus MN, Wen B, Maeda M, Shinn P, Ferrer-Alegre M, Conlon KD, Bamford RN, O'Shea JJ, Thomas C, Waldmann TAProc. Natl. Acad. Sci. U.S.A. , (112), 12480-5, 2015. Article Pubmed Adult T-cell leukemia (ATL) develops in individuals infected with human T-cell lymphotropic virus-1 (HTLV-1). Presently there is no curative therapy for ATL. HTLV-1-encoded protein Tax (transactivator from the X-gene region) up-regulates Bcl-xL (B-cell lymphoma-extra large) expression and activates interleukin-2 (IL-2), IL-9, and IL-15 autocrine/paracrine systems, resulting in amplified JAK/STAT signaling. Inhibition of JAK signaling reduces cytokine-dependent ex vivo proliferation of peripheral blood mononuclear cells (PBMCs) from ATL patients in smoldering/chronic stages. Currently, two JAK inhibitors are approved for human use. In this study, we examined activity of multiple JAK inhibitors in ATL cell lines. The selective JAK inhibitor ruxolitinib was examined in a high-throughput matrix screen combined with >450 potential therapeutic agents, and Bcl-2/Bcl-xL inhibitor navitoclax was identified as a strong candidate for multicomponent therapy. The combination was noted to strongly activate BAX (Bcl-2-associated X protein), effect mitochondrial depolarization, and increase caspase 3/7 activities that lead to cleavage of PARP (poly ADP ribose polymerase) and Mcl-1 (myeloid cell leukemia 1). Ruxolitinib and navitoclax independently demonstrated modest antitumor efficacy, whereas the combination dramatically lowered tumor burden and prolonged survival in an ATL murine model. This combination strongly blocked ex vivo proliferation of five ATL patients' PBMCs. These studies provide support for a therapeutic trial in patients with smoldering/chronic ATL using a drug combination that inhibits JAK signaling and antiapoptotic protein Bcl-xL.
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A small molecule that inhibits OGT activity in cells.Ortiz-Meoz RF, Jiang J, Lazarus MB, Orman M, Janetzko J, Fan C, Duveau D, Tan ZW, Thomas C, Walker SACS Chem. Biol. , (10), 1392-7, 2015. Article Pubmed O-GlcNAc transferase (OGT) is an essential mammalian enzyme that regulates numerous cellular processes through the attachment of O-linked N-acetylglucosamine (O-GlcNAc) residues to nuclear and cytoplasmic proteins. Its targets include kinases, phosphatases, transcription factors, histones, and many other intracellular proteins. The biology of O-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 O-GlcNAcylation in several mammalian cell lines without qualitatively altering cell surface N- or O-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.
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High-throughput combinatorial screening identifies drugs that cooperate with ibrutinib to kill activated B-cell-like diffuse large B-cell lymphoma cells.Mathews Griner LA, Guha R, Shinn P, Young RM, Keller JM, Liu D, Goldlust IS, Yasgar A, McKnight C, Boxer MB, Duveau D, Jiang J, Michael S, Mierzwa T, Huang W, Walsh MJ, Mott BT, Patel P, Leister W, Maloney DJ, LeClair C, Rai Bantukallu G, Jadhav A, Peyser BD, Austin C, Martin SE, Simeonov A, Ferrer-Alegre M, Staudt LM, Thomas CProc. Natl. Acad. Sci. U.S.A. , (111), 2349-54, 2014. Article Pubmed The clinical development of drug combinations is typically achieved through trial-and-error or via insight gained through a detailed molecular understanding of dysregulated signaling pathways in a specific cancer type. Unbiased small-molecule combination (matrix) screening represents a high-throughput means to explore hundreds and even thousands of drug-drug pairs for potential investigation and translation. Here, we describe a high-throughput screening platform capable of testing compounds in pairwise matrix blocks for the rapid and systematic identification of synergistic, additive, and antagonistic drug combinations. We use this platform to define potential therapeutic combinations for the activated B-cell-like subtype (ABC) of diffuse large B-cell lymphoma (DLBCL). We identify drugs with synergy, additivity, and antagonism with the Bruton's tyrosine kinase inhibitor ibrutinib, which targets the chronic active B-cell receptor signaling that characterizes ABC DLBCL. Ibrutinib interacted favorably with a wide range of compounds, including inhibitors of the PI3K-AKT-mammalian target of rapamycin signaling cascade, other B-cell receptor pathway inhibitors, Bcl-2 family inhibitors, and several components of chemotherapy that is the standard of care for DLBCL.
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Elucidation of the structural basis of interaction of the BCR-ABL kinase inhibitor, nilotinib (Tasigna) with the human ABC drug transporter P-glycoprotein.Shukla S, Chufan EE, Singh S, Skoumbourdis AP, Kapoor K, Boxer MB, Duveau D, Thomas C, Talele TT, Ambudkar SVLeukemia , (28), 961-4, 2014. Article Pubmed |
Structure-activity relationship studies and biological characterization of human NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase inhibitors.Duveau D, Yasgar A, Wang Y, Hu X, Kouznetsova J, Brimacombe K, Jadhav A, Simeonov A, Thomas C, Maloney DJBioorg. Med. Chem. Lett. , (24), 630-5, 2014. Article Pubmed The structure-activity relationship (SAR) study of two chemotypes identified as inhibitors of the human NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (HPGD, 15-PGDH) was conducted. Top compounds from both series displayed potent inhibition (IC50 <50 nM), demonstrate excellent selectivity towards HPGD and potently induce PGE2 production in A549 lung cancer and LNCaP prostate cancer cells.
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The importance of the trifluoromethyl group in the polypharmacological profile of nilotinib was investigated. Molecular editing of nilotinib led to the design, synthesis and biological evaluation of analogues where the trifluoromethyl group was replaced by a proton, fluorine and a methyl group. While these analogues were less active than nilotinib toward Abl, their activity toward Kit was comparable, with the monofluorinated analogue being the most active. Docking of nilotinib and of analogues 2a-c to the binding pocket of Abl and of Kit showed that the lack of shape complementarity in Kit is compensated by the stabilizing effect from its juxtamembrane region.
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