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miR-196b target screen reveals mechanisms maintaining leukemia stemness with therapeutic potential.Meyer SE, Muench DE, Rogers AM, Newkold TJ, Orr E, O'Brien E, Perentesis JP, Doench JG, Lal A, Morris P, Thomas C, Lieberman J, McGlinn E, Aronow BJ, Salomonis N, Grimes HLJ. Exp. Med. , (215), 2115-2136, 2018. Article Pubmed We have shown that antagomiR inhibition of miRNA miR-21 and miR-196b activity is sufficient to ablate MLL-AF9 leukemia stem cells (LSC) in vivo. Here, we used an shRNA screening approach to mimic miRNA activity on experimentally verified miR-196b targets to identify functionally important and therapeutically relevant pathways downstream of oncogenic miRNA in MLL-r AML. We found Cdkn1b (p27Kip1) is a direct miR-196b target whose repression enhanced an embryonic stem cell-like signature associated with decreased leukemia latency and increased numbers of leukemia stem cells in vivo. Conversely, elevation of p27Kip1 significantly reduced MLL-r leukemia self-renewal, promoted monocytic differentiation of leukemic blasts, and induced cell death. Antagonism of miR-196b activity or pharmacologic inhibition of the Cks1-Skp2-containing SCF E3-ubiquitin ligase complex increased p27Kip1 and inhibited human AML growth. This work illustrates that understanding oncogenic miRNA target pathways can identify actionable targets in leukemia.
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MEK inhibition induces MYOG and remodels super-enhancers in RAS-driven rhabdomyosarcoma.Yohe ME, Gryder BE, Shern JF, Song YK, Chou HC, Sindiri S, Mendoza A, Patidar R, Zhang X, Guha R, Butcher D, Isanogle KA, Robinson CM, Luo X, Chen JQ, Walton A, Awasthi P, Edmondson EF, Difilippantonio S, Wei JS, Zhao K, Ferrer-Alegre M, Thomas C, Khan JSci Transl Med , (10), 2018. Article Pubmed The RAS isoforms are frequently mutated in many types of human cancers, including PAX3/PAX7 fusion-negative rhabdomyosarcoma. Pediatric RMS arises from skeletal muscle progenitor cells that have failed to differentiate normally. The role of mutant RAS in this differentiation blockade is incompletely understood. We demonstrate that oncogenic RAS, acting through the RAF-MEK [mitogen-activated protein kinase (MAPK) kinase]-ERK (extracellular signal-regulated kinase) MAPK effector pathway, inhibits myogenic differentiation in rhabdomyosarcoma by repressing the expression of the prodifferentiation myogenic transcription factor, MYOG. This repression is mediated by ERK2-dependent promoter-proximal stalling of RNA polymerase II at the MYOG locus. Small-molecule screening with a library of mechanistically defined inhibitors showed that RAS-driven RMS is vulnerable to MEK inhibition. MEK inhibition with trametinib leads to the loss of ERK2 at the MYOG promoter and releases the transcriptional stalling of MYOG expression. MYOG subsequently opens chromatin and establishes super-enhancers at genes required for late myogenic differentiation. Furthermore, trametinib, in combination with an inhibitor of IGF1R, potently decreases rhabdomyosarcoma cell viability and slows tumor growth in xenograft models. Therefore, this combination represents a potential therapeutic for RAS-mutated rhabdomyosarcoma.
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Ketamine and Ketamine Metabolite Pharmacology: Insights into Therapeutic Mechanisms.Zanos P, Moaddel R, Morris P, Riggs LM, Highland JN, Georgiou P, Pereira EFR, Albuquerque EX, Thomas C, Zarate CA, Gould TDPharmacol. Rev. , (70), 621-660, 2018. Article Pubmed Ketamine, a racemic mixture consisting of (S)- and (R)-ketamine, has been in clinical use since 1970. Although best characterized for its dissociative anesthetic properties, ketamine also exerts analgesic, anti-inflammatory, and antidepressant actions. We provide a comprehensive review of these therapeutic uses, emphasizing drug dose, route of administration, and the time course of these effects. Dissociative, psychotomimetic, cognitive, and peripheral side effects associated with short-term or prolonged exposure, as well as recreational ketamine use, are also discussed. We further describe ketamine's pharmacokinetics, including its rapid and extensive metabolism to norketamine, dehydronorketamine, hydroxyketamine, and hydroxynorketamine (HNK) metabolites. Whereas the anesthetic and analgesic properties of ketamine are generally attributed to direct ketamine-induced inhibition of N-methyl-D-aspartate receptors, other putative lower-affinity pharmacological targets of ketamine include, but are not limited to, γ-amynobutyric acid (GABA), dopamine, serotonin, sigma, opioid, and cholinergic receptors, as well as voltage-gated sodium and hyperpolarization-activated cyclic nucleotide-gated channels. We examine the evidence supporting the relevance of these targets of ketamine and its metabolites to the clinical effects of the drug. Ketamine metabolites may have broader clinical relevance than was previously considered, given that HNK metabolites have antidepressant efficacy in preclinical studies. Overall, pharmacological target deconvolution of ketamine and its metabolites will provide insight critical to the development of new pharmacotherapies that possess the desirable clinical effects of ketamine, but limit undesirable side effects.
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Identification of Combinations of Approved Drugs With Synergistic Activity Against Ebola Virus in Cell Cultures.Dyall J, Nelson EA, DeWald LE, Guha R, Hart BJ, Zhou H, Postnikova E, Logue J, Vargas WM, Gross R, Michelotti J, Deiuliis N, Bennett RS, Crozier I, Holbrook MR, Morris P, Klumpp-Thomas C, McKnight C, Mierzwa T, Shinn P, Glass PJ, Johansen LM, Jahrling PB, Hensley LE, Olinger GG, Thomas C, White JMJ. Infect. Dis. , 2018. Article Pubmed Background: A need to develop therapeutics to treat Ebola virus disease patients in remote and resource-challenged settings remains in the wake of the 2013-2016 epidemic in West Africa. Toward this goal, we screened drugs under consideration as treatment options and other drugs of interest, most being small molecules approved by the Food and Drug Administration. Drugs demonstrating in vitro antiviral activity were advanced for evaluation in combinations because of advantages often provided by drug cocktails.
Methods: Drugs were screened for blockade of Ebola virus infection in cultured cells. Twelve drugs were tested in all (78 pair-wise) combinations, and 3 were tested in a subset of combinations.
Results: Multiple synergistic drug pairs emerged, with the majority comprising 2 entry inhibitors. For the pairs of entry inhibitors studied, synergy was demonstrated at the level of virus entry into host cells. Highly synergistic pairs included aripiprazole/piperacetazine, sertraline/toremifene, sertraline/bepridil, and amodiaquine/clomiphene.
Conclusions: Our study shows the feasibility of identifying pairs of approved drugs that synergistically block Ebola virus infection in cell cultures. We discuss our findings in terms of the theoretic ability of these or alternate combinations to reach therapeutic levels. Future research will assess selected combinations in small-animal models of Ebola virus disease.
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A multiprotein supercomplex controlling oncogenic signalling in lymphoma.Phelan JD, et al.Nature , 2018. Article Pubmed B cell receptor (BCR) signaling has emerged as a therapeutic target in B cell lymphomas, but inhibiting this pathway in diffuse large B cell lymphoma (DLBCL) has benefited only a subset of patients1. Gene expression profiling identified two major DLBCL subtypes, known as germinal center (GC) B cell-like (GCB) and activated B cell-like (ABC)2,3, with inferior outcomes following immunochemotherapy in ABC. Autoantigens drive BCR-dependent activation of NF-κB in ABC DLBCL through a kinase cascade of SYK, BTK and PKCβ to promote the assembly of the CARD11-BCL10-MALT1 (CBM) adapter complex that recruits and activates IκB kinase (IKK)4-6. Genome sequencing revealed gain-of-function mutations targeting the CD79A and CD79B BCR subunits and the Toll-like receptor (TLR) signaling adapter MYD885,7, with MYD88L265P being the most prevalent isoform. In a clinical trial, the BTK inhibitor, ibrutinib, produced responses in 37% of ABC cases1. The most striking response rate (80%) was observed in tumors with both CD79B and MYD88L265P mutations, but how these mutations cooperate to promote dependence on BCR signaling remains unclear. Herein, we used genome-wide CRISPR-Cas9 screening and functional proteomics to understand the molecular basis of exceptional clinical responses to ibrutinib. We discovered a new mode of oncogenic BCR signaling in ibrutinib-responsive cell lines and biopsies, coordinated by a multiprotein supercomplex formed by MYD88, TLR9, and the BCR (My-T-BCR). The My-T-BCR co-localizes with mTOR on endolysosomes, where it drives pro-survival NF-κB and mTOR signaling. Inhibitors of BCR and mTOR signaling cooperatively decreased My-T-BCR supercomplex formation and function, providing mechanistic insight into their synergistic toxicity for My-T-BCR+ DLBCL cells. My-T-BCR complexes characterized ibrutinib-responsive malignancies and distinguished ibrutinib responders from non-responders. Our data provide a roadmap for the rational deployment of oncogenic signaling inhibitors in molecularly-defined subsets of DLBCL.
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Pharmacological and genomic profiling of neurofibromatosis type 1 plexiform neurofibroma-derived schwann cells.Ferrer-Alegre M, Gosline SJC, Stathis M, Zhang X, Guo X, Guha R, Ryman DA, Wallace MR, Kasch-Semenza L, Hao H, Ingersoll R, Mohr D, Thomas C, Verma S, Guinney J, Blakeley JOSci Data , (5), 180106, 2018. Article Pubmed Neurofibromatosis type I (NF1) is an autosomal dominant genetic condition characterized by peripheral nervous system tumors (PNSTs), including plexiform neurofibromas (pNFs) that cause nerve dysfunction, deformity, pain damage to adjacent structures, and can undergo malignant transformation. There are no effective therapies to prevent or treat pNFs. Drug discovery efforts are slowed by the 'benign' nature of the Schwann cells that are the progenitor cells of pNF. In this work we characterize a set of pNF-derived cell lines at the genomic level (via SNP Arrays, RNAseq, and Whole Exome- Sequencing), and carry out dose response-based quantitative high-throughput screening (qHTS) with a collection of 1,912 oncology-focused compounds in a 1536-well microplate cell proliferation assays. Through the characterization and screening of NF1-/-, NF1+/+ and NF1+/- Schwann cell lines, this resource introduces novel therapeutic avenues for the development for NF1 associated pNF as well as all solid tumors with NF1 somatic mutations. The integrated data sets are openly available for further analysis at http://www.synapse.org/pnfCellCulture.
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Functional screening of FGFR4-driven tumorigenesis identifies PI3K/mTOR inhibition as a therapeutic strategy in rhabdomyosarcoma.McKinnon T, Venier R, Yohe M, Sindiri S, Gryder BE, Shern JF, Kabaroff L, Dickson B, Schleicher K, Chouinard-Pelletier G, Menezes S, Gupta A, Zhang X, Guha R, Ferrer-Alegre M, Thomas C, Wei Y, Davani D, Guidos CJ, Khan J, Gladdy RAOncogene , 2018. Article Pubmed Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma and outcomes have stagnated, highlighting a need for novel therapies. Genomic analysis of RMS has revealed that alterations in the receptor tyrosine kinase (RTK)/RAS/PI3K axis are common and that FGFR4 is frequently mutated or overexpressed. Although FGFR4 is a potentially druggable receptor tyrosine kinase, its functions in RMS are undefined. This study tested FGFR4-activating mutations and overexpression for the ability to generate RMS in mice. Murine tumor models were subsequently used to discover potential therapeutic targets and to test a dual PI3K/mTOR inhibitor in a preclinical setting. Specifically, we provide the first mechanistic evidence of differential potency in the most common human RMS mutations, V550E or N535K, compared to FGFR4wtoverexpression as murine myoblasts expressing FGFR4V550Eundergo higher rates of cellular transformation, engraftment into mice, and rapidly form sarcomas that highly resemble human RMS. Murine tumor cells overexpressing FGFR4V550Ewere tested in an in vitro dose-response drug screen along with human RMS cell lines. Compounds were grouped by target class, and potency was determined using average percentage of area under the dose-response curve (AUC). RMS cells were highly sensitive to PI3K/mTOR inhibitors, in particular, GSK2126458 (omipalisib) was a potent inhibitor of FGFR4V550Etumor-derived cell and human RMS cell viability. FGFR4V550E-overexpressing myoblasts and tumor cells had low nanomolar GSK2126458 EC50values. Mass cytometry using mouse and human RMS cell lines validated GSK2126458 specificity at single-cell resolution, decreasing the abundance of phosphorylated Akt as well as decreasing phosphorylation of the downstream mTOR effectors 4ebp1, Eif4e, and S6. Moreover, PI3K/mTOR inhibition also robustly decreased the growth of RMS tumors in vivo. Thus, by developing a preclinical platform for testing novel therapies, we identified PI3K/mTOR inhibition as a promising new therapy for this devastating pediatric cancer.
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Characterization of clinically used oral antiseptics as quadruplex-binding ligands.Calabrese D, Zlotkowski K, Alden S, Hewitt WM, Connelly CM, Wilson RM, Gaikwad S, Chen L, Guha R, Thomas C, Mock BA, Schneekloth JSNucleic Acids Res. , 2018. Article Pubmed Approaches to characterize the nucleic acid-binding properties of drugs and druglike small molecules are crucial to understanding the behavior of these compounds in cellular systems. Here, we use a Small Molecule Microarray (SMM) profiling approach to identify the preferential interaction between chlorhexidine, a widely used oral antiseptic, and the G-quadruplex (G4) structure in the KRAS oncogene promoter. The interaction of chlorhexidine and related drugs to the KRAS G4 is evaluated using multiple biophysical methods, including thermal melt, fluorescence titration and surface plasmon resonance (SPR) assays. Chlorhexidine has a specific low micromolar binding interaction with the G4, while related drugs have weaker and/or less specific interactions. Through NMR experiments and docking studies, we propose a plausible binding mode driven by both aromatic stacking and groove binding interactions. Additionally, cancer cell lines harbouring oncogenic mutations in the KRAS gene exhibit increased sensitivity to chlorhexidine. Treatment of breast cancer cells with chlorhexidine decreases KRAS protein levels, while a KRAS gene transiently expressed by a promoter lacking a G4 is not affected. This work confirms that known ligands bind broadly to G4 structures, while other drugs and druglike compounds can have more selective interactions that may be biologically relevant.
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Keap1 loss promotes Kras-driven lung cancer and results in dependence on glutaminolysis.Romero R, Sayin VI, Davidson SM, Bauer MR, Singh SX, LeBoeuf SE, Karakousi TR, Ellis DC, Bhutkar A, Sánchez-Rivera FJ, Subbaraj L, Martinez B, Bronson RT, Prigge JR, Schmidt EE, Thomas C, Goparaju C, Davies A, Dolgalev I, Heguy A, Allaj V, Poirier JT, Moreira AL, Rudin CM, Pass HI, Vander Heiden MG, Jacks T, Papagiannakopoulos TNat. Med. , (23), 1362-1368, 2017. Article Pubmed Treating KRAS-mutant lung adenocarcinoma (LUAD) remains a major challenge in cancer treatment given the difficulties associated with directly inhibiting the KRAS oncoprotein. One approach to addressing this challenge is to define mutations that frequently co-occur with those in KRAS, which themselves may lead to therapeutic vulnerabilities in tumors. Approximately 20% of KRAS-mutant LUAD tumors carry loss-of-function mutations in the KEAP1 gene encoding Kelch-like ECH-associated protein 1 (refs. 2, 3, 4), a negative regulator of nuclear factor erythroid 2-like 2 (NFE2L2; hereafter NRF2), which is the master transcriptional regulator of the endogenous antioxidant response. The high frequency of mutations in KEAP1 suggests an important role for the oxidative stress response in lung tumorigenesis. Using a CRISPR-Cas9-based approach in a mouse model of KRAS-driven LUAD, we examined the effects of Keap1 loss in lung cancer progression. We show that loss of Keap1 hyperactivates NRF2 and promotes KRAS-driven LUAD in mice. Through a combination of CRISPR-Cas9-based genetic screening and metabolomic analyses, we show that Keap1- or Nrf2-mutant cancers are dependent on increased glutaminolysis, and this property can be therapeutically exploited through the pharmacological inhibition of glutaminase. Finally, we provide a rationale for stratification of human patients with lung cancer harboring KRAS/KEAP1- or KRAS/NRF2-mutant lung tumors as likely to respond to glutaminase inhibition.
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Activation of the NRF2 antioxidant program generates an imbalance in central carbon metabolism in cancer.Sayin VI, LeBoeuf SE, Singh SX, Davidson SM, Biancur D, Guzelhan BS, Alvarez SW, Wu WL, Karakousi TR, Zavitsanou AM, Ubriaco J, Muir A, Karagiannis D, Morris P, Thomas C, Possemato R, Vander Heiden MG, Papagiannakopoulos TElife , (6), 2017. Article Pubmed During tumorigenesis, the high metabolic demand of cancer cells results in increased production of reactive oxygen species. To maintain oxidative homeostasis, tumor cells increase their antioxidant production through hyperactivation of the NRF2 pathway, which promotes tumor cell growth. Despite the extensive characterization of NRF2-driven metabolic rewiring, little is known about the metabolic liabilities generated by this reprogramming. Here, we show that activation of NRF2, in either mouse or human cancer cells, leads to increased dependency on exogenous glutamine through increased consumption of glutamate for glutathione synthesis and glutamate secretion by xc(-) antiporter system. Together, this limits glutamate availability for the tricarboxylic acid cycle and other biosynthetic reactions creating a metabolic bottleneck. Cancers with genetic or pharmacological activation of the NRF2 antioxidant pathway have a metabolic imbalance between supporting increased antioxidant capacity over central carbon metabolism, which can be therapeutically exploited.
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