Browsing by Author "Wells, Clark D."

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    Bidirectional regulation of YAP and ALDH1A1
    (2015-08-10) Martien, Matthew F.; Wells, Clark D.; Hurley, Thomas D.; Quilliam, Lawrence A.
    Breast cancer is the second leading cause of cancer death for women in the United States. Approximately, 1 in 5 women will recur with cancer within 10 years of completing treatment and recent publications have suggested that breast cancer stem cells confer resistance to therapy. These reports highlight aldehyde dehydrogenase 1A1 (ALDH1A1) and Yes-associated protein (YAP) as a biomarker and key mediator of the stem cell phenotype respectively. To further understand how YAP and ALDH1A1 facilitate chemoresistance, this study investigated how ALDH1A1 specific inhibition affected YAP activity and growth of basal-like breast cancer cells, which are enriched in cancer stem cells. Intriguingly, attenuation of growth by ALDH1A1 inhibition was observed when cells were plated on a reconstituted basement membrane. Further, the inhibition of cell growth correlated with cytosolic retention of YAP and a reduction in YAP signaling. In a complementary analysis, the overexpression of YAP correlated with an increased level of ALDH1A1 transcript. Results from this study indicate a novel mechanism by which basal-like breast cancer cells utilize YAP to maintain the stem cell phenotype and also suggest ALDH1A1 as a potential therapeutic target for breast cancer therapy.
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    A Cell-Based Model to Study Factors that Drive Diffuse Astrocytoma Development
    (2016-08) Folck, Anthony F.; Wells, Clark D.; Goebl, Mark G.; Quilliam, Lawrence A.
    Secondary gliomas are an incurable form of brain cancer that are diagnosed in people at a median age of 45 years. Next-generation sequencing has found that secondary glioblastomas have a distinct genetic profile from the more common primary glioblastomas, which are diagnosed in people typically over the age of 60. Over 80% of secondary gliomas contain an IDH1R132H mutation, resulting in neomorphic mutations, which catalyze isocitrate to the oncometabolite D-2-hydroxyglutarate (2-HG) instead of alpha-ketoglutarate (α-KG). As 2-HG accumulates, it induces a hypermethylator phenotype that prevents the cells from differentiating. Acquisition of additional mutations in tumor suppressors such as p53 and/or ATRX likely leads to tumor initiation. Work in the Wells Laboratory has found that loss of the HIPPO adaptor protein AmotL1 is also associated with increased malignancy. AmotL1 inhibits the transcriptional co-activator YAP to suppress both cell growth and migration. To understand the molecular events leading to secondary glioma development, this thesis developed a series of astrocyte cell lines that carry IDH1 and/or p53 mutations. These lines were then studied in 2D and 3D cell culture systems to identify changes that are associated with early secondary glial tumors. Work was also carried out to enable screens for small molecules that can be tested on these cell lines.
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    Combination therapy in a xenograft model of glioblastoma: enhancement of the antitumor activity of temozolomide by an MDM2 antagonist
    (American Association of Neurological Surgeons, 2017-02) Wang, Haiyan; Cai, Shanbao; Bailey, Barbara J.; Saadatzadeh, M. Reza; Ding, Jixin; Tonsing-Carter, Eva; Georgiadis, Taxiarchis M.; Gunter, T. Zachary; Long, Eric C.; Minto, Robert E.; Gordon, Kevin R.; Sen, Stephanie E.; Cai, Wenjing; Eitel, Jacob A.; Waning, David L.; Bringman, Lauren R.; Wells, Clark D.; Murray, Mary E.; Sarkaria, Jann N.; Gelbert, Lawrence M.; Jones, David R.; Cohen-Gadol, Aaron A.; Mayo, Lindsey D.; Shannon, Harlan E.; Pollok, Karen E.; Pediatrics, School of Medicine
    OBJECTIVE Improvement in treatment outcome for patients with glioblastoma multiforme (GBM) requires a multifaceted approach due to dysregulation of numerous signaling pathways. The murine double minute 2 (MDM2) protein may fulfill this requirement because it is involved in the regulation of growth, survival, and invasion. The objective of this study was to investigate the impact of modulating MDM2 function in combination with front-line temozolomide (TMZ) therapy in GBM. METHODS The combination of TMZ with the MDM2 protein-protein interaction inhibitor nutlin3a was evaluated for effects on cell growth, p53 pathway activation, expression of DNA repair proteins, and invasive properties. In vivo efficacy was assessed in xenograft models of human GBM. RESULTS In combination, TMZ/nutlin3a was additive to synergistic in decreasing growth of wild-type p53 GBM cells. Pharmacodynamic studies demonstrated that inhibition of cell growth following exposure to TMZ/nutlin3a correlated with: 1) activation of the p53 pathway, 2) downregulation of DNA repair proteins, 3) persistence of DNA damage, and 4) decreased invasion. Pharmacokinetic studies indicated that nutlin3a was detected in human intracranial tumor xenografts. To assess therapeutic potential, efficacy studies were conducted in a xenograft model of intracranial GBM by using GBM cells derived from a recurrent wild-type p53 GBM that is highly TMZ resistant (GBM10). Three 5-day cycles of TMZ/nutlin3a resulted in a significant increase in the survival of mice with GBM10 intracranial tumors compared with single-agent therapy. CONCLUSIONS Modulation of MDM2/p53-associated signaling pathways is a novel approach for decreasing TMZ resistance in GBM. To the authors' knowledge, this is the first study in a humanized intracranial patient-derived xenograft model to demonstrate the efficacy of combining front-line TMZ therapy and an inhibitor of MDM2 protein-protein interactions.
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    Contributions of Angiomotin-Like-1 on Astrocytic Morphology: Potential Roles in Regulating Connexin-43-Based Astrocytic Gap Junctions, Remodeling the Actin Cytoskeleton and Influencing Cellular Polarity
    (2019-10) Downing, Nicholas Frederick; Wells, Clark D.; Goebl, Mark G.; Quilliam, Lawrence A.
    Glioblastoma is a lethal cancer that arises from support cells in the nervous system and kills around 20,000 people in the United States each year. While much is known about the highly malignant primary glioblastoma, the natural history of lower grade glioma (LGG) is less understood. While the majority of LGGs are initiated by a mutation in isocitrate dehydrogenase, the events leading to their malignant progression into a grade IV tumor are not known. Analysis of primary tumor sample data has revealed that low transcript levels of Angiomotin-like-1 (AmotL1) strongly associate with poor outcomes of patients with these cancers. Follow-up RNA-sequencing of human embryonic astrocytes with AmotL1 silencing revealed the downregulation of many transcripts that encode proteins mediating gap junctions (GJ) between astrocytes, especially connexin-43 (Cx43). Cx43 protein oligomerizes to form functional channels comprising the astrocytic GJ. AmotL1 knockdown through RNA interference decreases Cx43 transcript and protein levels while increasing its distribution to GJs. This suggests increased GJ formation and intercellular communication, as similar localization patterns are observed in differentiated astrocytes. Astrocytes with AmotL1 knockdown also display a pronounced pancake-like morphology, suggesting that the actin cytoskeleton is affected. Imaging reveals that cells with reduced AmotL1 have characteristic losses in both stress fibers and focal actin under the cell body but notable increases in cortical F-actin. Consistent with previous studies, AmotL1 may promote increases in the number and thickness of F-actin fibers. Because actin binding to related angiomotins is inhibited by phosphorylation from the LATs kinases, I define the effects of expressing wildtype AmotL1 versus mutants that mimic or prevent phosphorylation by LATs1/2. Interestingly, expression of AmotL1 S262D in combination with NEDD4-1, a ubiquitin ligase, results in a profound loss of actin stress fibers. Dependence on NEDD4-1 suggests that this phenotype is due to the induced degradation of proteins that promote F-actin, e.g. RhoA. These results directly support a model in which phosphorylated AmotL1 specifically inhibits F-actin formation as opposed to unphosphorylated AmotL1 which is known to promote stress fiber formation. Thus, in addition to regulating polarity and YAP/TAZ transcriptional co-activators, AmotL1 plays major functions in dictating cellular F-actin dynamics.
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    Defining the Roles of Various Lysines and Arginines in Amot Lipid Binding
    (Office of the Vice Chancellor for Research, 2014-04-11) Hall, L'eCelia; Kimble-Hill, Ann C.; Wells, Clark D.; Hurley, Thomas
    One of the defining traits of cancerous cells is proliferation. The focus of this study is on the proliferation of mammary cells. As an adaptor protein, the Amot membrane binding event is key to the localization and sorting of proteins responsible for cellular differentiation, proliferation, and migration. The Amot coiled-coil homology domain (ACCH) is a lipid-binding domain responsible for cholesterol affinity and binding to endothelial membranes. Our working hypothesis is that the ability to modulate Amot lipid-binding will lead to means to prevent ductal cell hyperplasia progression into breast cancer tumors. We will determine which residues are responsible for lipid-binding by changing positively charged lysine and arginine into uncharged or negatively charged amino acids. Approximately 40 of these mutations have been screened using a liposome binding assay which mimics how the protein binds with the cell membrane by using an in vitro mixture of lipids similar to that seen in endothelial cells. Forster resonance energy transfer (FRET) was used to confirm significant decreases in lipid binding of ACCH mutants selected from the liposome binding assay, as energy transfer only occurs when the tyrosines in the protein and the Dansylated liposome are in close proximity to each other. In order to saturate the binding affinity of the mutants, the liposomes will be combined with cholesterol in increasing amounts. It has been found that Amot protein is concentrated in areas of PI with higher levels of cholesterol. This will provide a target for the ACCH domain to associate with in the membrane. Mutants deemed important from this study will then be transformed into human cells to study their effects on cell polarity, signal transduction, cell shape, and cellular proliferation.
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    The effects of verteporfin on non-small cell lung cancer
    (2016-08) Ackerman, Todd R., Jr; Quilliam, Lawrence A.; Wells, Clark D.; Goebl, Mark G.
    Non-small cell lung cancer (NSCLC) accounts for 85% of lung cancers and is the leading cause of cancer death in the Unites States. Better treatments must be devised in order to improve the prognosis of this disease. Verteporfin, an FDA approved drug, has recently been reported to downregulate a potential core pathway of NSCLC, the Hippo pathway. The pathway consists of a kinase cascade to control the transcriptional coactivators YAP and TAZ. When these transcriptional coactivators lack phosphorylation of key residues, they are able to translocate into the nucleus and bind to the TEAD member of transcription factors. This augments transcription for genes responsible for proliferation, survival, and stem maintenance. In this study, we report that verteporfin limits proliferation and survival of NSCLC and may potentially be a viable treatment option. Inhibition of cell survival dose-dependently correlated with inhibition of YAP-TEAD transcription target CTGF. We also report the covalent homo-oligomerization of p62, a prominent protein involved with autophagy, with the introduction of verteporfin into NSCLC cells.
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    Exploring the Existing Drug Space for Novel pTyr Mimetic and SHP2 Inhibitors
    (American Chemical Society, 2015-07-09) He, Rongjun; Yu, Zhi-Hong; Zhang, Ruo-Yu; Wu, Li; Gunawan, Andrea M.; Lane, Brandon S.; Shim, Joong S.; Zeng, Li-Fan; He, Yantao; Chen, Lan; Wells, Clark D.; Liu, Jun O.; Zhang, Zhong-Yin; Department of Biochemistry & Molecular Biology, IU School of Medicine
    Protein tyrosine phosphatases (PTPs) are potential therapeutic targets for many diseases. Unfortunately, despite considerable drug discovery efforts devoted to PTPs, obtaining selective and cell permeable PTP inhibitors remains highly challenging. We describe a strategy to explore the existing drug space for previously unknown PTP inhibitory activities. This led to the discovery of cefsulodin as an inhibitor of SHP2, an oncogenic phosphatase in the PTP family. Crystal structure analysis of SHP2 interaction with cefsulodin identified sulfophenyl acetic amide (SPAA) as a novel phosphotyrosine (pTyr) mimetic. A structure-guided and SPAA fragment-based focused library approach produced several potent and selective SHP2 inhibitors. Notably, these inhibitors blocked SHP2-mediated signaling events and proliferation in several cancer cell lines. Thus, SPAA may serve as a new platform for developing chemical probes for other PTPs.
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    Functional Insights Into Oncogenic Protein Tyrosine Phosphatases By Mass Spectrometry
    (2012-12) Walls, Chad Daniel; Zhang, Zhong-Yin; Wang, Mu; Wells, Clark D.; Zhang, Jian-Ting
    Phosphatase of Regenerating Liver 3 (PRL3) is suspected to be a causative factor toward cellular metastasis when overexpressed. To date, the molecular basis for PRL3 function remains an enigma, justifying the use of 'shot-gun'-style phosphoproteomic strategies to define the PRL3-mediated signaling network. On the basis of aberrant Src tyrosine kinase activation following ectopic PRL3 expression, phosphoproteomic data reveal a signal transduction network downstream of a mitogenic and chemotactic PDGF (α and β), Eph (A2, B3, B4), and Integrin (β1 and β5) receptor array known to be utilized by migratory mesenchymal cells during development and acute wound healing in the adult animal. Tyrosine phosphorylation is present on a multitude of signaling effectors responsible for Rho-family GTPase, PI3K-Akt, Jak-STAT3, and Ras-ERK1/2 pathway activation, linking observations made by the field as a whole under Src as a primary signal transducer. Our phosphoproteomic data paint the most comprehensive picture to date of how PRL3 drives pro-metastatic molecular events through Src activation. The Src-homology 2 (SH2) domain-containing tyrosine phosphatase 2 (SHP2), encoded by the Ptpn11 gene, is a bona-fide proto-oncogene responsible for the activation of the Ras/ERK1/2 pathway following mitogen stimulation. The molecular basis for SHP2 function is pTyr-ligand-mediated alleviation of intramolecular autoinhibition by the N-terminal SH2 domain (N-SH2 domain) upon the PTP catalytic domain. Pathogenic mutations that reside within the interface region between the N-SH2 and PTP domains are postulated to weaken the autoinhibitory interaction leading to SHP2 catalytic activation in the open conformation. Conversely, a subset of mutations resides within the catalytic active site and cause catalytic impairment. These catalytically impaired SHP2 mutants potentiate the pathogenesis of LEOPARD-syndrome (LS), a neuro-cardio-facial-cutaneous (NCFC) syndrome with very similar clinical presentation to related Noonan syndrome (NS), which is known to be caused by gain-of-function (GOF) SHP2 mutants. Here we apply hydrogen-deuterium exchange mass spectrometry (H/DX-MS) to provide direct evidence that LS-associated SHP2 mutations which cause catalytic impairment also weaken the autoinhibitory interaction that the N-SH2 domain makes with the PTP domain. Our H/DX-MS study shows that LS-SHP2 mutants possess a biophysical property that is absolutely required for GOF-effects to be realized, in-vivo.
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    HE AMOT FAMILY OF PROTEINS BINDS AND ACTIVATES NEDD4 FAMILY LIGASES TO PROMOTE THE UBIQUITINATION OF LATS AND YAP
    (Office of the Vice Chancellor for Research, 2012-04-13) Adler, Jacob J.; Heller, Brigitte L.; Tuchek, Chad A.; Ingham, Robert J.; Wells, Clark D.
    Amot adaptor proteins bind and integrate signaling that controls cell po-larity and growth. All three Amot family members (Amot, AmotL1 and AmotL2) directly bind YAP; a transcriptional co-activator that controls the expression of genes involved in organ homeostasis and cell growth. Preven-tion of nuclear accumulation of YAP by either sequestration or degradation in the cytosol abolishes its transcriptional functions and is a major mechanism for growth arrest in response to cellular differentiation. This is mainly thought to be regulated by phosphorylation of YAP by the Hippo kinases LATS1/2. Recently, binding by the Amot proteins was also found to inhibit YAP by sequestering it in the cytosol through both LATS dependent and in-dependent mechanisms. This study identifies a novel mechanism whereby Amot proteins control YAP activation in a Hippo independent mechanism by coupling it to ubiquitination by Nedd4 family ligases. Amot proteins mediate the coupling of Nedd4 ligases with YAP by simultaneously binding both pro-teins via multiple PY motifs that are recognized by WW domains in both YAP and Nedd4. Binding of Nedd4 by Amot is also shown to relieve the auto-inhibition of its ligase activity. This may be a direct consequence of binding Amot or from being re-targeted in cells by Amot proteins to endosomes. Im-portantly, Amot induced ubiquitination of YAP by Nedd4 proteins is shown to enhance the residence of YAP in the nucleus and in YAP activated transcrip-tion. Taken together our data suggest that Amot couples Nedd4 family ubiq-uitin ligases with the transcriptional co-activator YAP to drive the ubiquitination and activation of YAP.
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    Identification of Novel Small Molecule Inhibitors of YAP-TEAD Binding within the Hippo Signaling Pathway
    (2020-09) Thompson, Abigail Fisk; Wells, Clark D.; Georgiadis, Millie M.; Goebl, Mark G.; Johnson, Steven M.
    The Hippo signaling pathway controls organ size by regulating cell proliferation, apoptosis, and cell differentiation. The Hippo pathway ultimately regulates the concentration of the coactivator yes-associated protein 1 (YAP1) in the nucleus, which binds the transcription factor TEA domains (TEAD), activating genes related to cell proliferation. In several cancers, increased YAP1 activity is linked to increased cellular proliferation, de-differentiation and survival to drive tumor progression and spreading. The development of inhibitors against YAP-TEAD binding are consequently a research topic of much interest. While several inhibitors of the YAP-TEAD interaction are reported, none possess both high specificity and anti-tumor activity. The work described here is based upon a collaboration with AtomWise, Inc to find a best in class competitive inhibitor of YAP-TEAD binding. AtomWise completed a computational screen of ten million compounds for potential binding to a TEAD pocket that is essential for interaction with YAP1. This master’s thesis encompasses both intracellular and biochemical validation of these compounds. Initially, a luciferase YAP/TEAD dependent reporter assay was used to identify compounds with potential intracellular activity. This data was compared with the action of these compounds on the metabolic activity (MTT assay) across three cancer cell lines. Three sulfonamide-based compounds, 4, 22, and 59 were identified as top 10 YAP/TEAD inhibitors. Six additional compounds were synthesized that combined specific moieties in these three compounds. Dose response curves for inhibition of TEAD reporter activity showed that compounds 22 and 59 exhibited both high inhibition and low IC50 values relative to the derivatives. Further, compound 22 also significantly reduced the levels of CTGF transcript (measured by RT-qPCR), a surrogate measure for endogenous YAP1: TEAD activity. Preliminary fluorescent polarization data also suggests that compound 22 inhibits the binding of a 5(6)-FAM labeled YAP1 peptide (residues 58-74) to a purified TEAD fragment. In this work, compounds 22 and 59 are found to inhibit TEAD dependent transcription and the growth of multiple cancer cell lines. In addition, promising preliminary biochemical data indicates that compound 22 may inhibit the interaction of YAP1 with TEAD.