• Applications of Quantitative Proteomics and Phosphoproteomics to Study the Development of Resistance to Targeted Therapy in Cancer

      Awasthi, Shivangi; Shapiro, Paul, Ph.D.; Goodlett, David Robinson, 1960-; Guha, Udayan (2018)
      Targeted inhibition of protein kinases is a major approach to treat cancer. However, the effectiveness of kinase inhibitors is limited due to intrinsic and acquired resistance mechanisms that promote the progression and survival of cancer cells. The objective of this dissertation is to use liquid chromatography coupled to mass spectrometry (LC MS) based quantitative proteomics to identify potential biomarkers of resistance and response to molecularly targeted therapies in cutaneous melanoma and lung adenocarcinoma in vitro. For the first part of this thesis, I conducted a proteomic analysis of the acquired drug resistance to extracellular signal-regulated kinase (ERK1/2) pathway inhibitors in a melanoma cell line model. A combination of immunoblot assays, global label-free bottom-up proteomics, phosphoproteomics and pathway analysis was used to characterize the differential protein expression in drug resistant melanoma cells. Examination of the quantitative data pointed to an invasive and metastatic phenotypic signature in the resistant cells. We also identified and verified the overexpression of β-catenin and Caveolin-1 (CAV-1) in MEK1/2 and ERK1/2 inhibitor resistant cells. These findings suggest that these proteins have a role in the development of resistance and may represent novel targets for co-therapy. For the second part of this thesis, I have utilized a multiple reaction monitoring (MRM) based targeted proteomic technique to verify previously identified potential biomarkers of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) response in lung adenocarcinoma. Published global phosphoproteomic data were used to select a list of phosphotyrosine peptides (pY) and MRM based relative and absolute quantitative methods were developed to measure their expression in TKI sensitive and resistant lung adenocarcinoma cells. Modified immuno-MRM assays were optimized using heavy labelled synthetic peptide standards which identified the targets with good reproducibility and repeatability. The results indicated that of the 11 chosen sites, EGFR-pY1197 can be used as potential biomarker of EGFR TKI sensitivity, regardless of the EGFR TKI used. Overall these data advance our understanding of the mechanisms of targeted therapy resistance and highlight candidate biomarkers of resistance and sensitivity.
    • Exploiting Vulnerabilities in Cancers with Activated Extracellular Signal-Regulated Kinase (ERK1/2)

      Martinez, Ramon; Shapiro, Paul, Ph.D.; 0000-0002-0050-3172 (2021)
      Constitutively active extracellular signal–regulated kinase (ERK) 1/2 signaling promotes cancer cell proliferation and survival. ERK1/2 pathway inhibitors are important therapies for treating many cancers, however, acquired resistance to most protein kinase inhibitors limit their ability to provide durable responses. Few studies have looked at the adaptive proteome responses of BRAF/MEK dual inhibitor resistant cells, and fewer still have established the utility of allosteric inhibitors targeting the ERK2 protein. The overall goal of the current study was to identify signaling mechanisms of therapeutic resistance and further investigate a previously identified inhibitor of melanoma cell growth with putative activity with the protein ERK2. To test this, we hypothesize that vulnerable targets can be identified for therapeutic intervention, and test if alternative inhibitors exhibit clinical potential for melanoma treatment in two specific aims. In Aim 1, we characterized the global protein changes happening in constitutively-activeERK1/2 melanoma cells models that developed resistance to BRAF (PLX4032) and MEK1/2 (AZD6244) inhibitors using mass spectrometry. We additionally identified putative targets for treatment and analyzed the potential for a metabolic inhibitor, niclosamide. In Aim 2, studies were focused on elucidating the structure-activity relationship and binding mechanism of a ERK2-specific inhibitor developed in house, SF-3-030. We identified chemical features required for biologic activity and global effects on gene and protein levels in A375 melanoma cells containing mutant BRAF(V600E). We then identified the mechanism of action of the inhibitor in preventing melanoma growth using mass spectrometry analysis. Overall, these studies help to elucidate how cells overcome currently used clinical therapies, what vulnerable markers can be identified and used for therapeutic intervention, and that inhibitors alternative to the clinical standard can show potential for melanoma treatment.
    • Identification of potential compounds that selectively inhibit ERK-substrate interactions

      Chen, Fengming; Shapiro, Paul, Ph.D. (2009)
      The extracellular signal-regulated kinase (ERK1 and ERK2) proteins play an important role in the proliferation and survival of many types of cancer cells. However, no specific inhibitors of ERK proteins are currently available. Drug discovery efforts focusing on kinase inhibitors target the ATP binding site and the catalytic domain. However, the conserved features of the ATP binding site among protein kinases make identifying a selective inhibitor difficult. Therefore, it is important to develop ATP-independent inhibitors that block the functions of active ERK2 in cancer cells. Different from traditional methods which screen compounds from large chemical libraries, we used the 3D structure of active ERK2 and computer aided drug design (CADD) to identify novel compounds that selectively prevent ERK-substrate interactions. ATP-independent ERK docking domain inhibitors were initially selected in silico, and then were characterized by in vitro cell based assays. The results indicated that several test compounds bind to ERK2, and inhibit ERK substrate-specific phosphorylation and cancer cell proliferation. Subsequently, real time binding interactions between ERK2 and substrates were measured using surface plasmon resonance (SPR). Effects of an identified ERK2 inhibitor, compound 76, on substrate interactions were evaluated. Lastly, Caenorhabditis elegans (C. elegans) was chosen as a research model in order to examine the potency of ERK2 inhibitors in a whole organism. C. elegans is a well characterized organism that is inexpensive and easy to maintain in the laboratory. More importantly, C. elegans has the MPK-1 protein, which is homologous to human ERK2, known to be required for vulva formation and egg laying. It was shown that preincubation of C. elegans eggs or larvae with several compounds causes inhibition of egg laying. This effect was due to compound 76 inhibition of proper vulva cell fusion during development. In conclusion, CADD and biological assays can be used to identify lead compounds that are selective for active ERK2 and its interactions with substrates involved in cancer cell proliferation and normal physiological functions.
    • Novel Extracellular Signal-Regulated Kinase (ERK) Targeted Inhibitors Effects on Apoptotic Signaling: A Potential Cancer Therapy

      Boston, Sarice Renee; Shapiro, Paul, Ph.D. (2011)
      The extracellular signal-regulated kinase 1 and 2 (ERK 1/2) proteins potently mediate cell proliferation and survival signals via substrate phosphorylation events. Aberrant activation of ERK due to mutations in upstream activators such as B-Raf is a hallmark of a variety of cancers. Thus, inhibition of substrates involved in these mechanisms provides a promising target for anti-cancer therapies. Small molecules designed to interfere with five putative binding sites on ERK2, which are potentially involved in substrate interactions, were developed using computer-aided drug design (CADD) and assessed for their ability to inhibit ERK-mediated signaling events. The inhibitors identified successfully inhibited cell proliferation and survival signals as demonstrated by inhibition of cell cycle progression and/or the induction of apoptosis in cell based assays. Evaluation of the mechanism of action of these ERK-targeted inhibitors revealed inhibitory effects on ERK mediated phosphorylation of p90Rsk-1, which plays a role in cell growth and survival as well as caspase-9, which is involved in the activation of the intrinsic apoptotic pathway resulting in the induction of apoptosis. Additionally, the ERK-targeted inhibitors appeared to enhance chemotherapeutic drugs by inducing a cytostatic effect through selective inhibition of p90Rsk-1 phosphorylation and cyclin D1 expression indicative of a G1-phase arrest in a melanoma model harboring a B-Raf mutation. These studies suggest that selective inhibition of ERK functions is an alternative approach to complete ERK pathway inhibition and can be used to enhance the sensitivity of melanoma cells to chemotherapeutics drugs potentially reducing toxicity to normal cells and the development of drug resistance.
    • Overcoming Drug Resistance in BRaf Mutated Melanoma Cells

      Samadani, Ramin; Shapiro, Paul, Ph.D. (2015)
      Targeted inhibition of the ERK1/2 signaling pathway has received much attention after the transient success of the BRaf inhibitor, vemurafenib, in metastatic melanoma patients with the BRafV600E mutation. Approximately 50% of melanomas have an activating mutation to BRaf which drives cancer cell proliferation and survival through the ERK1/2 pathway. Therapies targeting BRaf or its downstream kinase MEK1/2 have demonstrated very promising initial clinical results. However, these treatments invariably lead to drug resistance through the activation of alternate pathways or mutations that circumvent the blockade and reactivate ERK1/2. Current efforts are centered on the use of combination therapies as well as the direct inhibition of ERK1/2 itself. The purpose of these studies is to characterize putative substrate-selective ERK inhibitors and examine their effects in various clinically relevant models of drug resistance. Specifically melanoma cell lines resistant to BRaf and MEK inhibitors, PLX-4032 and AZD-6244 respectively, were generated to test the efficacy of these novel compounds in cell viability assays. Furthermore, the potency of these molecules was evaluated in drug resistant cell lines that overexpress MAP3K8 (the gene encoding COT/Tpl2). The data show that these putative substrate-selective ERK inhibitors preferentially inhibit proliferation of cell lines with activated ERK1/2 signaling. In addition, these novel molecularly targeted therapies are equipotent in drug resistant versus non-resistant melanoma cells. Given these compounds' selectivity for BRaf mutated melanoma cells and their efficacy in drug resistance models, we submit these novel chemical entities as potential lead candidates for new salvage therapies in patients who have become resistant to currently available treatments. Moreover, these ATP-independent inhibitors target only select ERK1/2 functions and may be inherently less susceptible to developing their own drug resistance as compared to complete ablation of all ERK1/2 signaling as seen with ATP-competitive inhibitors.
    • Studies on Extracellular Signal-Regulated Kinase-1/2 (ERK1/2) Function and Identification of Small Molecule Inhibitors that Selectively Target ERK1/2 Regulation of Activator Protein-1 (AP-1) Function in Cancer Cell Proliferation

      Zhang, Jun; Shapiro, Paul, Ph.D. (2014)
      Excessive activation of the Raf/MEK/ERK pathway participates in the pathogenesis of numerous human tumors. In this pathway, extracellular signal regulated kinase 1/2 (ERK1/2) occupy a unique position on which signals from various membrane receptors converge. Therefore, ERK1/2 have been appealing targets for the development of anticancer drugs. In the recent years, careful examination of ATP binding site of protein kinases coupled with structure-based drug design methods has largely facilitated development of specific protein kinase inhibitors. The crystal structures of ERK2 with ATP complex refined to 2.3 Å have been reported previously. However, the details of interactions between ERK2 and ATP binding pocket were not certain because of low resolution. We have obtained high resolution crystals and refined the structures of the apoenzyme, ATP-bound ERK2, ADP-bound ERK2 to 1.8, 1.7 and 1.8 Å respectively. We then described the identification of a novel lead compound with a thienyl benzenesulfonate scaffold that targets the F-domain Recruitment site (FRS) of ERK1/2. Our crystal structure data suggested that this compound interacted with ERK2 in the vicinity of the FRS. Biological analyses further showed that this compound, along with a few structurally similar analogs derived from computational methods based on information provided by the crystal structure, preferentially inhibited F-site containing substrates that form the activator protein-1 (AP-1) transcription factor complex including c-Fos, Fra1, and FosB. Cell viability analysis revealed that melanoma cell lines that harbor constitutively activated ERK1/2 driven by activating B-Raf mutants were more sensitive to growth inhibition by these compounds. Lastly, we discovered a novel mechanism by which ERK1/2 activation regulates the phosphorylation of protein kinase C (PKC) isoforms PKCβII/δ. Our studies demonstrated that growth factor mediated ERK1/2 activation regulated PKCβII/δ activity as inhibition of ERK1/2 affected PKC substrate phosphorylation. Moreover, we provide evidence that inhibition of ERK1/2 reduced cell migration through a mechanism involving regulation of PKCβII. Thus, these data indicate that ERK1/2 proteins act as upstream regulators of PKC proteins and that inhibition of this regulation may have utility in preventing cancer cell migration and metastasis.
    • Study of the Interactions Between Mitogen-Activated Protein Kinases and Small Molecule Inhibitors of Substrate Proteins Using Fluorescence Spectroscopy

      Nevels, Kerrick; Shapiro, Paul, Ph.D. (2011)
      The extracellular-signal-regulated kinase (ERK) proteins belong to the mitogen-activated protein kinase (MAPK) family. In addition to participating in important cell signaling pathways involved in normal cellular functions, unregulated activation of the ERK pathway promotes the proliferation and survival of cancer cells. As such, targeted inhibition of the ERK pathway is a goal in the development of new anti-cancer drugs. Given the importance of the ERK pathway in regulating normal cellular processes, we sought to identify small molecules that can selectively inhibit ERK interactions with protein substrates that promote cancer cell proliferation, but preserve ERK functions in normal cells. To accomplish this, computer-aided drug design (CADD) was used to identify low molecular weight compounds that are predicted to interact with specific substrate docking sites on ERK2. In addition, chemical modifications were made to explore structure-activity relationships. The results of the studies demonstrate how steady-state fluorescence spectroscopy methods can be used to rapidly identify ERK-targeted compounds, determine binding affinities, and evaluate the specificity for the ERK proteins as compared to the structurally related MAPK proteins.
    • Targeting the Activator Protein-1 Complex to Inhibit Airway Smooth Muscle Cell Hyperproliferation in Asthma

      Defnet, Amy Elizabeth; Shapiro, Paul, Ph.D.; Kane, Maureen A. (2021)
      Hyperproliferation of airway smooth muscle (ASM) cells leads to increased ASM mass causing airway obstruction in inflammatory diseases such as asthma. Currently, there are no effective therapies to modulate ASM cell proliferation that contributes to debilitating bronchoconstriction in severe asthmatics. Previous studies suggest that activator protein-1 (AP-1) transcription factor expression is upregulated in airway cells in asthma and inhibition of AP-1 could mitigate the hyperproliferation of ASM cells. AP-1 activity has been shown to be enhanced by upstream extracellular signal-regulated kinase (ERK1/2) signaling or antagonized by retinoic acid receptor (RAR)-mediated signaling. The overall goal of the current study was to evaluate the therapeutic potential of a combination therapy of an ERK1/2 inhibitor and RAR agonist to modulate AP-1 complex formation and activation. Aim 1 studies tested the hypothesis that a novel function-selective ERK1/2 inhibitor, referred to as SF-3-030, would mitigate off-target toxicity while regulating platelet-derived growth factor (PDGF) induced AP-1 activity and ASM cell proliferation. In Aim 2 studies we evaluated the role of retinoids in controlling AP-1 complex formation and identified a RARγ isoform-specific agonist, CD1530, as a potential therapeutic option for inhibition of AP-1 activity and ASM cell hyperproliferation. Aim 3 studies determined whether a polypharmacological approach of combining ERK1/2 inhibition and RAR agonism to target two different aspects of the AP-1 complex activation and formation would have an additive effect in preventing ASM hyperproliferation. Overall, these studies help further our understanding of how AP-1 signaling causes the hyperproliferation of ASM cells while elucidating possible therapeutic treatment options through ERK1/2 inhibition and RAR agonism.