Publications by authors named "Forest M White"

Tyrosine phosphorylation of metabolic enzymes is an evolutionarily conserved posttranslational modification that facilitates rapid and reversible modulation of enzyme activity, localization, or function. Despite the high abundance of tyrosine phosphorylation events detected on metabolic enzymes in high-throughput mass spectrometry-based studies, functional characterization of tyrosine phosphorylation sites has been limited to a subset of enzymes. Since tyrosine phosphorylation is dysregulated across human diseases, including cancer, understanding the consequences of metabolic enzyme tyrosine phosphorylation events is critical for informing disease biology and therapeutic interventions.

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Background: Intratumoral heterogeneity (ITH) and subclonal antigen expression blunt antitumor immunity and are associated with poor responses to immune-checkpoint blockade immunotherapy (ICB) in patients with cancer. The underlying mechanisms however thus far remained elusive, preventing the design of novel treatment approaches for patients with high ITH tumors.

Methods: We developed a mouse model of lung adenocarcinoma with defined expression of different neoantigens (NeoAg), enabling us to analyze how these impact antitumor T-cell immunity and to study underlying mechanisms.

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Vaccines and immunotherapies that target peptide-major histocompatibility complexes (peptide-MHCs) have the potential to address multiple unmet medical needs in cancer and infectious disease. Designing vaccines and immunotherapies to target peptide-MHCs requires accurate identification of target peptides in infected or cancerous cells or tissue, and may require absolute or relative quantification to identify abundant targets and measure changes in presentation under different treatment conditions. Internal standard parallel reaction monitoring (also known as 'SureQuant') can be used to validate and/or quantify MHC peptides previously identified by using untargeted methods such as data-dependent acquisition.

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Vaccine-elicited T cell responses can contribute to immune protection against emerging infectious disease risks such as antimicrobials-resistant (AMR) microbial pathogens and viruses with pandemic potential, but rapidly identifying appropriate targets for T cell priming vaccines remains challenging. Mass spectrometry (MS) analysis of peptides presented on major histocompatibility complexes (MHCs) can identify potential targets for protective T cell responses in a proteome-wide manner. However, pathogen-derived peptides are outnumbered by self peptides in the MHC repertoire and may be missed in untargeted MS analyses.

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Article Synopsis
  • Coordination of cellular signaling and adaptive metabolism is crucial for energy balance and homeostasis, with phosphorylation being a key regulatory mechanism for metabolic networks.
  • The study categorizes phosphorylation sites on metabolic enzymes, finding that many are located near functional areas and emphasizing ones on oxidoreductases, particularly phosphotyrosine sites linked to enzyme function.
  • Using a high fat diet model, the research uncovers sex-specific changes in metabolic regulation and identifies specific phosphotyrosine sites that predict metabolic responses, revealing how they influence enzyme activity and metabolic pathways.
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A major contributor to poor sensitivity to anti-cancer kinase inhibitor therapy is drug-induced cellular adaptation, whereby remodeling of signaling and gene regulatory networks permits a drug-tolerant phenotype. Here, we resolve the scale and kinetics of critical subcellular events following oncogenic kinase inhibition and preceding cell cycle re-entry, using mass spectrometry-based phosphoproteomics and RNA sequencing to capture molecular snapshots within the first minutes, hours, and days of BRAF kinase inhibitor exposure in a human -mutant melanoma model of adaptive therapy resistance. By enriching specific phospho-motifs associated with mitogenic kinase activity, we monitored the dynamics of thousands of growth- and survival-related protein phosphorylation events under oncogenic BRAF inhibition and drug removal.

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Unlabelled: Cyclin-dependent kinase 9 (CDK9) coordinates signaling events that regulate RNA polymerase II (Pol II) pause-release states. It is an important co-factor for transcription factors, such as MYC, that drive aberrant cell proliferation when their expression is deregulated. CDK9 modulation offers an approach for attenuating dysregulation in such transcriptional programs.

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Article Synopsis
  • Glioblastoma (GBM) is a challenging brain cancer with poor outcomes, and understanding its tumor microenvironment could improve treatment effectiveness.
  • The study adapts stereotactic biopsies to gather multi-omics data from GBM patients, revealing detailed insights into the tumor and immune response.
  • Results indicate that stereotactic needle biopsies can provide high-quality samples for comprehensive analysis, aiding in monitoring treatment responses and furthering research.
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Phosphorylation of connexin 43 (Cx43) is an important regulatory mechanism of gap junction (GJ) function. Cx43 is modified by several kinases on over 15 sites within its ~140 amino acid-long C terminus (CT). Phosphorylation of Cx43CT on S255, S262, S279, and S282 by ERK has been widely documented in several cell lines, by many investigators.

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Triple negative breast cancer (TNBC) is a heterogeneous group of tumors which lack estrogen receptor, progesterone receptor, and HER2 expression. Targeted therapies have limited success in treating TNBC, thus a strategy enabling effective targeted combinations is an unmet need. To tackle these challenges and discover individualized targeted combination therapies for TNBC, we integrated phosphoproteomic analysis of altered signaling networks with patient-specific signaling signature (PaSSS) analysis using an information-theoretic, thermodynamic-based approach.

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Background: The GL261 and CT2A syngeneic tumor lines are frequently used as immunocompetent orthotopic mouse models of human glioblastoma (huGBM) but demonstrate distinct differences in their responses to immunotherapy.

Methods: To decipher the cell-intrinsic mechanisms that drive immunotherapy resistance in CT2A-luc and to define the aspects of human cancer biology that these lines can best model, we systematically compared their characteristics using whole exome and transcriptome sequencing, and protein analysis through immunohistochemistry, Western blot, flow cytometry, immunopeptidomics, and phosphopeptidomics.

Results: The transcriptional profiles of GL261-luc2 and CT2A-luc tumors resembled those of some huGBMs, despite neither line sharing the essential genetic or histologic features of huGBM.

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Tyrosine phosphorylation regulates signaling network activity downstream of receptor tyrosine kinase (RTK) activation. Receptor protein tyrosine phosphatases (RPTPs) serve to dephosphorylate RTKs and their proximal adaptor proteins, thus serving to modulate RTK activity. While the general function of RPTPs is well understood, the direct and indirect substrates for each RPTP are poorly characterized.

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Increased fibrotic extracellular matrix (ECM) deposition promotes tumor invasion, which is the first step of the metastatic cascade. Yet, the underlying mechanisms are poorly understood as conventional studies of tumor cell migration are often performed in 2D cultures lacking the compositional and structural complexity of native ECM. Moreover, these studies frequently focus on select candidate pathways potentially overlooking other relevant changes in cell signaling.

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Receptor tyrosine kinase (RTK)-targeted therapies are often effective but invariably limited by drug resistance. A major mechanism of acquired resistance involves "bypass" switching to alternative pathways driven by non-targeted RTKs that restore proliferation. One such RTK is AXL whose overexpression, frequently observed in bypass resistant tumors, drives both cell survival and associated malignant phenotypes such as epithelial-to-mesenchymal (EMT) transition and migration.

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Damage that affects large volumes of skeletal muscle tissue can severely impact health, mobility, and quality-of-life. Efforts to restore muscle function by implanting tissue engineered muscle grafts at the site of damage have demonstrated limited restoration of force production. Various forms of mechanical and biochemical stimulation have been shown to have a potentially beneficial impact on graft maturation, vascularization, and innervation.

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Engineered cytokine-based approaches for immunotherapy of cancer are poised to enter the clinic, with IL-12 being at the forefront. However, little is known about potential mechanisms of resistance to cytokine therapies. We found that orthotopic murine lung tumors were resistant to systemically delivered IL-12 fused to murine serum albumin (MSA, IL12-MSA) because of low IL-12 receptor (IL-12R) expression on tumor-reactive CD8+ T cells.

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Background: Although the epidermal growth factor receptor (EGFR) is a frequent oncogenic driver in glioblastoma (GBM), efforts to therapeutically target this protein have been largely unsuccessful. The present preclinical study evaluated the novel EGFR inhibitor WSD-0922.

Methods: We employed flank and orthotopic patient-derived xenograft models to characterize WSD-0922 and compare its efficacy to erlotinib, a potent EGFR inhibitor that failed to provide benefit for GBM patients.

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In a recent issue of , Zecha et al. present decryptM, an approach aimed at defining the mechanisms of action of anti-cancer therapeutics through systems-level analysis of protein post-translational modifications (PTMs). By using a broad range of concentrations, decryptM generates drug response curves for each detected PTM, enabling identification of drug effects at different therapeutic doses.

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Article Synopsis
  • - The study investigates the role of specific phosphorylation sites on the epidermal growth factor receptor (EGFR) and their effects on cellular behavior, addressing a gap in understanding their contributions to signaling outcomes.
  • - Researchers used cell lines with targeted mutations in the EGFR to analyze the network's stability and uncover previously unknown connections to EGFR signaling.
  • - Findings reveal important signaling nodes related to cell responses such as migration, proliferation, and receptor trafficking, suggesting that similar methods could uncover new insights in less-explored receptor tyrosine kinases (RTKs).
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CD8+ T cell recognition of ()-specific peptides presented on major histocompatibility complex class I (MHC-I) contributes to immunity to tuberculosis (TB), but the principles that govern presentation of antigens on MHC-I are incompletely understood. In this study, mass spectrometry (MS) analysis of the MHC-I repertoire of -infected primary human macrophages reveals that substrates of 's type VII secretion systems (T7SS) are overrepresented among -derived peptides presented on MHC-I. Quantitative, targeted MS shows that ESX-1 activity is required for presentation of peptides derived from both ESX-1 substrates and ESX-5 substrates on MHC-I, consistent with a model in which proteins secreted by multiple T7SSs access a cytosolic antigen processing pathway via ESX-1-mediated phagosome permeabilization.

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Central to successful cancer immunotherapy is effective T cell antitumor immunity. Multiple targeted immunotherapies engineered to invigorate T cell-driven antitumor immunity rely on identifying the repertoire of T cell antigens expressed on the tumor cell surface. Mass spectrometry-based survey of such antigens ("immunopeptidomics") combined with other omics platforms and computational algorithms has been instrumental in identifying and quantifying tumor-derived T cell antigens.

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Combining multiple therapeutic strategies in NRAS/BRAF mutant melanoma-namely MEK/BRAF kinase inhibitors, immune checkpoint inhibitors (ICIs), and targeted immunotherapies-may offer an improved survival benefit by overcoming limitations associated with any individual therapy. Still, optimal combination, order, and timing of administration remains under investigation. Here, we measure how MEK inhibition (MEKi) alters anti-tumor immunity by utilizing quantitative immunopeptidomics to profile changes in the peptide major histocompatibility molecules (pMHC) repertoire.

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Neoadjuvant chemotherapy (NAC) remains the cornerstone of the treatment for triple negative breast cancer (TNBC), with the goal of complete eradication of disease. However, for patients with residual disease after NAC, recurrence and mortality rates are high and the identification of novel therapeutic targets is urgently needed. We quantified tyrosine phosphorylation (pTyr)-mediated signaling networks in chemotherapy sensitive (CS) and resistant (CR) TNBC patient-derived xenografts (PDX), to gain novel therapeutic insights.

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The RNA binding protein ELAVL4/HuD regulates the translation and splicing of multiple Alzheimer's disease (AD) candidate genes. We generated ELAVL4 knockout (KO) human induced pluripotent stem cell-derived neurons to study the effect that ELAVL4 has on AD-related cellular phenotypes. ELAVL4 KO significantly increased the levels of specific APP isoforms and intracellular phosphorylated tau, molecular changes that are related to the pathological hallmarks of AD.

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Immunosurveillance of cancer requires the presentation of peptide antigens on major histocompatibility complex class I (MHC-I) molecules. Current approaches to profiling of MHC-I-associated peptides, collectively known as the immunopeptidome, are limited to in vitro investigation or bulk tumour lysates, which limits our understanding of cancer-specific patterns of antigen presentation in vivo. To overcome these limitations, we engineered an inducible affinity tag into the mouse MHC-I gene (H2-K1) and targeted this allele to the KrasTrp53 mouse model (KP/KStrep).

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