Validation and quantification of peptide antigens presented on MHCs using SureQuant.

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.

MEK inhibition enhances presentation of targetable MHC-I tumor antigens in mutant melanomas.

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.

Dendritic cell-mediated cross presentation of tumor-derived peptides is biased against plasma membrane proteins.

Background: For effective tumor elimination, cytotoxic CD8 T cells must recognize tumor-derived antigens presented on class I major histocompatibility complex (MHC-I). Despite a general association between the expression of immunogenic antigens, typically neoantigens, and response to immunotherapy, the majority of patients lack strong endogenous responses to most putative neoantigens due to mechanisms that are not well understood. Cytotoxic CD8 T-cell responses are induced by dendritic cells (DCs) cross-presenting tumor-derived peptides on MHC-I.

Activation of STAT3 through combined SRC and EGFR signaling drives resistance to a mitotic kinesin inhibitor in glioblastoma.

Inhibitors of the mitotic kinesin Kif11 are anti-mitotics that, unlike vinca alkaloids or taxanes, do not disrupt microtubules and are not neurotoxic. However, development of resistance has limited their clinical utility. While resistance to Kif11 inhibitors in other cell types is due to mechanisms that prevent these drugs from disrupting mitosis, we find that in glioblastoma (GBM), resistance to the Kif11 inhibitor ispinesib works instead through suppression of apoptosis driven by activation of STAT3.

Deciphering the immunopeptidome in vivo reveals new tumour antigens.

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).

Absolute quantification of tumor antigens using embedded MHC-I isotopologue calibrants.

Absolute quantification measurements (copies per cell) of peptide major histocompatibility complex (pMHC) antigens are necessary to inform targeted immunotherapy drug design; however, existing methods for absolute quantification have critical limitations. Here, we present a platform termed SureQuant-IsoMHC, utilizing a series of pMHC isotopologues and internal standard-triggered targeted mass spectrometry to generate an embedded multipoint calibration curve to determine endogenous pMHC concentrations for a panel of 18 tumor antigens. We apply SureQuant-IsoMHC to measure changes in expression of our target panel in a melanoma cell line treated with a MEK inhibitor and translate this approach to estimate antigen concentrations in melanoma tumor biopsies.

Quantitative Consequences of Protein Carriers in Immunopeptidomics and Tyrosine Phosphorylation MS Analyses.

Utilizing a protein carrier in combination with isobaric labeling to "boost" the signal of other low-level samples in multiplexed analyses has emerged as an attractive strategy to enhance data quantity while minimizing protein input in mass spectrometry analyses. Recent applications of this approach include pMHC profiling and tyrosine phosphoproteomics, two applications that are often limited by large sample requirements. While including a protein carrier has been shown to increase the number of identifiable peptides in both applications, the impact of a protein carrier on quantitative accuracy remains to be thoroughly explored, particularly in relevant biological contexts where samples exhibit dynamic changes in abundance across peptides.

High-Density, Targeted Monitoring of Tyrosine Phosphorylation Reveals Activated Signaling Networks in Human Tumors.

Tyrosine phosphorylation (pTyr) plays a pivotal role in signal transduction and is commonly dysregulated in cancer. As a result, profiling tumor pTyr levels may reveal therapeutic insights critical to combating disease. Existing discovery and targeted mass spectrometry-based methods used to monitor pTyr networks involve a tradeoff between broad coverage of the pTyr network, reproducibility in target identification across analyses, and accurate quantification.

Multiplexed relative and absolute quantitative immunopeptidomics reveals MHC I repertoire alterations induced by CDK4/6 inhibition.

Peptides bound to class I major histocompatibility complexes (MHC) play a critical role in immune cell recognition and can trigger an antitumor immune response in cancer. Surface MHC levels can be modulated by anticancer agents, altering immunity. However, understanding the peptide repertoire's response to treatment remains challenging and is limited by quantitative mass spectrometry-based strategies lacking normalization controls.

Rebalancing Protein Homeostasis Enhances Tumor Antigen Presentation.

Purpose: Despite the accumulation of extensive genomic alterations, many cancers fail to be recognized as "foreign" and escape destruction by the host immune system. Immunotherapies designed to address this problem by directly stimulating immune effector cells have led to some remarkable clinical outcomes, but unfortunately, most cancers fail to respond, prompting the need to identify additional immunomodulatory treatment options. We elucidated the effect of a novel treatment paradigm using sustained, low-dose HSP90 inhibition and in syngeneic mouse models using genetic and pharmacologic tools.

Microtubule-Based Control of Motor-Clutch System Mechanics in Glioma Cell Migration.

Microtubule-targeting agents (MTAs) are widely used chemotherapy drugs capable of disrupting microtubule-dependent cellular functions, such as division and migration. We show that two clinically approved MTAs, paclitaxel and vinblastine, each suppress stiffness-sensitive migration and polarization characteristic of human glioma cells on compliant hydrogels. MTAs influence microtubule dynamics and cell traction forces by nearly opposite mechanisms, the latter of which can be explained by a combination of changes in myosin motor and adhesion clutch number.

Functional Genomics Approach Identifies Novel Signaling Regulators of TGFα Ectodomain Shedding.

Ectodomain shedding of cell-surface precursor proteins by metalloproteases generates important cellular signaling molecules. Of importance for disease is the release of ligands that activate the EGFR, such as TGFα, which is mostly carried out by ADAM17 [a member of the A-disintegrin and metalloprotease (ADAM) domain family]. EGFR ligand shedding has been linked to many diseases, in particular cancer development, growth and metastasis, as well as resistance to cancer therapeutics.

Fabric Force Sensors for the Clinical Breast Examination Simulator.

Sensor enabled simulators may help in training and assessing clinical skill. Their are imitations on the locations current sensors can be placed without interfering with the clinical examination. In this study novel fabric force sensors were developed and tested.

Mechanosignaling through YAP and TAZ drives fibroblast activation and fibrosis.

Pathological fibrosis is driven by a feedback loop in which the fibrotic extracellular matrix is both a cause and consequence of fibroblast activation. However, the molecular mechanisms underlying this process remain poorly understood. Here we identify yes-associated protein (YAP) (homolog of drosophila Yki) and transcriptional coactivator with PDZ-binding motif (TAZ) (also known as Wwtr1), transcriptional effectors of the Hippo pathway, as key matrix stiffness-regulated coordinators of fibroblast activation and matrix synthesis.

A simplified culture system to examine soluble factor interactions between mammalian cells.

Interactions between different cell types play critical roles in normal development and disease, but remain challenging to analyse. Here, a co-culture system is described that overcomes many of the limitations of existing methods, is simple to construct and modify, and is compatible with standard cellular and molecular assays.