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) molecules1-5. 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 vivo6. To overcome these limitations, we engineered an inducible affinity tag into the mouse MHC-I gene (H2-K1) and targeted this allele to the KrasLSL-G12D/+Trp53fl/fl mouse model (KP/KbStrep)7. This approach enabled us to precisely isolate MHC-I peptides from autochthonous pancreatic ductal adenocarcinoma and from lung adenocarcinoma (LUAD) in vivo. In addition, we profiled the LUAD immunopeptidome from the alveolar type 2 cell of origin up to late-stage disease. Differential peptide presentation in LUAD was not predictable by mRNA expression or translation efficiency and is probably driven by post-translational mechanisms. Vaccination with peptides presented by LUAD in vivo induced CD8+ T cell responses in naive mice and tumour-bearing mice. Many peptides specific to LUAD, including immunogenic peptides, exhibited minimal expression of the cognate mRNA, which prompts the reconsideration of antigen prediction pipelines that triage peptides according to transcript abundance8. Beyond cancer, the KbStrep allele is compatible with other Cre-driver lines to explore antigen presentation in vivo in the pursuit of understanding basic immunology, infectious disease and autoimmunity.

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. These data reveal a collection of tumor antigens whose presentation levels are selectively augmented following therapy, including several epitopes present at over 1,000 copies per cell. We leveraged the tunable abundance of MEKi-modulated antigens by targeting four epitopes with pMHC-specific T cell engagers and antibody drug conjugates, enhancing cell killing in tumor cells following MEK inhibition. These results highlight drug treatment as a means to enhance immunotherapy efficacy by targeting specific upregulated pMHCs and provide a methodological framework for identifying, quantifying, and therapeutically targeting additional epitopes of interest.

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 MS2 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. Here, we describe two sets of analyses comparing MS2-based quantitation using a 20× protein carrier in pMHC analyses and a high (~100×) and low (~9×) protein carrier in pTyr analyses, using CDK4/6 inhibitors and EGF stimulation to drive dynamic changes in the immunopeptidome and phosphoproteome, respectively. In both applications, inclusion of a protein carrier resulted in an increased number of MHC peptide or phosphopeptide identifications, as expected. At the same time, quantitative accuracy was adversely affected by the presence of the protein carrier, altering interpretation of the underlying biological response to perturbation. Moreover, for tyrosine phosphoproteomics, the presence of high levels of protein carrier led to a large number of missing values for endogenous phosphopeptides, leading to fewer quantifiable peptides relative to the "no-boost" condition. These data highlight the unique limitations and future experimental considerations for both analysis types and provide a framework for assessing quantitative accuracy in protein carrier experiments moving forward.

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. YAP and TAZ are prominently expressed in fibrotic but not healthy lung tissue, with particularly pronounced nuclear expression of TAZ in spindle-shaped fibroblastic cells. In culture, both YAP and TAZ accumulate in the nuclei of fibroblasts grown on pathologically stiff matrices but not physiologically compliant matrices. Knockdown of YAP and TAZ together in vitro attenuates key fibroblast functions, including matrix synthesis, contraction, and proliferation, and does so exclusively on pathologically stiff matrices. Profibrotic effects of YAP and TAZ operate, in part, through their transcriptional target plasminogen activator inhibitor-1, which is regulated by matrix stiffness independent of transforming growth factor-ß signaling. Immortalized fibroblasts conditionally expressing active YAP or TAZ mutant proteins overcome soft matrix limitations on growth and promote fibrosis when adoptively transferred to the murine lung, demonstrating the ability of fibroblast YAP/TAZ activation to drive a profibrotic response in vivo. Together, these results identify YAP and TAZ as mechanoactivated coordinators of the matrix-driven feedback loop that amplifies and sustains fibrosis.

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. We hypothesized that cross presentation may form an unappreciated source of bias in the induction of cytotoxic T-cell responses. METHODS: We used stable isotope labeling of amino acids combined with immunopeptidomics to distinguish cross-presented from endogenous MHC-I peptides on DCs. To test impacts on T-cell activation, we targeted the model antigen SIINFEKL to specific subcellular compartments in tumor cells, which were used as sources for cross presentation to T cells. In vitro observations were validated using DNA and RNA sequencing data from two cohorts of patients with melanoma undergoing checkpoint blockade therapy. We used a novel quantitative mass spectrometry approach to measure the levels of model antigen on cross-presenting DCs following various means of tumor cell death. RESULTS: DCs exhibited a strong bias for cross-presenting peptides derived from cytoplasmic proteins and against those from plasma membrane proteins, which was confirmed using the model antigen SIINFEKL. In patients with melanoma, the proportion of membrane-derived neoantigens was correlated with reduced survival and failure to respond to therapy. Quantification of cross-presented SIINFEKL revealed that the mode of cell death could overcome DCs' bias against plasma membrane proteins. CONCLUSIONS: Cross presentation of cellular antigens by DCs may impose constraints on the range of peptides available to activate CD8+ T cells that have previously gone unappreciated. The share of neoantigens arising from membrane-derived sources may render some tumors less immunogenic due to inefficient cross presentation. These observations carry important implications for the encounter and intracellular processing of cellular antigens by DCs and merit further clinical studies for their therapeutic potential in stratifying patient populations and design of vaccine-based therapies.Sorry this seems to be the only funciton that works yes I confirm TBF, LES and FC are joint first authors. Please that away the line TFB and FC contributed equally. thanks!!

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. This form of resistance requires dual phosphorylation of STAT3 residues Y705 and S727, mediated by SRC and epidermal growth factor receptor (EGFR), respectively. Simultaneously inhibiting SRC and EGFR reverses this resistance, and combined targeting of these two kinases in vivo with clinically available inhibitors is synergistic and significantly prolongs survival in ispinesib-treated GBM-bearing mice. We thus identify a translationally actionable approach to overcoming Kif11 inhibitor resistance that may work to block STAT3-driven resistance against other anti-cancer therapies as well.

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. To address these limitations, we developed a targeted approach, termed "SureQuant pTyr," coupling low input pTyr enrichment with a panel of isotopically labeled internal standard peptides to guide data acquisition of low-abundance tyrosine phosphopeptides. SureQuant pTyr allowed for reliable quantification of several hundred commonly dysregulated pTyr targets with high quantitative accuracy, improving the robustness and usability of targeted mass spectrometry assays. We established the clinical applicability of SureQuant pTyr by profiling pTyr signaling levels in human colorectal tumors using minimal sample input, characterizing patient-specific oncogenic-driving mechanisms. While in some cases pTyr profiles aligned with previously reported proteomic, genomic, and transcriptomic molecular characterizations, we highlighted instances of new insights gained using pTyr characterization and emphasized the complementary nature of pTyr measurements with traditional biomarkers for improving patient stratification and identifying therapeutic targets. The turn-key nature of this approach opens the door to rapid and reproducible pTyr profiling in research and clinical settings alike and enables pTyr-based measurements for applications in precision medicine. SIGNIFICANCE: SureQuant pTyr is a mass spectrometry-based targeted method that enables sensitive and selective targeted quantitation of several hundred low-abundance tyrosine phosphorylated peptides commonly dysregulated in cancer, including oncogenic signaling networks.

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. We describe an experimental platform that leverages recombinant heavy isotope-coded peptide MHCs (hipMHCs) and multiplex isotope tagging to quantify peptide repertoire alterations using low sample input. HipMHCs improve quantitative accuracy of peptide repertoire changes by normalizing for variation across analyses and enable absolute quantification using internal calibrants to determine copies per cell of MHC antigens, which can inform immunotherapy design. Applying this platform in melanoma cell lines to profile the immunopeptidome response to CDK4/6 inhibition and interferon-γ - known modulators of antigen presentation - uncovers treatment-specific alterations, connecting the intracellular response to extracellular immune presentation.

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.Experimental Design: We elucidated the effect of a novel treatment paradigm using sustained, low-dose HSP90 inhibition in vitro and in syngeneic mouse models using genetic and pharmacologic tools. Profiling of treatment-associated tumor cell antigens was performed using immunoprecipitation followed by peptide mass spectrometry. RESULTS: We show that sustained, low-level inhibition of HSP90 both amplifies and diversifies the antigenic repertoire presented by tumor cells on MHC-I molecules through an IFNγ-independent mechanism. In stark contrast, we find that acute, high-dose exposure to HSP90 inhibitors, the only approach studied in the clinic to date, is broadly immunosuppressive in cell culture and in patients with cancer. In mice, chronic non-heat shock-inducing HSP90 inhibition slowed progression of colon cancer implants, but only in syngeneic animals with intact immune function. Addition of a single dose of nonspecific immune adjuvant to the regimen dramatically increased efficacy, curing a subset of mice receiving combination therapy. CONCLUSIONS: These highly translatable observations support reconsideration of the most effective strategy for targeting HSP90 to treat cancers and suggest a practical approach to repurposing current orally bioavailable HSP90 inhibitors as a new immunotherapeutic strategy.See related commentary by Srivastava and Callahan, p. 6277.

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. Excessive EGFR ligand release can outcompete therapeutic EGFR inhibition or the inhibition of other growth factor pathways by providing bypass signaling via EGFR activation. Drugging metalloproteases directly have failed clinically because it indiscriminately affected shedding of numerous substrates. It is therefore essential to identify regulators for EGFR ligand cleavage. Here, integration of a functional shRNA genomic screen, computational network analysis, and dedicated validation tests succeeded in identifying several key signaling pathways as novel regulators of TGFα shedding in cancer cells. Most notably, a cluster of genes with NFκB pathway regulatory functions was found to strongly influence TGFα release, albeit independent of their NFκB regulatory functions. Inflammatory regulators thus also govern cancer cell growth-promoting ectodomain cleavage, lending mechanistic understanding to the well-known connection between inflammation and cancer.Implications: Using genomic screens and network analysis, this study defines targets that regulate ectodomain shedding and suggests new treatment opportunities for EGFR-driven cancers. Mol Cancer Res; 16(1); 147-61. ©2017 AACR.

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. Our results support a microtubule-dependent signaling-based model for controlling traction forces through a motor-clutch mechanism, rather than microtubules directly relieving tension within F-actin and adhesions. Computational simulations of cell migration suggest that increasing protrusion number also impairs stiffness-sensitive migration, consistent with experimental MTA effects. These results provide a theoretical basis for the role of microtubules and mechanisms of MTAs in controlling cell migration.

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. These sensors are soft and flexible and undetectable when placed in different locations in the simulator. Five sensors were added to our current sensor enabled breast simulator. Eight participants performed the clinical breast examination on the simulator and documented their findings. There was a significant relationship for both clinical breast examination time (r(6) = 0.99, p < 0.001) and average force (r(6) = 0.92, p < 0.005) between our current sensors and the new fabric sensors. In addition the senors were not noticed by the participants. These new sensors provide new methods to measure and assess clinical skill and performance.

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.