SUMMARY Poorly immunogenic tumor cells evade host immunity and grow even in the presence of an intact immune system, but the complex mechanisms regulating tumor immunogenicity have not been elucidated. Here, we discovered an unexpected role of the Hippo pathway in suppressing anti-tumor immunity. We demonstrate that, in three different murine syngeneic tumor models (B16, SCC7, and 4T1), loss of the Hippo pathway kinases LATS1/2 (large tumor suppressor 1 and 2) in tumor cells inhibits tumor growth. Tumor regression by LATS1/2 deletion requires adaptive immune responses, and LATS1/2 deficiency enhances tumor vaccine efficacy. Mechanistically, LATS1/2-null tumor cells secrete nucleic-acid-rich extracellular vesicles, which induce a type I interferon response via the Toll-like receptors-MYD88/TRIF pathway. LATS1/2 deletion in tumors thus improves tumor immunogenicity, leading to tumor destruction by enhancing anti-tumor immune responses. Our observations uncover a key role of the Hippo pathway in modulating tumor immunogenicity and demonstrate a proof of concept for targeting LATS1/2 in cancer immunotherapy.

The Consortium for Top-Down Proteomics (www.topdownproteomics.org) launched the present study to assess the current state of top-down mass spectrometry (TD MS) and middle-down mass spectrometry (MD MS) for characterizing monoclonal antibody (mAb) primary structures, including their modifications. To meet the needs of the rapidly growing therapeutic antibody market, it is important to develop analytical strategies to characterize the heterogeneity of a therapeutic product's primary structure accurately and reproducibly. The major objective of the present study is to determine whether current TD/MD MS technologies and protocols can add value to the more commonly employed bottom-up (BU) approaches with regard to confirming protein integrity, sequencing variable domains, avoiding artifacts, and revealing modifications and their locations. We also aim to gather information

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Histones are the primary protein component of chromatin and are involved in virtually all DNA‐templated processes. Histones are abundantly post‐translationally modified by a variety of chromatin‐modifying machinery. These post‐translational modifications (PTMs) are recognized by a range of “reader” proteins, which recruit additional proteins to specific locations on chromatin and impart precise and powerful effects on gene regulation. Each PTM typically exerts a positive or negative effect on transcription, and recent studies have shown that histone PTMs function in a combinatorial histone code: that is, histone PTMs function in combination to exert precise DNA‐templated regulation. Thus, there is a need to identify and understand proteoforms, or unambiguously defined single protein molecules with all combinations of modifications. Top‐down proteomics is currently the only viable approach for identifying and quantitating histone proteoforms, and mass spectrometry instruments have become sufficiently powerful to perform these quantitative analyses in a robust and high‐throughput fashion. These recent innovations have enabled new experimental directions in chromatin research but have also introduced temporal and other constraints. This has led us to develop the protocols described here, which increase throughput, reduce sample requirements, and maintain robust quantitation. Although originally designed for high‐throughput quantitative top‐down proteomics, the protocols described here are useful for a wide range of chromatin biology applications. Starting with small amounts of cells or tissue, we describe two basic protocols for exceptionally rapid and efficient nuclei isolation, acid extraction of histones, and high‐performance liquid chromatography fractionation of histones into histone families. We additionally describe the quantitative top‐down proteomic analysis of histone H4 proteoforms. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Nuclei isolation and acid extraction of histones from mammalian cells in culture/tissues Basic Protocol 2: HPLC fractionation of histones and histone H4 HPLC‐MS/MS Support Protocol: Preparation of intact H3 histone tails by Glu‐C digestion

Urine as a true non-invasive sampling source holds great potential for biomarker discovery. While approximately 2000 proteins can be detected by mass spectrometry in urine from healthy people, the amount of these proteins vary considerably. A systematic evaluation of a large number of samples is needed to determine the range of the variations. Current biomarker studies often measure limited number of urine samples in the discovery phase, which makes it difficult to determine whether proteins differentially expressed between control and disease groups represent actual difference, or are just physiological variations among the individuals, leads to failures in the validation phase with the increased sample numbers. Here, we report a streamlined workflow with capacity of measuring 8 urine proteomes per day at the coverage of > 1500 proteins. With this workflow, we evaluated variations in 497 urine proteomes from 167 healthy donors, establishing reference intervals (RIs) that covered urine protein variations. We demonstrated that RIs could be used to monitor physiological changes by detecting transient outlier proteins. Furthermore, we provided a RIs-based algorithm for biomarker discovery and validation to screen for diseases such as cancer. This study provided a proof-of-principle workflow for the use of urine proteome for health monitoring and disease screening.