KDM5B promotes immune evasion by recruiting SETDB1 to silence retroelements.

Tumours use various strategies to evade immune surveillance1,2. Immunotherapies targeting tumour immune evasion such as immune checkpoint blockade have shown considerable efficacy on multiple cancers3,4 but are ineffective for most patients due to primary or acquired resistance5-7. Recent studies showed that some epigenetic regulators suppress anti-tumour immunity2,8-12, suggesting that epigenetic therapies could boost anti-tumour immune responses and overcome resistance to current immunotherapies. Here we show that, in mouse melanoma models, depletion of KDM5B-an H3K4 demethylase that is critical for melanoma maintenance and drug resistance13-15-induces robust adaptive immune responses and enhances responses to immune checkpoint blockade. Mechanistically, KDM5B recruits the H3K9 methyltransferase SETDB1 to repress endogenous retroelements such as MMVL30 in a demethylase-independent manner. Derepression of these retroelements activates cytosolic RNA-sensing and DNA-sensing pathways and the subsequent type-I interferon response, leading to tumour rejection and induction of immune memory. Our results demonstrate that KDM5B suppresses anti-tumour immunity by epigenetic silencing of retroelements. We therefore reveal roles of KDM5B in heterochromatin regulation and immune evasion in melanoma, opening new paths for the development of KDM5B-targeting and SETDB1-targeting therapies to enhance tumour immunogenicity and overcome immunotherapy resistance.

Peer Teaching as Bioinformatics Training Strategy: Incentives, Challenges, and Benefits.

Bioinformatics is essential for basic and clinical research. Peer-to-peer (P2P) teaching was used to respond to the bioinformatics training needs at a research-intensive institution. In addition to the data collected from the workshops, personal experiences of the teachers were used to understand incentives, challenges, and benefits of P2P teaching. Developing communication skills such as confidence in teaching, explaining complex concepts, and better understanding of topics benefited P2P teachers. Lack of time and classroom management were identified as major challenges. Hence, P2P teaching can be beneficial not only for bioinformatics trainees but also as a professional development opportunity for peer teachers.

Tofacitinib Treatment and Molecular Analysis of Cutaneous Sarcoidosis.

There is evidence that Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling plays a role in the pathogenesis of sarcoidosis. We treated a patient with cutaneous sarcoidosis with the JAK inhibitor tofacitinib; the patient had not previously had a response to medications and had not received systemic glucocorticoids. This treatment resulted in clinical and histologic remission of her skin disease. Sequencing of RNA and immunohistochemical examination of skin-lesion samples obtained from the patient before and during therapy and immunohistochemical testing of lesion samples obtained from other patients with cutaneous sarcoidosis support a role for JAK-STAT signaling in cutaneous sarcoidosis. (Funded by the Ranjini and Ajay Poddar Resource Fund for Dermatologic Diseases Research and others.).

scFTD-seq: freeze-thaw lysis based, portable approach toward highly distributed single-cell 3' mRNA profiling.

Cellular barcoding of 3' mRNAs enabled massively parallel profiling of single-cell gene expression and has been implemented in droplet and microwell based platforms. The latter further adds the value for compatibility with low input samples, optical imaging, scalability, and portability. However, cell lysis in microwells remains challenging despite the recently developed sophisticated solutions. Here, we present scFTD-seq, a microchip platform for performing single-cell freeze-thaw lysis directly toward 3' mRNA sequencing. It offers format flexibility with a simplified, widely adoptable workflow that reduces the number of preparation steps and hands-on time, with the quality of data and cost per sample matching that of the state-of-the-art scRNA-seq platforms. Freeze-thaw, known as an unfavorable lysis method resulting in possible RNA fragmentation, turns out to be fully compatible with 3' scRNA-seq. We applied it to the profiling of circulating follicular helper T cells implicated in systemic lupus erythematosus pathogenesis. Our results delineate the heterogeneity in the transcriptional programs and effector functions of these rare pathogenic T cells. As scFTD-seq decouples on-chip cell isolation and library preparation, we envision it to allow sampling at the distributed sites including point-of-care settings and downstream processing at centralized facilities, which should enable wide-spread adoption beyond academic laboratories.

Janus kinase inhibition induces disease remission in cutaneous sarcoidosis and granuloma annulare.

BACKGROUND: Sarcoidosis and granuloma annulare (GA) are cutaneous granulomatous disorders that can be difficult to treat. There is evidence of underlying Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway activation in sarcoidosis, suggesting that JAK inhibition might be effective. OBJECTIVE: To evaluate treatment with tofacitinib, a JAK inhibitor, in patients with recalcitrant sarcoidosis and GA. METHODS: A prospective evaluation of tofacitinib in 4 consecutive patients with recalcitrant cutaneous sarcoidosis (n = 3) and generalized GA (n = 1) was conducted. Immunohistochemical analysis of skin biopsy specimens from other patients with sarcoidosis (n = 21) and GA (n = 17) was performed to characterize patterns of JAK-STAT pathway activation. RESULTS: Tofacitinib resulted in a mean improvement in the baseline Cutaneous Sarcoidosis Activity and Morphology Instrument and Granuloma Annulare Scoring Index scores of 96% (standard deviation, 2%). Histologic resolution of disease was documented in all patients (3 out of 3) who had skin biopsies while receiving therapy. Constitutive STAT1 and STAT3 activation was observed in both sarcoidosis and GA, albeit in different patterns. Signal regulatory protein α may explain the differences in JAK-STAT signaling between sarcoidosis and GA. LIMITATIONS: The study is limited by the small number of participants. CONCLUSIONS: Tofacitinib resulted in dramatic improvement in 4 patients with cutaneous sarcoidosis and GA. Larger studies are underway to better understand this effect.

Mutational landscape of uterine and ovarian carcinosarcomas implicates histone genes in epithelial-mesenchymal transition.

Carcinosarcomas (CSs) of the uterus and ovary are highly aggressive neoplasms containing both carcinomatous and sarcomatous elements. We analyzed the mutational landscape of 68 uterine and ovarian CSs by whole-exome sequencing. We also performed multiregion whole-exome sequencing comprising two carcinoma and sarcoma samples from six tumors to resolve their evolutionary histories. The results demonstrated that carcinomatous and sarcomatous elements derive from a common precursor having mutations typical of carcinomas. In addition to mutations in cancer genes previously identified in uterine and ovarian carcinomas such as TP53, PIK3CA, PPP2R1A, KRAS, PTEN, CHD4, and BCOR, we found an excess of mutations in genes encoding histone H2A and H2B, as well as significant amplification of the segment of chromosome 6p harboring the histone gene cluster containing these genes. We also found frequent deletions of the genes TP53 and MBD3 (a member with CHD4 of the nucleosome remodeling deacetylase complex) and frequent amplification of chromosome segments containing the genes PIK3CA, TERT, and MYC Stable transgenic expression of H2A and H2B in a uterine serous carcinoma cell line demonstrated that mutant, but not wild-type, histones increased expression of markers of epithelial-mesenchymal transition (EMT) as well as tumor migratory and invasive properties, suggesting a role in sarcomatous transformation. Comparison of the phylogenetic relationships of carcinomatous and sarcomatous elements of the same tumors demonstrated separate lineages leading to these two components. These findings define the genetic landscape of CSs and suggest therapeutic targets for these highly aggressive neoplasms.

Discovery of a Structurally Unique Small Molecule that Inhibits Protein Synthesis.

Identifying and characterizing natural products and synthetic small molecules that inhibit biochemical processes such as ribosomal translation can lead to novel sources of molecular probes and therapeutics. The search for new antibiotics has been invigorated by the increasing burden of drug-resistant bacteria and has identified many clinically essential prokaryote-specific ribosome inhibitors. However, the current cohort of antibiotics is limited with regards to bacterial resistance mechanisms because of structural similarity within classes. From a high-throughput screen for translation inhibitors, we discovered a new compound, T6102, which inhibits bacterial protein synthesis in vitro, inhibits bacterial growth of Bacillus subtilis in vivo, and has a chemical structure that appears to be unique among known classes of translation-inhibiting antibiotics. T6102's unique structure compared to current clinically-utilized antibiotics makes it an exciting new candidate for the development of next-generation antibiotics.

Crystal structure of an insect antifreeze protein and its implications for ice binding.

Antifreeze proteins (AFPs) help some organisms resist freezing by binding to ice crystals and inhibiting their growth. The molecular basis for how these proteins recognize and bind ice is not well understood. The longhorn beetle Rhagium inquisitor can supercool to below -25 °C, in part by synthesizing the most potent antifreeze protein studied thus far (RiAFP). We report the crystal structure of the 13-kDa RiAFP, determined at 1.21 Å resolution using direct methods. The structure, which contains 1,914 nonhydrogen protein atoms in the asymmetric unit, is the largest determined ab initio without heavy atoms. It reveals a compressed ß-solenoid fold in which the top and bottom sheets are held together by a silk-like interdigitation of short side chains. RiAFP is perhaps the most regular structure yet observed. It is a second independently evolved AFP type in beetles. The two beetle AFPs have in common an extremely flat ice-binding surface comprising regular outward-projecting parallel arrays of threonine residues. The more active, wider RiAFP has four (rather than two) of these arrays between which the crystal structure shows the presence of ice-like waters. Molecular dynamics simulations independently reproduce the locations of these ordered crystallographic waters and predict additional waters that together provide an extensive view of the AFP interaction with ice. By matching several planes of hexagonal ice, these waters may help freeze the AFP to the ice surface, thus providing the molecular basis of ice binding.

Diagnosis of Occult and Obscure Gastrointestinal Bleeding.

Occult and obscure bleeding are challenging conditions to manage; however, recent advances in gastroenterology and endoscopy have improved our diagnostic and therapeutic capabilities. Obscure gastrointestinal (GI) bleeding is an umbrella category of bleeding of unknown origin that persists or recurs after endoscopic evaluation of the entire bowel fails to reveal a bleeding source. This review details the evaluation of patients with occult and obscure GI bleeding and offers diagnostic algorithms. The treatment of GI bleeding depends on the type and location of the bleeding lesion and an overview of how to manage these conditions is presented.

Expression, purification, crystallization and preliminary crystallographic studies of Rhagium inquisitor antifreeze protein.

Antifreeze proteins (AFPs) are a specialized evolutionary adaptation of a variety of bacteria, fish, arthropods and other organisms to inhibit ice-crystal growth for survival in harsh subzero environments. The recently reported novel hyperactive AFP from Rhagium inquisitor (RiAFP) is the second distinct type of AFP in beetles and its structure could reveal important molecular insights into the evolution of AFPs. For this purpose, RiAFP was overexpressed in Escherichia coli, purified and crystallized at 293 K using a combination of 23% PEG 3350 and 0.2 M ammonium sulfate as a precipitant. X-ray diffraction data were collected to 1.3 Šresolution using a synchrotron-radiation source. The crystals belonged to the trigonal space group P3(1)21 (or P3(2)21), with unit-cell parameters a = b = 46.46, c = 193.21 Å.

Isotope tracing reveals distinct substrate preference in murine melanoma subtypes with differing anti-tumor immunity.

BACKGROUND: Research about tumor "metabolic flexibility"-the ability of cells to toggle between preferred nutrients depending on the metabolic context-has largely focused on obesity-associated cancers. However, increasing evidence for a key role for nutrient competition in the tumor microenvironment, as well as for substrate regulation of immune function, suggests that substrate metabolism deserves reconsideration in immunogenic tumors that are not strongly associated with obesity. METHODS: We compare two murine models: immunologically cold YUMM1.7 and immunologically-hot YUMMER1.7. We utilize stable isotope and radioisotope tracer-based metabolic flux studies as well as gas and liquid chromatography-based metabolomics analyses to comprehensively probe substrate preference in YUMM1.7 and YUMMER1.7 cells, with a subset of studies on the impact of available metabolites across a panel of five additional melanoma cell lines. We analyze bulk RNA-seq data and identify increased expression of amino acid and glucose metabolism genes in YUMMER1.7. Finally, we analyze melanoma patient RNA-seq data to identify potential prognostic predictors rooted in metabolism. RESULTS: We demonstrate using stable isotope tracer-based metabolic flux studies as well as gas and liquid chromatography-based metabolomics that immunologically-hot melanoma utilizes more glutamine than immunologically-cold melanoma in vivo and in vitro. Analyses of human melanoma RNA-seq data demonstrate that glutamine transporter and other anaplerotic gene expression positively correlates with lymphocyte infiltration and function. CONCLUSIONS: Here, we highlight the importance of understanding metabolism in non-obesity-associated cancers, such as melanoma. This work advances the understanding of the correlation between metabolism and immunogenicity in the tumor microenvironment and provides evidence supporting metabolic gene expression as potential prognostic factors of melanoma progression and may inform investigations of adjunctive metabolic therapy in melanoma. TRIAL REGISTRATION: Deidentified data from The Cancer Genome Atlas were analyzed.

Magnetic poly(lactide-co-glycolide) and cellulose particles for MRI-based cell tracking.

Biodegradable, superparamagnetic microparticles and nanoparticles of poly(lactide-co-glycolide) (PLGA) and cellulose were designed, fabricated, and characterized for magnetic cell labeling. Monodisperse nanocrystals of magnetite were incorporated into microparticles and nanoparticles of PLGA and cellulose with high efficiency using an oil-in-water single emulsion technique. Superparamagnetic cores had high magnetization (72.1 emu/g). The resulting polymeric particles had smooth surface morphology and high magnetite content (43.3 wt % for PLGA and 69.6 wt % for cellulose). While PLGA and cellulose nanoparticles displayed highest r 2* values per millimole of iron (399 sec(-1) mM(-1) for cellulose and 505 sec(-1) mM(-1) for PLGA), micron-sized PLGA particles had a much higher r 2* per particle than either. After incubation for a month in citrate buffer (pH 5.5), magnetic PLGA particles lost close to 50% of their initial r 2* molar relaxivity, while magnetic cellulose particles remained intact, preserving over 85% of their initial r 2* molar relaxivity. Lastly, mesenchymal stem cells and human breast adenocarcinoma cells were magnetically labeled using these particles with no detectable cytotoxicity. These particles are ideally suited for noninvasive cell tracking in vivo via MRI and due to their vastly different degradation properties, offer unique potential for dedicated use for either short (PLGA-based particles) or long-term (cellulose-based particles) experiments.

Topical arginase inhibition decreases growth of cutaneous squamous cell carcinoma.

Cutaneous squamous cell carcinomas (cSCC) are among the most commonly diagnosed malignancies, causing significant morbidity and mortality. Tumor-associated macrophage (TAM) expression of arginase is implicated in tumor progression, and therapeutic use of arginase inhibitors has been studied in various cancers. However, investigating potential cSCC immunotherapies including arginase inhibition in pre-clinical models is hampered by the lack of appropriate tumor models in immunocompetent mice. PDV is a cSCC cell line derived from chemical carcinogenesis of mouse keratinocytes. PDVC57 cells were derived from a PDV tumor in C57BL/6 (B6) mice. Unlike PDV, PDVC57 tumors grow consistently in B6 mice, and have increased TAMs, decreased dendritic and T cell intra-tumor infiltration. Arginase inhibition in cSCC tumors using Nω-hydroxy-nor-arginine (nor-NOHA) reduced tumor growth in B6 mice but not immunodeficient Rag1-deficient mice. nor-NOHA administration increased dendritic and T cell tumor-infiltration and PD-1 expression. The combination of nor-NOHA and anti-PD-1 therapy with nivolumab enhanced anti-PD-1 therapeutic efficacy. This study demonstrates the therapeutic potential of transcutaneous arginase inhibition in cSCC. A competent immune microenvironment is required for tumor growth inhibition using this arginase inhibitor. Synergistic co-inhibition of tumor growth in these results, supports further examination of transcutaneous arginase inhibition as a therapeutic modality for cSCC.

PD-L1 methylation regulates PD-L1 expression and is associated with melanoma survival.

The aim of this study is to determine the significance of programmed death ligand 1 (PD-L1 or CD274) methylation in relation to PD-L1 expression and survival in melanoma. Despite the clinical importance of therapies targeting the PD-1/PD-L1 immune checkpoint in melanoma, factors regulating PD-L1 expression, including epigenetic mechanisms, are not completely understood. In this study, we examined PD-L1 promoter methylation in relation to PD-L1 expression and overall survival in melanoma patients. Our results suggest that DNA methylation regulates PD-L1 expression in melanoma, and we identify the key methylated CpG loci in the PD-L1 promoter, establish PD-L1 methylation as an independent survival prognostic factor, provide proof of concept for altering PD-L1 expression by hypomethylating agents, and uncover that PD-L1 methylation is associated with an interferon signaling transcriptional phenotype. Based on our findings, measuring and altering PD-L1 promoter DNA methylation may have potential prognostic and therapeutic applications in melanoma.

Myeloid-targeted immunotherapies act in synergy to induce inflammation and antitumor immunity.

Eliciting effective antitumor immune responses in patients who fail checkpoint inhibitor therapy is a critical challenge in cancer immunotherapy, and in such patients, tumor-associated myeloid cells and macrophages (TAMs) are promising therapeutic targets. We demonstrate in an autochthonous, poorly immunogenic mouse model of melanoma that combination therapy with an agonistic anti-CD40 mAb and CSF-1R inhibitor potently suppressed tumor growth. Microwell assays to measure multiplex protein secretion by single cells identified that untreated tumors have distinct TAM subpopulations secreting MMP9 or cosecreting CCL17/22, characteristic of an M2-like state. Combination therapy reduced the frequency of these subsets, while simultaneously inducing a separate polyfunctional inflammatory TAM subset cosecreting TNF-α, IL-6, and IL-12. Tumor suppression by this combined therapy was partially dependent on T cells, and on TNF-α and IFN-γ. Together, this study demonstrates the potential for targeting TAMs to convert a "cold" into an "inflamed" tumor microenvironment capable of eliciting protective T cell responses.

UV-induced somatic mutations elicit a functional T cell response in the YUMMER1.7 mouse melanoma model.

Human melanomas exhibit relatively high somatic mutation burden compared to other malignancies. These somatic mutations may produce neoantigens that are recognized by the immune system, leading to an antitumor response. By irradiating a parental mouse melanoma cell line carrying three driver mutations with UVB and expanding a single-cell clone, we generated a mutagenized model that exhibits high somatic mutation burden. When inoculated at low cell numbers in immunocompetent C57BL/6J mice, YUMMER1.7 (Yale University Mouse Melanoma Exposed to Radiation) regresses after a brief period of growth. This regression phenotype is dependent on T cells as YUMMER1.7 tumors grow significantly faster in immunodeficient Rag1-/- mice and C57BL/6J mice depleted of CD4 and CD8 T cells. Interestingly, regression can be overcome by injecting higher cell numbers of YUMMER1.7, which results in tumors that grow without effective rejection. Mice that have previously rejected YUMMER1.7 tumors develop immunity against higher doses of YUMMER1.7 tumor challenge. In addition, escaping YUMMER1.7 tumors are sensitive to anti-CTLA-4 and anti-PD-1 therapy, establishing a new model for the evaluation of immune checkpoint inhibition and antitumor immune responses.