Potent GCN2 Inhibitor Capable of Reversing MDSC-Driven T Cell Suppression Demonstrates In Vivo Efficacy as a Single Agent and in Combination with Anti-Angiogenesis Therapy.

General control nonderepressible 2 (GCN2) protein kinase is a cellular stress sensor within the tumor microenvironment (TME), whose signaling cascade has been proposed to contribute to immune escape in tumors. Herein, we report the discovery of cell-potent GCN2 inhibitors with excellent selectivity against its closely related Integrated Stress Response (ISR) family members heme-regulated inhibitor kinase (HRI), protein kinase R (PKR), and (PKR)-like endoplasmic reticulum kinase (PERK), as well as good kinome-wide selectivity and favorable PK. In mice, compound 39 engages GCN2 at levels ≥80% with an oral dose of 15 mg/kg BID. We also demonstrate the ability of compound 39 to alleviate MDSC-related T cell suppression and restore T cell proliferation, similar to the effect seen in MDSCs from GCN2 knockout mice. In the LL2 syngeneic mouse model, compound 39 demonstrates significant tumor growth inhibition (TGI) as a single agent. Furthermore, TGI mediated by anti-VEGFR was enhanced by treatment with compound 39 demonstrating the complementarity of these two mechanisms.

Bifidobacterium breve and Bifidobacterium longum Attenuate Choline-Induced Plasma Trimethylamine N-Oxide Production by Modulating Gut Microbiota in Mice.

Atherosclerosis is the main cause of myocardial infarction and stroke, and the morbidity and mortality rates of cardiovascular disease are among the highest of any disease worldwide. Excessive plasma trimethylamine-N-oxide (TMAO), an intestinal metabolite, promotes the development of atherosclerosis. Therefore, effective measures for reducing plasma TMAO production can contribute to preventing atherosclerosis. Probiotics are living microorganisms that are beneficial to the human body, and some of them can attenuate plasma TMAO production. To explore the effects of probiotic supplementation on plasma TMAO in choline-fed mice, we intragastrically administered eight strains of Bifidobacterium breve and eight strains of Bifidobacterium longum to mice for 6 weeks. B. breve Bb4 and B. longum BL1 and BL7 significantly reduced plasma TMAO and plasma and cecal trimethylamine concentrations. However, hepatic flavin monooxygenase (FMO) activity, flavin-containing monooxygenase 3 (FMO3), farnesoid X receptor (FXR) protein expression and TMAO fractional excretion were not significantly affected by Bifidobacterium supplementation. The treatment of Bifidobacterium strains modulated the abundances of several genera such as Ruminococcaceae UCG-009, Ruminococcaceae UCG-010, which belong to the Firmicutes that has been reported with cut gene clusters, which may be related to the reduction in intestinal TMA and plasma TMAO. Additionally, a reduction in Ruminococcaceae indicates a reduction in circulating glucose and lipids, which may be another pathway by which Bifidobacterium strains reduce the risk of atherosclerosis. The effect of Bifidobacterium strains on Bacteroides also suggests a relationship between the abundance of this genus and TMA concentrations in the gut. Therefore, the mechanism underlying these changes might be gut microbiota regulation. These Bifidobacterium strains may have therapeutic potential for alleviating TMAO-related diseases.

Lactobacillus paracasei CCFM1229 and Lactobacillus rhamnosus CCFM1228 Alleviated Depression- and Anxiety-Related Symptoms of Chronic Stress-Induced Depression in Mice by Regulating Xanthine Oxidase Activity in the Brain.

Depression is a common mood disorder that affects around 350 million people worldwide. We studied the effect of supplementation with Lactobacillus strains for the treatment of depression. Except for control group (n = 8), C57BL/6J mice were treated with Lactobacillus during six weeks of chronic unpredictable stress (depression group: n = 9, Lactobacillus intervention group: n = 7). L. paracasei CCFM1229 and L. rhamnosus CCFM1228 significantly reduced depressive behaviour in the forced swimming test and tail suspension test, significantly reduced anxiety behaviour in the open field test, and reduced anxiety behaviour in the marble burying test and light/dark box test. L. paracasei CCFM1229 and L. rhamnosus CCFM1228 significantly increased the brain serotonin and brain-derived neurotrophic factor concentrations, and CCFM1229 significantly decreased the serum corticosterone concentration, all of which are closely associated with the relief of depressive symptoms. Furthermore, CCFM1229 and CCFM1228 were shown to regulate purine metabolism in mice, as indicated by decreases in brain xanthine oxidase activity and an increase in liver adenosine deaminase activity. Anxiety- and depression-related indicators were significantly associated with xanthine oxidase activity in the cerebral cortex. The strains CCFM1229 and CCFM1228 reduced anxiety- and depression-related behaviour in a mouse model of chronic stress-induced depression, which may be achieved by regulating the activity of brain xanthine oxidase.

EBV+ tumors exploit tumor cell-intrinsic and -extrinsic mechanisms to produce regulatory T cell-recruiting chemokines CCL17 and CCL22.

The Epstein-Barr Virus (EBV) is involved in the etiology of multiple hematologic and epithelial human cancers. EBV+ tumors employ multiple immune escape mechanisms, including the recruitment of immunosuppressive regulatory T cells (Treg). Here, we show some EBV+ tumor cells express high levels of the chemokines CCL17 and CCL22 both in vitro and in vivo and that this expression mirrors the expression levels of expression of the EBV LMP1 gene in vitro. Patient samples from lymphoblastic (Hodgkin lymphoma) and epithelial (nasopharyngeal carcinoma; NPC) EBV+ tumors revealed CCL17 and CCL22 expression of both tumor cell-intrinsic and -extrinsic origin, depending on tumor type. NPCs grown as mouse xenografts likewise showed both mechanisms of chemokine production. Single cell RNA-sequencing revealed in vivo tumor cell-intrinsic CCL17 and CCL22 expression combined with expression from infiltrating classical resident and migratory dendritic cells in a CT26 colon cancer mouse tumor engineered to express LMP1. These data suggest that EBV-driven tumors employ dual mechanisms for CCL17 and CCL22 production. Importantly, both in vitro and in vivo Treg migration was effectively blocked by a novel, small molecule antagonist of CCR4, CCR4-351. Antagonism of the CCR4 receptor may thus be an effective means of activating the immune response against a wide spectrum of EBV+ tumors.

An in vitro screening method for probiotics with antidepressant-like effect using the enterochromaffin cell model.

Certain probiotics can regulate the host's neurobehavioral function through the microbiota-gut-brain axis. However, screening these probiotics is mainly carried out in animal models, and is costly and inefficient. Herein, a putative enterochromaffin cell line (RIN14B) was used as an in vitro pre-screening model; 30 bacterial strains were tested for bacteria-stimulated tryptophan hydroxylase 1 gene (Tph1) expression and 5-hydroxytryptophan/5-hydroxytryptamine secretion. All strains were further validated for their neurobehavioral effects in chronic stress-induced depressive mice. Using partial least squares (PLS) modeling of in vitro and in vivo datasets, we found that the level of Tph1 mRNA in RIN14B significantly correlated with the performance of a forced swim test and sucrose preference test, and serum corticosterone level in chronically stressed mice. Four strains were identified as the best candidates among 30 strains using principal component analysis on all in vivo measures, and unsurprisingly, three of them could enhance Tph1 expression in RIN14B, which further proved that the RIN14B-based screening method (especially the detection of bacteria-stimulated Tph1 mRNA) has good predictive validity and screening efficiency for the strain's antidepressant-like capacity. Collectively, this study provides a novel in vitro method for screening probiotics (or other related bioproducts) with antidepressant-like potential.

Construction of Bi/phthalocyanine manganese nanocomposite for trimodal imaging directed photodynamic and photothermal therapy mediated by 808 nm light.

The current conventional photo-therapeutic agents often show low therapy efficacy because of their single treatment model, the limited penetration depth of excitation light and hypoxia in the tumor microenvironment (TME). Herein, a new type of phthalocyanine manganese (MnPcE4) photosensitizer with strong NIR absorption was designed and fabricated for the first time, and was used to modify pure Bi nanomaterials to obtain an intelligent multifunctional Bi/MnPcE4 nanocomposites. The Mn2+ in the Bi/MnPcE4 nanocomposite could catalyze H2O2 to generate O2, thus helping to overcome TME hypoxia and enhancing the photodynamic therapy (PDT) efficacy. Further, the nanocomposites showed excellent T1-weighted MRI performance. Our novel use of a pure metal Bi core, offers lower toxicity, higher CT imaging performance, and a photothermal therapy (PTT) effect triggered by 808 nm near infrared (NIR) laser. Moreover, in vivo fluorescence imaging (in vivo FL) vividly showed that the nanocomposite rapidly accumulates in tumor sites due to the enhanced permeability and retention (EPR) effect and metabolized in the organs. The presence of Bi enables the use of these nanocomposites as a CT contrast agent, and the Mn content enables them to be used in MRI. This triple imaging ability implies that our nanocomposites have a high potential for use in imaging directed tumor therapy.

Upconversion-mediated ZnFe2O4 nanoplatform for NIR-enhanced chemodynamic and photodynamic therapy.

ZnFe2O4, a semiconductor catalyst with high photocatalytic activity, is ultrasensitive to ultraviolet (UV) light and tumor H2O2 for producing reactive oxygen species (ROS). Thereby, ZnFe2O4 can be used for photodynamic therapy (PDT) from direct electron transfer and the newly defined chemodynamic therapy (CDT) from the Fenton reaction. However, UV light has confined applicability because of its high phototoxicity, low penetration, and speedy attenuation in the biotissue. Herein, an upconversion-mediated nanoplatform with a mesoporous ZnFe2O4 shell was developed for near-infrared (NIR) light enhanced CDT and PDT. The nanoplatform (denoted as Y-UCSZ) was comprised of upconversion nanoparticles (UCNPs), silica shell, and mesoporous ZnFe2O4 shell and was synthesized through a facile hydrothermal method. The UCNPs can efficiently transfer penetrable NIR photons to UV light, which can activate ZnFe2O4 for producing singlet oxygen thus promoting the Fenton reaction for ROS generation. Besides, Y-UCSZ possesses enormous internal space, which is highly beneficial for housing DOX (doxorubicin, a chemotherapeutic agent) to realize chemotherapy. Moreover, the T 2-weighted magnetic resonance imaging (MRI) effect from Fe3+ and Gd3+ ions in combination with the inherent upconversion luminescence (UCL) imaging and computed tomography (CT) from the UCNPs makes an all-in-one diagnosis and treatment system. Importantly, in vitro and in vivo assays authenticated excellent biocompatibility of the PEGylated Y-UCSZ (PEG/Y-UCSZ) and high anticancer effectiveness of the DOX loaded PEG/Y-UCSZ (PEG/Y-UCSZ&DOX), indicating its potential application in the cancer treatment field.

Targeted Genomic Profiling of Acral Melanoma.

BACKGROUND: Acral melanoma is a rare type of melanoma that affects world populations irrespective of skin color and has worse survival than other cutaneous melanomas. It has relatively few single nucleotide mutations without the UV signature of cutaneous melanomas, but instead has a genetic landscape characterized by structural rearrangements and amplifications. BRAF mutations are less common than in other cutaneous melanomas, and knowledge about alternative therapeutic targets is incomplete. METHODS: To identify alternative therapeutic targets, we performed targeted deep-sequencing on 122 acral melanomas. We confirmed the loss of the tumor suppressors p16 and NF1 by immunohistochemistry in select cases. RESULTS: In addition to BRAF (21.3%), NRAS (27.9%), and KIT (11.5%) mutations, we identified a broad array of MAPK pathway activating alterations, including fusions of BRAF (2.5%), NTRK3 (2.5%), ALK (0.8%), and PRKCA (0.8%), which can be targeted by available inhibitors. Inactivation of NF1 occurred in 18 cases (14.8%). Inactivation of the NF1 cooperating factor SPRED1 occurred in eight cases (6.6%) as an alternative mechanism of disrupting the negative regulation of RAS. Amplifications recurrently affected narrow loci containing PAK1 and GAB2 (n = 27, 22.1%), CDK4 (n = 27, 22.1%), CCND1 (n = 24, 19.7%), EP300 (n = 20, 16.4%), YAP1 (n = 15, 12.3%), MDM2 (n = 13, 10.7%), and TERT (n = 13, 10.7%) providing additional and possibly complementary therapeutic targets. Acral melanomas with BRAFV600E mutations harbored fewer genomic amplifications and were more common in patients with European ancestry. CONCLUSION: Our findings support a new, molecularly based subclassification of acral melanoma with potential therapeutic implications: BRAFV600E mutant acral melanomas with characteristics similar to nonacral melanomas that could benefit from BRAF inhibitor therapy, and non-BRAFV600E mutant acral melanomas. Acral melanomas without BRAFV600E mutations harbor a broad array of therapeutically relevant alterations. Expanded molecular profiling would increase the detection of potentially targetable alterations for this subtype of acral melanoma.

The tumor suppressor BAP1 cooperates with BRAFV600E to promote tumor formation in cutaneous melanoma.

The deubiquitinating enzyme BAP1 is mutated in a hereditary cancer syndrome with a high risk of mesothelioma and melanocytic tumors. Here, we show that Bap1 deletion in melanocytes cooperates with the constitutively active, oncogenic form of BRAF (BRAFV600E ) and UV to cause melanoma in mice, albeit at very low frequency. In addition, Bap1-null melanoma cells derived from mouse tumors are more aggressive and colonize and grow at distant sites more than their wild-type counterparts. Molecularly, Bap1-null melanoma cell lines have increased DNA damage measured by γH2aX and hyperubiquitination of histone H2a. Therapeutically, these Bap1-null tumors are completely responsive to BRAF- and MEK-targeted therapies. Therefore, BAP1 functions as a tumor suppressor and limits tumor progression in melanoma.

Human tumor genomics and zebrafish modeling identify SPRED1 loss as a driver of mucosal melanoma.

Melanomas originating from mucosal surfaces have low mutation burden, genomic instability, and poor prognosis. To identify potential driver genes, we sequenced hundreds of cancer-related genes in 43 human mucosal melanomas, cataloging point mutations, amplifications, and deletions. The SPRED1 gene, which encodes a negative regulator of mitogen-activated protein kinase (MAPK) signaling, was inactivated in 37% of the tumors. Four distinct genotypes were associated with SPRED1 loss. Using a rapid, tissue-specific CRISPR technique to model these genotypes in zebrafish, we found that SPRED1 functions as a tumor suppressor, particularly in the context of KIT mutations. SPRED1 knockdown caused MAPK activation, increased cell proliferation, and conferred resistance to drugs inhibiting KIT tyrosine kinase activity. These findings provide a rationale for MAPK inhibition in SPRED1-deficient melanomas and introduce a zebrafish modeling approach that can be used more generally to dissect genetic interactions in cancer.

Mitochondrial control through nutritionally regulated global histone H3 lysine-4 demethylation.

Histone demethylation by Jumonji-family proteins is coupled with the decarboxylation of α-ketoglutarate (αKG) to yield succinate, prompting hypotheses that their activities are responsive to levels of these metabolites in the cell. Consistent with this paradigm we show here that the Saccharomyces cerevisiae Jumonji demethylase Jhd2 opposes the accumulation of H3K4me3 in fermenting cells only when they are nutritionally manipulated to contain an elevated αKG/succinate ratio. We also find that Jhd2 opposes H3K4me3 in respiratory cells that do not exhibit such an elevated αKG/succinate ratio. While jhd2∆ caused only limited gene expression defects in fermenting cells, transcript profiling and physiological measurements show that JHD2 restricts mitochondrial respiratory capacity in cells grown in non-fermentable carbon in an H3K4me-dependent manner. In association with these phenotypes, we find that JHD2 limits yeast proliferative capacity under physiologically challenging conditions as measured by both replicative lifespan and colony growth on non-fermentable carbon. JHD2's impact on nutrient response may reflect an ancestral role of its gene family in mediating mitochondrial regulation.

Evolutionary transformation of rod photoreceptors in the all-cone retina of a diurnal garter snake.

Vertebrate retinas are generally composed of rod (dim-light) and cone (bright-light) photoreceptors with distinct morphologies that evolved as adaptations to nocturnal/crepuscular and diurnal light environments. Over 70 years ago, the "transmutation" theory was proposed to explain some of the rare exceptions in which a photoreceptor type is missing, suggesting that photoreceptors could evolutionarily transition between cell types. Although studies have shown support for this theory in nocturnal geckos, the origins of all-cone retinas, such as those found in diurnal colubrid snakes, remain a mystery. Here we investigate the evolutionary fate of the rods in a diurnal garter snake and test two competing hypotheses: (i) that the rods, and their corresponding molecular machinery, were lost or (ii) that the rods were evolutionarily modified to resemble, and function, as cones. Using multiple approaches, we find evidence for a functional and unusually blue-shifted rhodopsin that is expressed in small single "cones." Moreover, these cones express rod transducin and have rod ultrastructural features, providing strong support for the hypothesis that they are not true cones, as previously thought, but rather are modified rods. Several intriguing features of garter snake rhodopsin are suggestive of a more cone-like function. We propose that these cone-like rods may have evolved to regain spectral sensitivity and chromatic discrimination as a result of ancestral losses of middle-wavelength cone opsins in early snake evolution. This study illustrates how sensory evolution can be shaped not only by environmental constraints but also by historical contingency in forming new cell types with convergent functionality.

Identification and evolution of the silkworm helitrons and their contribution to transcripts.

In this study, we developed a structure-based approach to identify Helitrons in four lepidopterans and systematically analysed Helitrons in the silkworm genome. We found that the content of Helitrons varied greatly among genomes. The silkworm genome harboured 67,555 Helitron-related sequences that could be classified into 21 families and accounted for ≈ 4.23% of the genome. Thirteen of the families were new. Three families were putatively autonomous and included the replication initiator motif and helicase domain. The silkworm Helitrons were widely and randomly distributed in the genome. Most Helitron families radiated within the past 2 million years and experienced a single burst of expansion. These Helitron families captured 3724 gene fragments and contributed to at least 1.4% of the silkworm full-length cDNAs, suggesting important roles of Helitrons in the evolution of the silkworm genes. In addition, we found that some new Helitrons were generated by combinations of other Helitrons. Overall, the results presented in this study provided insights into the generation and evolution of Helitron transposons and their contribution to transcripts.

Timing of transcriptional quiescence during gametogenesis is controlled by global histone H3K4 demethylation.

Gametes are among the most highly specialized cells produced during development. Although gametogenesis culminates in transcriptional quiescence in plants and animals, regulatory mechanisms controlling this are unknown. Here, we confirm that gamete differentiation in the single-celled yeast Saccharomyces cerevisiae is accompanied by global transcriptional shutoff following the completion of meiosis. We show that Jhd2, a highly conserved JARID1-family histone H3K4 demethylase, activates protein-coding gene transcription in opposition to this programmed transcriptional shutoff, sustaining the period of productive transcription during spore differentiation. Moreover, using genome-wide nucleosome, H3K4me, and transcript mapping experiments, we demonstrate that JHD2 globally represses intergenic noncoding transcription during this period. The widespread transcriptional defects of JHD2 mutants are associated with precocious differentiation and the production of stress-sensitive spores, demonstrating that Jhd2 regulation of the global postmeiotic transcriptional program is critical for the production of healthy meiotic progeny.