Mucosal Genes Encoding Clock, Inflammation and Their Mutual Regulators Are Disrupted in Pediatric Patients with Active Ulcerative Colitis.

Patients with active ulcerative colitis (UC) display a misalignment of the circadian clock, which plays a vital role in various immune functions. Our aim was to characterize the expression of clock and inflammation genes, and their mutual regulatory genes in treatment-naïve pediatric patients with UC. Using the Inflammatory Bowel Disease Transcriptome and Metatranscriptome Meta-Analysis (IBD TaMMA) platform and R algorithms, we analyzed rectal biopsy transcriptomic data from two cohorts (206 patients with UC vs. 20 healthy controls from the GSE-109142 study, and 43 patients with UC vs. 55 healthy controls from the GSE-117993 study). We compared gene expression levels and correlation of clock genes (BMAL1, CLOCK, PER1, PER2, CRY1, CRY2), inflammatory genes (IκB, IL10, NFκB1, NFκB2, IL6, TNFα) and their mutual regulatory genes (RORα, RORγ, REV-ERBα, PGC1α, PPARα, PPARγ, AMPK, SIRT1) in patients with active UC and healthy controls. The clock genes BMAL1, CLOCK, PER1 and CRY1 and the inflammatory genes IκB, IL10, NFκB1, NFκB2, IL6 and TNFα were significantly upregulated in patients with active UC. The genes encoding the mutual regulators RORα, RORγ, PGC1α, PPARα and PPARγ were significantly downregulated in patients with UC. A uniform pattern of gene expression was found in healthy controls compared to the highly variable expression pattern in patients with UC. Among the healthy controls, inflammatory genes were positively correlated with clock genes and they all showed reduced expression. The difference in gene expression levels was associated with disease severity and endoscopic score but not with histological score. In patients with active UC, clock gene disruption is associated with abnormal mucosal immune response. Disrupted expression of genes encoding clock, inflammation and their mutual regulators together may play a role in active UC.

Systematic proximal mapping of the classical RAD51 paralogs unravel functionally and clinically relevant interactors for genome stability.

Homologous recombination (HR) plays an essential role in the maintenance of genome stability by promoting the repair of cytotoxic DNA double strand breaks (DSBs). More recently, the HR pathway has emerged as a core component of the response to replication stress, in part by protecting stalled replication forks from nucleolytic degradation. In that regard, the mammalian RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3) have been involved in both HR-mediated DNA repair and collapsed replication fork resolution. Still, it remains largely obscure how they participate in both processes, thereby maintaining genome stability and preventing cancer development. To gain better insight into their contribution in cellulo, we mapped the proximal interactome of the classical RAD51 paralogs using the BioID approach. Aside from identifying the well-established BCDX2 and CX3 sub-complexes, the spliceosome machinery emerged as an integral component of our proximal mapping, suggesting a crosstalk between this pathway and the RAD51 paralogs. Furthermore, we noticed that factors involved RNA metabolic pathways are significantly modulated within the BioID of the classical RAD51 paralogs upon exposure to hydroxyurea (HU), pointing towards a direct contribution of RNA processing during replication stress. Importantly, several members of these pathways have prognostic potential in breast cancer (BC), where their RNA expression correlates with poorer patient outcome. Collectively, this study uncovers novel functionally relevant partners of the different RAD51 paralogs in the maintenance of genome stability that could be used as biomarkers for the prognosis of BC.

An Exercise-Induced Metabolic Shield in Distant Organs Blocks Cancer Progression and Metastatic Dissemination.

Exercise prevents cancer incidence and recurrence, yet the underlying mechanism behind this relationship remains mostly unknown. Here we report that exercise induces the metabolic reprogramming of internal organs that increases nutrient demand and protects against metastatic colonization by limiting nutrient availability to the tumor, generating an exercise-induced metabolic shield. Proteomic and ex vivo metabolic capacity analyses of murine internal organs revealed that exercise induces catabolic processes, glucose uptake, mitochondrial activity, and GLUT expression. Proteomic analysis of routinely active human subject plasma demonstrated increased carbohydrate utilization following exercise. Epidemiologic data from a 20-year prospective study of a large human cohort of initially cancer-free participants revealed that exercise prior to cancer initiation had a modest impact on cancer incidence in low metastatic stages but significantly reduced the likelihood of highly metastatic cancer. In three models of melanoma in mice, exercise prior to cancer injection significantly protected against metastases in distant organs. The protective effects of exercise were dependent on mTOR activity, and inhibition of the mTOR pathway with rapamycin treatment ex vivo reversed the exercise-induced metabolic shield. Under limited glucose conditions, active stroma consumed significantly more glucose at the expense of the tumor. Collectively, these data suggest a clash between the metabolic plasticity of cancer and exercise-induced metabolic reprogramming of the stroma, raising an opportunity to block metastasis by challenging the metabolic needs of the tumor. SIGNIFICANCE: Exercise protects against cancer progression and metastasis by inducing a high nutrient demand in internal organs, indicating that reducing nutrient availability to tumor cells represents a potential strategy to prevent metastasis. See related commentary by Zerhouni and Piskounova, p. 4124.

Cross-species identification of cancer resistance-associated genes that may mediate human cancer risk.

Cancer is a predominant disease across animals. We applied a comparative genomics approach to systematically characterize genes whose conservation levels correlate positively (PC) or negatively (NC) with cancer resistance estimates across 193 vertebrates. Pathway analysis reveals that NC genes are enriched for metabolic functions and PC genes in cell cycle regulation, DNA repair, and immune response, pointing to their corresponding roles in mediating cancer risk. We find that PC genes are less tolerant to loss-of-function (LoF) mutations, are enriched in cancer driver genes, and are associated with germline mutations that increase human cancer risk. Their relevance to cancer risk is further supported via the analysis of mouse functional genomics and cancer mortality of zoo mammals' data. In sum, our study describes a cross-species genomic analysis pointing to candidate genes that may mediate human cancer risk.

Multi-omics data integration analysis identifies the spliceosome as a key regulator of DNA double-strand break repair.

DNA repair by homologous recombination (HR) is critical for the maintenance of genome stability. Germline and somatic mutations in HR genes have been associated with an increased risk of developing breast (BC) and ovarian cancers (OvC). However, the extent of factors and pathways that are functionally linked to HR with clinical relevance for BC and OvC remains unclear. To gain a broader understanding of this pathway, we used multi-omics datasets coupled with machine learning to identify genes that are associated with HR and to predict their sub-function. Specifically, we integrated our phylogenetic-based co-evolution approach (CladePP) with 23 distinct genetic and proteomic screens that monitored, directly or indirectly, DNA repair by HR. This omics data integration analysis yielded a new database (HRbase) that contains a list of 464 predictions, including 76 gold standard HR genes. Interestingly, the spliceosome machinery emerged as one major pathway with significant cross-platform interactions with the HR pathway. We functionally validated 6 spliceosome factors, including the RNA helicase SNRNP200 and its co-factor SNW1. Importantly, their RNA expression correlated with BC/OvC patient outcome. Altogether, we identified novel clinically relevant DNA repair factors and delineated their specific sub-function by machine learning. Our results, supported by evolutionary and multi-omics analyses, suggest that the spliceosome machinery plays an important role during the repair of DNA double-strand breaks (DSBs).

Current gene panels account for nearly all homologous recombination repair-associated multiple-case breast cancer families.

It was hypothesized that variants in underexplored homologous recombination repair (HR) genes could explain unsolved multiple-case breast cancer (BC) families. We investigated HR deficiency (HRD)-associated mutational signatures and second hits in tumor DNA from familial BC cases. No candidates genes were associated with HRD in 38 probands previously tested negative with gene panels. We conclude it is unlikely that unknown HRD-associated genes explain a large fraction of unsolved familial BC.

PAX8 plays an essential antiapoptotic role in uterine serous papillary cancer.

Endometrial carcinoma (EC) is the fourth-most common cancer in women in the United States, and generally carries a favorable prognosis. However, about 10% of EC patients have a rare and aggressive form, uterine serous papillary carcinoma (USPC), which carries a much higher mortality rate. The developmental transcription factor PAX8 is expressed in nearly 100% of USPCs. We show that PAX8 plays a critical antiapoptotic role in USPC and this role is established via transcriptional activation of two aberrant signaling pathways. First, PAX8 positively regulates mutated p53, and missense p53 mutations have an oncogenic gain of function effect. Second, PAX8 directly transcriptionally regulates p21, in a p53-independent manner, and p21 acquires a growth promoting role that is mediated via cytoplasmic localization of the protein. We propose that mutated p53 and cytoplasmic p21 can independently mediate the pro-proliferative role of PAX8 in USPC. In addition, we performed a genome-wide transcriptome analysis to detect pathways that are regulated by PAX8, and propose that metabolism and HIF-1alpha -related pathways are potential candidates for mediating the role of PAX8 in USPC. Taken together our findings demonstrate for the first time that PAX8 is an essential lineage marker in USPC, and suggest its mechanism of action.

Alternative Splicing of the Inhibitory Immune Checkpoint Receptor SLAMF6 Generates a Dominant Positive Form, Boosting T-cell Effector Functions.

SLAMF6 is a homotypic receptor of the Ig-superfamily associated with progenitor-exhausted T cells. Here we show that in humans, SLAMF6 has three splice isoforms involving its V-domain. Although the canonical receptor inhibited T-cell activation through SAP recruitment, the short isoform SLAMF6Δ17-65 had a strong agonistic effect. The costimulatory action depended on protein phosphatase SHP1 and led to a cytotoxic molecular profile mediated by the expression of TBX21 and RUNX3. Patients treated with immune checkpoint blockade showed a shift toward SLAMF6Δ17-65 in peripheral blood T cells. We developed splice-switching antisense oligonucleotides (ASO) designed to target the relevant SLAMF6 splice junction. Our ASOs enhanced SLAMF6Δ17-65 expression in human tumor-infiltrating lymphocytes and improved their capacity to inhibit human melanoma in mice. The yin-yang relationship of SLAMF6 splice isoforms may represent a balancing mechanism that could be exploited to improve cancer immunotherapy.

Reversal of diet-induced hepatic steatosis by peripheral CB1 receptor blockade in mice is p53/miRNA-22/SIRT1/PPARα dependent.

OBJECTIVE: The endocannabinoid (eCB) system is increasingly recognized as being crucially important in obesity-related hepatic steatosis. By activating the hepatic cannabinoid-1 receptor (CB1R), eCBs modulate lipogenesis and fatty acid oxidation. However, the underlying molecular mechanisms are largely unknown. METHODS: We combined unbiased bioinformatics techniques, mouse genetic manipulations, multiple pharmacological, molecular, and cellular biology approaches, and genomic sequencing to systematically decipher the role of the hepatic CB1R in modulating fat utilization in the liver and explored the downstream molecular mechanisms. RESULTS: Using an unbiased normalized phylogenetic profiling analysis, we found that the CB1R evolutionarily coevolves with peroxisome proliferator-activated receptor-alpha (PPARα), a key regulator of hepatic lipid metabolism. In diet-induced obese (DIO) mice, peripheral CB1R blockade (using AM6545) induced the reversal of hepatic steatosis and improved liver injury in WT, but not in PPARα-/- mice. The antisteatotic effect mediated by AM6545 in WT DIO mice was accompanied by increased hepatic expression and activity of PPARα as well as elevated hepatic levels of the PPARα-activating eCB-like molecules oleoylethanolamide and palmitoylethanolamide. Moreover, AM6545 was unable to rescue hepatic steatosis in DIO mice lacking liver sirtuin 1 (SIRT1), an upstream regulator of PPARα. Both of these signaling molecules were modulated by the CB1R as measured in hepatocytes exposed to lipotoxic conditions or treated with CB1R agonists in the absence/presence of AM6545. Furthermore, using microRNA transcriptomic profiling, we found that the CB1R regulated the hepatic expression, acetylation, and transcriptional activity of p53, resulting in the enhanced expression of miR-22, which was found to specifically target SIRT1 and PPARα. CONCLUSIONS: We provide strong evidence for a functional role of the p53/miR-22/SIRT1/PPARα signaling pathway in potentially mediating the antisteatotic effect of peripherally restricted CB1R blockade.

Titanium Tackles the Endoplasmic Reticulum: A First Genomic Study on a Titanium Anticancer Metallodrug.

PhenolaTi is an advanced non-toxic anticancer chemotherapy; this inert bis(phenolato)bis(alkoxo) Ti(IV) complex demonstrates the intriguing combination of high and wide efficacy with no detected toxicity in animals. Here we unravel the cellular pathways involved in its mechanism of action by a first genome study on Ti(IV)-treated cells, using an attuned RNA sequencing-based available technology. First, phenolaTi induced apoptosis and cell-cycle arrest at the G2/M phase in MCF7 cells. Second, the transcriptome of the treated cells was analyzed, identifying alterations in pathways relating to protein translation, DNA damage, and mitochondrial eruption. Unlike for common metallodrugs, electrophoresis assay showed no inhibition of DNA polymerase activity. Reduced in vitro cytotoxicity with added endoplasmic reticulum (ER) stress inhibitor supported the ER as a putative cellular target. Altogether, this paper reveals a distinct ER-related mechanism by the Ti(IV) anticancer coordination complex, paving the way for wider applicability of related techniques in mechanistic analyses of metallodrugs.

MYORG Mutations: a Major Cause of Recessive Primary Familial Brain Calcification.

PURPOSE OF REVIEW: Until recently, the gene associated with the recessive form of familial brain calcification (PFBC, Fahr disease) was unknown. MYORG, a gene that causes recessive PFBC was only recently discovered and is currently the only gene associated with a recessive form of this disease. Here, we review the radiological and clinical findings in adult MYORG mutation homozygous and heterozygous individuals. RECENT FINDINGS: MYORG was shown to be the cause of a large fraction of recessive cases of PFBC in patients of different ethnic populations. Pathogenic mutations include inframe insertions and deletions in addition to nonsense and missense mutations that are distributed throughout the entire MYORG coding region. Homozygotes have extensive brain calcification in all known cases, whereas in some carriers of heterozygous mutation, punctuated calcification of the globus pallidus is demonstrated. The clinical spectrum in homozygotes ranges from the lack of neurological symptoms to severe progressive neurological syndrome with bulbar and cerebellar signs, parkinsonism and other movement disorders, and cognitive impairments. Heterozygotes are clinically asymptomatic. MYORG is a transmembrane protein localized to the endoplasmic reticulum and is mainly expressed in astrocytes. While the biochemical pathways of the protein are still unknown, information from its evolution profile across hundreds of species (phylogenetic profiling) suggests a role for MYORG in regulating ion homeostasis via its glycosidase domain. MYORG mutations are a major cause for recessive PFBC in different world populations. Future studies are required in order to reveal the cellular role of the MYORG protein.

A high-throughput screening and computation platform for identifying synthetic promoters with enhanced cell-state specificity (SPECS).

Cell state-specific promoters constitute essential tools for basic research and biotechnology because they activate gene expression only under certain biological conditions. Synthetic Promoters with Enhanced Cell-State Specificity (SPECS) can be superior to native ones, but the design of such promoters is challenging and frequently requires gene regulation or transcriptome knowledge that is not readily available. Here, to overcome this challenge, we use a next-generation sequencing approach combined with machine learning to screen a synthetic promoter library with 6107 designs for high-performance SPECS for potentially any cell state. We demonstrate the identification of multiple SPECS that exhibit distinct spatiotemporal activity during the programmed differentiation of induced pluripotent stem cells (iPSCs), as well as SPECS for breast cancer and glioblastoma stem-like cells. We anticipate that this approach could be used to create SPECS for gene therapies that are activated in specific cell states, as well as to study natural transcriptional regulatory networks.

Mapping global and local coevolution across 600 species to identify novel homologous recombination repair genes.

The homologous recombination repair (HRR) pathway repairs DNA double-strand breaks in an error-free manner. Mutations in HRR genes can result in increased mutation rate and genomic rearrangements, and are associated with numerous genetic disorders and cancer. Despite intensive research, the HRR pathway is not yet fully mapped. Phylogenetic profiling analysis, which detects functional linkage between genes using coevolution, is a powerful approach to identify factors in many pathways. Nevertheless, phylogenetic profiling has limited predictive power when analyzing pathways with complex evolutionary dynamics such as the HRR. To map novel HRR genes systematically, we developed clade phylogenetic profiling (CladePP). CladePP detects local coevolution across hundreds of genomes and points to the evolutionary scale (e.g., mammals, vertebrates, animals, plants) at which coevolution occurred. We found that multiscale coevolution analysis is significantly more biologically relevant and sensitive to detect gene function. By using CladePP, we identified dozens of unrecognized genes that coevolved with the HRR pathway, either globally across all eukaryotes or locally in different clades. We validated eight genes in functional biological assays to have a role in DNA repair at both the cellular and organismal levels. These genes are expected to play a role in the HRR pathway and might lead to a better understanding of missing heredity in HRR-associated cancers (e.g., heredity breast and ovarian cancer). Our platform presents an innovative approach to predict gene function, identify novel factors related to different diseases and pathways, and characterize gene evolution.

MYORG is associated with recessive primary familial brain calcification.

Objective: To investigate the genetic basis of the recessive form of primary familial brain calcification and study pathways linking a novel gene with known dominant genes that cause the disease. Methods: Whole exome sequencing and Sanger-based segregation analysis were used to identify possible disease causing mutations. Mutation pathogenicity was validated by structural protein modeling. Functional associations between the candidate gene, MYORG, and genes previously implicated in the disease were examined through phylogenetic profiling. Results: We studied nine affected individuals from two unrelated families of Middle Eastern origin. The median age of symptom onset was 29.5 years (range 21-57 years) and dysarthria was the most common presenting symptom. We identified in the MYORG gene, a homozygous c.1233delC mutation in one family and c.1060_1062delGAC mutation in another. The first mutation results in protein truncation and the second in deletion of a highly conserved aspartic acid that is likely to disrupt binding of the protein with its substrate. Phylogenetic profiling analysis of the MYORG protein sequence suggests co-evolution with a number of calcium channels as well as other proteins related to regulation of anion transmembrane transport (False Discovery Rate, FDR < 10-8) and with PDCD6IP, a protein interacting with PDGFR ß which is known to be involved in the disease. Interpretation: MYORG mutations are linked to a recessive form of primary familial brain calcification. This association was recently described in patients of Chinese ancestry. We suggest the possibility that MYORG mutations lead to calcification in a PDGFR ß-related pathway.

SHLD2/FAM35A co-operates with REV7 to coordinate DNA double-strand break repair pathway choice.

DNA double-strand breaks (DSBs) can be repaired by two major pathways: non-homologous end-joining (NHEJ) and homologous recombination (HR). DNA repair pathway choice is governed by the opposing activities of 53BP1, in complex with its effectors RIF1 and REV7, and BRCA1. However, it remains unknown how the 53BP1/RIF1/REV7 complex stimulates NHEJ and restricts HR to the S/G2 phases of the cell cycle. Using a mass spectrometry (MS)-based approach, we identify 11 high-confidence REV7 interactors and elucidate the role of SHLD2 (previously annotated as FAM35A and RINN2) as an effector of REV7 in the NHEJ pathway. FAM35A depletion impairs NHEJ-mediated DNA repair and compromises antibody diversification by class switch recombination (CSR) in B cells. FAM35A accumulates at DSBs in a 53BP1-, RIF1-, and REV7-dependent manner and antagonizes HR by limiting DNA end resection. In fact, FAM35A is part of a larger complex composed of REV7 and SHLD1 (previously annotated as C20orf196 and RINN3), which promotes NHEJ and limits HR Together, these results establish SHLD2 as a novel effector of REV7 in controlling the decision-making process during DSB repair.

Mutated MITF-E87R in Melanoma Enhances Tumor Progression via S100A4.

Melanoma, a melanocyte origin neoplasm, is the most lethal type of skin cancer, and incidence is increasing. Several familial and somatic mutations have been identified in the gene encoding the melanocyte lineage master regulator, MITF; however, the neoplastic mechanisms of these mutant MITF variants are mostly unknown. Here, by performing unbiased analysis of the transcriptomes in cells expressing mutant MITF, we identified calcium-binding protein S100A4 as a downstream target of MITF-E87R. By using wild-type and mutant MITF melanoma lines, we found that both endogenous wild-type and MITF-E87R variants occupy the S100A4 promoter. Remarkably, whereas wild-type MITF represses S100A4 expression, MITF-E87R activates its transcription. The opposite effects of wild-type and mutant MITF result in opposing cellular phenotypes, because MITF-E87R via S100A4 enhanced invasion and reduced adhesion in contrast to wild-type MITF activity. Finally, we found that melanoma patients with altered S100A4 expression have poor prognosis. These data show that a change in MITF transcriptional activity from repression to activation of S100A4 that results from a point mutation in MITF alters melanoma invasive ability. These data suggest new opportunities for diagnosis and treatment of metastatic melanoma.

Synthetic RNA-Based Immunomodulatory Gene Circuits for Cancer Immunotherapy.

Despite its success in several clinical trials, cancer immunotherapy remains limited by the rarity of targetable tumor-specific antigens, tumor-mediated immune suppression, and toxicity triggered by systemic delivery of potent immunomodulators. Here, we present a proof-of-concept immunomodulatory gene circuit platform that enables tumor-specific expression of immunostimulators, which could potentially overcome these limitations. Our design comprised de novo synthetic cancer-specific promoters and, to enhance specificity, an RNA-based AND gate that generates combinatorial immunomodulatory outputs only when both promoters are mutually active. These outputs included an immunogenic cell-surface protein, a cytokine, a chemokine, and a checkpoint inhibitor antibody. The circuits triggered selective T cell-mediated killing of cancer cells, but not of normal cells, in vitro. In in vivo efficacy assays, lentiviral circuit delivery mediated significant tumor reduction and prolonged mouse survival. Our design could be adapted to drive additional immunomodulators, sense other cancers, and potentially treat other diseases that require precise immunological programming.

Interactions of Melanoma Cells with Distal Keratinocytes Trigger Metastasis via Notch Signaling Inhibition of MITF.

The most critical stage in initiation of melanoma metastasis is the radial to vertical growth transition, yet the triggers of this transition remain elusive. We suggest that the microenvironment drives melanoma metastasis independently of mutation acquisition. Here we examined the changes in microenvironment that occur during melanoma radial growth. We show that direct contact of melanoma cells with the remote epidermal layer triggers vertical invasion via Notch signaling activation, the latter serving to inhibit MITF function. Briefly, within the native Notch ligand-free microenvironment, MITF, the melanocyte lineage master regulator, binds and represses miR-222/221 promoter in an RBPJK-dependent manner. However, when radial growth brings melanoma cells into contact with distal differentiated keratinocytes that express Notch ligands, the activated Notch intracellular domain impairs MITF binding to miR-222/221 promoter. This de-repression of miR-222/221 expression triggers initiation of invasion. Our findings may direct melanoma prevention opportunities via targeting specific microenvironments.

High-resolution mapping reveals a conserved, widespread, dynamic mRNA methylation program in yeast meiosis.

N(6)-methyladenosine (m(6)A) is the most ubiquitous mRNA base modification, but little is known about its precise location, temporal dynamics, and regulation. Here, we generated genomic maps of m(6)A sites in meiotic yeast transcripts at nearly single-nucleotide resolution, identifying 1,308 putatively methylated sites within 1,183 transcripts. We validated eight out of eight methylation sites in different genes with direct genetic analysis, demonstrated that methylated sites are significantly conserved in a related species, and built a model that predicts methylated sites directly from sequence. Sites vary in their methylation profiles along a dense meiotic time course and are regulated both locally, via predictable methylatability of each site, and globally, through the core meiotic circuitry. The methyltransferase complex components localize to the yeast nucleolus, and this localization is essential for mRNA methylation. Our data illuminate a conserved, dynamically regulated methylation program in yeast meiosis and provide an important resource for studying the function of this epitranscriptomic modification.

Human disease locus discovery and mapping to molecular pathways through phylogenetic profiling.

Genes with common profiles of the presence and absence in disparate genomes tend to function in the same pathway. By mapping all human genes into about 1000 clusters of genes with similar patterns of conservation across eukaryotic phylogeny, we determined that sets of genes associated with particular diseases have similar phylogenetic profiles. By focusing on those human phylogenetic gene clusters that significantly overlap some of the thousands of human gene sets defined by their coexpression or annotation to pathways or other molecular attributes, we reveal the evolutionary map that connects molecular pathways and human diseases. The other genes in the phylogenetic clusters enriched for particular known disease genes or molecular pathways identify candidate genes for roles in those same disorders and pathways. Focusing on proteins coevolved with the microphthalmia-associated transcription factor (MITF), we identified the Notch pathway suppressor of hairless (RBP-Jk/SuH) transcription factor, and showed that RBP-Jk functions as an MITF cofactor.