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Tissue fibrosis is a progressive pathological process with excessive deposition of extracellular matrix proteins (ECM). Myofibroblasts, identified by alpha-smooth muscle actin (αSMA) expression, play an important role in tissue fibrosis by producing ECM. Here, we found that the Wnt antagonist Dickkopf1 (DKK1) induced gene expressions associated with inflammation and fibrosis in lung fibroblasts. We demonstrated that genetic deletion of LRP6, a receptor for Wnt ligands and DKK1, in αSMA-expressing cells using Acta2-cre Lrp6 fl/fl (Lrp6 AKO ) mice abrogated the bleomycin (BLM)-induced lung inflammation and fibrosis phenotype, suggesting an important role for LRP6 in modulating inflammation and fibrotic processes in the lung. Our results highlight the crucial role of LRP6 in fibroblasts in regulating inflammation and fibrosis upon BLM-induced lung injury., (© 2025 The Author(s). FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Three-dimensional (3D) chromatin interactions, such as enhancer-promoter interactions (EPIs), loops, topologically associating domains (TADs), and A/B compartments, play critical roles in a wide range of cellular processes by regulating gene expression. Recent development of chromatin conformation capture technologies has enabled genome-wide profiling of various 3D structures, even with single cells. However, current catalogs of 3D structures remain incomplete and unreliable due to differences in technology, tools, and low data resolution. Machine learning methods have emerged as an alternative to obtain missing 3D interactions and/or improve resolution. Such methods frequently use genome annotation data (ChIP-seq, DNAse-seq, etc.), DNA sequencing information (k-mers and transcription factor binding site (TFBS) motifs), and other genomic properties to learn the associations between genomic features and chromatin interactions. In this review, we discuss computational tools for predicting three types of 3D interactions (EPIs, chromatin interactions, and TAD boundaries) and analyze their pros and cons. We also point out obstacles to the computational prediction of 3D interactions and suggest future research directions., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Chemotherapy-induced peripheral neuropathy (CIPN) is a prevalent and severe side effect affecting cancer patients undergoing paclitaxel treatment. Growing evidence underscores the pivotal role of calcitonin-related peptide (CGRP) in the development of CIPN. Repeated administration of paclitaxel induces alterations in CGRP release from sensory neurons within the dorsal root ganglia (DRG). The density of the CGRP receptor is most prominent in the dorsal horn of the spinal cord, where it overlaps with the distribution of CGRP. However, the impact of chemotherapy treatment on expression of the CGRP receptor in the spinal cord remains unclear, as well as the potential therapeutic benefits of a CGRP receptor antagonist in an animal model of CIPN. Using a mouse model of paclitaxel-induced mechanical hypersensitivity, we show upregulation of Calcitonin receptor-like receptor ( Calcrl ) mRNA expression in the spinal cord, an event that occurred in association with upregulation of the H3K4 methyltransferase MLL2 . This effect of repeated paclitaxel administration was also linked to an increase in the recruitment of MLL2, thereby enhancing levels of the active mark H3K4me2 at the Calcrl promoter. Furthermore, administration of the CGRP receptor antagonist BIBN4096 mitigated mechanical and cold hypersensitivity in paclitaxel-treated mice. Together, these observations suggest the CGRP receptor in the spinal cord as a potential target for reducing paclitaxel-induced neuropathic pain in animal models., Competing Interests: Declaration of conflicting interestsThe author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: J.G.-M. has as sponsor research contract with Terran Biosciences.

Hepatic xenobiotic metabolism and transport decline with age, while intact xenobiotic metabolism is associated with longevity. However, few studies have examined the genome-wide impact of epigenetic aging on these processes. We used reduced representation bisulfite sequencing (RRBS) to map DNA methylation changes in liver DNA from mice ages 4 and 24 months. We identified several thousand age-associated differentially methylated sites (a-DMS), many of which overlapped genes encoding Phase I and Phase II drug metabolizing enzymes, in addition to ABC and SLC classes of transporters. Notable genes harboring a-DMS were Cyp1a2, Cyp2d9, and Abcc2 that encode orthologs of the human drug metabolizing enzymes CYP1A2 and CYP2D6, and the multidrug resistance protein 2 (MRP2) transporter. Cyp2d9 hypermethylation with age was significantly associated with reduced gene expression, while Abcc2 expression was unchanged with age. Cyp1a2 lost methylation with age while, counterintuitively, its expression also reduced with age. We hypothesized that age-related dysregulation of the hepatic transcriptional machinery caused down-regulation of genes despite age-related hypomethylation. Bioinformatic analysis of hypomethylated a-DMS in our sample found them to be highly enriched for hepatic nuclear factor 4 alpha (HNF4α) binding sites. HNF4α promotes Cyp1a2 expression and is downregulated with age, which could explain the reduction in Cyp1a2 expression. Overall, our study supports the broad impact of epigenetic aging on xenobiotic metabolism and transport. Future work should evaluate the interplay between hepatic nuclear receptor function and epigenetic aging. These results may have implications for studies of longevity and healthy aging., (© 2024. The Author(s).)

Systemic lupus erythematosus is a remitting relapsing autoimmune disease characterized by autoantibody production and multi-organ involvement. T cell epigenetic dysregulation plays an important role in the pathogenesis of lupus. We have previously demonstrated upregulation of the key epigenetic regulator EZH2 in CD4 + T cells isolated from lupus patients. To further investigate the role of EZH2 in the pathogenesis of lupus, we generated a tamoxifen-inducible CD4 + T cell Ezh2 conditional knockout mouse on the MRL/ lpr lupus-prone background. We demonstrate that Ezh2 deletion abrogates lupus-like disease and prevents T cell differentiation. Single-cell analysis suggests impaired T cell function and activation of programmed cell death pathways in EZH2-deficient mice. Ezh2 deletion in CD4 + T cells restricts TCR clonal repertoire and prevents kidney-infiltrating effector CD4 + T cell expansion and tubulointerstitial nephritis, which has been linked to end-stage renal disease in patients with lupus nephritis., Competing Interests: None of the authors report any relevant financial conflict of interest. X.Z. is currently employed as a scientist at The Jackson Laboratory., (© 2024 The Author(s).)

Background: Breast cancer's complex transcriptional landscape requires an improved understanding of cellular diversity to identify effective treatments. The study of genetic variations among breast cancer subtypes at single-cell resolution has potential to deepen our insights into cancer progression., Methods: In this study, we amalgamate single-cell RNA sequencing data from patient tumours and matched lymph metastasis, reduction mammoplasties, breast cancer patient-derived xenografts (PDXs), PDX-derived organoids (PDXOs), and cell lines resulting in a diverse dataset of 117 samples with 506 719 total cells. These samples encompass hormone receptor positive (HR+), human epidermal growth factor receptor 2 positive (HER2+), and triple-negative breast cancer (TNBC) subtypes, including isogenic model pairs. Herein, we delineated similarities and distinctions across models and patient samples and explore therapeutic drug efficacy based on subtype proportions., Results: PDX models more closely resemble patient samples in terms of tumour heterogeneity and cell cycle characteristics when compared with TNBC cell lines. Acquired drug resistance was associated with an increase in basal-like cell proportions within TNBC PDX tumours as defined with SCSubtype and TNBCtype cell typing predictors. All patient samples contained a mixture of subtypes; compared to primary tumours HR+ lymph node metastases had lower proportions of HER2-Enriched cells. PDXOs exhibited differences in metabolic-related transcripts compared to PDX tumours. Correlative analyses of cytotoxic drugs on PDX cells identified therapeutic efficacy was based on subtype proportion., Conclusions: We present a substantial multimodel dataset, a dynamic approach to cell-wise sample annotation, and a comprehensive interrogation of models within systems of human breast cancer. This analysis and reference will facilitate informed decision-making in preclinical research and therapeutic development through its elucidation of model limitations, subtype-specific insights and novel targetable pathways., Key Points: Patient-derived xenografts models more closely resemble patient samples in tumour heterogeneity and cell cycle characteristics when compared with cell lines. 3D organoid models exhibit differences in metabolic profiles compared to their in vivo counterparts. A valuable multimodel reference dataset that can be useful in elucidating model differences and novel targetable pathways., (© 2024 The Author(s). Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.)

Ethanol's anxiolytic actions contribute to increased consumption and the development of Alcohol Use Disorder (AUD). Our laboratory previously identified genetic loci contributing to the anxiolytic-like properties of ethanol in BXD recombinant inbred mice, derived from C57BL/6J (B6) and DBA/2J (D2) progenitor strains. That work identified Ninein ( Nin ) as a candidate gene underlying ethanol's acute anxiolytic-like properties in BXD mice. Nin has a complex exonic content with known alternative splicing events that alter cellular distribution of the NIN protein. We hypothesize that strain-specific differences in Nin alternative splicing contribute to changes in Nin gene expression and B6/D2 strain differences in ethanol anxiolysis. Using quantitative reverse-transcriptase PCR to target specific Nin splice variants, we identified isoform-specific exon expression differences between B6 and D2 mice in prefrontal cortex, nucleus accumbens and amygdala. We extended this analysis using deep RNA sequencing in B6 and D2 nucleus accumbens samples and found that total Nin expression was significantly higher in D2 mice. Furthermore, exon utilization and alternative splicing analyses identified eight differentially utilized exons and significant exon-skipping events between the strains, including three novel splicing events in the 3' end of the Nin gene that were specific to the D2 strain. Additionally, we document multiple single nucleotide polymorphisms in D2 Nin exons that are predicted to have deleterious effects on protein function. Our studies provide the first in-depth analysis of Nin alternative splicing in brain and identify a potential genetic mechanism altering Nin expression and function between B6 and D2 mice, thus possibly contributing to differences in the anxiolytic-like properties of ethanol between these strains. This work adds novel information to our understanding of genetic differences modulating ethanol actions on anxiety that may contribute to the risk for alcohol use disorder., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Gnatowski, Jurmain, Dozmorov, Wolstenholme and Miles.)

Low-density lipoprotein receptor-related protein 6 (LRP6) is a receptor for Wnt ligands. Tissue fibrosis is a progressive pathological process with excessive extracellular matrix proteins (ECM) deposition. Myofibroblasts, identified by alpha-smooth muscle actin (αSMA) expression, play an important role in tissue fibrosis by producing ECM production. Here we found that Wnt antagonist Dickkopf1 (DKK1) induced gene expressions associated with inflammation and fibrosis in lung fibroblasts. We demonstrated that genetic deletion of LRP6 in αSMA-expressing cells using Acta2 -cre Lrp6 fl/fl ( Lrp6 AKO ) mice abrogated bleomycin (BLM)-induced lung inflammation and fibrosis phenotype, suggesting an important role of LRP6 in modulating inflammation and fibrotic processes in the lung. Our results highlight the crucial role of LRP6 in fibroblasts in regulating inflammation and fibrosis upon BLM-induced lung injury.

Synovial Sarcoma (SS) is driven by the SS18::SSX fusion oncoprotein and is ultimately refractory to therapeutic approaches. SS18::SSX alters ATP-dependent chromatin remodeling BAF (mammalian SWI/SNF) complexes, leading to the degradation of canonical (cBAF) complex and amplified presence of an SS18::SSX-containing non-canonical BAF (ncBAF or GBAF) that drives an SS-specific transcription program and tumorigenesis. We demonstrate that SS18::SSX activates the SUMOylation program and SSs are sensitive to the small molecule SAE1/2 inhibitor, TAK-981. Mechanistically, TAK-981 de-SUMOylates the cBAF subunit SMARCE1, stabilizing and restoring cBAF on chromatin, shifting away from SS18::SSX-ncBAF-driven transcription, associated with DNA damage and cell death and resulting in tumor inhibition across both human and mouse SS tumor models. TAK-981 synergized with cytotoxic chemotherapy through increased DNA damage, leading to tumor regression. Targeting the SUMOylation pathway in SS restores cBAF complexes and blocks the SS18::SSX-ncBAF transcriptome, identifying a therapeutic vulnerability in SS, positioning the in-clinic TAK-981 to treat SS., Competing Interests: Additional Declarations: There is NO Competing Interest. DECLARATION OF INTERESTS A.C.F. is a consultant and equity holder in Treeline Biosciences and has previously served as a scientific advisor for AbbVie and has received research funding from IDP Pharma. K.V. receives support from AstraZeneca. R.L. is an inventor of a provisional patent on targeted killing of EBV-positive cancer cells by CRISPR/dCas9-mediated EBV reactivation. S.B. is Consultant for Caris Lifescience and has received honoraria from SpringWorks for an educational lecture. K.V. receives support from AstraZeneca with no relationship to the present study. The authors declare that these listed activities have no relationship to the present study.

Systemic lupus erythematosus is a remitting relapsing autoimmune disease characterized by autoantibody production and multi-organ involvement. T cell epigenetic dysregulation plays an important role in the pathogenesis of lupus. We have previously demonstrated upregulation of the key epigenetic regulator EZH2 in CD4+ T cells isolated from lupus patients. To further investigate the role of EZH2 in the pathogenesis of lupus, we generated a tamoxifen-inducible CD4+ T cell Ezh2 conditional knockout mouse on the MRL/lpr lupus-prone background. We demonstrate that Ezh2 deletion abrogates lupus-like disease and prevents T cell differentiation. Single-cell analysis suggests impaired T cell function and activation of programed cell death pathways in EZH2-deficient mice. Ezh2 deletion in CD4+ T cells restricts TCR clonal repertoire and prevents kidney-infiltrating effector CD4+ T cell expansion and tubulointerstitial nephritis, which has been linked to end-stage renal disease in patients with lupus nephritis.

Synovial Sarcoma (SS) is driven by the SS18::SSX fusion oncoprotein. and is ultimately refractory to therapeutic approaches. SS18::SSX alters ATP-dependent chromatin remodeling BAF (mammalian SWI/SNF) complexes, leading to the degradation of canonical (cBAF) complex and amplified presence of an SS18::SSX-containing non-canonical BAF (ncBAF or GBAF) that drives an SS-specific transcription program and tumorigenesis. We demonstrate that SS18::SSX activates the SUMOylation program and SSs are sensitive to the small molecule SAE1/2 inhibitor, TAK-981. Mechanistically, TAK-981 de-SUMOylates the cBAF subunit SMARCE1, stabilizing and restoring cBAF on chromatin, shifting away from SS18::SSX-ncBAF-driven transcription, associated with DNA damage and cell death and resulting in tumor inhibition across both human and mouse SS tumor models. TAK-981 synergized with cytotoxic chemotherapy through increased DNA damage, leading to tumor regression. Targeting the SUMOylation pathway in SS restores cBAF complexes and blocks the SS18::SSX-ncBAF transcriptome, identifying a therapeutic vulnerability in SS, positioning the in-clinic TAK-981 to treat SS.

Genome-wide association studies (GWAS) of psychiatric disorders (PD) yield numerous loci with significant signals, but often do not implicate specific genes. Because GWAS risk loci are enriched in expression/protein/methylation quantitative loci (e/p/mQTL, hereafter xQTL), transcriptome/proteome/methylome-wide association studies (T/P/MWAS, hereafter XWAS) that integrate xQTL and GWAS information, can link GWAS signals to effects on specific genes. To further increase detection power, gene signals are aggregated within relevant gene sets (GS) by performing gene set enrichment (GSE) analyses. Often GSE methods test for enrichment of "signal" genes in curated GS while overlooking their linkage disequilibrium (LD) structure, allowing for the possibility of increased false positive rates. Moreover, no GSE tool uses xQTL information to perform mendelian randomization (MR) analysis. To make causal inference on association between PD and GS, we develop a novel MR GSE (MR-GSE) procedure. First, we generate a "synthetic" GWAS for each MSigDB GS by aggregating summary statistics for x-level (mRNA, protein or DNA methylation (DNAm) levels) from the largest xQTL studies available) of genes in a GS. Second, we use synthetic GS GWAS as exposure in a generalized summary-data-based-MR analysis of complex trait outcomes. We applied MR-GSE to GWAS of nine important PD. When applied to the underpowered opioid use disorder GWAS, none of the four analyses yielded any signals, which suggests a good control of false positive rates. For other PD, MR-GSE greatly increased the detection of GO terms signals (2,594) when compared to the commonly used (non-MR) GSE method (286). Some of the findings might be easier to adapt for treatment, e.g., our analyses suggest modest positive effects for supplementation with certain vitamins and/or omega-3 for schizophrenia, bipolar and major depression disorder patients. Similar to other MR methods, when applying MR-GSE researchers should be mindful of the confounding effects of horizontal pleiotropy on statistical inference.

Three-Dimensional (3D) chromatin interactions, such as enhancer-promoter interactions (EPIs), loops, Topologically Associating Domains (TADs), and A/B compartments play critical roles in a wide range of cellular processes by regulating gene expression. Recent development of chromatin conformation capture technologies has enabled genome-wide profiling of various 3D structures, even with single cells. However, current catalogs of 3D structures remain incomplete and unreliable due to differences in technology, tools, and low data resolution. Machine learning methods have emerged as an alternative to obtain missing 3D interactions and/or improve resolution. Such methods frequently use genome annotation data (ChIP-seq, DNAse-seq, etc.), DNA sequencing information (k-mers, Transcription Factor Binding Site (TFBS) motifs), and other genomic properties to learn the associations between genomic features and chromatin interactions. In this review, we discuss computational tools for predicting three types of 3D interactions (EPIs, chromatin interactions, TAD boundaries) and analyze their pros and cons. We also point out obstacles of computational prediction of 3D interactions and suggest future research directions., Competing Interests: Conflict of Interest. None.

Background and Aims: The oncogene Melanoma differentiation associated gene-9/syndecan binding protein (MDA-9/SDCBP) is overexpressed in many cancers, promoting aggressive, metastatic disease. However, the role of MDA-9 in regulating hepatocellular carcinoma (HCC) has not been well studied., Approach and Results: To unravel the function of MDA-9 in HCC, we generated and characterized a transgenic mouse with hepatocyte-specific overexpression of MDA-9 (Alb/MDA-9). Compared with wild-type (WT) littermates, Alb/MDA-9 mice demonstrated significantly higher incidence of N-nitrosodiethylamine/phenobarbital-induced HCC, with marked activation and infiltration of macrophages. RNA sequencing (RNA-seq) in naive WT and Alb/MDA-9 hepatocytes identified activation of signaling pathways associated with invasion, angiogenesis, and inflammation, especially NF-κB and integrin-linked kinase signaling pathways. In nonparenchymal cells purified from naive livers, single-cell RNA-seq showed activation of Kupffer cells and macrophages in Alb/MDA-9 mice versus WT mice. A robust increase in the expression of Secreted phosphoprotein 1 (Spp1/osteopontin) was observed upon overexpression of MDA-9. Inhibition of NF-κB pathway blocked MDA-9-induced Spp1 induction, and knock down of Spp1 resulted in inhibition of MDA-9-induced macrophage migration, as well as angiogenesis., Conclusions: Alb/MDA-9 is a mouse model with MDA-9 overexpression in any tissue type. Our findings unravel an HCC-promoting role of MDA-9 mediated by NF-κB and Spp1 and support the rationale of using MDA-9 inhibitors as a potential treatment for aggressive HCC., (Copyright © 2023 American Association for the Study of Liver Diseases.)

There is a critical need to identify new therapeutic vulnerabilities in pancreatic ductal adenocarcinoma (PDAC). Transcriptional co-regulators C-terminal binding proteins (CtBP) 1 and 2 are highly overexpressed in human PDAC, and CRISPR-based homozygous deletion of Ctbp2 in a mouse PDAC cell line (CKP) dramatically decreased tumor growth, reduced metastasis, and prolonged survival in orthotopic mouse allografts. Transcriptomic profiling of tumors derived from CKP vs. Ctbp2-deleted CKP cells (CKP/KO) revealed significant downregulation of the EGFR-superfamily receptor Erbb3, the heterodimeric signaling partner for both EGFR and ErbB2. Compared with CKP cells, CKP/KO cells also demonstrated reduced Erbb2 expression and did not activate downstream Akt signaling after stimulation of Erbb3 by its ligand neuregulin-1. ErbB3 expression in human PDAC cell lines was similarly dependent on CtBP2 and depletion of ErbB3 in a human PDAC cell line severely attenuated growth, demonstrating the critical role of ErbB3 signaling in maintaining PDAC cell growth. Sensitivity to the ErbB2-targeted tyrosine kinase inhibitor lapatinib, but not the EGFR-targeted agent erlotinib, varied in proportion to the level of ErbB3 expression in mouse and human PDAC cells, suggesting that an ErBb2 inhibitor can effectively leverage CtBP2-driven transcriptional activation of physiologic ErbB2/3 expression and signaling in PDAC cells for therapeutic benefit., (© 2023. The Author(s).)

Diverse subpopulations of astrocytes tile different brain regions to accommodate local requirements of neurons and associated neuronal circuits. Nevertheless, molecular mechanisms governing astrocyte diversity remain mostly unknown. We explored the role of a zinc finger transcription factor Yin Yang 1 (YY1) that is expressed in astrocytes. We found that specific deletion of YY1 from astrocytes causes severe motor deficits in mice, induces Bergmann gliosis, and results in simultaneous loss of GFAP expression in velate and fibrous cerebellar astrocytes. Single cell RNA-seq analysis showed that YY1 exerts specific effects on gene expression in subpopulations of cerebellar astrocytes. We found that although YY1 is dispensable for the initial stages of astrocyte development, it regulates subtype-specific gene expression during astrocyte maturation. Moreover, YY1 is continuously needed to maintain mature astrocytes in the adult cerebellum. Our findings suggest that YY1 plays critical roles regulating cerebellar astrocyte maturation during development and maintaining a mature phenotype of astrocytes in the adult cerebellum., (© 2023 The Authors. GLIA published by Wiley Periodicals LLC.)

African-American (AA)/Black hepatocellular carcinoma (HCC) patients have increased incidence and decreased survival rates compared to non-Hispanic (White) patients, the underlying molecular mechanism of which is not clear. Analysis of existing RNA-sequencing (RNA-seq) data in The Cancer Genome Atlas (TCGA) and in-house RNA-sequencing of 14 White and 18 AA/Black HCC patients revealed statistically significant activation of type I interferon (IFN-I) signaling pathway in AA/Black patients. A four-gene signature of IFN-stimulated genes (ISGs) showed increased expression in AA/Black HCC tumors versus White. HCC is a disease of chronic inflammation, and IFN-Is function as pro-inflammatory cytokines. We tested efficacy of ginger extract (GE), a dietary compound known for anti-inflammatory properties, on HCC cell lines derived from White (HepG2), AA/Black (Hep3B and O/20) and Asian (HuH-7) patients. GE exhibited a significantly lower IC 50 on Hep3B and O/20 cells than on HepG2 and HuH-7 cells. The GE treatment inhibited the activation of downstream mediators of IFN-I signaling pathways and expression of ISGs in all four HCC cells. Our data suggest that ginger can potentially attenuate IFN-I-mediated signaling pathways in HCC, and cells from AA/Black HCC patients may be more sensitive to ginger. AA/Black HCC patients might benefit from a holistic diet containing ginger.

Objectives: EZH2 regulates B cell development and differentiation. We previously demonstrated increased EZH2 expression in peripheral blood mononuclear cells from lupus patients. The goal of this study was to evaluate the role of EZH2 expression in B cells in the pathogenesis of lupus., Methods: We generated an MRL/lpr mouse with floxed Ezh2, which was crossed with CD19-Cre mice to examine the effect of B cell EZH2 deficiency in MRL/lpr lupus-prone mice. Differentiation of B cells was assessed using flow cytometry. Single-cell RNA sequencing and single-cell B cell receptor sequencing were performed. In vitro B cell culture with an X-box binding protein 1 (XBP1) inhibitor was performed. EZH2 and XBP1 messenger RNA levels in CD19+ B cells isolated from lupus patients and healthy controls were analyzed., Results: We show that Ezh2 deletion in B cells significantly decreased autoantibody production and improved glomerulonephritis. B cell development was altered in the bone marrow and spleen of EZH2-deficient mice. Differentiation of germinal center B cells and plasmablasts was impaired. Single-cell RNA sequencing showed that XBP1, a key transcription factor in B cell development, is down-regulated in the absence of EZH2. Inhibiting XBP1 in vitro impairs plasmablast development similar to EZH2 deficiency in mice. Single-cell B cell receptor RNA sequencing revealed defective immunoglobulin class-switch recombination in EZH2-deficient mice. In human lupus B cells, we observed a strong correlation between EZH2 and XBP1 messenger RNA expression levels., Conclusion: EZH2 overexpression in B cells contributes to disease pathogenesis in lupus., (© 2023 American College of Rheumatology.)

Breast cancer alone accounts for the majority of cancer deaths among women, with the most commonly diagnosed subtype being estrogen receptor positive (ER+). Survival has greatly improved for patients with ER+ breast cancer, due in part to the development of antiestrogen compounds, such as tamoxifen. While treatment of the primary disease is often successful, as many as 30% of patients will experience recurrence and metastasis, mainly due to developed endocrine therapy resistance. In this study, we discovered two tamoxifen combination therapies, with simeprevir and VX-680, that reduce the tumor burden in animal models of ER+ breast cancer more than either compound or tamoxifen alone. Additionally, these tamoxifen combinations reduced the expression of HER2, a hallmark of tamoxifen treatment, which can facilitate acquisition of a treatment-resistant phenotype. These combinations could provide clinical benefit by potentiating tamoxifen treatment in ER+ breast cancer.

Motivation: Enrichment analysis is a widely utilized technique in genomic analysis that aims to determine if there is a statistically significant association between two sets of genomic features. To conduct this type of hypothesis testing, an appropriate null model is typically required. However, the null distribution that is commonly used can be overly simplistic and may result in inaccurate conclusions., Results: bootRanges provides fast functions for generation of block bootstrapped genomic ranges representing the null hypothesis in enrichment analysis. As part of a modular workflow, bootRanges offers greater flexibility for computing various test statistics leveraging other Bioconductor packages. We show that shuffling or permutation schemes may result in overly narrow test statistic null distributions and over-estimation of statistical significance, while creating new range sets with a block bootstrap preserves local genomic correlation structure and generates more reliable null distributions. It can also be used in more complex analyses, such as accessing correlations between cis-regulatory elements (CREs) and genes across cell types or providing optimized thresholds, e.g. log fold change (logFC) from differential analysis., Availability and Implementation: bootRanges is freely available in the R/Bioconductor package nullranges hosted at https://bioconductor.org/packages/nullranges., (© The Author(s) 2023. Published by Oxford University Press.)

Motivation: Deriving biological insights from genomic data commonly requires comparing attributes of selected genomic loci to a null set of loci. The selection of this null set is non-trivial, as it requires careful consideration of potential covariates, a problem that is exacerbated by the non-uniform distribution of genomic features including genes, enhancers, and transcription factor binding sites. Propensity score-based covariate matching methods allow the selection of null sets from a pool of possible items while controlling for multiple covariates; however, existing packages do not operate on genomic data classes and can be slow for large data sets making them difficult to integrate into genomic workflows., Results: To address this, we developed matchRanges, a propensity score-based covariate matching method for the efficient and convenient generation of matched null ranges from a set of background ranges within the Bioconductor framework., Availability and Implementation: Package: https://bioconductor.org/packages/nullranges, Code: https://github.com/nullranges, Documentation: https://nullranges.github.io/nullranges., (© The Author(s) 2023. Published by Oxford University Press.)

Summary: Exclusion regions are sections of reference genomes with abnormal pileups of short sequencing reads. Removing reads overlapping them improves biological signal, and these benefits are most pronounced in differential analysis settings. Several labs created exclusion region sets, available primarily through ENCODE and Github. However, the variety of exclusion sets creates uncertainty which sets to use. Furthermore, gap regions (e.g. centromeres, telomeres, short arms) create additional considerations in generating exclusion sets. We generated exclusion sets for the latest human T2T-CHM13 and mouse GRCm39 genomes and systematically assembled and annotated these and other sets in the excluderanges R/Bioconductor data package, also accessible via the BEDbase.org API. The package provides unified access to 82 GenomicRanges objects covering six organisms, multiple genome assemblies, and types of exclusion regions. For human hg38 genome assembly, we recommend hg38.Kundaje.GRCh38_unified_blacklist as the most well-curated and annotated, and sets generated by the Blacklist tool for other organisms., Availability and Implementation: https://bioconductor.org/packages/excluderanges/. Package website: https://dozmorovlab.github.io/excluderanges/., (© The Author(s) 2023. Published by Oxford University Press.)

Changes in the three-dimensional (3D) structure of the genome are an emerging hallmark of cancer. Cancer-associated copy number variants and single nucleotide polymorphisms promote rewiring of chromatin loops, disruption of topologically associating domains (TADs), active/inactive chromatin state switching, leading to oncogene expression and silencing of tumor suppressors. However, little is known about 3D changes during cancer progression to a chemotherapy-resistant state. We integrated chromatin conformation capture (Hi-C), RNA-seq, and whole-genome sequencing obtained from triple-negative breast cancer patient-derived xenograft primary tumors (UCD52) and carboplatin-resistant samples and found increased short-range (< 2 Mb) interactions, chromatin looping, formation of TAD, chromatin state switching into a more active state, and amplification of ATP-binding cassette transporters. Transcriptome changes suggested the role of long-noncoding RNAs in carboplatin resistance. Rewiring of the 3D genome was associated with TP53, TP63, BATF, FOS-JUN family of transcription factors and led to activation of aggressiveness-, metastasis- and other cancer-related pathways. Integrative analysis highlighted increased ribosome biogenesis and oxidative phosphorylation, suggesting the role of mitochondrial energy metabolism. Our results suggest that 3D genome remodeling may be a key mechanism underlying carboplatin resistance., (© 2023. The Author(s).)

Basal-like triple-negative breast cancer (TNBC) tumor cells are difficult to eliminate due to resistance mechanisms that promote survival. While this breast cancer subtype has low PIK3CA mutation rates when compared to estrogen receptor-positive (ER+) breast cancers, most basal-like TNBCs have an overactive PI3K pathway due to gene amplification or high gene expression. BYL-719 is a PIK3CA inhibitor that has been found to have low drug-drug interactions, which increases the likelihood that it could be useful for combinatorial therapy. Alpelisib (BYL-719) with fulvestrant was recently approved for treating ER+ breast cancer patients whose cancer had developed resistance to ER-targeting therapy. In these studies, a set of basal-like patient-derived xenograft (PDX) models was transcriptionally defined with bulk and single-cell RNA-sequencing and clinically actionable mutation profiles defined with Oncomine mutational profiling. This information was overlaid onto therapeutic drug screening results. BYL-719-based, synergistic two-drug combinations were identified with 20 different compounds, including everolimus, afatinib, and dronedarone, which were also found to be effective at minimizing tumor growth. These data support the use of these drug combinations towards cancers with activating PIK3CA mutations/gene amplifications or PTEN deficient/PI3K overactive pathways.

Reciprocal interactions between breast cancer cells and the tumor microenvironment (TME) are important for cancer progression and metastasis. We report here that the deletion or inhibition of sphingosine kinase 2 (SphK2), which produces sphingosine-1-phosphate (S1P), markedly suppresses syngeneic breast tumor growth and lung metastasis in mice by creating a hostile microenvironment for tumor growth and invasion. SphK2 deficiency decreased S1P and concomitantly increased ceramides, including C16-ceramide, in stromal fibroblasts. Ceramide accumulation suppressed activation of cancer-associated fibroblasts (CAF) by upregulating stromal p53, which restrained production of tumor-promoting factors to reprogram the TME and to restrict breast cancer establishment. Ablation of p53 in SphK2-deficient fibroblasts reversed these effects, enabled CAF activation and promoted tumor growth and invasion. These data uncovered a novel role of SphK2 in regulating non-cell-autonomous functions of p53 in stromal fibroblasts and their transition to tumor-promoting CAFs, paving the way for the development of a strategy to target the TME and to enhance therapeutic efficacy., Significance: Sphingosine kinase 2 (SphK2) facilitates the activation of stromal fibroblasts to tumor-promoting cancer-associated fibroblasts by suppressing host p53 activity, revealing SphK2 as a potential target to reprogram the TME., (©2022 American Association for Cancer Research.)

Nonalcoholic fatty liver disease is a major risk factor for hepatocellular carcinoma (HCC). Astrocyte elevated gene-1/Metadherin (AEG-1/MTDH) augments lipid accumulation (steatosis), inflammation, and tumorigenesis, thereby promoting the whole spectrum of this disease process. Targeting AEG-1 is a potential interventional strategy for nonalcoholic steatohepatitis (NASH) and HCC. Thus, proper understanding of the regulation of this molecule is essential. We found that AEG-1 is palmitoylated at residue cysteine 75 (Cys75). Mutation of Cys75 to serine (Ser) completely abolished AEG-1 palmitoylation. We identified ZDHHC6 as a palmitoyltransferase catalyzing the process in HEK293T cells. To obtain insight into how palmitoylation regulates AEG-1 function, we generated knock-in mice by CRISPR/Cas9 in which Cys75 of AEG-1 was mutated to Ser (AEG-1-C75S). No developmental or anatomical abnormality was observed between AEG-1-wild type (AEG-1-WT) and AEG-1-C75S littermates. However, global gene expression analysis by RNA-sequencing unraveled that signaling pathways and upstream regulators, which contribute to cell proliferation, motility, inflammation, angiogenesis, and lipid accumulation, were activated in AEG-1-C75S hepatocytes compared to AEG-1-WT. These findings suggest that AEG-1-C75S functions as dominant positive and that palmitoylation restricts oncogenic and NASH-promoting functions of AEG-1. We thus identify a previously unknown regulatory mechanism of AEG-1, which might help design new therapeutic strategies for NASH and HCC.

The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor, classically associated with the regulation of xenobiotic metabolism in response to environmental toxins. In recent years, transgenic rodent models have implicated AhR in aging and longevity. Moreover, several AhR ligands, such as resveratrol and quercetin, are compounds proven to extend the lifespan of model organisms. In this paper, we first review AhR biology with a focus on aging and highlight several AhR ligands with potential anti-aging properties. We outline how AhR-driven expression of xenobiotic metabolism genes into old age may be a key mechanism through which moderate induction of AhR elicits positive benefits on longevity and healthspan. Furthermore, via integration of publicly available datasets, we show that liver-specific AhR target genes are enriched among genes subject to epigenetic aging. Changes to epigenetic states can profoundly affect transcription factor binding and are a hallmark of the aging process. We suggest that the interplay between AhR and epigenetic aging should be the subject of future research and outline several key gaps in the current literature. Finally, we recommend that a broad range of non-toxic AhR ligands should be investigated for their potential to promote healthspan and longevity., Competing Interests: We report no potential conflict of interest.

Summary: CTCF (CCCTC-binding factor) is an 11-zinc-finger DNA binding protein which regulates much of the eukaryotic genome's 3D structure and function. The diversity of CTCF binding motifs has led to a fragmented landscape of CTCF binding data. We collected position weight matrices of CTCF binding motifs and defined strand-oriented CTCF binding sites in the human and mouse genomes, including the recent Telomere to Telomere and mm39 assemblies. We included selected experimentally determined and predicted CTCF binding sites, such as CTCF-bound cis-regulatory elements from SCREEN ENCODE. We recommend filtering strategies for CTCF binding motifs and demonstrate that liftOver is a viable alternative to convert CTCF coordinates between assemblies. Our comprehensive data resource and usage recommendations can serve to harmonize and strengthen the reproducibility of genomic studies utilizing CTCF binding data., Availability and Implementation: https://bioconductor.org/packages/CTCF. Companion website: https://dozmorovlab.github.io/CTCF/; Code to reproduce the analyses: https://github.com/dozmorovlab/CTCF.dev., Supplementary Information: Supplementary data are available at Bioinformatics Advances online., (© The Author(s) 2022. Published by Oxford University Press.)

The goals of this study were to identify transcriptomic changes that arise in basal-like breast cancer cells during the development of resistance to epidermal growth factor receptor inhibitors (EGFRi) and to identify drugs that are cytotoxic once EGFRi resistance occurs. Human patient-derived xenografts (PDXs) were grown in immunodeficient mice and treated with a set of EGFRi; the EGFRi erlotinib was selected for more expansive in vivo studies. Single-cell RNA sequencing was performed on mammary tumors from the basal-like PDX WHIM2 that was treated with vehicle or erlotinib for 9 weeks. The PDX was then subjected to long-term erlotinib treatment in vivo. Through serial passaging, an erlotinib-resistant subline of WHIM2 was generated. Bulk RNA-sequencing was performed on parental and erlotinib-resistant tumors. In vitro high-throughput drug screening with > 500 clinically used compounds was performed on parental and erlotinib-resistant cells. Previously published bulk gene expression microarray data from MMTV-Wnt1 tumors were contrasted with the WHIM2 PDX data. Erlotinib effectively inhibited WHIM2 tumor growth for approximately 4 weeks. Compared to untreated cells, single-cell RNA sequencing revealed that a greater proportion of erlotinib-treated cells were in the G1 phase of the cell cycle. Comparison of WHIM2 and MMTV-Wnt1 gene expression data revealed a set of 38 overlapping genes that were differentially expressed in the erlotinib-resistant WHIM2 and MMTV-Wnt1 tumors. Comparison of all three data types revealed five genes that were upregulated across all erlotinib-resistant samples: IL19, KLK7, LCN2, SAA1, and SAA2. Of these five genes, LCN2 was most abundantly expressed in triple-negative breast cancers, and its knockdown restored erlotinib sensitivity in vitro. Despite transcriptomic differences, parental and erlotinib-resistant WHIM2 displayed similar responses to the majority of drugs assessed for cytotoxicity in vitro. This study identified transcriptomic changes arising in erlotinib-resistant basal-like breast cancer. These data could be used to identify a biomarker or develop a gene signature predictive of patient response to EGFRi. Future studies should explore the predictive capacity of these gene signatures as well as how LCN2 contributes to the development of EGFRi resistance., (© 2022. The Author(s).)

Triple negative breast cancer (TNBC) accounts for over 30% of all breast cancer (BC)-related deaths, despite accounting for only 10% to 15% of total BC cases. Targeted therapy development has largely stalled in TNBC, underlined by a lack of traditionally druggable addictions like receptor tyrosine kinases (RTKs). Here, through full genome CRISPR/Cas9 screening of TNBC models, we have uncovered the sensitivity of TNBCs to the depletion of the ubiquitin-like modifier activating enzyme 1 (UBA1). Targeting UBA1 with the first-in-class UBA1 inhibitor TAK-243 induced unresolvable endoplasmic reticulum (ER)-stress and activating transcription factor 4 (ATF4)-mediated upregulation of proapoptotic NOXA, leading to cell death. c-MYC expression correlates with TAK-243 sensitivity and cooperates with TAK-243 to induce a stress response and cell death. Importantly, there was an order of magnitude greater sensitivity of TNBC lines to TAK-243 compared to normal tissue-derived cells. In five patient derived xenograft models (PDXs) of TNBC, TAK-243 therapy led to tumor inhibition or frank tumor regression. Moreover, in an intracardiac metastatic model of TNBC, TAK-243 markedly reduced metastatic burden, indicating UBA1 is a potential new target in TNBC expressing high levels of c-MYC., (© The Author(s) 2022. Published by Oxford University Press on behalf of National Academy of Sciences.)

Psychedelics, also known as classical hallucinogens, affect processes related to perception, cognition and sensory processing mostly via the serotonin 5-HT 2A receptor (5-HT 2A R). This class of psychoactive substances, which includes lysergic acid diethylamide (LSD), psilocybin, mescaline and the substituted amphetamine 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), is receiving renewed attention for their potential therapeutic properties as it relates to psychiatric conditions such as depression and substance use disorders. Current studies focused on the potentially clinical effects of psychedelics on human subjects tend to exclude sex as a biological variable. Much of the understanding of psychedelic pharmacology is derived from rodent models, but most of this preclinical research has only focused on male mice. Here we tested the effects of DOI on head-twitch behavior (HTR) - a mouse behavioral proxy of human psychedelic potential - in male and female mice. DOI elicited more HTR in female as compared to male C57BL/6J mice, a sex-specific exacerbated behavior that was not observed in 129S6/SvEv animals. Volinanserin (or M100907) - a 5-HT 2A R antagonist - fully prevented DOI-induced HTR in male and female C57BL/6J mice. Accumulation of inositol monophosphate (IP 1 ) in the frontal cortex upon DOI administration showed no sex-related effect in C57BL/6J mice. However, the pharmacokinetic properties of DOI differed among sexes - brain and plasma concentrations of DOI were lower 30 and 60 min after drug administration in female as compared to male C57BL/6J mice. Together, these results suggest strain-dependent and sex-related differences in the behavioral and pharmacokinetic profiles of the 5-HT 2A R agonist DOI in C57BL/6J mice, and support the importance of studying sex as a biological variable in preclinical psychedelic research., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Preclinical and clinical studies have evidenced that effective targeted therapy treatment against receptor tyrosine kinases (RTKs) in different solid tumor paradigms is predicated on simultaneous inhibition of both the PI3K and MEK intracellular signaling pathways. Indeed, re-activation of either pathway results in resistance to these therapies. Recently, oncogenic phosphatase SHP2 inhibitors have been developed with some now reaching clinical trials. To expand on possible indications for SHP099, we screened over 800 cancer cell lines covering over 25 subsets of cancer. We found HNSCC was the most sensitive adult subtype of cancer to SHP099. We found that, in addition to the MEK pathway, SHP2 inhibition blocks the PI3K pathway in sensitive HNSCC, resulting in downregulation of mTORC signaling and anti-tumor effects across several HNSCC mouse models, including an HPV+ patient-derived xenograft (PDX). Importantly, we found low levels of the RTK ligand epiregulin identified HNSCCs that were sensitive to SHP2 inhibitor, and, adding exogenous epiregulin mitigated SHP099 efficacy. Mechanistically, epiregulin maintained SHP2-GAB1 complexes in the presence of SHP2 inhibition, preventing downregulation of the MEK and PI3K pathways. We demonstrate HNSCCs were highly dependent on GAB1 for their survival and knockdown of GAB1 is sufficient to block the ability of epiregulin to rescue MEK and PI3K signaling. These data connect the sensitivity of HNSCC to SHP2 inhibitors and to a broad reliance on GAB1-SHP2, revealing an important and druggable signaling axis. Overall, SHP2 inhibitors are being heavily developed and may have activity in HNSCCs, and in particular those with low levels of epiregulin., Competing Interests: Conflict of Interest Disclosure: The work in the laboratory of C.H.B. is funded in part by Amgen and Novartis. A.C.F. has served as a scientific advisor for AbbVie and currently serves as a scientific advisor for Treeline Biosciences.

Obesity is an enormous global health problem, and obesity-induced nonalcoholic steatohepatitis (NASH) is contributing to a rising incidence and mortality for hepatocellular carcinoma (HCC). Increase in de novo lipogenesis and decrease in fatty acid β-oxidation (FAO) underlie hepatic lipid accumulation in NASH. Astrocyte-elevated gene-1/metadherin (AEG-1) overexpression contributes to both NASH and HCC. AEG-1 harbors an LXXLL motif through which it blocks activation of peroxisome proliferator activated receptor α (PPARα), a key regulator of FAO. To better understand the role of LXXLL motif in mediating AEG-1 function, using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology, we generated a mouse model (AEG-1-L24K/L25H) in which the LXXLL motif in AEG-1 was mutated to LXXKH. We observed increased activation of PPARα in AEG-1-L24K/L25H livers providing partial protection from high-fat diet-induced steatosis. Interestingly, even with equal gene dosage levels, compared with AEG-1-wild-type livers, AEG-1-L24K/L25H livers exhibited increase in levels of lipogenic enzymes, mitogenic activity and inflammation, which are attributes observed when AEG-1 is overexpressed. These findings indicate that while LXXLL motif favors steatotic activity of AEG-1, it keeps in check inflammatory and oncogenic functions, thus maintaining a homeostasis in AEG-1 function. AEG-1 is being increasingly appreciated as a viable target for ameliorating NASH and NASH-HCC, and as such, in-depth understanding of the functions and molecular attributes of this molecule is essential. Conclusion: The present study unravels the unique role of the LXXLL motif in mediating the balance between the metabolic and oncogenic functions of AEG-1., (© 2021 The Authors. Hepatology Communications published by Wiley Periodicals LLC on behalf of American Association for the Study of Liver Diseases.)

Triple-negative breast cancer (TNBC) is a subtype of breast cancer lacking targetable biomarkers. TNBC is known to be most aggressive and when metastatic is often drug-resistant and uncurable. Biomarkers predicting response to therapy improve treatment decisions and allow personalized approaches for patients with TNBC. This study explores sulfated glycosaminoglycan (sGAG) levels as a predictor of TNBC response to platinum therapy. sGAG levels were quantified in three distinct TNBC tumor models, including cell line-derived, patient-derived xenograft (PDX) tumors, and isogenic models deficient in sGAG biosynthesis. The in vivo antitumor efficacy of Triplatin, a sGAG-directed platinum agent, was compared in these models with the clinical platinum agent, carboplatin. We determined that >40% of TNBC PDX tissue microarray samples have high levels of sGAGs. The in vivo accumulation of Triplatin in tumors as well as antitumor efficacy of Triplatin positively correlated with sGAG levels on tumor cells, whereas carboplatin followed the opposite trend. In carboplatin-resistant tumor models expressing high levels of sGAGs, Triplatin decreased primary tumor growth, reduced lung metastases, and inhibited metastatic growth in lungs, liver, and ovaries. sGAG levels served as a predictor of Triplatin sensitivity in TNBC. Triplatin may be particularly beneficial in treating patients with chemotherapy-resistant tumors who have evidence of residual disease after standard neoadjuvant chemotherapy. More effective neoadjuvant and adjuvant treatment will likely improve clinical outcome of TNBC., (©2021 American Association for Cancer Research.)

Ferroptosis is an iron-dependent, oxidative form of cell death that is countered mainly by glutathione peroxidase 4 (GPX4) and the production of glutathione (GSH), which is formed from cysteine. The identification of the cancers that may benefit from pharmacological ferroptotic induction is just emerging. We recently demonstrated that inducing ferroptosis genetically or pharmacologically in MYCN -amplified neuroblastoma (NB) is a novel and effective way to kill these cells. MYCN increases iron metabolism and subsequent hydroxyl radicals through increased expression of the transferrin receptor 1 (TfR1) and low levels of the ferroportin receptor. To counter increased hydroxyl radicals, MYCN binds to the promoter of SLC3A2 (solute carrier family 3 member 2). SLC3A2 is a subunit of system Xc-, which is the cysteine-glutamate antiporter that exports glutamate and imports cystine. Cystine is converted to cysteine intracellularly. Here, we investigated other ways MYCN may increase cysteine levels. By performing metabolomics in a syngeneic NB cell line either expressing MYCN or GFP, we demonstrate that the transsulfuration pathway is activated by MYCN. Furthermore, we demonstrate that MYCN -amplified NB cell lines and tumors have higher levels of cystathionine beta-synthase (CBS), the rate-limiting enzyme in transsulfuration, which leads to higher levels of the thioether cystathionine ( R-S -(2-amino-2-carboxyethyl)-l-homocysteine). In addition, MYCN -amplified NB tumors have high levels of methylthioadenosine phosphorylase (MTAP), an enzyme that helps salvage methionine following polyamine metabolism. MYCN directly binds to the promoter of MTAP . We propose that MYCN orchestrates both enhanced cystine uptake and enhanced activity of the transsulfuration pathway to counteract increased reactive oxygen species (ROS) from iron-induced Fenton reactions, ultimately contributing to a ferroptosis vulnerability in MYCN -amplified neuroblastoma., Competing Interests: Conflict of Interest: A.C.F. has served as a paid consultant for AbbVie., (Copyright: © 2022 Floros et al.)

Motivation: Chromosome conformation capture technologies (Hi-C) revealed extensive DNA folding into discrete 3D domains, such as Topologically Associating Domains and chromatin loops. The correct binding of CTCF and cohesin at domain boundaries is integral in maintaining the proper structure and function of these 3D domains. 3D domains have been mapped at the resolutions of 1 kilobase and above. However, it has not been possible to define their boundaries at the resolution of boundary-forming proteins., Results: To predict domain boundaries at base-pair resolution, we developed preciseTAD, an optimized transfer learning framework trained on high-resolution genome annotation data. In contrast to current TAD/loop callers, preciseTAD-predicted boundaries are strongly supported by experimental evidence. Importantly, this approach can accurately delineate boundaries in cells without Hi-C data. preciseTAD provides a powerful framework to improve our understanding of how genomic regulators are shaping the 3D structure of the genome at base-pair resolution., Availability and Implementation: preciseTAD is an R/Bioconductor package available at https://bioconductor.org/packages/preciseTAD/., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2021. Published by Oxford University Press.)

Non-alcoholic steatohepatitis (NASH) is a major cause of chronic liver disease that can ultimately lead to cirrhosis and hepatocellular carcinoma. Although NASH is associated with excessive liver lipid accumulation, hepatocyte injury, inflammation, and fibrosis, its etiology remains incompletely understood. These can be characterized by determining transcriptional changes in specific genes previously found to be involved in these processes. As an inherently multifaceted disease, studies of NASH often require unbiased examination of major genes and pathways to identify the mechanisms involved in this disorder. To address this need, quantitative approaches such as mRNA-sequencing have been developed for the global assessment of gene expression. Here, we describe a protocol for bulk mRNA-sequencing that can be utilized for both liver samples and specific cell types isolated from the liver. This approach provides an important resource to further understand the molecular changes that occur during the development of NASH that can be utilized to design better therapeutic treatments., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Objectives: Phase II drug metabolism is poorly studied in advanced age and older adults may exhibit significant variability in their expression of phase II enzymes. We hypothesized that age-related changes to epigenetic regulation of genes involved in phase II drug metabolism may contribute to these effects., Methods: We examined published epigenome-wide studies of human blood and identified the SULT1A1 and UGT1A6 genes as the top loci showing epigenetic changes with age. To assess possible functional alterations with age in the liver, we assayed DNA methylation (5mC) and histone acetylation changes around the mouse homologs Sult1a1 and Ugt1a6 in liver tissue from mice aged 4-32 months., Results: Our sample shows a significant loss of 5mC at Sult1a1 (β = -1.08, 95% CI [-1.8, -0.2], SE = 0.38, P = 0.011), mirroring the loss of 5mC with age observed in human blood DNA at the same locus. We also detected increased histone 3 lysine 9 acetylation (H3K9ac) with age at Sult1a1 (β = 0.11, 95% CI [0.002, 0.22], SE = 0.05, P = 0.04), but no change to histone 3 lysine 27 acetylation (H3K27ac). Sult1a1 gene expression is significantly positively associated with H3K9ac levels, accounting for 23% of the variation in expression. We did not detect any significant effects at Ugt1a6., Conclusions: Sult1a1 expression is under epigenetic influence in normal aging and this influence is more pronounced for H3K9ac than DNA methylation or H3K27ac in this study. More generally, our findings support the relevance of epigenetics in regulating key drug-metabolizing pathways. In the future, epigenetic biomarkers could prove useful to inform dosing in older adults., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

An estimated 284,000 Americans will be diagnosed with breast cancer in 2021. Of these individuals, 15-20% have basal-like triple-negative breast cancer (TNBC), which is known to be highly metastatic. Chemotherapy is standard of care for TNBC patients, but chemoresistance is a common clinical problem. There is currently a lack of alternative, targeted treatment strategies for TNBC; this study sought to identify novel therapeutic combinations to treat basal-like TNBCs. For these studies, four human basal-like TNBC cell lines were utilized to determine the cytotoxicity profile of 1363 clinically-used drugs. Ten promising therapeutic candidates were identified, and synergism studies were performed in vitro. Two drug combinations that included KPT-330, an XPO1 inhibitor, were synergistic in all four cell lines. In vivo testing of four basal-like patient-derived xenografts (PDX) identified one combination, KPT-330 and GSK2126458 (a PI3K/mTOR inhibitor), that decreased tumor burden in mice significantly more than monotherapy with either single agent. Bulk and single-cell RNA-sequencing, immunohistochemistry, and analysis of published genomic datasets found that XPO1 was abundantly expressed in human basal-like TNBC cell lines, PDXs, and patient tumor samples. Within basal-like PDXs, XPO1 overexpression was associated with increased proliferation at the cellular level. Within patient datasets, XPO1 overexpression was correlated with greater rates of metastasis in patients with basal-like tumors. These studies identify a promising potential new combination therapy for patients with basal-like breast cancer., (Copyright © 2021. Published by Elsevier Inc.)

The EWSR1-FLI1 t(11;22)(q24;q12) translocation is the hallmark genomic alteration of Ewing sarcoma, a malignancy of the bone and surrounding tissue, predominantly affecting children and adolescents. Although significant progress has been made for the treatment of localized disease, patients with metastasis or who relapse after chemotherapy have less than a 30% five-year survival rate. EWS-FLI1 is currently not clinically druggable, driving the need for more effective targeted therapies. Treatment with the H3K27 demethylase inhibitor, GSK-J4, leads to an increase in H3K27me and a decrease in H3K27ac, a significant event in Ewing sarcoma because H3K27ac associates strongly with EWS-FLI1 binding at enhancers and promoters and subsequent activity of EWS-FLI1 target genes. We were able to identify targets of EWS-FLI1 tumorigenesis directly inhibited by GSK-J4. GSK-J4 disruption of EWS-FLI1-driven transcription was toxic to Ewing sarcoma cells and slowed tumor growth in patient-derived xenografts (PDX) of Ewing sarcoma. Responses were markedly exacerbated by cotreatment with a disruptor of RNA polymerase II activity, the CDK7 inhibitor THZ1. This combination together suppressed EWS-FLI1 target genes and viability of ex vivo PDX Ewing sarcoma cells in a synergistic manner. In PDX models of Ewing Sarcoma, the combination shrank tumors. We present a new therapeutic strategy to treat Ewing sarcoma by decreasing H3K27ac at EWS-FLI1 - driven transcripts, exacerbated by blocking phosphorylation of the C-terminal domain of RNA polymerase II to further hinder the EWS-FLI1-driven transcriptome., (©2021 American Association for Cancer Research.)

Synovial sarcoma (SS) is frequently diagnosed in teenagers and young adults and continues to be treated with polychemotherapy with variable success. The SS18-SSX gene fusion is pathognomonic for the disease, and high expression of the anti-apoptotic BCL-2 pathologically supports the diagnosis. As the oncogenic SS18-SSX fusion gene itself is not druggable, BCL-2 inhibitor-based therapies are an appealing therapeutic opportunity. Venetoclax, an FDA-approved BCL-2 inhibitor that is revolutionizing care in some BCL-2-expressing hematological cancers, affords an intriguing therapeutic possibility to treat SS. In addition, there are now dozens of venetoclax-based combination therapies in clinical trials in hematological cancers, attributing to the limited toxicity of venetoclax. However, preclinical studies of venetoclax in SS have demonstrated an unexpected ineffectiveness. In this study, we analyzed the response of SS to venetoclax and the underlying BCL-2 family biology in an effort to understand venetoclax treatment failure and find a therapeutic strategy to sensitize SS to venetoclax. We found remarkably depressed levels of the endogenous MCL-1 inhibitor, NOXA, in SS compared to other sarcomas. Expressing NOXA led to sensitization to venetoclax, as did the addition of the MCL-1 BH3 mimetic, S63845. Importantly, the venetoclax/S63845 combination induced tumor regressions in SS patient-derived xenograft (PDX) models. As a very close analog of S63845 (S64315) is now in clinical trials with venetoclax in AML (NCT03672695), the combination of MCL-1 BH3 mimetics and venetoclax should be considered for SS patients as a new therapy.

Background: Maternal smoking during pregnancy is related to altered DNA methylation in infant umbilical cord blood. The extent to which low levels of smoke exposure among nonsmoking pregnant women relates to offspring DNA methylation is unknown., Objective: This study sought to evaluate relationships between maternal prenatal plasma cotinine levels and DNA methylation in umbilical cord blood in newborns using the Infinium HumanMethylation 450K BeadChip., Methods: Participants from the Newborn Epigenetics Study cohort who reported not smoking during pregnancy had verified low levels of cotinine from maternal prenatal plasma (0 ng / mL to < 4  ng / mL ), and offspring epigenetic data from umbilical cord blood were included in this study ( n = 79 ). Multivariable linear regression models were fit to the data, controlling for cell proportions, age, race, education, and parity. Estimates represent changes in response to any 1 -ng / mL unit increase in exposure., Results: Multivariable linear regression models yielded 29,049 CpGs that were differentially methylated in relation to increases in cotinine at a 5% false discovery rate. Top CpGs were within or near genes involved in neuronal functioning ( PRKG1 , DLGAP2 , BSG ), carcinogenesis ( FHIT , HSPC157 ) and inflammation ( AGER ). Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses suggest cotinine was related to methylation of gene pathways controlling neuronal signaling, metabolic regulation, cell signaling and regulation, and cancer. Further, enhancers associated with transcription start sites were enriched in altered CpGs. Using an independent sample from the same study population ( n = 115 ), bisulfite pyrosequencing was performed with infant cord blood DNA for two genes within our top 20 hits ( AGER and PRKG1 ). Results from pyrosequencing replicated epigenome results for PRKG1 (cg17079497, estimate = - 1.09 , standard error  ( SE ) = 0.45 , p = 0.018 ) but not for AGER (cg09199225; estimate = - 0.16 , SE = 0.21 , p = 0.44 )., Discussion: Secondhand smoke exposure among nonsmoking women may alter DNA methylation in regions involved in development, carcinogenesis, and neuronal functioning. These novel findings suggest that even low levels of smoke exposure during pregnancy may be sufficient to alter DNA methylation in distinct sites of mixed umbilical cord blood leukocytes in pathways that are known to be altered in cord blood from pregnant active smokers. https://doi.org/10.1289/EHP8099.

Background: Sequencing of patient-derived xenograft (PDX) mouse models allows investigation of the molecular mechanisms of human tumor samples engrafted in a mouse host. Thus, both human and mouse genetic material is sequenced. Several methods have been developed to remove mouse sequencing reads from RNA-seq or exome sequencing PDX data and improve the downstream signal. However, for more recent chromatin conformation capture technologies (Hi-C), the effect of mouse reads remains undefined., Results: We evaluated the effect of mouse read removal on the quality of Hi-C data using in silico created PDX Hi-C data with 10% and 30% mouse reads. Additionally, we generated 2 experimental PDX Hi-C datasets using different library preparation strategies. We evaluated 3 alignment strategies (Direct, Xenome, Combined) and 3 pipelines (Juicer, HiC-Pro, HiCExplorer) on Hi-C data quality., Conclusions: Removal of mouse reads had little-to-no effect on data quality as compared with the results obtained with the Direct alignment strategy. Juicer extracted more valid chromatin interactions for Hi-C matrices, regardless of the mouse read removal strategy. However, the pipeline effect was minimal, while the library preparation strategy had the largest effect on all quality metrics. Together, our study presents comprehensive guidelines on PDX Hi-C data processing., (© The Author(s) 2021. Published by Oxford University Press on behalf of GigaScience.)

MYCN is amplified in 20% to 25% of neuroblastoma, and MYCN -amplified neuroblastoma contributes to a large percent of pediatric cancer-related deaths. Therapy improvements for this subtype of cancer are a high priority. Here we uncover a MYCN-dependent therapeutic vulnerability in neuroblastoma. Namely, amplified MYCN rewires the cell through expression of key receptors, ultimately enhancing iron influx through increased expression of the iron import transferrin receptor 1. Accumulating iron causes reactive oxygen species (ROS) production, and MYCN -amplified neuroblastomas show enhanced reliance on the system Xc- cystine/glutamate antiporter for ROS detoxification through increased transcription of this receptor. This dependence creates a marked vulnerability to targeting the system Xc-/glutathione (GSH) pathway with ferroptosis inducers. This reliance can be exploited through therapy with FDA-approved rheumatoid arthritis drugs sulfasalazine (SAS) and auranofin: in MYCN -amplified, patient-derived xenograft models, both therapies blocked growth and induced ferroptosis. SAS and auranofin activity was largely mitigated by the ferroptosis inhibitor ferrostatin-1, antioxidants like N-acetyl-L-cysteine, or by the iron scavenger deferoxamine (DFO). DFO reduced auranofin-induced ROS, further linking increased iron capture in MYCN -amplified neuroblastoma to a therapeutic vulnerability to ROS-inducing drugs. These data uncover an oncogene vulnerability to ferroptosis caused by increased iron accumulation and subsequent reliance on the system Xc-/GSH pathway. SIGNIFICANCE: This study shows how MYCN increases intracellular iron levels and subsequent GSH pathway activity and demonstrates the antitumor activity of FDA-approved SAS and auranofin in patient-derived xenograft models of MYCN -amplified neuroblastoma., (©2021 American Association for Cancer Research.)

Adolescent exposure to chronic stress, a risk factor for mood disorders in adulthood, sensitizes the neuroinflammatory response to a subsequent immune challenge. We previously showed that chronic adolescent stress (CAS) in rats led to distinct patterns of neuroimmune priming in adult male and female rats. However, sex differences in the neuroimmune consequences of CAS and their underlying mechanisms are not fully understood. Here we hypothesized that biological sex would dictate differential induction of inflammation-related transcriptomic pathways and immune cell involvement (microglia activation and leukocyte presence) in the hippocampus of male and female rats with a history of CAS. Adolescent rats underwent CAS (six restraint and six social defeat episodes during postnatal days 38-49), and behavioral assessments were conducted in adolescence and adulthood. Neuroimmune measures were obtained following vehicle or a systemic lipopolysaccharide (LPS) challenge in adulthood. CAS led to increased time in the corners of the open field in adolescence. In males, CAS also increased social avoidance. As adults, CAS rats displayed an exaggerated enrichment of the nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) pathway and chemokine induction following LPS challenge, and increased number of perivascular CD45 + cells in the hippocampus. However, CAS females, but not males, showed exaggerated glucocorticoid receptor (GR) pathway enrichment and increased microglial complexity. These results provide further insight to the mechanisms by which peripheral immune events may influence neuroimmune responses differentially among males and females and further demonstrate the importance of adolescent stress in shaping adult responses.

Cell cycle control drives cancer progression and treatment response in high grade serous ovarian carcinoma (HGSOC). MYBL2 (encoding B-Myb), an oncogene with prognostic significance in several cancers, is highly expressed in most HGSOC cases; however, the clinical significance of B-Myb in this disease has not been well-characterized. B-Myb is associated with cell proliferation through formation of the MMB (Myb and MuvB core) protein complex required for transcription of mitotic genes. High B-Myb expression disrupts the formation of another transcriptional cell cycle regulatory complex involving the MuvB core, DREAM (DP, RB-like, E2F, and MuvB), in human cell lines. DREAM coordinates cell cycle dependent gene expression by repressing over 800 cell cycle genes in G0/G1. Here, we take a bioinformatics approach to further evaluate the effect of B-Myb expression on DREAM target genes in HGSOC and validate our cellular model with clinical specimens. We show that MYBL2 is highly expressed in HGSOC and correlates with expression of DREAM and MMB target genes in both The Cancer Genome Atlas (TCGA) as well as independent analyses of HGSOC primary tumors ( N = 52). High B-Myb expression was also associated with poor overall survival in the TCGA cohort and analysis by a DREAM target gene expression signature yielded a negative impact on survival. Together, our data support the conclusion that high expression of MYBL2 is associated with deregulation of DREAM/MMB-mediated cell cycle gene expression programs in HGSOC and may serve as a prognostic factor independent of its cell cycle role. This provides rationale for further, larger scale studies aimed to determine the clinical predictive value of the B-Myb gene expression signature for treatment response as well as patient outcomes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Iness, Rubinsak, Meas, Chaoul, Sayeed, Pillappa, Temkin, Dozmorov and Litovchick.)

Human epidermal growth factor receptor 2 gene (HER2) is focally amplified in approximately 20% of breast cancers. HER2 inhibitors alone are not effective, and sensitizing agents will be necessary to move away from a reliance on heavily toxic chemotherapeutics. We recently demonstrated that the efficacy of HER2 inhibitors is mitigated by uniformly low levels of the myeloid cell leukemia 1 (MCL-1) endogenous inhibitor, NOXA. Emerging clinical data have demonstrated that clinically advanced cyclin-dependent kinase (CDK) inhibitors are effective MCL-1 inhibitors in patients, and, importantly, well tolerated. We, therefore, tested whether the CDK inhibitor, dinaciclib, could block MCL-1 in preclinical HER2-amplified breast cancer models and therefore sensitize these cancers to dual HER2/EGFR inhibitors neratinib and lapatinib, as well as to the novel selective HER2 inhibitor tucatinib. Indeed, we found dinaciclib suppresses MCL-1 RNA and is highly effective at sensitizing HER2 inhibitors both in vitro and in vivo. This combination was tolerable in vivo. Mechanistically, liberating the effector BCL-2 protein, BAK, from MCL-1 results in robust apoptosis. Thus, clinically advanced CDK inhibitors may effectively combine with HER2 inhibitors and present a chemotherapy-free therapeutic strategy in HER2-amplified breast cancer, which can be tested immediately in the clinic.

High-risk (HR) human papillomaviruses are known causative agents in 5% of human cancers including cervical, ano-genital and head and neck carcinomas. In part, HR-HPV causes cancer by targeting host-cell tumor suppressors including retinoblastoma protein (pRb) and RB-like proteins p107 and p130. HR-HPV E7 uses a LxCxE motif to bind RB proteins, impairing their ability to control cell-cycle dependent transcription. E7 disrupts DREAM (Dimerization partner, RB-like, E2F and MuvB), a transcriptional repressor complex that can include p130 or p107, but not pRb, which regulates genes required for cell cycle progression. However, it is not known whether disruption of DREAM plays a significant role in HPV-driven tumorigenesis. In the DREAM complex, LIN52 is an adaptor that binds directly to p130 via an E7-like LxSxE motif. Replacement of the LxSxE sequence in LIN52 with LxCxE (LIN52-S20C) increases p130 binding and partially restores DREAM assembly in HPV-positive keratinocytes and human cervical cancer cells, inhibiting proliferation. Our findings demonstrate that disruption of the DREAM complex by E7 is an important process promoting cellular proliferation by HR-HPV. Restoration of the DREAM complex in HR-HPV positive cells may therefore have therapeutic benefits in HR-HPV positive cancers.