Detection of metabolic adaptation in a triple-negative breast cancer animal model with [18F]choline-PET imaging as a surrogate for drug resistance.

PURPOSE: Test the feasibility of an image-based method to identify taxane resistance in mouse bearing triple-negative breast cancer (TNBC) tumor xenografts. METHODS: Xenograft tumor-bearing mice from paclitaxel-sensitive and paclitaxel-resistant TNBC cells (MDA-MD-346) were generated by orthotopic injection into female NOD-SCID mice. When tumors reached 100-150 mm3, mice were scanned using [18F]choline PET/CT. Tumors were collected and sliced for autoradiography and immunofluorescence analysis. Quantitative data was analyzed accordingly. RESULTS: From fifteen mice scanned, five had taxane-sensitive cell line tumors of which two underwent taxol-based treatment. From the remaining 10 mice with taxane-resistant cell line tumors, four underwent taxol-based treatment. Only 13 mice had the tumor sample analyzed histologically. When normalized to the blood pool, both cell lines showed differences in metabolic uptake before and after treatment. CONCLUSIONS: Treated and untreated taxane-sensitive and taxane-resistant cell lines have different metabolic properties that could be leveraged before the start of chemotherapy.

Chromatin insulation orchestrates matrix metalloproteinase gene cluster expression reprogramming in aggressive breast cancer tumors.

BACKGROUND: Triple-negative breast cancer (TNBC) is an aggressive subtype that exhibits a high incidence of distant metastases and lacks targeted therapeutic options. Here we explored how the epigenome contributes to matrix metalloprotease (MMP) dysregulation impacting tumor invasion, which is the first step of the metastatic process. METHODS: We combined RNA expression and chromatin interaction data to identify insulator elements potentially associated with MMP gene expression and invasion. We employed CRISPR/Cas9 to disrupt the CCCTC-Binding Factor (CTCF) binding site on an insulator element downstream of the MMP8 gene (IE8) in two TNBC cellular models. We characterized these models by combining Hi-C, ATAC-seq, and RNA-seq with functional experiments to determine invasive ability. The potential of our findings to predict the progression of ductal carcinoma in situ (DCIS), was tested in data from clinical specimens. RESULTS: We explored the clinical relevance of an insulator element located within the Chr11q22.2 locus, downstream of the MMP8 gene (IE8). This regulatory element resulted in a topologically associating domain (TAD) boundary that isolated nine MMP genes into two anti-correlated expression clusters. This expression pattern was associated with worse relapse-free (HR = 1.57 [1.06 - 2.33]; p = 0.023) and overall (HR = 2.65 [1.31 - 5.37], p = 0.005) survival of TNBC patients. After CRISPR/Cas9-mediated disruption of IE8, cancer cells showed a switch in the MMP expression signature, specifically downregulating the pro-invasive MMP1 gene and upregulating the antitumorigenic MMP8 gene, resulting in reduced invasive ability and collagen degradation. We observed that the MMP expression pattern predicts DCIS that eventually progresses into invasive ductal carcinomas (AUC = 0.77, p < 0.01). CONCLUSION: Our study demonstrates how the activation of an IE near the MMP8 gene determines the regional transcriptional regulation of MMP genes with opposing functional activity, ultimately influencing the invasive properties of aggressive forms of breast cancer.

3-D chromatin conformation, accessibility, and gene expression profiling of triple-negative breast cancer.

OBJECTIVES: Triple-negative breast cancer (TNBC) is a highly aggressive breast cancer subtype with limited treatment options. Unlike other breast cancer subtypes, the scarcity of specific therapies and greater frequencies of distant metastases contribute to its aggressiveness. We aimed to find epigenetic changes that aid in the understanding of the dissemination process of these cancers. DATA DESCRIPTION: Using CRISPR/Cas9, our experimental approach led us to identify and disrupt an insulator element, IE8, whose activity seemed relevant for cell invasion. The experiments were performed in two well-established TNBC cellular models, the MDA-MB-231 and the MDA-MB-436. To gain insights into the underlying molecular mechanisms of TNBC invasion ability, we generated and characterized high-resolution chromatin interaction (Hi-C) and chromatin accessibility (ATAC-seq) maps in both cell models and complemented these datasets with gene expression profiling (RNA-seq) in MDA-MB-231, the cell line that showed more significant changes in chromatin accessibility. Altogether, our data provide a comprehensive resource for understanding the spatial organization of the genome in TNBC cells, which may contribute to accelerating the discovery of TNBC-specific alterations triggering advances for this devastating disease.

Transposable Elements Are Co-opted as Oncogenic Regulatory Elements by Lineage-Specific Transcription Factors in Prostate Cancer.

Transposable elements hold regulatory functions that impact cell fate determination by controlling gene expression. However, little is known about the transcriptional machinery engaged at transposable elements in pluripotent and mature versus oncogenic cell states. Through positional analysis over repetitive DNA sequences of H3K27ac chromatin immunoprecipitation sequencing data from 32 normal cell states, we report pluripotent/stem and mature cell state-specific "regulatory transposable elements." Pluripotent/stem elements are binding sites for pluripotency factors (e.g., NANOG, SOX2, OCT4). Mature cell elements are docking sites for lineage-specific transcription factors, including AR and FOXA1 in prostate epithelium. Expanding the analysis to prostate tumors, we identify a subset of regulatory transposable elements shared with pluripotent/stem cells, including Tigger3a. Using chromatin editing technology, we show how such elements promote prostate cancer growth by regulating AR transcriptional activity. Collectively, our results suggest that oncogenesis arises from lineage-specific transcription factors hijacking pluripotent/stem cell regulatory transposable elements. SIGNIFICANCE: We show that oncogenesis relies on co-opting transposable elements from pluripotent stem cells as regulatory elements altering the recruitment of lineage-specific transcription factors. We further discover how co-option is dependent on active chromatin states with important implications for developing treatment options against drivers of oncogenesis across the repetitive DNA. This article is featured in Selected Articles from This Issue, p. 2293.

The chromatin and single-cell transcriptional landscapes of CD4 T cells in inflammatory bowel disease link risk loci with a proinflammatory Th17 cell population.

Introduction: The imbalance between Th17 and regulatory T cells in inflammatory bowel diseases (IBD) promotes intestinal epithelial cell damage. In this scenario, T helper cell lineage commitment is accompanied by dynamic changes to the chromatin that facilitate or repress gene expression. Methods: Here, we characterized the chromatin landscape and heterogeneity of intestinal and peripheral CD4 T cellsfrom IBD patients using in house ATAC-Seq and single cell RNA-Seq libraries. Results: We show that chromatin accessibility profiles of CD4 T cells from inflamed intestinal biopsies relate to genes associated with a network of inflammatory processes. After integrating the chromatin profiles of tissue-derived CD4 T cells and in-vitro polarized CD4 T cell subpopulations, we found that the chromatin accessibility changes of CD4 T cells were associated with a higher predominance of pathogenic Th17 cells (pTh17 cells) in inflamed biopsies. In addition, IBD risk loci in CD4 T cells were colocalized with accessible chromatin changes near pTh17-related genes, as shown in intronic STAT3 and IL23R regions enriched in areas of active intestinal inflammation. Moreover, single cell RNA-Seq analysis revealed a population of pTh17 cells that co-expresses Th1 and cytotoxic transcriptional programs associated with IBD severity. Discussion: Altogether, we show that cytotoxic pTh17 cells were specifically associated with IBD genetic variants and linked to intestinal inflammation of IBD patients.

Human papillomavirus integration transforms chromatin to drive oncogenesis.

BACKGROUND: Human papillomavirus (HPV) drives almost all cervical cancers and up to 70% of head and neck cancers. Frequent integration into the host genome occurs predominantly in tumorigenic types of HPV. We hypothesize that changes in chromatin state at the location of integration can result in changes in gene expression that contribute to the tumorigenicity of HPV. RESULTS: We find that viral integration events often occur along with changes in chromatin state and expression of genes near the integration site. We investigate whether introduction of new transcription factor binding sites due to HPV integration could invoke these changes. Some regions within the HPV genome, particularly the position of a conserved CTCF binding site, show enriched chromatin accessibility signal. ChIP-seq reveals that the conserved CTCF binding site within the HPV genome binds CTCF in 4 HPV+ cancer cell lines. Significant changes in CTCF binding pattern and increases in chromatin accessibility occur exclusively within 100 kbp of HPV integration sites. The chromatin changes co-occur with out-sized changes in transcription and alternative splicing of local genes. Analysis of The Cancer Genome Atlas (TCGA) HPV+ tumors indicates that HPV integration upregulates genes which have significantly higher essentiality scores compared to randomly selected upregulated genes from the same tumors. CONCLUSIONS: Our results suggest that introduction of a new CTCF binding site due to HPV integration reorganizes chromatin state and upregulates genes essential for tumor viability in some HPV+ tumors. These findings emphasize a newly recognized role of HPV integration in oncogenesis.

Chronic hypoxia favours adoption to a castration-resistant cell state in prostate cancer.

Predicting and treating recurrence in intermediate-risk prostate cancer patients remains a challenge despite having identified genomic instability [1] and hypoxia [2, 3] as risk factors. This underlies challenges in assigning the functional impact of these risk factors to mechanisms promoting prostate cancer progression. Here we show chronic hypoxia (CH), as observed in prostate tumours [4], leads to the adoption of an androgen-independent state in prostate cancer cells. Specifically, CH results in prostate cancer cells adopting transcriptional and metabolic alterations typical of castration-resistant prostate cancer cells. These changes include the increased expression of transmembrane transporters for the methionine cycle and related pathways leading to increased abundance of metabolites and expression of enzymes related to glycolysis. Targeting of the Glucose Transporter 1 (GLUT1) identified a dependency on glycolysis in androgen-independent cells. Overall, we identified a therapeutically targetable weakness in chronic hypoxia and androgen-independent prostate cancer. These findings may offer additional strategies for treatment development against hypoxic prostate cancer.

A noncoding single-nucleotide polymorphism at 8q24 drives IDH1-mutant glioma formation.

Establishing causal links between inherited polymorphisms and cancer risk is challenging. Here, we focus on the single-nucleotide polymorphism rs55705857, which confers a sixfold greater risk of isocitrate dehydrogenase (IDH)-mutant low-grade glioma (LGG). We reveal that rs55705857 itself is the causal variant and is associated with molecular pathways that drive LGG. Mechanistically, we show that rs55705857 resides within a brain-specific enhancer, where the risk allele disrupts OCT2/4 binding, allowing increased interaction with the Myc promoter and increased Myc expression. Mutating the orthologous mouse rs55705857 locus accelerated tumor development in an Idh1R132H-driven LGG mouse model from 472 to 172 days and increased penetrance from 30% to 75%. Our work reveals mechanisms of the heritable predisposition to lethal glioma in ~40% of LGG patients.

A primary hierarchically organized patient-derived model enables in depth interrogation of stemness driven by the coding and non-coding genome.

Many cancers are organized as cellular hierarchies sustained by cancer stem cells (CSC), whose eradication is crucial for achieving long-term remission. Difficulties to isolate and undertake in vitro and in vivo experimental studies of rare CSC under conditions that preserve their original properties currently constitute a bottleneck for identifying molecular mechanisms involving coding and non-coding genomic regions that govern stemness. We focussed on acute myeloid leukemia (AML) as a paradigm of the CSC model and developed a patient-derived system termed OCI-AML22 that recapitulates the cellular hierarchy driven by leukemia stem cells (LSC). Through classical flow sorting and functional analyses, we established that a single phenotypic population is highly enriched for LSC. The LSC fraction can be easily isolated and serially expanded in culture or in xenografts while faithfully recapitulating functional, transcriptional and epigenetic features of primary LSCs. A novel non-coding regulatory element was identified with a new computational approach using functionally validated primary AML LSC fractions and its role in LSC stemness validated through efficient CRISPR editing using methods optimized for OCI-AML22 LSC. Collectively, OCI-AML22 constitutes a valuable resource to uncover mechanisms governing CSC driven malignancies.

Chromatin Variants Reveal the Genetic Determinants of Oncogenesis in Breast Cancer.

Breast cancer presents as multiple distinct disease entities. Each tumor harbors diverse cell populations defining a phenotypic heterogeneity that impinges on our ability to treat patients. To date, efforts mainly focused on genetic variants to find drivers of inter- and intratumor phenotypic heterogeneity. However, these efforts have failed to fully capture the genetic basis of breast cancer. Through recent technological and analytical approaches, the genetic basis of phenotypes can now be decoded by characterizing chromatin variants. These variants correspond to polymorphisms in chromatin states at DNA sequences that serve a distinct role across cell populations. Here, we review the function and causes of chromatin variants as they relate to breast cancer inter- and intratumor heterogeneity and how they can guide the development of treatment alternatives to fulfill the goal of precision cancer medicine.

PRMT inhibition induces a viral mimicry response in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype with the worst prognosis and few effective therapies. Here we identified MS023, an inhibitor of type I protein arginine methyltransferases (PRMTs), which has antitumor growth activity in TNBC. Pathway analysis of TNBC cell lines indicates that the activation of interferon responses before and after MS023 treatment is a functional biomarker and determinant of response, and these observations extend to a panel of human-derived organoids. Inhibition of type I PRMT triggers an interferon response through the antiviral defense pathway with the induction of double-stranded RNA, which is derived, at least in part, from inverted repeat Alu elements. Together, our results represent a shift in understanding the antitumor mechanism of type I PRMT inhibitors and provide a rationale and biomarker approach for the clinical development of type I PRMT inhibitors.

Cancer-associated chromatin variants uncover the oncogenic role of transposable elements.

The vast array of cell states found across human tissue arises from chromatin variants, which correspond to segments of the genome, known as DNA elements, adopting a different chromatin state over cell state transitions. Oncogenesis stems from alterations to the chromatin states over DNA elements that result in cancer-associated chromatin variants. Here, we review how cancer-associated chromatin variants call attention to repetitive DNA elements, and guide the functional characterization of transposable elements to decode their role in oncogenesis. We further discuss prevailing opportunities in the study of repetitive DNA elements to move towards the 'complete cancer genome' goal for precision medicine in oncology.

PRMT5 regulates ATF4 transcript splicing and oxidative stress response.

Protein methyltransferase 5 (PRMT5) symmetrically dimethylates arginine residues leading to regulation of transcription and splicing programs. Although PRMT5 has emerged as an attractive oncology target, the molecular determinants of PRMT5 dependency in cancer remain incompletely understood. Our transcriptomic analysis identified PRMT5 regulation of the activating transcription factor 4 (ATF4) pathway in acute myelogenous leukemia (AML). PRMT5 inhibition resulted in the expression of unstable, intron-retaining ATF4 mRNA that is detained in the nucleus. Concurrently, the decrease in the spliced cytoplasmic transcript of ATF4 led to lower levels of ATF4 protein and downregulation of ATF4 target genes. Upon loss of functional PRMT5, cells with low ATF4 displayed increased oxidative stress, growth arrest, and cellular senescence. Interestingly, leukemia cells with EVI1 oncogene overexpression demonstrated dependence on PRMT5 function. EVI1 and ATF4 regulated gene signatures were inversely correlated. We show that EVI1-high AML cells have reduced ATF4 levels, elevated baseline reactive oxygen species and increased sensitivity to PRMT5 inhibition. Thus, EVI1-high cells demonstrate dependence on PRMT5 function and regulation of oxidative stress response. Overall, our findings identify the PRMT5-ATF4 axis to be safeguarding the cellular redox balance that is especially important in high oxidative stress states, such as those that occur with EVI1 overexpression.

ecDNAs personify cancer gangsters.

Alterations to gene regulatory plexuses typify oncogenesis, and two recent studies from Hung, Yost, Xie et al. (Hung et al., 2021) and Yi et al. (2021) collectively reveal the competitive advantage of extrachromosomal DNAs (ecDNAs) to set their own rules to control transcription by clustering into ecDNA hubs through intermolecular interactions that forge hub-specific regulatory plexuses driving high-oncogene expression.

Mutations in Noncoding Cis-Regulatory Elements Reveal Cancer Driver Cistromes in Luminal Breast Cancer.

Whole-genome sequencing of primary breast tumors enabled the identification of cancer driver genes and noncoding cancer driver plexuses from somatic mutations. However, differentiating driver from passenger events among noncoding genetic variants remains a challenge. Herein, we reveal cancer-driver cis-regulatory elements linked to transcription factors previously shown to be involved in development of luminal breast cancers by defining a tumor-enriched catalogue of approximately 100,000 unique cis-regulatory elements from 26 primary luminal estrogen receptor (ER)+ progesterone receptor (PR)+ breast tumors. Integrating this catalog with somatic mutations from 350 publicly available breast tumor whole genomes, we uncovered cancer driver cistromes, defined as the sum of binding sites for a transcription factor, for ten transcription factors in luminal breast cancer such as FOXA1 and ER, nine of which are essential for growth in breast cancer with four exclusive to the luminal subtype. Collectively, we present a strategy to find cancer driver cistromes relying on quantifying the enrichment of noncoding mutations over cis-regulatory elements concatenated into a functional unit. IMPLICATIONS: Mapping the accessible chromatin of luminal breast cancer led to discovery of an accumulation of mutations within cistromes of transcription factors essential to luminal breast cancer. This demonstrates coopting of regulatory networks to drive cancer and provides a framework to derive insight into the noncoding space of cancer.

Alu insertion variants alter gene transcript levels.

Alu are high copy number interspersed repeats that have accumulated near genes during primate and human evolution. They are a pervasive source of structural variation in modern humans. Impacts that Alu insertions may have on gene expression are not well understood, although some have been associated with expression quantitative trait loci (eQTLs). Here, we directly test regulatory effects of polymorphic Alu insertions in isolation of other variants on the same haplotype. To screen insertion variants for those with such effects, we used ectopic luciferase reporter assays and evaluated 110 Alu insertion variants, including more than 40 with a potential role in disease risk. We observed a continuum of effects with significant outliers that up- or down-regulate luciferase activity. Using a series of reporter constructs, which included genomic context surrounding the Alu, we can distinguish between instances in which the Alu disrupts another regulator and those in which the Alu introduces new regulatory sequence. We next focused on three polymorphic Alu loci associated with breast cancer that display significant effects in the reporter assay. We used CRISPR to modify the endogenous sequences, establishing cell lines varying in the Alu genotype. Our findings indicate that Alu genotype can alter expression of genes implicated in cancer risk, including PTHLH, RANBP9, and MYC These data show that commonly occurring polymorphic Alu elements can alter transcript levels and potentially contribute to disease risk.

Reorganization of the 3D Genome Pinpoints Noncoding Drivers of Primary Prostate Tumors.

Prostate cancer is a heterogeneous disease whose progression is linked to genome instability. However, the impact of this instability on the noncoding genome and its three-dimensional organization to aid progression is unclear. Using primary benign and tumor tissue, we find a high concordance in higher-order three-dimensional genome organization. This concordance argues for constraints to the topology of prostate tumor genomes. Nonetheless, we identified changes in focal chromatin interactions, typical of loops bridging noncoding cis-regulatory elements, and showed how structural variants can induce these changes to guide cis-regulatory element hijacking. Such events resulted in opposing differential expression of genes found at antipodes of rearrangements. Collectively, these results argue that changes to focal chromatin interactions, as opposed to higher-order genome organization, allow for aberrant gene regulation and are repeatedly mediated by structural variants in primary prostate cancer. SIGNIFICANCE: This work showcases how the noncoding genome can be hijacked by focal insults to its three-dimensional organization that contribute to prostate cancer oncogenesis.

Copy-scAT: Deconvoluting single-cell chromatin accessibility of genetic subclones in cancer.

Single-cell epigenomic assays have tremendous potential to illuminate mechanisms of transcriptional control in functionally diverse cancer cell populations. However, application of these techniques to clinical tumor specimens has been hampered by the current inability to distinguish malignant from nonmalignant cells, which potently confounds data analysis and interpretation. Here, we describe Copy-scAT, an R package that uses single-cell epigenomic data to infer copy number variants (CNVs) that define cancer cells. Copy-scAT enables studies of subclonal chromatin dynamics in complex tumors like glioblastoma. By deploying Copy-scAT, we uncovered potent influences of genetics on chromatin accessibility profiles in individual subclones. Consequently, some genetic subclones were predisposed to acquire stem-like or more differentiated molecular phenotypes, reminiscent of developmental paradigms. Copy-scAT is ideal for studies of the relationships between genetics and epigenetics in malignancies with high levels of intratumoral heterogeneity and to investigate how cancer cells interface with their microenvironment.

TFEB-mediated endolysosomal activity controls human hematopoietic stem cell fate.

It is critical to understand how human quiescent long-term hematopoietic stem cells (LT-HSCs) sense demand from daily and stress-mediated cues and then transition into bioenergetically active progeny to differentiate and meet these cellular needs. However, the demand-adapted regulatory circuits of these early steps of hematopoiesis are largely unknown. Here we show that lysosomes, sophisticated nutrient-sensing and signaling centers, are regulated dichotomously by transcription factor EB (TFEB) and MYC to balance catabolic and anabolic processes required for activating LT-HSCs and guiding their lineage fate. TFEB-mediated induction of the endolysosomal pathway causes membrane receptor degradation, limiting LT-HSC metabolic and mitogenic activation, promoting quiescence and self-renewal, and governing erythroid-myeloid commitment. In contrast, MYC engages biosynthetic processes while repressing lysosomal catabolism, driving LT-HSC activation. Our study identifies TFEB-mediated control of lysosomal activity as a central regulatory hub for proper and coordinated stem cell fate determination.

CRISPRi screens reveal a DNA methylation-mediated 3D genome dependent causal mechanism in prostate cancer.

Prostate cancer (PCa) risk-associated SNPs are enriched in noncoding cis-regulatory elements (rCREs), yet their modi operandi and clinical impact remain elusive. Here, we perform CRISPRi screens of 260 rCREs in PCa cell lines. We find that rCREs harboring high risk SNPs are more essential for cell proliferation and H3K27ac occupancy is a strong indicator of essentiality. We also show that cell-line-specific essential rCREs are enriched in the 8q24.21 region, with the rs11986220-containing rCRE regulating MYC and PVT1 expression, cell proliferation and tumorigenesis in a cell-line-specific manner, depending on DNA methylation-orchestrated occupancy of a CTCF binding site in between this rCRE and the MYC promoter. We demonstrate that CTCF deposition at this site as measured by DNA methylation level is highly variable in prostate specimens, and observe the MYC eQTL in the 8q24.21 locus in individuals with low CTCF binding. Together our findings highlight a causal mechanism synergistically driven by a risk SNP and DNA methylation-mediated 3D genome architecture, advocating for the integration of genetics and epigenetics in assessing risks conferred by genetic predispositions.