Estrogen receptor 1 chromatin profiling in human breast tumors reveals high inter-patient heterogeneity with enrichment of risk SNPs and enhancer activity at most-conserved regions.

Estrogen Receptor 1 (ESR1; also known as ERα, encoded by ESR1 gene) is the main driver and prime drug target in luminal breast cancer. ESR1 chromatin binding is extensively studied in cell lines and a limited number of human tumors, using consensi of peaks shared among samples. However, little is known about inter-tumor heterogeneity of ESR1 chromatin action, along with its biological implications. Here, we use a large set of ESR1 ChIP-seq data from 70 ESR1+ breast cancers to explore inter-patient heterogeneity in ESR1 DNA binding to reveal a striking inter-tumor heterogeneity of ESR1 action. Of note, commonly shared ESR1 sites show the highest estrogen-driven enhancer activity and are most engaged in long-range chromatin interactions. In addition, the most commonly shared ESR1-occupied enhancers are enriched for breast cancer risk SNP loci. We experimentally confirm SNVs to impact chromatin binding potential for ESR1 and its pioneer factor FOXA1. Finally, in the TCGA breast cancer cohort, we can confirm these variations to associate with differences in expression for the target gene. Cumulatively, we reveal a natural hierarchy of ESR1-chromatin interactions in breast cancers within a highly heterogeneous inter-tumor ESR1 landscape, with the most common shared regions being most active and affected by germline functional risk SNPs for breast cancer development.

Epigenetic-focused CRISPR/Cas9 screen identifies (absent, small, or homeotic)2-like protein (ASH2L) as a regulator of glioblastoma cell survival.

BACKGROUND: Glioblastoma is the most common and aggressive primary brain tumor with extremely poor prognosis, highlighting an urgent need for developing novel treatment options. Identifying epigenetic vulnerabilities of cancer cells can provide excellent therapeutic intervention points for various types of cancers. METHOD: In this study, we investigated epigenetic regulators of glioblastoma cell survival through CRISPR/Cas9 based genetic ablation screens using a customized sgRNA library EpiDoKOL, which targets critical functional domains of chromatin modifiers. RESULTS: Screens conducted in multiple cell lines revealed ASH2L, a histone lysine methyltransferase complex subunit, as a major regulator of glioblastoma cell viability. ASH2L depletion led to cell cycle arrest and apoptosis. RNA sequencing and greenCUT&RUN together identified a set of cell cycle regulatory genes, such as TRA2B, BARD1, KIF20B, ARID4A and SMARCC1 that were downregulated upon ASH2L depletion. Mass spectrometry analysis revealed the interaction partners of ASH2L in glioblastoma cell lines as SET1/MLL family members including SETD1A, SETD1B, MLL1 and MLL2. We further showed that glioblastoma cells had a differential dependency on expression of SET1/MLL family members for survival. The growth of ASH2L-depleted glioblastoma cells was markedly slower than controls in orthotopic in vivo models. TCGA analysis showed high ASH2L expression in glioblastoma compared to low grade gliomas and immunohistochemical analysis revealed significant ASH2L expression in glioblastoma tissues, attesting to its clinical relevance. Therefore, high throughput, robust and affordable screens with focused libraries, such as EpiDoKOL, holds great promise to enable rapid discovery of novel epigenetic regulators of cancer cell survival, such as ASH2L. CONCLUSION: Together, we suggest that targeting ASH2L could serve as a new therapeutic opportunity for glioblastoma. Video Abstract.

Breast cancer risk SNPs converge on estrogen receptor binding sites commonly shared between breast tumors to locally alter estrogen signalling output.

Estrogen Receptor alpha (ERα) is the main driver and prime drug target in luminal breast. ERα chromatin binding is extensively studied in cell lines and a limited number of human tumors, using consensi of peaks shared among samples. However, little is known about inter-tumor heterogeneity of ERα chromatin action, along with its biological implications. Here, we use a large set of ERα ChIP-seq data from 70 ERα+ breast cancers to explore inter-patient heterogeneity in ERα DNA binding, to reveal a striking inter-tumor heterogeneity of ERα action. Interestingly, commonly-shared ERα sites showed the highest estrogen-driven enhancer activity and were most-engaged in long-range chromatin interactions. In addition, the most-commonly shared ERα-occupied enhancers were enriched for breast cancer risk SNP loci. We experimentally confirm SNVs to impact chromatin binding potential for ERα and its pioneer factor FOXA1. Finally, in the TCGA breast cancer cohort, we could confirm these variations to associate with differences in expression for the target gene. Cumulatively, we reveal a natural hierarchy of ERα-chromatin interactions in breast cancers within a highly heterogeneous inter-tumor ERα landscape, with the most-common shared regions being most active and affected by germline functional risk SNPs for breast cancer development.

Curcuphenol possesses an unusual histone deacetylase enhancing activity that counters immune escape in metastatic tumours.

Curcuphenol, a common component of the culinary spices, naturally found in marine invertebrates and plants, has been identified as a novel candidate for reversing immune escape by restoring expression of the antigen presentation machinery (APM) in invasive cancers, thereby resurrecting the immune recognition of metastatic tumours. Two synthetic curcuphenol analogues, were prepared by informed design that demonstrated consistent induction of APM expression in metastatic prostate and lung carcinoma cells. Both analogues were subsequently found to possess a previously undescribed histone deacetylase (HDAC)-enhancing activity. Remarkably, the H3K27ac ChIPseq analysis of curcuphenol-treated cells reveals that the induced epigenomic marks closely resemble the changes in genome-wide pattern observed with interferon-γ, a cytokine instrumental for orchestrating innate and adaptive immunity. These observations link dietary components to modifying epigenetic programs that modulate gene expression guiding poised immunity.

Differentiation of Peritubular Myoid-Like Cells from Human Induced Pluripotent Stem Cells.

Infertility affects 10-15% of couples, with half attributed to male factors. An improved understanding of the cell-type-specific dysfunction contributing to male infertility is needed to improve available therapies; however, human testicular tissues are difficult to obtain for research purposes. To overcome this, researchers have begun to use human induced pluripotent stem cells (hiPSCs) to generate various testis-specific cell types in vitro. Peritubular myoid cells (PTMs) are one such testicular cell type that serves a critical role in the human testis niche but, to date, have not been derived from hiPSCs. This study set forth to generate a molecular-based differentiation method for deriving PTMs from hiPSCs, mirroring in vivo patterning factors. Whole transcriptome profiling and quantitative polymerase chain reaction (qPCR) show that this differentiation method is sufficient to derive cells with PTM-like transcriptomes, including upregulation of hallmark PTM functional genes, secreted growth and matrix factors, smooth muscle, integrins, receptors, and antioxidants. Hierarchical clustering shows that they acquire transcriptomes similar to primary isolated PTMs, and immunostaining shows the acquisition of a smooth muscle phenotype. Overall, these hiPSC-PTMs will allow in vitro study of patient-specific PTM development and function in spermatogenesis and infertility.

Optimized high-throughput screening of non-coding variants identified from genome-wide association studies.

The vast majority of disease-associated single nucleotide polymorphisms (SNP) identified from genome-wide association studies (GWAS) are localized in non-coding regions. A significant fraction of these variants impact transcription factors binding to enhancer elements and alter gene expression. To functionally interrogate the activity of such variants we developed snpSTARRseq, a high-throughput experimental method that can interrogate the functional impact of hundreds to thousands of non-coding variants on enhancer activity. snpSTARRseq dramatically improves signal-to-noise by utilizing a novel sequencing and bioinformatic approach that increases both insert size and the number of variants tested per loci. Using this strategy, we interrogated known prostate cancer (PCa) risk-associated loci and demonstrated that 35% of them harbor SNPs that significantly altered enhancer activity. Combining these results with chromosomal looping data we could identify interacting genes and provide a mechanism of action for 20 PCa GWAS risk regions. When benchmarked to orthogonal methods, snpSTARRseq showed a strong correlation with in vivo experimental allelic-imbalance studies whereas there was no correlation with predictive in silico approaches. Overall, snpSTARRseq provides an integrated experimental and computational framework to functionally test non-coding genetic variants.

Extensive androgen receptor enhancer heterogeneity in primary prostate cancers underlies transcriptional diversity and metastatic potential.

Androgen receptor (AR) drives prostate cancer (PCa) development and progression. AR chromatin binding profiles are highly plastic and form recurrent programmatic changes that differentiate disease stages, subtypes and patient outcomes. While prior studies focused on concordance between patient subgroups, inter-tumor heterogeneity of AR enhancer selectivity remains unexplored. Here we report high levels of AR chromatin binding heterogeneity in human primary prostate tumors, that overlap with heterogeneity observed in healthy prostate epithelium. Such heterogeneity has functional consequences, as somatic mutations converge on commonly-shared AR sites in primary over metastatic tissues. In contrast, less-frequently shared AR sites associate strongly with AR-driven gene expression, while such heterogeneous AR enhancer usage also distinguishes patients' outcome. These findings indicate that epigenetic heterogeneity in primary disease is directly informative for risk of biochemical relapse. Cumulatively, our results illustrate a high level of AR enhancer heterogeneity in primary PCa driving differential expression and clinical impact.

Genetic determinants of chromatin reveal prostate cancer risk mediated by context-dependent gene regulation.

Many genetic variants affect disease risk by altering context-dependent gene regulation. Such variants are difficult to study mechanistically using current methods that link genetic variation to steady-state gene expression levels, such as expression quantitative trait loci (eQTLs). To address this challenge, we developed the cistrome-wide association study (CWAS), a framework for identifying genotypic and allele-specific effects on chromatin that are also associated with disease. In prostate cancer, CWAS identified regulatory elements and androgen receptor-binding sites that explained the association at 52 of 98 known prostate cancer risk loci and discovered 17 additional risk loci. CWAS implicated key developmental transcription factors in prostate cancer risk that are overlooked by eQTL-based approaches due to context-dependent gene regulation. We experimentally validated associations and demonstrated the extensibility of CWAS to additional epigenomic datasets and phenotypes, including response to prostate cancer treatment. CWAS is a powerful and biologically interpretable paradigm for studying variants that influence traits by affecting transcriptional regulation.

EPIKOL, a chromatin-focused CRISPR/Cas9-based screening platform, to identify cancer-specific epigenetic vulnerabilities.

Dysregulation of the epigenome due to alterations in chromatin modifier proteins commonly contribute to malignant transformation. To interrogate the roles of epigenetic modifiers in cancer cells, we generated an epigenome-wide CRISPR-Cas9 knockout library (EPIKOL) that targets a wide-range of epigenetic modifiers and their cofactors. We conducted eight screens in two different cancer types and showed that EPIKOL performs with high efficiency in terms of sgRNA distribution and depletion of essential genes. We discovered novel epigenetic modifiers that regulate triple-negative breast cancer (TNBC) and prostate cancer cell fitness. We confirmed the growth-regulatory functions of individual candidates, including SS18L2 and members of the NSL complex (KANSL2, KANSL3, KAT8) in TNBC cells. Overall, we show that EPIKOL, a focused sgRNA library targeting ~800 genes, can reveal epigenetic modifiers that are essential for cancer cell fitness under in vitro and in vivo conditions and enable the identification of novel anti-cancer targets. Due to its comprehensive epigenome-wide targets and relatively high number of sgRNAs per gene, EPIKOL will facilitate studies examining functional roles of epigenetic modifiers in a wide range of contexts, such as screens in primary cells, patient-derived xenografts as well as in vivo models.

Drug-Induced Epigenomic Plasticity Reprograms Circadian Rhythm Regulation to Drive Prostate Cancer toward Androgen Independence.

In prostate cancer, androgen receptor (AR)-targeting agents are very effective in various disease stages. However, therapy resistance inevitably occurs, and little is known about how tumor cells adapt to bypass AR suppression. Here, we performed integrative multiomics analyses on tissues isolated before and after 3 months of AR-targeting enzalutamide monotherapy from patients with high-risk prostate cancer enrolled in a neoadjuvant clinical trial. Transcriptomic analyses demonstrated that AR inhibition drove tumors toward a neuroendocrine-like disease state. Additionally, epigenomic profiling revealed massive enzalutamide-induced reprogramming of pioneer factor FOXA1 from inactive chromatin sites toward active cis-regulatory elements that dictate prosurvival signals. Notably, treatment-induced FOXA1 sites were enriched for the circadian clock component ARNTL. Posttreatment ARNTL levels were associated with patients' clinical outcomes, and ARNTL knockout strongly decreased prostate cancer cell growth. Our data highlight a remarkable cistromic plasticity of FOXA1 following AR-targeted therapy and revealed an acquired dependency on the circadian regulator ARNTL, a novel candidate therapeutic target. SIGNIFICANCE: Understanding how prostate cancers adapt to AR-targeted interventions is critical for identifying novel drug targets to improve the clinical management of treatment-resistant disease. Our study revealed an enzalutamide-induced epigenomic plasticity toward prosurvival signaling and uncovered the circadian regulator ARNTL as an acquired vulnerability after AR inhibition, presenting a novel lead for therapeutic development. See related commentary by Zhang et al., p. 2017. This article is highlighted in the In This Issue feature, p. 2007.

Androgen Receptor-Mediated Transcription in Prostate Cancer.

Androgen receptor (AR)-mediated transcription is critical in almost all stages of prostate cancer (PCa) growth and differentiation. This process involves a complex interplay of coregulatory proteins, chromatin remodeling complexes, and other transcription factors that work with AR at cis-regulatory enhancer regions to induce the spatiotemporal transcription of target genes. This enhancer-driven mechanism is remarkably dynamic and undergoes significant alterations during PCa progression. In this review, we discuss the AR mechanism of action in PCa with a focus on how cis-regulatory elements modulate gene expression. We explore emerging evidence of genetic variants that can impact AR regulatory regions and alter gene transcription in PCa. Finally, we highlight several outstanding questions and discuss potential mechanisms of this critical transcription factor.

Specific cannabinoids revive adaptive immunity by reversing immune evasion mechanisms in metastatic tumours.

Emerging cancers are sculpted by neo-Darwinian selection for superior growth and survival but minimal immunogenicity; consequently, metastatic cancers often evolve common genetic and epigenetic signatures to elude immune surveillance. Immune subversion by metastatic tumours can be achieved through several mechanisms; one of the most frequently observed involves the loss of expression or mutation of genes composing the MHC-I antigen presentation machinery (APM) that yields tumours invisible to Cytotoxic T lymphocytes, the key component of the adaptive cellular immune response. Fascinating ethnographic and experimental findings indicate that cannabinoids inhibit the growth and progression of several categories of cancer; however, the mechanisms underlying these observations remain clouded in uncertainty. Here, we screened a library of cannabinoid compounds and found molecular selectivity amongst specific cannabinoids, where related molecules such as Δ9-tetrahydrocannabinol, cannabidiol, and cannabigerol can reverse the metastatic immune escape phenotype in vitro by inducing MHC-I cell surface expression in a wide variety of metastatic tumours that subsequently sensitizing tumours to T lymphocyte recognition. Remarkably, H3K27Ac ChIPseq analysis established that cannabigerol and gamma interferon induce overlapping epigenetic signatures and key gene pathways in metastatic tumours related to cellular senescence, as well as APM genes involved in revealing metastatic tumours to the adaptive immune response. Overall, the data suggest that specific cannabinoids may have utility in cancer immunotherapy regimens by overcoming immune escape and augmenting cancer immune surveillance in metastatic disease. Finally, the fundamental discovery of the ability of cannabinoids to alter epigenetic programs may help elucidate many of the pleiotropic medicinal effects of cannabinoids on human physiology.

DNA binding alters ARv7 dimer interactions.

Androgen receptor (AR) splice variants are proposed to be a potential driver of lethal castration-resistant prostate cancer. AR splice variant 7 (ARv7) is the most commonly observed isoform and strongly correlates with resistance to second-generation anti-androgens. Despite this clinical evidence, the interplay between ARv7 and the highly expressed full-length AR (ARfl) remains unclear. In this work, we show that ARfl/ARv7 heterodimers readily form in the nucleus via an intermolecular N/C interaction that brings the four termini of the proteins in close proximity. Combining fluorescence resonance energy transfer and fluorescence recovery after photobleaching, we demonstrate that these heterodimers undergo conformational changes following DNA binding, indicating dynamic nuclear receptor interaction. Although transcriptionally active, ARv7 can only form short-term interactions with DNA at highly accessible high-occupancy ARfl binding sites. Dimerization with ARfl does not affect ARv7 binding dynamics, suggesting that DNA binding occupancy is determined by the individual protein monomers and not the homodimer or heterodimer complex. Overall, these biophysical studies reveal detailed properties of ARv7 dynamics as both a homodimer or heterodimer with ARfl.

Functional mapping of androgen receptor enhancer activity.

BACKGROUND: Androgen receptor (AR) is critical to the initiation, growth, and progression of prostate cancer. Once activated, the AR binds to cis-regulatory enhancer elements on DNA that drive gene expression. Yet, there are 10-100× more binding sites than differentially expressed genes. It is unclear how or if these excess binding sites impact gene transcription. RESULTS: To characterize the regulatory logic of AR-mediated transcription, we generated a locus-specific map of enhancer activity by functionally testing all common clinical AR binding sites with Self-Transcribing Active Regulatory Regions sequencing (STARRseq). Only 7% of AR binding sites displayed androgen-dependent enhancer activity. Instead, the vast majority of AR binding sites were either inactive or constitutively active enhancers. These annotations strongly correlated with enhancer-associated features of both in vitro cell lines and clinical prostate cancer samples. Evaluating the effect of each enhancer class on transcription, we found that AR-regulated enhancers frequently interact with promoters and form central chromosomal loops that are required for transcription. Somatic mutations of these critical AR-regulated enhancers often impact enhancer activity. CONCLUSIONS: Using a functional map of AR enhancer activity, we demonstrated that AR-regulated enhancers act as a regulatory hub that increases interactions with other AR binding sites and gene promoters.

Generation of TRAIL-resistant cell line models reveals distinct adaptive mechanisms for acquired resistance and re-sensitization.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces tumor cell-specific apoptosis, making it a prime therapeutic candidate. However, many tumor cells are either innately TRAIL-resistant, or they acquire resistance with adaptive mechanisms that remain poorly understood. In this study, we generated acquired TRAIL resistance models using multiple glioblastoma (GBM) cell lines to assess the molecular alterations in the TRAIL-resistant state. We selected TRAIL-resistant cells through chronic and long-term TRAIL exposure and noted that they showed persistent resistance both in vitro and in vivo. Among known TRAIL-sensitizers, proteosome inhibitor Bortezomib, but not HDAC inhibitor MS-275, was effective in overcoming resistance in all cell models. This was partly achieved through upregulating death receptors and pro-apoptotic proteins, and downregulating major anti-apoptotic members, Bcl-2 and Bcl-xL. We showed that CRISPR/Cas9 mediated silencing of DR5 could block Bortezomib-mediated re-sensitization, demonstrating its critical role. While overexpression of Bcl-2 or Bcl-xL was sufficient to confer resistance to TRAIL-sensitive cells, it failed to override Bortezomib-mediated re-sensitization. With RNA sequencing in multiple paired TRAIL-sensitive and TRAIL-resistant cells, we identified major alterations in inflammatory signaling, particularly in the NF-κB pathway. Inhibiting NF-κB substantially sensitized the most resistant cells to TRAIL, however, the sensitization effect was not as great as what was observed with Bortezomib. Together, our findings provide new models of acquired TRAIL resistance, which will provide essential tools to gain further insight into the heterogeneous therapy responses within GBM tumors. Additionally, these findings emphasize the critical importance of combining proteasome inhibitors and pro-apoptotic ligands to overcome acquired resistance.

Androgen receptor-binding sites are highly mutated in prostate cancer.

Androgen receptor (AR) signalling is essential in nearly all prostate cancers. Any alterations to AR-mediated transcription can have a profound effect on carcinogenesis and tumor growth. While mutations of the AR protein have been extensively studied, little is known about those somatic mutations that occur at the non-coding regions where AR binds DNA. Using clinical whole genome sequencing, we show that AR binding sites have a dramatically increased rate of mutations that is greater than any other transcription factor and specific to only prostate cancer. Demonstrating this may be common to lineage-specific transcription factors, estrogen receptor binding sites were also found to have elevated rate of mutations in breast cancer. We provide evidence that these mutations at AR binding sites, and likely other related transcription factors, are caused by faulty repair of abasic sites. Overall, this work demonstrates that non-coding AR binding sites are frequently mutated in prostate cancer and can impact enhancer activity.

Systematic characterization of chromatin modifying enzymes identifies KDM3B as a critical regulator in castration resistant prostate cancer.

Androgen deprivation therapy (ADT) is the standard care for prostate cancer (PCa) patients who fail surgery or radiotherapy. While initially effective, the cancer almost always recurs as a more aggressive castration resistant prostate cancer (CRPC). Previous studies have demonstrated that chromatin modifying enzymes can play a critical role in the conversion to CRPC. However, only a handful of these potential pharmacological targets have been tested. Therefore, in this study, we conducted a focused shRNA screen of chromatin modifying enzymes previously shown to be involved in cellular differentiation. We found that altering the balance between histone methylation and demethylation impacted growth and proliferation. Of all genes tested, KDM3B, a histone H3K9 demethylase, was found to have the most antiproliferative effect. These results were phenocopied with a KDM3B CRISPR/Cas9 knockout. When tested in several PCa cell lines, the decrease in proliferation was remarkably specific to androgen-independent cells. Genetic rescue experiments showed that only the enzymatically active KDM3B could recover the phenotype. Surprisingly, despite the decreased proliferation of androgen-independent cell no alterations in the cell cycle distribution were observed following KDM3B knockdown. Whole transcriptome analyses revealed changes in the gene expression profile following loss of KDM3B, including downregulation of metabolic enzymes such as ARG2 and RDH11. Metabolomic analysis of KDM3B knockout showed a decrease in several critical amino acids. Overall, our work reveals, for the first time, the specificity and the dependence of KDM3B in CRPC proliferation.

Bromodomain inhibition of the coactivators CBP/EP300 facilitate cellular reprogramming.

Silencing of the somatic cell type-specific genes is a critical yet poorly understood step in reprogramming. To uncover pathways that maintain cell identity, we performed a reprogramming screen using inhibitors of chromatin factors. Here, we identify acetyl-lysine competitive inhibitors targeting the bromodomains of coactivators CREB (cyclic-AMP response element binding protein) binding protein (CBP) and E1A binding protein of 300 kDa (EP300) as potent enhancers of reprogramming. These inhibitors accelerate reprogramming, are critical during its early stages and, when combined with DOT1L inhibition, enable efficient derivation of human induced pluripotent stem cells (iPSCs) with OCT4 and SOX2. In contrast, catalytic inhibition of CBP/EP300 prevents iPSC formation, suggesting distinct functions for different coactivator domains in reprogramming. CBP/EP300 bromodomain inhibition decreases somatic-specific gene expression, histone H3 lysine 27 acetylation (H3K27Ac) and chromatin accessibility at target promoters and enhancers. The master mesenchymal transcription factor PRRX1 is one such functionally important target of CBP/EP300 bromodomain inhibition. Collectively, these results show that CBP/EP300 bromodomains sustain cell-type-specific gene expression and maintain cell identity.

Sulfated levan from Halomonas smyrnensis as a bioactive, heparin-mimetic glycan for cardiac tissue engineering applications.

Chemical derivatives of levan from Halomonas smyrnensis AAD6(T) with low, medium and high levels of sulfation were synthesized and characterized by FTIR and 2D-NMR. Sulfated levan samples were found to exhibit anticoagulation activity via the intrinsic pathway like heparin in a dose-dependent manner. Exceptionally high heparin equivalent activity of levan sulfate was shown to proceed via thrombin inhibition where decreased Factor Xa activity with increasing concentration was observed in antithrombin tests and above a certain concentration, levan sulfate showed a better inhibitor activity than heparin. In vitro experimental results were then verified in silico by docking studies using equilibrium structures obtained by molecular dynamic simulations and results suggested a sulfation dependent binding mechanism. With its high biocompatibility and heparin mimetic activity, levan sulfate can be considered as a suitable functional biomaterial to design biologically active, functionalized, thin films and engineered smart scaffolds for cardiac tissue engineering applications.