Biology subjects

Gertz, J.

Publications and source records attributed to Gertz, J..

At least 19 recordsLinked to original sources

bioRxiv2025

Identification of genomic features that uniquely impact estrogen receptor alpha binding and its effects on gene expression in endometrial cancer

Estrogen receptor alpha (ER) is an established oncogenic transcription factor in breast and endometrial cancer; however, more is known about the mechanisms controlling ER behavior in breast cancer, and therapies targeting ER have been much more successful in breast cancer. To address this disparity, we characterize the genomic features that control ER in endometrial cancer and determine to what extent these factors differ from those in breast cancer. We focus on the locations of estrogen response elements (EREs), ERs preferred DNA binding motif, throughout the human genome. To identify factors that predict ER genomic binding and effects on target gene expression, we apply machine learning to genomic data for each ERE in Ishikawa cells (ER-positive endometrial cancer) and T-47D cells (ER-positive breast cancer). Many of these factors, such as chromatin accessibility and histone modifications, are predictive of ER activity in both cell lines. However, the transcription factors that predict ER activity are cell type-specific, including FOXA1 and GATA3 in T-47D cells, and ETV4 and SOX17 in Ishikawa cells. In addition, the features that predict ER binding and effects on gene expression differ, with transcription at EREs in the absence of estrogen being predictive of ER regulatory activity. A CRISPR knockout screen in Ishikawa cells, and follow-up experiments, confirm the discovery that SOX17 controls ER activity in endometrial cancer cells. These results identify important genomic features of ER binding and regulatory activity and how these features differ between endometrial cancer and breast cancer cells.

genomics↗

bioRxiv2025

Characterizing intra- and inter-tumor heterogeneity in ovarian high-grade serous carcinoma subtypes using single-cell and spatial transcriptomics

Ovarian high-grade serous carcinoma (HGSC) is an aggressive ovarian cancer with a heterogeneous tumor microenvironment (TME). Advances in single-cell RNA sequencing (scRNA-seq) and spatially-resolved transcriptomics have enabled the study of complex TME. This study explores connections between molecular subtypes described from bulk transcriptomes and spatial domains characterized by distinct gene expression in HGSC and their variability between patients. We explored both intraand inter-tumor heterogeneity across 2D space and identified differing spatial patterns of gene expression pertaining to immune pathways and vasculature development. Functional characterization of tumor spaces revealed potentially shared cell states across molecular subtypes, while correlation analysis underscored subtype-specific spatial anti-colocalization between spots exhibiting antigen-presenting functions and B cell-mediated immunity. Lastly, we performed spatially-aware cell-cell communication analysis on the spatial samples and identified a molecular subtype specific difference in total signaling activity and heterogeneity in Midikine signaling between the differentiated subtype. Our results suggest that generating multiple tissue slices per patient might be necessary to enable comprehensive characterization of HGSC spatial transcriptomes.

genomics↗

bioRxiv2025

ZNF865 (BLST) Regulates Human Cell Senescence and DNA Damage

Senescence has been shown to contribute to the progression of aging related diseases including degenerative disc disease (DDD). However, the mechanisms regulating senescence in the intervertebral disc (IVD) and other tissues/diseases remain poorly understood. Recently, ZNF865 (BLST) was identified as a previously uncharacterized zinc finger protein, that regulates a wide array of genes related to protein processing, cell senescence and DNA damage repair. Here, we show that ZNF865 expression decreases with age and pathology in human and mouse IVD samples. Utilizing CRISPR-guided gene modulation, we show that ZNF865 is necessary for healthy cell function and is a critical protein in regulating senescence and DNA damage in IVD cells, with implications for a wide range of tissues and organs. We also demonstrate that downregulation of ZNF865 induces senescence in asymptomatic human nucleus pulposus (NP) cells. Conversely, restoration in degenerative NP cells mitigates senescence, SASP expression and DNA damage, enhances ECM anabolism, and restores chromatin accessibility and gene expression to a healthy state. In vivo, CRISPRi of ZNF865 induces disc degeneration and painful behaviors. Collectively, our findings establish ZNF865 as a novel regulator of genome stability and a potential therapeutic target for mediating senescence/DNA damage in aging related diseases.

bioengineering↗

bioRxiv2024

EstroGene2.0: A multi-omic database of response to estrogens, ER-modulators, and resistance to endocrine therapies in breast cancer

Endocrine therapies targeting the estrogen receptor (ER/ESR1) are the cornerstone to treat ER-positive breast cancers patients, but resistance often limits their effectiveness. Understanding the molecular mechanisms is thus key to optimize the existing drugs and to develop new ER-modulators. Notable progress has been made although the fragmented way data is reported has reduced their potential impact. Here, we introduce EstroGene2.0, an expanded database of its precursor 1.0 version. EstroGene2.0 focusses on response and resistance to endocrine therapies in breast cancer models. Incorporating multi-omic profiling of 361 experiments from 212 studies across 28 cell lines, a user-friendly browser offers comprehensive data visualization and metadata mining capabilities (https://estrogeneii.web.app/). Taking advantage of the harmonized data collection, our follow-up meta-analysis revealed substantial diversity in response to different classes of ER-modulators including SERMs, SERDs, SERCA and LDD/PROTAC. Notably, endocrine resistant models exhibit a spectrum of transcriptomic alterations including a contra-directional shift in ER and interferon signaling, which is recapitulated clinically. Furthermore, dissecting multiple ESR1-mutant cell models revealed the different clinical relevance of genome-edited versus ectopic overexpression model engineering and identified high-confidence mutant-ER targets, such as NPY1R. These examples demonstrate how EstroGene2.0 helps investigate breast cancers response to endocrine therapies and explore resistance mechanisms.

cancer biology↗

bioRxiv2024

Estrogen-induced chromatin looping changes identify a subset of functional regulatory elements

Transcriptional enhancers can regulate individual or multiple genes through long-range three-dimensional (3D) genome interactions, and these interactions are commonly altered in cancer. Yet, the functional relationship between changes in 3D genome interactions associated with regulatory regions and differential gene expression appears context-dependent. In this study, we used HiChIP to capture changes in 3D genome interactions between active regulatory regions of endometrial cancer cells in response to estrogen treatment and uncovered significant differential long-range interactions strongly enriched for estrogen receptor (ER) bound sites (ERBS). The ERBS anchoring differential chromatin loops with either a genes promoter or distal regions were correlated with larger transcriptional responses to estrogen compared to ERBS not involved in differential 3D genome interactions. To functionally test this observation, CRISPR- based Enhancer-i was used to deactivate specific ERBS, which revealed a wide range of effects on the transcriptional response to estrogen. However, these effects are only subtly and not significantly stronger for ERBS in differential chromatin loops. In addition, we observed an enrichment of 3D genome interactions between the promoters of estrogen upregulated genes and found that looped promoters can work together cooperatively. Overall, our work reveals that estrogen treatment causes large changes in 3D genome structure in endometrial cancer cells; however, these changes are not required for a regulatory region to contribute to an estrogen transcriptional response.

cancer biology↗

bioRxiv2024

3D genomic analysis reveals novel enhancer-hijacking caused by complex structural alterations that drive oncogene overexpression

Cancer genomes are composed of many complex structural alterations on chromosomes and extrachromosomal DNA (ecDNA), making it difficult to identify non-coding enhancer regions that are hijacked to activate oncogene expression. Here, we describe a 3D genomics-based analysis called HAPI (Highly Active Promoter Interactions) to characterize enhancer hijacking. HAPI analysis of HiChIP data from 34 cancer cell lines identified enhancer hijacking events that activate both known and potentially novel oncogenes such as MYC, CCND1, ETV1, CRKL, and ID4. Furthermore, we found enhancer hijacking among multiple oncogenes from different chromosomes, often including MYC, on the same complex amplicons such as ecDNA. We characterized a MYC-ERBB2 chimeric ecDNA, in which ERBB2 heavily hijacks MYCs enhancers. Notably, CRISPRi of the MYC promoter led to increased interaction of ERBB2 with MYC enhancers and elevated ERBB2 expression. Our HAPI analysis tool provides a robust strategy to detect enhancer hijacking and reveals novel insights into oncogene activation.

cancer biology↗

bioRxiv2023

ASPSCR1-TFE3 reprograms transcription by organizing enhancer loops around hexameric VCP/p97

The t(X,17) chromosomal translocation, generating the ASPSCR1-TFE3 fusion oncoprotein, is the singular genetic driver of alveolar soft part sarcoma (ASPS) and some Xp11-rearranged renal cell carcinomas (RCC), frustrating efforts to identify therapeutic targets for these rare cancers. Proteomic analysis showed that VCP/p97, an AAA+ ATPase with known segregase function, was strongly enriched in co-immunoprecipitated nuclear complexes with ASPSCR1-TFE3. We demonstrate that VCP is a likely obligate co-factor of ASPSCR1-TFE3, one of the only such fusion oncoprotein co-factors identified in cancer biology. Specifically, VCP co-distributed with ASPSCR1-TFE3 across chromatin in association with enhancers genome-wide. VCP presence, its hexameric assembly, and its enzymatic function orchestrated the oncogenic transcriptional signature of ASPSCR1-TFE3, by facilitating assembly of higher-order chromatin conformation structures as demonstrated by HiChIP. Finally, ASPSCR1-TFE3 and VCP demonstrated co-dependence for cancer cell proliferation and tumorigenesis in vitro and in ASPS and RCC mouse models, underscoring VCPs potential as a novel therapeutic target.

cancer biology↗

bioRxiv2023

Deconvolution reveals compositional differences in high-grade serous ovarian cancer subtypes

Ovarian cancer is a deadly disease with few effective therapies. The most common form is high-grade serous ovarian cancer (HGSOC). Transcriptomic subtypes of HGSOC have shown promise in characterizing tumor heterogeneity and are associated with survival. Gene expression signatures for the subtypes suggest variation in stromal cell types in the tumor microenvironment (TME). Here, we characterize the TME composition of HGSOC on a population scale by performing deconvolution on bulk transcriptomic data. We use comprehensive cell type profiles from 164 HGSOC tumor samples from two independent reference datasets, in order to compare cell type proportions across and within bulk transcriptomic datasets, and assess their alignment to the subtypes proposed by The Cancer Genome Atlas. We also assess the relationship between tumor composition and clinical outcomes. Our results suggest that HGSOC transcriptomic subtypes are driven by TME composition, specifically fibroblast and immune cell content, and we propose a modified HGSOC subtype model informed by cell composition.

bioinformatics↗

bioRxiv2023

Cis-regulatory control of transcriptional timing and noise in response to estrogen

Cis-regulatory elements control transcription levels, temporal dynamics, and cell-cell variation or transcriptional noise. However, the combination of regulatory features that control these different attributes is not fully understood. Here, we used single cell RNA-seq during an estrogen treatment time course and machine learning to identify predictors of expression timing and noise. We find that genes with multiple active enhancers exhibit faster temporal responses. We verified this finding by showing that manipulation of enhancer activity changes the temporal response of estrogen target genes. Analysis of transcriptional noise uncovered a relationship between promoter and enhancer activity, with active promoters associated with low noise and active enhancers linked to high noise. Finally, we observed that co-expression across single cells is an emergent property associated with chromatin looping, timing, and noise. Overall, our results indicate a fundamental tradeoff between a genes ability to quickly respond to incoming signals and maintain low variation across cells.

genomics↗

bioRxiv2022

Estrogen receptor alpha mutations regulate gene expression and cell growth in breast cancer through microRNAs

Estrogen receptor (ER) mutations occur in up to 30% of metastatic ER-positive breast cancers. Recent data has shown that ER mutations impact the expression of thousands of genes not typically regulated by wildtype ER. While the majority of these altered genes can be explained by constant activity of mutant ER or genomic changes such as altered ER binding and chromatin accessibility, as much as 33% remain unexplained, indicating the potential for post-transcriptional effects. Here we explored the role of microRNAs in mutant ER-driven gene regulation and identified several microRNAs that are dysregulated in ER mutant cells. These differentially regulated microRNAs target a significant portion of mutant-specific genes involved in key cellular processes. When the activity of microRNAs is altered using mimics or inhibitors, significant changes are observed in gene expression and cellular proliferation related to mutant ER. An in-depth evaluation of miR-301b led us to discover an important role for PRKD3 in the proliferation of ER mutant cells. Our findings show that microRNAs contribute to mutant ER gene regulation and cellular effects in breast cancer cells.

cancer biology↗

bioRxiv2022

Characterization of a novel estrogen- and progesterone-responsive endometrial cancer cell line: HCI-EC-23

Most endometrial cancers express the hormone receptor estrogen receptor alpha (ER) and are driven by excess estrogen signaling. However, evaluation of the estrogen response in endometrial cancer cells has been limited by the availability of hormonally responsive in vitro models, with one cell line, Ishikawa, being used in most studies. Here, we describe a novel, adherent endometrioid endometrial cancer (EEC) cell line model, HCI-EC-23. We show that HCI-EC-23 retains ER expression and that ER functionally responds to estrogen induction over a range of passages. We also demonstrate that this cell line retains paradoxical activation of ER by tamoxifen, which is also observed in Ishikawa and is consistent with clinical data. The mutational landscape shows that HCI-EC-23 is mutated at many of the commonly altered genes in EEC, has relatively few copy-number alterations, and is microsatellite instable high (MSI-high). In vitro proliferation of HCI-EC-23 is strongly reduced upon combination estrogen and progesterone treatment. HCI-EC-23 exhibits strong estrogen dependence for tumor growth in vivo and tumor size is reduced by combination estrogen and progesterone treatment. Molecular characterization of estrogen induction in HCI-EC-23 revealed hundreds of estrogen-responsive genes that significantly overlapped with those regulated in Ishikawa. Analysis of ER genome binding identified similar patterns in HCI-EC-23 and Ishikawa, although ER exhibited more bound sites in Ishikawa. This study demonstrates that HCI-EC-23 is an estrogen- and progesterone-responsive cell line model that can be used to study the hormonal aspects of endometrial cancer.

cancer biology↗

bioRxiv2022

TXNIP loss expands Myc-dependent transcriptional programs by increasing Myc genomic binding

c-Myc protooncogene places a demand on glucose uptake to drive glucose-dependent biosynthetic pathways. To achieve this demand, c-Myc protein (Myc henceforth) drives the expression of glucose transporters and represses the expression of Thioredoxin Interacting Protein (TXNIP), which is a potent negative regulator of glucose uptake. A Mychigh/TXNIPlow gene signature is clinically significant as it correlates with poor clinical prognosis in Triple-Negative Breast Cancer (TNBC) but not in other subtypes of breast cancer. To better understand how TXNIP function contributes to the aggressive behavior of TNBC, we generated TXNIP null MDA-MB-231 (231:TKO) cells for our study. We show here that TXNIP loss drives a transcriptional program that resembles those driven by Myc and increases global Myc genome occupancy. TXNIP loss allows Myc to invade the promoters and enhancers of target genes that are potentially relevant to cell transformation. Together, these findings suggest that TXNIP is a broad repressor of Myc genomic binding. The increase in Myc genomic binding in the 231:TKO cells expands the Myc-dependent transcriptome we identified in parental MDA-MB-231 cells. This expansion of Myc-dependent transcription following TXNIP loss occurs without an apparent increase in Mycs intrinsic capacity to activate transcription and without increasing Myc levels. Together, our findings suggest that TXNIP loss mimics Myc overexpression, connecting Myc genomic binding and transcriptional programs to the metabolic signals that control TXNIP expression.

cancer biology↗

bioRxiv2022

Allele-specific gene regulation, phenotypes, and therapeutic vulnerabilities in estrogen receptor alpha mutant endometrial cancer

Activating estrogen receptor alpha (ER) mutations are present in primary endometrial and metastatic breast cancers, promoting estrogen-independent activation of the receptor. Functional characterizations in breast cancer have established unique molecular and phenotypic consequences of the receptor, yet the impact of ER mutations in endometrial cancer has not been fully explored. In this study, we used CRISPR-Cas9 to model the clinically prevalent ER-Y537S mutation and compared results to ER-D538G to discover allele-specific differences between ER mutations in endometrial cancer. We found that constitutive activity of mutant ER resulted in changes in the expression of thousands of genes, stemming from combined alterations to ER binding and chromatin accessibility. The unique gene expression programs resulted in ER mutant cells developing increased cancer associated phenotypes, including migration, invasion, anchorage independent growth, and growth in vivo. To uncover potential treatment strategies, we identified ER associated proteins via Rapid Immunoprecipitation and Mass Spectrometry of Endogenous Proteins (RIME) and interrogated two candidates, CDK9 and NCOA3. Inhibition of these regulatory proteins resulted in decreased growth and migration, representing potential novel treatment strategies for ER mutant endometrial cancer. ImplicationsThis study provides insight into mutant ER activity in endometrial cancer and identifies potential therapies for women with ER mutant endometrial cancer. STATEMENT OF SIGNIFICANCEActivating estrogen receptor alpha (ER) mutations promote ligand-independent activity of the receptor. This study evaluates ER-Y537S and ER-D538G mutations in primary endometrial cancer, revealing their effects on gene regulation and cancer-associated phenotypes. By identifying ER associated proteins, we also uncover potential novel treatments for women with ER mutant endometrial cancer.

cancer biology↗

bioRxiv2021

A breast cancer patient-derived xenograft and organoid platform for drug discovery and precision oncology

Model systems that recapitulate the complexity of human tumors and the reality of variable treatment responses are urgently needed to better understand cancer biology and to develop more effective cancer therapies. Here we report development and characterization of a large bank of patient-derived xenografts (PDX) and matched organoid cultures from tumors that represent some of the greatest unmet needs in breast cancer research and treatment. These include endocrine-resistant, treatment-refractory, and metastatic breast cancers and, in some cases, multiple tumor collections from the same patients. The models can be grown long-term with high fidelity to the original tumors. We show that development of matched PDX and PDX-derived organoid (PDxO) models facilitates high-throughput drug screening that is feasible and cost-effective, while also allowing in vivo validation of results. Our data reveal consistency between drug screening results in organoids and drug responses in breast cancer PDX. Moreover, we demonstrate the feasibility of using these patient-derived models for precision oncology in real time with patient care, using a case of a triple negative breast cancer with early metastatic recurrence as an example. Our results uncovered an FDA-approved drug with high efficacy against the models. Treatment with the PDxO-directed therapy resulted in a complete response for the patient and a progression-free survival period more than three times longer than her previous therapies. This work provides valuable new methods and resources for functional precision medicine and drug development for human breast cancer. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=189 HEIGHT=200 SRC="FIGDIR/small/433268v2_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@134d5cdorg.highwire.dtl.DTLVardef@1c0a419org.highwire.dtl.DTLVardef@556a4forg.highwire.dtl.DTLVardef@1b68c9c_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2021

Hotspot ESR1 mutations are multimodal and contextual drivers of breast cancer metastasis

Constitutively active estrogen receptor- (ER/ESR1) mutations have been identified in approximately one third of ER+ metastatic breast cancer. Although these mutations are known mediators of endocrine resistance, their potential role in promoting metastatic disease has not yet been mechanistically addressed. In this study, we show the presence of ESR1 mutations exclusively in distant, but not local recurrences. In concordance with transcriptomic profiling of ESR1 mutant tumors, genome-edited Y537S and D538G cell models have a reprogrammed cell adhesive gene network via alterations in desmosome/gap junction genes and the TIMP3/MMP axis, which functionally confers enhanced cell-cell contacts while decreased cell-ECM adhesion. Context-dependent migratory phenotypes revealed co-targeting of Wnt and ER as vulnerability. Mutant ESR1 exhibits non-canonical regulation of several metastatic pathways including secondary transactivation and de novo FOXA1-driven chromatin remodeling. Collectively, our data supports evidence for ESR1 mutation-driven metastases and provides insight for future preclinical therapeutic strategies. SignificanceContext and allele-dependent transcriptome and cistrome reprogramming in genome-edited ESR1 mutation cell models elicit diverse metastatic phenotypes, including but not limited to alterations in cell adhesion and migration. The gain-of-function mutations can be pharmacologically targeted, and thus may be key components of novel therapeutic treatment strategies for ER-mutant metastatic breast cancer.

cancer biology↗

bioRxiv2020

ESR1 mutant breast cancers show elevated basal cytokeratins and immune activation

Estrogen receptor alpha (ER/ESR1) is mutated in 30-40% of endocrine resistant ER-positive (ER+) breast cancer. ESR1 mutations cause ligand-independent growth and increased metastasis in vivo and in vitro. Despite the distinct clinical features and changes in therapeutic response associated with ESR1 mutations, there are no data about their potential role in intrinsic subtype switching. Applying four luminal and basal gene set pairs, ESR1 mutant cell models and clinical samples showed a significant enrichment of basal subtype markers. Among them, the six basal cytokeratins (BCKs) were the most enriched genes. Induction of BCKs was independent of ER binding and instead associated with chromatin reprogramming centered around a progesterone receptor-orchestrated topological associated domain at the KRT14/16/17 genomic region. Unexpectedly, high BCK expression in ER+ primary breast cancer is associated with good prognosis, and these tumors show enriched activation of a number of immune pathways, a distinctive feature shared with ESR1 mutant tumors. S100A8 and S100A9 were among the most highly induced immune mediators shared between high-BCKs ER+ and ESR1 mutant tumors, and single-cell RNA-seq analysis inferred their involvement in paracrine crosstalk between epithelial and stromal cells. Collectively, these observations demonstrate that ESR1 mutant tumors gain basal features with induction of basal cytokeratins via epigenetic mechanisms in rare subpopulation of cells. This is associated with increased immune activation, encouraging additional studies of immune therapeutic vulnerabilities in ESR1 mutant tumors.

cancer biology↗

bioRxiv2020

Genetic demultiplexing of pooled single-cell RNA-sequencing samples in cancer facilitates effective experimental design

BackgroundPooling cells from multiple biological samples prior to library preparation within the same single-cell RNA sequencing experiment provides several advantages, including lower library preparation costs and reduced unwanted technological variation, such as batch effects. Computational demultiplexing tools based on natural genetic variation between individuals provide a simple approach to demultiplex samples, which does not require complex additional experimental procedures. However, these tools have not been evaluated in cancer, where somatic variants, which could differ between cells from the same sample, may obscure the signal in natural genetic variation. ResultsHere, we performed in silico benchmark evaluations by combining raw sequencing reads from multiple single-cell samples in high-grade serous ovarian cancer, which has a high copy number burden, and lung adenocarcinoma, which has a high tumor mutational burden. Our results confirm that genetic demultiplexing tools can be effectively deployed on cancer tissue using a pooled experimental design, although high proportions of ambient RNA from cell debris reduce performance. ConclusionsThis strategy provides significant cost savings through pooled library preparation. To facilitate similar analyses at the experimental design phase, we provide freely accessible code and a reproducible Snakemake workflow built around the best-performing tools found in our in silico benchmark evaluations, available at https://github.com/lmweber/snp-dmx-cancer.

bioinformatics↗

bioRxiv2020

Estrogen receptor alpha mutations in breast cancer cells cause gene expression changes through constant activity and through secondary effects

While breast cancer patients with tumors that express estrogen receptor (ER) generally respond well to hormone therapies that block ER activity, a significant number of patients relapse. Approximately 30% of these recurrences harbor activating mutations in the ligand binding domain (LBD) of ER, which have been shown to confer ligand-independent function. However, much is still unclear regarding the effect of mutant ER beyond its estrogen independence. To investigate the molecular effects of mutant ER, we developed multiple isogenic ER mutant cell lines for the most common LBD mutations, Y537S and D538G. These mutations induced differential expression of thousands of genes, the majority of which were mutant allele-specific and were not observed upon estrogen treatment of wildtype cells. These mutant-specific genes showed consistent differential expression across ER mutant lines developed in other laboratories. Wildtype cells with long-term estrogen exposure only exhibited some of these transcriptional changes, suggesting that mutant ER causes novel regulatory effects that are not simply due to constant activity. While ER mutations exhibited minor effects on ER genomic binding, with the exception of ligand independence, ER mutations conferred substantial differences in chromatin accessibility. Mutant ER was bound to approximately a quarter of mutant-enriched accessible regions that were enriched for other DNA binding factors including FOXA1, CTCF, and OCT1. Overall, our findings indicate that mutant ER causes several consistent effects on gene expression, both indirectly and through constant activity.

cancer biology↗