Chromatin Landscape Research Articles

FOXK1 regulates Wnt signalling to promote cardiogenesis.

Congenital heart disease (CHD) is the most common genetic birth defect, which has considerable morbidity and mortality. We focused on deciphering key regulators that govern cardiac progenitors and cardiogenesis. FOXK1 is a forkhead/winged helix transcription factor known to regulate cell cycle kinetics and is restricted to mesodermal progenitors, somites, and heart. In the present study, we define an essential role for FOXK1 during cardiovascular development. We used the mouse embryoid body system to differentiate control and Foxk1 KO embryonic stem cells into mesodermal, cardiac progenitor cells and mature cardiac cells. Using flow cytometry, immunohistochemistry, cardiac beating, transcriptional and chromatin immunoprecipitation quantitative polymerase chain reaction assays, bulk RNA sequencing (RNAseq) and assay for transposase-accessible chromatin using sequencing (ATACseq) analyses, FOXK1 was observed to be an important regulator of cardiogenesis. Flow cytometry analyses revealed perturbed cardiogenesis in Foxk1 KO embryoid bodies (EBs). Bulk RNAseq analysis at two developmental stages showed a significant reduction of the cardiac molecular program in Foxk1 KO EBs compared to the control EBs. ATACseq analysis during EB differentiation demonstrated that the chromatin landscape nearby known important regulators of cardiogenesis was significantly relaxed in control EBs compared to Foxk1 KO EBs. Furthermore, we demonstrated that in the absence of FOXK1, cardiac differentiation was markedly impaired by assaying for cardiac Troponin T expression and cardiac contractility. We demonstrate that FOXK1 is an important regulator of cardiogenesis by repressing the Wnt/β-catenin signalling pathway and thereby promoting differentiation. These results identify FOXK1 as an essential transcriptional and epigenetic regulator of cardiovascular development. Mechanistically, FOXK1 represses Wnt signalling to promote the development of cardiac progenitor cells.

Abstract 6049: Exogenous serine promotes cancer stem cells in oral squamous cell carcinoma

Abstract Cancer development and progression is associated with metabolic reprogramming of tumor cells required to meet their proliferative, bioenergetic and survival challenges. Some metabolites, including a non-essential amino acid, serine, have been shown to play a role in tumorigenesis by promoting aggressive cell states, such as cancer stem cells (CSCs). Previous studies from our laboratory showed that the nuclear branch of the Wnt/β-catenin signaling pathway, the β-catenin/CBP/MLL1 axis, enhanced CSC states during oral squamous cell carcinoma (OSCC) evolution through H3K4 trimethylation (H3K4me3) and epigenetic remodeling of the chromatin landscape. Given that metabolism has also been shown to modulate cell plasticity via epigenomic modifications, we have aimed to decode the relationship between serine synthesis and cell identity in OSCC. We have shown that under serine starvation conditions, OSCC cells upregulate steady-state mRNA and protein levels of serine synthesis pathway enzymes. The latter is associated with an increase in the by-product, alpha-ketoglutarate (αKG), a co-substrate for nuclear αKG-dependent dioxygenases, which demethylate histone marker H3K27me3 and de-repress differentiation-associated genes. Furthermore, increased production of αKG is associated with downregulation of the β-catenin/CBP/MLl1 complex and CSC-associated H3K4me3. In this study, we used human OSCC cell lines CAL27 and HSC3, derived from a primary tongue tumor and a metastatic site, respectively. Each cell line was cultured in either complete medium or serine starvation medium. Serine starvation conditions led to a downregulation of H3K27me3 along with H3K4me3, promoting a switch from cancer stem cell to differentiation-enhancing chromatin landscape. The observed changes in cell plasticity were further investigated through tumorsphere formation, a surrogate assay for cancer stem cells. We found that OSCC CAL27 and HSC3 cells readily formed tumorspheres replete with stem cell markers BMI1, KRT14 and SOX2, under non-adhesive conditions when grown in complete media, supporting our findings that utilization of exogenous serine enables OSCC cells to maintain stemness. Our findings suggest that a switch from exogenous serine uptake to the endogenous serine biosynthesis promotes OSCC cell differentiation concomitant with the loss of CSC identity. Citation Format: Stacy Ann Jankowski, Nina C. Hardy, Maria A. Kukuruzinska. Exogenous serine promotes cancer stem cells in oral squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6049.

Abstract 3564: SNAI2 enhances oncogenesis in malignant peripheral nerve sheath tumors

Abstract Background: MPNSTs are highly aggressive soft tissue sarcomas. Recurrent genetic mutations have been linked to its tumorigenesis, including the loss of functional polycomb repressive complex 2 (PRC2). This results in an altered transcription landscape characterized by the global loss of methylation on histone H3 lysine 27 (H3K27me3) and increased super-enhancer (SE) -associated histone acetylation. We have previously identified SNAI2 as a core SE-driven transcription factor (TF) that is essential for the growth of MPNST. Although SNAI2 is known to be critical for oncogenic processes, including epithelial-to-mesenchymal transition (EMT) and dedifferentiation, its role in maintaining MPNST survival through transcriptomic and epigenetic regulation has not been explored. Methods: Using CRISPR mediated knockout (KO), we targeted the SNAI2 DNA binding domain and confirmed KO via western blot, followed by in vitro phenotypic characterization, including 2D growth assays, anchorage-independent colony formation assays, and scratch assays. Additionally, an integrative analysis of SNAI2 genomic distribution and the transcriptomic effect of its KO was performed using chromatin immunoprecipitation coupled with DNA sequencing (ChIPseq) and RNA sequencing (RNAseq). Finally, an inducible re-expression system was employed to examine the effects on the chromatin landscape with SNAI2 rescue. Results: Genetic loss of SNAI2 slowed the growth of MPNST cells by 50% in comparison to the control. In vitro tumorigenic assays revealed that SNAI2 KO diminished the cells’ capacity in invasion but not in anchorage-independent cell growth. Analysis of the RNAseq results revealed that SNAI2 loss upregulated 790 genes and downregulated 1623 genes (log2fold change> 1, p<0.01), indicating a dominant transcriptional activation role. Gene Set Enrichment Analysis of these SNAI2-regulated genes revealed upregulation of hallmark Myc targets (normalized enrichment score (NES)=2.6, FDR=0.0), whilst genes involved in response to alpha interferon proteins were downregulated (NES=-1.87, FDR=0.002). Interestingly, genes defining EMT were upregulated (NES=1.6, FDR=0.006) in cells upon SNAI2 loss, contrasting its known repressive role in this biological process in other systems. Through integrative analysis of the RNAseq and ChIPseq data, we demonstrate SNAI2 binding to promoters of its regulated targets. Downstream targets of interest include CRELD1, a gene involved in cell adhesion, where SNAI2 KO caused loss of promoter binding and gene downregulation (p<0.001). Conclusion: SNAI2 is an essential SE-driven TF in MPNST cells. Transcriptomic and genomic profiling of SNAI2 through loss- and gain-of-function experiments revealed that it is a critical TF that is involved in key biological processes. Future efforts will focus on designing methods to employ targeted degradation of this essential TF and test preclinical efficacy in treating MPNST. Citation Format: Hilda Jafarah, Béga Murray, Jack Shern, Xiyuan Zhang. SNAI2 enhances oncogenesis in malignant peripheral nerve sheath tumors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3564.

Abstract 1138: Novel SMARCA2 degrading bifunctional molecules as therapeutics in SMARCA4 mutant lung cancer

Abstract Lung cancer is the top cause of cancer mortality. Despite recent advances, the majority of patients with lung cancer still lack effective therapeutic options, underscoring the dire need for additional treatment approaches. Genomic studies have identified frequent mutations in subunits of the SWI/SNF chromatin remodeling complex including SMARCA4 and ARID1A in non-small cell lung cancer with a frequency of up to 33% in advanced stage disease, making it the most frequently mutated complex in lung cancer. Recent reports, and our own data, have identified the paralogue SMARCA2 to be synthetic lethal to SMARCA4 suggesting SMARCA2 is a valuable therapeutic target. However, the discovery of selective inhibitors of SMARCA2 has been challenging. To overcome this hurdle, we have utilized iterative structure-activity relationship (SAR) studies to develop novel, potent and selective SMARCA2 degrading small molecules based on proteolysis targeting chimera (PROTAC) technology. We demonstrated that YD23, our lead SMARCA2 PROTAC, potently and selectively induces degradation of SMARCA2. Using global proteomic analysis and quantification of more than 8000 proteins, we showed that our SMARCA2 PROTAC is highly selective for SMARCA2. Importantly, we showed that YD23 selectively inhibits growth of SMARCA4 mutant lung cancer cells in vitro. Mechanistically, we demonstrated that YD23 reduces chromatin accessibility only in SMARCA4 deficient cells. In particular, YD23 profoundly decreased chromatin accessibility at enhancers of a number of genes including cell cycle and cell growth regulatory genes. Gene expression profiling and pathway analysis indicated that various cell cycle genes were downregulated by YD23 consistent with the reduced chromatin accessibility at their regulatory regions. In conclusion, our study provides a potent chemical probe for studying the synthetic lethal interaction between SMARCA2 and SMARCA4, dissect the chromatin and epigenetic landscape alterations and lay the foundation for future preclinical and clinical development of SMARCA2 degraders as therapeutics. Citation Format: Sasi Kotagiri, Nicholas Blazanin, Yuanxin Xi, Jing Wang, Yonathan Lissanu. Novel SMARCA2 degrading bifunctional molecules as therapeutics in SMARCA4 mutant lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1138.

Abstract 6289: Enhancer reprogramming by novel SMARCA2 degrading PROTACs underlies therapeutic utility in SMARCA4 mutant tumors

Abstract Lung cancer is the top cause of cancer mortality. Despite recent advances, the majority of patients with lung cancer still lack effective therapeutic options, underscoring the dire need for additional treatment approaches. Genomic studies have identified frequent mutations in subunits of the SWI/SNF chromatin remodeling complex including SMARCA4 and ARID1A in non-small cell lung cancer with a frequency of up to 33% in advanced stage disease, making it the most frequently mutated complex in lung cancer. Recent reports, as well as our own data, have identified the paralogue SMARCA2 to be synthetic lethal to SMARCA4 suggesting SMARCA2 is a valuable therapeutic target. However, the discovery of selective inhibitors of SMARCA2 has been challenging. To overcome this hurdle, we have utilized iterative structure-activity relationship (SAR) studies to develop novel, potent and selective SMARCA2 degrading small molecules based on proteolysis targeting chimera (PROTAC) technology. We demonstrated that YD23, our lead SMARCA2 PROTAC, potently and selectively induces degradation of SMARCA2. Mechanistically, we demonstrated that YD23 reduces chromatin accessibility only in SMARCA4-deficient cells with profoundly decreased chromatin accessibility at enhancer regions. Gene expression profiling and pathway analysis indicated that core cell cycle genes were downregulated by YD23 and is consistent with reduced chromatin accessibility at their regulatory regions. Furthermore, integrative analysis of RNA-seq and ATAC-seq revealed a strong correlation between gene expression and enhancer accessibility changes by YD23. In particular, we identified the mitotic regulator NEDD9 as a critical target in enhancers that are strongly reduced by YD23 in SMARCA4-deficient lung cancer cells. In conclusion, our study provides a promising chemical probe for studying the synthetic lethal interaction between SMARCA2 and SMARCA4, dissect the chromatin and epigenetic landscape alterations and lay the foundation for future preclinical and clinical development of SMARCA2 degraders as therapeutics. Citation Format: Sasi Kotagiri, Nicholas Blazanin, Yuanxin Xi, Jing Wang, Yonathan Lissanu. Enhancer reprogramming by novel SMARCA2 degrading PROTACs underlies therapeutic utility in SMARCA4 mutant tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6289.

Abstract 3558: Role of FLI portion of EWS::FLI in transcription regulation via modulation of chromatin 3D landscape in Ewing sarcoma

Abstract Ewing sarcoma is an aggressive bone-associated tumor currently treated with dose-intense chemotherapy, radiation, and surgery and it affects adolescents and young adults. The hallmark of Ewing sarcoma is a translocated fusion transcription factor named EWS::FLI that drives the oncogenic process. We hypothesize that FLI portion of EWS::FLI containing a crucial alpha-helix plays a novel role in transcription regulation of thousands of genes by modulating chromatin looping. The hypothesis is based on recent evidences from our lab: an alpha-helix immediately downstream of DNA binding domain as an important player in regulating transcriptional activity and that EWS::FLI has a substantial role in shaping the chromatin landscape of Ewing sarcoma cells. The objective of this study is to elucidate the mechanism underlying transcriptional regulation by FLI. We utilized knockdown/rescue experiments in which EWS::FLI was depleted with shRNA and replaced with constructs containing the ETS DNA Binding Domain (DBD) alone or DBD+ (DBD and 4th alpha-helix). The binding pattern and transcriptional regulation were assessed with CUT&Tag and RNA-Seq respectively. The extent of roles each of these construct play in organization, structure, and function of chromatin are being assessed using Micro-C technique, a variation of Hi-C with improved resolution, higher signal-to-noise ratio and more information on chromatin domain boundaries and chromatin looping. The DNA binding and genomic localization of EWS::FLI was unaltered by the deletion surrounding the DNA binding domain (which contains a 4th alpha-helix) in A673 and TTC466 cells. Despite this similarity in genomic localization and binding, the transcriptional output driven by EWS::FLI was significantly diminished by the deletion. With this current study, we hope to understand if the flanking region neighboring the DNA binding domain contributes to the chromatin architecture remodeling function of EWS::FLI and whether this function could be attributed to the transcriptional output differences in the DBD and DBD+ conditions. Citation Format: Ariunaa Bayanjargal, Cenny Taslim, Jesse Crow, Julia Selich-Anderson, Stephen L. Lessnick. Role of FLI portion of EWS::FLI in transcription regulation via modulation of chromatin 3D landscape in Ewing sarcoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3558.

Abstract 2122: The dual BRM/BRG1 (SMARCA2/4) inhibitor FHD-286 induces differentiation in preclinical models of AML

Abstract The BRG1/BRM-associated factors (BAF) complex (also known as mSWI/SNF) is a critical regulator of the chromatin landscape of the genome. By controlling chromatin accessibility, BAF regulates lineage-specific transcriptional programs, including those important for AML blast cell growth and survival. FHD-286 is a highly potent inhibitor of the BAF catalytic subunits BRM and BRG1 (SMARCA2/4), which demonstrates strong tumor growth inhibition in several AML CDX and PDX models. FHD-286 is being developed for the treatment of relapsed/refractory AML and MDS (see Foghorn website for current status).Investigation of FHD-286 in a broad panel of AML cell lines showed that BAF inhibition affects hematopoietic transcriptional programs important for blast cell self-renewal and identity, agnostic of mutational background. Similarly, FHD-286 demonstrated broad efficacy in ex vivo treatment of AML patient-derived samples from diverse genetic backgrounds, including those with difficult to treat mutational profiles, including mtNPM1, FLT3 ITD, and Inv(3) with EVI1 overexpression. Interestingly, while higher concentrations (≥90 nM) of FHD-286 predominantly induced cytoreduction, lower concentrations (≤30 nM) predominantly led to differentiation-like responses. To investigate this differentiation effect, we performed immunophenotyping of cell lines and primary AML samples following prolonged treatment with FHD-286. Extended treatment (7+ days) with relevant concentrations (5-20 nM) of FHD-286 led to time- and dose-dependent upregulation of the myeloid differentiation marker CD11b, and acquisition of monocyte/metamyelocyte morphology. CD11b+ cells expressed lower levels of the hematopoietic transcription factor PU.1, as well as proliferation and survival proteins Ki67, Myc and BCL2. Additionally, primary patient samples showed a loss of CD34 positivity in blasts and GMP-like cells after 7 days of ex vivo treatment, indicating a decrease in stemness in these populations. Finally, upregulation of CD11b coincided with decreased BRG1 protein, suggesting that immature blasts are characterized by high levels of BRG1. These results suggest that BAF functions to drive transcriptional programs required to maintain AML cells in an undifferentiated state, and that FHD-286 may inhibit AML cell growth by overcoming this differentiation block. Expanding on these findings, we have also demonstrated synergistic activity with multiple combination partners, including cytarabine and decitabine, in vitro. Elaboration of this in both CDX and PDX in vivo models also shows significant survival benefit in difficult to treat mutational backgrounds. Taken together, these findings suggest that FHD-286 is able to target blast progenitor populations that are heavily BRM/BRG1-dependent, and that combination with standard of care agents can achieve profound, mutationally agnostic antitumor activity in AML. Citation Format: Mike Collins, Astrid Thomsen, Ashley Gartin, Gabriel J. Sandoval, Ammar Adam, Sarah Reilly, Laure Delestre, Virginie Penard-Lacronique, Warren Fiskus, Kapil Bhalla, Stephane de Botton, Sam Agresta, Jessica Piel, Murphy Hentemann. The dual BRM/BRG1 (SMARCA2/4) inhibitor FHD-286 induces differentiation in preclinical models of AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2122.

Abstract 5375: Learned phenotypic embeddings enable scalable imputation of high-content molecular data elucidating prognostic chromatin signatures

Abstract Emerging high-content data modalities like functional genomics and spatial proteomics have enormous potential to reveal determinants of phenotypic plasticity that underlie variability in clinical outcomes, but to date these modalities are only collected in modestly sized research cohorts (< 200-400 patients), where we lack power to detect subtype-specific or prognostic signatures. To study intertumor heterogeneity on a much larger scale (>10,000 patients), we developed a machine learning framework based on self-supervised embeddings that allows scalable imputation of high-content data on large standard of care datasets. Our framework starts by learning a phenotypic embedding of tumor state based solely on H&E histology images, allowing the embedding to be trained on large patient cohorts regardless of availability of molecular covariates. It then learns to predict genomic or proteomic labels from the lower-dimensional phenotypic embeddings. This model can be used for imputation in much larger cohorts, where only clinical outcome and histology are available. To demonstrate our method, we use the TCGA ATAC-seq data, which is available for 400 patients across 23 cancer types. By learning self-supervised embeddings of histology, our framework was able to impute ATAC-seq for 5,000 peaks in 11,000 patients across 31 cancer types, with high accuracy in held out samples (R2 = 0.61). To our knowledge, this represents the broadest available pan-cancer chromatin landscape. The imputed ATAC-seq reveals a subset of peaks that are significantly associated with overall survival (OS) in multiple cancer types (e.g., Breast (BC) ATAC-only HR 1.75, p: 8.6E-3). Genes proximal to these peaks are strongly enriched for well characterized oncogenes, and also several novel genes with functions in cellular metabolism and chromatin remodeling whose expression is not known to be prognostic in our disease settings. Finally, we developed models to predict OS from H&E slide embeddings and from imputed ATAC-seq, both pan-cancer and in specific tumor types. Both models significantly outperform baseline stage and molecular subtype clinical risk predictors (e.g., BC baseline HR: 2.44, p: 3E-6 vs. embedding/imputed ATAC HR 3.78, 2E-9, p for improvement 2E-9) and, interestingly, we find that adding an ATAC-seq based risk score to an embedding-based risk score significantly improves disease-specific survival prediction (HCC embedding-only HR: 2.13, p: 8E-5 vs. HCC embedding/imputed ATAC HR: 2.65, p: 1E-6). This suggests that histopathology images are a rich source of prognostic information beyond that which is captured by traditional pathologist grading. Overall, our work highlights the ability to use self-supervised embeddings of histopathology to impute biological covariates on large, standard-of-care cohorts, empowering novel insights into disease mechanisms and patient outcome. Citation Format: Christopher S. Probert, Zachary R. McCaw, Navami Jain, Daphne Koller. Learned phenotypic embeddings enable scalable imputation of high-content molecular data elucidating prognostic chromatin signatures. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5375.

Abstract 5754: Determining the regulatory logic of breast cancer cells using single-cell multi-omics

Abstract Cancer cells rewire regulatory elements scattered throughout the genome (such as enhancers) to drive aberrant gene expression. Thus, deconvoluting the regulatory mechanisms that contribute to oncogenic gene expression in cancer cells is key to understanding tumor biology. To this end, we have charted the transcriptional and epigenetic landscape of breast cancer at single-cell resolution to quantitatively link variation in chromatin accessibility to gene expression across malignant and non-malignant cell types. Our comprehensive dataset profiles the chromatin landscape (scATAC-seq) in concert with the transcriptional profiles (scRNA-seq) of 4 breast cancer cell lines, 12 primary breast tumors, and 4 normal mammary reduction tissue specimens collected and processed immediately after surgical resection. This dataset, encompassing over 250,000 individual cells, allowed us to define the regulatory logic of cancer cells by 1) revealing how the epigenome underlies cellular heterogeneity of these tumors in comparison to normal mammary tissue, 2) defining how malignant cells hijack enhancer elements to drive key transcriptional programs in a subtype-specific manner, and 3) annotating which cancer-specific enhancer-to-gene connections portend a worse outcome in patients. Notably, we discovered that cancer cells acquire de novo non-coding enhancer elements to modulate hallmark cancer pathways that were previously hidden using bulk genomics approaches. This highlights the potential for cancer-specific enhancers to serve as markers with diagnostic and prognostic potential, or even serve as tractable targets for therapeutic intervention. Together these data enable the annotation of the cellular composition, transcriptional, and epigenetic landscape of breast tumors to help pinpoint clinically relevant mechanisms of tumorigenesis. Citation Format: Matthew J. Regner, Aatish Thennavan, Susana Garcia-Recio, Kamila Wisniewska, Philip M. Spanheimer, Joel S. Parker, Charles M. Perou, Hector L. Franco. Determining the regulatory logic of breast cancer cells using single-cell multi-omics. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5754.

Abstract 3891: Epigenetic rewiring promotes antiandrogen resistance and metastasis via heterogenous oncogenic drivers in prostate cancer

Abstract Prostate cancer (PCa) is the most diagnosed cancer among American men, which has traditionally been treated through hormone therapy. However, after hormone therapy many patients with PCa still develop a more aggressive stage of PCa, called metastasis castration resistant prostate cancer (mCRPC). The second- generation antiandrogens, such as enzalutamide or apalutamide, are used to competitively inhibit the androgen receptor (AR) signaling and achieved great clinical success. However, most of the mCRPC patients would develop resistance to the targeted therapy drugs within 6 months to 2 years after initial administration. Consequently, understanding the molecular mechanism of antiandrogen resistance, has become a critical endeavor to provide a greater benefit to patients. mCRPC is characterized by extensive heterogeneity of genomic copy number alterations, which may lead to antiandrogen resistance. TP53 and RB1 alterations have been reported to be dramatically enriched in mCRPC neuroendocrine cancer and we have previously found that inactivation of both TP53 and RB1 confers resistance to antiandrogen through lineage plasticity, where cancer cells can transdifferentiate from a luminal lineage to a mixture of basal and neuroendocrine lineages, which is no longer dependent on AR signaling. Despite these exciting discoveries, only 10% of patients have TP53 and RB1 loss and it is estimated that approximately 40% of patients develop antiandrogen resistance through an undiscovered mechanism.To identify more genomic alterations which confer antiandrogen resistance, we performed an in vivo library screening and identified some of the frequently depleted genes as top candidates mediating antiandrogen response, including the chromatin helicase DNA-binding factor (CHD1). Strikingly, the loss of CHD1 establishes an altered chromatin landscape and enables the activation of heterogenous resistant subclones to emerge, including clones with ectopic NR3C1/GR, POU3F2/BRN2, TBX2, and NR2F1. This work provided an innovative model to explain the dramatically increased tumor heterogeneity in therapy resistant prostate cancer and suggested potential druggable targets to overcome resistance. Building on those exciting results, we are now examining the underlying mechanism how the epigenetic rewiring in resistant tumor clones confer resistance and monitoring the evolution of those heterogenous resistant subclones. Furthermore, we are revealing the collaborative function of those four resistant driver genes in promoting metastasis to various distal organs through multidisplinary approaches including single cell transcriptomics and 3D organoid modeling. The completion of this study will not only add clarity to the mechanism of antiandrogen resistance but may also lead to the development of novel biomarker and therapeutic approaches to overcome resistance. Citation Format: Xiaong li, Su Deng, Julisa Gonzalez, Carla Rodriguez Tirado, Choushi Wang, Nickolas A. Johnson, Lauren Metang, Ping Mu. Epigenetic rewiring promotes antiandrogen resistance and metastasis via heterogenous oncogenic drivers in prostate cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3891.

Abstract 4730: ASCL1 activates neuronal stem cell-like lineage programming through the remodeling of the chromatin landscape in prostate cancer

Abstract Introduction: Second-generation AR pathway inhibitors such as Enzalutamide (ENZ) are highly effective in treating castration-resistant prostate cancer (CRPC). However, they play an important role in the emergence of AR-independent phenotypes, including treatment-induced neuroendocrine prostate cancer (tNEPC). Few genetic differences are observed between CRPC and tNEPC, suggesting an epigenetic dysregulation underlining this conversion. Method: To capture the evolution of CRPC to tNEPC, we measured changes in chromatin accessibility of CRPC cells upon exposure to ENZ and in tNEPC cell lines via ATACseq. We performed RNAseq from matched cell lines. We conducted ASCL1, H3K27me3 and EZH2 ChIPseq. Multi-omic analysis were performed integrating RNAseq, ATACseq, ChIPseq, RNA and protein expression. Result: We identified the motif of the transcription factor ASCL1 becoming accessible as early as 3 days, with continued enrichment at 10 days post ENZ-treatment and in NEPC cell-lines. ASCL1 expression and activity are significantly upregulated in NEPC cell lines and patient tumors. Using our unique model of tNEPC, we showed that knockdown of ASCL1 causes extensive chromatin reorganization leading to reduced expression of neuronal and plasticity markers and overall abolishment of the NEPC program. Loss of ASCL1 dysregulated Polycomb repressor complex 2 activity (loss of H3K27 methylation) via di-association of complex due to phosphorylation of EZH2 at T311 resulting in loss of EZH2 binding to the chromatin and likely cause of the H3K27 demethylation. Cell fractionation and confocal microscopy combined with ChIPseq data confirmed loss of EZH2 binding to the chromatin. We identified that loss of ASCL1, phenocopying EZH2 inhibition, reactivated luminal programming and potentially re-sensitizing tNEPC cells to further AR targeted therapies. Altogether, suggests that ASCL1 may drive early transcriptional and epigenetic reprogramming through the PRC2, facilitating the emergence and maintenance of tNEPC. Conclusion: Our research provides the centrality of epigenetic reprogramming in driving the uprising of a plastic phenotype in response to AR pathway inhibition and provides insight into the role of ASCL1 in early drug-induced epigenomic plasticity that supports this reprogramming toward androgen independence. We report that ASCL1 modulates the chromatin dynamics to support a plastic lineage by influencing neuronal stem cell-like regulatory networks. In the treatment-resistant, high plasticity state, inhibition of ASCL1 reverses the lineage to an epithelial-luminal state, providing potential for targeting these highly aggressive tumors. This work provides much-needed insight into ASCL1 function and dependency that together nominates ASCL1 as a bona fide clinical target. Citation Format: Shaghayegh Nouruzi, Dwaipayan Ganguli, Nakisa Tabrizian, Maxim Kobelev, Olena Sivak, Takeshi Namekawa, Alastair Davies, Amina Zoubeidi. ASCL1 activates neuronal stem cell-like lineage programming through the remodeling of the chromatin landscape in prostate cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4730.

Abstract 4743: Wnt/beta-catenin mediated epigenetic modifications drive age-dependent oral squamous cell carcinoma evolution

Abstract Oral Squamous Cell Carcinoma (OSCC) is a devastating disease associated with high morbidity, poor survival, and few therapeutic options. This malignancy is driven, in part, by epigenetic reprogramming of the chromatin landscape directed by β-catenin in complex with CREB-binding protein (CBP) and mixed lineage leukemia methyltransferase 1 (MLL1). The β-catenin/CBP/MLL1 complex promotes an open chromatin structure by enabling transcription of genes associated with cell plasticity, including cancer stem cells and cells with partial EMT (p-EMT) phenotypes. Growing evidence indicates that cell plasticity and cellular senescence are integral processes shared by cancer and aging. Given that the median age of OSCC diagnosis is 66 years, it is likely that aging promotes OSCC evolution to advanced disease. To examine the effects of aging on OSCC evolution, we adapted a syngeneic mouse model of tobacco-associated oral carcinogenesis. This model utilizes a mouse cell line, 4MOSC1, derived from 4-nitroquinoline-1 oxide- (4NQO)-induced tongue tumor which, when implanted into mouse tongues, generates tumors that recapitulate human OSCC mutanome. The growth of 4MOSC1 derived orthotopic tumors was studied in 6- and 80-week-old mice. Tumors were allowed to develop for 18 days, after which they were harvested and processed for single cell RNA sequencing, histopathology and immunofluorescence (IF) analyses. Results showed that tumors grew at a faster rate and to a greater overall size in old mice compared to their young cohorts. OSCC harvested from the old mice displayed statistically significant reduction in membranous E-cadherin concomitant with increased Cbp and H3K4me3 abundance, suggesting an age-associated upregulation of β-catenin/Cbp/Mll1 epigenetic activity. Increased expression of Bmi1, keratin 14, and podoplanin supported increased cell plasticity in OSCC from aged mice. Further, these tumors exhibited augmented cellular senescence, as judged by the disruption of lamin B1-associated nuclear membrane integrity, and by an increased cell population with markers of senescence from single cell sequencing analyses. These data suggest that aging is associated with increased β-catenin/Cbp/Mll1 epigenetic signaling, cell plasticity and cellular senescence, which collectively contribute to the evolution of OSCC. Citation Format: Emily R. Fisher, Anthony Spinella, Nina C. Hardy, Xaralabos Varelas, Manish Bais, Maria A. Kukuruzinska. Wnt/beta-catenin mediated epigenetic modifications drive age-dependent oral squamous cell carcinoma evolution. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4743.

Plasmodium falciparum gametocytes display global chromatin remodelling during sexual differentiation

BackgroundThe protozoan malaria parasite Plasmodium falciparum has a complex life cycle during which it needs to differentiate into multiple morphologically distinct life forms. A key process for transmission of the disease is the development of male and female gametocytes in the human blood, yet the mechanisms determining sexual dimorphism in these haploid, genetically identical sexual precursor cells remain largely unknown. To understand the epigenetic program underlying the differentiation of male and female gametocytes, we separated the two sexual forms by flow cytometry and performed RNAseq as well as comprehensive ChIPseq profiling of several histone variants and modifications.ResultsWe show that in female gametocytes the chromatin landscape is globally remodelled with respect to genome-wide patterns and combinatorial usage of histone variants and histone modifications. We identified sex specific differences in heterochromatin distribution, implicating exported proteins and ncRNAs in sex determination. Specifically in female gametocytes, the histone variants H2A.Z/H2B.Z were highly enriched in H3K9me3-associated heterochromatin. H3K27ac occupancy correlated with stage-specific gene expression, but in contrast to asexual parasites this was unlinked to H3K4me3 co-occupancy at promoters in female gametocytes.ConclusionsCollectively, we defined novel combinatorial chromatin states differentially organising the genome in gametocytes and asexual parasites and unravelled fundamental, sex-specific differences in the epigenetic code. Our chromatin maps represent an important resource for future understanding of the mechanisms driving sexual differentiation in P. falciparum.

Development's Inaugural Outstanding Paper Prize.

Development's Inaugural Outstanding Paper Prize.

Spurious intragenic transcription is a feature of mammalian cellular senescence and tissue aging.

Mammalian aging is characterized by the progressive loss of tissue function and increased risk for disease. Accumulation of senescent cells in aging tissues partly contributes to this decline, and targeted depletion of senescent cells in vivo ameliorates many age-related phenotypes. The fundamental molecular mechanisms responsible for the decline of cellular health and fitness during senescence and aging are largely unknown. In this study, we investigated whether chromatin-mediated loss of transcriptional fidelity, known to contribute to fitness and survival in yeast and worms, also occurs during human cellular senescence and mouse aging. Our findings reveal aberrant transcription initiation inside genes during senescence and aging that co-occurs with changes in the chromatin landscape. Interventions that alter these spurious transcripts have profound consequences on cellular health, primarily affecting intracellular signal transduction pathways. We propose that age-related spurious transcription promotes a noisy transcriptome and degradation of coherent transcriptional networks.

The Landscape of Accessible Chromatin and Developmental Transcriptome Maps Reveal a Genetic Mechanism of Skeletal Muscle Development in Pigs

The epigenetic regulation mechanism of porcine skeletal muscle development relies on the openness of chromatin and is also precisely regulated by transcriptional machinery. However, fewer studies have exploited the temporal changes in gene expression and the landscape of accessible chromatin to reveal the underlying molecular mechanisms controlling muscle development. To address this, skeletal muscle biopsy samples were taken from Landrace pigs at days 0 (D0), 60 (D60), 120 (D120), and 180 (D180) after birth and were then analyzed using RNA-seq and ATAC-seq. The RNA-seq analysis identified 8554 effective differential genes, among which ACBD7, TMEM220, and ATP1A2 were identified as key genes related to the development of porcine skeletal muscle. Some potential cis-regulatory elements identified by ATAC-seq analysis contain binding sites for many transcription factors, including SP1 and EGR1, which are also the predicted transcription factors regulating the expression of ACBD7 genes. Moreover, the omics analyses revealed regulatory regions that become ectopically active after birth during porcine skeletal muscle development after birth and identified 151,245, 53,435, 30,494, and 40,911 peaks. The enriched functional elements are related to the cell cycle, muscle development, and lipid metabolism. In summary, comprehensive high-resolution gene expression maps were developed for the transcriptome and accessible chromatin during postnatal skeletal muscle development in pigs.

Ornithine aminotransferase supports polyamine synthesis in pancreatic cancer.

There is a need to develop effective therapies for pancreatic ductal adenocarcinoma (PDA), a highly lethal malignancy with increasing incidence1 and poor prognosis2. Although targeting tumour metabolism has been the focus of intense investigation for more than a decade, tumour metabolic plasticity and high risk of toxicity have limited this anticancer strategy3,4. Here we use genetic and pharmacological approaches in human and mouse in vitro and in vivo models to show that PDA has a distinct dependence on de novo ornithine synthesis from glutamine. We find thatthis process, which is mediated through ornithine aminotransferase (OAT), supports polyamine synthesis and is required for tumour growth. This directional OAT activity is usually largely restricted to infancy and contrasts with the reliance of most adult normal tissues and other cancer types on arginine-derived ornithine for polyamine synthesis5,6. This dependency associates with arginine depletion in the PDA tumour microenvironment and is driven by mutant KRAS. ActivatedKRAS induces the expression of OAT and polyamine synthesis enzymes, leading to alterations in the transcriptome and open chromatin landscape in PDA tumour cells. The distinct dependence of PDA, but not normal tissue, on OAT-mediated de novo ornithine synthesis provides an attractive therapeutic window for treating patients with pancreatic cancer with minimal toxicity.

Transcriptomic and Chromatin Landscape Analysis Reveals That Involvement of Pituitary Level Transcription Factors Modulate Incubation Behaviors of Magang Geese.

The incubation behavior of geese seriously affects their egg production performance. Studies on incubation behavior have identified functional genes, but the regulatory architecture relationship between functional genes and chromatin accessibility remains poorly understood. Here, we present an integrated analysis of open chromatin profiles and transcriptome to identify the cis-regulatory element and their potential transcription factors involved in regulating incubation behavior in goose pituitary. Assay for transposase-accessible chromatin sequencing (ATAC-seq) revealed that open chromatin regions increased in the pituitary during the transition from incubation behavior to laying. We identified 920 significant differential accessible regions (DARs) in the pituitary. Compared to the laying stage, most DARs had higher chromatin accessibility in the brooding stage. Motif analysis of open DARs showed that the most significant transcription factor (TF) occupied sites predominantly enriched in motifs binding to the RFX family (RFX5, RFX2, and RFX1). While the majority of TF motifs enriched under sites of the nuclear receptor (NR) family (ARE, GRE, and PGR) in closed DARs at the incubation behavior stage. Footprint analysis indicated that the transcription factor RFX family exhibited higher binding on chromatin at the brooding stage. To further elucidate the effect of changes in chromatin accessibility on gene expression levels, a comparison of the transcriptome revealed 279 differentially expressed genes (DEGs). The transcriptome changes were associated with processes of steroid biosynthesis. By integrating ATAC-seq and RNA-seq, few DARs directly affect incubation behavior by regulating the transcription levels of genes. Five DAR-related DEGs were found to be closely related to maintaining the incubation behavior in geese. Footprinting analysis revealed a set of transcription factors (RFX1, RFX2, RFX3, RFX5, BHLHA15, SIX1, and DUX) which displayed the highest activity at the brooding stage. SREBF2 was predicted to be the unique differentially expressed transcription factor whose mRNA level was down-regulated and enriched in hyper-accessible regions of PRL in the broody stage. In the present study, we comprehensively profiled the transcriptome and chromatin accessibility in the pituitary related to incubation behavior. Our findings provided insight into the identification and analysis of regulatory elements in goose incubation behavior. The epigenetic alterations profiled here can help decipher the epigenetic mechanisms that contribute to the regulation of incubation behavior in birds.

Drug addiction unveils a repressive methylation ceiling in EZH2-mutant lymphoma.

Drug addiction, a phenomenon where cancer cells paradoxically depend on continuous drug treatment for survival, has uncovered cell signaling mechanisms and cancer codependencies. Here we discover mutations that confer drug addiction to inhibitors of the transcriptional repressor polycomb repressive complex 2 (PRC2) in diffuse large B-cell lymphoma. Drug addiction is mediated by hypermorphic mutations in the CXC domain of the catalytic subunit EZH2, which maintain H3K27me3 levels even in the presence of PRC2 inhibitors. Discontinuation of inhibitor treatment leads to overspreading of H3K27me3, surpassing a repressive methylation ceiling compatible with lymphoma cell survival. Exploiting this vulnerability, we show that inhibition of SETD2 similarly induces the spread of H3K27me3 and blocks lymphoma growth. Collectively, our findings demonstrate that constraints on chromatin landscapes can yield biphasic dependencies in epigenetic signaling in cancer cells. More broadly, we highlight how approaches to identify drug addiction mutations can be leveraged to discover cancer vulnerabilities.

SEACells infers transcriptional and epigenomic cellular states from single-cell genomics data

Metacells are cell groupings derived from single-cell sequencing data that represent highly granular, distinct cell states. Here we present single-cell aggregation of cell states (SEACells), an algorithm for identifying metacells that overcome the sparsity of single-cell data while retaining heterogeneity obscured by traditional cell clustering. SEACells outperforms existing algorithms in identifying comprehensive, compact and well-separated metacells in both RNA and assay for transposase-accessible chromatin (ATAC) modalities across datasets with discrete cell types and continuous trajectories. We demonstrate the use of SEACells to improve gene–peak associations, compute ATAC gene scores and infer the activities of critical regulators during differentiation. Metacell-level analysis scales to large datasets and is particularly well suited for patient cohorts, where per-patient aggregation provides more robust units for data integration. We use our metacells to reveal expression dynamics and gradual reconfiguration of the chromatin landscape during hematopoietic differentiation and to uniquely identify CD4 T cell differentiation and activation states associated with disease onset and severity in a Coronavirus Disease 2019 (COVID-19) patient cohort.