SMAD2/3-SMYD2 and developmental transcription factors cooperate with cell cycle inhibitors to guide tissue formation.

Tissue formation and organ homeostasis is achieved by precise coordination of proliferation and differentiation of stem cells and progenitors. While deregulation of these processes can result in degenerative disease or cancer, their molecular interplays remain unclear. Here we show that the switch of human pluripotent stem cell (hPSC) self-renewal to differentiation is associated with the induction of distinct cyclin dependent kinase inhibitors (CDKIs). In hPSCs, Activin/Nodal/TGFß signalling maintains CDKIs in a poised state via SMAD2/3-NANOG-OCT4-EZH2-SNON transcriptional complex. Upon gradual differentiation, CDKIs are induced by successive transcriptional complexes between SMAD2/3-SMYD2 and developmental regulators such as EOMES, thereby lengthening the G1 phase. This, in turn, induces SMAD2/3 transcriptional activity by blocking its linker phosphorylation. Such SMAD2/3-CDKI positive feedback loops drive the exit from pluripotency and stepwise cell fate specification that could be harnessed for producing cells for therapeutic applications. Our study uncovers fundamental mechanisms how cell fate specification is interconnected to cell cycle dynamics and provides insight to autonomous circuitries governing tissue self-formation.

The pRb/RBL2-E2F1/4-GCN5 axis regulates cancer stem cell formation and G0 phase entry/exit by paracrine mechanisms.

The lethality, chemoresistance and metastatic characteristics of cancers are associated with phenotypically plastic cancer stem cells (CSCs). How the non-cell autonomous signalling pathways and cell-autonomous transcriptional machinery orchestrate the stem cell-like characteristics of CSCs is still poorly understood. Here we use a quantitative proteomic approach for identifying secreted proteins of CSCs in pancreatic cancer. We uncover that the cell-autonomous E2F1/4-pRb/RBL2 axis balances non-cell-autonomous signalling in healthy ductal cells but becomes deregulated upon KRAS mutation. E2F1 and E2F4 induce whereas pRb/RBL2 reduce WNT ligand expression (e.g. WNT7A, WNT7B, WNT10A, WNT4) thereby regulating self-renewal, chemoresistance and invasiveness of CSCs in both PDAC and breast cancer, and fibroblast proliferation. Screening for epigenetic enzymes identifies GCN5 as a regulator of CSCs that deposits H3K9ac onto WNT promoters and enhancers. Collectively, paracrine signalling pathways are controlled by the E2F-GCN5-RB axis in diverse cancers and this could be a therapeutic target for eliminating CSCs.

BRD9-SMAD2/3 Orchestrates Stemness and Tumorigenesis in Pancreatic Ductal Adenocarcinoma.

BACKGROUND & AIMS: The dismal prognosis of pancreatic ductal adenocarcinoma (PDAC) is linked to the presence of pancreatic cancer stem-like cells (CSCs) that respond poorly to current chemotherapy regimens. The epigenetic mechanisms regulating CSCs are currently insufficiently understood, which hampers the development of novel strategies for eliminating CSCs. METHODS: By small molecule compound screening targeting 142 epigenetic enzymes, we identified that bromodomain-containing protein BRD9, a component of the BAF histone remodeling complex, is a key chromatin regulator to orchestrate the stemness of pancreatic CSCs via cooperating with the TGFß/Activin-SMAD2/3 signaling pathway. RESULTS: Inhibition and genetic ablation of BRD9 block the self-renewal, cell cycle entry into G0 phase and invasiveness of CSCs, and improve the sensitivity of CSCs to gemcitabine treatment. In addition, pharmacological inhibition of BRD9 significantly reduced the tumorigenesis in patient-derived xenografts mouse models and eliminated CSCs in tumors from pancreatic cancer patients. Mechanistically, inhibition of BRD9 disrupts enhancer-promoter looping and transcription of stemness genes in CSCs. CONCLUSIONS: Collectively, the data suggest BRD9 as a novel therapeutic target for PDAC treatment via modulation of CSC stemness.

KMT2A associates with PHF5A-PHF14-HMG20A-RAI1 subcomplex in pancreatic cancer stem cells and epigenetically regulates their characteristics.

Pancreatic cancer (PC), one of the most aggressive and life-threatening human malignancies, is known for its resistance to cytotoxic therapies. This is increasingly ascribed to the subpopulation of undifferentiated cells, known as pancreatic cancer stem cells (PCSCs), which display greater evolutionary fitness than other tumor cells to evade the cytotoxic effects of chemotherapy. PCSCs are crucial for tumor relapse as they possess 'stem cell-like' features that are characterized by self-renewal and differentiation. However, the molecular mechanisms that maintain the unique characteristics of PCSCs are poorly understood. Here, we identify the histone methyltransferase KMT2A as a physical binding partner of an RNA polymerase-associated PHF5A-PHF14-HMG20A-RAI1 protein subcomplex and an epigenetic regulator of PCSC properties and functions. Targeting the protein subcomplex in PCSCs with a KMT2A-WDR5 inhibitor attenuates their self-renewal capacity, cell viability, and in vivo tumorigenicity.

RBL2-E2F-GCN5 guide cell fate decisions during tissue specification by regulating cell-cycle-dependent fluctuations of non-cell-autonomous signaling.

The retinoblastoma family proteins (RBs) and E2F transcription factors are cell-autonomous regulators of cell-cycle progression, but they also impact fate choice in addition to tumor suppression. The range of mechanisms involved remains to be uncovered. Here, we show that RBs, particularly RBL2/p130, repress WNT ligands such as WNT4 and WNT8A, thereby directing ectoderm specification between neural crest to neuroepithelium. RBL2 achieves this function through cell-cycle-dependent cooperation with E2Fs and GCN5 on the regulatory regions of WNT loci, which direct neuroepithelial versus neural crest specification by temporal fluctuations of WNT/ß-catenin and DLL/NOTCH signaling activity. Thus, the RB-E2F bona fide cell-autonomous axis controls cell fate decisions, and RBL2 regulates field effects via WNT ligands. This reveals a non-cell-autonomous function of RBL2-E2F in stem cell and tissue progenitor differentiation that has broader implications for cell-cycle-dependent cell fate specification in organogenesis, adult stem cells, tissue homeostasis, and tumorigenesis.

BRD9-SMAD2/3 orchestrates stemness and tumorigenesis in pancreatic ductal adenocarcinoma.

The dismal prognosis of pancreatic ductal adenocarcinoma (PDAC) is linked to the presence of pancreatic cancer stem-like cells (CSCs) that respond poorly to current chemotherapy regimens. By small molecule compound screening targeting 142 epigenetic enzymes, we identified that bromodomain-containing protein BRD9, a component of the BAF histone remodelling complex, is a key chromatin regulator to orchestrate the stemness of pancreatic CSCs via cooperating with the TGFß/Activin-SMAD2/3 signalling pathway. Inhibition and genetic ablation of BDR9 block the self-renewal, cell cycle entry into G0 phase and invasiveness of CSCs, and improve the sensitivity of CSCs to gemcitabine treatment. In addition, pharmacological inhibition of BRD9 significantly reduced the tumorigenesis in patient-derived xenografts mouse models and eliminated CSCs in tumours from pancreatic cancer patients. Mechanistically, inhibition of BRD9 disrupts enhancer-promoter looping and transcription of stemness genes in CSCs. Collectively, the data suggest BRD9 as a novel therapeutic target for PDAC treatment via modulation of CSC stemness.

Chemically modified neoantigen-based immunotherapy for targeting KRASG12C-driven tumors.

The clinical efficacy and durability of KRASG12C-targeted therapies are limited by the development of resistance mechanisms. Here, we provide a review of recent KRASG12C-targeted therapy and immunotherapy-unifying strategies that utilize covalently modified peptide/MHC class I complexes as tumor-specific neoantigens to tag drug-resistant cancer cells for destruction with hapten-based immunotherapeutics.

Epigenetic and transcriptional regulations prime cell fate before division during human pluripotent stem cell differentiation.

Stem cells undergo cellular division during their differentiation to produce daughter cells with a new cellular identity. However, the epigenetic events and molecular mechanisms occurring between consecutive cell divisions have been insufficiently studied due to technical limitations. Here, using the FUCCI reporter we developed a cell-cycle synchronised human pluripotent stem cell (hPSC) differentiation system for uncovering epigenome and transcriptome dynamics during the first two divisions leading to definitive endoderm. We observed that transcription of key differentiation markers occurs before cell division, while chromatin accessibility analyses revealed the early inhibition of alternative cell fates. We found that Activator protein-1 members controlled by p38/MAPK signalling are necessary for inducing endoderm while blocking cell fate shifting toward mesoderm, and that enhancers are rapidly established and decommissioned between different cell divisions. Our study has practical biomedical utility for producing hPSC-derived patient-specific cell types since p38/MAPK induction increased the differentiation efficiency of insulin-producing pancreatic beta-cells.

Inside the stemness engine: Mechanistic links between deregulated transcription factors and stemness in cancer.

Cell identity is largely determined by its transcriptional profile. In tumour, deregulation of transcription factor expression and/or activity enables cancer cell to acquire a stem-like state characterised by capacity to self-renew, differentiate and form tumours in vivo. These stem-like cancer cells are highly metastatic and therapy resistant, thus warranting a more complete understanding of the molecular mechanisms downstream of the transcription factors that mediate the establishment of stemness state. Here, we review recent research findings that provide a mechanistic link between the commonly deregulated transcription factors and stemness in cancer. In particular, we describe the role of master transcription factors (SOX, OCT4, NANOG, KLF, BRACHYURY, SALL, HOX, FOX and RUNX), signalling-regulated transcription factors (SMAD, ß-catenin, YAP, TAZ, AP-1, NOTCH, STAT, GLI, ETS and NF-κB) and unclassified transcription factors (c-MYC, HIF, EMT transcription factors and P53) across diverse tumour types, thereby yielding a comprehensive overview identifying shared downstream targets, highlighting unique mechanisms and discussing complexities.

3D chromatin architecture and transcription regulation in cancer.

Chromatin has distinct three-dimensional (3D) architectures important in key biological processes, such as cell cycle, replication, differentiation, and transcription regulation. In turn, aberrant 3D structures play a vital role in developing abnormalities and diseases such as cancer. This review discusses key 3D chromatin structures (topologically associating domain, lamina-associated domain, and enhancer-promoter interactions) and corresponding structural protein elements mediating 3D chromatin interactions [CCCTC-binding factor, polycomb group protein, cohesin, and Brother of the Regulator of Imprinted Sites (BORIS) protein] with a highlight of their associations with cancer. We also summarise the recent development of technologies and bioinformatics approaches to study the 3D chromatin interactions in gene expression regulation, including crosslinking and proximity ligation methods in the bulk cell population (ChIA-PET and HiChIP) or single-molecule resolution (ChIA-drop), and methods other than proximity ligation, such as GAM, SPRITE, and super-resolution microscopy techniques.

Rewiring of 3D Chromatin Topology Orchestrates Transcriptional Reprogramming and the Development of Human Dilated Cardiomyopathy.

BACKGROUND: Transcriptional reconfiguration is central to heart failure, the most common cause of which is dilated cardiomyopathy (DCM). The effect of 3-dimensional chromatin topology on transcriptional dysregulation and pathogenesis in human DCM remains elusive. METHODS: We generated a compendium of 3-dimensional epigenome and transcriptome maps from 101 biobanked human DCM and nonfailing heart tissues through highly integrative chromatin immunoprecipitation (H3K27ac [acetylation of lysine 27 on histone H3]), in situ high-throughput chromosome conformation capture, chromatin immunoprecipitation sequencing, assay for transposase-accessible chromatin using sequencing, and RNA sequencing. We used human induced pluripotent stem cell-derived cardiomyocytes and mouse models to interrogate the key transcription factor implicated in 3-dimensional chromatin organization and transcriptional regulation in DCM pathogenesis. RESULTS: We discovered that the active regulatory elements (H3K27ac peaks) and their connectome (H3K27ac loops) were extensively reprogrammed in DCM hearts and contributed to transcriptional dysregulation implicated in DCM development. For example, we identified that nontranscribing NPPA-AS1 (natriuretic peptide A antisense RNA 1) promoter functions as an enhancer and physically interacts with the NPPA (natriuretic peptide A) and NPPB (natriuretic peptide B) promoters, leading to the cotranscription of NPPA and NPPB in DCM hearts. We revealed that DCM-enriched H3K27ac loops largely resided in conserved high-order chromatin architectures (compartments, topologically associating domains) and their anchors unexpectedly had equivalent chromatin accessibility. We discovered that the DCM-enriched H3K27ac loop anchors exhibited a strong enrichment for HAND1 (heart and neural crest derivatives expressed 1), a key transcription factor involved in early cardiogenesis. In line with this, its protein expression was upregulated in human DCM and mouse failing hearts. To further validate whether HAND1 is a causal driver for the reprogramming of enhancer-promoter connectome in DCM hearts, we performed comprehensive 3-dimensional epigenome mappings in human induced pluripotent stem cell-derived cardiomyocytes. We found that forced overexpression of HAND1 in human induced pluripotent stem cell-derived cardiomyocytes induced a distinct gain of enhancer-promoter connectivity and correspondingly increased the expression of their connected genes implicated in DCM pathogenesis, thus recapitulating the transcriptional signature in human DCM hearts. Electrophysiology analysis demonstrated that forced overexpression of HAND1 in human induced pluripotent stem cell-derived cardiomyocytes induced abnormal calcium handling. Furthermore, cardiomyocyte-specific overexpression of Hand1 in the mouse hearts resulted in dilated cardiac remodeling with impaired contractility/Ca2+ handling in cardiomyocytes, increased ratio of heart weight/body weight, and compromised cardiac function, which were ascribed to recapitulation of transcriptional reprogramming in DCM. CONCLUSIONS: This study provided novel chromatin topology insights into DCM pathogenesis and illustrated a model whereby a single transcription factor (HAND1) reprograms the genome-wide enhancer-promoter connectome to drive DCM pathogenesis.

Mechanisms of Cancer Cell Death: Therapeutic Implications for Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a type of cancer that is strongly associated with poor prognosis and short median survival times. In stark contrast to the progress seen in other cancer types in recent decades, discoveries of new treatments in PDAC have been few and far between and there has been little improvement in overall survival (OS). The difficulty in treating this disease is multifactorial, contributed to by late presentation, difficult access to primary tumour sites, an 'immunologically cold' phenotype, and a strong tendency of recurrence likely driven by cancer stem cell (CSC) populations. Furthermore, apparently contrasting roles of tumour components (such as fibrotic stroma) and intracellular pathways (such as autophagy and TGFß) have made it difficult to distinguish beneficial from detrimental drug targets. Despite this, progress has been made in the field, including the determination of mFOLFIRINOX as the standard-of-care adjuvant therapy and the discovery of KRASG12C mutant inhibitors. Moreover, new research, as outlined in this review, has highlighted promising new approaches including the targeting of the tumour microenvironment, enhancement of immunotherapies, epigenetic modulation, and destruction of CSCs.

Pancreatic Cancer Microenvironment and Cellular Composition: Current Understandings and Therapeutic Approaches.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal human solid tumors, despite great efforts in improving therapeutics over the past few decades. In PDAC, the distinct characteristic of the tumor microenvironment (TME) is the main barrier for developing effective treatments. PDAC TME is characterized by a dense stroma, cancer-associated fibroblasts, and immune cells populations that crosstalk to the subpopulations of neoplastic cells that include cancer stem cells (CSCs). The heterogeneity in TME is also exhibited in the diversity and dynamics of acellular components, including the Extracellular matrix (ECM), cytokines, growth factors, and secreted ligands to signaling pathways. These contribute to drug resistance, metastasis, and relapse in PDAC. However, clinical trials targeting TME components have often reported unexpected results and still have not benefited patients. The failures in those trials and various efforts to understand the PDAC biology demonstrate the highly heterogeneous and multi-faceted TME compositions and the complexity of their interplay within TME. Hence, further functional and mechanistic insight is needed. In this review, we will present a current understanding of PDAC biology with a focus on the heterogeneity in TME and crosstalk among its components. We also discuss clinical challenges and the arising therapeutic opportunities in PDAC research.

Extracellular vesicles in pancreatic cancer progression and therapies.

Pancreatic cancer (PC) is one of the leading causes of cancer-related death worldwide due to delayed diagnosis and limited treatments. More than 90% of all pancreatic cancers are pancreatic ductal adenocarcinoma (PDAC). Extensive communication between tumour cells and other cell types in the tumour microenvironment have been identified which regulate cancer hallmarks during pancreatic tumorigenesis via secretory factors and extracellular vesicles (EVs). The EV-capsuled factors not only facilitate tumour growth locally, but also enter circulation and reach distant organs to construct a pre-metastatic niche. In this review, we delineate the key factors in pancreatic ductal adenocarcinoma derived EVs that mediate different tumour processes. Also, we highlight the factors that are related to the crosstalk with cancer stem cells/cancer-initiating cells (CSC/CIC), the subpopulation of cancer cells that can efficiently metastasize and resist currently used chemotherapies. Lastly, we discuss the potential of EV-capsuled factors in early diagnosis and antitumour therapeutic strategies.

Mitochondrial nucleoid in cardiac homeostasis: bidirectional signaling of mitochondria and nucleus in cardiac diseases.

Metabolic function and energy production in eukaryotic cells are regulated by mitochondria, which have been recognized as the intracellular 'powerhouses' of eukaryotic cells for their regulation of cellular homeostasis. Mitochondrial function is important not only in normal developmental and physiological processes, but also in a variety of human pathologies, including cardiac diseases. An emerging topic in the field of cardiovascular medicine is the implication of mitochondrial nucleoid for metabolic reprogramming. This review describes the linear/3D architecture of the mitochondrial nucleoid (e.g., highly organized protein-DNA structure of nucleoid) and how it is regulated by a variety of factors, such as noncoding RNA and its associated R-loop, for metabolic reprogramming in cardiac diseases. In addition, we highlight many of the presently unsolved questions regarding cardiac metabolism in terms of bidirectional signaling of mitochondrial nucleoid and 3D chromatin structure in the nucleus. In particular, we explore novel techniques to dissect the 3D structure of mitochondrial nucleoid and propose new insights into the mitochondrial retrograde signaling, and how it regulates the nuclear (3D) chromatin structures in mitochondrial diseases.

ROS and TGFß: from pancreatic tumour growth to metastasis.

Transforming growth factor ß (TGFß) signalling pathway switches between anti-tumorigenic function at early stages of cancer formation and pro-tumorigenic effects at later stages promoting cancer metastasis. A similar contrasting role has been uncovered for reactive oxygen species (ROS) in pancreatic tumorigenesis. Down-regulation of ROS favours premalignant tumour development, while increasing ROS level in pancreatic ductal adenocarcinoma (PDAC) enhances metastasis. Given the functional resemblance, we propose that ROS-mediated processes converge with the spatial and temporal activation of TGFß signalling and thereby differentially impact early tumour growth versus metastatic dissemination. TGFß signalling and ROS could extensively orchestrate cellular processes and this concerted function can be utilized by cancer cells to facilitate their malignancy. In this article, we revisit the interplay of canonical and non-canonical TGFß signalling with ROS throughout pancreatic tumorigenesis and metastasis. We also discuss recent insight that helps to understand their conflicting effects on different stages of tumour development. These considerations open new strategies in cancer therapeutics.