bims-ectoca Biomed News
on Epigenetic control of tolerance in cancer
Issue of 2022‒05‒15
fifteen papers selected by
Ankita Daiya
BITS Pilani

  1. Cancers (Basel). 2022 May 03. pii: 2282. [Epub ahead of print]14(9):
      The Hippo pathway is one of the most important ones in mammals. Its key functions in cell proliferation, tissue growth, repair, and homeostasis make it the most crucial one to be controlled. Many means have been deployed for its regulation, since this pathway is not only composed of core regulatory components, but it also communicates with and regulates various other pathways, making this signalisation even more complex. Its role in cancer has been studied more and more over the past few years, and it presents YAP/TAZ as the major oncogenic actors. In this review, we relate how vital this pathway is for different organs, and how regulatory mechanisms have been bypassed to lead to cancerous states. Most studies present an upregulation status of YAP/TAZ, and urge the need to target them. A focus is made here on gastric carcinogenesis, its main dysregulations, and the major strategies adopted and tested to counteract Hippo pathway disbalance in this disease. Hippo pathway targeting can be achieved by various means, which are described in this review. Many studies have tested different potential molecules, which are detailed hereby. Though not all tested in gastric cancer, they could represent a real interest.
    Keywords:  CD44; LIF; TAZ; YAP; cancer stem cells; cancer therapy; gastric cancer; hippo; verteporfin
  2. Cells. 2022 May 05. pii: 1560. [Epub ahead of print]11(9):
      Three-dimensional genome organization represents an additional layer in the epigenetic regulation of gene expression. Active transcription controlled by enhancers or super-enhancers has been extensively studied. Enhancers or super-enhancers can recruit activators or co-activators to activate target gene expression through long-range chromatin interactions. Chromatin interactions and phase separation play important roles in terms of enhancer or super-enhancer functioning. Silencers are another major type of cis-regulatory element that can mediate gene regulation by turning off or reducing gene expression. However, compared to active transcription, silencer studies are still in their infancy. This review covers the current knowledge of human silencers, especially the roles of chromatin interactions and phase separation in silencers. This review also proposes future directions for human silencer studies.
    Keywords:  chromatin interactions; phase separation; silencers
  3. FASEB J. 2022 May;36 Suppl 1
      RNA binding proteins (RBPs) regulate all aspects of RNA biogenesis from transcription, splicing, translation to degradation, and they have a critical role in cellular homeostasis and functional diversity. Recent studies have indicated that altered expressions of RBPs are associated with many human diseases ranging from neurologic disorders to cancer. The transcriptional coregulator yes-associated protein 1 (YAP1), a critical nuclear effector of the mammalian Hippo pathway, regulates cell fate, cell contact, metabolism, and developmental processes. This study aims to demonstrate a link between YAP1 and nucleophosmin 1 (NPM1) protein. NPM1 is an RNA-binding protein that regulates many cellular activities, including ribosome biogenesis, RNA processing, chromatin remodeling, DNA repair, and genomic stability. Using proteomics approaches, we identified NPM1 from YAP1 protein complexes of androgen-responsive human cancer cells. Our proximity ligation assay demonstrated that YAP1 and NPM1 physically interacted with each other. The interaction between YAP1 and NPM1 occurred in cell nuclei and was regulated by androgen hormone signaling. In addition, our GST-pulldown assay demonstrated that NPM1 formed a protein complex with the proline-rich domain of YAP1. Furthermore, our enhanced RNA interactome capture (eRIC) assay showed that androgen also regulated the interaction of RBPs to polyA+ mRNA within the cell. Consistent with this observation, our eRIC assay combined with the mass spectrometry method enabled identifying distinct RBP patterns in human cancer cells that are genetically related but phenotypically different. These observations indicate that global alterations of RBPs under changing environmental conditions may have important roles in cellular physiology and disease biology.
  4. FASEB J. 2022 May;36 Suppl 1
      Macromolecular crowding within the nucleus contributes to heterogeneity in chromatin packing and thus plays an important role in the regulation of gene expression. Nuclear crowding models predict that a change in chromatin structure will affect gene accessibility and transcription, which may have important functional consequences in cellular stress response. Since these studies have focused on ovarian adenocarcinoma, we aim to determine whether this theory can be extended to other types of cancer. Using differential gene expression analysis on bulk RNA-sequencing data and partial wave spectroscopic (PWS) microscopy imaging of colon and mutant ovarian cancer cell lines, we see that the impact of crowding and chromatin packing on transcription can be extended to other cancer types. Comparison of these results with predictions from the chromatin packing-macromolecular crowding model (CPMC), which relates physical changes in chromatin structure to changes in gene expression patterns, further showed that the complex bidirectional response of transcription to crowding is not cell-line specific. Additionally, we show that the transcriptional response changes when the cellular stressor is a chemotherapeutic agent rather than a benign ion modulating compound. The results have important implications in understanding the role of chromatin structure in cellular stress response and drug resistance in cancer.
  5. FASEB J. 2022 May;36 Suppl 1
      Chromatin accessibility is the ability of nucleosomal DNA to be physically accessed by cellular biomachinery. Accessibility of chromatin is controlled by a slew of post translational modifications to histone proteins, DNA, and chromatin-binding factors. This growing field of research has become increasingly important as chromatin accessibility has a large effect on transcription and gene regulation. Gene and cell cycle regulation must be understood to properly study their misregulation which commonly occurs in most, if not all, cancers. Thus, understanding the organization and regulation of the chromatin state by post translational modifications plays an important role in understanding the dysfunction of cancerous cells. The most basic unit of chromatin is the nucleosome core particle. The nucleosome core particle is made up of an octamer that has two copies each of the 4 histone proteins H2A, H2B, H3, and H4 with DNA wrapping around the octamer itself. It is on the N-terminal tails of these histone proteins that many post-translational modifications take place the regulate the chromatin state. In this study, the effect of histone lysine acetylation on the DNA-histone core interaction will be assessed by its thermal stability and accessibility of nucleosomal DNA to Pst1 Digestion. We propose to assay singly acetylated mononucleosomes at the following sites: H2A: K5; H2B: K5, K12, K15, and K20; H3: K9, K14, K18, K23, K27, K36, K37, K64, K56, K79, K115, and K122; H4: K5, K8, K12, and K16. We predict that, as previously shown with nu'-H3K18ac and nu'-H3K36ac acetylation at lysine sites will facilitate unwrapping of nucleosomal DNA.
  6. FASEB J. 2022 May;36 Suppl 1
      Enhancers control cell type-specific gene expression and are marked by H3K4me1. Active enhancers are further marked by H3K27ac. We identified MLL3/MLL4 as major H3K4me1 methyltransferases and CBP/p300 as the H3K27 acetyltransferases in mammalian cells (EMBO J 2011; eLife 2013). During differentiation of adipocytes, myocytes and ES cells, MLL3/MLL4 co-localize with lineage-determining transcription factors (LDTFs), CBP/p300, Brd4 and MED1 on active enhancers. MLL3/MLL4 are required for enhancer activation, enhancer-promoter interaction, cell type-specific gene expression, and cell differentiation (eLife 2013; PNAS 2016; Nat Comm 2017). MLL3/MLL4 control cell differentiation by orchestrating CBP/p300-mediated enhancer activation (NAR 2017). Ectopic expression of H3.3K4M, an inhibitor of H3K4 methylation, or deletion of the enzymatic SET domain, destabilizes MLL3/MLL4 proteins, prevents enhancer activation, and impairs cell differentiation and tissue development (NAR 2019). Using proteomic approaches, we found that endogenous MLL4 complex associates with SWI/SNF chromatin remodeling complex components in cells. Using adipogenesis as a model system, we establish an interdependent relationship between SWI/SNF complex BAF and MLL4 in promoting cell type-specific enhancer activation by LDTFs, which rectifies seemingly conflicting results from previous studies (Nat Comm 2021). We showed that MLL4 protein, rather than MLL4-mediated H3K4me1, controls p300 recruitment to enhancers during ES cell differentiation, suggesting that MLL4 regulates enhancer activation independent of H3K4me1 (PNAS 2016). We have generated enzyme-dead MLL3/MLL4 double knockin ES cells and mice by CRISPR. Initial analyses indicate that MLL3/MLL4 regulate ES cell differentiation and mouse embryonic development through both enzymatic activity-dependent and -independent mechanisms.
  7. J Med Chem. 2022 May 07.
      Enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase that can change the expression of downstream target genes by catalyzing the trimethylation of lysine 27 of histone H3 (H3K27me3). Studies have found that EZH2 is highly expressed in a variety of tumor tissues and is closely related to the occurrence, development, invasion, and metastasis of tumors; therefore, EZH2 is becoming a new molecular target in antitumor therapy. Tazemetostat (EPZ-6438) was approved in 2020 as the first inhibitor targeting catalytic EZH2 for the treatment of epithelioid sarcoma. In addition, a variety of EZH2 inhibitors are being investigated in basic and clinical research for the treatment of tumors, and encouraging results have been obtained. This article systematically reviews the research progress on EZH2 inhibitors and proteolysis targeting chimera (PROTAC)-based EZH2 degradation agents with a focus on their design strategies, structure-activity relationships (SARs), and safety and clinical manifestations.
  8. Trends Genet. 2022 May 08. pii: S0168-9525(22)00077-4. [Epub ahead of print]
      Single-cell transcriptomic approaches have revolutionised the study of complex biological systems, with the routine measurement of gene expression in thousands of cells enabling construction of whole-organism cell atlases. However, the transcriptome is just one layer amongst many that coordinate to define cell type and state and, ultimately, function. In parallel with the widespread uptake of single-cell RNA-seq (scRNA-seq), there has been a rapid emergence of methods that enable multiomic profiling of individual cells, enabling parallel measurement of intercellular heterogeneity in the genome, epigenome, transcriptome, and proteomes. Linking measurements from each of these layers has the potential to reveal regulatory and functional mechanisms underlying cell behaviour in healthy development and disease.
    Keywords:  epigenomics; genomics; multiomics; single-cell; transcriptomics
  9. FASEB J. 2022 May;36 Suppl 1
      Pancreatic cancer is one of the deadliest cancers with 5-year survival rate of ~ 10 percent. Late diagnosis, early metastasis, and resistance to therapeutics necessitate a better treatment approach for pancreatic cancer. Alterations in epigenetic profile through DNA and histone modifications enable tumor growth and metastasis. Evading the control of growth suppressors that regulate cell cycle is also a major hallmark of cancer pathophysiology. We aimed to study a combination therapeutic targeting histone deacetylase (HDAC) and key cell cycle regulator cyclin-dependent kinase (CDK) in pancreatic cancer. We used panobinostat, a pan-HDAC inhibitor approved for multiple myeloma and abemaciclib, an FDA-approved CDK4/6 inhibitor for breast cancer. Our preliminary in vitro data in pancreatic cancer cells depicts synergistic cytotoxicity when treated in combination. In addition, combination treatment resulted in a significant decrease in proliferation and clonogenic survival of pancreatic cancer cells. Our mechanistic studies on the effects of this potent combination on histone modifications, gene expression, and cell cycle regulation will provide insights on drug synergy in pancreatic cancer cells. The results of our in vitro studies show that combining drugs that pharmacologically target two critical aspects of pancreatic cancer proliferation has a high potential for treating this lethal disease and as such merits further investigation.
  10. FASEB J. 2022 May;36 Suppl 1
      The MLL family histone methyltransferases deposit mono-, di-, tri-methylation of histone H3 lysine 4 (H3K4me). Epigenomic studies highlight the discrete distribution of H3K4me3 and H3K4me1 at gene promoters and distal enhancers, respectively. However, how this is achieved remains unclear. We have performed single particle cryo-EM studies for the MLL1 core complex on the nucleosome core particles (NCP). We revealed a surprisingly dynamic nature of the MLL1 complex on the NCP. We show that DPY30 and the intrinsically disordered regions (IDRs) of ASH2L work together to restrict the rotational dynamics of the MLL1 complex, which is necessary for dramatic increase of processivity and activity of the MLL1 complex. The DPY30 and ASH2L-IDR dependent regulation applies to all members of the MLL/SET1 family enzymes. We further show that DPY30 is causal for de novo establishment of H3K4me3 in cells and its preferred localization at gene promoters may be the primary reason for selective enrichment of H3K4me3. Our study provides a new paradigm of how discrete H3K4me state is regulated on chromatin.
  11. Cell Rep. 2022 May 10. pii: S2211-1247(22)00567-8. [Epub ahead of print]39(6): 110800
      Tumors are heterogeneous cellular environments with entwined metabolic dependencies. Here, we use a tumor transcriptome deconvolution approach to profile the metabolic states of cancer and non-cancer (stromal) cells in bulk tumors of 20 solid tumor types. We identify metabolic genes and processes recurrently altered in cancer cells across tumor types, highlighting pan-cancer upregulation of deoxythymidine triphosphate (dTTP) production. In contrast, the tryptophan catabolism rate-limiting enzymes IDO1 and TDO2 are highly overexpressed in stroma, raising the hypothesis that kynurenine-mediated suppression of antitumor immunity may be predominantly constrained by the stroma. Oxidative phosphorylation is the most upregulated metabolic process in cancer cells compared to both stromal cells and a large atlas of cancer cell lines, suggesting that the Warburg effect may be less pronounced in cancer cells in vivo. Overall, our analysis highlights fundamental differences in metabolic states of cancer and stromal cells inside tumors and establishes a pan-cancer resource to interrogate tumor metabolism.
    Keywords:  CP: Cancer; CP: Metabolism; metabolism; tumor microenvironment
  12. Matrix Biol. 2022 May 10. pii: S0945-053X(22)00070-1. [Epub ahead of print]
      OBJECTIVE: Increased matrix stiffness is sensed by the collagen-binding receptor tyrosine kinase discoidin domain receptor 1 (DDR1). We have previously shown that DDR1 stimulates a positive feedback loop to increase its own expression in vascular smooth muscle cells (VSMCs). The transcriptional co-factors YAP/TAZ are stiffness sensing molecules that have not previously been investigated in DDR1 signaling. Here, we test the hypothesis that DDR1 signals through YAP/TAZ to auto-regulate its own expression.APPROACH AND RESULTS: We used vascular smooth muscle cells (VSMCs) from wild-type and DDR1 knockout mice stimulated with collagen and/or substrates of different stiffness. We show that DDR1 controls YAP/TAZ nuclear localization and activity, whereas knockdown of YAP/TAZ attenuates DDR1 expression. In response to increased substrate stiffness, collagen stimulation, or RhoA activation, YAP/TAZ translocate to the nucleus and bind to chromatin. Finally, collagen stimulation promotes increased YAP/TAZ association with the Ddr1 promoter.
    CONCLUSIONS: These findings reveal the mechanism by which DDR1 regulates YAP/TAZ activity which can then mediate positive feedback regulation of DDR1 expression by promoting transcription of the DDR1 gene.
    Keywords:  YAP/TAZ; collagen; discoidin domain receptor 1; smooth muscle; stiffness
  13. Cancer Res. 2022 May 10. pii: canres.2395.2021. [Epub ahead of print]
      Identifying biomarkers predictive of cancer cell response to drug treatment constitutes one of the main challenges in precision oncology. Recent large-scale cancer pharmacogenomic studies have opened new avenues of research to develop predictive biomarkers by profiling thousands of human cancer cell lines at the molecular level and screening them with hundreds of approved drugs and experimental chemical compounds. Many studies have leveraged these data to build predictive models of response using various statistical and machine learning methods. However, a common pitfall to these methods is the lack of interpretability as to how they make predictions, hindering the clinical translation of these models. To alleviate this issue, we used the recent logic modeling approach to develop a new machine learning pipeline that explores the space of bimodally expressed genes in multiple large in vitro pharmacogenomic studies and builds multivariate, nonlinear, yet interpretable logic-based models predictive of drug response. The performance of this approach was showcased in a compendium of the three largest in vitro pharmacogenomic data sets to build robust and interpretable models for 101 drugs that span 17 drug classes with high validation rates in independent datasets. These results along with in vivo and clinical validation, support a better translation of gene expression biomarkers between model systems using bimodal gene expression.
  14. FASEB J. 2022 May;36 Suppl 1
      Anticancer drug response is determined by genetic and epigenetic mechanisms. To identify the epigenetic regulators of anticancer drug response, we conducted a chemical epigenetics screen using chemical probes that target different epigenetic modulators. In this screen, we tested 31 epigenetic probes in combination with 14 mechanistically diverse anticancer agents and measured the viability in A549 lung adenocarcinoma cells by the Resazurin assay. We identified 6 epigenetic probes that significantly potentiated the cytotoxicity of TAK-243, a first-in-class ubiquitin-activating enzyme (UBA1) inhibitor evaluated in several solid and hematologic malignancies. These probes include TP-472, GSK-864, A-196, UNC1999, SGC-CBP30 and PFI-4, and target BRD9/7, mutant IDH1, SUV420H1/H2, EZH2/H1, p300/CBP and BRPF1B, respectively. To validate the screen results, we assessed the viability after combination of TAK-243 and the identified probes and observed 4- to 30-fold potentiation of TAK-243 cytotoxicity in myeloma cells. Moreover, we tested the identified probes in 13 additional cell lines and observed profound potentiation. Upon combination with a panel of anticancer agents, epigenetic probes did not potentiate their cytotoxicity suggesting the observed effects are selective for TAK-243. In contrast to epigenetic probes, negative chemical controls did not have a significant impact on TAK-243 cytotoxicity. As assessed by immunoblotting, potentiation of TAK-243 cytotoxicity was associated with reduced ubiquitylation and induction of apoptosis. Using the cellular thermal shift assay (CETSA), UBA1 displayed increased engagement with TAK-243, suggesting the epigenetic probes enhanced intracellular drug accumulation. Mechanistically, the epigenetic probes exerted their potentiation by inhibiting the efflux transporter ABCG2 without inducing significant changes in ubiquitylation pathways or ABCG2 expression levels. Additionally, the identified probes shared significant chemical scaffold similarities with TAK-243. Based on the screen results, we developed a cell-based assay that exploits TAK-243 and ABCG2-overexpressing cells to reliably quantify the ABCG2-inhibitory activity of novel compounds. In conclusion, our study identifies epigenetic probes that profoundly potentiate TAK-243 cytotoxicity through off-target ABCG2 inhibition. We also provide experimental evidence of the inability of negative controls to exclude a subset of off-target effects of chemical probes. Finally, we have developed a robust cell-based assay that can quantitatively evaluate ABCG2 inhibition by drug candidates.
  15. FASEB J. 2022 May;36 Suppl 1
      Core binding factor beta (CBFβ) functions as a binding partner to the RUNX family of DNA binding transcription factors (RUNX1-3) and acts as a transcriptional co-activator by allosterically increasing their DNA binding. According to The Cancer Genome Atlas Program (TCGA) and Genotype-Tissue Expression (GTEx) datasets, high CBFβ expression is correlated with poor disease-free and overall survival across 17 cancer types, including sarcomas. Of particular interest to osteosarcoma (OS) research is the interaction of CBFβ with RUNX2, the master regulator of bone growth and differentiation, also dysregulated in aggressive OS and implicated in chemoresistance. Recent research supports a non-canonical role of CBFβ as a regulator of protein translation initiation. In breast cancer cells, this translation-associated activity occurs via interactions with heterogeneous nuclear ribonucleoprotein K (hnRNPK) and allows CBFβ to influence translation of hundreds of mRNA transcripts. Binding of CBFβ to hnRNPK or RUNX1 appears mutually exclusive, suggesting possible competition between these two roles. We hypothesized that CBFβ plays a role in the translation of RUNX2 and RUNX2-target gene mRNAs in OS and that disruption of this role may result in an antitumor effect. The intertwined nature of the two roles of CBFβ hampers study of each in OS. Thus, we aimed to decouple them to identify which presents the most viable therapeutic target in OS. Using CRISPR/Cas9 we generated a CBFβ knockout (KO) U2OS cell line and evaluated RUNX2 protein and mRNA levels via western blot (WB) and qRT-PCR, respectively. Cycloheximide chase assay and proteasome inhibition were used to evaluate RUNX2 stability and degradation, respectively. Interactions between CBFβ and RUNX2 or hnRNPK were assessed via co-immunoprecipitation. Site-directed mutagenesis (SDM) of CBFβ and transfection of U2OS CBFβ KO cells with FLAG tagged CBFβ mutants was used to prevent RUNX2-CBFβ interaction. WB was used to evaluate changes in subcellular CBFβ localization. Global, as well as protein-specific, changes in de novo protein synthesis were evaluated using the Click-IT system. Finally, in silico modeling was used to design a peptide that mimics a 10 amino acid sequence on RUNX2 in the CBFβ binding interface, which was then evaluated in vitro using a recombinant RUNX2-CBFβ pulldown assay. Our results demonstrate that loss of CBFβ leads to reduced RUNX2 protein expression without changes in RUNX2 mRNA levels. RUNX2 protein half-life is not reduced in CBFβ nor does proteasome inhibition rescue RUNX2 protein levels. CBFβ interacts with hnRNPK in OS cells, and through this binding may perform a role in oncoprotein translation in OS, specifically modulating protein levels of RUNX2. SDM of CBFβ identified important residues for RUNX2 binding, which when targeted via a peptide resulted in a nearly 50% reduction in CBFβ and RUNX2 interaction. Ribosome footprinting combined with RNAseq and immunoprecipitation-mass spectrometry utilizing WT U2OS, CBFβ mutants and peptide-inhibited cells will provide further information on the translational role of CBFβ and potential therapeutic targets in OS.