Rapid Reprogramming Research Articles

Rapid Transcriptional Reprogramming Triggered by Alteration of the Carbon/Nitrogen Balance Has an Impact on Energy Metabolism in Nostoc sp. PCC 7120.

Nostoc (Anabaena) sp. PCC 7120 is a filamentous cyanobacterial species that fixes N2 to nitrogenous compounds using specialised heterocyst cells. Changes in the intracellular ratio of carbon to nitrogen (C/N balance) is known to trigger major transcriptional reprogramming of the cell, including initiating the differentiation of vegetative cells to heterocysts. Substantial transcriptional analysis has been performed on Nostoc sp. PCC 7120 during N stepdown (low to high C/N), but not during C stepdown (high to low C/N). In the current study, we shifted the metabolic balance of Nostoc sp. PCC 7120 cultures grown at 3% CO2 by introducing them to atmospheric conditions containing 0.04% CO2 for 1 h, after which the changes in gene expression were measured using RNAseq transcriptomics. This analysis revealed strong upregulation of carbon uptake, while nitrogen uptake and metabolism and early stages of heterocyst development were downregulated in response to the shift to low CO2. Furthermore, gene expression changes revealed a decrease in photosynthetic electron transport and increased photoprotection and reactive oxygen metabolism, as well a decrease in iron uptake and metabolism. Differential gene expression was largely attributed to change in the abundances of the metabolites 2-phosphoglycolate and 2-oxoglutarate, which signal a rapid shift from fluent photoassimilation to glycolytic metabolism of carbon after transition to low CO2. This work shows that the C/N balance in Nostoc sp. PCC 7120 rapidly adjusts the metabolic strategy through transcriptional reprogramming, enabling survival in the fluctuating environment.

Regulatory and evolutionary adaptation of yeast to acute lethal ethanol stress.

The yeast Saccharomyces cerevisiae has been the subject of many studies aimed at understanding mechanisms of adaptation to environmental stresses. Most of these studies have focused on adaptation to sub-lethal stresses, upon which a stereotypic transcriptional program called the environmental stress response (ESR) is activated. However, the genetic and regulatory factors that underlie the adaptation and survival of yeast cells to stresses that cross the lethality threshold have not been systematically studied. Here, we utilized a combination of gene expression profiling, deletion-library fitness profiling, and experimental evolution to systematically explore adaptation of S. cerevisiae to acute exposure to threshold lethal ethanol concentrations-a stress with important biotechnological implications. We found that yeast cells activate a rapid transcriptional reprogramming process that is likely adaptive in terms of post-stress survival. We also utilized repeated cycles of lethal ethanol exposure to evolve yeast strains with substantially higher ethanol tolerance and survival. Importantly, these strains displayed bulk growth-rates that were indistinguishable from the parental wild-type strain. Remarkably, these hyper-ethanol tolerant strains had reprogrammed their pre-stress gene expression states to match the likely adaptive post-stress response in the wild-type strain. Our studies reveal critical determinants of yeast survival to lethal ethanol stress and highlight potentially general principles that may underlie evolutionary adaptation to lethal stresses in general.

ALU non-B-DNA conformations, flipons, binary codes and evolution.

ALUs contribute to genetic diversity by altering DNA's linear sequence through retrotransposition, recombination and repair. ALUs also have the potential to form alternative non-B-DNA conformations such as Z-DNA, triplexes and quadruplexes that alter the read-out of information from the genome. I suggest here these structures enable the rapid reprogramming of cellular pathways to offset DNA damage and regulate inflammation. The experimental data supporting this form of genetic encoding is presented. ALU sequence motifs that form non-B-DNA conformations under physiological conditions are called flipons. Flipons are binary switches. They are dissipative structures that trade energy for information. By efficiently targeting cellular machines to active genes, flipons expand the repertoire of RNAs compiled from a gene. Their action greatly increases the informational capacity of linearly encoded genomes. Flipons are programmable by epigenetic modification, synchronizing cellular events by altering both chromatin state and nucleosome phasing. Different classes of flipon exist. Z-flipons are based on Z-DNA and modify the transcripts compiled from a gene. T-flipons are based on triplexes and localize non-coding RNAs that direct the assembly of cellular machines. G-flipons are based on G-quadruplexes and sense DNA damage, then trigger the appropriate protective responses. Flipon conformation is dynamic, changing with context. When frozen in one state, flipons often cause disease. The propagation of flipons throughout the genome by ALU elements represents a novel evolutionary innovation that allows for rapid change. Each ALU insertion creates variability by extracting a different set of information from the neighbourhood in which it lands. By elaborating on already successful adaptations, the newly compiled transcripts work with the old to enhance survival. Systems that optimize flipon settings through learning can adapt faster than with other forms of evolution. They avoid the risk of relying on random and irreversible codon rewrites.

Interferon-mediated Reprogramming of Intracellular Cholesterol Metabolism in Response to Secreted Bacterial Toxins

Abstract Macrophages undergo profound metabolic reprogramming in response to activation signals. However, the purpose and the mechanism of this reprogramming remains poorly understood. Cholesterol is an essential lipid molecule that modulates biochemical and biophysical properties of cellular membranes. Microbes have evolved strategies which specifically exploit the presence of membrane cholesterol to facilitate pathogenesis. Cholesterol-dependent cytolysin (CDC) are pore-forming toxins produced by gram-positive microorganisms that results in loss of membrane integrity, cellular dysfunction, and cell death. Herein, we delineate a unappreciated immune cell strategy that relies on rapid reprogramming of cholesterol homeostasis to avoid the deleterious effects CDCs. We show that interferon signals convey resistance to CDCs in myeloid cells by altering the availability of cholesterol in the plasma membrane (PM). Mechanistic studies reveal that resistance to CDCs occurs as a result of the movement of cholesterol from the PM to the endoplasmic reticulum (ER), and ultimately into an intracellular esterified pool. We also find that IFN-mediated inhibition of cholesterol synthesis is required to ensure maximal resistance to CDC challenge. Finally, we demonstrate that the IFN-mediated inhibition of cholesterol synthesis is dependent on production of the oxysterol, 25-hydroxycholesterol, and that the loss of CH25H renders mice more susceptible to CDC-mediated tissue damage. Together, these data provide evidence for remodeling of membrane lipid composition as a host defense strategy to bacterial toxins, and suggests that metabolic reprogramming of lipid architecture has significance beyond that of supporting immune function.

Roles of cell fusion, hybridization and polyploid cell formation in cancer metastasis.

Cell-cell fusion is a normal biological process playing essential roles in organ formation and tissue differentiation, repair and regeneration. Through cell fusion somatic cells undergo rapid nuclear reprogramming and epigenetic modifications to form hybrid cells with new genetic and phenotypic properties at a rate exceeding that achievable by random mutations. Factors that stimulate cell fusion are inflammation and hypoxia. Fusion of cancer cells with non-neoplastic cells facilitates several malignancy-related cell phenotypes, e.g., reprogramming of somatic cell into induced pluripotent stem cells and epithelial to mesenchymal transition. There is now considerable in vitro, in vivo and clinical evidence that fusion of cancer cells with motile leucocytes such as macrophages plays a major role in cancer metastasis. Of the many changes in cancer cells after hybridizing with leucocytes, it is notable that hybrids acquire resistance to chemo- and radiation therapy. One phenomenon that has been largely overlooked yet plays a role in these processes is polyploidization. Regardless of the mechanism of polyploid cell formation, it happens in response to genotoxic stresses and enhances a cancer cell’s ability to survive. Here we summarize the recent progress in research of cell fusion and with a focus on an important role for polyploid cells in cancer metastasis. In addition, we discuss the clinical evidence and the importance of cell fusion and polyploidization in solid tumors.

The mTOR-glycolytic pathway promotes T-cell immunobiology in oral lichen planus.

Oral lichen planus (OLP) is a T-cell-mediated inflammatory mucosal disease. T cells require rapid metabolic reprogramming for their effector functions after activation by immunologic stimuli. The mammalian target of rapamycin (mTOR) is a central player in the metabolic reprogramming and immune responses of T cells. The present study investigated the role of mTOR in the immunometabolism of OLP. mTOR and its direct target eukaryotic initiation factor 4E binding protein 1 (4E-BP1) were highly phosphorylated in peripheral T cells of OLP patients. Rapamycin-mediated blockage of mTOR activation restrained both T-cell proliferation and DNA synthesis, promoted apoptosis, and decreased Th1/Th2 and Th17/Treg ratios. Dual blockage of mTOR and phosphatidylinositol 3-kinase (PI3K) exerted stronger inhibition on T-cell immunobiology than selective repression of PI3K alone. Rapamycin also blocked the glycolytic pathway in T cells. Moreover, glucose-induced activation of mTOR-glycolytic pathway increased T-cell proliferation, DNA synthesis, and the Th17/Treg ratio, and decreased T-cell apoptosis. In contrast, inhibition of glycolysis by 2-Deoxy-d-glucose (2-DG) yielded the opposite effects on T-cell immunobiology by blocking the mTOR pathway. In conclusion, enhanced activation of the mTOR-glycolytic pathway promoted T-cell immunobiology, suggesting that dysregulation of immunometabolism might be associated with T-cell dysfunction in OLP.

Reprogramming and remodeling: transcriptional and epigenetic regulation of salicylic acid-mediated plant defense.

As a plant hormone, salicylic acid (SA) plays essential roles in plant defense against biotrophic and hemibiotrophic pathogens. Significant progress has been made in understanding the SA biosynthesis pathways and SA-mediated defense signaling networks in the past two decades. Plant defense responses involve rapid and massive transcriptional reprogramming upon the recognition of pathogens. Plant transcription factors and their co-regulators are critical players in establishing a transcription regulatory network and boosting plant immunity. A multitude of transcription factors and epigenetic regulators have been discovered, and their roles in SA-mediated defense responses have been reported. However, our understanding of plant transcriptional networks is still limited. As such, novel genomic tools and bioinformatic techniques will be necessary if we are to fully understand the mechanisms behind plant immunity. Here, we discuss current knowledge, provide an update on the SA biosynthesis pathway, and describe the transcriptional and epigenetic regulation of SA-mediated plant immune responses.

The landscape of tiered regulation of breast cancer cell metabolism

Altered metabolism is a hallmark of cancer, but little is still known about its regulation. In this study, we measure transcriptomic, proteomic, phospho-proteomic and fluxomics data in a breast cancer cell-line (MCF7) across three different growth conditions. Integrating these multiomics data within a genome scale human metabolic model in combination with machine learning, we systematically chart the different layers of metabolic regulation in breast cancer cells, predicting which enzymes and pathways are regulated at which level. We distinguish between two types of reactions, directly and indirectly regulated. Directly-regulated reactions include those whose flux is regulated by transcriptomic alterations (~890) or via proteomic or phospho-proteomics alterations (~140) in the enzymes catalyzing them. We term the reactions that currently lack evidence for direct regulation as (putative) indirectly regulated (~930). Many metabolic pathways are predicted to be regulated at different levels, and those may change at different media conditions. Remarkably, we find that the flux of predicted indirectly regulated reactions is strongly coupled to the flux of the predicted directly regulated ones, uncovering a tiered hierarchical organization of breast cancer cell metabolism. Furthermore, the predicted indirectly regulated reactions are predominantly reversible. Taken together, this architecture may facilitate rapid and efficient metabolic reprogramming in response to the varying environmental conditions incurred by the tumor cells. The approach presented lays a conceptual and computational basis for mapping metabolic regulation in additional cancers.

CAMP/EPAC Signaling Enables ETV2 to Induce Endothelial Cells with High Angiogenesis Potential.

Although the generation of ETV2-induced endothelial cells (iECs) from human fibroblasts serves as a novel therapeutic strategy in regenerative medicine, the process is inefficient, resulting in incomplete iEC angiogenesis. Therefore, we employed chromatin immunoprecipitation (ChIP) sequencing and identified molecular mechanisms underlying ETV2-mediated endothelial transdifferentiation to efficiently produce iECs retaining appropriate functionality in long-term culture. We revealed that the majority of ETV2 targets in human fibroblasts are related to vasculature development and signaling transduction pathways, including Rap1 signaling. From a screening of signaling pathway modulators, we confirmed that forskolin facilitated efficient and rapid iEC reprogramming via activation of the cyclic AMP (cAMP)/exchange proteins directly activated by cAMP (EPAC)/RAP1 axis. The iECs obtained via cAMP signaling activation showed superior angiogenesis invivo as well as invitro. Moreover, these cells could form aligned endothelium along the vascular lumen exvivo when seeded into decellularized liver scaffold. Overall, our study provided evidence that the cAMP/EPAC/RAP1 axis is required for the efficient generation of iECs with angiogenesis potential.

Duplication and diversification of trehalase confers evolutionary advantages on lepidopteran insects.

Gene duplication provides a major source of new genes for evolutionary novelty and ecological adaptation. However, the maintenance of duplicated genes and their relevance to adaptive evolution has long been debated. Insect trehalase (Treh) plays key roles in energy metabolism, growth, and stress recovery. Here, we show that the duplication of Treh in Lepidoptera (butterflies and moths) is linked with their adaptation to various environmental stresses. Generally, two Treh genes are present in insects: Treh1 and Treh2. We report three distinct forms of Treh in lepidopteran insects, where Treh1 was duplicated into two gene clusters (Treh1a and Treh1b). These gene clusters differ in gene expression patterns, enzymatic properties, and subcellular localizations, suggesting that the enzymes probably underwent sub- and/or neofunctionalization in the lepidopteran insects. Interestingly, selective pressure analysis provided significant evidence of positive selection on duplicate Treh1b gene in lepidopteran insect lineages. Most positively selected sites were located in the alpha-helical region, and several sites were close to the trehalose binding and catalytic sites. Subcellular adaptation of duplicate Treh1b driven by positive selection appears to have occurred as a result of selected changes in specific sequences, allowing for rapid reprogramming of duplicated Treh during evolution. Our results suggest that gene duplication of Treh and subsequent functional diversification could increase the survival rate of lepidopteran insects through various regulations of intracellular trehalose levels, facilitating their adaptation to diverse habitats. This study provides evidence regarding the mechanism by which gene family expansion can contribute to species adaptation through gene duplication and subsequent functional diversification.

Herpes simplex viral nucleoprotein creates a competitive transcriptional environment facilitating robust viral transcription and host shut off.

Herpes simplex virus-1 (HSV-1) replicates within the nucleus coopting the host's RNA Polymerase II (Pol II) machinery for production of viral mRNAs culminating in host transcriptional shut off. The mechanism behind this rapid reprogramming of the host transcriptional environment is largely unknown. We identified ICP4 as responsible for preferential recruitment of the Pol II machinery to the viral genome. ICP4 is a viral nucleoprotein which binds double-stranded DNA. We determined ICP4 discriminately binds the viral genome due to the absence of cellular nucleosomes and high density of cognate binding sites. We posit that ICP4's ability to recruit not just Pol II, but also more limiting essential components, such as TBP and Mediator, create a competitive transcriptional environment. These distinguishing characteristics ultimately result in a rapid and efficient reprogramming of the host's transcriptional machinery, which does not occur in the absence of ICP4.

Rapid Irreversible Transcriptional Reprogramming in Human Stem Cells Accompanied by Discordance between Replication Timing and Chromatin Compartment.

SummaryThe temporal order of DNA replication is regulated during development and is highly correlated with gene expression, histone modifications and 3D genome architecture. We tracked changes in replication timing, gene expression, and chromatin conformation capture (Hi-C) A/B compartments over the first two cell cycles during differentiation of human embryonic stem cells to definitive endoderm. Remarkably, transcriptional programs were irreversibly reprogrammed within the first cell cycle and were largely but not universally coordinated with replication timing changes. Moreover, changes in A/B compartment and several histone modifications that normally correlate strongly with replication timing showed weak correlation during the early cell cycles of differentiation but showed increased alignment in later differentiation stages and in terminally differentiated cell lines. Thus, epigenetic cell fate transitions during early differentiation can occur despite dynamic and discordant changes in otherwise highly correlated genomic properties.

Comparative analyses of the variation of the transcriptome and proteome of Rhodobacter sphaeroides throughout growth

BackgroundIn natural environments, bacteria must frequently cope with extremely scarce nutrients. Most studies focus on bacterial growth in nutrient replete conditions, while less is known about the stationary phase. Here, we are interested in global gene expression throughout all growth phases, including the adjustment to deep stationary phase.ResultsWe monitored both the transcriptome and the proteome in cultures of the alphaproteobacterium Rhodobacter sphaeroides, beginning with the transition to stationary phase and at different points of the stationary phase and finally during exit from stationary phase (outgrowth) following dilution with fresh medium. Correlation between the transcriptomic and proteomic changes was very low throughout the growth phases. Surprisingly, even in deep stationary phase, the abundance of many proteins continued to adjust, while the transcriptome analysis revealed fewer adjustments. This pattern was reversed during the first 90 min of outgrowth, although this depended upon the duration of the stationary phase. We provide a detailed analysis of proteomic changes based on the clustering of orthologous groups (COGs), and compare these with the transcriptome.ConclusionsThe low correlation between transcriptome and proteome supports the view that post-transcriptional processes play a major role in the adaptation to growth conditions. Our data revealed that many proteins with functions in transcription, energy production and conversion and the metabolism and transport of amino acids, carbohydrates, lipids, and secondary metabolites continually increased in deep stationary phase. Based on these findings, we conclude that the bacterium responds to sudden changes in environmental conditions by a radical and rapid reprogramming of the transcriptome in the first 90 min, while the proteome changes were modest. In response to gradually deteriorating conditions, however, the transcriptome remains mostly at a steady state while the bacterium continues to adjust its proteome. Even long after the population has entered stationary phase, cells are still actively adjusting their proteomes.

Local Diversification of Methicillin- Resistant Staphylococcus aureus ST239 in South America After Its Rapid Worldwide Dissemination.

The global spread of specific clones of methicillin-resistant Staphylococcus aureus (MRSA) has become a major public health problem, and understanding the dynamics of geographical spread requires worldwide surveillance. Over the past 20 years, the ST239 lineage of MRSA has been recognized as an emerging clone across the globe, with detailed studies focusing on isolates from Europe and Asia. Less is known about this lineage in South America, and, particularly, Brazil where it was the predominant lineage of MRSA in the early 1990s to 2000s. To gain a better understanding about the introduction and spread of ST239 MRSA in Brazil we undertook a comparative phylogenomic analysis of ST239 genomes, adding seven completed, closed Brazilian genomes. Brazilian ST239 isolates grouped in a subtree with those from South American, and Western, romance-language-speaking, European countries, here designated the South American clade. After an initial worldwide radiation in the 1960s and 1970s, we estimate that ST239 began to spread in South America and Brazil in approximately 1988. This clone demonstrates specific genomic changes that are suggestive of local divergence and adaptational change including agrC single-nucleotide polymorphisms variants, and a distinct pattern of virulence-associated genes (mainly the presence of the chp and the absence of sea and sasX). A survey of a geographically and chronologically diverse set of 100 Brazilian ST239 isolates identified this virulence genotype as the predominant pattern in Brazil, and uncovered an unexpectedly high prevalence of agr-dysfunction (30%). ST239 isolates from Brazil also appear to have undergone transposon (IS256) insertions in or near global regulatory genes (agr and mgr) that likely led to rapid reprogramming of bacterial traits. In general, the overall pattern observed in phylogenomic analyses of ST239 is of a rapid initial global radiation, with subsequent local spread and adaptation in multiple different geographic locations. Most ST239 isolates harbor the ardA gene, which we show here to have in vivo anti-restriction activity. We hypothesize that this gene may have improved the ability of this lineage to acquire multiple resistance genes and distinct virulence-associated genes in each local context. The allopatric divergence pattern of ST239 also may suggest strong selective pressures for specific traits in different geographical locations.

The Role of miRNAs in Drosophila melanogaster Male Courtship Behavior.

Drosophila melanogaster courtship, although stereotypical, continually changes based on cues received from the courtship subject. Such adaptive responses are mediated via rapid and widespread transcriptomic reprogramming, a characteristic now widely attributed to microRNAs (miRNAs), along with other players. Here, we conducted a large-scale miRNA knockout screen to identify miRNAs that affect various parameters of male courtship behavior. Apart from identifying miRNAs that impact male-female courtship, we observed that miR-957 mutants performed significantly increased male-male courtship and "chaining" behavior, whereby groups of males court one another. We tested the effect of miR-957 reduction in specific neuronal cell clusters, identifying miR-957 activity in Doublesex (DSX)-expressing and mushroom body clusters as an important regulator of male-male courtship interactions. We further characterized the behavior of miR-957 mutants and found that these males court male subjects vigorously, but do not elicit courtship. Moreover, they fail to lower courtship efforts toward females with higher levels of antiaphrodisiac pheromones. At the level of individual pheromones, miR-957 males show a reduced inhibitory response to both 7-Tricosene (7-T) and cis-vaccenyl acetate, with the effect being more pronounced in the case of 7-T. Overall, our results indicate that a single miRNA can contribute to the regulation of complex behaviors, including detection or processing of chemicals that control important survival strategies such as chemical mate-guarding, and the maintenance of sex- and species-specific courtship barriers.

SET domains and stress: uncovering new functions for yeast Set4.

Chromatin dynamics are central to the regulation of gene expression and genome stability, particularly in the presence of environmental signals or stresses that prompt rapid reprogramming of the genome to promote survival or differentiation. While numerous chromatin regulators have been implicated in modulating cellular responses to stress, gaps in our mechanistic understanding of chromatin-based changes during stress suggest that additional proteins are likely critical to these responses and the molecular details underlying their activities are unclear in many cases. We recently identified a role for the relatively uncharacterized SET domain protein Set4 in promoting cell survival during oxidative stress in Saccharomyces cerevisiae. Set4 is a member of the Set3 subfamily of SET domain proteins which are defined by the presence of a PHD finger and divergent SET domain sequences. Here, we integrate our new observations on the function of Set4 with known roles for other related family members, including yeast Set3, fly UpSET and mammalian proteins MLL5 and SETD5. We discuss outstanding questions regarding the molecular mechanisms by which these proteins control gene expression and their potential contributions to cellular responses to environmental stress.

Oncolytic adenovirus Delta-24-RGD induces a widespread glioma proteotype remodeling during autophagy

Adenovirus Delta-24-RGD has shown a remarkable efficacy in a phase I clinical trial for glioblastoma. Delta-24-RGD induces autophagy in glioma cells, however, the molecular derangements associated with Delta-24-RGD infection remains poorly understood. Here, proteomics was applied to characterize the glioma metabolic disturbances soon after Delta-24-RGD internalization and late in infection. Minutes post-infection, a rapid survival reprogramming of glioma cells was evidenced by an early c-Jun activation and a time-dependent dephosphorylation of multiple survival kinases. At 48 h post-infection (hpi), a severe intracellular proteostasis impairment was characterized, detecting differentially expressed proteins related to mRNA splicing, cytoskeletal organization, oxidative response, and inflammation. Specific kinase-regulated protein interactomes for Delta-24-RGD-modulated proteome revealed interferences with the activation dynamics of protein kinases C and A (PKC, PKA), tyrosine-protein kinase Src (c-Src), glycogen synthase kinase-3 (GSK-3) as well as serine/threonine-protein phosphatases 1 and 2A (PP1, PP2A) at 48hpi, in parallel with adenoviral protein overproduction. Moreover, the late activation of the nuclear factor kappa B (NF-κB) correlates with the extracellular increment of specific cytokines involved in migration, and activation of different inflammatory cells. Taken together, our integrative analysis provides further insights into the effects triggered by Delta-24-RGD in the modulation of tumor suppression and immune response against glioma. SignificanceThe current study provides new insights regarding the molecular mechanisms governing the glioma metabolism during Delta-24-RGD oncolytic adenoviral therapy. The compilation and analysis of intracellular and extracellular proteomics have led us to characterize: i) the cell survival reprogramming during Delta-24-RGD internalization, ii) the proteostatic disarrangement induced by Delta-24-RGD during the autophagic stage, iii) the protein interactomes for Delta-24-RGD-modulated proteome, iv) the regulatory effects on kinase dynamics induced by Delta-24-RGD late in infection, and v) the overproduction of multitasking cytokines upon Delta-24-RGD treatment. We consider that the quantitative molecular maps generated in this study may establish the foundations for the development of complementary adenoviral based-vectors to increase the potency against glioma.

Ex vivo human HSC expansion requires coordination of cellular reprogramming with mitochondrial remodeling and p53 activation

AbstractThe limited number of hematopoietic stem cells (HSCs) in umbilical cord blood (UCB) units restricts their use for stem cell transplantation. Ex vivo treatment of UCB-CD34+ cells with valproic acid (VPA) increases the number of transplantable HSCs. In this study, we demonstrate that HSC expansion is not merely a result of proliferation of the existing stem cells but, rather, a result of a rapid reprogramming of CD34+CD90− cells into CD34+CD90+ cells, which is accompanied by limited numbers of cell divisions. Beyond this phenotypic switch, the treated cells acquire and retain a transcriptomic and mitochondrial profile, reminiscent of primary HSCs. Single and bulk RNA-seq revealed a signature highly enriched for transcripts characteristic of primary HSCs. The acquisition of this HSC signature is linked to mitochondrial remodeling accompanied by a reduced activity and enhanced glycolytic potential. These events act in concert with a modest upregulation of p53 activity to limit the levels of reactive oxygen species (ROS). Inhibition of either glycolysis or p53 activity impairs HSC expansion. This study indicates that a complex interplay of events is required for effective ex vivo expansion of UCB-HSCs.

A cell identity switch allows residual BCC to survive Hedgehog pathway inhibition.

Despite the efficacy of Hedgehog pathway inhibitors in the treatment of basal cell carcinoma (BCC)1, residual disease persists in some patients and may contribute to relapse when treatment is discontinued2. Here, to study the effect of the Smoothened inhibitor vismodegib on tumour clearance, we have used a Ptch1-Trp53 mouse model of BCC3 and found that mice treated with vismodegib harbour quiescent residual tumours that regrow upon cessation of treatment. Profiling experiments revealed that residual BCCs initiate a transcriptional program that closely resembles that of stem cells of the interfollicular epidermis and isthmus, whereas untreated BCCs are more similar to the hair follicle bulge. This cell identity switch was enabled by a mostly permissive chromatin state accompanied by rapid Wnt pathway activation and reprogramming of super enhancers to drive activation of key transcription factors involved in cellular identity. Accordingly, treatment of BCC with both vismodegib and a Wnt pathway inhibitor reduced the residual tumour burden and enhanced differentiation. Our study identifies a resistance mechanism in which tumour cells evade treatment by adopting an alternative identity that does not rely on the original oncogenic driver for survival.

Abstract IA23: Enhancer remodeling in response to MEK inhibition in TNBC

Abstract In contrast to acquired resistance to targeted therapeutics via genomic changes, adaptive resistance in cancer involves rapid cellular reprogramming at a nongenomic level. In triple-negative breast cancer (TNBC) a robust transcriptional adaptation to MEK inhibition (MEKi) by the MEK inhibitor, trametinib, is observed in both TNBC patients and preclinical models. This transcriptional adaptation includes upregulation of bypass signaling molecules, including receptor tyrosine kinases (RTKs), to reactivate cellular proliferation programs in the presence of the initial drug challenge. Rather than attempting to antagonize RTK upregulation using a secondary kinase inhibitor in conjunction with the initial, primary targeted therapeutic, we have employed a strategy to broadly inhibit MEKi-induced transcriptional adaptation through the use of BET bromodomain inhibitors. BET protein family bromodomains bind to acetylated lysine moieties of histone subunits or transcription factors to regulate transcriptional elongation via recruitment of P-TEFb, a RNA polymerase II regulatory complex containing CDK9 and Cyclin T1. In TNBC we found the combination of MEKi + BET bromodomain inhibition (BETi) resulted in durable tumor cell growth inhibition not obtained with trametinib alone, nor with different kinase inhibitor combinations. Importantly, we observed in vivo MEKi + BETi synergy in orthotopic SUM159PT xenograft and T11 and 2225 mouse orthotopic syngeneic transplant models of TNBC. This synergism is not limited to TNBC, as we have shown that MEKi + BETi results in durable growth suppression in HER2+ breast cancer. A central question is the mechanism of BETi efficacy in the context of suppressing adaptation to targeted therapeutics. We asked if BRD4 function at enhancers is critical for adaptive transcription in response to MEK1/2 inhibition by trametinib. We found vast chromatin remodeling in the form of de novo enhancer formation and remodeling in response to trametinib in TNBC cells. Enhancers with pronounced BRD4 density and co-occupied with prototypical enhancer marks (H3K27ac, MED1, H3K4me1) were rapidly (1-4 h) formed genome-wide, including at sites proximal to receptor tyrosine kinase loci including DDR1, KDR, FGFR2, and PDGFRB, each influential in TNBC adaptive resistance. Trametinib-responsive enhancers were remodeled across the genome, but with BETi the total number of enhancers remained near baseline. In fact, BETi was able to disrupt enhancers seeded in response to trametinib. We observed correlation of transcriptional induction of genes proximal to the enhancer density induced by trametinib and a corresponding correlative decrease in transcript levels of the cognate genes of the seeded enhancers with combination MEKi+BETi treatment. Does the enhancer paradigm provide a potential source of pharmacologic targets for attenuating adaptive transcription beyond that of BRD4? In addition to BETi, we found that pharmacologic perturbation of CBP/p300 acetyltransferase, capable of depositing acetylation at H3K27 of enhancers, or core P-TEFb constituent CDK9 abrogated adaptive RTK upregulation elicited by MEKi. BRD4 directly associates with the transcriptional regulator JMJD6, a JmjC family demethylase, and, accordingly, the use of a pan-JmjC family demethylase inhibitor or siRNA targeting JMJD6 diminished RTK adaption. Similar response abrogation was achieved with depletion of the histone H3 lysine 36 methyltransferase NSD3 or by depleting CDK7, whose transcriptional regulatory activity is conferred by phosphorylation of its substrates including CDK9 and the carboxy-terminal domain (CTD) of RNA polymerase II. While small-molecule inhibitors are currently not yet available or are pre-phase I for many of these purported strategies to block adaptive resistance, our preclinical data suggest that protein complexes of drug-induced enhancers may be a largely untapped frontier of epigenetic targets to block adaptive bypass resistance to targeted therapies. Citation Format: Jon Zawistowski, Samantha Bevill, Gary L. Johnson. Enhancer remodeling in response to MEK inhibition in TNBC [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr IA23.