Disruption Of Gene Expression Research Articles

Cortical RORβ is required for layer 4 transcriptional identity and barrel integrity.

Retinoic acid-related orphan receptor beta (RORβ) is a transcription factor (TF) and marker of layer 4 (L4) neurons, which are distinctive both in transcriptional identity and the ability to form aggregates such as barrels in rodent somatosensory cortex. However, the relationship between transcriptional identity and L4 cytoarchitecture is largely unknown. We find RORβ is required in the cortex for L4 aggregation into barrels and thalamocortical afferent (TCA) segregation. Interestingly, barrel organization also degrades with age in wildtype mice. Loss of RORβ delays excitatory input and disrupts gene expression and chromatin accessibility, with down-regulation of L4 and up-regulation of L5 genes, suggesting a disruption in cellular specification. Expression and binding site accessibility change for many other TFs, including closure of neurodevelopmental TF binding sites and increased expression and binding capacity of activity-regulated TFs. Lastly, a putative target of RORβ, Thsd7a, is down-regulated without RORβ, and Thsd7a knock-out alone disrupts TCA organization in adult barrels.

Abstract NG04: Somatic mutations drive specific but reversible epigenetic heterogeneity states in AML

Abstract Cells from the same tumor can exhibit significant variation in their genomes, epigenomes, and transcriptomes, resulting in distinct cellular phenotypes. Such intra-tumor heterogeneity is a substrate for tumor evolution, e.g. generating cell clones with a growth advantage or chemotherapy resistance, and thus poses a key challenge in cancer therapy. Epigenetic heterogeneity is increasingly recognized as a critical feature of tumors that endows them with biological variability and hence options for subsets of cells to manifest selective growth and survival advantages. However, the source of epigenetic heterogeneity and its potential link to cancer-associated mutations remains unknown. Herein, we have addressed this topic in the context of acute myeloid leukemia, a disease that is usually fatal but that nonetheless is characterized by a relative paucity of somatic mutations. We hypothesize that genetic lesions drive epigenetic heterogeneity, which disrupts gene expression in tumor cell clones, and leads to fitness differences that can be selected for as the cancer evolves. We test the hypothesis in the following three questions about epigenetic heterogeneity. First, what is the source of epigenetic heterogeneity in cancer? We show for the first time that epigenetic clonality is directly linked to specific mutations. Surprisingly, epigenetic alleles tend to occur in specific patterns in patients with particular driver mutations, suggesting that leukemia oncoproteins can destabilize specific loci that are linked to hematopoietic stem cell transcription factors. However, we also identify a sizeable cohort of patients with mutations that do not drive epigenetic allele patterning. Instead these patients manifest extensive epigenetic clonal diversity that apparently evolves in a stochastic manner through natural selection, and which is linked with inferior clinical outcomes. Second, is epigenetic allele diversity a manifestation of transformed cells, or does it precede and hence potentially contribute to malignant transformation? Here we show that development of epigenetic clonality actually precedes prior to transformation and so is demonstrably caused by AML mutations. Notably, mutations in IDH are a potent inducer of epigenetic allele diversity. Third, is epigenetic clonality reversible with epigenetic therapy agents? Remarkably, we find that epigenetic targeted therapy can reverse both epigenetic allele diversity and the transcriptional deregulation that it causes. This may explain why it is that epigenetic therapy can prime tumors for treatment with chemotherapy drugs, by eliminating options for subpopulations of cancer cells to escape. In sum, we show for the first time that epigenetic clonality is directly linked to specific mutations, and epigenetic allele diversity precede and potentially contribute to malignant transformation. Furthermore, epigenetic clonality reversible with epigenetic therapy agents. Together, these findings support the rationale for future studies to rigorously track epigenetic heterogeneity over time in AML patients at relevant time points, and to determine if epiallele reduction associates with improved therapeutic response. Citation Format: Sheng Li. Somatic mutations drive specific but reversible epigenetic heterogeneity states in AML [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr NG04.

Molecular Crosstalk Between Circadian Rhythmicity and the Development of Neurodegenerative Disorders.

Neurodegenerative disorders have been shown to exhibit substantial interconnectedness with circadian rhythmicity. Alzheimer’s patients exhibit high degradation of the suprachiasmatic nucleus (SCN), the central endogenous circadian timekeeper, and Parkinson’s patients have highly disrupted peripheral clock gene expression. Disrupted sleep patterns are highly evident in patients with neurodegenerative diseases; fragmented sleep has been shown to affect tau-protein accumulation in Alzheimer’s patients, and rapid eye movement (REM) behavioral disorder is observed in a significant amount of Parkinson’s patients. Although numerous studies exist analyzing the mechanisms of neurodegeneration and circadian rhythm function independently, molecular mechanisms establishing specific links between the two must be explored further. Thus, in this review, we explore the possible intersecting molecular mechanisms between circadian rhythm and neurodegeneration, with a particular focus on Parkinson’s disease. We provide evidence for potential influences of E3 ligase and poly adenosine diphosphate (ADP-ribose) polymerase 1 (PARP1) activity on neurodegenerative pathology. The cellular stress and subsequent DNA damage signaling imposed by hyperactivity of these multiple molecular systems in addition to aberrant circadian rhythmicity lead to extensive protein aggregation such as α-synuclein pre-formed fibrils (α-Syn PFFs), suggesting a specific molecular pathway linking circadian rhythmicity, PARP1/E3 ligase activity, and Parkinson’s disease.

Speciation and changes in male gene expression in Drosophila.

It has long been acknowledged that changes in the regulation of gene expression may account for major organismal differences. However, we still do not fully understand how changes in gene expression evolve and how do such changes influence organisms' differences. We are even less aware of the impact such changes might have in restricting gene flow between species. Here, we focus on studies of gene expression and speciation in the Drosophila model. We review studies that have identified gene interactions in post-mating reproductive isolation and speciation, particularly those that modulate male gene expression. We also address studies that have experimentally manipulated changes in gene expression to test their effect in post-mating reproductive isolation. We highlight the need for a more in-depth analysis of the role of selection causing disrupted gene expression of such candidate genes in sterile/inviable hybrids. Moreover, we discuss the relevance to incorporate more routinely assays that simultaneously evaluate the potential effects of environmental factors and genetic background in modulating plastic responses in male genes and their potential role in speciation.

Gene expression changes in response to low temperatures in embryos of the kelp grouper, Epinephelus moara

The kelp grouper Epinephelus moara has high economic value and is popular in fisheries and aquaculture in China. In the previous study, we treated the embryos at 16–22 somite stage at 4 °C, −25.7 °C, −140 °C and −196 °C, and successfully obtained surviving embryos in each group. To better understand the molecular changes affected by the low temperatures, we conducted a comparative transcriptome analysis among embryos exposed at 4 °C for 30 min, embryos exposed at −25.7 °C for 30 min and the control group. qPCR assays were conducted for the validation. Signal transduction pathways were highly enriched for the differentially expressed genes. c-Fos, c-Jun, JunD, GADD45, involved in MAPK signaling pathway, were upregulated when embryos were treated at low temperatures. As immediate early genes, Egr-1a and b, and IER2, that respond quickly to the environment stress, their expression increased as well. Hsp70 showed similar expression pattern as immediate early genes. Meanwhile, transcription factors Sox, HES, TFIID, muscle movement and protein synthesis-related genes were downregulated. Taken together, our findings suggest that cooling disrupts gene expression patterns in E. moara embryos. The differentially expressed genes may be involved in cellular resistance against low temperatures, possibly through neural activation, apoptosis, proliferation, differentiation, cellular recovery and heat shock regulation. This study also provides transcriptome dataset of E. moara embryos exposed to cold temperatures for future studies focusing on the molecular effects of cryopreservation.

Prenatal ethanol exposure increases susceptibility to depression- and anxiety-like behavior in adult female offspring and its underlying mechanism

Epidemiological investigations have found that maternal alcohol intake increases the risk of mental illness in offspring. Our study investigated changes of depression- and anxiety-like behaviors in adult offspring caused by prenatal ethanol exposure (PEE) and explored the potential mechanism. After Wistar rats were intragastrically administered ethanol at a dose of 4 g/kg·d on the 9–20 t h days of pregnancy, the offspring were given 21 days of chronic unpredictable mild stress (CUMS) starting from the 9th week after birth. Before CUMS, the behavioral results showed that the PEE offspring appeared excited and anxious. After CUMS, the PEE offspring rats were more sensitive to the same intensity of stimulation, and then the behavioral disorders aggravated. In adult offspring from the PEE group, the intercellular space was enlarged in the hippocampus, and there was a loss of pyramidal cells. The expression of brain-derived neurotrophic factor (BDNF) decreased; the mRNA expression of the glucocorticoid receptor and synaptic plasticity-related genes decreased; the apoptosis-related genes expressed disrupted. In order to determine whether hippocampal injury and dysfunction resulted from ethanol directly or indirectly, we performed in vitro study. The outcome was accompanied by disrupted gene expression related to neurogenesis and synaptic plasticity. PEE increases the susceptibility of adult female offspring to depression- and anxiety-like behaviors, and its mechanism may be related to the toxic effects of ethanol, both directly and indirectly. The latter inhibits the hippocampal BDNF pathway, leading to the disruption of hippocampal neurogenesis, apoptosis and decreased synaptic plasticity.

Development of a tunable method to generate various three-dimensional microstructures by replenishing macromolecules such as extracellular matrix components and polysaccharides

Multicellular spheroids (spheroids) are expected to be a promising approach to mimic in vivo organ functions and cell microenvironments. However, conventional spheroids do not fully consider the existence of extracellular matrices (ECMs). In this study, we developed a tunable method for replenishing macromolecules, including ECM components and polysaccharides, into spheroids without compromising cell viability by injecting a microvolume cell suspension into a high density of methylcellulose dissolved in the culture medium. Adjusting the ECM concentration in the cell suspension enabled the generation of different three-dimensional microstructures, such as “ECM gel capsules”, which contained individually separated cells, and “ECM-loaded spheroids”, which had thin ECM layers between cells. ECM-loaded spheroids with a 30-fold dilution of Matrigel (0.3 mg/ml) showed significantly higher albumin secretion than control spheroids composed of Hep G2 or HuH-7 cells. Additionally, the expression levels of major CYP genes were decreased in ECM gel capsules with undiluted Matrigel (9 mg/ml) compared to those in control spheroids. However, 0.3 mg/ml Matrigel did not disrupt gene expression. Furthermore, cell polarity associated with tight junction proteins (ZO-1 and Claudin-1) and the transporter protein MRP2 was markedly induced by using 0.3 mg/ml Matrigel. Thus, high-performance three-dimensional tissues fabricated by this method are applicable to increasing the efficiency of drug screening and to regenerative medicine.

Voluntary exercise as a preventive strategy against aircraft noise induced cardio/cerebrovascular damage

BackgroundSeveral experimental studies found transportation noise to be associated with oxidative stress‐induced vascular damage, mediated by activation of the NADPH oxidase, uncoupling of endothelial nitric oxide synthase, and vascular infiltration with inflammatory cells. Furthermore, we could show that the AMP‐dependent‐Kinase (AMPK) is involved in the signaling processes leading to exercise‐mediated vascular protection. In the present study, we investigated the activation of AMPK during voluntary exercise as a preventive strategy.Methods and ResultsWild type (WT) mice were exposed to aircraft noise (maximum sound level of 85 dB(A), average sound pressure level of 72 dB(A) for 4 days after 6 weeks of voluntary exercise. We could show that 4 days noise exposure lead to a significant endothelial dysfunction accompanied by and significant increase in oxidative stress. The voluntary exercise training for 6 weeks resulted in a marked improvement of noise induced endothelium dysfunction and activation of ROS sources (NADPH oxidases). The beneficial effects of exercise training in the vasculature are accompanied by improvement of cerebral oxidative stress due to NADPH oxidases (NOX‐1, NOX‐2) and cerebral inflammatory responses (VCAM‐1, PAI‐1, COX‐2, MCP‐1). Transportation noise is disrupting gene expression patterns of circadian rhythm but we could show that exercise training is able to reestablish aortic circadian clock gene expression in noise exposed wild type mice.ConclusionWe here present novel data on exercise training as a protective strategy against negative effects of aircraft noise exposure by conferring AMPK activation. Our data suggest that exercise training might be an attractive mitigation strategy to protect the general population from noise‐induced cardiovascular damage. Especially patients at high cardiovascular risk could benefit from supervised exercise training to prevent additive cardio/cerebrovascular damage in response to transportation noise exposure.Support or Funding InformationGerman Research Foundation (Deutsche Forschungsgemeinschaft) DFG

Postnatal Ethanol Exposure Activates HDAC-Mediated Histone Deacetylation, Impairs Synaptic Plasticity Gene Expression and Behavior in Mice.

Alcohol consumption during pregnancy is widespread and contributes to pediatric neurological defects, including hippocampal and neocortex dysfunction, causing cognitive deficits termed fetal alcohol spectrum disorders. However, the critical mechanisms underlying these brain abnormalities remain poorly described. Using a postnatal ethanol exposure (PEE) animal model and pharmacological, epigenetic, synaptic plasticity-related and behavioral approaches, we discovered a novel persistent epigenetic mechanism of neurodegeneration in neonatal hippocampus and neocortex brain regions and of cognitive decline in adult animals. PEE, which activates caspase-3 (CC3, a neurodegeneration marker), enhanced histone deacetylase (HDAC1-HDAC3) levels and reduced histone 3 (H3) and 4 (H4) acetylation (ac) in mature neurons. PEE repressed the expression of several synaptic plasticity genes, such as brain-derived neurotrophic factor, C-Fos, early growth response 1 (Egr1), and activity-regulated cytoskeleton-associated protein (Arc). Detailed studies on Egr1 and Arc expression revealed HDAC enrichment at their promoter regions. HDAC inhibition with trichostatin A (TSA) before PEE rescued H3ac/H4ac levels and prevented CC3 formation. Antagonism/null mutation of cannabinoid receptor type-1 (CB1R) before PEE to inhibit CC3 production prevented Egr1 and Arc loss via epigenetic events. TSA administration before PEE prevented postnatal ethanol-induced loss of Egr1 and Arc expression and neurobehavioral defects in adult mice via epigenetic remodeling. In adult mice, 3-day TSA administration attenuated PEE-induced behavioral defects. These findings demonstrate that CB1R/HDAC-mediated epigenetic remodeling disrupts gene expression and is a critical step in fetal alcohol spectrum disorder-associated cognitive decline but is reversed by restoration of histone acetylation in the brain.

Genetic engineering of Hoxb8-immortalized hematopoietic progenitors - a potent tool to study macrophage tissue migration.

Tumor-associated macrophages (TAMs) are detrimental in most cancers. Controlling their recruitment is thus potentially therapeutic. We previously found that TAMs perform protease-dependent mesenchymal migration in cancer, while macrophages perform amoeboid migration in other tissues. Inhibition of mesenchymal migration correlates with decreased TAM infiltration and tumor growth, providing rationale for a new cancer immunotherapy specifically targeting TAM motility. To identify new effectors of mesenchymal migration, we produced ER-Hoxb8-immortalized hematopoietic progenitors (cells with estrogen receptor-regulated Hoxb8 expression), which show unlimited proliferative ability in the presence of estrogen. The functionality of macrophages differentiated from ER-Hoxb8 progenitors was compared to bone marrow-derived macrophages (BMDMs). They polarized into M1- and M2-orientated macrophages, generated reactive oxygen species (ROS), ingested particles, formed podosomes, degraded the extracellular matrix, adopted amoeboid and mesenchymal migration in 3D, and infiltrated tumor explants ex vivo using mesenchymal migration. We also used the CRISPR/Cas9 system to disrupt gene expression of a known effector of mesenchymal migration, WASP (also known as WAS), to provide a proof of concept. We observed impaired podosome formation and mesenchymal migration capacity, thus recapitulating the phenotype of BMDM isolated from Wasp-knockout mice. Thus, we validate the use of ER-Hoxb8-immortalized macrophages as a potent tool to investigate macrophage functionalities.

Enhancer Domains Predict Gene Pathogenicity and Inform Gene Discovery in Complex Disease

Non-coding transcriptional regulatory elements are critical for controlling the spatiotemporal expression of genes. Here, we demonstrate that the sizes and number of enhancers linked to a gene reflect its disease pathogenicity. Moreover, genes with redundant enhancer domains are depleted of cis-acting genetic variants that disrupt gene expression, and they are buffered against the effects of disruptive non-coding mutations. Our results demonstrate that dosage-sensitive genes have evolved a robustness to the disruptive effects of genetic variation by expanding their regulatory domains. This solves a puzzle about why genes associated with human disease are depleted of cis-eQTLs (cis-expression quantitative trait loci), suggesting that this relationship might complicate gene identification in causal genome-wide association studies (GWASs) using eQTL information, and establishes a framework for identifying non-coding regulatory variation with phenotypic consequences.

Optogenetic Control of Gene Expression Using Cryptochrome 2 and a Light-Activated Degron.

Optogenetic tools allow for use of light as an external input to control cellular processes. When applied to regulate the function of transcription factors, optogenetic approaches provide a tunable, reversible, and bidirectional method to control gene expression. Herein, we present a detailed method to induce gene expression in mammalian cells using the light dependent dimerization of cryptochrome 2 (CRY2) and CIB1 to complement a split transcription factor. We also describe a protocol to disrupt gene expression with light by fusing a dimeric transcription factor to CRY2. When combined with a light-induced degron attached to the gene product, this method allows for rapid modulation of target protein abundance.

Synergistic toxicity of microcystin-LR and Cu to zebrafish (Danio rerio)

Toxic cyanobacterial blooms often coincide with metal pollution in a freshwater environment because of surface run-off enriched with nutrients and metals. However, the joint toxic effects of cyanobacterial toxins and metals on aquatic animals remain unknown. In this study, single and joint toxic effects and mechanisms of microcystin-LR (MCLR) and copper (Cu) were investigated in the early development of zebrafish (Danio rerio). The LC5072-h values were 2.79 mg/L for MCLR and 3.23 mg/L for Cu. The sublethal concentrations of MCLR (≤600 μg/L) did not affect the normal development of zebrafish but increased its hatchability. Strong synergistic toxic effects were observed after co-exposure to MCLR and Cu at environmental concentrations (≤60 μg/L). The synergistic toxic effects of these two compounds could be attributed to the increased bioaccumulation of MCLR and Cu, which was mediated by MCLR transporters (e.g., oatp1d1 and oatp2b1) and Cu transporters (e.g., ctr1 and atp7a), in zebrafish. Such bioaccumulation caused oxidative stress, as suggested by the disrupted gene expression of anti-oxidative enzymes (e.g., Cu/Zn-SOD, Mn-SOD, and CAT). Our results revealed for the first time the synergistic toxic effects and potential toxic mechanism of MCLR–Cu in aquatic animals. These synergistic effects should be considered when assessing the ecological risk of toxic cyanobacterial blooms.

SFRP1 inhibited the epithelial ovarian cancer through inhibiting Wnt/β-catenin signaling.

Epithelial ovarian cancer is the most malignant gynecologic neoplasm accounting for 90% of the ovarian cancer patients. Researchers proved that epigenetic alterations could disrupt gene expression as often as genetic alterations. Secreted frizzed related protein (SFRP1), a Wnt antagonist, exerts a significant effect on ovarian cancer. The aim of this research was to investigate the effects and the mechanism of action of SFRP1 on epithelial ovarian cancer. Clinical specimens (including fallopian tubes epithelium from 60 epithelial ovarian cancer patients' and 20 healthy subjects who were undergoing surgical treatments), transgenic mice (overexpressing SFRP1 gene), and 4 epithelial ovarian cancer cell lines (including OVCAR4, SKOV3, COV644, TOV21G) were used in this study. Overexpression of SFRP1 in cells was carried out on OVCAR4 cells by transfection using Lipofectamine 2000. Gene transcription was analyzed by qRT-PCR. The methylation of SFRP1 gene was quantified by methylation-specific PCR. The level of protein expression was measured by Western blot or immunohistochemistry analysis. Cell proliferation was analyzed by CCK8 methods. The ability of cell migration and invasion were measured by wound healing assay and transwell assay. Abnormal expression level and hypermethylation status of SFRP1 were found in clinical epithelial ovarian cancer samples and cell lines. We observed that SFRP1 knockdown could promote proliferation, migration and invasion abilities of epithelial ovarian cancer cells. Additionally, we discovered a potential inhibitory effect of SFRP1 on Wnt/β-catenin signaling pathway in epithelial ovarian cancer cells. Furthermore, the anti-tumor effect of SFRP1 was tested in SFRP1 transgenic mice. SFRP1 inhibited epithelial ovarian cancer through inhibiting Wnt/β-catenin pathway, suggesting that SFRP1 could be considered as a potential therapeutic target in epithelial ovarian cancer.

Inducible SBDS Deficiency Impairs Bone Marrow Niche Function to Engraft Donor Hematopoietic Stem Cell after Transplantation

Hematopoietic stem cell (HSC) transplantation (HSCT) is required for curative therapy for patients with high-risk hematologic malignancies, and a number of non-malignant disorders including inherited bone marrow failure syndromes (iBMFS). Strategies to enhance bone marrow (BM) niche capacity to engraft donor HSC have the potential to improve HSCT outcome by decreasing graft failure rates and enabling reduction in conditioning intensity and regimen-associated complications. Several studies in animal models of iBMFS have demonstrated that BM niche dysfunction contributes to both the pathogenesis of iBMFS, as well as impaired graft function after HSCT. We hypothesize that such iBMFS mouse models are useful tools for discovering targetable niche elements critical for donor engraftment after HSCT. Here, we report the development of a novel mouse model of Shwachman-Diamond Syndrome (SDS) driven by conditional Sbds deletion, which demonstrates profound impairment of healthy donor hematopoietic engraftment after HSCT due to pathway-specific dysfunctional signaling within SBDS-deficient recipient niches. We first attempted to delete Sbds specifically in mature osteoblasts by crossing Sbdsfl/flmice with Col1a1Cre+mice. However, the Col1a1CreSbdsExc progenies are embryonic lethal at E12-E15 stage due to developmental musculoskeletal abnormalities. Alternatively, we generated an inducible SDS mouse model by crossing Sbdsfl/flmice with Mx1Cre+ mice, and inducing Sbds deletion in Mx1-inducible BM hematopoietic and osteolineage niche cells by polyinosinic-polycytidilic acid (pIpC) administration. Compared with Sbdsfl/flcontrols, Mx1CreSbdsExc mice develop significantly decreased platelet counts, an inverted peripheral blood myeloid/lymphoid cell ratio, and reduced long-term HSC within BM, consistent with stress hematopoiesis seen in BMF and myelodysplastic syndromes. To assess whether inducible SBDS deficiency impacts niche function to engraft donor HSC, we transplanted GFP+ wildtype donor BM into pIpC-treated Mx1CreSbdsExc mice and Sbdsfl/flcontrols after 1100 cGy of total body irradiation (TBI). Following transplantation, Mx1CreSbdsExc recipient mice exhibit significantly higher mortality than controls (Figure 1). The decreased survival was related to primary graft failure, as Mx1CreSbdsExc mice exhibit persistent BM aplasia after HSCT and decreased GFP+ reconstitution in competitive secondary transplantation assays. We next sought to identify the molecular and cellular defects within BM niche cells that contribute to the engraftment deficits in SBDS-deficient mice. We performed RNA-seq analysis on the BM stromal cells from irradiated Mx1CreSbdsExc mice versus controls, and the results revealed that SBDS deficiency in BM niche cells caused disrupted gene expression within osteoclast differentiation, FcγR-mediated phagocytosis, and VEGF signaling pathways. Multiplex ELISA assays showed that the BM niche of irradiated Mx1CreSbdsExc mice expresses lower levels of CXCL12, P-selectin and IGF-1, along with higher levels of G-CSF, CCL3, osteopontin and CCL9 than controls. Together, these results suggest that poor donor HSC engraftment in SBDS-deficient mice is likely caused by alterations in niche-mediated donor HSC homing/retention, bone metabolism, host monocyte survival, signaling within IGF-1 and VEGF pathways, and an increased inflammatory state within BM niches. Moreover, flow cytometry analysis showed that compared to controls, the BM niche of irradiated Mx1CreSbdsExc mice contained far fewer megakaryocytes, a hematopoietic cell component of BM niches that we previously demonstrated to be critical in promoting osteoblastic niche expansion and donor HSC engraftment. Taken together, our data demonstrated that SBDS deficiency in BM niches results in reduced capacity to engraft donor HSC. We have identified multiple molecular and cellular defects in the SBDS-deficient niche contributing to this phenotype. Such niche signaling pathway-specific deficits implicate these pathways as critical for donor engraftment during HSCT, and suggest their potential role as targets of therapeutic approaches to enhance donor engraftment and improve HSCT outcome in any condition for which HSCT is required for cure. Disclosures Olson: Merck: Membership on an entity's Board of Directors or advisory committees; Bluebird Bio: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Miltenyi: Honoraria.

Unravelling Heterogeneity of Stem and Progenitor Cells in Myeloid Neoplasms Through Single Cell Multi-omics

Intratumoural heterogeneity (ITH) underlies many of the challenges we face in cancer medicine, including therapy-resistance, disease progression/evolution and relapse after seemingly effective therapy. Distinct tumour cell subpopulations selectively evade therapy and drive disease-progression and technologies that reveal key aspects of ITH are therefore critical for the application of precision cancer medicine. Although bulk genomic analysis has without question provided many insights into genetic ITH, this approach faces a number of fundamental limitations: ITH in cancer occurs at many levels, not restricted to genetics (mutations) but also other factors, such as presence of cancer stem cells in some tumours. Furthermore, bulk genomic analysis reveals patterns of somatic mutations, but not their molecular consequences within distinct (and therapy-resistant) cancer subclones. Whilst many of the scientific questions relating to ITH have remained the same over many decades, our ability to address these questions has advanced dramatically not least because of advances in technology. Ultimately, as the unit of evolution and clonal selection by therapy in cancer is the cell, techniques that resolve heterogeneity at the single-cell level are ideally placed to unravel ITH and provide entirely new insights into cancer biology, with enormous potential to accelerate the development of new approaches to improve outcomes for patients. However, the lack of coverage across key mutation hotspots when studying cancers using single-cell RNA-sequencing techniques has precluded the correlation of genetic and transcriptional readouts from the same single cell, limiting their application to the study of tumors. To overcome such limitation, we developed TARGET-seq, a single cell multi-omic method for the high-sensitivity detection of mutations within single cells in parallel with whole transcriptome analysis. TARGET-seq achieved extremely low allelic dropout rates, allowing resolution of clonal hierarchies with over 98% accuracy, while obtaining unbiased high quality transcriptomes from the same single cell. We have applied TARGET-seq to the study of over ten thousand haematopoietic stem and progenitor cells (HSPCs) from JAK2-mutant myeloproliferative neoplasms. This analysis revealed a high degree of genetic heterogeneity, identifying both linear and branching patterns of clonal evolution. At the transcriptome level different genetic subclones showed distinct transcriptional signatures, indicating that each of them was molecularly distinct. Wild-type cells from MPN patients also showed disrupted gene expression as compared to cells from normal donors, upregulating molecular pathways associated with inflammation (TNFα, TGFβ and IFN signalling). This suggests cell-extrinsic effects disrupting gene expression in non-mutant cells, which has been shown to have prognostic significance and might underlie therapy response. Moreover, TARGET-seq analysis allowed us to identify putative biomarkers of JAK2V617F mutant cells, including novel therapeutic targets to selectively eradicate JAK2-mutant cells and importantly, potential candidates for antibody-based immunotherapy. Analysis of samples from MPN patients undergoing disease transformation to Acute Myeloid Leukemia (sAML) revealed striking patterns of clonal evolution in different immunophenotypically-defined cell types. We identified pre-leukemic and leukemic subclones emerging from hematopoietic stem cells rather than more mature progenitors, in contrast to evolution patterns in de novo AML, which might indicate different cancer stem cell reservoirs. In summary, TARGET-seq allowed us to identify distinct and biologically relevant molecular signatures of different genetic subclones of HSPCs in myeloproliferative neoplasms. TARGET-seq could also be broadly applied to the study of other types of tumours, providing a powerful tool for biomarker and therapeutic target discovery for precision medicine. Disclosures Mead: Bristol Myers-Squibb: Consultancy; Pfizer: Consultancy; Novartis: Consultancy, Honoraria, Other: Travel/accommodation expenses, Research Funding, Speakers Bureau; CTI: Honoraria, Research Funding; Celgene: Consultancy, Research Funding.

First-in-Human Assessment of Feasibility and Safety of Multiplexed Genetic Engineering of Autologous T Cells Expressing NY-ESO -1 TCR and CRISPR/Cas9 Gene Edited to Eliminate Endogenous TCR and PD-1 (NYCE T cells) in Advanced Multiple Myeloma (MM) and Sarcoma

Stadtmauer: Celgene: Consultancy; Takeda: Consultancy; Janssen: Consultancy; Amgen: Consultancy; Novartis: Consultancy, Research Funding; Tmunity: Research Funding; Abbvie: Research Funding. Cohen:Poseida Therapeutics, Inc.: Research Funding. Lacey:Novartis: Patents & Royalties: Patents related to CAR T cell biomarkers; Tmunity: Research Funding; Novartis: Research Funding. Melenhorst:Incyte: Research Funding; Novartis: Research Funding, Speakers Bureau; Parker Institute for Cancer Immunotherapy: Research Funding; Genentech: Speakers Bureau; Stand Up to Cancer: Research Funding; IASO Biotherapeutics, Co: Consultancy; Simcere of America, Inc: Consultancy; Shanghai Unicar Therapy, Co: Consultancy; Colorado Clinical and Translational Sciences Institute: Membership on an entity's Board of Directors or advisory committees; National Institutes of Health: Research Funding. Fraietta:Tmunity: Research Funding; Cabaletta: Research Funding; LEK Consulting: Consultancy. Mangan:amgen: Speakers Bureau; takeda: Speakers Bureau; celgene: Speakers Bureau; janssen: Speakers Bureau. Lancaster:novartis: Research Funding. Suhoski:novartis: Research Funding. Fesnak:Novartis: Research Funding. Young:novartis: Research Funding. Chew:tmunity: Other: Scientific Founder, Research Funding; novartis: Research Funding. Zhao:Tmunity: Membership on an entity's Board of Directors or advisory committees, Research Funding; novartis: Research Funding. Hwang:Novartis: Research Funding; Tmunity: Research Funding. Hexner:novartis: Research Funding. June:Novartis: Research Funding; Tmunity: Other: scientific founder, for which he has founders stock but no income, Patents & Royalties.

Cardiotoxicity and lung toxicity in male rats induced by long-term exposure to iron oxide and silver nanoparticles.

Engineered nanoparticles (NPs) have been increasingly used in numerous fields over the last decade. In particular, iron oxide NPs (Fe2O3NPs) and silver NPs (AgNPs) have contributed to the current increase in NP usage. However, the possible side effects of increased NP exposure remain not fully elucidated. The present study aimed to assess the toxic effects of Fe2O3NPs and AgNPs, both individually and in combination, on the heart and lungs of male rats. To evaluate the in vivo NP toxic effects, the experimental animals were orally administered with Fe2O3NPs (5 mg/kg) and/or AgNPs (50 mg/kg). Animals were treated every day for 79 days. The results demonstrated that at the molecular level, Fe2O3NPs and AgNPs caused marked DNA base oxidation as indicated by the elevated DNA content of 8-hydroxy-2−deoxyguanosine in the heart and lungs. Fe2O3NPs and/or AgNPs decreased paraoxonase 1, antioxidant enzymes, total antioxidant capacity, and reduced glutathione in heart and lung. A dose-dependent increase in production of creatine kinase, thiobarbituric acid-reactive substances, nitric oxide end products, tumor necrosis factor-α, interleukin-6 and lipid profiles was detected. Histological changes were also evident in heart and lung tissues. The two NPs demonstrated similar toxic effects for the majority of factors when co-supplemented. In conclusion, the present study identified that Fe2O3NPs and AgNPs, alone and in combination, induced cardiotoxicity and lung toxicity. Furthermore, findings demonstrated that there was a greater toxic effect due to administration of both NPs compared to individual administration. It was hypothesized that the toxic effects may be mediated through the induction of oxidative DNA damage, lipid peroxidation, shifting redox status, disrupted gene expression, and deregulation in cytokine production.

Exposure to heavy metal-contaminated sediments disrupts gene expression, lipid profile, and life history traits in the midge Chironomus riparius

Despite the concern about anthropogenic heavy metal accumulation, there remain few multi-level ecotoxicological studies to evaluate their effects in fluvial ecosystems. The toxicity of field-collected sediments exhibiting a gradient of heavy metal contamination (Cd, Pb, and Zn) was assessed in Chironomus riparius. For this purpose, larvae were exposed throughout their entire life cycle to these sediments, and toxic effects were measured at different levels of biological organization, from the molecular (lipidomic analysis and transcriptional profile) to the whole organism response (respiration rate, shape markers, and emergence rate). Alterations in the activity of relevant genes, as well as an increase of storage lipids and decrease in membrane fluidity, were detected in larvae exposed to the most contaminated sediments. Moreover, reduced larval and adult mass, decrease of larval respiration rate, and delayed emergence were observed, along with increased mentum and mandible size in larvae and decreased wing loading in adults. This study points out the deleterious effects of heavy metal exposure at various levels of biological organization and provides some clues regarding the mode of toxic action. This integrative approach provides new insights into the multi-level effects on aquatic insects exposed to heavy metal mixtures in field sediments, providing useful tools for ecological risk assessment in freshwater ecosystems.

Genomic instability reflected by elevated Alu retroelement copy-number among workers exposed to diesel engine exhaust

OPS 44: Occupational health studies with environmental implications, Room 411, Floor 4, August 27, 2019, 4:30 PM - 5:30 PM Background/Aim: Millions of transportation and industrial workers worldwide are exposed to diesel engine exhaust (DEE), an established lung carcinogen that contains DNA-damaging constituents. Alu retroelements are repetitive DNA sequences that can multiply, insert throughout the genome, and disrupt gene expression and genomic architecture. Site-specific DNA strand breaks activate Alu retrotransposition and altered Alu repeats have been linked to cancer and mortality risk. However, whether Alu retroelements are influenced by environmental pollutants is largely unexplored. In an occupational setting with high levels of DEE, we investigated associations between personal exposure and Alu copy-number. Methods: A cross-sectional study was conducted in China with 54 DEE-exposed male workers from an engine testing facility and 55 unexposed male controls. Personal air samples were measured for elemental carbon (EC), a DEE surrogate, using NIOSH Method 5040. Leukocyte DNA was extracted from blood samples and quantitative polymerase chain reaction was used to measure Alu copy-number relative to albumin (Alb) single gene copy-number. The unitless Alu/Alb ratio reflects the average Alu copy-number. Linear regression models were used to test for differences in Alu/Alb ratio between controls and DEE-exposed workers, and to evaluate exposure–response relationships across DEE-exposure groups (i.e., controls and EC-tertiles: 6.1–39.0, 39.1–54.5, and 54.6–107.7 µg/m3) adjusted for age and smoking status. Results: DEE-exposed workers had a higher Alu/Alb ratio than unexposed controls (p=0.03). Further, we found a positive exposure-response relationship with EC (p-trend=0.02). The Alu/Alb ratio was largest among DEE-exposed workers in the highest EC-tertile versus controls (1.12+/-0.08 SD versus 1.06+/-0.07 SD, p=0.01). Age, body mass index, smoking, and work years were not associated with Alu/Alb ratio. Conclusions: Our findings suggest that increased levels of DEE exposure may contribute to genomic instability, possibly reflected by Alu copy-number. Further studies are needed to evaluate the influence of environmental pollutants on Alu copy-number in relation to carcinogenesis.