Genes In Embryonic Stem Cells Research Articles

Identification of differentially expressed genes in mouse embryonic stem cell under hypoxia.

Under hypoxia, mouse embryonic stem cells (mESCs) lose the ability to self-renew and begin to differentiate through down-regulation of LIFR-STAT3 pathway via hypoxia-inducible factor-1α (HIF-1α). However, it remains largely unknown what kinds of factors are involved in hypoxia-induced differentiation of mESCs. This study aims to identify the differentially expressed genes (DEGs) in early differentiation of mESCs under hypoxia. Here we utilized a Genefishing techniqueTM to discover the new DEGs during hypoxia-induced early differentiation in CCE mESCs. Next, we investigated the role of DEGs using morphological observation, alkaline phosphatase (ALP) assay, STAT3 activation analysis, and biomarkers analysis for stemness. We detected 19 DEGs under hypoxia and performed cloning with sequencing in six genes. We confirmed the expression patterns of five DEGs including H2afz and GOT1 by realtime PCR assay. Among them, H2afz was significantly decreased under hypoxia, depending on HIF-1α. H2afz-overexpressing CCE mESCs maintained their ALP activity and stem cell markers (Nanog and Rex1), even in hypoxic condition. On the other hand, the early differentiation markers such as FGF5 and STAT5a, which had been increased in hypoxic conditions, were reduced by H2afz overexpression. We discovered that H2afz could be a new target gene that functions in hypoxia-induced differentiation in mESCs and have revealed that it is involved in maintaining the pluripotency of mESCs in the early stages of differentiation. These findings will provide insights into mechanisms of hypoxia-mediated differentiation of mESCs during early development.

Conditional surrender in one generation: determining the reproductive roles of mouse embryo lethal genes by embryo complementation.

The laboratory mouse is the most widely used animal model for studying the genetics and biology of mammalian development and reproduction. Embryonic stem cell (ESC) gene targeting technology, and the sophisticated genomic manipulations it allowed, was unique to this organism for a long period of time; this was a major factor in the mouse's rise to pre-eminence as a model system over the past three decades or so. The recent advent of CRISPR/Cas9 technology has democratized the application of genome editing to essentially all organisms. Nevertheless, the scientific infrastructure behind the mouse still makes it the organism of choice for studying molecular mechanisms of mammalian development, and for modeling human development and disease.

Xeno- and transgene-free reprogramming of mesenchymal stem cells toward the cells expressing neural markers using exosome treatments.

Neural stem cells (NSCs), capable of self-renew and differentiate into neural cells, hold promise for use in studies and treatments for neurological diseases. However, current approaches to obtain NSCs from a live brain are risky and invasive, since NSCs reside in the subventricular zone and the in the hippocampus dentate gyrus. Alternatively, mesenchymal stem cells (MSCs) could be a more available cell source due to their abundance in tissues and easier to access. However, MSCs are committed to producing mesenchymal tissue and are not capable of spontaneously differentiating into neural cells. Thus, the process of reprogramming of MSCs into neural cells to use in clinical and scientific settings has significantly impacted the advancement of regenerative medicine. Previously, our laboratory reported trans-differentiation of MSCs to neural cells through the induced pluripotent stem (iPS) cells state, which was produced by overexpression of the embryonic stem cell gene NANOG. In the current study, we demonstrate that treatment with exosomes derived from NSCs makes MSCs capable of expressing neural cell markers bypassing the generation of iPS cells. An epigenetic modifier, decitabine (5-aza-2'-deoxycytidine), enhanced the process. This novel Xeno and transgene-free trans-differentiation technology eliminates the issues associated with iPS cells, such as tumorigenesis. Thus, it may accelerate the development of neurodegenerative therapies and in vitro neurological disorder models for personalized medicine.

Regulation and Roles of the Nucleolus in Embryonic Stem Cells: From Ribosome Biogenesis to Genome Organization.

The nucleolus is the largest compartment of the eukaryotic cell's nucleus. It acts as a ribosome factory, thereby sustaining the translation machinery. The nucleolus is also the subnuclear compartment with the highest transcriptional activity in the cell, where hundreds of ribosomal RNA (rRNA) genes transcribe the overwhelming majority of RNAs. The structure and composition of the nucleolus change according to the developmental state. For instance, in embryonic stem cells (ESCs), rRNA genes display a hyperactive transcriptional state and open chromatin structure compared with differentiated cells. Increasing evidence indicates that the role of the nucleolus and rRNA genes might go beyond the control of ribosome biogenesis. One such role is linked to the genome architecture, since repressive domains are often located close to the nucleolus. This review highlights recent findings describing how the nucleolus is regulated in ESCs and its role in regulating ribosome biogenesis and genome organization for the maintenance of stem cell identity.

Two Functional Axes of Feedback-Enforced PRC2 Recruitment in Mouse Embryonic StemCells.

SummaryPolycomb Repressive Complex 2 (PRC2) plays an essential role in gene repression during development, catalyzing H3 lysine 27 trimethylation (H3K27me3). MTF2 in the PRC2.1 sub-complex, and JARID2 in PRC2.2, are central in core PRC2 recruitment to target genes in mouse embryonic stem cells (mESCs). To investigate how PRC2.1 and PRC2.2 cooperate, we combined Polycomb mutant mESCs with chemical inhibition of binding to H3K27me3. We find that PRC2.1 and PRC2.2 mediate two distinct paths for recruitment, which are mutually reinforced. Whereas PRC2.1 recruitment is mediated by MTF2 binding to DNA, JARID2-containing PRC2.2 recruitment is more dependent on PRC1. Both recruitment axes are supported by core subunit EED binding to H3K27me3, but EED inhibition exhibits a more pronounced effect in Jarid2 null cells. Finally, we show that PRC1 and PRC2 enhance reciprocal binding. Together, these data disentangle the interdependent interactions that are important for PRC2 recruitment.

Diphtheria toxin-mediated transposon-driven poly (A)-trapping efficiently disrupts transcriptionally silent genes in embryonic stem cells.

Random gene trapping is the application of insertional mutagenesis techniques that are conventionally used to inactivate protein-coding genes in mouse embryonic stem (ES) cells. Transcriptionally silent genes are not effectively targeted by conventional random gene trapping techniques, thus we herein developed an unbiased poly (A) trap (UPATrap) method using a Tol2 transposon, which preferentially integrated into active genes rather than silent genes in ES cells. To achieve efficient trapping at transcriptionally silent genes using random insertional mutagenesis in ES cells, we generated a new diphtheria toxin (DT)-mediated trapping vector, DTrap that removed cells, through the expression of DT that was induced by the promoter activity of the trapped genes, and selected trapped clones using the neomycin-resistance gene of the vector. We found that a double-DT, the dDT vector, dominantly induced the disruption of silent genes, but not active genes, and showed more stable integration in ES cells than the UPATrap vector. The dDT vector disrupted differentiated cell lineage genes, which were silent in ES cells, and labeled trapped clone cells by the expression of EGFP upon differentiation. Thus, the dDT vector provides a systematic approach to disrupt silent genes and examine the cellular functions of trapped genes in the differentiation of target cells and development.

The polycomb group protein PCGF6 mediates germline gene silencing by recruiting histone-modifying proteins to target gene promoters

Polycomb group (PcG) proteins are essential for maintenance of lineage fidelity by coordinating developmental gene expression programs. Polycomb group ring finger 6 (PCGF6) has been previously reported to repress expression of lineage-specific genes, especially germ cell-related genes in mouse embryonic stem cells (ESCs) via the noncanonical polycomb repressive complex PRC1.6. However, the molecular mechanism of this repression remains largely unknown. Here, using RNA-Seq, real-time RT-PCR, immunohistochemistry, immunoprecipitation, and ChIP analyses, we demonstrate that PCGF6 plays an essential role in embryonic development, indicated by the partially penetrant embryonic lethality in homozygous PCGF6 (Pcgf6-/-)-deficient mice. We also found that surviving Pcgf6-deficient mice exhibit reduced fertility. Using the Pcgf6-deficient mice, we observed that ablation of Pcgf6 in somatic tissues robustly derepresses germ cell-related genes. We further provide evidence that these genes are direct targets of PCGF6 in ESCs and that endogenous PCGF6 co-localizes with the histone-modifying proteins G9A histone methyltransferase (G9A)/G9a-like protein (GLP) and histone deacetylase 1/2 (HDAC1/2) on the promoters of the germ cell-related genes. Moreover, the binding of these proteins to their target genes correlated with methylation of Lys-9 of histone 3 and with the status of histone acetylation at these genes. Moreover, the recruitment of G9A/GLP and HDAC1/2 to target promoters depended on the binding of PCGF6. Our findings indicate that PCGF6 has a critical role in safeguarding lineage decisions and in preventing aberrant expression of germ cell-related genes.

TET1 Deficiency Impairs Morphogen-free Differentiation of Human Embryonic Stem Cells to Neuroectoderm

The TET family of 5-methylcytosine (5mC) dioxygenases plays critical roles in development by modifying DNA methylation. Using CRISPR, we inactivated the TET1 gene in H9 human embryonic stem cells (hESCs). Mutant H9 hESCs remained pluripotent, even though the level of hydroxymethylcytosine (5hmC) decreased to 30% of that in wild-type cells. Neural differentiation induced by dual SMAD inhibitors was not significantly affected by loss of TET1 activity. However, in a morphogen-free condition, TET1 deficiency significantly reduced the generation of NESTIN+SOX1+ neuroectoderm cells from 70% in wild-type cells to 20% in mutant cells. This was accompanied by a 20-fold reduction in the expression level of PAX6 and a significant decrease in the amount of 5hmC on the PAX6 promoter. Overexpression of the TET1 catalytic domain in TET1-deficient hESCs significantly increased 5hmC levels and elevated PAX6 expression during differentiation. Consistent with these in vitro data, PAX6 expression was significantly decreased in teratomas formed by TET1-deficient hESCs. However, TET1 deficiency did not prevent the formation of neural tube-like structures in teratomas. Our results suggest that TET1 deficiency impairs the intrinsic ability of hESCs to differentiate to neuroectoderm, presumably by decreasing the expression of PAX6, a key regulator in the development of human neuroectoderm.

Dppa2 and Dppa4 safeguard bivalent chromatin in order to establish a pluripotent epigenome.

Bivalent chromatin domains contain opposing histone modifications that assist cell lineage specification. Two studies report a role for Dppa2 and Dppa4 in the establishment of bivalency and the prevention of de novo DNA methylation at development-related genes in mouse embryonic stem cells.

Role of mitochondrial fission-related genes in mitochondrial morphology and energy metabolism in mouse embryonic stem cells

Mitochondria, the major organelles that produce energy for cell survival and function, dynamically change their morphology via fusion and fission, a process called mitochondrial dynamics. The details of the underlying mechanism of mitochondrial dynamics have not yet been elucidated. Here, we aimed to investigate the function of mitochondrial fission genes in embryonic stem cells (ESCs). To this end, we generated homozygous knockout ESC lines, namely, Fis1−/−, Mff−/−, and Dnm1l−/− ESCs, using the CRISPR-Cas9 system. Interestingly, the Fis1−/−, Mff−/−, and Dnm1l−/− ESCs showed normal morphology, self-renewal, and the ability to differentiate into all three germ layers in vitro. However, transmission electron microscopy showed a significant increase in the cytoplasm to nucleus ratio and mitochondrial elongation in Dnm1l−/− ESCs, which was due to incomplete fission. To assess the change in metabolic energy, we analyzed oxidative phosphorylation (OXPHOS), glycolysis, and the intracellular ATP concentration. The ESC knockout lines showed an increase in OXPHOS, decrease in glycolysis, and an increase in intracellular ATP concentration, which was related to mitochondrial elongation. In particular, the Dnm1l knockout most significantly affected mitochondrial morphology, energy metabolism, and ATP production in ESCs. Furthermore, RNA sequencing and gene ontology analysis showed that the differentially expressed genes in Mff-/- ESCs were distinct from those in Dnm1l−/- or Fis1−/− ESCs. In total, five metabolism-related genes, namely, Aass, Cdo1, Cyp2b23, Nt5e, and Pck2, were expressed in all three knockout ESC lines, and three of them were associated with regulation of ATP generation.

MMTR/Dmap1 Sets the Stage for Early Lineage Commitment of Embryonic Stem Cells by Crosstalk with PcG Proteins.

Chromatin remodeling, including histone modification, chromatin (un)folding, and nucleosome remodeling, is a significant transcriptional regulation mechanism. By these epigenetic modifications, transcription factors and their regulators are recruited to the promoters of target genes, and thus gene expression is controlled through either transcriptional activation or repression. The Mat1-mediated transcriptional repressor (MMTR)/DNA methyltransferase 1 (DNMT1)-associated protein (Dmap1) is a transcription corepressor involved in chromatin remodeling, cell cycle regulation, DNA double-strand break repair, and tumor suppression. The Tip60-p400 complex proteins, including MMTR/Dmap1, interact with the oncogene Myc in embryonic stem cells (ESCs). These proteins interplay with the stem cell-related proteome networks and regulate gene expressions. However, the detailed mechanisms of their functions are unknown. Here, we show that MMTR/Dmap1, along with other Tip60-p400 complex proteins, bind the promoters of differentiation commitment genes in mouse ESCs. Hence, MMTR/Dmap1 controls gene expression alterations during differentiation. Furthermore, we propose a novel mechanism of MMTR/Dmap1 function in early stage lineage commitment of mouse ESCs by crosstalk with the polycomb group (PcG) proteins. The complex controls histone mark bivalency and transcriptional poising of commitment genes. Taken together, our comprehensive findings will help better understand the MMTR/Dmap1-mediated transcriptional regulation in ESCs and other cell types.

Differential sequences and single nucleotide polymorphism of exosomal SOX2 DNA in cancer.

Glioblastoma multiforme (GBM) is the most common form of brain cancer, with an average life expectancy of fewer than two years post-diagnosis. We have previously reported that cancer cell originated exosomes, including GBM, have NANOG and NANOGP8 DNA associated with them. The exosomal NANOG DNA has certain differences as compared to its normal counterpart that are of immense importance as a potential cancer biomarker. NANOG has been demonstrated to play an essential role in the maintenance of embryonic stem cells, and its pseudogene, NANOGP8, is suggested to promote the cancer stem cell phenotype. Similarly, SOX2 is another stemness gene highly expressed in cancer stem cells with an intimate involvement in GBM progression and metastasis as well as promotion of tumorigenicity in Neuroblastoma (NB). Since exosomes are critical in intercellular communication with a role in dissipating hallmark biomolecules responsible for cancer, we conducted a detailed analysis of the association of the SOX2 gene with exosomes whose sequence modulations with further research and appropriate sample size can help to identify diagnostic markers for cancer. We have detected SOX2 DNA associated with exosomes and have identified some of the SNPs and nucleotide variations in the sequences from a GBM and SH-SY5Y sample. Although a further systematic investigation of exosomal DNA from GBM and NB patient’s blood is needed, finding of SOX2 DNA in exosomes in the current study may have value in clinical research. SOX2 is known to be misregulated in cancer cells by changes in miRNA function, such as SNPs in the binding sites. Our finding of cancer-specific SNPs in exosomal SOX2 DNA sequence may reflect those changes in the cancer stem cells as well as cancer cells. A series of our study on embryonic stem cell gene analysis in exosomal DNA may lead to a minimally invasive exosome-based diagnosis, and give us a key in understanding the mechanisms of cancer formation, progression, and metastasis.

Analytical distributions for detailed models of stochastic gene expression in eukaryotic cells

The stochasticity of gene expression presents significant challenges to the modeling of genetic networks. A two-state model describing promoter switching, transcription, and messenger RNA (mRNA) decay is the standard model of stochastic mRNA dynamics in eukaryotic cells. Here, we extend this model to include mRNA maturation, cell division, gene replication, dosage compensation, and growth-dependent transcription. We derive expressions for the time-dependent distributions of nascent mRNA and mature mRNA numbers, provided two assumptions hold: 1) nascent mRNA dynamics are much faster than those of mature mRNA; and 2) gene-inactivation events occur far more frequently than gene-activation events. We confirm that thousands of eukaryotic genes satisfy these assumptions by using data from yeast, mouse, and human cells. We use the expressions to perform a sensitivity analysis of the coefficient of variation of mRNA fluctuations averaged over the cell cycle, for a large number of genes in mouse embryonic stem cells, identifying degradation and gene-activation rates as the most sensitive parameters. Furthermore, it is shown that, despite the model's complexity, the time-dependent distributions predicted by our model are generally well approximated by the negative binomial distribution. Finally, we extend our model to include translation, protein decay, and auto-regulatory feedback, and derive expressions for the approximate time-dependent protein-number distributions, assuming slow protein decay. Our expressions enable us to study how complex biological processes contribute to the fluctuations of gene products in eukaryotic cells, as well as allowing a detailed quantitative comparison with experimental data via maximum-likelihood methods.

Chronic cypermethrin exposure alters mouse embryonic stem cell growth kinetics, induces Phase II detoxification response and affects pluripotency and differentiation gene expression

Worldwide uncontrolled use of synthetic pyrethroids contaminates water and soil leading to health hazards. Cypermethrin (CYP), the most used pyrethroid, induces detrimental effects on adults and embryos at different stages of development of several vertebrate species. In Mammals, CYP-induced alterations have been previously described in adult somatic cells and in post-implantation embryos. It remains unknown whether CYP has effects during pre-implantation development. Studies to access pre-implantation embryo toxicity are complicated by the restricted number of blastocysts that may be obtained, either in vivo or in vitro. Embryonic stem cells (ESCs) are an in vitro model study that overcomes these limitations, as millions of pluripotent cells are available to the analysis. Also, ESCs maintain the same pluripotency characteristics and differentiation capacity of the inner cell mass (ICM) present in the blastocyst, from which they derive. In this work, using mouse R1 ESCs, we studied CYP-induced cell death, ROS production, the activation of oxidative stress-related and detoxification responses and the population growth kinetics following 72 h exposure at the 0.3 mM LD50 dose. Also, the expression levels of pluripotency genes in exposed ESCs and of markers of the three germ layers after their differentiation into embryoid bodies (EBs) were determined. Two apoptotic waves were observed at 12-24 h and at 72 h. The increase of ROS production, at 24 h until the end of the culture period, was accompanied by the induction, at 48 h, of redox-related Cat, Sod1, Sod2, Gpx1 and Gpx4 genes. Up-regulation of Cyp1b1, but not of Cyp1a1, phase I gene was detected at 72 h and induction of Nqo1, Gsta1 and Ugt1a6 phase II genes began at 24 h exposure. The results show that exposed R1 ESCs activate oxidative stress-related and detoxification responses, although not sufficient, during the culture period tested, to warrant recovery of the growth rate observed in untreated cells. Also, CYP exposure altered the expression of Oct-4 and Nanog pluripotency genes in ESCs and, when differentiated into EBs, the expression of Fgf5, Brachyury and Foxa2, early markers of the ectoderm, mesoderm and endoderm germ layers, respectively. NIH/3T3 cells, a differentiated cell line of embryonic origin, were used for comparison.

The LncRNA HOTAIRM1 Promotes Tamoxifen Resistance by Mediating HOXA1 Expression in ER+ Breast Cancer Cells.

Breast cancer is one of the most commonly diagnosed cancers in women worldwide. Approximately 40% of patients with breast cancer acquire endocrine resistance following therapy with tamoxifen. Many explanations for the development of endocrine resistance have been put forward, one of them being the dysregulation of long non-coding RNAs (lncRNAs). The lncRNA HOTAIRM1, known to be involved in myelopoiesis as well as transcriptional regulation of the HOXA genes in embryonic stem cells, is also expressed in breast cancer cells. This study explored the molecular mechanisms of HOTAIRM1 involved in acquired tamoxifen resistance. We showed that HOTAIRM1 and HOXA1 are concurrently up-regulated in tamoxifen-resistant MCF7 (TAMR) cells. Knockdown of HOTAIRM1 down-regulated HOXA1 expression and restored sensitivity to tamoxifen. In addition, the knockdown of HOXA1 showed similar effects, suggesting that the HOTAIRM1/HOXA1 axis regulates tamoxifen resistance. Furthermore, we showed that HOTAIRM1 directly interacts with EZH2 and prevents the PRC2 complex from binding and depositing H3K27me3 on the putative promoter of HOXA1. Together, our findings suggest that HOXA1 and its neighboring lncRNA, HOTAIRM1, might serve as potential therapeutic targets for ER+ breast cancer patients who have acquired tamoxifen resistance.

H3K18ac Primes Mesendodermal Differentiation upon Nodal Signaling.

SummaryCellular responses to transforming growth factor β (TGF-β) depend on cell context. Here, we explored how TGF-β/nodal signaling crosstalks with the epigenome to promote mesendodermal differentiation. We find that expression of a group of mesendodermal genes depends on both TRIM33 and nodal signaling in embryoid bodies (EBs) but not in embryonic stem cells (ESCs). Only in EBs, TRIM33 binds these genes in the presence of expanded H3K18ac marks. Furthermore, the H3K18ac landscape at mesendodermal genes promotes TRIM33 recruitment. We reveal that HDAC1 binds to active gene promoters and interferes with TRIM33 recruitment to mesendodermal gene promoters. However, the TRIM33-interacting protein p300 deposits H3K18ac and further enhances TRIM33 recruitment. ATAC-seq data demonstrate that TRIM33 primes mesendodermal genes for activation by maintaining chromatin accessibility at their regulatory regions. Altogether, our study suggests that HDAC1 and p300 are key factors linking the epigenome through TRIM33 to the cell context-dependent nodal response during mesendodermal differentiation.

PRC2.1 and PRC2.2 Synergize to Coordinate H3K27 Trimethylation.

Polycomb repressive complex 2 (PRC2) is composed of EED, SUZ12, and EZH1/2 and mediates mono-, di-, and trimethylation of histone H3 at lysine 27. At least two independent subcomplexes exist, defined by their specific accessory proteins: PRC2.1 (PCL1-3, EPOP, and PALI1/2) and PRC2.2 (AEBP2 and JARID2). We show that PRC2.1 and PRC2.2 share the majority of target genes in mouse embryonic stem cells. The loss of PCL1-3 is sufficient to evict PRC2.1 from Polycomb target genes but only leads to a partial reduction of PRC2.2 and H3K27me3. Conversely, disruption of PRC2.2 function through the loss of either JARID2 or RING1A/B is insufficient to completely disrupt targeting of SUZ12 by PCLs. Instead, the combined loss of both PRC2.1 and PRC2.2 is required, leading to the global mislocalization of SUZ12. This supports a model in which the specific accessory proteins within PRC2.1 and PRC2.2 cooperate to direct H3K27me3 via both synergistic and independent mechanisms.

Introducing gene deletions by mouse zygote electroporation of Cas12a/Cpf1

CRISPR-associated (Cas) nucleases are established tools for engineering of animal genomes. These programmable RNA-guided nucleases have been introduced into zygotes using expression vectors, mRNA, or directly as ribonucleoprotein (RNP) complexes by different delivery methods. Whereas microinjection techniques are well established, more recently developed electroporation methods simplify RNP delivery but can provide less consistent efficiency. Previously, we have designed Cas12a-crRNA pairs to introduce large genomic deletions in the Ubn1, Ubn2, and Rbm12 genes in mouse embryonic stem cells (ESC). Here, we have optimized the conditions for electroporation of the same Cas12a RNP pairs into mouse zygotes. Using our protocol, large genomic deletions can be generated efficiently by electroporation of zygotes with or without an intact zona pellucida. Electroporation of as few as ten zygotes is sufficient to obtain a gene deletion in mice suggesting potential applicability of this method for species with limited availability of zygotes.

Identification and Characterization of Tumor-Initiating Cells in Multiple Myeloma.

Treatment failures in cancers, including multiple myeloma (MM), are most likely due to the persistence of a minor population of tumor-initiating cells (TICs), which are noncycling or slowly cycling and very drug resistant. Gene expression profiling and real-time quantitative reverse transcription polymerase chain reaction were employed to define genes differentially expressed between the side-population cells, which contain the TICs, and the main population of MM cells derived from 11 MM patient samples. Self-renewal potential was analyzed by clonogenicity and drug resistance of CD24+ MM cells. Flow cytometry (n = 60) and immunofluorescence (n = 66) were applied on MM patient samples to determine CD24 expression. Therapeutic effects of CD24 antibodies were tested in xenograft MM mouse models containing three to six mice per group. CD24 was highly expressed in the side-population cells, and CD24+ MM cells exhibited high expression of induced pluripotent or embryonic stem cell genes. CD24+ MM cells showed increased clonogenicity, drug resistance, and tumorigenicity. Only 10 CD24+ MM cells were required to develop plasmacytomas in mice (n = three of five mice after 27 days). The frequency of CD24+ MM cells was highly variable in primary MM samples, but the average of CD24+ MM cells was 8.3% after chemotherapy and in complete-remission MM samples with persistent minimal residual disease compared with 1.0% CD24+ MM cells in newly diagnosed MM samples (n = 26). MM patients with a high initial percentage of CD24+ MM cells had inferior progression-free survival (hazard ratio [HR] = 3.81, 95% confidence interval [CI] = 5.66 to 18.34, P < .001) and overall survival (HR = 3.87, 95% CI = 16.61 to 34.39, P = .002). A CD24 antibody inhibited MM cell growth and prevented tumor progression in vivo. Our studies demonstrate that CD24+ MM cells maintain the TIC features of self-renewal and drug resistance and provide a target for myeloma therapy.

Abstract 2571: Role of XRCC4 downregulation in bioflavonoid-induced chromosomal translocations

Abstract The faithful repair of DNA double strand breaks (DSBs) is crucial for genome integrity, and aberrant repair can result in chromosomal translocations, including those associated with infant leukemia. Evidence from our lab and others suggests that environmental and dietary compounds such as bioflavonoids have the potential to promote chromosomal translocations in utero. DSBs can be repaired by either classical non-homologous end joining (C-NHEJ) that is mostly accurate or by microhomology-mediated alternative end joining (Alt-EJ) that is more error prone and can cause chromosomal translocations. Ku70/80, Ligase 4 (LIG4), X-Ray Repair Cross-Complementing factor 4 (XRCC4), and XRCC4 like factor (XLF) are central to C-NHEJ. Poly ADP Ribose 1 (PARP 1) and C terminal binding protein interacting protein (CtIP) participate in Alt-EJ. CtIP favors Alt-EJ pathway by promoting the resection of DNA ends to uncover microhomologies supporting the formation of large deletions and chromosomal translocations. Studies suggest that loss of efficient activity in the C-NHEJ repair pathway may promote the use of the other more error-prone pathway such as Alt-EJ. The purpose of the study is to understand the relationship between bioflavonoid exposure, induction of DNA repair proteins, and repair pathway choice to understand the underlying mechanisms of bioflavonoid-induced infant leukemias. Embryonic stem (ES) cells were exposed to genistein 1 hr at doses 0μM-100μM based on determined LD50 values. Following exposure, cells were recovered 4hr, 24hr and 48 hr, followed by protein extraction and Western blotting. Protein levels of γH2AX, Ku70, XRCC4, XLF, CtIP and PARP-1 were measured by densitometry and analyzed using one way ANOVA. CRISPR-Cas9 was used to downregulate C-NHEJ DNA repair protein XRCC4. XRCC4-/- cells will subsequently be exposed to genistein, recovered, followed by protein extraction and Western blotting. Protein levels of γH2AX, Ku70, XRCC4, XLF, CtIP and PARP-1 will measured by densitometry. Protein levels before and after gene editing of XRCC4 will be compared and analyzed using two way ANOVA. We have downregulated the XRCC4 gene in ES cells and anticipate that following exposure to genistein in the absence of XRCC4, we will detect elevated chromosomal translocation frequency in those stem cells by stimulating the Alt-EJ pathway. This research will be highly impactful in understanding how bioflavonoid-induced DNA damage is repaired by cells and whether these compounds promote genome instability analogous to that observed in infant leukemia. Citation Format: Anindita Ghosh, Christine A. Richardson. Role of XRCC4 downregulation in bioflavonoid-induced chromosomal translocations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2571.