Eunjee Lee

Genome-wide CRISPR-Cas9 Screens Reveal Loss of Redundancy between PKMYT1 and WEE1 in Glioblastoma Stem-like Cells.

To identify therapeutic targets for glioblastoma (GBM), we performed genome-wide CRISPR-Cas9 knockout (KO) screens in patient-derived GBM stem-like cells (GSCs) and human neural stem/progenitors (NSCs), non-neoplastic stem cell controls, for genes required for their in vitro growth. Surprisingly, the vast majority GSC-lethal hits were found outside of molecular networks commonly altered in GBM and GSCs (e.g., oncogenic drivers). In vitro and in vivo validation of GSC-specific targets revealed several strong hits, including the wee1-like kinase, PKMYT1/Myt1. Mechanistic studies demonstrated that PKMYT1 acts redundantly with WEE1 to inhibit cyclin B-CDK1 activity via CDK1-Y15 phosphorylation and to promote timely completion of mitosis in NSCs. However, in GSCs, this redundancy is lost, most likely as a result of oncogenic signaling, causing GBM-specific lethality.

Application of magnetic nanoparticle for controlled tissue assembly and tissue engineering

Abstract Magnetic nanoparticles have been subjected to extensive studies in the past few decades owing to their promising potentials in biomedical applications. The versatile intrinsic properties of magnetic nanoparticles enable their use in many biomedical applications. Recently, magnetic nanoparticles were utilized to control the cell’s function. In addition, intracellular delivery of magnetic nanoparticles allowed cell’s positioning by appropriate use of magnetic field and created cellular cluster. Furthermore, magnetic nanoparticles have been utilized to assemble more complex tissue structures than those that are achieved by conventional scaffold-based tissue engineering strategies. This review addresses recent work in the use magnetic nanoparticle for controlled tissue assembly and complex tissue formation.

Targeting the SIN3A-PF1 interaction inhibits epithelial to mesenchymal transition and maintenance of a stem cell phenotype in triple negative breast cancer.

Triple negative breast cancer (TNBC) is characterized by a poorly differentiated phenotype and limited treatment options. Aberrant epigenetics in this subtype represent a potential therapeutic opportunity, but a better understanding of the mechanisms contributing to the TNBC pathogenesis is required. The SIN3 molecular scaffold performs a critical role in multiple cellular processes, including epigenetic regulation, and has been identified as a potential therapeutic target. Using a competitive peptide corresponding to the SIN3 interaction domain of MAD (Tat-SID), we investigated the functional consequences of selectively blocking the paired amphipathic α-helix (PAH2) domain of SIN3. Here, we report the identification of the SID-containing adaptor PF1 as a factor required for maintenance of the TNBC stem cell phenotype and epithelial-to-mesenchymal transition (EMT). Tat-SID peptide blocked the interaction between SIN3A and PF1, leading to epigenetic modulation and transcriptional downregulation of TNBC stem cell and EMT markers. Importantly, Tat-SID treatment also led to a reduction in primary tumor growth and disseminated metastatic disease in vivo. In support of these findings, knockdown of PF1 expression phenocopied treatment with Tat-SID both in vitro and in vivo. These results demonstrate a critical role for a complex containing SIN3A and PF1 in TNBC and provide a rational for its therapeutic targeting.

Inferred miRNA activity identifies miRNA-mediated regulatory networks underlying multiple cancers

MOTIVATION MicroRNAs (miRNAs) play a key role in regulating tumor progression and metastasis. Identifying key miRNAs, defined by their functional activities, can provide a deeper understanding of biology of miRNAs in cancer. However, miRNA expression level cannot accurately reflect miRNA activity. RESULTS We developed a computational approach, ActMiR, for identifying active miRNAs and miRNA-mediated regulatory mechanisms. Applying ActMiR to four cancer datasets in The Cancer Genome Atlas (TCGA), we showed that (i) miRNA activity was tumor subtype specific; (ii) genes correlated with inferred miRNA activities were more likely to enrich for miRNA binding motifs; (iii) expression levels of these genes and inferred miRNA activities were more likely to be negatively correlated. For the four cancer types in TCGA we identified 77-229 key miRNAs for each cancer subtype and annotated their biological functions. The miRNA-target pairs, predicted by our ActMiR algorithm but not by correlation of miRNA expression levels, were experimentally validated. The functional activities of key miRNAs were further demonstrated to be associated with clinical outcomes for other cancer types using independent datasets. For ER(-)/HER2(-) breast cancers, we identified activities of key miRNAs let-7d and miR-18a as potential prognostic markers and validated them in two independent ER(-)/HER2(-) breast cancer datasets. Our work provides a novel scheme to facilitate our understanding of miRNA. In summary, inferred activity of key miRNA provided a functional link to its mediated regulatory network, and can be used to robustly predict patients survival. AVAILABILITY AND IMPLEMENTATION the software is freely available at http://research.mssm.edu/integrative-network-biology/Software.html. CONTACT jun.zhu@mssm.edu SUPPLEMENTARY INFORMATION Supplementary data are available at Bioinformatics online.

A functional genomics predictive network model identifies regulators of inflammatory bowel disease

A major challenge in inflammatory bowel disease (IBD) is the integration of diverse IBD data sets to construct predictive models of IBD. We present a predictive model of the immune component of IBD that informs causal relationships among loci previously linked to IBD through genome-wide association studies (GWAS) using functional and regulatory annotations that relate to the cells, tissues, and pathophysiology of IBD. Our model consists of individual networks constructed using molecular data generated from intestinal samples isolated from three populations of patients with IBD at different stages of disease. We performed key driver analysis to identify genes predicted to modulate network regulatory states associated with IBD, prioritizing and prospectively validating 12 of the top key drivers experimentally. This validated key driver set not only introduces new regulators of processes central to IBD but also provides the integrated circuits of genetic, molecular, and clinical traits that can be directly queried to interrogate and refine the regulatory framework defining IBD.

MODMatcher: multi-omics data matcher for integrative genomic analysis.

Errors in sample annotation or labeling often occur in large-scale genetic or genomic studies and are difficult to avoid completely during data generation and management. For integrative genomic studies, it is critical to identify and correct these errors. Different types of genetic and genomic data are inter-connected by cis-regulations. On that basis, we developed a computational approach, Multi-Omics Data Matcher (MODMatcher), to identify and correct sample labeling errors in multiple types of molecular data, which can be used in further integrative analysis. Our results indicate that inspection of sample annotation and labeling error is an indispensable data quality assurance step. Applied to a large lung genomic study, MODMatcher increased statistically significant genetic associations and genomic correlations by more than two-fold. In a simulation study, MODMatcher provided more robust results by using three types of omics data than two types of omics data. We further demonstrate that MODMatcher can be broadly applied to large genomic data sets containing multiple types of omics data, such as The Cancer Genome Atlas (TCGA) data sets.

Efficient myogenic commitment of human mesenchymal stem cells on biomimetic materials replicating myoblast topography

Recent developments in stem cell technologies have demonstrated human mesenchymal stem cells (hMSCs) as a possible cell source for cell‐based therapies and regenerative medicine applications. Self‐renewal and differentiation abilities of hMSCs have enabled hMSCs to be applied in regeneration of musculoskeletal tissue. hMSCs are able to myogenically differentiate via various approaches; however, the most efficient method has not been developed. Here, we describe the efficient commitment of hMSCs to the myogenic lineage on biomimetic substrates replicating myoblast topography. We have created a tissue culture platform that replicates the micro‐and nanoscale topography of fully differentiated skeletal myoblasts. Using UV‐assisted capillary force lithography, an optically transparent cellular model of fully differentiated myoblasts was developed using a UV curable poly(urethane acrylate) resin, which was fabricated and employed as a cell‐culture substrate for the myogenic pattern of hMSCs. When hMSCs were cultured and differentiated on these biomimetic patterns, cells followed the underlying myoblast pattern and more efficiently committed to myogenic fate. These results demonstrate that myogenic potentials of hMSCs are highly depended on the micro‐ and nanoscale topographical cues. Furthermore, the described tissue culture platform can be used in larger culture settings with consistent results and easily applied to other lineage of hMSCs.

Chondroitin Sulfate-Based Biomineralizing Surface Hydrogels for Bone Tissue Engineering

Chondroitin sulfate (CS) is the major component of glycosaminoglycan in connective tissue. In this study, we fabricated methacrylated PEGDA/CS-based hydrogels with varying CS concentration (0, 1, 5, and 10%) and investigated them as biomineralizing three-dimensional scaffolds for charged ion binding and depositions. Due to its negative charge from the sulfate group, CS exhibited an osteogenically favorable microenvironment by binding charged ions such as calcium and phosphate. Particularly, ion binding and distribution within negatively charged hydrogel was dependent on CS concentration. Furthermore, CS dependent biomineralizing microenvironment induced osteogenic differentiation of human tonsil-derived mesenchymal stem cells in vitro. Finally, when we transplanted PEGDA/CS-based hydrogel into a critical sized cranial defect model for 8 weeks, 10% CS hydrogel induced effective bone formation with highest bone mineral density. This PEGDA/CS-based biomineralizing hydrogel platform can be utilized for in situ bone formation in addition to being an investigational tool for in vivo bone mineralization and resorption mechanisms.

Induced myogenic commitment of human chondrocytes via non-viral delivery of minicircle DNA

Lineage conversion from one somatic cell type to another is an attractive approach for deriving specific therapeutic cell generation. In order to bypass inducing pluripotent stage, transdifferentiation/direct conversion technologies have been recently developed. We report the development of a direct conversion methodology in which cells are transdifferentiated through a plastic intermediate state induced by exposure to non-integrative minicircle DNA (MCDNA)-based reprogramming factors, followed by differentiation into myoblasts. In order to increase the MCDNA delivery efficiency, reprogramming factors were delivered into the chondrocytes via electroporation followed by poly (β-amino esters) (PBAE) transfection. We used this approach to convert human chondrocytes to myoblast, and with treatment of SB-431542, an inhibitor of the activin receptor-like kinase receptors, to enhance myogenic commitment. Differentiated cells exhibited expression of myogenic markers such as MyoD and Myog. This methodology for direct lineage conversion from chondrocytes to myoblast represents a novel non-viral Method to convert hard-to-transfect cells to other lineage.

Extracellular matrix-immobilized nanotopographical substrates for enhanced myogenic differentiation

Extracellular matrix (ECM) components such as fibronectin (FN) and laminin (LMN) play prominent roles in controlling cellular behaviors. Many attempts have been made to explore cellular behaviors on combinatorial ECM arrays in high-throughput systems. However these studies were limited to physical adsorption of ECM, which does not guarantee a lasting effect of ECM in vitro. Here, we demonstrate ECM immobilization on polyurethane acrylate (PUA) substrate fabricated in 24 well-plate platforms to effectively differentiate C2C12. Our study demonstrate that co-immobilization of FN and LMN was found to enhance myogenic differentiation of C2C12 cells compared to single immobilization of either FN or LMN alone. Furthermore, utilizing nano-imprint lithography technique, 300 nm and 5 µm line-patterned substrates were fabricated on 24-well plates. FN and LMN co-immobilized substrates with line-patterns additionally provided the directionality for mimicking musculoskeletal structure and enhanced the myogenic differentiation.