CRISPR-Cas9-Mediated Bioluminescent Tagging of Endogenous Proteins by Fluorescent Protein-Assisted Cell Sorting.

Oncogenic fusion genes are attractive therapeutic targets because of their tumor-specific expression and central "driver" roles in various human cancers. However, oncogenic fusions involving transcription factors such as PAX3-FOXO1 in alveolar fusion gene-positive rhabdomyosarcoma (FP-RMS) have been difficult to inhibit due to the apparent lack of tractable drug-like binding sites comparable to that recognized by Gleevec (imatinib mesylate) on the BCR-ABL1 tyrosine kinase fusion protein. Toward the identification of novel small molecules that selectively target PAX3-FOXO1, we used CRISPR-Cas9-mediated knock-in to append the pro-luminescent HiBiT tag onto the carboxy terminus of the endogenous PAX3-FOXO1 fusion protein in two human FP-RMS cell lines (RH4 and SCMC).

KDM3B inhibitors disrupt the oncogenic activity of PAX3-FOXO1 in fusion-positive rhabdomyosarcoma.

Fusion-positive rhabdomyosarcoma (FP-RMS) is an aggressive pediatric sarcoma driven primarily by the PAX3-FOXO1 fusion oncogene, for which therapies targeting PAX3-FOXO1 are lacking. Here, we screen 62,643 compounds using an engineered cell line that monitors PAX3-FOXO1 transcriptional activity identifying a hitherto uncharacterized compound, P3FI-63. RNA-seq, ATAC-seq, and docking analyses implicate histone lysine demethylases (KDMs) as its targets.

Neopetrotaurines A-C, Isoquinoline Alkaloids with an Unprecedented Taurine Bridge from the Sponge sp.

Neopetrotaurines A-C (-), unusual alkaloids possessing two isoquinoline-derived moieties that are linked via a unique taurine bridge, were isolated from a sp. marine sponge. These new compounds have proton-deficient structural scaffolds that are difficult to unambiguously assign using only conventional 2- and 3-bond H-C and H-N heteronuclear correlation data.

Functional genetic variants of the GATA4 gene promoter in acute myocardial infarction.

Coronary artery disease (CAD), including acute myocardial infarction (AMI), is a common complex disease; however, the genetic causes remain largely unknown. Recent epidemiological investigations indicated that the incidence of CAD in patients with congenital heart diseases is markedly higher than that observed in healthy controls. It was therefore hypothesized that the dysregulated expression of cardiac developmental genes may be involved in CAD development.

Correlating Chemical Sensitivity with Low Level Activation of Mechanotransduction Pathways in Hematologic Malignancies.

Large-scale screening has revealed that human hematopoietic cancer cell lines are generally more sensitive to various classes of drugs than cell lines established from solid tumors. A detailed examination of data in the Cancer Therapeutics Response Portal (http://portals.broadinstitute.

Increased expression of the tight junction protein TJP1/ZO-1 is associated with upregulation of TAZ-TEAD activity and an adult tissue stem cell signature in carfilzomib-resistant multiple myeloma cells and high-risk multiple myeloma patients.

Tight junction protein 1 (TJP1) has recently been proposed as a biomarker to identify multiple myeloma (MM) patients most likely to respond to bortezomib- and carfilzomib-based proteasome inhibitor regimens. Herein we report increased expression of during the adaptive response mediating carfilzomib resistance in the LP-1/Cfz MM cell line. Moreover, increased expression delineated a subset of relapsed/refractory MM patients on bortezomib-based therapy sharing an LP-1/Cfz-like phenotype characterized by activation of interacting transcriptional effectors of the Hippo signaling cascade (TAZ and TEAD1) and an adult tissue stem cell signature.

Fluorescent Proteins for Flow Cytometry.

Fluorescent proteins have become standard tools for cell and molecular biologists. The color palette of fluorescent proteins spans the ultraviolet, visible, and near-infrared spectrum. Utility of fluorescent proteins has been greatly facilitated by the availability of compact and affordable solid state lasers capable of providing various excitation wavelengths.

Genetic analysis of the ATG16L1 gene promoter in sporadic Parkinson's disease.

Parkinson's disease (PD) is a common and progressive neurodegenerative disease in which the majority of cases arise sporadically. Sporadic PD is caused by the interactions of genetic and environmental factors. To date, genetic causes for sporadic PD remain largely unknown.

Novel and functional ATG12 gene variants in sporadic Parkinson's disease.

Parkinson's disease (PD) is a common and progressive neurodegenerative disease, including familial and sporadic cases. To date, genetic causes for sporadic PD, majority of PD cases, remain largely unknown. Accumulating evidence indicates that dysfunctional autophagy, a highly conserved cellular process, is involved in the PD pathogenesis.

Noncanonical SQSTM1/p62-Nrf2 pathway activation mediates proteasome inhibitor resistance in multiple myeloma cells via redox, metabolic and translational reprogramming.

Multiple Myeloma (MM) is a B-cell malignancy characterized by the accumulation of clonal plasma cells in the bone marrow, with drug resistance being a major cause of therapeutic failure. We established a carfilzomib-resistant derivative of the LP-1 MM cell line (LP-1/Cfz) and found that the transcription factor NF-E2 p45-related factor 2 (Nrf2; gene symbol NFE2L2) contributes to carfilzomib resistance. The mechanism of Nrf2 activation involved enhanced translation of Nrf2 as well as its positive regulator, the autophagy receptor sequestosome 1 (SQSTM1)/p62.

KLF4-SQSTM1/p62-associated prosurvival autophagy contributes to carfilzomib resistance in multiple myeloma models.

Multiple myeloma (MM) is an incurable clonal plasma cell malignancy. Because of a high rate of immunoglobulin synthesis, the endoplasmic reticulum of MM cells is subjected to elevated basal levels of stress. Consequently, proteasome inhibitors, which exacerbate this stress by inhibiting ubiquitin-proteasome-mediated protein degradation, are an important new class of chemotherapeutic agents being used to combat this disease.

Novel and functional ABCB1 gene variant in sporadic Parkinson's disease.

Parkinson's disease (PD) is a common progressive neurodegenerative disease. Most cases of PD are sporadic, which is caused by interaction of genetic and environmental factors. To date, genetic causes for sporadic PD remain largely unknown.

Identification of an ABCB1 (P-glycoprotein)-positive carfilzomib-resistant myeloma subpopulation by the pluripotent stem cell fluorescent dye CDy1.

Multiple myeloma (MM) is characterized by the malignant expansion of differentiated plasma cells. Although many chemotherapeutic agents display cytotoxic activity toward MM cells, patients inevitably succumb to their disease because the tumor cells become resistant to the anticancer drugs. The cancer stem cell hypothesis postulates that a small subpopulation of chemotherapy-resistant cancer cells is responsible for propagation of the tumor.

The DN2 Myeloid-T (DN2mt) Progenitor is a Target Cell for Leukemic Transformation by the TLX1 Oncogene.

Introduction: Inappropriate activation of the TLX1 (T-cell leukemia homeobox 1) gene by chromosomal translocation is a recurrent event in human T-cell Acute Lymphoblastic Leukemia (T-ALL). Ectopic expression of TLX1 in murine bone marrow progenitor cells using a conventional retroviral vector efficiently yields immortalized cell lines and induces T-ALL-like tumors in mice after long latency.

Methods: To eliminate a potential contribution of retroviral insertional mutagenesis to TLX1 immortalizing and transforming function, we incorporated the TLX1 gene into an insulated self-inactivating retroviral vector.

A novel and functional variant within the ATG5 gene promoter in sporadic Parkinson's disease.

Parkinson's disease (PD) is one of the most common neurodegenerative diseases. Majority of PD are sporadic, for which genetic causes remain largely unknown. Alpha-synuclein, the main component of Lewy bodies, plays a central role in the PD pathogenesis.

Genetic analysis of the ATG7 gene promoter in sporadic Parkinson's disease.

Parkinson's disease (PD) is the second most common neurodegenerative disease. The majority of PD cases are sporadic, for which genetic causes and underlying molecular mechanisms remain largely unclear. Autophagy, a highly conserved cellular process that governs the breakdown of long-lived proteins and organelles, has been involved in the degradation of α-synuclein (α-Syn), the main component of Lewy bodies.

An Integrated Bioinformatics and Computational Biology Approach Identifies New BH3-Only Protein Candidates.

In this study, we utilized an integrated bioinformatics and computational biology approach in search of new BH3-only proteins belonging to the BCL2 family of apoptotic regulators. The BH3 (BCL2 homology 3) domain mediates specific binding interactions among various BCL2 family members. It is composed of an amphipathic α-helical region of approximately 13 residues that has only a few amino acids that are highly conserved across all members.

Flow cytometry of fluorescent proteins.

Fluorescent proteins are now a critical tool in all areas of biomedical research. In this article, we review the techniques required to use fluorescent proteins for flow cytometry, concentrating specifically on the excitation and emission requirements for each protein, and the specific equipment required for optimal use.

Apoptotic role of IKK in T-ALL therapeutic response.

Despite considerable progress in the treatment of T cell acute lymphoblastic leukemia (T-ALL), it is still the highest risk malignancy among ALL. The outcome of relapsed patients remains dismal. The pro-survival role of NOTCH1 and NFκB in T-ALL is well documented; also, both factors were reported to be predictive of relapse.

Factor VIII delivered by haematopoietic stem cell-derived B cells corrects the phenotype of haemophilia A mice.

The main impediments to clinical application of haematopoietic stem cell (HSC) gene therapy for treatment of haemophilia A are the bone marrow transplant-related risks and the potential for insertional mutagenesis caused by retroviral vectors. To circumvent these limitations, we have adapted a non-myeloablative conditioning regimen and directed factor VIII (FVIII) protein synthesis to B lineage cells using an insulated lentiviral vector containing an immunoglobulin heavy chain enhancer-promoter. Transplantation of lentiviral vector-modified HSCs resulted in therapeutic levels of FVIII in the circulation of all transplanted mice for the duration of the study (six months).

Lentiviral fluorescent protein expression vectors for biotinylation proteomics.

In vivo biotinylation tagging, based on a method in which a protein of interest is tagged with a peptide that is biotinylated in vivo by coexpression of Escherichia coli BirA biotin ligase, has been successfully used for the isolation of protein-protein and protein-DNA complexes in mammalian cells. We describe a modification of this methodology in which cells stably expressing the tagged gene of interest and the BirA gene can be selected by fluorescence-activated cell sorting (FACS). We recently implemented this approach to isolate and characterize proteins associated with TLX1, a homeodomain transcription factor with leukemogenic function.