Browsing by Author "Motea, Edward"

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    Identification and Characterization of TONSL as an Immortalizing Oncogene
    (2024-11) Khatpe, Aditi Sanjay; Nakshatri, Harikrishna; Nephew, Kenneth; Motea, Edward; Cornett, Evan; Corson, Timothy
    The global issue of exponentially increasing breast cancer cases necessitates investigating the early genomic aberrations leading to tumorigenesis. To address this, we employed our unique biobank of healthy breast tissue to generate an isogeneic cell line model system, comprising primary breast epithelial cells and their immortalized counterparts. By comparing the genetic alterations between these cell types, we discovered that TONSL upregulation is one of the early events during breast tumorigenesis. TONSL is a Tonsoku-like DNA repair protein located on chromosome 8q24.3. Our findings reveal that TONSL is an immortalizing oncogene, capable of transforming primary breast epithelial cells in conjunction with defined oncogenes, resulting in Estrogen Receptor-positive breast adenocarcinomas. Furthermore, we observed that TONSL-amplified breast cancer cells are dependent on TONSL for tumor growth, and these TONSLHigh cells and tumors exhibit an upregulated homologous recombination DNA repair pathway, which may contribute to chemotherapy resistance. It is noteworthy that higher levels of TONSL protein in primary breast cancers, particularly in ER+ breast cancers, are associated with poor outcomes. Approximately 20% of breast cancers display recurring genomic amplification involving chromosome 8q24.3, with TONSL amplification potentially being the initial hit leading to tumorigenesis. In an attempt to target these TONSL/chr. 8q24.3 amplified breast tumors, we observed that breast cancer cells with this amplification are sensitive to CBL0137 – a TONSL-FACT complex inhibitor, both in vitro and in vivo. TONSL interacts with multiple proteins and functions in multiple cellular processes. To study the TONSL specific interactome, we performed immunoprecipitation with a TONSL antibody using protein lysates from TONSL-immortalized primary breast epithelial cells, followed by mass spectrometry analysis of the immunoprecipitates. Our results identified several proteins selectively enriched with the TONSL antibody, with the most significant being ETS variant transcription factor 6 (ETV6). ETV6 is known to play a role as a transcriptional repressor during embryonic development and hematopoiesis. Further studies on TONSL/chromosome 8q24.3 amplification will contribute to our understanding of breast tumor initiation, progression, and metastasis processes, as well as facilitate the development of novel therapeutic agents targeting the TONSL interactome.
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    Small Molecule Inhibitors of GroEL That Disrupt Active Replication of Mycobacterium Tuberculosis and ESKAPE Bacteria
    (2022-07) Tepper, Katelyn; Johnson, Steven M.; Georgiadis, Millie; Motea, Edward; Absalon, Sabrina
    Globally, millions of people die every year due to complications involving infections from antibiotic-resistant bacteria. Of these infections, the most common organisms are Mycobacterium tuberculosis (Mtb) and a group of bacteria known as the ESKAPE pathogens (an acronym that stands for Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, Enterobacter species). Unfortunately, as the need for antibiotics increases, industrial antibiotic development programs are drying up. However, unique antibiotic candidates targeting new pathways may be better for addressing antibacterial resistance. A target that is currently not the focus of any drug on the market is the bacterial GroEL chaperonin system. GroEL chaperonins are complex, oligomeric proteins that are upregulated in the cell under stressful conditions and prevent the misfolding and aggregation of other proteins. All bacteria have one homolog that performs protein folding functions – such is the case for E. coli and the ESKAPE bacteria – while others, like M. tuberculosis, contain additional GroEL isoforms that appear to perform non-canonical functions that are not well understood. The canonical isoforms are essential for survival under all conditions; thus, these chaperonins represent excellent targets for antibiotic development. This study aimed to identify inhibitors of GroEL in the ESKAPE bacteria and Mtb from a library of compounds with known antibiotic properties that was provided by the Medicines for Malaria Venture. Using two orthogonal assays that assess GroEL activity via its refolding of denatured enzymes Malate Dehydrogenase and Rhodanase, 37 inhibitors of E. coli GroEL were identified. Of these, 33 were examined in dose response testing in in vitro biochemical and cell viability assays. Compound 23 stood out in potency for inhibiting GroEL functions and actively-replicating Mtb bacteria, and thus a small panel of analogs were evaluated to develop structure-activity relationships (SAR) and study their mechanism. Two cysteine residues were identified as covalently modified by compound 23 and one of the lead analogs, giving insight into inhibitory sites on GroEL. Another lead analog bearing a nitrofuran moiety exhibited inhibition of actively-replicating E. coli, S. aureus, and Mtb bacteria. Importantly, this study identified new classes of GroEL inhibitors to explore for optimization as antibacterial candidates.