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Warden, C.

Publications and source records attributed to Warden, C..

3 recordsLinked to original sources

Kinetic Inference Resolves Epigenetic Mechanism of Drug Resistance in Melanoma

Drug-induced dedifferentiation towards a drug-tolerant persister state is a common mechanism cancer cells exploit to escape therapies, posing a significant obstacle to sustained therapeutic efficacy. The dynamic coordination of epigenomic and transcriptomic programs at the early-stage of drug exposure, which initiates and orchestrates these reversible dedifferentiation events, remains largely unexplored. Here we employ high-temporal-resolution multi-omics profiling, information-theoretic approaches, and dynamic system modeling to probe these processes in BRAF-mutant melanoma models and patient specimens. We uncover a hysteretic transition trajectory of melanoma cells in response to oncogene inhibition and subsequent release, driven by the sequential operation of two tightly coupled transcriptional waves, which orchestrate genome-scale chromatin state reconfiguration. Modeling of the transcriptional wave interactions predicts NF-{kappa}B/RelA-driven chromatin remodeling as the underlying mechanism of cell-state dedifferentiation, a finding we validate experimentally. Our results identify critical RelA-target genes that are epigenetically modulated to drive this process, establishing a quantitative epigenome gauge to measure cell-state plasticity in melanomas, which supports the potential use of drugs targeting epigenetic machineries to potentiate oncogene inhibition. Extending our investigation to other cancer models, we identify oxidative stress-mediated NF-{kappa}B/RelA activation as a common mechanism driving cellular transitions towards drug-tolerant persister states, revealing a novel and pivotal role for the NF-{kappa}B signaling axis in linking cellular oxidative stress to cancer progression.

cancer biology↗

A human embryonic stem cell model of Aβ-dependent chronic progressive neurodegeneration

We describe construction and phenotypic analysis of a human embryonic stem cell model of progressive A{beta}-dependent neurodegeneration (ND) with potential relevance to Alzheimers disease (AD). We modified one allele of the normal APP locus to directly express a secretory form of A{beta}40 or A{beta}42, eliminating the need for amyloidogenic APP proteolysis. Following neuronal differentiation edited cell lines specifically accumulate aggregated/oligomeric A{beta}, exhibit a synaptic deficit and have an abnormal accumulation of endolysosomal vesicles. Edited cultures progress to a stage of overt ND. All phenotypes appear at earlier culture times for A{beta}42 relative to A{beta}40. Whole transcriptome RNA-Seq analysis identified 23 up and 70 down regulated genes (DEGs) with similar directional fold change but larger absolute values in the A{beta}42 samples suggesting common underlying pathogenic mechanisms. Pathway/annotation analysis suggested that down regulation of extracellular matrix and cilia functions are significantly overrepresented. This cellular model could be useful for uncovering mechanisms directly linking A{beta} to neuronal death and as a tool to screen for new therapeutic agents that slow or prevent human ND.

neuroscience↗

Identification of a Novel Neutralizing and Two Non-Neutralizing Epitopes on Epstein-Barr Virus gp350 Protein

Prevention of Epstein-Barr virus (EBV) primary infection has focused on generating neutralizing antibodies (nAbs) targeting the major envelope glycoprotein gp350/220 (gp350). To date, eight gp350 epitopes have been identified, but only one has elicited nAbs. In this study, we generated 23 hybridomas that produced anti-gp350 antibodies. We compared the candidate anti-gp350 antibodies to nAb-72A1 by: (1) testing their ability to detect gp350 using ELISA, flow cytometry, and immunoblot; (2) sequencing their heavy and light chain complementarity-determining regions (CDRs); (3) measuring the ability of each monoclonal antibody (mAb) to neutralize EBV infection in vitro; and (4) mapping the gp350 amino acids bound by the mAbs using RepliTope peptide microarrays. Eight antibodies recognized both denatured and non-denatured gp350, whereas five failed to react with denatured gp350 but recognized native gp350, suggesting they recognized conformational epitope(s). Sequence analysis of the heavy and light chain variable regions of the hybridomas identified 15 as mAbs with novel CDR regions unique from those of nAb-72A1. Seven of the new mAbs neutralized EBV in vitro, with HB20 and HB17 reducing EBV infection by 40% and >60%, and >30% and 80%, at 10 g/ml and 50 g/ml, respectively. Epitope mapping identified nine epitopes and defined their core residues, including two unique immunodominant epitopes, 253TPIPGTGYAYSLRLTPRPVSRFL253 and 875LLLLVMADCAFRRNLSTSHTYTTPPY899, and a novel nAb epitope 381GAFASNRTFDIT392. This study provides comprehensive in vitro mapping of the exact residues defining nine epitopes of EBV gp350. Our findings will inform novel strategies to design optimal EBV vaccines capable of conferring broader protection against the virus.\n\nImportanceNeutralizing antibodies (nAbs) directed against Epstein-Barr virus envelope glycoprotein gp350/220 (gp350) are generated in humans upon infection or immunization, and are thought to prevent neonatal infection. However, clinical use of exogenous nAbs (passive immunization) is limited to a single study using the only well-characterized nAb, 72A1. The gp350 ectodomain contains at least eight unique B-cell binding epitopes; two of these epitopes are recognized by nAb-72A1. The exact amino acid residues of the other six epitopes and their role in generating nAbs has not been elucidated. We used our 15 newly generated and fully characterized monoclonal antibodies and a peptide-overlapping RepliTope array to provide a comprehensive map of the core amino acid residues that define epitopes of gp350 and to understand their role in generating nAbs. These results will inform design of better-targeted gp350 peptide vaccines that contain only protective epitopes, which will focus the B-cell response to produce predominantly nAbs.

immunology↗