Biology subjects

Kosarek, N. N.

Publications and source records attributed to Kosarek, N. N..

4 recordsLinked to original sources

bioRxiv2026

Host DNA repair factors empower a mechanism of antiviral nucleoside analog resistance

How host functions affect resistance to antiviral drugs is poorly understood. Ganciclovir, a chain-terminating nucleoside analog, is a first-line therapy against human cytomegalovirus, a widespread herpesvirus that causes life-threatening disease in immunocompromised individuals and newborns. Ganciclovir resistance, which is caused by mutations that affect the viral kinase, UL97 and/or the viral polymerase, UL54, can cause treatment failures. Among these mutations, those reducing the exonuclease activity of the viral DNA polymerase permit ganciclovir incorporation without chain termination. However, the fate of DNA strands containing the incorporated nucleotide analog is unknown. We show here that template DNA containing ganciclovir fails to support DNA synthesis of the complementary strand by exonuclease-mutant polymerase. Moreover, while DNA synthesis and ganciclovir incorporation are limited in drug-treated fibroblasts infected by virus with wild-type polymerase, an exonuclease-resistant mutant virus can better synthesize full-length genomes and incorporate substantially more ganciclovir into DNA. Notably, ganciclovir is lost from DNA when drug is removed, suggesting that ganciclovir-containing templates are repaired. We identify the host nucleotide excision repair component, XPA, and the repair enzyme, polymerase kappa, as each being necessary for mutant virus ganciclovir resistance and polymerase kappa as being required for the mutants cidofovir resistance, demonstrating a role for host DNA repair machinery in a mechanism of antiviral resistance. We propose a model for this mechanism, which has relevance for at least one other antiviral drug and likely other nucleoside analog therapeutics, and highlights the participation of host DNA repair machinery during human cytomegalovirus DNA replication. IMPORTANCENucleoside analogues such as ganciclovir, which is a leading drug for preventing and treating human cytomegalovirus, are a critical defense against viral diseases, but antiviral resistance often results in treatment failures. This study reveals a critical role for host DNA repair in a mechanism of resistance to ganciclovir, and identifies at least one specific repair pathway that permits viral DNA synthesis in the presence of ganciclovir, defining a mechanism by which cellular DNA repair pathways conspire to enable antiviral drug resistance. This mechanism is relevant to at least one other antiviral drug and may apply to other antiviral and anticancer agents. The study also showcases the participation of host DNA repair machinery during human cytomegalovirus DNA synthesis.

microbiology↗

bioRxiv2025

Anti-CD206 CAR T Cell Treatment Restores Fibrosis-Induced Loss of Dermal White Adipose Tissue

Fibrosis drives pathology in the chronic autoimmune disease systemic sclerosis (SSc), which has the highest case fatality rate of any systemic autoimmune disease with no validated biomarkers or curative treatments. Our prior work has shown that CD206+ macrophages and dermal fibroblasts engage in cooperative mechanisms of inflammatory and fibrotic activation in SSc. Here, we designed a targeted immunotherapeutic approach to eliminate CD206+ macrophages using chimeric antigen receptor (CAR) T cells. We demonstrate that systemic delivery of a single dose of anti-CD206 CAR T cells restores dermal white adipose tissue (DWAT) in vivo. Notably, loss of subcutaneous fat is a well-recognized but poorly understood aspect of SSc pathogenesis that precedes the development of fibrosis and is driven by changes in lineage commitment of adipose-derived stem cells (ADSCs). Using snRNA-seq and a newly-developed in vitro co-culture model, we report that CD206high macrophages mediate ADSC shift from adipocytic to fibrotic activation in part through an IL-6-dependent mechanism. This report implicates a novel function for macrophages in the regulation of early SSc pathogenesis and is the first to establish the therapeutic efficacy of using CAR T cell immunotherapy to target macrophages in the treatment of SSc skin disease.

synthetic biology↗

bioRxiv2024

Single-cell epigenomic dysregulation of Systemic Sclerosis fibroblasts via CREB1/EGR1 axis in self-assembled human skin equivalents

Systemic sclerosis (SSc) is an autoimmune disease characterized by skin fibrosis, internal organ involvement and vascular dropout. We previously developed and phenotypically characterized an in vitro 3D skin-like tissue model of SSc, and now analyze the transcriptomic (scRNA-seq) and epigenetic (scATAC-seq) characteristics of this model at single-cell resolution. SSc 3D skin-like tissues were fabricated using autologous fibroblasts, macrophages, and plasma from SSc patients or healthy control (HC) donors. SSc tissues displayed increased dermal thickness and contractility, as well as increased -SMA staining. Single-cell transcriptomic and epigenomic analyses identified keratinocytes, macrophages, and five populations of fibroblasts (labeled FB1 - 5). Notably, FB1 APOE-expressing fibroblasts were 12-fold enriched in SSc tissues and were characterized by high EGR1 motif accessibility. Pseudotime analysis suggests that FB1 fibroblasts differentiate from a TGF-{beta}1-responsive fibroblast population and ligand-receptor analysis indicates that the FB1 fibroblasts are active in macrophage crosstalk via soluble ligands including FGF2 and APP. These findings provide characterization of the 3D skin-like model at single cell resolution and establish that it recapitulates subsets of fibroblasts and macrophage phenotypes observed in skin biopsies.

genomics↗

bioRxiv2023

CDDO-Methyl Ester Inhibits BRAF Inhibitor Resistance and Remodels the Myeloid Compartment in BRAF-mutant Melanoma

Approximately 50% of advanced melanomas harbor activating BRAFV600E mutations that are sensitive to BRAF inhibition. However, the duration of the response to BRAF inhibitors (BRAFi) has been limited due to the development of acquired resistance, which is preceded by recruitment of immunosuppressive myeloid cells and regulatory T cells (Tregs). While the addition of MAPK/ERK kinase 1 inhibitors (MEKi) prolongs therapeutic response to BRAF inhibition, most patients still develop resistance. Using a BrafV600E/+/Pten-/- graft mouse model of melanoma, we now show that the addition of the methyl ester of the synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (C-Me) to the BRAFi vemurafenib analog PLX4720 at resistance significantly reduces tumor burden. Dual treatment remodels the BRAFi resistant-tumor microenvironment (TME), reducing infiltration of Tregs and tumor associated macrophages (TAMs), and attenuates immunosuppressive cytokine production. For the first time, we characterize myeloid populations using scRNA-seq in BRAFi-resistant tumors and demonstrate that restoration of therapeutic response is associated with significant changes in immune-activated myeloid subset representation. Collectively, these studies suggest that C-Me inhibits acquired resistance to BRAFi. Use of C-Me in combination with other therapies may both inhibit melanoma growth and enhance therapeutic responsiveness more broadly.

immunology↗