Biology subjects

Nettles, K. W.

Publications and source records attributed to Nettles, K. W..

8 recordsLinked to original sources

bioRxiv2026

Multi-region biopsies and patient-derived neurosphere cultures reveal spatial divergence in glioblastoma.

Intratumor heterogeneity (ITH) is one of the main reasons for the lack of effective targeted therapies for glioblastoma (GBM). Imaging-guided surgical navigation allows for tumor-wide sampling to account for variation across distant regions of the tumor, but typical drug screening is performed on cell lines derived from a single biopsy and does not account for GBM heterogeneity. Here we profiled matching MRI-guided multi-region primary tumor biopsies from 6 GBM cases (n=40 biopsies) and corresponding neurosphere cultures (n=30) derived from these spatially distinct tumor samples. We found that in vitro cultures derived from distinct regions of the same tumor display divergent phenotypes, proliferative capacity and ability to accumulate 5-aminolevulinic acid, used to visualize cancer cells during surgery. The differential drug response of the multi-region neurospheres remains linked to the gene expression of the original tumor biopsies. Thus, studies with multiregion-derived neurospheres are essential to faithfully model GBM ITH for therapeutic testing. KEY POINTSO_LIMulti-region biopsy-derived neurospheres represent distinct spatial locations in the GBM tumor. C_LIO_LICultures derived from different regions of the tumor retain phenotypic diversity. C_LIO_LIParental biopsy phenotype predicts drug response better than to in vitro phenotype. C_LI IMPORTANCE OF THE STUDYCell lines developed from spatially distinct regions of glioblastoma capture its intratumor heterogeneity. We show that while the transcriptional output of these cell lines is not connected to their spatial origin, their drug response can be linked to it. Thus, spatial heterogeneity reflected in our neurosphere collection provides a new paradigm for drug screening in these highly heterogenous and difficult to treat tumors.

cancer biology↗

bioRxiv2026

Selective Glucocorticoid Receptor Modulators of Immune Checkpoint Function

Glucocorticoids (GCs) coordinate immunity, inflammation, and metabolism through allosteric regulation of the glucocorticoid receptor (GR) transcription factor. GCs are indispensable anti-inflammatory drugs yet linking specific ligand-receptor structural states to specific biological outcomes has remained a major barrier to designing safer, more selective therapies. Using structure-based design, we developed selective glucocorticoid receptor modulators (SGRMs) of immune function by extending a steroidal scaffold from the ligand-binding pocket into an adjacent solvent channel. These SGRMs suppressed T cell pro-inflammatory cytokines and promoted differentiation of memory precursor T cells while showing minimal induction of M2 macrophage polarization or T cell checkpoint proteins PD-1 and CTLA-4, all key targets of immunotherapy. Molecular dynamics simulations revealed that solvent-channel substituents function as a lever arm to drive dynamic oscillations in the steroid core, thereby allosterically tuning GR activity states. Systematic perturbation of immune cells with a graded series of ligands enabled a ligand perturbation with machine learning (LPML) framework to map coregulated responses across cell types and identified effector T cell gene networks tightly coupled with immune checkpoint induction. This approach outlines a general strategy for decoding the logic of allosteric drug action, enabling the rational design of SGRMs with tailored immunomodulatory profiles.

systems biology↗

bioRxiv2024

Asymmetric Allostery in Estrogen Receptor-α Homodimers Drives Responses to the Ensemble of Estrogens in the Hormonal Milieu

The estrogen receptor- (ER) is thought to function only as a homodimer, but responds to a variety of environmental, metazoan, and therapeutic estrogens at sub-saturating doses, supporting binding mixtures of ligands as well as dimers that are only partially occupied. Here, we present a series of flexible ER ligands that bind to receptor dimers with individual ligand poses favoring distinct receptor conformations --receptor conformational heterodimers--mimicking the binding of two different ligands. Molecular dynamics simulations showed that the pairs of different ligand poses changed the correlated motion across the dimer interface to generate asymmetric communication between the dimer interface, the ligands, and the surface binding sites for epigenetic regulatory proteins. By examining binding of the same ligand in crystal structures of ER in the agonist versus antagonist conformers, we also showed that these allosteric signals are bidirectional. The receptor conformer can drive different ligand binding modes to support agonist versus antagonist activity profiles, a revision of ligand binding theory that has focused on unidirectional signaling from ligand to the coregulator binding site. We also observed differences in the allosteric signals between ligand and coregulator binding sites in the monomeric versus dimeric receptor, and when bound by two different ligands, states that are physiologically relevant. Thus, ER conformational heterodimers integrate two different ligand-regulated activity profiles, representing new modes for ligand-dependent regulation of ER activity. SignificanceThe estrogen receptor- (ER) regulates transcription in response to a hormonal milieu that includes low levels of estradiol, a variety of environmental estrogens, as well as ER antagonists such as breast cancer anti-hormonal therapies. While ER has been studied as a homodimer, the variety of ligand and receptor concentrations in different tissues means that the receptor can be occupied with two different ligands, with only one ligand in the dimer, or as a monomer. Here, we use X-ray crystallography and molecular dynamics simulations to reveal a new mode for ligand regulation of ER activity whereby sequence-identical homodimers can act as functional or conformational heterodimers having unique signaling characteristics, with ligand-selective allostery operating across the dimer interface integrating two different signaling outcomes.

biophysics↗

bioRxiv2022

The SARS-CoV-2 spike protein binds and modulates estrogen receptors

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein binds angiotensin-converting enzyme 2 (ACE2) at the cell surface, which constitutes the primary mechanism driving SARS-CoV-2 infection. Molecular interactions between the transduced S and endogenous proteins likely occur post-infection, but such interactions are not well understood. We used an unbiased primary screen to profile the binding of full-length S against >9,000 human proteins and found significant S-host protein interactions, including one between S and human estrogen receptor alpha (ER). After confirming this interaction in a secondary assay, we used bioinformatics, supercomputing, and experimental assays to identify a highly conserved and functional nuclear receptor coregulator (NRC) LXD-like motif on the S2 subunit and an S-ER binding mode. In cultured cells, S DNA transfection increased ER cytoplasmic accumulation, and S treatment induced ER-dependent biological effects and ACE2 expression. Noninvasive multimodal PET/CT imaging in SARS-CoV-2-infected hamsters using [18F]fluoroestradiol (FES) localized lung pathology with increased ER lung levels. Postmortem experiments in lung tissues from SARS-CoV-2-infected hamsters and humans confirmed an increase in cytoplasmic ER expression and its colocalization with S protein in alveolar macrophages. These findings describe the discovery and characterization of a novel S-ER interaction, imply a role for S as an NRC, and are poised to advance knowledge of SARS-CoV-2 biology, COVID-19 pathology, and mechanisms of sex differences in the pathology of infectious disease.

immunology↗

bioRxiv2020

Dual-Mechanism Estrogen Receptor Inhibitors Reveal an Ensemble of Binding Modes for Robust Breast Cancer Antagonism

Efforts to improve estrogen receptor-a (ER)-targeted therapies in breast cancer have relied upon a single mechanism, with ligands having a single side chain on the ligand core that extends outward to determine antagonism of breast cancer growth. Here, we describe inhibitors with two ER-targeting moieties, one of which uses an alternate structural mechanism to generate full antagonism, freeing the side chain to independently determine other critical properties of the ligands. By combining two molecular targeting approaches into a single ER ligand, we have generated antiestrogens that function through new mechanisms and structural paradigms to achieve antagonism. These dual-mechanism ER inhibitors (DMERIs) cause alternate, non-canonical structural perturbations of the receptor ligand-binding domain (LBD) to drive antagonism of proliferation in ER-positive breast cancer cells and in allele-specific resistance models. Solution structural and coregulator peptide binding analyses with DMERIs highlight marked differences from current standard-of-care, single-mechanism antiestrogens. These findings uncover an enhanced flexibility of the ER LBD through which it can access non-consensus conformational modes in response to DMERI binding, broadly and effectively suppressing ER activity. Significance StatementTo address the unmet clinical need for effectively suppressing estrogen receptor (ER) activity with both de novo resistance and in advanced ER-positive breast cancers that are resistant to standard-of-care antiestrogens, we have developed dual-mechanism ER inhibitors (DMERIs) that employ two distinct ER targeting moieties. These DMERI elicited non-canonical structural perturbations of the receptor ligand-binding domain and stabilized multiple antagonist sub-states within the dimer to generate highly efficacious antagonism of proliferation in ER-positive breast cancer cells and in allele-specific resistance models. This work reveals new conformational modes by which the activity of ER can be effectively suppressed to block breast cancer proliferation.

cancer biology↗

bioRxiv2020

Activation of the Crtc2/Creb1 transcriptional network in skeletal muscle enhances weight loss during intermittent fasting

The Creb-Regulated Transcriptional Coactivator (Crtc) family of transcriptional coregulators drive Creb1-mediated transcription effects on metabolism in many tissues, but the in vivo effects of Crtc2/Creb1 transcription on skeletal muscle metabolism are not known. Skeletal muscle-specific overexpression of Crtc2 (Crtc 2 mice) induced greater mitochondrial activity, metabolic flux capacity for both carbohydrates and fats, improved glucose tolerance and insulin sensitivity, and increased oxidative capacity, supported by upregulation of key metabolic genes. Crtc2 overexpression led to greater weight loss during alternate day fasting (ADF), selective loss of fat rather than lean mass, supported by maintenance of higher energy expenditure during the fast. ADF downregulated most of the mitochondrial electron transport genes, and other regulators of mitochondrial function, that were substantially reversed by Crtc2-driven transcription. Glucocorticoids acted with AMPK to drive atrophy and mitophagy, which was reversed by Crtc2/Creb1 signaling. Crtc2/Creb1-mediated signaling coordinates metabolic adaptations in skeletal muscle that explain how Crtc2/Creb1 regulates metabolism and weight loss.

physiology↗

bioRxiv2020

Chemical Systems Biology Reveals Mechanisms of Dissociated Glucocorticoid Receptor Signaling

Glucocorticoids display remarkable anti-inflammatory activity, but their use is limited by on-target adverse effects including insulin resistance and skeletal muscle atrophy. We used a chemical systems biology approach, Ligand Class Analysis (LCA), to examine ligands designed to modulate glucocorticoid receptor activity through distinct structural mechanisms. These ligands displayed diverse activity profiles, providing the variance required to identify target genes and coregulator interactions that were highly predictive of their effects on myocyte glucose disposal and protein balance. Their anti-inflammatory effects were linked to glucose disposal but not muscle atrophy. This approach also predicted selective modulation in vivo, identifying compounds that were muscle sparing or anabolic for protein balance and mitochondrial potential. LCA defined the mechanistic links between the ligand-receptor interface and ligand-driven physiological outcomes, a general approach that can be applied to any ligand-regulated allosteric signaling system.

pharmacology and toxicology↗

bioRxiv2018

The SERM/SERD Bazedoxifene Disrupts ESR1 Helix 12 to Overcome Acquired Hormone Resistance in Breast Cancer Cells

Acquired resistance to endocrine therapy remains a significant clinical burden for breast cancer patients. Somatic mutations in the ESR1 (estrogen receptor alpha (ER) gene ligand-binding domain (LBD) represent a recognized mechanism of acquired resistance. Antiestrogens with improved efficacy versus tamoxifen might overcome the resistant phenotype in ER+ breast cancers. Bazedoxifene (BZA) is a potent antiestrogen that is clinically approved for use in hormone replacement therapies. We find BZA possesses improved inhibitory potency against the Y537S and D538G ER mutants compared to tamoxifen and has additional inhibitory activity in combination with the CDK4/6 inhibitor palbociclib. In addition, comprehensive biophysical and structural biology studies show that BZAs selective estrogen receptor degrading (SERD) properties that override the stabilizing effects of the Y537S and D538G ER mutations.\n\nSignificanceBazedoxifene (BZA) is a potent orally available antiestrogen that is clinically approved for use in hormone replacement therapy (DUAVEE). We explore the efficacy of BZA to inhibit activating somatic mutants of ER that can arise in metastatic breast cancers after prolonged exposure to aromatase inhibitors or tamoxifen therapy. Breast cancer cell line, biophysical, and structural data show that BZA disrupts helix 12 of the ER ligand binding domain to achieve improved potency against Y537S and D538G somatic mutants compared to 4-hydroxytamoxifen.

cancer biology↗