Biology subjects

Thorne, C. A.

Publications and source records attributed to Thorne, C. A..

11 recordsLinked to original sources

bioRxiv2026

Ceramide Synthases Regulate Myristate-Induced Intestinal IRE1α Activation

Background & AimsHigh-fat diets (HFDs) are a major modifiable risk factor for intestinal health. Current research focuses primarily on palmitate (C16:0); however, myristate (C14:0, rich in dairy products) has been minimally investigated. HFDs increase ceramide generation which drives endoplasmic reticulum (ER) stress; with both sphingolipids and ER stress being key contributors to intestinal biology. Whether different fatty acids uniquely impact sphingolipid metabolism and ER stress in intestinal biology has not been well defined. MethodsHuman colon epithelial cells were utilized to determine the role of ceramide synthases (CerS) 5 and 6 on myristate-induced ER stress using pharmacologic inhibitors and siRNA. Intestinal epithelial cell specific CerS5 and/or CerS6 knockout mice of both sexes were fed a control, high milk-fat, or high lard-fat diet for 16 weeks. Cells and colon tissues were analyzed for lipids, mRNA, and protein. ResultsMyristate treatment increased C14:0-ceramide and induced IRE1-dependent ER stress. Inhibition of CerS suppressed these effects, yet knockdown of CerS5/6, the primary enzymes generating C14:0-ceramide, unexpectedly exacerbated IRE1 activation both in vitro and in vivo, potentially due to depletion of dihydro(dh)sphingosine. ConclusionsCerS are required for myristate-induced IRE1 activation and restoration of the sphingoid base pool provides partial protection from intestinal ER stress. SYNOPSISThis study identifies a new mechanism linking dietary fats to intestinal cell stress. Ceramide synthases drive ER stress triggered by myristate, a dairy-derived fat, while restoring sphingoid bases partially protects cells, revealing a new role for sphingolipids in shaping intestinal responses to diet. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=193 SRC="FIGDIR/small/728542v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@d66503org.highwire.dtl.DTLVardef@181bffaorg.highwire.dtl.DTLVardef@162f675org.highwire.dtl.DTLVardef@22a511_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2026

Preservation of Human Colonic Stem Cells Requires an ERK Dynamics Checkpoint Mediated by AKT

Colonic stem cells reside in a microenvironment enriched in epidermal growth factor, which is essential for their survival and can activate both PI3K-AKT and MAPK-ERK pathways. This predicts co-activation of both pathways within the growth factor-high stem cell compartment at the base of crypts. However, in patient-derived human colonic organoids and normal human tissue, stem cells maintain robust AKT activity while suppressing ERK signaling despite active EGFR engagement. As stem cells differentiate, they activate pulsatile ERK signaling, which is essential for migration, survival, and maintenance of barrier function. We show that AKT-dependent phosphorylation of RAF-1 at serine 259 establishes a post-receptor checkpoint that maintains ERK temporal dynamics in stem cells. Acute activation of ERK in stem cells triggers rapid global differentiation. Disruption of the ERK checkpoint via mutation of serine 259 leads to sustained AKT and ERK co-activation in stem cells. Unlike ERK/AKT coactivation driven by apoptosis, co-activation in the stem cell compartment results in the emergence of a neoplastic, architecturally disorganized cell population dominating the cell fate profile. Incredibly, introducing brief ERK pulses through AKT inhibition or ERK activation triggers re-differentiation of neoplastic cells. Consistent with duration-dependent MAPK encoding principles, these data demonstrate that regardless of baseline signaling amplitude, ERK signaling dynamics are epistatic to total kinase signaling load in human colonic stem cells. SIGNIFICANCEStem cells must balance self-renewal and differentiation while remaining responsive to continuous mitogenic stimulation to preserve tissue homeostasis. When self-renewal is impaired, wound healing and barrier integrity decline, whereas loss of proper differentiation drives tumorigenesis. Our findings demonstrate that this balance in the human colon is achieved through temporal control of kinase signaling rather than modulation of ligand availability. By establishing an AKT-dependent ERK dynamics checkpoint, colonic stem cells suppress differentiation-inducing ERK pulses while maintaining growth factor responsiveness. These results identify kinase dynamics as a fundamental determinant of epithelial homeostasis and suggest that subtle alterations in these dynamics may destabilize tissue organization during regeneration or chronic inflammation. Temporal encoding of kinase activity thus represents a central organizing principle in human stem cell biology.

cell biology↗

bioRxiv2026

Developmental Synchrony of Retinal Waves, Apoptosis, and Angiogenesis in Postnatal Retina

Postnatal mouse retinal development is a multi-faceted process involving the coordinated interaction of spontaneous neural activity as retinal waves, vascular plexus growth, and programmed cell death. While these processes are known to interact at a coarse scale, the specific mechanisms integrating them have remained elusive. Using large-scale, widefield calcium imaging, high-density multielectrode array recordings, single cell RNA-seq, and immunohistochemistry, we characterise a tightly aligned centrifugal expansion pattern during retinal development. This pattern is common to stage II retinal wave onsets, vascular development, Heme oxygenase-1 (Hmox1) expressing microglia, apoptotic cell markers, and a novel set of auto-fluorescent cluster complexes (ACCs) identified in this study. Apoptotic cells are known to upregulate functional Pannexin1 (PANX-1) hemichannels. These voltage-gated channels release purinergic molecules which act as "eat me" signals to neighbouring microglia. PANX-1 hemichannel blockade with the drug probenecid results in a profound decrease in spontaneous wave frequency and strength, suggesting that retinal waves are indeed triggered by these apoptotic cells. Taken together, our observations suggest that spontaneous waves are initially triggered in hotspots by hyperactive apoptotic RGCs in unvascularised retinal areas. These apoptotic cells release purinergic molecules via PANX-1 hemichannels, leading to wave generation. This hyperactivity leads to local hypoxic conditions, which, coupled with high extracellular ATP concentrations, promotes angiogenesis. Once blood vessels reach a particular hotspot, ATP release activates Hmox1 positive microglia, which engulf the dying RGCs, creating the auto-fluorescent clusters. Herein, we present a unified mechanism linking causally linking early neural activity, programmed cell death and angiogenesis in the mammalian retina.

neuroscience↗

bioRxiv2025

Dyr726, a brain-penetrant inhibitor of PI3Kα, Type III receptor tyrosine kinases, and WNT signalling

The vast majority of clinical small molecule multi-kinase inhibitors (mKI) report abject failures in targeting cancers with high stem cell contents like high-grade glioma and colorectal cancers. The FDA-approved mKIs to date ablate receptor tyrosine kinase signaling but do not target the paradoxical WNT signaling which is a key survival driver for the self-renewing cancer stem cells. The WNT pathway enhances cancer plasticity and triggers relapse of highly heterogenous tumours. Using de novo synthesis and structure-activity-relationship (SAR) studies with blood-brain-barrier (BBB) penetrant mKI scaffolds, we designed a highly potent and selective small molecule inhibitor of PI3K, PDGFR/KIT, and the WNT pathway denoted Dyr726. Dyr726 is superior to clinical mKIs and inhibits PI3K-AKT-mTOR and WNT-pathway signaling at multiple nodes thereby impeding proliferation, invasion, and tumour growth. Phospho-proteomic, structural, and target engagement analyses, combined with in vitro, in vivo efficacy, and pharmacokinetic studies reveal that Dyr726 is a brain-penetrant small molecule which effectively reduces tumour volume and extends survival of murine orthotopic models. Our current work establishes a first-in-class brain penetrant small molecule mKI which simultaneously antagonize the PI3K-AKT-mTOR and WNT pathways in preclinical cancer stem cell cultures, adult and pediatric primary organoids, and orthotopic murine models with positive efficacy in combination with clinical standard of care.

cancer biology↗

bioRxiv2024

Cell-based high-content approach for SARS-CoV-2 neutralization identifies unique monoclonal antibodies and PI3K pathway inhibitors.

The sudden rise of the SARS-CoV-2 virus and the delay in the development of effective therapeutics to mitigate it made evident a need for ways to screen for compounds that can block infection and prevent further pathogenesis and spread. Yet, identifying effective drugs efficacious against viral infection and replication with minimal toxicity for the patient can be difficult. Monoclonal antibodies were shown to be effective, yet as the SARS-CoV-2 mutated, these antibodies became ineffective. Small molecule antivirals were identified using pseudovirus constructs to recapitulate infection in non-human cells, such as Vero E6 cells. However, the impact was limited due to poor translation of these compounds in the clinical setting. This is partly due to the lack of similarity of screening platforms to the in vivo physiology of the patient and partly because drugs effective in vitro showed dose-limiting toxicities. In this study, we performed two high-throughput screens in human lung adenocarcinoma cells with authentic SARS-CoV-2 virus to identify both monoclonal antibodies that neutralize the virus and clinically useful kinase inhibitors to block the virus and prioritize minimal host toxicity. Using high-content imaging combined with single-cell and multidimensional analysis, we identified antibodies and kinase inhibitors that reduce virus infection without affecting the host. Our screening technique uncovered novel antibodies and overlooked kinase inhibitors (i.e. PIK3i, mTORi, multiple RTKi) that could be effective against SARS-CoV-2 virus. Further characterization of these molecules will streamline the repurposing of compounds for the treatment of future pandemics and uncover novel mechanisms viruses use to hijack and infect host cells.

pharmacology and toxicology↗

bioRxiv2023

SGRN: A Cas12a-driven Synthetic Gene Regulatory Network System

Gene regulatory networks, which control gene expression patterns in development and in response to stimuli, use regulatory logic modules to coordinate inputs and outputs. One example of a regulatory logic module is the gene regulatory cascade (GRC), where a series of transcription factor genes turn on in order. Synthetic biologists have derived artificial systems that encode regulatory rules, including GRCs. Furthermore, the development of single-cell approaches has enabled the discovery of gene regulatory modules in a variety of experimental settings. However, the tools available for validating these observations remain limited. Based on a synthetic GRC using DNA cutting-defective Cas9 (dCas9), we designed and implemented an alternative synthetic GRC utilizing DNA cutting-defective Cas12a (dCas12a). Comparing the ability of these two systems to express a fluorescent reporter, the dCas9 system was initially more active, while the dCas12a system was more streamlined. Investigating the influence of individual components of the systems identified nuclear localization as a major driver of differences in activity. Improving nuclear localization for the dCas12a system resulted in 1.5-fold more reporter-positive cells and a 15-fold increase in reporter intensity relative to the dCas9 system. We call this optimized system the "Synthetic Gene Regulatory Network" (SGRN, pronounced "sojourn").

synthetic biology↗

bioRxiv2022

Genetically Engineered Human Pituitary Corticotroph Tumor Organoids Exhibit Divergent Responses To Glucocorticoid Receptor Modulators

Cushings disease (CD) is a serious endocrine disorder attributed to an ACTH-secreting pituitary neuroendocrine tumor (PitNET) that subsequently causes chronic hypercortisolemia. PitNET regression has been reported following treatment with the investigational selective glucocorticoid receptor (GR) modulator relacorilant, but the mechanisms behind that effect remain unknown. Human PitNET organoid models were generated from induced human pluripotent stem cells (iPSCs) or fresh tissue obtained from CD patient PitNETs (hPITOs). Genetically engineered iPSC derived organoids were used to model the development of corticotroph PitNETs expressing USP48 (iPSCUSP48) or USP8 (iPSCUSP8) somatic mutations. Organoids were treated with the GR antagonist mifepristone or the GR modulator relacorilant with or without somatostatin receptor (SSTR) agonists pasireotide or octreotide. In iPSCUSP48 and iPSCUSP8 cultures, mifepristone induced the predominant expression of SSTR2 with a concomitant increase in ACTH secretion and tumor cell proliferation. Relacorilant predominantly induced SSTR5 expression and tumor cell apoptosis with minimal ACTH induction. Hedgehog signaling mediated the induction of SSTR2 and SSTR5 in response to mifepristone and relacorilant. Relacorilant sensitized PitNET organoid responsiveness to pasireotide. Therefore, our study identified the potential therapeutic use of relacorilant in combination with somatostatin analogs and demonstrated the advantages of relacorilant over mifepristone, supporting its further development for use in the treatment of CD patients. HIGHLIGHTSO_LICushing disease (CD) is a serious endocrine disorder caused by an adrenocorticotropic hormone (ACTH)-secreting pituitary neuroendocrine tumor (PitNET) that leads to chronic hypercortisolemia C_LIO_LIMifepristone (Korlym(R)), a non-selective glucocorticoid receptor (GR) antagonist, is an approved treatment for patients with Cushing disease, and competes with the binding of cortisol to the GR as well as the binding of progesterone to the progesterone receptor. C_LIO_LIRelacorilant is an investigational selective GR modulator in development for the treatment of Cushing syndrome that, unlike mifepristone, does not bind to the other hormone receptors. C_LIO_LIUnlike mifepristone, relacorilant does not significantly raise systemic cortisol levels, and cases of PitNET regression with relacorilant have been reported. However, the mechanisms behind these clinical differences remained unknown. C_LIO_LIPitNET organoids were generated from: 1) CRISPR-Cas9 gene editing of patient iPSCs, and 2) CD patient corticotroph PitNETs (hPITOs) and used to compare the diverse effects of mifepristone and relacorilant in a human-relevant model that recapitulates the PitNET microenvironment in vitro. C_LIO_LIMifepristone and relacorilant have different effects on the induction of somatostatin receptor (SSTR) SSTR2 and SSTR5 expression, ACTH secretion and PitNET organoid proliferation and apoptosis. C_LI BRIEF COMMENTARYO_ST_ABSBackgroundC_ST_ABSCushings disease (CD), a serious endocrine disorder caused by an adrenocorticotropic hormone (ACTH)-secreting pituitary neuroendocrine tumor (PitNET) leads to chronic hypercortisolemia. Approved for the treatment for CD, Mifepristone (Korlym(R)) is a non-selective glucocorticoid receptor (GR) antagonist with additional competitive binding with progesterone for the progesterone receptor. Relacorilant, an investigational selective GR modulator in development for the treatment of CD, does not bind to the other hormone receptors. Translational SignificancePatient-derived PitNET organoids recapitulate the tumor microenvironment in vitro. PitNET organoids revealed the advantages of relacorilant over mifepristone, supporting its further development for use in the treatment of CD.

physiology↗

bioRxiv2022

Live-Cell Imaging in Human Colonic Monolayers Reveals Erk Waves Limit the Stem Cell Compartment to Maintain Epithelial Homeostasis

The establishment and maintenance of different cellular compartments in tissues is a universal requirement across all metazoans. Maintaining the correct ratio of cell types in time and space allows tissues to form patterned compartments and perform complex functions. Patterning is especially evident in the human colon, where tissue homeostasis is maintained by stem cells in crypt structures that balance proliferation and differentiation. Here we developed a 2D patient derived organoid (PDO) screening platform to study tissue patterning and kinase pathway dynamics in single cells across hundreds of conditions. Using this system, we discovered that waves of Erk signaling induced by apoptotic cells play a critical role in maintaining tissue patterning and homeostasis. If Erk is activated acutely across all cells instead of in wavelike patterns, then tissue patterning, and stem cell maintenance are lost. Conversely, if Erk activity is inhibited, then stem cells become unrestricted and expand dramatically. This work demonstrates that the colonic epithelium requires coordinated Erk signaling dynamics to maintain patterning and tissue homeostasis. Our work reveals how Erk can antagonize stem cells yet support cell replacement and the function of the gut.

cell biology↗

bioRxiv2022

Feedback in the β-catenin destruction complex imparts bistability and cellular memory

Wnt ligands are considered classical morphogens, for which the strength of the cellular response is proportional to the concentration of the ligand. Herein, we show an emergent property of bistability arising from feedback among the Wnt destruction complex proteins that target the key transcriptional co-activator {beta}-catenin for degradation. Using biochemical reconstitution, we identified positive feedback between the scaffold protein Axin and the kinase GSK3. Theoretical modeling of this feedback between Axin and GSK3 predicted that the activity of the destruction complex exhibits bistable behavior. We experimentally confirmed these predictions by demonstrating that cellular cytoplasmic {beta}-catenin concentrations exhibit an "all-or-none" response with sustained memory (hysteresis) of the signaling input. This bistable behavior was transformed into a graded response and memory was lost through inhibition of GSK3. These findings provide a mechanism for establishing decisive, switch-like cellular response and memory upon Wnt pathway stimulation. One Sentence SummaryPositive feedback within the {beta}-catenin destruction complex gives rise to bistability and memory in response to Wnt stimulation, imparting signal transduction accuracy and insulation.

cell biology↗

bioRxiv2021

Convergent antibody responses to the SARS-CoV-2 spike protein in convalescent and vaccinated individuals

Unrelated individuals can produce genetically similar clones of antibodies, known as public clonotypes, which have been seen in responses to different infectious diseases as well as healthy individuals. Here we identify 37 public clonotypes in memory B cells from convalescent survivors of SARS-CoV-2 infection or in plasmablasts from an individual after vaccination with mRNA-encoded spike protein. We identified 29 public clonotypes, including clones recognizing the receptor-binding domain (RBD) in the spike protein S1 subunit (including a neutralizing, ACE2-blocking clone that protects in vivo), and others recognizing non-RBD epitopes that bound the heptad repeat 1 region of the S2 domain. Germline-revertant forms of some public clonotypes bound efficiently to spike protein, suggesting these common germline-encoded antibodies are preconfigured for avid recognition. Identification of large numbers of public clonotypes provides insight into the molecular basis of efficacy of SARS-CoV-2 vaccines and sheds light on the immune pressures driving the selection of common viral escape mutants.

immunology↗

bioRxiv2020

In silico identification and validation of inhibitors of the interaction between neuropilin receptor 1 and SARS-CoV-2 Spike protein

Neuropilin-1 (NRP-1) is a multifunctional transmembrane receptor for ligands that affect developmental axonal growth and angiogenesis. In addition to a role in cancer, NRP-1 is a reported entry point for several viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal agent of coronavirus disease 2019 (COVID-19). The furin cleavage product of SARS-CoV-2 Spike protein takes advantage of the vascular endothelial growth factor A (VEGF-A) binding site on NRP-1 which accommodates a polybasic stretch ending in a C-terminal arginine. This site has long been a focus of drug discovery efforts for cancer therapeutics. We recently showed that interruption of the VEGF-A/NRP-1 signaling pathway ameliorates neuropathic pain and hypothesize that interference of this pathway by SARS-CoV-2 spike protein interferes with pain signaling. Here, we report hits from a small molecule and natural product screen of nearly 0.5 million compounds targeting the VEGF-A binding site on NRP-1. We identified nine chemical series with lead- or drug-like physico-chemical properties. Using an ELISA, we demonstrate that six compounds disrupt VEGF-A-NRP-1 binding more effectively than EG00229, a known NRP-1 inhibitor. Secondary validation in cells revealed that almost all tested compounds inhibited VEGF-A triggered VEGFR2 phosphorylation. Two compounds displayed robust inhibition of a recombinant vesicular stomatitis virus protein that utilizes the SARS-CoV-2 Spike for entry and fusion. These compounds represent a first step in a renewed effort to develop small molecule inhibitors of the VEGF-A/NRP-1 signaling for the treatment of neuropathic pain and cancer with the added potential of inhibiting SARS-CoV-2 virus entry.

neuroscience↗