Biology subjects

Stauffer, S. R.

Publications and source records attributed to Stauffer, S. R..

8 recordsLinked to original sources

bioRxiv2026

CpG island density predicts CBP/p300 dependency across 3D chromatin clusters

RNA Polymerase II (Pol2) organizes transcription through higher-ordered chromatin clusters that integrate promoter and enhancer interactions to coordinate gene expression. Nevertheless, the features that distinguish unique classes of Pol2-mediated clusters remain to be defined. Here, we identify two distinct classes of Pol2-mediated clusters: one enriched for CpG islands, promoter-promoter interactions, and housekeeping gene expression, and another characterized by high CBP/p300 occupancy, enhancer-promoter looping, and lineage-defining (LD) transcriptional programs. Acute inhibition of CBP/p300 catalytic activity leads to rapid loss of acetylation at enhancers and preferential downregulation of LD genes, resulting in impaired cellular proliferation and activation of apoptotic programs. Integrative machine learning modeling reveals that cluster strength, RNA half-life, and CpG island content as strong predictors of genes sensitive to CBP/p300 inhibition. Together, these findings clarify enhancer-addiction and vulnerability to CBP/p300 inhibition.

cancer biology↗

bioRxiv2025

Fragment-Based Development of NSP14 Exonuclease Inhibitors Confounded by Batch-to-Batch Variability

Point mutations in the exonuclease (ExoN) site of non-structural protein 14 (NSP14) compromise the fitness of betacoronaviruses like SARS-CoV-2, implicating NSP14 ExoN inhibition as an antiviral strategy. However, there are no advanced compounds that inhibit NSP14s ExoN activity. Building upon the reported crystal structures of two fragments bound to NSP14s ExoN site, we identified a series of 3,5-disubsituted pyrazoles that bound to and inhibited NSP14 ExoN. However, upon resynthesis, we discovered that these putative leads were false positives, perhaps due to contaminating divalent cations which potently inhibit NSP14 ExoN. Our results provide a cautionary tale to the field about the sensitivity of NSP14 to divalent cations and illustrate the challenges associated with directly targeting the NSP14 ExoN site via fragment merging.

biochemistry↗

bioRxiv2025

MCL1 may not mediate chemoresistance

The anti-apoptotic BCL2 family member MCL1 is overexpressed in many cancers and has been linked to chemoresistance. Unlike other BCL2 family members, MCL1 displays both well-defined mitochondrial anti-apoptotic activities and also emerging nuclear functions. Prior reports suggest that MCL1 enters the nucleus during chemotherapy and promotes chemoresistance by influencing cell cycle progression and DNA repair. These nuclear roles of MCL1, however, remain poorly characterized. Using a newly validated monoclonal antibody across several cell lines and treatments, we find no evidence that MCL1 enhances chemoresistance or preferentially accumulates in the nucleus after drug exposure. Proximity biotinylation identified novel nuclear MCL1 interactors but did not recover previously reported DNA repair or cell cycle partners. Thus, while MCL1 does reach the nucleus and interact with nuclear proteins, our data do not support a role for MCL1 in chemoresistance. Further work is needed to clarify the functional significance of nuclear MCL1. SIGNFIGANCEPrevious studies have implicated MCL1 in promoting chemoresistance via interactions with DNA repair machinery in the nucleus. Using a validated, monoclonal anti-MCL1 antibody, we were unable to replicate these data. We report that MCL1 neither confers chemoresistance, translocates to the nucleus during chemotherapy treatment, nor interacts with DNA repair proteins in live cancer cells.

cell biology↗

bioRxiv2025

Development and Characterization of Triazole-Based WDR5 Inhibitors for the Treatment of Glioblastoma

Glioblastoma (GBM) cancer stem cells (CSCs) contribute to tumor recurrence, treatment resistance, and dismal clinical outcomes. Genetic and pharmacological evidence suggests that the nuclear scaffolding protein WD-repeat containing protein 5 (WDR5) is a therapeutic vulnerability of the CSC population. However, previously reported WDR5 inhibitors display low permeability and are unable to penetrate the blood-brain barrier (BBB), limiting their utility in GBM. Herein, we report the structure-guided development of a novel series of triazole-based WDR5 WIN-site inhibitors designed to increase passive brain penetration. We identified triazole-based WDR5 inhibitors that are potent, passively permeable, and in some cases more brain penetrant than other scaffolds. We phenotypically assessed our novel WDR5 inhibitors in a panel of patient-derived CSC models and uncovered unique WDR5-regulated metabolic genes in GBM. We also evaluated their antiproliferative activity against CSCs both in vitro and in vivo. Finally, to identify novel combination opportunities, we screened a 2,100-compound chemical probe library and identified that the ATAD2 inhibitor BAY-850 synergizes with WDR5 inhibitors to enhance CSC killing. Our work diversifies the chemical matter targeting WDR5, clarifies the in vitro consequences of WIN-site inhibition in CSCs, and encourages the future development of next-generation WDR5 inhibitors with the potential to achieve in vivo efficacy in the brain.

cancer biology↗

bioRxiv2025

Mechanism and Structure-Guided Optimization of SLC1A1/EAAT3-Selective Inhibitors in Kidney Cancer

Renal Cell Carcinomas (RCCs) depend metabolically on the trimeric sodium-coupled aspartate and glutamate transporter, SLC1A1/EAAT3; however, pharmacologically targeting SLC1A1 is challenging. We determined a cryo-EM structure of human SLC1A1 bound to compound 3e, a recently described SLC1A1-selective bicyclic imidazo[1,211]pyridine-3-amine (BIA) inhibitor. 3e binds a membrane-embedded allosteric pocket accessible only in the apo state, when SLC1A1 is unbound to substrate and sodium, and likely prevents sodium and substrate binding. Moreover, by wedging between the trimerization domain and the substrate-binding transport domain, alongside a cholesterol moiety from the lipid bilayer, 3e blocks SLC1A1s elevator-like movements that are essential for transport. Mutations in this binding pocket abolish 3e interaction and counteract 3es cytotoxicity in RCC cells, confirming on-target activity and explaining SLC1A1 selectivity. A structure-guided medicinal chemistry effort yielded two new SLC1A1-selective BIA derivatives, PBJ1 and PBJ2, with enhanced cytotoxicity resulting from the inhibition of SLC1A1-dependent aspartate, glutamate, and cysteine metabolism.

cancer biology↗

bioRxiv2024

ISGylation of the SARS-CoV-2 N protein by HERC5 impedes N oligomerization and thereby viral RNA synthesis

Interferon (IFN)-stimulated gene 15 (ISG15), a ubiquitin-like protein, is covalently conjugated to host (immune) proteins such as MDA5 and IRF3 in a process called ISGylation, thereby limiting the replication of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, whether SARS-CoV-2 proteins can be directly targeted for ISGylation remains elusive. In this study, we identified the nucleocapsid (N) protein of SARS-CoV-2 as a major substrate of ISGylation catalyzed by the host E3 ligase HERC5; however, N ISGylation is readily removed through de-ISGylation by the papain-like protease (PLpro) activity of NSP3. Mass spectrometry analysis identified that the N protein undergoes ISGylation at four lysine residues (K266, K355, K387 and K388), and mutational analysis of these sites in the context of a SARS-CoV-2 replicon (N-4KR) abolished N ISGylation and alleviated ISGylation-mediated inhibition of viral RNA synthesis. Furthermore, our results indicated that HERC5 targets preferentially phosphorylated N protein for ISGylation to regulate its oligomeric assembly. These findings reveal a novel mechanism by which the host ISGylation machinery directly targets SARS-CoV-2 proteins to restrict viral replication and illuminate how an intricate interplay of host (HERC5) and viral (PLpro) enzymes coordinates viral protein ISGylation and thereby regulates virus replication.

microbiology↗

bioRxiv2023

The SLC1A1/EAAT3 Dicarboxylic Amino Acid Transporter is an Epigenetically Dysregulated Nutrient Carrier that Sustains Oncogenic Metabolic Programs

Inactivation of pVHL tumor suppressor in clear cell Renal Cell Carcinoma (ccRCC) increases the abundance of Histone H3 lysine 27 acetylation (H3K27ac). We hypothesized that H3K27ac, a marker of transcriptional activation, drives the expression of critical oncogenes in ccRCC. Using H3K27ac ChIP-Seq; RNA-Seq; an in vivo positive selection screen; cell-based functional studies; and clinical validations; here, we report the identification of the SLC1A1/EAAT3 aspartate (Asp) and glutamate (Glu) transporter as a ccRCC oncogene. pVHL loss promotes SLC1A1 expression in a HIF-independent manner. Importantly, SLC1A1 inactivation depletes Asp/Glu-derived metabolites, impedes ccRCC growth both in vitro and in vivo, and sensitizes ccRCCs to metabolic therapeutics (e.g., glutaminase blockers). Finally, in human ccRCC biospecimens, higher SLC1A1 expression is associated with metastatic disease and clusters with elevated expression of other solute carriers, but not HIF/Hypoxia pathways. Altogether, our studies identify a HIF-independent metabolic hub in ccRCC and credential SLC1A1 as an actionable ccRCC oncogene. STATEMENT OF SIGNIFICANCETargeting chronic HIF activation underlies many therapeutic strategies in ccRCC; but, unfortunately, is not curative. SLC1A1, instead, represents a HIF-independent ccRCC dependency, which is targetable alone and together with other antimetabolites, such as glutaminase inhibitors. These observations identify an actionable metabolic program that functions independent of HIF in ccRCC.

cancer biology↗

bioRxiv2021

The WRAD complex represents a therapeutically exploitable target for cancer stem cells in glioblastoma

Glioblastomas (GBMs) are heterogeneous, treatment-resistant tumors that are driven by populations of cancer stem cells (CSCs). Despite their importance for tumor growth, few molecular mechanisms critical for CSC population maintenance have been exploited for therapeutic development. We developed a spatially resolved loss-of-function screen in GBM patient-derived organoids to identify essential epigenetic regulators in the SOX2-enriched, therapy resistant niche and identified WDR5 as indispensable for this population. WDR5 is a component of the WRAD complex, which promotes SET1-family-mediated Lys4 methylation of histone H3, associated with positive regulation of transcription. In GBM CSC models, WDR5 inhibitors blocked WRAD complex assembly and reduced H3K4 trimethylation and expression of genes involved in CSC-relevant oncogenic pathways. H3K4me3 peaks lost with WDR5 inhibitor treatment occurred disproportionally on POU transcription factor motifs, including the POU5F1(OCT4)::SOX2 motif. We incorporated a SOX2/OCT4 motif driven GFP reporter system into our CSC cell models and found that WDR5 inhibitor treatment diminished reporter activity. Further, WDR5 inhibitor treatment altered the stem cell state, disrupting CSC in vitro growth and self-renewal as well as in vivo tumor growth. These findings highlight the role of WDR5 and the WRAD complex in maintaining the CSC state and provide a rationale for therapeutic development of WDR5 inhibitors for GBM and other advanced cancers. SignificanceIn this study, we perform an epigenetic-focused functional genomics screen in glioblastoma organoids and identify WDR5 as an essential epigenetic regulator in the SOX2-enriched, therapy resistant cancer stem cell niche. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=135 HEIGHT=200 SRC="FIGDIR/small/461125v2_ufig1.gif" ALT="Figure 1"> View larger version (60K): org.highwire.dtl.DTLVardef@bc5c5eorg.highwire.dtl.DTLVardef@545a2borg.highwire.dtl.DTLVardef@1ebd3c9org.highwire.dtl.DTLVardef@19d5d30_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗