Biology subjects

Bedford, M. T.

Publications and source records attributed to Bedford, M. T..

11 recordsLinked to original sources

bioRxiv2026

Acquired resistance to the PRMT5 inhibitor confers collateral sensitivity to MEK inhibition in MTAP-null non-small cell lung cancer

Protein arginine methyltransferase 5 (PRMT5) is a synthetic lethal target in methylthioadenosine phosphorylase-deleted (MTAP-null) cancers. Second-generation MTA-cooperative PRMT5 inhibitors preferentially target MTAP-null cells while largely sparing MTAP-wildtype (MTAP-WT) cells, thereby improving tumor selectivity over first-generation PRMT5 inhibitors. Despite encouraging efficacy and safety signals in early clinical studies, the modest objective response rates (ORRs) observed with these inhibitors suggest that intrinsic or acquired resistance may limit their clinical benefit. Here, we investigated mechanisms of acquired resistance to the MTA-cooperative PRMT5 inhibitor BMS-986504/MRTX1719 in MTAP-null non-small cell lung cancer (NSCLC) cells and sought to identify therapeutic vulnerabilities that emerge upon resistance. Using multiple in vitro-derived resistant models, we found that acquired resistance was not fully explained by alterations in PRMT5 activity or reduced MTA levels. Instead, resistance was associated with collateral sensitivity to MEK inhibition and enrichment of MAPK-related transcriptional programs. Together, these findings identify MEK inhibition as an actionable collateral vulnerability in MTAP-null NSCLC cells that acquire resistance to PRMT5 inhibition.

cancer biology↗

bioRxiv2025

Transient Poly(ADP-Ribose) Triggers FUS Condensation Hysteresis via a Prion-Like Mechanism

Hysteresis--where a system retains memory of a transient stimulus--is common in signaling but can also arise in intracellular organization. DNA repair foci, a type of biomolecular condensate, are initiated by the short-lived noncanonical nucleic acid poly(ADP-ribose) (PAR). PAR recruits proteins with prion-like domains (PrLDs), such as Fused in Sarcoma (FUS), and initiates their condensation, which persists even after PAR degradation. How FUS transitions from PAR-dependent to PAR-independent condensation remains unclear. Here, we show that PAR binding triggers a conformational switch in FUS, enabling sustained condensation. PAR binds to the C-terminal arginine-rich region of FUS, displacing intramolecular contacts, and exposing the N-terminal PrLD. This conformational opening allows PrLD interactions in trans, stabilizing condensates independently of PAR. FUS thus undergoes a regulated, nucleated conformational conversion--reminiscent of classical prions. This mechanism implies a paradigm of nucleic acid-induced conformational memory that may underlie hysteresis in intracellular organization in health and disease. HIGHLIGHTS[bullet] PAR-initiated FUS condensation follows a bi-modular mechanism involving both FUS termini. [bullet]Simulations predict and experiments confirm PAR disrupts FUS intramolecular contacts. [bullet]Upon condensation, FUS adopts a conformation with its N-terminus open for interactions. [bullet]N-terminal interactions maintain FUS condensation when PAR degrades during DNA repair.

molecular biology↗

bioRxiv2025

Mechanistic Insights into Proteomic Mutation-Phenotype Linkages from Tiling Mutagenesis Screens

High-throughput mutagenesis screens are powerful tools for mapping mutations to phenotypes. However, deciphering the molecular mechanisms that link mutations to phenotypic outcomes remains a significant challenge. Here, we present ProTiler-Mut, a versatile computational framework that harnesses tiling mutagenesis screens, which introduce variants across entire protein sequences, to facilitate investigation of mutation-to-phenotype associations at multiple levels, including individual residues, protein substructures, and protein-protein interactions (PPIs). As demonstrated through our analyses of base editing (BE) screens targeting DNA Damage Response (DDR) proteins and T cell regulators, ProTiler-Mut provides novel insights into the mutation-phenotype linkages, including: i) refined classification of mutation that reveals separation-of-function (SOF) category beyond the conventional binary classification of loss-of-function (LOF) and gain-of-function (GOF); ii) definition of phenotype-associated hotspot substructures that enable the inference of the function of unscreened pathogenic mutations; and iii) identification of phenotype-associated PPIs disrupted by functional mutations. Through ProTiler-Mut analyses, we identified a substructure harboring pathogenic GOF mutations that disrupt interactions between the kinases MAPK1 and RSK1, leading to MAPK1 activation and elevated expression of the immune checkpoint receptor PD-1. Furthermore, we demonstrate the applicability of ProTiler-Mut to various mutagenesis screening platforms, highlighting its broad utility and generalizability.

bioinformatics↗

bioRxiv2024

Affinity purification mass spectrometry characterization of the interactome of receptor tyrosine kinase proline rich motifs in cancer

Receptor tyrosine kinase (RTK) overexpression is linked to the development and progression of multiple cancers. RTKs are classically considered to initiate cytoplasmic signalling pathways via ligand-induced tyrosine phosphorylation, however recent evidence points to a second tier of signalling contingent on interactions mediated by the proline-rich motif (PRM) regions of non-activated RTKs. The presence of PRMs on the C-termini of >40% of all RTKs and the abundance of PRM-binding proteins encoded by the human genome suggests that there is likely to be a large number of previously unexplored interactions which add to the RTK intracellular interactome. Here, we explore the RTK PRM interactome and its potential significance using affinity purification mass spectrometry and in silico enrichment analyses. Peptides comprising PRM-containing C-terminal tail regions of EGFR, FGFR2 and HER2 were used as bait to affinity purify bound proteins from different cancer cell line lysates. 490 unique interactors were identified, amongst which proteins with metabolic, homeostatic and migratory functions were overrepresented. This suggests that PRMs from RTKs may sustain a diverse interactome in cancer cells. Since RTK overexpression is common in cancer RTK PRM-derived signalling may be an important, but as yet underexplored, contributor to negative cancer outcomes including resistance to kinase inhibitors.

cell biology↗

bioRxiv2022

Genome-wide analysis of dendritic cell differentiation

Dendritic cells (DCs) are immune sentinel cells that comprise antigen-presenting conventional DCs (cDCs) and cytokine-producing plasmacytoid DCs (pDCs). Cytokine Flt3 ligand (Flt3L) supports the proliferation of hematopoietic progenitors, and is also necessary and sufficient for DC differentiation. Here we characterized the spontaneous differentiation of a Flt3L-dependent murine progenitor cell line into pDCs and "myeloid" cDCs (cDC2s), and interrogated it using a genome-wide CRISPR/Cas9 dropout screen. The screen revealed multiple regulators of DC differentiation including the glycosylphosphatidylinositol transamidase complex, the Nieman-Pick type C cholesterol transporter and arginine methyltransferase Carm1; the role of Carm1 in pDC and cDC2 differentiation was confirmed by conditional targeting in vivo. We also found that negative regulators of mTOR signaling, including the subunits of TSC and GATOR1 complexes, restricted progenitor growth but enabled DC differentiation. The results provide a comprehensive forward genetic analysis of DC differentiation, and help explain how the opposing processes of proliferation and differentiation could be driven by the same cytokine.

immunology↗

bioRxiv2021

Molecular basis for SPINDOC-Spindlin1 engagement and its role in transcriptional inhibition

Spindlin1 is a transcriptional coactivator with three Tudor-like domains, of which the first and second Tudors are engaged in histone methylation readout, while the function of the third Tudor is largely unknown. Recent studies revealed that the transcriptional co-activator activity of Spindlin1 could be attenuated by SPIN*DOC. Here we solved the crystal structure of SPIN*DOC-Spindlin1 complex, revealing that a hydrophobic motif, DOCpep3 (256-281), of SPIN*DOC interacts with Tudor 3 of Spindlin1 and completes its {beta}-barrel fold. Massive hydrophobic contacts and hydrogen bonding interactions ensure a high affinity DOCpep3-Spindlin1 engagement with a binding Kd of 30 nM. Interestingly, we characterized two more K/R-rich motifs of SPIN*DOC, DOCpep1 (187-195) and DOCpep2 (228-239), which bind to Spindlin1 at lower affinities with Kd values of 78 M and 31 M, respectively. Structural and binding studies revealed that DOCpep1 and DOCpep2 competitively bind to the aromatic cage of Spindlin1 Tudor 2 that is responsible for H3K4me3 readout. Although DOCpep3-Spindlin1 engagement is compatible with histone readout, an extended SPIN*DOC fragment containing DOCpep1 and DOCpep2 inhibits histone or TCF4 binding by Spindin1 due to introduced competition. This inhibitory effect is more pronounced for weaker binding targets but not for strong ones such as H3 "K4me3-K9me3" bivalent mark. Our RT-qPCR experiment showed that the removal of the hydrophobic motif or the K/R-rich region compromised the inhibitory effects of SPIN*DOC on Spindlin1-mediated transcriptional activation. In sum, here we revealed multivalent engagement between SPIN*DOC and Spindlin1, in which a hydrophobic motif acts as the primary binding site for stable SPIN*DOC-Spindlin1 association, while two more neighboring K/R-rich motifs further modulate the target selectivity of Spindlin1 via competitive inhibition, therefore attenuating the transcriptional co-activator activities of Spindlin1 through affecting its chromatin association.

biophysics↗

bioRxiv2021

REPROGRAMMING CBX8-PRC1 FUNCTION WITH A POSITIVE ALLOSTERIC MODULATOR

ABSTRACTCanonical targeting of Polycomb Repressive Complex 1 (PRC1) to repress developmental genes is mediated by cell type-specific, paralogous chromobox (CBX) proteins (CBX2, 4, 6, 7 and 8). Based on their central role in silencing and their misregulation associated with human disease including cancer, CBX proteins are attractive targets for small molecule chemical probe development. Here, we have used a quantitative and target-specific cellular assay to discover a potent positive allosteric modulator (PAM) of CBX8. The PAM activity of UNC7040 antagonizes H3K27me3 binding by CBX8 while increasing interactions with nucleic acids and participation in variant PRC1. We show that treatment with UNC7040 leads to efficient PRC1 chromatin eviction, loss of silencing and reduced proliferation across different cancer cell lines. Our discovery and characterization of UNC7040 not only revealed the most cellularly potent CBX8-specific chemical probe to date, but also corroborates a mechanism of polycomb regulation by non-histone lysine methylated interaction partners.

synthetic biology↗

bioRxiv2021

Angiomotin Counteracts the Negative Regulatory Effect of Host WWOX on Viral PPxY-Mediated Egress

Filoviridae family members Ebola (EBOV) and Marburg (MARV) viruses and Arenaviridae family member Lassa virus (LASV) are emerging pathogens that can cause hemorrhagic fever and high rates of mortality in humans. A better understanding of the interplay between these viruses and the host will inform about the biology of these pathogens, and may lead to the identification of new targets for therapeutic development. Notably, expression of the filovirus VP40 and LASV Z matrix proteins alone drives assembly and egress of virus-like particles (VLPs). The conserved PPxY Late (L) domain motifs in the filovirus VP40 and LASV Z proteins play a key role in the budding process by mediating interactions with select host WW-domain containing proteins that then regulate virus egress and spread. To identify the full complement of host WW-domain interactors, we utilized WT and PPxY mutant peptides from EBOV and MARV VP40 and LASV Z proteins to screen an array of GST-WW-domain fusion proteins. We identified WW domain-containing oxidoreductase (WWOX) as a novel PPxY-dependent interactor, and we went on to show that full-length WWOX physically interacts with eVP40, mVP40 and LASV Z to negatively regulate egress of VLPs and of a live VSV/Ebola recombinant virus (M40). Interestingly, WWOX is a versatile host protein that regulates multiple signaling pathways and cellular processes via modular interactions between its WW-domains and PPxY motifs of select interacting partners, including host angiomotin (AMOT). Notably, we demonstrated recently that expression of endogenous AMOT not only positively regulates egress of VLPs, but also promotes egress and spread of live EBOV and MARV. Toward the mechanism of action, we show that the competitive and modular interplay among WWOX-AMOT-VP40/Z regulates VLP and M40 virus egress. Thus, WWOX is the newest member of an emerging group of host WW-domain interactors (e.g. BAG3; YAP/TAZ) that negatively regulate viral egress. These findings further highlight the complex interplay of virus-host PPxY/WW-domain interactions and their potential impact on the biology of both the virus and the host during infection. Author SummaryFiloviruses (Ebola [EBOV] and Marburg [MARV]) and arenavirus (Lassa virus; LASV) are zoonotic, emerging pathogens that cause outbreaks of severe hemorrhagic fever in humans. A fundamental understanding of the virus-host interface is critical for understanding the biology of these viruses and for developing future strategies for therapeutic intervention. Here, we identified host WW-domain containing protein WWOX as a novel interactor with VP40 and Z, and showed that WWOX inhibited budding of VP40/Z virus-like particles (VLPs) and live virus in a PPxY/WW-domain dependent manner. Our findings are important to the field as they expand the repertoire of host interactors found to regulate PPxY-mediated budding of RNA viruses, and further highlight the competitive interplay and modular virus-host interactions that impact both the virus lifecycle and the host cell.

microbiology↗

bioRxiv2020

SARS-CoV-2 Envelope (E) Protein Interacts with PDZ-Domain-2 of Host Tight Junction Protein ZO1

Newly emerged SARS-CoV-2 is the cause of an ongoing global pandemic leading to severe respiratory disease in humans. SARS-CoV-2 targets epithelial cells in the respiratory tract and lungs, which can lead to amplified chloride secretion and increased leak across epithelial barriers, contributing to severe pneumonia and consolidation of the lungs as seen in many COVID-19 patients. There is an urgent need for a better understanding of the molecular aspects that contribute to SARS-CoV-2-induced pathogenesis and for the development of approaches to mitigate these damaging pathologies. The multifunctional SARS-CoV-2 Envelope (E) protein contributes to virus assembly/egress, and as a membrane protein, also possesses viroporin channel properties that may contribute to epithelial barrier damage, pathogenesis, and disease severity. The extreme C-terminal (ECT) sequence of E also contains a putative PDZ-domain binding motif (PBM), similar to that identified in the E protein of SARS-CoV-1. Here, we screened an array of GST-PDZ domain fusion proteins using either a biotin-labeled WT or mutant ECT peptide from the SARS-CoV-2 E protein. Notably, we identified a singular specific interaction between the WT E peptide and the second PDZ domain of human Zona Occludens-1 (ZO1), one of the key regulators of TJ formation/integrity in all epithelial tissues. We used homogenous time resolve fluorescence (HTRF) as a second complementary approach to further validate this novel modular E-ZO1 interaction. We postulate that SARS-CoV-2 E interacts with ZO1 in infected epithelial cells, and this interaction may contribute, in part, to tight junction damage and epithelial barrier compromise in these cell layers leading to enhanced virus spread and severe respiratory dysfunction that leads to morbidity. Prophylactic/therapeutic intervention targeting this virus-host interaction may effectively reduce airway barrier damage and mitigate virus spread.

microbiology↗

bioRxiv2020

Transcriptomic and proteomic regulation through abundant, dynamic, and independent arginine methylation by Type I and Type II PRMTs

Arginine methylation is essential for both cellular viability and development and is also dysregulated in cancer. PRMTs catalyze the post translational monomethylation (Rme1/MMA, catalyzed by Type I-III), asymmetric (Rme2a/ADMA, Type I enzymes)-, or symmetric (Rme2s/SDMA, Type II enzymes) dimethylation of arginine. Despite many studies, a thorough integration of PRMT enzyme substrate determination and proteomic and transcriptomic consequences of inhibiting Type I and II PRMTs is lacking. To characterize cellular substrates for Type I (Rme2a) and Type II (Rme2s) PRMTs, human A549 lung adenocarcinoma cells were treated with either Type I (MS023) or Type II (GSK591) inhibitors. Using total proteome hydrolysis, we developed a new mass spectrometry approach to analyze total arginine and lysine content. We showed that Rme1 was a minor population ([~]0.1% of total arginine), Rme2a was highly abundant ([~]1.1%), and Rme2s was intermediate ([~]0.4%). While Rme2s was mostly eliminated by GSK591 treatment, total Rme1 and Rme2a were more resistant to perturbation. To quantitatively characterize substrate preferences of the major enzymes PRMT1, PRMT4(CARM1), and PRMT5, we used oriented peptide array libraries (OPAL) in methyltransferase assays. We demonstrated that while PRMT5 tolerates aspartic acid residues in the substrate, PRMT1 does not. Importantly, PRMT4 methylated previously uncharacterized hydrophobic motifs. To integrate our studies, we performed PTMScan on PRMT-inhibited A549 cells and enriched for methylated arginine containing tryptic peptides. For detection of highly charged peptides, a method to analyze the samples using electron transfer dissociation was developed. Proteomic analysis revealed distinct methylated species enriched in nuclear function, RNA-binding, intrinsically disordered domains, and liquid-liquid phase separation. Parallel studies with proteomics and RNA-Seq revealed distinct, but ontologically overlapping, consequences to PRMT inhibition. Overall, we demonstrate a wider PRMT substrate diversity and methylarginine functional consequence than previously shown.

molecular biology↗

bioRxiv2019

De novo Identification of Essential Protein Domains from CRISPR/Cas9 Tiling-sgRNA Knockout Screens

High-throughput CRISPR/Cas9 knockout screens using a tiling-sgRNA design permit in situ evaluation of protein domain function. To facilitate de novo identification of essential protein domains from such screens, we developed ProTiler, a computational method for the robust mapping of CRISPR knockout hyper-sensitive (CKHS) regions, which refers to the protein regions that are associated with strong sgRNA dropout effect in the screens. We used ProTiler to analyze a published CRISPR tiling screen dataset, and identified 175 CKHS regions in 83 proteins. Of these CKHS regions, more than 80% overlapped with annotated Pfam domains, including all of the 15 known drug targets in the dataset. ProTiler also revealed unannotated essential domains, including the N-terminus of the SWI/SNF subunit SMARCB1, which we validated experimentally. Surprisingly, the CKHS regions were negatively correlated with phosphorylation and acetylation sites, suggesting that protein domains and post-translational modification sites have distinct sensitivities to CRISPR/Cas9 mediated amino acids loss.

bioinformatics↗