Biology subjects

Ly, D.

Publications and source records attributed to Ly, D..

11 recordsLinked to original sources

bioRxiv2026

Targeting CBL ubiquitin ligase activation to downregulate tyrosine kinase signalling

The CBL E3 ubiquitin ligase is a critical regulator of tyrosine kinase (TK) signalling. CBL activity is regulated by a feedback loop in which an active TK phosphorylates CBL, relieving its autoinhibited conformation, and allowing ubiquitination of substrates, including TKs, leading to their degradation. Binding of the Src Like Adapter Protein 2 (SLAP2) to CBL can also activate autoinhibited CBL and promote substrate ubiquitination. Using an engineered CBL mutant in the SLAP2 binding interface, termed RE CBL, that mimics activation by SLAP2 binding, we characterized the cellular functions of this CBL activation mechanism. Comparison of wildtype and RE CBL interactomes using MiniTurboID showed extensive and overlapping interaction networks, with a discrete subset of proteins, including the known substrate, epidermal growth factor receptor (EGFR), as well as endocytic factors such as EPS15, in higher abundance with RE CBL compared to wildtype. Consistent with these observations, RE CBL interacted more readily with EGFR, enhanced EGFR internalization, and attenuated downstream signalling compared to WT. In Cbl null hematopoietic cells, RE CBL expression reduced sensitivity to cytokines IL-3 and GM-CSF, and decreased activation of the Src-family kinase Lyn. Furthermore, we optimized and conducted a small molecule screen to identify a group of structurally related compounds that, like SLAP2 binding, promoted CBL activation in vitro. Together these findings provide proof of concept for targeting CBL activity to downregulate TK signalling.

biochemistry↗

bioRxiv2026

Exploiting HLA-II Promiscuity via Peptide Terminal Overhang Recognition for Pan-Allelic and Tumor-Selective AML Immunotherapy

Antibodies targeting peptides presented by human leukocyte antigen (HLA) molecules expand the therapeutic landscape by enabling recognition of intracellular antigens. While most efforts have focused on allele-restricted peptides presented by HLA class I (HLA-I), HLA class II (HLA-II) epitopes remain underexplored despite their potential for promiscuous presentation. Acute myeloid leukemia (AML) is characterized by high expression of both HLA-II and the myeloid lineage antigen myeloperoxidase (MPO). Here, we identified an MPO-derived epitope (MPO100-132) that is promiscuously presented by multiple HLA-II molecules. We generated a MPO100-132-specific antibody (146D5) that recognizes the N-terminal overhang of this peptide independent of specific HLA contacts, enabling pan-allelic recognition. Engineered into bispecific T cell engagers (BiTEs), this antibody mediated robust cytotoxicity against primary AML samples across diverse HLA-II backgrounds. Crucially, 146D5-based BiTEs selectively spared normal myeloid cells, indicating that the MPO100-132 peptide, derived from the MPO propeptide, was functionally undetectable in normal myeloid cells, providing a significant safety window. In vivo, the MPO-targeting BiTE demonstrated potent antitumor activity and prolonged survival in AML xenograft models. Our findings identify peptide terminal overhangs as an actionable class of antibody targets and introduce a strategy to exploit HLA-II promiscuity for broadly applicable HLA-dependent but allele-agnostic immunotherapies.

immunology↗

bioRxiv2025

Genome-wide screening reveals producer-cell modifications that improve virus-like particle production and delivery potency

Engineered virus-like particles (eVLPs) are promising vehicles for transient delivery of gene editing agents. While extensive particle engineering has yielded efficient eVLPs, it remains underexplored whether engineering the cells used to produce eVLPs could further improve eVLP properties. We developed a genome-wide screening approach to systematically investigate how genetic perturbations in producer cells influence eVLP production. This approach generates eVLPs loaded with guide RNAs that identify the genetic perturbation in the cell that produced a particular particle; the abundance of each guide RNA in eVLPs therefore reflects how the corresponding genetic perturbation influences eVLP production or cargo loading. We applied this approach to identify several genes that regulate eVLP cargo expression and loading into particles during the production process. Leveraging these insights, we engineered producer cells that support increased eVLP cargo packaging and a 2- to 9-fold increase in eVLP delivery potency across several cargo, particle, and target-cell types in cultured cells and in mice. Our findings suggest the potential of producer-cell engineering as a useful strategy for improving the utility of eVLPs and related delivery methods.

synthetic biology↗

bioRxiv2025

Peptide-Specific CAR T cells Recognize WT1 Promiscuously Presented by Diverse HLA Class II Alleles

Chimeric antigen receptor (CAR) technology has revolutionized B-cell malignancy treatment by enabling T cells to effectively recognize and target cancer-specific surface antigens. However, CAR T cells show limited efficacy against other blood cancers and solid tumors due to challenges in identifying suitable surface targets. Here, we present a novel approach to CAR development, targeting the intracellular Wilms tumor 1 (WT1) oncoprotein, cross-presented by surface HLA-class II (HLA-II) alleles. WT1-CAR T cells, derived from an antibody raised solely against a WT1 peptide, recognized the WT1330-348 peptide promiscuously presented by 18 out of 20 tested HLA-II alleles, overcoming traditional HLA restrictions. WT1-CAR T cells specifically recognized leukemic cells in a WT1 and HLA-II-dependent manner and mediated an antitumor response in vitro and in vivo. This innovative approach to CAR T cell development transcends traditional HLA restrictions and offers a promising therapeutic option to a wide and genetically diverse patient population. Statement of significanceThis study describes a novel CAR T therapy approach leveraging the distinctive and shared characteristic of HLA-II-peptide binding promiscuity, enabling targeting of the intracellular oncoprotein WT1 presented across diverse HLA-II families. Our study demonstrates a viable framework for designing CAR T therapies that benefit genetically diverse patient populations.

immunology↗

bioRxiv2025

Sequence based prediction of cell type specific microRNA binding and mRNA degradation for therapeutic discovery

MicroRNAs and RNA binding proteins are crucial elements of post-transcriptional gene regulation, which governs the fate of mRNA molecules in the cell. However, the landscape of these regulatory interactions, particularly across different mammalian cell types, remains underexplored. We describe REPRESS, a deep learning model that predicts cell-type-specific microRNA binding and mRNA degradation directly from RNA sequence. REPRESS was trained on AGO2-CLIP, miR-eCLIP and Degradome-Seq data profiling millions of microRNA binding and mRNA degradation sites across multiple cell types in human and mouse. It reveals biology that other state-of-the-art methods did not, such as identifying repressive non-canonical miRNA target sites and decoding the regulatory effects of sequence context and miRNA binding site multiplicity. REPRESS outperforms other advanced methods and neural architectures on a comprehensive suite of seven orthogonal tasks, including identifying genetic variants that affect microRNA binding, predicting out-of-distribution data from massively parallel reporter assays, and predicting canonical and non-canonical miRNA mediated repression. To demonstrate the general utility of REPRESS, we show that it provides insights into novel biology and the design of RNA therapeutics. Code is available at : https://github.com/deepgenomics/repress

genomics↗

bioRxiv2023

Recent evolution of large offspring size and post-fertilization nutrient provisioning in swordtails

Organisms have evolved diverse reproductive strategies that impact the probability that their offspring survive to adulthood. Here, we describe divergence in reproductive strategy between two closely related species of swordtail fish (Xiphophorus). Swordtail fish and their relatives have evolved viviparity: they have internal fertilization and give birth to fully developed fry. We find that one species, X. malinche, which lives in high-elevation environments, has evolved larger offspring than its closest relative X. birchmanni and dwarfs the offspring size of other species in the genus. The larger fry of X. malinche are more resilient to starvation than their X. birchmanni relatives, hinting that the evolution of large offspring size may be an adaptation to the particularly challenging environments in which X. malinche are born. We find evidence that X. malinche achieves larger offspring size in part by continuing to provision their offspring over the course of embryonic development after fertilization, the first time this process has been documented in the Xiphophorus genus. Moreover, we observe differential regulation in the ovary of genes associated with maternal nutrient provisioning in other species that use this reproductive strategy. Intriguingly, these reproductive differences may drive an asymmetric hybrid incompatibility, since X. birchmanni mothers pregnant with F1 embryos give birth to premature and stillborn fry at an exceptionally high rate.

evolutionary biology↗

bioRxiv2023

An RNA foundation model enables discovery of disease mechanisms and candidate therapeutics

Accurately modeling and predicting RNA biology has been a long-standing challenge, bearing significant clinical ramifications for variant interpretation and the formulation of tailored therapeutics. We describe a foundation model for RNA biology, "BigRNA", which was trained on thousands of genome-matched datasets to predict tissue-specific RNA expression, splicing, microRNA sites, and RNA binding protein specificity from DNA sequence. Unlike approaches that are restricted to missense variants, BigRNA can identify pathogenic non-coding variant effects across diverse mechanisms, including polyadenylation, exon skipping and intron retention. BigRNA accurately predicted the effects of steric blocking oligonucleotides (SBOs) on increasing the expression of 4 out of 4 genes, and on splicing for 18 out of 18 exons across 14 genes, including those involved in Wilson disease and spinal muscular atrophy. We anticipate that BigRNA and foundation models like it will have widespread applications in the field of personalized RNA therapeutics.

bioinformatics↗

bioRxiv2023

Uncovering structural features that control substrate specificity in a Lactobacillus chlorogenic acid esterase

The structural determinants of chlorogenic acid esterase (CE) substrate specificity are poorly understood. Here, we establish how a Lactobacillus helveticus CE selects for its substrate, chlorogenic acid (CGA). We determine that a Lys residue in an extended loop over the active site imparts substrate specificity by hydrogen bonding to CGA. Mutation of the Lys residue abolishes CGA specificity. Comparison with other bacterial CEs reveals that the extended loop is not conserved. However, the hydrogen bonding functionality to CGA is preserved thanks to other residues. Structural comparison with ferulic acid esterases (FAEs), a related enzyme class, shows that CEs feature a more restricted active site, reflecting the fact that they hydrolyze smaller substrates compared to FAEs.

biochemistry↗

bioRxiv2020

Prophage exotoxins enhance colonization fitness in epidemic scarlet fever-causing Streptococcus pyogenes

The re-emergence of scarlet fever poses a new global public health threat. The capacity of North-East Asian serotype M12 (emm12) Streptococcus pyogenes (group A Streptococcus, GAS) to cause scarlet fever has been linked epidemiologically to the presence of novel prophages, including prophage {Phi}HKU.vir encoding the secreted superantigens SSA and SpeC and the DNase Spd1. Here we report the comprehensive molecular characterization of {Phi}HKU.vir-encoded exotoxins. We demonstrate that streptolysin O (SLO)-induced glutathione efflux from host cellular stores is a previously unappreciated GAS virulence mechanism that promotes SSA release and activity, representing the first description of a thiol-activated bacterial superantigen. Spd1 is required for optimal growth in human blood, confers resistance to neutrophil killing, and degrades neutrophil extracellular traps (NETs). Investigating single, double and triple isogenic knockout mutants of the {Phi}HKU.vir-encoded exotoxins, we find that SpeC and Spd1 act synergistically to facilitate nasopharyngeal colonization in a mouse model. These results offer insight into the etiology and pathogenesis of scarlet fever-causing GAS mediated by phage {Phi}HKU.vir exotoxins.

microbiology↗

bioRxiv2020

Streptococcus pyogenes M1T1 variants induce an inflammatory phenotype in neutrophils

Invasive infections due to Group A Streptococcus (GAS) advance rapidly causing tissue degradation and unregulated inflammation. Neutrophils are the primary immune cells that respond to GAS. The neutrophil response to GAS was characterised in response to two M1T1 isolates; 5448 and animal passaged variant 5448AP. Neutrophil co-incubation with 5448AP allowed proliferation of GAS while it also lowered the production of reactive oxygen species by neutrophils when compared with 5448. Infection with both strains invoked neutrophil death, however apoptosis was reduced in response to 5448AP. Both strains induced neutrophil caspase-1 activation and caspase-4 expression in vitro, with caspase-1 activation detected in vivo. Thus, GAS infection of neutrophils corresponds to increased caspase-1 activity and caspase-4 expression, consistent with inflammasome activation and pyroptosis. GAS infections that promote an inflammatory neutrophil phenotype may contribute to increased inflammation yet ineffective bacterial eradication, contributing to the speed and severity of invasive GAS infections.

microbiology↗

bioRxiv2018

Non-driver somatic alteration burden confers good prognosis in non-small cell lung cancer

BackgroundGenomic profiling of patient tumors has linked somatic driver mutations to survival outcomes of non-small cell lung cancer (NSCLC) patients, especially for those receiving targeted therapies. However, it remains unclear whether specific non-driver mutations have any prognostic utility.\n\nMethodsWhole exomes and transcriptomes were measured from NSCLC xenograft models of patients with diverse clinical outcomes. Penalised regression analysis was performed to identify a set of 865 genes associated with patient survival. The number of somatic copy number aberrations, point mutations and associated expression changes within the 865 genes were used to stratify independent NSCLC patient populations, filtered for chemotherapy naive and early-stage. In-depth genomic analysis and functional testing was conducted on the genomic alterations to understand their effect on improving survival.\n\nResultsHigh burden of somatic alterations are associated with longer disease-free survival (HR=0.153, P=1.48x10-4) in NSCLC patients. When somatic alterations burden was integrated with gene expression, we were able to predict prognosis in three independent patient datasets. Patients with high alteration burden could be further stratified based on the presence of immunogenic mutations, revealing another subgroup of patients with even better prognosis (85% with >5 years survival), and associated with cytotoxic T-cell expression. In addition, 95% of these 865 genes lack documented activity relevant to cancer, but are in pathways regulating cell proliferation, motility and immune response were implicated.\n\nConclusionOur results demonstrate that non-driver somatic alterations may influence the outcome of cancer patients by increasing beneficial immune response and inhibiting processes associated to tumorigenesis.

cancer biology↗