Biology subjects

Yong, C.

Publications and source records attributed to Yong, C..

7 recordsLinked to original sources

bioRxiv2026

Performance of the 10X Genomics Flex Single-Cell Sequencing Assay and its Application to Overcome Challenges in Clinical Trial Samples

BackgroundSingle-cell RNA sequencing (scRNA-seq) has become essential for understanding disease biology, yet its application in clinical trials is often limited by logistical challenges associated with the handling of biospecimens. Newly developed protocols aim to address these limitations by enabling profiling of fixed tissue. These new solutions need to be benchmarked against well-established protocols to assess their performance and suitability for clinical research. ResultsWe systematically compared different scRNA-seq protocols in a set of samples commonly analysed in clinical trials. The 10X Genomics GEM-X Flex Gene Expression assay (GEM-X Flex), in combination with the "chop-fix" preprocessing protocol, demonstrated superior performance to the standard GEM-X Universal Gene Expression solution when applied to both primary tumor tissue fragments and FFPE blocks. Moreover, the quality of the data obtained from GEM-X Flex applied to FFPE blocks outperformed that of single-nuclei RNA sequencing (snRNA-seq) from frozen biopsies, more robustly capturing the biological signals associated with the mechanism of action of a drug evaluated in an internal clinical trial. ConclusionsGEM-X Flex generates reliable, comprehensive transcriptomic data from both fixed tissue and clinical biopsies. By overcoming some of the limitations of fresh and frozen tissue analysis, this protocol offers a robust solution for the broad implementation of scRNA-seq in clinical trials.

genomics↗

bioRxiv2026

Human lung explants as a predictive platform for evaluating the on-target, off-tumor toxicity of T cell bispecifics

T cell bispecifics (TCBs) are potent immunotherapies with proven efficacy in hematological cancers but limited success in solid tumors, where on-target, off-tumor toxicity has restricted their therapeutic index. Clinical discontinuation of TCB formulations against epithelial cell adhesion molecule (EpCAM TCB, Solitomab) and preclinical termination of a TCB against folate receptor alpha (FolR1-TCB) highlight the urgent need for predictive preclinical models to evaluate such toxicities. Conventional models, including cell lines and organoids, lack the immune and stromal complexity of the tumor microenvironment (TME), and while patient-derived explants preserve native tissue architecture and heterogeneity, quantification of TCB-mediated target cell killing has not been previously achieved in this system. Here, we establish a human lung explant model to assess TCB activity in matched tumor and normal adjacent tissues. Using a multi-modal approach combining flow cytometry, cytokine profiling, and multiplexed immunofluorescence, we show that EpCAM and FolR1 TCBs drive robust T cell activation and, critically, quantifiable epithelial cell killing above background levels. Normal lung explants exhibited consistently higher levels of killing than tumor counterparts, reflecting the on-target, off-tumor toxicities observed preclinically and clinically with the tested TCBs. In contrast, colon explants displayed poor viability ex vivo, limiting their suitability for assessing TCB-induced killing despite measurable immune activation. Our findings establish lung explants as a predictive and clinically relevant preclinical model that uniquely enables simultaneous quantification of TCB-mediated T cell activation and target cell killing. This model captures key features of TCB mechanism of action and recapitulates clinically observed toxicities, supporting its application for preclinical evaluation of TCB drug candidates and advancing the development of immunotherapies for solid tumors.

cancer biology↗

bioRxiv2025

Mechanisms of resistance to VHL loss-induced genetic and pharmacological vulnerabilities

The von Hippel-Lindau tumor suppressor (VHL) is a component of a ubiquitin ligase complex that normally controls cellular responses to hypoxia. Endogenous VHL is also utilized by proteolysis-targeting chimera (PROTAC) protein degraders, a promising class of anti-cancer agents. VHL is broadly essential for cell proliferation, yet it is a key tumor suppressor in renal cell carcinoma. To understand the functional consequences of VHL loss, and to identify targeted approaches for the elimination of VHL null cells, we have used genome-wide CRISPR-Cas9 screening in human renal epithelial cells. We find that, upon VHL loss, the HIF1A/ARNT complex is the central inhibitor of cellular fitness, suppressing mitochondrial respiration, and that VHL null cells show HIF1A-dependent molecular vulnerabilities that can be targeted pharmacologically. Combined VHL/HIF1A inactivation in breast and esophageal cancer cells can also provide resistance to ARV-771, a VHL-based bromodomain degrader that has anti-cancer activity. HIF1A stabilization can thus provide opportunities for early intervention in neoplastic VHL clones, and the VHL-HIF1A axis may be relevant for the development of resistance to the emerging class of PROTAC-based cancer therapies.

cancer biology↗

bioRxiv2024

Double crossed? Structural and computational studies of an unusual crosslinked heme in Methylococcus capsulatus cytochrome P460

Cytochromes P460 oxidise hydroxylamine within the nitrogen cycle and contain as their active site an unusual catalytic c-type heme where the porphyrin is cross-linked to the protein via a lysine residue in addition to the canonical cross links from cysteine residues. Understanding how enzymes containing P460 heme oxidise hydroxylamine into either nitrous oxide or nitric oxide has implications for climate change. Interestingly the P460 containing hydroxylamine oxidoreductase utilises a tyrosine cross link to heme and performs similar chemistry. Previous crystal structures of cytochrome P460 from Nitrosomonas europaea (NeP460) clearly show the existence of a single crosslink between the Nz atom of lysine and the heme porphyrin with mutagenesis studies indicating roles for the crosslink in positioning a proton transfer residue and/or influencing the distortion of the heme. Here we describe the evidence for a novel double cross link between lysine and heme in the cytochrome P460 from Methylococcus capsulatus (Bath). In order to understand the complexities of this enzyme system we applied high resolution structural biology approaches at synchrotron and XFEL sources paired with crystal spectroscopies. Linked to this we carried out QM/MM simulations that enabled the prediction of electronic absorption spectra providing a crucial validation to linking simulations and experimental structures. Our work demonstrates the feasibility of a double crosslink in McP460 and provides an opportunity to investigate how simulations can interact with experimental structures.

biochemistry↗

bioRxiv2022

In-vivo characterization of glutamine metabolism identifies therapeutic targets in clear cell renal cell carcinoma

Targeting metabolic vulnerabilities has been proposed as a therapeutic strategy in renal cell carcinoma (RCC). Here, we analyzed metabolism in patient-derived xenografts (tumorgrafts) from diverse forms of RCC. Tumorgrafts from VHL-mutant clear cell RCC (ccRCC) retained metabolic features of human ccRCC and engage in oxidative and reductive glutamine metabolism. We used several approaches to suppress glutamine metabolism and test the effect on tumor growth. Genetic silencing of isocitrate dehydrogenase-1 or -2 impaired reductive labeling of TCA cycle intermediates and suppressed tumor growth. Glutaminase inhibition resulted in modest growth suppression and variable effects on glutamine metabolism in vivo. Infusions with [amide-15N]glutamine revealed persistent amidotransferase activity during glutaminase inhibition, and blocking these activities with the amidotransferase inhibitor JHU-083 also reduced tumor growth. We conclude that ccRCC tumorgrafts catabolize glutamine via multiple pathways, perhaps explaining why it has been challenging to achieve therapeutic responses in patients by inhibiting glutaminase. TeaserGlutamine fuels the TCA cycle and amidotransferase pathways in clear cell renal cell carcinoma.

cancer biology↗

bioRxiv2022

HIRA loss transforms FH-deficient cells

Fumarate Hydratase (FH) is a mitochondrial enzyme that catalyses the reversible hydration of fumarate to malate in the TCA cycle. Germline mutations of FH lead to HLRCC, a cancer syndrome characterised by a highly aggressive form of renal cancer(1). Although HLRCC tumours metastasise rapidly, FH-deficient mice develop premalignant cysts in the kidneys, rather than carcinomas (2). How Fh1-deficient cells overcome these tumour suppressive events during transformation is unknown. Here, we perform a genome-wide CRISPR/Cas9 screen to identify genes that, when ablated, enhance the proliferation of Fh1-deficient cells. We found that the depletion of HIRA enhances proliferation and invasion of Fh1-deficient cells in vitro and in vivo. Mechanistically, Hira loss enables the activation of MYC and its target genes, increasing nucleotide metabolism specifically in Fh1-deficient cells, independent of its histone chaperone activity. These results are instrumental for understanding mechanisms of tumorigenesis in HLRCC and the development of targeted treatments for patients.

cancer biology↗

bioRxiv2020

Locally-invasive, castrate-resistant prostate cancer in a Pten/Trp53 double knockout mouse model of prostate cancer monitored with non-invasive bioluminescent imaging

Here we have improved an existing mouse model of prostate cancer based on prostate-specific deletion of Pten and Trp53 by incorporating a Cre-activatable luciferase reporter. By coupling the deletion of those genes to the activation of a luciferase reporter, we were able to monitor tumor burden non-invasively over time. We show that, consistent with previous reports, deletion of both Pten and Trp53 on a C57BL/6 background accelerates tumor growth and results in both the loss of androgen receptor expression and castrate resistant tumors as compared with loss of Pten alone. Loss of Trp53 results in the development of sarcomatoid histology and the expression of markers of epithelial-to-mesenchymal transition Zeb1 and vimentin, with kinetics and penetrance dependent on whether one or both alleles of Trp53 were deleted. Homozygous deletion of Trp53 and Pten resulted in uniformly lethal disease by 25 weeks. While we were able to detect locally invasive disease in the peritoneal cavity in aggressive tumors from the double knockout mice, we were unable to detect lymphatic or hematogenous metastatic disease in lymph nodes or at distant sites.

cancer biology↗