Biology subjects

Blackburn, J. S.

Publications and source records attributed to Blackburn, J. S..

8 recordsLinked to original sources

bioRxiv2025

A live cell biosensor protocol for high-resolution screening of therapy-resistant cancer cells

The Genetically Encoded Death Indicator (GEDI) is a ratiometric, dual-fluorescence biosensor that enables real-time detection of cell death through calcium influx. Originally developed for use in neurodegeneration models, GEDI can be applied to cancer cells to quantify therapy-induced death at single-cell resolution. This protocol details how to generate GEDI-expressing cancer cell lines, empirically determine stress-induced GEDI thresholds using radiation or chemotherapeutic agents, and perform time-resolved imaging and image analysis to track cell fate. This workflow is optimized for high-throughput drug and radiation screening in heterogeneous populations and is especially useful for identifying chemo- and radio-resistant subclones. Key limitations include the need for empirical GEDI threshold calibration for each treatment condition and careful standardization of imaging parameters. The protocol outputs include GEDI ratio values, single-cell time-of-death annotations, and whole-cell morphological data in parallel, which can be linked to downstream applications such as FACS-based isolation of live or dying subpopulations, transcriptomic profiling of resistant clones, or in vivo validation using xenografts or organotypic slice culture. Associated contentio.protocol DOI: dx.doi.org/10.17504/protocols.io.eq2ly4d7qlx9/v1

cancer biology↗

bioRxiv2025

Phosphatase activity is dispensable for PRL-3-mediated oncogenesis and tumor progression

Phosphatase of Regenerating Liver 3 (PRL-3) is frequently upregulated in cancer and associated with poor prognosis, yet its oncogenic mechanism remains unresolved. Although traditionally studied for its phosphatase activity, PRL-3 also engages in protein-protein interactions via its catalytic site, notably binding the CNNM magnesium transporters, rendering these functions mutually exclusive. The commonly used C104S mutant disrupts both activities, confounding interpretations of prior studies. To dissect PRL-3s distinct functions, we employed mutants selectively deficient in phosphatase activity (C104D) or CNNM binding (R138E). In zebrafish models of acute lymphoblastic leukemia (ALL) and rhabdomyosarcoma (RMS), as well as in human cancer cell lines, both wild-type PRL-3 and C104D enhanced tumor initiation, growth, and dissemination, while R138E had no effect. These findings indicate that PRL-3 promotes cancer via non-catalytic mechanisms. To support therapeutic development, we established an in vitro FRET-based assay to screen for inhibitors of the PRL-3:CNNM interaction and validated a cyclic peptide as a positive control. By demonstrating that PRL-3 enhances cancer progression independently of its catalytic activity, this study shifts focus toward targeting its binding functions as a therapeutic strategy. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=158 SRC="FIGDIR/small/654016v1_ufig1.gif" ALT="Figure 1"> View larger version (57K): org.highwire.dtl.DTLVardef@41de8dorg.highwire.dtl.DTLVardef@7a3bbdorg.highwire.dtl.DTLVardef@e800d2org.highwire.dtl.DTLVardef@18df51a_HPS_FORMAT_FIGEXP M_FIG C_FIG Statement of significanceThis study demonstrates that PRL-3 drives cancer initiation and progression through a phosphatase-independent mechanism across diverse models, highlighting the need to therapeutically target its non-catalytic protein interactions in future cancer treatment strategies.

cancer biology↗

bioRxiv2025

Small Extracellular Vesicles from Radioresistant H3K27M-Pediatric Diffuse Midline Glioma Cells Modulate Tumor Phenotypes and Radiation Response

Pediatric diffuse midline gliomas with the Histone 3 lysine 27-to-methionine mutation (H3K27M-pDMG) are aggressive brain tumors characterized by intrinsic resistance to radiation therapy, the current standard of care. These tumors exhibit significant intratumoral heterogeneity, with distinct subclonal populations likely contributing to therapy resistance. Emerging evidence suggests that small extracellular vesicles (sEV) mediate oncogenic signaling within glioma stem cell populations, yet their role under radiation-induced stress remains poorly understood. In this study, we characterized sEV uptake dynamics among H3K27M-pDMG tumor cells, identified key sEV surface proteins, and demonstrated that sEVs derived from radioresistant (RR) H3K27M-pDMG cells confer radioprotective effects on radiosensitive tumor cells. Molecular profiling revealed that RR-sEVs carry proteins, microRNAs (miRNAs), and metabolites associated with glycolysis, oxidative phosphorylation, and DNA repair. Upon uptake, RR-sEVs reprogrammed recipient cells by altering gene expression and metabolic pathways, and enhancing DNA repair and survival following radiation exposure. These findings provide insights into the role of sEV-mediated intratumoral communication as a contributor to radiation resistance in H3K27M-pDMG and suggest potential therapeutic strategies to disrupt this process and enhance radiation efficacy.

cancer biology↗

bioRxiv2023

Zebrafish Drug Screening Identifies Erlotinib as an Inhibitor of Wnt/β-Catenin Signaling and Self-Renewal in T-cell Acute Lymphoblastic Leukemia

The Wnt/{beta}-catenin pathways significance in cancer initiation, progression, and stem cell biology underscores its therapeutic potential, yet clinical application of Wnt inhibitors remains limited due to challenges posed by off-target effects and complex crosstalk with other pathways. In this study, we leveraged the zebrafish model to perform a robust and rapid drug screening of 773 FDA-approved compounds to identify Wnt/{beta}-catenin inhibitors with minimal toxicity. Utilizing zebrafish expressing a Wnt reporter, we identified several drugs that suppressed Wnt signaling without compromising zebrafish development. The efficacy of the top hit, Erlotinib, extended to human cells, where it blocked Wnt/{beta}-catenin signaling downstream of the destruction complex. Notably, Erlotinib treatment reduced self-renewal in human T-cell Acute Lymphoblastic Leukemia cells, which are known to rely on active {beta}-catenin signaling for maintenance of leukemia-initiating cells. Erlotinib also reduced leukemia-initiating cell frequency and delayed disease formation in zebrafish models. This study underscores zebrafishs translational potential in drug discovery and repurposing, and highlights a new use for Erlotinib as a Wnt inhibitor for cancers driven by aberrant Wnt/{beta}-catenin signaling. HighlightsO_LIZebrafish-based drug screening offers an inexpensive and robust platform for identifying compounds with high efficacy and low toxicity in vivo. C_LIO_LIErlotinib, an Epidermal Growth Factor Receptor (EGFR) inhibitor, emerged as a potent and promising Wnt inhibitor with effects in both zebrafish and human cell-based Wnt reporter assays. C_LIO_LIThe identification of Erlotinib as a Wnt inhibitor underscores the value of repurposed drugs in developing targeted therapies to disrupt cancer stemness and improve clinical outcomes C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=42 SRC="FIGDIR/small/555200v1_ufig1.gif" ALT="Figure 1"> View larger version (14K): org.highwire.dtl.DTLVardef@150e0a0org.highwire.dtl.DTLVardef@cb3455org.highwire.dtl.DTLVardef@d8d8a8org.highwire.dtl.DTLVardef@780535_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2021

Long-Read Sequencing of the Zebrafish Genome Reorganizes Genomic Architecture

Nanopore sequencing technology has revolutionized the field of genome biology with its ability to generate extra-long reads that can resolve regions of the genome that were previously inaccessible to short-read sequencing platforms. Although long-read sequencing has been used to resolve several vertebrate genomes, a nanopore-based zebrafish assembly has not yet been released. Over 50% of the zebrafish genome consists of difficult to map, highly repetitive, low complexity elements that pose inherent problems for short-read sequencers and assemblers. We used nanopore sequencing to improve upon and resolve the issues plaguing the current zebrafish reference assembly (GRCz11). Our long-read assembly improved the current resolution of the reference genome by identifying 1,697 novel insertions and deletions over 1Kb in length and placing 106 previously unlocalized scaffolds. We also discovered additional sites of retrotransposon integration previously unreported in GRCz11 and observed their expression in adult zebrafish under physiologic conditions, implying they have active mobility in the zebrafish genome and contribute to the ever-changing genomic landscape.

genomics↗

bioRxiv2020

Development and validation of nanobodies specific to the oncogenic phosphatase Protein Tyrosine Phosphatase 4A3 (PTP4A3 or PRL-3)

Phosphatase of Regenerating Liver-3 (PRL-3) is associated with cancer progression and metastasis in various solid tumors and leukemias. The mechanisms that drive PRL-3s oncogenic functions are not well understood, in part due to a lack of research tools available to study this protein. In particular, small molecules do not exhibit binding specificity for PRL-3 over highly homologous family members PRL-1 and PRL-2, and antibodies directed against PRL-3 are limited by assay type. We have begun to address these issues by developing alpaca-derived single domain antibodies, or nanobodies, targeting PRL-3 with a KD of 30-300 nM and no activity towards PRL-1 and PRL-2. Hydrogen deuterium exchange mass spectrometry (HDX-MS) and co-immunoprecipitation with a known PRL-3 substrate showed the nanobodies bind PRL-3 outside of the active site, meaning they can be used to study PRL-3 interaction with binding partners. The nanobodies were also specific to PRL-3 over other PRLs in immunoprecipitation and immunofluorescence experiments in human cancer cells that overexpressed the PRL family. We found that N-terminal tags on PRL-3, such as GFP and FLAG, changed PRL-3 localization compared to untagged protein, indicating that the nanobodies may provide new insights into PRL-3 trafficking and function. The anti-PRL-3 nanobodies represent an important expansion of the research tools available to study PRL-3 function and can be used to define the role of PRL-3 in cancer progression.

biochemistry↗

bioRxiv2020

A screen of FDA-approved drugs identifies inhibitors of Protein Tyrosine Phosphatase 4A3 (PTP4A3 or PRL-3)

Protein tyrosine phosphatase 4A3 (PTP4A3 or PRL-3) is highly expressed in a variety of cancers, where it promotes tumor cell migration and metastasis leading to poor prognosis. Despite its clinical significance, there are currently no viable options to target PRL-3 in vivo. Here, we screened 1,443 FDA-approved drugs for their ability to inhibit the activity of the PRL phosphatase family. We identified five specific inhibitors for PRL-3 as well as one selective inhibitor of PRL-2. Additionally, we found nine drugs that broadly and significantly suppressed PRL activity. Two of these broad PRL inhibitors, Salirasib and Candesartan blocked PRL-3-induced migration in human embryonic kidney (HEK) cells with no negative impact on cell viability. Both drugs prevented migration of PRL-3 expressing human colorectal cancer cells to a similar degree as the research-grade PRL inhibitor, Thienopyridone, and are selective towards PRLs over other phosphatases. In silico modeling revealed that Salirasib binds a putative allosteric site near the WPD loop of PRL-3, while Candesartan binds a potentially novel targetable site adjacent to the CX5R motif. Inhibitor binding at either of these sites is predicted to trap PRL-3 in a closed conformation, preventing substrate binding and inhibiting function.

cancer biology↗

bioRxiv2019

In vivo imaging defines vascular interplay in the development of lymphocytic leukemia in zebrafish models

The vascular microenvironment at the primary tumor site is represented by functionally diverse types of vessels that contribute to metabolic supply, trafficking/dissemination of tumor cells, and delivery of chemotherapeutics. However, the role of leukemia-associated vascular networks in leukemia progression remains poorly understood. We utilized a MYC-induced T cell leukemia model in zebrafish to assess the involvement of vascular endothelial growth factor A-dependent (VEGFR2+), VEGFA-independent vasculature (VEGFR2-), and lymphatics in the initial stages of leukemia cell dissemination using intravital microscopy. Leukemogenesis underwent sequential progression, from initial successive emigration of single leukemic cells from the thymus towards the kidney marrow, followed by collective migration in a dorsal-lateral direction with scattered migration from the ventral thymus towards the aortic arches, before streaming towards the rostral kidney area. Trafficking appeared independent of VEGFR2+ vasculature but was closely associated with VEGFR2-microvessels, and, interestingly, leukemic cells did not utilize lymphatics during initial dissemination. Overall, leukemic cells appeared to utilize the same routes as normal T cell progenitors during development but in a reverse order, and primarily recruited VEGFR2-microvessels during the initial stages of progression. Ultimately, interfering with leukemia cell migration by targeting vascular networks may represent a new therapeutic strategy to control leukemia progression.

cancer biology↗