Biology subjects

Pasquale, E. B.

Publications and source records attributed to Pasquale, E. B..

8 recordsLinked to original sources

bioRxiv2025

Automation of high-throughput workflow for arrayed CRISPR activation library screening

CRISPR-mediated gene activation (CRISPRa) is among the most efficient and reliable strategies for mimicking sustained activation of endogenous promoters and their corresponding genes at physiological levels. By leveraging guide-RNA (gRNA) library design, CRISPRa screens can be applied on a whole-genome scale and are compatible with both arrayed and pooled formats, depending on assay requirements. Compared with conventional arrayed CRISPRa libraries that use single or dual gRNAs and often require multiple gRNA candidates per target, a recently developed CRISPRa library (termed T. gonfio) incorporates four tandem gRNAs per lentivector per target, thereby reducing library complexity and representing the smallest arrayed genome-wide CRISPRa library. To streamline genome-wide arrayed CRISPRa screening, this study developed a high-throughput automated workflow using the Biomek i7 Hybrid liquid-handling platform, integrated with multiple peripheral instruments. The workflow comprises three pipelines: lentiviral library transduction, cell library passaging, and assay processing. These pipelines together establish and maintain the transduced cell library for extended screening times. This enables assay processing at desired extended time points and improves the likelihood of identifying phenotypes that require longer time to develop, making the workflow suitable even for rapidly proliferating cell models. In a pilot arrayed screen using a T. gonfio mini-library targeting kinases and phosphatases, activation of the EPHA2 receptor promoter induced a growth reduction phenotype in the HEK293 cell model. This phenotype was recapitulated in a parallel pooled CRISPRa screen using the same mini-library and further validated in a co-culture assay.

cancer biology↗

bioRxiv2025

Frequent EPHA2 receptor mutations in cholangiocarcinoma disrupt receptor forward signaling supporting a tumor suppressor role

EPHA2 is a receptor tyrosine kinase highly expressed in many cancers. By analyzing cancer patient databases for mutations in the EPHA2 coding sequence, we found that cholangiocarcinoma (a hepatobiliary cancer with dismal prognosis) exhibits a uniquely high incidence of EPHA2 mutations. To deUine the functional signiUicance of these mutations, we generated representative EPHA2 mutants and monitored major receptor autophosphorylation sites as indicative of kinase activity-dependent signal transduction (known as forward signaling). We found that missense mutations in the ligand-binding domain abrogate ligand binding and ligand-induced EPHA2 tyrosine phosphorylation, while most missense mutations in the kinase domain abrogate kinase activity. We detected less pronounced effects of missense mutations in other domains, which vary depending on the phosphosite, suggesting that these EPHA2 mutations might differentially affect (or bias) different downstream signaling pathways. Other EPHA2 mutations introduce early stop codons and encode receptor truncated forms lacking all or part of the kinase domain. We also found that an EPHA2 secreted truncated form, a transmembrane truncated form, and a full-length kinase inactive mutant can inhibit tyrosine phosphorylation of co-expressed EPHA2 wild-type. Taken together, these data suggest that mutations interfering with EPHA2 forward signaling facilitate cholangiocarcinoma development. We indeed obtained evidence that an EPHA2 kinase inactive mutant, but not EPHA2 wild-type, can induce proliferative masses consistent with well differentiated cholangiocarcinoma in a validated mouse model of cholangiocarcinogenesis. Taken together, our Uindings suggest that EPHA2 is a driver gene in cholangiocarcinoma and that its forward signaling has tumor suppressor activity.

biochemistry↗

bioRxiv2024

The pro-tumoral and anti-tumoral roles of EphA4 on T regulatory cells and tumor associated macrophages during HNSCC tumor progression.

Head and Neck Squamous Cell Carcinoma (HNSCC) is a deadly cancer with poor response to targeted therapy, largely driven by an immunosuppressive tumor microenvironment (TME). Here we examine the immune-modulatory role of the receptor tyrosine kinase EphA4 in HNSCC progression. Within the TME, EphA4 is primarily expressed on regulatory T cells (Tregs) and macrophages. In contrast ephrinB2, an activating ligand of EphA4, is expressed in tumor blood vessels. Using genetically engineered mouse models, we show that EphA4 expressed in Tregs promotes tumor growth, whereas EphA4 expressed in monocytes inhibits tumor growth. In contrast, ephrinB2 knockout in blood vessels reduces both intratumoral Tregs and macrophages. A novel specific EphA4 inhibitor, APY-d3-PEG4, reverses the accelerated tumor growth we had previously reported with EphB4 cancer cell knockout. EphA4 knockout in macrophages not only enhanced their differentiation into M2 macrophage but also increased Treg suppressive activity. APY-d3-PEG4 reversed the accelerated growth seen in the EphA4 knockout of monocytes but conferred no additional benefit when EphA4 was knocked out on Tregs. Underscoring an EphA4-mediated interplay between Tregs and macrophages, we found that knockout of EphA4 in Tregs not only decreases their activation but also reduces tumor infiltration of pro-tumoral M2 macrophages. These data identify Tregs as a primary target of APY-d3-PEG4 and suggest a role for Tregs in regulating macrophage conversion. These data also support the possible anti-cancer therapeutic value of bispecific peptides or antibodies capable of promoting EphA4 blockade in Tregs but not macrophages. SignificanceEphA4 in regulatory T cells has a pro-tumoral effect while EphA4 in macrophages plays an anti-tumoral role underscoring the necessity of developing biologically rational therapeutics.

cancer biology↗

bioRxiv2024

Manipulating the EphB4-ephrinB2 axis to reduce metastasis in HSNCC

The EphB4-ephrinB2 signaling axis has been heavily implicated in metastasis across numerous cancer types. Our emerging understanding of the dichotomous roles that EphB4 and ephrinB2 play in head and neck squamous cell carcinoma (HNSCC) poses a significant challenge to rational drug design. We find that EphB4 knockdown in cancer cells enhances metastasis in preclinical HNSCC models by augmenting immunosuppressive cells like T regulatory cells (Tregs) within the tumor microenvironment. EphB4 inhibition in cancer cells also amplifies their ability to metastasize through increased expression of genes associated with epithelial mesenchymal transition and hallmark pathways of metastasis. In contrast, vascular ephrinB2 knockout coupled with radiation therapy (RT) enhances anti-tumor immunity, reduces Treg accumulation into the tumor, and decreases metastasis. Notably, targeting the EphB4-ephrinB2 signaling axis with the engineered EphB4 ligands EFNB2-Fc-His and Fc-TNYL-RAW-GS reduces local tumor growth and distant metastasis in a preclinical model of HNSCC. Our data suggest that targeted inhibition of vascular ephrinB2 while avoiding inhibition of EphB4 in cancer cells could be a promising strategy to mitigate HNSCC metastasis.

cancer biology↗

bioRxiv2022

The efficacy of EphA2 tyrosine phosphorylation increases with EphA2 oligomer size

The receptor tyrosine kinase (RTK) EphA2 is expressed in epithelial and endothelial cells and controls the assembly of cell-cell junctions. EphA2 has also been implicated in many diseases, including cancer. Unlike most RTKs, which signal predominantly as dimers, EphA2 readily forms higher order oligomers upon ligand binding. Here we investigated if a correlation exists between EphA2 signaling properties and the size of the EphA2 oligomers induced by multiple ligands, including the widely used ephrinA1-Fc ligand, the soluble monomeric m-ephrinA1, and novel engineered peptide ligands. We used Fluorescence Intensity Fluctuation (FIF) spectrometry to characterize the EphA2 oligomer populations induced by the different ligands. Interestingly, we found that different monomeric and dimeric ligands induce EphA2 oligomers with widely different size distributions. Comparison of FIF brightness distribution parameters and EphA2 signaling parameters reveals that the efficacy of EphA2 phosphorylation on tyrosine 588, which is indicative of receptor activation, correlates with EphA2 mean oligomer size. However, other characteristics, such as the efficacy of AKT inhibition and ligand bias coefficients, appear to be independent of EphA2 oligomer size. This work highlights the utility of FIF in RTK signaling research and demonstrates a quantitative correlation between the architecture of EphA2 signaling complexes and signaling features.

biophysics↗

bioRxiv2022

Plasticity of transmembrane helix interactions in EphA2 dimers and oligomers

Lateral interactions can stabilize different EphA2 receptor assemblies in the plasma membrane in response to different ligands. Here we use two fluorescent techniques, Forster Resonance Energy Transfer (FRET) and Fluorescence Intensity Fluctuations (FIF) spectrometry, to investigate how mutations in the EphA2 transmembrane (TM) helix affect the association between full-length EphA2 molecules in the absence of ligand and in the presence of three ligands: ephrinA1-Fc, m-ephrinA1, and the YSA peptide. The EphA2 mutations we studied have been previously characterized in the context of the isolated EphA2 TM helix. Working with full-length EphA2, we observed modest effects of the mutations on receptor-receptor interaction. Our data do not support the currently accepted model of a switch between two discrete TM helix dimerization motifs corresponding to active or inactive receptor states. Instead, we propose that different dimeric/oligomeric arrangements of the EphA2 extracellular region couple to an ensemble of TM helix dimer interfaces. Plasticity in the arrangements of receptor tyrosine kinase TM helices in active dimers and oligomers may serve to facilitate the cross-phosphorylation of multiple tyrosines in different positions of the intracellular regions.

biophysics↗

bioRxiv2021

Direct quantification of ligand-induced lipid and protein microdomains with distinctive signaling properties

Lipid rafts are known as highly ordered lipid domains that are enriched in saturated lipids such as the ganglioside GM1. While lipid rafts are believed to exist in cells and to serve as signaling platforms through their enrichment in signaling components, they have never been directly observed in the plasma membrane without treatments that artificially cluster GM1 into large lattices. Here we report that microscopic GM1-enriched domains can form, without lipid cross-linking, in the plasma membrane of live mammalian cells expressing the EphA2 receptor tyrosine kinase in response to its ligand ephrinA1-Fc. The GM1-enriched microdomains form concomitantly with EphA2-enriched microdomains, but only partially co-localize with them. To gain insight into how plasma membrane heterogeneity controls signaling, we quantify the degree of EphA2 segregation and study initial EphA2 signaling steps in both EphA2-enriched and EphA2-depleted domains. By measuring dissociation constants, we demonstrate that EphA2 oligomerization is the same in EphA2-enriched and -depleted domains. However, EphA2 interacts preferentially with its downstream effector SRC in EphA2-depleted domains. The ability to induce microscopic GM1-enriched domains in live cells using a ligand for a transmembrane receptor will give us unprecedented opportunities to study the biology of lipid rafts.

biophysics↗

bioRxiv2021

Allosteric Regulation of the EphA2 Receptor Intracellular Region by Serine/Threonine Kinases

Eph receptor tyrosine kinases play a key role in cell-cell communication. However, lack of structural information on the entire multi-domain intracellular region of any Eph receptor has hindered detailed understanding of their signaling mechanisms. Here, we use an integrative structural biology approach combining X-ray crystallography, small-angle X-ray scattering and hydrogen-deuterium exchange mass spectrometry, to gain the first insights into the structure and dynamics of the entire EphA2 intracellular region. EphA2 promotes cancer malignancy through a poorly understood non-canonical form of signaling that depends on serine/threonine phosphorylation of the linker connecting the EphA2 kinase and SAM domains. We uncovered two distinct molecular mechanisms that may function in concert to mediate the effects of linker phosphorylation through an orchestrated allosteric regulatory network. The first involves a shift in the equilibrium between a "closed" configuration of the EphA2 intracellular region and an "open" more extended configuration induced by the accumulation of phosphorylation sites in the linker. This implies that cooperation of multiple serine/threonine kinase signaling networks is necessary to promote robust EphA2 non-canonical signaling. The second involves allosteric rearrangements in the kinase domain and juxtamembrane segment induced by phosphorylation of some linker residues, suggesting a link between EphA2 non-canonical signaling and canonical signaling through tyrosine phosphorylation. Given the key role of EphA2 in cancer malignancy, this new knowledge can inform therapeutic strategies.

biophysics↗