A Multipronged Unbiased Strategy Guides the Development of an anti-EGFR/EPHA2 Bispecific Antibody for Combination Cancer Therapy
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PROVIDER: PRJNA721399 | ENA |
REPOSITORIES: ENA
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Project description:Purpose: Accumulating analyses of pro-oncogenic molecular mechanisms triggered a rapid development of targeted cancer therapies. Although many of these treatments produce impressive initial responses, eventual resistance onset is practically unavoidable. One of the main approaches for preventing this refractory condition relies on the implementation of combination therapies. This includes dual-specificity reagents that affect both of their targets with a high level of selectivity. Unfortunately, selection of target combinations for these treatments is often confounded by limitations in our understanding of tumor biology. Here, we describe and validate a multipronged unbiased strategy for predicting optimal co-targets for bispecific therapeutics. Experimental design: Our strategy integrates ex-vivo genome-wide loss of function screening, BioID interactome profiling and gene expression analysis of patient data to identify the best fit co-targets. Final validation of selected target combinations is done in tumorsphere cultures and xenograft models. Results: Integration of our experimental approaches unambiguously pointed towards EGFR and EPHA2 tyrosine kinase receptors as molecules of choice for co-targeting in multiple tumor types. Following this lead, we generated a human bispecific anti-EGFR/EPHA2 antibody that, as predicted, very effectively suppresses tumor growth, compared to its prototype anti-EGFR therapeutic antibody, cetuximab. Conclusion: Our work not only presents a new bispecific antibody with a high potential for being developed into clinically-relevant biologics, but more importantly, successfully validates a novel unbiased strategy for selecting biologically optimal target combinations. This is of a significant translational relevance, as such multifaceted unbiased approaches are likely to augment the development of effective combination therapies for cancer treatment.
2023-02-23 | GSE171920 | GEO
Project description:Recent progress in dissecting molecular mechanisms essential for the survival and propagation of cancer cells triggered rapid development of targeted therapies. Although many of these therapies produce impressive initial responses in tumor suppression, the onset of resistance is practically unavoidable. One of the main approaches for preventing this refractory condition relies on the development of effective combination therapies. Unfortunately, selection of optimal targets for combinatorial treatments is often confounded by limitations in our understanding of tumor biology. Here, we describe an unbiased comprehensive strategy that combines ex-vivo shRNA-based genome wide screening, proteomic profiling using BioID and patient tumor sequencing data analyses to overcome current challenges in identifying the best fit co-targeting agents. Integrating these strategies, we selected EGFR and EPHA2 receptors as molecules of choice for co-targeting in cancer cells, and generated a bispecific fully humanized anti-EGFR/EPHA2 antibody with druggable bioanalytical properties that very effectively suppresses tumor growth compared to its prototype therapeutic anti-EGFR antibody, cetuximab. Despite the overactivation of EGFR signaling in multiple malignancies, the therapeutic applicability of cetuximab is limited due to frequent resistance and could benefit from being used in a combination with another targeted compound. Our analysis indicates that EGFR and EPHA2 expression positively correlate in various cancer types and thus, are available for co-targeting in most tumors. Therefore, our work not only presents an efficient unbiased strategy for selecting optimal targets for combination therapies, but also describes a novel bispecific antibody, which has a high potential for being developed into a new clinically-relevant biologics effective in a broad variety of malignancies.
2023-01-27 | PXD023034 | Pride
Project description:RNA-seq analysis of sorted EPHA2-WT and EPHA2-KO tumor cells from subcutaneously implanted mouse pancreatic tumors
2019-08-31 | GSE120208 | GEO
Project description:We used Crispr/Cas9 technology to establish a homozygous clone of EphA2-SE deletion in tumor cells. Wild-type cells (WT) and homozygous cloned cells (EphA2-SE-/-) were selected for high-throughput data detection.
2021-03-18 | GSE159353 | GEO
Project description:Transcriptional profiling of EPHA2-WT and EPHA2-KO pancreatic tumor cells
| PRJNA491946 | ENA
Project description:Purpose: To understand the relationship between ciliogenesis and autophagy in the corneal epithelium. Methods: siRNAs for EphA2 or PLD1 were used to inhibit protein expression in vitro. Morpholino-anti-EphA2 was used to knockdown EphA2 in Xenopus skin. An EphA2 knockout mouse was used to conduct loss of function studies. Autophagic vacuoles were visualized by contrast light microscopy. Autophagy flux, was measured by LC3 turnover and p62 protein levels. Immunostaining and confocal microscopy were conducted to visualize cilia in cultured cells and in vivo. Results: Loss of EphA2 (i) increased corneal epithelial thickness by elevating proliferative potential in wing cells, (ii) reduced the number of ciliated cells, (iii) increased large hollow vacuoles, that could be rescued by BafA1; (iv) inhibited autophagy flux and (v) increased GFP-LC3 puncta in the mouse corneal epithelium. This indicated a role for EphA2 in stratified epithelial assembly via regulation of proliferation as well as a positive role in both ciliogenesis and end-stage autophagy. Inhibition of PLD1, an EphA2 interacting protein that is a critical regulator of end-stage autophagy, reversed the accumulation of vacuoles, and the reduction in the number of ciliated cells due to EphA2 depletion, suggesting EphA2 regulation of both end-stage autophagy and ciliogenesis via PLD1. PLD1 mediated rescue of ciliogenesis by EphA2 depletion was blocked by BafA1, placing autophagy between EphA2 signaling and regulation of ciliogenesis. Conclusion: Our findings demonstrate a novel role for EphA2 in regulating both autophagy and ciliogenesis, processes that are essential for proper corneal epithelial homeostasis. Purpose: To understand the relationship between ciliogenesis and autophagy in the corneal epithelium. Methods: siRNAs for EphA2 or PLD1 were used to inhibit protein expression in vitro. Morpholino-anti-EphA2 was used to knockdown EphA2 in Xenopus skin. An EphA2 knockout mouse was used to conduct loss of function studies. Autophagic vacuoles were visualized by contrast light microscopy. Autophagy flux, was measured by LC3 turnover and p62 protein levels. Immunostaining and confocal microscopy were conducted to visualize cilia in cultured cells and in vivo. Results: Loss of EphA2 (i) increased corneal epithelial thickness by elevating proliferative potential in wing cells, (ii) reduced the number of ciliated cells, (iii) increased large hollow vacuoles, that could be rescued by BafA1; (iv) inhibited autophagy flux and (v) increased GFP-LC3 puncta in the mouse corneal epithelium. This indicated a role for EphA2 in stratified epithelial assembly via regulation of proliferation as well as a positive role in both ciliogenesis and end-stage autophagy. Inhibition of PLD1, an EphA2 interacting protein that is a critical regulator of end-stage autophagy, reversed the accumulation of vacuoles, and the reduction in the number of ciliated cells due to EphA2 depletion, suggesting EphA2 regulation of both end-stage autophagy and ciliogenesis via PLD1. PLD1 mediated rescue of ciliogenesis by EphA2 depletion was blocked by BafA1, placing autophagy between EphA2 signaling and regulation of ciliogenesis. Conclusion: Our findings demonstrate a novel role for EphA2 in regulating both autophagy and ciliogenesis, processes that are essential for proper corneal epithelial homeostasis.
2021-05-19 | GSE174649 | GEO
Project description:We ued CRISPR/Cas9 to knock out EphA2 gene in PC3 cells, and overexpress EphA2 gene in Du145 cells with overexpressed plasmids to explore the underlying mechanisms of EphA2 function in castration-resistant prostate cancer(CRPC) and clinical relevance, providing more evidence for EphA2 as a potential therapeutic target for CRPC.
2024-05-20 | GSE267508 | GEO
Project description:Genetic variations in ephrin type-A receptor 2 (EPHA2) have been associated with inherited and age-related forms of cataract in humans. Here we have characterized the eye lens phenotype and transcript profile of germline Epha2 knock-in mutant mice homozygous for either a missense variant associated with age-related cataract in humans (Epha2-Q722) or a novel insertion-deletion mutation (Epha2-indel722) that were both located within the tyrosine-kinase domain of EPHA2. Whole-mount confocal imaging of clear lenses from Epha2-indel722 mice on a fluorescent reporter background revealed misalignment of epithelial-to-fiber cell meridional-rows at the lens equator and severe disturbance of Y-suture formation at the lens poles, whereas, Epha2-Q722 lenses displayed mild disturbance of posterior sutures. Immunofluorescent labeling showed that EPHA2 was mostly localized to lens fiber cell membranes with some sub-membrane localization observed in Epha2-Q722 lenses and diffuse membrane and perinuclear localization in Epha2-indel722 lenses. Immunoprecipitation/blotting studies indicated that EPHA2 formed strong complexes with Src kinase but not with catenin beta 1 or cadherin 2 and was mostly serine phosphorylated in the lens. RNA-sequencing analysis revealed differential expression of several cytoskeleton-associated genes in Epha2-mutant and Epha2-null lenses including strong downregulation of Lgsn and Clic5. Collectively, our data suggest that mutations within the tyrosine-kinase domain of EPHA2 result in lens cell patterning defects and dysregulated expression of several cytoskeletal proteins.
2021-08-05 | GSE181358 | GEO
Project description:Ewing sarcoma (ES) is the second most common bone malignancy affecting children and young adults with poor prognosis due to high metastasis incidence. Our group previously described that EphA2, a tyrosine kinase receptor, promotes angiogenesis in ES cells via ligand-dependent signaling. EphA2 ligand-independent activity, controlled upon phosphorylation at S897 (p-EphA2S897), has been linked to metastasis in several malignancies. Here, we stablish a correlation between ES cells aggressiveness and p-EphA2S897. Moreover, stable overexpression of EphA2 in low EphA2 expression ES cells enhanced proliferation and migration, but not a nonphosphorylable mutant (S987A). Consistently, silencing of EphA2 reduced tumorigenicity, migration and invasion in vitro, and lung metastasis incidence in experimental and spontaneous metastasis assays in vivo. A gene expression microarray revealed the implication of EphA2 in cell signaling, cellular movement and survival. Altogether, our results suggest that p-EphA2S897 correlates with aggressiveness in ES, so blocking its function may be a promising treatment.
2018-04-11 | GSE106480 | GEO
Project description:RNA-seq data for EphA2 knockdown (KD) in Huh7 cells
2021-02-25 | GSE141880 | GEO