Gametogenesis is dependent on intercellular communication facilitated by stable intercellular bridges connecting developing germ cells. During Drosophila oogenesis, intercellular bridges (referred to as ring canals; RCs) have a dynamic actin cytoskeleton that drives their expansion to a diameter of 10 μm. Although multiple proteins have been identified as components of RCs, we lack a basic understanding of how RC proteins interact together to form and regulate the RC cytoskeleton. Thus, here, we optimized a procedure for proximity-dependent biotinylation in live tissue using the APEX enzyme to interrogate the RC interactome. APEX was fused to four different RC components (RC-APEX baits) and 55 unique highconfidence prey were identified. The RC-APEX baits produced almost entirely distinct interactomes that included both known RC proteins and uncharacterized proteins. A proximity ligation assay was used to validate close-proximity interactions between the RC-APEX baits and their respective prey. Furthermore, an RNA interference screen revealed functional roles for several high-confidence prey genes in RC biology. These findings highlight the utility of enzymecatalyzed proximity labeling for protein interactome analysis in live tissue and expand our understanding of RC biology.

Gametogenesis is dependent on intercellular communication facilitated by stable intercellular bridges connecting developing germ cells. During Drosophila oogenesis, intercellular bridges (referred to as ring canals) have a dynamic actin cytoskeleton that drives their expansion to a diameter of 10µm. While multiple proteins have been identified as components of ring canals (RCs), we lack a basic understanding of how RC proteins interact together to form and regulate the RC cytoskeleton. We optimized a procedure for proximity-dependent biotinylation in live tissue using the APEX enzyme to interrogate the RC interactome. APEX was fused to four different RC components (RC-APEX baits) and 55 unique high-confidence preys were identified. The RC-APEX baits produced almost entirely distinct interactomes that included both known RC proteins as well as uncharacterized proteins. The proximity ligation assay was used to validate close-proximity interactions between the RC-APEX baits and their respective preys. Further, an RNAi screen revealed functional roles for several high-confidence prey genes in RC biology. These findings highlight the utility of enzyme-catalyzed proximity labeling for protein interactome analysis in live tissue and expand our understanding of RC biology. Summary StatementHere, we optimized a procedure for enzyme-catalyzed proximity labeling in live Drosophila ovary tissue to interrogate the Drosophila ring canal protein interactome.

During endocytosis, molecules are internalized by the cell through the invagination of the plasma membrane. Endocytosis is required for proper cell function and for normal development in Drosophila. One component of the endocytic pathway is the retromer complex, which recycles transmembrane proteins to other parts of the cell such as the plasma membrane and the trans-Golgi network. Previous studies have shown that mutations to the retromer complex result in developmental defects in Drosophila. In humans, retromer dysfunction has been implicated in Alzheimer's and Parkinson's disease, but little is known about the role of the retromer complex in Drosophila oogenesis. In the current project, we examined the role of the retromer protein Vps26 in oogenesis by characterizing the phenotype of vps26 germline clones. Immunofluorescence was used to visualize the expression of membrane proteins and vesicular trafficking markers in mutant egg chambers. We find that vps26 germline clones exhibit a signaling defect between the germline cells and follicle cells indicated by an increase in LysoTracker staining of the border cells in the mutants. We show that this signaling defect in vps26 mutants may be the result of impaired Notch signaling based on the misexpression of multiple proteins in the Notch signaling pathway in vps26 mutants.

Lung cancer is the leading cause of cancer-related death worldwide and affects over 2 million people each year. In lung adenocarcinoma (LUAD), somatic activating mutations of epidermal growth factor receptor (EGFR) occur in approximately 15% of patients. The first-line therapy for patients with EGFR-mutant LUAD is administration of osimertinib, an EGFR tyrosine kinase inhibitor (TKI). While many patients’ tumors initially respond to osimertinib treatment, resistance inevitably develops in most cases, which is a major challenge that hinders treatment efficacy. One common mechanism of resistance to osimertinib is the amplification of oncogenes, such as MET, HER2, and RET. Despite the high prevalence of oncogene amplifications in resistant tumors, the mechanisms that control oncogene amplification and transcription are poorly understood. Recent work has shown that oncogenes are frequently amplified on extrachromosomal DNA (ecDNA), which are circularized and highly amplified DNA sequences that are present in at least 14% of human cancers. Additional studies have implicated ecDNA amplification in the acquisition of resistance to TKIs, yet the mechanisms through which ecDNA is regulated are largely unknown. In the present study, we have identified a novel epigenetic factor that promotes the acquisition of TKI resistance through the organization and transcription of oncogene amplicons, some of which occur on ecDNA. To determine whether this factor binds to DNA, we used and found that it is enriched at the promoters of amplified oncogenes. Further, we performed protein immunoprecipitation followed by mass spectrometry and identified multiple candidate interacting factors, such as histone readers and components of the cohesin machinery. Depletion of this epigenetic factor prevents the acquisition of TKI resistance and leads to reduced transcription of oncogene amplicons. Thus, we propose a novel mechanism that promotes the acquisition of TKI resistance via the spatial organization and transcription of oncogene amplicons. Citation Format: Rebecca M. Starble, Eric G. Sun, Tyler B. Jensen, Rana Gbyli, Ning Sun, Andrew Z. Xiao. A novel epigenetic factor is required for the acquisition of TKI resistance in lung adenocarcinoma via the regulation of oncogene amplicons. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr B016.