Abstract 5768: Proximity labeling with molecular signal sensing of oncogenic PIK3CA

Abstract

Abstract PIK3CA mutations are major oncogenic drivers for oncogenesis. Mutant PIK3CA continuously activates the PI3K pathway, transmitting oncogenic signals within the transformed cells. Despite decades of efforts focused on targeting oncogenic PIK3CA for cancer therapy, the precise molecular and functional machinery remains incomplete due to the molecular complexity of the PI3K pathway. Mapping of proximity proteins in the oncogenic PI3K pathway and identifying mutant signals will signify a deep understanding of oncogenesis and a broad range of therapeutic applications. In this study, we hypothesize that PIK3CA mutant cancer cells establish a unique molecular program networking with distinct proteins, which differs from that of cancer cells with wild-type (WT) PIK3CA. To test this hypothesis, we have developed a novel bioengineering approach to decode protein-protein interactions of oncogenic PIK3CA and identify key molecular partners, potentially providing therapeutic vulnerabilities in cancer cells. First, we applied the proximity labeling technique, based on the fusion of a promiscuous protein ligase to a targeting protein of interest (POI), featuring proximity-dependent biotinylation of interacting and neighboring proteins. The promiscuous biotin ligases, BioID and BioID2, were utilized to integrate WT PIK3CA or gain-of-function hotspot mutations in at E545K or H1047R residues. To optimize the labeling protocol and biotinylation conditions, the BioID and BioID2 plasmids were fused with each POI, incorporating multiple combinations of protein tags, including myc, HA, and red fluorescence protein. Lamin A was used as the positive control, and a total of 15 plasmids were constructed in pcDNA3.1 mammalian expression vector. The sizes of the plasmids and the gene sequences were validated by PCR amplification and Sanger sequencing. Next, we employed a programmable nuclease, the prokaryotic Argonaute (pAgo), assisted by fluorescence resonance energy transfer technology, to detect oncogenic signals derived from mutant PIK3CA in cell-free conditions. To test our methods, we engineered two cancer cell lines (DHSA-1426 and COSB) derived from naturally occurring hemangiosarcoma in dogs, to induce PIK3CA H1047R mutations using CRISPR/Cas9. Our technical approach enables temporal and spatial molecular sensing of oncogenic PIK3CA and its functional interactome in living cancer cells by integrating high-resolution molecular mapping with a pAgo-based detection system. Our ongoing work involves the standardization and optimization of proximity labeling for oncogenic PIK3CA across ontogenetically distinct cancer cells. Furthermore, we will determine the sensitivity and specificity of the integrated approach to achieve proximity labeling with programmable detection of molecular signals from oncogenic PIK3CA. Citation Format: Rong Li, Donghee Lee, Md Abdullah, Jong Hyuk Kim. Proximity labeling with molecular signal sensing of oncogenic PIK3CA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5768.