Abstract A37: Mapping KRAS signaling pathways using the Mammalian-Membrane Two-Hybrid (MaMTH) assay to elucidate novel therapeutic targets
Notice bibliographique
Résumé
Abstract Background: KRAS is a well-established cancer driver. Clinically successful therapeutics have not yet been developed despite its disease burden, suggesting that KRAS-driven cancers are more complicated than previously thought. Aim: Our goal is to identify the global changes in KRAS interaction patterns that occur in disease states. This is accomplished by mapping the dynamic interactome of both WT and oncogenic KRAS isoforms using the Mammalian Membrane Two-Hybrid (MaMTH) assay, which is suitable for identification of protein interactors of virtually any integral membrane or membrane-associated protein. Methods: To probe the protein-protein interactions (PPIs) of KRAS, we used the Mammalian Membrane Two-Hybrid (MaMTH) assay, a split-ubiquitin-based two-hybrid system that can be applied to any cell line. This assay is suited for PPI detection using both low- to-medium throughput array and high-throughput large-scale PPI screening. We generated stably expressing “bait”-tagged KRAS constructs in HEK293T cells that contained the MaMTH reporter system. KRAS baits, namely KRAS-WT, -G12D, -G12V, and -Q61H, were characterized using signaling assays as well as within the MaMTH system using “prey”-tagged CRAF interaction control, Western blotting, and immunofluorescence techniques. Currently, we are performing unbiased screening for PPIs of KRAS using the human ORFeome (hORFeome) library consisting of approximately 13,000 fully sequenced human ORFs. Results: We first validated compatibility of KRAS in the MaMTH assay through several means. Oncogenic KRAS variants showed increased interaction signal with CRAF (a known KRAS binding partner) compared to KRAS-WT. This corresponded with upregulated MAPK pathway activation by oncogenic KRAS, as determined from increased pERK levels via Western blotting. KRAS bait expression levels were similar across all variants, suggesting that these findings were not due to differential expression of KRAS isotypes. Additionally, we confirmed that the KRAS bait correctly localizes to the plasma membrane, consistent with previous literature. After establishing KRAS compatibility with the MaMTH assay, we have begun unbiased screening of the hORFeome against KRAS baits in the WT, G12D, G12V, and Q61H isoforms. Conclusions: We have characterized KRAS compatibility with the MaMTH assay. Currently, we are performing large screening protocols for high-throughput detection of PPIs of KRAS using MaMTH. Citation Format: Ingrid Claudia Grozavu, Jamie Snider, Anna Lyakisheva, Igor Stagljar. Mapping KRAS signaling pathways using the Mammalian-Membrane Two-Hybrid (MaMTH) assay to elucidate novel therapeutic targets [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A37.
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Comment cette classification a été obtenuedéplier
Prédiction distillée sur la base complète
Imitation des enseignantsNi prévalence calibrée, ni vérité terrain. Validation humaine à venir. Apprise à partir de 10 348 étiquettes directes de Codex et de 10 348 étiquettes directes de Gemma. Le mode candidate est l'union des têtes enseignantes seuillées; le consensus est leur intersection. Ces sorties portent le statut machine_predicted_unvalidated et ne sont ni des étiquettes humaines ni des étiquettes directes de modèles de pointe.
Scores Codex et Gemma par catégorie
| Catégorie | Codex | Gemma |
|---|---|---|
| Métarecherche | 0,002 | 0,000 |
| Méta-épidémiologie (sens strict) | 0,000 | 0,000 |
| Méta-épidémiologie (sens large) | 0,000 | 0,000 |
| Bibliométrie | 0,000 | 0,001 |
| Études des sciences et des technologies | 0,000 | 0,000 |
| Communication savante | 0,000 | 0,000 |
| Science ouverte | 0,001 | 0,001 |
| Intégrité de la recherche | 0,000 | 0,001 |
| Charge utile insuffisante (le modèle a refusé de juger) | 0,000 | 0,000 |
Scores machine (provisoires)
Les deux têtes enseignantes du modèle étudiant, lues sur ce travail. Un score ordonne la base pour la relecture; il n'affirme jamais une catégorie, et le statut de validation accompagne chaque rangée tel quel.
Scores de référence d'un modèle non mature (critères de maturité non atteints, 7 itérations). Un score ordonne; il n'affirme jamais une catégorie.
score_only:v0-immature-baseline · tel quel depuis la passe de notation : score_only signifie que le nombre peut ordonner les travaux, et qu'aucune étiquette de catégorie n'en découleClassification
machine, non validéePrédiction automatique; un appel candidat d’une seule tête enseignante, pas un consensus.
Le détail, modèle par modèle et score par score, se trouve en fin de page sous « Comment cette classification a été obtenue ».