bioluminescence-resonance-energy-transfer-bret-based-assay-for

Bioluminescence Resonance Energy Transfer (BRET)-Based Assay for Measuring Interactions of CRAF with 14-3-3 Proteins in Live Cells

CRAF is a primary effector of RAS GTPases and plays a critical role in the tumorigenesis of several KRAS-driven cancers. In addition, CRAF is a hotspot ...

Medical University of South Carolina

National Cancer Institute-Frederick

RAS genes are the most commonly mutated oncogenes in human cancers. Despite tremendous efforts over the past several decades, however, RAS-specific inhibitors remain elusive. Thus, targeting RAS remains a highly sought-after goal of cancer research. Previously, we have reported a new approach to inhibit RAS-dependent signaling and transformation in vitro by targeting the α4-α5 dimerization interface with a novel RAS-specific monobody termed NS1. Expression of NS1 inhibits oncogenic K-RAS and H-RAS signaling and transformation in vitro. Here, we evaluated the efficacy of targeting RAS dimerization as an approach to inhibit tumor formation in vivo. Using a doxycycline (DOX)-regulated NS1 expression system, we demonstrate that DOX-induced NS1 inhibited oncogenic K-RAS-driven tumor growth in vivo. Furthermore, we observed context-specific effects of NS1 on RAS-mediated signaling in 2D vs 3D growth conditions. Finally, our results highlight the potential therapeutic efficacy of targeting the α4-α5 dimerization interface as an approach to inhibit RAS-driven tumors in vivo.

Targeting the α4-α5 dimerization interface of K-RAS inhibits tumor formation in vivo.

Additionally, a further error has been corrected on page five in the sentence: Subsequent improvements to the chemistry of these lead compounds have resulted in the most recent interaction, with ARS-1620, which demonstrates selective inhibition of K-RAS(G12C) mutant tumor models in vivo [23]. The word 'with' has been removed from this sentence to ensure the correct meaning is communicated, such that the sentence now is: Subsequent improvements to the chemistry of these lead compounds have resulted in the most recent interaction, ARS-1620, which demonstrates selective inhibition of K-RAS(G12C) mutant tumor models in vivo [23].

Correction: Targeting the α4-α5 dimerization interface of K-RAS inhibits tumor formation in vivo.

RAS guanosine triphosphatases (GTPases) are mutated in nearly 20% of human tumors, making them an attractive therapeutic target. Following our discovery that nucleotide-free RAS (apo RAS) regulates cell signaling, we selectively target this state as an approach to inhibit RAS function. Here, we describe the R15 monobody that exclusively binds the apo state of all three RAS isoforms in vitro, regardless of the mutation status, and captures RAS in the apo state in cells. R15 inhibits the signaling and transforming activity of a subset of RAS mutants with elevated intrinsic nucleotide exchange rates (i.e., fast exchange mutants). Intracellular expression of R15 reduces the tumor-forming capacity of cancer cell lines driven by select RAS mutants and KRAS(G12D)-mutant patient-derived xenografts (PDXs). Thus, our approach establishes an opportunity to selectively inhibit a subset of RAS mutants by targeting the apo state with drug-like molecules.

Article	Year
Targeting the α4-α5 dimerization interface of K-RAS inhibits tumor formation in vivo. Oncogene\| PubMed ID: 30573767	2019
Correction: Targeting the α4-α5 dimerization interface of K-RAS inhibits tumor formation in vivo. Oncogene\| PubMed ID: 30710144	2019
Identification of the nucleotide-free state as a therapeutic vulnerability for inhibition of selected oncogenic RAS mutants. Cell reports\| PubMed ID: 35139380	2022

Russell Spencer-Smith

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