Battling Cancer with an NRAS Mutant-specific Monobody

The RAS GTPase isoforms HRAS, KRAS, and NRAS regulate crucial intracellular signaling cascades that control cell proliferation and survival (Karnoub and Weinberg); furthermore, they represent some of the most frequently mutated oncogenes in human cancers. We currently lack targeted therapies for NRAS-mutated cancers, which include melanoma, multiple myeloma, and colorectal cancer (Prior et al. (a) and Prior et al. (b)) even though recent years have seen advancements in the development of anti-RAS therapeutics. Overall, the description of clinical trials of KRAS inhibitors in non-small cell lung cancer (Jänne et al. and Skoulidis et al.) and pre-clinical reports of non-covalent, selective mutant-specific and pan-KRAS inhibitors (Wang et al. and Kim et al.) have provided some encouragement.

As an alternative to small-molecule RAS inhibitors, researchers from the laboratories of Shohei Koide (NYU) and John P. O’Bryan (Medical University of South Carolina) have developed “monobodies” that interact with and inhibit specific RAS isoforms or mutants (Spencer-Smith et al. (a), Khan et al. (a), and Spencer-Smith et al. (b)). Monobodies - synthetic binding proteins constructed employing a fibronectin type III domain as a molecular scaffold (Hantschel et al.) – represent a simple and robust alternative to antibodies for creating target-binding proteins.

While the selective targeting of NRAS by monobodies faced challenges associated with molecular recognition, a recent study from the Koide and O´Bryan laboratories reported in Oncogene describes a newly developed inhibitory NRAS monobody as a novel “weapon” in the battle against cancer. This study describes how “Mb24” can bind to GDP/GTP-bound states of NRAS, inhibit NRAS-mediated signaling, and form a component of a genetically encoded NRAS-specific degrader (Whaby et al. (a)). Does the development of this new, highly specific monobody now offer a means of efficiently battling NRAS mutant cancers?

The development of Mb24 employed established methods that combine phage and yeast display technologies (Khan et al. (b), Teng et al., and Koide et al.), positive (GDP/GTP-bound NRAS) and negative (GDP/GTP-bound HRAS and KRAS) selection methodologies, and the confirmation of specificity in immortalized human cells, where Mb24 selectively co-precipitated with two different NRAS mutants. Whaby et al. next demonstrated that Mb24 specifically inhibited ERK/MAPK signaling mediated by epidermal growth factor-stimulated wild-type NRAS and oncogenic NRAS mutants thanks to the implementation of two cell models - the “RASless” HEK 293 model cellular system (Cuevas-Navarro et al.) and the Flp-In FRT system (generates cells that stably express single RAS isoforms). Next, the authors confirmed that Mb24 inhibited the biological activity of oncogenic NRAS mutants, finding that the induced expression of Mb24 in NRAS mutant cancer cells (but not wild-type NRAS cancer cells) effectively decreased ERK/MAPK phosphorylation (indicating reduced signaling activity) and significantly reduced anchorage-independent cell growth and cell migration (suggesting the loss of cancer cell-like characteristics).

Analysis of the mechanisms controlling Mb24-driven NRAS inhibition evaluated the effect of Mb24 on crucial protein:protein interactions occurring in the RAS/MAPK signaling pathway in live cells thanks to the application of NanoBiT technology (Whaby et al. (b)). This approach discounted NRAS interactions with CRAF but suggested that Mb24 can disrupt oncogenic CRAF:BRAF heterodimerization and inhibit NRAS by sterically interfering with nanoclustering at the plasma membrane to inhibit NRAS-stimulated RAF dimerization and activation.

Finally, the authors aimed to increase the utility of Mb24 by deploying this specific monobody as a “warhead” that prompted the targeted proteasomal degradation of mutant NRAS as a potential anti-cancer approach (Teng et al., Akkapeddi et al., and Lim et al.). To do so, the authors genetically fused Mb24 to a truncated version of the VHL E3 ligase (Teng et al.) and revealed that Mb24 could target VHL E3 ligase activity to NRAS, which supported the proteasome-dependent degradation. Overall, the authors provide evidence that the VHL-Mb24 fusion protein represents a new weapon that can target NRAS mutant cancers.

Overall, this first report of Mn24 as a highly specific and effective “weapon” highlights a new avenue for the development of NRAS-selective therapeutics that will significantly aid in the battle against certain types of cancer.

Active Motif can help in the battle against cancer with a range of RAS GTPase-related products, which include the Ras GTPase Chemi ELISA Kit for the rapid and sensitive analysis of activated Ras GTPase and the KRAS In-well Lysis ELISA Kit for the rapid, sensitive, and highly specific quantification of active KRAS.

For more details on how an NRAS mutant-specific monobody may represent a new tool in the battle against cancer, see Oncogene, October 2024.

This article was originally published on the Active Motif blog.