RAS GTPases are important mediators of oncogenesis in humans. However, pharmacological inhibition of RAS has proved challenging. Here, we describe a functionally critical region of RAS located outside the effector lobe that can be targeted for inhibition. We developed a synthetic binding protein (monobody), termed NS1, that bound with high affinity to both GTP- and GDP-bound states of H- and K-RAS but not N-RAS. NS1 potently inhibited growth factor signaling and oncogenic H- and K-RAS-mediated signaling and transformation but did not block oncogenic N-RAS, BRAF or MEK1. NS1 bound the α4-β6-α5 region of RAS disrupting RAS dimerization/nanoclustering, which in turn blocked CRAF:BRAF heterodimerization and activation. These results establish the importance of the α4-β6-α5 interface in RAS-mediated signaling and define a previously unrecognized site in RAS for inhibiting RAS function.

Highlights d C-Raf binds all Ras proteins equivalently, but B-Raf exhibits selectivity for K-Ras d Raf N-terminal segments and Ras HVR sequences determine binding preferences d C-Raf is critical for downstream transmission of H-Ras-driven signaling d Events that increase B-Raf/C-Raf dimerization augment the B-Raf/H-Ras interaction

RAS GTPases (H-, K-, and N-RAS) are the most frequently mutated oncoprotein family in human cancer. However, the relatively smooth surface architecture of RAS and its picomolar affinity for nucleotide have given rise to the assumption that RAS is an "undruggable" target. Recent advancements in drug screening, molecular modeling, and a greater understanding of RAS function have led to a resurgence in efforts to pharmacologically target this challenging foe. This review focuses on the state of the art of RAS inhibition, the approaches taken to achieve this goal, and the challenges of translating these discoveries into viable therapeutics.

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 reported a new approach to inhibit RAS-dependent signaling and transformation in vitro through 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 .