Probing RAS Function with Monobodies

Abstract

RAS is frequently mutated in human cancers with nearly 20% of all cancers harboring mutations in one of three RAS isoforms (KRAS, HRAS, or NRAS). Furthermore, RAS proteins are critical oncogenic drivers of tumorigenesis. As such, RAS has been a prime focus for development of targeted cancer therapeutics. Although RAS is viewed by many as undruggable, the recent development of allele-specific covalent inhibitors to KRAS(G12C) has provided significant hope for the eventual pharmacological inhibition of RAS (Ostrem et al., Nature 503(7477):548-551, 2013; Patricelli et al., Cancer Discov 6(3):316-329, 2016; Janes et al., Cell 172(3):578-589.e17, 2018; Canon et al., Nature 575(7781):217-223, 2019; Hallin et al., Cancer Discov 10(1):54-71, 2020). Indeed, these (G12C)-specific inhibitors have elicited promising responses in early phase clinical trials (Canon et al., Nature 575(7781):217-223, 2019; Hallin et al., Cancer Discov 10(1):54-71, 2020). Despite this success in pharmacologically targeting KRAS(G12C), the remaining RAS mutants lack readily tractable chemistries for development of covalent inhibitors. Thus, alternative approaches are needed to develop broadly efficacious RAS inhibitors. We have utilized Monobody (Mb) technology to identify vulnerabilities in RAS that can potentially be exploited for development of novel RAS inhibitors. Here, we describe the methods used to isolate RAS-specific Mbs and to define their inhibitory activity.

Keywords: Cell signaling; HEK293; Monobody; NIH/3T3; PEI; RAS; RAS foci; Soft agar assays; Transfection; Tumorigenesis; Xenograft tumor assays.

Figure 1.. Monobody analysis strategy.

Outline of strategy to isolate RAS-specific Mbs using phage display selection followed by affinity maturation in yeast display format. Isolated RAS Mbs are then subcloned into mammalian expression vectors for transient and stable expression as well as chemically regulated expression in various human tumor lines. The described analyses are not meant to be exhaustive but rather a standard approach for initial characterization of RAS-specific Mb clones. Additional analyses may be performed to further define the mechanism of action of a particular Mb.

Figure 2.. Analysis of Mb binding to RAS.

HEK293 cells were transiently transfected with expression constructs encoding the indicated HRAS mutants along with Mb expression constructs encoding NS1 or a novel RAS-specific Mb, Mb-A. Samples were processed as described in Section 3.3.

Figure 3.. Cell Signaling Assays.

Mb expression constructs were co-transfected with oncogenic HRAS(Q61L) or oncogenic MEK(DD) along with MYC-tagged ERK. Samples were processed as described in Section 3.4b. CFP-NS1 inhibited ERK activation by oncogenic HRAS(Q61L) but not MEK(DD).

Figure 4.. NIH/3T3 focus formation assay.

NIH/3T3 cells were transfected with oncogenic HRAS(Q61L) or BRAF(V600E) along with CFP, CFP-NS1 of CFP-Mb-A. Cells were stained to visualize foci as described in Section 3.6.

Figure 5.. Analysis of RAS-specific Mb effects in human tumor cells.

The pancreatic cancer cell line CFPAC-1 was infected with a DOX-inducible expression construct encoding CFP-NS1 and then stable selected in antibiotic-containing media to generate a polyclonal cell line, CFPAC-1^NS1. (A) CFPAC-1^NS1 cells were plated in duplicate wells and then treated with DOX for 72 hrs. Lysates were then analyzed by Western blot for Mb expression (top panel) and ERK activation (bottom 4 panels). Vinculin was used as a normalization control on blots for pERK or total ERK. (B) CFPAC-1^NS1 cells were plated in soft agar as described in Section 3.9