Using cells devoid of RAS proteins as tools for drug discovery

Abstract

Mutational activation of RAS proteins occurs in nearly 30% of all human tumors. To date direct pharmacological inhibition of RAS oncoproteins has not been possible. As a consequence, current strategies are focusing on the development of inhibitors that target those kinases acting downstream of RAS proteins, including those of the RAF/MEK/ERK and PI3K/AKT pathways. Most of these inhibitors have undesired off-target effects that mask the potential therapeutic effect of blocking their targeted kinases. To facilitate the screening of selective inhibitors, we have generated lines of mouse embryonic fibroblasts that lack endogenous Ras proteins. These cells proliferate due to ectopic expression of either Ras oncoproteins that selectively activate the Raf/Mek/Erk pathway such as H-Ras(G12V/D38E) or constitutively active kinases such as B-Raf and Mek1. These cell lines were exposed to inhibitors against the RAF, MEK, and AKT kinases as well as inhibitors of other kinases known to crosstalk with RAS signaling such as JNK and p38. Amongst all compounds tested, only the MEK inhibitors U0126 and PD0325901, showed the expected specificity pattern. Yet, PD0325901, but not U0126, was able to inhibit a cell line lacking Ras proteins that owed its proliferative properties to loss of p53. Thus, suggesting unexpected off-target activities for this compound. The use of cell lines whose proliferative properties exclusively depend on selective targets provide a novel strategy to analyze the specificity of selective inhibitors designed against molecular targets implicated in human cancer.

Multivalent Small-Molecule Pan-RAS Inhibitors

“…To further examine the potential RAS-dependent lethality of 3144, we used a previously reported mouse embryo fibroblast (MEF) cell line in which murine Hras and Nras had been deleted, and only Kras remained, flanked by loxP sites; a tamoxifen-inducible Cre recombinase was also expressed in these Kras lox/lox , Hras −/− , Nras −/− RERTn ert/ert cells (Urosevic et al, 2009). We found that these cells were sensitive to the lethality of compound 3144, as expected (IC 50 = 3.8 μM, Figure 3B); however, excision of Kras from these cells using tamoxifen, and introduction of membrane-targeted BRAF V600E -CAAX resulted in resistance to 3144 (Figure 3B), indicating a degree of Kras -dependent lethality.…”

Ras palmitoylation is necessary for N-Ras activation and signal propagation in growth factor signalling

“…To test this hypothesis we employed an experimental system that allowed us to study signal transduction by ectopic N-Ras variants in the absence of confounding signals by endogenous Ras proteins. H-Ras − / − , N-Ras − / − , K-Ras lox/lox , RERT ert/ert MEFs (from here on K-Ras lox/lox ), a mouse embryonic cell line engineered and characterized by Barbacid and co-workers [38,39], carries knockout H-Ras and N-Ras alleles, floxed K-Ras loci and inducible Cre recombinase. Recombinase induction with 4-OHT leads to excision of the K-Ras alleles, rendering the K-Ras lox/lox MEFs devoid of all three Ras proteins.…”

Ras activation of Erk restores impaired tonic BCR signaling and rescues immature B cell differentiation

“…As Erk is directly activated by the mitogen-activated protein kinase (MAPK) kinase (MEK), we used the specific MEK inhibitor U0126 (Favata et al, 1998;Urosevic et al, 2009) to prevent Erk activation in the in vitro B cell differentiation assay. As shown in Fig.…”