Targeting RAS Membrane Association: Back to the Future for Anti-RAS Drug Discovery?

Abstract

RAS proteins require membrane association for their biologic activity, making this association a logical target for anti-RAS therapeutics. Lipid modification of RAS proteins by a farnesyl isoprenoid is an obligate step in that association, and is an enzymatic process. Accordingly, farnesyltransferase inhibitors (FTI) were developed as potential anti-RAS drugs. The lack of efficacy of FTIs as anticancer drugs was widely seen as indicating that blocking RAS membrane association was a flawed approach to cancer treatment. However, a deeper understanding of RAS modification and trafficking has revealed that this was an erroneous conclusion. In the presence of FTIs, KRAS and NRAS, which are the RAS isoforms most frequently mutated in cancer, become substrates for alternative modification, can still associate with membranes, and can still function. Thus, FTIs failed not because blocking RAS membrane association is an ineffective approach, but because FTIs failed to accomplish that task. Recent findings regarding RAS isoform trafficking and the regulation of RAS subcellular localization have rekindled interest in efforts to target these processes. In particular, improved understanding of the palmitoylation/depalmitoylation cycle that regulates RAS interaction with the plasma membrane, endomembranes, and cytosol, and of the potential importance of RAS chaperones, have led to new approaches. Efforts to validate and target other enzymatically regulated posttranslational modifications are also ongoing. In this review, we revisit lessons learned, describe the current state of the art, and highlight challenging but promising directions to achieve the goal of disrupting RAS membrane association and subcellular localization for anti-RAS drug development. Clin Cancer Res; 21(8); 1819-27. ©2015 AACR. See all articles in this CCR Focus section, "Targeting RAS-Driven Cancers."

Figure 1

Membrane targeting sequences of RAS proteins. Top: the RAS on/off switch that is broken in oncogenically mutated RAS and fails to turn off from the active, GTP-bound state that interacts with effectors (E) to transmit downstream signals. Since membrane association is required for proper effector interaction, interfering with membrane targeting can impair signal transmission, like unwiring an electrical switch to prevent it from carrying current. Bottom: ribbon diagram of the four RAS proteins, which are 90% similar throughout their G domains that bind the guanine nucleotides, regulators and effectors (including switch regions SI, SII), but differ greatly at their C-terminal membrane targeting domains. The latter consist of a carboxyterminal CAAX tetrapeptide motif (pink boxes) with an invariant cysteine that is the site of farnesylation, and an upstream hypervariable region (yellow boxes) that include the “second signals” of one (NRAS) or two (HRAS, KRAS4A) palmitoylatable cysteines or clusters of positively charged (polybasic) residues (PBR), as well as “third signals” comprised of the surrounding residues. KRAS4B has a stretch of six contiguous lysines and no palmitoylatable cysteine, whereas KRAS4A has a hybrid motif of both a bipartite PBR and a palmitoylatable cysteine. Numbers refer to amino acid residues. Asterisks indicate sites of mutational hotspots at G12, G13 and Q61. Dots above the ribbon mark each 10 amino acid stretch. Brown bars in the ribbon mark sites of sequence variation. P, phosphorylation of KRAS4B at Serine181.

Figure 2

RAS trafficking pathway. Nascent RAS proteins leaving the polysome are rapidly modified by farnesyltransferase (FTase), which attaches a C15 farnesyl isoprenoid lipid to the cysteine of the CAAX motif. This provides them sufficient affinity for the endoplasmic reticulum (ER), where they are further modified by RAS-converting CAAX endopeptidase 1 (RCE1)-catalyzed proteolytic removal of the AAX residues, and by reversible isoprenylcysteine carboxylmethyltransferase (ICMT)-catalyzed carboxylmethylation of the now terminal farnesylated cysteine residue. By preventing the first and obligate step, FTase inhibitors (FTIs) prevent all three of these modifications. In the presence of FTIs, KRAS and NRAS are alternatively prenylated by geranylgeranyltransferase I (GGTase I), which attaches a C20 geranylgeranyl isoprenoid that allows the same subsequent processing steps. RAS trafficking to the inner leaflet of the plasma membrane (PM) requires a second membrane-targeting element that dictates the pathway it will take to the PM. NRAS and HRAS have one or two cysteine residues, respectively, that undergo reversible acylation by a Golgi-resident protein acyltransferase (PAT) to promote their trafficking to the PM. Rapid deacylation by an acyl protein thioesterase (APT1/2) frees them up to be re-acylated and trafficked back to the PM. The nonpalmitoylated pool of APT in the cytosol is the active form, and is in dynamic equilibrium with a palmitoylated pool on the Golgi. KRAS4B, which has no palmitoylatable cysteine but a stretch of 6 lysines (polybasic region) does not go to the Golgi but trafficks to the more directly to the PM, where it binds by virtue of its electrostatic charge. KRAS4A, which has a hybrid motif of a palmitoylated cysteine and a bifurcated polybasic region, undergoes an intermediate form of trafficking. Phosphodiesterase-6δ (PDE6δ) recognizes the farnesyl isoprenoid and solubilizes nonpalmitoylated RAS proteins from any compartment, thereby promoting their availability for restoration to the PM; deltarasin blocks this interaction. Not pictured: other chaperone proteins that guide the lipidated RAS proteins between and within membrane regions. Each enzyme depicted has been a target for drug discovery.