Targeting KRAS mutant cancers by preventing signaling transduction in the MAPK pathway

Highlights

• KRAS genes are the most commonly mutated oncogenes in cancer.

• Therapeutic strategies for targeting KRAS mutant cancers have proven to be difficult.

• Advances have been made when targeting the plasma membrane localization process.

• Farnesyltransferase and allosteric inhibitors have both advanced to human clinical trials.

Abstract

KRAS genes are the most commonly mutated oncogenes in cancer. Unfortunately, effective therapeutic strategies for targeting KRAS mutant cancers have proven to be difficult to obtain. A key reason for this setback is due to the lack of success direct KRAS mutant inhibitors have received. Researchers have turned their efforts away from targeting the KRAS nucleotide-binding site directly and towards targeting other areas of the MAPK signaling pathway to block KRAS function. Researchers found that inhibiting enzymes and protein-protein interactions involved in the MAPK signaling pathway inhibit the activation of KRAS mutant therefore can lead to a potential therapeutic for KRAS mutated cancers. Throughout the past two decades, various indirect inhibitors have been designed and tested. EGFR and MEK inhibitors have presented with less success; however, significant advances have been made when targeting the plasma membrane localization process and the allosteric site of KRAS mutant. Farnesyltransferase and allosteric inhibitors have both advanced to human clinical trials. This comprehensive review presents the most recent developments of direct and indirect KRAS mutant inhibitors. This review summarizes published data on the inhibitory and anti-cancer activity of compounds that target KRAS activation as well as highlights the most promising strategies for targeting KRAS mutant cancers.

Introduction

Cancer is one of the deadliest diseases of the 21st century. Globally, one in six individuals die of cancer, making it the second leading cause of death. In 2018, an estimated 9.6 million people died of cancer [1]. The most common cancers are lung (2.09 million cases), breast (2.09 million cases), and colorectal (1.80 million cases) cancer; however, this list is in no way exhaustive [1]. Cancer can develop in any part of the body and is simply defined by an abnormal and rapid growth of cells that permeate beyond regular cell limits. This abnormal growth of cells is often called a tumor. The vast number of cases and deaths due to cancer have motivated numerous studies and clinical trials in order to develop appropriate medications and procedures to combat this merciless disease. However, in order to treat and potentially cure cancer, its biological properties, mechanisms, and causes must be well understood. Genetic mutations that alter the proper expression and function of genes and proteins that are vital to cell growth, proliferation, and differentiation have been identified as the predominant cause of most cancers [2]. Mutations in the RAS gene family have most commonly been associated with the development of cancerous tumors [2]. The RAS gene family consists of three genes: KRAS, HRAS, and NRAS [2]. Mutations of the KRAS are prevalent among the top 3 deadliest types of cancers in the United States: pancreatic (95%), colorectal (45%), and lung (35%). Mutations in KRAS genes cause the hyperactivation of cell proliferation, which promotes the growth of tumors. The prevention of KRAS gene mutations would halt excessive cell proliferation, thus stopping the development of tumors. To prevent further KRAS gene mutation, the inhibition of KRAS mutant protein has been targeted as a potential therapeutic for cancer. Difficulty in finding a reliable KRAS mutant cancer therapy had emerged when targeting KRAS mutant protein directly. In this review, we aim to identify and discuss past and current efforts to target KRAS mutations by preventing KRAS function. Additionally, we aim to devise a guide for researchers when exploring strategies for finding a therapeutic for KRAS mutations.

RAS proteins are small GTP-binding proteins that play a major role in regulating cellular responses such as cell growth, proliferation, differentiation, and migration. RAS proteins are monomeric globular proteins comprised of 189 residues [3]. The secondary structure consists of a central beta-sheet composed of 6 beta-strands, 5 alpha helices, and a few loops. RAS proteins consist of two domains: a G-domain, which binds to guanosine, and a carboxyl terminal hypervariable region. The G-domain consists of a P-loop, Switch I, and Switch II region which are all in close proximity to the nucleotide-binding site and promotes further binding (Fig. 1) [4]. The Switch I and II regions are crucial in facilitating the activation of RAS protein specifically in the GTP/GDP cycle [5].

RAS proteins are members of a larger family of GTPase enzymes, which are essential in the extracellular signaling transduction of diverse cellular responses. RAS protein acts as a “molecular switch” that is activated when growth factors bind to extracellular receptors which induce nucleotide exchange from GDP to GTP [6]. RAS exists in two states: the active state which is bound to GTP and the inactive state which is bound to GDP. The activated Son of Sevenless protein (SOS), allows for the exchange of GDP to GTP on RAS, therefore promoting RAS to be in its active or in its “on” state [5]. The Switch I and II regions undergo a conformational change during the cycling between the active state and the inactive state [5]. This conformational change mediates the functionality of RAS protein acting as a “molecular switch”. The intrinsic hydrolysis of the GTP returns RAS to its inactive or in its “off” state (Fig. 2) [7]. The GTPase-activating proteins (GAPs) accelerate this hydrolysis process and controls the signaling pathway by slowing the production of the active-state of RAS [7].

The active RAS activates a series of serine/threonine-specific protein kinases, which in turn activate enzymatic substrates via phosphorylation. This creates an activated MAPK, thus activating transcription factors that bind to a specific DNA sequence, resulting in transcription [6]. This process is known as the mitogen-activated protein kinase (MAPK) pathway (Fig. 3). The RAS protein is crucial for this process to occur. Regulating this pathway is vital for the normal and healthy expression of the RAS gene.

The MAPK pathway, originally known as extracellular-signal-regulated kinase (ERK) pathway, plays a significant role in regulating gene expression in eukaryotic cells. This signaling pathway controls cellular processes such as cell growth, proliferation, differentiation, and migration [3]. The MAPK signaling pathway begins with the activation of a receptor tyrosine kinase (RTK) which is embedded in the plasma membrane. The RTK is activated by the binding of an extracellular ligand; most commonly with growth factors such as the epidermal growth factor (EGF) and transforming growth factor alpha (TGF-$\alpha$) [8]. The EGF binds to a receptor tyrosine kinase, specifically to the epidermal growth factor receptor (EGFR). This interaction leads to the dimerization of two EGFRs and in turn cross-phosphorylation [9].

Once the EGFR are phosphorylated, the receptor is bound by an adaptor molecule, specifically the growth factor receptor-bound protein 2 (Grb-2) [9]. The Grb-2 is made up of one SH2 domain and two SH3 domains. The SH2 domain binds to the tyrosine phosphorylated sequence on the EGFR and the SH3 domains bind to the SOS protein, a guanine nucleotide exchange factor (GEF) [9]. The SOS is fundamental in the activation of RAS protein. The SOS binds to RAS and opens up the nucleotide-binding site, allowing GDP to escape and GTP to enter in its place and bind [6]. The RAS proteins cycle between an active GTP-bound state and an inactive GDP-bound state. RAS proteins possess a slow intrinsic GTPase activity for the hydrolysis of GTP to GDP, a reaction enhanced by GAPs [6]. This regulates the production of activated RAS and slows the signal transduction pathway.

The activated RAS binds to and activates Rapidly Accelerated Fibrosarcoma (RAF), a serine/threonine-specific protein kinase. This interaction causes a cascade of serine/threonine-specific proteins to be activated. The RAF protein phosphorylates a MAPK kinase (MEK), which further phosphorylates MAPK. The phosphorylated MAPK then enters the nucleus, activating the transcription factors that bind to DNA sequences and initiating the transcription of genes in involved in cell proliferation, differentiation, migration, and survival [10].

The major RAS isoforms are encoded by three genes: KRAS, HRAS, and NRAS. These RAS genes are named after their place of discovery [3]. The KRAS and HRAS gene are oncogenes which were originally identified in Kristen rat sarcoma virus and Harvey rat sarcoma virus, respectively. The NRAS gene is an oncogene which was primarily found in human neuroblastoma cells [3]. The KRAS gene is localized on the short arm (p) of chromosome 12 at position 12.1. This gene consists of 6 exons and yields two alternate splice variants resulting in KRAS4A and KRAS4B isoforms. The KRAS4A isoform includes all the coding regions in the mRNA transcript whereas the KRAS4B excludes exon 6 [3]. The HRAS gene is localized on the short arm (p) of chromosome 11 at position 15.5. The HRAS gene also encodes two different isoforms: p19 HRAS and p21 HRAS. The mRNA structures of these alternative sequences differ due to the absence of IDX exon (intron-D-exon). The predominant isoform, p21 HRAS, excludes the IDX exon while the rare isoform, p19 HRAS, includes it. Since the p21 HRAS isoform is the most common isoform it is widely referred to in literature as simply the HRAS isoform [3]. Lastly, the NRAS gene is localized on chromosome 1 at position 13.2. The NRAS gene has only one isoform [3].

The three human RAS genes encode four small guanosine triphosphatases: KRAS4A, KRAS4B, HRAS, and NRAS. The primary structure of the KRAS4A, KRAS4B, HRAS, and NRAS proteins are immensely similar to one another. The first 172 residues of the four RAS proteins constitute the G-domain, which shares 92–98% sequence identity [11]. The G-domain is divided into two regions: an effector lobe (residues 1–86) which is identical among the RAS proteins and an allosteric lobe (residues 87–172) which has slight differences [11]. Next in the sequence is the carboxyl terminal hypervariable region (HVR) which differs tremendously among the RAS proteins. The HVR region consist of the final 17 residues. This region is the most divergent region of the RAS proteins. The HVR holds a terminal CAAX motif (cysteine, aliphatic, aliphatic, any amino acid) (Fig. 4). The CAAX box anchors the RAS proteins into the active site and directs post-translational modification [11].

All four RAS proteins undergo prenylation at the CAAX [6]. Protein prenylation is a post-translational lipid modification (PTM) process of adding either farnesyl or geranylgeranyl isoprenoids to the cysteine residue of the CAAX. The prenylation process of KRAS4A, KRAS4B, and NRAS can be modified by either farnesyltransferase or geranylgeranyltransferase-1 [12]. This allows a sense of flexibility given that one enzyme is inhibited. However, this is not true for the HRAS CAAX cysteine which is only modified by the farnesyltransferase [12]. Once the CAAX cysteine of the RAS protein undergoes prenylation, the –AAX is cleaved by RAS Converting CAAX Endopeptidase 1 (RCE1). This process is followed by the methylation of the alpha-carboxyl group on the following cysteine. The carboxyl methylation step is completed by isoprenylcysteine carboxymethyltransferase (ICMT). After these PTMs, the RAS protein becomes more hydrophobic allowing for the attachment of the protein to the plasma or nuclear membrane (Fig. 5) [13]. To ensure plasma membrane localization, there is also a ‘second attachment source’ to promote another hydrophobic anchor to the membrane. The ‘second attachment source’ is the palmitoylation of cysteine of the hypervariable region (165–185 residues) [14]. KRAS4B is the only RAS protein that does not contain a second cysteine in the HVR region as a second source of localization, therefore KRAS4B anchors to the plasma membrane through electrostatic interactions at the polylysine region of the HVR [14]. This is very crucial in the MAPK signaling pathway. The RAS protein must be attached to the cell membrane for the signaling pathway to occur. This is important when looking into potential targets to prevent signal transduction.

Although all four RAS proteins have similar sequence structure and function, the mutated RAS protein demonstrates different biological characteristics. It has been reported that the KRAS, HRAS, and NRAS genes are the most frequently mutated oncogenes in human cancer. The most recurrent sites of oncogenic mutation for RAS are found at the nucleic acid sequence that corresponds to residue glycine 12, glycine 13, and glutamine 61. Glycine 12 and 13 are found in the P-loop of RAS protein whereas glutamine 61 is found in Switch II region [3]. The majority of KRAS and NRAS mutations occur at the nucleic acid sequence that encodes for residue 12 and 61, respectively. The KRAS4A and KRAS4B isoforms are predominately mutated in solid malignant cancers such as pancreatic, colorectal, and lung cancer. NRAS mutations are mostly present in melanoma and myeloid leukemia. Furthermore, the most prominent mutation in KRAS and NRAS is G12D and Q61R, respectively [3] (Table 1). The HRAS mutation occurs in bladder cancer and are found at either residue 12, 13, or 61. The most prominent mutation in HRAS is G13R [3].

The mutation-induced substitution of glycine 12 or 13 by any amino acid other than proline disrupts RAS inactivation, thus disrupting the regulation of RAS-GTP active form. RAS inactivation is mediated by GAPs which accelerates the hydrolysis process of RAS-GTP active form back to RAS-GDP inactive form. The key residue of GAP-mediated catalysis is the “arginine finger”. The “arginine finger” of GAPs is adjacent to glycine 12 and 13 of the RAS protein [15]. The mutated glycine 12 or 13 generates steric interference with the “arginine finger” and therefore impairs the intrinsic and GAP-mediated catalysis causing the hydrolysis rate of RAS-GTP active form to decrease immensely. The hyperactivation of RAS-GTP active form causes an overexpression of the mutated gene thus causing an immense cell growth production and tumor formation. Mutated glutamine 61 also causes serious blockage of GAP-mediated hydrolysis. Glutamine 61 aligns the nucleophilic water molecule and stabilizes the transition state of the hydrolysis reaction [15]. Mutations at these three common sites decrease GAP-mediated GTPase activity leading to disruption of the MAPK signaling pathway and non-regulated gene expression.

Due to the immense cell growth and tumor formation set by RAS mutant proteins, much work has focused on targeting the inhibition of these mutant proteins as a therapeutic approach in human cancer. The KRAS protein is the main target of inhibition since it is the most frequently mutated isoform found in human cancer. It is reported that 83% of all RAS mutation are KRAS mutations. The KRAS mutations are prevalent among the top three deadliest cancer types in the United States: pancreatic, colorectal, and lung cancer. It is reported that 95% of pancreatic, 45% of colorectal, and 35% of lung tumors possess KRAS mutants [14]. Numerous strategies have been employed to explore the prevention of signaling transduction in order to block KRAS function in tumor formation.