Progress on Ras/MAPK Signaling Research and Targeting in Blood and Solid Cancers

Abstract

The mitogen-activated protein kinase (MAPK) pathway, consisting of the Ras-Raf-MEK-ERK signaling cascade, regulates genes that control cellular development, differentiation, proliferation, and apoptosis. Within the cascade, multiple isoforms of Ras and Raf each display differences in functionality, efficiency, and, critically, oncogenic potential. According to the NCI, over 30% of all human cancers are driven by Ras genes. This dysfunctional signaling is implicated in a wide variety of leukemias and solid tumors, both with and without viral etiology. Due to the strong evidence of Ras-Raf involvement in tumorigenesis, many have attempted to target the cascade to treat these malignancies. Decades of unsuccessful experimentation had deemed Ras undruggable, but recently, the approval of Sotorasib as the first ever KRas inhibitor represents a monumental breakthrough. This advancement is not without novel challenges. As a G12C mutant-specific drug, it also represents the issue of drug target specificity within Ras pathway; not only do many drugs only affect single mutational profiles, with few pan-inhibitor exceptions, tumor genetic heterogeneity may give rise to drug-resistant profiles. Furthermore, significant challenges in targeting downstream Raf, especially the BRaf isoform, lie in the paradoxical activation of wild-type BRaf by BRaf mutant inhibitors. This literature review will delineate the mechanisms of Ras signaling in the MAPK pathway and its possible oncogenic mutations, illustrate how specific mutations affect the pathogenesis of specific cancers, and compare available and in-development treatments targeting the Ras pathway.

Keywords: ATLL; ERK; MEK; Ras signaling; leukemia; solid tumors; viral oncogenesis.

Figure 1

The MAPK cascade. Once a ligand binds the tyrosine kinase receptor, it self-phosphorylates [18]. This creates binding sites for Shc and Shp2. GRB2 can associate with either and then recruit SOS [19,20]. SOS is a guanine exchange factor for Ras and induces the exchange of GDP for GTP [21]. Now active Ras will dimerize and bind Raf [21]. After activating Raf, GTPase activating proteins (GAP) will hydrolyze the GTP to GDP to return Ras to its resting inactive state [22]. The active Raf dimers will recruit MEK [23], which then activates ERK [3]. ERK interacts with Importin 7 at the nuclear envelope to facilitate its entry through the nuclear pore complex into the nucleus [24,25]. Once inside, it phosphorylates multiple transcription factors to alter gene expression in the cell and induce proliferation and survival [26].

Figure 2

Post translational farnesylation of Ras protein. The first modification is prenylation, preferentially performed by farnesyl transferase (FTase) [44]. This is initiated after recognition of the CaaX box on Ras’s C-terminus by FTase. Alternatively, the KRas-4B and NRas isoforms can be acted on by geranylgeranyl transferase 1 (GGTase1) if FTase is inhibited [43,45]. The farnesyl and geranylgeranyl moieties add enough hydrophobicity to enable Ras insertion into the endoplasmic reticulum membrane. Ras converting enzyme (Rce1) performs a final cleavage of the CaaX residues before isoprenylcysteine carboxyl methyltransferase (Icmt) adds a carboxymethyl group [44]. The final processing and transfer to the plasma membrane is isoform specific. Due to the farnesyl tail and a five amino acid sequence motif (Lys- Ser- Lys-Thr-Lys) in the C-terminus region, KRas-4B is directly chaperoned to the membrane by phosphodiesterase delta (PDEδ) [43,46,47]. Lacking the necessary motif, all other isoforms enter the Golgi apparatus for reversible palmitoylation by palmitoyl transferase. From the Golgi apparatus, HRas and NRas are trafficked to the plasma membrane on motile vesicles [48]. KRas-4A is trafficked by a poorly understood Golgi- independent route depending on mitochondrial function and class C vacuolar protein sorting (vps) proteins [49]. Afterwards, all isoforms associate with the membrane through their respective two-point anchors: the farnesyl modification and polybasic region of six lysines for KRas4b and the palmitoyl and farnesyl modifications for the other isoforms [50].

Figure 3

The roles of src homology region 2 domain-containing phosphatase 2 (Shp2) in the Ras-Raf-MEK-ERK pathway. Shp2 is a GRB2 scaffolding protein that anchors it to tyrosine kinase receptors [56]. Additionally, Shp2 dephosphorylates Sprouty family proteins to release sequestered GRB2 molecules [57,58]. It also dephosphorylates Ras docking sites of RASA, a Ras GTPase activating protein (Ras-GAP) that accelerates the hydrolysis of Ras bound GTP [59,60]. This allows more active Ras-GTP molecules to accumulate instead of being converted to inactive Ras-GDP. After being recruited by Gab1, Shp2 can also dephosphorylate the binding sites of Paxillin and the Csk binding protein/phosphoprotein associated with glycosphingolipid-enriched microdomains (Cbp/PAG). This prevents the docking of C-terminal Src kinase (Csk) on Src family kinases (SFKs) and inhibits its activity by phosphorylating Tyr527 [61,62,63,64]. When active, SFKs initiate a signaling pathway through phospholipase C γ (PLCγ), diacylglycerol (DAG) and calcium. This results in the recruitment of the Ras guanine nucleotide exchange factor RasGRP1, directing it to the Golgi to activate intracellular Ras molecules [65,66].

Figure 4

G protein-coupled receptor subunits Gα_q and Gβγ_i interaction with the Ras-Raf pathway. G-proteins are heterotrimeric guanine nucleotide binding proteins with α-, β-, and γ-subunits. When a ligand binds to the extracellular portion of the receptor, it confers a guanine nucleotide exchange factor confirmation that induces the α-subunit to exchange its bound GDP to GTP. This causes the α-subunit to disassociate from the receptor and βγ- subunit. Both the α and βγ subunits effect changes in the cell, before the α subunit hydrolyzes the GTP and returns the receptor complex to its inactive state [94,95,107,108]. Both α_q and βγ_i activate phospholipase C-β (PLCβ) to create the second messengers of diacylglycerol (DAG) and inositol-1,4,5-triphosphate (IP3) through the hydrolyzation of phosphatidylinositol 4,5-bisphosphate (PIP2) [109]. DAG activates PKC, which directly phosphorylates and activates Ras proteins [110,111]. IP3 stimulates the calmodulin pathway and Pyk2 kinase by way of inducing calcium release from the endoplasmic reticulum [112]. The resulting phosphorylation provides the base for Shc anchoring and recruitment of Ras’ guanine exchange factor complex [113]. Both DAG and IP3 play a role in allosterically controlling CalDAG-GEF1, a guanine exchange factor for Rap1 [94,114]. Once Rap1 has exchanged its bound GDP for GTP, it can activate BRaf in the place of Ras [115]. Furthermore, βγ_i activates phosphoinositide 3-kinase (PI3K), which augments cell signaling from tyrosine kinase receptors to increase Dynamin II [116], an additional anchor for Shc and Ras’ GEF complex [117,118,119].

Figure 5

G-protein-coupled receptor subunits Gαs and Gαi interaction with the Ras-Raf pathway. When activated by a ligand, the GPCR is induced to exchange its bound GDP for GTP, freeing the α subunit to act on the cell. α_s acts on adenylyl cyclase (AC) to increase the production of the second messenger cAMP from ATP [120]. In turn, cAMP activates Epac-1 [121] and protein kinase A [120]. Epac-1 and C3G, a downstream molecule from PKA, are guanine-nucleotide exchange factors for Rap-1 and induce the exchange of GDP to GTP to activate it [121,122]. Both activate Rap-1, which can modulate the activity of BRaf [115]. Protein Kinase A has other mechanisms of action as well. It can directly prevent the activation of CRaf through phosphorylation. Both activate Rap-1, which can modulate the activity of BRaf [115]. Protein kinase A has other mechanisms of action as well. It can directly prevent the activation of CRaf through phosphorylation [123]. It also activates a Ras GEF, RasGRF1, to start the MAPK cascade in certain cells [124]. In contrast, αi inhibits adenylyl cyclase and produces opposite effects. It also activates a Ras GEF, RasGRF1, to start the MAPK cascade in certain cells [124]. In contrast, αi inhibits adenylyl cyclase and produces opposite effects.