Targeting RAS phosphorylation in cancer therapy: Mechanisms and modulators

Abstract

RAS, a member of the small GTPase family, functions as a binary switch by shifting between inactive GDP-loaded and active GTP-loaded state. RAS gain-of-function mutations are one of the leading causes in human oncogenesis, accounting for ∼19% of the global cancer burden. As a well-recognized target in malignancy, RAS has been intensively studied in the past decades. Despite the sustained efforts, many failures occurred in the earlier exploration and resulted in an 'undruggable' feature of RAS proteins. Phosphorylation at several residues has been recently determined as regulators for wild-type and mutated RAS proteins. Therefore, the development of RAS inhibitors directly targeting the RAS mutants or towards upstream regulatory kinases supplies a novel direction for tackling the anti-RAS difficulties. A better understanding of RAS phosphorylation can contribute to future therapeutic strategies. In this review, we comprehensively summarized the current advances in RAS phosphorylation and provided mechanistic insights into the signaling transduction of associated pathways. Importantly, the preclinical and clinical success in developing anti-RAS drugs targeting the upstream kinases and potential directions of harnessing allostery to target RAS phosphorylation sites were also discussed.

Keywords: ABL, Abelson; APC, adenomatous polyposis coli; Allostery; CK1, casein kinase 1; CML, chronic myeloid leukemia; ER, endoplasmic reticulum; GAPs, GTPase-activating proteins; GEFs, guanine nucleotide exchange-factors; GSK3, glycogen synthase kinase 3; HVR, hypervariable region; IP3R, inositol trisphosphate receptors; LRP6, lipoprotein-receptor-related protein 6; OMM, outer mitochondrial membrane; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; PPIs, protein−protein interactions; Phosphorylation; Protein kinases; RAS; RIN1, RAB-interacting protein 1; SHP2, SRC homology 2 domain containing phosphatase 2; SOS, Son of Sevenless; STK19, serine/threonine-protein kinase 19; TKIs, tyrosine kinase inhibitors; Undruggable.

Graphical abstract

Figure 1

Proposed model of RAS^pTyr4 ubiquitination by Rabex-5. Phosphorylation on RAS Tyr4 promotes ubiquitination of RAS-GDP and RAS-GTP. Tyr4 phosphorylation is potentially mediated by JAK2, SRC, and/or EGFR according to experimental observations.

Figure 2

Model of RAS Tyr32/Tyr64 phosphorylation and its activity. (A) Impact of phosphorylation on the RAS GTPase cycle. SRC phosphorylates Tyr32 and Tyr64 of K-RAS, reduces GEF- and GAP-mediated nucleotide exchange and hydrolysis, and causes the accumulation of phosphorylated RAS-GTP. K-RAS^pY32,pY64 has diminished affinity for RAF, thus shifting the RAS GTPase cycle to the ‘Dark State’. SHP2 dephosphorylates RAS and reactivate the RAS GTPase cycle. (B) Wild-type and mutant RAS activity shift model mediated by upstream signal, SRC, and SHP2. Therapeutic inhibition of SHP2 disrupts the dephosphorylation of both wild-type and oncogenic K-RAS, shifting the equilibrium of RAS GTPase towards the silence ‘Dark State’.

Figure 3

Crystal structure of RAS and its regulatory proteins SOS and GAP. (A) Front view of RAS‒SOS complex (PDB ID 1BKD). Switch I and Switch II of RAS are shown in pink and marine, respectively, while the αH helix of SOS is shown in cyan. Yellow dashed lines represent hydrogen-bond interactions. RAS Tyr32 forms hydrogen bonds with Asn944 of SOS, promoting the insertion of SOS helix αH into the RAS nucleotide-binding domain. (B) Back view of RAS‒SOS complex. RAS Tyr64 forms a hydrogen bond with SOS Gly931 of helix αH. (C) Proposed conformation of RAS Switch I and Switch II in intrinsic GTP hydrolysis (PDB ID 4G0N). Tyr32 binds to a bridging water molecule (depicted as red spheres), thus stabilizes GTP γ-phosphate and facilitates the attack by another water molecule. (D) Crystal structure of RAS‒GAP complex (PDB ID 1WQ1). RAS Tyr64 interacts with GAP Leu902, thereby facilitates the GAP-catalyzed GTP hydrolysis.

Figure 4

Small molecule SHP2 inhibitors. (A) II-B08, orthosteric inhibitor; (B) 11a-1, orthosteric inhibitor; (C) GS493, orthosteric inhibitor; (D) SHP099, allosteric inhibitor.

Figure 5

Small molecule inhibitors targeting STK19 kinase. (A) ZT-12-037-01; (B) chelidonine.

Figure 6

(A) Model of H-RAS/RIN1/ABL ternary complex. The C-terminal of RIN1 binds to H-RAS while its N-terminal binds to ABL. Activated ABL phosphorylates H-RAS on Tyr137. (B) Crystal structure of wild-type H-RAS in complex with GTP analog GppNHp (PDB ID 3K8Y). Switch II, helix 3, and helix 4 are shown in green. Gln61 is stabilized in a precatalytic conformation (R state) by a water-mediated hydrogen-bond network. Waters and residues that participate in the network are depicted as red spheres and sticks, respectively. Yellow dash lines represent hydrogen-bond interactions. (C) Crystal structure of H-RAS^Y137E (cyan) (PDB ID 4XVQ) superimposed on H-RAS^WT (green). Mutated residue Glu137 forms hydrogen bonds with Arg97 and Lys101, dragging Arg97 deeper into the hydrophobic core of H-RAS. (D) Crystal structure of H-RAS^Y137F (magenta, PDB ID 4XVR) superimposed on H-RAS^WT (green). The orientations of R97 and F137 side-chains showed no significant differences with wild-type H-RAS.

Figure 7

Clinical approved small molecule BCR-ABL inhibitors. (A) Imatinib; (B) nilotinib; (C) dasatinib; (D) bosutinib; (E) ponatinib.

Figure 8

Model of phosphorylation-induced H-RAS degradation. GSK3β mediates H-RAS to phosphorylate Thr144 and Thr147, followed by polyubiquitylation via β-TrCP and degradation via 26S proteasome. GSK3β-mediated phosphorylation is inhibited by WNT3 and is facilitated by Axin and APC.

Figure 9

WNT/β-catenin pathway inhibitors. (A) PORCN inhibitor LGK974; (B) PORCN inhibitor ETC-159; (C) Tankyrase inhibitor NVP-TNKS656.

Figure 10

Model of phosphorylation-induced K-RAS4B redistribution and cell apoptosis. Protein kinase C (PKC) phosphorylates K-RAS4B on Ser181 of hypervariable region (HVR) and induces K-RAS4B to translocate towards ER, Golgi, and OMM. K-RAS4B^pSer181 attenuates BCL-X_L/IP3R-mediated calcium transfer from ER to mitochondria, thus promoting cell apoptosis.

Figure 11

Small molecules targeting PKC activity. (A) Bryostatin-1; (B) Edelfosine; (C) Bisindolylmaleimide (BIM); (D) Gö6983.