Targeting the RAS/RAF/MAPK pathway for cancer therapy: from mechanism to clinical studies

Abstract

Metastatic dissemination of solid tumors, a leading cause of cancer-related mortality, underscores the urgent need for enhanced insights into the molecular and cellular mechanisms underlying metastasis, chemoresistance, and the mechanistic backgrounds of individuals whose cancers are prone to migration. The most prevalent signaling cascade governed by multi-kinase inhibitors is the mitogen-activated protein kinase (MAPK) pathway, encompassing the RAS-RAF-MAPK kinase (MEK)-extracellular signal-related kinase (ERK) pathway. RAF kinase is a primary mediator of the MAPK pathway, responsible for the sequential activation of downstream targets, such as MEK and the transcription factor ERK, which control numerous cellular and physiological processes, including organism development, cell cycle control, cell proliferation and differentiation, cell survival, and death. Defects in this signaling cascade are associated with diseases such as cancer. RAF inhibitors (RAFi) combined with MEK blockers represent an FDA-approved therapeutic strategy for numerous RAF-mutant cancers, including melanoma, non-small cell lung carcinoma, and thyroid cancer. However, the development of therapy resistance by cancer cells remains an important barrier. Autophagy, an intracellular lysosome-dependent catabolic recycling process, plays a critical role in the development of RAFi resistance in cancer. Thus, targeting RAF and autophagy could be novel treatment strategies for RAF-mutant cancers. In this review, we delve deeper into the mechanistic insights surrounding RAF kinase signaling in tumorigenesis and RAFi-resistance. Furthermore, we explore and discuss the ongoing development of next-generation RAF inhibitors with enhanced therapeutic profiles. Additionally, this review sheds light on the functional interplay between RAF-targeted therapies and autophagy in cancer.

Fig. 1

Historical events of the discovery and development of the RAS/RAF/MAPK pathway in health and diseases. The journey of the MAPK signal cascade commenced in 1960s with the groundbreaking discovery of the viral RAS gene. Subsequently, in 1992, the identification of RAF as both an upstream kinase activator of MEK and a RAS effector marked significant milestones. These pivotal findings culminated in the comprehensive definition of the entire MAPK signaling pathway. Over time, the MAPK signal emerged as a critical components in the development of therapeutic strategies for combating cancer. Each of these major milestones in the RAS/RAF/MAPK discovery is represented within its respective box. This figure was created with BioRender.com

Fig. 2

Mitogen-activated protein kinase (MAPK) cascades and their physiological functions. All cascades consist of three-layered core-signaling pathways in which each kinase is consecutively activated, and MAPK components are highly conserved. The first layer consists of MAPK kinase kinases (MAPKKKs or MEKKs), which are activated by stimuli and phosphorylate and activate MAPK kinases (MAPKKs or MEKs). MAPKKs are dual-specificity kinases that can phosphorylate threonine or tyrosine residues to activate the terminal serine/threonine MAPK, leading to the activation of multiple cytoplasmic and nuclear proteins involved in various biological functions. This figure was created with BioRender.com

Fig. 3

Structure and activation mechanism of RAS and RAF kinase in the RAS/RAF/MAPK signal cascade. a RAS upstream components. Various mitogens including TGF-α, EGF, VEGF, and PDGF-β bind to their own receptors and lead to RAS activation and subsequent stimulation of the MAPK pathway. b GTPase cycle. GEFs stimulates the transition of inactive RAS-GDP to active RAS-GTP, enabling to transmit the proliferation and differentiation signals through its downstream effectors. Subsequently, the active RAS can be quickly deactivated by the action of GAPs. c RAS domain. The effector lobe (1–86 a.a.), allosteric lobe (87–165 a.a.), and HVR (167–188/189 a.a.) are all parts of the structure of RAS proteins. The effector lobe contains switches I (30–40 a.a.) and II (60–76 a.a.) are involved in effector binding and GEF or GAP binding, respectively. d RAF domain structure. RAF proteins consist of three conserved regions (CR1, CR2, and CR3) or two functional domains: an N-terminal regulatory domain and a C-terminal catalytic domain. e RAF dimerization. In the absence of cellular stimulation, RAF tends to exist in the monomeric, autoinhibited state. Upon stimulation by RAS-GTP, the autoinhibitory domain is released, freeing the inactive kinase domain to form homo- or heterodimers (with kinase suppressor of RAS [KSR]). Dimerization triggers mutual phosphorylation of the dimer components, fully activating the kinase. Phosphorylation and activation of target proteins, such as MEK1 and MEK2, propagates the MAPK cascade, leading to ERK1/ERK2 activation. This figure was created with BioRender.com

Fig. 4

RAF signaling regulation by accessory proteins. a Accessory proteins consist of anchoring proteins, docking proteins, adapter proteins, and scaffold proteins. Anchoring proteins bind to membrane kinases and other effectors, whereas adapter proteins link receptor kinases with guanine exchange factors (GEFs). Docking proteins connect active receptors with multiple effectors. Scaffold proteins offer a signaling platform for the spatial regulation of the mitogen-activated protein kinase (MAPK) pathway. b CRAF kinase inhibitor protein (RKIP) is a tumor suppressor. RKIP, an intrinsic RAF kinase inhibitor, is associated with many malignant features, including metastasis and chemotherapy resistance, through the regulation of oncogenic mediators and signaling axes, such as NF-kB, YY1, MAPK28, STAT3, NRF2, and AKT29. Arrows and bars indicate stimulating and inhibiting signals, respectively. This figure was created with BioRender.com

Fig. 5

Kinase-independent regulation of RAF-interacting signaling. Three RAF effector proteins, Bcl-2 agonist of cell death (BAD), apoptosis signal-regulating kinase 1 (ASK1), and mammalian Ste20-like kinase 2 (MST2), are kinase-independent negative regulators of apoptosis. RAF can enhance cell cycle progression via the extracellular signal-related kinase (ERK) pathway, and RAF can regulate the cell cycle in a kinase-independent manner. RAF interacts with polo-like kinase 1 (PLK1) and Aurora kinase A (Aurora-A). During cell migration, RAF also functions as a spatial regulator of Rho-associated kinase (ROK)-α, a downstream effector of RHO, in a kinase-independent manner by inhibiting ROK-α activity. Several additional RAF substrates, such as NF-κB, Vimentin, Snail, and Keratin, are associated with cytoskeleton organization. This figure was created with BioRender.com

Fig. 6

The crosstalk of RAS/RAF/MAPK with other pathways. a Crosstalk between the RAS/RAF/MAPK, PI3K/mTOR or MAT2/Hippo signaling pathways. The RAS/RAF/MAPK signal collaborates within its own cascade and interfaces with the PI3K/mTOR pathway, where its influence is MAPK-dependent. Conversely, the MST-2/Hippo pathway operates independently of MAPK activity but relies on the presence of RAF for its functionality. b Functional interactions between autophagy and mitogen-activated protein kinase (MAPK) signaling during the epithelial-to-mesenchymal transition (EMT). The MAPK pathway can be activated by canonical receptor tyrosine kinases (RTKs) and through Smad-independent activation by transforming growth factor-beta (TGF-β). Both signals activate a typical RAS–RAF–MAPK cascade, stimulating the EMT process. During the metastasis process, cancer cells migrate from a primary site to a secondary site facing many stressors, and therefore metabolic and cellular alterations are necessary to overcome these stressors. c The role of autophagy in tumorigenesis. During early tumorigenesis, autophagy acts as a tumor suppressor. However, autophagy drives tumor growth, progression, and metastasis by enhancing migration, invasion, EMT, and metabolic tolerance during advanced stages of cancer, allowing cancer cells to evade RAFi therapy. Arrows and bars indicate stimulating and inhibiting signals, respectively. This figure was created with BioRender.com

Fig. 7

RAS/RAF/MAPK-targeted therapy in BRAF-mutated malignancies. a Functional classifications of BRAF mutations. In Class I, BRAF mutants (e.g., V600E) transmit signals via a monomeric form independent of RAS activation, leading to increased extracellular signal-related kinase (ERK) activation. In Class II, BRAF mutants (e.g., K601E) are RAS-independent, forming mutant–mutant BRAF dimers. RAF inhibitors (RAFi), such as Vemurafenib and Dabrafenib, block both Class I and II RAF kinases. In Class III, mutant BRAF (e.g., D287H, V459L) exhibits increased RAS binding and heterodimer formation between mutant BRAF and wild-type CRAF. MEK inhibitors (MEKi), such as Trametinib or Cobimetinib, show an additive effect when combined with RAFi for cancer treatment. b RAS/RAF/MAPK-targeted therapies. Specific Inhibitors targeting the RAS/RAF/MAPK pathway represent as each group of action: EGFR agonists (EGFRi), RAS inhibitors (RASi), RAF inhibitors (RAFi), MEK inhibitors (MEKi), and ERK inhibitors (ERKi). Arrows and bars indicate stimulating and inhibiting signals, respectively. This figure was created with BioRender.com

Fig. 8

Mechanistic basis of RAF inhibitor (RAFi) resistance. a RAFi resistance in RAF-mutant cancer. BRAF^V600E–mutant cancers are characterized by hyperactive extracellular signal-related kinase (ERK) signaling during early disease, resulting in negative feedback inhibition of upstream signaling. RAS-GTP expression is minimal, but monomeric BRAF^V600E responds to signals. Under these conditions, RAFi inhibits ERK signaling. Subsequent treatment with Vemurafenib suppresses ERK-dependent negative feedback, restoring receptor tyrosine kinase (RTK) signaling. Despite the presence of RAFi, RTK signal restoration elevates RAS-GTP levels, drives the formation of RAFi-insensitive RAF dimers, and reactivates ERK signaling. Over time, negative feedback pathways are partially restored, and a new steady-state condition featuring reactivated ERK signaling develops. b RAFi resistance mechanisms. RAFi resistance promotes significant RAF dimerization through growth factors or RTK activation, NRAS mutation (NRAS Q61), NF1 loss, expression of RAF splice variants (p61), or overexpression of BRAF or CRAF. Reactivation of ERK signaling and RAFi resistance can also develop in a RAF dimerization–independent manner involving MEK mutations or RAF bypass activation by COT (an ERK kinase kinase). This figure was created with BioRender.com

Fig. 9

The possible therapeutic strategies for overcome resistance to RAS/RAF/MAPK inhibitors. a Alternative approaches to overcome MAPK inhibitor (RAFi) resistance. b Targeting autophagy. In cancer cells, autophagy-related genes are functionally and physically associated with mitogen-activated protein kinase (MAPK)-targeted therapy and cancer resistance induced by MAPK signaling inhibitors (e.g., RAFi, MEKi). The regulatory mechanisms involved in autophagy induction and the mediators that regulate autophagy are described in each rectangle. Arrows and bars indicate stimulating and inhibiting signals, respectively. This figure was created with BioRender.com