Activation Of Raf-1 Research Articles

Phloretin Alleviates Arsenic Trioxide-Induced Apoptosis of H9c2 Cardiomyoblasts via Downregulation in Ca2+/Calcineurin/NFATc Pathway and Inflammatory Cytokine Release.

Arsenic trioxide (ATO) is among the first-line chemotherapeutic drugs for treating acute promyelocytic leukemia patients, but its clinical use is hampered due to cardiotoxicity. The present investigation unveils the mechanism underlying ATO-induced oxidative stress that promotes calcineurin (a ubiquitous Ca2+/calmodulin-dependent serine/threonine phosphatase expressed only during sustained Ca2+ elevation) expression, inflammatory cytokine release and apoptosis in H9c2 cardiomyoblasts, and its possible modulation with phloretin (PHL, an antioxidant polyphenol present in apple peel). ATO caused Ca2+ overload resulting in elevated expression of calcineurin and its downstream transcriptional effector NFATc causing the release of cytokines such as IL-2, IL-6, MCP-1, IFN-γ, and TNF-α in H9c2 cardiomyoblast. There was a visible increase in the nuclear fraction of NF-κB and ROS-mediated apoptotic cell death. The expression levels of cardiac-specific genes (troponin, desmin, and caveolin-3) and genes of the apoptotic signaling pathway (BCL-2, BAX, IGF1, AKT, ERK1, -2, RAF1, and JNK) in response to ATO and PHL were studied. The putative binding mode and the potential ligand-target interactions of PHL with calcineurin using docking software (Autodock and iGEMDOCKv2) showed the high binding affinity of PHL to calcineurin. PHL co-treatment significantly reduced Ca2+ influx and normalized the expression of calcineurin, NFATc, NF-κB, and other cytokines. PHL co-treatment resulted in activation of BCL-2, IGF1, AKT, RAF1, ERK1, and ERK2 and inhibition of BAX and JNK. Overall, these results revealed that PHL has a protective effect against ATO-induced apoptosis and we propose calcineurin as a druggable target for the interaction of PHL in ATO cardiotoxicity in H9c2 cells.

The next-generation pan-RAF inhibitor, KIN-2787, is active in class II and class III BRAF mutant models.

3116 Background: Oncogenic BRAF gene alterations, leading to aberrantly activated BRAF monomers (Class I mutations) or dimers (Class II and Class III mutations), are observed in approximately 6% of all human cancers. First-generation BRAF inhibitors targeting Class I BRAF mutants, including dabrafenib, encorafenib, and vemurafenib, provide significant clinical benefit to patients with BRAF V600 mutation-driven melanoma and select solid tumors as monotherapies or in combination with other targeted therapies. The currently approved BRAF inhibitors have not, however, proven to be effective in patients with Class II or III BRAF alterations which account for a large proportion (34%) of BRAF mutations. KIN-2787 is an orally available, potent and selective small molecule pan-RAF inhibitor specifically designed to inhibit Class II and III BRAF dimers, in addition to Class I mutants. Methods: The efficacy and tolerability of the pan-RAF inhibitor, KIN-2787, was evaluated in vitro and in vivo in Class I, II, and III BRAF mutation-driven human cancer models. Results: In biochemical assays, KIN-2787 showed low nanomolar to picomolar potency against RAF1, BRAF, and ARAF (IC50 0.06-3.46 nM) with minimal activity towards non-RAF kinases. In cell-based assays, KIN-2787 inhibited RAF activity, as measured by inhibition of downstream ERK phosphorylation (pERK), across multiple BRAF mutant cancer cell lines. Class II and III BRAF mutant cell lines were the most responsive when treated with KIN-2787 (IC50 < 50 nM); 19- and 7-fold more sensitive compared to cells harboring wild-type BRAF, respectively. Dose-dependent inhibition of A-375 (Class I), BxPC-3 (Class II), and WM3629 (Class III) BRAF mutant human xenograft tumor growth was attained with daily KIN-2787 treatment and was well-tolerated. A trend towards greater tumor responses was observed with twice daily (BID) compared to once daily (QD) dosing of KIN-2787; however, the two dosing regimens led to similar tumor growth inhibition (TGI) and regressions (mean TGI up to 101-118%; p ≤0.0001) at equivalent total daily doses. Furthermore, KIN-2787 led to a significant in vivo pharmacodynamic response using either regimen, however, prolonged target coverage, as measured by pERK, was achieved with BID dosing. The impact of KIN-2787 treatment on additional biomarkers, including transcriptional changes and MAPK pathway modulation in cell-based models and patient-derived samples, will be presented at the meeting. Conclusions: KIN-2787 is a next-generation pan-RAF inhibitor with pronounced in vitro and in vivo activity against human cancers driven by Class II and III BRAF mutations. A phase 1 dose escalation and expansion clinical trial evaluating the safety and efficacy of KIN-2787 monotherapy in patients with advanced or metastatic solid tumors harboring BRAF gene alterations, including Class II and III mutations, is expected to initiate in 2021.

Parallel Rap1>RalGEF>Ral and Ras signals sculpt the C. elegans nervous system

Ras is the most commonly mutated oncogene in humans and uses three oncogenic effectors: Raf, PI3K, and RalGEF activation of Ral. Understanding the importance of RalGEF>Ral signaling in cancer is hampered by the paucity of knowledge about their function in animal development, particularly in cell movements. We found that mutations that disrupt function of RalGEF or Ral enhance migration phenotypes of mutants for genes with established roles in cell migration. We used as a model the migration of the canal associated neurons (CANs), and validated our results in HSN cell migration, neurite guidance, and general animal locomotion. These functions of RalGEF and Ral are specific to their control of Ral signaling output rather than other published functions of these proteins. In this capacity Ral functions cell autonomously as a permissive developmental signal. In contrast, we observed Ras, the canonical activator of RalGEF>Ral signaling in cancer, to function as an instructive signal. Furthermore, we unexpectedly identified a function for the close Ras relative, Rap1, consistent with activation of RalGEF>Ral. These studies define functions of RalGEF>Ral, Rap1 and Ras signaling in morphogenetic processes that fashion the nervous system. We have also defined a model for studying how small GTPases partner with downstream effectors. Taken together, this analysis defines novel molecules and relationships in signaling networks that control cell movements during development of the nervous system.

Interactome Profile of Visceral Adipose Tissue in Obesity Links Key Genes to Cancer Pathogenesis

Obesity increases the risk of the development of several malignancies. The visceral adipose tissue (VAT) depot is one of the pivotal contributors behind the obesity-related pathogenetic mechanisms. In this study, we analyzed the differential gene expression profile in the VAT of obese children using two Gene Expression Omnibus datasets. GSE29718 and GSE9624 were sorted and 68 common differentially expressed genes (DEG) with fold change 1.5 upregulation or downregulation (cutoff |logFC|≥0.58496) were obtained. Gene ontology and functional enrichment and protein-protein interaction (PPI) network for the DEG were analyzed in Search Tool for the Retrieval of Interacting Genes (STRING), which revealed 37 biological processes, 3 cellular components, and 1 molecular function to be significantly associated. Reactome pathway analysis showed the DEG to be involved in- one carbon pool by folate, glycine degradation, transcriptional regulation by TP53, ERK inactivation, G1/S-specific transcription, Fanconi anemia pathway, beta-catenin phosphorylation cascade, RAF activation, and negative regulation of the MAPK pathway. The PPI network was set with a minimum interaction score of 0.400 and a maximum of 10 interactions, and it was significantly enriched (p-value 0.047) with 66 nodes and 46 edges. Target prediction was performed using miRNet. Several miRNA, including hsa-miR-1-3p, hsa-let-7b-5p, hsa-miR-16-5p, hsa-miR-27a-3p and hsa-miR-34a-5p were part of the mRNA-miRNA interaction network. Using the CytoHubba plugin in Cytoscape, the top 10 hub genes from the PPI network were discovered. Thymidine phosphorylase (TYMP) and dihydrofolate reductase (DHFR), essential components of nucleic acid metabolism, have been shown to be involved in angiogenesis and endothelial cell growth, and correlated to p53 mutations, respectively. Protein phosphatase 2, regulatory subunit A & regulatory subunit B (PPP2R1A and PPP2R1B) mutations are involved in ovarian, endometrial, lung and colorectal cancers. HLA-DQA1 mutation is involved cervical cancer, and it is involved in increased immune sensitivity and liver damage in breast cancer patients. The RAB7Ab and RAB7-interacting lysosomal protein (RILP) are regulators of endo-lysosomal trafficking and suppresses breast cancer cell invasion. To conclude, this study identifies several genes and their regulatory pathways in VAT which may contribute to the increased risk of cancer pathogenesis in obese individuals.

A Growth Inhibitory PKCα‐ERK Signaling Pathway In the Intestine/Colon

Background Increasing evidence points to growth inhibitory and tumor suppressive roles of theprotein kinase C (PKC) family of serine/threonine kinases. The conventional PKC isozyme, PKCα, has anti-proliferative and antitumor effects in the epithelium of the intestine and colon. Activation of PKCα in intestinal cells induces a program of cell cycle withdrawal that involves downregulation of the pro-proliferative proteins cyclin D1 and Id1 and upregulation of the cyclin-dependent kinase inhibitor p21Cip1. These growth inhibitory effects of PKCα are dependent on activation of the MEK-ERK pathway, a signaling axis with established pro-proliferative effects in normal intestinal cells and in colon cancer. The objective of this study is to define the novel PKCα-induced growth suppressive MEK-ERK signaling cascade identified in intestinal epithelial cells. Hypothesis We hypothesize that PKCα modulates the activity of unique signaling intermediates to activate growth inhibitory ERK signaling. We will establish the point at which PKCα intersects the RAS-RAF-MEK-ERK pathway to induce growth arrest, identify the factors that link PKCα to the ERK pathway, and define effects that determine antiproliferative vs proliferative ERK signaling in intestinal cells. Results We have determined that PKCα activates RAS, RAF, MEK, and ERK in intestinal epithelial cells. The requirement for each of these pathway components in PKCα-mediated modulation of cyclin D1, Id1, and p21Cip1 and cell cycle withdrawal was confirmed using genetic approaches and inhibition of RAS, RAF, MEK, and ERK activity with the pharmacological agents Salirasib, LY3009120, PD0325901, and SCH772984, respectively. PKCα activation was shown to induce dimerization/activation of all three members of the RAF family (ARAF, BRAF and CRAF) and combinatorial knockdown indicated redundancy between these proteins. siRNA-mediated knockdown excluded a role for the RAF-MEK-ERK scaffolding protein KSR1. Analysis of the roles of Ras guanine nucleotide exchange factors identified at least two growth inhibitory pathways downstream of PKCα. Knockdown of the RasGRP isoform, RasGRP3, but not RasGRP1 or 2, inhibited the ability of PKCα activation to upregulate p21Cip1. However, RasGRP3 knockdown had no effect on PKCα-induced downregulation of cyclin D1 or Id1. Consistent with these findings, knockdown of RasGRP3 only partially inhibited PKCα-induced growth arrest. Based on evidence that SOS1 phosphorylation status is altered by PKCα signaling, we are currently exploring the potential involvement of SOS1 in the growth-inhibitory effects of PKCα. Conclusion PKCα activates RAS upstream of RAF, MEK and ERK to induce cell cycle arrest in intestinal cells. Activation of growth inhibitory ERK signaling by PKCα involves at least two mechanisms, one of which is RasGRP3-dependent and leads to p21Cip1 induction. Ongoing studies using siRNA knockdown strategies are exploring the role of Ras-GEFs such as RasGRP3, SOS1 and SOS2 in mediating PKCα-induced activation of RAS, to identify distinctive effects related to growth inhibitory versus growth promoting ERK signaling.

Raf promotes dimerization of the Ras G‐domain with increased allosteric connections

Dimerization is a critical regulatory step throughout the MAPK signaling pathway. While dimerization of the Raf kinase domain is widely accepted and occurs even in the absence of Ras, Ras dimerization has been contested for several decades, but has been recently been shown to be essential for Raf activation. Our work shows that the Ras binding domain of Raf (Raf-RBD) promotes robust Ras dimerization on supported lipid bilayers and, to a lesser extent, in solution as confirmed via size-exclusion chromatography coupled to small-angle x-ray scattering. Community network analyses of molecular dynamics simulations of the dimer of the Ras/Raf-RBD complex reveal extensive allosteric connections unifying Raf-RBD into a single allosteric network and bridging the two Raf-RBD D113 residues, located within the predicted galectin scaffold protein binding site and 85 Å apart, at opposite ends of the complex. Our findings suggest that Ras/Raf-RBD binding and dimerization occur simultaneously at the membrane to yield a high-affinity signaling complex. We propose a higher order macromolecular assembly comprising Ras/Raf/galectin complexes for tightly regulated and synchronized signaling through the MAPK pathway.

Raf promotes dimerization of the Ras G-domain with increased allosteric connections

Ras dimerization is critical for Raf activation. Here we show that the Ras binding domain of Raf (Raf-RBD) induces robust Ras dimerization at low surface densities on supported lipid bilayers and, to a lesser extent, in solution as observed by size exclusion chromatography and confirmed by SAXS. Community network analysis based on molecular dynamics simulations shows robust allosteric connections linking the two Raf-RBD D113 residues located in the Galectin scaffold protein binding site of each Raf-RBD molecule and 85 Å apart on opposite ends of the dimer complex. Our results suggest that Raf-RBD binding and Ras dimerization are concerted events that lead to a high-affinity signaling complex at the membrane that we propose is an essential unit in the macromolecular assembly of higher order Ras/Raf/Galectin complexes important for signaling through the Ras/Raf/MEK/ERK pathway.

43P Pre-clinical evaluation of potent and orally bioavailable next-generation inhibitors targeting the family of mutants that drive oncogenic BRAF dimer formation

The canonical BRAF V600E (class I) mutation is a potent oncogene which is uniquely active as a RAS-independent monomer, and which has been successfully targeted by several FDA-approved inhibitors. While active against monomeric BRAF V600E, these first generation BRAF inhibitors induce paradoxical activation of RAS-driven BRAF dimers in cells expressing wild-type RAF, and this can lead to secondary malignancies. More recently, numerous non-canonical BRAF oncogenic mutations including BRAF-fusions have been described as oncogenes that drive RAS-independent (class II) or RAS-dependent (class III) dimers. These non-canonical dimeric BRAF oncogenes are resistant to the first-generation drugs, effective only against the monomeric BRAF V600E mutation. Discovery of an inhibitor directed against the family of dimeric BRAF oncogenic mutations which avoids paradoxical activation is a major unmet need. We applied our proprietary Mutation-Allostery-Pharmacology (MAP) platform technology developed by Black Diamond Therapeutics to identify and validate a group of previously uncharacterized non-canonical oncogenic class II and class III BRAF mutation clusters. We further demonstrate that this ensemble of both novel and previously validated non-canonical oncogenic BRAF mutants can form the basis of a differentiated drug discovery program aimed at identifying small molecules that potently and selectively target this family of dimeric BRAF mutations. Herein, we describe a series of small molecule inhibitors with potent anti-proliferative activity directed against tumor cells harboring dimer-inducing BRAF oncogenic mutations and which are devoid of paradoxical RAF activation. Leading exemplars of BDTX compounds are orally available inhibitors that achieve target engagement of BRAF dimer oncogenes in vivo and robust anti-tumor efficacy in xenograft models in mice. These data support continued development of rationally designed molecules targeting a broad range of non-canonical BRAF dimer-promoting mutations to extend the prospect of precision medicine in patients with BRAF mutant tumors.

KRAS interaction with RAF1 RAS-binding domain and cysteine-rich domain provides insights into RAS-mediated RAF activation

The first step of RAF activation involves binding to active RAS, resulting in the recruitment of RAF to the plasma membrane. To understand the molecular details of RAS-RAF interaction, we present crystal structures of wild-type and oncogenic mutants of KRAS complexed with the RAS-binding domain (RBD) and the membrane-interacting cysteine-rich domain (CRD) from the N-terminal regulatory region of RAF1. Our structures reveal that RBD and CRD interact with each other to form one structural entity in which both RBD and CRD interact extensively with KRAS. Mutations at the KRAS-CRD interface result in a significant reduction in RAF1 activation despite only a modest decrease in binding affinity. Combining our structures and published data, we provide a model of RAS-RAF complexation at the membrane, and molecular insights into RAS-RAF interaction during the process of RAS-mediated RAF activation.

Crystal Structure Reveals the Full Ras:Raf Interface and Advances Mechanistic Understanding of Raf Activation

The interaction between Ras and Raf-kinase through the Ras-binding (RBD) and cysteine-rich domains (CRD) of Raf is essential for signaling through the mitogen-activated protein kinase (MAPK) pathway, yet the molecular mechanism leading to Raf activation has remained elusive. We present the 2.8 Å crystal structure of the HRas/CRaf-RBD_CRD complex showing the Ras/Raf interface as a continuous surface on Ras. in the Ras dimer, with helices roughly perpendicular to the membrane, the CRD is located between the two Ras protomers and far from the membrane, where its dynamic nature in the Ras binding pocket is expected to accommodate BRaf and CRaf heterodimers. Our structure and its analysis by MD simulations, combined with work in the literature, result in a molecular model in which Ras binding is involved in the release of Raf autoinhibition while the Ras/Raf complex dimerizes to promote a platform for signal amplification, with Raf-CRD poised to have direct and allosteric effects on both the Ras active site and the dimerization interface.

Alliance/CALGB 80802: Impact of hepatitis C (HCV) on doxorubicin (DO) + sorafenib (S) versus S in patients (pts) with advanced hepatocellular carcinoma (aHCC).

325 Background: Alliance/CALGB 80802 randomized phase III trial evaluated DO+S vs. S in pts with aHCC, and showed no improvement in median OS. Multi-drug resistant pathway mitigation by the Ras/Raf/MEK/ERK pathway and bFGF-mediated activation of Raf-1 promotes the formation of antiapoptotic Raf-1 and ASK1 complex, induced by anthracyclines. S efficiently blocks NS5A-recruited c-Raf mediated HCV replication and viral gene expression. Once inhibited by S, VEGF expression of HepG2 may limit HCV cellular entry. Release of Raf-1-Ask-1 dimer and inhibition of Raf-1 via S putatively differ in the presence or absence of DO. We hypothesize treatment with S reduces HCV titer levels (TL) and influence pts’ outcome. Methods: In 80802 HCV pts, TL were evaluated in both arms at baseline and post-baseline at Day 1 of Cycles 2, 3, and every 2 cycles and at progression or discontinuation of therapy. HCV undetectable (HCV-UN) levels were defined as < 50 copies/mL. TL were evaluated in relation to OS and PFS. HCV RNA levels were measured by TaqMan PCR and by genotype. Results: Of 356 pts, 83 were HCV+ with more Black/African American (25/50 = 50%) vs. White (54/239 = 23%) or other race groups (4/67 = 6%) (p < 0.0001). HCV titer data were available on 54 pts (S: 28, DO+S: 26). At baseline, 12 pts (S: 7, DO+S: 5) were HCV-UN, and post-baseline HCV TL did not significantly differ between treatment arms; one patient in each arm went from detectable (HCV-D) to HCV-UN. Post-baseline, 40 pts were HCV-D vs. 14 who were HCV-UN (S+DO: 8, S: 6 pts). Except for the two pts who became HCV-UN, baseline HCV-D vs. HCV-UN titers was similar to that status post-baseline. PFS and OS between HCV-D and HCV-UN both at baseline and post-baseline are delineated in the table. Conclusions: We observed that S did not influence HCV TL. Pts treated with DO+S vs. S had worse PFS if they had HCV-UN, and further that higher levels of HCV titers at baseline were associated with significantly improved PFS. Given the small sample size, these findings warrant further prospective evaluation. Support: U10CA180821, U10CA180882, U24CA196171; Bayer, Bristol-Myers-Squibb, and Sanofi. https://acknowledgments.alliancefound.org . Clinical trial information: NCT01015833. [Table: see text]

PI3K Driver Mutations: A Biophysical Membrane-Centric Perspective.

Ras activates its effectors at the membrane. Active PI3Kα and its associated kinases/phosphatases assemble at membrane regions enriched in signaling lipids. In contrast, the Raf kinase domain extends into the cytoplasm and its assembly is away from the crowded membrane surface. Our structural membrane-centric outlook underscores the spatiotemporal principles of membrane and signaling lipids, which helps clarify PI3Kα activation. Here we focus on mechanisms of activation driven by PI3Kα driver mutations, spotlighting the PI3Kα double (multiple) activating mutations. Single mutations can be potent, but double mutations are stronger: their combination is specific, a single strong driver cannot fully activate PI3K, and two weak drivers may or may not do so. In contrast, two strong drivers may successfully activate PI3K, where one, for example, H1047R, modulates membrane interactions facilitating substrate binding at the active site (km) and the other, for example, E542K and E545K, reduces the transition state barrier (ka), releasing autoinhibition by nSH2. Although mostly unidentified, weak drivers are expected to be common, so we ask here how common double mutations are likely to be and why PI3Kα with double mutations responds effectively to inhibitors. We provide a structural view of hotspot and weak driver mutations in PI3Kα activation, explain their mechanisms, compare these with mechanisms of Raf activation, and point to targeting cell-specific, chromatin-accessible, and parallel (or redundant) pathways to thwart the expected emergence of drug resistance. Collectively, our biophysical outlook delineates activation and highlights the challenges of drug resistance.

The mechanism of Raf activation through dimerization.

Raf, a threonine/serine kinase in the Raf/MEK/ERK pathway, regulates cell proliferation. Raf's full activation requires dimerization. Aberrant activation through dimerization is an important therapeutic target. Despite its clinical importance, fundamental questions, such as how the side-to-side dimerization promotes the OFF-to-ON transition of Raf's kinase domain and how the fully activated ON-state kinase domain is stabilized in the dimer for Raf signaling, remain unanswered. Herein, we decipher an atomic-level mechanism of Raf activation through dimerization, clarifying this enigma. The mechanism reveals that the replacement of intramolecular π–π stacking by intermolecular π–π stacking at the dimer interface releases the structural constraint of the αC-helix, promoting the OFF-to-ON transition. During the transition, the inhibitory hydrophobic interactions were disrupted, making the phosphorylation sites in A-loop approach the HRD motif for cis-autophosphorylation. Once fully activated, the ON-state kinase domain can be stabilized by a newly identified functional N-terminal basic (NtB) motif in the dimer for Raf signaling. This work provides atomic level insight into critical steps in Raf activation and outlines a new venue for drug discovery against Raf dimerization.

Synergistic anti-tumor efficacy of oncolytic influenza viruses and B7-H3 immune- checkpoint inhibitors against IC-resistant lung cancers

ABSTRACT Non-small cell lung cancers (NSCLCs) establish a highly immunosuppressive tumor microenvironment supporting cancer growth. To interfere with cancer-mediated immunosuppression, selective immune-checkpoint inhibitors (ICIs) have been approved as a standard-of-care treatment for NSCLCs. However, the majority of patients poorly respond to ICI-based immunotherapies. Oncolytic viruses are amongst the many promising immunomodulatory treatments tested as standalone therapy or in combination with ICIs to improve therapeutic outcome. Previously, we demonstrated the oncolytic and immunomodulatory efficacy of low-pathogenic influenza Aviruses (IAVs) against NSCLCs in immunocompetent transgenic mice with a lung-specific overexpression of active Raf kinase (Raf-BxB). IAV infection not only resulted in significant primary virus-induced oncolysis, but also caused a functional reversion of tumor-associated macrophages (TAMs) comprising additional anti-cancer activity. Here we show that NSCLCs as well as TAMs and cytotoxic immune cells overexpress IC molecules of the PD-L2/PD-1 and B7-H3 signaling axes. Thus, we aimed to combine oncolytic IAV-infection with ICIs to exploit the benefits of both anti-cancer approaches. Strikingly, IAV infection combined with the novel B7-H3 ICI led to increased levels of M1-polarized alveolar macrophages and increased lung infiltration by cytotoxic T lymphocytes, which finally resulted in significantly improved oncolysis of about 80% of existing tumors. In contrast, application of clinically approved α-PD-1 IC antibodies alone or in combination with oncolytic IAV did not provide additional oncolytic or immunomodulatory efficacy. Thus, individualized therapy with synergistically acting oncolytic IAV and B7-H3 ICI might be an innovative future approach to target NSCLCs that are resistant to approved ICIs in patients.

Inhibition of RAF dimers: it takes two to tango.

The RAS-regulated RAF–MEK1/2–ERK1/2 pathway promotes cell proliferation and survival and RAS and BRAF proteins are commonly mutated in cancer. This has fuelled the development of small molecule kinase inhibitors including ATP-competitive RAF inhibitors. Type I and type I½ ATP-competitive RAF inhibitors are effective in BRAFV600E/K-mutant cancer cells. However, in RAS-mutant cells these compounds instead promote RAS-dependent dimerisation and paradoxical activation of wild-type RAF proteins. RAF dimerisation is mediated by two key regions within each RAF protein; the RKTR motif of the αC-helix and the NtA-region of the dimer partner. Dimer formation requires the adoption of a closed, active kinase conformation which can be induced by RAS-dependent activation of RAF or by the binding of type I and I½ RAF inhibitors. Binding of type I or I½ RAF inhibitors to one dimer partner reduces the binding affinity of the other, thereby leaving a single dimer partner uninhibited and able to activate MEK. To overcome this paradox two classes of drug are currently under development; type II pan-RAF inhibitors that induce RAF dimer formation but bind both dimer partners thus allowing effective inhibition of both wild-type RAF dimer partners and monomeric active class I mutant RAF, and the recently developed “paradox breakers” which interrupt BRAF dimerisation through disruption of the αC-helix. Here we review the regulation of RAF proteins, including RAF dimers, and the progress towards effective targeting of the wild-type RAF proteins

Mechanisms of Ras Membrane Organization and Signaling: Ras Rocks Again.

Ras is the most frequently mutated oncogene and recent drug development efforts have spurred significant new research interest. Here we review progress toward understanding how Ras functions in nanoscale, proteo-lipid signaling complexes on the plasma membrane, called nanoclusters. We discuss how G-domain reorientation is plausibly linked to Ras-nanoclustering and -dimerization. We then look at how these mechanistic features could cooperate in the engagement and activation of RAF by Ras. Moreover, we show how this structural information can be integrated with microscopy data that provide nanoscale resolution in cell biological experiments. Synthesizing the available data, we propose to distinguish between two types of Ras nanoclusters, an active, immobile RAF-dependent type and an inactive/neutral membrane anchor-dependent. We conclude that it is possible that Ras reorientation enables dynamic Ras dimerization while the whole Ras/RAF complex transits into an active state. These transient di/oligomer interfaces of Ras may be amenable to pharmacological intervention. We close by highlighting a number of open questions including whether all effectors form active nanoclusters and whether there is an isoform specific composition of Ras nanocluster.

Dimerization Induced by C-Terminal 14-3-3 Binding Is Sufficient for BRAF Kinase Activation.

The Ras-RAF-MEK-ERK signaling axis, commonly mutated in human cancers, is highly regulated to prevent aberrant signaling in healthy cells. One of the pathway modulators, 14-3-3, a constitutive dimer, induces RAF dimerization and activation by binding to a phosphorylated motif C-terminal to the RAF kinase domain. Recent work has suggested that a C-terminal "DTS" region in BRAF is necessary for this 14-3-3-mediated activation. We show that the catalytic activity and ATP binding affinity of the BRAF:14-3-3 complex is insensitive to the presence or absence of the DTS, while the ATP sites of both BRAF molecules are identical and available for binding. We also present a crystal structure of the apo BRAF:14-3-3 complex showing that the DTS is not required to attain the catalytically active conformation of BRAF. Rather, BRAF dimerization induced by 14-3-3 is the key step in activation, allowing the active BRAF:14-3-3 tetramer to achieve catalytic activity comparable to the constitutively active oncogenic BRAF V600E mutant.

Tumor regression and resistance mechanisms upon CDK4 and RAF1 inactivation in KRAS/P53 mutant lung adenocarcinomas

KRAS mutant lung adenocarcinomas remain intractable for targeted therapies. Genetic interrogation of KRAS downstream effectors, including the MAPK pathway and the interphase CDKs, identified CDK4 and RAF1 as the only targets whose genetic inactivation induces therapeutic responses without causing unacceptable toxicities. Concomitant CDK4 inactivation and RAF1 ablation prevented tumor progression and induced complete regression in 25% of KRAS/p53-driven advanced lung tumors, yet a significant percentage of those tumors that underwent partial regression retained a population of CDK4/RAF1-resistant cells. Characterization of these cells revealed two independent resistance mechanisms implicating hypermethylation of several tumor suppressors and increased PI3K activity. Importantly, these CDK4/RAF1-resistant cells can be pharmacologically controlled. These studies open the door to new therapeutic strategies to treat KRAS mutant lung cancer, including resistant tumors.

KRASQ61H Preferentially Signals through MAPK in a RAF Dimer-Dependent Manner in Non–Small Cell Lung Cancer

Assembly of RAS molecules into complexes at the cell membrane is critical for RAS signaling. We previously showed that oncogenic KRAS codon 61 mutations increase its affinity for RAF, raising the possibility that KRASQ61H, the most common KRAS mutation at codon 61, upregulates RAS signaling through mechanisms at the level of RAS assemblies. We show here that KRASQ61H exhibits preferential binding to RAF relative to PI3K in cells, leading to enhanced MAPK signaling in in vitro models and human NSCLC tumors. X-ray crystallography of KRASQ61H:GTP revealed that a hyperdynamic switch 2 allows for a more stable interaction with switch 1, suggesting that enhanced RAF activity arises from a combination of absent intrinsic GTP hydrolysis activity and increased affinity for RAF. Disruption of KRASQ61H assemblies by the RAS oligomer-disrupting D154Q mutation impaired RAF dimerization and altered MAPK signaling but had little effect on PI3K signaling. However, KRASQ61H oligomers but not KRASG12D oligomers were disrupted by RAF mutations that disrupt RAF-RAF interactions. KRASQ61H cells show enhanced sensitivity to RAF and MEK inhibitors individually, whereas combined treatment elicited synergistic growth inhibition. Furthermore, KRASQ61H tumors in mice exhibited high vulnerability to MEK inhibitor, consistent with cooperativity between KRASQ61H and RAF oligomerization and dependence on MAPK signaling. These findings support the notion that KRASQ61H and functionally similar mutations may serve as predictive biomarkers for targeted therapies against the MAPK pathway. SIGNIFICANCE: These findings show that oncogenic KRASQ61H forms a cooperative RAS-RAF ternary complex, which renders RAS-driven tumors vulnerable to MEKi and RAFi, thus establishing a framework for evaluating RAS biomarker-driven targeted therapies.

Abstract 1088: Elucidating the regulation of delayed-early gene targets of sustained MAPK signaling

Abstract RAS and its downstream effector, BRAF, are commonly mutated proto-oncogenes in many types of human cancer. Mutationally activated RAS or BRAF signal through the MEK→ERK MAP kinase (MAPK) pathway to regulate key cancer cell hallmarks such as cell division cycle progression, reduced programmed cell death, and enhanced cell motility. Amongst the list of RAS/RAF-regulated genes are those encoding integrins, alpha-beta heterodimeric transmembrane proteins that regulate cell adhesion to the extracellular matrix. Altered integrin expression has been linked to the acquisition of more aggressive behavior by melanoma, lung, and breast cancer cells leading to diminished survival of cancer patients. We have previously documented the ability of the RAS-activated MAPK pathway to induce the expression of ITGB3 encoding integrin β3 in several different cell types. RAS/RAF-mediated induction of ITGB3 mRNA requires sustained, high-level activation of RAF→MEK→ERK signaling mediated by oncogene activation and is classified as “delayed-early”, in that it is sensitive to the protein synthesis inhibitor cycloheximide. However, to date, the regulatory mechanisms that allow for induced ITGB3 downstream of sustained, high-level activation of MAPK signaling remains obscure. We have identified over 300 DEGs, including those expressing additional cell surface proteins, that display similar regulatory characteristics as ITGB3. We use integrin β3 as a model to test our hypothesis that there is a different mechanism of regulation for delayed-early genes (DEG) compared to the canonical regulation of Immediate-Early genes. There are three regions in the chromatin upstream of the ITGB3 that become more accessible during RAF activation. We are relating the chromatin changes seen during RAF activation to active enhancer histone marks. To elucidate the essential genes of this regulation process, we are employing the use of a genome-wide CRISPR knockout screen. The work presented from this abstract will help elucidate the regulatory properties of oncogenic progression in BRAF mutated cancers that could lead to the identification of biomarkers. Citation Format: Kali J. Dale, Martin McMahon. Elucidating the regulation of delayed-early gene targets of sustained MAPK signaling [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1088.