Raf Kinase Research Articles

Abstract P3113: RAF 'Paradox Breaker' Inhibitor, PLX8394, Enhances Inflammation And Apoptosis In Murine Hypertension In Vivo

Introduction: The ERK1/2 cascade, activated by RAF kinases, is a key pathway for cardiac remodelling and cytoprotection. Due to activating oncogenic mutations in BRAF, small molecule RAF inhibitors have been developed. However, owing to inhibitor resistance or paradoxical ERK1/2 pathway activation, a new generation of ‘paradox breaking’ drugs are currently in clinical trials. Our previous work characterised RAF targeting in hypertension, where it is instrumental in driving cardiac remodelling. Here we explored whether use of the RAF paradox breakers (e.g. PLX8394) are viable modalities for targeting RAF in hypertension. Hypothesis: We hypothesize that RAF ‘paradox breakers’ will protect cardiac function by inhibiting maladaptive hypertensive remodelling. Methods & Results: To assess the role of RAF inhibition in cardiac adaptation to hypertension in vivo , C57Bl/6J mice were treated with angiotensin-II (AngII; 0.8mg/kg/d, 7d n=8;) without/with 5mg/kg/d PLX8394 (n=11). PLX8394 alone had no detrimental effect on cardiac function or remodelling. AngII promoted cardiac hypertrophy, by increasing left ventricular (LV) wall thickness (WT) and decreasing LV internal diameter (ID; assessed by echocardiography). PLX8394 had no impact on AngII-induced hypertrophy (WT:ID, p=0.45), or abundance of AngII-induced hypertrophic Nppa (p=0.77) or Nppb (p=0.49) mRNAs. Additionally, PLX8394 did not affect AngII-induced perivascular fibrosis (p=0.69; picrosirius red histology) or moderate Col1a1 (p=0.22) and Col3a1 (p=0.82) mRNAs. However, PLX8394 use in AngII-hypertension was pro-inflammatory, with increased pro-inflammatory cytokines ( Il1b , p=0.02; Il6 , p=0.008) and the inflammatory cell marker CD45 (p=0.001) mRNAs; a response echoed in hearts labelled by immunohistochemistry for CD45 (p=0.02). Moreover, western blotting revealed elevated PARP cleavage (p=0.005), indicating increased apoptosis with PLX8394 use. Conclusion: Despite no adverse effect on cardiac function or global hypertensive remodelling at 7 days, RAF inhibition by PLX8394 appears to be pro-inflammatory and pro-apoptotic. Thus, long-term use of RAF ‘paradox breakers’ in hypertensive individuals may be cardiotoxic and promote adverse cardiac events.

ERK2 signaling regulates cell-cell adhesion of epithelial cells and enhances growth factor-induced cell scattering

The ERK signaling pathway, consisting of core protein kinases Raf, MEK and effector kinases ERK1/2, regulates various biological outcomes such as cell proliferation, differentiation, apoptosis, or cell migration. Signal transduction through the ERK signaling pathway is tightly controlled at all levels of the pathway. However, it is not well understood whether ERK pathway signaling can be modulated by the abundance of ERK pathway core kinases. In this study, we investigated the effects of low-level overexpression of the ERK2 isoform on the phenotype and scattering of cuboidal MDCK epithelial cells growing in discrete multicellular clusters. We show that ERK2 overexpression reduced the vertical size of lateral membranes that contain cell-cell adhesion complexes. Consequently, ERK2 overexpressing cells were unable to develop cuboidal shape, remained flat with increased spread area and intercellular adhesive contacts were present only on the basal side. Interestingly, ERK2 overexpression was not sufficient to increase phosphorylation of multiple downstream targets including transcription factors and induce global changes in gene expression, namely to increase the expression of pro-migratory transcription factor Fra1. However, ERK2 overexpression enhanced HGF/SF-induced cell scattering as these cells scattered more rapidly and to a greater extent than parental cells. Our results suggest that an increase in ERK2 expression primarily reduces cell-cell cohesion and that weakened intercellular adhesion synergizes with upstream signaling in the conversion of the multicellular epithelium into single migrating cells. This mechanism may be clinically relevant as the analysis of clinical data revealed that in one type of cancer, pancreatic adenocarcinoma, ERK2 overexpression correlates with a worse prognosis.

S-palmitoylation of PCSK9 induces sorafenib resistance in liver cancer by activating the PI3K/AKT pathway.

Sorafenib is currently the first-line treatment for advanced hepatocellular carcinoma (HCC). However, sorafenib resistance remains a significant challenge. Aberrant AKT signaling activation is a crucial mechanism driving sorafenib resistance in HCC. Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a vital role in antitumor immune responses. In this study, we demonstrate that aberrant PCSK9 upregulation promotes cell proliferation and sorafenib resistance in HCC by inducing AKT-S473 phosphorylation. After palmitoylation at cysteine 600, the binding affinity between PCSK9 and tensin homolog (PTEN) is dramatically increased, inducing lysosome-mediated PTEN degradation and subsequent AKT activation. We identify zinc finger DHHC-type palmitoyltransferase 16 (ZDHHC16) as a palmitoyltransferase that promotes PCSK9 palmitoylation at cysteine 600. We also develop a biologically active PCSK9-derived peptide that competitively inhibits PCSK9 palmitoylation, suppressing AKT phosphorylation and augmenting the antitumor effects of sorafenib in HCC.

Sodium Ferulate Inhibits Rat Cardiomyocyte Hypertrophy Induced by Angiotensin II Through Enhancement of Endothelial Nitric Oxide Synthase/Nitric Oxide/Cyclic Guanosine Monophosphate Signaling Pathway.

Sodium ferulate (SF) is the sodium salt of ferulic acid, which is one of the effective components of Angelica sinensis and Lignsticum chuanxiong , and plays an important role in protecting the cardiovascular system. In this study, myocardial hypertrophy was induced by angiotensin II 0.1 μmol/L in neonatal Sprague-Dawley rat ventricular myocytes. Nine groups were designed, that is, normal, normal administration, model, L-arginine (L-arg 1000 μmol/L), SF (50, 100, 200 μmol/L) group, and N G -nitro-L-arg-methyl ester 1500 μmol/L combined with SF 200 μmol/L or L-arg 1000 μmol/L group, respectively. Cardiomyocyte hypertrophy was confirmed by observing histological changes and measurements of cell diameter, protein content and atrial natriuretic factor, and β-myosin heavy chain levels of the cells. Notably, SF could inhibit significantly myocardial hypertrophy of neonatal rat cardiomyocytes in a concentration-dependent manner without producing cytotoxicity, and the levels of nitric oxide, NO synthase (NOS), endothelial NOS, and cyclic guanosine monophosphate were increased, but the level of cyclic adenosine monophosphate was decreased in cardiomyocytes. Simultaneously, levels of protein kinase C beta, Raf-1, and extracellular regulated protein kinase 1/2 (ERK1/2) were downregulated, whereas levels of mitogen-activated protein kinase phosphatase-1 were significantly upregulated. All the beneficial effects of SF were blunted by N G -nitro-L-arg-methyl ester. Overall, these findings reveal that SF can inhibit angiotensin II-induced myocardial hypertrophy of neonatal rat cardiomyocytes, which is closely related to activation of endothelial NOS/NO/cyclic guanosine monophosphate, and inhibition of protein kinase C and mitogen-activated protein kinase signaling pathways.

Differential regulations of abscisic acid-induced desiccation tolerance and vegetative dormancy by group B3 Raf kinases in liverworts.

Phytohormone abscisic acid (ABA) plays a key role in stomata closure, osmostress acclimation, and vegetative and embryonic dormancy. Group B3 Raf protein kinases (B3-Rafs) serve as positive regulators of ABA and osmostress signaling in the moss Physcomitrium patens and the angiosperm Arabidopsis thaliana. While P. patens has a single B3-Raf called ARK, specific members of B3-Rafs among six paralogs regulate ABA and osmostress signaling in A. thaliana, indicating functional diversification of B3-Rafs in angiosperms. However, we found that the liverwort Marchantia polymorpha, belonging to another class of bryophytes, has three paralogs of B3-Rafs, MpARK1, MpARK2, and MpARK3, with structural variations in the regulatory domains of the polypeptides. By reporter assays of the P. patens ark line and analysis of genome-editing lines of M. polymorpha, we found that these B3-Rafs are functionally redundant in ABA response, with respect to inhibition of growth, tolerance to desiccation and expression of stress-associated transcripts, the majority of which are under the control of the PYR/PYL/RCAR-like receptor MpPYL1. Interestingly, gemmae in gemma cups were germinating only in mutant lines associated with MpARK1, indicating that dormancy in the gametophyte is controlled by a specific B3-Raf paralog. These results indicated not only conservation of the role of B3-Rafs in ABA and osmostress response in liverworts but also functional diversification of B3-Rafs, which is likely to have occurred in the early stages of land plant evolution.

A Negative Regulatory Role for RKIP in Breast Cancer Immune Response.

Simple SummaryBreast cancer is the second most common cancer in women worldwide. Regulation of breast cancer metastasis remains an elusive phenomenon. Elucidating the mechanistic pathway of metastatic signaling may identify targets for regulating cancer metastatic potential. Raf-1 kinase inhibitor protein (RKIP) has been shown to negatively regulate signaling pathways involved in cancer progression and metastasis. RKIP may suppress metastasis of breast cancer cells by downregulating elements of the immune system. Raf-1 kinase inhibitor protein was first identified as a negative regulator of the Raf signaling pathway. Subsequently, it was shown to have a causal role in containing cancer progression and metastasis. Early studies suggested that RKIP blocks cancer progression by inhibiting the Raf-1 pathway. However, it is not clear if the RKIP tumor and metastasis suppression function involve other targets. In addition to the Raf signaling pathway, RKIP has been found to modulate several other signaling pathways, affecting diverse biological functions including immune response. Recent advances in medicine have identified both positive and negative roles of immune response in cancer initiation, progression and metastasis. It is possible that one way that RKIP exerts its effect on cancer is by targeting an immune response mechanism. Here, we provide evidence supporting the causal role of tumor and metastasis suppressor RKIP in downregulating signaling pathways involved with immune response in breast cancer cells and discuss its potential ramification on cancer therapy.

Asynchronous Reciprocal Coupling of Martini 2.2 Coarse-Grained and CHARMM36 All-Atom Simulations in an Automated Multiscale Framework.

The appeal of multiscale modeling approaches is predicated on the promise of combinatorial synergy. However, this promise can only be realized when distinct scales are combined with reciprocal consistency. Here, we consider multiscale molecular dynamics (MD) simulations that combine the accuracy and macromolecular flexibility accessible to fixed-charge all-atom (AA) representations with the sampling speed accessible to reductive, coarse-grained (CG) representations. AA-to-CG conversions are relatively straightforward because deterministic routines with unique outcomes are achievable. Conversely, CG-to-AA conversions have many solutions due to a surge in the number of degrees of freedom. While automated tools for biomolecular CG-to-AA transformation exist, we find that one popular option, called Backward, is prone to stochastic failure and the AA models that it does generate frequently have compromised protein structure and incorrect stereochemistry. Although these shortcomings can likely be circumvented by human intervention in isolated instances, automated multiscale coupling requires reliable and robust scale conversion. Here, we detail an extension to Multiscale Machine-learned Modeling Infrastructure (MuMMI), including an improved CG-to-AA conversion tool called sinceCG. This tool is reliable (∼98% weakly correlated repeat success rate), automatable (no unrecoverable hangs), and yields AA models that generally preserve protein secondary structure and maintain correct stereochemistry. We describe how the MuMMI framework identifies CG system configurations of interest, converts them to AA representations, and simulates them at the AA scale while on-the-fly analyses provide feedback to update CG parameters. Application to systems containing the peripheral membrane protein RAS and proximal components of RAF kinase on complex eight-component lipid bilayers with ∼1.5 million atoms is discussed in the context of MuMMI.

Mechanisms of isoform-specific residue influence on GTP-bound HRas, KRas, and NRas

HRas, KRas, and NRas are GTPases with a common set of effectors that control many cell-signaling pathways, including proliferation through Raf kinase. Their G-domains are nearly identical in sequence, with a few isoform-specific residues that have an effect on dynamics and biochemical properties. Here, we use accelerated molecular dynamics (aMD) simulations consistent with solution x-ray scattering experiments to elucidate mechanisms through which isoform-specific residues associated with each Ras isoform affects functionally important regions connected to the active site. HRas-specific residues cluster in loop 8 to stabilize the nucleotide-binding pocket, while NRas-specific residues on helix 3 directly affect the conformations of switch I and switch II. KRas, the most globally flexible of the isoforms, shows greatest fluctuations in the switch regions enhanced by a KRas-specific residue in loop 7 and a highly dynamic loop 8 region. The analysis of isoform-specific residue effects on Ras proteins is supported by NMR experiments and is consistent with previously published biochemical data.

Antagonism of N/OFQ attenuates externalization of β1-adrenergic receptor and ventricular arrhythmias in acute myocardial ischemia rat model

Nociceptin/orphanin FQ (N/OFQ) and adrenergic activations play roles in promoting cardiac arrhythmia in acute myocardial ischemia but whether N/OFQ and β1-adrenergic activities interact and how they interact in the arrhythmogenesis are still unknown. We designed this study to investigate the potential interaction of N/OFQ and β1-adrenergic activities and the underlying mechanism in arrhythmogenesis in acute myocardial ischemia. Ventricular arrhythmia was evaluated in anaesthetized rats following permanent coronary artery occlusion (CAO), in presence and absence of UFP-101 (a selective antagonist of N/OFQ receptor). The changes of β1-adrenergic receptor (β1-AR) in plasma membrane of cardiomyocytes were quantitatively evaluated and the relations with the alterations of phosphorylated Raf kinase inhibitor protein (p-RKIP) and phosphorylated connexin 43 (p-Cx43) were investigated. The ventricular arrhythmia was 59% less in the animals pre-treated with UFP-101 than the placebo-treated control (difference of means = −2.41; 95% confidence interval (CI) −2.84 to −1.99; P < 0.001). Meanwhile, p-RKIP and membrane β1-AR in the myocardium were downregulated by 59% and 24%, respectively (p-RKIP: difference of means = −6.91; 95% CI -8.38 to −5.45; P < 0.001; membrane β1-AR difference of means = −27.06; 95% CI -29.89 to −24.23; P < 0.001). Artificial upregulation of RKIP by didymin significant increased β1-AR in plasma membrane of the cardiomyocytes in the animals prone to ventricular arrhythmia. The findings may suggest that activation of N/OFQ receptor in acute myocardial ischemia induces upregulation of p-RKIP, externalization of β1-adrenergic receptor and downregulation of p-Cx43 in the cardiomyocytes, which promotes ventricular arrhythmia.

Structural basis for SHOC2 modulation of RAS signalling

The RAS–RAF pathway is one of the most commonly dysregulated in human cancers1–3. Despite decades of study, understanding of the molecular mechanisms underlying dimerization and activation4 of the kinase RAF remains limited. Recent structures of inactive RAF monomer5 and active RAF dimer5–8 bound to 14-3-39,10 have revealed the mechanisms by which 14-3-3 stabilizes both RAF conformations via specific phosphoserine residues. Prior to RAF dimerization, the protein phosphatase 1 catalytic subunit (PP1C) must dephosphorylate the N-terminal phosphoserine (NTpS) of RAF11 to relieve inhibition by 14-3-3, although PP1C in isolation lacks intrinsic substrate selectivity. SHOC2 is as an essential scaffolding protein that engages both PP1C and RAS to dephosphorylate RAF NTpS11–13, but the structure of SHOC2 and the architecture of the presumptive SHOC2–PP1C–RAS complex remain unknown. Here we present a cryo-electron microscopy structure of the SHOC2–PP1C–MRAS complex to an overall resolution of 3 Å, revealing a tripartite molecular architecture in which a crescent-shaped SHOC2 acts as a cradle and brings together PP1C and MRAS. Our work demonstrates the GTP dependence of multiple RAS isoforms for complex formation, delineates the RAS-isoform preference for complex assembly, and uncovers how the SHOC2 scaffold and RAS collectively drive specificity of PP1C for RAF NTpS. Our data indicate that disease-relevant mutations affect complex assembly, reveal the simultaneous requirement of two RAS molecules for RAF activation, and establish rational avenues for discovery of new classes of inhibitors to target this pathway.

Surface plasmon-enhanced electrochemiluminescence of P, N-doped carbon dots for ultrasensitive detection of BRAF gene

Carbon dots (CDs) have shown enormous potential as electrochemiluminescence (ECL) probes for biosensing and bioimaging applications due to their ease of synthesis and modification, high biocompatibility, stable optical and electrochemical properties. However, the mild ECL emission of CDs often restricts their practical applications. Herein, an ECL biosensor for mutant BRAF gene determination was proposed based on the surface plasmon resonance-enhanced ECL (SPR-ECL) between asymmetrically modified amine-gold nanodendrites (am-AuNDs) and phosphorus (P) and nitrogen (N) co-doped CDs (PNCDs). Compared to the N-doped CDs (NCDs), PNCDs exhibited an 8-fold enhanced ECL because of the larger atom size and higher electron-donating ability of P atom compared to N. Due to the surface plasmon resonance effect of am-AuNDs and proper distance controlled by catalytic hairpin assembly (CHA), the ECL signal of PNCDs was remarkably improved based on a nonradiative energy transfer between PNCDs and the am-AuNDs. The as-constructed SPR-ECL biosensor performed well for the detection of mutant B-type Raf kinase (BRAF) gene with high sensitivity, stability and accuracy in the range from 0.5 pM to 2000 pM with a limit of detection (LOD) of 0.34 pM. This work would open a new avenue to boost the ECL performance of carbon nanomaterials for practical applications, especially for bioanalysis and diagnostics.

Abstract 2677: JZP815, a potent and selective pan-RAF inhibitor, demonstrates efficacy in RAF and RAS mutant tumor pre-clinical models

Abstract Activation of the mitogen-activated protein kinase (MAPK) pathway by oncogenic mutations in RAS and RAF is a frequent driver of human cancer. Targeting specific components of the pathway is a precise and rational route to deliver benefits to cancer patients with high unmet therapeutic needs. The RAF kinase proteins (ARAF, BRAF, CRAF) are core pathway components which mediate MAPK signaling. Mutations in these critical signaling proteins leads to constitutive activation of the MAPK pathway and tumor growth. Using state of the art screening methodologies and medicinal chemistry we discovered and developed a next generation pan-RAF kinase inhibitor, JZP815, which potently inhibits wild-type and mutant RAF kinases in cell-free and cell-based assays. JZP815 is a selective RAF kinase inhibitor that suppresses all 3 RAF kinase family members at low-to-sub nanomolar potencies in cell-free assays. Moreover, in tumor cells JZP815 does not induce significant paradoxical pathway activation, observed with approved 1st generation BRAF-selective inhibitors, while demonstrating equivalent potencies for MAPK pathway inhibition driven by either mutant RAF monomers or RAS-induced RAF dimers in tumor cells. JZP815 significantly inhibited tumor growth as a single agent in multiple mouse xenograft solid tumor models harboring RAS and/or BRAF mutations. Furthermore, JZP815’s favorable pharmacokinetic profile sustained on-target pathway pharmacodynamic responses in a predictable dose and time dependent manner. JZP815 demonstrated enhanced activity when combined with inhibitors of other MAPK pathway components in both class 2 and class 3 mutant BRAF patient-derived tumor cells ex vivo, and KRAS mutant NSCLC and CRC xenografts in vivo. In summary, JZP815 is a potent and selective pan-RAF inhibitor that is orally bioavailable in multiple preclinical species with a well-behaved safety profile and pharmacokinetic properties predicted to provide drug exposure required for target engagement in humans, and therefore suitable for advancing into first-in-human trials. Citation Format: Robert Hauptschein, Simon Woodcock, Andrew Belfield, Helen Mason, Dorottya Keppel, Svetlana Markova, Kyuri Kim, Shane Roller, Caroline Phillips, Clifford D. Jones, Robin C. Humphreys. JZP815, a potent and selective pan-RAF inhibitor, demonstrates efficacy in RAF and RAS mutant tumor pre-clinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2677.

Abstract 3596: RM-036, a first-in-class, orally-bioavailable, Tri-Complex covalent KRASG12D(ON) inhibitor, drives profound anti-tumor activity in KRASG12D mutant tumor models

Abstract KRASG12D mutant cancers represent a significant unmet medical need with 55,000 new diagnoses annually in the US. The KRASG12D mutation occurs commonly in multiple tumor histotypes, including about 20%, 29% and 17% of KRAS mutant colorectal, pancreatic, and non-small cell lung cancers, respectively. However, there are no directly targeted inhibitors of KRASG12D in clinical development. Mutant RAS proteins exist predominantly in the GTP-bound RAS(ON) state, leading to excessive downstream signaling via interaction with effectors such as RAF kinases. Highly selective inhibitors that covalently target the KRASG12C(OFF) state and inhibit conversion to KRASG12C(ON) have been shown to be active and well tolerated in the clinic, and we have described a novel, covalent tri-complex KRASG12C(ON) inhibitor that has robust anti-tumor activity and is well tolerated in preclinical studies. Developing a covalent inhibitor of KRASG12D presents several challenges. The Asp residue in KRASG12D is significantly less reactive toward electrophiles than is Cys. And most reported covalent chemical warheads capable of reacting with Asp exhibit high nonspecific reactivity. The intrinsic GTP hydrolysis rate of KRASG12D is about 2.6-fold lower than that of KRASG12C, further biasing the cellular KRASG12D pool to the RAS(ON) state and emphasizing the importance of targeting the KRASG12D(ON) state for maximal suppression of this oncogenic driver. Based on these considerations we designed RM-036, a potent, selective, orally bioavailable, covalent KRASG12D(ON) inhibitor with attractive drug-like properties. It forms a tri-complex between the abundant intracellular chaperone cyclophilin A (CypA) and the active state of KRASG12D, positioning the warhead relative to KRASG12D and enabling selective covalent engagement of Asp. In cellular models, RM-036 exhibited efficient and selective covalent binding to KRASG12D, with no detectable reactivity toward the adjacent Asp residue in cells with the KRASG13D mutation. RM-036 showed low off-target reactivity in cell-based proteomic screens. RM-036 potently inhibited growth and induced apoptosis in KRASG12D mutant cancer cells in vitro but not in BRAFV600E-dependent cells. RM-036, as a single agent, produced deep, durable, and dose-dependent suppression of tumor RAS pathway activation with covalent target engagement in vivo following repeat oral administration. Profound tumor regressions, including complete regressions, were observed in various KRASG12D mutant xenograft models upon treatment with RM-036. All treatments regimens tested with RM-036 were well tolerated in vivo. These preclinical findings provide a strong foundation for advancing RM-036 toward clinical evaluation in patients with tumors bearing the KRASG12D mutation. Citation Format: John E. Knox, Jingjing Jiang, G. Leslie Burnett, Yang Liu, Caroline E. Weller, Zhican Wang, Laura McDowell, Shelby L. Steele, Shook Chin, Kang Jye Chou, Fang Wang, Mengqi Zhong, Elena S. Koltun, David Wildes, Mallika Singh, Adrian L. Gill, Jacqueline A. Smith. RM-036, a first-in-class, orally-bioavailable, Tri-Complex covalent KRASG12D(ON) inhibitor, drives profound anti-tumor activity in KRASG12D mutant tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3596.

Abstract 5460: Preclinical characterization of QLH11906, a novel pan-RAF inhibitor

Abstract Background: The type 1/2 BRAF inhibitors (e.g., vemurafenib) have been used for patients with BRAFV600E-mutant tumors. But the application of the inhibitors was restricted to a few types of tumors (e.g., melanoma). Common adverse effects such as pruritus and skin papilloma have limited the usage. It could be attributed to the paradoxical activation of the MAPK pathway. The paradoxical activation also leads to tumor promotion in BRAF wild-type melanoma and skin toxicity in 30%-70% patients with KRAS mutations. Therefore, development of type II pan-RAF inhibitors which avoid MAPK paradoxical activation is necessary. Methods: The selective pan-RAF inhibitor QLH11906 was tested on A/B/C RAF kinases and a representative kinase panel. Both 2D and 3D proliferation studies were conducted for cell lines with BRAF and various KRAS mutations, followed by both immunoprecipitation and ERK phosphorylation assays to elucidate the mode of action. Combination studies with MEK inhibitors were implemented in both KRAS-mutant cell lines and BRAF/KRAS-mutant patient derived organoids (PDO). Single-agent and combination studies were also carried out in vivo on both BRAF/KRAS-mutant cell line-derived xenograft (CDX) and KRAS-mutant patient-derived xenograft (PDX) models. Results: QLH11906 inhibited wild-type A/B/C RAF and BRAFV600E with the IC50 of 4.9, 1.9, 0.5, and 1.1 nM, respectively, and showed no inhibition at 1 μM in a selectivity panel of 28 kinases except for DDR1 and DDR2. Under 2D culture condition, QLH11906 inhibited proliferation of various BRAF/KRAS-mutant cell lines (IC50 300-1700 nM) and suppressed ERK phosphorylation (IC50 20-600 nM). In 3D cultures, QLH11906 showed higher activity in proliferation suppression in both HCT116 (KRASG13D, IC50 24 nM 3D vs 1186 nM 2D) and Calu-6 (KRASQ61K, IC50 85 nM 3D vs 1411 nM 2D). In immunoprecipitation studies with HCT116 and Calu-6 cells, both BRAF homodimers and BRAF/CRAF heterodimers could be detected under the treatment of QLH11906. QLH11906 showed synergistic effect when combined with MEK inhibitors trametinib or cobimetinib in KRAS-mutant cell lines, including A549 (KRASG12S), Calu-6, HCT116, NCIH2122 (KRASG12C) and Panc1005 (KRASG12D), and 77%-99% tumor suppression was reached at the expected clinical Ctrough concentrations. Synergistic effects with trametinib were observed in PDO models including pancreatic cancers (KRASG12D and KRASG12V), colon cancers (KRASG12D and KRASG13D) and lung cancers (KRASG12V). QLH11906 also promoted tumor shrinkage in vivo as a single agent in Calu-6, HCT116, and Colo-205 (BRAFV600E) CDX models or combined with trametinib in PDX models (KRASG12C lung cancers and KRASG12C colon cancers) Conclusion: QLH11906 is a highly selective pan-RAF inhibitor with promising antitumor activity both in vitro and in vivo. It is hopeful to be a therapeutic agent for BRAF and KRAS-mutant tumor patients. Citation Format: Zhe Cheng, Fei Chen, Xile Liu, Cong Gao, Linchao Jia, Yuanzhi Lao. Preclinical characterization of QLH11906, a novel pan-RAF inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5460.

Abstract 1599: Genes subject to changes in heterogeneity can function as regulators of metastasis

Abstract Tumor cell heterogeneity has been strongly implicated in metastatic progression of solid tumors such as triple-negative breast cancer (TNBC), leading to resistance and recurrence. Raf Kinase Inhibitory Protein (RKIP or PEBP1) effectively suppresses metastasis in TNBC but is lost in many patients. We hypothesize that RKIP works in part by reducing the variability of gene expression in TNBC cells. To test whether RKIP reduces overall transcriptional heterogeneity, we compared the variation in gene expression between individual cells in metastatic (low RKIP) versus non-metastatic (high RKIP) TNBC tumors. Single-cell RNA-sequencing revealed transcriptional heterogeneity was decreased with RKIP overexpression. Many cancer regulatory genes were more homogeneous and up-regulated in the RKIP-expressing cells. Among these enriched and homogenously expressed genes is KMT5C, a histone methyltransferase that facilitates epigenetic transcriptional repression. Cell, mouse, and clinical studies suggest KMT5C expression blocks invasion, extravasation, and colonization of tumor cells, consistent with a role as a novel suppressor of metastasis. These studies reveal the importance of genes involved in heterogeneity as potential regulators of metastatic progression in cancer. Citation Format: Dongbo Yang, Christopher Dann, Andrea Valdespino, Lydia Robinson-Mailman, Mengjie Chen, Sebastian Pott, Marsha Rosner. Genes subject to changes in heterogeneity can function as regulators of metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1599.

Abstract 355: Molecular characterization of BRAF V600E CRC in response to Src-targeted therapy

Abstract Oncogenic mutations in B-type Raf Kinase (BRAF) (particularly V600E) are present in 7-10% of metastatic colorectal cancer (mCRC), conferring resistance to conventional therapies and a poor prognosis. Src Family Kinases (SFKs) play a relevant role in CRC, where Src activation is associated with an aggressive phenotype and a worse prognosis; and could be involved in chemoresistance mechanisms. Dasatinib is a potent Src inhibitor with demonstrated anti-proliferative activity, although deeper knowledge is needed. Thus, we aimed to explore the role of Src in BRAF V600E CRC and to investigate this tumor subgroup in response to Dasatinib. SRC activation was evaluated by Western Blot. We generated Knock-Out (KO) Src cell lines by CRISPR/Cas9 system and stable Src overexpression (OE) by lentivirus. Cell viability was analysed by crystal violet and MTS assay. Phospho-kinase proteins screening was performed using the Proteome Profiler Human Phospho-Kinase Array kit. Expression levels of Src and phospho-Src (p-Src) displayed an inverse correlation with IC50 values for Dasatinib in a panel of 7 CRC cell lines including BRAF V600E (HT29, COLO201, RKO, LS411N) and BRAF WT (DIFI, HCA7 and LIM1215), and these doses were able to inhibit Src activation in all cell lines. Focusing on HT29 cells, inhibition of p-Src occurs in a dose-dependent manner and in shortened periods of treatment. Dasatinib showed a significant inhibition of proliferation and migration on HT29. Moreover, stable Src-OE HT29 cells showed higher clonogenicity, migration and proliferation capacities, as well as higher resistance to Dasatinib. Conversely, HT29 Src KO cells were more sensitive. In line with those results, silencing Src in other BRAF V600E cells (RKO and LS411N) also resulted in a remarkable reduction in clonogenicity and migration abilities. Xenograft mouse model of SRC depletion showed that the growth of SRC KO tumors was significantly slower than WT tumors. Phospho-kinase array quantification in basal and Src-OE HT29 cells showed up- and downregulated proteins related to classical tumorigenic signaling pathways such as MAPK (JNK1/2, MSK), PI3K/Akt (Akt, TOR, GSK3), and others. Further studies to explore resistance mechanisms mediated by Src/SFKs are required. To conclude, lower p-Src expression at baseline is related to a higher resistance to Dasatinib in all CRC cell lines. Dasatinib showed a significant anti-proliferative activity in a subset of CRC cell lines and regulates the activity of Src in a time and dose-dependent manner. Modified expression of Src (OE and KO) in BRAF V600E CRC cells significantly regulates clonogenicity, proliferation and migration abilities in vitro. Src overexpression is responsible of the acquired resistance to Dasatinib and regulates several pathways that could be involved in the tumorigenesis of BRAF V600E CRC, although further studies to explore resistance mechanisms mediated by Src/SFKs are required. Citation Format: Beatriz Rubio Cuesta, Beatriz Gil Calderón, Patricia LLamas Granda, Jacinto Sarmentero, Carlos Carretero Puche, María del Carmen Riesco, Rocío García Carbonero, Beatriz Soldevilla. Molecular characterization of BRAF V600E CRC in response to Src-targeted therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 355.

Abstract LB521: Antitumor activity of potent RAF inhibitors in solid tumors with activated RAS-RAF axis

Abstract FDA approved three RAF inhibitors for the treatment of tumors containing BRAFV600 mutations, but one of the major drawbacks of these type I RAF inhibitors is to activate MAPK signaling pathway, instead of inhibiting signaling, which is referred to as paradoxical activation. Such undesired paradoxical activation not only leads to renewed tumor growth but also spurs additional cancer growth in non-cancerous wild-type BRAF tissue. Plus, these first-generation RAF inhibitors targeting BRAFV600 mutants are unable to inhibit oncogenic RAF dimers. This has led to the development of type II RAF inhibitors such as belvarafenib and day101 to block the activity of multiple forms of RAF while avoiding paradoxical activation. Two lead-like stage compounds 1 and 2 were specifically designed as type II RAF inhibitors to have activity across RAF isoforms including BRAFV600E, BRAF wild-type, and CRAF, showing higher potency than competitors. Compounds 1 and 2 potently inhibited the growth of BRAFV600E melanoma cells and NRAS or KRAS mutant cancer cells. Compounds 1 and 2 promoted the formation of BRAF/CRAF heterodimers by directly binding to the RAF kinase domain like other type II RAF inhibitors and inhibited phosphorylation of downstream effectors MEK and ERK in a dose-dependent manner in RAS mutant cancer cells, suggesting less paradoxical activation liability. Compound 2 showed superior on-target inhibitory activity for BRAFV600E, BRAF wild-type, and CRAF than belvarafenib through RAF immunoprecipitation (IP) kinase assay. Inhibition of the RAF downstream signaling was also confirmed by quantifying the level of phospho-ERK in NRAS or KRAS mutant cancer cells. In HCT116 (KRASG13D) subcutaneous xenograft model, compound 1 showed tumor growth inhibition efficacy, suggesting a potential to address RAS mutant driven- as well as BRAFV600 mutant driven tumors. Also, combination treatment with MEK inhibitor and/or immune checkpoint inhibitor would further improve the therapeutic activity and expand target indication for unmet medical needs. Citation Format: Kwangwoo Hwang, SeoHyun Jo, Jieun Choi, Ga-young Choi, Jiseon Choi, Ji-Hye Kwon, Dong-Guk Shin, Jiyeon Kim, Se-Hyuk Kim, Haelee Kim, Ha Yeon Cho, Jung Beom Son, Nam Doo Kim, Hwan Geun Choi, Daekwon Kim, Sunghwan Kim. Antitumor activity of potent RAF inhibitors in solid tumors with activated RAS-RAF axis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB521.

Abstract CT248: Design and rationale of a first in human (FIH) phase 1/1b study evaluating KIN-2787, a potent and highly selective pan-RAF inhibitor, in adult patients with BRAF- and NRAS-mutation positive solid tumors

Abstract Background: BRAFV600 alterations (BRAF Class (Cl) I alterations) encode a highly active monomeric oncogenic kinase that can be targeted by approved RAF kinase inhibitors (RAFi) in patients (pts) with melanoma (MEL), NSCLC, colorectal &amp; anaplastic thyroid cancers. Cl II and III represent molecularly distinct BRAF alterations driving dimer-dependent signaling. Cl II alterations include single nucleotide variants (SNVs), indels and gene fusions that induce BRAF homodimer formation, leading to oncogenic kinase activation. Cl III alterations, typically SNVs, are low activity alterations that promote RAF dimerization and RAS-dependent oncogenic MAPK pathway signaling. Approved BRAFi have limited clinical activity in solid tumors driven by BRAF Cl II or III alterations, highlighting the urgency to develop effective targeted therapies for these patients. Combinations of a RAFi and a MEK inhibitor (MEKi) show promising results in early clinical trials. KIN-2787 is a potent, highly selective type II pan-RAFi designed to target both monomeric &amp; dimeric forms of the altered BRAF kinase and minimize paradoxical activation, a liability often observed with other RAFi that can adversely impact tolerability &amp; require addition of a MEKi to suppress pathway activation. KIN-2787 has favorable pharmaceutical properties, achieves substantial systemic exposures in pre-clinical toxicology studies, &amp; induces regressions in human cancer xenograft models driven by BRAF Cl I, II, or III alterations. Combinations of KIN-2787 with a MEKi show promising activity in preclinical models. Methods: This is a FIH, multicenter, non-randomized Ph1 study (NCT04913285) of KIN-2787 in adult pts with BRAF mutant advanced &amp; metastatic solid tumors (AMST). KIN-2787 will be given orally bid continuously in 28-day cycles until drug intolerance or disease progression. Planned sample size is approx. 155 pts in 2 parts: Part A is a dose-escalation to MTD open to pts with AMST driven by either BRAF genomic alterations (Cl I, II or III ) or by NRAS. Part A1 assesses single agent KIN-2787; Part A2 assesses KIN-2787 in combination w/binimetinib. Part B will evaluate a selected dose of KIN-2787 in 3 cohorts of pts (MEL, NSCLC, or other AMST), each driven by BRAF Cl II or III alterations. Standard Ph1 enrollment criteria are required (ECOG PS 0-2, normal organ function, prior receipt of standard treatment or medical judgment that such is not appropriate). Pts may have measurable or evaluable disease. Key exclusion criteria include known active brain metastases, prior BRAF-, MEK-, or MAPK inhibitor therapy (unless for FDA approved indications), HBV/HCV seropositivity. Primary endpoints are safety/tolerability (Part A), and preliminary antitumor activity: objective response rate, disease control rate, duration of response, &amp; duration of stable disease (Part B). Secondary objectives include pharmacokinetic and pharmacodynamic assessments including measures of MAPK signaling inhibition. Enrollment began in August 2022. Trial is open globally. Citation Format: Meredith McKean, Cesar A. Perez, Shumei Kato, Alexander I. Spira, Yan Xing, Hao Xie, Michael Millward, Anthony Olszanski, Ken Kobayashi, Nichol Miller, Paul Severson, Betty Tam, Caro Unger. Design and rationale of a first in human (FIH) phase 1/1b study evaluating KIN-2787, a potent and highly selective pan-RAF inhibitor, in adult patients with BRAF- and NRAS-mutation positive solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT248.

Abstract 5775: Landscape of BRAF and RAF1 fusions identified by next-generation sequencing in a Chinese multi-cancer retrospective analysis

Abstract Background: The RAF family includes three members, ARAF, BRAF, and RAF1/CRAF. These kinases function downstream of RAS as part of the MAPK (RAF-MEK-ERK) signaling cascade. The BRAF gene is mutated in ∼7% of cancers, whereas ARAF and RAF1 mutations are rare. Gene fusions in BRAF or RAF1 fusions, which activate the MAPK pathway, can be oncogenic drivers in a variety of cancer types and represent potential targets for targeted therapy. Here we focused on the prevalence of BRAF and RAF1 fusions in a multi-cancer cohort for patients (pts) whom might benefit from MEK/RAF inhibitors in clinical development. Methods: Genomic profiling was performed on samples from 29,102 lung cancer pts, 676 melanoma pts, 392 colorectal cancer pts, 1,463 gastric cancer pts, 1,014 sarcoma pts, 1,079 liver cancer pts, 215 gallbladder carcinoma pts, 297 ovarian cancer pts, 46 tongue cancer pts, 62 thymic carcinoma pts, 6 hemangioma pts, and 890 pancreatic cancer pts using next-generation sequencing (NGS), which covers all exons of BRAF and RAF1. The fusion patterns were analyzed. Results: A total of 5 RAF1 and 114 BRAF fusions were identified in approximately 0.34% (120/35,242) of the patients in this Chinese multi-cancer cohort. The positive rates of RAF1 fusions were 0.0034% (1/29,102) in lung cancer pts, 0.44% (3/676) in melanoma pts, and 0.10% (1/1,014) in sarcoma pts. The fusion partners of RAF1 were LYRM4 in lung cancer, LMCD1 and USP20 in melanoma, and FMR1 in sarcoma. RAF1 fusions involving the intact in-frame RAF1 kinase domain were LYRM4-RAF1, LMCD1-RAF1, and USP20-RAF1, which might be targeted with MEK/RAF inhibitors. The BRAF fusion-positive rates were 0.27% (80/29,102) in lung cancer pts, 1.18% (8/676) in melanoma pts, 2.81% (11/392) in colorectal cancer pts, 0.41% (6/1,463) in gastric cancer pts, 0.20% (2/1,014) in sarcoma pts, 0.19% (2/1,079) in liver cancer pts, 0.47% (1/215) in gallbladder carcinoma pts, 0.34% (1/297) in ovarian cancer pts, 2.17% (1/46) in tongue cancer pts, 1.61% (1/62) in thymic carcinoma pts, 16.67% (1/6) in hemangioma pts, and 0.11% (1/890) in pancreatic cancer pts. The most common 5’partners of BRAF were TRIM24 (4 cases) and SND1 (3 cases). Whereas the most common 3’partners of BRAF were BAIAP2L1 (5 cases) and TRIM24 (4 cases). Furthermore, 57 BRAF fusions contained intact BRAF kinase domain and nearly half of BRAF fusion-positive patients might be sensitive to MEK inhibitor. The most frequent location of the breakpoints occurred in intron 8 of the BRAF gene, followed by intron 7. However, the sensitivity of these fusions to MEK/RAF inhibitors in different cancers needs to be further studied. Conclusions: This study revealed the molecular features of BRAF and RAF1 rearrangements in Chinese cancer patients, which could help to identify therapeutic targets and personalize therapy management, leading to improved patient outcomes. Citation Format: Tao Li, Xin Zhang, Yanling Niu, Tonghui Ma. Landscape of BRAF and RAF1 fusions identified by next-generation sequencing in a Chinese multi-cancer retrospective analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5775.

PP2 protects from keratin mutation-associated liver injury and filament disruption via SRC kinase inhibition in male but not female mice.

Hepatocyte keratin polypeptides 8/18 (K8/K18) are unique among intermediate filaments proteins (IFs) in that their mutation predisposes to, rather than causes, human disease. Mice that overexpress human K18 R90C manifest disrupted hepatocyte keratin filaments with hyperphosphorylated keratins and predisposition to Fas-induced liver injury. We hypothesized that high-throughput screening will identify compounds that protect the liver from mutation-triggered predisposition to injury. Using A549 cells transduced with a lentivirus K18 construct and high-throughput screening, we identified the SRC-family tyrosine kinases inhibitor, PP2, as a compound that reverses keratin filament disruption and protects from apoptotic cell death caused by K18 R90C mutation at this highly conserved arginine. PP2 also ameliorated Fas-induced apoptosis and liver injury in male but not female K18 R90C mice. The PP2 male selectivity is due to its lower turnover in male versus female livers. Knockdown of SRC but not another kinase target of PP2, protein tyrosine kinase 6, in A549 cells abrogated the hepatoprotective effect of PP2. Phosphoproteomic analysis and validation showed that the protective effect of PP2 associates with Ser/Thr but not Tyr keratin hypophosphorylation, and differs from the sex-independent effect of the Ser/Thr kinase inhibitor PKC412. Inhibition of RAF kinase, a downstream target of SRC, by vemurafenib had a similar protective effect to PP2 in A549 cells and male K18 R90C mice. PP2 protects, in a male-selective manner, keratin mutation-induced mouse liver injury by inhibiting SRC-triggered downstream Ser/Thr phosphorylation of K8/K18, which is phenocopied by RAF kinase inhibitor vemurafenib. The PP2/vemurafenib-associated findings, and their unique mechanisms of action, further support the potential role of select kinase inhibition as therapeutic opportunities for keratin and other IF-associated human diseases.