Modelling vemurafenib resistance in melanoma reveals a strategy to forestall drug resistance.

Mutational activation of BRAF is the most prevalent genetic alteration in human melanoma, with ≥50% of tumours expressing the BRAF(V600E) oncoprotein. Moreover, the marked tumour regression and improved survival of late-stage BRAF-mutated melanoma patients in response to treatment with vemurafenib demonstrates the essential role of oncogenic BRAF in melanoma maintenance. However, as most patients relapse with lethal drug-resistant disease, understanding and preventing mechanism(s) of resistance is critical to providing improved therapy. Here we investigate the cause and consequences of vemurafenib resistance using two independently derived primary human melanoma xenograft models in which drug resistance is selected by continuous vemurafenib administration. In one of these models, resistant tumours show continued dependency on BRAF(V600E)→MEK→ERK signalling owing to elevated BRAF(V600E) expression. Most importantly, we demonstrate that vemurafenib-resistant melanomas become drug dependent for their continued proliferation, such that cessation of drug administration leads to regression of established drug-resistant tumours. We further demonstrate that a discontinuous dosing strategy, which exploits the fitness disadvantage displayed by drug-resistant cells in the absence of the drug, forestalls the onset of lethal drug-resistant disease. These data highlight the concept that drug-resistant cells may also display drug dependency, such that altered dosing may prevent the emergence of lethal drug resistance. Such observations may contribute to sustaining the durability of the vemurafenib response with the ultimate goal of curative therapy for the subset of melanoma patients with BRAF mutations.

Imidazo[1,2-a]pyridin-6-yl-benzamide analogs as potent RAF inhibitors.

A series of imidazo[1,2-a]pyridin-6-yl-benzamide analogs was designed as inhibitors of B-RAFV600E. Medicinal chemistry techniques were employed to explore the SAR for this series and improve selectivity versus P38 and VEGFR2.

Inhibition of tumorigenesis driven by different Wnt proteins requires blockade of distinct ligand-binding regions by LRP6 antibodies.

Disregulated Wnt/beta-catenin signaling has been linked to various human diseases, including cancers. Inhibitors of oncogenic Wnt signaling are likely to have a therapeutic effect in cancers. LRP5 and LRP6 are closely related membrane coreceptors for Wnt proteins. Using a phage-display library, we identified anti-LRP6 antibodies that either inhibit or enhance Wnt signaling. Two classes of LRP6 antagonistic antibodies were discovered: one class specifically inhibits Wnt proteins represented by Wnt1, whereas the second class specifically inhibits Wnt proteins represented by Wnt3a. Epitope-mapping experiments indicated that Wnt1 class-specific antibodies bind to the first propeller and Wnt3a class-specific antibodies bind to the third propeller of LRP6, suggesting that Wnt1- and Wnt3a-class proteins interact with distinct LRP6 propeller domains. This conclusion is further supported by the structural functional analysis of LRP5/6 and the finding that the Wnt antagonist Sclerostin interacts with the first propeller of LRP5/6 and preferentially inhibits the Wnt1-class proteins. We also show that Wnt1 or Wnt3a class-specific anti-LRP6 antibodies specifically block growth of MMTV-Wnt1 or MMTV-Wnt3 xenografts in vivo. Therapeutic application of these antibodies could be limited without knowing the type of Wnt proteins expressed in cancers. This is further complicated by our finding that bivalent LRP6 antibodies sensitize cells to the nonblocked class of Wnt proteins. The generation of a biparatopic LRP6 antibody blocks both Wnt1- and Wnt3a-mediated signaling without showing agonistic activity. Our studies provide insights into Wnt-induced LRP5/6 activation and show the potential utility of LRP6 antibodies in Wnt-driven cancer.

Precision Oncology Comes of Age: Designing Best-in-Class Small Molecules by Integrating Two Decades of Advances in Chemistry, Target Biology, and Data Science.

Small-molecule drugs have enabled the practice of precision oncology for genetically defined patient populations since the first approval of imatinib in 2001. Scientific and technology advances over this 20-year period have driven the evolution of cancer biology, medicinal chemistry, and data science. Collectively, these advances provide tools to more consistently design best-in-class small-molecule drugs against known, previously undruggable, and novel cancer targets. The integration of these tools and their customization in the hands of skilled drug hunters will be necessary to enable the discovery of transformational therapies for patients across a wider spectrum of cancers. SIGNIFICANCE: Target-centric small-molecule drug discovery necessitates the consideration of multiple approaches to identify chemical matter that can be optimized into drug candidates. To do this successfully and consistently, drug hunters require a comprehensive toolbox to avoid following the "law of instrument" or Maslow's hammer concept where only one tool is applied regardless of the requirements of the task. Combining our ever-increasing understanding of cancer and cancer targets with the technological advances in drug discovery described below will accelerate the next generation of small-molecule drugs in oncology.

STX-478, a Mutant-Selective, Allosteric PI3Kα Inhibitor Spares Metabolic Dysfunction and Improves Therapeutic Response in PI3Kα-Mutant Xenografts.

Phosphoinositide 3-kinase α (PIK3CA) is one of the most mutated genes across cancers, especially breast, gynecologic, and head and neck squamous cell carcinoma tumors. Mutations occur throughout the gene, but hotspot mutations in the helical and kinase domains predominate. The therapeutic benefit of isoform-selective PI3Kα inhibition was established with alpelisib, which displays equipotent activity against the wild-type and mutant enzyme. Inhibition of wild-type PI3Kα is associated with severe hyperglycemia and rash, which limits alpelisib use and suggests that selectively targeting mutant PI3Kα could reduce toxicity and improve efficacy. Here we describe STX-478, an allosteric PI3Kα inhibitor that selectively targets prevalent PI3Kα helical- and kinase-domain mutant tumors. STX-478 demonstrated robust efficacy in human tumor xenografts without causing the metabolic dysfunction observed with alpelisib. Combining STX-478 with fulvestrant and/or cyclin-dependent kinase 4/6 inhibitors was well tolerated and provided robust and durable tumor regression in ER+HER2- xenograft tumor models. SIGNIFICANCE: These preclinical data demonstrate that the mutant-selective, allosteric PI3Kα inhibitor STX-478 provides robust efficacy while avoiding the metabolic dysfunction associated with the nonselective inhibitor alpelisib. Our results support the ongoing clinical evaluation of STX-478 in PI3Kα-mutated cancers, which is expected to expand the therapeutic window and mitigate counterregulatory insulin release. See related commentary by Kearney and Vasan, p. 2313. This article is featured in Selected Articles from This Issue, p. 2293.

RAF-Mutant Melanomas Differentially Depend on ERK2 Over ERK1 to Support Aberrant MAPK Pathway Activation and Cell Proliferation.

Half of advanced human melanomas are driven by mutant BRAF and dependent on MAPK signaling. Interestingly, the results of three independent genetic screens highlight a dependency of BRAF-mutant melanoma cell lines on BRAF and ERK2, but not ERK1. ERK2 is expressed higher in melanoma compared with other cancer types and higher than ERK1 within melanoma. However, ERK1 and ERK2 are similarly required in primary human melanocytes transformed with mutant BRAF and are expressed at a similar, lower amount compared with established cancer cell lines. ERK1 can compensate for ERK2 loss as seen by expression of ERK1 rescuing the proliferation arrest mediated by ERK2 loss (both by shRNA or inhibition by an ERK inhibitor). ERK2 knockdown, as opposed to ERK1 knockdown, led to more robust suppression of MAPK signaling as seen by RNA-sequencing, qRT-PCR, and Western blot analysis. In addition, treatment with MAPK pathway inhibitors led to gene expression changes that closely resembled those seen upon knockdown of ERK2 but not ERK1. Together, these data demonstrate that ERK2 drives BRAF-mutant melanoma gene expression and proliferation as a function of its higher expression compared with ERK1. Selective inhibition of ERK2 for the treatment of melanomas may spare the toxicity associated with pan-ERK inhibition in normal tissues. IMPLICATIONS: BRAF-mutant melanomas overexpress and depend on ERK2 but not ERK1, suggesting that ERK2-selective inhibition may be toxicity sparing.

LXH254, a Potent and Selective ARAF-Sparing Inhibitor of BRAF and CRAF for the Treatment of MAPK-Driven Tumors.

PURPOSE: Targeting RAF for antitumor therapy in RAS-mutant tumors holds promise. Herein, we describe in detail novel properties of the type II RAF inhibitor, LXH254. EXPERIMENTAL DESIGN: LXH254 was profiled in biochemical, in vitro, and in vivo assays, including examining the activities of the drug in a large panel of cancer-derived cell lines and a comprehensive set of in vivo models. In addition, activity of LXH254 was assessed in cells where different sets of RAF paralogs were ablated, or that expressed kinase-impaired and dimer-deficient variants of ARAF. RESULTS: We describe an unexpected paralog selectivity of LXH254, which is able to potently inhibit BRAF and CRAF, but has less activity against ARAF. LXH254 was active in models harboring BRAF alterations, including atypical BRAF alterations coexpressed with mutant K/NRAS, and NRAS mutants, but had only modest activity in KRAS mutants. In RAS-mutant lines, loss of ARAF, but not BRAF or CRAF, sensitized cells to LXH254. ARAF-mediated resistance to LXH254 required both kinase function and dimerization. Higher concentrations of LXH254 were required to inhibit signaling in RAS-mutant cells expressing only ARAF relative to BRAF or CRAF. Moreover, specifically in cells expressing only ARAF, LXH254 caused paradoxical activation of MAPK signaling in a manner similar to dabrafenib. Finally, in vivo, LXH254 drove complete regressions of isogenic variants of RAS-mutant cells lacking ARAF expression, while parental lines were only modestly sensitive. CONCLUSIONS: LXH254 is a novel RAF inhibitor, which is able to inhibit dimerized BRAF and CRAF, as well as monomeric BRAF, while largely sparing ARAF.

Design and Discovery of N-(3-(2-(2-Hydroxyethoxy)-6-morpholinopyridin-4-yl)-4-methylphenyl)-2-(trifluoromethyl)isonicotinamide, a Selective, Efficacious, and Well-Tolerated RAF Inhibitor Targeting RAS Mutant Cancers: The Path to the Clinic.

Direct pharmacological inhibition of RAS has remained elusive, and efforts to target CRAF have been challenging due to the complex nature of RAF signaling, downstream of activated RAS, and the poor overall kinase selectivity of putative RAF inhibitors. Herein, we describe 15 (LXH254, Aversa, R.; et al. Int. Patent WO2014151616A1, 2014), a selective B/C RAF inhibitor, which was developed by focusing on drug-like properties and selectivity. Our previous tool compound, 3 (RAF709; Nishiguchi, G. A.; et al. J. Med. Chem. 2017, 60, 4969), was potent, selective, efficacious, and well tolerated in preclinical models, but the high human intrinsic clearance precluded further development and prompted further investigation of close analogues. A structure-based approach led to a pyridine series with an alcohol side chain that could interact with the DFG loop and significantly improved cell potency. Further mitigation of human intrinsic clearance and time-dependent inhibition led to the discovery of 15. Due to its excellent properties, it was progressed through toxicology studies and is being tested in phase 1 clinical trials.

Distinct Transcriptional Programming Drive Response to MAPK Inhibition in BRAF V600-Mutant Melanoma Patient-Derived Xenografts.

Inhibitors targeting BRAF and its downstream kinase MEK produce robust response in patients with advanced BRAF V600-mutant melanoma. However, the duration and depth of response vary significantly between patients; therefore, predicting response a priori remains a significant challenge. Here, we utilized the Novartis collection of patient-derived xenografts to characterize transcriptional alterations elicited by BRAF and MEK inhibitors in vivo, in an effort to identify mechanisms governing differential response to MAPK inhibition. We show that the expression of an MITF-high, "epithelial-like" transcriptional program is associated with reduced sensitivity and adaptive response to BRAF and MEK inhibitor treatment. On the other hand, xenograft models that express an MAPK-driven "mesenchymal-like" transcriptional program are preferentially sensitive to MAPK inhibition. These gene-expression programs are somewhat similar to the MITF-high and -low phenotypes described in cancer cell lines, but demonstrate an inverse relationship with drug response. This suggests a discrepancy between in vitro and in vivo experimental systems that warrants future investigations. Finally, BRAF V600-mutant melanoma relies on either MAPK or alternative pathways for survival under BRAF and MEK inhibition in vivo, which in turn predicts their response to further pathway suppression using a combination of BRAF, MEK, and ERK inhibitors. Our findings highlight the intertumor heterogeneity in BRAF V600-mutant melanoma, and the need for precision medicine strategies to target this aggressive cancer.

Hyperactivation of MAPK Signaling Is Deleterious to RAS/RAF-mutant Melanoma.

The most frequent genetic alterations in melanoma are gain-of-function (GOF) mutations in BRAF, which result in RAF-MEK-ERK signaling pathway addiction. Despite therapeutic success of RAF and MEK inhibitors in treating BRAFV600-mutant tumors, a major challenge is the inevitable emergence of drug resistance, which often involves reactivation of the MAPK pathway. Interestingly, resistant tumors are often sensitive to drug withdrawal, suggesting that hyperactivation of the MAPK pathway is not tolerated. To further characterize this phenomenon, isogenic models of inducible MAPK hyperactivation in BRAFV600E melanoma cells were generated by overexpression of ERK2. Using this model system, supraphysiologic levels of MAPK signaling led to cell death, which was reversed by MAPK inhibition. Furthermore, complete tumor regression was observed in an ERK2-overexpressing xenograft model. To identify mediators of MAPK hyperactivation-induced cell death, a large-scale pooled shRNA screen was conducted, which revealed that only shRNAs against BRAF and MAP2K1 rescued loss of cell viability. This suggested that no single downstream ERK2 effector was required, consistent with pleiotropic effects on multiple cellular stress pathways. Intriguingly, the detrimental effect of MAPK hyperactivation could be partially attributed to secreted factors, and more than 100 differentially secreted proteins were identified. The effect of ERK2 overexpression was highly context dependent, as RAS/RAF mutant but not RAS/RAF wild-type melanoma were sensitive to this perturbation. IMPLICATIONS: This vulnerability to MAPK hyperactivation raises the possibility of novel therapeutic approaches for RAS/RAF-mutant cancers.

Tumor Intrinsic Efficacy by SHP2 and RTK Inhibitors in KRAS-Mutant Cancers.

KRAS, an oncogene mutated in nearly one third of human cancers, remains a pharmacologic challenge for direct inhibition except for recent advances in selective inhibitors targeting the G12C variant. Here, we report that selective inhibition of the protein tyrosine phosphatase, SHP2, can impair the proliferation of KRAS-mutant cancer cells in vitro and in vivo using cell line xenografts and primary human tumors. In vitro, sensitivity of KRAS-mutant cells toward the allosteric SHP2 inhibitor, SHP099, is not apparent when cells are grown on plastic in 2D monolayer, but is revealed when cells are grown as 3D multicellular spheroids. This antitumor activity is also observed in vivo in mouse models. Interrogation of the MAPK pathway in SHP099-treated KRAS-mutant cancer models demonstrated similar modulation of p-ERK and DUSP6 transcripts in 2D, 3D, and in vivo, suggesting a MAPK pathway-dependent mechanism and possible non-MAPK pathway-dependent mechanisms in tumor cells or tumor microenvironment for the in vivo efficacy. For the KRASG12C MIAPaCa-2 model, we demonstrate that the efficacy is cancer cell intrinsic as there is minimal antiangiogenic activity by SHP099, and the effects of SHP099 is recapitulated by genetic depletion of SHP2 in cancer cells. Furthermore, we demonstrate that SHP099 efficacy in KRAS-mutant models can be recapitulated with RTK inhibitors, suggesting RTK activity is responsible for the SHP2 activation. Taken together, these data reveal that many KRAS-mutant cancers depend on upstream signaling from RTK and SHP2, and provide a new therapeutic framework for treating KRAS-mutant cancers with SHP2 inhibitors.

Allele-Specific Mechanisms of Activation of MEK1 Mutants Determine Their Properties.

Mutations at multiple sites in MEK1 occur in cancer, suggesting that their mechanisms of activation might be different. We analyzed 17 tumor-associated MEK1 mutants and found that they drove ERK signaling autonomously or in a RAS/RAF-dependent manner. The latter are sensitive to feedback inhibition of RAF, which limits their functional output, and often cooccur with RAS or RAF mutations. They act as amplifiers of RAF signaling. In contrast, another class of mutants deletes a hitherto unrecognized negative regulatory segment of MEK1, is RAF- and phosphorylation-independent, is unaffected by feedback inhibition of upstream signaling, and drives high ERK output and transformation in the absence of RAF activity. Moreover, these RAF-independent mutants are insensitive to allosteric MEK inhibitors, which preferentially bind to the inactivated form of MEK1. All the mutants are sensitive to an ATP-competitive MEK inhibitor. Thus, our study comprises a novel therapeutic strategy for tumors driven by RAF-independent MEK1 mutants.Significance: Mutants with which MEK1 mutants coexist and their sensitivity to inhibitors are determined by allele-specific properties. This study shows the importance of functional characterization of mutant alleles in single oncogenes and identifies a new class of MEK1 mutants, insensitive to current MEK1 inhibitors but treatable with a new ATP-competitive inhibitor. Cancer Discov; 8(5); 648-61. ©2018 AACR.See related commentary by Maust et al., p. 534This article is highlighted in the In This Issue feature, p. 517.

Antitumor Properties of RAF709, a Highly Selective and Potent Inhibitor of RAF Kinase Dimers, in Tumors Driven by Mutant RAS or BRAF.

Resistance to the RAF inhibitor vemurafenib arises commonly in melanomas driven by the activated BRAF oncogene. Here, we report antitumor properties of RAF709, a novel ATP-competitive kinase inhibitor with high potency and selectivity against RAF kinases. RAF709 exhibited a mode of RAF inhibition distinct from RAF monomer inhibitors such as vemurafenib, showing equal activity against both RAF monomers and dimers. As a result, RAF709 inhibited MAPK signaling activity in tumor models harboring either BRAFV600 alterations or mutant N- and KRAS-driven signaling, with minimal paradoxical activation of wild-type RAF. In cell lines and murine xenograft models, RAF709 demonstrated selective antitumor activity in tumor cells harboring BRAF or RAS mutations compared with cells with wild-type BRAF and RAS genes. RAF709 demonstrated a direct pharmacokinetic/pharmacodynamic relationship in in vivo tumor models harboring KRAS mutation. Furthermore, RAF709 elicited regression of primary human tumor-derived xenograft models with BRAF, NRAS, or KRAS mutations with excellent tolerability. Our results support further development of inhibitors like RAF709, which represents a next-generation RAF inhibitor with unique biochemical and cellular properties that enables antitumor activities in RAS-mutant tumors.Significance: In an effort to develop RAF inhibitors with the appropriate pharmacological properties to treat RAS mutant tumors, RAF709, a compound with potency, selectivity, and in vivo properties, was developed that will allow preclinical therapeutic hypothesis testing, but also provide an excellent probe to further unravel the complexities of RAF kinase signaling. Cancer Res; 78(6); 1537-48. ©2018 AACR.

Phase I Dose-Escalation and -Expansion Study of the BRAF Inhibitor Encorafenib (LGX818) in Metastatic BRAF-Mutant Melanoma.

Purpose: Encorafenib, a selective BRAF inhibitor (BRAFi), has a pharmacologic profile that is distinct from that of other clinically active BRAFis. We evaluated encorafenib in a phase I study in patients with BRAFi treatment-naïve and pretreated BRAF-mutant melanoma.Experimental Design: The pharmacologic activity of encorafenib was first characterized preclinically. Encorafenib monotherapy was then tested across a range of once-daily (50-700 mg) or twice-daily (75-150 mg) regimens in a phase I, open-label, dose-escalation and -expansion study in adult patients with histologically confirmed advanced/metastatic BRAF-mutant melanoma. Study objectives were to determine the maximum tolerated dose (MTD) and/or recommended phase II dose (RP2D), characterize the safety and tolerability and pharmacokinetic profile, and assess the preliminary antitumor activity of encorafenib.Results: Preclinical data demonstrated that encorafenib inhibited BRAF V600E kinase activity with a prolonged off-rate and suppressed proliferation and tumor growth of BRAF V600E-mutant melanoma models. In the dose-escalation phase, 54 patients (29 BRAFi-pretreated and 25 BRAFi-naïve) were enrolled. Seven patients in the dose-determining set experienced dose-limiting toxicities. Encorafenib at a dose of 300 mg once daily was declared the RP2D. In the expansion phase, the most common all-cause adverse events were nausea (66%), myalgia (63%), and palmar-plantar erythrodysesthesia (54%). In BRAFi-naïve patients, the overall response rate (ORR) and median progression-free survival (mPFS) were 60% and 12.4 months [95% confidence interval (CI), 7.4-not reached (NR)]. In BRAFi-pretreated patients, the ORR and mPFS were 22% and 1.9 months (95% CI, 0.9-3.7).Conclusions: Once-daily dosing of single-agent encorafenib had a distinct tolerability profile and showed varying antitumor activity across BRAFi-pretreated and BRAFi-naïve patients with advanced/metastatic melanoma. Clin Cancer Res; 23(18); 5339-48. ©2017 AACR.

Design and Discovery of N-(2-Methyl-5'-morpholino-6'-((tetrahydro-2H-pyran-4-yl)oxy)-[3,3'-bipyridin]-5-yl)-3-(trifluoromethyl)benzamide (RAF709): A Potent, Selective, and Efficacious RAF Inhibitor Targeting RAS Mutant Cancers.

RAS oncogenes have been implicated in >30% of human cancers, all representing high unmet medical need. The exquisite dependency on CRAF kinase in KRAS mutant tumors has been established in genetically engineered mouse models and human tumor cells. To date, many small molecule approaches are under investigation to target CRAF, yet kinase-selective and cellular potent inhibitors remain challenging to identify. Herein, we describe 14 (RAF709) [ Aversa , Biaryl amide compounds as kinase inhibitors and their preparation . WO 2014151616, 2014 ], a selective B/C RAF inhibitor, which was developed through a hypothesis-driven approach focusing on drug-like properties. A key challenge encountered in the medicinal chemistry campaign was maintaining a balance between good solubility and potent cellular activity (suppression of pMEK and proliferation) in KRAS mutant tumor cell lines. We investigated the small molecule crystal structure of lead molecule 7 and hypothesized that disruption of the crystal packing would improve solubility, which led to a change from N-methylpyridone to a tetrahydropyranyl oxy-pyridine derivative. 14 proved to be soluble, kinase selective, and efficacious in a KRAS mutant xenograft model.

Inhibition of prenylated KRAS in a lipid environment.

RAS mutations lead to a constitutively active oncogenic protein that signals through multiple effector pathways. In this chemical biology study, we describe a novel coupled biochemical assay that measures activation of the effector BRAF by prenylated KRASG12V in a lipid-dependent manner. Using this assay, we discovered compounds that block biochemical and cellular functions of KRASG12V with low single-digit micromolar potency. We characterized the structural basis for inhibition using NMR methods and showed that the compounds stabilized the inactive conformation of KRASG12V. Determination of the biophysical affinity of binding using biolayer interferometry demonstrated that the potency of inhibition matches the affinity of binding only when KRAS is in its native state, namely post-translationally modified and in a lipid environment. The assays we describe here provide a first-time alignment across biochemical, biophysical, and cellular KRAS assays through incorporation of key physiological factors regulating RAS biology, namely a negatively charged lipid environment and prenylation, into the in vitro assays. These assays and the ligands we discovered are valuable tools for further study of KRAS inhibition and drug discovery.

Discovery of a Selective and Potent Inhibitor of Mitogen-Activated Protein Kinase-Interacting Kinases 1 and 2 (MNK1/2) Utilizing Structure-Based Drug Design.

The discovery of a highly potent and selective small molecule inhibitor 9 for in vitro target validation of MNK1/2 kinases is described. The aminopyrazine benzimidazole series was derived from an HTS hit and optimized by utilization of a docking model, conformation analysis, and binding pocket comparison against antitargets.

Ligand-independent EPHA2 signaling drives the adoption of a targeted therapy-mediated metastatic melanoma phenotype.

UNLABELLED: Many patients with BRAF inhibitor resistance can develop disease at new sites, suggesting that drug-induced selection pressure drives metastasis. Here, we used mass spectrometry-based phosphoproteomic screening to uncover ligand-independent EPHA2 signaling as an adaptation to BRAF inhibitor therapy that led to the adoption of a metastatic phenotype. The EPHA2-mediated invasion was AKT-dependent and readily reversible upon removal of the drug as well as through PI3K and AKT inhibition. In xenograft models, BRAF inhibition led to the development of EPHA2-positive metastases. A retrospective analysis of patients with melanoma on BRAF inhibitor therapy showed that 68% of those failing therapy develop metastases at new disease sites, compared with 35% of patients on dacarbazine. Further IHC staining of melanoma specimens taken from patients on BRAF inhibitor therapy as well as metastatic samples taken from patients failing therapy showed increased EPHA2 staining. We suggest that inhibition of ligand-independent EPHA2 signaling may limit metastases associated with BRAF inhibitor therapy. SIGNIFICANCE: This study provides evidence that BRAF inhibition promotes the adoption of a reversible, therapy-driven metastatic phenotype in melanoma. The cotargeting of ligand-independent EPHA2 signaling and BRAF may be one strategy to prevent the development of therapy-mediated disease at new sites.

Discovery of RAF265: A Potent mut-B-RAF Inhibitor for the Treatment of Metastatic Melanoma.

Abrogation of errant signaling along the MAPK pathway through the inhibition of B-RAF kinase is a validated approach for the treatment of pathway-dependent cancers. We report the development of imidazo-benzimidazoles as potent B-RAF inhibitors. Robust in vivo efficacy coupled with correlating pharmacokinetic/pharmacodynamic (PKPD) and PD-efficacy relationships led to the identification of RAF265, 1, which has advanced into clinical trials.

Molecular pathways: response and resistance to BRAF and MEK inhibitors in BRAF(V600E) tumors.

The RAS-RAF-MEK (MAP-ERK kinase)-ERK (extracellular signal-regulated kinase) pathway plays a central role in driving proliferation, survival, and metastasis signals in tumor cells, and the prevalence of oncogenic mutations in RAS and BRAF and upstream nodes makes this pathway the focus of significant oncology drug development efforts. This focus has been justified by the recent success of BRAF and MEK inhibitors in prolonging the lives of patients with BRAF(V600E/K)-mutant melanoma. Although it is disappointing that cures are relatively rare, this should not detract from the value of these agents to patients with cancer and the opportunity they provide in allowing us to gain a deeper understanding of drug response and resistance. These insights have already provided the basis for the evaluation of alternative dosing regimens and combination therapies in patients with melanoma.