RESUMEN
The type II class of RAF inhibitors currently in clinical trials paradoxically activate BRAF at subsaturating concentrations. Activation is mediated by induction of BRAF dimers, but why activation rather than inhibition occurs remains unclear. Using biophysical methods tracking BRAF dimerization and conformation, we built an allosteric model of inhibitor-induced dimerization that resolves the allosteric contributions of inhibitor binding to the two active sites of the dimer, revealing key differences between type I and type II RAF inhibitors. For type II inhibitors the allosteric coupling between inhibitor binding and BRAF dimerization is distributed asymmetrically across the two dimer binding sites, with binding to the first site dominating the allostery. This asymmetry results in efficient and selective induction of dimers with one inhibited and one catalytically active subunit. Our allosteric models quantitatively account for paradoxical activation data measured for 11 RAF inhibitors. Unlike type II inhibitors, type I inhibitors lack allosteric asymmetry and do not activate BRAF homodimers. Finally, NMR data reveal that BRAF homodimers are dynamically asymmetric with only one of the subunits locked in the active αC-in state. This provides a structural mechanism for how binding of only a single αC-in inhibitor molecule can induce potent BRAF dimerization and activation.
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Inhibidores de Proteínas Quinasas , Multimerización de Proteína , Proteínas Proto-Oncogénicas B-raf , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/química , Regulación Alostérica/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/metabolismo , Multimerización de Proteína/efectos de los fármacos , Humanos , Conformación Proteica , Unión Proteica , Modelos MolecularesRESUMEN
Rapidly accelerated fibrosarcoma (ARAF, BRAF, CRAF) kinase is central to the MAPK pathway (RAS-RAF-MEK-ERK). Inactive RAF kinase is believed to be monomeric, autoinhibited, and cytosolic, while activated RAF is recruited to the membrane via RAS-GTP, leading to the relief of autoinhibition, phosphorylation of key regulatory sites, and dimerization of RAF protomers. Although it is well known that active and inactive BRAF have differential phosphorylation sites that play a crucial role in regulating BRAF, key details are still missing. In this study, we report the characterization of a novel phosphorylation site, BRAFS732 (equivalent in CRAFS624), located in proximity to the C-terminus binding motif for the 14-3-3 scaffolding protein. At the C terminus, 14-3-3 binds to BRAFpS729 (CRAFpS621) and enhances RAF dimerization. We conducted mutational analysis of BRAFS732A/E and CRAFS624A/E and revealed that the phosphomimetic SâE mutant decreases 14-3-3 association and RAF dimerization. In normal cell signaling, dimerized RAF phosphorylates MEK1/2, which is observed in the phospho-deficient SâA mutant. Our results suggest that phosphorylation and dephosphorylation of this site fine-tune the association of 14-3-3 and RAF dimerization, ultimately impacting MEK phosphorylation. We further characterized the BRAF homodimer and BRAF:CRAF heterodimer and identified a correlation between phosphorylation of this site with drug sensitivity. Our work reveals a novel negative regulatory role for phosphorylation of BRAFS732 and CRAFS624 in decreasing 14-3-3 association, dimerization, and MEK phosphorylation. These findings provide insight into the regulation of the MAPK pathway and may have implications for cancers driven by mutations in the pathway.
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The role of BRAF in tumor initiation has been established, however, the precise mechanism of autoinhibition has only been illustrated recently by several structural studies. These structures uncovered the basis by which the regulatory domains engage in regulating the activity of BRAF kinase domain, which lead to a more complete picture of the regulation cycle of RAF kinases. Small molecule BRAF inhibitors developed specifically to target BRAFV600E have proven effective at inhibiting the most dominant BRAF mutant in melanomas, but are less potent against other BRAF mutants in RAS-driven diseases due to paradoxical activation of the MAPK pathway. A variety of new generation inhibitors that do not show paradoxical activation have been developed. Alternatively, efforts have begun to develop inhibitors targeting the dimer interface of BRAF. A deeper understanding of BRAF regulation together with more diverse BRAF inhibitors will be beneficial for drug development in RAF or RASdriven cancers.
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Melanoma , Proteínas Proto-Oncogénicas B-raf , Humanos , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Línea Celular Tumoral , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/química , Melanoma/tratamiento farmacológico , Melanoma/genética , Melanoma/metabolismo , MutaciónRESUMEN
Current experimental and clinical data are inadequate to conclusively predict the oncogenicity of uncommon BRAF mutants and their sensitivity towards kinase inhibitors. Therefore, the present study aims at estimating sensitivity profiles of uncommon lung cancer specific BRAF mutations towards clinically approved as well as experimental therapeutics based on computationally derived direct binding energies. Based on the data derived from cBioportal, BRAF mutants displayed significant mutual exclusivity with KRAS and EGFR mutants indicating them as potential drivers in lung cancer. Predicted sensitivity of BRAF-V600E conformed to published experimental and clinical data thus validating the usefulness of computational approach. The BRAF-V600K displayed higher sensitivity to most inhibitors as compared to that of the BRAF-V600E. All the uncommon mutants displayed higher sensitivity than both the wild type and BRAF-V600E towards PLX 8394 and LSN3074753. While V600K, G469R and N581S displayed favorable sensitivity profiles to most inhibitors, V600L/M, G466A/E/V and G469A/V displayed resistance profiles to a variable degree. Notably, molecular dynamic (MD) simulation revealed that increased number of interactions caused enhanced sensitivity of G469R and N581S towards sorafenib. RAF kinase inhibitors were further classified into two groups as per their selectivity (Group I: BRAF-V600E-selective and Group II: CRAF-selective) based on which potential mutation-wise combinations of RAF kinase inhibitors were proposed to overcome resistance. Based on computational inhibitor sensitivity profiles, appropriate treatment strategies may be devised to prevent or overcome secondary drug resistance in lung cancer patients with uncommon mutations.
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Neoplasias Pulmonares , Proteínas Proto-Oncogénicas B-raf , Línea Celular Tumoral , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Mutación , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Sorafenib/farmacología , Sorafenib/uso terapéuticoRESUMEN
The development of RAF inhibitors targeting cancers with wild type RAF kinase and/or RAS mutation has been challenging due to the paradoxical activation of the RAS-RAF-MEK-ERK cascade following RAF inhibitor treatment. Herein is the discovery and optimization of a series of RAF inhibitors with a novel spiro structure. The most potent spiro molecule 9 showed excellent in vitro potency against b/c RAF enzymes and RAS mutant H358 cancer cells with minimal paradoxical RAF signaling activation. Compound 9 also exhibited good drug-like properties as demonstrated by in vitro cytochrome P450 (CYP), liver microsome stability (LMS) data and moderate oral pharmacokinetics (PK) profiles in rat and mouse.
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Neoplasias , Compuestos de Espiro , Animales , Línea Celular Tumoral , Sistema de Señalización de MAP Quinasas , Ratones , Mutación , Inhibidores de Proteínas Quinasas/química , Proteínas Proto-Oncogénicas B-raf , Proteínas Proto-Oncogénicas p21(ras)/genética , Ratas , Compuestos de Espiro/farmacologíaRESUMEN
The RAS-RAF-MEK-ERK signaling pathway plays a key role to regulate multiple cellular functions. Acquired resistance to the first-generation RAF inhibitors that only targeted the bRAFV600E mutation prompted the need for a new generation of RAF inhibitors to target cancers bearing mutant RAS and wild type RAF activity by inhibition of paradoxical activation. Starting from the company's previously reported RAF inhibitor 1, extensive drug potency and drug-like properties optimizations led to the discovery of molecule 33 (SHR902275) with greatly improved in vitro potency and solubility. Molecule 33 exhibited good DMPK (Drug Metabolism and Pharmacokinetics) properties, excellent permeability, and outstanding mouse/rat oral PK. It was further evaluated in an in vivo RAS mutant Calu6 xenograft mouse model and demonstrated dose dependent efficacy. To achieve high exposure in a toxicity study, pro-drug 48 was also explored.
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Antineoplásicos/farmacología , Descubrimiento de Drogas , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Ratones , Ratones Desnudos , Modelos Moleculares , Estructura Molecular , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Proteínas Proto-Oncogénicas B-raf/metabolismo , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/metabolismo , Relación Estructura-ActividadRESUMEN
ATP-competitive inhibitors have been developed as promising anti-cancer agents. However, drug-resistance frequently occurs, and the underlying mechanisms are not fully understood. Here, we show that the activation of c-Src and its downstream phosphorylation cascade can be paradoxically induced by Src-targeted and RTK-targeted kinase inhibitors. We reveal that inhibitor binding induces a conformational change in c-Src, leading to the association of the active form c-Src with focal adhesion kinase (FAK). Reduction of the inhibitor concentration results in the dissociation of inhibitors from the c-Src-FAK complex, which allows c-Src to phosphorylate FAK and initiate FAK-Grb2-mediated Erk signaling. Furthermore, a drug-resistant mutation in c-Src, which reduces the affinity of inhibitors for c-Src, converts Src inhibitors into facilitators of cell proliferation by enhancing the phosphorylation of FAK and Erk in c-Src-mutated cells. Our data thus reveal paradoxical enhancement of cell growth evoked by target-based kinase inhibitors, providing potentially important clues for the future development of effective and safe cancer treatment.
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Resistencia a Antineoplásicos , Familia-src Quinasas/metabolismo , Animales , Secuencia de Bases , Dasatinib/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Adhesiones Focales/efectos de los fármacos , Adhesiones Focales/metabolismo , Proteína Adaptadora GRB2/metabolismo , Humanos , Ligandos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Células MCF-7 , Modelos Biológicos , Mutación/genética , Unión Proteica/efectos de los fármacos , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Xenopus , Dominios Homologos src , Familia-src Quinasas/antagonistas & inhibidores , Familia-src Quinasas/genéticaRESUMEN
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.
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Inhibidores de Proteínas Quinasas/farmacología , Quinasas raf/antagonistas & inhibidores , Animales , Antineoplásicos/farmacología , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Multimerización de Proteína/efectos de los fármacos , Multimerización de Proteína/fisiología , Estructura Secundaria de Proteína/efectos de los fármacos , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/fisiología , Quinasas raf/química , Quinasas raf/metabolismoRESUMEN
ERK5 is a protein kinase that also contains a nuclear localisation signal and a transcriptional transactivation domain. Inhibition of ERK5 has therapeutic potential in cancer and inflammation and this has prompted the development of ERK5 kinase inhibitors (ERK5i). However, few ERK5i programmes have taken account of the ERK5 transactivation domain. We have recently shown that the binding of small molecule ERK5i to the ERK5 kinase domain stimulates nuclear localisation and paradoxical activation of its transactivation domain. Other kinase inhibitors paradoxically activate their intended kinase target, in some cases leading to severe physiological consequences highlighting the importance of mitigating these effects. Here, we review the assays used to monitor ERK5 activities (kinase and transcriptional) in cells, the challenges faced in development of small molecule inhibitors to the ERK5 pathway, and classify the molecular mechanisms of paradoxical activation of protein kinases by kinase inhibitors.
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Inhibidores Enzimáticos/farmacología , Regulación Enzimológica de la Expresión Génica , Proteína Quinasa 7 Activada por Mitógenos/antagonistas & inhibidores , Adenosina Difosfato/química , Adenosina Trifosfato/química , Animales , Núcleo Celular/metabolismo , Glutatión/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Inflamación , Factores de Transcripción MEF2/metabolismo , Modelos Moleculares , Fosforilación , Conformación Proteica , Dominios Proteicos , Factor de Transcripción AP-1/metabolismo , Activación TranscripcionalRESUMEN
Class 3 mutations in B-Raf proto-oncogene, Ser/Thr kinase (BRAF), that result in kinase-impaired or kinase-dead BRAF have the highest mutation frequency in BRAF gene in lung adenocarcinoma. Several studies have reported that kinase-dead BRAF variants amplify mitogen-activated protein kinase (MAPK) signaling by dimerizing with and activating WT C-Raf proto-oncogene, Ser/Thr kinase (CRAF). However, the structural and functional principles underlying their activation remain elusive. Herein, using cell biology and various biochemical approaches, we established that variant BRAFD594G, a kinase-dead representative of class 3 mutation-derived BRAF variants, has a higher dimerization potential as compared with WT BRAF. Molecular dynamics simulations uncovered that the D594G substitution orients the αC-helix toward the IN position and extends the activation loop within the kinase domain, shifting the equilibrium toward the active, dimeric conformation, thus priming BRAFD594G as an effective allosteric activator of CRAF. We found that B/CRAF heterodimers are the most thermodynamically stable RAF dimers, suggesting that RAF heterodimers, and not homodimers, are the major players in determining the amplitude of MAPK signaling in cells. Additionally, we show that BRAFD594G:CRAF heterodimers bypass autoinhibitory P-loop phosphorylation, which might contribute to longer duration of MAPK pathway signaling in cancer cells. Last, we propose that the dimer interface of the BRAFD594G:CRAF heterodimer may represent a promising target in the design of novel anticancer therapeutics.
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Sistema de Señalización de MAP Quinasas , Mutación/genética , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas 14-3-3/metabolismo , Adenosina Trifosfato/metabolismo , Dominio Catalítico , Línea Celular , Humanos , Enlace de Hidrógeno , Modelos Biológicos , Fosforilación , Multimerización de Proteína , Estabilidad Proteica , Estructura Secundaria de Proteína , Proteolisis , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas B-raf/química , Proteínas Proto-Oncogénicas c-raf/metabolismoRESUMEN
The RAS/RAF/MEK/ERK (MAPK) signaling cascade is essential for cell inter- and intra-cellular communication, which regulates fundamental cell functions such as growth, survival, and differentiation. The MAPK pathway also integrates signals from complex intracellular networks in performing cellular functions. Despite the initial discovery of the core elements of the MAPK pathways nearly four decades ago, additional findings continue to make a thorough understanding of the molecular mechanisms involved in the regulation of this pathway challenging. Considerable effort has been focused on the regulation of RAF, especially after the discovery of drug resistance and paradoxical activation upon inhibitor binding to the kinase. RAF activity is regulated by phosphorylation and conformation-dependent regulation, including auto-inhibition and dimerization. In this review, we summarize the recent major findings in the study of the RAS/RAF/MEK/ERK signaling cascade, particularly with respect to the impact on clinical cancer therapy.
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Antineoplásicos/farmacología , Inhibidores Enzimáticos/farmacología , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Transducción de Señal/efectos de los fármacos , Quinasas raf/antagonistas & inhibidores , Proteínas ras/antagonistas & inhibidores , Animales , Humanos , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Neoplasias/metabolismo , Quinasas raf/metabolismo , Proteínas ras/metabolismoRESUMEN
BRAF fusions are detected in numerous neoplasms, but their clinical management remains unresolved. We identified six melanoma lines harboring BRAF fusions representative of the clinical cases reported in the literature. Their unexpected heterogeneous responses to RAF and MEK inhibitors could be categorized upon specific features of the fusion kinases. Higher expression level correlated with resistance, and fusion partners containing a dimerization domain promoted paradoxical activation of the mitogen-activated protein kinase (MAPK) pathway and hyperproliferation in response to first- and second-generation RAF inhibitors. By contrast, next-generation αC-IN/DFG-OUT RAF inhibitors blunted paradoxical activation across all lines and had their therapeutic efficacy further increased in vitro and in vivo by combination with MEK inhibitors, opening perspectives in the clinical management of tumors harboring BRAF fusions.
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Resistencia a Antineoplásicos/genética , Melanoma/patología , Proteínas de Fusión Oncogénica/metabolismo , Proteínas Proto-Oncogénicas B-raf/genética , Animales , Dimerización , Resistencia a Antineoplásicos/efectos de los fármacos , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Melanoma/genética , Ratones , Ratones Desnudos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas de Fusión Oncogénica/antagonistas & inhibidores , Proteínas de Fusión Oncogénica/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Vemurafenib/farmacología , Proteínas ras/genética , Proteínas ras/metabolismoRESUMEN
The dimerization-driven paradoxical activation of RAF proto-oncogene Ser/Thr kinase (RAF) is the predominant cause of drug resistance and toxicity in cancer therapies with RAF inhibitors. The scaffold protein 14-3-3, which binds to the RAF C terminus, is essential for RAF activation under physiological conditions, but the molecular basis is unclear. Here we investigated whether and how 14-3-3 regulates the dimerization-driven transactivation of the RAF isoform CRAF by RAF inhibitors and affects drug resistance and toxicity by virtue of the dominant role of CRAF in these processes. We demonstrated that 14-3-3 enhances the dimerization-driven transactivation of CRAF by stabilizing CRAF dimers. Further, we identified AMP-activated protein kinase (AMPK) and CRAF itself as two putative kinases that redundantly phosphorylate CRAF's C terminus and thereby control its association with 14-3-3. Next, we determined whether the combinatory inhibition of AMPK and CRAF could overcome the paradoxical effect of RAF inhibitors. We found that the AMPK inhibitor (AMPKi) not only blocked the RAF inhibitor-driven paradoxical activation of ERK signaling and cellular overgrowth in Ras-mutated cancer cells by blocking phosphorylation of Ser-621 in CRAF but also reduced the formation of drug-resistant clones of BRAFV600E-mutated cancer cells. Last, we investigated whether 14-3-3 binding to the C terminus of CRAF is required for CRAF catalytic activity and observed that it was dispensable in vivo Altogether, our study unravels the molecular mechanism by which 14-3-3 regulates dimerization-driven RAF activation and identified AMPKi as a potential agent to counteract drug resistance and adverse effects of RAF inhibitors in cancer therapies.
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Adenilato Quinasa/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-raf/antagonistas & inhibidores , Serina/metabolismo , Proteínas 14-3-3/metabolismo , Línea Celular Tumoral , Dimerización , Células HEK293 , Humanos , Fosforilación , Unión Proteica , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-raf/química , Proteínas Proto-Oncogénicas c-raf/metabolismo , Serina/química , Transducción de SeñalRESUMEN
Clostridium perfringens toxin TpeL belongs to the family of large clostridial glycosylating toxins. The toxin causes N-acetylglucosaminylation of Ras proteins at threonine35 thereby inactivating the small GTPases. Here, we show that all main types of oncogenic Ras proteins (H-Ras, K-Ras and N-Ras) are modified by the toxin in vitro and in vivo. Toxin-catalyzed modification of Ras was accompanied by inhibition of the MAP kinase pathway. Importantly, TpeL inhibited the paradoxical activation of the MAP kinase pathway induced by the BRAF inhibitor Vemurafenib in the human melanoma cell line SBCL2. The toxin also blocked Ras signaling in a zebrafish embryo model expressing oncogenic H-RasG12V, resulting in a reduction of melanocyte number. By using the binding and translocation component of anthrax toxin (protective antigen), the glucosyltransferase domain of TpeL was effectively introduced into target cells that were not sensitive to native TpeL toxin. To reach a higher specificity towards cancer cells, a chimeric TpeL toxin was engineered that possessed the knob region of adenovirus serotype 35 fiber, which interacts with CD46 of target cells frequently overexpressed in cancer cells. The chimeric TpeL fusion toxin efficiently inhibited Ras and MAP kinases in human pancreatic cancer Capan-2 cells, which were insensitive to the wild-type toxin. The data reveal that TpeL and TpeL-related immunotoxins provide a new toolset as Ras-inactivating agents.
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BRAF inhibitors (BRAFi) are standard of care for the treatment of BRAF V600 mutation-driven metastatic melanoma, but can lead to paradoxical activation of the mitogen-activated protein kinase (MAPK) signalling pathway. This can result in the promotion of precancerous lesions and secondary neoplasms, mainly (but not exclusively) associated with pre-existing mutations in RAS genes. We previously reported a patient with synchronous BRAF-mutated metastatic melanoma and BRAF wt /KRAS G12D-metastatic colorectal cancer (CRC), whose CRC relapsed and progressed when treated with the BRAF inhibitor dabrafenib (GSK2118436). We used tissue from the resected CRC metastasis to derive a cell line, LM-COL-1, which directly and reliably mimicked the clinical scenario including paradoxical activation of the MAPK signalling pathway resulting in increased cell proliferation upon dabrafenib treatment. Novel BRAF inhibitors (PLX8394 and PLX7904), dubbed as "paradox breakers", were developed to inhibit V600 mutated oncogenic BRAF without causing paradoxical MAPK pathway activation. In this study we used our LM-COL-1 model alongside multiple other CRC cell lines with varying mutational backgrounds to demonstrate and confirm that the paradox breaker PLX8394 retains on-target inhibition of mutated BRAF V600 without paradoxically promoting MAPK signalling.
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Adenocarcinoma/tratamiento farmacológico , Neoplasias del Colon/tratamiento farmacológico , Compuestos Heterocíclicos con 2 Anillos/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Sulfonamidas/farmacología , Adenocarcinoma/metabolismo , Antineoplásicos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Neoplasias del Colon/metabolismo , Humanos , Indoles/farmacología , Melanoma/tratamiento farmacológico , Melanoma/genética , Melanoma/patología , Mutación , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , VemurafenibRESUMEN
Patients with Neurofibromatosis type 1 (NF1) and Neurofibromatosis type 2 (NF2) are predisposed to tumors of the nervous system. NF1 patients predominantly develop neurofibromas, and Malignant Peripheral Nerve Sheath Tumors (MPNST) while NF2 patients develop schwannomas and meningiomas. Here we quantified the drug sensitivities of NF1 and NF2 tumor cell lines in a high throughput platform. The platform contained a comprehensive collection of inhibitors of MEK, RAF, RAS, farnesyl transferase, PAK and ERK, representative drugs against many other cancer pathways including Wnt, Hedgehog, p53, EGF, HDAC, as well as classical cytotoxic agents recommended for treating MPNST, such as doxorubicin and etoposide. We profiled seven NF1-associated MPNST cell lines (ST88-14, ST88-3, 90-8, sNF02.2, T265, S462TY, SNF96.2), one sporadic MPNST cell line (STS26), one schwannoma from a NF2 patient (HEI193), one NF2-deficient malignant meningioma (KT21-MG-Luc5D), one mouse NF2 schwannoma (SC4) and one sporadic rat schwannoma (RT4-67 or RT4). NF1 cells were primarily distinguished from NF2 cells and the sporadic MPNST cell line by their sensitivity to MEK and ERK inhibitors, and to a smaller extent their sensitivity to BH3 mimetics and farnesyl transferase inhibitors. The platform was highly successful in predicting the effects of clinical trials for Neurofibromas.
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Protein kinases are versatile molecule switches that govern functional processes in signal transduction networks and regulate fundamental biological processes of cell cycle and organism development. The continuous growth of biological information and a remarkable breath of structural, genetic, and pharmacological studies on protein kinase genes have significantly advanced our knowledge of the kinase activation, drug binding and allosteric mechanisms underlying kinase regulation and interactions in signaling cascades.. Structural and biochemical studies of the genetic and molecular determinants of protein kinases binding with inhibitors have been the cornerstone of drug discovery efforts in clinical oncology leading to proliferation of effective anticancer therapies. Recent advances in understanding allosteric regulation of protein kinases have fueled unprecedented efforts aiming in the discovery of targeted and allosteric kinase inhibitors that can combat cancer mutants and are at the forefront of the precision medicine initiative in oncology. Despite diversity of regulatory scenarios underlying kinase functions, dimerization-driven activation is a common mechanism of allosteric regulation that is shared by many protein kinase families, most notably ErbB and BRAF kinases that play a central role in growth factor signaling and human disease. In this review, we focused on structural, biochemical and computational studies of the ErbB and BRAF kinases and discuss how diversity of the structural landscape for these kinase genes and dimerization- dependent mechanisms of their regulation can be leveraged in the design and discovery of kinase inhibitors and allosteric modulators of kinase activation. The lessons from this analysis could inform discovery of specific targeted therapies and robust drug combinations for cancer treatment.
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Descubrimiento de Drogas , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Regulación Alostérica/efectos de los fármacos , Humanos , Estructura Molecular , Inhibidores de Proteínas Quinasas/química , Bibliotecas de Moléculas Pequeñas/químicaRESUMEN
The Reproducibility Project: Cancer Biology seeks to address growing concerns about reproducibility in scientific research by conducting replications of selected experiments from a number of high-profile papers in the field of cancer biology. The papers, which were published between 2010 and 2012, were selected on the basis of citations and Altmetric scores (Errington et al., 2014). This Registered Report describes the proposed replication plan of key experiments from 'RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth' by Hatzivassiliou and colleagues, published in Nature in 2010 (Hatzivassiliou et al., 2010). Hatzivassiliou and colleagues examined the paradoxical response of RAF-WT tumors to treatment with RAF inhibitors. The key experiments being replicated include Figure 1A, in which the original authors demonstrated that treatment of a subset of BRAF(WT) tumor cell lines with RAF small molecule inhibitors resulted in an increase in cell viability, Figure 2B, which reported that RAF inhibitor activation of the MAPK pathway was dependent on CRAF but not BRAF, and Figure 4A, where the dimerization of BRAF and CRAF was modulated by the RAF inhibitor PLX4720, but not GDC-0879. The Reproducibility Project: Cancer Biology is a collaboration between the Center for Open Science and Science Exchange, and the results of the replications will be published by eLife.
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Proliferación Celular , Neoplasias/patología , Inhibidores de Proteínas Quinasas/metabolismo , Transducción de Señal , Quinasas raf/antagonistas & inhibidores , Quinasas raf/metabolismo , Línea Celular Tumoral , Humanos , Modelos Biológicos , Reproducibilidad de los ResultadosRESUMEN
Keratoacanthoma (KA) are common but exceptional benign tumors, often appearing on sun-exposed areas of light skinned people and showing spontaneous resolution. The goal of this study was to review existing literature, to point out the etiological complexity of KA biology and to answer the controversial debate if or not KA is a distinct entity or a variant of squamous cell carcinoma (SCC). Relying on recent results, we highlight that KA is an individual lesion with a unique molecular signature caused by alterations in the TGFß signalling pathway. These recent findings will help to understand the nature of KA and to develop new reliable diagnostic tools, simplifying the discrimination of the histologically similar KA and SCC.
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Queratoacantoma , Enfermedades de la Piel , Carcinoma de Células Escamosas/diagnóstico , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/efectos de la radiación , Hibridación Genómica Comparativa , Diagnóstico Diferencial , Progresión de la Enfermedad , Predisposición Genética a la Enfermedad , Humanos , Queratoacantoma/diagnóstico , Queratoacantoma/etiología , Queratoacantoma/genética , Queratoacantoma/metabolismo , Queratoacantoma/patología , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiología , Neoplasias Inducidas por Radiación/química , Neoplasias Inducidas por Radiación/diagnóstico , Neoplasias Inducidas por Radiación/genética , Neoplasias Inducidas por Radiación/patología , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/fisiología , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/deficiencia , Receptores de Factores de Crecimiento Transformadores beta/genética , Receptores de Factores de Crecimiento Transformadores beta/fisiología , Transducción de Señal , Enfermedades de la Piel/diagnóstico , Enfermedades de la Piel/etiología , Enfermedades de la Piel/genética , Enfermedades de la Piel/metabolismo , Enfermedades de la Piel/patología , Neoplasias Cutáneas/diagnóstico , Luz Solar/efectos adversos , Factor de Crecimiento Transformador beta/fisiología , Rayos Ultravioleta/efectos adversosRESUMEN
Drug resistance is a major obstacle to the success of EGFR-targeted therapy. We recently studied the mechanism by which a small subset of EGFR mutant lung cancer cells remains viable after EGFR inhibition. We found that this drug-tolerant subpopulation develops because EGFR inhibition prevents AKT activity and thus inactivates Ets-1 function. In this article, we discuss how changes in intrinsic cell signaling after EGFR inhibition open a new avenue to drug resistance in NSCLCs, and comment on combined TKI and MEK inhibitor treatment to reduce the probability of emergent resistance to EGFR TKIs.