RESUMEN
BRAF serves as a gatekeeper of the RAS/RAF/MEK/ERK pathway, which plays a crucial role in homeostasis. Since aberrant signalling of this axis contributes to cancer and other diseases, it is tightly regulated by crosstalk with the PI3K/AKT/mTOR pathway and ERK mediated feedback loops. For example, ERK limits BRAF signalling through phosphorylation of multiple residues. One of these, T401, is widely considered as an ERK substrate following acute pathway activation by growth factors. Here, we demonstrate that prominent T401 phosphorylation (pT401) of endogenous BRAF is already observed in the absence of acute stimulation in various cell lines of murine and human origin. Importantly, the BRAF/RAF1 inhibitor naporafenib, the MEK inhibitor trametinib and the ERK inhibitor ulixertinib failed to reduce pT401 levels in these settings, supporting an alternative ERK-independent pathway to T401 phosphorylation. In contrast, the mTOR inhibitor torin1 and the dual-specific PI3K/mTOR inhibitor dactolisib significantly suppressed pT401 levels in all investigated cell types, in both a time and concentration dependent manner. Conversely, genetic mTOR pathway activation by oncogenic RHEB (Q64L) and mTOR (S2215Y and R2505P) mutants substantially increased pT401, an effect that was reverted by dactolisib and torin1 but not by trametinib. We also show that shRNAmir mediated depletion of the mTORC1 complex subunit Raptor significantly enhanced the suppression of T401 phosphorylation by a low torin1 dose, while knockdown of the mTORC2 complex subunit Rictor was less effective. Using mass spectrometry, we provide further evidence that torin1 suppresses the phosphorylation of T401, S405 and S409 but not of other important regulatory phosphorylation sites such as S446, S729 and S750. In summary, our data identify the mTOR axis and its inhibitors of (pre)clinical relevance as novel modulators of BRAF phosphorylation at T401.
Asunto(s)
Proteínas Proto-Oncogénicas B-raf , Serina-Treonina Quinasas TOR , Fosforilación/efectos de los fármacos , Humanos , Serina-Treonina Quinasas TOR/metabolismo , Animales , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas Proto-Oncogénicas B-raf/genética , Ratones , Transducción de Señal/efectos de los fármacos , Células HEK293 , Pirimidinonas/farmacología , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Piridonas/farmacología , NaftiridinasRESUMEN
Melanocytes derived from neural crest cells harbor the BRAF V600E mutation, which is the predominant driver of nevus formation in humans. This mutation leads to malignant cell proliferation and subsequent cell cycle arrest, culminating in oncogene-induced senescence and nevus development. Nevertheless, emerging evidence has highlighted the heterogeneity of cellular senescence markers in BRAF V600E-induced senescent melanocytes. Moreover, the capacity of melanocytes within nevi to regain their proliferative ability raises questions about the molecular mechanisms by which BRAF V600E, via the mitogen-activated protein kinase signaling pathway, triggers nevus formation. This study provides an overview and discussion of the molecular mechanisms underpinning BRAF V600E-induced melanocyte nevus formation and the relevant animal models employed for their elucidation. It also highlights the significance of elucidating dynamic changes in cytoplasmic and nuclear substrates that interact with phosphorylated extracellular signal-regulated protein kinases 1 and 2 and underscores the value of using targeted BRAF V600E animal models created through gene editing technologies.
Asunto(s)
Nevo , Proteínas Proto-Oncogénicas B-raf , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Humanos , Animales , Nevo/genética , Nevo/metabolismo , Melanocitos/metabolismo , Mutación , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/metabolismoRESUMEN
The coordination between kinases and phosphatases is crucial for regulating the phosphorylation levels of essential signaling molecules. Methods enabling precise control of kinase activities are valuable for understanding the kinase functions and for developing targeted therapies. Here, we use the abscisic acid (ABA)-induced proximity system to reversibly control kinase signaling by recruiting phosphatases. Using this method, we found that the oncogenic tyrosine kinase BCR::ABL1 can be inhibited by recruiting various cytoplasmic phosphatases. We also discovered that the oncogenic serine/threonine kinase BRAF(V600E), which has been reported to bypass phosphorylation regulation, can be positively regulated by protein phosphatase 1 (PP1) and negatively regulated by PP5. Additionally, we observed that the dual-specificity kinase MEK1 can be inhibited by recruiting PP5. This suggests that bifunctional molecules capable of recruiting PP5 to MEK or RAF kinases could be promising anticancer drug candidates. Thus, the ABA-induced dephosphorylation method enables rapid screening of phosphatases to precisely control kinase signaling.
Asunto(s)
Ácido Abscísico , Transducción de Señal , Fosforilación , Transducción de Señal/efectos de los fármacos , Humanos , Ácido Abscísico/metabolismo , Ácido Abscísico/farmacología , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas Proto-Oncogénicas B-raf/genética , MAP Quinasa Quinasa 1/metabolismo , Proteínas de Fusión bcr-abl/metabolismo , Proteínas de Fusión bcr-abl/genética , Proteína Fosfatasa 1/metabolismo , Proteína Fosfatasa 1/genéticaRESUMEN
Herein, we described the rational drug design and synthesis of a series of 5-amino-4-fluoro-1H-benzo[d]imidazole-6-carboxamide derivatives that inhibit MEK and RAF kinases. The detailed screening cascades revealed that 16b was a preferred compound, which might act like a "clamp" to stabilize the MEK/RAF complex, thereby effectively inhibiting MEK1, BRAF, and BRAFV600E with IC50 values of 28, 3, and 3 nM, respectively. 16b possessed an excellent selectivity over other 312 human-related kinases at 1 µM. In vitro, 16b showed potent antiproliferative activities against MIA PaCa-2 (G12C KRAS), HCT116 (G13D KRAS), and C26 (G12D KRAS) cells with IC50 values of 0.011, 0.079, and 0.096 µM, respectively. CoIP experiments demonstrated that 16b could induce MEK/RAF complex formation. Most importantly, in the C26 syngeneic colorectal and HCT116 mice xenograft tumor models, 16b demonstrated tumor growth inhibition of 70 and 93%, respectively, suggesting that 16b may be a promising MEK/RAF complex inhibitor and worthy of further development.
Asunto(s)
Antineoplásicos , Proliferación Celular , Diseño de Fármacos , Inhibidores de Proteínas Quinasas , Humanos , Animales , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Antineoplásicos/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/química , Ratones , Proliferación Celular/efectos de los fármacos , Relación Estructura-Actividad , Línea Celular Tumoral , Quinasas raf/antagonistas & inhibidores , Quinasas raf/metabolismo , Bencimidazoles/farmacología , Bencimidazoles/síntesis química , Bencimidazoles/química , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/metabolismo , Ensayos de Selección de Medicamentos Antitumorales , Ensayos Antitumor por Modelo de Xenoinjerto , Ratones DesnudosRESUMEN
The current targeted therapy for BRAFV600E-mutant lung cancer consists of a dual blockade of RAF/MEK kinases often combining dabrafenib/trametinib (D/T). This regimen extends survival when compared to single-agent treatments, but disease progression is unavoidable. By using whole-genome CRISPR screening and RNA sequencing, we characterize the vulnerabilities of both persister and D/T-resistant cellular models. Oxidative stress together with concomitant induction of antioxidant responses is boosted by D/T treatment. However, the nature of the oxidative damage, the choice of redox detoxification systems, and the resulting therapeutic vulnerabilities display stage-specific differences. Persister cells suffer from lipid peroxidation and are sensitive to ferroptosis upon GPX4 inhibition in vivo. Biomarkers of lipid peroxidation are detected in clinical samples following D/T treatment. Acquired alterations leading to mitogen-activated protein kinase (MAPK) reactivation enhance cystine transport to boost GPX4-independent antioxidant responses. Similarly to BRAFV600E-mutant melanoma, histone deacetylase (HDAC) inhibitors decrease D/T-resistant cell viability and extend therapeutic response in vivo.
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Adenocarcinoma del Pulmón , Resistencia a Antineoplásicos , Inhibidores de Histona Desacetilasas , Neoplasias Pulmonares , Fosfolípido Hidroperóxido Glutatión Peroxidasa , Proteínas Proto-Oncogénicas B-raf , Humanos , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Adenocarcinoma del Pulmón/genética , Adenocarcinoma del Pulmón/tratamiento farmacológico , Adenocarcinoma del Pulmón/patología , Adenocarcinoma del Pulmón/metabolismo , Fosfolípido Hidroperóxido Glutatión Peroxidasa/metabolismo , Fosfolípido Hidroperóxido Glutatión Peroxidasa/genética , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/metabolismo , Línea Celular Tumoral , Animales , Inhibidores de Histona Desacetilasas/farmacología , Ferroptosis/efectos de los fármacos , Ferroptosis/genética , Ratones , Estrés Oxidativo/efectos de los fármacos , Oximas/farmacología , Imidazoles/farmacología , Piridonas/farmacología , Pirimidinonas/farmacología , Peroxidación de Lípido/efectos de los fármacos , Mutación/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Papillary thyroid carcinoma (PTC) is the most common type of thyroid malignancies worldwide. Oncogenic transcription factors (TFs) drive transcriptional reprogramming and tumorigenesis. The myc-associated zinc finger protein (MAZ) is one of the Myc family TFs. The role of MAZ in PTC pathogenesis is still largely unknown. Here, we report that MAZ profoundly promotes proliferation of PTC cells ex vivo and in vivo through activating MAPK signaling. We firstly profiled gene expression of PTC cells after silencing of MAZ. BRAF, KRAS and LOC547 were identified as important target genes of TF MAZ. In particular, TF MAZ bound to the promoters of BRAF or KRAS and significantly increased their transcription and expression levels. Meanwhile, MAZ could noticeably elevate LOC547 transcription and expression as a TF. High levels of LOC547 relocated ACTN4 protein from the nucleus to the cytosol, improved protein-protein interactions between ACTN4 and EGFR in the cytosol, enhanced ERK1/2 phosphorylation, activated the MAPK signaling and, thus, promoted PTC progression. Our data identify a previously underappreciated MAZ-controlled transcriptional reprogram and ERK1/2 activation via BRAF, KRAS and LOC547. Our data illustrate that activation of the MAZ-controlled axis promotes thyroid tumorigenesis. These insights would advance our knowledge of the role of TFs in cancer development and highlight the potential of TFs as future targets for treatments against cancers.
Asunto(s)
Proliferación Celular , Progresión de la Enfermedad , Regulación Neoplásica de la Expresión Génica , Proteínas Proto-Oncogénicas B-raf , Proteínas Proto-Oncogénicas p21(ras) , Cáncer Papilar Tiroideo , Neoplasias de la Tiroides , Factores de Transcripción , Humanos , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/patología , Neoplasias de la Tiroides/metabolismo , Cáncer Papilar Tiroideo/genética , Cáncer Papilar Tiroideo/patología , Cáncer Papilar Tiroideo/metabolismo , Línea Celular Tumoral , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Animales , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Ratones , Sistema de Señalización de MAP Quinasas , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/genética , Ratones DesnudosRESUMEN
The classical mitogen-activated protein kinase (MAPK) signaling pathway, the Ras/Raf/MEK (mitogen-activated protein kinase/ERK kinase)/ERK protein kinase cascade, is a conserved cascade that regulates cell growth, differentiation, and proliferation. The significance of BRAF in cancer was established with the discovery of cancer-activating mutations in BRAF in several human tumors in 2002. Currently, BRAF is recognized as a driver mutation that affects cancer phenotypes in different ways, making it an important therapeutic target for cancer. BRAF-selective inhibitors have shown promise in clinical trials involving patients with metastatic melanoma. However, resistance mechanisms to BRAF inhibitors therapy have resulted in short-lived therapeutic responses. Further in-depth research is imperative to explore resistance mechanisms that oppose the effectiveness of BRAF inhibitors. Metabolic reprogramming has emerging role in BRAF-mutant cancers. In particular, mitochondrial metabolism and its closely related signaling pathways mediated by mitochondria have become recognized as potential new targets for treating BRAF-mutant cancers. This review, examines the progress in understanding BRAF mutations in cancer, the clinicopathological correlation of BRAF inhibitors, and recent advances in mitochondrial metabolism, mitochondrial dynamics and mitochondrial mediated death in BRAF-mutant cancer. This review will inform future cancer research and lay the foundation for novel treatment combinations of BRAF-mutant cancers.
Asunto(s)
Mitocondrias , Terapia Molecular Dirigida , Mutación , Neoplasias , Inhibidores de Proteínas Quinasas , Proteínas Proto-Oncogénicas B-raf , Humanos , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/metabolismo , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/genética , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/patología , Neoplasias/metabolismo , Terapia Molecular Dirigida/métodos , Inhibidores de Proteínas Quinasas/uso terapéutico , Inhibidores de Proteínas Quinasas/farmacología , Animales , Antineoplásicos/uso terapéutico , Antineoplásicos/farmacologíaRESUMEN
Mutant BRAF is a critical oncogenic driver in melanoma, making it an attractive therapeutic target. However, the success of targeted therapy using BRAF inhibitors vemurafenib and dabrafenib has been limited due to development of resistance, restricting their clinical efficacy. A prior knowledge of resistance mechanisms to BRAFi or any cancer drug can lead to development of drugs that overcome resistance thus improving clinical outcomes. In vitro cellular models are powerful systems that can be utilized to mimic and study resistance mechanisms. In this study, we employed a multi-omics approach to characterize a panel of BRAF mutant melanoma cell lines to develop and systematically characterize BRAFi persister and resistant cells using exome sequencing, proteomics and phosphoproteomics. Our datasets revealed frequently observed intrinsic and acquired, genetic and non-genetic mechanisms of BRAFi resistance that have been studied in patients who developed resistance. In addition, we identified proteins that can be potentially targeted to overcome BRAFi resistance. Overall, we demonstrate that in vitro systems can be utilized not only to predict resistance mechanisms but also to identify putative therapeutic targets.
Asunto(s)
Resistencia a Antineoplásicos , Melanoma , Inhibidores de Proteínas Quinasas , Proteómica , Proteínas Proto-Oncogénicas B-raf , Humanos , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/metabolismo , Melanoma/tratamiento farmacológico , Melanoma/genética , Melanoma/metabolismo , Melanoma/patología , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Proteómica/métodos , Inhibidores de Proteínas Quinasas/farmacología , Línea Celular Tumoral , Mutación , Vemurafenib/farmacología , Oximas/farmacología , Multiómica , ImidazolesRESUMEN
BACKGROUND: BRAF (v-raf murine sarcoma viral oncogene homolog B1)/MEK (mitogen-activated protein kinase kinase) inhibitors are used for melanoma treatment. Unfortunately, patients treated with this combined therapy develop resistance to treatment quite quickly, but the mechanisms underlying this phenomenon are not yet fully understood. Here, we report and characterize two melanoma cell lines (WM9 and Hs294T) resistant to BRAF (vemurafenib) and MEK (cobimetinib) inhibitors. METHODS: Cell viability was assessed via the XTT test. The level of selected proteins as well as activation of signaling pathways were evaluated using Western blotting. The expression of the chosen genes was assessed by RT-PCR. The distribution of cell cycle phases was analyzed by flow cytometry, and confocal microscopy was used to take photos of spheroids. The composition of cytokines secreted by cells was determined using a human cytokine array. RESULTS: The resistant cells had increased survival and activation of ERK kinase in the presence of BRAF/MEK inhibitors. The IC50 values for these cells were over 1000 times higher than for controls. Resistant cells also exhibited elevated activation of AKT, p38, and JNK signaling pathways with increased expression of EGFR, ErbB2, MET, and PDGFRß receptors as well as reduced expression of ErbB3 receptor. Furthermore, these cells demonstrated increased expression of genes encoding proteins involved in drug transport and metabolism. Resistant cells also exhibited features of epithelial-mesenchymal transition and cancer stem cells as well as reduced proliferation rate and elevated cytokine secretion. CONCLUSIONS: In summary, this work describes BRAF/MEK-inhibitor-resistant melanoma cells, allowing for better understanding the underlying mechanisms of resistance. The results may thus contribute to the development of new, more effective therapeutic strategies.
Asunto(s)
Azetidinas , Resistencia a Antineoplásicos , Melanoma , Piperidinas , Inhibidores de Proteínas Quinasas , Proteínas Proto-Oncogénicas B-raf , Vemurafenib , Humanos , Melanoma/patología , Melanoma/genética , Melanoma/tratamiento farmacológico , Melanoma/metabolismo , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/metabolismo , Azetidinas/farmacología , Línea Celular Tumoral , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Inhibidores de Proteínas Quinasas/farmacología , Piperidinas/farmacología , Vemurafenib/farmacología , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Supervivencia Celular/efectos de los fármacosRESUMEN
Protein kinases act as central molecular switches in the control of cellular functions. Alterations in the regulation and function of protein kinases may provoke diseases including cancer. In this study we investigate the conformational states of such disease-associated kinases using the high sensitivity of the kinase conformation (KinCon) reporter system. We first track BRAF kinase activity conformational changes upon melanoma drug binding. Second, we also use the KinCon reporter technology to examine the impact of regulatory protein interactions on LKB1 kinase tumor suppressor functions. Third, we explore the conformational dynamics of RIP kinases in response to TNF pathway activation and small molecule interactions. Finally, we show that CDK4/6 interactions with regulatory proteins alter conformations which remain unaffected in the presence of clinically applied inhibitors. Apart from its predictive value, the KinCon technology helps to identify cellular factors that impact drug efficacies. The understanding of the structural dynamics of full-length protein kinases when interacting with small molecule inhibitors or regulatory proteins is crucial for designing more effective therapeutic strategies.
Asunto(s)
Conformación Proteica , Humanos , Proteínas Proto-Oncogénicas B-raf/química , Proteínas Proto-Oncogénicas B-raf/metabolismo , Unión Proteica , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/química , Proteínas Quinasas/metabolismo , Proteínas Quinasas/química , Melanoma/tratamiento farmacológico , Melanoma/metabolismo , Quinasas de la Proteína-Quinasa Activada por el AMP , Línea Celular TumoralRESUMEN
Mutant BRAFV600E is one of the most common oncogenic drivers in metastatic melanoma. While first generation BRAFV600E inhibitors are capable of controlling tumors systemically, they are unable to adequately treat tumors that have metastasized to the brain due to insufficient penetration across the blood-brain barrier (BBB). Through a combination of structure-based drug design (SBDD) and the optimization of physiochemical properties to enhance BBB penetration, we herein report the discovery of the brain-penetrant BRAFV600E inhibitor PF-07284890 (ARRY-461). In mice studies, ARRY-461 proved to be highly brain-penetrant and was able to drive regressions of A375 BRAFV600E tumors implanted both subcutaneously and intracranially. Based on compelling preclinical safety and efficacy studies, ARRY-461 was progressed into a Phase 1 A/B clinical trial (NCT04543188).
Asunto(s)
Antineoplásicos , Inhibidores de Proteínas Quinasas , Proteínas Proto-Oncogénicas B-raf , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/metabolismo , Humanos , Animales , Ratones , Inhibidores de Proteínas Quinasas/farmacocinética , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/uso terapéutico , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Femenino , Barrera Hematoencefálica/metabolismo , Encéfalo/metabolismo , Melanoma/tratamiento farmacológico , Melanoma/patología , Relación Estructura-Actividad , Ratas , Ratones Desnudos , Ensayos Antitumor por Modelo de Xenoinjerto , MasculinoRESUMEN
BACKGROUND AND AIM: Colorectal cancer (CRC) is considered one of the most common cancers in the world. Serrated polyps were found to be precursor lesions for CRC. BRAF mutation (V600E) has been strongly linked to the development of these lesions. No previous study concerning BRAF immunohistochemical expression in serrated polyps- was done in Oman. The primary objective of our study was to assess the prevalence of BRAF (V600E) mutation in serrated colorectal polyps in the Omani population. The secondary objectives were to assess the prevalence of serrated polyps and their characteristic features: type, site and size as well as the relationship between BRAF (V600E) mutation and polyp type, site and size. MATERIALS AND METHODS: Ninety-one hyperplastic polyps (HP) (76.5%), 24 sessile serrated lesions (SSL) (20.2%) and 4 cases of tubular adenomas with low grade dysplasia (3.4%) were studied for BRAF (V600E) immunohistochemical expression. No case of traditional serrated adenoma (TSA) was present. Control cases of craniopharyngioma and papillary thyroid carcinoma were included. RESULTS: BRAF (V600E) IHC was positive in 63 of the HP polyps (69.2%), 13 SSLs (54.2%) and none of the adenomatous polyps. The majority of positive polyps (75.0%) were ≤5 mm in size, 17.9% were 5-10 mm and 7.1% were ≥10 mm in size. The majority of BRAF (V600E) positive polyps (68.1 %) were in the distal colon and 31.9 % were in the proximal colon. The majority of positive cases for BRAF (V600E) were showing multiple polyps (61.8 %). None of the tubular adenomas showed any BRAF (V600E) positivity. CONCLUSION: Serrated polyps are now well known for their potential to develop CRC. Immunohistochemistry is an easy and reproducible way to detect BRAF (V600E) mutation. Our study showed there is high prevalence (64.3%) of BRAF mutation in serrated polyps in the Omani population. The majority of these polyps- were HP and SSL; and ≤5 mm in size and located in the distal colon.
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Adenoma , Pólipos del Colon , Neoplasias Colorrectales , Mutación , Proteínas Proto-Oncogénicas B-raf , Humanos , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Femenino , Masculino , Omán , Pólipos del Colon/genética , Pólipos del Colon/patología , Pólipos del Colon/metabolismo , Persona de Mediana Edad , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/metabolismo , Adulto , Adenoma/genética , Adenoma/patología , Adenoma/metabolismo , Centros de Atención Terciaria , Pronóstico , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Anciano , Estudios de Seguimiento , Estudios de Casos y Controles , Cáncer Papilar Tiroideo/genética , Cáncer Papilar Tiroideo/patología , Cáncer Papilar Tiroideo/metabolismo , Lesiones Precancerosas/genética , Lesiones Precancerosas/patología , Lesiones Precancerosas/metabolismo , Adulto Joven , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/patología , Neoplasias de la Tiroides/metabolismo , Técnicas para Inmunoenzimas , Hiperplasia/genética , Hiperplasia/patología , Hiperplasia/metabolismo , Carcinoma Papilar/genética , Carcinoma Papilar/patología , Carcinoma Papilar/metabolismoRESUMEN
Malignant melanoma, an aggressive skin cancer with a poor prognosis, frequently features BRAFV600E mutation resulting in activation of the MAPK pathway and melanocyte proliferation and survival. BRAFV600E inhibitors like vemurafenib and dabrafenib have enhanced patient survival, yet drug resistance remains a significant challenge. We investigated the role of the ERK5 pathway in BRAFV600E melanoma cells and cells with acquired resistance to PLX4720 (vemurafenib) and dabrafenib. In BRAFV600E melanoma, ERK5 inhibition minimally affected viability compared to ERK1/2 inhibition. In vemurafenib-resistant cells, ERK5 inhibition alone didn't impact viability or restore drug sensitivity to vemurafenib. However, in dabrafenib-resistant cells, ERK5 inhibition reduced viability and enhanced the anti-proliferative effect of MEK1/2 inhibition. Targeting the ERK5 pathway may represent a therapeutic opportunity in dabrafenib-resistant melanoma.
Asunto(s)
Resistencia a Antineoplásicos , Imidazoles , Melanoma , Proteína Quinasa 7 Activada por Mitógenos , Oximas , Proteínas Proto-Oncogénicas B-raf , Vemurafenib , Oximas/farmacología , Humanos , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Melanoma/tratamiento farmacológico , Melanoma/genética , Melanoma/patología , Melanoma/metabolismo , Imidazoles/farmacología , Vemurafenib/farmacología , Resistencia a Antineoplásicos/genética , Resistencia a Antineoplásicos/efectos de los fármacos , Línea Celular Tumoral , Proteína Quinasa 7 Activada por Mitógenos/metabolismo , Proteína Quinasa 7 Activada por Mitógenos/genética , Indoles/farmacología , Sulfonamidas/farmacología , Proliferación Celular/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Neoplasias Cutáneas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , MutaciónRESUMEN
BACKGROUND: BRAF inhibitors are widely employed in the treatment of melanoma with the BRAF V600E mutation. However, the development of resistance compromises their therapeutic efficacy. Diverse genomic and transcriptomic alterations are found in BRAF inhibitor resistant melanoma, posing a pressing need for convergent, druggable target that reverse therapy resistant tumor with different resistance mechanisms. METHODS: CRISPR-Cas9 screens were performed to identify novel target gene whose inhibition selectively targets A375VR, a BRAF V600E mutant cell line with acquired resistance to vemurafenib. Various in vitro and in vivo assays, including cell competition assay, water soluble tetrazolium (WST) assay, live-dead assay and xenograft assay were performed to confirm synergistic cell death. Liquid Chromatography-Mass Spectrometry analyses quantified polyamine biosynthesis and changes in proteome in vemurafenib resistant melanoma. EIF5A hypusination dependent protein translation and subsequent changes in mitochondrial biogenesis and activity were assayed by O-propargyl-puromycin labeling assay, mitotracker, mitoSOX labeling and seahorse assay. Bioinformatics analyses were used to identify the association of polyamine biosynthesis with BRAF inhibitor resistance and poor prognosis in melanoma patient cohorts. RESULTS: We elucidate the role of polyamine biosynthesis and its regulatory mechanisms in promoting BRAF inhibitor resistance. Leveraging CRISPR-Cas9 screens, we identify AMD1 (S-adenosylmethionine decarboxylase 1), a critical enzyme for polyamine biosynthesis, as a druggable target whose inhibition reduces vemurafenib resistance. Metabolomic and proteomic analyses reveal that polyamine biosynthesis is upregulated in vemurafenib-resistant cancer, resulting in enhanced EIF5A hypusination, translation of mitochondrial proteins and oxidative phosphorylation. We also identify that sustained c-Myc levels in vemurafenib-resistant cancer are responsible for elevated polyamine biosynthesis. Inhibition of polyamine biosynthesis or c-Myc reversed vemurafenib resistance both in vitro cell line models and in vivo in a xenograft model. Polyamine biosynthesis signature is associated with poor prognosis and shorter progression free survival after BRAF/MAPK inhibitor treatment in melanoma cohorts, highlighting the clinical relevance of our findings. CONCLUSIONS: Our findings delineate the molecular mechanisms involving polyamine-EIF5A hypusination-mitochondrial respiration pathway conferring BRAF inhibitor resistance in melanoma. These targets will serve as effective therapeutic targets that can maximize the therapeutic efficacy of existing BRAF inhibitors.
Asunto(s)
Resistencia a Antineoplásicos , Factor 5A Eucariótico de Iniciación de Traducción , Melanoma , Mutación , Factores de Iniciación de Péptidos , Poliaminas , Proteínas Proto-Oncogénicas B-raf , Proteínas de Unión al ARN , Vemurafenib , Humanos , Melanoma/tratamiento farmacológico , Melanoma/genética , Melanoma/metabolismo , Melanoma/patología , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Resistencia a Antineoplásicos/genética , Animales , Poliaminas/metabolismo , Ratones , Factores de Iniciación de Péptidos/metabolismo , Factores de Iniciación de Péptidos/genética , Línea Celular Tumoral , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Vemurafenib/farmacología , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Ensayos Antitumor por Modelo de Xenoinjerto , Sistemas CRISPR-Cas , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Lisina/análogos & derivadosRESUMEN
Single-agent TAS102 (trifluridine/tipiracil) and regorafenib are FDA-approved treatments for metastatic colorectal cancer (mCRC). We previously reported that regorafenib combined with a fluoropyrimidine can delay disease progression in clinical case reports of multidrug-resistant mCRC patients. We hypothesized that the combination of TAS102 and regorafenib may be active in CRC and other gastrointestinal (GI) cancers and may in the future provide a treatment option for patients with advanced GI cancer. We investigated the therapeutic effect of TAS102 in combination with regorafenib in preclinical studies employing cell culture, colonosphere assays that enrich for cancer stem cells, and in vivo. TAS102 in combination with regorafenib has synergistic activity against multiple GI cancers in vitro including colorectal and gastric cancer, but not liver cancer cells. TAS102 inhibits colonosphere formation and this effect is potentiated by regorafenib. In vivo anti-tumor effects of TAS102 plus regorafenib appear to be due to anti-proliferative effects, necrosis and angiogenesis inhibition. Growth inhibition by TAS102 plus regorafenib occurs in xenografted tumors regardless of p53, KRAS or BRAF mutations, although more potent tumor suppression was observed with wild-type p53. Regorafenib significantly inhibits TAS102-induced angiogenesis and microvessel density in xenografted tumors, as well inhibits TAS102-induced ERK1/2 activation regardless of RAS or BRAF status in vivo. TAS102 plus regorafenib is a synergistic drug combination in preclinical models of GI cancer, with regorafenib suppressing TAS102-induced increase in microvessel density and p-ERK as contributing mechanisms. The TAS102 plus regorafenib drug combination may be further tested in gastric and other GI cancers.
Asunto(s)
Combinación de Medicamentos , Sinergismo Farmacológico , Neoplasias Gastrointestinales , Mutación , Células Madre Neoplásicas , Neovascularización Patológica , Compuestos de Fenilurea , Proteínas Proto-Oncogénicas B-raf , Proteínas Proto-Oncogénicas p21(ras) , Piridinas , Pirrolidinas , Factor de Transcripción STAT3 , Timina , Trifluridina , Uracilo , Ensayos Antitumor por Modelo de Xenoinjerto , Humanos , Trifluridina/farmacología , Compuestos de Fenilurea/farmacología , Animales , Piridinas/farmacología , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Neoplasias Gastrointestinales/tratamiento farmacológico , Neoplasias Gastrointestinales/genética , Neoplasias Gastrointestinales/patología , Neoplasias Gastrointestinales/metabolismo , Uracilo/farmacología , Uracilo/análogos & derivados , Ratones , Factor de Transcripción STAT3/metabolismo , Factor de Transcripción STAT3/genética , Timina/farmacología , Línea Celular Tumoral , Pirrolidinas/farmacología , Pirrolidinas/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , AngiogénesisRESUMEN
The MAPK signaling pathway with BRAF mutations has been shown to drive the pathogenesis of 40-60% of melanomas. Inhibitors of this pathway's BRAF and MEK components are currently used to treat these malignancies. However, responses to these treatments are not always successful. Therefore, identifying noninvasive biomarkers to predict treatment responses is essential for personalized medicine in melanoma. Using noninvasive 1H magnetic resonance spectroscopy (1H MRS), we previously showed that BRAF inhibition reduces lactate and alanine tumor levels in the early stages of effective therapy and could be considered as metabolic imaging biomarkers for drug response. The present work demonstrates that these metabolic changes observed by 1H MRS and those assessed by 31P MRS are also found in preclinical human melanoma models treated with MEK inhibitors. Apart from 1H and 31P MRS, additional supporting in vitro biochemical analyses are described. Our results indicate significant early metabolic correlations with response levels to MEK inhibition in the melanoma models and are consistent with our previous study of BRAF inhibition. Given these results, our study supports the potential clinical utility of noninvasive MRS to objectively image metabolic biomarkers for the early prediction of melanoma's response to MEK inhibition.
Asunto(s)
Melanoma , Metabolómica , Inhibidores de Proteínas Quinasas , Melanoma/metabolismo , Melanoma/tratamiento farmacológico , Melanoma/patología , Humanos , Metabolómica/métodos , Línea Celular Tumoral , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Espectroscopía de Resonancia Magnética/métodos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Espectroscopía de Protones por Resonancia Magnética/métodosRESUMEN
Cancer remains a leading cause of death worldwide, often resulting from uncontrolled growth in various organs. Protein kinase inhibitors represent an important class of targeted cancer therapies. Recently, the kinases BRAF and VEGFR-2 have shown synergistic effects on tumor progression. Seeking to develop dual BRAF/VEGFR-2 inhibitors, we synthesized 18 amino-benzothiazole derivatives with structural similarities to reported dual inhibitors. Four compounds-4a, 4f, 4l, and 4r-demonstrated remarkable cytotoxicity, with IC50 values ranging from 3.58 to 15.36 µM, against three cancer cell lines. Furthermore, these compounds showed IC50 values of 38.77-66.22 µM in the case of a normal cell line, which was significantly safer than the reference, sorafenib. Subsequent investigation revealed that compound 4f exhibited the capacity to inhibit the BRAF and VEGFR-2 enzymes, with IC50 values similar to sorafenib (0.071 and 0.194 µM, respectively). Moreover, compound 4f caused G2-M- and S-phase cycle arrest. Molecular modeling demonstrated binding patterns compatible with inhibition for both targets, where 4f exerted the critical interactions in the BRAF site and interacted in the VEGFR-2 site in a manner akin to sorafenib, demonstrating affinity similar to dabrafenib.
Asunto(s)
Antineoplásicos , Benzotiazoles , Proliferación Celular , Simulación del Acoplamiento Molecular , Inhibidores de Proteínas Quinasas , Proteínas Proto-Oncogénicas B-raf , Tiadiazoles , Receptor 2 de Factores de Crecimiento Endotelial Vascular , Receptor 2 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Humanos , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/síntesis química , Benzotiazoles/química , Benzotiazoles/farmacología , Benzotiazoles/síntesis química , Tiadiazoles/química , Tiadiazoles/farmacología , Tiadiazoles/síntesis química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Diseño de Fármacos , Relación Estructura-Actividad , Sorafenib/farmacología , Sorafenib/química , Estructura Molecular , Simulación por Computador , Ensayos de Selección de Medicamentos AntitumoralesRESUMEN
Activating mutations in the mitogen-activated protein kinase (MAPK) pathway represent driver alterations governing tumorigenesis, metastasis, and therapy resistance. MAPK activation predominantly occurs through genomic alterations in RAS and BRAF. BRAF is an effector kinase that functions downstream of RAS and propagates this oncogenic activity through MEK and ERK. Across cancers, BRAF alterations include gain-of-function mutations, copy-number alterations, and structural rearrangements. In cancer patients, BRAF-targeting precision therapeutics are effective against Class I BRAF alterations (p.V600 hotspot mutations) in tumors such as melanomas, thyroid cancers, and colorectal cancers. However, numerous non-Class I BRAF inhibitors are also in development and have been explored in some cancers. Here we discuss the diverse forms of BRAF alterations found in human cancers and the strategies to inhibit them in patients harboring cancers of distinct origins.
Asunto(s)
Terapia Molecular Dirigida , Neoplasias , Inhibidores de Proteínas Quinasas , Proteínas Proto-Oncogénicas B-raf , Humanos , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/metabolismo , Neoplasias/genética , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Neoplasias/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Mutación , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Animales , Sistema de Señalización de MAP Quinasas/genéticaRESUMEN
Metastatic cutaneous melanoma is a fatal skin cancer. Resistance to targeted and immune therapies limits the benefits of current treatments. Identifying and adding anti-resistance agents to current treatment protocols can potentially improve clinical responses. Myocardin-related transcription factor (MRTF) is a transcriptional coactivator whose activity is indirectly regulated by actin and the Rho family of GTPases. We previously demonstrated that development of BRAF inhibitor (BRAFi) resistance frequently activates the Rho/MRTF pathway in human and mouse BRAFV600E melanomas. In clinical trials, pretreatment with BRAFi reduces the benefit of immune therapies. We aimed to test the efficacy of concurrent treatment with our MRTF pathway inhibitor CCG-257081 and anti-PD1 in vivo and to examine its effects on the melanoma immune microenvironment. Because MRTF pathway activation upregulates the expression of immune checkpoint inhibitor genes/proteins, we asked whether CCG-257081 can improve the response to immune checkpoint blockade. CCG-257081 reduced the expression of PDL1 in BRAFi-resistant melanoma cells and decreased surface PDL1 levels on both BRAFi-sensitive and -resistant melanoma cells. Using our recently described murine vemurafenib-resistant melanoma model, we found that CCG-257081, in combination with anti-PD1 immune therapy, reduced tumor growth and increased survival. Moreover, anti-PD1/CCG-257081 co-treatment increased infiltration of CD8+ T cells and B cells into the tumor microenvironment and reduced tumor-associated macrophages. Here, we propose CCG-257081 as an anti-resistance and immune therapy-enhancing anti-melanoma agent.
Asunto(s)
Antígeno B7-H1 , Resistencia a Antineoplásicos , Inhibidores de Puntos de Control Inmunológico , Melanoma , Proteínas Proto-Oncogénicas B-raf , Neoplasias Cutáneas , Microambiente Tumoral , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Animales , Melanoma/tratamiento farmacológico , Melanoma/metabolismo , Melanoma/genética , Melanoma/patología , Ratones , Humanos , Antígeno B7-H1/antagonistas & inhibidores , Antígeno B7-H1/metabolismo , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/patología , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/metabolismo , Microambiente Tumoral/efectos de los fármacos , Línea Celular Tumoral , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/metabolismo , Transactivadores/metabolismo , Transactivadores/genética , Femenino , Transducción de Señal/efectos de los fármacos , Proteínas de Unión al GTP rho/metabolismoRESUMEN
The oncogenic role of KRAS in colorectal cancer (CRC) progression is well-established. Despite this, identifying effective therapeutic targets for KRAS-mutated CRC remains a significant challenge. This study identifies pyruvate dehydrogenase phosphatase catalytic subunit 1 (PDP1) as a previously unrecognized yet crucial regulator in the progression of KRAS mutant CRC. A substantial upregulation of PDP1 expression is observed in KRAS mutant CRC cells and tissues compared to wild-type KRAS samples, which correlates with poorer prognosis. Functional experiments elucidate that PDP1 accelerates the malignance of KRAS mutant CRC cells, both in vitro and in vivo. Mechanistically, PDP1 acts as a scaffold, enhancing BRAF and MEK1 interaction and activating the MAPK signaling, thereby promoting CRC progression. Additionally, transcription factor KLF5 is identified as the key regulator for PDP1 upregulation in KRAS mutant CRC. Crucially, targeting PDP1 combined with MAPK inhibitors exhibits an obvious inhibitory effect on KRAS mutant CRC. Overall, PDP1 is underscored as a vital oncogenic driver and promising therapeutic target for KRAS mutant CRC.