RESUMO
Being a component of the Ras/Raf/MEK/ERK signaling pathway crucial for cellular responses, the VRAF murine sarcoma viral oncogene homologue B1 (BRAF) kinase has emerged as a promising target for anticancer drug discovery due to oncogenic mutations that lead to pathway hyperactivation. Despite the discovery of several small-molecule BRAF kinase inhibitors targeting oncogenic mutants, their clinical utility has been limited by challenges such as off-target effects and suboptimal pharmacological properties. This study focuses on identifying miniprotein inhibitors for the oncogenic V600E mutant BRAF, leveraging their potential as versatile drug candidates. Using a structure-based de novo design approach based on binding affinity to V600E mutant BRAF and hydration energy, 39 candidate miniprotein inhibitors comprising three helices and 69 amino acids were generated from the substructure of the endogenous ligand protein (14-3-3). Through in vitro binding and kinase inhibition assays, two miniproteins (63 and 76) were discovered as novel inhibitors of V600E mutant BRAF with low-micromolar activity, with miniprotein 76 demonstrating a specific impediment to MEK1 phosphorylation in mammalian cells. These findings highlight miniprotein 76 as a potential lead compound for developing new cancer therapeutics, and the structural features contributing to its biochemical potency against V600E mutant BRAF are discussed in detail.
Assuntos
Antineoplásicos , Desenho de Fármacos , Descoberta de Drogas , Inibidores de Proteínas Quinases , Proteínas Proto-Oncogênicas B-raf , Humanos , Antineoplásicos/farmacologia , Antineoplásicos/química , Descoberta de Drogas/métodos , Modelos Moleculares , Mutação , Fosforilação/efeitos dos fármacos , Ligação Proteica , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/química , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Relação Estrutura-AtividadeRESUMO
Kinase-targeted therapies have the potential to improve the survival of patients with cancer. However, the cancer-specific spectrum of kinase alterations exhibits distinct functional properties and requires mutation-oriented drug treatments. Besides post-translational modifications and diverse intermolecular interactions of kinases, it is the distinct disease mutation which reshapes full-length kinase conformations, affecting their activity. Oncokinase mutation profiles differ between cancer types, as it was shown for BRAF in melanoma and non-small-cell lung cancers. Here, we present the target-oriented application of a kinase conformation (KinCon) reporter platform for live-cell measurements of autoinhibitory kinase activity states. The bioluminescence-based KinCon biosensor allows the tracking of conformation dynamics of full-length kinases in intact cells and real time. We show that the most frequent BRAF cancer mutations affect kinase conformations and thus the engagement and efficacy of V600E-specific BRAF inhibitors (BRAFi). We illustrate that the patient mutation harboring KinCon reporters display differences in the effectiveness of the three clinically approved BRAFi vemurafenib, encorafenib, and dabrafenib and the preclinical paradox breaker PLX8394. We confirmed KinCon-based drug efficacy predictions for BRAF mutations other than V600E in proliferation assays using patient-derived lung cancer cell lines and by analyzing downstream kinase signaling. The systematic implementation of such conformation reporters will allow to accelerate the decision process for the mutation-oriented RAF-kinase cancer therapy. Moreover, we illustrate that the presented kinase reporter concept can be extended to other kinases which harbor patient mutations. Overall, KinCon profiling provides additional mechanistic insights into full-length kinase functions by reporting protein-protein interaction (PPI)-dependent, mutation-specific, and drug-driven changes of kinase activity conformations.
Assuntos
Neoplasias Pulmonares/tratamento farmacológico , Conformação Proteica/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/genética , Células A549 , Carbamatos/química , Carbamatos/farmacologia , Compostos Heterocíclicos com 2 Anéis/farmacologia , Humanos , Imidazóis/química , Imidazóis/farmacologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Mutação/efeitos dos fármacos , Oximas/química , Oximas/farmacologia , Fosfotransferases/antagonistas & inibidores , Fosfotransferases/ultraestrutura , Inibidores de Proteínas Quinases/química , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/genética , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/ultraestrutura , Sulfonamidas/química , Sulfonamidas/farmacologia , Vemurafenib/química , Vemurafenib/farmacologiaRESUMO
Pleomorphic xanthoastrocytoma (PXA) is an uncommon, long-term epilepsy associated tumor of young adults. Its pigmented variant is exceedingly rare, with only five previously reported cases on record. We report the sixth case of pigmented PXA in a 24-year-old lady presenting with long-standing seizures. The MRI revealed a solid cystic lesion located in the right medial temporal lobe. Histopathologically, the superficially located tumor showed typical features of PXA with melanin-laden astrocytic component and was negative for V600E-mutant BRAF. The histogenesis is discussed.
Assuntos
Astrocitoma/diagnóstico , Astrocitoma/etiologia , Neoplasias Encefálicas/etiologia , Epilepsia/complicações , Adulto , Lobectomia Temporal Anterior , Astrocitoma/cirurgia , Neoplasias Encefálicas/diagnóstico por imagem , Neoplasias Encefálicas/cirurgia , Feminino , Humanos , Imageamento por Ressonância Magnética , Melaninas , Mutação , PigmentaçãoRESUMO
The general transcription factor E2F1 reportedly functions in a protumorigenic manner in several cancer models. We show that the genetic context of cancer cells influence E2F1's role to impede the protumorigenic role. Thirty to fifty percent of melanoma patients carry mutant BRAF with about 90% of mutant BRAF melanomas being V600E mutation. Tissue microarrays from melanoma patients were used to establish an association between E2F1 and BRAFV600E . We show for the first time that low E2F1 levels in BRAFV600E melanomas are associated with lymph node metastasis. Genetic manipulation of E2F1 in BRAFV600E and BRAFwt cells were used to determine its role in malignant melanoma progression by examining effects on migration and invasion. E2F1-mediated negative regulation of myosin light chain kinase (MYLK) increased migration and invasion in BRAFV600E cells by phosphorylating myosin light chain and increased stress fiber formation. We show that E2F1 inhibits extracellular signal-regulated kinase (ERK) activation in BRAFV600E cells and provide evidence for a negative feedback loop between E2F1 and ERK in these cells. This study shows for the first time that E2F1 has a cancer protective role in oncogenic BRAF-activated melanoma cells and that loss of E2F1 can allow disease progression through a novel mechanism of E2F1-mediated MYLK regulation. This study has implications for oncogenic BRAF-activated tumors and resistance to targeted oncogenic BRAF therapy.
Assuntos
Movimento Celular/genética , Fator de Transcrição E2F1/genética , Melanoma/genética , Melanoma/patologia , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Metástase Linfática/genética , Metástase Linfática/patologia , Sistema de Sinalização das MAP Quinases/genética , Mutação/genética , Fosforilação/genética , Proteínas Proto-Oncogênicas B-raf/genética , Melanoma Maligno CutâneoRESUMO
This study utilized a microfluidic mixer for the sample pretreatment of cell extracts for target protein quantification by mass spectrometers, including protein immunoprecipitation and protein enzymatic digestion. The time of sample pretreatment was reduced and thus the throughput of quantitative mutant proteins was increased by using the proposed method. Whole cell lysates of the cancer cell line HT-29 with gene mutations were used as the sample. The target protein BRAF was immunoprecipitated using magnetic beads in a pneumatic micromixer. Purified protein was then eluted and digested by trypsin in another two micromixers to yield peptide fragments in the solution. Using stable isotope-labeled standard as the internal control, wild-type and mutant BRAF proteins were quantified using mass spectrometry, which could be used for cancer screening. Compared with conventional methods in which protein immunoprecipitation lasts overnight, the micromixer procedure takes only 1 h, likely improving the throughput of mutant BRAF protein quantification by mass spectrometry. Graphical abstract Three micromixers were used to reduce the sample pretreatment time of cell extracts for target protein quantification by mass spectrometers, including protein immunoprecipitation, protein elution, and protein enzymatic digestion.
Assuntos
Imunoprecipitação/instrumentação , Dispositivos Lab-On-A-Chip , Proteínas Proto-Oncogênicas B-raf/análise , Desenho de Equipamento , Células HT29 , Humanos , Mutação , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/genética , Proteólise , Proteínas Proto-Oncogênicas B-raf/genética , Espectrometria de Massas em Tandem/métodos , Tripsina/químicaRESUMO
BACKGROUND/AIM: Recent evidence suggests that melanoma patients treated with BRAF inhibitors experience radiosensitization with an increased frequency of side-effects. This could also imply increased effectiveness when treating melanoma. MATERIALS AND METHODS: To test whether the BRAF inhibitors dabrafenib and vemurafenib together with ionizing radiation more effectively inhibit melanoma cells, primary human melanoma tumor cell lines expressing wild-type (WT) or mutant V600E BRAF were analyzed by cell survival, cell death, and cell-cycle testing. RESULTS: All melanoma cell lines examined were radioresistant in these assays. BRAF inhibitor treatment alone suppressed cell survival more effectively than radiation in all the mutant V600E BRAF cell lines, and vemurafenib, but not dabrafenib, also inhibited cell survival in the WT BRAF cell lines at clinically relevant concentrations. However, when cells were treated with BRAF inhibitor followed by radiation, there was no increased effect on the suppression of cell survival. Vemurafenib induced more necrosis than radiation in most melanoma cell lines, irrespective of BRAF status, but this effect was not additive with the combination treatment. BRAF inhibitors and radiation had variable, but independent effects on the induction of cell-cycle arrest. CONCLUSION: These results suggest that BRAF inhibitors and ionizing radiation do not act synergistically to inhibit the growth of primary human melanoma cells.
Assuntos
Imidazóis/farmacologia , Indóis/farmacologia , Oximas/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Sulfonamidas/farmacologia , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Sobrevivência Celular/efeitos da radiação , Relação Dose-Resposta a Droga , Relação Dose-Resposta à Radiação , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos da radiação , Humanos , Melanoma/genética , Melanoma/metabolismo , Melanoma/patologia , Mutação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Células Tumorais Cultivadas , VemurafenibRESUMO
Patient-derived tumor xenograft (PDTX) mouse models were used to discover new therapies for naïve and drug resistant BRAFV600E -mutant melanoma. Tumor histology, oncogenic protein expression, and antitumor activity were comparable between patient and PDTX-matched models thereby validating PDTXs as predictive preclinical models of therapeutic response in patients. PDTX models responsive and non-responsive to BRAF/MEK standard of care (SOC) therapy were used to identify efficacious combination therapies. One such combination includes a CDK4/6 inhibitor that blocks cell cycle progression. The rationale for this is that the retinoblastoma protein (pRb) is 95% wildtype in BRAF mutant melanoma. We discovered that 77/77 stage IV metastatic melanoma tissues were positive for inactive phosphorylated pRb (pRb-Ser780). Rb is hyperphosphorylated and inactivated by CDK4/6:cyclin D1 and when restored to its hypophosphorylated active form blocks cell cycle progression. The addition of a CDK4/6 inhibitor to SOC therapy was superior to SOC. Importantly, triple therapy in an upfront treatment and salvage therapy setting provided sustained durable response. We also showed that CDK4/6 blockade resensitized drug resistant melanoma to SOC therapy. Durable response was associated with sustained suppression of pRb-Ser780. Thus, reactivation of pRb may prove to be a clinical biomarker of response and the mechanism responsible for durable response. In light of recent clinical trial data using this triple therapy against BRAFV600E -mutant melanoma, our findings demonstrating superior and prolonged durable response in PDTX models portend use of this therapeutic strategy against naïve and SOC resistant BRAFV600E -mutant metastatic melanoma coupled with pRB-Ser780 as a biomarker of response.
RESUMO
Mutant serine/threonine protein kinase B-Raf (BRAF) protein is expressed in over half of all melanoma tumors. Although BRAF inhibitors (BRAFi) elicit rapid anti-tumor responses in the majority of patients with mutant BRAF melanoma, the tumors inevitably relapse after a short time. We hypothesized that polyamines are essential for tumor survival in mutant BRAF melanomas. These tumors rely on both polyamine biosynthesis and an upregulated polyamine transport system (PTS) to maintain their high intracellular polyamine levels. We evaluated the effect of a novel arylpolyamine (AP) compound that is cytotoxic upon cellular entry via the increased PTS activity of melanoma cells with different BRAF mutational status. Mutant BRAF melanoma cells demonstrated greater PTS activity and increased sensitivity to AP compared to wild type BRAF (BRAFWT) melanoma cells. Treatment with an inhibitor of polyamine biosynthesis, α-difluoromethylornithine (DFMO), further upregulated PTS activity in mutant BRAF cells and increased their sensitivity to AP. Furthermore, viability assays of 3D spheroid cultures of mutant BRAF melanoma cells demonstrated greater resistance to the BRAFi, PLX4720, compared to 2D monolayer cultures. However, co-treatment with AP restored the sensitivity of melanoma spheroids to PLX4720. These data indicate that mutant BRAF melanoma cells are more dependent on the PTS compared to BRAFWT melanoma cells, resulting in greater sensitivity to the PTS-targeted cytotoxic AP compound.
RESUMO
Oncogenic mutations of BRAF occur in approximately 10% of colon cancers and are associated with their resistance to clinically available therapeutic drugs and poor prognosis of the patients. Here we report that colon cancer cells with mutant BRAF are also resistant to the heat shock protein 90 (HSP90) inhibitor AUY922, and that this is caused by rebound activation of ERK and Akt. Although AUY922 triggered rapid reduction in ERK and Akt activation in both wild-type and mutant BRAF colon cancer cells, activation of ERK and Akt rebounded shortly in the latter leading to resistance of the cells to AUY922-induced apoptosis. Reactivation of ERK was associated with the persistent expression of mutant BRAF, which, despite being a client of HSP90, was only partially degraded by AUY922, whereas reactivation of Akt was related to the activity of the HSP90 co-chaperone, cell division cycle 37 (CDC37), in that knockdown of CDC37 inhibited Akt reactivation in mutant colon cancer cells treated with AUY922. In support, as a HSP90 client protein, Akt was only diminished by AUY922 in wild-type but not mutant BRAF colon cancer cells. Collectively, these results reveal that reactivation of ERK and Akt associated respectively with the activity of mutant BRAF and CDC37 renders mutant BRAF colon cancer cells resistant to AUY922, with implications of co-targeting mutant BRAF and/or CDC37 and HSP90 in the treatment of mutant BRAF colon cancers.
Assuntos
Neoplasias do Colo/metabolismo , Resistencia a Medicamentos Antineoplásicos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Isoxazóis/química , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Resorcinóis/química , Apoptose , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Chaperoninas/metabolismo , Neoplasias do Colo/tratamento farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Mutação , Proteínas Proto-Oncogênicas B-raf/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Targeted therapies for mutant BRAF metastatic melanoma are effective but not curative due to acquisition of resistance. PI3K signaling is a common mediator of therapy resistance in melanoma; thus, the need for effective PI3K inhibitors is critical. However, testing PI3K inhibitors in adherent cultures is not always reflective of their potential in vivo. To emphasize this, we compared PI3K inhibitors of different specificity in two- and three-dimensional (2D, 3D) melanoma models and show that drug response predictions gain from evaluation using 3D models. Our results in 3D demonstrate the anti-invasive potential of PI3K inhibitors and that drugs such as PX-866 have beneficial activity in physiological models alone and when combined with BRAF inhibition. These assays finally help highlight pathway effectors that could be involved in drug response in different environments (e.g. p4E-BP1). Our findings show the advantages of 3D melanoma models to enhance our understanding of PI3K inhibitors.
Assuntos
Melanoma/patologia , Modelos Biológicos , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Animais , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Colágeno/farmacologia , Gonanos/farmacologia , Indóis/farmacologia , Melanoma/enzimologia , Camundongos Endogâmicos NOD , Mutação/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/patologia , Sulfonamidas/farmacologia , Microambiente Tumoral/efeitos dos fármacosRESUMO
BRAF inhibitors have revolutionized treatment of mutant BRAF metastatic melanomas. However, resistance develops rapidly following BRAF inhibitor treatment. We have found that BRAF-mutant melanoma cell lines are more sensitive than wild-type BRAF cells to the small molecule tyrosine kinase inhibitor dovitinib. Sensitivity is associated with inhibition of a series of known dovitinib targets. Dovitinib in combination with several agents inhibits growth more effectively than either agent alone. These combinations inhibit BRAF-mutant melanoma and colorectal carcinoma cell lines, including cell lines with intrinsic or selected BRAF inhibitor resistance. Hence, combinations of dovitinib with second agents are potentially effective therapies for BRAF-mutant melanomas, regardless of their sensitivity to BRAF inhibitors.