ABSTRACT
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.
Subject(s)
Azetidines , Drug Resistance, Neoplasm , Melanoma , Piperidines , Protein Kinase Inhibitors , Proto-Oncogene Proteins B-raf , Vemurafenib , Humans , Melanoma/pathology , Melanoma/genetics , Melanoma/drug therapy , Melanoma/metabolism , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Proto-Oncogene Proteins B-raf/metabolism , Azetidines/pharmacology , Cell Line, Tumor , Drug Resistance, Neoplasm/drug effects , Drug Resistance, Neoplasm/genetics , Protein Kinase Inhibitors/pharmacology , Piperidines/pharmacology , Vemurafenib/pharmacology , Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitors , Mitogen-Activated Protein Kinase Kinases/metabolism , Cell Survival/drug effectsABSTRACT
OBJECTIVE: Targeted therapy (TT) with BRAF/MEK inhibitors has emerged as a potential treatment in papillary craniopharyngiomas (PCPs). However, standardized data on large cohorts are lacking. Our study aimed to assess real-life efficacy and safety of BRAF/MEK inhibition in patients with PCPs. DESIGN: Retrospective French multicenter study involving BRAF V600E-mutated PCP patients, treated with BRAF/MEK inhibitor combination dabrafenib and trametinib, from April 2019 to July 2023. METHODS: Objective response and clinical and safety outcomes were assessed after 3 months and at the last available follow-up during TT. RESULTS: Sixteen patients (8 females, mean age 50.5 ± 15.75 years), receiving either neoadjuvant therapy (NEO) for non-resectable tumors (n = 6), post-surgical adjuvant therapy (ADJ; n = 8), or palliative therapy (PAL) following failure of multimodal treatment (n = 2), were included.At the last follow-up (mean 7.6 ± 5.3 months), 12 patients showed subtotal response, 3 exhibited partial response, and 1 maintained stable disease. Mean volume reduction was 88.9 ± 4.4%, 73.3 ± 23.4%, and 91.8 ± 4.3% in the NEO, ADJ, and PAL groups, respectively.Targeted therapy resolved headaches in 5/5 patients and visual impairment in 6/9; 2/3 patients had improved neurological symptoms, 1/4 presented weight loss, and 2/14 recovered endocrine function.Targeted therapy was well-tolerated in 62.5% of cases; adverse events led to treatment discontinuation in 5 patients and definitive discontinuation in 3 cases. CONCLUSIONS: In this study, 94% of patients showed partial response or better to TT. Adverse events were acceptable. Further research is needed to establish standardized protocols; however, these results advocate for a NEO approach in invasive PCPs.
Subject(s)
Craniopharyngioma , Oximes , Pituitary Neoplasms , Proto-Oncogene Proteins B-raf , Pyridones , Pyrimidinones , Humans , Female , Male , Middle Aged , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Proto-Oncogene Proteins B-raf/genetics , Adult , Retrospective Studies , Craniopharyngioma/drug therapy , Pyridones/therapeutic use , Pyridones/administration & dosage , Pyridones/adverse effects , Aged , Pituitary Neoplasms/drug therapy , Pyrimidinones/therapeutic use , Pyrimidinones/administration & dosage , Pyrimidinones/adverse effects , Oximes/therapeutic use , Oximes/administration & dosage , Oximes/adverse effects , Cohort Studies , Protein Kinase Inhibitors/therapeutic use , Protein Kinase Inhibitors/administration & dosage , Protein Kinase Inhibitors/adverse effects , Molecular Targeted Therapy/methods , Imidazoles/therapeutic use , Imidazoles/administration & dosage , Imidazoles/adverse effects , Treatment Outcome , Antineoplastic Combined Chemotherapy Protocols/therapeutic useABSTRACT
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.
Subject(s)
Drug Resistance, Neoplasm , Eukaryotic Translation Initiation Factor 5A , Melanoma , Mutation , Peptide Initiation Factors , Polyamines , Proto-Oncogene Proteins B-raf , RNA-Binding Proteins , Vemurafenib , Humans , Melanoma/drug therapy , Melanoma/genetics , Melanoma/metabolism , Melanoma/pathology , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins B-raf/metabolism , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Drug Resistance, Neoplasm/genetics , Animals , Polyamines/metabolism , Mice , Peptide Initiation Factors/metabolism , Peptide Initiation Factors/genetics , Cell Line, Tumor , RNA-Binding Proteins/metabolism , RNA-Binding Proteins/genetics , Vemurafenib/pharmacology , Proto-Oncogene Proteins c-myc/metabolism , Proto-Oncogene Proteins c-myc/genetics , Xenograft Model Antitumor Assays , CRISPR-Cas Systems , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Lysine/analogs & derivativesABSTRACT
INTRODUCTION: BRAF V600E substitution predicts sensitivity of a cancer to BRAF inhibitor therapy. The mutation is rarely found in soft-tissue sarcomas. Here we describe a case of undifferentiated spindle cell sarcoma showing primary insensitivity to standard chemotherapy and pronounced but non-sustained response to BRAF/MEK inhibitors at recurrence. CASE PRESENTATION: A 13-year-old girl was diagnosed with low-grade spindle cell sarcoma of pelvic localization, BRAF exon 15 double-mutated: c.1799T>A p.V600E and c.1819T>A p.S607T in cis-position. The tumor showed resistance to CWS-based first-line chemotherapy and was treated surgically by radical resection. Seven months after surgery the patient developed metastatic relapse with abdominal carcinomatosis. Combined targeted therapy with BRAF/MEK inhibitors afforded complete response in 1 month and was continued, though complicated by severe side effects (fever, rash) necessitating 1-2 week toxicity breaks. After 4 months from commencement the disease recurred and anti-BRAF/MEK regimen consolidation was unsuccessful. Intensive salvation chemotherapy was ineffective. Empirical immunotherapy afforded a transient partial response giving way to fatal progression with massive, abdominal cocoon-complicated peritoneal carcinomatosis. CONCLUSION: This is the first report of spindle cell sarcoma BRAF V600E/S607T double-mutated, responding to a combination of B-Raf and MEK inhibitors. Despite the low histological grade and radical surgical treatment of the tumor at primary manifestation, the disease had aggressive clinical course and the response to BRAF/MEK targeted therapy at recurrence was complete but nondurable. Empirical use of pembrolizumab provided no unambiguous evidence on the clinical relevance of immunotherapy in protein kinase -rearranged spindle cell tumors.
Subject(s)
Exons , Mutation , Protein Kinase Inhibitors , Proto-Oncogene Proteins B-raf , Sarcoma , Humans , Female , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Adolescent , Sarcoma/genetics , Sarcoma/drug therapy , Sarcoma/pathology , Protein Kinase Inhibitors/therapeutic use , Protein Kinase Inhibitors/pharmacology , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitorsABSTRACT
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.
Subject(s)
Molecular Targeted Therapy , Neoplasms , Protein Kinase Inhibitors , Proto-Oncogene Proteins B-raf , Humans , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Proto-Oncogene Proteins B-raf/metabolism , Neoplasms/genetics , Neoplasms/drug therapy , Neoplasms/pathology , Neoplasms/metabolism , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Mutation , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Animals , MAP Kinase Signaling System/geneticsABSTRACT
Tovorafenib (OJEMDA™) is a once-weekly oral, selective, brain-penetrant, type II RAF kinase inhibitor being developed by Day One Biopharmaceuticals, Inc., under a license from Takeda Oncology, for the treatment of paediatric low-grade glioma (pLGG) and solid tumours. Most pLGGs harbour alterations in the MAPK pathway, such as a BRAF mutation or BRAF fusion, which result in aberrant intracellular signalling. Tovorafenib is an inhibitor of mutant BRAF V600E, wild-type BRAF and wild-type CRAF kinases and BRAF fusions. In April 2024, tovorafenib received its first approval in the USA for the treatment of patients aged ≥ 6 months with relapsed or refractory pLGGs harbouring a BRAF fusion or rearrangement, or BRAF V600 mutation. It received accelerated approval for this indication based on the response rate and duration of response achieved in this population in the ongoing, pivotal, phase 2 FIREFLY-1 study. Clinical development of tovorafenib is underway in numerous countries worldwide. This article summarizes the milestones in the development of tovorafenib leading to this first approval for relapsed or refractory pLGG with an activating BRAF alteration.
Subject(s)
Drug Approval , Glioma , Proto-Oncogene Proteins B-raf , Humans , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Proto-Oncogene Proteins B-raf/genetics , Glioma/drug therapy , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Antineoplastic Agents/therapeutic use , Antineoplastic Agents/pharmacology , Child , Brain Neoplasms/drug therapy , Mutation , Phenylurea Compounds/therapeutic use , Phenylurea Compounds/pharmacologyABSTRACT
Melanoma metabolism can be reprogrammed by activating BRAF mutations. These mutations are present in up to 50% of cutaneous melanomas, with the most common being V600E. BRAF mutations augment glycolysis to promote macromolecular synthesis and proliferation. Prior to the development of targeted anti-BRAF therapies, these mutations were associated with accelerated clinical disease in the metastatic setting. Combination BRAF and MEK inhibition is a first line treatment option for locally advanced or metastatic melanoma harboring targetable BRAF mutations. This therapy shows excellent response rates but these responses are not durable, with almost all patients developing resistance. When BRAF mutated melanoma cells are inhibited with targeted therapies the metabolism of those cells also changes. These cells rely less on glycolysis for energy production, and instead shift to a mitochondrial phenotype with upregulated TCA cycle activity and oxidative phosphorylation. An increased dependence on glutamine utilization is exhibited to support TCA cycle substrates in this metabolic rewiring of BRAF mutated melanoma. Herein we describe the relevant core metabolic pathways modulated by BRAF inhibition. These adaptive pathways represent vulnerabilities that could be targeted to overcome resistance to BRAF inhibitors. This review evaluates current and future therapeutic strategies that target metabolic reprogramming in melanoma cells, particularly in response to BRAF inhibition.
Subject(s)
Melanoma , Proto-Oncogene Proteins B-raf , Humans , Melanoma/drug therapy , Melanoma/metabolism , Melanoma/genetics , Melanoma/pathology , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Proto-Oncogene Proteins B-raf/genetics , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Mutation , Skin Neoplasms/drug therapy , Skin Neoplasms/pathology , Skin Neoplasms/metabolism , Skin Neoplasms/genetics , Glycolysis/drug effectsABSTRACT
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.
Subject(s)
Antineoplastic Agents , Benzothiazoles , Cell Proliferation , Molecular Docking Simulation , Protein Kinase Inhibitors , Proto-Oncogene Proteins B-raf , Thiadiazoles , Vascular Endothelial Growth Factor Receptor-2 , Vascular Endothelial Growth Factor Receptor-2/antagonists & inhibitors , Vascular Endothelial Growth Factor Receptor-2/metabolism , Humans , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Proto-Oncogene Proteins B-raf/metabolism , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/chemical synthesis , Benzothiazoles/chemistry , Benzothiazoles/pharmacology , Benzothiazoles/chemical synthesis , Thiadiazoles/chemistry , Thiadiazoles/pharmacology , Thiadiazoles/chemical synthesis , Cell Line, Tumor , Cell Proliferation/drug effects , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemistry , Antineoplastic Agents/chemical synthesis , Drug Design , Structure-Activity Relationship , Sorafenib/pharmacology , Sorafenib/chemistry , Molecular Structure , Computer Simulation , Drug Screening Assays, AntitumorABSTRACT
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).
Subject(s)
Antineoplastic Agents , Protein Kinase Inhibitors , Proto-Oncogene Proteins B-raf , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Proto-Oncogene Proteins B-raf/metabolism , Humans , Animals , Mice , Protein Kinase Inhibitors/pharmacokinetics , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/therapeutic use , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemistry , Antineoplastic Agents/therapeutic use , Cell Line, Tumor , Female , Blood-Brain Barrier/metabolism , Brain/metabolism , Melanoma/drug therapy , Melanoma/pathology , Structure-Activity Relationship , Rats , Mice, Nude , Xenograft Model Antitumor Assays , MaleABSTRACT
The randomized METIMMOX trial (NCT03388190) examined if patients with previously untreated, unresectable abdominal metastases from microsatellite-stable (MSS) colorectal cancer (CRC) might benefit from potentially immunogenic, short-course oxaliplatin-based chemotherapy alternating with immune checkpoint blockade (ICB). Three of 38 patients assigned to this experimental treatment had metastases from BRAF-mutant MSS-CRC, in general a poor-prognostic subgroup explored here. The ≥70-year-old females presented with ascending colon adenocarcinomas with intermediate tumor mutational burden (6.2-11.8 mutations per megabase). All experienced early disappearance of the primary tumor followed by complete response of all overt metastatic disease, resulting in progression-free survival as long as 20-35 months. However, they encountered recurrence at previously unaffected sites and ultimately sanctuary organs, or as intrahepatic tumor evolution reflected in the terminal loss of initially induced T-cell clonality in liver metastases. Yet, the remarkable first-line responses to short-course oxaliplatin-based chemotherapy alternating with ICB may offer a novel therapeutic option to a particularly hard-to-treat MSS-CRC subgroup.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols , Colorectal Neoplasms , Immune Checkpoint Inhibitors , Oxaliplatin , Proto-Oncogene Proteins B-raf , Humans , Oxaliplatin/therapeutic use , Oxaliplatin/administration & dosage , Female , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , Immune Checkpoint Inhibitors/therapeutic use , Immune Checkpoint Inhibitors/pharmacology , Aged , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Mutation , Microsatellite Instability/drug effects , Treatment Outcome , Aged, 80 and overABSTRACT
Metastatic melanoma is among the most enigmatic advanced cancers to clinically manage despite immense progress in the way of available therapeutic options and historic decreases in the melanoma mortality rate. Most patients with metastatic melanoma treated with modern targeted therapies (for example, BRAFV600E/K inhibitors) and/or immune checkpoint blockade (for example, anti-programmed death 1 therapy) will progress, owing to profound tumor cell plasticity fueled by genetic and nongenetic mechanisms and dichotomous host microenvironmental influences. Here we discuss the determinants of tumor heterogeneity, mechanisms of therapy resistance and effective therapy regimens that hold curative promise.
Subject(s)
Drug Resistance, Neoplasm , Melanoma , Humans , Melanoma/drug therapy , Melanoma/genetics , Tumor Microenvironment/drug effects , Immune Checkpoint Inhibitors/therapeutic use , Immune Checkpoint Inhibitors/pharmacology , Molecular Targeted Therapy/methods , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Skin Neoplasms/drug therapy , Skin Neoplasms/geneticsABSTRACT
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.
Subject(s)
Melanoma , Metabolomics , Protein Kinase Inhibitors , Melanoma/metabolism , Melanoma/drug therapy , Melanoma/pathology , Humans , Metabolomics/methods , Cell Line, Tumor , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Proto-Oncogene Proteins B-raf/metabolism , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Magnetic Resonance Spectroscopy/methods , Mitogen-Activated Protein Kinases/metabolism , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Proton Magnetic Resonance Spectroscopy/methodsABSTRACT
Personalized cancer therapeutics bring directed treatment options to patients based on their tumor's genetic signature. Unfortunately, tumor genomes are remarkably adaptable, and acquired resistance through gene mutation frequently occurs. Identifying mutations that promote resistance within drug-treated patient populations can be cost, resource, and time intensive. Accordingly, base editing, enabled by Cas9-deaminase domain fusions, has emerged as a promising approach for rapid, large-scale gene variant screening in situ. Here, we adapt and optimize a conditional activation-induced cytidine deaminase (AID)-dead Cas9 (dCas9) system, which demonstrates greater heterogeneity of edits with an expanded footprint compared to the most commonly utilized cytosine base editor, BE4. In combination with a custom single guide RNA (sgRNA) library, we identify individual and compound variants in epidermal growth factor receptor (EGFR) and v-raf murine sarcoma viral oncogene homolog B1 (BRAF) that confer resistance to established EGFR inhibitors. This system and analytical pipeline provide a simple, highly scalable platform for cis or trans drug-modifying variant discovery and for uncovering valuable insights into protein structure-function relationships.
Subject(s)
Drug Resistance, Neoplasm , ErbB Receptors , Humans , Drug Resistance, Neoplasm/genetics , ErbB Receptors/metabolism , ErbB Receptors/genetics , ErbB Receptors/antagonists & inhibitors , Cell Line, Tumor , Gene Editing/methods , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins B-raf/metabolism , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , CRISPR-Cas Systems/genetics , Mutation/genetics , MutagenesisABSTRACT
BACKGROUND/AIM: Melanoma, a variant of skin cancer, presents the highest mortality rates among all skin cancers. Despite advancements in targeted therapies, immunotherapies, and tissue culture techniques, the absence of an effective early treatment model remains a challenge. This study investigated the impact of dabrafenib on both 2D and 3D cell culture models with distinct molecular profiles. MATERIALS AND METHODS: We developed a high-throughput workflow enabling drug screening on spheroids. Our approach involved cultivating 2D and 3D cultures derived from normal melanocytes and metastatic melanoma cells, treating them with dabrafenib and conducting viability, aggregation, migration, cell cycle, and apoptosis assays. RESULTS: Dabrafenib exerted multifaceted influences, particularly on migration at concentrations of 10 and 25 µM. It induced a decrease in cell viability, impeded cellular adhesion to the matrix, inhibited cellular aggregation and spheroid formation, arrested the cell cycle in the G1 phase, and induced apoptosis. CONCLUSION: These results confirm the therapeutic potential of dabrafenib in treating melanoma with the BRAF V600E mutation and that 3D models are validated models to study the potential of new molecules for therapeutic purposes. Furthermore, our study underscores the relevance of 3D models in simulating physiological in vivo microenvironments, providing insights into varied treatment responses between normal and tumor cells.
Subject(s)
Apoptosis , Cell Movement , Cell Survival , Imidazoles , Melanoma , Oximes , Proto-Oncogene Proteins B-raf , Spheroids, Cellular , Humans , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Cell Culture Techniques , Cell Culture Techniques, Three Dimensional/methods , Cell Cycle/drug effects , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Drug Screening Assays, Antitumor/methods , Imidazoles/pharmacology , Melanoma/drug therapy , Melanoma/pathology , Melanoma/genetics , Oximes/pharmacology , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Spheroids, Cellular/drug effectsABSTRACT
The molecular pathogenesis of thyroid carcinoma is well studied and of importance for the treatment of advanced stages. Differentiated, poorly differentiated and anaplastic carcinomas originate in the follicular cells, while medullary carcinomas derive from the Ccells. The prognosis of differentiated thyroid carcinoma is generally very favourable after surgery and radioiodine therapy. Where tumours progress and lose the ability to enrich iodine, curative treatment is usually not possible. A strategy of watchful waiting is often appropriate. Activating mutations in BRAF or gene fusions of RET and NTRK provide opportunities for targeted therapies. These may be applied with the aim of restoring iodine uptake (redifferentiation). In the absence of molecular therapy targets, multityrosine kinase inhibitors (MKI) are the therapy of choice. If anaplastic thyroid carcinoma is suspected, rapid diagnostic workup including molecular pathology is warranted. Surgery where possible and radiochemotherapy are essential components of therapy. In the presence of a BRAF mutation, inhibition of BRAF and MEK is effective, even if it is not approved in Germany. Where molecular targets are lacking, combination therapy with the MKI lenvatinib and immune checkpoint inhibition is highly effective. Mutations in RET are present in the vast majority of cases of medullary thyroid carcinoma. In aggressive advanced disease, selective RET inhibition has recently been approved as first-line therapy and often leads to an objective response and long-lasting disease stabilisation. In summary, thyroid carcinomas are among the tumour entities for which molecularly targeted therapies can be used most frequently. The involvement of specialised centres is advisable.
Subject(s)
Thyroid Neoplasms , Humans , Molecular Targeted Therapy/methods , Mutation , Phenylurea Compounds/therapeutic use , Prognosis , Protein Kinase Inhibitors/therapeutic use , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Proto-Oncogene Proteins c-ret/genetics , Proto-Oncogene Proteins c-ret/antagonists & inhibitors , Quinolines , Thyroid Neoplasms/therapy , Thyroid Neoplasms/genetics , Thyroid Neoplasms/pathology , Thyroid Neoplasms/drug therapyABSTRACT
Approved BRAF inhibitors have shown limited clinical benefit due to recurrent disease progression. In a recent Cancer Discovery paper, Yaeger et al. show that a next-generation BRAF inhibitor, PF-07799933, has widespread therapeutic activity in experimental models and patients who were refractory to treatment with approved BRAF inhibitors.
Subject(s)
Drug Resistance, Neoplasm , Protein Kinase Inhibitors , Proto-Oncogene Proteins B-raf , Humans , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Proto-Oncogene Proteins B-raf/genetics , Drug Resistance, Neoplasm/drug effects , Drug Resistance, Neoplasm/genetics , Protein Kinase Inhibitors/therapeutic use , Protein Kinase Inhibitors/pharmacology , Animals , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Melanoma/drug therapy , Melanoma/pathology , Melanoma/genetics , Melanoma/immunology , MutationABSTRACT
Targeted therapies against mutant BRAF are effectively used in combination with MEK inhibitors (MEKi) to treat advanced melanoma. However, treatment success is affected by resistance and adverse events (AEs). Approved BRAF inhibitors (BRAFi) show high levels of target promiscuity, which can contribute to these effects. The blood vessel lining is in direct contact with high plasma concentrations of BRAFi, but effects of the inhibitors in this cell type are unknown. Hence, we aimed to characterize responses to approved BRAFi for melanoma in the vascular endothelium. We showed that clinically approved BRAFi induced a paradoxical activation of endothelial MAPK signaling. Moreover, phosphoproteomics revealed distinct sets of off-targets per inhibitor. Endothelial barrier function and junction integrity were impaired upon treatment with vemurafenib and the next-generation dimerization inhibitor PLX8394, but not with dabrafenib or encorafenib. Together, these findings provide insights into the surprisingly distinct side effects of BRAFi on endothelial signaling and functionality. Better understanding of off-target effects could help to identify molecular mechanisms behind AEs and guide the continued development of therapies for BRAF-mutant melanoma.
Subject(s)
Melanoma , Protein Kinase Inhibitors , Proto-Oncogene Proteins B-raf , Signal Transduction , Vemurafenib , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Proto-Oncogene Proteins B-raf/metabolism , Humans , Protein Kinase Inhibitors/pharmacology , Melanoma/drug therapy , Melanoma/metabolism , Signal Transduction/drug effects , Vemurafenib/pharmacology , Oximes/pharmacology , Sulfonamides/pharmacology , Endothelium, Vascular/drug effects , Endothelium, Vascular/metabolism , Imidazoles/pharmacology , Endothelial Cells/drug effects , Endothelial Cells/metabolism , MAP Kinase Signaling System/drug effects , Carbamates/pharmacology , Human Umbilical Vein Endothelial Cells/drug effects , Human Umbilical Vein Endothelial Cells/metabolism , Cell Line, Tumor , MutationABSTRACT
PURPOSE OF REVIEW: Given the rapid development of multiple targeted and immune therapies for patients with advanced melanoma, it can be challenging to select a therapy based on currently available data. This review aims to provide an overview of frontline options for metastatic melanoma, with practical guidance for selecting a treatment regimen. RECENT FINDINGS: Recently reported data from randomized trials suggests that the majority of patients with unresectable melanoma should receive a PD-1 checkpoint inhibitor as part of their first line therapy, irrespective of BRAF mutation status. Additional data also suggests that combination immunotherapies result in improved outcomes compared to single agent, albeit at the cost of increased toxicity, though to date no biomarker exists to help guide treatment selection. As the number therapeutic options continue to grow for patients with advanced melanoma, there is likely to be a continued focus on combination strategies. Defining the optimal treatment approach in order to maximize efficacy while minimizing toxicity remains an area of active investigation.
Subject(s)
Immune Checkpoint Inhibitors , Melanoma , Humans , Melanoma/drug therapy , Melanoma/pathology , Immune Checkpoint Inhibitors/therapeutic use , Immunotherapy/methods , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Neoplasm Metastasis , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Proto-Oncogene Proteins B-raf/genetics , Skin Neoplasms/drug therapy , Skin Neoplasms/pathology , Randomized Controlled Trials as TopicABSTRACT
PURPOSE OF REVIEW: This report highlights several of the recent therapeutic advancements in the treatment of BRAF-mutant tumors, discusses the most common adverse events observed with BRAF-targeted agents, and suggests strategies to manage and mitigate treatment-related toxicities. RECENT FINDINGS: BRAF and MEK inhibitors represent a significant advancement in the treatment of BRAF-mutated malignancies with data across tumor types demonstrating the anti-tumor efficacy of dual MAPK inhibition. Although these agents have a reasonable toxicity profile, variable side effects across organ systems can develop. The discovery of activating BRAF mutations and subsequent development of BRAF and MEK inhibitors has transformed the treatment algorithms of BRAF-mutant malignancies. With increased application of these targeted regimens, identification and prompt management of their unique adverse events are crucial.
Subject(s)
Neoplasms , Protein Kinase Inhibitors , Proto-Oncogene Proteins B-raf , Humans , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Proto-Oncogene Proteins B-raf/genetics , Protein Kinase Inhibitors/adverse effects , Protein Kinase Inhibitors/therapeutic use , Neoplasms/drug therapy , Mutation , Antineoplastic Agents/adverse effects , Antineoplastic Agents/therapeutic use , Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitors , Molecular Targeted Therapy/adverse effectsABSTRACT
Melanoma cells express high levels of CD73 that produce extracellular immunosuppressive adenosine. Changes in the CD73 expression occur in response to tumor environmental factors, contributing to tumor phenotype plasticity and therapeutic resistance. Previously, we have observed that CD73 expression can be up-regulated on the surface of melanoma cells in response to nutritional stress. Here, we explore the mechanism by which melanoma cells release soluble CD73 under low nutrient availability and whether this might be affected by agents targeting the proto-oncogene B-Raf (BRAF). We found that starved melanoma cells can release high levels of CD73, able to convert AMP into adenosine, and this activity is abrogated by selective CD73 inhibitors, APCP or PSB-12489. The release of CD73 from melanoma cells is mediated by the matrix metalloproteinase MMP-9. Indeed, MMP-9 inhibitors significantly reduce the levels of CD73 released from the cells, while its surface levels increase. Of relevance, melanoma cells, harboring an activating BRAF mutation, upon treatment with dabrafenib or vemurafenib, show a strong reduction of CD73 cell expression and reduced levels of CD73 released into the extracellular space. Conversely, melanoma cells resistant to dabrafenib show high expression of membrane-bound CD73 and soluble CD73 released into the culture medium. In summary, our data indicate that CD73 is released from melanoma cells. The expression of CD73 is associated with response to BRAF inhibitors. Melanoma cells developing resistance to dabrafenib show increased expression of CD73, including soluble CD73 released from cells, suggesting that CD73 is involved in acquiring resistance to treatment.