eIF4F controls ERK MAPK signaling in melanomas with BRAF and NRAS mutations.

The eIF4F translation initiation complex plays a critical role in melanoma resistance to clinical BRAF and MEK inhibitors. In this study, we uncover a function of eIF4F in the negative regulation of the rat sarcoma (RAS)/rapidly accelerated fibrosarcoma (RAF)/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) signaling pathway. We demonstrate that eIF4F is essential for controlling ERK signaling intensity in treatment-naïve melanoma cells harboring BRAF or NRAS mutations. Specifically, the dual-specificity phosphatase DUSP6/MKP3, which acts as a negative feedback regulator of ERK activity, requires continuous production in an eIF4F-dependent manner to limit excessive ERK signaling driven by oncogenic RAF/RAS mutations. Treatment with small-molecule eIF4F inhibitors disrupts the negative feedback control of MAPK signaling, leading to ERK hyperactivation and EGR1 overexpression in melanoma cells in vitro and in vivo. Furthermore, our quantitative analyses reveal a high spare signaling capacity in the ERK pathway, suggesting that eIF4F-dependent feedback keeps the majority of ERK molecules inactive under normal conditions. Overall, our findings highlight the crucial role of eIF4F in regulating ERK signaling flux and suggest that pharmacological eIF4F inhibitors can disrupt the negative feedback control of MAPK activity in melanomas with BRAF and NRAS activating mutations.

Ursolic acid interaction with transcription factors BRAF, V600E, and V600K: a computational approach towards new potential melanoma treatments.

CONTEXT: Melanoma is one of the cancers with the highest mortality rate for its ability to metastasize. Several targets have undergone investigation for the development of drugs against this pathology. One of the main targets is the kinase BRAF (RAF, rapidly accelerated fibrosarcoma). The most common mutation in melanoma is BRAFV600E and has been reported in 50-90% of patients with melanoma. Due to the relevance of the BRAFV600E mutation, inhibitors to this kinase have been developed, vemurafenib-OMe and dabrafenib. Ursolic acid (UA) is a pentacyclic triterpene with a privileged structure, the pentacycle scaffold, which allows to have a broad variety of biological activity; the most studied is its anticancer capacity. In this work, we reported the interaction profile of vemurafenib-OMe, dabrafenib, and UA, to define whether UA has binding capacity to BRAFWT, BRAFV600E, and BRAFV600K. Homology modeling of BRAFWT, V600E, and V600K; molecular docking; and molecular dynamics simulations were carried out and interactions and residues relevant to the binding of the inhibitors were obtained. We found that UA, like the inhibitors, presents hydrogen bond interactions, and hydrophobic interactions of van der Waals, and π-stacking with I463, Q530, C532, and F583. The ΔG of ursolic acid in complex with BRAFV600K (- 63.31 kcal/mol) is comparable to the ΔG of the selective inhibitor dabrafenib (- 63.32 kcal/mol) in complex to BRAFV600K and presents a ΔG like vemurafenib-OMe with BRAFWT and V600E. With this information, ursolic acid could be considered as a lead compound for design cycles and to optimize the binding profile and the selectivity towards mutations for the development of new selective inhibitors for BRAFV600E and V600K to new potential melanoma treatments. METHODS: The homology modeling calculations were executed on the public servers I-TASSER and ROBETTA, followed by molecular docking calculations using AutoGrid 4.2.6, AutoDockGPU 1.5.3, and AutoDockTools 1.5.6. Molecular dynamics and metadynamics simulations were performed in the Desmond module of the academic version of the Schrödinger-Maestro 2020-4 program, utilizing the OPLS-2005 force field. Ligand-protein interactions were evaluated using Schrödinger-Maestro program, LigPlot + , and PLIP (protein-ligand interaction profiler). Finally, all of the protein figures presented in this article were made in the PyMOL program.

BRAF Modulates the Interplay Between Cell-Cell and Cell-Extracellular Matrix Adhesions in PECAM-1-Mediated Mechanotransduction.

Mechanotransduction, the process of how cells sense and convert mechanical stimuli into biochemical response, is crucial in the migration of leukocytes or cancer cells through the endothelium during inflammation or metastasis. Migrating cells exert forces on the endothelium through cell surface adhesion molecules, such as platelet endothelial adhesion molecule PECAM-1, and this is essential for a successful transmigration. To study PECAM-1-mediated mechanotransduction, we applied PECAM-1-antibody-coated magnetic beads and exerted about 40 pN force on the endothelial monolayer. We show that force increases cell-ECM adhesion in the cell center and is accompanied by the opening of cell-cell junctions. Upon depletion of the MEK/ERK kinase, BRAF force increases cell-ECM adhesion both at the cell periphery and in the cell center, but this does not result in the opening of cell-cell junctions. Decreasing cell-ECM adhesion in BRAF-depleted cells through FAK inhibition results in the remodeling of cell-cell junctions. Force-induced increase in cell-ECM adhesion in the cell center correlates with the activation of the transcriptional cofactor Yes-associated protein (YAP). Furthermore, the induced activation of YAP through LATS inhibition prevents junctional remodeling in control cells. Thus, the activation of YAP might determine the strength of cell-cell junctions during PECAM-1-mediated mechanotransduction.

Matched three-dimensional organoids and two-dimensional cell lines of melanoma brain metastases mirror response to targeted molecular therapy.

Despite advances in the treatment paradigm for patients with metastatic melanoma, melanoma brain metastasis (MBM) continues to represent a significant treatment challenge. The study of MBM is limited, in part, by shortcomings in existing preclinical models. Surgically eXplanted Organoids (SXOs) are ex vivo, three-dimensional cultures prepared from primary tissue samples with minimal processing that recapitulate genotypic and phenotypic features of parent tumors without an artificial extracellular scaffold. MBM SXOs were created by a novel protocol incorporating techniques for establishing glioma and cutaneous melanoma organoids. A BRAFV600K-mutant and BRAF-wildtype MBM sample were collected directly from the operating room. Organoids were cultured in an optimized culture medium without an artificial extracellular scaffold. Concurrently, matched patient-derived cell lines were created. Organoid growth was observed within 3-4 weeks, and MBM SXOs retained histological features of the parent tissue, including pleomorphic epithelioid cells with abundant cytoplasm, large nuclei, focal melanin accumulation, and strong SOX10 positivity. After sufficient growth, organoids could be manually parcellated to increase the number of replicates. Matched SXOs and cell lines demonstrated sensitivity to BRAF and MEK inhibitors. Further study using SXOs may improve the translational relevance of preclinical studies and enable the study of the metastatic melanoma tumor microenvironment.

Strongly ROS-Correlated, Time-Dependent, and Selective Antiproliferative Effects of Synthesized Nano Vesicles on BRAF Mutant Melanoma Cells and Their Hyaluronic Acid-Based Hydrogel Formulation.

Cutaneous metastatic melanoma (CMM) is the most aggressive form of skin cancer with a poor prognosis. Drug-induced secondary tumorigenesis and the emergency of drug resistance worsen an already worrying scenario, thus rendering urgent the development of new treatments not dealing with mutable cellular processes. Triphenyl phosphonium salts (TPPSs), in addiction to acting as cytoplasmic membrane disruptors, are reported to be mitochondria-targeting compounds, exerting anticancer effects mainly by damaging their membranes and causing depolarization, impairing mitochondria functions and their DNA, triggering oxidative stress (OS), and priming primarily apoptotic cell death. TPP-based bola amphiphiles are capable of self-forming nanoparticles (NPs) with enhanced biological properties, as commonly observed for nanomaterials. Already employed in several other biomedical applications, the per se selective potent antibacterial effects of a TPP bola amphiphile have only recently been demonstrated on 50 multidrug resistant (MDR) clinical superbugs, as well as its exceptional and selective anticancer properties on sensitive and MDR neuroblastoma cells. Here, aiming at finding new molecules possibly developable as new treatments for counteracting CMM, the effects of this TPP-based bola amphiphile (BPPB) have been investigated against two BRAF mutants CMM cell lines (MeOV and MeTRAV) with excellent results (even IC50 = 49 nM on MeOV after 72 h treatment). With these findings and considering the low cytotoxicity of BPPB against different mammalian non-tumoral cell lines and red blood cells (RBCs, selectivity indexes up to 299 on MeOV after 72 h treatment), the possible future development of BPPB as topical treatment for CMM lesions was presumed. With this aim, a biodegradable hyaluronic acid (HA)-based hydrogel formulation (HA-BPPB-HG) was prepared without using any potentially toxic crosslinking agents simply by dispersing suitable amounts of the two ingredients in water and sonicating under gentle heating. HA-BPPB-HA was completely characterized, with promising outcomes such as high swelling capability, high porosity, and viscous elastic rheological behavior.

Treatment resistance to melanoma therapeutics on a single cell level.

Therapy targeting the BRAF-MEK cascade created a treatment revolution for patients with BRAF mutant advanced melanoma. Unfortunately, 80% patients treated will progress by 5 years follow-up. Thus, it is imperative we study mechanisms of melanoma progression and therapeutic resistance. We created a scRNA (single cell RNA) atlas of 128,230 cells from 18 tumors across the treatment spectrum, discovering melanoma cells clustered strongly by transcriptome profiles of patients of origins. Our cell-level investigation revealed gains of 1q and 7q as likely early clonal events in metastatic melanomas. By comparing patient tumors and their derivative cell lines, we observed that PD1 responsive tumor fraction disappears when cells are propagated in vitro. We further established three anti-BRAF-MEK treatment resistant cell lines using three BRAF mutant tumors. ALDOA and PGK1 were found to be highly expressed in treatment resistant cell populations and metformin was effective in targeting the resistant cells. Our study suggests that the investigation of patient tumors and their derivative lines is essential for understanding disease progression, treatment response and resistance.

Phenylephrine Enhances the Mitogenic Effect of S-Allyl-L-cysteine on Primary Cultured Hepatocytes through Protein Kinase C-Induced B-Raf Phosphorylation.

The co-mitogenic effects of the α1-adrenoceptor agonist phenylephrine on S-allyl-L-cysteine (SAC)-induced hepatocyte proliferation were examined in primary cultures of adult rat hepatocytes. The combination of phenylephrine (10-10-10-6 M) and SAC (10-6 M) exhibited a significant dose-dependent increase in the number of hepatocyte nuclei and viable cells compared to SAC alone. This combination also increased the progression of hepatocyte nuclei into the S-phase. The potentiating effect of phenylephrine on SAC-induced cell proliferation was counteracted by prazosin (an α1-adrenergic receptor antagonist) and GF109203X (selective protein kinase C (PKC) inhibitor). In addition, PMA (direct PKC activator) potentiated the proliferative effects of SAC similarly to phenylephrine. In essence, these findings suggest that PKC activity plays a crucial role in enhancing SAC-induced cell proliferation. Moreover, the effects of phenylephrine on SAC-induced Ras activity, Raf phosphorylation, and extracellular signal-regulated kinase 2 (ERK2) phosphorylation were investigated. Phenylephrine (or PMA) in combination with SAC did not augment Ras activity, but further increased ERK2 phosphorylation and its upstream B-Raf phosphorylation. These results indicate that PKC activation, triggered by stimulating adrenergic α1 receptors, further amplifies SAC-induced cell proliferation through enhanced ERK2 phosphorylation via increased B-Raf-specific phosphorylation in primary cultured hepatocytes.

The mTOR pathway controls phosphorylation of BRAF at T401.

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.

Sex-dependent effects in the aged melanoma tumor microenvironment influence invasion and resistance to targeted therapy.

There is documented sex disparity in cutaneous melanoma incidence and mortality, increasing disproportionately with age and in the male sex. However, the underlying mechanisms remain unclear. While biological sex differences and inherent immune response variability have been assessed in tumor cells, the role of the tumor-surrounding microenvironment, contextually in aging, has been overlooked. Here, we show that skin fibroblasts undergo age-mediated, sex-dependent changes in their proliferation, senescence, ROS levels, and stress response. We find that aged male fibroblasts selectively drive an invasive, therapy-resistant phenotype in melanoma cells and promote metastasis in aged male mice by increasing AXL expression. Intrinsic aging in male fibroblasts mediated by EZH2 decline increases BMP2 secretion, which in turn drives the slower-cycling, highly invasive, and therapy-resistant melanoma cell phenotype, characteristic of the aged male TME. Inhibition of BMP2 activity blocks the emergence of invasive phenotypes and sensitizes melanoma cells to BRAF/MEK inhibition.

In vivo vulnerabilities to GPX4 and HDAC inhibitors in drug-persistent versus drug-resistant BRAFV600E lung adenocarcinoma.

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.

Decoding the Molecular Mechanisms of BRAF V600E-Induced Nevi Formation.

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.

Design, Synthesis, and Biological Evaluation of 5-Amino-4-fluoro-1H-benzo[d]imidazole-6-carboxamide Derivatives as Novel and Potential MEK/RAF Complex Inhibitors Based on the "Clamp" Strategy.

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.

Characterization of two melanoma cell lines resistant to BRAF/MEK inhibitors (vemurafenib and cobimetinib).

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.

Reversible control of kinase signaling through chemical-induced dephosphorylation.

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.

Role of mitochondria and potential of mitochondria-targeted therapy in BRAF mutant cancer: A review.

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.

Multi-omics analysis delineates resistance mechanisms associated with BRAF inhibition in melanoma cells.

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.

Impact of protein and small molecule interactions on kinase conformations.

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.

MAZ promotes thyroid cancer progression by driving transcriptional reprogram and enhancing ERK1/2 activation.

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.

BRAFV600E/p-ERK/p-DRP1(Ser616) Promotes Tumor Progression and Reprogramming of Glucose Metabolism in Papillary Thyroid Cancer.

Background: Papillary thyroid cancer (PTC) with the BRAFV600E mutation is associated with a poorer prognosis. BRAF inhibitors may demonstrate limited efficacy due to emerging drug resistance. The Warburg effect may have cancer therapeutic implications. It is not known if the BRAFV600E mutation is associated with altered glucose metabolism in PTC. Methods: This study examined the effect of BRAFV600E and dynamin-related protein 1 (DRP1) on various cellular processes in PTC cells, including cell proliferation, migration, invasion, mitochondrial fission, glucose metabolism, reactive oxygen species (ROS) generation, and apoptosis. We used RT-qPCR to assess the expression of key glycolytic enzymes in thyroid cancer tissues. Additionally, the regulatory interaction between BRAFV600E and DRP1 was investigated through Western blot and immunohistochemical staining. We further evaluated the impact of DRP1 in PTC and the inhibitory effects of dabrafenib and 2-deoxy-d-glucose (2-DG) in vitro and in vivo. Results: We found that the BRAFV600E mutation significantly augments aerobic glycolysis while suppressing oxidative phosphorylation in PTC. We identified the BRAFV600E/p-ERK/p-DRP1(Ser616) signaling pathway as a critical mediator in PTC progression. First, the BRAFV600E/p-ERK/p-DRP1(Ser616) signaling pathway enhances cell proliferation by upregulating hexokinase 2 expression and thereby increasing aerobic glycolysis. Second, it inhibits apoptosis by promoting mitochondrial fission and reducing ROS levels. Moreover, we demonstrated that the combination therapy of 2-DG and dabrafenib markedly impedes the progression of BRAFV600E-positive PTC. Conclusion: The BRAFV600E/p-ERK/p-DRP1(Ser616) signaling pathway plays a pivotal role in glucose metabolism reprogramming, contributing to the aggressiveness and progression of BRAFV600E-positive PTC. Our findings suggest that a combined therapeutic approach using 2-DG and dabrafenib has the potential to improve the outcome of PTC patients with BRAFV600E.

Histopathologic, genomic, transcriptomic, and functional characteristics of eight melanocytic tumors with BRAF fusions showing stronger MAPK pathway activation compared to BRAF V600E tumors.

BACKGROUND: Activating BRAF gene alterations are central to melanocytic tumor pathogenesis. A small, emerging subset of melanocytic tumors driven by BRAF fusions has distinct therapeutic implications and has been described to have Spitzoid morphology patterns. However, such morphological patterns do not encompass all cases, and little is known about the functional molecular events. MATERIALS AND METHODS: We conducted a retrospective search through our molecular archives to identify melanocytic tumors with BRAF fusions. We reviewed clinical, histopathological, and genomic features. We further explored transcriptomic and protein-level findings. RESULTS: Histopathologic patterns varied, with many cases without a distinctive pattern. We identified novel and diverse BRAF gene fusion partners. Differential transcriptomic analysis between low-risk BRAF fusion tumors and reference BRAF V600E tumors showed no differentially expressed genes. However, quantitatively stronger MAPK pathway activation of BRAF fusion tumors over BRAF V600E tumors was demonstrated by statistically significant stronger staining of p-ERK immunohistochemistry. Gene-specific RNA analysis shows comparable BRAF transcript levels between the two groups. DISCUSSION AND CONCLUSION: The quantitatively stronger activation of the MAPK pathway of BRAF fusion tumors, instead of qualitatively different transcriptomes, may account for the morphology difference from conventional BRAF V600E tumors. BRAF fusions likely act through dysregulated protein function rather than RNA upregulation related to the characteristics of the fusion partners.