Mutant Melanoma Cells Research Articles

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.

Effects of TET2-mediated methylation reconstruction on A2058 melanoma cell sensitivity to matrix stiffness in a 3D culture system

Matrix stiffness is a crucial factor in the tumor microenvironment, impacting tumor progression and development. TET2 is vital for epigenetic regulation in melanoma and is significantly reduced in advanced melanomas compared with nevi and thin melanomas. However, it is unclear how TET2 mediates the effect of matrix stiffness on melanoma cells. This study utilized A2058 cell lines and prepared different stiffness collagen hydrogels to evaluate TET2 overexpression (TET2OE) and mutant (TET2M) melanoma cells' activity, proliferation, and invasion. A2058 melanoma cells' viability and invasion decreased with increased matrix stiffness, with TET2OE cells experiencing a more significant impact than TET2M cells. Methylation analysis revealed that TET2 determines gene methylation levels, influencing cell-ECM interactions. Transcriptome analysis confirmed that TET2 promotes matrix stiffness's effect on melanoma cell fate. This research provides promising directions and opportunities for melanoma treatment.

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.

Abstract 3078: Role of HuR in metabolic reprogramming of BRAF mutant melanoma treated with dabrafenib

Abstract Introduction: Oncogenic BRAF mutations are present in about 50% of melanoma and are clinically targetable by BRAF inhibitors such as dabrafenib. While BRAF inhibitors show high rates of clinical response, that response is transient and resistance inevitably develops. Previously we have demonstrated that BRAF inhibitors induce a metabolic shift towards the mitochondrial oxidative phosphorylation. We have shown that inhibition of wild type isocitrate dehydrogenase 1 (wtIDH1), a vital anaplerotic and anti-ROS enzyme, demonstrates synergy with dabrafenib in vitro and in vivo. IDH1 expression has been found in pancreatic cancer to be posttranscriptionally regulated by the RNA-binding and stress response protein, HuR (ELAVL1) under nutrient and chemotherapy stress. We hypothesize that HuR functions as a survival mechanism in melanoma under BRAF inhibition, regulating metabolic reprogramming including the expression of IDH1. Methods: The human BRAF V600E mutant melanoma cell line, A375, was used for all experiments. HuR and IDH1 expression was suppressed by siRNA oilgos through lipofectamine transfection, as validated by immunoblot analysis. The bioenergetic assay was conducted using Seahorse XFp mini extracellular analyzer to measure the oxygen consumption rate (OCR) under nutrient stress, both with and without dabrafenib exposure. The impact of HuR on IDH1 expression was further explored using qPCR and Immunoblot. Ivosidenib was used for pharmacologic inhibition of wtIDH as previously described. Results: Mitochondrial respiration (OCR) increased in parental A375 BRAF mutant melanoma cells following nutrient stress with low glucose conditions akin to the tumor microenvironment. OCR was further increased after dabrafenib exposure. Immunoblot analysis revealed that dabrafenib treated cells at 6-hour and 12-hour time points displayed HuR translocation from nucleus to cytoplasm, as detected in cytoplasmic lysates. Transient HuR silencing blocked the adaptive OCR response to both low glucose and BRAF inhibition. This suggests that HuR is involved in the metabolic response to acute BRAF inhibition in BRAF mutant melanoma. HuR cytoplasmic translocation is associated with increased IDH1 expression 24 hours after dabrafenib exposure. However, there is no increase in IDH1 levels under nutrient or treatment stress in HuR knockdown cells, indicating that IDH1 is regulated by HuR. Transient IDH1 knockdown, as well as pharmacologic IDH1 inhibition, blocks the OCR response of melanoma cells to BRAF inhibition and low glucose. This indicates that IDH1 is important in the HuR metabolic response to targeted therapy. Conclusion: We find that BRAF mutant melanoma cells adapt to acute dabrafenib exposure via HuR mediated expression of IDH1 to upregulate mitochondrial metabolism. This demonstrates a targeted therapy induced metabolic vulnerability that can be exploited by wtIDH1 inhibition. Citation Format: Alexander Loftus, Mehrdad Zarei, Hallie Graor, Omid Hajihassani, Christina Boutros, Jordan M. Winter, Luke D. Rothermel. Role of HuR in metabolic reprogramming of BRAF mutant melanoma treated with dabrafenib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3078.

Abstract 3008: Loss of Rab25 accompanied by NRAS activation cooperatively mediates the tumorigenic transformation in melanoma

Abstract Background: Ras oncogene activation is a common and often essential event in cancer development, including melanoma. Ras mutation is present in 20-30% of melanomas and remains an undrugged target. NRAS mutation is the most frequent Ras isoform activated in melanoma, with more than 80% cases of Q61 substitutions, and is closely associated with tumor aggressiveness. Rab25 is a ubiquitously expressed small GTPase protein with a context-dependent role in cancer pathophysiology. Its role remains undiscovered in the case of melanoma. Rab25 deep deletion can be seen in portions of melanoma in databases such as the Cancer Genome Atlas and Cbioportal. Interestingly, Rab25 has a consensus sequence homologous with the binding site of Q61 mutant Ras. We propose that the loss of Rab25 in melanocytes accompanied by Ras oncogenic activation are significant events in melanoma development, and restoration of Rab25 can potentially inhibit constitutively activated N-RAS; Rab25 serves as a tumor suppressor in melanoma genesis. Objectives: To discover the role of Rab25 in melanoma, we studied Rab25 expression via mRNA and protein levels in different melanoma cell lines and screened biopsy-proven melanoma samples through immunohistochemistry in 61 different melanoma sections. Further, to see the tumor suppressive mechanism of Rab25 in vitro, we transduced selected cell lines Rab25. Results: There is an 87% and 62.7% reduction in Rab25 mRNA level in NRASQ61R(SKMEL-2) and NRASQ61L type (WM1366) melanoma cell lines, respectively, also reflected in Rab25 protein level, and statistically significant at p-value <0.005, using student’s T-test. Further, Rab25, prominent in adjacent normal tissues, was significantly lost in biopsy-proven melanoma tissues, with an H-score ranging from 13.28 to 91.77% and 0.00 to-11.24 % for normal skin tissue and melanoma section, respectively, and a negative correlation of -0.350, indicating a promising association of Rab25 loss with the progression of melanoma. Conclusion: There is a loss of Rab25 in NRAS mutant melanoma with cancer progression. We saw a significant increase in Rab25 expression among the transduced sublines of NRAS mutant melanoma cell lines at the mRNA level, which will be further assessed through western blotting, cell proliferation assay, trans well migration assay, soft-agar colony formation assay to confirm NRAS mutant melanoma cell behavior. We will attempt to deliver a liposomally encapsulated six-peptide sequence to therapeutically inhibit the mutant NRAS. Citation Format: Samikshya Kandel, Krishna Rao. Loss of Rab25 accompanied by NRAS activation cooperatively mediates the tumorigenic transformation in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3008.

Abstract 2900: BRAFi-induced epigenetic switch drives the reprogramming of resistant cancer-associated fibroblasts

Abstract Cancer-associated fibroblasts (CAFs) are a potential target for optimizing therapeutic strategies against melanoma. However, a major unknown still exists in CAFs, which is how CAFs maintain and even reinforce their functions in drug therapy and influence melanoma cell drug sensitivity. Yes-associated protein 1 (YAP1) is the master regulator that shuttles between the cytoplasm and nucleus in CAFs. Apparently, proteins associated with nuclear YAP1 in CAFs when treated with BRAF inhibitor (BRAFi) can potentially influence tumor drug response. Here we unveil that CAFs require the YAP1 function to proliferate, migrate, remodel the cytoskeletal machinery and matrix, and promote cancer cell invasion. Ablating YAP1 in CAFs increases the response of BRAF-mutant melanoma cells to BRAFi and MEK inhibitor (MEKi) in vivo and in vitro. Using an approach termed RIME (Rapid Immunoprecipitation Mass Spectrometry), we purified nuclear YAP1 and identified protein arginine methyl transferase 1 (PRMT1) as a major YAP1 binding partner in BRAFi-treated CAFs. Binding PRMT1 to YAP1 was confirmed by Co-immunoprecipitation and proximity ligation assay, and YAP1 deficiency in CAFs diminishes the nuclear accumulation of PRMT1, suggesting BRAFi-induced nuclear translocation of PRMT1 in CAFs requires the formation of YAP1 and PRMT1 complex. Silencing PRMT1 expression in CAFs not only deprives them of their ability to proliferate and to induce BRAF mutant melanoma cell resistance to BRAFi and MEKi. PRMT1 is a type I R-methyltransferase that mediates arginine methylation in human cells, which was known to be recruited to chromatin and exert its enzymatic activity to methylate Arg3 of histone H4 (H4R3). Using Histone H4 Modification Multiple Assay, we observed a global increase in Histone H4 methylation, especially H4R3 methylation, in CAFs in response to BRAFi stimulation, suggesting increased chromatin unfolding and accessibility and transcription activation. When YAP1 or PRMT1 expression was silenced, H4R3 methylation was notably suppressed, indicating BRAFi-induced H4R3 methylation was indeed driven by the YAP1 and PRMT1 signaling axis. In conclusion, our data disclose a novel BRAFi-induced YAP1-PRMT1-mediated epigenetic mechanism that reprograms CAFs through H4R3 methylation-directed transcriptional activation to reinforce their drug-resistant functions and modulate melanoma cell drug sensitivity. Moreover, the discovery of PRMT1 as a key player in the intricate molecular mechanisms that govern epigenetic regulation in CAFs in response to BRAFi treatment suggest PRMT1 as a potential CAF target in cancer therapy. Citation Format: Yao Xiao, Linli Zhou, Yuhang Zhang. BRAFi-induced epigenetic switch drives the reprogramming of resistant cancer-associated fibroblasts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2900.

Dataset: Impact of β-Galactosylceramidase Overexpression on the Protein Profile of Braf(V600E) Mutated Melanoma Cells

β-Galactosylceramidase (GALC) is a lysosomal enzyme involved in sphingolipid metabolism by removing β-galactosyl moieties from β-galactosyl ceramide and β-galactosyl sphingosine. Previous observations have shown that GALC exerts a pro-oncogenic activity in human melanoma. Here, the impact of GALC overexpression on the proteomic landscape of BRAF-mutated A2058 and A375 human melanoma cell lines was investigated by liquid chromatography–tandem mass spectrometry analysis of the cell extracts. The results indicate that GALC overexpression causes the upregulation/downregulation of 172/99 proteins in GALC-transduced cells when compared to control cells. Gene ontology categorization of up/down-regulated proteins indicates that GALC may modulate the protein landscape in BRAF-mutated melanoma cells by affecting various biological processes, including RNA metabolism, cell organelle fate, and intracellular redox status. Overall, these data provide further insights into the pro-oncogenic functions of the sphingolipid metabolizing enzyme GALC in human melanoma.

USP18 enhances the resistance of BRAF-mutated melanoma cells to vemurafenib by stabilizing cGAS expression to induce cell autophagy

This study aims to discern the possible molecular mechanism of the effect of ubiquitin-specific peptidase 18 (USP18) on the resistance to BRAF inhibitor vemurafenib in BRAF V600E mutant melanoma by regulating cyclic GMP-AMP synthase (cGAS). The cancer tissues of BRAF V600E mutant melanoma patients before and after vemurafenib treatment were collected, in which the protein expression of USP18 and cGAS was determined. A BRAF V600E mutant human melanoma cell line (A2058R) resistant to vemurafenib was constructed with its viability, apoptosis, and autophagy detected following overexpression and depletion assays of USP18 and cGAS. Xenografted tumors were transplanted into nude mice for in vivo validation. Bioinformatics analysis showed that the expression of cGAS was positively correlated with USP18 in melanoma, and USP18 was highly expressed in melanoma. The expression of cGAS and USP18 was up-regulated in cancer tissues of vemurafenib-resistant patients with BRAF V600E mutant melanoma. Knockdown of cGAS inhibited the resistance to vemurafenib in A2058R cells and the protective autophagy induced by vemurafenib in vitro. USP18 could deubiquitinate cGAS to promote its protein stability. In vivo experimentations confirmed that USP18 promoted vemurafenib-induced protective autophagy by stabilizing cGAS protein, which promoted resistance to vemurafenib in BRAF V600E mutant melanoma cells. Collectively, USP18 stabilizes cGAS protein expression through deubiquitination and induces autophagy of melanoma cells, thereby promoting the resistance to vemurafenib in BRAF V600E mutant melanoma.

A multi-omics integrative approach unravels novel genes and pathways associated with senescence escape after targeted therapy in NRAS mutant melanoma

Therapy Induced Senescence (TIS) leads to sustained growth arrest of cancer cells. The associated cytostasis has been shown to be reversible and cells escaping senescence further enhance the aggressiveness of cancers. Chemicals specifically targeting senescent cells, so-called senolytics, constitute a promising avenue for improved cancer treatment in combination with targeted therapies. Understanding how cancer cells evade senescence is needed to optimise the clinical benefits of this therapeutic approach. Here we characterised the response of three different NRAS mutant melanoma cell lines to a combination of CDK4/6 and MEK inhibitors over 33 days. Transcriptomic data show that all cell lines trigger a senescence programme coupled with strong induction of interferons. Kinome profiling revealed the activation of Receptor Tyrosine Kinases (RTKs) and enriched downstream signaling of neurotrophin, ErbB and insulin pathways. Characterisation of the miRNA interactome associates miR-211-5p with resistant phenotypes. Finally, iCell-based integration of bulk and single-cell RNA-seq data identifies biological processes perturbed during senescence and predicts 90 new genes involved in its escape. Overall, our data associate insulin signaling with persistence of a senescent phenotype and suggest a new role for interferon gamma in senescence escape through the induction of EMT and the activation of ERK5 signaling.

Abstract 2591: Role of Rab25 in Melanoma and its potential targeting

Abstract Background: RAB25 is a protein that belongs to the RAB11 subfamily located in the lq22 locus of chromosome and is ubiquitously expressed in cells of the gastrointestinal mucosa, ileum, and kidney. Its role in tumorigenesis is variable but is unclear in the case of melanoma progression. Our previous studies showed that loss of RAB25 has a special role in mediating the tumorigenic transformation of human mammary epithelial cells. Its loss particularly cooperated with mutant Ras isoforms to promote transformation in vitro. We are now examining the role of RAB25 in melanomas as melanomas exhibit a high rate of N-Ras mutations. We hypothesize that the presence of NRAS types of mutation and expression of RAB25 are mutually exclusive as RAB25 functions are a tumor suppressor to inhibit this mutation. Methods: Different NRAS mutant melanoma cell lines (SKMEL-2, SKMEL-28, WM1366 and WM266-4) were examined forRab25 expression through qRT-PCR and Western Blotting. The positive control was MCF cell lines and MDA-MB-231 was considered as negative control. Immunohistochemistry was done with biopsy proven melanoma tissue samples and normal adjacent tissue to observe the trend of Rab25 expression. Keratinocytes and fibroblasts in the tissue samples were considered as internal controls.Results: We found a significant decrease in Rab25 expression in NRAS mutant melanoma cell lines compared to Raf mutants and primary melanocytes. The reduction was prominent at both the RNA and protein level. RAB25 expression was also reduced in melanoma tumors as compared to surrounding tissue. Conclusion: RAB25 loss may indeed cooperate with NRAS mutation to promote transformation in melanomas. Further studies including overexpression experiment in the cell line model followed by migration assay, invasion assay, and in vivo nude mouse experiments will provide further confirmation of our hypothesis. Citation Format: Samikshya Kandel, Krishna Rao. Role of Rab25 in Melanoma and its potential targeting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2591.

Erianin suppresses constitutive activation of MAPK signaling pathway by inhibition of CRAF and MEK1/2

Constitutive activation of RAS-RAF-MEK-ERK signaling pathway (MAPK pathway) frequently occurs in many cancers harboring RAS or RAF oncogenic mutations. Because of the paradoxical activation induced by a single use of BRAF or MEK inhibitors, dual-target RAF and MEK treatment is thought to be a promising strategy. In this work, we evaluated erianin is a novel inhibitor of CRAF and MEK1/2 kinases, thus suppressing constitutive activation of the MAPK signaling pathway induced by BRAF V600E or RAS mutations. KinaseProfiler enzyme profiling, surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), cellular thermal shift assay, computational docking, and molecular dynamics simulations were utilized to screen and identify erianin binding to CRAF and MEK1/2. Kinase assay, luminescent ADP detection assay, and enzyme kinetics assay were investigated to identify the efficiency of erianin in CRAF and MEK1/2 kinase activity. Notably, erianin suppressed BRAF V600E or RAS mutant melanoma and colorectal cancer cell by inhibiting MEK1/2 and CRAF but not BRAF kinase activity. Moreover, erianin attenuated melanoma and colorectal cancer in vivo. Overall, we provide a promising leading compound for BRAF V600E or RAS mutant melanoma and colorectal cancer through dual targeting of CRAF and MEK1/2.

Niclosamide inhibits epithelial-mesenchymal transition with apoptosis induction in BRAF/ NRAS mutated metastatic melanoma cells

Malignant melanoma is considered a deadly aggressive form of skin cancer that frequently metastasizes to various distal organs, which harbors mutations of the BRAF or NRAS which occur in 30 to 50% of melanoma patients. The growth factors secreted by melanoma cells contribute to tumor angiogenesis with the acquisition of metastatic potential by epithelial-mesenchymal transition (EMT) and drive melanoma growth toward a more aggressive form. Niclosamide (NCL) is an FDA-approved anthelmintic drug and is reported to have strong anti-cancer properties against various solid and liquid tumors. Its role in BRAF or NRAS mutated cells is unknown. In this context, we uncovered the role of NCL in impeding malignant metastatic melanoma in vitro in SK-MEL-2 and SK-MEL-28 cell lines. We found that NCL induces significant ROS generation and apoptosis through a series of molecular mechanisms, such as depolarization of mitochondrial membrane potential, arresting the cell cycle at the sub G1 phase with a significant increase in the DNA cleavage via topoisomerase II in both cell lines. We also found that NCL potently inhibited metastasis, which was examined by scratch wound assay, Additionally, we found that NCL inhibits the most important markers involved in the EMT signaling cascade that are stimulated by TGF-β such as N-cadherin, Snail, Slug, Vimentin, α-SMA and p-Smad 2/3. This work provides useful insights into the mechanism of NCL in BRAF/NRAF mutant melanoma cells via inhibition of molecular signaling events involved in EMT signaling, and apoptosis induction.

Discovery of a Novel ATP-Competitive MEK Inhibitor DS03090629 that Overcomes Resistance Conferred by BRAF Overexpression in BRAF-Mutated Melanoma.

Patients with melanoma with activating BRAF mutations (BRAF V600E/K) initially respond to combination therapy of BRAF and MEK inhibitors. However, their clinical efficacy is limited by acquired resistance, in some cases driven by amplification of the mutant BRAF gene and subsequent reactivation of the MAPK pathway. DS03090629 is a novel and orally available MEK inhibitor that inhibits MEK in an ATP-competitive manner. In both in vitro and in vivo settings, potent inhibition of MEK by DS03090629 or its combination with the BRAF inhibitor dabrafenib was demonstrated in a mutant BRAF-overexpressing melanoma cell line model that exhibited a higher MEK phosphorylation level than the parental cell line and then became resistant to dabrafenib and the MEK inhibitor trametinib. DS03090629 also exhibited superior efficacy against a melanoma cell line-expressing mutant MEK1 protein compared with dabrafenib and trametinib. Biophysical analysis revealed that DS03090629 retained its affinity for the MEK protein regardless of its phosphorylation status, whereas the affinity of trametinib declined when the MEK protein was phosphorylated. These results suggest that DS03090629 may be a novel therapeutic option for patients who acquire resistance to the current BRAF- and MEK-targeting therapies.

Silencing FOXP2 reverses vemurafenib resistance in BRAFV600E mutant papillary thyroid cancer and melanoma cells

BackgroundVemurafenib (VEM) is a commonly used inhibitor of papillary thyroid cancer (PTC) and melanoma with the BRAFV600E mutation; however, acquired resistance is unavoidable. The present study aimed to identify a potential target to reverse resistance.Materials and methodsA VEM-resistant PTC cell line (B-CPAP/VR) was established by gradually increasing the drug concentration, and a VEM-resistant BRAFV600E melanoma cell line (A375/VR) was also established. RNA sequencing and bioinformatics analyses were conducted to identify dysregulated genes and construct a transcription factor (TF) network. The role of a potential TF, forkhead box P2 (FOXP2), verified by qRT-PCR, was selected for further confirmation.ResultsThe two resistant cell lines were tolerant of VEM and displayed higher migration and colony formation abilities (p < 0.05). RNA sequencing identified 9177 dysregulated genes in the resistant cell lines, and a TF network consisting of 13 TFs and 44 target genes was constructed. Alterations in FOXP2 expression were determined to be consistent between the two VEM-resistant cell lines. Finally, silencing FOXP2 resulted in an increase in drug sensitivity and significant suppression of the migration and colony formation abilities of the two resistant cell lines (p < 0.05).ConclusionsThe present study successfully established two VEM-resistant cell lines and identified a potential target for VEM-resistant PTC or melanoma.

Raman spectroscopy combined with deep learning for rapid detection of melanoma at the single cell level

Melanoma is an aggressive and metastatic skin cancer caused by genetic mutations in melanocytes, and its incidence is increasing year by year. Understanding the gene mutation information of melanoma cases is very important for its precise treatment. The current diagnostic methods for melanoma include radiological, pharmacological, histological, cytological and molecular techniques, but the gold standard for diagnosis is still pathological biopsy, which is time consuming and destructive. Raman spectroscopy is a rapid, sensitive and nondestructive detection method. In this study, a total of 20,000 Surface-enhanced Raman scattering (SERS) spectra of melanocytes and melanoma cells were collected using a positively charged gold nanoparticles planar solid SERS substrate, and a classification network system based on convolutional neural networks (CNN) was constructed to achieve the classification of melanocytes and melanoma cells, wild-type and mutant melanoma cells and their drug resistance. Among them, the classification accuracy of melanocytes and melanoma cells was over 98%. Raman spectral differences between melanocytes and melanoma cells were analyzed and compared, and the response of cells to antitumor drugs were also evaluated. The results showed that Raman spectroscopy provided a basis for the medication of melanoma, and SERS spectra combined with CNN classification model realized classification of melanoma, which is of great significance for rapid diagnosis and identification of melanoma.

Guanine inhibits the growth of human glioma and melanoma cell lines by interacting with GPR23.

Guanine-based purines (GBPs) exert numerous biological effects at the central nervous system through putative membrane receptors, the existence of which is still elusive. To shed light on this question, we screened orphan and poorly characterized G protein-coupled receptors (GPRs), selecting those that showed a high purinoreceptor similarity and were expressed in glioma cells, where GBPs exerted a powerful antiproliferative effect. Of the GPRs chosen, only the silencing of GPR23, also known as lysophosphatidic acid (LPA) 4 receptor, counteracted GBP-induced growth inhibition in U87 cells. Guanine (GUA) was the most potent compound behind the GPR23-mediated effect, acting as the endpoint effector of GBP antiproliferative effects. Accordingly, cells stably expressing GPR23 showed increased sensitivity to GUA. Furthermore, while GPR23 expression was low in a hypoxanthine-guanine phosphoribosyl-transferase (HGPRT)-mutated melanoma cell line showing poor sensitivity to GBPs, and in HGPRT-silenced glioma cells, GPR23-induced expression in both cell types rescued GUA-mediated cell growth inhibition. Finally, binding experiments using [3H]-GUA and U87 cell membranes revealed the existence of a selective GUA binding (KD = 29.44 ± 4.07 nM; Bmax 1.007 ± 0.035 pmol/mg prot) likely to GPR23. Overall, these data suggest GPR23 involvement in modulating responses to GUA in tumor cell lines, although further research needs to verify whether this receptor mediates other GUA effects.

Combining fiber optical tweezers and Raman spectroscopy for rapid identification of melanoma.

Cutaneous melanoma is a skin tumor with a high degree of malignancy and fatality rate, the incidence of which has increased in recent years. Therefore, a rapid and sensitive diagnostic technique of melanoma cells is urgently needed. In this paper, we present a new approach using fiber optical tweezers to manipulate melanoma cells to measure their Raman spectra. Then, combined with Principal Component Analysis and Support Vector Machines (PCA-SVM) classification model, to achieve the classification of common mutant, wild-type and drug-resistant melanoma cells. A total of 150 Raman spectra of 30 cells were collected from mutant, wild-type and drug-resistant melanoma cell lines, and the classification accuracy was 92%, 94%, 97.5%, respectively. These results suggest that the study of tumor cells based on fiber optical tweezers and Raman spectroscopy is a promising method for early and rapid identification and diagnosis of tumor cells.

The A to I editing landscape in melanoma and its relation to clinical outcome

ABSTRACT RNA editing refers to non-transient RNA modifications that occur after transcription and prior to translation by the ribosomes. RNA editing is more widespread in cancer cells than in non-transformed cells and is associated with tumorigenesis of various cancer tissues. However, RNA editing can also generate neo-antigens that expose tumour cells to host immunosurveillance. Global RNA editing in melanoma and its relevance to clinical outcome currently remain poorly characterized. The present study compared RNA editing as well as gene expression in tumour cell lines from melanoma patients of short or long metastasis-free survival, patients relapsing or not after immuno- and targeted therapy and tumours harbouring BRAF or NRAS mutations. Overall, our results showed that NTRK gene expression can be a marker of resistance to BRAF and MEK inhibition and gives some insights of candidate genes as potential biomarkers. In addition, this study revealed an increase in Adenosine-to-Inosine editing in Alu regions and in non-repetitive regions, including the hyperediting of the MOK and DZIP3 genes in relapsed tumour samples during targeted therapy and of the ZBTB11 gene in NRAS mutated melanoma cells. Therefore, RNA editing could be a promising tool for identifying predictive markers, tumour neoantigens and targetable pathways that could help in preventing relapses during immuno- or targeted therapies.

Cytotoxicity, acute and sub-chronic toxicities of the fruit extract of Tetrapleura tetraptera (Schumm. & Thonn.) Taub. (Fabaceae)

BackgroundTetrapleura tetraptera is a medicinal spice traditionally used to treat cancer, diabetes, and several other ailments. This study analyzed the cytotoxicity of the dichloromethane methanol extract of T. tetraptera fruits (TTF) and its constituents. The toxicity profile of the TTF extract was also evaluated in rats.MethodsThe Cytotoxicity of this extract was evaluated using the resazurin reduction assay (RRA). Acute and sub-chronic toxicity studies were performed according to the protocol described by the Organisation for Economic Cooperation, and Development (OECD). Hematological, serum, and urine biochemical parameters, as well as histological sections of the liver and kidney, were also evaluated based on standard methods.ResultsThe TTF extract, compound 5, and the reference drug doxorubicin were active in all 9 tested cancer cell lines. The recorded IC50 ranged from 18.32 μM (against B16-F1 murine melanoma cells) to 36.18 μM (against SKMel-505 BRAF wildtype melanoma cells) for TTF, from 10.02 μM (towards MaMel-80a BRAF-V600E homozygous mutant melanoma cells) to 31.73 μM (against SKMel-28 BRAF-V600E homozygous mutant melanoma cells) for compound 5, and from 0.22 μM (against B16-F1 cells) to 9.39 μM (against SKMel-505 cells) for doxorubicin. The study of acute toxicity test showed that the lethal dose (LD50) of this extract was greater than 5000 mg/kg body weight. In the sub-chronic toxicity studies, variations were observed in some biochemical parameters, especially at higher doses.ConclusionTTF and its most active compound (5) are found to be potential cytotoxic agents, meanwhile, TTF was safe when given a single oral dose of 5000 mg/kg. However, caution is necessary in case of prolonged oral administration due to potential alterations of renal function at high doses (> 1000 mg/kg).

Abstract 2674: Antitumor activity of KIN-2787, a next-generation pan-RAF inhibitor, in preclinical models of human RAF/RAS mutant melanoma

Abstract Background: In the US in 2021, invasive melanoma will account for an estimated 106,000 new cases and &amp;gt; 7,000 deaths. Somatic mutations that activate the MAPK signaling pathway are a leading cause of melanoma with 50% harboring oncogenic BRAF alterations and another 20% with activating NRAS mutations. Of note, NRAS mutant melanoma has been shown to be dependent upon RAF signaling via CRAF dimers for downstream activation of MEK/ERK. While targeted therapies are approved for V600 (Class I, monomer-driven) BRAF mutant melanoma, no approved targeted therapy exists for patients with melanoma driven by Class II or Class III dimer-dependent BRAF alterations or NRAS mutations. KIN-2787 is a novel, orally available, potent, and selective pan-RAF inhibitor designed to be effective in RAF-dependent cancers, including all classes of BRAF alterations, by targeting mutant BRAF monomers and RAF dimers, regardless of isoform. Methods: KIN-2787 activity was assessed by suppression of downstream MAPK pathway signaling and subsequent cell growth inhibition in a panel of human melanoma cell lines. In vivo KIN-2787 efficacy was evaluated in BRAF and NRAS mutant melanoma cell- and patient-derived xenograft models. Results: KIN-2787 cellular activity was measured by inhibition of ERK phosphorylation across a panel of melanoma cell lines, including those harboring Class I BRAF alterations, Class II and III BRAF alterations, NRAS mutations, KRAS mutations, and wild type RAF/RAS. In contrast to vemurafenib, an approved BRAF inhibitor with activity limited to Class I BRAF alterations, KIN-2787 was active across all classes of BRAF mutant melanoma cells (EC50 values &amp;lt; 100 nM). NRAS and KRAS mutant cell lines were moderately responsive to KIN-2787 inhibition. Melanoma cells expressing wild type RAS/RAF were the least sensitive to MAPK pathway inhibition by KIN-2787. KIN-2787 also inhibited cell proliferation in BRAF and NRAS mutant melanoma in 2D and 3D cell cultures. Daily KIN-2787 treatment resulted in significant tumor growth inhibition in human melanoma xenograft models bearing Class I, II and III BRAF alterations as well as NRAS mutations and was associated with MAPK pathway suppression. Additionally, KIN-2787 was efficacious in a pre-/post-treatment melanoma PDX pair in which the original tumor was Class I BRAF V600E but acquired a Class II BRAF kinase domain duplication upon progression on dabrafenib + trametinib. Details from the above findings will be presented at the meeting. Conclusions: KIN-2787 is a next-generation, pan-RAF inhibitor with in vitro and in vivo activity against human melanoma driven by BRAF and/or NRAS mutations. Data supports KIN-2787 use in acquired BRAF dimer-dependent resistance to BRAF+MEK inhibitor therapy. A Phase I dose escalation and expansion clinical trial evaluating the safety and efficacy of KIN-2787 is ongoing (NCT04913285). Citation Format: Nichol L. G. Miller, Tim S. Wang, Paul Severson, Ping Jiang, Michelle Perez, Noel Timple, Toufike Kanouni, Aleksandra Franovic, Eric S. Martin, Eric Murphy. Antitumor activity of KIN-2787, a next-generation pan-RAF inhibitor, in preclinical models of human RAF/RAS mutant melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2674.