Uveal melanoma cell lines Mel270 and 92.1 exhibit a mesenchymal phenotype and sensitivity to the cytostatic effects of transforming growth factor beta in vitro.

Purpose: Uveal melanoma (UM) is a deadly cancer with limited therapeutic options. At advanced stages, UM cells metastasize almost exclusively into the liver, where targeting metastatic UM cells remain a clinical challenge. Transforming growth factor beta (TGF-ß) exhibits a functional duality in cancer, with one arm limiting tumor growth at an early stage and the second arm promoting metastasis at an advanced stage, notably by inducing an epithelial-to-mesenchymal transition. Thus, we hypothesized that targeting the TGF-ß pathway could be relevant in the treatment of metastatic UM. Methods: In this study, we first characterized the pseudoepithelial/mesenchymal phenotype of UM cell lines Mel270 and 92.1. We then treated the cell lines with TGF-ß to evaluate their responsiveness to the cytokine and to characterize the functional impact of TGF-ß on their cell viability. Results: The results demonstrated, first, that the UM cell lines exhibited a mesenchymal phenotype and responded to TGF-ß treatment in vitro and, second, that TGF-ß promoted a cytostatic effect on the UM cell lines. Conclusions: Our findings indicate that UM cells are sensitive to the two arms of TGF-ß signaling, which suggests that targeting the TGF-ß pathway could be challenging in UM and would require a precise selection of patients in which only the prometastatic arm of TGF-ß is activated.

Quercetin Impairs the Growth of Uveal Melanoma Cells by Interfering with Glucose Uptake and Metabolism.

Monosomy 3 in uveal melanoma (UM) increases the risk of lethal metastases, mainly in the liver, which serves as the major site for the storage of excessive glucose and the metabolization of the dietary flavonoid quercetin. Although primary UMs with monosomy 3 exhibit a higher potential for basal glucose uptake, it remains unknown as to whether glycolytic capacity is altered in such tumors. Herein, we initially analyzed the expression of n = 151 genes involved in glycolysis and its interconnected branch, the "pentose phosphate pathway (PPP)", in the UM cohort of The Cancer Genome Atlas Study and validated the differentially expressed genes in two independent cohorts. We also evaluated the effects of quercetin on the growth, survival, and glucose metabolism of the UM cell line 92.1. The rate-limiting glycolytic enzyme PFKP was overexpressed whereas the ZBTB20 gene (locus: 3q13.31) was downregulated in the patients with metastases in all cohorts. Quercetin was able to impair proliferation, viability, glucose uptake, glycolysis, ATP synthesis, and PPP rate-limiting enzyme activity while increasing oxidative stress. UMs with monosomy 3 display a stronger potential to utilize glucose for the generation of energy and biomass. Quercetin can prevent the growth of UM cells by interfering with glucose metabolism.

Combination of Molecule-Targeted Therapy and Photodynamic Therapy Using Nanoformulated Verteporfin for Effective Uveal Melanoma Treatment.

Uveal melanoma (UM) is the most common primary ocular malignancy in adults and has high mortality. Recurrence, metastasis, and therapeutic resistance are frequently observed in UM, but no beneficial systemic therapy is available, presenting an urgent need for developing effective therapeutic drugs. Verteporfin (VP) is a photosensitizer and a Yes-Associated Protein (YAP) inhibitor that has been used in clinical practice. However, VP's lack of tumor targetability, poor biocompatibility, and relatively low treatment efficacy hamper its application in UM management. Herein, we developed a biocompatible CD44-targeting hyaluronic acid nanoparticle (HANP) carrying VP (HANP/VP) to improve UM treatment efficacy. We found that HANP/VP showed a stronger inhibitory effect on cell proliferation than that of free VP in UM cells. Systemic delivery of HANP/VP led to targeted accumulation in the UM-tumor-bearing mouse model. Notably, HANP/VP mediated photodynamic therapy (PDT) significantly inhibited UM tumor growth after laser irradiation compared with no treatment or free VP treatment. Consistently, in HANP/VP treated tumors after laser irradiation, the tumor proliferation and YAP expression level were decreased, while the apoptotic tumor cell and CD8+ immune cell levels were elevated, contributing to effective tumor growth inhibition. Overall, the results of this preclinical study showed that HANP/VP is an effective nanomedicine for tumor treatment through PDT and inhibition of YAP in the UM tumor mouse model. Combining phototherapy and molecular-targeted therapy offers a promising approach for aggressive UM management.

Impact of Nintedanib and Anti-Angiogenic Agents on Uveal Melanoma Cell Behavior.

Purpose: The purpose of this study was to investigate the direct impact of the combined angiokinase inhibitor nintedanib as well as the anti-angiogenic agents ranibizumab, bevacizumab, and aflibercept on the primary uveal melanoma (UM) cell line Mel270 and liver metastasis UM cell line OMM2.5. Methods: The metabolic activity, viability, and oxidative stress levels were analyzed by the Thiazolyl Blue Tetrazolium Bromide (MTT), LIVE/DEAD, and reactive oxygen species (ROS) assays. Expression of intracellular VEGF-A165 and VEGF receptor-2 was detected by immunofluorescent staining. The secretion of VEGF-A165 into the cell culture supernatants was evaluated by VEGF-A165 ELISA. Results: Nintedanib, at a concentration of 1 µg/mL, resulted in a median reduction of metabolic activity (for Mel270 of approximately 38% and for OMM2.5 of 46% compared to the untreated control) without exerting toxicity in either cell line, whereas the other 3 substances did not result in any changes (which also means that none of the 4 substances led to an increased cell death). Moreover, nintedanib (1 µg/mL) induced oxidative stress in the Mel270 by approximately 1.2 to 1.5-fold compared to the untreated control, but not the OMM2.5 cells. Conclusions: Nintedanib could suppress the growth of UM cells in a concentration-dependent manner. The metastatic UM cell line OMM2.5 was not sensitive to the pro-oxidant activity of nintedanib. This study was the first to investigate nintedanib in the context of UM. We propose further investigation of this substance to elucidate its effects on this tumor entity with the hope of identifying advantageous therapeutic options for future adjuvant tumor therapies.

CAR-T cell therapy targeting surface expression of TYRP1 to treat cutaneous and rare melanoma subtypes.

A major limitation to developing chimeric antigen receptor (CAR)-T cell therapies for solid tumors is identifying surface proteins highly expressed in tumors but not in normal tissues. Here, we identify Tyrosinase Related Protein 1 (TYRP1) as a CAR-T cell therapy target to treat patients with cutaneous and rare melanoma subtypes unresponsive to immune checkpoint blockade. TYRP1 is primarily located intracellularly in the melanosomes, with a small fraction being trafficked to the cell surface via vesicular transport. We develop a highly sensitive CAR-T cell therapy that detects surface TYRP1 in tumor cells with high TYRP1 overexpression and presents antitumor activity in vitro and in vivo in murine and patient-derived cutaneous, acral and uveal melanoma models. Furthermore, no systemic or off-tumor severe toxicities are observed in an immunocompetent murine model. The efficacy and safety profile of the TYRP1 CAR-T cell therapy supports the ongoing preparation of a phase I clinical trial.

Discovery of Darovasertib (NVP-LXS196), a Pan-PKC Inhibitor for the Treatment of Metastatic Uveal Melanoma.

Uveal melanoma (UM) is the most common primary intraocular malignancy in the adult eye. Despite the aggressive local management of primary UM, the development of metastases is common with no effective treatment options for metastatic disease. Genetic analysis of UM samples reveals the presence of mutually exclusive activating mutations in the Gq alpha subunits GNAQ and GNA11. One of the key downstream targets of the constitutively active Gq alpha subunits is the protein kinase C (PKC) signaling pathway. Herein, we describe the discovery of darovasertib (NVP-LXS196), a potent pan-PKC inhibitor with high whole kinome selectivity. The lead series was optimized for kinase and off target selectivity to afford a compound that is rapidly absorbed and well tolerated in preclinical species. LXS196 is being investigated in the clinic as a monotherapy and in combination with other agents for the treatment of uveal melanoma (UM), including primary UM and metastatic uveal melanoma (MUM).

Ultrasound-enhanced nano catalyst with ferroptosis-apoptosis combined anticancer strategy for metastatic uveal melanoma.

Uveal melanoma is the most common primary ocular tumor owing to its highly invasive and metastatic characteristics. Currently, standard clinical treatment has an unsatisfied curative effect due to the lack of an effective approach to inhibit the tumor metastasis. Therefore, it is necessary to develop a new strategy that can both restraint local tumors and suppress the ocular tumor metastasis. Herein, we developed ultrasound-responsive nanoparticles (FeP NPs) that can both hinder the growth of in situ ocular tumor and prevent the tumor metastasis through the ferroptosis-apoptosis combined-anticancer strategy. The FeP NPs were assembling by stimulating gallic acid-Fe (III) and paclitaxel, then could be internalized into tumor cells under the cooperative effect of ultrasound, which further activates the intracellular Fenton reaction and generates high reactive oxygen species levels, ultimately leading to mitochondrial damage, lipid per-oxidation, and apoptosis. The FeP NPs can efficiently inhibit the tumor growth in an orthotopic uveal melanoma model. More importantly, the level of the promoting-metastatic factor nerve growth factor receptor (NGFR) secreted by cancer cells is significantly reduced, further limits cancer metastasis to the cervical lymph node and finally inhibits lung metastasis of uveal melanoma. We believe that these designed ultrasound-enhanced nanoparticles possess potential clinical application for preventing the regeneration and metastasis of uveal melanoma.

Globe Salvage and Vision Preservation by Neoadjuvant Darovasertib and Crizotinib in Uveal Melanoma.

PURPOSE: To report the effective use of neoadjuvant darovasertib and crizotinib in a patient with a large uveal melanoma (UM) in his only functional eye. DESIGN: Case report. SUBJECTS: One patient with T4b UM. INTERVENTION: Neoadjuvant darovasertib and crizotinib, followed by plaque brachytherapy. MAIN OUTCOME MEASURES: Objective tumor response and conversion from planned enucleation to placement of fovea- and optic nerve-sparing plaque brachytherapy. RESULTS: A patient with a history of left eye blindness from retinal artery occlusion presented with rapidly declining right eye vision due to a primary UM measuring 18 mm in maximal diameter and 16.5 mm in maximal thickness. To salvage vision, neoadjuvant treatment was initiated using darovasertib and crizotinib. After 6 months of neoadjuvant treatment, which included intraocular lens replacement for tumor-associated cataract, the tumor regressed to 14.1 mm in maximal diameter and 2.6 mm in maximal thickness, enabling treatment with plaque brachytherapy rather than enucleation. CONCLUSIONS: The combination of darovasertib and crizotinib for UM is an effective neoadjuvant strategy that warrants further investigation as an approach to improve visual outcomes from the treatment of primary UM. FINANCIAL DISCLOSURE: The other authors have no proprietary or commercial interest in any materials discussed in this article.

Design, synthesis and evaluation of quinazoline derivatives as Gαq/11 proteins inhibitors against uveal melanoma.

Uveal melanoma (UM) represents the predominant ocular malignancy among adults, exhibiting high malignancy and proclivity for liver metastasis. GNAQ and GNA11 encoding Gαq and Gα11 proteins are key genes to drive UM, making the selective inhibition of Gαq/11 proteins to be a potential therapeutic approach for combating UM. In this study, forty-six quinazoline derivatives were designed, synthesized, and assessed for their ability to inhibit Gαq/11 proteins and UM cells. Compound F33 emerged as the most favorable candidate, and displayed moderate inhibitory activity against Gαq/11 proteins (IC50 = 9.4 µM) and two UM cell lines MP41 (IC50 = 6.7 µM) and 92.1 (IC50 = 3.7 µM). Being a small molecule inhibitor of Gαq/11 proteins, F33 could effectively suppress the activation of downstream signaling pathways in a dose-dependent manner, and significantly inhibits UM in vitro.F33 represents a promising lead compound for developing therapeutics for UM by targeting Gαq/11 proteins.

Tebentafusp as a Promising Drug for the Treatment of Uveal Melanoma.

Uveal melanoma (UM) is the most common primary intraocular malignancy in adults and commonly occurs in the Caucasian population. The malignancy involves the uvea of the eye, which includes the iris, ciliary body, and choroid. The etiology of UM is still not well understood, but age is a risk factor. Symptoms include blurred vision, redness of the eye, floaters, dark spots, a change in the size of the pupil, and loss of vision. The location, shape, and size of the tumor are important for therapeutic purposes. Treating metastasis is always a challenge in UM cases. In cases of lung metastasis, the survival rate decreases. Treatment includes surgery, laser therapy, immunotherapy, hormone therapy, and chemotherapy. Recently, in 2022, the United States Food and Drug Administration (FDA) approved the drug tebentafusp. Tebentafusp was developed to target the most common HLA complex in humans. The present review discusses the indications for the use of a new drug tebentafusp, its mechanism of action, dose, pharmacokinetics, results of clinical trials conducted, and adverse effects like cytokine release syndrome. Hence, tebentafusp is the first T cell receptor (TCR) therapeutic drug that could be considered for the treatment of UM.

Kinome profiling identifies MARK3 and STK10 as potential therapeutic targets in uveal melanoma.

Most uveal melanoma cases harbor activating mutations in either GNAQ or GNA11. Despite activation of the mitogen-activated protein kinase (MAPK) signaling pathway downstream of Gαq/11, there are no effective targeted kinase therapies for metastatic uveal melanoma. The human genome encodes numerous understudied kinases, also called the "dark kinome". Identifying additional kinases regulated by Gαq/11 may uncover novel therapeutic targets for uveal melanoma. In this study, we treated GNAQ-mutant uveal melanoma cell lines with a Gαq/11 inhibitor, YM-254890, and conducted a kinase signaling proteomic screen using multiplexed-kinase inhibitors followed by mass spectrometry. We observed downregulated expression and/or activity of 22 kinases. A custom siRNA screen targeting these kinases demonstrated that knockdown of microtubule affinity regulating kinase 3 (MARK3) and serine/threonine kinase 10 (STK10) significantly reduced uveal melanoma cell growth and decreased expression of cell cycle proteins. Additionally, knockdown of MARK3 but not STK10 decreased ERK1/2 phosphorylation. Analysis of RNA-sequencing and proteomic data showed that Gαq signaling regulates STK10 expression and MARK3 activity. Our findings suggest an involvement of STK10 and MARK3 in the Gαq/11 oncogenic pathway and prompt further investigation into the specific roles and targeting potential of these kinases in uveal melanoma.

High-throughput chemogenetic drug screening reveals PKC-RhoA/PKN as a targetable signaling vulnerability in GNAQ-driven uveal melanoma.

Uveal melanoma (UM) is the most prevalent cancer of the eye in adults, driven by activating mutation of GNAQ/GNA11; however, there are limited therapies against UM and metastatic UM (mUM). Here, we perform a high-throughput chemogenetic drug screen in GNAQ-mutant UM contrasted with BRAF-mutant cutaneous melanoma, defining the druggable landscape of these distinct melanoma subtypes. Across all compounds, darovasertib demonstrates the highest preferential activity against UM. Our investigation reveals that darovasertib potently inhibits PKC as well as PKN/PRK, an AGC kinase family that is part of the "dark kinome." We find that downstream of the Gαq-RhoA signaling axis, PKN converges with ROCK to control FAK, a mediator of non-canonical Gαq-driven signaling. Strikingly, darovasertib synergizes with FAK inhibitors to halt UM growth and promote cytotoxic cell death in vitro and in preclinical metastatic mouse models, thus exposing a signaling vulnerability that can be exploited as a multimodal precision therapy against mUM.

Three-Year Overall Survival with Tebentafusp in Metastatic Uveal Melanoma.

BACKGROUND: Tebentafusp, a T-cell receptor-bispecific molecule that targets glycoprotein 100 and CD3, is approved for adult patients who are positive for HLA-A*02:01 and have unresectable or metastatic uveal melanoma. The primary analysis in the present phase 3 trial supported a long-term survival benefit associated with the drug. METHODS: We report the 3-year efficacy and safety results from our open-label, phase 3 trial in which HLA-A*02:01-positive patients with previously untreated metastatic uveal melanoma were randomly assigned in a 2:1 ratio to receive tebentafusp (tebentafusp group) or the investigator's choice of therapy with pembrolizumab, ipilimumab, or dacarbazine (control group), with randomization stratified according to the lactate dehydrogenase level. The primary end point was overall survival. RESULTS: At a minimum follow-up of 36 months, median overall survival was 21.6 months in the tebentafusp group and 16.9 months in the control group (hazard ratio for death, 0.68; 95% confidence interval, 0.54 to 0.87). The estimated percentage of patients surviving at 3 years was 27% in the tebentafusp group and 18% in the control group. The most common treatment-related adverse events of any grade in the tebentafusp group were rash (83%), pyrexia (76%), pruritus (70%), and hypotension (38%). Most tebentafusp-related adverse events occurred early during treatment, and no new adverse events were observed with long-term administration. The percentage of patients who discontinued treatment because of adverse events continued to be low in both treatment groups (2% in the tebentafusp group and 5% in the control group). No treatment-related deaths occurred. CONCLUSIONS: This 3-year analysis supported a continued long-term benefit of tebentafusp for overall survival among adult HLA-A*02:01-positive patients with previously untreated metastatic uveal melanoma. (Funded by Immunocore; IMCgp100-202 ClinicalTrials.gov number, NCT03070392; EudraCT number, 2015-003153-18.).

Tumor Shape-Specific Brachytherapy Implants by 3D-Printing, Precision Radioactivity Painting, and Biomedical Imaging.

In brachytherapy (BT), or internal radiation therapy, cancer is treated by radioactive implants. For instance, episcleral plaques (EPs) for the treatment of uveal melanoma, are designed according to generic population approximations. However, more personalized implants can enhance treatment precision through better adjustment of dose profiles to the contours of cancerous tissues. An original approach integrating biomedical imaging, 3D printing, radioactivity painting, and biomedical imaging, is developed as a workflow for the development of tumor shape-specific BT implants. First, computer-aided design plans of EP are prepared according to guidelines prescribed by the Collaborative Ocular Melanoma Study protocol. Polyetheretherketone (PEEK), a high-performance polymer suitable for permanent implants, is used to 3D-print plaques and the geometrical accuracy of the printed design is evaluated by imaging. The possibility to modulate the dose distribution in a tridimensional manner is demonstrated by painting the inner surfaces of the EPs with radioactive 103Pd, followed by dose profile measurements. The possibility to modulate dose distributions generated by these 3D-printed plaques through radioactivity painting is therefore confirmed. Ex vivo surgical tests on human eyeballs are performed as an assessment of manipulation ease. Overall, this work provides a solution for the fabrication of tumor-specific radioactive implants requiring higher dose precision.

Uveal melanoma: In the era of new treatments.

Uveal melanoma (UM), also known as choroidal melanoma, is the leading adult intraocular tumor worldwide, affecting mainly Caucasian populations. The last decade has seen an improvement in the outcome of these tumors at the localized stage, in favor of conservative treatment of the eye, notably with new radioactive treatment techniques. Despite optimal management, half of the patients will become metastatic, with liver involvement in 90% of cases. The prognosis is pejorative and considers clinical, tumor anatomy, histological and molecular parameters. This review provides a broad overview of the different therapeutic options for the management of localized or metastatic UM disease, with recently updated data. Despite the known limited efficacy of chemotherapy and immune checkpoint inhibitors (ICI), we discuss the first results of combined immunotherapies, the arrival of a new first-in-class immunomodulatory treatment Tebentafusp, in HLA-A*02:01 patients, avenues of research into targeted anti-tyrosine kinase therapies, and the growing use of ctDNA to guide treatment prescription.

Single-cell sequencing in primary intraocular tumors: understanding heterogeneity, the microenvironment, and drug resistance.

Retinoblastoma (RB) and uveal melanoma (UM) are the most common primary intraocular tumors in children and adults, respectively. Despite continued increases in the likelihood of salvaging the eyeball due to advancements in local tumor control, prognosis remains poor once metastasis has occurred. Traditional sequencing technology obtains averaged information from pooled clusters of diverse cells. In contrast, single-cell sequencing (SCS) allows for investigations of tumor biology at the resolution of the individual cell, providing insights into tumor heterogeneity, microenvironmental properties, and cellular genomic mutations. SCS is a powerful tool that can help identify new biomarkers for diagnosis and targeted therapy, which may in turn greatly improve tumor management. In this review, we focus on the application of SCS for evaluating heterogeneity, microenvironmental characteristics, and drug resistance in patients with RB and UM.

Role of immune checkpoint inhibitors in metastatic uveal melanoma: a single-center retrospective cohort study.

BACKGROUND: Uveal melanoma is an orphan malignancy with very limited data on treatment options in metastatic setting. METHODS: In this single-center retrospective study, we describe real-world epidemiological and survival data on 121 metastatic uveal melanoma (MUM) patients registered in our institution. As a large tertiary referral center, almost 30% of all diagnoses in the Flemish region of Belgium were covered. Primarily, we determined whether introduction of immune checkpoint inhibitors (ICI) led to improved overall survival (OS) in MUM patients. Secondarily, response rates to ICI were assessed and we evaluated whether first-line ICI could be a valid alternative to liver-directed therapy (LDT) in liver-only disease. RESULTS: The initially perceived 10.8 months survival benefit from treatment with ICI disappeared after correction for immortality bias. By analyzing treatment type as time-varying covariate on OS, no significant benefit of ICI over other systemic therapies (HR = 0.771) or best supportive care (BSC) (HR = 0.780) was found. Also comparison of the pre-ICI versus ICI era showed no OS improvement after introduction of ICI in our center (p = 0.7994). Only liver-directed and local oligometastatic approaches were associated with a lower chance of mortality when compared to ICI (p = 0.0025), other systemic therapies (p = 0.0001) and BSC (p = 0.0003), yet without correction for selection bias. We reported overall response rates on ICI ranging from 8-15% and we found some support for neoadjuvant strategies with ICI resulting in remission or downsizing, allowing oligometastatic approaches later on. In first-line liver-only disease, median real-world progression-free survival and OS did not significantly differ between patients treated with LDT or ICI upfront (p = 0.2930 and p = 0.5461 respectively). CONCLUSION: Although we documented responses to ICI, our analyses do not demonstrate an OS benefit of ICI over alternative treatment strategies for MUM. However, local treatment options, whether liver-directed or for oligometastatic disease, may be beneficial and should be considered.

Selamectin increases cisplatin sensitivity by inhibiting cisplatin-resistant genes expression and autophagy in uveal melanoma.

Cisplatin resistance is the main reason for uveal melanoma (UM) treatment failure. Thus, developing strategy that increasing cisplatin sensitivity is needed. In this study, we performed drug repositioning analysis with the Connectivity Map database using a panel of previously identified cisplatin sensitivity-associated genes and cisplatin resistance-associated genes as the signature and obtained the antiparasitic drug selamectin. We demonstrated that the selamectin and cisplatin combination showed a synergistic effect on inhibiting UM cell growth. Experiments in tumor-bearing nude mice further showed that selamectin and cisplatin have synergistic effects in reducing tumor growth. Previous studies have linked increased autophagy with tumor resistance to chemotherapy. We found that selamectin inhibited the expression of the autophagy-related gene ATG9B, thus reducing autophagy. The cisplatin resistance-associated genes PDGFRB, DUSP1, MAST1 and IL11 were significantly downregulated in UM cells treated with selamectin. In summary, our study shows that selamectin enhanced the sensitivity of UM to cisplatin, through the mechanism of inhibiting cisplatin resistance-associated gene expression and autophagy. These findings may provide a new strategy for the treatment of UM.