Human Uveal Melanoma Cell Lines Research Articles

Chimeric Antigen Receptor T Cell with an Inducible Caspase 9 Suicide Gene Eradicates Uveal Melanoma Liver Metastases via B7-H3 Targeting

Uveal melanoma (UM) is the most common intraocular malignant tumor. Despite successful treatment of the primary tumor, about 50% of patients will recur with systemic disease for which there are no effective treatment strategies. Here we investigated the preclinical efficacy of a chimeric antigen receptor (CAR) T cell-based immunotherapy targeting B7-H3. B7-H3 expression on primary and metastatic human UM samples and cell lines was assessed by RNA sequencing, flow cytometry, and immunohistochemistry. Antitumor activity of CAR T cells targeting B7-H3 was tested in vitro with UM cell lines, metastatic UM patient-derived organotypic tumor spheroids (PDOTS), and in immunodeficient and humanized murine models. B7-H3 is expressed at high levels on >95% UM tumor cells in vitro and in vivo. We generated a B7-H3 CAR with an inducible caspase-9 (iCas9) suicide gene controlled by the chemical inducer of dimerization AP1903, which effectively kills UM cells in vitro and eradicates UM liver metastases in murine models. Delivery of iCas9.B7-H3 CAR T cells in experimental models of UM liver metastases demonstrates a durable anti-tumor response, even upon tumor re-challenge or in the presence of a significant metastatic disease burden. We demonstrate effective iCas9.B7-H3 CAR T cell elimination in vitro and in vivo in response to AP1903. Our studies demonstrate more effective tumor suppression with iCas9.B7-H3 CAR T cells as compared to a B7-H3-targeted humanized monoclonal antibody. These studies support a phase I clinical trial with iCas9.B7-H3 CAR T cells to treat patients with metastatic UM.

Mass Spectrometry-Based Profiling of Histone Post-Translational Modifications in Uveal Melanoma Tissues, Human Melanocytes, and Uveal Melanoma Cell Lines - A Pilot Study.

Epigenetic alterations in uveal melanoma (UM) are still neither well characterized, nor understood. In this pilot study, we sought to provide a deeper insight into the possible role of epigenetic alterations in the pathogenesis of UM and their potential prognostic relevance. To this aim, we comprehensively profiled histone post-translational modifications (PTMs), which represent epigenetic features regulating chromatin accessibility and gene transcription, in UM formalin-fixed paraffin-embedded (FFPE) tissues, control tissues, UM cell lines, and healthy melanocytes. FFPE tissues of UM (n = 24), normal choroid (n = 4), human UM cell lines (n = 7), skin melanocytes (n = 6), and uveal melanocytes (n = 2) were analyzed through a quantitative liquid chromatography-mass spectrometry (LC-MS) approach. Hierarchical clustering showed a clear separation with several histone PTMs that changed significantly in a tumor compared to normal samples, in both tissues and cell lines. In addition, several acetylations and H4K20me1 showed lower levels in BAP1 mutant tumors. Some of these changes were also observed when we compared GNA11 mutant tumors with GNAQ tumors. The epigenetic profiling of cell lines revealed that the UM cell lines MP65 and UPMM1 have a histone PTM pattern closer to the primary tissues than the other cell lines analyzed. Our results suggest the existence of different histone PTM patterns that may be important for diagnosis and prognosis in UM. However, further analyses are needed to confirm these findings in a larger cohort. The epigenetic characterization of a panel of UM cell lines suggested which cellular models are more suitable for epigenetic investigations.

FOXD1 expression-based prognostic model for uveal melanoma

FOXD1, a new member of the FOX transcription factor family, serves as a mediator and biomarker for cell reprogramming. But its contribution to prognosis of uveal melanoma (UVM) is unclear. This study demonstrated that FOXD1 might promote tumor growth and invasion, because FOXD1 expression was negatively correlated with overall survival, progression-free survival, and disease-specific survival in UVM patients. This conjecture was verified in cell culture with human uveal melanoma cell line (MUM2B) as model cells. Additionally, the biological mechanisms of FOXD1 based on FOXD1-related genomic spectrum, molecular pathways, tumor microenvironment, and drug treatment sensitivity were examined using The Cancer Genome Atlas (TCGA) database, aiming to reasonably explain why FOXD1 leads to poor prognosis of UVM. On these bases, a novel tumor prognostic model was established using the FOXD1-related immunomodulators TMEM173, TNFRSF4, TNFSF13, and ULBP1, which will enable the stratification of disease seriousness and clinical treatment for patients.

Suicide-Gene-Modified Extracellular Vesicles of Human Primary Uveal Melanoma in Future Therapies.

Extracellular vesicles secreted from uveal melanoma (UM) cells are involved in the establishment of the premetastatic niche and display transforming potential for the formation of metastases, preferentially in the liver. In this study, we cultivated human primary UM cells and uveal melanoma-associated fibroblasts in vitro to be transduced by infection with a retrovirus containing the suicide gene-fused yeast cytosine deaminase::uracil phospho-ribosyl transferase (yCD::UPRT). A homogenous population of yCD::UPRT-UM cells with the integrated provirus expressed the gene, and we found it to continuously secrete small extracellular vesicles (sEVs) possessing mRNA of the suicide gene. The yCD::UPRT-UM-sEVs were internalized by tumor cells to the intracellular conversion of the prodrug 5-fluorocytosine (5-FC) to the cytotoxic drug 5-fluorouracil (5-FU). The host range of the yCD::UPRT-UM-sEVs was not limited to UMs only. The yCD::UPRT-UM-sEVs inhibited the growth of the human cutaneous melanoma cell line A375 and uveal melanoma cell line MP38, as well as other primary UMs, to various extents in vitro. The yCD::UPRT-UM-sEVs hold the therapeutic and prophylactic potential to become a therapeutic drug for UM. However, the use of yCD::UPRT-UM-sEVs must first be tested in animal preclinical studies.

Genome-wide CRISPR-Cas9 screening to identify CDS2 as an essential gene in uveal melanoma.

e21585 Background: Despite low rates of local recurrence in primary uveal melanomas following radical treatment, approximately half of all patients will develop metastatic disease. There are limited effective systemic therapeutic options and survival outcomes remain poor. Genome-wide CRISPR-Cas9 knockout screening enables appropriate molecular targets and pathways that underlie cancer development to be identified. Methods: Genome-wide single gRNA CRISPR knockout screens were performed using Human Improved Genome-wide Knockout CRISPR Library v1.1. Eight human uveal melanoma cell lines were screened for 14 days to identify gene dependencies. Prior to screening, all cell lines underwent RNA-Seq to quantify gene expression. CRISPR screen hits were validated using single gRNA competitive growth assays. In vitro functional assessment of CDS2 knockout was performed using inducible CDS2 knockout isogenic cell lines in colony forming assays and Annexin V apoptosis assays. To determine the effects of CDS2 loss on phosphoinositide synthesis, quantification of phosphatidic acid and phosphoinositides was performed using HPLC-MS. To test the association between CDS2 essentiality and CDS1 expression across all cancer types, we analysed gene essentiality data from Project Score and Project Achilles with gene expression data obtained from Cell Model Passports and the DepMap download portal (22Q4). Cell lines with CDS1 expression log2(TPM+1) < 1 were annotated as low-expressors, and the rest were annotated as high-expressors. Single group data were assessed using Student's t test. Results: We identified CDS2, which encodes enzymes that maintain phosphoinositide levels during PLC signalling, to be an essential gene in 7 out of 8 screened cell lines. All cell lines were found to have low expression of its paralogous gene, CDS1. Doxycycline-inducible CDS2 knockout clones were generated from cell lines MP41 and OMM2.5. Reduced colony formation, with a significant decrease in surviving fraction was observed after doxycycline compared with treatment with a DMSO control in both cell lines (P < 0.0001). Apoptotic and dead cells increased significantly when doxycycline was added in MP41(13.6 ± 0.9% to 47.4 ± 3.3%; P= 0.0006) and OMM2.5 (3.7 ± 0.4% to 60.4± 0.7%, P< 0.0001). CDS2 knockout resulted in an increase in the precursor phosphatidic acid, and a significant reduction of the phosphoinositides PI (MP41, P= 0.018; OMM2.5, P= 0.0005) and PIP (MP41, P= 0.033; OMM2.5, P= 0.006). CDS2 was more essential in CDS1 low-expressors in both Project Score ( P< 0.0001) and Project Achilles ( P< 0.0001) datasets, compared with CDS1 high-expressors. Conclusions: Disruption of CDS2 results in reduced cellular fitness in uveal melanoma. Given the synthetic lethal relationship between CDS1 and CDS2, in the context of low CDS1 gene expression as observed in uveal melanomas and in other tumour types, CDS2 is a potential novel therapeutic target.

Comparison of Fucoidans from Saccharina latissima Regarding Age-Related Macular Degeneration Relevant Pathomechanisms in Retinal Pigment Epithelium.

Fucoidans from brown algae are described as anti-inflammatory, antioxidative, and antiangiogenic. We tested two Saccharina latissima fucoidans (SL-FRO and SL-NOR) regarding their potential biological effects against age-related macular degeneration (AMD). Primary porcine retinal pigment epithelium (RPE), human RPE cell line ARPE-19, and human uveal melanoma cell line OMM-1 were used. Cell survival was assessed in tetrazolium assay (MTT). Oxidative stress assays were induced with erastin or H2O2. Supernatants were harvested to assess secreted vascular endothelial growth factor A (VEGF-A) in ELISA. Barrier function was assessed by measurement of trans-epithelial electrical resistance (TEER). Protectin (CD59) and retinal pigment epithelium-specific 65 kDa protein (RPE65) were evaluated in western blot. Polymorphonuclear elastase and complement inhibition assays were performed. Phagocytosis of photoreceptor outer segments was tested in a fluorescence assay. Secretion and expression of proinflammatory cytokines were assessed with ELISA and real-time PCR. Fucoidans were chemically analyzed. Neither toxic nor antioxidative effects were detected in ARPE-19 or OMM-1. Interleukin 8 gene expression was slightly reduced by SL-NOR but induced by SL-FRO in RPE. VEGF secretion was reduced in ARPE-19 by SL-FRO and in RPE by both fucoidans. Polyinosinic:polycytidylic acid induced interleukin 6 and interleukin 8 secretion was reduced by both fucoidans in RPE. CD59 expression was positively influenced by fucoidans, and they exhibited a complement and elastase inhibitory effect in cell-free assay. RPE65 expression was reduced by SL-NOR in RPE. Barrier function of RPE was transiently reduced. Phagocytosis ability was slightly reduced by both fucoidans in primary RPE but not in ARPE-19. Fucoidans from Saccharina latissima, especially SL-FRO, are promising agents against AMD, as they reduce angiogenic cytokines and show anti-inflammatory and complement inhibiting properties; however, potential effects on gene expression and RPE functions need to be considered for further research.

Properties of Cephalopod Skin Ommochromes to Inhibit Free Radicals, and the Maillard Reaction and Retino-Protective Mechanisms in Cellular Models Concerning Oxidative Stress, Angiogenesis, and Inflammation

Ommochromes are pigments of invertebrates that exhibit oxidative stress protection. The aim of this study was to investigate ommochromes extracted from cephalopod’s skin for their ability to inhibit age-related-macular degeneration (AMD)-related factors such as H2O2-induced and iron-dependent oxidative stress (ferroptosis and erastin), accumulation of advanced glycation end-products (AGEs), as well as vascular endothelial growth factor (VEGF), and inflammatory cytokines (interleukin 6 and interleukin 8) secretion. As cell systems, we used primary porcine retinal pigment epithelium (RPE), human retinal pigment epithelium cell line ARPE-19 and uveal melanoma cell line OMM-1. In vitro, ommochromes produced an antiglycation effect by the inhibition of fructosylation reaction. The ommochromes showed protective effects against erastin- induced cell death in ARPE-19. In addition, in long-term stimulation (7 days) ommochromes decreased constitutively secreted VEGF, as well as interleukin 6 and interleukin 8 induced by Poly I:C in primary RPE. No relevant effects were detected in OMM-1 cells. The effects are dependent on the cell system, time of exposition, and concentration. This substance is of interest for further research concerning age-related macular degeneration.

Spheroid-induced heterogeneity and plasticity of uveal melanoma cells

PurposeThe mechanism underlying cancer heterogeneity and plasticity remains elusive, in spite of the fact that multiple hypotheses have been put forward. We intended to clarify this heterogeneity in uveal melanoma (UM) by looking for evidence of cancer stem cell involvement and a potential role of ZEB1 in cancer cell plasticity.MethodsSpheroids derived from human UM cells as well as xenograft tumors in nude mice were dissected for signs of heterogeneity and plasticity. Two human UM cell lines were studied: the epithelioid type C918 cell line and the spindle type OCM1 cell line. We knocked down ZEB1 in both cell lines to investigate its involvement in the regulation of stem-like cell formation and vascularization by qRT-PCR, immunohistochemistry, flow cytometry, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assays.ResultsWe found that a small side population (SP) in OCM1 showed stem cell-like properties such as heterogeneity, remote dissemination and nuclear dye exclusion after spheroid formation in vitro. ZEB1 regulated UM stem cell generation indirectly by promoting cell proliferation to form large size tumors in vivo and spheroid in vitro, and directly by binding to stemness genes such as TERT and ABCB1. In addition, we found that ZEB1 participates in vasculogenic mimicry system formation through the regulation of CD34 and VE-cadherin expression.ConclusionsFrom our data we conclude that cancer stem cells may contribute to UM heterogeneity and plasticity and that ZEB1 may play a regulatory role in it.

Elaiophylin Inhibits Tumorigenesis of Human Uveal Melanoma by Suppressing Mitophagy and Inducing Oxidative Stress via Modulating SIRT1/FoxO3a Signaling.

Uveal melanoma (UM) is the most common primary intraocular tumor in adults, which is associated with poor prognosis. Up to 50% of UM patients develop metastasis. Therapeutics that have proven effective in cutaneous melanoma have little success in treating UM, possibly due to its low mutational burden. Therefore, new drug therapies are highly desired for UM. Our in vitro studies showed that Elaiophylin, a late-stage autophagy inhibitor, exhibited an outstanding anticancer activity in human UM cell lines and human UM primary cells through suppressing mitophagy, inducing oxidative stress and leading to autophagic cell death. Our mechanistic study revealed that Elaiophylin exerted its effect by down-regulating SIRT1 and thus influencing deacetylation and mitochondrial localization of FoxO3a. In our confirmatory experiments, SRT1720, a SIRT1 specific activator, could attenuate Elaiophylin-induced inhibition of mitophagy and elevation of oxidative stress, and such effects was partly reversed by FoxO3a knockdown. Our further in vivo studies showed that Elaiophylin dramatically inhibited tumor growth in the human UM xenograft mouse model, which was accompanied with a decreased SIRT1 expression. Thus, the current study is the first to demonstrate that Elaiophylin has a potent anti-cancer effect against UM, which activity is possibly mediated through regulating SIRT1-FoxO3a signaling axis. And Elaiophylin may be a new and promising drug candidate to treat human UM.

Animal Models of Uveal Melanoma.

Animal models are crucial for the study of tumorigenesis and therapies in oncology research. Though rare, uveal melanoma (UM) is the most common intraocular tumor and remains one of the most lethal cancers. Given the limitations of studying human UM cells in vitro, animal models have emerged as excellent platforms to investigate disease onset, progression, and metastasis. Since Greene’s initial studies on hamster UM, researchers have dramatically improved the array of animal models. Animals with spontaneous tumors have largely been replaced by engrafted and genetically engineered models. Inoculation techniques continue to be refined and expanded. Newer methods for directed mutagenesis have formed transgenic models to reliably study primary tumorigenesis. Human UM cell lines have been used to generate rapidly growing xenografts. Most recently, patient-derived xenografts have emerged as models that closely mimic the behavior of human UM. Separate animal models to study metastatic UM have also been established. Despite the advancements, the prognosis has only recently improved for UM patients, especially in patients with metastases. There is a need to identify and evaluate new preclinical models. To accomplish this goal, it is important to understand the origin, methods, advantages, and disadvantages of current animal models. In this review, the authors present current and historic animal models for the experimental study of UM. The strengths and shortcomings of each model are discussed and potential future directions are explored.

PRRX1 Is a Novel Prognostic Biomarker and Facilitates Tumor Progression Through Epithelial-Mesenchymal Transition in Uveal Melanoma.

Uveal melanoma (UM) is the most common primary intraocular malignancy in adults. UM develops and is sustained by inflammation and immunosuppression from the tumor microenvironment (TME). This study sought to identify a reliable TME-related biomarker that could provide survival prediction and new insight into therapy for UM patients. Based on clinical characteristics and the RNA-seq transcriptome data of 80 samples from The Cancer Genome Atlas (TCGA) database, PRRX1 as a TME- and prognosis-related gene was identified using the ESTIMATE algorithm and the LASSO–Cox regression model. A prognostic model based on PRRX1 was constructed and validated with a Gene Expression Omnibus (GEO) dataset of 63 samples. High PRRX1 expression was associated with poorer overall survival (OS) and metastasis-free survival (MFS) in UM patients. Comprehensive results of the prognostic analysis showed that PRRX1 was an independent and reliable predictor of UM. Then the results of immunological characteristics demonstrated that higher expression of PRRX1 was accompanied by higher expression of immune checkpoint genes, lower tumor mutation burden (TMB), and greater tumor cell infiltration into the TME. Gene set enrichment analysis (GSEA) showed that high PRRX1 expression correlated with angiogenesis, epithelial–mesenchymal transition (EMT), and inflammation. Furthermore, downregulation of PRRX1 weakened the process of EMT, reduced cell invasion and migration of human UM cell line MuM-2B in vitro. Taken together, these findings indicated that increased PRRX1 expression is independently a prognostic factor of poorer OS and MFS in patients with UM, and that PRRX1 promotes malignant progression of UM by facilitating EMT, suggesting that PRRX1 may be a potential target for UM therapy.

Uveal melanoma-associated mutations in PLCβ4 are constitutively activating and promote melanocyte proliferation and tumorigenesis.

Activating mutations in Gαq/11 proteins are frequent in uveal melanoma, the most common eye cancer arising from the uveal tract. A small proportion of uveal melanomas have a D630Y mutation in phospholipase C β4 (PLCβ4), an effector of Gαq/11. Here, we found that the D630Y mutation in PLCβ4 results in a high level of constitutive PLCβ4 activity. Mutations at the corresponding position in other PLC isoforms also resulted in constitutive activity, revealing an unrecognized mechanism underlying PLC activation. In cultured human uveal melanoma cell lines, inhibition of PLC suppressed proliferation in Gαq/11-dependent cells. Furthermore, we found that PLCβ4(D630Y) mediated proliferation in cutaneous melanocytes and the growth of melanomas in mice. These results are consistent with PLCβ4(D630Y) driving oncogenic signaling downstream of Gαq/11.

543 The high expression of pro-apoptotic BCL2 family members in uveal melanomas contribute to their sensitivity to MCL1 inhibitors

Uveal Melanoma (UM), a rare melanoma of the eye, has a poor survival prognosis and lacks any FDA approved drug treatments; thus, there is an urgent need for treatments. UM does not have BRAF-V600 mutations commonly found in cutaneous melanoma (CM). BH3 mimetics are a novel class of drugs that mimic pro-apoptotic BCL2 family members. In hematological cancers, venetoclax (BCL2 inhibitor) is approved and S64315 (MCL1 inhibitor/MCL1i) is in clinical trials. This study aims to examine the efficacy of various BH3 mimetics in UM and to determine the underlining mechanism for the differing sensitivity we found between UM and CM. We used in vitro assays (viability, IncuCyte live cell imaging, immunoblot, sphere formation, shRNA techniques), in vivo mouse models, and bioinformatics analyses of the TCGA database. We found that treatments of MCL1i alone at nM doses were highly effective in reducing viability, inhibiting cell proliferation, and activating caspase 3/7 in human UM cell lines (p<0.01), but not CM lines. MCL1i also significantly reduced tumor growth in vivo (p<0.05). In addition, UM lines displayed higher endogenous expression of pro-apoptotic proteins BMF, PUMA and BAD, compared to CM lines, and blocking BRAF/MAPK signaling in CM with MEKi increased their expression. Further, knock down of BMF, PUMA, and BAD partially protected UM against MCL1i (p<0.05). Results indicated that the lack of activating mutations in BRAF/MAPK signaling in UM was the cause for greater pro-apoptotic protein expression. Moreover, TCGA data also showed a higher expression in UM vs. CM, indicating the clinical relevance of our finding. However, MCL1i alone was ineffective at killing the UM stem-like cells, but a combination with venetoclax synergistically killed UM stem-like cells, inhibited their capacity to self-renew, and reduced tumor growth in vivo (p<0.01). In summary, our data indicated MCL1i, alone or in combination with venetoclax, are promising treatments for UM.

Combined Inhibition of Gαq and MEK Enhances Therapeutic Efficacy in Uveal Melanoma.

All uveal melanoma and a fraction of other melanoma subtypes are driven by activation of the G-protein alpha-q (Gαq) pathway. Targeting these melanomas has proven difficult despite advances in the molecular understanding of key driver signaling pathways in the disease pathogenesis. Inhibitors of Gαq have shown promising preclinical results, but their therapeutic activity in distinct Gαq mutational contexts and in vivo have remained elusive. We used an isogenic melanocytic cellular system to systematically examine hotspot mutations in GNAQ (e.g., G48V, R183Q, Q209L) and CYSLTR2 (L129Q) found in human uveal melanoma. This cellular system and human uveal melanoma cell lines were used in vitro and in in vivo xenograft studies to assess the efficacy of Gαq inhibition as a single agent and in combination with MEK inhibition. We demonstrate that the Gαq inhibitor YM-254890 inhibited downstream signaling and in vitro growth in all mutants. In vivo, YM-254890 slowed tumor growth but did not cause regression in human uveal melanoma xenografts. Through comprehensive transcriptome analysis, we observed that YM-254890 caused inhibition of the MAPK signaling with evidence of rebound by 24 hours and combination treatment of YM-254890 and a MEK inhibitor led to sustained MAPK inhibition. We further demonstrated that the combination caused synergistic growth inhibition in vitro and tumor shrinkage in vivo. These data suggest that the combination of Gαq and MEK inhibition provides a promising therapeutic strategy and improved therapeutic window of broadly targeting Gαq in uveal melanoma.See related commentary by Neelature Sriramareddy and Smalley, p. 1217.

Parthenolide Enhances Cisplatin Sensitivity in Uveal Melanoma by Regulated Mitogen-Activated Protein Kinase Signal Pathway

To investigate parthenolide (PTL)’s effect to cisplatin (DDP) sensitivity in uveal melanoma and to show the underlying mechanism. Human uveal melanoma cell line M23 was split into the control group, PTL treatment group, DDP treatment group, PTL + DDP treatment group, DDP + Mitogen-activated protein kinase (MAPK) signal inhibitor (SB203580) treatment group, and PTL + DDP + MAPK signal activator (anisomycin) treatment group. CCK-8 test was conducted to detect cell viability, flow cytometry was utilized for cell apoptosis detection, and western blot was utilized to determine the phosphorylation of p38MAPK and JNK. Compared to the control group, PLT treatment group M23 cell activity, and expression of p-p38MAPK, p-JNK were reduced notably (P &lt; 0.05). Meanwhile, these indices of DDP treatment group were lower than of the control group but higher than of the PTL treatment group. In the PTL + DDP treatment group M23 cell activity and expression level of p-p38MAPK, p-JNK was significantly lower than in the PTL and DDP treatment groups (P &lt; 0.05). Inhibition of MAPK signal pathway increased the DDP effect to proliferation inhibition and apoptosis promotion. In contrast, MAPK signal pathway activator treatment alleviates PTL + DDP treatment effect on M23 cell line apoptosis and proliferation. Parthenolide (PTL) increased cisplatin (DDP) sensitivity in uveal melanoma through the inhibition of MAPK signal pathway.

Abstract 1890: Chloroquine synergizes with MEK1/2 targeted therapy through dual YAP and lysosomal inhibition in GNAQ/11 mutant uveal melanoma

Abstract GNAQ and GNA11 (GNAQ/11) mutations are found in less than 2% of all melanoma, but more than 80% of uveal melanoma. Mutations in these Gα proteins lead to constitutive activation of multiple oncogenic pathways, including MAPK (RAF-&amp;gt;MEK1/2-&amp;gt;ERK1/2) and YAP signaling. Metastatic uveal melanoma is refractory to all forms of pharmacologic treatment, such as FDA-approved targeted therapies inhibiting MEK1/2 (i.e. trametinib and binimetinib). We show that combining MEK1/2 inhibitors with 4-aminoquinoline antimalarials, chloroquine or hydroxychloroquine, resulted in synergistic and apoptosis-mediated cytotoxicity in GNAQ/11 mutant uveal melanoma cell lines. Interestingly, in contrast to our previous work in pancreatic and other RAS-driven cancers, the lysosomotropic role of chloroquine was not sufficient to promote cytotoxicity with MEK1/2 inhibitors, as neither lysosome inhibition with Bafilomycin A1 nor autophagy-specific and macropinocytosis-specific inhibition yielded enhanced cell death in combination with MEK1/2 inhibition. We then found that chloroquine prevented nuclear localization of the transcriptional coactivator, YAP, suggesting a novel mechanism of chloroquine. YAP inhibition combined with MEK1/2 inhibition enhanced cell death only in the presence of Bafilomycin A1. Gα-specific inhibition (inhibiting YAP and MAPK) combined with Bafilomycin A1 yielded similar results. This implies that the ability of chloroquine to inhibit both YAP signaling and lysosome function is required for promoting cell death in the presence of MEK1/2 inhibition. For in vivostudies, we utilized a hepatic colonization model using luciferized human metastatic uveal melanoma cell lines, OMM2.5 and OMM1. Daily treatment of trametinib with hydroxychloroquine in combination resulted in delayed tumor growth and increased overall survival compared to either treatment as monotherapy or chemotherapy. These findings were also recapitulated in an immunocompetent mouse model in which immortalized mouse melanocytes (Melan-A) with either a GNAQ or GNA11 activating mutation were implanted into syngeneic C57BL/6 mice. Our findings identify a novel mechanism of chloroquine and suggest a potentially effective strategy combining two FDA-approved drugs for the treatment of metastatic uveal melanoma. Citation Format: Amanda Truong, Michael Scherzer, Conan Kinsey, John Michael Sanchez, Jae Hyuk Yoo, Jackson Richards, Donghan Shin, Phaedra Ghazi, Michael Onken, Kendall Blumer, Shannon Odelberg, Martin McMahon. Chloroquine synergizes with MEK1/2 targeted therapy through dual YAP and lysosomal inhibition in GNAQ/11 mutant uveal melanoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1890.

Potential Onco-Suppressive Role of miR122 and miR144 in Uveal Melanoma through ADAM10 and C-Met Inhibition.

Uveal melanoma (UM) is a rare tumor of the eye that leads to deadly metastases in about half of the patients. ADAM10 correlates with c-Met expression in UM and high levels of both molecules are related to the development of metastases. MiR122 and miR144 modulate ADAM10 and c-Met expression in different settings. We hypothesized a potential onco-suppressive role for miR122 and miR144 through modulation of ADAM10 and c-Met in UM. We analyzed the UM Cancer Genome Atlas data portal (TCGA) dataset, two other cohorts of primary tumors and five human UM cell lines for miR122 and miR144 expression by miR microarray, RT-qPCR, Western blotting, miR transfection and luciferase reporter assay. Our results indicate that miR122 and miR144 are expressed at low levels in the UM cell lines and in the TCGA UM dataset and were down-modulated in a cohort of seven UM samples, compared to normal choroid. Both miR122 and miR144 directly targeted ADAM10 and c-Met. Overexpression of miR122 and miR144 led to reduced expression of ADAM10 and c-Met in the UM cell lines and impaired cell proliferation, migration, cell cycle and shedding of c-Met ecto-domain. Our results show that miR122 and miR144 display an onco-suppressive role in UM through ADAM10 and c-Met modulation.

Generation of a Liver Orthotopic Human Uveal Melanoma Xenograft Platform in Immunodeficient Mice.

In recent decades, subcutaneously implanted patient-derived xenograft tumors or cultured human cell lines have been increasingly recognized as more representative models to study human cancers in immunodeficient mice than traditional established human cell lines in vitro. Recently, orthotopically implanted patient-derived tumor xenograft (PDX) models in mice have been developed to better replicate features of patient tumors. A liver orthotopic xenograft mouse model is expected to be a useful cancer research platform, providing insights into tumor biology and drug therapy. However, liver orthotopic tumor implantation is generally complicated. Here we describe our protocols for the orthotopic implantation of patient-derived liver-metastatic uveal melanoma tumors. We cultured human liver metastatic uveal melanoma cell lines into immunodeficient mice. The protocols can result in consistently high technical success rates using either a surgical orthotopic implantation technique with chunks of patient-derived uveal melanoma tumor or a needle injection technique with cultured human cell line. We also describe protocols for CT scanning to detect interior liver tumors and for re-implantation techniques using cryopreserved tumors to achieve re-engraftment. Together, these protocols provide a better platform for liver orthotopic tumor mouse models of liver metastatic uveal melanoma in translational research.

LncRNA PVT1 knockdown affects proliferation and apoptosis of uveal melanoma cells by inhibiting EZH2.

To detect the expression of long non-coding ribonucleic acid (lncRNA) plasmacytoma variant translocation gene 1 (PVT1) in uveal melanoma (UM) tissues, and to investigate its influence on the proliferation and apoptosis of UM cells as well as its mechanism. 40 cases of UM tissues and 40 cases of adjacent tissues surgically resected in our hospital from October 2015 to April 2018 were collected. The expression level of lncRNA PVT1 in these tissues was determined by Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Stable knockdown of lncRNA PVT1 was constructed in human UM cell line OCM-1 using small interfering RNA (siRNA). The impact of lncRNA PVT1 on UM cell proliferation was detected by Cell Counting kit-8 (CCK-8) and colony formation assay. Flow cytometry was applied to measure the apoptotic level of UM cells in the blank control group and lncRNA PVT1 knockdown group. Meanwhile, the expression level of enhancer of zeste homologue 2 (EZH2) was determined by Western blotting. The expression level of lncRNA PVT1 in UM tissues was remarkably higher than that in the adjacent tissues (p<0.05). UM cell proliferation was notably repressed after lncRNA PVT1 knockdown by siRNA. Flow cytometry results indicated that the number of apoptotic UM cells in lncRNA PVT1 knockdown group significantly increased compared with that in the blank control group (p<0.05). The protein expression of EZH2 was suppressed after lncRNA PVT1 knockdown (p<0.05). LncRNA PVT1 knockdown in UM cells can repress the proliferation of UM cells and promote their apoptosis by regulating EZH2 expression.

Experimental therapy of doxorubicin resistant human uveal melanoma with targeted cytotoxic luteinizing hormone-releasing hormone analog (AN-152)

BackgroundCytotoxic analogs of LHRH (luteinizing hormone-releasing hormone) can be successfully used for the treatment of hormone-dependent cancers such as prostatic, ovarian, endometrial, but our knowledge about their effect on hormone-independent cancers such as human uveal melanoma (UM) is limited. Previously, we have demonstrated that 46% of UM express full-length LHRH receptors. This finding has led us to further examine the mechanism of action of LHRH receptor based targeted therapies in this malignancy. AimsIn the present study we investigated the cellular uptake of doxorubicin (DOX) and cytotoxic LHRH analog AN-152 (AEZS-108, zoptarelin doxorubicin) on human UM cell lines (OCM3) and its DOX resistant form OCM3DOX320 by confocal laser scanning microscopy. The LHRH receptor expression was characterized by RT-PCR and immunocytochemistry. ResultsWe were able to establish a new, stable and DOX resistant human UM cell line OCM3DOX320. Our results demonstrated the expression of splice variants and isoforms of receptor for LHRH in OCM3 UM cell line and its doxorubicin resistant form OCM3DOX320.It has been revealed by MTT assay that AN-152 inhibited cell proliferation in a dose dependent manner in OCM3DOX320 cells. Furthermore, receptor-mediated uptake of AN-152 was demonstrated using confocal laser scanning microscopy in both cell line. ConclusionsOur results suggest that the antiproliferative effect of AN-152 can be detected even if only LHRH receptor isoforms are expressed. Our study also demonstrates the LHRH receptor-mediated uptake of AN-152 in DOX resistant OCM3DOX320 cells. Our experiments provide new insights into a potential targeted therapy of UM and give further details about the accumulation of AN-152 in hormone-independent DOX-resistant cells expressing splice variants of the LHRH receptors.