Machine Learning Methods for Gene Selection in Uveal Melanoma.

Uveal melanoma (UM) is the most common primary intraocular malignancy with a limited five-year survival for metastatic patients. Limited therapeutic treatments are currently available for metastatic disease, even if the genomics of this tumor has been deeply studied using next-generation sequencing (NGS) and functional experiments. The profound knowledge of the molecular features that characterize this tumor has not led to the development of efficacious therapies, and the survival of metastatic patients has not changed for decades. Several bioinformatics methods have been applied to mine NGS tumor data in order to unveil tumor biology and detect possible molecular targets for new therapies. Each application can be single domain based while others are more focused on data integration from multiple genomics domains (as gene expression and methylation data). Examples of single domain approaches include differentially expressed gene (DEG) analysis on gene expression data with statistical methods such as SAM (significance analysis of microarray) or gene prioritization with complex algorithms such as deep learning. Data fusion or integration methods merge multiple domains of information to define new clusters of patients or to detect relevant genes, according to multiple NGS data. In this work, we compare different strategies to detect relevant genes for metastatic disease prediction in the TCGA uveal melanoma (UVM) dataset. Detected targets are validated with multi-gene score analysis on a larger UM microarray dataset.

Interdependence of Molecular Lesions That Drive Uveal Melanoma Metastasis.

The metastatic risk of uveal melanoma (UM) is defined by a limited number of molecular lesions, somatic mutations (SF3B1 and BAP1), and copy number alterations (CNA): monosomy of chromosome 3 (M3), chr8q gain (8q), chr6p gain (6p), yet the sequence of events is not clear. We analyzed data from three datasets (TCGA-UVM, GSE27831, GSE51880) with information regarding M3, 8q, 6p, SF3B1, and BAP1 status. We confirm that BAP1 mutations are always associated with M3 in high-risk patients. All other features (6p, 8q, M3, SF3B1 mutation) were present independently from each other. Chr8q gain was frequently associated with chr3 disomy. Hierarchical clustering of gene expression data of samples with different binary combinations of aggressivity factors shows that patients with 8q|M3, BAP1|M3 form one cluster enriched in samples that developed metastases. Patients with 6p combined with either 8q or SF3B1 are mainly represented in the other, low-risk cluster. Several gene expression events that show a non-significant association with outcome when considering single features become significant when analyzing combinations of risk features indicating additive action. The independence of risk factors is consistent with a random risk model of UM metastasis without an obligatory sequence.

Whole-Exome Sequencing and cfDNA Analysis Uncover Genetic Determinants of Melanoma Therapy Response in a Real-World Setting.

Although several studies have explored the molecular landscape of metastatic melanoma, the genetic determinants of therapy resistance are still largely unknown. Here, we aimed to determine the contribution of whole-exome sequencing and circulating free DNA (cfDNA) analysis in predicting response to therapy in a consecutive real-world cohort of 36 patients, undergoing fresh tissue biopsy and followed during treatment. Although the underpowered sample size limited statistical analysis, samples from non-responders had higher copy number variations and mutations in melanoma driver genes compared to responders in the BRAF V600+ subset. In the BRAF V600- subset, Tumor Mutational Burden (TMB) was twice that in responders vs. non-responders. Genomic layout revealed commonly known and novel potential intrinsic/acquired resistance driver gene variants. Among these, RAC1, FBXW7, GNAQ mutations, and BRAF/PTEN amplification/deletion were present in 42% and 67% of patients, respectively. Both Loss of Heterozygosity (LOH) load and tumor ploidy were inversely associated with TMB. In immunotherapy-treated patients, samples from responders showed higher TMB and lower LOH and were more frequently diploid compared to non-responders. Secondary germline testing and cfDNA analysis proved their efficacy in finding germline predisposing variants carriers (8.3%) and following dynamic changes during treatment as a surrogate of tissue biopsy, respectively.

Ataxia-Telangiectasia Mutated Loss of Heterozygosity in Melanoma.

ATM germline pathogenic variants were recently found enriched in high-risk melanoma patients. However, ATM loss of heterozygosity (LOH) has never been investigated in melanoma and, therefore, a causal association with melanoma development has not been established yet. The purpose of this study was to functionally characterize 13 germline ATM variants found in high-risk melanoma patients-and classified by in silico tools as pathogenic, uncertain significance, or benign-using multiple assays evaluating ATM/pATM expression and/or LOH in melanoma tissues and cell lines. We assessed ATM status by Immunohistochemistry (IHC), Western Blot, Whole-Exome Sequencing/Copy Number Variation analysis, and RNA sequencing, supported by Sanger sequencing and microsatellite analyses. For most variants, IHC results matched those obtained with in silico classification and LOH analysis. Two pathogenic variants (p.Ser1135_Lys1192del and p.Ser1993ArgfsTer23) showed LOH and complete loss of ATM activation in melanoma. Two variants of unknown significance (p.Asn358Ile and p.Asn796His) showed reduced expression and LOH, suggestive of a deleterious effect. This study, showing a classic two-hit scenario in a well-known tumor suppressor gene, supports the inclusion of melanoma in the ATM-related cancer spectrum.

Increased Arginase1 expression in tumor microenvironment promotes mammary carcinogenesis via multiple mechanisms.

Arginine metabolism plays a significant role in regulating cell function, affecting tumor growth and metastatization. To study the effect of the arginine-catabolizing enzyme Arginase1 (ARG1) on tumor microenvironment, we generated a mouse model of mammary carcinogenesis by crossbreeding a transgenic mouse line overexpressing ARG1 in macrophages (FVBArg+/+) with the MMTV-Neu mouse line (FVBNeu+/+). This double transgenic line (FVBArg+/-;Neu+/+) showed a significant shortening in mammary tumor latency, and an increase in the number of mammary nodules. Transfer of tumor cells from FVBNeu+/+ into either FVB wild type or FVBArg+/+ mice resulted in increase regulatory T cells in the tumor infiltrate, suggestive of an impaired antitumor immune response. However, we also found increased frequency of tumor stem cells in tumors from FVBArg+/-;Neu+/+ transgenic compared with FVBNeu+/+ mice, suggesting that increased arginine metabolism in mammary tumor microenvironment may supports the cancer stem cells niche. We provide in vivo evidence of a novel, yet unexploited, mechanism through which ARG1 may contribute to tumor development.

Recombinant IL-21 and anti-CD4 antibodies cooperate in syngeneic neuroblastoma immunotherapy and mediate long-lasting immunity.

IL-21 is an immune-enhancing cytokine, which showed promising results in cancer immunotherapy. We previously observed that the administration of anti-CD4 cell-depleting antibody strongly enhanced the anti-tumor effects of an IL-21-engineered neuroblastoma (NB) cell vaccine. Here, we studied the therapeutic effects of a combination of recombinant (r) IL-21 and anti-CD4 monoclonal antibodies (mAb) in a syngeneic model of disseminated NB. Subcutaneous rIL-21 therapy at 0.5 or 1 µg/dose (at days 2, 6, 9, 13 and 15 after NB induction) had a limited effect on NB development. However, coadministration of rIL-21 at the two dose levels and a cell-depleting anti-CD4 mAb cured 28 and 70 % of mice, respectively. Combined immunotherapy was also effective if started 7 days after NB implant, resulting in a 30 % cure rate. Anti-CD4 antibody treatment efficiently depleted CD4(+) CD25(high) Treg cells, but alone had limited impact on NB. Combination immunotherapy by anti-CD4 mAb and rIL-21 induced a CD8(+) cytotoxic T lymphocyte response, which resulted in tumor eradication and long-lasting immunity. CD4(+) T cells, which re-populated mice after combination immunotherapy, were required for immunity to NB antigens as indicated by CD4(+) T cell depletion and re-challenge experiments. In conclusion, these data support a role for regulatory CD4(+) T cells in a syngeneic NB model and suggest that rIL-21 combined with CD4(+) T cell depletion reprograms CD4(+) T cells from immune regulatory to anti-tumor functions. These observations open new perspectives for the use of IL-21-based immunotherapy in conjunction with transient CD4(+) T cell depletion, in human metastatic NB.

Cerivastatin Synergizes with Trametinib and Enhances Its Efficacy in the Therapy of Uveal Melanoma.

BACKGROUND: Metastatic uveal melanoma (MUM) is a highly aggressive, therapy-resistant disease. Driver mutations in Gα-proteins GNAQ and GNA11 activate MAP-kinase and YAP/TAZ pathways of oncogenic signalling. MAP-kinase and MEK-inhibitors do not significantly block MUM progression, likely due to persisting YAP/TAZ signalling. Statins inhibit YAP/TAZ activation by blocking the mevalonate pathway, geranyl-geranylation, and subcellular localisation of the Rho-GTPase. We investigated drugs that affect the YAP/TAZ pathway, valproic acid, verteporfin and statins, in combination with MEK-inhibitor trametinib. METHODS: We established IC50 values of the individual drugs and monitored the effects of their combinations in terms of proliferation. We selected trametinib and cerivastatin for evaluation of cell cycle and apoptosis. Synergism was detected using isobologram and Chou-Talalay analyses. The most synergistic combination was tested in vivo. RESULTS: Synergistic concentrations of trametinib and cerivastatin induced a massive arrest of proliferation and cell cycle and enhanced apoptosis, particularly in the monosomic, BAP1-mutated UPMM3 cell line. The combined treatment reduced ERK and AKT phosphorylation, increased the inactive, cytoplasmatic form of YAP and significantly impaired the growth of UM cells with monosomy of chromosome 3 in NSG mice. CONCLUSION: Statins can potentiate the efficacy of MEK inhibitors in the therapy of UM.

Guadecitabine increases response to combined anti-CTLA-4 and anti-PD-1 treatment in mouse melanoma in vivo by controlling T-cells, myeloid derived suppressor and NK cells.

BACKGROUND: The combination of Programmed Cell Death 1 (PD-1) and Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4) blockade has dramatically improved the overall survival rate for malignant melanoma. Immune checkpoint blockers (ICBs) limit the tumor's immune escape yet only for approximately a third of all tumors and, in most cases, for a limited amount of time. Several approaches to overcome resistance to ICBs are being investigated among which the addition of epigenetic drugs that are expected to act on both immune and tumor cells. Guadecitabine, a dinucleotide prodrug of a decitabine linked via phosphodiester bond to a guanosine, showed promising results in the phase-1 clinical trial, NIBIT-M4 (NCT02608437). METHODS: We used the syngeneic B16F10 murine melanoma model to study the effects of immune checkpoint blocking antibodies against CTLA-4 and PD-1 in combination, with and without the addition of Guadecitabine. We comprehensively characterized the tumor's and the host's responses under different treatments by flow cytometry, multiplex immunofluorescence and methylation analysis. RESULTS: In combination with ICBs, Guadecitabine significantly reduced subcutaneous tumor growth as well as metastases formation compared to ICBs and Guadecitabine treatment. In particular, Guadecitabine greatly enhanced the efficacy of combined ICBs by increasing effector memory CD8+ T cells, inducing effector NK cells in the spleen and reducing tumor infiltrating regulatory T cells and myeloid derived suppressor cells (MDSC), in the tumor microenvironment (TME). Guadecitabine in association with ICBs increased serum levels of IFN-γ and IFN-γ-induced chemokines with anti-angiogenic activity. Guadecitabine led to a general DNA-demethylation, in particular of sites of intermediate methylation levels. CONCLUSIONS: These results indicate Guadecitabine as a promising epigenetic drug to be added to ICBs therapy.

Prognostic Role of Soluble and Extracellular Vesicle-Associated PD-L1, B7-H3 and B7-H4 in Non-Small Cell Lung Cancer Patients Treated with Immune Checkpoint Inhibitors.

The treatment of non-small cell lung cancer (NSCLC) has changed dramatically with the advent of immune checkpoint inhibitors (ICIs). Despite encouraging results, their efficacy remains limited to a subgroup of patients. Circulating immune checkpoints in soluble (s) form and associated with extracellular vesicles (EVs) represent promising markers, especially in ICI-based therapeutic settings. We evaluated the prognostic role of PD-L1 and of two B7 family members (B7-H3, B7-H4), both soluble and EV-associated, in a cohort of advanced NSCLC patients treated with first- (n = 56) or second-line (n = 126) ICIs. In treatment-naïve patients, high baseline concentrations of sPD-L1 (>24.2 pg/mL) were linked to worse survival, whereas high levels of sB7-H3 (>0.5 ng/mL) and sB7-H4 (>63.9 pg/mL) were associated with better outcomes. EV characterization confirmed the presence of EVs positive for PD-L1 and B7-H3, while only a small portion of EVs expressed B7-H4. The comparison between biomarker levels at the baseline and in the first radiological assessment under ICI-based treatment showed a significant decrease in EV-PD-L1 and an increase in EV-B7H3 in patients in the disease response to ICIs. Our study shows that sPD-L1, sB7-H3 and sB7-H4 levels are emerging prognostic markers in patients with advanced NSCLC treated with ICIs and suggests potential EV involvement in the disease response to ICIs.

In uveal melanoma Gα-protein GNA11 mutations convey a shorter disease-specific survival and are more strongly associated with loss of BAP1 and chromosomal alterations than Gα-protein GNAQ mutations.

BACKGROUND AND AIM OF THE STUDY: Mutations in the Gα-genes GNAQ and GNA11 are found in 85-90% of uveal melanomas (UM). Aim of the study is to understand whether the mutations in both genes differentially affect tumor characteristics and outcome and if so, to identify potential mechanisms. METHODS: We analyzed the association between GNAQ and GNA11 mutations with disease-specific survival, gene expression profiles, and cytogenetic alterations in 219 UMs. We used tandem-affinity-purification, mass spectrometry and immunoprecipitation to identify protein interaction partners of the two G-proteins and analyzed their impact on DNA-methylation. RESULTS: GNA11 mutation was associated with: i) an increased frequency of loss of BRCA1-associated protein 1 (BAP1) expression (p = 0.0005), ii) monosomy of chromosome 3 (p < 0.001), iii) amplification of chr8q (p = 0.038), iv) the combination of the latter two (p = 0.0002), and inversely with v) chr6p gain (p = 0.003). Our analysis also showed a shorter disease-specific survival of GNA11-mutated cases as compared to those carrying a GNAQ mutation (HR = 1.97 [95%CI 1.12-3.46], p = 0.02). GNAQ and GNA11 encoded G-proteins have different protein interaction partners. Specifically, the Tet Methylcytosine Dioxygenase 2 (TET2), a protein that is involved in DNA demethylation, physically interacts with the GNAQ protein but not with GNA11, as confirmed by immunoprecipitation analyses. High-risk UM cases show a clearly different DNA-methylation pattern, suggesting that a different regulation of DNA methylation by the two G-proteins might convey a different risk of progression. CONCLUSIONS: GNA11 mutated uveal melanoma has worse prognosis and is associated with high risk cytogenetic, mutational and molecular tumor characteristics that might be determined at least in part by differential DNA-methylation.

A Circulating Risk Score, Based on Combined Expression of Exo-miR-130a-3p and Fibrinopeptide A, as Predictive Biomarker of Relapse in Resectable Non-Small Cell Lung Cancer Patients.

To date, the 5-year overall survival rate of 60% for early-stage non-small cell lung cancer (NSCLC) is still unsatisfactory. Therefore, reliable prognostic factors are needed. Growing evidence shows that cancer progression may depend on an interconnection between cancer cells and the surrounding tumor microenvironment; hence, circulating molecules may represent promising markers of cancer recurrence. In order to identify a prognostic score, we performed in-depth high-throughput analyses of plasma circulating markers, including exosomal microRNAs (Exo-miR) and peptides, in 67 radically resected NSCLCs. The miRnome profile selected the Exo-miR-130a-3p as the most overexpressed in relapsed patients. Peptidome analysis identified four progressively more degraded forms of fibrinopeptide A (FpA), which were depleted in progressing patients. Notably, stepwise Cox regression analysis selected Exo-miR-130a-3p and the greatest FpA (2-16) to build a score predictive of recurrence, where high-risk patients had 18 months of median disease-free survival. Moreover, in vitro transfections showed that higher levels of miR-130a-3p lead to a deregulation of pathways involved in metastasis and angiogenesis, including the coagulation process and metalloprotease increase which might be linked to FpA reduction. In conclusion, by integrating circulating markers, the identified risk score may help clinicians predict early-stage NSCLC patients who are more likely to relapse after primary surgery.

Inhibition of MDR1 activity and induction of apoptosis by analogues of nifedipine and diltiazem: an in vitro analysis.

We report herein the reversal of multidrug resistance-1 (MDR1) in A2780/DX3 cells by the two nifedipine-like compounds 1 and 2 that are part of a library of 1,4-dihydropyridines (1,4-DHPs) calcium-channel modulators bearing in C-4 a different substituted imidazo[2,1-b]thiazole system. By methylthiazol tetrazolium (MTT) assay, cytofluorimetry, and fluorescence microscopy we evaluated their ability to reverse MDR in our cell system. Moreover, together with compound 3 (the diltiazem-like 8-(4-chlorophenyl)-5-methyl-8-[(2Z)-pent-2-en-1-yloxy]-8H-[1,2,4]oxadiazolo[3,4-c][1,4]thiazin-3-one) we analyzed their ability to potentiate the triggering of apoptosis after exposure to doxorubicin, through the nuclear morphological analysis after 4',6-diamidino-2-phenylindole (DAPI), the fluorescein isothiocyanate (FITC)-Annexin-V/propidium iodide (PI) staining and the caspase activity determination. Our results demonstrate that compounds 1 and 2, at concentrations showing a very low (5%) or absent inhibition of cell proliferation, in combination with doxorubicin enhance its antiproliferative activity (from 30% to 54% IC(50) reduction) in A2780/DX3 cells through an increase of doxorubicin intracellular accumulation. These compounds together with compound 3, which has already been demonstrated to act as a potent inhibitor of MDR1 function, were also able to significantly potentiate the activation of the apoptosis machinery triggered by the exposure to doxorubicin. In conclusion, our results identify two new molecules structurally related to the calcium-channel blocker nifedipine, but characterized by a very low LTCC blockers activity, able to potentiate the antiproliferative and apoptotic activities of doxorubicin through an increase of its intracellular concentration likely caused by the inhibition of MDR1 function.

The recognition of facial emotions in spinocerebellar ataxia patients.

Patients with cerebellar lesions present some affective and cognitive disorders, defining a peculiar pattern of cognitive impairment, so-called cerebellar cognitive affective syndrome. This pattern has been confirmed in many genotypes of spinocerebellar ataxias (SCA), a group of genetically defined pathologies characterized by the degeneration of the cerebellum and its connections. Recently, in SCA patients, some authors focused the interest on social cognition evidencing an impairment of theory of mind and basic emotion recognition by verbal material. The recognition of emotions in faces is an essential component of social cognition; therefore, we assessed this ability in SCA patients, expanding the study from the basic verbal emotions to the basic and social visual emotion recognition. We assessed facial emotion recognition using two basic and social emotion tasks in a group of SCA patients together with a complete clinical and neuropsychological evaluation. We compared results with the performance of a control group. We demonstrated a significant difference between patients and controls both in basic and social emotion recognition, although we found a specific impairment only for social emotions. The deficit was not correlated to clinical and demographic features. The cognitive and psychological profile did not explain the impairment in emotion recognition. This result supports the hypothesis that the impairment in social emotion recognition could be specifically related to a defect in the corticocerebellar network.

Uveal Melanoma Metastasis.

Uveal melanoma (UM) is characterized by relatively few, highly incident molecular alterations and their association with metastatic risk is deeply understood. Nevertheless, this knowledge has so far not led to innovative therapies for the successful treatment of UM metastases or for adjuvant therapy, leaving survival after diagnosis of metastatic UM almost unaltered in decades. The driver mutations of UM, mainly in the G-protein genes GNAQ and GNA11, activate the MAP-kinase pathway as well as the YAP/TAZ pathway. At present, there are no drugs that target the latter and this likely explains the failure of mitogen activated kinase kinase inhibitors. Immune checkpoint blockers, despite the game changing effect in cutaneous melanoma (CM), show only limited effects in UM probably because of the low mutational burden of 0.5 per megabase and the unavailability of antibodies targeting the main immune checkpoint active in UM. The highly pro-tumorigenic microenvironment of UM also contributes to therapy resistance. However, T-cell redirection by a soluble T-cell receptor that is fused to an anti-CD3 single-chain variable fragment, local, liver specific therapy, new immune checkpoint blockers, and YAP/TAZ specific drugs give new hope to repeating the success of innovative therapy obtained for CM.

Therapeutic Implications of Tumor Microenvironment in Lung Cancer: Focus on Immune Checkpoint Blockade.

In the last decade, the treatment of non-small cell lung cancer (NSCLC) has been revolutionized by the introduction of immune checkpoint inhibitors (ICI) directed against programmed death protein 1 (PD-1) and its ligand (PD-L1), or cytotoxic T lymphocyte antigen 4 (CTLA-4). In spite of these improvements, some patients do not achieve any benefit from ICI, and inevitably develop resistance to therapy over time. Tumor microenvironment (TME) might influence response to immunotherapy due to its prominent role in the multiple interactions between neoplastic cells and the immune system. Studies investigating lung cancer from the perspective of TME pointed out a complex scenario where tumor angiogenesis, soluble factors, immune suppressive/regulatory elements and cells composing TME itself participate to tumor growth. In this review, we point out the current state of knowledge involving the relationship between tumor cells and the components of TME in NSCLC as well as their interactions with immunotherapy providing an update on novel predictors of benefit from currently employed ICI or new therapeutic targets of investigational agents. In first place, increasing evidence suggests that TME might represent a promising biomarker of sensitivity to ICI, based on the presence of immune-modulating cells, such as Treg, myeloid derived suppressor cells, and tumor associated macrophages, which are known to induce an immunosuppressive environment, poorly responsive to ICI. Consequently, multiple clinical studies have been designed to influence TME towards a pro-immunogenic state and subsequently improve the activity of ICI. Currently, the mostly employed approach relies on the association of "classic" ICI targeting PD-1/PD-L1 and novel agents directed on molecules, such as LAG-3 and TIM-3. To date, some trials have already shown promising results, while a multitude of prospective studies are ongoing, and their results might significantly influence the future approach to cancer immunotherapy.

IL-27 Mediates PD-L1 Expression and Release by Human Mesothelioma Cells.

Malignant mesothelioma (MM) is a rare tumor with an unfavorable prognosis. MM genesis involves asbestos-mediated local inflammation, supported by several cytokines, including IL-6. Recent data showed that targeting PD-1/PD-L1 is an effective therapy in MM. Here, we investigated the effects of IL-6 trans-signaling and the IL-6-related cytokine IL-27 on human MM cells in vitro by Western blot analysis of STAT1/3 phosphorylation. The effects on PD-L1 expression were tested by qRT-PCR and flow-cytometry and the release of soluble (s)PD-L1 by ELISA. We also measured the concentrations of sPD-L1 and, by multiplexed immunoassay, IL-6 and IL-27 in pleural fluids obtained from 77 patients in relation to survival. IL-27 predominantly mediates STAT1 phosphorylation and increases PD-L1 gene and surface protein expression and sPD-L1 release by human MM cells in vitro. IL-6 has limited activity, whereas a sIL-6R/IL-6 chimeric protein mediates trans-signaling predominantly via STAT3 phosphorylation but has no effect on PD-L1 expression and release. IL-6, IL-27, and sPD-L1 are present in pleural fluids and show a negative correlation with overall survival, but only IL-27 shows a moderate albeit significant correlation with sPD-L1 levels. Altogether these data suggest a potential role of IL-27 in PD-L1-driven immune resistance in MM.

How to Make Immunotherapy an Effective Therapeutic Choice for Uveal Melanoma.

Uveal melanoma (UM), though a rare form of melanoma, is the most common intraocular tumor in adults. Conventional therapies of primary tumors lead to an excellent local control, but 50% of patients develop metastases, in most cases with lethal outcome. Somatic driver mutations that act on the MAP-kinase pathway have been identified, yet targeted therapies show little efficacy in the clinics. No drugs are currently available for the G protein alpha subunitsGNAQ and GNA11, which are the most frequent driver mutations in UM. Drugs targeting the YAP-TAZ pathway that is also activated in UM, the tumor-suppressor gene BRCA1 Associated Protein 1 (BAP1) and the Splicing Factor 3b Subunit 1 gene (SF3B1) whose mutations are associated with metastatic risk, have not been developed yet. Immunotherapy is highly effective in cutaneous melanoma but yields only poor results in the treatment of UM: anti-PD-1 and anti-CTLA-4 blocking antibodies did not meet the expectations except for isolated cases. Here, we discuss how the improved knowledge of the tumor microenvironment and of the cross-talk between tumor and immune cells could help to reshape anti-tumor immune responses to overcome the intrinsic resistance to immune checkpoint blockers of UM. We critically review the dogma of low mutational load, the induction of immune-suppressive cells, and the expression of alternative immune checkpoint molecules. We argue that immunotherapy might still be an option for the treatment of UM.

Transient depletion of CD4(+) T cells augments IL-21-based immunotherapy of disseminated neuroblastoma in syngeneic mice.

IL-21 is a member of the IL-2 cytokine family, produced by CD4+ T cells. We previously showed that immunotherapy (IT) with IL-21-transduced neuroblastoma cells (Neuro2a/IL-21) cured 33% of syngeneic mice bearing systemic NB. Here, we studied whether the removal of Treg cells could potentiate the therapeutic efficacy of Neuro2a/IL-21 vaccine. The administration of anti-CD25 mAb, which targets Treg cells, slightly potentiated the effect of vaccine IT (50% cure rate), but anti-CD4 mAb had a more potent effect leading to 80% cure rate. Anti-CD25 mAb, indeed, only partially depleted CD4+CD25+FoxP3+ Treg cells, whereas anti-CD4 mAb was more effective in this respect, leading to 90% depletion of Treg cells. In mice receiving vaccine+anti-CD4 mAb, which developed systemic immunity to NB, CD4+ T cells counts completely recovered in 90 days. Depletion of CD8+ T cells abrogated the effect of the combined IT, indicating a predominant role of these cells in driving the immune response. In addition, CD8+ T cells from cured mice coinjected with Neuro2a/parental cells (pc) in NOD-SCID mice completely inhibited tumor growth. Spleen cells from mice receiving Neuro2a/IL-21 vaccination showed increased expression of IFN-alpha2, -beta1 and -gamma mRNA. Moreover, mice receiving vaccine therapy alone or vaccine+anti-CD4 mAb showed increased IFN-gamma serum levels and IFN-gamma-producing CD8+ T cells were found in spleen cells. In conclusion, anti-CD4 mAb potentiated IL-21-based IT by removing Treg cells and/or their precursors and other potentially immune-suppressive CD4+ cell subsets, thus allowing the development of an IL-21-driven CD8+ T cell response, which mediates NB rejection.

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.

Targeted Therapy of Uveal Melanoma: Recent Failures and New Perspectives.

Among Uveal Melanoma (UM) driver mutations, those involving GNAQ or GNA11 genes are the most frequent, while a minor fraction of tumors bears mutations in the PLCB4 or CYSLTR2 genes. Direct inhibition of constitutively active oncoproteins deriving from these mutations is still in its infancy in UM, whereas BRAFV600E-targeted therapy has obtained relevant results in cutaneous melanoma. However, UM driver mutations converge on common downstream signaling pathways such as PKC/MAPK, PI3K/AKT, and YAP/TAZ, which are presently considered as actionable targets. In addition, BAP1 loss, which characterizes UM metastatic progression, affects chromatin structure via histone H2A deubiquitylation that may be counteracted by histone deacetylase inhibitors. Encouraging results of preclinical studies targeting signaling molecules such as MAPK and PKC were unfortunately not confirmed in early clinical studies. Indeed, a general survey of all clinical trials applying new targeted and immune therapy to UM displayed disappointing results. This paper summarizes the most recent studies of UM-targeted therapies, analyzing the possible origins of failures. We also focus on hyperexpressed molecules involved in UM aggressiveness as potential new targets for therapy.