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Uveal melanoma is the most common intraocular tumor in adults, representing approximately 5% of all melanoma cases. Up to 50% of uveal melanoma patients develop metastases that are resistant to most of the commonly used antineoplastic treatments. Virtually all uveal melanoma tumors harbor activating mutations in GNAQ or GNA11 , encoding Gαq and Gα11, respectively. Constant activity of these proteins causes deregulation of multiple downstream signaling pathways including PKC, MAPK and YAP1/TAZ. While the importance of YAP1 signaling for the proliferation of uveal melanoma has recently been demonstrated, much less is known about the paralog of YAP1 transcriptional coactivator, named TAZ; however, similar to YAP1, TAZ is expected to be a therapeutic target in uveal melanoma. We performed a small-scale drug screen to discover a compound synergistically inhibiting uveal melanoma proliferation/survival in combination with YAP1/TAZ inhibition. We found that the combination of genetic depletion of YAP1/TAZ together with Mcl-1 inhibition demonstrates a synergistic inhibitory effect on the viability of uveal melanoma cell lines. Similarly, indirect attenuation of the YAP1/TAZ signaling pathway with an inhibitor of the mevalonate pathway, that is, the geranyl-geranyl transferase inhibitor GGTI-298, synergizes with Mcl-1 inhibition. This combination could be potentially used as a treatment for metastatic uveal melanoma., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc.)

Uveal melanoma (UM) has a high risk to progress to metastatic disease with a median survival of 3.9 months after metastases detection, as metastatic UM responds poorly to conventional and targeted chemotherapy and is largely refractory to immunotherapy. Here, we present a patient-derived zebrafish UM xenograft model mimicking metastatic UM. Cells isolated from Xmm66 spheroids derived from metastatic UM patient material were injected into 2 days-old zebrafish larvae resulting in micro-metastases in the liver and caudal hematopoietic tissue. Metastasis formation could be reduced by navitoclax and more efficiently by the combinations navitoclax/everolimus and flavopiridol/quisinostat. We obtained spheroid cultures from 14 metastatic and 10 primary UM tissues, which were used for xenografts with a success rate of 100%. Importantly, the ferroptosis-related genes GPX4 and SLC7A11 are negatively correlated with the survival of UM patients (TCGA: n = 80; Leiden University Medical Centre cohort: n = 64), ferroptosis susceptibility is correlated with loss of BAP1, one of the key prognosticators for metastatic UM, and ferroptosis induction greatly reduced metastasis formation in the UM xenograft model. Collectively, we have established a patient-derived animal model for metastatic UM and identified ferroptosis induction as a possible therapeutic strategy for the treatment of UM patients., (© 2023. The Author(s).)

Uveal melanoma (UM) is a rare malignant cancer of the eye, with up to 50% of patients dying from metastasis, for which no effective treatment is available. Due to the rarity of the disease, there is a great need to harness the limited material available from primary tumors and metastases for advanced research and preclinical drug screening. We established a platform to isolate, preserve, and transiently recover viable tissues, followed by the generation of spheroid cultures derived from primary UM. All assessed tumor-derived samples formed spheroids in culture within 24 h and stained positive for melanocyte-specific markers, indicating the retention of their melanocytic origin. These short-lived spheroids were only maintained for the duration of the experiment (7 days) or re-established from frozen tumor tissue acquired from the same patient. Intravenous injection of fluorescently labeled UM cells derived from these spheroids into zebrafish yielded a reproducible metastatic phenotype and recapitulated molecular features of the disseminating UM. This approach allowed for the experimental replications required for reliable drug screening (at least 2 individual biological experiments, with n > 20). Drug treatments with navitoclax and everolimus validated the zebrafish patient-derived model as a versatile preclinical tool for screening anti-UM drugs and as a preclinical platform to predict personalized drug responses.

Uveal Melanoma (UM) is a rare and malignant intraocular tumor with dismal prognosis. Even if radiation or surgery permit an efficient control of the primary tumor, up to 50% of patients subsequently develop metastases, mainly in the liver. The treatment of UM metastases is challenging and the patient survival is very poor. The most recurrent event in UM is the activation of Gαq signaling induced by mutations in GNAQ/11. These mutations activate downstream effectors including protein kinase C (PKC) and mitogen-activated protein kinases (MAPK). Clinical trials with inhibitors of these targets have not demonstrated a survival benefit for patients with UM metastasis. Recently, it has been shown that GNAQ promotes YAP activation through the focal adhesion kinase (FAK). Pharmacological inhibition of MEK and FAK showed remarkable synergistic growth-inhibitory effects in UM both in vitro and in vivo. In this study, we have evaluated the synergy of the FAK inhibitor with a series of inhibitors targeting recognized UM deregulated pathways in a panel of cell lines. The combined inhibition of FAK and MEK or PKC had highly synergistic effects by reducing cell viability and inducing apoptosis. Furthermore, we demonstrated that these combinations exert a remarkable in vivo activity in UM patient-derived xenografts. Our study confirms the previously described synergy of the dual inhibition of FAK and MEK and identifies a novel combination of drugs (FAK and PKC inhibitors) as a promising strategy for therapeutic intervention in metastatic UM.

Purpose: Uveal melanoma (UM) is considered a rare disease; yet, it is the most common intraocular malignancy in adults. Although the primary tumor may be efficiently managed, more than 50% of patients with UM develop distant metastases. The mortality at the first year after diagnosis of metastatic UM has been estimated at 81%, and the poor prognosis has not improved in the past years due to the lack of effective therapies., Methods: In order to search for novel therapeutic possibilities for metastatic UM, we performed a small-scale screen of targeted drug combinations. We verified the targets of the tested compounds by western blotting and PCR and clarified the mechanism of action of the selected combinations by caspase 3 and 7 activity assay and flow cytometry. The best two combinations were tested in a mouse patient-derived xenograft (PDX) UM model as putative therapeutics for metastatic UM., Results: Combinations of the multitarget drug trabectedin with either the CK2/CLK double-inhibitor CX-4945 (silmitasertib) or the c-MET/TAM (TYRO3, Axl, MERTK) receptor inhibitors foretinib and cabozantinib demonstrated synergistic effects and induced apoptosis (relative caspase 3 and 7 activity increased up to 20.5-fold in UM cell lines). In the case of the combination of foretinib and cabozantinib, inhibition of the TAM receptors, but not c-Met, was essential to inhibit the growth of UM cells. Monotreatment with trabectedin inhibited tumor growth by 42%, 49%, and 35% in the MM26, MM309, and MM339 PDX mouse models, respectively., Conclusions: Trabectedin alone or in combination with cabozantinib inhibited tumor growth in PDX UM mouse models. Blocking of MERTK, rather than TYRO3, activity inhibited UM cell growth and synergized with trabectedin.

The tumor suppressor protein p53 has an important role in cell-fate determination. In cancer cells, the activity of p53 is frequently repressed by high levels of MDMX and/or MDM2. MDM2 is a ubiquitin ligase whose activity results in ubiquitin- and proteasome-dependent p53 degradation, while MDMX inhibits p53-activated transcription by shielding the p53 transactivation domain. Interestingly, the oncogenic functions of MDMX appear to be more wide-spread than inhibition of p53. The present study aimed to elucidate the MDMX-controlled transcriptome. Therefore, we depleted MDMX with four distinct shRNAs from a high MDMX expressing uveal melanoma cell line and determined the effect on the transcriptome by RNAseq. Biological function analyses indicate the inhibition of the cell cycle regulatory genes and stimulation of cell death activating genes upon MDMX depletion. Although the inhibition of p53 activity clearly contributes to the transcription regulation controlled by MDMX, it appeared that the transcriptional regulation of multiple genes did not only rely on p53 expression. Analysis of gene regulatory networks indicated a role for Forkhead box (FOX) transcription factors. Depletion of FOXO proteins partly prevented the transcriptional changes upon MDMX depletion. Furthermore, depletion of FOXO proteins relatively diminished the growth inhibition upon MDMX knockdown, although the knockdown of the FOXO transcription factors also reduces cell growth. In conclusion, the p53-independent oncogenic functions of MDMX could be partially explained by its regulation of FOXO activity., Competing Interests: The authors declare no conflicts of interest.

Metastatic prostate cancer is a lethal disease in patients incapable of responding to therapeutic interventions. Invasive prostate cancer spread is caused by failure of the normal anti-cancer defense systems that are controlled by the tumour suppressor protein, p53. Upon mutation, p53 malfunctions. Therapeutic strategies to directly re-empower the growth-restrictive capacities of p53 in cancers have largely been unsuccessful, frequently because of a failure to discriminate responses in diseased and healthy tissues. Our studies sought alternative prostate cancer drivers, intending to uncover new treatment targets. We discovered the oncogenic potency of MDM4 in prostate cancer cells, both in the presence and absence of p53 and also its mutation. We uncovered that sustained depletion of MDM4 is growth inhibitory in prostate cancer cells, involving either apoptosis or senescence, depending on the cell and genetic context. We identified that the potency of MDM4 targeting could be potentiated in prostate cancers with mutant p53 through the addition of a first-in-class small molecule drug that was selected as a p53 reactivator and has the capacity to elevate oxidative stress in cancer cells to drive their death., Competing Interests: The authors declare no conflict of interest.

Currently, no systemic treatment is approved as the standard of care for metastatic uveal melanoma (UM). mTOR has been evaluated as a drug target in UM. However, one of the main limitations is dose reduction due to adverse effects. The combination of everolimus with another targeted agent would allow the reduction of the dose of a single drug, thus widening the therapeutic window. In our study, we aimed to identify a synergistic combination with everolimus in order to develop a novel treatment option for metastatic UM. We exploited CRISPR-Cas9 synthetic lethality screening technology to search for an efficient combination. IGF1R and PRKDC and several other genes were identified as hits in the screen. We investigated the effect of the combination of everolimus with the inhibitors targeting IGF1R and DNA-PKcs on the survival of UM cell lines. These combinations synergistically slowed down cell growth but did not induce apoptosis in UM cell lines. These combinations were tested on PDX UM in an in vivo model, but we could not detect tumor regression. However, we could find significant activity of the dual DNA-PKcs/mTOR inhibitor CC-115 on PDX UM in the in vivo model.

Reactivation of dormant cancer cells can lead to cancer relapse, metastasis, and patient death. Dormancy is a nonproliferative state and is linked to late relapse and death. No targeted therapy is currently available to eliminate dormant cells, highlighting the need for a deeper understanding and reliable models. Here, we thoroughly characterize the dormant D2.OR and ZR-75-1, and proliferative D2A1 breast cancer cell line models in vivo and/or in vitro, and assess if there is overlap between a dormant and a senescent phenotype. We show that D2.OR but not D2A1 cells become dormant in the liver of an immunocompetent model. In vitro, we show that D2.OR and ZR-75-1 cells in response to a 3D environment or serum-free conditions are growth-arrested in G1, of which a subpopulation resides in a 4NG1 state. The dormancy state is reversible and not associated with a senescence phenotype. This will aid future research on breast cancer dormancy., (© 2021. The Author(s).)

In Uveal Melanoma (UM), an inflammatory phenotype is strongly associated with the development of metastases and with chromosome 3/BAP1 expression loss. As an increased expression of several Histone Deacetylases (HDACs) was associated with loss of chromosome 3, this suggested that HDAC expression might also be related to inflammation. We analyzed HDAC expression and the presence of leukocytes by mRNA expression in two sets of UM (Leiden and TCGA) and determined the T lymphocyte fraction through ddPCR. Four UM cell lines were treated with IFNγ (50IU, 200IU). Quantitative PCR (qPCR) was used for mRNA measurement of HDACs in cultured cells. In both cohorts (Leiden and TCGA), a positive correlation occurred between expression of HDACs 1, 3 and 8 and the presence of a T-cell infiltrate, while expression of HDACs 2 and 11 was negatively correlated with the presence of tumor-infiltrating macrophages. Stimulation of UM cell lines with IFNγ induced an increase in HDACs 1, 4, 5, 7 and 8 in two out of four UM cell lines. We conclude that the observed positive correlations between HDAC expression and chromosome 3/BAP1 loss may be related to the presence of infiltrating T cells.

Targeting the MAPK signaling pathway has transformed the treatment of metastatic melanoma. CRISPR-Cas9 genetic screens provide a genome-wide approach to uncover novel genetic dependencies that might serve as therapeutic targets. Here, we analyzed recently reported CRISPR-Cas9 screens comparing data from 28 melanoma cell lines and 313 cell lines of other tumor types in order to identify fitness genes related to melanoma. We found an average of 1,494 fitness genes in each melanoma cell line. We identified 33 genes, inactivation of which specifically reduced the fitness of melanoma. This set of tumor type-specific genes includes established melanoma fitness genes as well as many genes that have not previously been associated with melanoma growth. Several genes encode proteins that can be targeted using available inhibitors. We verified that genetic inactivation of DUSP4 and PPP2R2A reduces the proliferation of melanoma cells. DUSP4 encodes an inhibitor of ERK, suggesting that further activation of MAPK signaling activity through its loss is selectively deleterious to melanoma cells. Collectively, these data present a resource of genetic dependencies in melanoma that may be explored as potential therapeutic targets., (© 2020 The Authors. Pigment Cell & Melanoma Research published by John Wiley & Sons Ltd.)

The treatment of uveal melanoma (UM) metastases or adjuvant treatment may imply immunological approaches or chemotherapy. It is to date unknown how epigenetic modifiers affect the expression of immunologically relevant targets, such as the HLA Class I antigens, in UM. We investigated the expression of HDACs and the histone methyl transferase EZH2 in a set of 64 UMs, using an Illumina HT12V4 array, and determined whether a histone deacetylase (HDAC) inhibitor and EZH2 inhibitor modified the expression of HLA Class I on three UM cell lines. Several HDACs (HDAC1, HDAC3, HDAC4, and HDAC8) showed an increased expression in high-risk UM, and were correlated with an increased HLA expression. HDAC11 had the opposite expression pattern. While in vitro tests showed that Tazemetostat did not influence cell growth, Quisinostat decreased cell survival. In the three tested cell lines, Quisinostat increased HLA Class I expression at the protein and mRNA level, while Tazemetostat did not have an effect on the cell surface HLA Class I levels. Combination therapy mostly followed the Quisinostat results. Our findings indicate that epigenetic drugs (in this case an HDAC inhibitor) may influence the expression of immunologically relevant cell surface molecules in UM, demonstrating that these drugs potentially influence immunotherapy.

Background: Immunotherapy of cancer is successful but tumor regression often is incomplete and followed by escape. Understanding the mechanisms underlying this acquired resistance will aid the development of more effective treatments., Methods: We exploited a mouse model where tumor-specific therapeutic vaccination results in tumor regression, followed by local recurrence and resistance. In depth studies on systemic, local and tumor intrinsic changes were performed with flow and mass cytometry, immunohistochemistry, transcriptomics and several perturbation studies with inhibitors or agonistic antibodies in mice. Main findings were recapitulated in vaccinated patients., Results: Full tumor regression and cure of tumor-bearing mice is dependent on the magnitude of the vaccine-induced T-cell response. Recurrence of tumors did not involve classical immune escape mechanisms, such as antigen-presentation alterations, immune checkpoint expression, resistance to killing or local immune suppression. However, the recurrent tumors displayed a changed transcriptome with alterations in p53, tumor necrosis factor-α and transforming growth factor-β signaling pathways and they became immunologically cold. Remarkably, ex vivo cell-sorted recurrent tumors, directly reinjected in naïve hosts retained their resistance to vaccination despite a strong infiltration with tumor-specific CD8 + T cells, similar to that of vaccine-responsive tumors. The influx of inflammatory mature myeloid effector cells in the resistant tumors, however, was impaired and this turned out to be the underlying mechanisms as restoration of inflammatory myeloid cell infiltration reinstated the sensitivity of these refractory tumors to vaccination. Notably, impaired myeloid cell infiltration after vaccination was also associated with vaccine resistance in patients., Conclusion: An immunotherapy-induced disability of tumor cells to attract innate myeloid effector cells formed a major mechanism underlying immune escape and acquired resistance. These data not only stresses the importance of myeloid effector cells during immunotherapy but also demands for new studies to harness their tumoricidal activities., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

The Mdm4 (alias MdmX) oncoprotein, like its paralogue and interaction partner Mdm2, antagonizes the tumor suppressor p53. p53-independent roles of the Mdm proteins are emerging, and we have reported the ability of Mdm2 to modify chromatin and to support DNA replication by suppressing the formation of R-loops (DNA/RNA-hybrids). We show here that the depletion of Mdm4 in p53-deficient cells compromises DNA replication fork progression as well. Among various deletion mutants, only full-length Mdm4 was able to support DNA replication fork progression. Co-depletion of Mdm4 and Mdm2 further impaired DNA replication, and the overexpression of each partially compensated for the other's loss. Despite impairing replication, Mdm4 depletion only marginally hindered cell proliferation, likely due to compensation through increased firing of replication origins. However, depleting Mdm4 sensitized p53-/- cells to the nucleoside analog gemcitabine, raising the future perspective of using Mdm4 inhibitors as chemosensitizers. Mechanistically, Mdm4 interacts with members of the Polycomb Repressor Complexes and supports the ubiquitination of H2A, thereby preventing the accumulation of DNA/RNA-hybrids. Thus, in analogy to previously reported activities of Mdm2, Mdm4 enables unperturbed DNA replication through the avoidance of R-loops.

A proportion of patients diagnosed with melanoma has a positive family history. Despite increasing knowledge on the genes responsible for familial clustering, the genetic basis in the majority of the families with an inherited predisposition to melanoma remains to be clarified. To identify novel melanoma-susceptibility genes, we applied whole-exome sequencing on DNA from two members of a family with four melanoma cases, not explained by established high penetrance melanoma-susceptibility genes. Whole-exome sequencing identified 10 rare, co-segregating, predicted deleterious missense gene variants. Subsequent co-segregation analysis revealed that only variants in the DOT1L (R409H) and the SLCO4C1 (P597A) genes were present in the other two affected members of this family. DOT1L is a methyltransferase that methylates histone H3 lysine 79 (H3K79). It is involved in maintenance of genomic stability, since mutations in the DOT1L gene have been previously reported to compromise the removal of ultraviolet photoproducts in ultraviolet-irradiated melanocytes, thereby enhancing malignant transformation. We hypothesized that the presence of DOT1L R409H variant might be associated with an increased risk of melanoma, since we found co-segregation of the DOT1L mutation in all four melanoma-affected family members. However, this missense variant did neither lead to detectable loss-of-heterozygosity nor reduction of histone methyltransferase activity in melanoma samples from mutation carriers nor altered ultraviolet-survival of mouse embryonic stem cells containing an engineered homozygous DOT1L R409H mutation. Although functional analysis of this rare co-segregating variant did not reveal compromised histone methyltransferase activity and ultraviolet exposure sensitivity, the role of DOT1L as melanoma susceptibility gene deserves further study.

Uveal melanoma (UM) is the most common primary intraocular malignancy in adults, and gives rise to metastases in 50% of cases. The presence of an inflammatory phenotype is a well-known risk factor for the development of metastases. This inflammatory phenotype is characterized by the presence of high numbers of lymphocytes and macrophages, and a high expression of the HLA Class I and II antigens. An abnormal expression of HLA Class I may influence cytotoxic T lymphocyte (CTL) as well as Natural Killer (NK) cell responses. We provide a comprehensive review regarding the inflammatory phenotype in UM and the expression of locus- and allele-specific HLA Class I and of Class II antigens in primary UM and its metastases. Furthermore, we describe the known regulators and the role of genetics (especially chromosome 3 and BRCA-Associated Protein 1 (BAP1 status)), and, last but not least, the effect of putative therapeutic treatments on HLA expression., Competing Interests: The authors declare no conflict of interest.

One of the characteristics of prognostically infaust uveal melanoma (UM) is an inflammatory phenotype, which is characterized by high numbers of infiltrating T cells and macrophages, and a high HLA Class I expression. We wondered how this inflammation is regulated, and considered that one of the most important regulators of inflammation, the NFkB pathway, might play a role. We analyzed 64 UM samples for expression of HLA Class I, its regulators, and of members of the NFkB transcription family, using an Illumina HT12V4 array. HLA Class I expression and infiltrating immune cells were also determined by immunohistochemical staining. Information was obtained regarding chromosome status by Affymetrix Nsp array. Our analysis shows that expression of NFkB1, NFkB2 and RELB positively correlates with the level of HLA Class I expression and the number of infiltrating T cells and macrophages, while SPP1 and PPARγ are negatively correlated. Increased levels of NFkB1 and NFkB2 and decreased levels of SPP1 and PPARγ are seen in Monosomy 3/BAP1-negative tumors. This is also the case in non-inflammatory UM, indicating that our observation not only involves infiltrating leukocytes but the tumor cells themselves. We report that the NFkB pathway is associated with inflammation and HLA Class I expression in UM, and is upregulated when BAP1 expression is lost.

Expression of DNA repair genes was studied in uveal melanoma (UM) in order to identify genes that may play a role in metastases formation. We searched for genes that are differentially expressed between tumors with a favorable and unfavorable prognosis. Gene-expression profiling was performed on 64 primary UM from the Leiden University Medical Center (LUMC), Leiden, The Netherlands. The expression of 121 genes encoding proteins involved in DNA repair pathways was analyzed: a total of 44 genes differed between disomy 3 and monosomy 3 tumors. Results were validated in a cohort from Genoa and Paris and the The Cancer Genome Atlas (TCGA) cohort. Expression of the PRKDC , WDR48 , XPC , and BAP1 genes was significantly associated with clinical outcome after validation. PRKDC was highly expressed in metastasizing UM ( p < 0.001), whereas WDR48 , XPC , and BAP1 were lowly expressed ( p < 0.001, p = 0.006, p = 0.003, respectively). Low expression of WDR48 and XPC was related to a large tumor diameter ( p = 0.01 and p = 0.004, respectively), and a mixed/epithelioid cell type ( p = 0.007 and p = 0.03, respectively). We conclude that the expression of WDR48 , XPC , and BAP1 is significantly lower in UM with an unfavorable prognosis, while these tumors have a significantly higher expression of PRKDC . Pharmacological inhibition of DNA-PKcs resulted in decreased survival of UM cells. PRKDC may be involved in proliferation, invasion and metastasis of UM cells. Unraveling the role of DNA repair genes may enhance our understanding of UM biology and result in the identification of new therapeutic targets.

Despite much progress in our understanding of uveal melanoma (UM) over the past decades, this rare tumour is still often misclassified. Although UM, like other melanomas, is very probably derived from melanocytes, it is drastically different from cutaneous melanoma and most other melanoma subtypes in terms of epidemiology, aetiology, biology and clinical features, including an intriguing metastatic hepatotropism. UM carries distinctive prognostic chromosome alterations, somatic mutations and gene expression profiles, allowing an active tailored surveillance strategy and dedicated adjuvant clinical trials. There is no standard systemic treatment for disseminated UM at present. In contrast to cutaneous melanoma, UMs are not BRAF -mutated, thus curtailing the use of B-Raf inhibitors. Although these tumours are characterised by some immune infiltrates, immune checkpoint inhibitors are rarely effective, possibly due to a low mutation burden. UM patients across the world not only face rare cancer-related issues (e.g., specific management strategies, access to information and to expert centres), but also specific UM problems, which can be exacerbated by the common misconception that it is a subtype of cutaneous melanoma. As a European Consortium dedicated to research on UM and awareness on the disease, "UM Cure 2020" participants urge medical oncologists, pharmaceutical companies, and regulatory agencies to acknowledge UM as a melanoma with specific issues, in order to accelerate the development of new therapies for patients., Competing Interests: M.R. and S.P. received research grants from Bristol-Myers Squibb (BMS) and Merck Sharp & Dohme. E.K. has consulting/advisory relationships with BMS, Novartis, Roche, Amgen, Pierre-Fabre (honoraria paid to institution). She received research grants from BMS.

Malignant melanoma of the conjunctiva (CM) is an uncommon but potentially deadly disorder. Many malignancies show an increased activity of the epigenetic modifier enhancer of zeste homolog 2 (EZH2). We studied whether EZH2 is expressed in CM, and whether it may be a target for therapy in this malignancy. Immunohistochemical analysis showed that EZH2 protein expression was absent in normal conjunctival melanocytes and primary acquired melanosis, while EZH2 was highly expressed in 13 (50%) of 26 primary CM and seven (88%) of eight lymph node metastases. Increased expression was positively associated with tumour thickness (p =0.03). Next, we targeted EZH2 with specific inhibitors (GSK503 and UNC1999) or depleted EZH2 by stable shRNA knockdown in three primary CM cell lines. Both pharmacological and genetic inactivation of EZH2 inhibited cell growth and colony formation and influenced EZH2-mediated gene transcription and cell cycle profile in vitro. The tumour suppressor gene p21/CDKN1A was especially upregulated in CM cells after EZH2 knockdown in CM cells. Additionally, the potency of GSK503 against CM cells was monitored in zebrafish xenografts. GSK503 profoundly attenuated tumour growth in CM xenografts at a well-tolerated concentration. Our results indicate that elevated levels of EZH2 are relevant to CM tumourigenesis and progression, and that EZH2 may become a potential therapeutic target for patients with CM. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland., (© 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.)

Uveal melanoma (UM) is the most frequent ocular cancer in adults, accounting for ~5% of the total melanoma incidence. Although the primary tumor is well treatable, patients frequently develop metastases for which no curative therapy exists. Highly activated protein kinase C (PKC) is a common feature of UM and has shown potential as therapeutic intervention for UM patients. Unfortunately, PKC inhibition as single treatment appears to have only limited clinical benefit. Combining PKC inhibition with activation of p53, which is rarely mutated in UM, by MDM2 inhibitors has shown promising results in vitro and in vivo. However, clinical studies have shown strong adverse effects of MDM2 inhibition. Therefore, we investigated alternative approaches to achieve similar anticancer effects, but with potentially less adverse effects. We studied the potential of targeting MDMX, an essential p53 inhibitor during embryonal development but less universally expressed in adult tissues compared with MDM2. Therefore, targeting MDMX is predicted to have less adverse effects in patients. Depletion of MDMX, like the pharmacological activation of p53, inhibits the survival of UM cells, which is enhanced in combination with PKC inhibition. Also pan-PKC inhibitors elicit adverse effects in patients. As the PKC family consists of 10 different isoforms, it could be hypothesized that targeting a single PKC isoform would have less adverse effects compared with a pan-PKC inhibitor. Here we show that specifically depleting PKCδ inhibits UM cell growth, which can be further enhanced by p53 reactivation. In conclusion, our data show that the synergistic effects of p53 activation by MDM2 inhibition and broad spectrum PKC inhibition on survival of UM cells can also largely be achieved by the presumably less toxic combination of depletion of MDMX and targeting a specific PKC isoform, PKCδ.

Epithelioid hemangioma is a locally aggressive vascular neoplasm, found in bones and soft tissue, whose cause is currently unknown, but may involve oncogene activation. FOS is one of the earliest viral oncogenes to be characterized, and normal cellular FOS forms part of the activator protein 1 (AP-1) transcription factor complex, which plays a pivotal role in cell growth, differentiation, and survival as well as the DNA damage response. Despite this, a causal link between aberrant FOS function and naturally occurring tumors has not yet been established. Here, we describe a thorough molecular and biochemical analysis of a mutant FOS protein we identified in these vascular tumors. The mutant protein lacks a highly conserved helix consisting of the C-terminal four amino acids of FOS, which we show is indispensable for fast, ubiquitin-independent FOS degradation via the 20S proteasome. Our work reveals that FOS stimulates endothelial sprouting and that perturbation of normal FOS degradation could account for the abnormal vessel growth typical of epithelioid hemangioma. To the best of our knowledge, this is the first functional characterization of mutant FOS proteins found in tumors., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: Ewing sarcoma is an aggressive, highly metastatic primary bone and soft tissue tumor most frequently occurring in the bone of young adolescents. Patients, especially those diagnosed with a metastatic disease, have a poor overall survival. Chemokine receptor CXCR4 has a key pro-tumorigenic role in the tumor microenvironment of Ewing sarcoma and has been suggested to be involved in the increased metastatic propensity. Earlier studies on CXCR4 protein expression in Ewing sarcoma yielded contradictory results when compared to CXCR4 RNA expression studies. Previously, we demonstrated that CXCR4 expression could be detected in vivo using the fluorescently tagged CXCR4-specific peptide MSAP-Ac-TZ14011. Therefore, we studied the membranous CXCR4 expression in Ewing sarcoma cell lines using MSAP-Ac-TZ14011., Methods: The CXCR4 membrane expression levels were studied in EWS cell lines by flow cytometry using the hybrid peptide MSAP-Ac-TZ14011 and were correlated to CXCR4 RNA expression levels. The measurements were compared to levels detected using the CXCR4 antibody ab2074 under various cell preparation conditions. In addition, the staining patterns were analyzed by confocal fluorescence microscopy over time., Results: The hybrid peptide MSAP-Ac-TZ14011 levels showed a strong and better correlation of CXCR4 membrane expression with the CXCR4 RNA expression levels than observed with the anti-CXCR4 antibody ab2074. With the hybrid peptide MSAP-Ac-TZ14011 using live cell confocal microscopy CXCR4 membrane staining and internalization was detected and the signal intensity correlated well with CXCR4 mRNA expression levels., Conclusions: The fluorescently labeled CXCR4 targeting peptide-based method provides a reliable alternative to antibody staining to study the CXCR4 membrane expression in live cells using either flow cytometry or live cell fluorescence microscopy. The fluorescently tagged CXCR4 targeting peptide could enable in vivo detection of CXCR4 expression in Ewing sarcoma which may help to stratify cases for anti-CXCR4 therapy.

Purpose: Conjunctival melanoma (CM) is a rare but lethal form of cancer. Similar to cutaneous melanoma, CM frequently carries activating mutations in BRAF and NRAS . We studied whether CM as well as conjunctival benign and premalignant melanocytic lesions express targets in the mitogen-activated protein kinase (MAPK) and AKT pathways, and whether specific inhibitors can suppress CM growth in vitro ., Methods: 131 conjunctival lesions obtained from 129 patients were collected. The presence of BRAF V600E mutation and expression of phosphorylated (p)-ERK and p-AKT were assessed by immunohistochemistry. We studied cell proliferation, phosphorylation, cell cycling and apoptosis in three CM cell lines using two BRAF inhibitors (Vemurafenib and Dabrafenib), a MEK inhibitor (MEK162) and an AKT inhibitor (MK2206)., Results: The BRAF V600E mutation was present in 19% of nevi and 26% of melanomas, but not in primary acquired melanosis (PAM). Nuclear and cytoplasmic p-ERK and p-AKT were expressed in all conjunctival lesions. Both BRAF inhibitors suppressed growth of both BRAF mutant CM cell lines, but only one induced cell death. MEK162 and MK2206 inhibited proliferation of CM cells in a dose-dependent manner, and the combination of these two drugs led to synergistic growth inhibition and cell death in all CM cell lines., Conclusion: ERK and AKT are constitutively activated in conjunctival nevi, PAM and melanoma. While BRAF inhibitors prohibited cell growth, they were not always cytotoxic. Combining MEK and AKT inhibitors led to more growth inhibition and cell death in CM cells. The combination may benefit patients suffering from metastatic conjunctival melanoma., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflicts of interest.

The MDMX protein was identified as a p53-interacting protein with a strong similarity to MDM2. Like Mdm2, Mdmx expression is essential for curbing p53 activity during embryonic development, indicating nonredundant functions of Mdmx and Mdm2. There is now a large body of evidence indicating that cancers frequently up-regulate MDMX expression as a means to dampen p53 tumor-suppressor function. Importantly, MDMX also shows p53-independent oncogenic functions. These data make MDMX an attractive therapeutic target for cancer therapy. Here, we summarize the mechanisms used by cancer cells to increase MDMX expression and promising pharmacological strategies to target MDMX in cancer-in particular, the recent findings that antisense oligonucleotides (ASOs) can be used to efficiently modulate MDMX messenger RNA (mRNA) splicing., (Copyright © 2016 Cold Spring Harbor Laboratory Press; all rights reserved.)

Chemokine receptor CXCR4 is involved in tumor growth, angiogenesis and metastasis. Its function is regulated in many ways and one of them is alternative splicing. We identified two novel coding splice variants (CXCR4-3 and CXCR4-4) of CXCR4 in Ewing sarcoma (EWS) cell lines by whole transcriptome sequencing and validated these with reverse transcriptase- PCR and Sanger sequencing. The novel splice variants were expressed at RNA level in Ewing sarcoma samples and in other tumor cell lines and placenta, but not in lung. Due to inclusion of an additional exon the new isoforms have a 70 and 33 amino acid elongation of the N-terminal end of CXCR4. For validation at protein and functional level, the identified isoforms and normal CXCR4 were cloned into an EYFP tagged vector and ectopically expressed in HEK293T cell line and EWS cell line A673. Of the novel isoforms CXCR4-3 showed cell membrane localization and a functional response after addition of CXCR4 ligand CXCL12a. CXCR4-4 showed strong cytoplasmic accumulation and no response to ligand treatment. The role of the newly discovered isoforms in CXCR4 signaling is likely to be limited. Our data stresses the importance of functional validation of newly identified isoforms., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Mdm4, a protein related to the ubiquitin-ligase Mdm2, is an essential inhibitor of tumor suppressor protein p53. In both human and mouse cells, the Mdm4 gene encodes two major transcripts: one encodes the full-length oncoprotein (designated below as Mdm4-FL), whereas the other, resulting from a variant splicing that skips exon 6, encodes the shorter isoform Mdm4-S. Importantly, increased Mdm4-S mRNA levels were observed in several human cancers, and correlated with poor survival. However, the role of Mdm4-S in cancer progression remains controversial, because the Mdm4-S protein appeared to be a potent p53 inhibitor when overexpressed, but the splice variant also leads to a decrease in Mdm4-FL expression. To unambiguously determine the physiological impact of the Mdm4-S splice variant, we generated a mouse model with a targeted deletion of the Mdm4 exon 6, thereby creating an obligatory exon skipping. The mutant allele (Mdm4(ΔE6)) prevented the expression of Mdm4-FL, but also led to increased Mdm4-S mRNA levels. Mice homozygous for this allele died during embryonic development, but were rescued by a concomitant p53 deficiency. Furthermore in a hypomorphic p53(ΔP/ΔP) context, the Mdm4(ΔE6) allele led to p53 activation and delayed the growth of oncogene-induced tumors. We next determined the effect of Mdm4(+/ΔE6) heterozygosity in a hypermorphic p53(+/Δ31) genetic background, recently shown to be extremely sensitive to Mdm4 activity. Mdm4(+/ΔE6) p53(+/Δ31) pups were born, but suffered from aplastic anemia and died before weaning, again indicating an increased p53 activity. Our results demonstrate that the main effect of a skipping of Mdm4 exon 6 is not the synthesis of the Mdm4-S protein, but rather a decrease in Mdm4-FL expression. These and other data suggest that increased Mdm4-S mRNA levels might correlate with more aggressive cancers without encoding significant amounts of a potential oncoprotein. Hypotheses that may account for this apparent paradox are discussed.

Although murine xenograft models for human uveal melanoma (UM) are available, they are of limited utility for screening large compound libraries for the discovery of new drugs. We need new preclinical models which can efficiently evaluate drugs that can treat UM metastases. The zebrafish embryonic model is ideal for drug screening purposes because it allows the investigation of potential antitumor properties of drugs within 1 week. The optical transparency of the zebrafish provides unique possibilities for live imaging of fluorescence-labelled cancer cells and their behavior. In addition, the adaptive immune response, which is responsible for the rejection of transplanted material, is not yet present in the early stages of fish development, and systemic immunosuppression is therefore not required to allow growth of tumor cells. We studied the behavior of UM cells following injection into zebrafish embryos and observed different phenotypes. We also analyzed cell migration, proliferation, formation of micrometastasis and interaction with the host microenvironment. Significant differences were noted between cell lines: cells derived from metastases showed more migration and proliferation than cells derived from the primary tumors. The addition of the c-Met inhibitor crizotinib to the water in which the larvae were kept reduced the migration and proliferation of UM cells expressing c-Met. This indicates the applicability of the zebrafish xenografts for testing novel inhibitory compounds and provides a fast and sensitive in vivo vertebrate model for preclinical drug screening to combat UM.

TP53 gene mutation is associated with poor prognosis in breast cancer, but additional biomarkers that can further refine the impact of the p53 pathway are needed to achieve clinical utility. In this study, we evaluated a role for the HDMX-S/FL ratio as one such biomarker, based on its association with other suppressor mutations that confer worse prognosis in sarcomas, another type of cancer that is surveilled by p53. We found that HDMX-S/FL ratio interacted with p53 mutational status to significantly improve prognostic capability in patients with breast cancer. This biomarker pair offered prognostic utility that was comparable with a microarray-based prognostic assay. Unexpectedly, the utility tracked independently of DNA-damaging treatments and instead with different tumor metastasis potential. Finally, we obtained evidence that this biomarker pair might identify patients who could benefit from anti-HDM2 strategies to impede metastatic progression. Taken together, our work offers a p53 pathway marker, which both refines our understanding of the impact of p53 activity on prognosis and harbors potential utility as a clinical tool., (©2015 American Association for Cancer Research.)

The p53 tumor suppressor protein is primarily known for its important role in tumor suppression. In addition, p53 affects tumor cell migration, invasion, and epithelial-mesenchymal transition (EMT); processes also regulated by the transforming growth factor-β (TGF-β) signaling pathway. Here, we investigated the role of p53 in breast tumor cell invasion, migration, and EMT and examined the interplay of p53 with TGF-β3 in these processes. MCF-10A1 and MCF-10CA1a breast cancer cells were treated with Nutlin-3 and TGF-β3, and the effects on tumor cell migration and invasion were studied in transwell and 3D spheroid invasion assays. The effects of Nutlin-3 and TGF-β3 on EMT were examined in NMuMG cells. To identify genes involved in TGF-β-induced invasion that are modulated by p53, a Human Tumor Metastasis-specific RT-PCR array was performed. Verification of EPHB2 regulation by TGF-β3 and p53 was performed on breast cancer tumor cell lines. We demonstrate that p53 inhibits basal and TGF-β3-induced invasion, migration, and EMT in normal breast epithelial and breast cancer cells. Pharmacological activation of p53 inhibited induction of several TGF-β3 targets involved in TGF-β3-induced tumor cell invasion, i.e., matrix metallo proteinase (MMP)2, MMP9, and integrin β 3 . The ephrin-type B receptor 2 (EPHB2) gene was identified as a new TGF-β target important for TGF-β3-mediated invasion and migration, whose transcriptional activation by TGF-β3 is also inhibited by p53. The results show an intricate interplay between p53 and TGF-β3 whereby p53 inhibits the TGF-β3-induced expression of genes, e.g., EPHB2, to impede tumor cell invasion and migration.

Purpose: Uveal melanoma (UM) is fatal in up to 50% of patients because of liver metastases that are refractory to therapies currently available. While murine xenograft models for human uveal melanoma are available, they have limited utility for screening large compound libraries in drug discovery studies. Therefore, new robust preclinical models are needed that can efficiently evaluate drug efficacy for treatment of this malignancy., Methods: Uveal melanoma cell lines generated from primary tumors (92.1, Mel270) and metastases (OMM2.3, OMM2.5, OMM1) were injected into the yolk of 2-day-old zebrafish embryos. After 6 days, proliferation and active migration was quantified via automated confocal image analysis. To determine the suitability of this xenotransplantation model for drug testing, drugs with three different activities (dasatinib, quisinostat, and MLN-4924) were added to the water of uveal melanoma-engrafted embryos., Results: All tested UM cell lines proliferated and migrated in the embryos; significant differences could be discerned between cell lines: Cells derived from metastases showed more migration and proliferation than cells derived from the primary tumors, and provided preclinical models for drug testing. Addition of the Src-inhibitor dasatinib in the water of engrafted embryos reduced proliferation and migration of high Src-expressing 92.1 cells, but did not affect low Src-expressing metastatic OMM2.3 cells. Two experimental anticancer drugs, quisinostat (a histone deacetylase inhibitor) and MLN-4924 (neddylation pathway inhibitor), blocked migration and proliferation of 92.1 and OMM2.3., Conclusions: We established a zebrafish xenograft model of human uveal melanoma with demonstrated applicability for screening large libraries of compounds in drug discovery studies., (Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.)

Translocations involving ETS-transcription factors, most commonly leading to the EWSR1-FLI1 fusion protein, are the hallmark of Ewing sarcoma. Despite knowledge of this driving molecular event, an effective therapeutic strategy is lacking. To test potential treatment regimes, we established a novel Ewing sarcoma zebrafish engraftment model allowing time-effective, dynamic quantification of Ewing sarcoma progression and tumour burden in vivo, applicable for screening of single and combined compounds. In Ewing sarcoma the tumour-suppressor gene TP53 is commonly found to be wild-type, thus providing an attractive target for treatment. Here, we study TP53 wild-type (EW7, CADO-ES1 and TC32) and TP53-deleted (SK-N-MC) Ewing sarcoma cell lines to investigate the potentiating effect of p53 reactivation by Nutlin-3 on treatment with YK-4-279 to block transcriptional activity of EWSR1-FLI1 protein. Blocking EWSR1-FLI1 transcriptional activity reduced Ewing sarcoma tumour cell burden irrespective of TP53 status. We show that simultaneous YK-4-279 treatment with Nutlin-3 to stabilize p53 resulted in an additive inhibition of TP53 wild-type Ewing sarcoma cell burden, whilst not affecting TP53-deleted Ewing sarcoma cells. Improved inhibition of proliferation and migration by combinatorial treatment was confirmed in vivo by zebrafish engraftments. Mechanistically, both compounds together additively induced apoptosis of tumour cells in vivo by engaging distinct pathways. We propose reactivation of the p53 pathway in combination with complementary targeted therapy by EWSR1-FLI1 transcriptional activity disruption as a valuable strategy against p53 wild-type Ewing sarcoma., (Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Introduction: The majority of human sarcomas, particularly soft tissue sarcomas, are relatively resistant to traditional cytotoxic therapies. The proof-of-concept study by Ray-Coquard et al., using the Nutlin human double minute (HDM)2-binding antagonist RG7112, has recently opened a new chapter in the molecular targeting of human sarcomas., Areas Covered: In this review, the authors discuss the challenges and prospective remedies for minimizing the significant haematological toxicities of the cis-imidazole Nutlin HDM2-binding antagonists. Furthermore, they also chart the future direction of the development of p53-reactivating (p53-RA) drugs in 12q13-15 amplicon sarcomas and as potential chemopreventative therapies against sarcomagenesis in germ line mutated TP53 carriers. Drawing lessons from the therapeutic use of Imatinib in gastrointestinal tumours, the authors predict the potential pitfalls, which may lie in ahead for the future clinical development of p53-RA agents, as well as discussing potential non-invasive methods to identify the development of drug resistance., Expert Opinion: Medicinal chemistry strategies, based on structure-based drug design, are required to re-engineer cis-imidazoline Nutlin HDM2-binding antagonists into less haematologically toxic drugs. In silico modelling is also required to predict toxicities of other p53-RA drugs at a much earlier stage in drug development. Whether p53-RA drugs will be therapeutically effective as a monotherapy remains to be determined.

Purpose of Review: Targeted therapy of malignant melanoma recently experienced remarkable advances with gene mutation-based therapies with signaling pathway inhibitors (kinase inhibitors). The treatments prolong patients' survival, but in general resistance is acquired and progression of disease occurs. Therefore, additional therapeutic targets are desperately needed., Recent Findings: The p53 tumor suppressor gene is rarely mutated in melanoma, but its functional attenuation is needed for tumor development. Recently, it was found that the essential p53 inhibitor Mdmx is very frequently overexpressed in melanoma. Mdmx displays both p53-dependent and p53-independent oncogenic effects needed for melanoma growth, Summary: Current melanoma therapy based upon kinase inhibitors shows robust initial clinical effect, but the duration of effect is limited. Inactivation of Mdmx in melanoma inhibits tumor growth also of kinase-inhibitor-resistant tumors. An observed synergistic effect of kinase-inhibition and Mdmx targeting can lead to better and more durable treatment of melanoma patients.

Unrepaired DNA double-strand breaks (DSBs) cause genetic instability that leads to malignant transformation or cell death. Cells respond to DSBs with the ordered recruitment of signaling and repair proteins to the sites of DNA lesions. Coordinated protein SUMOylation and ubiquitylation have crucial roles in regulating the dynamic assembly of protein complexes at these sites. However, how SUMOylation influences protein ubiquitylation at DSBs is poorly understood. We show herein that Rnf4, an E3 ubiquitin ligase that targets SUMO-modified proteins, accumulates in DSB repair foci and is required for both homologous recombination (HR) and non-homologous end joining repair. To establish a link between Rnf4 and the DNA damage response (DDR) in vivo, we generated an Rnf4 allelic series in mice. We show that Rnf4-deficiency causes persistent ionizing radiation-induced DNA damage and signaling, and that Rnf4-deficient cells and mice exhibit increased sensitivity to genotoxic stress. Mechanistically, we show that Rnf4 targets SUMOylated MDC1 and SUMOylated BRCA1, and is required for the loading of Rad51, an enzyme required for HR repair, onto sites of DNA damage. Similarly to inactivating mutations in other key regulators of HR repair, Rnf4 deficiency leads to age-dependent impairment in spermatogenesis. These findings identify Rnf4 as a critical component of the DDR in vivo and support the possibility that Rnf4 controls protein localization at DNA damage sites by integrating SUMOylation and ubiquitylation events.

Conventional high-grade osteosarcoma is the most common primary bone malignancy. Although altered expression of the p53 inhibitor HDMX (Mdmx/Mdm4) is associated with cancer risk, progression, and outcome in other tumor types, little is known about its role in osteosarcoma. High expression of the Hdmx splice variant HDMX-S relative to the full-length transcript (the HDMX-S/HDMX-FL ratio) correlates with reduced HDMX protein expression, faster progression, and poorer survival in several cancers. Here, we show that the HDMX-S/HDMX-FL ratio positively correlates with less HDMX protein expression, faster metastatic progression, and a trend to worse overall survival in osteosarcomas. We found that the HDMX-S/HDMX-FL ratio associated with common somatic genetic lesions connected with p53 inhibition, such as p53 mutation and HDM2 overexpression in osteosarcoma cell lines. Interestingly, this finding was not limited to osteosarcomas as we observed similar associations in breast cancer and a variety of other cancer cell lines, as well as in tumors from patients with soft tissue sarcoma. The HDMX-S/HDMX-FL ratio better defined patients with sarcoma with worse survival rates than p53 mutational status. We propose a novel role for alternative splicing of HDMX, whereby it serves as a mechanism by which HDMX protein levels are reduced in cancer cells that have already inhibited p53 activity. Alternative splicing of HDMX could, therefore, serve as a more effective biomarker for p53 pathway attenuation in cancers than p53 gene mutation., (©2012 AACR.)

Senescence is thought to play an important role in the progressive age-related decline in tissue integrity and concomitant diseases, but not much is known about the complex interplay between upstream regulators and downstream effectors. We profiled whole genome gene expression of non-stressed and rotenone-stressed human fibroblast strains from young and oldest old subjects, and measured senescence associated β-gal activity. Microarray results identified gene sets involved in carbohydrate metabolism, Wnt/β-catenin signaling, the cell cycle, glutamate signaling, RNA-processing and mitochondrial function as being differentially regulated with chronological age. The most significantly differentially regulated mRNA corresponded to the p16 gene. p16 was then investigated using qPCR, Western blotting and immunocytochemistry. In conclusion, we have identified cellular pathways that are differentially expressed between fibroblast strains from young and old subjects., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Reactivation of the p53 tumor-suppressor protein by small molecules like Nutlin-3 and RITA (reactivation of p53 and induction of tumor cell apoptosis) is a promising strategy for cancer therapy. The molecular mechanisms involved in the responses to RITA remain enigmatic. Several groups reported the induction of a p53-dependent DNA damage response. Furthermore, the existence of a p53-dependent S-phase checkpoint has been suggested, involving the checkpoint kinase Chk1. We have recently shown synergistic induction of apoptosis by RITA in combination with Nutlin-3, and we observed concomitant Chk2 phosphorylation. Therefore, we investigated whether Chk2 contributes to the cellular responses to RITA. Strikingly, the induction of apoptosis seemed entirely Chk2 dependent. Transcriptional activity of p53 in response to RITA required the presence of Chk2. A partial rescue of apoptosis observed in Noxa knockdown cells emphasized the relevance of p53 transcriptional activity for RITA-induced apoptosis. In addition, we observed an early p53- and Chk2-dependent block of DNA replication upon RITA treatment. Replicating cells seemed more prone to entering RITA-induced apoptosis. Furthermore, the RITA-induced DNA damage response, which was not a secondary effect of apoptosis induction, was strongly attenuated in cells lacking p53 or Chk2. In conclusion, we identified Chk2 as an essential mediator of the cellular responses to RITA.

The prognosis of patients with uveal melanoma is poor. Because of the limited efficacy of current treatments, new therapeutic strategies need to be developed. Because p53 mutations are uncommon in uveal melanoma, reactivation of p53 may be used to achieve tumor regression. We investigated the use of combination therapies for intraocular melanoma, based on the p53 activators Nutlin-3 and reactivation of p53 and induction of tumor cell apoptosis (RITA) and the topoisomerase I inhibitor Topotecan. Nutlin-3 treatment induced p53-dependent growth inhibition in human uveal melanoma cell lines. The sensitivity to Nutlin-3 of the investigated cell lines did not correlate with basal Hdm2 or Hdmx levels. Nutlin-3 synergized with RITA and Topotecan to induce apoptosis in uveal melanoma cell lines and short-term cultures. Drug synergy correlated with enhanced induction of p53-Ser46 phosphorylation, which was attenuated by ATM inhibition. Nutlin-3 and Topotecan also significantly delayed tumor growth in vivo in a murine B16F10 model for ocular melanoma. Combination treatment appeared to inhibit tumor growth slightly more efficient than either drug alone. Nutlin-3, RITA and Topotecan lead to comparable p53 activation and growth inhibition under normoxia and hypoxia. Treatment with Nutlin-3 or RITA had no effect on HIF-1α induction by hypoxia, whereas the combination of these two drugs did inhibit hypoxia-induced HIF-1α. Also Topotecan, alone or in combination with Nutlin-3, reduced HIF-1α protein levels, suggesting that a certain level of DNA damage response is required for p53-mediated downregulation of HIF-1α. In conclusion, combination treatments based on small-molecule-induced p53 activation may have clinical potential for uveal melanoma.

The p53 tumor suppressor pathway is inactivated in cancer either via direct mutation or via deregulation of upstream regulators or downstream effectors. P53 mutations are rare in uveal melanoma. Here we investigated the role of the p53 inhibitor Hdmx in uveal melanoma. We found Hdmx over-expression in a subset of uveal melanoma cell lines and fresh-frozen tumor samples. Hdmx depletion resulted in cell-line dependent growth inhibition, apparently correlating with differential Hdm2 levels. Surprisingly, p53 knockdown hardly rescued cell cycle arrest and apoptosis induction upon Hdmx knockdown, whereas it effectively prevented growth suppression induced by the potent p53 activator Nutlin-3. In addition, two compounds inhibiting Hdmx function or expression, SAH-p53-8 and XI-011, also elicited a growth inhibitory effect in a partly p53-independent manner. These findings suggest a novel, growth-promoting function of Hdmx that does not rely on its ability to inhibit p53. We provide evidence for a contribution of p27 protein induction to the observed p53-independent G1 arrest in response to Hdmx knockdown. In conclusion, our study establishes the importance of Hdmx as an oncogene in a subset of uveal melanomas and widens the spectrum of its function beyond p53 inhibition.

Inactivation of the p53 tumour suppressor, either by mutation or by overexpression of its inhibitors Hdm2 and HdmX is the most frequent event in cancer. Reactivation of p53 by targeting Hdm2 and HdmX is therefore a promising strategy for therapy. However, Hdm2 inhibitors do not prevent inhibition of p53 by HdmX, which impedes p53-mediated apoptosis. Here, we show that p53 reactivation by the small molecule RITA leads to efficient HdmX degradation in tumour cell lines of different origin and in xenograft tumours in vivo. Notably, HdmX degradation occurs selectively in cancer cells, but not in non-transformed cells. We identified the inhibition of the wild-type p53-induced phosphatase 1 (Wip1) as the major mechanism important for full engagement of p53 activity accomplished by restoration of the ataxia telangiectasia mutated (ATM) kinase-signalling cascade, which leads to HdmX degradation. In contrast to previously reported transactivation of Wip1 by p53, we observed p53-dependent repression of Wip1 expression, which disrupts the negative feedback loop conferred by Wip1. Our study reveals that the depletion of both HdmX and Wip1 potentiates cell death due to sustained activation of p53. Thus, RITA is an example of a p53-reactivating drug that not only blocks Hdm2, but also inhibits two important negative regulators of p53 - HdmX and Wip1, leading to efficient elimination of tumour cells.