Novel Treatments of Uveal Melanoma Identified with a Synthetic Lethal CRISPR/Cas9 Screen

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

A NF-ĸB-Activin A signaling axis enhances prostate cancer metastasis.

Metastasis is a main cause of death in prostate cancer (PCa). To dissect the molecular cues from cancer cell-microenvironment interaction that drive metastatic cascade, bone metastatic PCa cells were intravenously implanted into zebrafish embryos and mice tibia forming metastatic lesions. Transcriptomic analysis showed an elevated expression of stemness genes, pro-inflammatory cytokines and TGF-β family member Activin A in the cancer cells at metastatic onset in both animal models. Consistently, analysis of clinical datasets revealed that the expression of Activin A is specifically elevated in metastases and correlates with poor prognosis in stratified high-risk PCa patients. It is further unveiled that the microenvironment induced Activin A expression by NF-κB activation. The elevated level of Activin A enhanced the invasive ALDHhi CSC-like phenotypes and PCa proliferation by activation of Smad and ERK1/2 signaling driving metastasis. Suppression of Activin A or Activin receptor significantly reduced the CSC-like subpopulation, invasion, metastatic growth, and bone lesion formation in zebrafish and mice xenografts, suggesting a functional role of NF-κB-dependent Activin A in PCa metastasis. Overall, our study demonstrates that human PCa cells can display a comparable response with the microenvironment in zebrafish and mice xenografts. Combining both animal models, we uncovered the microenvironment-dependent activin signaling as an essential driver in PCa metastasis with therapeutic potential

Correlation of breast cancer susceptibility loci with patient characteristics, metastasis-free survival, and mRNA expression of the nearest genes

To understand the biology of low-risk breast cancer alleles, and to investigate whether these loci also contribute to disease progression that was once established, we examined the association of SNPs tagging the low-risk breast cancer loci in or near FGFR2, LSP1, MAP3K1, H19, TOX3, POU5F1P1, MYC, and 2q35, with clinical, pathological characteristics, prognosis, and mRNA expression of the nearest genes. Tumor DNA samples of 2,480 breast cancer patients were available. Out of this cohort, 1,290 patients with lymph-node negative disease who did not receive adjuvant systemic therapy, the SNP status was associated with metastasis-free survival (MFS). In 1,401 patients, the mRNA expression levels of FGFR2, LSP1, MAP3K1, H19, TOX3, POU5F1P1, and MYC were determined and correlated with SNP genotypes. The SNP rs2981582 in FGFR2 was significantly associated with positive ER and PgR status (P < 0.001 and P = 0.003, respectively). No other significant associations with patient or tumor characteristics were observed. Only rs2107425 near H19 was significantly associated with shorter MFS in uni- and multi-variate analysis (HR: 1.53, CI: 1.12-2.08, P = 0.006 and HR: 1.59, CI: 1.16-2.20, P = 0.004, respectively), with the more aggressive minor allele displaying a recessive trait. The minor allele of SNP rs3803662 located near the TOX3 gene was associated with lower mRNA expression of this gene. In conclusion, except for the association of rs13283662 with TOX3 gene expression indicating a tumor suppressor role of TOX3, our findings suggest that breast cancer low-risk loci generally do not affect expression of the nearest gene in breast tumor tissue. Also the prognosis of patients is largely not affected by low-risk breast cancer loci except for the SNP near H19. How, this SNP affects prognosis warrants further study as it does not operate through altering H19 mRNA expression

Zebrafish Patient-Derived Xenograft Model as a Preclinical Platform for Uveal Melanoma Drug Discovery

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

Patient-derived zebrafish xenografts of uveal melanoma reveal ferroptosis as a drug target

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

Overexpression of EZH2 in conjunctival melanoma offers a new therapeutic target

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

Innovative approaches to establish and characterize primary cultures: an ex vivo 3D system and the zebrafish model

Patient-derived specimens are an invaluable resource to investigate tumor biology. However, in vivo studies on primary cultures are often limited by the small amount of material available, while conventional in vitro systems might alter the features and behavior that characterize cancer cells. We present our data obtained on primary dedifferentiated liposarcoma cells cultured in a 3D scaffold-based system and injected into a zebrafish model. Primary cells were characterized in vitro for their morphological features, sensitivity to drugs and biomarker expression, and in vivo for their engraftment and invasiveness abilities. The 3D culture showed a higher enrichment in cancer cells than the standard monolayer culture and a better preservation of liposarcoma-associated markers. We also successfully grafted primary cells into zebrafish, showing their local migratory and invasive abilities. Our work provides proof of concept of the ability of 3D cultures to maintain the original phenotype of ex vivo cells, and highlights the potential of the zebrafish model to provide a versatile in vivo system for studies with limited biological material. Such models could be used in translational research studies for biomolecular analyses, drug screenings and tumor aggressiveness assays