Multicellular species environmental DNA (eDNA) research constrained by overfocus on mitochondrial DNA.

Environmental DNA (eDNA) is becoming an established tool across the biological and medical sciences. Despite the evident successes and wide adoption of eDNA approaches, some fundamental questions remain. For instance, there is almost a dogma in the field around the superiority of mitochondrial DNA for use in eDNA studies, however robust comparison with nuclear eDNA is widely lacking. The dominance of mitochondrial-based eDNA for animal and plant studies appears to be largely settled, despite a widespread lack of rigorous nuclear eDNA testing. Outside of the source organism the protections conferred on eDNA by the cell, mitochondrial and nuclear membranes are poorly understood, including the contribution of each to eDNA persistence and degradation. Utilizing shotgun sequencing to unbiasedly assess the level of nuclear and mitochondrial eDNA across samples, we reveal stark differences in nuclear versus mitochondrial eDNA persistence and abundance. By focusing too heavily on mitochondrial DNA alone the field is underutilizing eDNA's full potential.

Green Turtle Fibropapillomatosis: Tumor Morphology and Growth Rate in a Rehabilitation Setting.

Fibropapillomatosis (FP) is a neoplastic disease most often found in green turtles (Chelonia mydas). Afflicted turtles are burdened with potentially debilitating tumors concentrated externally on the soft tissues, plastron, and eyes and internally on the lungs, kidneys, and the heart. Clinical signs occur at various levels, ranging from mild disease to severe debilitation. Tumors can both progress and regress in affected turtles, with outcomes ranging from death due to the disease to complete regression. Since its official description in the scientific literature in 1938, tumor growth rates have been rarely documented. In addition, FP tumors come in two very different morphologies; yet, to our knowledge, there have been no quantified differences in growth rates between tumor types. FP tumors are often rugose in texture, with a polypoid to papillomatous morphology, and may or may not be pedunculated. In other cases, tumors are smooth, with a skin-like surface texture and little to no papillose structures. In our study, we assessed growth-rate differences between rugose and smooth tumor morphologies in a rehabilitation setting. We measured average biweekly tumor growth over time in green turtles undergoing rehabilitation at the University of Florida Whitney Laboratory Sea Turtle Hospital in St. Augustine, Florida, and compared growth between rugose and smooth tumors. Our results demonstrate that both rugose and smooth tumors follow a similar active growth progression pattern, but rugose tumors grew at significantly faster rates (p = 0.013) than smooth ones. We also documented regression across several examined tumors, ranging from -0.19% up to -10.8% average biweekly negative growth. Our study offers a first-ever assessment of differential growth between tumor morphologies and an additional diagnostic feature that may lead to a more comprehensive understanding and treatment of the disease. We support the importance of tumor morphological categorization (rugose versus smooth) being documented in future FP hospital- and field-based health assessments.

Inadvertent human genomic bycatch and intentional capture raise beneficial applications and ethical concerns with environmental DNA.

The field of environmental DNA (eDNA) is advancing rapidly, yet human eDNA applications remain underutilized and underconsidered. Broader adoption of eDNA analysis will produce many well-recognized benefits for pathogen surveillance, biodiversity monitoring, endangered and invasive species detection, and population genetics. Here we show that deep-sequencing-based eDNA approaches capture genomic information from humans (Homo sapiens) just as readily as that from the intended target species. We term this phenomenon human genetic bycatch (HGB). Additionally, high-quality human eDNA could be intentionally recovered from environmental substrates (water, sand and air), holding promise for beneficial medical, forensic and environmental applications. However, this also raises ethical dilemmas, from consent, privacy and surveillance to data ownership, requiring further consideration and potentially novel regulation. We present evidence that human eDNA is readily detectable from 'wildlife' environmental samples as human genetic bycatch, demonstrate that identifiable human DNA can be intentionally recovered from human-focused environmental sampling and discuss the translational and ethical implications of such findings.

Divergent sensory and immune gene evolution in sea turtles with contrasting demographic and life histories.

Sea turtles represent an ancient lineage of marine vertebrates that evolved from terrestrial ancestors over 100 Mya. The genomic basis of the unique physiological and ecological traits enabling these species to thrive in diverse marine habitats remains largely unknown. Additionally, many populations have drastically declined due to anthropogenic activities over the past two centuries, and their recovery is a high global conservation priority. We generated and analyzed high-quality reference genomes for the leatherback (Dermochelys coriacea) and green (Chelonia mydas) turtles, representing the two extant sea turtle families. These genomes are highly syntenic and homologous, but localized regions of noncollinearity were associated with higher copy numbers of immune, zinc-finger, and olfactory receptor (OR) genes in green turtles, with ORs related to waterborne odorants greatly expanded in green turtles. Our findings suggest that divergent evolution of these key gene families may underlie immunological and sensory adaptations assisting navigation, occupancy of neritic versus pelagic environments, and diet specialization. Reduced collinearity was especially prevalent in microchromosomes, with greater gene content, heterozygosity, and genetic distances between species, supporting their critical role in vertebrate evolutionary adaptation. Finally, diversity and demographic histories starkly contrasted between species, indicating that leatherback turtles have had a low yet stable effective population size, exhibit extremely low diversity compared with other reptiles, and harbor a higher genetic load compared with green turtles, reinforcing concern over their persistence under future climate scenarios. These genomes provide invaluable resources for advancing our understanding of evolution and conservation best practices in an imperiled vertebrate lineage.

Proteasomal down-regulation of the proapoptotic MST2 pathway contributes to BRAF inhibitor resistance in melanoma.

The RAS-RAF-MEK-ERK pathway is hyperactivated in most malignant melanomas, and mutations in BRAF or NRAS account for most of these cases. BRAF inhibitors (BRAFi) are highly efficient for treating patients with BRAFV600E mutations, but tumours frequently acquire resistance within a few months. Multiple resistance mechanisms have been identified, due to mutations or network adaptations that revive ERK signalling. We have previously shown that RAF proteins inhibit the MST2 proapoptotic pathway in a kinase-independent fashion. Here, we have investigated the role of the MST2 pathway in mediating resistance to BRAFi. We show that the BRAFV600E mutant protein, but not the wild-type BRAF protein, binds to MST2 inhibiting its proapoptotic signalling. Down-regulation of MST2 reduces BRAFi-induced apoptosis. In BRAFi-resistant cell lines, MST2 pathway proteins are down-regulated by ubiquitination and subsequent proteasomal degradation rendering cells refractory to MST2 pathway-induced apoptosis. Restoration of apoptosis can be achieved by increasing MST2 pathway protein expression using proteasome inhibitors. In summary, we show that the MST2 pathway plays a role in the acquisition of BRAFi resistance in melanoma.

Detection and population genomics of sea turtle species via noninvasive environmental DNA analysis of nesting beach sand tracks and oceanic water.

Elusive aquatic wildlife, such as endangered sea turtles, are difficult to monitor and conserve. As novel molecular and genetic technologies develop, it is possible to adapt and optimize them for wildlife conservation. One such technology is environmental (e)DNA - the detection of DNA shed from organisms into their surrounding environments. We developed species-specific green (Chelonia mydas) and loggerhead (Caretta caretta) sea turtle probe-based qPCR assays, which can detect and quantify sea turtle eDNA in controlled (captive tank water and sand samples) and free ranging (oceanic water samples and nesting beach sand) settings. eDNA detection complemented traditional in-water sea turtle monitoring by enabling detection even when turtles were not visually observed. Furthermore, we report that high throughput shotgun sequencing of eDNA sand samples enabled sea turtle population genetic studies and pathogen monitoring, demonstrating that noninvasive eDNA techniques are viable and efficient alternatives to biological sampling (e.g., biopsies and blood draws). Genetic information was obtained from sand many hours after nesting events, without having to observe or interact with the target individual. This greatly reduces the sampling stress experienced by nesting mothers and emerging hatchlings, and avoids sacrificing viable eggs for genetic analysis. The detection of pathogens from sand indicates significant potential for increased wildlife disease monitoring capacity and viral variant surveillance. Together, these results demonstrate the potential of eDNA approaches to ultimately help understand and conserve threatened species such as sea turtles.

Fibropapillomatosis and Chelonid Alphaherpesvirus 5 Infection in Kemp's Ridley Sea Turtles (Lepidochelys kempii).

Fibropapillomatosis (FP), a debilitating, infectious neoplastic disease, is rarely reported in endangered Kemp's ridley sea turtles (Lepidochelys kempii). With this study, we describe FP and the associated chelonid alphaherpesvirus 5 (ChHV5) in Kemp's ridley turtles encountered in the United States during 2006-2020. Analysis of 22 case reports of Kemp's ridley turtles with FP revealed that while the disease was mild in most cases, 54.5% were adult turtles, a reproductively valuable age class whose survival is a priority for population recovery. Of 51 blood samples from tumor-free turtles and 12 tumor samples from turtles with FP, 7.8% and 91.7%, respectively, tested positive for ChHV5 DNA via quantitative polymerase chain reaction (qPCR). Viral genome shotgun sequencing and phylogenetic analysis of six tumor samples show that ChHV5 sequences in Kemp's ridley turtles encountered in the Gulf of Mexico and northwestern Atlantic cluster with ChHV5 sequences identified in green (Chelonia mydas) and loggerhead (Caretta caretta) sea turtles from Hawaii, the southwestern Atlantic Ocean, and the Caribbean. Results suggest an interspecific, spatiotemporal spread of FP among Kemp's ridley turtles in regions where the disease is enzootic. Although FP is currently uncommon in this species, it remains a health concern due to its uncertain pathogenesis and potential relationship with habitat degradation.

Genotype data not consistent with clonal transmission of sea turtle fibropapillomatosis or goldfish schwannoma.

Recent discoveries of transmissible cancers in multiple bivalve species suggest that direct transmission of cancer cells within species may be more common than previously thought, particularly in aquatic environments. Fibropapillomatosis occurs with high prevalence in green sea turtles ( Chelonia mydas) and the geographic range of disease has increased since fibropapillomatosis was first reported in this species. Widespread incidence of schwannomas, benign tumours of Schwann cell origin, reported in aquarium-bred goldfish (Carassius auratus), suggest an infectious aetiology. We investigated the hypothesis that cancers in these species arise by clonal transmission of cancer cells. Through analysis of polymorphic microsatellite alleles, we demonstrate concordance of host and tumour genotypes in diseased animals. These results imply that the tumours examined arose from independent oncogenic transformation of host tissue and were not clonally transmitted. Further, failure to experimentally transmit goldfish schwannoma via water exposure or inoculation suggest that this disease is unlikely to have an infectious aetiology.

Evolutionary Comparisons of Chelonid Alphaherpesvirus 5 (ChHV5) Genomes from Fibropapillomatosis-Afflicted Green (Chelonia mydas), Olive Ridley (Lepidochelys olivacea) and Kemp's Ridley (Lepidochelys kempii) Sea Turtles.

The spreading global sea turtle fibropapillomatosis (FP) epizootic is threatening some of Earth's ancient reptiles, adding to the plethora of threats faced by these keystone species. Understanding this neoplastic disease and its likely aetiological pathogen, chelonid alphaherpesvirus 5 (ChHV5), is crucial to understand how the disease impacts sea turtle populations and species and the future trajectory of disease incidence. We generated 20 ChHV5 genomes, from three sea turtle species, to better understand the viral variant diversity and gene evolution of this oncogenic virus. We revealed previously underappreciated genetic diversity within this virus (with an average of 2035 single nucleotide polymorphisms (SNPs), 1.54% of the ChHV5 genome) and identified genes under the strongest evolutionary pressure. Furthermore, we investigated the phylogeny of ChHV5 at both genome and gene level, confirming the propensity of the virus to be interspecific, with related variants able to infect multiple sea turtle species. Finally, we revealed unexpected intra-host diversity, with up to 0.15% of the viral genome varying between ChHV5 genomes isolated from different tumours concurrently arising within the same individual. These findings offer important insights into ChHV5 biology and provide genomic resources for this oncogenic virus.

A Chemo-Genomic Approach Identifies Diverse Epigenetic Therapeutic Vulnerabilities in MYCN-Amplified Neuroblastoma.

Although a rare disease, neuroblastoma accounts for the highest proportion of childhood cancer deaths. There is a lack of recurrent somatic mutations in neuroblastoma embryonal tumours, suggesting a possible role for epigenetic alterations in driving this cancer. While an increasing number of reports suggest an association of MYCN with epigenetic machinery, the mechanisms of these interactions are poorly understood in the neuroblastoma setting. Utilising chemo-genomic approaches we revealed global MYCN-epigenetic interactions and identified numerous epigenetic proteins as MYCN targets. The epigenetic regulators HDAC2, CBX8 and CBP (CREBBP) were all MYCN target genes and also putative MYCN interactors. MYCN-related epigenetic genes included SMARCs, HDACs, SMYDs, BRDs and CREBBP. Expression levels of the majority of MYCN-related epigenetic genes showed predictive ability for neuroblastoma patient outcome. Furthermore, a compound library screen targeting epigenetic proteins revealed broad susceptibility of neuroblastoma cells to all classes of epigenetic regulators, belonging to families of bromodomains, HDACs, HATs, histone methyltransferases, DNA methyltransferases and lysin demethylases. Ninety-six percent of the compounds reduced MYCN-amplified neuroblastoma cell viability. We show that the C646 (CBP-bromodomain targeting compound) exhibits switch-like temporal and dose response behaviour and is effective at reducing neuroblastoma viability. Responsiveness correlates with MYCN expression, with MYCN-amplified cells being more susceptible to C646 treatment. Thus, exploiting the broad vulnerability of neuroblastoma cells to epigenetic targeting compounds represents an exciting strategy in neuroblastoma treatment, particularly for high-risk MYCN-amplified tumours.

Environmental DNA monitoring of oncogenic viral shedding and genomic profiling of sea turtle fibropapillomatosis reveals unusual viral dynamics.

Pathogen-induced cancers account for 15% of human tumors and are a growing concern for endangered wildlife. Fibropapillomatosis is an expanding virally and environmentally co-induced sea turtle tumor epizootic. Chelonid herpesvirus 5 (ChHV5) is implicated as a causative virus, but its transmission method and specific role in oncogenesis and progression is unclear. We applied environmental (e)DNA-based viral monitoring to assess viral shedding as a direct means of transmission, and the relationship between tumor burden, surgical resection and ChHV5 shedding. To elucidate the abundance and transcriptional status of ChHV5 across early, established, regrowth and internal tumors we conducted genomics and transcriptomics. We determined that ChHV5 is shed into the water column, representing a likely transmission route, and revealed novel temporal shedding dynamics and tumor burden correlations. ChHV5 was more abundant in the water column than in marine leeches. We also revealed that ChHV5 is latent in fibropapillomatosis, including early stage, regrowth and internal tumors; higher viral transcription is not indicative of poor patient outcome, and high ChHV5 loads predominantly arise from latent virus. These results expand our knowledge of the cellular and shedding dynamics of ChHV5 and can provide insights into temporal transmission dynamics and viral oncogenesis not readily investigable in tumors of terrestrial species.

Molecular characterization of a marine turtle tumor epizootic, profiling external, internal and postsurgical regrowth tumors.

Sea turtle populations are under threat from an epizootic tumor disease (animal epidemic) known as fibropapillomatosis. Fibropapillomatosis continues to spread geographically, with prevalence of the disease also growing at many longer-affected sites globally. However, we do not yet understand the precise environmental, mutational and viral events driving fibropapillomatosis tumor formation and progression.Here we perform transcriptomic and immunohistochemical profiling of five fibropapillomatosis tumor types: external new, established and postsurgical regrowth tumors, and internal lung and kidney tumors. We reveal that internal tumors are molecularly distinct from the more common external tumors. However, they have a small number of conserved potentially therapeutically targetable molecular vulnerabilities in common, such as the MAPK, Wnt, TGFß and TNF oncogenic signaling pathways. These conserved oncogenic drivers recapitulate remarkably well the core pan-cancer drivers responsible for human cancers. Fibropapillomatosis has been considered benign, but metastatic-related transcriptional signatures are strongly activated in kidney and established external tumors. Tumors in turtles with poor outcomes (died/euthanized) have genes associated with apoptosis and immune function suppressed, with these genes providing putative predictive biomarkers.Together, these results offer an improved understanding of fibropapillomatosis tumorigenesis and provide insights into the origins, inter-tumor relationships, and therapeutic treatment for this wildlife epizootic.

Perspectives on the expansion of human precision oncology and genomic approaches to sea turtle fibropapillomatosis.

Our recent Communications Biology research article revealed the genomic drivers and therapeutic vulnerabilities of sea turtle fibropapillomatosis tumors. Fibropapillomatosis is a debilitating tumorous disease afflicting populations of green sea turtles globally. While a virus is involved in the development of this disease, it is increasingly understood that the key trigger is linked to anthropogenic disturbances of the environment. The specific environmental co-trigger(s) has yet to be functionally confirmed. Here we outline the next steps required to advance our understanding of this enigmatic disease, to enable us to more effectively clinically combat it and to ultimately tackle its environmental co-trigger to halt and hopefully reverse the spread of fibropapillomatosis.

Sea turtle fibropapilloma tumors share genomic drivers and therapeutic vulnerabilities with human cancers.

Wildlife populations are under intense anthropogenic pressures, with the geographic range of many species shrinking, dramatic reductions in population numbers and undisturbed habitats, and biodiversity loss. It is postulated that we are in the midst of a sixth (Anthropocene) mass extinction event, the first to be induced by human activity. Further, threatening vulnerable species is the increased rate of emerging diseases, another consequence of anthropogenic activities. Innovative approaches are required to help maintain healthy populations until the chronic underlying causes of these issues can be addressed. Fibropapillomatosis in sea turtles is one such wildlife disease. Here, we applied precision-medicine-based approaches to profile fibropapillomatosis tumors to better understand their biology, identify novel therapeutics, and gain insights into viral and environmental triggers for fibropapillomatosis. We show that fibropapillomatosis tumors share genetic vulnerabilities with human cancer types, revealing that they are amenable to treatment with human anti-cancer therapeutics.

A Brain-Derived Neurotrophic Factor Mimetic Is Sufficient to Restore Cone Photoreceptor Visual Function in an Inherited Blindness Model.

Controversially, histone deacetylase inhibitors (HDACi) are in clinical trial for the treatment of inherited retinal degeneration. Utilizing the zebrafish dye ucd6 model, we determined if treatment with HDACi can rescue cone photoreceptor-mediated visual function. dye exhibit defective visual behaviour and retinal morphology including ciliary marginal zone (CMZ) cell death and decreased photoreceptor outer segment (OS) length, as well as gross morphological defects including hypopigmentation and pericardial oedema. HDACi treatment of dye results in significantly improved optokinetic (OKR) (~43 fold, p < 0.001) and visualmotor (VMR) (~3 fold, p < 0.05) responses. HDACi treatment rescued gross morphological defects and reduced CMZ cell death by 80%. Proteomic analysis of dye eye extracts suggested BDNF-TrkB and Akt signaling as mediators of HDACi rescue in our dataset. Co-treatment with the TrkB antagonist ANA-12 blocked HDACi rescue of visual function and associated Akt phosphorylation. Notably, sole treatment with a BDNF mimetic, 7,8-dihydroxyflavone hydrate, significantly rescued dye visual function (~58 fold increase in OKR, p < 0.001, ~3 fold increase in VMR, p < 0.05). In summary, HDACi and a BDNF mimetic are sufficient to rescue retinal cell death and visual function in a vertebrate model of inherited blindness.

Retinoic acid and TGF-ß signalling cooperate to overcome MYCN-induced retinoid resistance.

BACKGROUND: Retinoid therapy is widely employed in clinical oncology to differentiate malignant cells into their more benign counterparts. However, certain high-risk cohorts, such as patients with MYCN-amplified neuroblastoma, are innately resistant to retinoid therapy. Therefore, we employed a precision medicine approach to globally profile the retinoid signalling response and to determine how an excess of cellular MYCN antagonises these signalling events to prevent differentiation and confer resistance. METHODS: We applied RNA sequencing (RNA-seq) and interaction proteomics coupled with network-based systems level analysis to identify targetable vulnerabilities of MYCN-mediated retinoid resistance. We altered MYCN expression levels in a MYCN-inducible neuroblastoma cell line to facilitate or block retinoic acid (RA)-mediated neuronal differentiation. The relevance of differentially expressed genes and transcriptional regulators for neuroblastoma outcome were then confirmed using existing patient microarray datasets. RESULTS: We determined the signalling networks through which RA mediates neuroblastoma differentiation and the inhibitory perturbations to these networks upon MYCN overexpression. We revealed opposing regulation of RA and MYCN on a number of differentiation-relevant genes, including LMO4, CYP26A1, ASCL1, RET, FZD7 and DKK1. Furthermore, we revealed a broad network of transcriptional regulators involved in regulating retinoid responsiveness, such as Neurotrophin, PI3K, Wnt and MAPK, and epigenetic signalling. Of these regulators, we functionally confirmed that MYCN-driven inhibition of transforming growth factor beta (TGF-ß) signalling is a vulnerable node of the MYCN network and that multiple levels of cross-talk exist between MYCN and TGF-ß. Co-targeting of the retinoic acid and TGF-ß pathways, through RA and kartogenin (KGN; a TGF-ß signalling activating small molecule) combination treatment, induced the loss of viability of MYCN-amplified retinoid-resistant neuroblastoma cells. CONCLUSIONS: Our approach provides a powerful precision oncology tool for identifying the driving signalling networks for malignancies not primarily driven by somatic mutations, such as paediatric cancers. By applying global omics approaches to the signalling networks regulating neuroblastoma differentiation and stemness, we have determined the pathways involved in the MYCN-mediated retinoid resistance, with TGF-ß signalling being a key regulator. These findings revealed a number of combination treatments likely to improve clinical response to retinoid therapy, including co-treatment with retinoids and KGN, which may prove valuable in the treatment of high-risk MYCN-amplified neuroblastoma.

Precision wildlife medicine: applications of the human-centred precision medicine revolution to species conservation.

The current species extinction crisis is being exacerbated by an increased rate of emergence of epizootic disease. Human-induced factors including habitat degradation, loss of biodiversity and wildlife population reductions resulting in reduced genetic variation are accelerating disease emergence. Novel, efficient and effective approaches are required to combat these epizootic events. Here, we present the case for the application of human precision medicine approaches to wildlife medicine in order to enhance species conservation efforts. We consider how the precision medicine revolution, coupled with the advances made in genomics, may provide a powerful and feasible approach to identifying and treating wildlife diseases in a targeted, effective and streamlined manner. A number of case studies of threatened species are presented which demonstrate the applicability of precision medicine to wildlife conservation, including sea turtles, amphibians and Tasmanian devils. These examples show how species conservation could be improved by using precision medicine techniques to determine novel treatments and management strategies for the specific medical conditions hampering efforts to restore population levels. Additionally, a precision medicine approach to wildlife health has in turn the potential to provide deeper insights into human health and the possibility of stemming and alleviating the impacts of zoonotic diseases. The integration of the currently emerging Precision Medicine Initiative with the concepts of EcoHealth (aiming for sustainable health of people, animals and ecosystems through transdisciplinary action research) and One Health (recognizing the intimate connection of humans, animal and ecosystem health and addressing a wide range of risks at the animal-human-ecosystem interface through a coordinated, collaborative, interdisciplinary approach) has great potential to deliver a deeper and broader interdisciplinary-based understanding of both wildlife and human diseases.

Integrative Genome-Scale Analysis Identifies Epigenetic Mechanisms of Transcriptional Deregulation in Unfavorable Neuroblastomas.

The broad clinical spectrum of neuroblastoma ranges from spontaneous regression to rapid progression despite intensive multimodal therapy. This diversity is not fully explained by known genetic aberrations, suggesting the possibility of epigenetic involvement in pathogenesis. In pursuit of this hypothesis, we took an integrative approach to analyze the methylomes, transcriptomes, and copy number variations in 105 cases of neuroblastoma, complemented by primary tumor- and cell line-derived global histone modification analyses and epigenetic drug treatment in vitro We found that DNA methylation patterns identify divergent patient subgroups with respect to survival and clinicobiologic variables, including amplified MYCN Transcriptome integration and histone modification-based definition of enhancer elements revealed intragenic enhancer methylation as a mechanism for high-risk-associated transcriptional deregulation. Furthermore, in high-risk neuroblastomas, we obtained evidence for cooperation between PRC2 activity and DNA methylation in blocking tumor-suppressive differentiation programs. Notably, these programs could be re-activated by combination treatments, which targeted both PRC2 and DNA methylation. Overall, our results illuminate how epigenetic deregulation contributes to neuroblastoma pathogenesis, with novel implications for its diagnosis and therapy. Cancer Res; 76(18); 5523-37. ©2016 AACR.

Wnt signalling is a bi-directional vulnerability of cancer cells.

Wnt signalling is involved in the formation, metastasis and relapse of a wide array of cancers. However, there is ongoing debate as to whether activation or inhibition of the pathway holds the most promise as a therapeutic treatment for cancer, with conflicting evidence from a variety of tumour types. We show that Wnt/ß-catenin signalling is a bi-directional vulnerability of neuroblastoma, malignant melanoma and colorectal cancer, with hyper-activation or repression of the pathway both representing a promising therapeutic strategy, even within the same cancer type. Hyper-activation directs cancer cells to undergo apoptosis, even in cells oncogenically driven by ß-catenin. Wnt inhibition blocks proliferation of cancer cells and promotes neuroblastoma differentiation. Wnt and retinoic acid co-treatments synergise, representing a promising combination treatment for MYCN-amplified neuroblastoma. Additionally, we report novel cross-talks between MYCN and ß-catenin signalling, which repress normal ß-catenin mediated transcriptional regulation. A ß-catenin target gene signature could predict patient outcome, as could the expression level of its DNA binding partners, the TCF/LEFs. This ß-catenin signature provides a tool to identify neuroblastoma patients likely to benefit from Wnt-directed therapy. Taken together, we show that Wnt/ß-catenin signalling is a bi-directional vulnerability of a number of cancer entities, and potentially a more broadly conserved feature of malignant cells.