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It is unclear if different genetic drivers in papillary thyroid cancer (PTC) confer different phenotypic tumor behavior leading to more aggressive disease. We hypothesized that RET-driven cancers are more aggressive.We reviewed records of consecutive patients treated for newly diagnosed PTC at this single institution from 2015 to 2016. Tumor samples from these patients were genotyped to identify RET-translocated, BRAFOf the 327 patients who underwent initial surgery for PTC during the study period, 192 (58.7%) had BRAFPatients with RET-driven PTCs had higher rates of extrathyroidal extension, multifocal disease, and distant metastases than patients whose tumors had BRAF

Background Characterization of circulating tumor DNA (ctDNA) has been integrated into clinical practice. Although labs have standardized validation procedures to develop single locus tests, the efficacy of on‐site plasma‐based next‐generation sequencing (NGS) assays still needs to be proved. Materials and Methods In this retrospective study, we profiled DNA from matched tissue and plasma samples from 75 patients with cancer. We applied an NGS test that detects clinically relevant alterations in 33 genes and microsatellite instability (MSI) to analyze plasma cell‐free DNA (cfDNA). Results The concordance between alterations detected in both tissue and plasma samples was higher in patients with metastatic disease. The NGS test detected 77% of sequence alterations, amplifications, and fusions that were found in metastatic samples compared with 45% of those alterations found in the primary tumor samples (p = .00005). There was 87% agreement on MSI status between the NGS test and tumor tissue results. In three patients, MSI‐high ctDNA correlated with response to immunotherapy. In addition, the NGS test revealed an FGFR2 amplification that was not detected in tumor tissue from a patient with metastatic gastric cancer, emphasizing the importance of profiling plasma samples in patients with advanced cancer. Conclusion Our validation experience of a plasma‐based NGS assay advances current knowledge about translating cfDNA testing into clinical practice and supports the application of plasma assays in the management of oncology patients with metastatic disease. With an in‐house method that minimizes the need for invasive procedures, on‐site cfDNA testing supplements tissue biopsy to guide precision therapy and is entitled to become a routine practice. Implications for Practice This study proposes a solution for decentralized liquid biopsy testing based on validation of a next‐generation sequencing (NGS) test that detects four classes of genomic alterations in blood: sequence mutations (single nucleotide substitutions or insertions and deletions), fusions, amplifications, and microsatellite instability (MSI). Although there are reference labs that perform single‐site comprehensive liquid biopsy testing, the targeted assay this study validated can be established locally in any lab with capacity to offer clinical molecular pathology assays. To the authors' knowledge, this is the first report that validates evaluating an on‐site plasma‐based NGS test that detects the MSI status along with common sequence alterations encountered in solid tumors., This report describes the concordance between circulating tumor DNA and matched tumor tissue molecular profiles, discusses interpretation of observed discordant data, and illustrates applications through clinical cases. It is the first known report to evaluate the performance of an on‐site plasma‐based next‐generation sequencing test to detect microsatellite instability status along with common sequence alterations in the context of its clinical utility and therapeutic applications in precision oncology.

Prenatal alcohol exposure (PAE) affects at least 10% of newborns globally and leads to the development of fetal alcohol spectrum disorders (FASDs). Despite its high incidence, there is no consensus on the implications of PAE on metabolic disease risk in adults. Here, we describe a cohort of adults with FASDs that had an increased incidence of metabolic abnormalities, including type 2 diabetes, low HDL, high triglycerides, and female-specific overweight and obesity. Using a zebrafish model for PAE, we performed population studies to elucidate the metabolic disease seen in the clinical cohort. Embryonic alcohol exposure (EAE) in male zebrafish increased the propensity for diet-induced obesity and fasting hyperglycemia in adulthood. We identified several consequences of EAE that may contribute to these phenotypes, including a reduction in adult locomotor activity, alterations in visceral adipose tissue and hepatic development, and persistent diet-responsive transcriptional changes. Taken together, our findings define metabolic vulnerabilities due to EAE and provide evidence that behavioral changes and primary organ dysfunction contribute to resultant metabolic abnormalities.

Hepatic cysts are fluid-filled lesions in the liver that are estimated to occur in 5% of the population. They may cause hepatomegaly and abdominal pain. Progression to secondary fibrosis, cirrhosis, or cholangiocarcinoma can lead to morbidity and mortality. Previous studies of patients and rodent models have associated hepatic cyst formation with increased proliferation and fluid secretion in cholangiocytes, which are partially due to impaired primary cilia. Congenital hepatic cysts are thought to originate from faulty bile duct development, but the underlying mechanisms are not fully understood. In a forward genetic screen, we identified a zebrafish mutant that develops hepatic cysts during larval stages. Cyst formation in these mutants is not due to changes in biliary cell proliferation, bile secretion, or impairment of primary cilia. Instead, time-lapse live imaging data showed that the mutant biliary cells failed to form interconnecting bile ducts because of defects in motility and protrusive activity. Accordingly, immunostaining revealed an excessive and disorganized actin and microtubule cytoskeleton in the mutant biliary cells. By whole-genome sequencing, we determined that the cystic phenotype in the mutant was caused by a missense mutation in the furinb gene which encodes a proprotein convertase. The mutation alters Furinb localization and causes endoplasmic reticulum (ER) stress. The cystic phenotype could be suppressed by treatment with the ER stress inhibitor 4-phenylbutyric acid and exacerbated by treatment with the ER stress inducer tunicamycin. The mutant livers also exhibited increased mTOR signaling and treatment with the mTOR inhibitor rapamycin partially blocked cyst formation by reducing ER stress. Our study has established a novel vertebrate model for studying hepatic cystogenesis and illustrated the role of ER stress in the disease pathogenesis.

Genetic alterations in RET lead to activation of ERK and AKT signaling and are associated with hereditary and sporadic thyroid cancer and lung cancer. Highly selective RET inhibitors have recently entered clinical use after demonstrating efficacy in treating patients with diverse tumor types harboring RET gene rearrangements or activating mutations. In order to understand resistance mechanisms arising after treatment with RET inhibitors, we performed a comprehensive molecular and genomic analysis of a patient with RET-rearranged thyroid cancer. Using a combination of drug screening and proteomic and biochemical profiling, we identified an adaptive resistance to RET inhibitors that reactivates ERK signaling within hours of drug exposure. We found that activation of FGFR signaling is a mechanism of adaptive resistance to RET inhibitors that activates ERK signaling. Combined inhibition of FGFR and RET prevented the development of adaptive resistance to RET inhibitors, reduced cell viability, and decreased tumor growth in cellular and animal models of CCDC6-RET–rearranged thyroid cancer.

Oncogenomic studies indicate that copy number variation (CNV) alters genes involved in tumor progression; however, identification of specific driver genes affected by CNV has been difficult, as these rearrangements are often contained in large chromosomal intervals among several bystander genes. Here, we addressed this problem and identified a CNV-targeted oncogene by performing comparative oncogenomics of human and zebrafish melanomas. We determined that the gene encoding growth differentiation factor 6 (GDF6), which is the ligand for the BMP family, is recurrently amplified and transcriptionally upregulated in melanoma. GDF6-induced BMP signaling maintained a trunk neural crest gene signature in melanomas. Additionally, GDF6 repressed the melanocyte differentiation gene MITF and the proapoptotic factor SOX9, thereby preventing differentiation, inhibiting cell death, and promoting tumor growth. GDF6 was specifically expressed in melanomas but not melanocytes. Moreover, GDF6 expression levels in melanomas were inversely correlated with patient survival. Our study has identified a fundamental role for GDF6 and BMP signaling in governing an embryonic cell gene signature to promote melanoma progression, thus providing potential opportunities for targeted therapy to treat GDF6-positive cancers.

Kinase inhibitors are a critical tool for cancer treatment, but their efficacy is limited by resistance mechanisms. In thyroid and lung cancer RET gene rearrangements result in constitutive MAPK pathway activation and drive malignancy. While treatment with new selective RET inhibitors has been associated with significant clinical responses, a majority of patients experience a partial response or disease stabilization as their best clinical outcome. Resistance to RET inhibitors has emerged as a clinical problem requiring new treatment strategies. Using a combination of drug screening, proteomic, and biochemical profiling we identified a strategy to overcome adaptive resistance to RET inhibitors in human thyroid cancer cell lines and vertebrate animal models. The identification of alternative signaling pathways that reactivates ERK signaling as a mechanism of resistance to RET inhibitors provides an opportunity to anticipate resistance to selective RET inhibitors and use combination therapy that leads to more significant and durable anti-tumor responses in patients with RET rearranged cancers. Citation Format: Renuka Raman, Jacques Villefranc, Timothy Ullmann, Jessica Thiesmeyer, Viviana Anelli, Chantal Pauli, Rohan Bareja, Kenneth Wha Eng, Princesca Dorsaint, David Wilkes, Shaham Beg, Reid Shaw, Michael Churchill, Naveen Gumpeni, Theresa Scognamiglio, Mark Rubin, Carla Grandori, Juan Mosquera, Juan Mosquera, Juan Mosquera, Noah Dephoure, Andrea Sboner, Olivier Elemento, Yariv Houvras. Uncovering the mechanism of adaptive resistance to RET inhibitors in RET rearranged thyroid cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1434.

Kasheta et al. report the identification of T reg–like cells in zebrafish, a means to track and live-image these cells, and foxp3a-deficient mutants that display lymphoproliferation, severe inflammation, and other hallmarks of T reg deficiency syndromes., Regulatory T (T reg) cells are a specialized sublineage of T lymphocytes that suppress autoreactive T cells. Functional studies of T reg cells in vitro have defined multiple suppression mechanisms, and studies of T reg–deficient humans and mice have made clear the important role that these cells play in preventing autoimmunity. However, many questions remain about how T reg cells act in vivo. Specifically, it is not clear which suppression mechanisms are most important, where T reg cells act, and how they get there. To begin to address these issues, we sought to identify T reg cells in zebrafish, a model system that provides unparalleled advantages in live-cell imaging and high-throughput genetic analyses. Using a FOXP3 orthologue as a marker, we identified CD4-enriched, mature T lymphocytes with properties of T reg cells. Zebrafish mutant for foxp3a displayed excess T lymphocytes, splenomegaly, and a profound inflammatory phenotype that was suppressed by genetic ablation of lymphocytes. This study identifies T reg–like cells in zebrafish, providing both a model to study the normal functions of these cells in vivo and mutants to explore the consequences of their loss.

Pancreatic ductal adenocarcinoma is an aggressive cancer with limited treatment options(1). Approximately 10% of cases exhibit familial predisposition, but causative genes are not known in most families(2). We perform whole-genome sequence analysis in a family with multiple cases of pancreatic ductal adenocarcinoma and identify a germline truncating mutation in the member of the RAS oncogene family-like (3) (RABL3) gene. Heterozygous rabl3 mutant zebrafish show increased susceptibility to cancer formation. Transcriptomic and mass spectrometry approaches implicate RABL3 in RAS pathway regulation and identify an interaction with RAP1GDS1 (SmgGDS), a chaperone regulating prenylation of RAS GTPases(3). Indeed, the truncated mutant RABL3 protein accelerates KRAS prenylation and requires RAS proteins to promote cell proliferation. Finally, evidence in patient cohorts with developmental disorders implicates germline RABL3 mutations in RASopathy syndromes. Our studies identify RABL3 mutations as a target for genetic testing in cancer families and uncover a mechanism for dysregulated RAS activity in development and cancer.

Dermal invasion is a hallmark of malignant melanoma. Although the molecular alterations that drive the progression of primary melanoma to metastatic disease have been studied extensively, the early progression of noninvasive primary melanoma to an invasive state is poorly understood. To elucidate the mechanisms underlying the transition from radial to vertical growth, the first step in melanoma invasion, we developed a zebrafish melanoma model in which constitutive activation of ribosomal protein S6 kinase A1 drives tumor invasion. Transcriptomic analysis of ribosomal protein S6 kinase A1–activated tumors identified metabolic changes, including up-regulation of genes associated with oxidative phosphorylation. Vertical growth phase human melanoma cells show higher oxygen consumption and preferential utilization of glutamine compared to radial growth phase melanoma cells. Peroxisome proliferator activated receptor γ coactivator (PGC)-1α, has been proposed as a master regulator of tumor oxidative phosphorylation. In human primary melanoma specimens, PGC1α protein expression was found to be positively associated with increased tumor thickness and expression of the proliferative marker Ki-67 and the reactive oxygen species scavenger receptor class A member 3. PGC1α depletion modulated cellular processes associated with primary melanoma growth and invasion, including oxidative stress. These results support a role for PGC1α in mediating glutamine-driven oxidative phosphorylation to facilitate the invasive growth of primary melanoma.

9089 Background: RET fusions are targetable oncogenic drivers in 1–2% of non-small cell lung cancer (NSCLC). ARROW (NCT03037385) supported the US FDA approval of pralsetinib, a highly potent oral selective RET inhibitor for RET-altered NSCLC and thyroid cancer. Here, we present updated results for a larger population of patients with RET fusion–positive NSCLC enrolled in ARROW. Methods: ARROW is a phase 1/2 open-label study conducted at 84 sites in 13 countries. Phase 2 expansion cohorts included patients with RET fusion–positive NSCLC. Initially, all treatment-naïve patients were not candidates for platinum-based therapy, a requirement removed by protocol amendment in July 2019. Primary objectives are overall response rate (ORR; blinded independent central review [BICR] per RECIST v1.1), assessed for patients with baseline measurable disease, and safety. Results: Updated analyses were completed as of Nov 6, 2020 (data cut-off), for patients who initiated pralsetinib 400 mg QD by May 22, 2020 (enrollment cut-off). Efficacy results, including analyses for treatment-naïve patients enrolled after eligibility criteria were revised to allow candidates for platinum-based therapy, are shown in the Table. Conclusions: Pralsetinib showed rapid, potent, and durable clinical activity in patients with RET fusion-positive NSCLC (regardless of prior therapies), including poor prognosis patients not eligible for platinum-based therapy. Overall, pralsetinib was well-tolerated. These data highlight the need for RET testing early in the course of disease to identify candidates who may benefit from treatment with pralsetinib. Clinical trial information: NCT03037385. [Table: see text]

Beginning with discovery of a new genetic cause of familial pancreatic ductal adenocarcinoma (PDAC), this work uncovers a mechanism for regulating KRAS intracellular trafficking with implications for hereditary cancer, sporadic cancer, and RASopathy syndromes. PDAC is one of the deadliest solid cancers, with limited treatment options despite intensive research efforts. Familial predisposition to PDAC is thought to occur in ~10% of cases, but causative genes have not been identified in most of these families. Uncovering the genetic basis for PDAC susceptibility has immediate prognostic implications for families and can provide precious mechanistic clues to PDAC pathogenesis. Here, we perform whole-genome sequence analysis in a family with five cases of PDAC and identify a germline nonsense mutation in the member of RAS oncogene family-like 3 (RABL3) gene that has never before been directly associated with hereditary cancer. The truncated mutant allele (RABL3_p.Ser36*) co-segregates with cancer occurrence. To evaluate the contribution of the RABL3 mutant allele in hereditary cancer, we generated rabl3 heterozygous mutant zebrafish and found increased susceptibility to cancer formation in two independent cancer models. Complementary unbiased approaches implicate RABL3 in RAS pathway regulation. RNA-Seq and genome-set enrichment analysis of juvenile rabl3 mutants reveals a KRAS upregulation signature. Furthermore, affinity-purification mass spectrometry for proteins associated with RABL3 or RABL3_p.Ser36* identifies Rap1 GTPase-GDP Dissociation Stimulator 1 (RAP1GDS1, SmgGDS), a chaperone that regulates prenylation of RAS GTPases. Indeed, in vitro studies demonstrate that RABL3_p.Ser36* accelerates KRAS prenylation, and this impact is lost in the absence of H/N/KRAS proteins. Whereas heterozygous rabl3 mutant zebrafish exhibit cancer predisposition, homozygous rabl3 mutant zebrafish develop severe craniofacial, skeletal, and growth defects consistent with human RASopathies, and these defects are partially rescued with the MEK inhibitor trametinib. Finally, we identify additional germline mutations in RABL3 that impact RAS activity in vivo and have a significant burden in a cohort of patients with developmental disorders, suggesting a role in undiagnosed RASopathies. Moreover, RABL3 is upregulated in multiple human PDAC cell lines and knockdown abrogates proliferation, consistent with a broader role for RABL3 in PDAC. The discovered causative RABL3 germline mutation provides new diagnostic opportunities for genetic testing in other cancer families and uncovers an alternative mechanism for dysregulated RAS signaling in development and cancer. This abstract is also being presented as Poster A12. Citation Format: Sahar Nissim, Ignaty Leshchiner, Joseph Mancias, Matthew Greenblatt, Ophélia Maertens, Christopher Cassa, Jill Rosenfeld, Andrew Cox, John Hedgepeth, Julia Wucherpfennig, Andrew Kim, Jake Henderson, Patrick Gonyo, Anthony Brandt, Ellen Lorimer, Bethany Unger, Gad Getz, Shamil Sunyaev, Wade Harper, Karen Cichowski, Alec Kimmelman, Yariv Houvras, Sapna Syngal, Carol Williams, Wolfram Goessling. Mutations in RABL3 alter KRAS prenylation and are associated with hereditary pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr PR02.

CRISPR/Cas9 has become a powerful tool for genome editing in zebrafish that permits the rapid generation of loss of function mutations and the knock-in of specific alleles using DNA templates and homology directed repair (HDR). We examined the efficiency of synthetic, chemically modified gRNAs and demonstrate induction of indels and large genomic deletions in combination with recombinant Cas9 protein. We developed an in vivo genetic assay to measure HDR efficiency and we utilized this assay to test the effect of altering template design on HDR. Utilizing synthetic gRNAs and linear dsDNA templates, we successfully performed knock-in of fluorophores at multiple genomic loci and demonstrate transmission through the germline at high efficiency. We demonstrate that synthetic HDR templates can be used to knock-in bacterial nitroreductase (ntr) to facilitate lineage ablation of specific cell types. Collectively, our data demonstrate the utility of combining synthetic gRNAs and dsDNA templates to perform homology directed repair and genome editing in vivo.

The Hippo pathway and its nuclear effector Yap regulate organ size and cancer formation. While many modulators of Hippo activity have been identified, little is known about the Yap target genes that mediate these growth effects. Here, we show that yap−/− mutant zebrafish exhibit defects in hepatic progenitor potential and liver growth due to impaired glucose transport and nucleotide biosynthesis. Transcriptomic and metabolomic analyses reveal that Yap regulates expression of glucose transporter glut1, causing decreased glucose uptake and use for nucleotide biosynthesis in yap−/− mutants, and impaired glucose tolerance in adults. Nucleotide supplementation improves Yap deficiency phenotypes, indicating functional importance of glucose-fueled nucleotide biosynthesis. Yap-regulated glut1 expression and glucose uptake are conserved in mammals, suggesting that stimulation of anabolic glucose metabolism is an evolutionarily conserved mechanism by which the Hippo pathway controls organ growth. Together, our results reveal a central role for Hippo signaling in glucose metabolic homeostasis., National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (Grant P30DK034854), National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (Grant 1R01DK090311), National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (Grant R24OD017870), National Institutes of Health (U.S.) (Grant P30DK034854), National Institutes of Health (U.S.) (Grant 1R01DK090311), National Institutes of Health (U.S.) (Grant 1R01DK105198), National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (Grant 1R01DK105198), National Institutes of Health (U.S.) (Grant R24OD017870), National Institute of General Medical Sciences (NIGMS) (Grant T32GM007753), NHMRC (Grant 1146558), National Cancer Institute (U.S.) (Grant 5P01CA120964), National Cancer Institute (U.S.) (Grant 5P30CA006516)

CRISPR/Cas9 has become a powerful tool for genome editing in zebrafish that permits the rapid generation of loss of function mutations and the knock-in of specific alleles using DNA templates and homology directed repair (HDR). We compared synthetic, chemically modified sgRNAs to in vitro transcribed sgRNAs and demonstrate the increased activity of synthetic sgRNAs in combination with recombinant Cas9 protein. We developed an in vivo genetic assay to measure HDR efficiency and we utilized this assay to optimize the design of synthetic DNA templates to promote HDR. Utilizing these principles, we successfully performed knock-in of fluorophores at multiple genomic loci and demonstrate transmission through the germline at high efficiency. We demonstrate that synthetic HDR templates can be used to knock-in bacterial nitroreductase (ntr) to facilitate lineage ablation of specific cell types. Collectively, our data demonstrate the utility of combining synthetic sgRNAs and dsDNA templates to perform homology directed repair and genome editing in vivo.

The two major melanoma histologic subtypes, superficial spreading and nodular melanomas, differ in their speed of dermal invasion but converge biologically once they invade and metastasize. Herein, we tested the hypothesis that distinct molecular alterations arising in primary melanoma cells might persist as these tumors progress to invasion and metastasis. Ribosomal protein S6 kinase, 90 kDa, polypeptide 1 (RSK1; official name RPS6KA1) was significantly hyperactivated in human melanoma lines and metastatic tissues derived from nodular compared with superficial spreading melanoma. RSK1 was constitutively phosphorylated at Ser-380 in nodular but not superficial spreading melanoma and did not directly correlate with BRAF or MEK activation. Nodular melanoma cells were more sensitive to RSK1 inhibition using siRNA and the pharmacological inhibitor BI-D1870 compared with superficial spreading cells. Gene expression microarray analyses revealed that RSK1 orchestrated a program of gene expression that promoted cell motility and invasion. Differential overexpression of the prometastatic matrix metalloproteinase 8 and tissue inhibitor of metalloproteinases 1 in metastatic nodular compared with metastatic superficial spreading melanoma was observed. Finally, using an in vivo zebrafish model, constitutive RSK1 activation increased melanoma invasion. Together, these data reveal a novel role for activated RSK1 in the progression of nodular melanoma and suggest that melanoma originating from different histologic subtypes may be biologically distinct and that these differences are maintained as the tumors invade and metastasize.

© 2016, National Academy of Sciences. All rights reserved. Selenium, an essential micronutrient known for its cancer prevention properties, is incorporated into a class of selenocysteine-containing proteins (selenoproteins). Selenoprotein H (SepH) is a recently identified nucleolar oxidoreductase whose function is not well understood. Here we report that seph is an essential gene regulating organ development in zebrafish. Metabolite profiling by targeted LC-MS/MS demonstrated that SepH deficiency impairs redox balance by reducing the levels of ascorbate and methionine, while increasing methionine sulfoxide. Transcriptome analysis revealed that SepH deficiency induces an inflammatory response and activates the p53 pathway. Consequently, loss of seph renders larvae susceptible to oxidative stress and DNA damage. Finally, we demonstrate that seph interacts with p53 deficiency in adulthood to accelerate gastrointestinal tumor development. Overall, our findings establish that seph regulates redox homeostasis and suppresses DNA damage. We hypothesize that SepH deficiency may contribute to the increased cancer risk observed in cohorts with low selenium levels.

Thyroid cancer is common, yet the sequence of alterations that promote tumor formation are incompletely understood. Here, we describe a novel model of thyroid carcinoma in zebrafish that reveals temporal changes due to BRAFV600E. Through the use of real-time in vivo imaging, we observe disruption in thyroid follicle structure that occurs early in thyroid development. Combinatorial treatment using BRAF and MEK inhibitors reversed the developmental effects induced by BRAFV600E. Adult zebrafish expressing BRAFV600E in thyrocytes developed invasive carcinoma. We identified a gene expression signature from zebrafish thyroid cancer that is predictive of disease-free survival in patients with papillary thyroid cancer. Gene expression studies nominated TWIST2 as a key effector downstream of BRAF. Using CRISPR/Cas9 to genetically inactivate a TWIST2 orthologue, we suppressed the effects of BRAFV600E and restored thyroid morphology and hormone synthesis. These data suggest that expression of TWIST2 plays a role in an early step of BRAFV600E-mediated transformation.

Over a decade has elapsed since the first genetically-engineered zebrafish cancer model was described. During this time remarkable progress has been made. Sophisticated genetic tools have been built to generate oncogene expressing cancers and characterize multiple models of solid and blood tumors. These models have led to unique insights into mechanisms of tumor initiation and progression. New drug targets have been identified, particularly through the functional analysis of cancer genomes. Now in the second decade, zebrafish cancer models are poised for even faster growth as they are used in high-throughput genetic analyses to elucidate key mechanisms underlying critical cancer phenotypes.

Cancers contain a wide diversity of cell types that are defined by differentiation states, genetic mutations and altered epigenetic programmes that impart functional diversity to individual cells. Elevated tumour cell heterogeneity is linked with progression, therapy resistance and relapse. Yet, imaging of tumour cell heterogeneity and the hallmarks of cancer has been a technical and biological challenge. Here we develop optically clear immune-compromised rag2E450fs (casper) zebrafish for optimized cell transplantation and direct visualization of fluorescently labelled cancer cells at single-cell resolution. Tumour engraftment permits dynamic imaging of neovascularization, niche partitioning of tumour-propagating cells in embryonal rhabdomyosarcoma, emergence of clonal dominance in T-cell acute lymphoblastic leukaemia and tumour evolution resulting in elevated growth and metastasis in BRAFV600E-driven melanoma. Cell transplantation approaches using optically clear immune-compromised zebrafish provide unique opportunities to uncover biology underlying cancer and to dynamically visualize cancer processes at single-cell resolution in vivo., Direct visualisation of heterogeneous cell populations in live animals has been challenging. Here, the authors optimize cell transplantation into optically clear immune-deficient zebrafish, and use intravital imaging to track and to assess functional diversity of individual cancer cells in vivo.

Over a decade has elapsed since the first genetically-engineered zebrafish cancer model was described. During this time remarkable progress has been made. Sophisticated genetic tools have been built to generate oncogene expressing cancers and characterize multiple models of solid and blood tumors. These models have led to unique insights into mechanisms of tumor initiation and progression. New drug targets have been identified, particularly through the functional analysis of cancer genomes. Now in the second decade, zebrafish cancer models are poised for even faster growth as they are used in high-throughput genetic analyses to elucidate key mechanisms underlying critical cancer phenotypes.

Captive zebrafish (Danio rerio) exhibit a limited repertoire of mating behaviors, likely due to the somewhat unnatural environment of aquaria. Observations in their natural habitat led us to believe that a depth gradient within the mating setup would positively affect fish mating. By tilting the tank to produce a depth gradient, we observed novel behaviors along with a preference for oviposition in the shallow area. Although we did not see an increase in the likelihood of a pair of fish to mate, we did see an increase in the embryo output in both adults and juveniles. In the adults, tilting led to a significant increase in embryo production (436 +/- 35 tilted vs. 362 +/- 34 untilted; p < 0.05). A similar effect was seen in juvenile fish as they progressed through sexual maturity. These results suggest that tilting of mating cages in the laboratory setting will lead to demonstrable improvements in embryo production for zebrafish researchers, and highlights the possibility of other manipulations to increase fecundity.

Genomic instability is a fundamental feature of human cancer often resulting from impaired genome maintenance. In prostate cancer, structural genomic rearrangements are a common mechanism driving tumorigenesis. However, somatic alterations predisposing to chromosomal rearrangements in prostate cancer remain largely undefined. Here, we show that SPOP, the most commonly mutated gene in primary prostate cancer modulates DNA double strand break (DSB) repair, and that SPOP mutation is associated with genomic instability. In vivo, SPOP mutation results in a transcriptional response consistent with BRCA1 inactivation resulting in impaired homology-directed repair (HDR) of DSB. Furthermore, we found that SPOP mutation sensitizes to DNA damaging therapeutic agents such as PARP inhibitors. These results implicate SPOP as a novel participant in DSB repair, suggest that SPOP mutation drives prostate tumorigenesis in part through genomic instability, and indicate that mutant SPOP may increase response to DNA-damaging therapeutics. DOI: http://dx.doi.org/10.7554/eLife.09207.001, eLife digest Prostate cancer is the most common type of cancer in men in the UK and USA. Cancers develop when cells in the body acquire genetic mutations that allow the cells to grow rapidly and form a mass known as a tumor. Prostate cancer cells from different individuals can carry different genetic mutations, which affects whether the disease progresses and how the tumors respond to medical treatments. This genetic variety arises in cancer cells partly from a phenomenon known as genomic instability, in which DNA mutations accumulate due to defects in DNA repair. Genetic studies of biopsies taken from human prostate cancers have shown that genomic instability causes chromosomes—the structures in which the cell's DNA is organized—to break and then be stuck back together haphazardly. As a result, fragments of chromosomes can end up in the wrong position, be duplicated, or be lost altogether. All of these mutations could spur on the growth of the tumor. However, it is currently not clear why some prostate cancers are more genomically unstable than others, or what exactly causes this instability. Boysen, Barbieri et al. studied prostate cancer cells taken from patients before they started medical treatment. The experiments show that the cancer cells with high levels of genomic instability also often had mutations in a gene that encodes a protein called SPOP. These mutations occur in about 10 percent of men with prostate cancer and appear early in the development of the tumors. Next, they studied the SPOP protein in zebrafish (which is nearly identical to human SPOP), as well as in mouse and human cells. The experiments show that SPOP normally helps the cell to accurately repair DNA that has been damaged. Mutations in SPOP change the DNA repair process, which lead to genomic instability by increasing the likelihood that broken chromosomes will be stuck back together incorrectly. Further experiments tested drugs known as PARP inhibitors on mouse and human prostate cancer cells. The drugs, which have been recently tested successfully in patients with prostate cancer, block a different method of DNA repair that operates separately to the one that involves SPOP. When both of these pathways were inactivated—one by the SPOP mutation, the other by the drug—the cancer cells died more quickly. Therefore, men that are diagnosed with types of prostate cancer in which the gene that encodes SPOP is mutated might benefit from treatment with PARP inhibitors or other therapies that affect DNA repair. DOI: http://dx.doi.org/10.7554/eLife.09207.002

The Hippo pathway is an important regulator of organ size and tumorigenesis. It is unclear, however, how Hippo signalling provides the cellular building blocks required for rapid growth. Here, we demonstrate that transgenic zebrafish expressing an activated form of the Hippo pathway effector Yap1 (also known as YAP) develop enlarged livers and are prone to liver tumour formation. Transcriptomic and metabolomic profiling identify that Yap1 reprograms glutamine metabolism. Yap1 directly enhances glutamine synthetase (glul) expression and activity, elevating steady-state levels of glutamine and enhancing the relative isotopic enrichment of nitrogen during de novo purine and pyrimidine biosynthesis. Genetic or pharmacological inhibition of GLUL diminishes the isotopic enrichment of nitrogen into nucleotides, suppressing hepatomegaly and the growth of liver cancer cells. Consequently, Yap-driven liver growth is susceptible to nucleotide inhibition. Together, our findings demonstrate that Yap1 integrates the anabolic demands of tissue growth during development and tumorigenesis by reprogramming nitrogen metabolism to stimulate nucleotide biosynthesis.

Multiplexing samples in sequencing experiments is a common approach to maximize information yield while minimizing cost. In most cases the number of samples that are multiplexed is determined by financial consideration or experimental convenience, with limited understanding on the effects on the experimental results. Here we set to examine the impact of multiplexing ChIP-seq experiments on the ability to identify a specific epigenetic modification. We performed peak detection analyses to determine the effects of multiplexing. These include false discovery rates, size, position and statistical significance of peak detection, and changes in gene annotation. We found that, for histone marker H3K4me3, one can multiplex up to 8 samples (7 IP + 1 input) at ~21 million single-end reads each and still detect over 90% of all peaks found when using a full lane for sample (~181 million reads). Furthermore, there are no variations introduced by indexing or lane batch effects and importantly there is no significant reduction in the number of genes with neighboring H3K4me3 peaks. We conclude that, for a well characterized antibody and, therefore, model IP condition, multiplexing 8 samples per lane is sufficient to capture most of the biological signal.

In Journal of Clinical Oncology, Wells et al present the results of the ZETA (Zactima Efficacy in Thyroid Cancer Assessment) trial, a phase III randomized, double-blind, placebo-controlled trial of vandetanib in patients with sporadic and hereditary medullary thyroid cancer (MTC). Vandetanib (formerly ZD6474) is a small-molecule tyrosine kinase inhibitor of RET, epidermal growth factor receptor (EGFR), and vascular endothelial growth factor receptor (VEGFR) kinases. In ZETA, patients with advanced MTC who were randomly assigned to treatment with vandetanib had significantly longer progression-free survival than those randomly assigned to treatment with placebo. The results of the ZETA trial led the US Food and Drug Administration to approve vandetanib for treatment of symptomatic or progressive MTC earlier this year. This is the first drug approved for MTC and the first TKI approved for any thyroid cancer indication. Thyroid cancer has a rising incidence, with an estimated 45,000 new cases diagnosed in the United States in 2011. MTC accounts for 3% to 5% of total thyroid cancer cases. In 10% to 20% of MTCs, a germline mutation in RET causes the hereditary syndrome of multiple endocrine neoplasia type 2A or 2B (MEN2A/B), which is variably associated with pheochromocytoma and parathyroid hyperplasia. MEN2 syndromes are characterized by strong correlations between mutant RET genotype and clinical phenotype, and treatment decisions are genotype driven. For example, mutations in RET codon 634 commonly lead to both MTC and pheochromocytoma, whereas mutations in codon 883 lead to MTC but rarely to pheochromocytoma. In 80% to 90% of MTCs, the disease arises sporadically in C cells. A majority of sporadic MTCs are caused by the M918T mutation. RET is a receptor tyrosine kinase that binds glial cell line– derived neurotrophic factor (GFR ) receptors bound to ligands of the glial cell line– derived neurotrophic factor (GDNF) family (Fig 1). Activation of RET has been shown to signal through multiple pathways, including Ras/extracellular signal-regulated kinase, phosphotidylinositol-3-kinase, SRC, phospholipase C , Jun-N terminal kinase, and signal transducer and activator of transcription 3. Both sporadic and hereditary MTCs arise from gain of function, activating mutations in RET. Mutations in the extracellular coding region (exons 8, 10, and 11) lead to ligand-independent dimerization and phosphorylation. Mutations in the intracellular kinase domain (exons 13 to 16) activate enzymatic activity and in some cases seem to alter substrate specificity. In some cases of hereditary MTC, mutations in RET cannot be identified, raising the possibility of mutations in other yet to be identified genes. RET function is required for development of sympathetic neurons in the enteric nervous system, and loss of function mutations in RET are a cause of aganglionic megacolon in Hirschprung disease. RET is subject to recurrent translocations in 10% to 30% of papillary thyroid cancers (PTCs). RET is translocated with several partner proteins, most commonly CCDC6 (PTC1) and NCOA4 (PTC3). Individuals exposed to ionizing radiation are at risk for developing PTC with RET translocations. Translocation of RET has been shown to result in constitutive activation of the kinase domain and transformation of papillary thyrocytes. Treatment of PTC cell lines harboring RET/PTC1 rearrangements with vandetanib induced growth arrest in vitro. Vandetanib also inhibited growth in vitro and tumorigenicity in vivo performed with NIH-3T3 cells transformed with RET/PTC3. These studies suggest that vandetanib may have a clinical role in the treatment of patients with RET/PTC-rearranged PTCs. A phase II study of vandetanib in differentiated thyroid cancer has been conducted, but the results have not yet been published. Further clinical investigation might also explore the role of vandetanib as a preventative therapy for patients at high risk of developing PTC postradiation exposure. Are the antitumor effects of vandetanib the result of RET inhibition or inhibition of EGFR, VEGFR, or other kinases? There are in vitro data suggesting that EGFR may be an important target in MTC, suggesting that the dual inhibition of RET and EGFR may contribute to the efficacy of vandetanib. Yet, in a phase II study of the EGFR inhibitor gefitinib in thyroid cancer, no objective responses were observed (four patients with MTC). It is also possible that inhibition of VEGFR2 contributes to the antitumor activity of vandetanib by an antiangiogenic effect. Genetic studies will be required to dissect these possibilities. MTC is associated with hypersecretion of calcitonin, carcinoembryonic antigen, and other neuropeptide hormones. In patients with MTC, elevated calcitonin often causes a secretory diarrhea proportional to the degree of elevation. In ZETA, treatment with vandetanib was associated with dramatic reductions of calcitonin (biochemical response rate: vandetanib, 69% v placebo, 3%), yet it was still associated with diarrhea (diarrhea any grade: vandetanib, 56% v placebo, 26%). Because adverse event data capture the peak, it is difficult to know what the trend in diarrhea was with treatment. Reported stool frequency was not significantly different between vandetanib and placebo. Additional studies may shed more light on the magnitude of symptom improvement with vandetanib treatment. JOURNAL OF CLINICAL ONCOLOGY U N D E R S T A N D I N G T H E P A T H W A Y VOLUME 30 NUMBER 2 JANUARY 1

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest solid cancers with limited treatment options despite intensive research efforts. Familial predisposition to PDAC is thought to occur in ~10% of cases, but causative genes have not been identified in most of these families. Uncovering the genetic basis for PDAC susceptibility has immediate prognostic implications for families and can provide precious mechanistic clues to PDAC pathogenesis. Here, we perform whole-genome sequence analysis in a family with high incidence of PDAC and identify a germline nonsense mutation in the member of RAS oncogene family-like 3 (RABL3) gene that has never before been directly associated with hereditary cancer. The truncated mutant allele (RABL3_p.Ser36*) co-segregates with cancer occurrence. To evaluate the contribution of the RABL3 mutant allele in hereditary cancer, we generated rabl3 heterozygous mutant zebrafish and found increased susceptibility to cancer formation in two independent cancer models. Complementary unbiased approaches implicate RABL3 in RAS pathway regulation. RNA-Seq and genome-set enrichment analysis of juvenile rabl3 mutants reveals a KRAS upregulation signature. Furthermore, affinity-purification mass-spectrometry for proteins associated with RABL3 or RABL3_p.Ser36* identifies Rap1 GTPase-GDP Dissociation Stimulator 1 (RAP1GDS1, SmgGDS), a chaperone that regulates prenylation of RAS GTPases. Indeed, in vitro studies demonstrate that RABL3_p.Ser36* accelerates KRAS prenylation, and this impact is lost in the absence of H/N/KRAS proteins. Whereas heterozygous rabl3 mutant zebrafish exhibit cancer predisposition, homozygous rabl3 mutant zebrafish develop severe craniofacial, skeletal, and growth defects consistent with human RASopathies, and these defects are partially rescued with the MEK inhibitor trametinib. Our findings support a gain-of-function rather than a null function typically associated with premature protein truncations. The discovered causative RABL3 germline mutation provides new diagnostic opportunities for genetic testing in other cancer families and uncovers an alternative mechanism for dysregulated RAS signaling in development and cancer. Note: This abstract was not presented at the meeting. Citation Format: Sahar Nissim, Ignaty Leshchiner, Joseph D. Mancias, Matthew B. Greenblatt, Ophélia Maertens, Christopher A. Cassa, Jill A. Rosenfeld, Andrew G. Cox, John Hedgepeth, Julia Wücherpfennig, Andrew J. Kim, Jake E. Henderson, Patrick Gonyo, Anthony Brandt, Ellen Lorimer, Bethany Unger, Jeremy W. Prokop, Jeremy W. Heidel, Xiao-Xu Wang, Chinedu I. Ukaegbu, Gad Getz, Shamil R. Sunyaev, J. Wade Harper, Karen Cichowski, Alec C. Kimmelman, Yariv Houvras, Sapna Syngal, Carol Williams, Wolfram Goessling. Mutations in RABL3 alter RAS prenylation and are associated with hereditary pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4272. doi:10.1158/1538-7445.AM2017-4272

BRCA1, a breast and ovarian cancer susceptibility gene, encodes a 220-kDa protein whose precise biochemical function remains unclear. BRCA1 contains an N-terminal RING finger that mediates protein-protein interaction. The C-terminal domain of BRCA1 (BRCT) can activate transcription and interacts with RNA polymerase holoenzyme. Using the yeast two-hybrid system, we identified an interaction between the BRCA1 RING finger and ATF1, a member of the cAMP response element-binding protein/activating transcription factor (CREB/ATF) family. We demonstrate that BRCA1 and ATF1 can physically associate in vitro, in yeast, and in human cells. BRCA1 stimulated transcription from a cAMP response element reporter gene in transient transfections. BRCA1 also stimulated transcription from a natural promoter, that of tumor necrosis factor-α, in a manner dependent on the integrity of the cAMP response element. These results implicate BRCA1 in transcriptional activation of ATF1 target genes, some of which are involved in the transcriptional response to DNA damage.

Much of the predisposition to hereditary breast and ovarian cancer has been attributed to inherited defects in the BRCA1 tumour-suppressor gene. The nuclear protein BRCA1 has the properties of a transcription factor, and can interact with the recombination and repair protein RAD51. Young women with germline alterations in BRCA1 develop breast cancer at rates 100-fold higher than the general population, and BRCA1-null mice die before day 8 of development. However, the mechanisms of BRCA1-mediated growth regulation and tumour suppression remain unknown. Here we show that BRCA1 transactivates expression of the cyclin-dependent kinase inhibitor p21WAF1/CIP1 in a p53-independent manner, and that BRCA1 inhibits cell-cycle progression into the S-phase following its transfection into human cancer cells. BRCA1 does not inhibit S-phase progression in p21-/- cells, unlike p21+/+ cells, and tumour-associated, transactivation-deficient mutants of BRCA1 are defective in both transactivation of p21 and cell-cycle inhibition. These data suggest that one mechanism by which BRCA1 contributes to cell-cycle arrest and growth suppression is through the induction of p21.

A phase 1 study was initiated to determine the safety, potential effectiveness, and maximal tolerated dose and recommended phase 2 dose of efatutazone and paclitaxel in anaplastic thyroid cancer.Patients received efatutazone (0.15, 0.3, or 0.5 mg) orally twice daily and then paclitaxel every 3 weeks. Patient tolerance and outcomes were assessed, as were serum efatutazone pharmacokinetics.Ten of 15 patients were women. Median age was 59 years. Seven patients received 0.15 mg of efatutazone, 6 patients received 0.3 mg, and 2 patients received 0.5 mg. One patient receiving 0.3 mg of efatutazone had a partial response from day 69 to day 175; 7 patients attained stable disease. Median times to progression were 48 and 68 days in patients receiving 0.15 mg of efatutazone and 0.3 mg of efatutazone, respectively; corresponding median survival was 98 vs 138 days. The median peak efatutazone blood level was 8.6 ng/mL for 0.15-mg dosing vs 22.0 ng/mL for 0.3-mg twice daily dosing. Ten patients had grade 3 or greater adverse events (Common Terminology Criteria for Adverse Events), with 2 of these (anemia and edema) related to efatutazone. Thirteen events of edema were reported in 8 patients, with 2 of grade 3 or greater. Eight patients had ≥1 serious adverse event, with 1 of these (anemia) attributed to efatutazone and 1 (anaphylactic reaction) related to paclitaxel. The maximal tolerated dose was not achieved. Angiopoietin-like 4 was induced by efatutazone in tissue biopsy samples of 2 patients.Efatutazone and paclitaxel in combination were safe and tolerated and had biologic activity.

Genomic studies of human cancers have yielded a wealth of information about genes that are altered in tumors1,2,3. A challenge arising from these studies is that many genes are altered, and it can be difficult to distinguish genetic alterations that drove tumorigenesis from that those arose incidentally during transformation. To draw this distinction it is beneficial to have an assay that can quantitatively measure the effect of an altered gene on tumor initiation and other processes that enable tumors to persist and disseminate. Here we present a rapid means to screen large numbers of candidate melanoma modifiers in zebrafish using an autochthonous tumor model4 that encompasses steps required for melanoma initiation and maintenance. A key reagent in this assay is the miniCoopR vector, which couples a wild-type copy of the mitfa melanocyte specification factor to a Gateway recombination cassette into which candidate melanoma genes can be recombined5. The miniCoopR vector has a mitfa rescuing minigene which contains the promoter, open reading frame and 3'-untranslated region of the wild-type mitfa gene. It allows us to make constructs using full-length open reading frames of candidate melanoma modifiers. These individual clones can then be injected into single cell Tg(mitfa:BRAFV600E);p53(lf);mitfa(lf)zebrafish embryos. The miniCoopR vector gets integrated by Tol2-mediated transgenesis6 and rescues melanocytes. Because they are physically coupled to the mitfa rescuing minigene, candidate genes are expressed in rescued melanocytes, some of which will transform and develop into tumors. The effect of a candidate gene on melanoma initiation and melanoma cell properties can be measured using melanoma-free survival curves, invasion assays, antibody staining and transplantation assays.