Alison M. Taylor

Zebrafish Tumor Assays: The State of Transplantation

Tumor transplant studies are important tools for studying cancer biology in a model organism. Transplantation is especially important for assaying tumor cell malignancy and migration capabilities, and is critical for identifying putative cancer stem cell populations. In this review, we discuss the current state of tumor transplantation studies performed in the zebrafish. We address several zebrafish-specific considerations for development of the transplant assay, including choosing recipient animals, transplant methods, and post-transplant observation. We also examine how the zebrafish is an advantageous model for transplantation, particularly with development of the translucent fish. Transplantation has already been critical for characterizing zebrafish models of leukemia, rhabdomyosarcoma, and melanoma. With further development of imaging techniques and other tools, zebrafish tumor transplantation will continue to contribute to our understanding of tumor cell biology.

Whole-exome sequencing and functional studies identify RPS29 as a novel gene mutated in multicase Diamond-Blackfan anemia families

Diamond-Blackfan anemia (DBA) is a cancer-prone inherited bone marrow failure syndrome. Approximately half of DBA patients have a germ-line mutation in a ribosomal protein gene. We used whole-exome sequencing to identify disease-causing genes in 2 large DBA families. After filtering, 1 nonsynonymous mutation (p.I31F) in the ribosomal protein S29 (RPS29[AUQ1]) gene was present in all 5 DBA-affected individuals and the obligate carrier, and absent from the unaffected noncarrier parent in 1 DBA family. A second DBA family was found to have a different nonsynonymous mutation (p.I50T) in RPS29. Both mutations are amino acid substitutions in exon 2 predicted to be deleterious and resulted in haploinsufficiency of RPS29 expression compared with wild-type RPS29 expression from an unaffected control. The DBA proband with the p.I31F RPS29 mutation had a pre-ribosomal RNA (rRNA) processing defect compared with the healthy control. We demonstrated that both RPS29 mutations failed to rescue the defective erythropoiesis in the rps29(-/-) mutant zebra fish DBA model. RPS29 is a component of the small 40S ribosomal subunit and essential for rRNA processing and ribosome biogenesis. We uncovered a novel DBA causative gene, RPS29, and showed that germ-line mutations in RPS29 can cause a defective erythropoiesis phenotype using a zebra fish model.

Small molecule screening in zebrafish: Swimming in potential drug therapies

Phenotype‐driven chemical genetic screens in zebrafish have become a proven approach for both dissection of developmental mechanisms and discovery of potential therapeutics. A library of small molecules can be arrayed into multiwell plates containing zebrafish embryos. The embryo becomes a whole organism in vivo bioassay that can produce a phenotype upon treatment. Screens have been performed that are based simply on the morphology of the embryo. Other screens have scored complex phenotypes using whole mount in situ hybridization, fluorescent transgenic reporters, and even tracking of embryo movement. The availability of many well‐characterized zebrafish mutants has also enabled the discovery of chemical suppressors of genetic phenotypes. Importantly, the application of chemical libraries that already contain FDA‐approved drugs has allowed the rapid translation of hits from zebrafish chemical screens to clinical trials. WIREs Dev Biol 2012, 1:459–468. doi: 10.1002/wdev.37

The genetic heterogeneity and mutational burden of engineered melanomas in zebrafish models

BackgroundMelanoma is the most deadly form of skin cancer. Expression of oncogenic BRAF or NRAS, which are frequently mutated in human melanomas, promote the formation of nevi but are not sufficient for tumorigenesis. Even with germline mutated p53, these engineered melanomas present with variable onset and pathology, implicating additional somatic mutations in a multi-hit tumorigenic process.ResultsTo decipher the genetics of these melanomas, we sequence the protein coding exons of 53 primary melanomas generated from several BRAFV600E or NRASQ61K driven transgenic zebrafish lines. We find that engineered zebrafish melanomas show an overall low mutation burden, which has a strong, inverse association with the number of initiating germline drivers. Although tumors reveal distinct mutation spectrums, they show mostly C > T transitions without UV light exposure, and enrichment of mutations in melanogenesis, p53 and MAPK signaling. Importantly, a recurrent amplification occurring with pre-configured drivers BRAFV600E and p53-/- suggests a novel path of BRAF cooperativity through the protein kinase A pathway.ConclusionThis is the first analysis of a melanoma mutational landscape in the absence of UV light, where tumors manifest with remarkably low mutation burden and high heterogeneity. Genotype specific amplification of protein kinase A in cooperation with BRAF and p53 mutation suggests the involvement of melanogenesis in these tumors. This work is important for defining the spectrum of events in BRAF or NRAS driven melanoma in the absence of UV light, and for informed exploitation of models such as transgenic zebrafish to better understand mechanisms leading to human melanoma formation.

Drug discovery for Diamond-Blackfan anemia using reprogrammed hematopoietic progenitors

A stem cell reprogramming approach enables disease modeling and drug discovery for a genetic blood disorder and uncovers a candidate therapeutic. Inducing autophagy to improve anemia Diamond-Blackfan anemia (DBA) is a rare blood disorder characterized by insufficient red blood cell production that is treated with corticosteroids and transfusion therapy. To identify additional therapeutics for DBA, Doulatov et al. performed a chemical screen with hematopoietic progenitor cells derived from iPSCs from two DBA patients with RPS19 and RPL5 genetic mutations. The autophagy inducing small-molecule SMER28 rescued erythroid differentiation in an autophagy factor ATG5-dependent manner in iPSC-derived patient cells, in zebrafish models of DBA, and in several mouse models. These results demonstrate the utility of iPSC-based screens for drug discovery for rare blood disorders and identify SMER28 and the autophagy pathway as promising targets for DBA therapy. Diamond-Blackfan anemia (DBA) is a congenital disorder characterized by the failure of erythroid progenitor differentiation, severely curtailing red blood cell production. Because many DBA patients fail to respond to corticosteroid therapy, there is considerable need for therapeutics for this disorder. Identifying therapeutics for DBA requires circumventing the paucity of primary patient blood stem and progenitor cells. To this end, we adopted a reprogramming strategy to generate expandable hematopoietic progenitor cells from induced pluripotent stem cells (iPSCs) from DBA patients. Reprogrammed DBA progenitors recapitulate defects in erythroid differentiation, which were rescued by gene complementation. Unbiased chemical screens identified SMER28, a small-molecule inducer of autophagy, which enhanced erythropoiesis in a range of in vitro and in vivo models of DBA. SMER28 acted through autophagy factor ATG5 to stimulate erythropoiesis and up-regulate expression of globin genes. These findings present an unbiased drug screen for hematological disease using iPSCs and identify autophagy as a therapeutic pathway in DBA.

Modeling Diamond Blackfan anemia in the zebrafish.

Diamond Blackfan anemia (DBA) is a rare congenital anemia, with more than 50% of patients having mutations in a ribosomal protein. Evidence suggests that both translation and p53 activation play roles in mediating the hematopoietic phenotype. The reason for erythroid specificity of DBA is unclear. Several zebrafish models of DBA have been generated, and these models have already provided key information about disease pathogenesis. The zebrafish model is particularly amenable for studying blood development, allows for advanced imaging techniques, can be manipulated genetically, and is useful for high-throughput screening. By applying zebrafish approaches to the existing DBA models, we will be able to better understand the role of the ribosomal protein mutation in DBA and develop better treatments for this disease.

Somatic Superenhancer Duplications and Hotspot Mutations Lead to Oncogenic Activation of the KLF5 Transcription Factor

The Krüppel-like family of transcription factors plays critical roles in human development and is associated with cancer pathogenesis. Krüppel-like factor 5 gene (KLF5) has been shown to promote cancer cell proliferation and tumorigenesis and to be genomically amplified in cancer cells. We recently reported that the KLF5 gene is also subject to other types of somatic coding and noncoding genomic alterations in diverse cancer types. Here, we show that these alterations activate KLF5 by three distinct mechanisms: (i) Focal amplification of superenhancers activates KLF5 expression in squamous cell carcinomas; (ii) Missense mutations disrupt KLF5-FBXW7 interactions to increase KLF5 protein stability in colorectal cancer; (iii) Cancer type-specific hotspot mutations within a zinc-finger DNA binding domain of KLF5 change its DNA binding specificity and reshape cellular transcription. Utilizing data from CRISPR/Cas9 gene knockout screening, we reveal that cancer cells with KLF5 overexpression are dependent on KLF5 for their proliferation, suggesting KLF5 as a putative therapeutic target.Significance: Our observations, together with previous studies that identified oncogenic properties of KLF5, establish the importance of KLF5 activation in human cancers, delineate the varied genomic mechanisms underlying this occurrence, and nominate KLF5 as a putative target for therapeutic intervention in cancer. Cancer Discov; 8(1); 108-25. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 1.

Genome-scale analysis identifies paralog lethality as a vulnerability of chromosome 1p loss in cancer

Functional redundancy shared by paralog genes may afford protection against genetic perturbations, but it can also result in genetic vulnerabilities due to mutual interdependency1–5. Here, we surveyed genome-scale short hairpin RNA and CRISPR screening data on hundreds of cancer cell lines and identified MAGOH and MAGOHB, core members of the splicing-dependent exon junction complex, as top-ranked paralog dependencies6–8. MAGOHB is the top gene dependency in cells with hemizygous MAGOH deletion, a pervasive genetic event that frequently occurs due to chromosome 1p loss. Inhibition of MAGOHB in a MAGOH-deleted context compromises viability by globally perturbing alternative splicing and RNA surveillance. Dependency on IPO13, an importin-β receptor that mediates nuclear import of the MAGOH/B-Y14 heterodimer9, is highly correlated with dependency on both MAGOH and MAGOHB. Both MAGOHB and IPO13 represent dependencies in murine xenografts with hemizygous MAGOH deletion. Our results identify MAGOH and MAGOHB as reciprocal paralog dependencies across cancer types and suggest a rationale for targeting the MAGOHB-IPO13 axis in cancers with chromosome 1p deletion.Analysis of paralog gene pairs using data from loss-of-function genetic screens in cancer cells identifies MAGOH and MAGOHB as reciprocal paralog dependencies across cancer types.