Cancer comprises a set of devastating diseases that cause significant morbidity and mortality. My laboratory has utilized the zebrafish as a model for studying cancer. Our zebrafish melanoma model was constructed by overexpression of the human BRAFV600 allele (mutated in about 70% of human melanomas) in combination with p53 deficiency: we developed an expression cloning strategy to examine the effect of specific genes on melanoma formation. In one example, we found the driver gene on human chromosome 1 that is amplified in 30 percent of melanoma. SETDB1, an H3K9 trimethylase, enhanced tumorigenesis in our melanoma model. SETDB1 is an epigenetic regulator that suppresses gene expression and promotes melanoma formation and invasion. We have since successfully undertaken a large-scale overexpression screen for other chromatin factors that accelerate melanoma in vivo. Here we propose to study the mechanism of action for SATB2, along with its family member SATB1 (SATB1 protein expression correlates to the stage of human melanoma), and HDGF. We will purify the SATB2 and HDGF protein complexes in human melanoma cells, and study components of the complex by gene knockdown with morpholinos and by overexpression in zebrafish. Our laboratory has also recently documented that leflunomide (LEF), known to inactivate DHODH (an enzyme required for pyrimidine synthesis), is capable of suppressing melanoma formation and, in combination with a BRAF inhibitor, lead to substantial reduction of melanoma growth. We have rapidly proceeded to a clinical trial that tests the combination therapy of a BRAF inhibitor and LEF as a new therapy for metastatic melanoma. The mechanism that underlies LEF-associated suppression of melanoma involves a block of transcriptional elongation of neural crest genes. Our study of the regulation of transcriptional pausing in melanoma cells revealed the involvement of the HEXIM complex, which is composed of RNA and proteins, and which sequesters the kinase P-TEFb that phosphorylates POLII to allow transcriptional elongation to occur. Inactivation of the HEXIM complex rescues the effects of LEF treatment of zebrafish embryos. Informatics studies have uncovered that HEXIM is substantially downregulated in a variety of melanomas. Preliminary data suggest that HEXIM overexpression can suppress melanoma formation in our model. We plan to express a constitutively active and dominant negative HEXIM mutant and evaluate the effect on melanoma formation. We have also initiated a chemical screen to find factors that alter transcriptional pausing in melanoma. Chemicals that rescue LEF could be helpful in determining the mechanism of action by which a DHODH inhibitor leads to a transcription elongation defect. We anticipate that results of the proposed experiments will have a broad impact on our understanding of the basic biology of melanoma, including epigenetic and transcriptional mechanisms, and will rapidly translate to new clinical trials on patients with melanoma.

Public Health Relevance

The zebrafish is an excellent animal model for studying how cancers develop, and developing targeted therapies for cancers. We have used this model to find new genes that participate in human melanoma and to derive new therapies that work by blocking how genes are turned on or off. We now plan to find other small molecules that can block transcription in melanoma cells, and to uncover additional cancer genes that play a role in the formation of melanoma (and perhaps also of other cancers).

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
2R01CA103846-11
Application #
8437367
Study Section
Cancer Molecular Pathobiology Study Section (CAMP)
Program Officer
Mietz, Judy
Project Start
2003-07-03
Project End
2018-02-28
Budget Start
2013-03-01
Budget End
2014-02-28
Support Year
11
Fiscal Year
2013
Total Cost
$368,865
Indirect Cost
$158,085
Name
Children's Hospital Boston
Department
Type
DUNS #
076593722
City
Boston
State
MA
Country
United States
Zip Code
02115
Kaufman, Charles K; Mosimann, Christian; Fan, Zi Peng et al. (2016) A zebrafish melanoma model reveals emergence of neural crest identity during melanoma initiation. Science 351:aad2197
Dang, Michelle; Henderson, Rachel E; Garraway, Levi A et al. (2016) Long-term drug administration in the adult zebrafish using oral gavage for cancer preclinical studies. Dis Model Mech 9:811-20
Hagedorn, Elliott J; Cillis, Jennifer L; Curley, Caitlyn R et al. (2016) Generation of Parabiotic Zebrafish Embryos by Surgical Fusion of Developing Blastulae. J Vis Exp :
Tan, Justin L; Fogley, Rachel D; Flynn, Ryan A et al. (2016) Stress from Nucleotide Depletion Activates the Transcriptional Regulator HEXIM1 to Suppress Melanoma. Mol Cell 62:34-46
Anderson, Heidi; Patch, Taylor C; Reddy, Pavankumar N G et al. (2015) Hematopoietic stem cells develop in the absence of endothelial cadherin 5 expression. Blood 126:2811-20
Ablain, Julien; Durand, Ellen M; Yang, Song et al. (2015) A CRISPR/Cas9 vector system for tissue-specific gene disruption in zebrafish. Dev Cell 32:756-64
Gjini, Evisa; Mansour, Marc R; Sander, Jeffry D et al. (2015) A zebrafish model of myelodysplastic syndrome produced through tet2 genomic editing. Mol Cell Biol 35:789-804
Kong, Yawei; Grimaldi, Michael; Curtin, Eugene et al. (2014) Neural crest development and craniofacial morphogenesis is coordinated by nitric oxide and histone acetylation. Chem Biol 21:488-501
Albacker, Colleen E; Storer, Narie Y; Langdon, Erin M et al. (2013) The histone methyltransferase SUV39H1 suppresses embryonal rhabdomyosarcoma formation in zebrafish. PLoS One 8:e64969
Yen, Jennifer; White, Richard M; Wedge, David C et al. (2013) The genetic heterogeneity and mutational burden of engineered melanomas in zebrafish models. Genome Biol 14:R113

Showing the most recent 10 out of 27 publications