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
Wattrus, Samuel J; Zon, Leonard I (2018) Stem cell safe harbor: the hematopoietic stem cell niche in zebrafish. Blood Adv 2:3063-3069
McConnell, Alicia M; Mito, Jeffrey K; Ablain, Julien et al. (2018) Neural crest state activation in NRAS driven melanoma, but not in NRAS-driven melanocyte expansion. Dev Biol :
Yu, Yong; Schleich, Kolja; Yue, Bin et al. (2018) Targeting the Senescence-Overriding Cooperative Activity of Structurally Unrelated H3K9 Demethylases in Melanoma. Cancer Cell 33:322-336.e8
Ablain, Julien; Xu, Mengshu; Rothschild, Harriet et al. (2018) Human tumor genomics and zebrafish modeling identify SPRED1 loss as a driver of mucosal melanoma. Science 362:1055-1060
van Rooijen, Ellen; Fazio, Maurizio; Zon, Leonard I (2017) From fish bowl to bedside: The power of zebrafish to unravel melanoma pathogenesis and discover new therapeutics. Pigment Cell Melanoma Res 30:402-412
Fazio, Maurizio; Zon, Leonard I (2017) Fishing for answers in precision cancer medicine. Proc Natl Acad Sci U S A 114:10306-10308
Ciarlo, Christie; Kaufman, Charles K; Kinikoglu, Beste et al. (2017) A chemical screen in zebrafish embryonic cells establishes that Akt activation is required for neural crest development. Elife 6:
Fazio, Maurizio; Avagyan, Serine; van Rooijen, Ellen et al. (2017) Efficient Transduction of Zebrafish Melanoma Cell Lines and Embryos Using Lentiviral Vectors. Zebrafish 14:379-382
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
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

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