There are two major ongoing projects in our lab.
1. Use a chemical suppressor screen to identify novel pathways involved in leukemogenesis
AML1-ETO is the fusion product of t(8;21) chromosomal translocation that can be found in approximately 12% of the acute myelogenous leukemia (AML) patients. AML1-ETO, as many other leukemic oncogenes, causes dysregulation of hematopoietic differentiation. Current treatments against AML rely heavily on non-specific cytotoxic drugs that kill proliferating cells, resulting in unbearable side effects, and yet most of the patients relapse after a brief remission.
Compared to conventional cytotoxic therapeutics, targeted cancer therapies hold great promise to be more effective and less toxic. Thus, we generated a zebrafish model of AML by inducing AML1-ETO expression in zebrafish embryos, and showed that AML1-ETO exerts AML-like hematopoietic differentiation defects within just hours. We have developed a chemical suppressor screen using this zebrafish model of AML. Several classes of chemical suppressors have been identified in the screen and they are currently being evaluated for therapeutic potential using human cells and mouse models of AML.
Figure 1: A chemical suppressor screen conducted in the zebrafish model of AML1-ETO identifies a potential role for COX-2 in AML leukemogenesis. (a) Expression of AML1-ETO results in a hematopoietic differentiation defect, suppressing the erythroid cell fate in zebrafish embryos. This effect can be readily detected using the erythroid cell marker, gata1. Each panel shows five zebrafish embryos per well in a 96-well screening plate after gata1 staining. (b) We found that COX-2 inhibitors reverse the hematopoietic defect caused by AML1-ETO, pointing to potential therapeutic benefits of COX inhibitors for treating AML.
2. Use customized zinc finger nucleases (ZFNs) to generate targeted mutations in zebrafish
In collaboration with the Peterson lab at the Cardiovascular Research Center and the Joung lab in the Department of Pathology at MGH, we have created a number of zebrafish mutant lines using the ZFN technology. Customized ZFNs comprise a common endonuclease domain and a DNA-binding domain that is engineered to bind to a specific DNA sequence. ZFN-induced DNA cleavage at the target site often results in the introduction of small insertions/deletions, causing mutations specifically in the target gene.
We are currently using these zebrafish mutants to understand iron homeostasis, arterial-venous fate determination, and neurological/psychiatric disease mechanisms. By exploiting the unique capabilities of the zebrafish for disease modeling and small molecule screening, these zebrafish mutant lines will provide powerful tools for dissecting signaling pathways involved in a broad range of biological processes and may point to promising new therapeutic approaches for treating human diseases.
Figure 2: The ZFN technology facilitates the creation of human disease models in zebrafish for the discovery of novel therapeutic agents. First, we engineer gene-specific zinc-finger nucleases (ZFNs) for generating zebrafish mutants of interest. Second, we identify zebrafish mutant lines that manifest specific disease phenotypes similar to humans. Third, zebrafish mutant embryos are subjected to large-scale chemical screening to identify compounds that can modify the disease phenotypes. Lastly, we identify the compounds’ underlying mechanisms of action, which may provide new insights into the development of therapeutic agents.
Foley JE, Maeder ML, Pearlberg J, Joung JK, Peterson RT, Yeh JR. Targeted mutagenesis in zebrafish using customized zinc-finger nucleases. Nat. Protoc. 2009;4:1855-1867.
Yeh JR, Munson KM, Elagib KE, Goldfarb AN, Sweetser DA, Peterson RT. Discovering chemical modifiers of oncogene-regulated hematopoietic differentiation. Nat. Chem. Biol. 2009;5:236-243.
Yeh JR and Peterson RT. Novel Wnt antagonists target porcupine and Axin. Nat. Chem. Biol. 2009;5:74-75
Foley JE*, Yeh JR*, Maeder ML, Reyon D, Sander JD, Peterson RT, Joung JK. Rapid mutation of endogenous zebrafish genes using zinc finger nucleases made by Oligomerized Pool Engineering (OPEN). PLos One 2009;4:e4348.
Sachidanandan C, Yeh JR, Peterson QP, Peterson RT. Identification of a novel retinoid by small molecule screening with zebrafish embryos. PLoS ONE 2008;3:e1947
Dayyani F, Wang J, Yeh JR, Ahn EY, Tobey E, Zhang DE, Berstein ID, Peterson RT, Sweetser DA. Loss of TLE1 and TLE4 from the del(9q) commonly deleted region in AML cooperates with AML1-ETO to affect myeloid cell proliferation and survival. Blood 2008;111:4338-4347.
Yeh JR, Munson K, Chao YL, Peterson QP, MacRae CA, Peterson RT. AML1-ETO reprograms hematopoietic cell fate by down-regulating scl expression. Development 2008;135:401-410.
Yeh JR, Ju R, Brdlik CM, Zhang W, Zhang Y, Shotwell JD and Crews, CM. Targeted disruption of methionine aminopeptidase 2 results in an embryonic gastrulation defect and endothelial cell growth arrest. Proc Natl Acad Sci USA 2006;103:10379-84.
Zhang Y, Yeh JR, Mara A, Ju R, Hines JF, Cirone P, Griesbach HL, Schneider I, Slusarski DC, Holley SA and Crews CM. A chemical and genetic approach to the mode of action of fumagillin. Chem Biol 2006;13: 1001-9.
Yeh JR, and Crews CM. Chemical genetics: adding to the developmental biology toolbox. Dev. Cell 2003;5:11-19
Yeh JR, Mohan R and Crews CM. The antiangiogenic agent TNP-470 requires p53 and p21Cip/Waf for endothelial cell growth arrest. Proc Natl Acad Sci USA 2000;97:12782-12787.
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