Cancer Target Discovery in Zebrafish
Pathway rewiring and genetic liabilities in cancer
One of the most fascinating aspects of cancer biology is the drastic change in cellular physiology and extensive pathway rewiring that accompanies the acquisition of oncogenic mutations. Such aberrant rewiring, while necessary to accommodate uncontrolled proliferation and metastatic behavior, might come at a cost for cancer cells, in the form of discrete biologic liabilities. We hypothesize that targeting these cancer-specific vulnerabilities will lead to system collapse and highly selective cancer cell death. In turn, targeting these discrete liabilities may lead to safer and more effective cancer therapies.
Conceptually, a molecular liability in cancer can be refined to a gene whose inactivation modifies the phenotype afforded by the cancer mutation (say, to convert a survival advantage into an apoptotic fate), but has otherwise little or no effect in the presence of wild-type copies of the cancer gene, that is, in healthy normal cells throughout the body. Unfortunately, the lack of an adequate model system in which to identify such genetic modifiers on a systematic basis has long hindered the discovery of rational targets in cancer.
Zebrafish as a model for cancer target discovery
To identify cancer liabilities, we use the zebrafish system, an experimental model that offers the high-throughput capacity and genetic tractability of flies or worms, within a vertebrate system that also faithfully recapitulates the core pathways of human oncogenesis. We recently discovered that zebrafish embryos deficient in the p53 tumor suppressor gene are hyperdependent on the Chk1 protein kinase for survival after radiation-induced DNA damage. Genetic or pharmacologic inhibition of Chk1 triggers a novel ATM/ATR-caspase-2 pathway that bypasses apoptotic blocks conferred by p53 loss or overexpression of the BCL2 oncoprotein. Importantly, this pathway is conserved in mammals and can be triggered by Chk1 inhibitors to restore radiation sensitivity in otherwise treatment-resistant human cancer cells. This study has illustrated how zebrafish genetics can identify cancer liabilities, and by doing so, can identify rational targets for pharmacologic attack in human cancer.
Beyond drug discovery: dissecting the mechanism of action
A core project the lab is to pursue the analysis of the Chk1-regulated apoptotic pathway. Why are p53 mutant cells addicted to Chk1? What is the molecular circuitry underlying the novel apoptotic pathway restrained by the kinase? In particular, what are the genes that act upstream or downstream of the caspase-2 protease? We are addressing these questions by screening for mutations, morpholinos, or drugs that enhance or suppress the lethal effects of Chk1 inhibition in p53 mutant zebrafish embryos, thus identifying new players in the pathway. A subset of these novel components might define novel targets for activating the pathway in tumors, as well as biomarkers for predicting or assessing pathway activity in patients. In pilot screens in zebrafish and cultured cell lines, we have identified five new pathway components whose functional analysis is underway.
Defining novel targets through zebrafish synthetic lethality screens
In a second project, we are searching for additional cancer liabilities through a novel approach that applies the concept of 'synthetic lethality' to in vivo cancer target discovery. Two genes are said to be synthetically lethal if mutation of either alone is viable but simultaneous mutation of both genes leads to death. This concept provides an attractive framework to identify anticancer targets, because targeting a gene synthetically lethal to a cancer lesion should be deadly to cancer cells but otherwise harmless to normal cells. The zebrafish embryo offers an ideal system in which to identify synthetic lethal interactors of cancer genes, because cell survival versus death can be monitored throughout the intact body in real time, within large clutches of isogenic animals that differ only at selected loci. We are screening candidate genes and small-molecule libraries for synthetic lethal interactors of p53, PTEN, RAS, and other commonly altered cancer genes. We recently developed the necessary assays and obtained proof of principle for the approach by identifying chk1 as a synthetic lethal interactor of the Fanconi anemia DNA repair protein, fancd2.