Methods for identifying anti-cancer drug targets

Details Methods for identifying anti-cancer drug targets Methods for identifying anti-cancer drug targets

2000-01-20

2003-03-11

Ketter, James (Department: 1632)

Multicellular living organisms and unmodified parts thereof and

Method of using a transgenic nonhuman animal in an in vivo...

C800S008000, C800S013000

Reexamination Certificate

active

06531644

ABSTRACT:

FIELD OF THE INVENTION
The invention concerns genetic engineering and screening methods useful for the identification of gene targets for anti-cancer agents.
BACKGROUND OF THE INVENTION
Cancer is a complex and devastating group of diseases that kills one in five adults in developing countries. Although cancers arise from a wide variety of cells and tissues in the body, there are unifying features of this group of diseases. Cancer is predominantly a genetic disease, resulting from the accumulation of mutations that promote clonal selection of cells that exhibit uncontrolled growth and division. For example, by the time a tumor reaches a palpable size of about one centimeter, it has already undergone about thirty cell doublings, has a mass of approximately one gram, and contains about one billion malignant cells. The result of such uncontrolled growth of tumor cells is the formation of disorganized tissue that compromises the function of normal organs, ultimately threatening the life of the patient. Obviously, methods for prevention, early detection and effective treatment of cancer are of paramount importance.
The past twenty years of research on the mechanistic basis of carcinogenesis have resulted in a revolution in our understanding of the molecular nature of genetic changes that initiate tumor formation. Specific genes have been identified that are frequently mutated in tumor cells, many of which have been grouped into two main classes termed oncogenes and tumor suppressor genes. A few key genes have been identified that are very commonly mutated in a large number of different tumors, such as the oncogene ras and the tumor suppressor genes p53 and Rb. Furthermore, genes that are mutated in tumor cells tend to have functions that cluster in one of the following categories: DNA repair, chromosomal integrity, cell cycle control, growth factor signaling, apoptosis, differentiation, angiogenesis, immune response, and cell migration. Thus, it is clear that there are specific mutations in certain genes that distinguish cancer cells from normal cells.
Despite the fundamental significance of these discoveries, they have not been paralleled by the development of highly selective drugs to treat cancer. This lag in the development of practical therapeutic applications from these discoveries is due to several factors. An ideal chemotherapeutic must selectively kill or block the proliferation of tumor cells without having a deleterious effect on normal growing cells in the body. Most of the genetic alterations found in tumors cells that distinguish them from normal cells are either gain-of-function mutations in oncogenes, which result in increased expression or activity of the gene product, or loss-of-function mutations in tumor suppressor genes, which result in underexpression or lack of activity of the gene product. The protein products of oncogenes having gain-of-function mutations are technically difficult drug targets, due to the lack of effective strategies to selectively inhibit solely the excessive activity of the protein in tumor cells, without deleteriously affecting necessary levels of protein activity in normal cells. Conversely, tumor suppressor genes with loss-of-function mutations are also problematic as drug targets, as it is technically very difficult to develop small molecule drugs that restore the function of a missing or defective protein.
Thus, there is a need for systematic methods to identify highly selective drugs and their cognate targets for killing or inhibiting the proliferation of cancer cells by exploiting the specific genetic alterations that characterize tumor cells. Genetic screening in model organisms offers one possible solution to this challenge. Large-scale, systematic genetic screens in model organisms provide a technically feasible strategy for functionally analyzing nearly all genes and gene products within an organism that relate to a physiological process of interest, and are robust and efficient enough to identify extremely rare genetic mutations. This approach has been used routinely to dissect physiologically important pathways in a number of genetically facile species including the baker's yeast
Saccharomyces cerevisiae
, the nematode
Caenorhabditis elegans
, the fruit fly
Drosophila melanogaster
, the zebrafish
Danio rerio
, and the mouse
Mus musculus
. With respect to using these model organisms for analyzing processes that relate to human disease, each model organism has its own advantages and disadvantages which generally reflect a balance of technical ease of manipulation versus direct relevance to human genetics and physiology. Factors affecting technical ease of use in each system include generation time, cost of growth and maintenance, genome size, and availability of tools for genetic engineering, mutagenesis, gene mapping, and gene cloning. Consequently, the unicellular yeast
S. cerevisiae
offers perhaps the greatest technical facility for genetic screens with a short generation time of only 2 hours, a haploid phase of the life cycle, and a small genome size less than {fraction (1/100)} that of human; however, this system suffers from the fact that baker's yeast is a unicellular organism and many genes and pathways involved in intracellular communication, differentiation, and growth control in humans are completely absent in
S. cerevisiae
. Conversely, as a mammal the mouse is clearly the most similar model organism in genome organization and physiology to human, but suffers from that fact that growth, maintenance, and manipulation of mice is relatively cumbersome, time consuming and expensive. Accordingly, the invertebrate animal model organisms,
C. elegans
and Drosophila, have found favor for large scale genetic screens because they have provided an especially effective comprise between ease of manipulation and functional relevance to human physiology.
Beyond the issues of the technical feasibility of performing large scale genetic screens with model organisms, properly designed genetic screening strategies provide an efficient and logically rigorous method to identify ideal drug targets. Most drugs act by specifically inhibiting the activity of the target proteins with which they associate. And, most mutations generated by mutagenesis in genetic screens are loss-of-function mutations which reduce the expression or activity of the protein products of those genes. Thus, it follows that a loss-of-function mutation can be considered a surrogate for the effect of a drug that specifically inhibits the activity of the protein product of that gene; and further a loss-of-function mutation in a gene which produces a phenotype in vivo that mimics a desired therapeutic effect therefore identifies as a potential drug target the protein product of that mutant gene. So, the challenge in using genetic screens to identify novel drug targets for a particular disease is to carefully design the screen such that the desired loss-of-function mutations, which simulate the ideal therapeutic effect of a drug in vivo, can be readily and efficiently selected by virtue of a specific, easily scored phenotype.
In fact, large scale genetic screens in model organisms have been extensively employed to dissect genetic and biochemical pathways that relate to fundamental aspects of cancer biology. For example, genetic analysis of yeast has proven to be a very valuable approach to identify genes and proteins involved in DNA repair (Friedberg, Micrbiol Rev (1988) 52:70) and control of the cell cycle (Hartwell, J. Cell Biol. (1980) 85:811-822). Genetic analysis in the nematode
C. elegans
has led to important discoveries regarding growth factor signaling, for example through the ras pathway (Kayne and Stemberg, Curr Opin Genet Dev (1995) 5:38-43), and factors involved in controlling apoptosis (Ellis and Horvitz, Cell (1986) 44:817-829). Similarly, large scale genetic screens in the fruit fly Drosophila have also led to the discovery of novel components of cancer associated signal transduction pathways, including the ras (Karim et al., Genetics (1

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