A Genome-Wide Knockout Screen to Identify Genes Involved in Acquired Carboplatin Resistance
Abstract
Rationale and Objective: Carboplatin (abbreviated CBDCA) is one of most effective drugs for the treatment of ovarian cancer, and almost all patients receive this drug as part of initial therapy. However, almost all patients become resistant to the drug as their therapy progresses. We know that this is the result of genetic changes in the tumor, but what genes are involved is unknown. It is the overall goal of this project to identify the genes that when disabled by mutation in human ovarian carcinoma cells result in resistance to CBDCA. It is our hypothesis that this can be most efficiently accomplished by using a novel and very powerful technique called CRISPR-cas9 that allows us to disable individual genes in ovarian cancer cells in culture. A library of retroviruses has now been created that whose elements express the components of the CRISPR-cas9 system and can simultaneously target >18,080 genes. This library (called the GeCKO library) can be used to disable a different gene in each of thousands of tumor cells using a pooled approach. The disabling process inserts a genetic bar code into the DNA so that the disabled gene can be identified from the pool using DNA sequencing. Treatment of the population of tumor cells with CBDCA will result in the survival of only those cells in which a disabling mutation results in resistance. DNA sequencing of the surviving population can then disclose the identity of the genes of interest. Central Problem in Ovarian Cancer Addressed by This Project: While most patients initially respond to therapy containing CBDCA, the tumor gradually becomes resistant and the majority of patients usually relapse and die of their disease despite continued treatment. While prior studies have established that resistance is largely the result of genetic changes, we do not know what genes are involved and thus we are unable to develop strategies to prevent the emergence of resistance or overcome it once it becomes established. The long-term goal of this effort is to find ways of preventing the development of resistance during treatment or reversing it once established. How Research Is Relevant to Ovarian Cancer Research Program (OCRP) Vision and Mission: The OCRP definition of innovative ideas includes those that will provide new paradigms, insights, technologies, or application that have the potential to improve the treatment of individuals with ovarian cancer. This project fulfills each of these criteria. By using a novel and extraordinarily powerful new technique, it will provide new basic information about the genes involved in CBDCA resistance. This will permit investigation of the mechanisms by which these genes control drug responsiveness, which will lead to new insights and new hypotheses and paradigms of how the tumor cells learn to become resistance to treatment. It is our hope that this understanding will eventually result in the development of new strategies for preventing the emergence of resistance or overcoming it once established. Impact: This is a high-risk, high-reward project. The successful identification of CBDCA resistance genes will open up new lines of investigation and provide basic information about the fundamentals of how resistance to CBDCA emerges. This information is absolutely essential to the development of novel strategies for the more effective treatment of this disease with CBDCA. Since CBCDA is also extensively used for the treatment of lung, breast, head and neck, skin, testicular, and a variety of other cancers, any information discovered in our study of ovarian cancer cells is likely to be immediately applicable to the management of CBDCA resistance in these other tumors as well. In some cases, the mutation of a gene that causes resistance to one drug results in hypersensitivity to another drug, a concept called synthetic lethality. Identification of the genes that when disabled result in resistance to CBDCA will permit us to then test
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Apr 04, 2016
- Source ID
- W81XWH1510063
Entities
People
- Stephen Howell
Organizations
- United States Army
- University of California, San Diego