RECENTLY FUNDED DEVELOPMENTAL RESEARCH PROJECTS

 

Identification of Targeted Therapies for Ovarian Cancers by Functional Genomics

Carla Grandori, MD, PhD and Masafumi Toyoshima, MD, PhD
Fred Hutchinson Cancer Research Center

Ovarian cancer is the most lethal gynecologic malignancy of the female reproductive system and the fifth leading cause of cancer death in women (1). Genetic changes such as p53 mutation, PIK3CA amplification and KRAS mutation occur with different frequencies in each histological subtype, indicating that distinct pathways may drive each subtype. Recently, amplification and/or overexpression of c-MYC has also been observed at high frequency in ovarian cancers (2). Although ovarian cancers are sensitive to a combination of platinum and taxane-based chemotherapy, development of multidrug resistance, which currently limits the efficacy of the chemotherapy, is frequently observed (3). Therefore, new effective therapies for ovarian cancer are urgently needed. In this proposal, we plan to identify new therapeutic targets for ovarian cancer by a functional genomics approach.

Specific Aim 1. To identify new therapeutic targets for MYC-overexpressed ovarian cancer. (Focused siRNA and chemical screening of MYC overexpressed versus non-overexpressed ovarian cancer cell lines): We previously identified a set of genes that exhibited synthetic lethality with MYC overexpression and validated a subset of these genes in neuroblastoma cells with MYCN amplification. We hypothesize that synthetic lethality genes with MYC overexpression exist in ovarian cancer cells and that the synthetic lethality can be exploited for the treatment of ovarian cancer. We plan to identify genes whose function is essential for the survival of ovarian cancer cells with MYC overexpression by performing a pilot siRNA screen focused on human kinases.

Specific Aim 2. To identify new therapeutic targets for MDR1 overexpressing chemo-resistant ovarian cancer. (Focused siRNA and chemical screening of chemosensitive and resistant isogenic ovarian cancer cell lines): We propose to utilize a functional genomic approach to screen isogenic chemosensitive and resistantovarian cancer cells lines to identify genes that modulate the response to paclitaxel in MDR-overexpressing cells. For both Aim1) and 2), we plan to utilize a library of siRNAs that targets the entire human kinome, as this gene set is most likely to identify druggable targets. Also, to identify potential rational combination treatments, we will perform a parallel chemical screen in both sensitive and resistant lines utilizing a recently acquired collection of oncology FDA approved drugs (NCI 88 oncology drugs).

Expected Outcome: These aims will identify druggable genes and pathways for molecularly defined ovarian cancer cells as well as for chemoresistant ovarian cancer. The results will also indicate rational combination treatments and biomarkers to aid therapeutic choices for molecularly defined patient populations. The success of this approach will also provide the justification for future genome scale screens and/or the application of this approach to other subtypes of ovarian cancers.

 

 

A New Molecular Strategy for Overcoming Chemotherapy Resistance in Cancer

Nelson Teng, MD, PhD, and Paul Wender, PhD
Stanford University

Efficiency of chemotherapy in ovarian cancer is an important issue. The first line therapy is usually a combination of a platinum compound (e.g. cisplatin or carboplatin) and a taxane (e.g. paclitaxel or docetaxel) used as adjuvant therapy after cytoreductive surgery. While the initial response is impressive (e.g. 70-80% for patients with stage III and IV), most patients recur. Recurrence within 6 months is thought to be associated with tumors that are intrinsically resistant to platinum/taxanes (refractory/resistant tumors), while later recurrence is thought to be associated with de novo developed resistance. Currently, it is impossible to predict the initial response; a surrogate marker (platinum-free interval - the time from the end of primary platinum-based chemotherapy to recurrence) is only useful to predict response to the second round of platinum-based therapy and cannot identify patients with refractory/resistant tumors before the failure of initial therapy. Thus, a biomarker for prediction of initial platinum sensitivity can be expected to improve outcomes.

Different sensitivity to therapy is linked to differences in gene activity, so expression patterns can reveal resistant and sensitive tumors. Measuring expression by RNA level is technically challenging in part because RNA is chemically unstable, so that negative result might reflect RNA degradation rather than lack of expression. DNA is a much more stable substrate, so a DNA-based assay can be practical if it provides information similar to expression profiling. Such an assay is indeed possible because a chemical modification – DNA methylation in gene promoters – correlates well with silencing of corresponding genes, so that densely methylated promoters correlate with lack of expression. Methylation can be detected in multiple promoters of the same sample to generate a DNA methylation profile, which will reflect gene silencing and thus serve as a mirror image of the expression profile. We hypothesize that this link can be used to differentiate between resistant and sensitive tumors and to predict outcome of the first line of chemotherapy.

We developed a new method to examine DNA methylation simultaneously in many (currently 56) cancer-related promoters within a single specimen. Using DNA from ovarian cancer and normal ovarian tissues we demonstrated that differences in the methylation profile could be used for tumor detection. Methylation differences were also observed in cell-free plasma DNA, suggesting that DNA methylation in blood can be used for patient monitoring.

In this project we propose to determine differences in DNA methylation profiles of ovarian cancer patients before treatment with platinum and taxanes, and six months after completion of treatment using cell-free plasma DNA. These profiles will then be compared to clinical outcomes and a testable methylation biomarker for platinum resistance will be established. Successful completion of this proof-of-principle study will open the way for creating a minimally invasive test for treatment monitoring and prediction of resistance to platinum.

 

T Cell Receptor Diversity in Ovarian Cancer

Cassian Yee, MD, Edus Warren, MD, and Harlan Robbins, PhD
Fred Hutchinson Cancer Research Center

A means to comprehensively profile the diversity of T cell responses in patients with ovarian cancer would be desirable for both understanding the nature of the extant tumor immune repertoire and for determining whether an immune repertoire signature exists that can be predictive of productive immune responses. The recent development of massively parallel high-throughput DNA sequencing technology (in this case the Illumina Solexa) now provides a strategy for direct measurement of TCR diversity through deep sequencing of CDR3 regions. A single sequencing run on the Illumina (Solexa) Genome Analyzer can generate between 40-60 million clonal sequence reads that are 35-40 nucleotides in length and span the hypervariable regions of the TCRB VDJ or coding regions. With the ability now to evaluate the repertoire with unprecedented level of complexity, the ideal use of this strategy may ultimately be to arrive at a TCR signature that represents early tumor immune detection – i.e. an immunologic profile that provides a means of early detection for patients at risk for ovarian cancer, prognostic information for a patient with ovarian cancer to predict survival or response to treatment (e.g. tumor vaccination). However, such an endeavor is beyond the scope of this pilot project and will require very large numbers of patient and control samples. We propose instead to examine the very specific question of whether the response to a prototypic ovarian-tumor-associated antigen, NY-ESO-1 can used as a model to predict antigen-specific responsiveness by examining the immune repertoire of patients with ovarian cancer.