Breast cancer is the second most common cancer among women in the United States. According to the Centers for Disease Control and Prevention, in 2010, more than 200,000 women were diagnosed with breast cancer, and nearly 41,000 women died from it. A promising approach to treating breast and other cancers is combination therapy, in which drugs with different actions are given together to enhance their anti-cancer effects. These investigators are using small interfering RNAs (siRNAs), which selectively and systematically inhibit the activity of genes, to look for genes in breast cancer cells that can be targeted in combination with an existing class of cancer drugs, called Topoisomerase 1 poisons (TOP1). This research could lead to the development of new drugs to be used in combination with TOP1 poisons for treating breast and other cancers.
Combination therapy is an effective and promising approach toward the treatment of cancer. The investigators are conducting large-scale RNAi screens for novel targets that synergize with TOP1 inhibitors. TOP1-targeted drugs (topotecan and irinotecan) represent an important class of drugs for the treatment of colorectal, ovarian, lung and pediatric cancers. The Pommier laboratory at the National Cancer Institute recently discovered a novel class of non-camptothecin TOP1 inhibitors, the indenoisoquinolines. The indenoisoquinolines have overcome limitations of the camptothecins in pre-clinical models and are among the most advanced anticancer drugs in the non-camptothecin TOP1 inhibitor class. Two indenoisoquinolines are in clinical trials at the NIH Clinical Center in Bethesda, Maryland.
The aim of this project is to use a large-scale siRNA screen to uncover novel pathways that determine the efficacy of TOP1 inhibitors. The investigators are using the triple-negative breast cancer cell line MDA-MB231 cells (previously validated as very effective for siRNA screen) and the clinically-relevant indenoisoquinoline TOP1 inhibitor NSC 725776 (LMP-776) and NSC 724998 (LMP-400) for the high-throughput screen at the NCATS Chemical Genomics Center. Hit validation is being performed using experiments in additional cell lines, and specificity is being determined by evaluating active genes in the context of non-TOP1 inhibitors (e.g., taxol and etoposide). These efforts will help discover new genomic biomarkers to predict the activity of TOP1 inhibitors and personalize therapies in cancer patients. Identification of new pathways may also lead to the development of novel therapies using small molecules or biologicals for the treatment of cancers in combination with TOP1 inhibitors.
National Cancer Institute
Yves Pommier, Ph.D.
Natasha Caplen, Ph.D.
National Center for Advancing Translational Sciences
Madhu Lal-Nag, Ph.D.
Public Health Impact
This study will reveal determinants of TOP1 poison efficacy, providing potential biomarkers for personalized medicine and identifying putative targets for combination therapies.
Martin SE, Wu ZH, Gehlhaus K, et al. RNAi Screening Identifies TAK1 as a Potential Target for the Enhanced Efficacy of Topoisomerase Inhibitors. Curr Cancer Drug Targets, 2011;11(8):976-986.
Zhang YW, Jones TL, Martin SE, et al. Implication of Checkpoint Kinase-Dependent Up-regulation of Ribonucleotide Reductase R2 in DNA Damage Response. J Biol Chem, 2009;284(27):18085-18095.
Among other pathways and targets, these efforts have identified TAK1 and other related genes as important regulators of TOP1 poison-mediated apoptosis.
Screening Protocol to Identify Determinants of TOP1 Poison Activity in Breast Cancer Cells
After optimization of transfection and assay conditions, a druggable genome siRNA library targeting H 7,000 human genes (4 siRNAs per gene) was screened both in the absence and presence of TOP1 poisons. Transfections were performed in MDA-MB-231 breast cancer cells. Knockdown was allowed to proceed for 48 hours before treatment with TOP1 poison for an additional 72 hours. Informatic analysis of this data identified more than 100 putative sensitizers. Rigorous follow-up resulted in the confirmation of numerous confident hits, including known DNA repair related genes such as ATR. Ongoing studies are being performed with validated targets in additional cell lines and in the context of non-TOP1 inhibitors (e.g., taxol and etoposide).
Follow-up dose response analysis with several siRNAs confirms that the down-regulation of ATR significantly enhances TOP1 poison activity in MDA-MB-231 breast cancer cells.