Synthetic Lethal Sirna Screening To Identify Novel Combinational Therapies With Topotecan In Neuroblastoma.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Despite advances in multimodal treatment, the outcome of neuroblastoma (NB) is still often fatal for children with advanced-stage disease. The topoisomerase-1 inhibitor topotecan is currently a mainstay for both up-front and salvage regimens for NB patients. In order to improve efficacy of single agent therapy, we used high throughput loss of function siRNA screens to identify genes whose inhibition sensitizes cell lines to topotecan treatment. The translation of these silenced-gene-drug interactions into potent synergistic drug-drug combinations for cancer therapy is the ultimate goal. We screened with the QIAgen human druggable genome kit containing 13910 siRNAs targeting 6878 genes in four NB cell lines, two MYCN-amplified (IMR5 and IMR32) and two non-amplified (SKNAS and NBEB) lines. To reduce the false positive rate mainly due to off-target binding of the seed region of siRNAs (2nd -8th base) to multiple gene transcripts, we performed common seed analysis to eliminate siRNA exhibiting this effect. We then ranked the siRNAs by their activity applying the Redundant siRNA activity (RSA) algorithm and identified 150 genes whose silencing caused a significant decrease in cell survival in combination with topotecan in any of the four cell lines. This list was further filtered to contain only curated drug targets for potential drug combination therapies. Furthermore, Haystack analysis allowed us to predict target genes based on the siRNA off-target effects in our screens. Using this method, 39 additional transcripts whose 3’-UTR regions contain off-target binding sites for the seeds of multiple, active siRNAs were identified. Additionally, we picked transcription factors predicted by Ingenuity Pathway Analysis (IPA) to regulate our target genes. These hits were verified with three additional siRNAs per gene. In 12-point drug dosage response screens, we evaluated 634 siRNAs targeting 213 genes for significant decrease of the IC50 concentration of topotecan. As expected, the prime target genes for therapeutical intervention are within the DNA double-strand breakage repair pathway. In addition, we also identified other biologically interesting targets ranking high on the list. Based on this hit list, we are now selecting the appropriate drugs targeting a particular gene or an affiliated pathway to test them for synergy with topotecan in the four cell lines. Furthermore, expression profiles are generated for the cell lines in the presence of a low and a high topotecan concentration to examine the biology behind the killing mechanism of the drug. A good correlation of an up-regulated expression with a strong sensitizing effect in the siRNA screens will further validate the significance of our target genes. Citation Format: Dominik Bogen, Eugen C. Buehler, Pinar Tuzmen, Rajesh Patidar, David Azorsa, Young K. Song, Hongling Liao, Catherine A. Tolman, Xinyu Wen, Jianbin N. He, Scott E. Martin, Jun S. Wei, Javed Khan. Synthetic lethal siRNA screening to identify novel combinational therapies with Topotecan in neuroblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4398. doi:10.1158/1538-7445.AM2013-4398