Whole Genome Rnai Screen Identifies Proteasome Inhibition As A Strategy For Nsclc Radiosensitization

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background: Each year, approximately 60,000 patients in the US are diagnosed with locally advanced non-small cell lung carcinoma (NSCLC). The majority is treated with a combination of radiation therapy (RT), chemotherapy +/− surgery. Despite best available therapy, over a third of patients fail locoregionally. The addition of concurrent chemotherapy to RT adds only about a 5% absolute survival benefit. Novel radiosensitizers may improve the therapeutic index of RT. Materials and Methods: We performed whole genome pooled RNAi screens using the Hannon-Elledge library of 74,705 distinct shRNAs directed against over 18,000 genes, in two NSCLC cell lines A549 and NCI-H460. After stable transduction, we passaged the lines for 12 population doublings, and extracted DNA both before and after passaging to compare the representation of each shRNA at each time point. We defined top hits as those genes for which multiple shRNAs decreased in representation by at least 50% during passaging, signifying silencings that were detrimental to proliferation. Results: The top hit of our screen was PSMA1, a subunit of the 20S proteasome; out of six shRNAs, between four and six scored in the two lines in screens performed in triplicate. Bortezomib, an inhibitor of the 20S proteasome, has shown activity against NSCLC in preclinical studies and Phase I/II clinical trials. We observed that bortezomib results in synergistic cytotoxicity in cells treated with fractionated ionizing radiation (IR) in vitro. 10 nM bortezomib resulted in 46% clonogenic survival of unirradiated A549 cells, but only 29% survival of cells treated with 1 Gy x 3 daily fractions compared to cells treated with IR alone. Similar results were observed with MG-132 and with NCI-H460. In soft agar anchorage-independent growth assays, bortezomib resulted in less than 1% colony formation following 1 Gy x 3 daily fractions compared to cells treated with IR alone. In vivo experiments are underway with subcutaneous xenografts in nude mice treated with concurrent CT-guided conformal RT. A retrospective analysis of 443 patients with profiled lung adenocarcinomas showed worse overall survival among patients with tumors showing high proteasome gene expression (median OS 4.2 vs. 6.2 y, 5-year OS 47.5 vs. 61.0%, log rank p = 0.04). Conclusions: Proteasome inhibition emerged as the top drug target of an RNAi screen designed to identify targets with sustained activity over multiple cell generations. Its synergistic impact on NSCLC viability when coupled with fractionated RT should be further explored in preclinical studies and early clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1448. doi:1538-7445.AM2012-1448