Background: One strategy to circumvent problems with conventional chemotherapy is to develop drugs against more specific cancer targets. Another is to use molecularly targeted agent (MTA) combinations to circumvent tumor resistance and increase the therapeutic index. Such synergistically lethal (SL) MTA combinations, however, are not easily predicted based on our rudimentary knowledge of cancer biology and drug action mechanisms. Method: In the first strategy, our aim was to identify genes modulating proliferation and survival in leukemic cell lines (K562, Jurkat, and Raji) using a pooled lentiviral library expressing 27,500 shRNA targeting 5,043 human pathway-associated genes. Cells were transduced by the viral library and collected at several time points. Bar-codes were amplified from genomic DNA and sequenced (Illumina GAIIx). In the second strategy, we have adapted the same approach to combinatorially screen shRNA sequences targeting 40 DNA Damage and Repair (DDR) genes to discover additive and synergistic combinations that generate a synthetic-lethal phenotype. Human mammary epithelial cancer (HMEC) cells were transduced with a 27K shRNA SL DDR lentiviral library comprised of a redundant set of 16 binary shRNA constructs for each possible gene-gene combination (1,600). The library-transduced HMEC cells were grown for ten days, then bar-codes amplified and sequenced (Illumina HiSeq2000). Results: The viability screen with leukemic cell lines identified more than 250 essential genes for each panel of cells. Subsequent validation using single shRNA-expressing constructs showed that in each screen, about 80% of shRNAs identified did indeed lead to cell death when transduced in cells. Analysis of the identified essential genes for known biological interactions revealed several non-random clusters of interacting proteins that provide some insight into signaling pathways and protein networks specific to these cancers. The SL screen in HMEC cells identified 10 SL shRNA pair candidates including known SL shRNA pair PARP1/BRCA1. Additional analysis of lethal combinations indicated redundant, complementary, and compensatory responses in cancer cells. Conclusion: We believe that newly discovered hematopoietic-specific genes represent potentially novel drug targets. Moreover, they can be used to develop and establish both novel cancer targeted therapies and myeloablative conditioning regimens with decreased toxicity. Based on SL screen results in HMEC cells, we believe that comprehensive experimental annotation of SL gene-gene interactions in a wider range of cancer and normal cells will not only predict the most promising synergistic lethal combinations but also allow the development of a new generation of multi-specific, highly effective anti-cancer therapeutics with unique mechanisms of action.

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 876. doi:1538-7445.AM2012-876