B162
Viability screens in cancer cells with pooled shRNA libraries identify potential therapeutic targets and synthetic lethal interactions
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, genes modulating proliferation and survival in oncogenic cells have been identified using pooled lentiviral-based libraries expressing many thousands of shRNAs. In the second strategy, we have adapted the approach to combinatorially screen shRNA sequences targeting hundreds of genes to discover additive and synergistic combinations that generate a synthetic-lethal phenotype.
Results
A viability assay with leukemic cell lines transduced with pooled shRNA libraries targeting thousands of genes identified a few hundred 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. Analysis of the lethal combinations from the SL screen 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, 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.
Acknowledgements
Karim Hyder and Paul Diehl, for poster and figures.