interactions in human cells. This method assists the systematic assessment of the impact of cancer aberrations on proliferation in response to a collection of FAK signaling drugs. Here, we present the approach and use it to query a 70 × 87 drug-gene interaction matrix in breast cancer cells, which allowed the interrogation of over 6 thousand drug-gene pairs. In addition to several previously identified drug-gene interactions, we report a novel mechanism of resistance to PI3K inhibitors, which are currently in clinical trials 20. This is of particular importance given the large fraction of breast tumors with activating mutations in the PI3K pathway 21.
RESULTS A platform for combinatorial fitness screens The first step in building a platform to multiplex large numbers of combinations of genetic and chemical perturbations was to develop a sensitive and quantitative method using molecular barcodes to allow the identification of populations of cells carrying specific genetic modifications within a complex FTY720 S1P Receptor inhibitor mixture. Molecular barcodes are short nontranscribed stretches of DNA, which when integrated into the genomic DNA of a cell line introduce a molecular beacon that can be selectively quantified by PCR. In a mixed population of cells, each containing a unique barcode, the relative number of cells containing a particular vector can therefore be determined by quantification of the barcodes. By pairing genetic modifications of cells with these barcodes, the cellular fitness upon drug treatment can be followed in a multiplexed fashion.
Thus, we first generated one hundred lentiviral vectors carrying unique molecular barcodes flanked by common primer sites for efficient delivery into human cells. We used an isogenic cell line approach to identify the effect of individual genetic changes on cell growth in response to a specific drug, and bypass the difficulty of Muellner et al. Page 2 Nat Chem Biol. Author manuscript, available in PMC 2012 May 1. UKPMC Funders Group Author Manuscript UKPMC Funders Group Author Manuscript comparing heterogeneous cell lines with their multitudes of genetic changes14. Individual genetic modifications were introduced into cells with the same genetic background using overexpression and RNA interference. To systematically analyze the effects of a drug library on this heterogeneous population of cells, each unique barcode was then paired with one genetic modification, so that the cellular fitness upon drug treatment could be followed in a multiplexed fashion.
To quantify the barcodes we used the hybridization-based Luminex xMAP technology, which uses a set of fluorescent microspheres coupled to antisense DNA barcodes that are analyzed by flow cytometry 22. Advantages of this methodology over massive parallel sequencing are that it is fast and the cost per sample is independent of the size of the experiment, making the method highly flexible and affordable. Briefly, barcodes were amplified from genomic DNA by PCR, fluorescently labeled and hybridized to microspheres that are coupled to the antisense barcode sequence. Subsequent analysis of the beads then reveals the relative abundance of each barcode.
We subjected the screening platform to specific tests to determine its reliability and power for identifying drug-gene interactions. The typical dynamic range and linearity of the barcode detection extended over two orders of magnitude and the relative signals were maintained upon reamplification, indicating limited PCR bias Furthermore, the method was highly robust as illustrated by the high correlation coefficients of both technical and biological replicates. Because the quantification method is hybridization-based, we needed to exclude any crosshybridization of barcode sequences as this could obscure the detection of indiv