Lentiviral ORF Screen for Drug Resistance

The arrayed, lentiviral ORF screen was performed as previously described26 (link). Effects of individual ORFs on drug resistance were determined by measuring differential viability (ratio of raw viability in 1 µM PLX4720: control) and subsequent normalization to an assay-specific positive control, MEK1^DD. Secondary screens were performed with the top nine candidate ORFs in 96-well format in A375 and SKMEL28 cells. Prioritization was accomplished via generation of a GI₅₀ for each ORF across a multi-point PLX4720 concentration range in both cell lines. The effects of identified resistance ORFs on MAPK pathway activation were demonstrated using both biochemical and cell biological approaches. Cell line copy number data was obtained as previously described27 (link). Detailed descriptions of all procedures are included in Materials and Methods.

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Johannessen C.M., Boehm J.S., Kim S.Y., Thomas S.R., Wardwell L., Johnson L.A., Emery C.M., Stransky N., Cogdill A.P., Barretina J., Caponigro G., Hieronymus H., Murray R.R., Salehi-Ashtiani K., Hill D.E., Vidal M., Zhao J.J., Yang X., Alkan O., Kim S., Harris J.L., Wilson C.J., Myer V.E., Finan P.M., Root D.E., Roberts T.M., Golub T., Flaherty K.T., Dummer R., Weber B., Sellers W.R., Schlegel R., Wargo J.A., Hahn W.C, & Garraway L.A. (2010). COT/MAP3K8 drives resistance to RAF inhibition through MAP kinase pathway reactivation. Nature, 468(7326), 968-972.

Publication 2010

Assay Biological Cell Cell line Drug resistance Orfs Plx4720

Corresponding Organization :

Other organizations : Massachusetts Institute of Technology, Broad Institute, Harvard University, Dana-Farber Cancer Institute, Massachusetts General Hospital, Genomics Institute of the Novartis Research Foundation, University of Zurich

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Variable analysis

independent variables

ORFs (individual)
PLX4720 concentration range

dependent variables

Differential viability (ratio of raw viability in 1 µM PLX4720: control)
GI50 for each ORF across a multi-point PLX4720 concentration range
MAPK pathway activation

control variables

Assay-specific positive control, MEK1DD

controls

Positive control: MEK1DD
Negative control: Not explicitly mentioned

Annotations

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