University of Texas MD Anderson Cancer Center (UT-MDACC): Systematic Functional Annotation of Somatic Mutations in Cancer
The CTD2 Center at the University of Texas MD Anderson Cancer Center utilized a functional annotation of mutations and fusions found in human cancers using two cell models, Ba/F3 (murine pro-B suspension cells) and MCF10A (human non-tumorigenic mammary epithelial cells).
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To assess the function of the mutation candidates, two growth-factor-dependent cell models, Ba/F3 and MCF10A cell models, were used. Both cell types stop proliferating and die in the absence of the required factor(s). The rationale is that a “driver” mutation will confer survival and proliferation advantages to the cells in the absence of required growth factor(s), but “passenger” mutations will not. The mutation candidates were put into both cell models with various types of controls. First, two experimental negative controls (GFP, mCherry, or Luciferase) and three experimental positive controls (PIK3CA wild-type, M1043I, and H1047R) with different activities (i.e. wild-type < M1043I < H1047R) served as technical controls to check if the experiments performed well. Second, mutations and their corresponding wild-type counterparts were assessed in parallel in the same experiment, the latter of which determined the basal activity of the genes in the cell models. Third, for selected genes, silent and literature-reported driver mutations were used as additional controls to determine the basal and activated activities of the genes, respectively. In total, 1,049 mutations were tested in batches, with up to 33 mutations per batch. In each experiment (i.e. batch), the set of five experimental controls (two negative and three positive) and corresponding wild-type clones were included. Additional silent and gene-specific positive mutations were also included, if available.
For each experiment, pHAGE constructs of mutants and wild-type genes were freshly prepared from a single colony and used for generating lentivirus for Ba/F3 and MCF10A transduction. Lentivirus was generated in the LentiX-293T cells by transfecting the pHAGE and two packaging plasmids (psPAX2 and pMD2.G). The medium of the transfected cells was refreshed at 16 hours post-transfection. The virus was harvested at three days post-transfection by filtering with 0.45 µM filter. Ba/F3 cells (0.6 million cells) were transduced by spinoculation (centrifugal inoculation) at 1000× g for three hours in the presence of polybrene (final concentration: 8 µg/ml). After spinning, cells were resuspended in the Ba/F3 assay medium in a 24-well plate format. For MCF10A cells, 5,000 cells were seeded into 96-well plates one day before transduction and transduced by spinoculation at 906× g for two hours in the presence of polybrene (final concentration: 2.7 µg/ml). The medium was refreshed after spinoculation with the MCF10A assay medium. Transduced cells were incubated at 37°C for three weeks. Cell viability of Ba/F3 and MCF10A cells were measured at four time points (at intervals of three or four days) during the three-week assay period. The functional annotations of mutations were based on a comparison to the corresponding wild-type clones.
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For questions, please contact Prahlad Ram.