University of texas md anderson cancer center characterization of pik3r1 neomorphic mutations office of cancer genomics endometrial biopsy side effects

A high-throughput BaF3 differential cytotoxicity screen was used to examine whether specific mutations in PIK3R1 alter sensitivity towards a collection of 145 compounds targeting major signaling pathways. The normally interleukin 3 (IL3)-dependent BaF3 cells were rendered IL3-independent by stable expression of the mutants. Importantly, growth inhibition or cell death induced by inhibitors can be rescued by exogenous IL3, providing an internal control, or “counterscreen” for the cytotoxic activity of the compounds. The day before treatment, stable construct expressing BaF3 cells were seeded in 96-well plates in medium with or without IL3. Cells were treated with DMSO or inhibitors (1 nM to 10 μM) in the presence or absence of IL3 for 72 hours. Cell viability was determined using PrestoBlue (Promega, Madison, WI).

Two independent experiments, each in duplicate, were performed. Some of these compounds were also tested in a panel of parental endometrial or ovarian cancer cells.

To determine whether pathway activation correlates with drug sensitivity, reverse-phase protein arrays (RPPA) were performed with the BaF3 stable cells in the RPPA Core Facility in MD Anderson Cancer Center. Protein lysates were arrayed on nitrocellulose-coated slides, which were then probed with validated primary antibodies plus biotin-conjugated secondary antibodies. The signal obtained was amplified and visualized. The slides were scanned, analyzed, and quantified to generate spot intensity. Each dilution curve was fitted with a logistic model (“Supercurve Fitting” developed by the Department of Bioinformatics and Computational Biology in MD Anderson Cancer Center, “”). This fits a single curve using all the samples (i.e., dilution series) on a slide with the signal intensity as the response variable and the dilution steps are independent variable. The fitted curve is plotted with the signal intensities both observed and fitted on the y-axis and the log2-concentration of proteins on the x-axis for diagnostic purposes. The protein concentrations of each set of slides were then normalized by median polish, which was corrected across samples by the linear expression values using the median expression levels of all antibody experiments to calculate a loading correction factor for each sample.

Endometrial cancer cells stably expressing the PIK3R1 mutations were subjected to in vitro functional assays to assess the tumorigenic effects of the mutations and to investigate whether the effects could be blocked by inhibitors. The assays measured cell proliferation indicated by BrdU incorporation and cell apoptosis indicated by DNA fragmentation.

The assay was used to evaluate the antitumor effect of inhibitors in vivo. All animal experiments were approved by MDACC’s Institutional Animal Care and Use Committee. Animal care was followed according to Institutional guidelines. Endometrial cancer cells or parental OVK18 ovarian cancer cells were injected subcutaneously into female athymic (nu/nu) mice (National Cancer Institute, Fredrick, MD). Treatments began on day 21 post-implantation. Tumor measurements were taken and total tumor volume was estimated. Antitumor effects are expressed as tumor growth inhibition (TGI; treated versus control), calculated by the equation, %TGI=100 − [100 × (T − T0)/(C − C0)].