University of Texas MD Anderson Cancer Center (complete)

Characterization of PIK3R1 Neomorphic Mutations

The goal of this project was to functionally characterize the most frequent mutation of the PIK3R1 gene and to explore potential therapeutic approaches to target the aberration.

Experimental Approaches

Cytotoxicity Screen

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.

Reverse-Phase Protein Lysate Microarray (RPPA)

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, “https://bioinformatics.mdanderson.org/”). 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.

BaF3 Cell Viability Assay

This assay was used to determine the oncogenic potential of the aberration. Plasmid constructs for expression of the mutants were introduced into BaF3 cells by electroporation. PrestoBlue was then added to assess cell viability.

In vitro Functional Assays

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.

In vivo Tumorigenicity Assay

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)].

High-throughput Screening Identifying Driving Mutations in Endometrial Cancer

Recent advances in next-generation sequencing technology have enabled the unprecedented characterization of a full spectrum of somatic alterations in cancer genomes. Given the large numbers of somatic mutations typically detected by this approach, a key challenge in the downstream analysis is to distinguish “drivers” that functionally contribute to tumorigenesis from “passengers” that occur as the consequence of genomic instability. For this study, the CTD² Center at the University of Texas MD Anderson Cancer Center implemented a systems biology approach to identify driver cancer genes in endometrial cancers.

Experimental Approaches

Whole exome sequencing was performed on 13 endometrial cancers along with matched normal samples to detect somatic alterations with high precision and sensitivity. For validated mutated genes, bioinformatics prioritization was combined with high throughput screening of shRNA and overexpression constructs in a highly sensitive BaF3 cell viability assay to identify candidate driver cancer genes. The BaF3 cell line is a murine bone marrow-derived pro-B-cell line that depends on interleukin-3 (IL-3) for proliferation, but readily becomes IL3-independent in the presence of an oncogene or oncogenic event.

Phenotypic Examination of PIK3CA Allelic Series Using MCF10A Cell Sensor Platform

The CTD² Center at the University of Texas MD Anderson Cancer Center utilized an established and operational MCF10A normal breast epithelial cell model to assess the ability of candidate driver aberrations to promote cell grow in anchorage-independent conditions (soft agar assay) and proliferate in the absence of insulin and epidermal growth factor (EGF). This “sensor” cell platform was used to functionally annotate 29 mutations occurring in PIK3CA, which is commonly mutated in breast and other cancer types, including frequent “hotspot” events (e.g., E545K and H1047R) and other mutations occurring less frequently across the cancer patient population.

Experimental Approaches

Allelic series construction and cell line production: To efficiently engineer PIK3CA expression vectors, they utilized a high-throughput mutagenesis and molecular barcoding (HiTMMoB) platform that permitted simultaneous completion of site-directed mutagenesis and recombination-based cloning of wild-type and mutant PIK3CA cDNAs into the pLenti6.3 V5/DEST (Life Technologies) lentiviral expression vector.  Lentivirus encoding these alleles was used to stably infect MCF10A cells for entry into anchorage-independent growth and proliferation assays as outlined below. 

Anchorage-independent growth assays: Soft agar assays were performed on 6-well plates in triplicate for cells transduced with pLenti6.3 V5/DEST-PIK3CA virus encoding individual wild-type or mutant alleles. Cells were selected for 5 days with 5.0 μg/μl blastocidin, and 1 × 10⁴ cells were mixed thoroughly in cell growth medium containing 0.4% SeaKem LE agarose (Fisher Scientific) in RPMI plus 10% FBS, followed by plating onto bottom agarose prepared with 0.65% agarose in RPMI plus 10% FBS. Each well was allowed to solidify and subsequently covered in 1 ml RPMI plus 10% FBS plus P/S, which was refreshed every 4 days. Colonies were stained with 0.05% (wt/vol) iodonitrotetrazolium chloride (Sigma) and scanned at 1200 dpi using a flatbed scanner for colony quantitation. 

Proliferation assays: Proliferation assays without growth factors (-insulin, -EGF or –insulin alone) were performed on 96-well opaque tissue culture plates. MCF10A cells expressing individual wild-type and mutant PIK3CA alleles were trypsinized and counted. Cells (300 per well) were resuspended in MEGM media (Lonza) with or without growth factors for daily serial time course growth measurements using Cell-Titer-Glo (Promega) following the manufacturer’s recommendations. Values were normalized to Day 0 and then average of all negative controls to calculate fold change at the indicated time points. 

Ba/F3 IL-less survival assays: Ba/F3 lines were created by cloning PIK3CA variants into pHAGE—DEST-IRES-GFP vector and then infecting Ba/F3 cells by lentiviruses encoding individual wild-type or mutant alleles.  Ba/F3 IL-less survival assays were performed by removing medium after viral infection and culturing cells in low IL3 medium (1:10000 dilution) in duplicates. Cell viability was measured after 7 days using Cell-Titer-Glo (Promega) following the manufacturer’s recommendations. 

Tumor formation assays: For PIK3CA  variant tumorigenesis competition assays,1X10⁵ cells from each of the HMLER cell lines expressing GFP, PIK3CA^WT, eight PIK3CA constructs with silent mutations and twenty rare PIK3CA mutants were pooled and re-suspended in a 1:1 solution of Hank’s balanced salts and Matrigel (BD Bioscience) and then subcutaneously injected into female nude animals (Harlan, Indianapolis, IN). Tumors were collected at the tumor burden limit described in our IACUC-approved animal protocol at Baylor College of Medicine. Then gDNA obtained and barcode regions were PCR amplified using T3 and V5 primers. After PCR product purification, samples were analyzed by IonTorrent PGM sequencing machine.

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).

Experimental Approaches

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.

Informer Drug Library Screening Against Patient-derived PDGFRA Mutations

The goal is to identify therapeutic liabilities across a spectrum of PDGFRA mutations found in cancer patients.

Experimental Approaches

The day before treatment, BaF3 cells stably expressing PDGFRA WT or mutants (1×10⁴ cells/100µL) were seeded in 96-well plates in 5% FBS medium with or without IL-3. To test the drug sensitivity of PDGFRA Y288C in low-serum conditions, BaF3 cells expressing PDGFRA Y288C were seeded at a density of 3×10⁴ cells/100µL in 1% FBS medium with or without IL-3. Cells were treated with DMSO or inhibitors (13 nM to 10 µM) in the presence or absence of IL-3 for 72 hr. Cell viability was determined using CellTiter-Blue at 530-nm excitation and 604-nm emission wavelengths, respectively. Each treatment was performed in triplicate.