Discovery of drug-resistant and drug-sensitizing mutations in the oncogenic PI3K isoform p110 alpha

Abstract

p110 alpha (PIK3CA) is the most frequently mutated kinase in human cancer, and numerous drugs targeting this kinase are currently in preclinical development or early-stage clinical trials. Clinical resistance to protein kinase inhibitors frequently results from point mutations that block drug binding; similar mutations in p110 alpha are likely, but currently none have been reported. Using a S. cerevisiae screen against a structurally diverse panel of PI3K inhibitors, we have identified a potential hotspot for resistance mutations (I800), a drug-sensitizing mutation (L814C), and a surprising lack of resistance mutations at the "gatekeeper" residue. Our analysis further reveals that clinical resistance to these drugs may be attenuated by using multitargeted inhibitors that simultaneously inhibit additional PI3K pathway members.

Figure 1. Rescue of PI3K-induced growth inhibition in S. cerevisiae by selective PI3K inhibitors

A: Three-fold serial dilutions of AFS92 and YRP1 yeast strains containing the pURA3-2μ-GAL1 plasmid expressing the indicated p110α-CAAX mutants were spotted onto agar plates of SD −URA media containing glucose, galactose, or galactose with 5 μM PI-103. All plates were incubated at 30°C for 2 days except the YRP1 strains grown on galactose, which were incubated for 6 days. B: Reverse halo assays with YRP1-pURA3-2μ-GAL1-p110αH1047R-CAAX grown on SD −URA +Galactose media, each plate spotted with 10 μl of a 10 mM DMSO stock of PI3K inhibitor. C: Schematic overview of the S. cerevisiae growth inhibition screen used to identify drug-resistant and drug-sensitized p110α mutants.

Figure 2. p110α gatekeeper mutants have reduced catalytic activity

A: Sequence alignment of p110α with protein kinases that display clinical resistance mutations at the gatekeeper position B: ATP binding sites of Abl (PDB code 2G1T) and p110α (PDB code 2RD0). ATP from p110γ co-crystal (PDB code 1E8X) was overlaid onto the apo p110α structure by structural alignment. The gatekeeper residue is colored red and labeled in both structures. C: Six-fold serial dilutions of YRP1-pURA3-2μ-GAL1-p110αH1047R-CAAX strains with the indicated p110α mutations were spotted onto agar plates of SD −URA media containing either glucose or galactose. The plates were incubated at 30°C for 2 days (glucose) and 6 days (galactose). D: Distribution of residues at the gatekeeper position in the human PI3K and protein kinase families.

Figure 3. Affinity pocket residues chosen for saturation mutagenesis

A: p110α active site, with the adenine pocket colored green and the affinity pocket colored red. The p110α apo structure (PDB code 2RD0) was overlaid with ATP (PDB code 1E8X) and PIK-90 (PDB code 2CHX) from p110γ co-crystal structures. B: p110α affinity pocket residues chosen for saturation mutagenesis, shown on the p110α structure (PDB code 2RD0). C: Sequence alignment of the human PI3K family. Affinity pocket residues chosen for saturation mutagenesis are indicated by boxes.

Figure 4. Growth inhibition screen of p110α mutant libraries in S. cerevisiae

A: YRP1-pURA3-2μ-GAL1-p110αH1047R-CAAX mutant library pinned onto SD −URA +Galactose and grown for 5 days at 30°C, and the distribution of colony sizes for that plate, relative to the same array plated on SD −URA +Glucose and grown for 3 days at 30°C (not shown). B: Representative image of pooled active clones from YRP1-pURA3-2μ-GAL1-p110αH1047R-CAAX mutant libraries, pinned onto SD −URA +Galactose with either DMSO control or 5 μM PI-103 and grown for 5 days at 30°C, and the corresponding distributions of colony sizes, relative to the same array plated on SD −URA +Glucose and grown for 3 days at 30°C (not shown). C: Ten-fold serial dilutions of YRP1-pURA3-2μ-GAL1-p110αH1047R-CAAX strains with the indicated p110α mutations spotted onto SD −URA +Galactose media containing the indicated PI3K inhibitors and grown for 5 days at 30°C.

Figure 5. Characterization of yeast screen hits by mammalian expression and in vitro kinase assay

A: The indicated Myc-tagged p110αH1047R mutants were immunoprecipitated from HEK-293T cells and assayed for PI3K activity. Data are represented as mean ± SEM. B: In vitro IC₅₀ values were determined for each p110α mutant against the indicated PI3K inhibitors at 10 μM ATP. Changes in inhibitor sensitivity are shown as the ratio of mutant IC₅₀ / WT IC₅₀. The label N.D. (not determined) indicates that the data from the performed dose response experiment were insufficient to generate an IC₅₀ curve.

Figure 6. Yeast screen hits I800L, I800M, and L814C confer EGF-independent growth to MCF10A cells, and maintain altered inhibitor sensitivities in vivo

A: MCF10A cell lines expressing the indicated p110α mutants were cultured in growth media lacking EGF, and monitored for growth by Alamar Blue assay. Data are represented as mean ± SEM. B and C: Growth of the I800L, I800M, and L814C MCF10A cell lines in media lacking EGF was monitored as in Fig. 6A in the presence of the indicated PI3K inhibitors. Data are represented as mean ± SEM.

Figure 7. Yeast screen hits I800L, I800M, and L814C produce EGF-independent Akt phosphorylation in MCF10A cells, which maintains altered inhibitor sensitivities

A: MCF10A cell lines expressing the indicated p110α mutants were cultured in growth media lacking EGF for 24 hours, and then treated with the indicated combinations of normal growth media (G. M.) and 30 μM PI-103. After 1 hour, the cells were lysed and subject to western blot analysis with the indicated antibodies. B: The indicated MCF10A cell lines were cultured in growth media lacking EGF for 24 hours, and then treated for 1 hour with serial dilutions of the indicated PI3K inhibitors, after which the cells were lysed and subjected to western blot analysis with the indicated antibodies. The PI3K inhibitor concentrations are indicated in μM.

Figure 8. Tolerance to mutation in the p110α affinity pocket

A: Colony size distributions for 384 colony arrays of the indicated p110α mutant libraries, grown in the YRP1 strain on SD −URA +Galactose media as described in Fig. 4A. B: Tolerance to mutation as calculated by Σ(1-x)² from the distributions in Fig. 8A, where x equals relative colony size. These values were converted into heat map values and are shown on the p110α crystal structure (PDB code 2RD0) with the PI3K inhibitor PIK-90 from the p110γ co-crystal structure (PDB code 2CHX) overlaid by structural alignment.