<i>PIK3CA</i> missense mutations promote glioblastoma pathogenesis, but do not enhance targeted PI3K inhibition

<div><p>Background</p><p>Glioblastoma (GBM) is the most common adult primary brain tumor. Multimodal treatment is empiric and prognosis remains poor. Recurrent <i>PIK3CA</i> missense mutations (<i>PIK3CA</i>^<i>mut</i>) in GBM are restricted to three functional domains: adaptor binding (ABD), helical, and kinase. Defining how these mutations influence gliomagenesis and response to kinase inhibitors may aid in the clinical development of novel targeted therapies in biomarker-stratified patients.</p><p>Methods</p><p>We used normal human astrocytes immortalized via expression of hTERT, E6, and E7 (NHA). We selected two <i>PIK3CA</i>^<i>mut</i> from each of 3 mutated domains and induced their expression in NHA with (NHA^RAS) and without mutant <i>RAS</i> using lentiviral vectors. We then examined the role of <i>PIK3CA</i>^<i>mut</i> in gliomagenesis <i>in vitro</i> and in mice, as well as response to targeted PI3K (PI3Ki) and MEK (MEKi) inhibitors <i>in vitro</i>.</p><p>Results</p><p><i>PIK3CA</i>^<i>mut</i>, particularly helical and kinase domain mutations, potentiated proximal PI3K signaling and migration of NHA and NHA^RAS<i>in vitro</i>. Only kinase domain mutations promoted NHA colony formation, but both helical and kinase domain mutations promoted NHA^RAS tumorigenesis <i>in vivo</i>. <i>PIK3CA</i>^<i>mut</i> status had minimal effects on PI3Ki and MEKi efficacy. However, PI3Ki/MEKi synergism was pronounced in NHA and NHA^RAS harboring ABD or helical mutations.</p><p>Conclusion</p><p><i>PIK3CA</i>^<i>mut</i> promoted differential gliomagenesis based on the mutated domain. While <i>PIK3CA</i>^<i>mut</i> did not influence sensitivity to single agent PI3Ki, they did alter PI3Ki/MEKi synergism. Taken together, our results demonstrate that a subset of <i>PIK3CA</i>^<i>mut</i> promote tumorigenesis and suggest that patients with helical domain mutations may be most sensitive to dual PI3Ki/MEKi treatment.</p></div

Helical and kinase <i>PIK3CA</i>^<i>mut</i> potentiate cellular transformation and tumorigenesis.

<p>Only H1047R increased colony formation compared to parental (*, P = 0.03) and <i>PIKCA</i>^<i>WT</i> (ǂ, P = 0.04) NHA (<b>A</b>). H1047R did not affect colony formation of NHA^RAS (P = 0.5). Orthotopic xenografts of GFP, <i>PIK3CA</i>^<i>WT</i>, and <i>PIK3CA</i>^<i>mut</i> NHA^RAS (<b>BC</b>). Median survival of mice with R88Q, E542K, or H1047R <i>PIK3CA</i>^<i>mut</i> NHA^RAS was decreased compared to GFP control tumors (*, P≤0.003). E542K and H1047R <i>PIK3CA</i>^<i>mut</i> also decreased survival compared to <i>PIK3CA</i>^<i>WT</i> (ǂ, P≤0.002) and R88Q <i>PIK3CA</i>^<i>mut</i> (P<0.0001). Fold changes in median survival relative to GFP and <i>PIK3CA</i>^<i>WT</i> NHA^RAS are shown as heatmaps.</p

MEKi inhibits growth and ablates MAPK regardless of <i>PIK3CA</i>^<i>mut</i> status.

<p>Selumetinib IC₅₀ were similar regardless of <i>PIK3CA</i>^<i>mut</i> status in NHA (<b>A</b>), but slightly higher in most <i>PIK3CA</i>^<i>mut</i> NHA^RAS compared to parental cells (*, P≤0.03) (<b>B</b>) (<b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200014#pone.0200014.s011" target="_blank">S11 Fig</a></b>). Fold changes in IC₅₀ relative to parental and <i>PIK3CA</i>^<i>WT</i> lines are shown as heatmaps. Representative immunoblots of control and <i>PIK3CA</i>^<i>mut</i> NHA (<b>C</b>) and NHA^RAS (<b>F</b>) treated with selumetinib for 24 h. Immunoblot quantification (<b>DEGH</b>) demonstrated dose-dependent decreases in MAPK in NHA (<b>D</b>) and NHA^RAS (<b>G</b>) lines. Although proximal PI3K was induced in control and <i>PIK3CA</i>^<i>mut</i> NHA (<b>E</b>), it was only potentiated in GFP and parental NHA^RAS (<b>H</b>) (<b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200014#pone.0200014.s012" target="_blank">S12 Fig</a></b>). Western blots were performed either 1 or 2 times per experiment (Mean = 1.8).</p

<i>PIK3CA</i>^<i>mut</i> potentiate proliferation and migration <i>in vitro</i>.

<p>MTS assays showed that <i>PIK3CA</i>^<i>WT</i> and all <i>PIK3CA</i>^<i>mut</i> decreased doubling times of NHA (<b>A</b>), but not NHA^RAS (<b>B</b>) (*, P≤0.02 vs parental, <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200014#pone.0200014.s005" target="_blank">S5A and S5B Fig</a></b>). <i>PIK3CA</i>^<i>mut</i>, except C90Y, decreased doubling times compared to <i>PIK3CA</i>^<i>WT</i> NHA (^ǂ, P≤0.03). Growth rates were analyzed by comparing k values. Error bars are 95% confidence intervals. <i>PIK3CA</i>^<i>WT</i> and <i>PIK3CA</i>^<i>mut</i>, except C90Y, increased migration of both NHA (<b>C</b>) and NHA^RAS (<b>D</b>) (*, P≤0.04, <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200014#pone.0200014.s005" target="_blank">S5C and S5D Fig</a></b>). E542K and H1047R also potentiated migration compared to <i>PIK3CA</i>^<i>WT</i> NHA and NHA^RAS (^ǂ, P≤0.005). Fold changes in doubling times and migration rates relative to parental and <i>PIK3CA</i>^<i>WT</i> lines are shown as heatmaps.</p

PI3Ki/MEKi synergism <i>in vitro</i> is influenced by <i>PIK3CA</i>^<i>mut</i> and mutant <i>RAS</i>.

<p>Buparlisib and selumetinib inhibited growth and were synergistic in control and PIK3CA^mut NHA (<b>A</b>) and NHA^RAS (<b>B</b>). BLISS showed that synergy was most pronounced with high nanomolar buparlisib and low micromolar/high nanomolar selumetinib in NHA lines. In contrast, synergistic concentrations in NHA^RAS lines were generally most pronounced at both low micromolar buparlisib and selumetinib.</p