<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

Selectivity, Cocrystal Structures, and Neuroprotective Properties of Leucettines, a Family of Protein Kinase Inhibitors Derived from the Marine Sponge Alkaloid Leucettamine B

DYRKs (dual specificity, tyrosine phosphorylation regulated kinases) and CLKs (cdc2-like kinases) are implicated in the onset and development of Alzheimer’s disease and Down syndrome. The marine sponge alkaloid leucettamine B was recently identified as an inhibitor of DYRKs/CLKs. Synthesis of analogues (leucettines) led to an optimized product, leucettine L41. Leucettines were cocrystallized with DYRK1A, DYRK2, CLK3, PIM1, and GSK-3β. The selectivity of L41 was studied by activity and interaction assays of recombinant kinases and affinity chromatography and competition affinity assays. These approaches revealed unexpected potential secondary targets such as CK2, SLK, and the lipid kinase PIKfyve/Vac14/Fig4. L41 displayed neuroprotective effects on glutamate-induced HT22 cell death. L41 also reduced amyloid precursor protein-induced cell death in cultured rat brain slices. The unusual multitarget selectivity of leucettines may account for their neuroprotective effects. This family of kinase inhibitors deserves further optimization as potential therapeutics against neurodegenerative diseases such as Alzheimer’s disease