GS-1101 selective inhibition of PI3Kδ inhibits Akt phosphorylation. (A) PI3K isoform expression is confirmed in HL cell lines. Proteins from 106 cells were separated by SDS-PAGE, transferred onto PVDF membranes, and analyzed by immunoblotting using antibodies specific for the α, β, δ, and γ isoforms. Purified recombinant PI3K proteins were used as controls (data not shown). Antiactin antibodies were used to assess equal loading of the samples. (B) Expression of PI3K isoforms in HL tumor samples. The number and percentage of cases positive for each isoform are shown. The intensity of expression is indicated as negative (−), weak (+), or strong (++). PI3Kα and δ were the isoforms more frequently expressed in tumor samples; they were detected in 97.2% and 80.6% of the cases, respectively. Strong expression of PI3Kα and δ was detected in 37.5% and 26.4% of cases, respectively. Protein expression was scored as −, +, or ++ depending on the staining signal intensity. Expression of each isoform in the Hodgkin RS cells was compared with that seen in positive control; if higher or equal, then expression of each p110 isoform was considered ++; and if lower, then expression was considered + (supplemental Figure 1). (C) GS-1101 selective inhibition of PI3Kδ reduces constitutive Akt phosphorylation in HL cell lines. Serum-starved cells were incubated for 2 hours with GS-1101. Proteins in cell lysates were resolved by SDS-PAGE electrophoresis, transferred onto PVDF membranes, and probed with appropriate antibodies. (D) GS-1101 selective inhibition of PI3Kδ overcomes phosphorylation of Akt in HL cell lines cocultured with HS-5 stromal cells. HL cells were cultured for 24 hours at 37°C with or without HS-5 stromal cells and GS-1101. Proteins from HL cell lysates were resolved by SDS-PAGE electrophoresis, transferred onto PVDF membranes, and probed with appropriate antibodies. Each bar histogram is an average of 4 independent experiments. *P < .001 (t test).
