PI3K inhibition during T-cell culture–enhanced TCR and MEK/ERK signaling with alterations in epigenetic regulators that promote T-cell stemness. T cells from patients with CLL (n = 9) were cultured with or without 300 nM duvelisib administered to cultures every 3 days and were harvested after 3, 9, or 15 days of culture. Cells were stimulated with anti-CD3/CD28 beads on days 1 and 9 of culture, with day-9 samples harvested 90 minutes after restimulation with anti-CD3/CD28 beads and addition of duvelisib. Hypothesis-generating NanoString analysis was first performed and followed-up by confirmatory western blot experiments. (A) Differential expression of metabolically relevant genes in duvelisib-cultured cells compared with control cells at day 9 of culture. Changes in messenger RNA levels for 102 genes met Benjamini-Hochberg (BH) false-discovery thresholds (red). The 5 genes with the greatest fold increase or decrease are labeled. (B) Pathway scores based on gene expression levels for TCR, costimulatory, MAPK, and mTOR signaling pathways. Western blot of cell lysates probing proteins related to proliferative pathways (C) and epigenetic regulatory pathways (D). Quantifications of select western blots (SIRT1, FOXO1, and TCF1/7) normalized to control (E) with ancillary flow cytometric analysis of frequencies of TCF1/7 expressing cells (F) at day 15 of culture. Results reproduced across 3 patients with CLL with quantitative results shown in the supplemental data (supplemental Figure 5). *P < .05; **P ≤ .01; ***P ≤ .001.