AREG activates PI3K/AKT/mTOR pathway and compromises HSC quiescence. (A) Stroma-derived AREG activates PI3K/AKT/mTOR pathway in WT Lin^–c-kit⁺ cells. WT Lin^–c-kit⁺ cells were cocultured with BM CD45^–LepR⁺ cells from LepR-Cre;Brca2^+/+ or LepR-Cre;Brca2^fl/fl mice in the presence of IgG, anti-AREG, or cetuximab for 5 days. Immunoblotting was then performed using 150 000 cells per lane to analyze phosphorylated (p) EGFR and total EGFR; p-AKT and total AKT; p-mTOR and total mTOR; and β-actin. (B) Schematic presentation of the experimental design. LepR-Cre;Brca2^+/+ and LepR-Cre;Brca2^fl/fl mice were treated with 3 doses of IgG, anti-AREG, or cetuximab (50 μg each; every other day). WBMCs were isolated from mice 120 minutes after the last treatment for flow cytometry analysis of PI3K/AKT/mTOR activation in SLAM cells or HSC quiescence. (C) Anti-AREG or cetuximab suppresses PI3K/AKT/mTOR activation. LepR-Cre;Brca2^+/+ and LepR-Cre;Brca2^fl/fl mice were treated with IgG, anti-AREG, or cetuximab, as described in panel B, followed by flow cytometry analysis for phosphorylation of EGFR, AKT, and mTOR in SLAM cells 120 minutes later. Representative histogram (top) and quantifications (bottom) are shown (n = 6). (D-E) Anti-AREG or cetuximab treatment improves HSC quiescence and limits HSC proliferation. LepR-Cre;Brca2^+/+ and LepR-Cre;Brca2^fl/fl mice were treated with IgG, anti-AREG, or cetuximab, as described in panel B, followed by flow cytometry analysis for HSC quiescence by Hoechst/PY (D) or HSC proliferation by 5-bromo-2′-deoxyuridine or 4′,6-diamidino-2-phenylindole (E); n = 6 to 8. Results are presented as mean ± standard deviation of 3 independent experiments. Two-way ANOVA was performed to compare between genotypes (Brca2^+/+ vs Brca2^fl/fl) and treatments (IgG, α-AREG, or cetuximab), followed by t tests for comparing interested groups in panels C-E. ∗P < .05; ∗∗P < .01. KO, knockout; MFI, mean fluorescence intensity.