The PIM1 inhibitor, SGI-1776, inhibits proliferation and induces apoptosis of leukemia cells. (A) Survival curve of TgERG leukemia cells of 2 TgERG mice treated with escalating doses of SGI-1776 for 3 days (mean ± SEM, n = 4). (B-C) Dose-dependent apoptosis of TgERG leukemia cells treated with SGI-1776 for 24 hours. (B) Flow cytometry analysis of cells stained with Annexin V and propidium iodide 24 hours after treatment with either vehicle or 5 µM SGI-1776. (C) Summary of the percentage of Annexin V–positive cells as a function of SGI-1776 dose (mean ± SEM, n = 3) (D) PIM1 expression levels by quantitative RT-PCR (normalized to β-actin) in human leukemia cell lines expressing high (CMY) low (CMK) or no ERG (Jurkat, mean ± SEM, n = 3). (E) Survival curve of CMK, CMY, and Jurkat cells treated with escalating doses of SGI-1776 for 2 days (mean ± SEM, n = 4, 1-way analysis of variance P = .026). (F) Percentage of Annexin V–positive CMK, CMY, and Jurkat cells treated for 24 hours with escalating doses of SGI-1776 (mean ± SEM, n = 3). (G-H) Pim1 knockdown inhibits growth of TgERG leukemia cells. TgERG leukemia cells were transfected with Pim1 siRNA and 3 days later collected and used for either protein extraction to determine PIM1 levels (G, vertical lines indicate repositioned gel lanes) or counted to determine live cell number (H, mean ± SEM, n = 3). (I) Combined high expression of ERG and PIM1 confers a poor prognosis in AML patients. Kaplan-Meier plots comparing survival in 4 groups of human AML⁴⁴  classified by PIM1 and ERG expression (see supplemental Figure 8). Significance levels (log-rank P values) are given with respect to the PIM1/ERG high subpopulation.