Figure 3.

Acute myeloid leukemia drives an IL-3-mediated overexpression of BCL2 in non-malignant Lin^- Sca1⁺ CD117⁺ which is reversed by IL-3 neutralization, and restores neutrophil and monocyte counts in combination with venetoclax in acute myeloid leukemia-engrafted mice. (A) Schematic depicting potential mechanisms for BCL2 transcription in non-malignant Lin^- Sca1⁺ CD117⁺ (LSK). (B) 5x10⁴ LSK were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin streptomycin (PenStrep), and treated with 100 ng/mL IL-3, IL-6, IL-1β or TNF for 24 hours (hr). Real-time qPCR assessed BCL2 gene expression in treated LSK (N=4) compared to non-treated LSK (N=4). (C) 5x10⁴ LSK were next cultured with MN1-conditioned media and treated with 5 mg/mL anti-IL-3 (IL-3 neutralizing antibody MP2-8F8; Bio X Cell, NH, USA) for 48 hr. Real-time qPCR assessed BCL2 gene expression in LSK cultured with MN1-conditioned media and anti-IL-3 antibody (N=6), LSK cultured with MN1-conditioned media only (N=6) and LSK-only controls (N=6). (D) 3x10⁵ MN1 and MEIS1/HOXA9 cells were treated with 5 μg/mL anti-IL-3 for 48 hr. Real-time qPCR assessed BCL2 gene expression in anti-IL-3-treated AML cells (N=5) compared to non-treated AML cells (N=6). (E) Experimental schematic. 2.5x10⁶ MN1 cells were injected into 12-14-week old male non-conditioned C57BL/6 mice. At day 10, 100 mg/kg venetoclax (Ven) was administered by oral gavage alongside 50 mg/kg anti-IL-3 by intraperitoneal injection, both daily for seven days. Peripheral blood (PB) was assessed by flow cytometry following sacrifice. (F) Cell counts for B cells, T cells, monocytes and neutrophils per mL of PB in MN1-engrafted mice treated with Ven plus anti-IL-3 antibody (N=7) compared to Ven only (N=7). All data in (B-D) and (F) are represented as median + interquartile range. *P<0.05, **P<0.01, ****P<0.0001 by Mann-Whitney U test or Kruskal-Wallis test for multiple comparisons.