Interleukin-4 Is a Negative Regulator of Acute Myeloid Leukemia Cells

Acute myeloid leukemia (AML) is the most common acute leukemia among adults and is associated with poor prognosis. Hence, there is a strong need to develop new therapeutic strategies towards improved treatments.

In this study, we performed an in vitro cytokine screen designed to identify selective negative regulators of leukemia cells. By mixing murine AML cells driven by the MLL-AF9 fusion gene in a dsRed transgenic background and non-dsRed normal bone marrow cells, we screened 114 murine recombinant proteins in a 96-well format and assessed their effect on cell expansion during 72 hours. Both the leukemia cells and the normal bone marrow cells were enriched for primitive cells by isolating C-kit positive cells.

In the screen, Interleukin 4 (IL-4) emerged as the top hit, as it caused the strongest depletion of leukemia cells while maintaining the normal bone marrow cells. Further validations demonstrated that the negative effect induced by IL-4 on leukemia proliferation was obtained even in the presence of cytokines such as IL-3, which alone strongly stimulates leukemia cell growth and survival. In contrast to the negative effects of IL-4 on leukemia cells, IL-4 alone was validated to be sufficient to maintain cell survival of normal bone marrow cells in short term cultures.

To assess the mechanisms by which IL-4 depletes leukemia cells, we carried out an apoptosis analysis upon stimulating the leukemia cells with IL-3 or a combination of IL-3 and IL-4. The percentage of late apoptotic cells was significantly increased by IL-4, suggesting that this cytokine pushes the cells into an apoptotic state.

To explore which signaling pathway is responsible for the IL-4-induced effect in leukemia cells, we performed phospho-flow analysis on Stat6, one of the main effectors downstream of the IL-4 receptor. IL-4 stimulation induced strong phosphorylation of Stat6, suggesting that this pathway may be responsible for the IL-4-induced effects in leukemic cells.

To address whether IL-4 stimulation of leukemia cells affects leukemia initiating cells, also termed leukemia stem cells, we next stimulated leukemia cells ex vivo for 3 days with IL-4, IL-3, or no cytokines, followed by transplantation of the cells to sublethally irradiated mice (n= 6 in all groups). Mice receiving IL-4 treated cells had a significant prolonged survival (p-value= 0.0003, median= 37 days) than those receiving cells treated with IL-3 or non-treated cells (median= 27 and 30 days, respectively). We next performed an in vivo treatment experiment with intra-peritoneal injections of IL-4 at a dose of 60 μg/Kg/day. Mice were treated daily for 10 days and sacrificed at day 13. Mice treated with IL-4 (n= 6) had lower percentage of MLL-AF9 cells both in blood (36±4.5%) and bone marrow (89±2,8%) compared to control mice (53±4.1% in blood, 96±0.6% in bone marrow, n= 6). We then treated mice with escalating doses of IL-4 (15, 30, and 60 μg/Kg/day, n= 5) and PBS as control (n= 7) and monitored their survival. Only the group receiving 60 μg/Kg/day had a significant prolonged survival (p-value= 0.0077, median= 26 days) when compared to the control group (median= 23 days).

In summary, these findings demonstrate that IL-4 is a negative regulator of AML cells in this murine MLL-AF9 leukemia model. Future experiments will explore the mechanisms underlying these effects and its relevance for human disease.