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. 2014 Nov;28(11):2257-60.
doi: 10.1038/leu.2014.212. Epub 2014 Jul 17.

Cytokines secreted by bone marrow stromal cells protect c-KIT mutant AML cells from c-KIT inhibitor-induced apoptosis

P M Gordon  1 , Stuart Dias  1 , D A Williams  1
Affiliations

Cytokines secreted by bone marrow stromal cells protect c-KIT mutant AML cells from c-KIT inhibitor-induced apoptosis

P M Gordon et al. Leukemia. 2014 Nov.
No abstract available

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Bone marrow stromal cell conditioned media attenuates the therapeutic effect of c-KIT inhibitors and down-regulates c-KIT expression
Kasumi-1 (A) and SKNO-1 (B) leukemia cells were treated with c-KIT inhibitors (1 μM) for 48 hours in either regular media, HS-5 bone marrow stromal cell line conditioned media, or conditioned media from primary bone marrow stromal cells from two independent donors (Lonza and Stem Cell Technologies) and assessed with the CellTiter-Glo viability assay (Promega). The data are normalized to the untreated controls and are the mean +/− SEM from three independent experiments. In (A), p ≥ 0.0001 for nilotinib in regular media when compared with all other conditioned medias. For imatinib, p ≥ 0.0001 for HS-5 conditioned media and p ≥ 0.05 for both primary bone marrow stromal cell conditioned medias. In (B), p ≥ 0.0001 for nilotinib and imatinib in regular media when compared with all other conditioned medias. Kasumi-1 (C) and SKNO-1 (D) cells were treated with c-KIT inhibitors (1 μM) in either regular or HS-5 conditioned media and cell number determined by trypan blue exclusion at 0, 24, and 48 hours. The data are the mean +/− SEM from three independent experiments. For both Kasumi-1 and SKNO-1 p<0.0001 when comparing nilotinib treated and untreated cells in regular media at 24 and 48 hours. (E) Western blot analysis of whole cell lysates prepared from Kasumi-1 cells grown in either regular (lanes 1–6) or HS-5 conditioned media (lanes 7–12) in the presence or absence of nilotinib. Blots were probed with antibodies specific to c-KIT (Cell Signaling Technology), phospho-c-KIT (Tyr719) (Cell Signaling Technology), and β-actin (Sigma). This is a representative blot from two independent experiments. (F) Cell surface expression of c-KIT in either regular media, HS-5 conditioned media, or regular media supplemented with SCF 20 ng/mL was determined by flow cytometry with an APC-conjugated c-KIT antibody (eBiosciences). The data are normalized to the regular media control and are the mean +/− SEM from three independent experiments. (G) Quantitative RT-PCR analysis of total RNA isolated from Kasumi-1 and SKNO-1 cells after culturing for 48 hours in either regular or HS-5 conditioned media. c-KIT mRNA levels were normalized to GAPDH. Reactions were performed in triplicate and the data are the mean +/− SEM from three independent experiments.
Figure 2
Figure 2. Cytokines attenuate the therapeutic effect of c-KIT inhibitors. (A)
The proliferation of Kasumi-1 leukemia cells was assessed with the CellTiter-Glo viability assay following treatment with the specified c-KIT inhibitor (1 μM) for 48 hours in regular media supplemented with 10 ng/mL G-CSF, SCF, or IL-6. *=p<0.05 (imatinib) and **=p<0.01 (nilotinib) when comparing regular and G-CSF supplemented media. (B) The proliferation of Kasumi-1 cells was assessed with the CellTiter-Glo viability assay following treatment with the specified c-KIT inhibitor (1 μM) for 48 hours in either regular media, regular media supplemented with a G-CSF receptor neutralizing antibody (1 μg/mL; R&D Systems), 0.05% HS-5 conditioned media, or 0.05% HS-5 conditioned media supplemented with a G-CSF receptor neutralizing antibody. The data are normalized to the untreated controls and are the mean +/− SEM from three independent experiments. For both imatinib and nilotinib p ≥ 0.001 when comparing HS-5 conditioned media against all other conditions. The SKNO-1 (C) and HMC1.1 (D) c-KIT dependent leukemia cells lines were treated with a c-KIT inhibitor (1 μM for SKNO-1 and 200 nM for HMC1.1) for 48 hours in regular media supplemented with 10 ng/mL G-CSF, SCF, GM-CSF, or IL-3 and assessed with the CellTiter-Glo viability assay. The data are normalized to the untreated controls and are the mean +/− SEM from three independent experiments. In (C), *=p ≥ 0.001 (imatinib) and **=p<0.05 (nilotinib) when comparing regular media and SCF supplemented media. In (D), *=p ≥ 0.0001 for both imatinib and nilotinib when comparing regular and HS-5 conditioned media. (E) Kasumi-1, SKNO-1, and HMC1.1 cells were stained with a PE conjugated anti-G-CSF receptor antibody (Biolegend) and assessed by flow cytometry. The data are the mean +/− SEM from three independent experiments.
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References

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