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

Leukemia volume 28, pages 2257–2260 (2014)Cite this article

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The identification of activating mutations in the receptor tyrosine kinase c-KIT in acute myelogenous leukemia (AML) coupled with the development of potent c-KIT inhibitors provides an approach for potentially improving survival in a disease where outcomes remain unsatisfactory.1 However, despite the in vitro sensitivity of c-KIT mutant cell lines to c-KIT inhibitors, primary resistance in patients remains a significant concern. Direct interactions between leukemia cells and bone marrow stromal cells in the hematopoietic microenvironment (HM) as well as soluble factors secreted by stromal cells can support leukemic cell proliferation and survival in the presence of cytotoxic and molecularly targeted therapies.2,3 Accordingly, defining the mechanisms by which the microenvironment contributes to leukemia growth and therapy resistance is essential for ensuring the success of c-KIT inhibitors, and other new therapies, as they advance from the laboratory to the clinic. This is particularly relevant for c-KIT mutated AML as a phase II clinical trial (NCT01830361) is currently underway to assess the efficacy of adding the c-KIT inhibitor midostaurin (PKC412) to standard therapy in patients with newly diagnosed c-KIT or FLT3-ITD mutated t(8;21) AML. In this study, we demonstrate that ex vivo modeling of components of the HM provides valuable insights into mechanisms of primary therapy resistance in c-KIT dependent AML.

To confirm that the effects of the drugs on apoptosis correlated with c-KIT inhibition, western blots for phospho-c-KIT and total c-KIT were performed on Kasumi-1 cells that had been treated with nilotinib for 4, 24 or 48 h in either regular or HS-5-conditioned media (Figure 1e). Nilotinib treatment in regular media diminished c-KIT phosphorylation relative to the total c-KIT protein levels at all time-points, consistent with a rapid and sustained c-KIT inhibition. In contrast, although nilotinib still inhibited the phosphorylation of c-KIT in HS-5-conditioned media, the level of total c-KIT protein was also significantly diminished relative to regular media in the absence of nilotinib (Figure 1e). In agreement, the level of c-KIT cell surface expression, as assessed with an allophycocyanin-conjugated c-KIT antibody and flow cytometry, was reduced in both the Kasumi-1 and SKNO-1 cell lines in HS-5-conditioned media (Figure 1f). The presence of stem cell factor (SCF), the ligand for c-KIT in HS-5-conditioned media, likely contributes to c-KIT downregulation as c-KIT is rapidly internalized and degraded following ligand binding6 (Figure 1f). However, in addition to the effects of HS-5-conditioned media on c-KIT protein stability, Kasumi-1 and SKNO-1 cells cultured in HS-5-conditioned media also exhibited significantly decreased c-KIT mRNA levels (Figure 1g). Together, these data suggest that the cause of c-KIT downregulation in HS-5-conditioned media is likely multifactorial and occurs at both transcriptional and post-translational level.

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Acknowledgements

We thank Drs Stegmaier and Butterfield for kindly providing the SKNO-1 and HMC1.1 cells lines, respectively. We also thank Maria Suarez and Natasha Rossi for administrative assistance and members of the Williams laboratory for helpful discussions and suggestions. PMG is supported by NIH Grant 5K08CA154782-02. DAW is supported by 5R01DK062757, 5R01CA113969-08 and the William Lawrence and Blanche Hughes Foundation.

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  1. P M Gordon

    Present address: 2Current Address: Division of Pediatric Hematology/Oncology and Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA,

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  1. Division of Hematology/Oncology, Boston Children’s Hospital and Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School and Harvard Stem Cell Institute, Boston, MA, USA

    P M Gordon, Stuart Dias & D A Williams

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Gordon, P., Dias, S. & Williams, D. Cytokines secreted by bone marrow stromal cells protect c-KIT mutant AML cells from c-KIT inhibitor-induced apoptosis. Leukemia 28, 2257–2260 (2014). https://doi.org/10.1038/leu.2014.212

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