Abstract
Recurrent homozygous CBL-inactivating mutations in myeloid malignancies decrease ubiquitin ligase activity that inactivates SRC family kinases (SFK) and receptor tyrosine kinases (RTK). However, the most important SFK and RTK affected by these mutations, and hence, the most important therapeutic targets, have not been clearly characterized. We compared SFK and RTK pathway activity and inhibitors in acute myeloid leukemia cell lines containing homozygous R420Q mutation (GDM-1), heterozygous deletion (MOLM13) and wild-type (WT) CBL (THP1, U937). As expected with CBL loss, GDM-1 displayed high KIT expression and granulocyte-macrophage colony-stimulating factor (GM-CSF) hypersensitivity. Ectopic expression of WT CBL decreased GDM-1 proliferation but not cell lines with WT CBL. GDM-1, but not the other cell lines, was highly sensitive to growth inhibition by dasatinib (dual SFK and RTK inhibitor, LD50 50 nM); there was less or no selective inhibition of GDM-1 growth by sunitinib (RTK inhibitor), imatinib (ABL, KIT inhibitor), or PP2 (SFK inhibitor). Phosphoprotein analysis identified phosphorylation targets uniquely inhibited by dasatinib treatment of GDM-1, including a number of proteins in the KIT and GM-CSF receptor pathways (for example, KIT Tyr721, STAT3 Tyr705). In conclusion, the promiscuous effects of CBL loss on SFK and RTK signaling appear to be best targeted by dual SFK and RTK inhibition.
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Acknowledgements
This work was supported in part by RO1HL-082983, U54 RR019391 (JPM and MAS), K24 HL-077522, DOD-MP048018 (MAM), and by a grant from AA&MDS International Foundation and Robert Duggan Charitable Fund (JPM). The THP-1 cell line was kindly provided by Professor S P Whitman, the Ohio State University. NKM-1 cells were kindly provided by Dr Akihiro Abe of the Department of Hematology, Nagoya University, School of Medicine. Granulocyte colony-stimulating factor, granulocyte macrophage colony-stimulating factor and erythropoietin were obtained from Amgen. Dasatinib was obtained from Bristol-Myers Squibb.
Author Contributions
H Makishima designed the study, collected patient data, performed experiments and wrote the manuscript; Y Sugimoto, HS, MJC and KPN performed experiments; H Muramatsu collected patient data, CO and Y Saunthararajah edited the manuscript; JPM designed the study, collected patient data and wrote the manuscript.
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Makishima, H., Sugimoto, Y., Szpurka, H. et al. CBL mutation-related patterns of phosphorylation and sensitivity to tyrosine kinase inhibitors. Leukemia 26, 1547–1554 (2012). https://doi.org/10.1038/leu.2012.7
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DOI: https://doi.org/10.1038/leu.2012.7
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