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Synergistic anti-leukemic activity of imatinib in combination with a small molecule Grb2 SH2 domain binding antagonist

Leukemia volume 28, pages 948–951 (2014)Cite this article

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The mechanisms underlying imatinib resistance in chronic myelogenous leukemia (CML) include BCR-ABL gene rearrangement, overexpression of Bcl-Abl tyrosine kinase fusion protein and activation of Bcr-Abl-independent pathways such as those of Src kinases.1, 2 Although impressive therapeutic responses have been achieved with imatinib treatment, many patients fail to respond optimally due to innate or acquired resistance.1, 2 The available treatments for these patients are limited and improved treatment options are urgently needed. In patients with Philadelphia chromosome-positive CML, tyrosyl phosphorylation of the Bcr-Abl oncoprotein enables binding to the Src homology-2 (SH2) domain of the adapter protein growth factor receptor-bound 2 (Grb2), thereby activating downstream signaling pathways, including the Ras/MAPK pathway, controlling cell proliferation.3 Grb2 mutants missing one of its two SH3 domains impair cell transformation by Bcr-Abl, highlighting the importance of Grb2 interactors, such as SOS1, in oncogenic signaling.4 These findings also suggest that Grb2 binding antagonists may provide an effective alternative or adjunct therapeutic strategy for CML patients resistant to imatinib.

Flow cytometry experiments were performed to further characterize synergistic inhibition of K562 cell proliferation by the combination of imatinib and TB03. Analysis using annexin V–fluorescein isothiocyanate/propidium iodide double staining showed that treatment with either imatinib or TB03 alone did not affect apoptosis relative to the vehicle control group, but the combination of these two agents increased apoptosis by >50% (20% vs 36%; Figure 1d), indicating functional complementation of distinct pro-apoptotic effects. Flow cytometry analysis using CellTrace Violet indicated that vehicle-treated K562 cells divided eight times in 72 h. Treatment with 0.25 or 2.5 μM imatinib alone resulted in 0.04% and 0.03% of cells reaching the eighth cell division, respectively, and treatment with 2 μM TB03 alone resulted in 0.08% of cells reaching the eighth cell division (Figure 1e). Combined imatinib/TB03 treatments further reduced the number of cells reaching eight divisions: 0.02% for 2 μM TB03 plus 0.25 μM imatinib and 0.01% for 2 μM TB03 plus 2.5 μM imatinib; the latter combination represents an eightfold reduction in cell survival (Figure 1e). Short-term analysis of cell cycle progression using Vybrant DyeCycle Green showed that imatinib treatment increased the G0/G1 population while decreasing the S and G2/M phase populations, consistent with cell cycle suppression (Figure 1f). Treatment with TB03 alone had little effect, but combined TB03/imatinib treatment increased the number of cells in G0/G1 in a dose-dependent manner, with proportionately fewer cells reaching S and G2/M. The increased number of cells in sub-G1 phase with imatinib treatment relative to control (1.5% vs 4.7%) may reflect pro-apoptotic effects. Consistent with the complementation of apoptosis observed by annexin staining, TB03 plus imatinib further increased the number of cells in this phase in a dose-dependent manner, from 4.7 to 7% for imatinib:TB03 at a ratio 0.5:6.25 μM and from 4.7 to 11% for imatinib:TB03 at 0.5:12.5 μM (Figure 1f). These results reinforce the hypothesis that TB03 alone inhibits only cell cycle progression, but enhances imatinib-induced apoptosis and cell cycle suppression, resulting in synergistic inhibition of K562 proliferation.

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Acknowledgements

This work was supported by The Children’s Leukemia Research Association, Inc. (a.k.a. National Leukemia Research Association) and by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.

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  1. The Ohio State University Comprehensive Cancer Center, Columbus, OH, USA

    M Zhang, Z Luo, H Liu, C M Croce & T R Burke Jr

  2. Chemical Biology Laboratory, Molecular Discovery Program, National Cancer Institute, Frederick National Laboratory for Cancer Research, Frederick, MD, USA

    T R Burke Jr

  3. Urologic Oncology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA

    D P Bottaro

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Zhang, M., Luo, Z., Liu, H. et al. Synergistic anti-leukemic activity of imatinib in combination with a small molecule Grb2 SH2 domain binding antagonist. Leukemia 28, 948–951 (2014). https://doi.org/10.1038/leu.2013.323

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