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. 2013 Sep;12(9):1738-48.
doi: 10.1158/1535-7163.MCT-13-0049-T. Epub 2013 Jul 3.

Targeting protein tyrosine phosphatase SHP2 for the treatment of PTPN11-associated malignancies

Bing Yu  1 , Wei Liu, Wen-Mei Yu, Mignon L Loh, Shawn Alter, Olgun Guvench, Alexander D Mackerell Jr, Li-Da Tang, Cheng-Kui Qu
Affiliations

Targeting protein tyrosine phosphatase SHP2 for the treatment of PTPN11-associated malignancies

Bing Yu et al. Mol Cancer Ther. 2013 Sep.

Abstract

Activating mutations in PTPN11 (encoding SHP2), a protein tyrosine phosphatase (PTP) that plays an overall positive role in growth factor and cytokine signaling, are directly associated with the pathogenesis of Noonan syndrome and childhood leukemias. Identification of SHP2-selective inhibitors could lead to the development of new drugs that ultimately serve as treatments for PTPN11-associated diseases. As the catalytic core of SHP2 shares extremely high homology to those of SHP1 and other PTPs that play negative roles in cell signaling, to identify selective inhibitors of SHP2 using computer-aided drug design, we targeted a protein surface pocket that is adjacent to the catalytic site, is predicted to be important for binding to phosphopeptide substrates, and has structural features unique to SHP2. From computationally selected candidate compounds, #220-324 effectively inhibited SHP2 activity with an IC50 of 14 μmol/L. Fluorescence titration experiments confirmed its direct binding to SHP2. This active compound was further verified for its ability to inhibit SHP2-mediated cell signaling and cellular function with minimal off-target effects. Furthermore, mouse myeloid progenitors with the activating mutation (E76K) in PTPN11 and patient leukemic cells with the same mutation were more sensitive to this inhibitor than wild-type cells. This study provides evidence that SHP2 is a "druggable" target for the treatment of PTPN11-associated diseases. As the small-molecule SHP2 inhibitor identified has a simple chemical structure, it represents an ideal lead compound for the development of novel anti-SHP2 drugs. Mol Cancer Ther; 12(9); 1738-48. ©2013 AACR.

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Conflict of interest statement

Competing financial interests: The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. #220–324 selectively inhibited the phosphatase activity of SHP2
(A) Chemical structure of #220–324. (B) Phosphatase assays were carried out using the indicated phosphatases as enzymes and the phospho-insulin receptor peptide as a substrate in the presence of various concentrations of #220–324, as described in Materials and Methods. Experiments were performed three times with similar results obtained. Representative results from one experiment are shown. (C) PTP activities of the SHP2 PTP domain, WT full length SHP2, and full length SHP2 E76K were determined in the absence or presence of #220–324 at the indicated concentrations using the in vitro phosphatase assay. Experiments were repeated three times. Similar results were obtained in each. Data shown are mean±S.E.M. from one experiment. (D) Inhibitory effects of #220–324 (25 μM) on the enzymatic activities of the SHP2 PTP domain, WT SHP2, and SHP2 E76K were determined using the phospho-insulin receptor peptide or pNPP as the substrates. Experiments were repeated three times. Similar results were obtained in each. Data shown are mean±S.E.M. from one experiment. (E) Fluorescence titration of SHP2 was performed by increasing the concentrations of #220–324 while maintaining the SHP2 protein concentration. The fluorescence is plotted against the log concentration in mol/L (Log [M]) for the test compound. Experiments were repeated twice. Similar results were obtained in each. Data shown are mean±S.E.M. from one experiment.
Figure 2
Figure 2. #220–324 inhibited SHP2-mediated cell signaling and cellular proliferation
(A) Ba/F3 cells were cultured in IL-3 (1.0 ng/mL) containing medium supplemented with #220–324 at the indicated concentrations or control DMSO. Cell numbers were determined 72 hrs later using the One Solution Cell Proliferation Assay kit. Experiments were performed three times with similar results obtained. Data shown are mean±S.E.M. from one experiment. (B) The cell cycle of Ba/F3 cells was analyzed 24 hrs after the treatment of #220–324 at the indicated concentrations. Percentages of the cells at G1, S, and G2/M phases were quantified by flow cytometric analyses. Experiments were repeated three times. Results shown are mean±S.E.M. from one experiment. (C) Ba/F3 cells were deprived of IL-3 overnight. Cells were treated with #220–324 (30 μM) for 3 hrs and then stimulated with IL-3 (2.0 ng/mL) for the indicated times. Whole cell lysates were prepared. The levels of p-Erk, p-Akt, p-Stat5, p-Jak2 were determined by immunoblotting analysis. Blots were striped and reprobed with anti-Erk, anti-Akt, and anti-Jak2, and anti-Stat5 antibodies to check for protein loading. Experiments were repeated three times. Representative results from one experiment are shown.
Figure 3
Figure 3. #220–324 had minimal effects in PTPN11 knockout cells
PTPN11 knockout (PTPN11Fl/Fl/Ad-Cre+) and WT (PTPN11+/+/Ad-Cre+) MEFs were cultured in DMEM with 10% FBS and #220–324 at the indicated concentrations for 72 hrs (A) or at 20 μM for the indicated periods of time (B). DMSO-treated cells were included as negative controls. Cell numbers were determined using the One Solution Cell Proliferation Assay kit. Experiments were performed twice with similar results. Data shown are mean±S.E.M. from one experiment.
Figure 4
Figure 4. Mutant MEFs and myeloid progenitors with the PTPN11E76K/+ mutation were more sensitive to #220–324 than WT cells
(A) WT and PTPN11E76K/+ MEFs were cultured in 10% FBS-containing DMEM supplemented with #220–324 at the indicated concentrations. DMSO-treated cells were included as negative controls. Cell numbers were determined using the One Solution Cell Proliferation Assay kit 72 hrs later. Experiments were repeated three times. Similar results were obtained in each. Data shown are mean±S.E.M. from one experiment. (B) Bone marrow cells (2×104 cells/mL) harvested from PTPN11E76K/+/Mx1-Cre+ and PTPN11+/+/Mx1-Cre+ mice were plated in methylcellulose medium containing GM-CSF (1.0 ng/mL) and #220–324 at the indicated concentrations or control DMSO. Colonies were enumerated 7 days later and normalized against the number of colonies derived from WT control cells without #220–324 treatment. Representative results from two independent experiments are shown. Data are presented as mean±S.E.M. (C) Bone marrow cells (5×104 cells/mL) harvested from PTPN11E76K/+/Mx1-Cre+ mice were plated in methylcellulose medium (without GM-CSF) supplemented with #220–324 at the indicated concentrations or control DMSO. Colonies derived in the cytokine-free medium were enumerated 7 days later and normalized against the number of colonies derived from the cells without #220–324 treatment. Representative results from two independent experiments are shown. Data are presented as mean±S.E.M.
Figure 5
Figure 5. Lung cancer cells with the PTPN11N58S/+ mutation were more sensitive to #220–324 than those without the PTPN11N58S/+ mutation
Human lung cancer cell lines H596 and H661 were cultured in DMEM supplemented with 10% FBS and #220–324 at the indicated concentrations for 72 hrs (A) or the cells were treated with #220–324 at 20 μM for the indicate times (B). DMSO-treated cells were included as negative controls. Cell numbers were determined using the One Solution Cell Proliferation Assay kit. Experiments were repeated three times. Similar results were obtained in each. Data are presented as mean±S.E.M. from one representative experiment.
Figure 6
Figure 6. #220–324 abrogated the growth of JMML myeloid progenitors with PTPN11E76K/+ mutation
(A) Splenocytes (2×104 cells/mL) from a JMML patient with the PTPN11E76K/+ mutation were plated in methylcellulose medium containing GM-CSF (1.0 ng/mL) and #220–324 at the indicated concentrations or control DMSO. Colonies were enumerated 14 days later and normalized against the number of colonies derived from the cells without #220–324 treatment. Three patient samples were tested in three independent experiments. Similar results were obtained in each. Data are presented as mean±S.E.M. from one patient sample. (B) Splenocytes from a JMML patient with the PTPN11E76K/+ mutation were cultured in RPMI 1640 medium containing GM-CSF (1.0 ng/mL) and #220–324 at the indicated concentrations or control DMSO. Total cell numbers were determined and normalized against the number of the cells in the untreated group. Three patient samples were tested in three independent experiments. Similar results were obtained in each. Data are presented as mean±S.E.M. from one patient sample. (C) Apheresis peripheral blood cells (2×104 cells/mL) from normal donors were plated in methylcellulose medium containing GM-CSF (1.0 ng/mL) and #220–324 at the indicated concentrations. Colonies were enumerated 14 days later and normalized against the number of colonies derived from the cells treated with control DMSO. Three samples were tested in three independent experiments. Similar results were obtained in each. Data are presented as mean±S.E.M. from one sample.
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References

    1. Xu D, Qu CK. Protein tyrosine phosphatases in the JAK/STAT pathway. Front Biosci. 2008;13:4925–32. - PMC - PubMed
    1. Chan G, Kalaitzidis D, Neel BG. The tyrosine phosphatase Shp2 (PTPN11) in cancer. Cancer Metastasis Rev. 2008;27:179–92. - PubMed
    1. Tonks NK. Protein tyrosine phosphatases: from genes, to function, to disease. Nat Rev Mol Cell Biol. 2006;7:833–46. - PubMed
    1. Zhao R, Fu X, Teng L, Li Q, Zhao ZJ. Blocking the function of tyrosine phosphatase SHP-2 by targeting its Src homology 2 domains. J Biol Chem. 2003;278:42893–8. - PubMed
    1. Pawson T. Specificity in signal transduction: from phosphotyrosine-SH2 domain interactions to complex cellular systems. Cell. 2004;116:191–203. - PubMed

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