The non-receptor protein tyrosine phosphatase SHP2, encoded by PTPN11, has an important role in signal transduction downstream of growth factor receptor signalling and was the first reported oncogenic tyrosine phosphatase1. Activating mutations of SHP2 have been associated with developmental pathologies such as Noonan syndrome and are found in multiple cancer types, including leukaemia, lung and breast cancer and neuroblastoma1,2,3,4,5. SHP2 is ubiquitously expressed and regulates cell survival and proliferation primarily through activation of the RAS–ERK signalling pathway2,3. It is also a key mediator of the programmed cell death 1 (PD-1) and B- and T-lymphocyte attenuator (BTLA) immune checkpoint pathways6,7. Reduction of SHP2 activity suppresses tumour cell growth and is a potential target of cancer therapy8,9. Here we report the discovery of a highly potent (IC50 = 0.071 μM), selective and orally bioavailable small-molecule SHP2 inhibitor, SHP099, that stabilizes SHP2 in an auto-inhibited conformation. SHP099 concurrently binds to the interface of the N-terminal SH2, C-terminal SH2, and protein tyrosine phosphatase domains, thus inhibiting SHP2 activity through an allosteric mechanism. SHP099 suppresses RAS–ERK signalling to inhibit the proliferation of receptor-tyrosine-kinase-driven human cancer cells in vitro and is efficacious in mouse tumour xenograft models. Together, these data demonstrate that pharmacological inhibition of SHP2 is a valid therapeutic approach for the treatment of cancers.
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Use of the IMCA-CAT beamline 17-ID at the Advanced Photon Source was supported by the companies of the Industrial Macromolecular Crystallography Association through a contract with Hauptman-Woodward Medical Research Institute. Use of the Advanced Photon Source was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under contract number DE-AC02-06CH11357.
All authors performed the work herein as employees of the Novartis Institutes for Biomedical Research, Inc.
Reviewer Information Nature thanks B. Neel and the other anonymous reviewer(s) for their contribution to the peer review of this work.
Extended data figures and tables
a, Cells expressing dox-inducible SHP2 shRNA in various RTK-amplified cancer cells were generated including SUM52 (FGFR2), KATOIII (FGFR2), MDA-MB-468, KYSE520 (EGFR), NCI-H2190 (HER2), NCI-H2228 (EML4-ALK). b, Stable clones of MDA-MB-231 (KRASG13D) and A2058 BRAFV600E) cancer cells were established as controls. Cells were treated with dox, and colony formation was measured after 11 days by crystal violet staining. c, Western blot showing the expression of SHP2, p-ERK and ERK in the presence (+) or absence (−) of dox in MDA-MB-468 SHP2-depleted cells stably expressing either GFP, wild-type HA–SHP2 or HA–SHP2C459S. d, Western blot of SHP2, p-ERK and ERK in the presence (+) or absence (−) of dox in SHP2-depleted SUM52 cells expressing vector control or HA–KRASG12V. In c and d, dox treatment induces depletion of endogenous SHP2 protein and simultaneously expression of the exogenous proteins GFP, wild-type HA–SHP2, HA–SHP2C459S or HA–KRASG12V.
a, Western blot of SHP2, p-ERK and ERK in SHP2-depleted SUM52 cells stably expressing vehicle, wild-type SHP2 or HA–SHP2C459S. Note, four lanes corresponding to an unrelated study were removed from the image. All lanes originated from the same gel at the same exposure. b, Colony formation of SHP2-depleted SUM52 cells stably expressing vehicle, wild-type SHP2 or HA–SHP2C459S. SHP2 knockdown and SHP2 variant re-expression perform using dox treatment. Colony formation was monitored after 11 days with crystal violet staining. c, Phosphatase activity of SHP2-depleted SUM52 cells stably expressing HA–SHP2 or HA–SHP2C459S. Cells were treated with dox for 3 days. SHP2 protein was immunoprecipitated from cell lysates and phosphatase activity was measured using DiFMUP assay. Data are presented as mean ± s.d. (n = 3).
Extended Data Figure 3 Thermodynamic characterization of the SHP099–SHP2 binding complex, comparison of SHP2 and SHP1’s allosteric pocket and characterization of SHP099-resistant SHP2 mutants.
a, Isothermal titration calorimetry of SHP099 binding to SHP2. SHP099 binds stoichiometrically to SHP2 with a dissociation constant measured at 73 ± 15 nM. b, Structural differences in the central tunnel between SHP1 and SHP2. Ribbon representation of SHP2 (multi-colour) and SHP1 (grey) X-ray structures in the closed conformation. The PTP (tan) and N-SH2 (green) domain overlay well (r.m.s.d. < 1.5 Å), however the C-SH2 (blue) domain has a significantly different orientation. c, Surface representation of SHP2–SHP099 co-crystal structure. d, Surface representation of SHP1 with SHP099 modelled on the basis of the SHP2 superimposition. Central tunnel is significantly larger in SHP1 owing to a change in orientation of the C-SH2 domain. This change repositions the linker between the two SH2 domains removing several key interactions, highlighted by residue Arg109 in SHP1 and by Arg111 in SHP2 (equivalent residues). e, Biochemical activity of wild-type SHP2, SHP2Q257L and SHP2T253M/Q257L. SHP2 activity was determined using DiFMUP in the presence of various concentrations of 2P-IRS-1. Data points along the line represent the mean of two replicate values. SHP2Q257L and SHP2T253M/Q257L retain activity regulation and 2P-IRS-1 activation potential comparable to wild-type SHP2 but are 18- and <1,000-fold less sensitive to SHP099 inhibition.
a, Western blot of SHP2, p-ERK, ERK and p-AKT from KYSE520, MDA-MB-468 or A2058 cells treated with SHP099 (1, 3, 10 μM). Note, three lanes corresponding to an unrelated study were removed from the image. All lanes originated from the same gel at the same exposure. b, Colony formation of KYSE520, MDA-MB-468, and A2058 in the presence of SHP099. Colony formation was measured after 11 days of SHP099 treatment by crystal violet staining. c, SHP099 inhibitory activity against cell lines KYSE520 (EGFR-amplified), MV-411(FLT3-ITD), MOLM-13 (FLT3-ITD), Kasumi (c-Kit altered) and negative control A2058 (BRAFV600E) treated with SHP099 concentration varying from 0.0046 to 20 μM. Cellular viability was measured using CellTiter-Glo. Data presented as mean ± s.d. (n = 3). d, Comparison of SHP099 activity with Lapatinib in a panel of 26 colorectal cell lines. SHP099 sensitivity correlates with sensitivity to Lapatinib, a potent tyrosine kinase inhibitor against Her1/2 and EGFR. RAS- and BRAF-mutated cell lines are shown in blue. Cellular viability was measured using CellTiter-Glo. The corresponding data and cell line genotypes are included in Supplementary Information Table 2.
a, Western blot of SHP2 and GAPDH in KYSE520 xenograft lysates following 14 days of dox treatment (+). b, Response of p-ERK in tumour xenograft lysates following 14 days of dox treatment. c, Antitumour efficacy of SHP099 administered orally for 14 consecutive days at the doses and schedules indicated. Data are plotted as the treatment mean ± s.e.m. (n = 9). d, Body weight of mice bearing subcutaneous KYSE520 xenografts and administered either SHP099 (100 mg per kg daily), erlotinib (80 mg per kg daily) or vehicle for 14 consecutive days. Data are presented as treatment mean ± s.e.m. (n = 7). e, Body weight of mice bearing orthotopic primary tumour derived xenografts and administered an oral gavage of SHP099 at 75 mg per kg daily. Data are presented as treatment mean ± s.e.m. (n = 7). f, The mouse spleen weight measurement of mice bearing the orthotopic AML xenograft model following 34 days of once-daily dosing with SHP099 at 75 mg per kg. Data are presented as treatment mean ± s.e.m. (n = 7).
This file contains the raw data for Figure 3e and Extended Data Figures 1a-d, 2a, 4a and 5a. (PDF 1634 kb)
This file shows the SHP099 inhibitory activity against hematopoietic cell lines. Data used to generate Figure 3b. (XLSX 10 kb)
This file shows the Inhibition of colorectal cancer cell lines proliferation by SHP099 or Lapatinib. Data used to generate extended data figure 4d. (XLSX 9 kb)
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Chen, YN., LaMarche, M., Chan, H. et al. Allosteric inhibition of SHP2 phosphatase inhibits cancers driven by receptor tyrosine kinases. Nature 535, 148–152 (2016). https://doi.org/10.1038/nature18621
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