Structural reorganization of SHP2 by oncogenic mutations and implications for oncoprotein resistance to allosteric inhibition

Abstract

Activating mutations in PTPN11, encoding the cytosolic protein tyrosine phosphatase SHP2, result in developmental disorders and act as oncogenic drivers in patients with hematologic cancers. The allosteric inhibitor SHP099 stabilizes the wild-type SHP2 enzyme in an autoinhibited conformation that is itself destabilized by oncogenic mutations. Here, we report the impact of the highly activated and most frequently observed mutation, E76K, on the structure of SHP2, and investigate the effect of E76K and other oncogenic mutations on allosteric inhibition by SHP099. SHP2^E76K adopts an open conformation but can be restored to the closed, autoinhibited conformation, near-identical to the unoccupied wild-type enzyme, when complexed with SHP099. SHP099 inhibitory activity against oncogenic SHP2 variants in vitro and in cells scales inversely with the activating strength of the mutation, indicating that either oncoselective or vastly more potent inhibitors will be necessary to suppress oncogenic signaling by the most strongly activating SHP2 mutations in cancer.

Fig. 1

The open conformation of SHP2 E76K is closed by SHP099. a Basal structure of autoinhibited SHP2^WT (PDB:2SHP) with the N-SH2 domain displayed in green, C-SH2 in blue, and PTP in beige. b Structure of SHP2^E76K (1–525) reveals a 120° rotation of the C-SH2 domain, relocation of the N-SH2 domain to a PTP surface opposite the active site, and a solvent-exposed catalytic pocket. Insets show select N-SH2•PTP interdomain contacts and interactions between the C-SH2 and PTP domains. c Cartoon illustrating the domain movements that occur in SHP2^E76K upon adopting the open conformation. E128 and D94 are chosen as arbitrary reference points to illustrate the effect of the 120° rotation. d Open conformation of SHP1 (PDB:3PS5) is similar to that of SHP2^E76K. e Interaction of SHP099 with SHP2^E76K rescues the autoinhibited conformation. Insets show select interactions of SHP099 with SHP2^E76K and SHP2^WT, in addition to differential orientations of residue 76 in SHP2^E76K bound to SHP099 and in SHP2^WT bound to SHP099 (White, PDB:5EHR)

Fig. 2

Small-angle X-ray scattering (SAXS) analysis of SHP2 proteins. a SHP2 protein SAXS intensity profiles across scattering vector q. SHP2^WT is shown in red and SHP2^E76K in violet. b p(r) functions of SHP2^WT and SHP2^E76K. c Kratky analysis of SAXS data. d Fitting of the X-ray structure of SHP2^WT (PDB:2SHP) into the SAXS envelope of SHP2^WT. e Fitting of the X-ray structure of SHP2^E76K into the SAXS envelope

Fig. 3

SHP2 cancer mutations enhance basal activity and response to phospholigands. a Sites of oncogenic mutations of SHP2 mapped onto the autoinhibited structure of SHP2 (PDB: 2SHP), which localize to the N-SH2:PTP interdomain interface. The N-SH2 domain of SHP2 is displayed in blue, C-SH2 in green, PTP in beige, and cancer mutations in red. b Normalized basal activity of SHP2^WT and cancer mutants (5 nM WT, 1 nM F285S, 0.2 nM G60V, 0.1 nM D61V & S502P, and 0.05 nM E76K & PTP), plotted as a function of DiFMUP concentration. c Enzymatic activity of SHP2 analogs (0.05 nM) in the presence of DiFMUP (200 μM) plotted as a function of the concentration of a synthetic IRS-1-derived bisphosphorylated peptide (p-IRS-1, SLNY(p)IDLDLVK-dPEG₈-LSTY(p)ASINFQK). d Enzymatic activity of various SHP2 proteins (0.05 nM) plotted as a function of DiFMUP concentration in the presence of saturating (10 μM) p-IRS-1. Data points are presented as mean ± standard deviation (n = 3)

Fig. 4

Allosteric inhibition of SHP2 is influenced by cancer mutations and activating ligands in vitro. a Analysis of the effect of p-IRS-1 on the effectiveness of SHP099 inhibition of DiFMUP dephosphorylation by wild-type and mutant SHP2 proteins. b SHP099 dose–response curves for inhibition of DiFMUP dephosphorylation by wild-type and oncogenic SHP2 proteins (0.1 nM) in the presence of a fixed concentration of p-IRS-1 (10 nM). c SHP099 IC₅₀ values plotted as a function of p-IRS-1 peptide concentration for wild-type SHP2 protein (red) and for a range of different oncogenic SHP2 mutants. Data points are presented as mean ± standard deviation (n = 2)

Fig. 5

Establishment of PTPN11-null U2OS (U2OS^{PTPN11−/−/−}) cells for SHP2 rescue experiments. a CRISPR guide, shown 5′-to-3′, designed to target exon 3 of PTPN11. PTPN11 is displayed as 16 exons, which correspondingly encode N-SH2 (green), C-SH2 (blue), and PTP (beige) domains. Allele sequences of U2OS^{PTPN11−/−/−} (null) cells are compared with the sequence of wild-type SHP2. b Western blot showing SHP2 expression levels in wild-type U2OS cells (U2OS), PTPN11-null cells (null), and null cells transfected with full-length SHP2^WT. c Western blot analysis showing SHP099 reduces pErk levels in wild-type U2OS cells. d Western blot analysis showing SHP099 does not reduce pErk levels in null cells. e Western blot analysis showing pErk levels in null cells transfected with SHP2^WT are sensitive to SHP099. f Densitometry quantification of SHP099-dependent pErk reductions, in wild-type U2OS and PTPN11-null cells re-expressing SHP2^WT, derived from the experiments shown in panels c–e. The left-most data point of each graph refers to pErk levels at 0 μM SHP099 in each respective cell line, which was used to normalize data in the quantification analysis. Data points are presented as mean ± standard error of the mean

Fig. 6

Oncogenic mutants of SHP2 negatively impact allosteric inhibition in SHP2-deficient U2OS cells. a Western blot quantification of SHP2 expression levels in wild-type U2OS cells (U2OS) and in SHP2-deficient U2OS cells (PTPN11-null) in the presence and absence of expression of SHP2 oncogenic variants. b Western blot analysis showing relative pErk levels in wild-type, SHP2-deficient, and SHP2-rescued U2OS cells. c Western blot determination of pErk levels in SHP2-deficient U2OS cells, in the presence and absence of the SHP2 allosteric inhibitor SHP099. d Quantification of pErk levels in SHP2-deficient U2OS cells expressing SHP2^WT and oncogenic mutants upon titration of SHP099. Data points are presented as mean ± standard error of the mean