Targeted Degradation of the Oncogenic Phosphatase SHP2

Abstract

SHP2 is a protein tyrosine phosphatase that plays a critical role in the full activation of the Ras-MAPK pathway upon stimulation of receptor tyrosine kinases, which are frequently amplified or mutationally activated in human cancer. In addition, activating mutations in SHP2 result in developmental disorders and hematologic malignancies. Several allosteric inhibitors have been developed for SHP2 and are currently in clinical trials. Here, we report the development and evaluation of a SHP2 PROTAC created by conjugating RMC-4550 with pomalidomide using a PEG linker. This molecule is highly selective for SHP2, induces degradation of SHP2 in leukemic cells at submicromolar concentrations, inhibits MAPK signaling, and suppresses cancer cell growth. SHP2 PROTACs serve as an alternative strategy for targeting ERK-dependent cancers and are useful tools alongside allosteric inhibitors for dissecting the mechanisms by which SHP2 exerts its oncogenic activity.

Figure 1.. Structure of SHP2 protein in complex with RMC-4550.

(A) Chemical structure of RMC-4550 and X-ray crystal structure of SHP2 in complex with RMC-4550 (PDB code 7RCT). Surface representation of SHP2 in complex with RMC-4550 bound in the central tunnel formed at the interface of N-SH2 (green), C-SH2 (blue) and PTP (wheat) domains. (B) Chemical structures of RMC-4550-based PROTAC candidates, R1–1C, R1–3C and R1–5C.

Figure 2.. SHP2 degradation induced by RMC-4550-based PROTACs.

(A) Inhibition of SHP2-F285S- or PTP-mediated DIFMUP dephosphorylation by R1–1C, R1–3C, R1–5C and RMC-4550. MV4;11 cells were treated with increasing doses of R1–3C (B), R1–1C or R1–5C (C) for 24 h and subjected to Western blotting using SHP2, GAPDH and β-actin antibodies. Quantification of band intensities on the gels are shown below the blots.

Figure 3.. Cell-based evaluation of R1–5C.

(A) Time course of SHP2 degradation by R1–5C (100 nM) in MV4;11 cells. Immunoblotting with SHP2 and β-actin antibodies. (B) CRBN^−/− and parental MOLT4 cells were treated with increasing doses of R1–5C for 24 h and subjected to Western blotting using SHP2, CRBN and β-actin antibodies. Quantification of band intensities on the gels are shown below the blots.

Figure 4.. Proteomics analysis showing selective SHP2 degradation by R1–5C.

(A-D) Scatterplots displaying relative fold-change in SHP2 abundance following treatment of MV4;11 cells with 100 nM R1–5C for 4 h (A), 8 h (B), 16 h (C) or 100 nM RMC-4550 (D). SHP2/PTPN11 is highlighted in red. Hits highlighted in blue in (C) and (D) indicate changes in abundance of proteins at 16 h time point due to secondary effects (such as transcriptional responses) of SHP2 degradation or inhibition. (E) Heatmap of the protein abundance changes in MV4;11 cells comparing treatment with 100 nM R1–1C (4 h and 16 h), 100 nM R1–3C (4 h and 16 h), 100 nM R1–5C (2 h, 4 h, 8 h and 16 h), 100 nM RMC-4550 (16 h) and 1 μM pomalidomide (5 h). The heatmap colors are scaled with red indicating a decrease in protein abundance (−2 log₂ FC) and blue indicating an increase (2 log₂ FC) in protein abundance.

Figure 5.. R1–5C inhibits MAPK signaling and suppresses cancer cell growth.

(A) Downregulation of DUSP6 transcript levels in KYSE-520 cells after 2 h and 16 h treatment with 200 nM R1–5C or DMSO carrier. DUSP6 mRNA was quantified by RT-qPCR using primer set B. (B and C) Cells were treated with 100 nM R1–5C, 100 nM RMC-4550 or DMSO carrier in triplicate and cell numbers were assessed at the indicated time points by automated cell counting (for MV4;11 cells) or CellTiter-Blue assay (for KYSE-520 cells).

Scheme 1.

Scheme 2.

Scheme 3.

Scheme 4.

Scheme 5.

Scheme 6.