Sinking the SHP That Drives Malignancy

Tyrosine kinases are enzymes that selectively phosphorylate specific tyrosine residues on diverse protein substrates and they play a pivotal role in signaling pathways that govern cell proliferation and survival. Receptor tyrosine kinases (RTKs) are transmembrane proteins that respond to extracellular stimuli and relay the signal to the internal milieu by phosphorylating targets that initiate a cascade of events. The activity of tyrosine kinases is tightly controlled and phosphorylation is thus counterbalanced by dephosphorylation, whereby the phosphate groups are removed by protein tyrosine phosphatases. Dysregulation of either of these functions can result in oncogenic transformation of cells, and this has led to research and development of inhibitors of these key enzymes. Tyrosine kinase inhibitors, such as those used to treat chronic myeloid leukemia, have been successful, but targeted therapy of phosphatases has lagged behind.

Src homology region 2 (SH2) –containing protein tyrosine phosphatase 2 (SHP2) is a nonreceptor phosphotyrosine phosphatase encoded by the PTPN11 gene. SHP2 is a ubiquitous enzyme that promotes activation of the RAS-ERK signaling pathway within cells. It is highly expressed in hematopoietic cells where it transduces signals from hematopoietic growth factors, and thus, it plays an essential role in hematopoietic cell development. Mutations in PTPN11 that activate SHP2 are implicated in numerous cancers, including leukemias, and it was the first identified proto-oncogene that encodes a tyrosine phosphatase.

To discover new therapeutic targets for cancer, Dr. Ying-Nan P. Chen and colleagues screened a panel of 250 cancer cell lines with a deep coverage, pooled, short-hairpin RNA library that targeted 7,500 genes. Cell lines that were dependent on RTKs showed marked sensitivity to SHP2 depletion. Additional growth and complementation experiments verified that SHP2 is essential for the survival of these cells and that p-ERK activation was the mechanism of action.

Structural features of SHP2 are known, and the researchers used this information to guide subsequent screening strategies focused on identifying SHP2 inhibitors. SHP2 contains two tandem SH2 domains (N-SH2 and C-SH2) at the N-terminal end of the protein; a catalytic phosphatase domain toward the C terminal; and a C terminal tail with a proline-rich motif and two tyrosine residues, which can be phosphorylated. The active site of the enzyme is tightly regulated by the N-SH2 domain. Autoinhibition occurs when N-SH2 binds the catalytic domain and changes the conformation to prevent access to the substrate. The enzyme is activated when docking proteins with appropriately spaced phosphotyrosine residues interact with N-SH2 and C-SH2 to expose the active site.

Catalytic inhibitors of SHP2 have been discovered but have not progressed to clinical use since they display low selectivity and potency, partly due to the highly solvated and polar nature of the catalytic pocket. The researchers therefore focused their attention on allosteric inhibitors to capitalize on the natural autoregulation of the enzyme. They partially activated recombinant SHP2 (residues 1-525) with a bis-phosphorylated peptide, 2P-IRS-1, and screened a library of 100,000 diverse compounds. Hits were prioritized by further screening against a truncated recombinant protein containing only the catalytic domain, as well as against a fully activated SHP2 molecule. Compounds that inhibited only the phosphatase domain were excluded, and further refinement led to the identification of SHP099, which was highly active with an IC₅₀ = 71nM. It also displayed high selectivity since it had no detectable activity against a panel of 21 phosphatases and 66 kinases and was also inactive against SHP1, the closest human homologue. To determine the mechanism of inhibition, the researchers conducted further biochemical studies and also solved the crystal structure of the SHP2-SHP099 complex. This revealed that SHP099 bound to the central tunnel formed at the interface of the N-SH2, C-SH2, and phosphatase domains, which stabilized the protein in the inactive conformation.

The next step was to test whether the compound could inhibit the enzyme within cells by screening several cell lines, including a panel of 71 hematopoietic cancer lines, against a range of SHP099 concentrations, and by measuring the effect on p-ERK. Cancer cells with known mutations in oncogenic RTKs or cytoplasmic tyrosine kinases were sensitive to SHP099 inhibition, whereas cells with RAS or BRAF mutations were not affected. These findings confirmed the link between RTK dependence and SHP sensitivity that was demonstrated in their short-hairpin RNA screen. Off-target effects may occur, and thus, inhibitor-resistant alleles were developed that had mutations in key residues, which would disrupt binding of SHP099 but maintain the integrity of the three-domain regulatory interface. Cell growth and inhibition of p-ERK were used as endpoints, and the data demonstrated that SHP099 inhibits MAPK signaling and proliferation in RTK-dependent cells in a direct, on-target manner.

In vivo testing of SHP099 using a subcutaneous xenograft model in mice showed marked dose-dependent inhibition of tumor growth, and the treatment was well tolerated. This was extended to an evaluation of an orthotopic human primary tumor-derived acute myeloid leukemia mouse model in which daily dosing virtually eradicated circulating human CD45+ leukemic cells and significantly reduced splenomegaly.