Wild-type SHP2 Research Articles

Effects of changes in SHP2 expression on liver fibrosis by influencing the apoptosis of hepatic stellate cells.

Accumulating research has revealed that src-homology domain 2-containing protein tyrosine phosphatase-2 (SHP2), an oncogenic protein tyrosine phosphatase, is associated with liver fibrosis. Currently, it is still unclear whether SHP2 affects liver fibrosis by influencing the apoptosis of hepatic stellate cells (HSC). In present study, we investigate effects of SHP2 expression changes on liver fibrosis, with special emphasis on the apoptosis of HSC. Using adenovirus vector, wild-type SHP2 gene and short hairpin RNA targeting SHP2 were introduced into rats with liver fibrosis and LX-2 cells in vitro. The expressions of type I and III collagen, pathological and functional changes, collagen deposition in rat liver and apoptosis of LX-2 cells were detected by immunohistochemical and HE staining, automated biochemical analyzer, Masson trichrome staining, and TUNEL. This study showed that overexpression of SHP2 exacerbated dysfunction, inflammatory damage, collagen deposition and increased expression of type I and III collagen in rat liver reduced apoptosis of LX-2 cells. On the contrary, low expression of SHP2 alleviated the aforementioned detection indicators of rats and promoted apoptosis of LX-2 cells. In conclusion, the downregulation of SHP2 expression alleviates liver fibrosis by inducing the apoptosis of HSC, while overexpressed SHP2 exacerbates liver fibrosis by inhibiting the apoptosis of HSC.

Ionizable networks mediate pH-dependent allostery in SH2 signaling proteins.

Transient intracellular pH dynamics 1 regulate mammalian proliferation 2,3 , migration 4 , and differentiation 5 . However, for many pH-dependent cell processes, the molecular mediators are unknown 6 . Prior work identified histidine residues as molecular switches in pH-sensitive proteins, but how other ionizable residues contribute to pH-dependent protein allostery is understudied. Here, we develop an in silico computational pipeline to identify putative pH-sensitive proteins and their molecular mechanisms. We first apply this pipeline to SHP2, a known pH-sensitive signaling protein with an uncharacterized molecular mechanism. We show wild-type SHP2 phosphatase activity is pH-sensitive in vitro and in cells, and mutation of identified H116 and E252 to non-titratable alanine residues abolishes pH-sensitive function. We also show that c-Src is a previously unrecognized pH-dependent kinase, and mutation of the identified ionizable network again abolishes pH-sensitive activity. Constant pH molecular dynamics simulations support a conserved allosteric mechanism of pH-dependent binding of inhibitory SH2 domains to the functional catalytic domains of SHP2 and c-Src. We apply our computational pipeline across SH2 domain-containing signaling proteins and identify evolutionarily conserved putative pH-sensing networks. Our results reveal that pH is an allosteric regulator of SH2 domain-containing signaling proteins providing insight into normal pH-dependent cell biology and diseases where pHi is dysregulated, such as cancer.

Monobody Inhibitor Selective to the Phosphatase Domain of SHP2 and its Use as a Probe for Quantifying SHP2 Allosteric Regulation

SHP2 is a phosphatase/adaptor protein that plays an important role in various signaling pathways. Its mutations are associated with cancers and developmental diseases. SHP2 contains a protein tyrosine phosphatase (PTP) and two SH2 domains. Selective inhibition of these domains has been challenging due to the multitude of homologous proteins in the proteome. Here, we developed a monobody, synthetic binding protein, that bound to and inhibited the SHP2 PTP domain. It was selective to SHP2 PTP over close homologs. A crystal structure of the monobody-PTP complex revealed that the monobody bound both highly conserved residues in the active site and less conserved residues in the periphery, rationalizing its high selectivity. Its epitope overlapped with the interface between the PTP and N-terminal SH2 domains that is formed in auto-inhibited SHP2. By using the monobody as a probe for the accessibility of the PTP active site, we developed a simple, nonenzymatic assay for the allosteric regulation of SHP2. The assay showed that, in the absence of an activating phospho-Tyr ligand, wild-type SHP2 and the “PTP-dead” C459E mutant were predominantly in the closed state in which the PTP active site is inaccessible, whereas the E76K and C459S mutants were in the open, active state. It also revealed that previously developed monobodies to the SH2 domains, ligands lacking a phospho-Tyr, weakly favored the open state. These results provide corroboration for a conformational equilibrium underlying allosteric regulation of SHP2, provide powerful tools for characterizing and controlling SHP2 functions, and inform drug discovery against SHP2.

Design and synthesis of improved active-site SHP2 inhibitors with anti-breast cancer cell effects

The Src homology containing phosphotyrosyl phosphatase 2 (SHP2) is a bona fide oncogene particularly in cancers driven by overexpression of receptor tyrosine kinases (RTKs). As such, there is a growing interest to target SHP2 in cancer. Based on these premises, several active site (type I) and allosteric site (type II) inhibitors have been developed, but no SHP2 targeting therapies have reached the clinic yet. In an effort to fill these gaps, we embarked on producing optimized versions of our parent active-site SHP2 inhibitor CNBDA. The objectives were to produce derivatives with increased inhibitory potential and improved selectivity. Accordingly, we designed derivatives around the CNBDA scaffold and predicted their binding property by in silico molecular modeling. Based on comparative differences in free energy of binding to the SHP2 versus the SHP1 active sites, ten were selected, chemically synthesized, and evaluated by NMR and mass spectroscopy for structural integrity. Among the ten derivatives, BPDA2 was found to be the most potent and highly selective compound, inhibiting the SHP2 enzyme activity with an IC50 of 92 nM when DiFMUP was used as a substrate and with an IC50 of 47 nM when pNPP was used as a substrate. Furthermore, enzyme kinetic analyses showed that BPDA2 is a competitive SHP2 inhibitor. Selectivity comparisons in a PTPase assay using DiFMUP as a substrate demonstrated that BPDA2 is more selective to SHP2 than to SHP1 and PTP1B by more than 369-fold and 442-fold, respectively. Evaluation with a cellular thermal shift assay (CETSA) confirmed that BPDA2 binds to wild-type SHP2 in a cellular context, and stabilizes it in solution. Treatment of cells with DBDA2 downregulates mitogenic and cell survival signaling and RTK expression in a concentration dependent manner. Furthermore, treatment of cells with BPDA2 suppresses anchorage independent growth and cancer stem cell properties of breast cancer cells. Overall, data described in this report show that BPDA2 is a more potent derivative of CNBDA with a highly improved selectivity for SHP2.

Abstract 3327: Discovery and characterization of the potent, allosteric SHP2 inhibitor GDC-1971 for the treatment of RTK/RAS driven tumors

Abstract The non-receptor protein tyrosine phosphatase SHP2 (PTPN11) plays an important role in the regulation of RAS/MAPK signal transduction downstream of growth factor receptor activation. Loss of SHP2 activity suppresses tumor cell growth, making SHP2 a potential target for cancer therapy. Here we report the discovery of GDC-1971 (formerly RLY-1971), a highly potent, selective, and orally bioavailable small-molecule SHP2 inhibitor that stabilizes SHP2 in a closed, auto-inhibited conformation. GDC-1971 potently inhibits both wild-type SHP2 (IC50 <1nM) and the E76K activating mutant (IC50 <250nM) in biochemical assays. In standard 2-dimensional and anchorage-independent growth conditions, GDC-1971 inhibits cellular proliferation in models harboring receptor tyrosine kinases (RTKs), SHP2, NF1, KRAS, or BRAF mutations in a dose-dependent manner. GDC-1971 potently inhibits the proliferation of cellular models harboring KRAS G12C or G12A mutations (median IC50 <80 nM) compared to models harboring other KRAS G12, G13 or Q61 mutations (median IC50 >1 uM), indicating a link between KRAS GTP hydrolysis and SHP2 dependency. Despite this trend, some non-KRAS G12C or G12A cell lines harboring other KRAS mutations responded to GDC-1971 in vitro, suggesting some heterogeneity in RTK/SHP2 signaling dependence in subsets of other KRAS mutants. In vivo, GDC-1971 demonstrates dose-dependent RAS/MAPK pathway inhibition and induces significant tumor-growth inhibition in human xenograft models harboring EGFR and KRAS alterations at continuous daily doses that are well tolerated. Given the reported role of SHP2 as a critical mediator of resistance to targeted therapies, we assessed the activity of GDC-1971 combinations in multiple contexts. We observed increased suppression of the MAPK signaling cascade and anti-proliferation synergy when combining GDC-1971 with EGFR, ALK, and KRAS G12C inhibitors in vitro. The observed in vitro synergy translated to dramatic anti-tumor growth effects in vivo. GDC-1971 in combination with the KRAS G12C covalent inhibitor GDC-6036 resulted in significant regressions at doses well below those required for single agent activity in a KRAS G12C-mutant NSCLC xenograft model. In rodent and dog toxicology studies, GDC-1971 is well tolerated at exposures above those required to induce regression in xenograft models. The biochemical and cellular potency and favorable pharmaceutical properties of GDC-1971 support the further clinical development in RTK/MAPK pathway altered tumors using continuous daily dosing alone and in combination with other targeted agents, including the KRAS-G12C inhibitor GDC-6036 (clinical trial NCT04449874). Citation Format: Bret Williams, Alexander Taylor, Olivia Orozco, Christopher Owen, Elizabeth Kelley, Andre Lescarbeau, Kelley Shortsleeves, Randy Kipp, Vy Nguyen, Erin Brophy, Jeremy Wilbur, Yong Tang, David Lanzetta, Nigel Waters, Sherri Smith, Fabrizio Giordanetto, Paul Maragakis, Jack Greismann, Lindsay Willmore, Eric Therrien, Yang Xiao, Marie Evangelista, Luca Gerosa, Eva Lin, Mark Merchant, Alfonso Arrazate, Emily Chan, Pablo Sáenz-López Larrocha, Stefan Chun, Thomas Hunsaker, Gauri Deshmukh, Christine M. Bowman, David E. Shaw, Mark Murcko, Mahesh Padval, W Patrick Walters, James Watters, Donald A. Bergstrom. Discovery and characterization of the potent, allosteric SHP2 inhibitor GDC-1971 for the treatment of RTK/RAS driven tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3327.

Abstract 3345: ERAS-601, a potent inhibitor of SHP2, synergistically enhances the activity of a FLT3 inhibitor, gilteritinib, in FLT3-mutated AML tumor models

Abstract ERAS-601 is a potent, small molecule allosteric inhibitor of wildtype SHP2, a non-receptor protein tyrosine phosphatase (PTP) encoded by the PTPN11 gene. ERAS-601 inhibits wildtype SHP2 biochemically with an IC50 of 4.6 nM and demonstrates selectivity across panels of 300 kinases and 12 phosphatases. SHP2 mediates upstream receptor tyrosine kinase (RTK) signaling via its phosphatase-mediated regulation of guanine nucleotide exchange factors (GEFs). ERAS-601 inhibits the SHP2 dependent cycling of KRAS from the inactive GDP-bound state to the active GTP-bound state and demonstrates anti-proliferative activity in FLT3-mutated human cancer cells. Gilteritinib is a second-generation FLT3 inhibitor and has demonstrated clinical activity, but emerging resistance limits the benefit of monotherapy. Here, we explored the combination of gilteritinib with ERAS-601 in FLT3 mutated AML nonclinical models. The combination of ERAS-601 with gilteritinib inhibits the oncogenic RAS/MAPK signaling as measured by pERK1/2 and synergistically inhibits the cellular viability of FLT3-mutated AML cells in vitro. The combination of ERAS-601 with gilteritinib achieves a more durable tumor growth inhibition than the respective gilteritinib and ERAS-601 monotherapies in vivo. These preclinical data support the clinical development of ERAS-601 in combination with gilteritinib in FLT3-altered AML. Citation Format: Leenus Martin, Roopal Patel, Jingchuan Zhang, Robin Nevarez, Taylor Congdon, Les Brail, Robert Shoemaker. ERAS-601, a potent inhibitor of SHP2, synergistically enhances the activity of a FLT3 inhibitor, gilteritinib, in FLT3-mutated AML tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3345.

Abstract 2671: ERAS-601, a potent allosteric inhibitor of SHP2, demonstrates anti-tumor activity in RAS/MAPK-driven tumor models

Abstract SHP2 is a non-receptor protein tyrosine phosphatase (PTP) encoded by the PTPN11 gene. SHP2 transduces upstream receptor tyrosine kinase (RTK) signaling to the RAS/MAPK pathway via its phosphatase-mediated regulation of guanine nucleotide exchange factors (GEFs). The modulation of GEF activity impacts the rate at which KRAS cycles from the inactive GDP-bound state to the active GTP-bound state. ERAS-601 is a potent, selective small molecule allosteric inhibitor of SHP2. ERAS-601 potently inhibits the wild type SHP2 protein with a biochemical IC50 of 4.6 nM. ERAS-601 is a selective SHP2 inhibitor and demonstrates no appreciable inhibition against any off-target kinase or phosphatase across panels of 300 kinases and 12 phosphatases. ERAS-601 inhibits the loading of active GTP-bound oncogenic RAS and inhibits RAS/MAPK pathway signaling as measured by pERK1/2 inhibition and DUSP6 mRNA. ERAS-601 demonstrates anti-proliferative activity across a panel of human cancer cell line models with oncogenic alterations in the RAS/MAPK pathway. In a mouse in vivo study, ERAS-601 achieves substantial systemic exposure and demonstrates inhibition of ERK1/2 phosphorylation and DUSP6 mRNA levels in the NCI-H358 xenograft model. ERAS-601 also inhibits tumor growth in multiple RAS/MAPK-driven CDX and PDX models that harbor EGFR, KRAS, BRAF Class III, and NF1LOF mutations. ERAS-601 is a potent and selective allosteric SHP2 inhibitor that demonstrates anti-tumor activity in vitro and in vivo and is currently being studied as a monotherapy in an ongoing Phase 1 clinical study in patients with advanced or metastatic solid tumors (FLAGSHP-1, NCT04670679). Citation Format: Leenus Martin, Roopal Patel, Jingchuan Zhang, Jennifer Yang, Robin Nevarez, Taylor Congdon, Les Brail, Robert Shoemaker. ERAS-601, a potent allosteric inhibitor of SHP2, demonstrates anti-tumor activity in RAS/MAPK-driven tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2671.

Abstract 2670: ERAS-601, a potent allosteric inhibitor of SHP2, synergistically enhances the efficacy of sotorasib/adagrasib and cetuximab in NSCLC, CRC, and HNSCC tumor models

Abstract ERAS-601 is a potent, small molecule allosteric inhibitor of wildtype SHP2, a non-receptor protein tyrosine phosphatase (PTP) encoded by the PTPN11 gene. ERAS-601 inhibits wild type SHP2 biochemically with an IC50 of 4.6 nM and demonstrates selectivity across panels of 300 kinases and 12 phosphatases. SHP2 mediates upstream receptor tyrosine kinase (RTK) signaling via its phosphatase-mediated regulation of guanine nucleotide exchange factors (GEFs). ERAS-601 inhibits the SHP2 dependent cycling of KRAS from the inactive GDP-bound state to the active GTP-bound state and demonstrates anti-proliferative activity in KRASG12C and EGFR amplified cell lines. The combination of upstream blockade of RAS-GTP cycling by ERAS-601 with inhibition of KRASG12C by a selective KRASG12C inhibitor synergistically inhibits cellular proliferation in multiple KRASG12C mutated human cancer cell lines. The combinations of ERAS-601 with KRASG12C inhibitors achieve tumor growth inhibition that is superior to the respective ERAS-601 and KRASG12C monotherapies in NSCLC and CRC CDX and PDX tumor models. Similarly, the combination of ERAS-601 with an EGFR antibody, cetuximab, inhibits oncogenic RAS/MAPK signaling as measured by pERK1/2 and enhances the anti-proliferative activity of cetuximab in RAS/RAF wild type CRC and HPV-negative HNSCC cell lines. The combination of ERAS-601 with cetuximab achieves tumor growth inhibition that is superior to respective ERAS-601 and cetuximab monotherapies in RAS/RAF wild type CRC and HPV-negative HNSCC CDX and PDX tumor models. These nonclinical data support the clinical development of ERAS-601 in combination with a KRASG12C inhibitor in NSCLC and CRC tumors, and ERAS-601 in combination with cetuximab in RAS/RAF wild type CRC as well as HPV-negative HNSCC tumors. Both combinations are being studied in ongoing clinical studies (HERKULES-2, NCT04959981; FLAGSHP-1, NCT04670679). Citation Format: Leenus Martin, Roopal Patel, Jingchuan Zhang, Robin Nevarez, Taylor Congdon, Les Brail, Robert Shoemaker. ERAS-601, a potent allosteric inhibitor of SHP2, synergistically enhances the efficacy of sotorasib/adagrasib and cetuximab in NSCLC, CRC, and HNSCC tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2670.

Establishment of Stable Cell Lines Representing Juvenile Myelomonocytic Leukemia with SHP2 Mutations

Protein tyrosine phosphatase SHP2 encoded by PTPN11 is a key regulator in growth factor and cytokine signaling. Overwhelming evidence suggests its vital role in hematopoietic stem cell function and hematopoiesis. As a bona fide proto-oncogene product, gain-of-function mutations of SHP2 cause hematological malignancies, most notably juvenile myelomonocytic leukemia (JMML) which bear somatic SHP2 mutations in 35% of cases. Numerous studies have utilized murine models to investigate the role of mutant SHP2 in hematopoiesis and leukemogenesis and successfully produced resembling myeloproliferative neoplasm (MPN) and even full-blown leukemia in recipient animals. However, mutant SHP2-transformed cell lines have not been generated. In the present study, we established oncogenic mutant SHP2-transformed cell lines from erythropoietin (EPO)-dependent HCD-57 erythroid leukemia cells. First, we generated recombinant retroviruses expressing SHP2-D61Y and SHP2-E76K, the two most common SHP2 mutants found in individuals with JMML, by using the pMSCV-IRES-GFP vector. We then infected HCD-57 cells with the recombinant retroviruses. Unlike the parent HCD-57 cells, the infected cells were able to grow in the absence of EPO as demonstrated by viable GFP-positive cells. We further performed semi-solid methylcellulose colony cultures and isolated single clones of EPO-independent HCD57 cells. The isolated clonal cells overexpressed mutant SHP2 and proliferate rapidly in the absence of EPO. In contrast, HCD57 cells infected with retroviruses expressing wild type SHP2 failed to survive in the absence of EPO, indicating only gain-of-function mutant forms of SHP2 have the cell-transformation capability. We also carried out parallel experiments with the pro-B Ba/F3 cell line that require interleukin 3 (IL3) for survival. Interestingly, over-expression of SHP2-D61Y and SHP2-E76K was not sufficient to give rise to IL3-indepdent Ba/F3 cells, suggesting that HCD57 cells have some unique properties making them susceptible for transformation by oncogenic SHP2 mutants. We further performed in vitro and in vivo characterization of transformed HCD57 cells. Cell signaling analyses revealed that both HCD57-SHP2-D61Y and HCD57-SHP2-E76Kcells exhibited aberrantly elevated levels of pERK and pAKT in the absence of cytokine stimulation, which was consistent with the notion that gain-of-function SHP2 mutants perturb growth control through deregulation of the Ras signaling pathway. Upon intravenous injection into immunodeficient mice, the SHP2 mutant-transformed HCD57 cells caused acute leukemia with markedly increased spleen. Finally, we screened a small molecule inhibitor library to identify compounds that may specifically target the SHP2 mutants. We found several tyrosine kinase inhibitors including dasatinib and trametinib potently inhibited HCD57-SHP2-D61Y and HCD57-SHP2-E76Kcells but not the parent HCD57 cells. At sub-micromolar concentrations, dasatinib and trametinib abolished elevated ERK and Akt activation caused by the SHP2 mutants. This study not only proves that gain-of function mutations of SHP2 are capable of fully transforming cells but also provides a unique cell system to study pathogenesis of SHP2 mutants and to identify specific inhibitors for drug development. DisclosuresNo relevant conflicts of interest to declare.

Molecular characterization of canine SHP2 mutants and anti-tumour effect of SHP2 inhibitor, SHP099, in a xenograft mouse model of canine histiocytic sarcoma.

Canine histiocytic sarcoma (HS) is an aggressive and highly metastatic neoplasm. Mutations in src homology 2 domain-containing phosphatase 2 (SHP2; encoded by PTPN11), which recently have been identified in canine HS tumour cells, could be attractive therapeutic targets for SHP099, an allosteric inhibitor of SHP2. Here, molecular characteristics of wild-type SHP2 and four SHP2 mutants (p.Ala72Gly, p.Glu76Gln, p.Glu76Ala and p.Gly503Val), including one that was newly identified in the present study, were investigated. Furthermore, in vivo effects of SHP099 on a HS cell line carrying SHP2 p.Glu76Ala were examined using a xenograft mouse model. While SHP2 Glu76 mutant cell lines and SHP2 wild-type/Gly503 mutant cell lines are highly susceptible and non-susceptible to SHP099, respectively, a cell line carrying the newly identified SHP2 p.Ala72Gly mutation exhibited moderate susceptibility to SHP099. Among recombinant wild-type protein and four mutant SHP2 proteins, three mutants (SHP2 p.Ala72Gly, p.Glu76Gln, p.Glu76Ala) were constitutively activated, while no activity was detected in wild-type SHP2 and SHP2 p.Gly503Val. Activities of these constitutively activated proteins were suppressed by SHP099; in particular, Glu76 mutants were highly sensitive. In the xenograft mouse model, SHP099 showed anti-tumour activity against a SHP2 p.Glu76Ala mutant cell line. Thus, there was heterogeneity in molecular characteristics among SHP2 mutants. SHP2 p.Glu76Ala and perhaps p.Glu76Gln, but not wild-type SHP2 or SHP2 p.Gly503Val, were considered to be oncogenic drivers targetable with SHP099 in canine HS. Further studies will be needed to elucidate the potential of SHP2 p.Ala72Gly as a therapeutic target of SHP099 in canine HS.

Activating Mutation of SHP2 Establishes a Tumorigenic Phonotype Through Cell-Autonomous and Non-Cell-Autonomous Mechanisms.

Gain-of-function mutation of SHP2 is a central regulator in tumorigenesis and cancer progression through cell-autonomous mechanisms. Activating mutation of SHP2 in microenvironment was identified to promote cancerous transformation of hematopoietic stem cell in non-autonomous mechanisms. It is interesting to see whether therapies directed against SHP2 in tumor or microenvironmental cells augment antitumor efficacy. In this review, we summarized different types of gain-of-function SHP2 mutations from a human disease. In general, gain-of-function mutations destroy the auto-inhibition state from wild-type SHP2, leading to consistency activation of SHP2. We illustrated how somatic or germline mutation of SHP2 plays an oncogenic role in tumorigenesis, stemness maintenance, invasion, etc. Moreover, the small-molecule SHP2 inhibitors are considered as a potential strategy for enhancing the efficacy of antitumor immunotherapy and chemotherapy. We also discussed the interconnection between phase separation and activating mutation of SHP2 in drug resistance of antitumor therapy.

Discriminating between competing models for the allosteric regulation of oncogenic phosphatase SHP2 by characterizing its active state

The Src-homology 2 domain containing phosphatase 2 (SHP2) plays a critical role in crucial signaling pathways and is involved in oncogenesis and in developmental disorders. Its structure includes two SH2 domains (N-SH2 and C-SH2), and a protein tyrosine phosphatase (PTP) domain. Under basal conditions, SHP2 is auto-inhibited, with the N-SH2 domain blocking the PTP active site. Activation involves a rearrangement of the domains that makes the catalytic site accessible, coupled to the association between the SH2 domains and cognate proteins containing phosphotyrosines. Several aspects of this transition are debated and competing mechanistic models have been proposed. A crystallographic structure of SHP2 in an active state has been reported (PDB code 6crf), but several lines of evidence suggests that it is not fully representative of the conformations populated in solution. To clarify the structural rearrangements involved in SHP2 activation, enhanced sampling simulations of the autoinhibited and active states have been performed, for wild type SHP2 and its pathogenic E76K variant. Our results demonstrate that the crystallographic conformation of the active state is unstable in solution, and multiple interdomain arrangements are populated, thus allowing association to bisphosphorylated sequences. Contrary to a recent proposal, activation is coupled to the conformational changes of the N-SH2 binding site, which is significantly more accessible in the active sate, rather than to the structure of the central β-sheet of the domain. In this coupling, a previously undescribed role for the N-SH2 BG loop emerged.

An allosteric interaction controls the activation mechanism of SHP2 tyrosine phosphatase

SHP2 is a protein tyrosine phosphatase (PTP) involved in multiple signaling pathways. Mutations of SHP2 can result in Noonan syndrome or pediatric malignancies. Inhibition of wild-type SHP2 represents a novel strategy against several cancers. SHP2 is activated by binding of a phosphopeptide to the N-SH2 domain of SHP2, thereby favoring dissociation of the N-SH2 domain and exposing the active site on the PTP domain. The conformational transitions controlling ligand affinity and PTP dissociation remain poorly understood. Using molecular simulations, we revealed an allosteric interaction restraining the N-SH2 domain into a SHP2-activating and a stabilizing state. Only ligands selecting for the activating N-SH2 conformation, depending on ligand sequence and binding mode, are effective activators. We validate the model of SHP2 activation by rationalizing modified basal activity and responsiveness to ligand stimulation of several N-SH2 variants. This study provides mechanistic insight into SHP2 activation and may open routes for SHP2 regulation.

Phase Separation of Disease-Associated SHP2 Mutants Underlies MAPK Hyperactivation

The non-receptor protein tyrosine phosphatase (PTP) SHP2, encoded by PTPN11, plays an essential role in RAS-mitogen-activated protein kinase (MAPK) signaling during normal development. It has been perplexing as to why both enzymatically activating and inactivating mutations in PTPN11 result in human developmental disorders with overlapping clinical manifestations. Here, we uncover a common liquid-liquid phase separation (LLPS) behavior shared by these disease-associated SHP2 mutants. SHP2 LLPS is mediated by the conserved well-folded PTP domain through multivalent electrostatic interactions and regulated by an intrinsic autoinhibitory mechanism through conformational changes. SHP2 allosteric inhibitors can attenuate LLPS of SHP2 mutants, which boosts SHP2 PTP activity. Moreover, disease-associated SHP2 mutants can recruit and activate wild-type (WT) SHP2 in LLPS to promote MAPK activation. These results not only suggest that LLPS serves as a gain-of-function mechanism involved in the pathogenesis of SHP2-associated human diseases but also provide evidence that PTP may be regulated by LLPS that can be therapeutically targeted.

Mechanism of activating mutations and allosteric drug inhibition of the phosphatase SHP2

Protein tyrosine phosphatase SHP2 functions as a key regulator of cell cycle control, and activating mutations cause several cancers. Here, we dissect the energy landscape of wild-type SHP2 and the oncogenic mutation E76K. NMR spectroscopy and X-ray crystallography reveal that wild-type SHP2 exchanges between closed, inactive and open, active conformations. E76K mutation shifts this equilibrium toward the open state. The previously unknown open conformation is characterized, including the active-site WPD loop in the inward and outward conformations. Binding of the allosteric inhibitor SHP099 to E76K mutant, despite much weaker, results in an identical structure as the wild-type complex. A conformational selection to the closed state reduces drug affinity which, combined with E76K’s much higher activity, demands significantly greater SHP099 concentrations to restore wild-type activity levels. The differences in structural ensembles and drug-binding kinetics of cancer-associated SHP2 forms may stimulate innovative ideas for developing more potent inhibitors for activated SHP2 mutants.

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

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. SHP2E76K 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.

Abstract 4877: Allosteric inhibition of SHP2 variants containing cancer-associated activating mutations

Abstract SHP2 (PTPN11) is a non-receptor protein tyrosine phosphatase and scaffold protein that functions in multiple signal transduction pathways. Genetic and pharmacologic evidence supports a role for SHP2 in driving the proliferation of cancer cells dependent upon a range of activated RTKs, certain RAS and BRAF mutations, and NF1 loss-of-function mutations. In addition, dominant activating mutations in PTPN11 fuel pathogenic RAS/MAPK signaling and underlie certain human RASopathies (40% of Noonan syndrome and 80% of LEOPARD syndrome) and cancers (35% of JMML and up to 5% of many other cancers). These mutants destabilize an autoinhibited conformation of SHP2, which can also be activated by binding to diphosphotyrosine motifs in vivo or to synthetic diphosphopeptides in vitro. Multiple selective, allosteric inhibitors that stabilize this autoinhibited state and potently inhibit wild-type (WT) SHP2 have recently been reported, but the impact of these inhibitors on mutant forms of the protein is less well established. Here we investigate the activity of a diverse set of over fifty allosteric SHP2 inhibitors on multiple cancer-associated activating mutants of SHP2, both in the presence and absence of an activating diphosphopeptide. The rank order of potencies of the inhibitors remains identical for all mutants, although a decrease in potency relative to WT is seen in some mutants, which is magnified further in the presence of activating peptide. These results conform to a simple equilibrium model, where inhibitors show a reduction in potency against activated mutants that is proportional to the energetic magnitude of the activating mutation. Surprisingly, some common activating mutations of SHP2 (e.g., D61G) have a relatively modest energetic effect, and exhibit similar sensitivity to allosteric inhibitors as WT SHP2. We follow up these biochemical results with cellular inhibition studies in HEK293 cells stably transfected with the respective SHP2 mutants. Collectively, these results suggest that potent allosteric SHP2 inhibitors will be effective at inhibiting the growth of cancers driven by a subset of activating mutations in SHP2. Citation Format: David Wildes, Naing Aay, Andreas Buckl, Daphne Hsieh, Ashutosh S. Jogalekar, Gert Kiss, Elena S. Koltun, Abby Marquez, Kevin T. Mellem, Jason Romero, Mae Saldajeno-Concar, Christopher J. Schulze, Chris M. Semko, Walter Won, Robert J. Nichols, Carlos Stahlhut, Christos Tzitzilonis, Adrian L. Gill, Jacqueline A. Smith. Allosteric inhibition of SHP2 variants containing cancer-associated activating mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4877.

Abstract 4879: Mutation selectivity of the allosteric SHP2 inhibitor SHP099

Abstract SHP2 (PTPN11) mediates oncogenic signaling and gain-of-function (GOF) SHP2 mutations are associated with leukemias and other types of cancer. Recently, SHP099 was identified as a novel allosteric SHP2 inhibitor that displayed high potency and selectivity to the wildtype SHP2 in vitro and inhibited protein tyrosine kinase oncogene-driven cancer cells. However, it was unclear whether SHP099 inhibits SHP2 mutants. Using SHP2 mutant-dependent TF-1 leukemia cells, we determined sensitivities of four common oncogenic SHP2 mutants to SHP099. SHP2D61Y-, SHP2A72V-, and SHP2E76K-expressing TF-1 cells were resistant, whereas SHP2E69K-expressing TF-1 cells were sensitive to SHP099 with a potency similar to that of cytokine-stimulated TF-1 cells expressing the wildtype SHP2. Consistently, SHP099 reduced active ERK1/2 and Bcl-XL and induced apoptosis of SHP2E69K-dependent TF-1 cells, but had no effects on the other SHP2 mutant cell lines. SHP2 knockout cells were unresponsive to SHP099, demonstrating specificity. Like the wildtype SHP2, SHP2E69K PTP was inhibited by SHP099 in vitro with a sub-micromolar IC50, whereas micromolar IC50s of SHP099 were observed against SHP2D61Y, SHP2A72V, and SHP2E76K. These results indicate that SHP2D61Y, SHP2A72V, and SHP2E76K mutants are resistant to SHP099 and that SHP2E69K is a SHP099-sensitive oncogenic mutant. Citation Format: Xiaojun Sun, Yuan Ren, Steven Gunawan, Peng Teng, Zhengming Chen, Harshani R. Lawrence, Jianfeng Cai, Nicholas J. Lawrence, Jie Wu. Mutation selectivity of the allosteric SHP2 inhibitor SHP099 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4879.

Abstract 2366: The tyrosine phosphatase, SHP-2, participates in regulating PD-L1 expression in non-small cell lung cancer

Abstract Immune checkpoint inhibitors, specifically those which target T cell surface receptor Programmed Cell Death-1 (PD-1) and its ligand, Programmed cell death ligand-1 (PD-L1), have demonstrated clinical benefit in patients with non-small cell lung cancer (NSCLC). Currently, tumor expression of PD-L1 serves as the FDA-approved companion diagnostic for response to anti-PD-1/PD-L1 therapy. However, a fraction of patients who express PD-L1 exhibit a response to treatment, and in some cases, patient tumors that do not express PD-L1 demonstrate response to therapy, making this a poor predictive biomarker. Further, the mechanism by which PD-L1 expression is controlled remains under-studied. In this study, we seek to determine the impact of the protein tyrosine phosphatase, SHP-2, on PD-L1 expression through Jak/STAT- mediated signaling in NSCLC cell lines. SHP-2 can harbor activating mutations which prevent auto-inhibition of the catalytic phosphatase domain, and that may be pro-tumorigenic in some tumors. To understand the impact of SHP-2 activity on PD-L1 expression, we first assessed activation of STAT3 in NSCLC tumor cells after treatment with IFNγ and other mitogens. We then transfected NSCLC cells with wildtype and hyperactive SHP-2 mutants or ablated SHP-2 with siRNA specific for SHP-2 and measured both STAT3 and PD-L1 expression. Cell lines A549 and H661 exhibit low endogenous expression of both SHP-2 and PD-L1, and thus were chosen for transfection with hyperactive SHP-2 mutants (E76K and G503V), which also altered PD-L1 expression by western blot analysis. Quantitative real-time PCR (qPCR) and western blot analysis revealed that siRNA ablation of SHP-2 led to increased PD-L1 expression in the H460 cell line. Cells transfected with the SHP-2 activating mutants will also be assessed alteration of cell proliferation. Finally, using data obtained from The Cancer Genome Atlas project, STAT3 and PD-L1 expression levels are being evaluated in NSCLC tumor samples in which SHP-2 is mutated (E76K or G503V). With these data, we expect to gain a more complete understanding of the role of SHP-2 within the tumor microenvironment and the extent to which it controls PD-L1 expression, leading to new drug targets or combinations of drugs and better predictive biomarkers of response to PD-1 and PD-L1 inhibitors. Citation Format: Keller Toral, Brent Harris, Jarrod Creameans, Penni Black. The tyrosine phosphatase, SHP-2, participates in regulating PD-L1 expression in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2366.

Redox Regulation of a Gain-of-Function Mutation (N308D) in SHP2 Noonan Syndrome.

SHP2 (Src homology 2 domain-containing protein tyrosine phosphatase 2; PTPN11) is a ubiquitous multidomain, nonreceptor protein tyrosine phosphatase (PTP) that plays an important role in diseases such as cancer, diabetes, and Noonan syndrome (NS). NS is one of the most common genetic disorders associated with congenital heart disease, and approximately half of the patients with Noonan syndrome have gain-of-function mutations in SHP2. One of the most common NS mutations is N308D. The activity of SHP2, like that of most PTPs, is reversibly inactivated by reactive oxygen species (ROS). However, the molecular basis of this inactivation and the consequences of NS-related mutations in PTPN11 on ROS-mediated inhibition are poorly understood. Here, we investigated the mechanistic and structural details of the reversible oxidation of the NS variant SHP2N308D. We show that SHP2N308D is more sensitive to oxidation when compared with wild-type SHP2. We also show that although the SHP2N308D catalytic domain can be reactivated by dithiothreitol as effectively as the wild-type, full-length SHP2N308D is only poorly reactivated by comparison. To understand the mechanism of oxidation at a molecular level, we determined the crystal structure of oxidized SHP2N308D. The structure shows that the catalytic Cys459 residue forms a disulfide bond with Cys367, which confirms that Cys367 functions as the “backdoor” cysteine in SHP2. Together, our data suggest that the reversible oxidation of SHP2 contributes negligibly, if at all, to the symptoms associated with NS.