SHP-1 Phosphatase Activity Research Articles

Shc1 cooperates with Frs2 and Shp2 to recruit Grb2 in FGF-induced lens development.

Fibroblast growth factor (FGF) signaling elicits multiple downstream pathways, most notably the Ras/MAPK cascade facilitated by the adaptor protein Grb2. However, the mechanism by which Grb2 is recruited to the FGF signaling complex remains unresolved. Here we showed that genetic ablation of FGF signaling prevented lens induction by disrupting transcriptional regulation and actin cytoskeletal arrangements, which could be reproduced by deleting the juxtamembrane region of the FGF receptor and rescued by Kras activation. Conversely, mutations affecting the Frs2-binding site on the FGF receptor or the deletion of Frs2 and Shp2 primarily impact later stages of lens vesicle development involving lens fiber cell differentiation. Our study further revealed that the loss of Grb2 abolished MAPK signaling, resulting in a profound arrest of lens development. However, disrupting the Grb2 binding site on Shp2 or abrogating Shp2 phosphatase activity only modestly influenced FGF signaling, whereas mutating the presumed Shp2 dephosphorylation site on Grb2 did not impede MAPK signaling in lens development, indicating that Shp2 is only partially responsible for Grb2 recruitment. In contrast, we observed that FGF signaling is required for the phosphorylation of the Grb2-binding sites on Shc1 and the deletion of Shc1 exacerbates the lens vesicle defect caused by Frs2 and Shp2 deletion. These results reveal that Shc1 collaborates with Frs2 and Shp2 to target Grb2 in FGF signaling.

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.

Abstract 1183: A first-in-class and SHP1 allosteric inhibitor, SB8091, shows a good efficacy and safety profile in preclinical models

Abstract Introduction: Src homology 2 domain-containing phosphatase 1 (SHP1) is a cytoplasmic protein tyrosine phosphatase (PTP) that is primarily expressed in hematopoietic cells and is commonly recognized as a negative regulator of multiple signaling pathways1. Previously, we showed that inhibition of the phosphatase activity of SHP1 by a novel SHP1 allosteric inhibitor, SB6299, results in the activation of innate and adaptive immune cells and enhanced anti-tumor immune responses2. Here, we report SB8091, an advanced SHP1 allosteric inhibitor with a promising initial safety profile and improved pharmacokinetic properties. Methods: SHP1 inhibitors were developed through a structure-based drug design approach, and optimized for cellular activity on target, in vitro ADME, and in vivo pharmacokinetic properties. Pharmacokinetic (PK) and preliminary toxicity studies were performed with mice. The lead series was assessed in in-vitro studies with mouse or human primary immune cells. In-vivo efficacy and pharmacodynamic data were generated using mouse syngeneic tumor models. Results: Structurally, SB8091 seemed to stabilize the autoinhibitory form of SHP1 as an allosteric inhibitor, in a nanomolar range and which can explain the outstanding selectivity compared to other PTP enzymes, including SHP2. In several human primary immune cell types, SB8091 significantly increased cytokine responses and demonstrated significantly enhanced activity in NK cells and macrophages against various cancer target cells. As a single agent, this compound exhibited inhibitory effects on tumor growth when administered orally, and the efficacy was further enhanced when co-administered with anti-PD1 antibodies in various syngeneic tumor models. Notably, this effect was associated with a pharmacodynamic response marked by increased levels of phosphorylated substrates regulated by SHP1. SB8091 exhibited favorable pharmacokinetic properties for oral administration, and SafetyScreen data showed a very low potential for off-target activity and drug-drug interactions. Additionally, during a 2-week repeat pilot toxicity study in mice, SB8091 demonstrated excellent tolerability even at high doses, indicating a high therapeutic index (TI > 100). Conclusion: Our advanced compound, SB8091, is a potent, highly selective, and orally bioavailable allosteric inhibitor of SHP1. It has demonstrated excellent efficacy as a monotherapy or in combination with immune checkpoint blockers, with favorable drug properties and safety profiles. Research to advance to the clinical stage is being conducted.

TGF-β antagonizes IFN-γ-mediated adaptive immune evasion via activation of the AKT-Smad3-SHP1 axis in lung adenocarcinoma.

IFN-γ-mediated signaling in tumor cells can induce immunosuppressive responses and cause tumor resistance to immunotherapy. Blocking TGF-β promotes T lymphocyte infiltration and turns immunologically cold tumors into hot tumors, thereby improving the efficacy of immunotherapy. Several studies have shown that TGF-β inhibits IFN-γ signaling in immune cells. We thus sought to determine whether TGF-β affects IFN-γ signaling in tumor cells and plays a role in the development of acquired resistance to immunotherapy. TGF-β stimulation of tumor cells increased SHP1 phosphatase activity in an AKT-Smad3-dependent manner, decreased IFN-γ-mediated tyrosine phosphorylation of JAK1/2 and STAT1, and suppressed the expression of STAT1-dependent immune evasion-related molecules, e.g., PD-L1, IDO1, HVEM, and Gal-9. In a lung cancer mouse model, dual blockade of TGF-β and PD-L1 led to superior antitumor activity and prolonged survival compared to anti-PD-L1 therapy alone. However, prolonged combined treatment resulted in tumor resistance to immunotherapy and increased expression of PD-L1, IDO1, HVEM, and Gal-9. Interestingly, after initial anti-PD-L1 monotherapy, dual TGF-β and PD-L1 blockade promoted both immune evasion gene expression and tumor growth compared to that in tumors treated with continuous PD-L1 monotherapy. Alternatively, treatment with JAK1/2 inhibitor following initial anti-PD-L1 therapy effectively suppressed tumor growth and downregulated immune evasion gene expression in tumors, indicating the involvement of IFN-γ signaling in immunotherapy resistance development. These results demonstrate an unappreciated effect of TGF-β on the development of IFN-γ-mediated tumor resistance to immunotherapy.

Abstract 3919: SHP2-DDX3X complexes contribute to translational control of CD274 mRNA in KRAS-active non-small cell lung cancer

Abstract Background: Immune checkpoint inhibitors (ICIs) have shifted the landscape in management for non-small cell lung cancer (NSCLC) and improve treatment outcomes. However, the 5-year overall survival rate of 23.2% for treatment-naïve patients with late-stage NSCLC on Pembrolizumab therapy can likely be improved. Clinical benefit is correlated with tumoral expression of PD-L1 and mutation burden. Many groups seek to identify other therapeutic targets that could improve response and overall survival. Ongoing work from our group demonstrated that SHP2, a tyrosine phosphatase, can negatively regulate PD-L1 expression in KRAS-active NSCLC, and we seek to understand the mechanism by which this occurs. SHP2 was ectopically expressed in H460 cells, immunoprecipitated and identification of interacting proteins was achieved by mass spectrometry. DDX3X, an RNA helicase, and eukaryotic translation initiation factor 1a (eIF-1a) were identified as high-ranking, potential partners of SHP2. We hypothesized that SHP2 complexes with DDX3X and eIF-1a on newly transcribed CD274 mRNA to negatively regulate PD-L1 expression. Materials and Methods: To confirm SHP2 interactions with DDX3X and eIF-1a, two pull-down assays were performed: 1. bacterially-expressed, his-tagged and immobilized proteins was used as bait for proteins contained in A549 and H460 extracts, and 2. immunoprecipitation of each endogenous protein in A549 and H460 cell extracts. Western blot analysis was performed to evaluate interacting proteins. To determine whether phosphatase activity of SHP2 is required for these interactions, immunoprecipitation was also carried out in the presence of allosteric inhibitors of SHP2 (SHP-099 and RMC-0455). SHP2 ablation was carried out with CRISPR and siRNA to examine PD-L1 expression, also determined by western blot assay. Results: His-tagged DDX3X from bacterial expression systems was able to capture SHP2 protein from both A549 cells and H460 cells, and vice versa with his-tagged SHP2. Immunoprecipitation of endogenous SHP2 was able to co-precipitate DDX3X and vice versa. Phosphatase activity of SHP2 was not required for complex formation nor does it change PD-L1 expression in A549 or H460 cells. Ablation of SHP2 leads to increased PD-L1 expression. Conclusion: A complex containing SHP2 and DDX3X may regulate the translation of CD274 in concert with eukaryotic initiation factors. Other groups have previously reported the control of translation of CD274 mRNA carried out at the 3’ UTR, and we believe this is novel evidence of a translation initiation complex monitoring the expression of CD274. Ribosome footprint and RNA immunoprecipitations are underway to address these questions. Citation Format: Palapoom Wongsaengpiboon. SHP2-DDX3X complexes contribute to translational control of CD274 mRNA in KRAS-active non-small cell lung cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3919.

Abstract 4012: Preclinical development of SHP2 allosteric inhibitor ICP-189

Abstract Background: SHP2 is a non-receptor protein tyrosine phosphatase which functions as a convergent signaling node downstream of multiple receptor tyrosine kinases (RTKs), and upstream of RAS to promote oncogenic signaling and tumor growth. SHP2 also mediates PD-1 immune blockade of T cells by binding to the immune receptor tyrosine-based inhibitory motif (ITIM) and immune receptor tyrosine-based switch motif (ITSM) of PD-1. SHP2 has long been considered as a plausible oncology drug target not only as an oncoprotein, but also as an important immune modulator. However, for decades, SHP2 was thought to be "undruggable" due to difficulties in targeting its catalytic site. Recent discovery of allosteric site of SHP2 has inspired a novel approach for SHP2 targeting. Here we report the development of ICP-189, which is a potent, highly selective, and orally bioavailable allosteric inhibitor of SHP2, demonstrated robust efficacies in various in vitro and in vivo RAS/MAPK pathway-driven models as mono-therapeutic agent or in combination with other anti-cancer drugs. Results: ICP-189 inhibits phosphatase activity of SHP2 with IC50 values < 5 nM. In a phosphatase profiling assay, ICP-189 efficiently inhibits the catalytic activity of SHP2, with no significant effects on 21 other tested tyrosine and serine/threonine phosphatases, indicating its high selectivity for SHP2. ICP-189 has demonstrated robust in vitro efficacies in a panel of tumor cell lines bearing activated RTK, RAS, NF1 loss-of-function, or BRAF class III mutations. It has also exhibited synergistic tumor killing effects in combination with EGFR, KRASG12C, MEK and CDK4/6 inhibitors. In a series of in vivo efficacy studies, ICP-189 treatment results in robust tumor growth control of MIA-PaCa-2, KYSE-520 and NCI-H358 xenografts. ICP-189 has also shown strong synergies in tumor killing when combined with MEK inhibitor trametinib, KRASG12C inhibitor sotorasib, EGFR inhibitor osimertinib in xenograft models; and when combined with anti-PD-1 antibody in EMT6 syngeneic tumors. The in vivo efficacy of ICP-189 is well accompanied by pharmacodynamic modulations, where ICP-189 exposure levels correlate with the inhibition of p-ERK and DUSP6 mRNA level in tumors. The pharmacokinetic parameters of ICP-189 are overall favorable, with high oral bioavailability. Nonclinical safety evaluations also exhibit acceptable drug tolerability in SD rats and Beagle dogs. Conclusions: ICP-189 is a novel allosteric inhibitor of SHP2 with broad-spectrum anti-tumor activities as a single agent or in combination with other targeted or immune modulating anti-cancer therapeutics. ICP-189 is now in phase I clinical trial in China and United States. Citation Format: Ruixia Liang, Yingxiang Gao, Fangfang Yang, Yingrui Han, Zuopeng Wang, Richard Liu, Charles Ying Wang, Jason Bin Zhang, Xiangyang Chen, Davy Xuesong Ouyang. Preclinical development of SHP2 allosteric inhibitor ICP-189. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4012.

Interfering B cell receptor signaling via SHP-1/p-Lyn axis shows therapeutic potential in diffuse large B-cell lymphoma

BackgroundDiffuse large B cell lymphoma (DLBCL) is an aggressive and molecularly heterogeneous non-Hodgkin’s lymphoma. The B cell receptor (BCR) signaling pathway in DLBCL emerges as a new drug target. Protein phosphatase SHP-1 negatively regulates several oncogenic tyrosine kinases and plays a tumor suppressive role.MethodsThe direct SHP-1 agonists were used to evaluate the potential therapeutic implication of SHP-1 in DLBCL. Immunohistochemical staining for SHP-1 was quantified by H-score. The SHP-1 phosphatase activity was determined using tyrosine phosphatase assay. In vitro studies, including MTT, western blot analysis and cell apoptosis, were utilized to examined biological functions of SHP-1.ResultsOral administration of SHP-1 agonist showed the potent anti-tumor effects compared to a selective Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib in mice bearing U2932 xenografts. SHP-1 agonist increased SHP-1 activity as well as downregulated p-Lyn in vivo. Here, we demonstrated that immunohistochemical staining for SHP-1 expression was positive in 76% of DLBCL samples. SHP-1 agonist exerted anti-proliferative and apoptotic effects compared with ibrutinib in DLBCL cells. Mechanistically, SHP-1 agonist decreased BCR signaling, especially p-Lyn, and led to apoptosis.ConclusionsThese data suggest that SHP-1 negatively regulates phosphorylation of Lyn, and targeting SHP-1/p-Lyn using SHP-1 agonist has therapeutic potential for treatment of DLBCL.

T cell-specific constitutive active SHP2 enhances T cell memory formation and reduces T cell activation.

Upon antigen recognition by the T cell receptor (TCR), a complex signaling network orchestrated by protein-tyrosine kinases (PTKs) and protein-tyrosine phosphatases (PTPs) regulates the transmission of the extracellular signal to the nucleus. The role of the PTPs Src-homology 2 (SH2) domain-containing phosphatase 1 (SHP1, Ptpn6) and Src-homology 2 (SH2) domain-containing phosphatase 2 (SHP2, Ptpn11) have been studied in various cell types including T cells. Whereas SHP1 acts as an essential negative regulator of the proximal steps in T cell signalling, the role of SHP2 in T cell activation is still a matter of debate. Here, we analyzed the role of the constitutively active SHP2-D61Y-mutant in T cell activation using knock-in mice expressing the mutant form Ptpn11D61Y in T cells. We observed reduced numbers of CD8+ and increased numbers of CD4+ T cells in the bone marrow and spleen of young and aged SHP2-D61Y-mutant mice as well as in Influenza A Virus (IAV)-infected mice compared to controls. In addition, we found elevated frequencies of effector memory CD8+ T cells and an upregulation of the programmed cell death protein 1 (PD-1)-receptor on both CD4+ and CD8+ T cells. Functional analysis of SHP2-D61Y-mutated T cells revealed an induction of late apoptosis/necrosis, a reduced proliferation and altered signaling upon TCR stimulation. However, the ability of D61Y-mutant mice to clear viral infection was not affected. In conclusion, our data indicate an important regulatory role of SHP2 in T cell function, where the effect is determined by the kinetics of SHP2 phosphatase activity and differs in the presence of the permanently active and the temporally regulated phosphatase. Due to interaction of SHP2 with the PD-1-receptor targeting the protein-tyrosine phosphatase might be a valuable tool to enhance T cell activities in immunotherapy.

Higher affinity binding alleles and copy number variation of inhibitory KIR2DL1 gene influence the immune surveillance in head and neck squamous cell carcinoma in the population of Assam, North-East India

Killer Ig like receptor (KIR) and the human leukocyte antigen (HLA) complex shows extensive polymorphism which has significance in context of disease association and evolution of immune responses. We have examined here the allelic diversity and copy number variation (CNV) of KIR2DL1 binding affinity with its cognate ligand HLA-C and its association with Head and Neck Squamous Cell Carcinoma (HNSCC) in a hospital based case-control study with 204 HNSCC patients and 225 non cancer participants in populations of Assam, North-East India. KIR2DL1*003 was seen at highest frequency of 79.1%. HLA-C*04 and HLA-C*07 were the most frequent HLA-C allotypes (28.5% and 25% respectively). The combined genotype of KIR2DL1*003-HLA-C2 was positively associated with HNSCC (p = 0.0152; OR = 1.9, 95%CI 1.118–2.534) and was more frequent in younger age group patients suggesting positive association with early onset of disease (p = 0.0008 OR = 2.0, 95% CI 1.157–2.363). HNSCC patients with KIR2DL1*003-HLA-C2 genotype had higher CNV of KIR2DL1 gene hinting at higher NK activation threshold which was supported by affinity binding of KIR2DL1 alleles with HLA-CW4 and by higher SHP2 phosphatase activity. Positive association of CNV with tumor proliferation, angiogenesis, cancer relapse and advance tumor stage suggested compromise of NK activation for anti-tumor response, consistent with downregulated cytokine response. In conclusion, our data suggested that higher affinity binding alleles together with higher CNV of inhibitory KIR2DL1 compromised immune surveillance in HNSCC and opens up the possibility of the use of KIR-HLA allotyping as prognostic markers.

The Discovery of Potent SHP2 Inhibitors with Anti-Proliferative Activity in Breast Cancer Cell Lines.

Despite available treatments, breast cancer is the leading cause of cancer-related death. Knowing that the tyrosine phosphatase SHP2 is a regulator in tumorigenesis, developing inhibitors of SHP2 in breast cells is crucial. Our study investigated the effects of new compounds, purchased from NSC, on the phosphatase activity of SHP2 and the modulation of breast cancer cell lines’ proliferation and viability. A combined ligand-based and structure-based virtual screening protocol was validated, then performed, against SHP2 active site. Top ranked compounds were tested via SHP2 enzymatic assay, followed by measuring IC50 values. Subsequently, hits were tested for their anti-breast cancer viability and proliferative activity. Our experiments identified three compounds 13030, 24198, and 57774 as SHP2 inhibitors, with IC50 values in micromolar levels and considerable selectivity over the analogous enzyme SHP1. Long MD simulations of 500 ns showed a very promising binding mode in the SHP2 catalytic pocket. Furthermore, these compounds significantly reduced MCF-7 breast cancer cells’ proliferation and viability. Interestingly, two of our hits can have acridine or phenoxazine cyclic system known to intercalate in ds DNA. Therefore, our novel approach led to the discovery of SHP2 inhibitors, which could act as a starting point in the future for clinically useful anticancer agents.

Phase separation ability and phosphatase activity of the SHP1-R360E mutant

SHP1 is a non-receptor protein tyrosine phosphatase that is widely expressed in hematopoietic cells such as white blood cells, neutrophils, and immune cells. SHP1 can regulate the occurrence and differentiation of immune cells and plays an important role as a tumor suppressor. Previous studies have suggested that SHP2, the homologous protein of phosphatase SHP1, can undergo liquid–liquid phase separation (LLPS). Therefore, in this study, we investigated if SHP1 is also capable of LLPS. To the best of our knowledge, our study is the first to reveal that SHP1 has the ability to undergo LLPS. In addition, we identified an important residue, SHP1-R360E, that can completely inhibit the LLPS ability of SHP1, but this mutation has no remarkable effect on SHP1's enzymatic activity. This allows us to explore the phosphatase activity and phase separation ability of SHP1 separately, providing a basis for future exploration of the phase separation mechanism of phosphatases.

Structure-based design, synthesis and biological evaluation of aminopyrazines as highly potent, selective, and cellularly active allosteric SHP2 inhibitors

Src homology-2-containing protein tyrosine phosphatase 2 (SHP2) encoded by the proto-oncogene PTPN11 is the first identified non-receptor protein tyrosine phosphatase. SHP2 dysregulation contributes to the pathogenesis of different cancers, making SHP2 a promising therapeutic target for cancer therapy. In this article, we report the structure-guided design based on the well-characterized SHP2 inhibitor SHP099, extensive structure-activity relationship studies (SARs) of aminopyrazines, biochemical characterization and cellular potency. These medicinal chemistry efforts lead to the discovery of the lead compound TK-453, which potently inhibits SHP2 (SHP2WT IC50 = 0.023 μM, ΔTm = 7.01 °C) in a reversible and noncompetitive manner. TK-453 exhibits high selectivity over SHP2PTP, SHP1 and PTP1B, and may bind at the “tunnel” allosteric site of SHP2 as SHP099. As the key pharmacophore, the aminopyrazine scaffold not only reorganizes the cationic-π stacking interaction with R111 via the novel hydrogen bond interaction between the S atom of thioether linker and T219, but also mediates a hydrogen bond with E250. In vitro studies indicate that TK-453 inhibits proliferation of HeLa, KYSE-70 and THP-1 cells moderately and induces apoptosis of Hela cells. Further mechanistic studies suggest that TK-453 can decrease the phosphorylation levels of AKT and Erk1/2 in HeLa and KYSE-70 cells. Collectively, TK-453 is a highly potent, selective, and cellularly active allosteric SHP2 inhibitor that modulates the phosphorylation of SHP2-mediated AKT and Erk cell signaling pathways by inhibiting the phosphatase activity of SHP2.

The clinicopathological and prognostic implications of tyrosine phosphatase SHP2 and ankyrin Hook1 gene expression in non- small cell lung cancer patients treated with gemcitabine plus platinum as first-line chemotherapy.

SHP2, a widely expressed phosphatase, which has been linked to the initiation, progression and prognosis of various malignancies. We previously identified a new SHP2 anchorage protein Hook1 in the alveolar II epithelial cells, and it is suggested that Hook1 is a novel endogenous suppressor molecule of SHP2 phosphatase activity. Nevertheless, few studies have been mentioned the clinicopathological and prognostic relevance of SHP2 and Hook1 expression in patients with non-small cell lung cancer (NSCLC). A total of 61 patients with NSCLC receiving gemcitabine plus carboplatin chemotherapy were studied. Immunohistochemical staining was conducted to determine the protein expression of SHP2 and Hook1. The relationships between gene expression and clinical and pathological factors, as well as prognosis, were evaluated. A significant correlation was observed between the SHP2 and Hook1 expression, with a total expression rate of 71.4%. The positive expression of Hook1 in adenocarcinoma and in stage IIIb-IV was higher than that in patients with squamous (73.5% vs. 31.2%, P=0.013) and I-IIIa (75% vs. 48.8%, P=0.05), suggesting that Hook1 might act as an indicator of NSCLC status. However, no significant correlation was observed between Hook1 expression and survival time (P>0.05). In contrast, patients with negative SHP2 expression had a higher survival rate (median overall survival 68 vs. 24 months, P=0.003). Cox multivariate regression analysis showed that SHP2 was an independent indicator for overall survival (P=0.009). The present study suggested that NSCLC patients with negative SHP2 expression could benefit from gemcitabine plus carboplatin chemotherapy, and further study is needed to confirm the prognostic value of SHP2 and Hook1.

Abstract 1039: Fragment-based drug discovery to identify small molecule allosteric inhibitors of SHP2

Abstract The ubiquitously expressed protein tyrosine phosphatase SHP2 is required for signalling downstream of receptor tyrosine kinases (RTKs) and plays a role in regulating many cellular processes. Recent advances have shown that genetic knockdown and pharmacological inhibition of SHP2 suppresses RAS/MAPK signalling and inhibits proliferation of RTK-driven cancer cell lines. SHP2 is now understood to act upstream of RAS and plays a role in KRAS-driven cancers, an area of research which is rapidly growing. Considering that RTK deregulation often leads to a wide range of cancers and the newly appreciated role of SHP2 in KRAS-driven cancers, SHP2 inhibitors are therefore a promising therapeutic approach. SHP2 contains two N-terminal tandem SH2 domains (N-SH2, C-SH2), a catalytic phosphatase domain and a C-terminal tail. SHP2 switches between “open” active and “closed” inactive forms due to autoinhibitory interactions between the N-SH2 domain and the phosphatase domain. Historically, phosphatases were deemed undruggable as there had been no advancements with active site inhibitors. We hypothesised that fragment screening would be highly applicable and amenable to this target to enable alternative means of inhibition through identification of allosteric binding sites. Here we describe the first reported fragment screen against SHP2. Using our fragment-based PyramidTM approach, screening was carried out on two constructs of SHP2; a closed autoinhibited C-terminal truncated form (phosphatase and both SH2 domains), as well as the phosphatase-only domain. A combination of screening methods such as X-ray crystallography and NMR were employed to identify fragment hits at multiple sites on SHP2, including the tunnel-like allosteric site reported by Chen et al, 2016. Initial fragment hits had affinities for SHP2 in the range of 1mM as measured by ITC. Binding of these hits was improved using structure-guided design to generate compounds which inhibit SHP2 phosphatase activity and are promising starting points for further optimization. Citation Format: Philip J. Day, Valerio Berdini, Juan Castro, Gianni Chessari, Thomas G. Davies, James E. Day, Satoshi Fukaya, Chris Hamlett, Keisha Hearn, Steve Hiscock, Rhian Holvey, Satoru Ito, Yasuo Kodama, Kenichi Matsuo, Yoko Nakatsuru, Nick Palmer, Amanda Price, Tadashi Shimamura, Jeffrey D.St. Denis, Nicola G. Wallis, Glyn Williams, Christopher N. Johnson. Fragment-based drug discovery to identify small molecule allosteric inhibitors of SHP2 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1039.

The YAP-Interacting Phosphatase SHP2 Can Regulate Transcriptional Coactivity and Modulate Sensitivity to Chemotherapy in Cholangiocarcinoma.

The Hippo pathway effector Yes-associated protein (YAP) is localized to the nucleus and transcriptionally active in a number of tumor types, including a majority of human cholangiocarcinomas. YAP activity has been linked to chemotherapy resistance and has been shown to rescue KRAS and BRAF inhibition in RAS/RAF-driven cancers; however, the underlying mechanisms of YAP-mediated chemoresistance have yet to be elucidated. Herein, we report that the tyrosine phosphatase SHP2 directly regulates the activity of YAP by dephosphorylating pYAPY357 even in the setting of RAS/RAF mutations, and that diminished SHP2 phosphatase activity is associated with chemoresistance in cholangiocarcinomas. A screen for YAP-interacting tyrosine phosphatases identified SHP2, and characterization of cholangiocarcinomas cell lines demonstrated an inverse relationship between SHP2 levels and pYAPY357. Human sequencing data demonstrated lower SHP2 levels in cholangiocarcinomas tumors as compared with normal liver. Cell lines with low SHP2 expression and higher levels of pYAPY357 were resistant to gemcitabine and cisplatin. In cholangiocarcinomas cells with high levels of SHP2, pharmacologic inhibition or genetic deletion of SHP2 increased YAPY357 phosphorylation and expression of YAP target genes, including the antiapoptotic regulator MCL1, imparting resistance to gemcitabine and cisplatin. In vivo evaluation of chemotherapy sensitivity demonstrated significant resistance in xenografts with genetic deletion of SHP2, which could be overcome by utilizing an MCL1 inhibitor. IMPLICATIONS: These findings demonstrate a role for SHP2 in regulating YAP activity and chemosensitivity, and suggest that decreased phosphatase activity may be a mechanism of chemoresistance in cholangiocarcinoma via a MCL1-mediated mechanism.

Retrograde nerve growth factor signaling abnormalities in familial dysautonomia.

Familial dysautonomia (FD) is the most prevalent form of hereditary sensory and autonomic neuropathy (HSAN). In FD, a germline mutation in the Elp1 gene leads to Elp1 protein decrease that causes sympathetic neuron death and sympathetic nervous system dysfunction (dysautonomia). Elp1 is best known as a scaffolding protein within the nuclear hetero-hexameric transcriptional Elongator protein complex, but how it functions in sympathetic neuron survival is very poorly understood. Here, we identified a cytoplasmic function for Elp1 in sympathetic neurons that was essential for retrograde nerve growth factor (NGF) signaling and neuron target tissue innervation and survival. Elp1 was found to bind to internalized TrkA receptors in an NGF-dependent manner, where it was essential for maintaining TrkA receptor phosphorylation (activation) by regulating PTPN6 (Shp1) phosphatase activity within the signaling complex. In the absence of Elp1, Shp1 was hyperactivated, leading to premature TrkA receptor dephosphorylation, which resulted in retrograde signaling failure and neuron death. Inhibiting Shp1 phosphatase activity in the absence of Elp1 rescued NGF-dependent retrograde signaling, and in an animal model of FD it rescued abnormal sympathetic target tissue innervation. These results suggest that regulation of retrograde NGF signaling in sympathetic neurons by Elp1 may explain sympathetic neuron loss and physiologic dysautonomia in patients with FD.

RUNX2/CBFB modulates the response to MEK inhibitors through activation of receptor tyrosine kinases in KRAS-mutant colorectal cancer

Intrinsic and acquired resistances are major hurdles preventing the effective use of MEK inhibitors for treatment of colorectal cancer (CRC). Some 35–45% of colorectal cancers are KRAS-mutant and their treatment remains challenging as these cancers are refractory to MEK inhibitor treatment, because of feedback activation of receptor tyrosine kinases (RTKs). We reported previously that loss of ERN1 sensitizes a subset of KRAS-mutant colon cancer cells to MEK inhibition. Here we show that the loss of RUNX2 or its cofactor CBFB can confer MEK inhibitor resistance in CRC cells. Mechanistically, we find that cells with genetically ablated RUNX2 or CBFB activate multiple RTKs, which coincides with high SHP2 phosphatase activity, a phosphatase that relays signals from the cell membrane to downstream pathways governing growth and proliferation. Moreover, we show that high activity of SHP2 is causal to loss of RUNX2-induced MEK inhibitor resistance, as a small molecule SHP2 inhibitor reinstates sensitivity to MEK inhibitor in RUNX2 knockout cells. Our results reveal an unexpected role for loss of RUNX2/CBFB in regulating RTK activity in colon cancer, resulting in reduced sensitivity to MEK inhibitors.

PTPN11 (SHP2) Is Indispensable for Growth Factors and Cytokine Signal Transduction During Bovine Oocyte Maturation and Blastocyst Development.

This study was aimed to investigate the role of SHP2 (Src-homology-2-containing phosphotyrosine phosphatase) in intricate signaling networks invoked by bovine oocyte to achieve maturation and blastocyst development. PTPN11 (Protein Tyrosine Phosphatase, non-receptor type 11) encoding protein SHP2, a positive transducer of RTKs (Receptor Tyrosine Kinases) and cytokine receptors, can play a significant role in bovine oocyte maturation and embryo development, but this phenomenon has not yet been explored. Here, we used different growth factors, cytokines, selective activator, and a specific inhibitor of SHP2 to ascertain its role in bovine oocyte developmental stages in vitro. We found that SHP2 became activated by growth factors and cytokines treatment and was highly involved in the activation of oocyte maturation and embryo development pathways. Activation of SHP2 triggered MAPK (mitogen-activated protein kinases) and PI3K/AKT (Phosphoinositide 3-kinase/Protein kinase B) signaling cascades, which is not only important for GVBD (germinal vesical breakdown) induction but also for maternal mRNA translation. Inhibition of phosphatase activity of SHP2 with PHPS1 (Phenylhydrazonopyrazolone sulfonate 1) reduced oocytes maturation as well as bovine blastocyst ICM (inner cell mass) volume. Supplementation of LIF (Leukemia Inhibitory Factor) to embryos showed an unconventional direct relation between p-SHP2 and p-STAT3 (Signal transducer and activator of transcription 3) for blastocyst ICM development. Other than growth factors and cytokines, cisplatin was used to activate SHP2. Cisplatin activated SHP2 modulate growth factors effect and combine treatment significantly enhanced quality and rate of developed blastocysts.

The prognostic significance of SHP2 and its binding protein Hook1 in non-small cell lung cancer.

BackgroundWe previously reported that Hook1 inhibits the phosphatase activity of SHP2 in the regulation of the epithelial-mesenchymal transition (EMT) in lung cancer. In this study, we performed a comprehensive analysis of SHP2 and Hook1 expression and relationships with the prognosis of patients with non-small cell lung cancer (NSCLC).Materials and methodsA total of 121 patients with NSCLC were included in this study. Expression of SHP2 and Hook1 was assessed by immunohistochemistry and Western blot analysis. The overall survival rate of NSCLC patients was analysed using Cox’s ratio hazard multivariate analysis and the log-rank test.ResultsIn tumour tissue specimens, positive expression rates of SHP2 proteins were 58.4% by immunohistochemical analysis. A significant correlation between expression of SHP2 and that of Hook1 was observed. Based on Western blot analysis, we found that Hook1 was downregulated and that SHP2 has a tendency to overexpress without statistical significance in NSCLC tissues compared with their levels in normal lung tissues. The median overall survival (OS) of NSCLC patients who presented low levels of SHP2 expression were better (40 vs 24 months, p=0.004) than those of patients who exhibited high levels of SHP2 expression. The results of multivariate analysis showed that the level of SHP2 expression was an independent prognostic factor for OS.ConclusionSHP2 might play an important role in NSCLC and has the potential to serve as a clinical biomarker or NSCLC.

Themis integrates T cell receptor and cytokine signals in CD8+ T cells

Abstract T cell receptor (TCR) signaling controls T cell development in the thymus and T cell functions in the periphery. Themis is a T-cell lineage-specific protein with a critical role in T cell development, where it regulates TCR signal strength though modulation of Shp1 phosphatase activity. The role of Themis in mature peripheral T cells is unknown. To address this, we generated Themis conditional knockout (cKO) mice, with post-selection Themis deletion mediated by distal Lck-Cre. The cKO mice have reduced CD8+ T cell numbers. Themis-deficient CD8+ T cells on the OT-I TCR transgenic background show normal functional responses to agonistic pMHC ligands of different affinities. However, Themis-deficient OT-I CD8+ T cells have reduced cell surface expression of CD5 and increased cell surface expression of CD8, suggesting decreased signal from self pMHC. Moreover, cKO OTI CD8+ T cells display severe reduction in lymphopenia-induced (homeostatic) proliferation in vivo, as well as reduction in proliferative responses to low affinity pMHC and pro-inflammatory cytokines in vitro. Signals from low affinity pMHC and cytokines synergistically induce phosphorylation of Akt, metabolic changes, and induction of c-Myc in CD8 T cells. This does not occur in the absence of Themis. Themis performs these functions through the phosphatase Shp1, as double deletion of Themis and Shp1 in peripheral T cells rescues the CD8+ T cell maintenance. This work shows a novel role for cytokines and very low affinity pMHC in altering cellular metabolism to drive CD8+ T cell proliferation. It also demonstrates that Themis and Shp1 act in concert to integrate T cell receptor and cytokine signals.