SH2 Domain-containing Phosphatase Research Articles

Design, Synthesis and Evaluation of Thioacetamide‐Tethered Thiadiazole‐1,2,4‐Triazole Hybrids as SHP2 Inhibitors for Cancer Therapy

AbstractSrc homology‐2 (SH2) domain‐containing phosphatase‐2 (SHP2), the first protooncogenic phosphatase is a key mediator in the development and progression of various cancers. Several allosteric sites have been identified in SHP2, inhibitors of which are being developed. In the current study, we have designed and synthesized a library of 21 thioacetamide‐tethered thiadiazole‐1,2,4‐triazole hybrids (compounds 16–36) and evaluated their in vitro SHP2 inhibitory potential. Compound 28 (N‐(5‐(benzo[d][1,3]dioxol‐5‐yl)‐1,3,4‐thiadiazol‐2‐yl)‐2‐((5‐(4‐methoxyphenyl)‐4H‐1,2,4‐triazol‐3‐yl)thio)acetamide) emerged as the most potent SHP2 inhibitor (IC50=0.318±0.001 μM) inhibiting the enzyme in a mixed to non‐competitive manner. In silico studies revealed that the lead inhibitor strongly binds to the tunnel allosteric site of SHP2. Further, cytotoxicity studies revealed that compound 28 caused death of SHP2‐driven MCF‐7 (GI50=37.02±0.25 μM) and U87MG cells (GI50=68.69±0.21 μM) in a dose‐dependent manner and inhibited MCF‐7 cell colony formation and migration. Flow cytometric analysis showed that it exerted its antiproliferative effect on U87MG cells by inducing early apoptosis (Q2 phase) and inhibiting cell cycle progression at the G1 and S phase. Compound 28 was shown to increase oxidative stress in the U87 cells by promoting ROS generation and loss of mitochondrial integrity. In summary, the present study produced a potent SHP2 inhibitor (compound 28) with a promising in vitro cytotoxicity profile, thus meriting further investigation.

SHP2 regulates GluA2 tyrosine phosphorylation required for AMPA receptor endocytosis and mGluR-LTD

Posttranslational modifications regulate the properties and abundance of synaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors that mediate fast excitatory synaptic transmission and synaptic plasticity in the central nervous system. During long-term depression (LTD), protein tyrosine phosphatases (PTPs) dephosphorylate tyrosine residues in the C-terminal tail of AMPA receptor GluA2 subunit, which is essential for GluA2 endocytosis and group I metabotropic glutamate receptor (mGluR)-dependent LTD. However, as a selective downstream effector of mGluRs, the mGluR-dependent PTP responsible for GluA2 tyrosine dephosphorylation remains elusive at Schaffer collateral (SC)-CA1 synapses. In the present study, we find that mGluR5 stimulation activates Src homology 2 (SH2) domain-containing phosphatase 2 (SHP2) by increasing phospho-Y542 levels in SHP2. Under steady-state conditions, SHP2 plays a protective role in stabilizing phospho-Y869 of GluA2 by directly interacting with GluA2 phosphorylated at Y869, without affecting GluA2 phospho-Y876 levels. Upon mGluR5 stimulation, SHP2 dephosphorylates GluA2 at Y869 and Y876, resulting in GluA2 endocytosis and mGluR-LTD. Our results establish SHP2 as a downstream effector of mGluR5 and indicate a dual action of SHP2 in regulating GluA2 tyrosine phosphorylation and function. Given the implications of mGluR5 and SHP2 in synaptic pathophysiology, we propose SHP2 as a promising therapeutic target for neurodevelopmental and autism spectrum disorders.

Tyrosine phosphatase PTPN11/SHP2 in solid tumors - bull's eye for targeted therapy?

Encoded by PTPN11, the Src-homology 2 domain-containing phosphatase 2 (SHP2) integrates signals from various membrane-bound receptors such as receptor tyrosine kinases (RTKs), cytokine and integrin receptors and thereby promotes cell survival and proliferation. Activating mutations in the PTPN11 gene may trigger signaling pathways leading to the development of hematological malignancies, but are rarely found in solid tumors. Yet, aberrant SHP2 expression or activation has implications in the development, progression and metastasis of many solid tumor entities. SHP2 is involved in multiple signaling cascades, including the RAS-RAF-MEK-ERK-, PI3K-AKT-, JAK-STAT- and PD-L1/PD-1- pathways. Although not mutated, activation or functional requirement of SHP2 appears to play a relevant and context-dependent dichotomous role. This mostly tumor-promoting and infrequently tumor-suppressive role exists in many cancers such as gastrointestinal tumors, pancreatic, liver and lung cancer, gynecological entities, head and neck cancers, prostate cancer, glioblastoma and melanoma. Recent studies have identified SHP2 as a potential biomarker for the prognosis of some solid tumors. Based on promising preclinical work and the advent of orally available allosteric SHP2-inhibitors early clinical trials are currently investigating SHP2-directed approaches in various solid tumors, either as a single agent or in combination regimes. We here provide a brief overview of the molecular functions of SHP2 and collate current knowledge with regard to the significance of SHP2 expression and function in different solid tumor entities, including cells in their microenvironment, immune escape and therapy resistance. In the context of the present landscape of clinical trials with allosteric SHP2-inhibitors we discuss the multitude of opportunities but also limitations of a strategy targeting this non-receptor protein tyrosine phosphatase for treatment of solid tumors.

Identification of new small molecule allosteric SHP2 inhibitor through pharmacophore-based virtual screening, molecular docking, molecular dynamics simulation studies, synthesis and in vitro evaluation

Src homology-2 (SH2) domain-containing phosphatase-2 (SHP2) is the first identified protooncogene and is a promising target for developing small molecule inhibitors as cancer chemotherapeutic agents. Pharmacophore-based virtual screening (PBVS) is a pharmacoinformatics methodology that employs physicochemical knowhow of the chemical space into the dynamic environs of computational technology to extract virtual molecular hits that are precise and promising for a drug target. In the current study, PBVS has been applied on EnamineTM Advanced Collection of 551,907 molecules by using a pharmacophore model developed upon SHP099 by Molecular Operating Environment (MOE) software to identify potential small molecule allosteric SHP2 inhibitors. Obtained 37 hits were further filtered through DruLiTo software for drug-likeness and PAINS remover which yielded 35 hits. These were subjected to molecular docking studies against the tunnel allosteric site of SHP2 (PDB ID: 5EHR) to screen them according to their binding affinity for the enzyme. Top 5 molecules having highest binding affinity for 5EHR were passed through an ADMET prediction screening and the top 2 hits (ligands 111675 and 546656) with the most favourable ADMET profile were taken for post screening molecular docking and MD simulation studies. From the protein-ligand interaction pattern, conformational stability and energy parameters, ligand 111675 (SHP2 Ki = 0.118 µM) resulted as the most active molecule. Further, the synthesis and in vitro evaluation of the lead compound 111675 unveiled its potent inhibitory activity (IC50 = 0.878 ± 0.008 µM) against SHP2. Communicated by Ramaswamy H. Sarma

From bench to bedside: current development and emerging trend of KRAS-targeted therapy.

Kirsten rat sarcoma 2 viral oncogene homolog (KRAS) is the most frequently mutated oncogene in human cancers with mutations predominantly occurring in codon 12. These mutations disrupt the normal function of KRAS by interfering with GTP hydrolysis and nucleotide exchange activity, making it prone to the GTP-bound active state, thus leading to sustained activation of downstream pathways. Despite decades of research, there has been no progress in the KRAS drug discovery until the groundbreaking discovery of covalently targeting the KRASG12C mutation in 2013, which led to revolutionary changes in KRAS-targeted therapy. So far, two small molecule inhibitors sotorasib and adagrasib targeting KRASG12C have received accelerated approval for the treatment of non-small cell lung cancer (NSCLC) harboring KRASG12C mutations. In recent years, rapid progress hasbeen achieved in the KRAS-targeted therapy field, especially the exploration of KRASG12C covalent inhibitors in other KRASG12C-positive malignancies, novel KRAS inhibitors beyond KRASG12C mutation or pan-KRAS inhibitors, and approaches to indirectly targeting KRAS. In this review, we provide a comprehensive overview of the molecular and mutational characteristics of KRAS and summarize the development and current status of covalent inhibitors targeting the KRASG12C mutation. We also discuss emerging promising KRAS-targeted therapeutic strategies, with a focus on mutation-specific and direct pan-KRAS inhibitors and indirect KRAS inhibitors through targeting the RAS activation-associated proteins Src homology-2 domain-containing phosphatase 2 (SHP2) and son of sevenless homolog 1 (SOS1), and shed light on current challenges and opportunities for drug discovery in this field.

Overcoming Immune Checkpoint Therapy Resistance with SHP2 Inhibition in Cancer and Immune Cells: A Review of the Literature and Novel Combinatorial Approaches.

SHP2 (Src Homology 2 Domain-Containing Phosphatase 2) is a protein tyrosine phosphatase widely expressed in various cell types. SHP2 plays a crucial role in different cellular processes, such as cell proliferation, differentiation, and survival. Aberrant activation of SHP2 has been implicated in multiple human cancers and is considered a promising therapeutic target for treating these malignancies. The PTPN11 gene and functions encode SHP2 as a critical signal transduction regulator that interacts with key signaling molecules in both the RAS/ERK and PD-1/PD-L1 pathways; SHP2 is also implicated in T-cell signaling. SHP2 may be inhibited by molecules that cause allosteric (bind to sites other than the active site and attenuate activation) or orthosteric (bind to the active site and stop activation) inhibition or via potent SHP2 degraders. These inhibitors have anti-proliferative effects in cancer cells and suppress tumor growth in preclinical models. In addition, several SHP2 inhibitors are currently in clinical trials for cancer treatment. This review aims to provide an overview of the current research on SHP2 inhibitors, including their mechanism of action, structure-activity relationships, and clinical development, focusing on immune modulation effects and novel therapeutic strategies in the immune-oncology field.

From Stem to Sternum: The Role of Shp2 in the Skeleton.

Src homology-2 domain-containing phosphatase 2 (SHP2) is a ubiquitously expressed phosphatase that is vital for skeletal development and maintenance of chondrocytes, osteoblasts, and osteoclasts. Study of SHP2 function in small animal models has led to insights in phenotypes observed in SHP2-mutant human disease, such as Noonan syndrome. In recent years, allosteric SHP2 inhibitors have been developed to specifically target the protein in neoplastic processes. These inhibitors are highly specific and have great potential for disease modulation in cancer and other pathologies, including bone disorders. In this review, we discuss the importance of SHP2 and related signaling pathways (e.g., Ras/MEK/ERK, JAK/STAT, PI3K/Akt) in skeletal development. We review rodent models of pathologic processes caused by germline mutations that activate SHP2 enzymatic activity, with a focus on the skeletal phenotype seen in these patients. Finally, we discuss SHP2 inhibitors in development and their potential for disease modulation in these genetic diseases, particularly as it relates to the skeleton.

Siglec-6 Signaling Uses Src Kinase Tyrosine Phosphorylation and SHP-2 Recruitment.

Preeclampsia is a pregnancy-specific disorder involving placental abnormalities. Elevated placental Sialic acid immunoglobulin-like lectin (Siglec)-6 expression has been correlated with preeclampsia. Siglec-6 is a transmembrane receptor, expressed predominantly by the trophoblast cells in the human placenta. It interacts with sialyl glycans such as sialyl-TN glycans as well as binds leptin. Siglec-6 overexpression has been shown to influence proliferation, apoptosis, and invasion in the trophoblast (BeWo) cell model. However, there is no direct evidence that Siglec-6 plays a role in preeclampsia pathogenesis and its signaling potential is still largely unexplored. Siglec-6 contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an ITIM-like motif in its cytoplasmic tail suggesting a signaling function. Site-directed mutagenesis and transfection were employed to create a series of Siglec-6 expressing HTR-8/SVneo trophoblastic cell lines with mutations in specific functional residues to explore the signaling potential of Siglec-6. Co-immunoprecipitation and inhibitory assays were utilized to investigate the association of Src-kinases and SH-2 domain-containing phosphatases with Siglec-6. In this study, we show that Siglec-6 is phosphorylated at ITIM and ITIM-like domains by Src family kinases. Phosphorylation of both ITIM and ITIM-like motifs is essential for the recruitment of phosphatases like Src homology region 2 containing protein tyrosine phosphatase 2 (SHP-2), which has downstream signaling capabilities. These findings suggest Siglec-6 as a signaling molecule in human trophoblasts. Further investigation is warranted to determine which signaling pathways are activated downstream to SHP-2 recruitment and how overexpression of Siglec-6 in preeclamptic placentas impacts pathogenesis.

Discovery of Novel Src Homology-2 Domain-Containing Phosphatase 2 and Histone Deacetylase Dual Inhibitors with Potent Antitumor Efficacy and Enhanced Antitumor Immunity.

Both Src homology-2 domain-containing phosphatase 2 (SHP2) and histone deacetylase (HDAC) are important oncoproteins and potential immunomodulators. In this study, we first observed a synergistic antiproliferation effect of an allosteric SHP2 inhibitor (SHP099) and HDAC inhibitor (SAHA) in MV4-11 cells. Inspired by this result, a series of SHP2/HDAC dual inhibitors were designed based on the pharmacophore fusion strategy. Among these inhibitors, the most potent compound 8t showed excellent inhibitory activities against SHP2 (IC50 = 20.4 nM) and HDAC1 (IC50 = 25.3 nM). In particular, compound 8t exhibited improved antitumor activities compared with those of SHP099 and SAHA in vitro and in vivo. Our study also indicated that treatment with 8t could trigger efficient antitumor immunity by activating T cells, enhancing the antigen presentation function and promoting cytokine secretion. To our knowledge, we report the first small molecular SHP2/HDAC dual inhibitor and demonstrate a new strategy for cancer immunotherapy.

Ajuforrestin A, an Abietane Diterpenoid from Ajuga ovalifolia var. calanthe, Induces A549 Cell Apoptosis by Targeting SHP2.

The Src-homology 2 domain-containing phosphatase 2 (SHP2), which is encoded by PTPN11, participates in many cellular signaling pathways and is closely related to various tumorigenesis. Inhibition of the abnormal activity of SHP2 by small molecules is an important part of cancer treatment. Here, three abietane diterpenoids, named compounds 1–3, were isolated from Ajuga ovalifolia var. calantha. Spectroscopic analysis was used to identify the exact structure of the compounds. The enzymatic kinetic experiment and the cellular thermal shift assay showed compound 2 selectively inhibited SHP2 activity in vitro. Molecular docking indicated compound 2 targeted the SHP2 catalytic domain. The predicted pharmacokinetic properties by SwissADME revealed that compound 2 passed the majority of the parameters of common drug discovery rules. Compound 2 restrained A549 proliferation (IC50 = 8.68 ± 0.96 μM), invasion and caused A549 cell apoptosis by inhibiting the SHP2–ERK/AKT signaling pathway. Finally, compound 2 (Ajuforrestin A) is a potent and efficacious SHP2 inhibitor and may be a promising compound for human lung epithelial cancer treatment.

SH2 Domain-Containing Phosphatase 2 Inhibition Attenuates Osteoarthritis by Maintaining Homeostasis of Cartilage Metabolism via the Docking Protein 1/Uridine Phosphorylase 1/Uridine Cascade.

Protein tyrosine kinases regulate osteoarthritis (OA) progression by activating a series of signal transduction pathways. However, the roles of protein tyrosine phosphatases (PTPs) in OA remain obscure. This study was undertaken to identify specific PTPs involved in OA and investigate their underlying mechanisms. The expression of 107 PTP genes in human OA cartilage was analyzed based on a single-cell sequencing data set. The enzyme activity of the PTP SH2 domain-containing phosphatase 2 (SHP-2) was detected in primary chondrocytes after interleukin-1β (IL-1β) treatment and in human OA cartilage. Mice subjected to destabilization of the medial meniscus (DMM) and IL-1β-stimulated mouse primary chondrocytes were treated with an SHP-2 inhibitor or celecoxib (a drug used for the clinical treatment of OA). The function of SHP-2 in OA pathogenesis was further verified in Aggrecan-CreERT ;SHP2flox/flox mice. The downstream protein expression profile and dephosphorylated substrate of SHP-2 were examined by tandem mass tag labeling-based global proteomic analysis and stable isotope labeling with amino acids in cell culture-labeled tyrosine phosphoproteomic analysis, respectively. SHP-2 enzyme activity significantly increased in human OA samples with serious articular cartilage injury and in IL-1β-stimulated mouse chondrocytes. Pharmacologic inhibition or genetic deletion of SHP-2 ameliorated OA progression. SHP-2 inhibitors dramatically reduced the expression of cartilage degradation-related genes and simultaneously promoted the expression of cartilage synthesis-related genes. Mechanistically, SHP-2 inhibition suppressed the dephosphorylation of docking protein 1 and subsequently reduced the expression of uridine phosphorylase 1 and increased the uridine level, thereby contributing to the homeostasis of cartilage metabolism. SHP-2 is a novel accelerator of the imbalance in cartilage homeostasis. Specific inhibition of SHP-2 may ameliorate OA by maintaining the anabolic-catabolic balance.

SLAMF3 and SLAMF4 are immune checkpoints that constrain macrophage phagocytosis of hematopoietic tumors.

The interaction of SIRPα with CD47 represents a major mechanism for preventing macrophage phagocytosis. However, CD47-independent mechanisms are poorly defined. Here, we report a critical role of SLAM family receptors (SFRs), ubiquitously expressed on hematopoietic cells and forming homotypic interactions, in constraining macrophage phagocytosis. We found that SFR deficiency triggered macrophage phagocytosis of hematopoietic cells, leading to severe rejection of donor hematopoietic graft in recipient mice. Specific SFR members, mainly SLAMF3 and SLAMF4, were identified as "don't eat me" receptors on macrophages. These receptors inhibited "eat me" signals, such as LRP1-mediated activation of mTOR and Syk, through SH2 domain-containing phosphatases. SFRs combined with, but were independent of, CD47 to mitigate macrophage phagocytosis, and the combined deletion of SFRs and CD47 resulted in hematopoietic cytopenia in mice. This SFR-mediated tolerance was compromised in patients with hemophagocytic lymphohistiocytosis, a syndrome characterized by inappropriate phagocytosis toward hematopoietic cells. Loss of SFRs potently elicited macrophage rejection of hematopoietic tumors. Deletion of SFRs also significantly enhanced the phagocytosis of CD19-positive hematopoietic targets by the macrophages expressing the chimeric CD19 antigen receptor. Therefore, SFR-mediated inhibition of macrophage phagocytosis is critical to hematopoietic homeostasis, and SFRs may represent previously unknown targets for tumor immunotherapy.

Discovery of 6-[(3S,4S)-4-Amino-3-methyl-2-oxa-8-azaspiro[4.5]decan-8-yl]-3-(2,3-dichlorophenyl)-2-methyl-3,4-dihydropyrimidin-4-one (IACS-15414), a Potent and Orally Bioavailable SHP2 Inhibitor.

Src homology 2 (SH2) domain-containing phosphatase 2 (SHP2) plays a role in receptor tyrosine kinase (RTK), neurofibromin-1 (NF-1), and Kirsten rat sarcoma virus (KRAS) mutant-driven cancers, as well as in RTK-mediated resistance, making the identification of small-molecule therapeutics that interfere with its function of high interest. Our quest to identify potent, orally bioavailable, and safe SHP2 inhibitors led to the discovery of a promising series of pyrazolopyrimidinones that displayed excellent potency but had a suboptimal in vivo pharmacokinetic (PK) profile. Hypothesis-driven scaffold optimization led us to a series of pyrazolopyrazines with excellent PK properties across species but a narrow human Ether-à-go-go-Related Gene (hERG) window. Subsequent optimization of properties led to the discovery of the pyrimidinone series, in which multiple members possessed excellent potency, optimal in vivo PK across species, and no off-target activities including no hERG liability up to 100 μM. Importantly, compound 30 (IACS-15414) potently suppressed the mitogen-activated protein kinase (MAPK) pathway signaling and tumor growth in RTK-activated and KRASmut xenograft models in vivo.

Strategy for Leukemia Treatment Targeting SHP-1,2 and SHIP.

Protein tyrosine phosphatases (PTPs) are modulators of cellular functions such as differentiation, metabolism, migration, and survival. PTPs antagonize tyrosine kinases by removing phosphate moieties from molecular signaling residues, thus inhibiting signal transduction. Two PTPs, SHP-1 and SHP-2 (SH2 domain-containing phosphatases 1 and 2, respectively) and another inhibitory phosphatase, SH2 domain-containing inositol phosphatase (SHIP), are essential for cell function, which is reflected in the defective phenotype of mutant mice. Interestingly, SHP-1, SHP-2, and SHIP mutations are identified in many cases of human leukemia. However, the impact of these phosphatases and their mutations regarding the onset and progression of leukemia is controversial. The ambiguity of the role of these phosphatases imposes challenges on the development of targeting therapies for leukemia. This fundamental problem, confronted by the expanding investigational field of leukemia, will be addressed in this review, which will include a discussion of the molecular mechanisms of SHP-1, SHP-2, and SHIP in normal hematopoiesis and their role in leukemia. Clinical development of leukemic therapies achieved by targeting these phosphatases will be addressed as well.

The tyrosine phosphatase SHP2 promotes proliferation and oxaliplatin resistance of colon cancer cells through AKT and ERK

The SH2 domain-containing phosphatase 2 (SHP2) is a widely expressed protein tyrosine phosphatase, and it is proposed to act as an oncogenic protein. SHP2 is also engaged in drug resistance of a variety of cancers. However, the role of SHP2 in the proliferation and drug resistance of colon cancer cells remains elusive. In this work we determined the effect of SHP2 expression on colon cancer cell proliferation and resistance to oxaliplatin (L-OHP), a commonly used drug in the clinic. Our results show that knockdown of SHP2 decreased and overexpression of SHP2 increased the proliferation of SW480 cells, respectively. Knockdown of SHP2 increased, and overexpression of SHP2 decreased apoptosis of the cells. We selected oxaliplatin-resistant SW480(SW480/L-OHP) and HCT116(HCT116/L-OHP) cells and found that the SHP2 protein level was raised in these drug-resistant cells. The upregulated SHP2 contributed to oxaliplatin resistance of the cells, as knockdown of SHP2 decreased the IC50 of oxaliplatin and abated proliferation and survival of SW480/L-OHP and HCT116/L-OHP cells in the presence of oxaliplatin. Also, SW480/L-OHP and HCT116/L-OHP cells had increased phosphorylation of AKT and ERK. Inhibition of AKT, ERK, or SHP2 sensitized SW480/L-OHP and HCT116/L-OHP cells to oxaliplatin. Our results indicate that SHP2 contributes oxaliplatin resistance through AKT and ERK. These results also suggest that SHP2-targeting is a potential strategy for overcoming oxaliplatin resistance of colon cancer cells.

SMURF1-mediated ubiquitylation of SHP-1 promotes cell proliferation and invasion of endometrial stromal cells in endometriosis.

BackgroundEndometriosis is a widespread benign gynecological disorder. The signal transducer and activator of transcription 3 (STAT3) signaling pathway plays an important role in the pathogenesis of endometriosis through regulating proliferation and invasion of endometrial stromal cells. Furthermore, the protein tyrosine phosphatase (PTP), SH2 domain-containing phosphatase 1 (SHP-1), negatively regulates STAT3 activation. However, regulation of the SHP-1-STAT3 pathway in the pathogenesis of endometriosis remains unclear.MethodsCell proliferation and invasion were assessed by Cell Counting Kit-8 (CCK-8) assay and Transwell analysis, respectively, to investigate the role and regulation of the SHP-1-STAT3 pathway in the proliferation and invasion of endometrial stromal cells. Expression of Smad ubiquitin regulatory factor 1 (SMURF1), SHP-1, matrix metalloproteinase 2 (MMP2), MMP9, STAT3, and phospho-STAT3 (p-STAT3) level in patients with endometriosis were measured by Western blotting and/or immunohistochemical staining. The interaction between SMURF1 and SHP-1 was investigated by co-immunoprecipitation and ubiquitylation analysis.ResultsThe present study demonstrated that downregulation of SHP-1 expression in patients with endometriosis was negatively correlated with SMURF1 expression. SMURF1, an E3 ubiquitin ligase, activated the STAT3 pathway via ubiquitylation and degradation of SHP-1. Furthermore, SMURF1 promoted cell proliferation and invasion of endometrial stromal cells by activating STAT3 signaling and expression of its downstream targets, MMP2 and MMP9, whereas SHP-1 demonstrated an inverse effect. Additionally, SHP-1 inhibited SMURF1-mediated cell invasion and proliferation of endometrial stromal cells.ConclusionsOur findings indicate that SMURF1-mediated ubiquitylation of SHP-1 regulates endometrial stromal cell proliferation and invasion during endometriosis.

FP12.03 SRC-Homology 2 Domain-Containing Phosphatase 2 (SHP2) in Resected Lung Adenocarcinoma (LUAD)

FP12.03 SRC-Homology 2 Domain-Containing Phosphatase 2 (SHP2) in Resected Lung Adenocarcinoma (LUAD)

Data-Driven Computational Modeling Identifies Determinants of Glioblastoma Response to SHP2 Inhibition.

Oncogenic protein tyrosine phosphatases have long been viewed as drug targets of interest, and recently developed allosteric inhibitors of SH2 domain-containing phosphatase-2 (SHP2) have entered clinical trials. However, the ability of phosphatases to regulate many targets directly or indirectly and to both promote and antagonize oncogenic signaling may make the efficacy of phosphatase inhibition challenging to predict. Here we explore the consequences of antagonizing SHP2 in glioblastoma, a recalcitrant cancer where SHP2 has been proposed as a useful drug target. Measuring protein phosphorylation and expression in glioblastoma cells across 40 signaling pathway nodes in response to different drugs and for different oxygen tensions revealed that SHP2 antagonism has network-level, context-dependent signaling consequences that affect cell phenotypes (e.g., cell death) in unanticipated ways. To map specific signaling consequences of SHP2 antagonism to phenotypes of interest, a data-driven computational model was constructed based on the paired signaling and phenotype data. Model predictions aided in identifying three signaling processes with implications for treating glioblastoma with SHP2 inhibitors. These included PTEN-dependent DNA damage repair in response to SHP2 inhibition, AKT-mediated bypass resistance in response to chronic SHP2 inhibition, and SHP2 control of hypoxia-inducible factor expression through multiple MAPKs. Model-generated hypotheses were validated in multiple glioblastoma cell lines, in mouse tumor xenografts, and through analysis of The Cancer Genome Atlas data. Collectively, these results suggest that in glioblastoma, SHP2 inhibitors antagonize some signaling processes more effectively than existing kinase inhibitors but can also limit the efficacy of other drugs when used in combination. SIGNIFICANCE: These findings demonstrate that allosteric SHP2 inhibitors have multivariate and context-dependent effects in glioblastoma that may make them useful components of some combination therapies, but not others.

Targeting SHP2 Sensitizes Papillary Thyroid Cancer Cells to MEK Inhibitors

Background: Pharmacologic targeting of components of the MAPK/ERK pathway in differentiated thyroid carcinoma (DTC) is often limited due to the development of adaptive resistance. However, the detailed mechanism of MEK inhibitor (MEKi) resistance is not fully understood. Methods: RNA-seq of the constructed MEKi-resistant DTC cell line BCPAP-R (compared with the BCPAP line) was performed to investigate the intrinsic mechanism of drug resistance. Colony formation assays, cell viability assays, and cell cycle analysis were performed, and various murine models, including xenograft models, long-term MEKi-treated models, and transgenic model were used to evaluate the treatment effect of combination therapy (SHP099 and selumetinib). Findings: MEKi-resistant models were constructed successfully, in which multiple receptor tyrosine kinases (RTKs) signaling pathways and Src-homology 2 domain-containing phosphatase 2 (SHP2) were activated in MEKi-resistant cells. Given the physiological role of SHP2 as the downstream target of many RTKs, we first found that blockade of SHP2 enhanced the sensitivity to MEKi in constructed MEKi-resistant thyroid cancer models (including constructed cell lines and a long-term MEKi-treated murine model). Interestingly, we also found that compared with MEKi treatment alone, MEKi in combination with an SHP2 inhibitor markedly suppressed the reactivation of the MEK/ERK pathway; thus, the addition of the SHP2 inhibitor significantly improved the anti-tumor effects of MEKi. The synergistic suppression of DTC upon treatment with both inhibitors was futher confirmed in xenograft models and transgenic models. Interpretation: RTKs activation leads to reactivation of the MAPK pathway and resistance to MEKi in DTC, which is reversed by SHP2 blockade. As a novel active inhibitor of SHP2, SHP099 in combination with selumetinib is a promising therapeutic approach for advanced DTC and MEKi-resistant one. Funding Statement: This work was partially supported by grants from the National Natural Science Foundation of China (GRANT no. 81872169), Tianjin Key Research and Development Program Science and Technology Support Key Projects (grant no. 17YFZCSY00690), and Tianjin Municipal Science and Technology Project (grant no. 19JCYBJC27400). Declaration of Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Ethics Approval Statement: All animal experiments were approved by the Tianjin Medical University Cancer Institute and the Hospital Animal Care and Use Committee and performed according to the IACUC protocol.

Expression of protein tyrosine phosphatases and Bombyx embryonic development

Protein phosphorylation is an integral component of signal transduction pathways within eukaryotic cells, and it is regulated by coordinated interactions between protein kinases and protein phosphatases. Our previous study demonstrated differential expressions of serine/threonine protein phosphatases (PP2A and calcineurin) between diapause and developing eggs in Bombyx mori. In the present study, we further investigated expression of protein tyrosine phosphatases (PTPs) in relation to the Bombyx embryonic development. An immunoblot analysis showed that eggs contained the proteins of the 51-kDa PTP 1B (PTP1B), the 55-kDa phosphatase and tensin homologue (PTEN), and the 70-kDa Src homology 2 (SH2) domain-containing phosphatase 2 (SHP2), which undergo differential changes between diapause and developing eggs. Protein level of PTP1B and PTEN in eggs whose diapause initiation was prevented by HCl gradually increased toward embryonic development. The protein level of SHP2 also showed a dramatic increase on days 7 and 8 after HCl treatment. However, protein levels of PTP1B, PTEN, and SHP2 in diapause eggs remained at low levels during the first 9 days after oviposition. These differential changing patterns in protein levels were further confirmed using both non-diapause eggs and eggs in which diapause had been terminated by chilling of diapausing eggs at 5 °C for 70 days and then were transferred to 25 °C. Direct determination of PTP enzymatic activities showed higher activities in developing eggs (HCl-treated eggs, non-diapause eggs, and chilled eggs) compared to those in diapause eggs. Examination of temporal changes in mRNA expression levels of PTP1B, PTEN, and SHP2 did not show significant differences between diapause eggs and HCl-treated eggs except high expression in SHP2 variant B during the later embryonic development in HCl-treated eggs. These results demonstrate that higher protein levels of PTP1B, PTEN, and SHP2 and increased tyrosine phosphatase enzymatic activities in developing eggs are likely related to embryonic development of B. mori.