Protein Tyrosine Phosphatase SHP-1 Research Articles

Shp2 contributes to the regulation of nuclear shape and cellular viscoelasticity in response to substrate spatial cues

Cell polarization can be guided by substrate topology through space constraints and adhesion induction, which are part of cellular mechanosensing pathways. Here, we demonstrated that protein tyrosine phosphatase Shp2 plays a crucial role in mediating the response of cells to substrate spatial cues. When compared to cells spreading on surfaces coated uniformly with fibronectin (FN), cells attached to 10 μm-width FN-strip micropattern (MP), which provides spatial cues for uniaxial spreading, exhibited elongated focal adhesions (FAs) and aligned stress fibers in the direction of the MP. As a result of uniaxial cell spreading, nuclei became elongated, dependent on ROCK-mediated actomyosin contractility. Additionally, intracellular viscoelasticity also increased. Shp2-deficient cells did not display elongated FAs mediated by MP, well-aligned stress fibers, or changes in nuclear shape and intracellular viscoelasticity. Overall, our data suggest that Shp2 is involved in regulating FAs and the actin cytoskeleton to modulate nuclear shape and intracellular physical properties in response to substrate spatial cues.

Promising natural products targeting protein tyrosine phosphatase SHP2 for cancer therapy.

The development of Src homology-2 domain containing protein tyrosine phosphatase-2 (SHP2) inhibitors is a hot spot in the research and development of antitumor drugs, which may induce immunomodulatory effects in the tumor microenvironment and participate in anti-tumor immune responses. To date, several SHP2 inhibitors have made remarkable progress and entered clinical trials for the treatment of patients with advanced solid tumors. Multiple compounds derived from natural products have been proved to influence tumor cell proliferation, apoptosis, migration and other cellular functions, modulate cell cycle and immune cell activation by regulating the function of SHP2 and its mutants. However, there is a paucity of information about their diversity, biochemistry, and therapeutic potential of targeting SHP2 in tumors. This review will provide the structure, classification, inhibitory activities, experimental models, and antitumor effects of the natural products. Notably, this review summarizes recent advance in the efficacy and pharmacological mechanism of natural products targeting SHP2 in inhibiting the various signaling pathways that regulate differentcancers and thus pave the way for further development of anticancer drugs targeting SHP2.

Fragment-Based Discovery of Allosteric Inhibitors of SH2 Domain-Containing Protein Tyrosine Phosphatase-2 (SHP2).

The ubiquitously expressed protein tyrosine phosphatase SHP2 is required for signaling downstream of receptor tyrosine kinases (RTKs) and plays a role in regulating many cellular processes. Genetic knockdown and pharmacological inhibition of SHP2 suppresses RAS/MAPK signaling and inhibit the proliferation of RTK-driven cancer cell lines. Here, we describe the first reported fragment-to-lead campaign against SHP2, where X-ray crystallography and biophysical techniques were used to identify fragments binding to multiple sites on SHP2. Structure-guided optimization, including several computational methods, led to the discovery of two structurally distinct series of SHP2 inhibitors binding to the previously reported allosteric tunnel binding site (Tunnel Site). One of these series was advanced to a low-nanomolar lead that inhibited tumor growth when dosed orally to mice bearing HCC827 xenografts. Furthermore, a third series of SHP2 inhibitors was discovered binding to a previously unreported site, lying at the interface of the C-terminal SH2 and catalytic domains.

Natural product-inspired molecules for covalent inhibition of SHP2 tyrosine phosphatase

Natural products have been playing indispensable roles in the development of lifesaving drug molecules. They are also valuable sources for covalent protein modifiers. However, they often are scarce in nature and have complex chemical structures, which are limiting their further biomedical development. Thus, natural product-inspired small molecules which still contain the essence of the parent natural products but are readily available and amenable for structural modification, are important and desirable in searching for lead compounds for various disease treatment. Inspired by the complex and diverse ent-kaurene diterpenoids with significant biological activities, we have created a synthetically accessible and focused covalent library by incorporating the bicyclo[3.2.1]octane α-methylene ketone, which is considered as the pharmacophore of ent-kaurene diterpenoids, as half of the structure, and replacing the other half with much less complex but more druglike scaffolds. From this library, we have identified and characterized selective covalent inhibitors of protein tyrosine phosphatase SHP2, an important anti-cancer therapeutic target. The success of this study demonstrated the importance of creating and evaluating natural product-inspired library as well as their application in targeting challenging disease targets.

Neuroimmune pathways involvement in neurodegeneration of R6/2 mouse model of Huntington's disease.

Mechanisms of tissue damage in Huntington's disease (HD) involve excitotoxicity, mitochondrial damage, and neuroinflammation, including microglia activation. CD47 is a membrane protein that interacts with the inhibitory immunoreceptor SIRPα. Engagement of SIRPα by CD47 provides a downregulatory signal that inhibits host cell phagocytosis, promoting a "don't-eat-me" signal. These proteins are involved in the immune response and are downmodulated in inflammatory diseases. The involvement of inflammation and of the inflammasome in HD has already been described. In this study, we focused on other factors that can be involved in the unregulated inflammatory response that accelerates and exacerbate the neurodegenerative process in HD. Our results show that CD47 on striatal neurons decreased in HD mice, while it increased in wild type mice with age. SIRPα, on the other hand, was present in neurons in the wild type and increases in the R6/2 mice at all stages. Recruitment of SIRPα and binding to CD47 promotes the activation through phosphorylating events of non-receptor protein tyrosine phosphatase SHP-1 and SHP-2 in neurons and microglia. SHP phosphatases are able to curb the activity of NLRP3 inflammasome thereby reducing the detrimental effect of neuroinflammation. Such activity is mediated by the inhibition (dephosphorylation) of the proteins signal transducer and activator of transcription (STAT). We found that activated SHP-1 was present in microglia and neurons of WT mice at 5 and 13 weeks, increasing with time; while in R6/2 it was not localized in neurons but only in microglia, where it decreases with time. Consequently, STAT1 was overexpressed in neurons of R6/2 mice, as an effect of lack of modulation by SHP-1. Thus, our results shed light on the pathophysiology of neuronal damage, on one hand, paving the way toward a modulation of signal transducer proteins by specific inhibitors to achieve neuroprotection in HD, on the other.

Discovery of 1H-pyrazolo[3,4-b]pyrazine derivatives as selective allosteric inhibitor of protein tyrosine phosphatase SHP2 for the treatment of KRASG12C-mutant non-small cell lung cancer

The high expression or mutation of SHP2 can induce cancer, so targeting SHP2 has become a new strategy for cancer treatment. In this study, we used the previously reported SHP2 allosteric inhibitor IACS-13909 as a lead drug for structural derivation and modification, and synthesized three SHP2 inhibitors. Among them, 1H-pyrazolo[3,4-b]pyrazine derivative 4b was a highly selective SHP2 allosteric inhibitor, with an IC50 value of 3.2 nM, and its inhibitory activity was 17.75 times than that of the positive control IACS-13909. The cell proliferation experiment detected that compound 4b would markedly inhibit the proliferation of various cancer cells. Interestingly, compound 4b was highly sensitive to KRASG12C-mutant non-small cell lung cancer NCI-H358 cells, with an IC50 value of 0.58 μM and its antiproliferative activity was 4.79 times than that of IACS-13909. Furthermore, the combination therapy of compound 4b and KRASG12C inhibitor sotorasib would play a strong synergistic effect against NCI-H358 cells. The western blot experiment detected that compound 4b markedly downregulated the phosphorylation levels of ERK and AKT in NCI-H358 cells. Molecular docking study predicted that compound 4b bound to the allosteric site of SHP2 and formed H-bond interactions with key residues Thr108, Glu110, Arg111, and Phe113. In summary, this study aims to provide new ideas for the development of SHP2 allosteric inhibitors for the treatment of KRASG12C mutant non-small cell lung cancer. Communicated by Ramaswamy H. Sarma

Hepatic protein tyrosine phosphatase Shp2 disruption mitigates the adverse effects of ethanol in the liver by modulating oxidative stress and ERK signaling

AimsChronic excessive alcohol intake is a significant cause of alcohol-associated liver disease (ALD), a leading contributor to liver-related morbidity and mortality. The Src homology phosphatase 2 (Shp2; encoded by Ptpn11) is a widely expressed protein tyrosine phosphatase that modulates hepatic functions, but its role in ALD is mostly uncharted. Main methodsHerein, we explore the effects of liver-specific Shp2 genetic disruption using the established chronic-plus-binge mouse model of ALD. Key findingsWe report that the hepatic Shp2 disruption had beneficial effects and partially ameliorated ethanol-induced injury, inflammation, and steatosis in the liver. Consistently, Shp2 deficiency was associated with decreased ethanol-evoked activation of extracellular signal-regulated kinase (ERK) and oxidative stress in the liver. Moreover, primary hepatocytes with Shp2 deficiency exhibited similar outcomes to those observed upon Shp2 disruption in vivo, including diminished ethanol-induced ERK activation, inflammation, and oxidative stress. Furthermore, pharmacological inhibition of ERK in primary hepatocytes mimicked the effects of Shp2 deficiency and attenuated oxidative stress caused by ethanol. SignificanceCollectively, these findings highlight Shp2 as a modulator of hepatic oxidative stress upon ethanol challenge and suggest the evaluation of this phosphatase as a potential therapeutic target for ALD.

Protein Tyrosine Phosphatase SHP2 in Macrophages Acts as an Antiatherosclerotic Regulator in Mice.

Macrophages have versatile roles in atherosclerosis. SHP2 (Src homology 2 containing protein tyrosine phosphatase 2) has been demonstrated to play a critical role in regulating macrophage activation. However, the mechanism of SHP2 regulation of macrophage function in an atherosclerotic microenvironment remains unknown. APOE (apolipoprotein E) or LDLR (low-density lipoprotein receptor) null mice treated with SHP099 were fed a Western diet for 8 weeks, while Shp2MKO:ApoE-/- or Shp2MKO:Ldlr-/- mice and exo-AAV8-SHP2E76K/ApoE-/- mice were fed a Western diet for 12 weeks. In vitro, levels of proinflammatory factors and phagocytic function were then studied in mouse peritoneal macrophages. RNA sequencing was used to identify PPARγ (peroxisome proliferative activated receptor γ) as the key downstream molecule. A PPARγ agonist was used to rescue the phenotypes observed in SHP2-deleted mice. Pharmacological inhibition and selective deletion in macrophages of SHP2 aggravated atherosclerosis in APOE and LDLR null mice with increased plaque macrophages and apoptotic cells. In vitro, SHP2 deficiency in APOE and LDLR null macrophages enhanced proinflammatory polarization and its efferocytosis was dramatically impaired. Conversely, the expression of gain-of-function mutation of SHP2 in mouse macrophages reduced atherosclerosis. The SHP2 agonist lovastatin repressesed macrophage inflammatory activation and enhanced efferocytosis. Mechanistically, RNA sequencing analysis identified PPARγ as a key downstream transcription factor. PPARγ was decreased in macrophages upon SHP2 deletion and inhibition. Importantly, PPARγ agonist decreased atherosclerosis in SHP2 knockout mice, restored efferocytotic defects, and reduced inflammatory activation in SHP2 deleted macrophages. PPARγ was decreased by the ubiquitin-mediated degradation upon SHP2 inhibition or deletion. Finally, we found that SHP2 was downregulated in atherosclerotic vessels. Overall, SHP2 in macrophages was found to act as an antiatherosclerotic regulator by stabilizing PPARγ in APOE/LDLR null mice.

Identification of GDC-1971 (RLY-1971), a SHP2 Inhibitor Designed for the Treatment of Solid Tumors.

Protein tyrosine phosphatase SHP2 mediates RAS-driven MAPK signaling and has emerged in recent years as a target of interest in oncology, both for treating with a single agent and in combination with a KRAS inhibitor. We were drawn to the pharmacological potential of SHP2 inhibition, especially following the initial observation that drug-like compounds could bind an allosteric site and enforce a closed, inactive state of the enzyme. Here, we describe the identification and characterization of GDC-1971 (formerly RLY-1971), a SHP2 inhibitor currently in clinical trials in combination with KRAS G12C inhibitor divarasib (GDC-6036) for the treatment of solid tumors driven by a KRAS G12C mutation.

A mAb to SIRPα downregulates the priming of naive CD4 + T cell in Primary immune thrombocytopenia

SIRPα is a transmembrane protein that binds the protein tyrosine phosphatases SHP-1 and SHP-2 through its cytoplasmic region and is abundantly expressed on monocytes, dendritic cells, and macrophages. Studies recently showed that SIRPα is essential for priming of CD4 + T cells by DCs and for development of Th17 cell-mediated autoimmune diseases. We have now further evaluated the importance of SIRPα and that of its ligand CD47 in primary immune thrombocytopenia (ITP). In this study, we show that there was a low expression state of SIRPα on the surface of monocytes. Treatment of cells culture from ITP patients with a mAb to SIRPα that blocks the binding of SIRPα to CD47 downregulated the ITP response. The abilities of monocytes from ITP patients to stimulate an allogenic MLR were reduced. The proliferation of, and production of IL-2, by CD4 + T cells from ITP patients were inhibited, the Treg cell numbers and the production of IL-10 pairs were upregulated, and the production of TGF-β not was inhibited, by a mAb to SIRPα. Moreover, a mAb to SIRPα, the expression of HLA-DR and CD86 were markedly inhibited and the expression of CD80 was slightly upregulated, on the surface of CD14 + monocytes from ITP patients as compared with healthy subjects. However, blockade of SIRPα increased the secretion of TLR-dependent cytokines TNF-α, IL-6 and IL-1β by PBMCs, which may be considered as a reserve in response to danger signals. These results suggest that SIRPα on monocytes is essential for the priming of naive T cells and the development of ITP. Therefore, SIRPα is a potential therapeutic target for ITP and other autoimmune diseases.

SHP-1 phosphatase acts as a coactivator of PCK1 transcription to control gluconeogenesis

We previously reported that the protein-tyrosine phosphatase SHP-1 (PTPN6) negatively regulates insulin signaling, but its impact on hepatic glucose metabolism and systemic glucose control remains poorly understood. Here, we use co-immunoprecipitation assays, chromatin immunoprecipitation sequencing, in silico methods, and gluconeogenesis assay, and found a new mechanism whereby SHP-1 acts as a co-activator for transcription of the phosphoenolpyruvate carboxykinase 1 (PCK1) gene to increase liver gluconeogenesis. SHP-1 is recruited to the regulatory regions of the PCK1 gene and interacts with RNA polymerase II. The recruitment of SHP-1 to chromatin is dependent on its association with the transcription factor signal transducer and activator of transcription 5 (STAT5). Loss of SHP-1 as well as STAT5 decrease RNA polymerase II recruitment to the PCK1 promoter and consequently PCK1 mRNA levels leading to blunted gluconeogenesis. This work highlights a novel nuclear role of SHP-1 as a key transcriptional regulator of hepatic gluconeogenesis adding a new mechanism to the repertoire of SHP-1 functions in metabolic control.

Deletion of protein tyrosine phosphatase SHP-1 restores SUMOylation of podocin and reverses the progression of diabetic kidney disease

Both clinical and experimental data suggest that podocyte injury is involved in the onset and progression of diabetic kidney disease (DKD). Although the mechanisms underlying the development of podocyte loss are not completely understood, critical structural proteins such as podocin play a major role in podocyte survival and function. We have reported that the protein tyrosine phosphatase SHP-1 expression increased in podocytes of diabetic mice and glomeruli of patients with diabetes. However, the invivo contribution of SHP-1 in podocytes is unknown. Conditional podocyte-specific SHP-1-deficient mice (Podo-SHP-1-/-) were generated to evaluate the impact of SHP-1 deletion at four weeks of age (early) prior to the onset of diabetes and after 20 weeks (late) of diabetes (DM; Ins2+/C96Y) on kidney function (albuminuria and glomerular filtration rate) and kidney pathology. Ablation of the SHP-1 gene specifically in podocytes prevented and even reversed the elevated albumin/creatinine ratio, glomerular filtration rate progression, mesangial cell expansion, glomerular hypertrophy, glomerular basement membrane thickening and podocyte foot process effacement induced by diabetes. Moreover, podocyte-specific deletion of SHP-1 at an early and late stage prevented diabetes-induced expression of collagen IV, fibronectin, transforming growth factor-β, transforming protein RhoA, and serine/threonine kinase ROCK1, whereas it restored nephrin, podocin and cation channel TRPC6 expression. Mass spectrometry analysis revealed that SHP-1 reduced SUMO2 post-translational modification of podocin while podocyte-specific deletion of SHP-1 preserved slit diaphragm protein complexes in the diabetic context. Thus, our data uncovered a new role of SHP-1 in the regulation of cytoskeleton dynamics and slit diaphragm protein expression/stability, and its inhibition preserved podocyte function preventing DKD progression.

Protein Tyrosine Phosphatase Non-Receptor 11 (PTPN11/Shp2) as a Driver Oncogene and a Novel Therapeutic Target in Non-Small Cell Lung Cancer (NSCLC)

PTPN11 encodes the SHP2 protein tyrosine phosphatase that activates the mitogen-activated protein kinase (MAPK) pathway upstream of KRAS and MEK. PTPN11/Shp2 somatic mutations occur frequently in Juvenile myelomonocytic leukaemia (JMML); however, the role of mutated PTPN11 in lung cancer tumourigenesis and its utility as a therapeutic target has not been fully addressed. We applied mass-spectrometry-based genotyping to DNA extracted from the tumour and matched the normal tissue of 356 NSCLC patients (98 adenocarcinomas (LUAD) and 258 squamous cell carcinomas (LUSC)). Further, PTPN11 mutation cases were identified in additional cohorts, including TCGA, Broad, and MD Anderson datasets and the COSMIC database. PTPN11 constructs harbouring PTPN11 E76A, A72D and C459S mutations were stably expressed in IL-3 dependent BaF3 cells and NSCLC cell lines (NCI-H1703, NCI-H157, NCI-H1299). The MAPK and PI3K pathway activation was evaluated using Western blotting. PTPN11/Shp2 phosphatase activity was measured in whole-cell protein lysates using an Shp2 assay kit. The Shp2 inhibitor (SHPi) was assessed both in vitro and in vivo in a PTPN11-mutated cell line for improved responses to MAPK and PI3K targeting therapies. Somatic PTPN11 hotspot mutations occurred in 4/98 (4.1%) adenocarcinomas and 7/258 (2.7%) squamous cells of 356 NSCLC patients. Additional 26 PTPN11 hotspot mutations occurred in 23 and 3 adenocarcinomas and squamous cell carcinoma, respectively, across the additional cohorts. Mutant PTPN11 significantly increased the IL-3 independent survival of Ba/F3 cells compared to wildtype PTPN11 (p < 0.0001). Ba/F3, NCI-H1703, and NCI-H157 cells expressing mutant PTPN11 exhibited increased PTPN11/Shp2 phosphatase activity and phospho-ERK1/2 levels compared to cells expressing wildtype PTPN11. The transduction of the PTPN11 inactivating mutation C459S into NSCLC cell lines led to decreased phospho-ERK, as well as decreased phospho-AKT in the PTPN11-mutated NCI-H661 cell line. NCI-H661 cells (PTPN11-mutated, KRAS-wild type) were significantly more sensitive to growth inhibition by the PI3K inhibitor copanlisib (IC50: 13.9 ± 4.7 nM) compared to NCI-H1703 (PTPN11/KRAS-wild type) cells (IC50: >10,000 nM). The SHP2 inhibitor, in combination with the PI3K targeting therapy copanlisib, showed no significant difference in tumour development in vivo; however, this significantly prevented MAPK pathway induction in vitro (p < 0.0001). PTPN11/Shp2 demonstrated the in vitro features of a driver oncogene and could potentially sensitize NSCLC cells to PI3K inhibition and inhibit MAPK pathway activation following PI3K pathway targeting.

PI3K inhibition circumvents resistance to SHP2 blockade in metastatic triple-negative breast cancer

The protein tyrosine phosphatase SHP2 activates oncogenic pathways downstream of most receptor tyrosine kinases (RTK) and has been implicated in various cancer types, including the highly aggressive subtype of triple-negative breast cancer (TNBC). Although allosteric inhibitors of SHP2 have been developed and are currently being evaluated in clinical trials, neither the mechanisms of the resistance to these agents, nor the means to circumvent such resistance have been clearly defined. The PI3K signaling pathway is also hyperactivated in breast cancer and contributes to resistance to anticancer therapies. When PI3K is inhibited, resistance also develops for example via activation of RTKs. We therefore assessed the effect of targeting PI3K and SHP2 alone or in combination in preclinical models of metastatic TNBC. In addition to the beneficial inhibitory effects of SHP2 alone, dual PI3K/SHP2 treatment decreased primary tumor growth synergistically, blocked the formation of lung metastases, and increased survival in preclinical models. Mechanistically, transcriptome and phospho-proteome analyses revealed that resistance to SHP2 inhibition is mediated by PDGFRβ-evoked activation of PI3K signaling. Altogether, our data provide a rationale for co-targeting of SHP2 and PI3K in metastatic TNBC.

Role of SHP2 (PTPN11) in glycoprotein VI-dependent thrombus formation: Improved platelet responsiveness by the allosteric drug SHP099 in Noonan syndrome patients

IntroductionThe protein tyrosine phosphatase SHP2 (PTPN11) is a negative regulator of glycoprotein VI (GPVI)-induced platelet signal under certain conditions. Clinical trials with derivatives of the allosteric drug SHP099, inhibiting SHP2, are ongoing as potential therapy for solid cancers. Gain-of-function mutations of the PTPN11 gene are observed in part of the patients with the Noonan syndrome, associated with a mild bleeding disorder. Assessment of the effects of SHP2 inhibition in platelets from controls and Noonan syndrome patients. Materials and methodsWashed human platelets were incubated with SHP099 and stimulated with collagen-related peptide (CRP) for stirred aggregation and flow cytometric measurements. Whole-blood microfluidics assays using a dosed collagen and tissue factor coating were performed to assess shear-dependent thrombus and fibrin formation. Effects on clot formation were evaluated by thromboelastometry. ResultsPharmacological inhibition of SHP2 did not alter GPVI-dependent platelet aggregation under stirring, but it enhanced integrin αIIbβ3 activation in response to CRP. Using whole-blood microfluidics, SHP099 increased the thrombus buildup on collagen surfaces. In the presence of tissue factor and coagulation, SHP099 increased thrombus size and reduced time to fibrin formation. Blood from PTPN11-mutated Noonan syndrome patients, with low platelet responsiveness, after ex vivo treatment with SHP099 showed a normalized platelet function. In thromboelastometry, SHP2 inhibition tended to increase tissue factor-induced blood clotting profiles with tranexamic acid, preventing fibrinolysis. ConclusionPharmacological inhibition of SHP2 by the allosteric drug SHP099 enhances GPVI-induced platelet activation under shear conditions with a potential to improve platelet functions of Noonan syndrome patients.

Abstract 3286: B-hCD94/hNKG2A mice provide a preclinical tool for evaluating therapeutic antibodies targeting human NKG2A

Abstract The immune cell checkpoint NKG2A and its coreceptor, CD94, are expressed by many circulating NK cells and by a subset of CD8 T cells in tumors. When CD94/NKG2A recognizes the HLA-E-peptide complex, the protein tyrosine phosphatase SHP-1 is recruited to the signaling synapse, resulting in the delivery of inhibitory signals to the effector cells and inhibition of their immune activities. In preclinical mouse models, blocking antibodies against NKG2A restores the reactivity of these effector cells, resulting in better suppression of tumor growth. Thus, NKG2A targeting is currently being evaluated in the clinic to evaluate tumor cell killing. To evaluate the in vivo efficacy of anti-NKG2A in mice, we successfully generated a humanized CD94/NKG2A double knock-in mouse strain (B-hCD94/hNKG2A mice), in which the mouse CD94 and NKG2A genes are replaced by the corresponding human CD94 and NKG2A genes in situ in cis, respectively. Human CD94 and NKG2A mRNA and proteins were exclusively detectable by flow cytometry in homozygous humanized mice, confirming successful generation of the model. This model was used to demonstrate in vivo efficacy of monalizumab analog in suppressing the growth of MC38 tumor cells expressing human HLA-E (B-hHLA-E MC38). Furthermore, dual blockade of monalizumab and PD-L1 further suppressed B-hHLA-E MC38 tumor growth. Together, these data indicate that this double humanized model is an effective tool for evaluating novel anti-human NKG2A therapies, and warrants the generation of a quadruple humanized model (B-hPD-1/hPD-L1/hCD94/hNKG2A mice) to further evaluate the synergy of drugs blocking each checkpoint. Citation Format: Chengzhang Shang, Fang He, Mari Kuraguchi, Jing Zhang, Qingcong Lin. B-hCD94/hNKG2A mice provide a preclinical tool for evaluating therapeutic antibodies targeting human NKG2A [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 3286.

Genetic dissection of mutual interference between two consecutive learning tasks in Drosophila.

Animals can continuously learn different tasks to adapt to changing environments and, therefore, have strategies to effectively cope with inter-task interference, including both proactive interference (Pro-I) and retroactive interference (Retro-I). Many biological mechanisms are known to contribute to learning, memory, and forgetting for a single task, however, mechanisms involved only when learning sequential different tasks are relatively poorly understood. Here, we dissect the respective molecular mechanisms of Pro-I and Retro-I between two consecutive associative learning tasks in Drosophila. Pro-I is more sensitive to an inter-task interval (ITI) than Retro-I. They occur together at short ITI (<20 min), while only Retro-I remains significant at ITI beyond 20 min. Acutely overexpressing Corkscrew (CSW), an evolutionarily conserved protein tyrosine phosphatase SHP2, in mushroom body (MB) neurons reduces Pro-I, whereas acute knockdown of CSW exacerbates Pro-I. Such function of CSW is further found to rely on the γ subset of MB neurons and the downstream Raf/MAPK pathway. In contrast, manipulating CSW does not affect Retro-I as well as a single learning task. Interestingly, manipulation of Rac1, a molecule that regulates Retro-I, does not affect Pro-I. Thus, our findings suggest that learning different tasks consecutively triggers distinct molecular mechanisms to tune proactive and retroactive interference.

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.

Potential clinical use of azacitidine and MEK inhibitor combination therapy in PTPN11-mutated juvenile myelomonocytic leukemia

Juvenile myelomonocytic leukemia (JMML) is a rare myeloproliferative neoplasm of childhood. The molecular hallmark of JMML is hyperactivation of the Ras/MAPK pathway with the most common cause being mutations in the gene PTPN11, encoding the protein tyrosine phosphatase SHP2. Current strategies for treating JMML include using the hypomethylating agent, 5-azacitidine (5-Aza) or MEK inhibitors trametinib and PD0325901 (PD-901), but none of these are curative as monotherapy. Utilizing an Shp2E76K/+ murine model of JMML, we show that the combination of 5-Aza and PD-901 modulates several hematologic abnormalities often seen in JMML patients, in part by reducing the burden of leukemic hematopoietic stem and progenitor cells (HSC/Ps). The reduced JMML features in drug-treated mice were associated with a decrease in p-MEK and p-ERK levels in Shp2E76K/+ mice treated with the combination of 5-Aza and PD-901. RNA-sequencing analysis revealed a reduction in several RAS and MAPK signaling-related genes. Additionally, a decrease in the expression of genes associated with inflammation and myeloid leukemia was also observed in Shp2E76K/+ mice treated with the combination of the two drugs. Finally, we report two patients with JMML and PTPN11 mutations treated with 5-Aza, trametinib, and chemotherapy who experienced a clinical response because of the combination treatment.

SHP-1 Protein Tyrosine Phosphatase Affects Early Postnatal Bone Development in Mice.

The Src homology region 2 domain-containing phosphatase-1 (SHP-1) is an intracellular tyrosine phosphatase that plays a negative regulatory role in immune cell signaling. Absent or diminished SHP-1 catalytic activity results in reduced bone mass with enhanced bone resorption. Here, we sought to investigate if Shp1 overexpression leads to increased bone mass and improved mechanical properties. Male and female wildtype (WT) and SHP1-transgenic (Tg) mice at 28, 56, and 84days of age were compared. We applied microcomputed tomography to assess femoral cortical bone geometry and trabecular architecture and 3-point mechanical bending to assess mid-diaphyseal structural and estimated material properties. Serum OPG, RANKL, P1NP, and CTX-1 concentrations were measured by enzyme-linked immunoassay. The majority of transgene effects were restricted to the 28-day-old mice. Trabecular bone volume per total volume, trabecular number, and connectivity density were greater in 28-day-old female SHP1-Tg mice when compared to WTs. SHP1-Tg female mice showed increased total and medullary areas, with no difference in cortical area and thickness. Cortical tissue mineral density was strongly genotype-dependent. Failure load, yield load, ultimate stress, and yield stress were all lower in 28-day-old SHP1-Tg females. In 28-day-old SHP1-Tg females, circulating levels of OPG and P1NP were higher and RANKL levels were lower than WT controls. Our study demonstrates a role for SHP-1 in early postnatal bone development; SHP-1 overexpression negatively impacted whole bone strength and material properties in females.