PTP Domain Research Articles

Characteristic Analysis of Adult Acute Myeloid Leukemia Patients with PTPN11 Gene Mutation

To investigate the incidence of PTPN11 gene mutation and its associated gene mutations in adult patients with acute myeloid leukemia (AML), and analyze its clinical characteristics. Second-generation sequencing and Sanger sequencing were used to detect 51 gene mutations, and multiplex-PCR was used to detect 41 fusion genes from 451 newly diagnosed adult AML patients admitted to Affiliated Hospital of Jiangnan University, Changzhou Second People's Hospital, Wuxi People's Hospital and Wuxi Second People's Hospital from January 2017 to July 2022. Among 451 primary adult AML patients, the PTPN11 gene mutation was detected in 34 cases, and the mutation rate was 7.5%. In the 34 patients, 37 PTPN11 alterations were found, which were exclusively missense mutations affecting residues located within the N-SH2 (31 cases) and PTP (6 cases) domains and clustered overwhelmingly in exon 3. The platelet count of PTPN11 mutation patients was 76.5(23.5, 119.0)×109/L, which was significantly higher than 41.0(22.0, 82.5)×109/L of wild-type patients (P < 0.05). While, there were no significant differences in sex, age, peripheral white blood cell count, hemoglobin, and bone marrow blast between PTPN11 mutation and wild-type patients (P >0.05). In FAB subtypes, PTPN11 mutations were mainly distributed in M5, followed by M2 and M4, less frequently in M3 and M6. There was no significant difference in the distribution of FAB subtypes between PTPN11 mutation and wild-type patients (P >0.05). A total of 118 AML patients were detected positive fusion gene, among which patients with PTPN11 mutations had a higher incidence of positive MLL-AF6 than wild-type ones (P < 0.01). 97.1% of 34 patients with PTPN11 mutations were accompanied by other mutations, in descending order, they were respectively NPM1 (38.2%), NRAS (32.4%), FLT3-ITD (32.4%), DNMT3A (32.4%) and KRAS (23.5%), etc . PTPN11 mutation has a certain incidence in AML patients and is clustered overwhelmingly in exon 3. ALL of them are exclusively missense mutations, and most often present in conjunction with NPM1 mutations. FAB typing of PTPN11 mutation is mostly manifested as M5 subtype, which is associated with higher platelet counts.

PTPN11variants in chronic myelomonocytic leukemia: Phenotypic and prognostic correlates.

6513 Background: PTPN11 (Ch12q24) is a proto-oncogene that codes for SHP2, a regulatory protein tyrosine phosphatase that impacts RAS/MAPK signaling. Germline variants have been described in juvenile myelomonocytic leukemia, while somatic variants have been associated with inferior survival in acute myeloid leukemia. PTPN11 variants are infrequent in chronic myelomonocytic leukemia (CMML), and their impact on disease phenotype and survival remains unclear. Methods: After IRB approval, 396 molecularly annotated CMML patients at Mayo Clinic were screened for somatic PTPN11 variants within 12 months of diagnosis while still in chronic phase disease. Overall (OS), acute myeloid leukemia (AML)-free survival (LFS), and predictors of survival were estimated using standard statistical measures. Results: 396 patients (median age 71 years; males 67%) with CMML were considered; pathogenic PTPN11 variants were seen in 12 (3%) with a median VAF of 20.5% (range: 4-50): A72T (n = 2), F285S (n = 2), and A72S (n = 2). Variants were most likely to be seen in the N-terminal SH2 domain (n = 7), followed by the PTP domain (n = 6), and the C-terminal SH2 domain (n = 1). PTPN11 variants clustered with CMML-2 (p = 0.02), higher absolute neutrophil count (p = 0.04), peripheral blood (p = 0.06) and bone marrow (p = 0.02) blast %, older age (p &lt;0.01), and were less likely to have SRSF2 comutations (p = 0.02). After a median follow-up of 18 months (range 0-193), 253 (64%) deaths and 68 (17%) AML transformations were recorded. PTPN11 variants negatively and independently impacted both OS (median 13 vs. 31 months; p &lt;0.01) and LFS (AML events: 42% (5/12) vs. 16% (63/384); p &lt;0.01). In age-adjusted multivariate analysis restricted to genetic risk factors, survival was negatively affected by abnormal karyotype and mutations involving PTPN11, DNMT3A, and SETBP1, and positively by TET2 mutations. All but SETBP1 retained significance in the presence of additional independent risk factors, namely male sex (p &lt;0.01) and leukocyte count (p &lt;0.01); HR in the latter analysis was 4.3 for PTPN11, 2.8 for DNMT3A, and 1.6 for abnormal karyotype. A similar analysis for LFS identified, as risk factors, PTPN11 (p = 0.01), DNMT3A (p &lt;0.01), and ASXL1 (p = 0.03) mutations, as well as bone marrow blast % (p &lt;0.01), absolute monocyte count ≥10 (p = 0.02), and immature circulating myeloid cells (p &lt;0.01). Conclusions: The current study identifies PTPN11 and DNMT3A mutations as independent risk factors for both OS and LFS in CMML. In regard to additional genetic risk factors, OS was positively affected by TET2 mutations and negatively by abnormal karyotype, while LFS was negatively affected by ASXL1 mutations.

Computational study on the binding mechanism of allosteric drug TNO155 inhibiting SHP2E76A.

E76A mutations of SHP2 have been reported to associate with genetic developmental diseases and cancers, and TNO155 is one of the effective inhibitors targeted to the allosteric site 1, which has already entered the clinical stage. However, the detailed binding mechanism between them still needs further clarification at micro-atomic level. In this study, the binding mechanism of TNO155 inhibiting SHP2E76A and the superiorities of TNO155 at binding affinity and dynamic interactive behavior with SHP2E76A were probed utilizing a series of computational drug design technologies. The results show that SHP2E76A forms tighter interaction with TNO155 compared to SHP099. SHP2E76A-TNO155 exhibits the largest electrostatic interaction among all complex systems, which can be manifested by the strong hydrogen bond interactions formed by two electrically charged residues, Arg111 and Glu250. Notably, in SHP2E76A-TNO155 system, Asp489 makes an additional substantial beneficial contribution. The E76A mutation brings stronger residue positive correlation and a larger conformation fluctuation between N-CH2 and PTP domains, resulting in tighter binding betweenTNO155and SHP2E76A. This study offers valuable insights for the further design and development of novel SHP2E76A allosteric inhibitors.

Allosterically activating SHP2 by oleanolic acid inhibits STAT3–Th17 axis for ameliorating colitis

Src homology 2 domain-containing tyrosine phosphatase 2 (SHP2) is an essential tyrosine phosphatase that is pivotal in regulating various cellular signaling pathways such as cell growth, differentiation, and survival. The activation of SHP2 has been shown to have a therapeutic effect in colitis and Parkinson's disease. Thus, the identification of SHP2 activators and a complete understanding of their mechanism is required. We used a two-step screening assay to determine a novel allosteric activator of SHP2 that stabilizes it in an open conformation. Oleanolic acid was identified as a suitable candidate. By binding to R362, K364, and K366 in the active center of the PTP domain, oleanolic acid maintained the active open state of SHP2, which facilitated the binding between SHP2 and its substrate. This oleanolic acid-activated SHP2 hindered Th17 differentiation by disturbing the interaction between STAT3 and IL-6Rα and inhibiting the activation of STAT3. Furthermore, via the activation of SHP2 and subsequent attenuation of the STAT3–Th17 axis, oleanolic acid effectively mitigated colitis in mice. This protective effect was abrogated by SHP2 knockout or administration of the SHP2 inhibitor SHP099. These findings underscore the potential of oleanolic acid as a promising therapeutic agent for treating inflammatory bowel diseases.

Single Ion Pair Is Essential for Stabilizing SHP2's Open Conformation.

Src-homology-2-domain-containing PTP-2 (SHP2) is a widely expressed signaling enzyme whose misregulation is associated with multiple human pathologies. SHP2's enzymatic activity is controlled by a conformational equilibrium between its autoinhibited ("closed") state and its activated ("open") state. Although SHP2's closed state has been extensively characterized, the putative structure of its open form has only been revealed in the context of a highly activated mutant (E76K), and no systematic studies of the biochemical determinants of SHP2's open-state stabilization have been reported. To identify amino-acid interactions that are critical for stabilizing SHP2's active state, we carried out a mutagenic study of residues that lie at potentially important interdomain interfaces of the open conformation. The open/closed equilibria of the mutants were evaluated, and we identified several interactions that contribute to the stabilization of SHP2's open state. In particular, our findings establish that an ion pair between glutamate 249 on SHP2's PTP domain and arginine 111 on an interdomain loop is the key determinant of SHP2's open-state stabilization. Mutations that disrupt the R111/E249 ion pair substantially shift SHP2's open/closed equilibrium to the closed state, even compared to wild-type SHP2's basal-state equilibrium, which strongly favors the closed state. To the best of our knowledge, the ion-pair variants uncovered in this study are the first known SHP2 mutants in which autoinhibition is augmented with respect to the wild-type protein. Such "hyperinhibited" mutants may provide useful tools for signaling studies that investigate the connections between SHP2 inhibition and the suppression of human disease progression.

Atomistic ensemble of active SHP2 phosphatase

SHP2 phosphatase plays an important role in regulating several intracellular signaling pathways. Pathogenic mutations of SHP2 cause developmental disorders and are linked to hematological malignancies and cancer. SHP2 comprises two tandemly-arranged SH2 domains, a catalytic PTP domain, and a disordered C-terminal tail. Under physiological, non-stimulating conditions, the catalytic site of PTP is occluded by the N-SH2 domain, so that the basal activity of SHP2 is low. Whereas the autoinhibited structure of SHP2 has been known for two decades, its active, open structure still represents a conundrum. Since the oncogenic mutant SHP2E76K almost completely populates the active, open state, this mutant has been extensively studied as a model for activated SHP2. By molecular dynamics simulations and accurate explicit-solvent SAXS curve predictions, we present the heterogeneous atomistic ensemble of constitutively active SHP2E76K in solution, encompassing a set of conformational arrangements and radii of gyration in agreement with experimental SAXS data.

Abstract C075: A first-in-class and highly selective SHP1 allosteric inhibitor exhibits robust anti-tumor immunity and synergizes with PD-1 blockade

Abstract Background: SHP1 is a member of the src homology 2 (SH2) domain-containing protein phosphatases. It contains two SH2 domains at the N-terminus, a catalytic domain and a C-terminal tail. SHP1 is primarily expressed in hematopoietic cells and commonly recognized as a negative regulator of multiple signaling pathways. It has been known that SHP1 interacts with proteins of the inhibitory-receptor superfamily containing immunoreceptor tyrosine-based inhibitory motifs. Methods: To screen for SHP1 allosteric inhibitors, we employed a DiFMUP high-throughput biochemical assay system. Compounds that only perturbed the activity of SHP1 was further optimized for cellular activity on target, in vitro ADME, and in vivo pharmacokinetic properties. X-ray crystallography has been introduced to determine the interaction between the inhibitor and the allosteric site of SHP1. We also evaluated the lead series in in-vitro studies with human primary immune cells and in-vivo efficacy studies in syngeneic mouse models. Results: We discovered a series of SHP1 allosteric inhibitors with IC50 values at the nanomolar level and high selectivity over the other PTP enzymes, including SHP2. Structurally, SHP1 adopts an autoinhibited conformation in its basal state, where the N-SH2 domain interacts with the PTP domain and blocks access to the catalytic site. X-ray crystallography reveals that our SHP1 inhibitors are bound to non-catalytic sites of the SHP1 inactive conformation. Among the compounds, SB6299 demonstrated potent cytokine responses in various human primary immune cell types. SB6299 significantly enhanced IL-2, IFN-γ, and TNF-α productions in human primary pan-T, NK, and monocyte cells, respectively. The cytokine release was confirmed as a result of selective SHP1 inhibition through the evaluation of SHP1 knockout in primary immune cells. Moreover, SB6299 showed dose responses in costimulatory molecules and cytokines (TNF-α and IL6) in human monocyte derived dendritic cells (DCs) and enhanced DC maturation markers such as CD80, CD86, and CD40. The anti-tumor efficacy of SB6299 was evaluated in the CT26 and MC38 syngeneic mouse models. Orally administered SB6299 showed tumor growth inhibition as a single agent, and the efficacy was further enhanced when administered in combination with an anti-PD1 antibody. Conclusions: 1. Our studies successfully demonstrate that SHP1 can be allosterically inhibited, allowing selectivity with other phosphatases, including SHP2. 2. SB6299 is the first-in-class orally available, potent, and selective SHP1 allosteric inhibitor. 3. Attenuation of SHP1 activity by SB6299 could promote cytokine secretion and cytotoxic effects in human primary immune cells. 4. Orally administered SB6299 induced tumor growth inhibition as a single agent and in combination with an anti-PD1 antibody. 5. Currently, more selective and potent inhibitors are being tested. Citation Format: Jun Gyu Kim, Ki Moon Ryu, Mi Yeon Jang, Jongho Cho, Dajeong Kim, Dae Hyeon Seong, Kyu Hwan Kim, Ik-Soo Jang, Jeong Yoon Shin, Hong Sik Han, Joon Yonug Hwang, Dae Young Lee, Chae Lim Ryu, Song Yi Lee, Tae-Dong Han, SeungMin Yang, Hyounmie Doh. A first-in-class and highly selective SHP1 allosteric inhibitor exhibits robust anti-tumor immunity and synergizes with PD-1 blockade [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C075.

Analysis of the clinical characteristics and prognosis of adult de novo acute myeloid leukemia (none APL) with PTPN11 mutations.

We discuss the clinical characteristics and prognostic significance of adult individuals with PTPN11 mutations who have developed acute myeloid leukemia (AML) (none acute promyelocytic leukemia). Next generation sequencing and Sanger sequencing were used to detect 51 gene mutations, and multiplex-PCR was used to detect 41 fusion genes from 232 de novo adult AML patients retrospectively. About 7.76% patients harbored PTPN11 mutations, 20 PTPN11 alterations were identified, all of which were missense mutations in the N-SH2 (n = 16) and PTP (n = 4) domains located in exon 3. Patients with PTPN11 mut had significantly higher platelet counts and hemoglobin levels (p < 0.001), which were mainly detected in M5 (n = 12, 66.67%, p < 0.001) subtype. Patients with MLL-AF6 positive showed a higher frequency of PTPN11 mut (p = 0.018) in the 118 AML cases. PTPN11 mut were accompanied by other mutations, which were NPM1 (44.44%), DNMT3A (38.89%), FLT3 (38.89%), and NRAS (17.2%). PTPN11 mut had a negative impact on the complete remission rate in M5 subtype patients (p < 0.001). However, no statistically significant effect on overall survival (OS) with PTPN11 mut patients in the whole cohort and age group (p > 0.05) was observed. Further analysis revealed no significant difference in OS among NPM1 mut/PTPN11 mut, NPM1 mut/PTPN11 wt, DNMT3A mut/PTPN11 mut, and DNMT3A mut/PTPN11 wt patients (p > 0.05). Multivariate analysis showed the proportion of bone marrow blasts ≥65.4% was a factor significantly affecting OS in PTPN11 mut patients (p = 0.043).

LAIR1 drives glioma progression by nuclear focal adhesion kinase dependent expressions of cyclin D1 and immunosuppressive chemokines/cytokines

Leukocyte-associated immunoglobulin-like receptor-1 (LAIR1), an immune receptor containing immunoreceptor tyrosine-based inhibiory motifs (ITIMs), has emerged as an attractive target for cancer therapy. However, the intrinsic function of LAIR1 in gliomas remains unclear. In this study, the poor prognosis of glioma patients and the malignant proliferation of glioma cells in vitro and in vivo were found to be closely correlated with LAIR1. LAIR1 facilitates focal adhesion kinase (FAK) nuclear localization, resulting in increased transcription of cyclin D1 and chemokines/cytokines (CCL5, TGFβ2, and IL33). LAIR1 specifically supports in the immunosuppressive glioma microenvironment via CCL5-mediated microglia/macrophage polarization. SHP2Q510E (PTP domain mutant) or FAKNLM (non-nuclear localizing mutant) significantly reversed the LAIR1-induced growth enhancement in glioma cells. In addition, LAIR1Y251/281F (ITIMs mutant) and SHP2Q510E mutants significantly reduced FAK nuclear localization, as well as CCL5 and cyclin D1 expression. Further experiments revealed that the ITIMs of LAIR1 recruited SH2-containing phosphatase 2 (SHP2), which then interacted with FAK and induced FAK nuclear localization. This study uncovered a critical role for intrinsic LAIR1 in facilitating glioma malignant progression and demonstrated a requirement for LAIR1 and SHP2 to enhance FAK nuclear localization.

Abstract 3845: Structural mechanisms of how PTEN mutations degrade function at the membrane and life expectancy of carriers of mutations in the human brain

Abstract PTEN negatively regulates PI3K/AKT/mTOR signaling through dephosphorylation of PIP3 to PIP2. PTEN dysfunction triggers disease phenotypes, ranging from cancer and PHTS to NDDs such as ASD. The most common PTEN mutations, nonsense, frameshift, and deletion/insertion, may cause premature termination of translation. PTEN has a considerable number of missense mutations that may result in loss of protein function through reduced catalytic activity and protein stability. To decipher mechanistic details of the mutations and structural features of the mutant proteins, comprehensive computational studies were performed for PTEN mutants at an anionic lipid bilayer containing PC, PS, PIP2, and PIP3. Six PTP mutations (Y68H, H93R, A126T, R130Q, G132D, R173C) and two C2 mutations (F241S, D252G) were considered. The PTP mutations are known to associate with cancer, PHTS, macrocephaly, and ASD, while the C2 mutations are exclusively related to macrocephaly and ASD. Our studies indicate that the PTEN mutants can effectively absorb the anionic lipid bilayer, similar to wild-type PTEN. However, Y68H significantly disrupts the core of PTP domain, reducing protein stability. H93R hijacks the substate PIP3, interrupting the catalytic site residues recruitment of the substate for catalysis. A126T, R130Q, and G132D in the P loop directly influence the dynamics of the loop, yielding a collapsed loop conformation. The collapsed P loop loses the interaction with the WPD loop, leading to dehydration in the catalytic site. R173C and D252G at the interface between two major domains disrupt the domain-domain interaction, allosterically biasing the P loop dynamics. However, F241S in C2 exhibits less effective allosteric connection to the catalytic site than that observed in the wild type. PCA of the sampled conformations found that the macrocephaly and ASD-related H93R and F241S are likely to sample the conformations present in the wild type. In contrast, the sampled conformations for the cancer and PHTS-related Y68H, A126T, and G132D are different from those for the wild type. These distinct structural features seem to correlate with mutation strength and timing of the expression of the genes that determine the cancer and NDD outcomes. The isoform expression data extracted from the BrainSpan indicate that exon 5 is impacted by NDD or non-NDD mutations. Interestingly, PTEN splicing isoforms that do not carry exon 5 are exclusively impacted by the NDD mutations, F241S and D252G. The increased life expectancy of PTEN variants carriers carrying exons 5 and 7 is highly correlated to increased lifetime risk for certain cancers. Our results underscore the detailed structural and functional mechanisms of PTEN with mutations at the membrane, and suggest that tissue-specific splicing isoform expression should be taken into consideration while predicting the effect of mutations in various diseases. Citation Format: Hyunbum Jang, Jiaye Chen, Lilia M. Iakoucheva, Ruth Nussinov. Structural mechanisms of how PTEN mutations degrade function at the membrane and life expectancy of carriers of mutations in the human brain. [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 3845.

Abstract 873: The mechanisms of SHP2 activation in cancer and neurodevelopmental disorder

Abstract The Src homology 2 domain-containing phosphatase 2 (SHP2) is a nonreceptor protein tyrosine phosphatase that regulates Ras/MAPK pathway, leading to cell proliferation. SHP2 mutations are commonly associated with human malignancies and neurodevelopmental disorders (NDDs). Somatic SHP2 mutations are associated with many types of cancer including sporadic juvenile myelomonocytic leukemia, acute myeloid leukemia, breast, gastric, lung, melanoma, and colorectal cancers, while heterozygous germline mutations are linked to NDDs such as Noonan and LEOPARD syndromes. Interestingly, different mutations at the same genomic loci of the gene result in different disease phenotypes, either cancer or NDDs. Patients with NDDs have a higher chance of developing cancer, suggesting a linkage between cancer and NDDs, and between their mutations. SHP2 consists of two SH2 domains (nSH2 and cSH2), a phosphatase (PTP) domain, and a flexible C-terminal tail. In autoinhibition, SHP2 is catalytically inactive, adopting a closed conformation in which the DE-loop of nSH2 blocks the catalytic site of PTP. The binding of nSH2 to the phosphotyrosine (pY)-motif relieve the autoinhibition via disruption of the nSH2-PTP interface. To depict mechanism of the mutation-associated activation of SHP2, extensive molecular dynamics simulations were performed for SHP2 protein with the mutations related to cancer and NDDs. The dynamics and conformational alterations of the wild type, cancer mutants (E76K, D61Y, D61V), and NDD mutants (E76D, D61G) of SHP2 were compared. Collective results showed that both cancer and NDD mutations weaken the nSH2-PTP interface, facilitating the SHP2 activation. Interestingly, cancer mutations induce stronger destruction at the nSH2-PTP interface than the NDD mutations. The PTP domain exhibits two pockets, orthosteric and allosteric pockets, at the interface with nSH2. In autoinhibition, the orthosteric pocket provides a binding site for D61 of nSH2, and the allosteric pocket accommodates E76 and A72 of nSH2. During the simulations, we observed that the mutations, E76K and E76D, reduce the interaction in the allosteric pocket, increasing the probability toward the open SHP2 conformation. This gradually weakens the interactions of D61 in the orthosteric pocket, promoting SH2 release and leading to SHP2 activation. The three mutations, D61Y, D61V, and D61G, directly disrupted the interaction in the orthosteric pocket, favoring the substrate binding. However, they do not optimize the nSH2 conformation for the pY binding and unlikely affect the interaction in the allosteric pocket. Our works explain the difference and similarity between the cancer and NDD mutations of SHP2 at the atomic level and provide deeper understanding of the mutation-induced activation of SHP2 for signal transduction through the Ras/MAPK pathway. Citation Format: Yonglan Liu, Mingzhen Zhang, Wengang Zhang, Hyunbum Jang, Ruth Nussinov. The mechanisms of SHP2 activation in cancer and neurodevelopmental disorder [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 873.

Identification of linderalactone as a natural inhibitor of SHP2 to ameliorate CCl4-induced liver fibrosis.

Liver fibrosis is characterised by the activation of hepatic stellate cells (HSCs) and matrix deposition. Accumulating evidence has revealed that the oncogenic protein tyrosine phosphatase Src homology 2 domain-containing phosphatase 2 (SHP2) acts as a therapeutic target of fibrosis. Although several SHP2 inhibitors have reached early clinical trials, there are currently no FDA-approved drugs that target SHP2. In this study, we aimed to identify novel SHP2 inhibitors from an in-house natural product library to treat liver fibrosis. Out of the screened 800 compounds, a furanogermacrane sesquiterpene, linderalactone (LIN), significantly inhibited SHP2 dephosphorylation activity in vitro. Cross-validated enzymatic assays, bio-layer interferometry (BLI) assays, and site-directed mutagenesis were used to confirm that LIN directly binds to the catalytic PTP domain of SHP2. In vivo administration of LIN significantly ameliorated carbon tetrachloride (CCl4)-induced HSC activation and liver fibrosis by inhibiting the TGFβ/Smad3 pathway. Thus, LIN or its derivatives could be considered potential therapeutic agents against SHP2-related diseases, such as liver fibrosis or NASH.

SHP-1 tyrosine phosphatase binding to c-Src kinase phosphor-dependent conformations: A comparative structural framework.

SHP-1 is a cytosolic tyrosine phosphatase that is primarily expressed in hematopoietic cells. It acts as a negative regulator of numerous signaling pathways and controls multiple cellular functions involved in cancer pathogenesis. This study describes the binding preferences of SHP-1 (pY536) to c-Srcopen (pY416) and c-Srcclose (pY527) through in silico approaches. Molecular dynamics simulation analysis revealed more conformational changes in c-Srcclose upon binding to SHP-1, as compared to its active/open conformation that is stabilized by the cooperative binding of the C-SH2 domain and C-terminal tail of SHP-1 to c-Src SH2 and KD. In contrast, c-Srcclose and SHP-1 interaction is mediated by PTP domain-specific WPD-loop (WPDXGXP) and Q-loop (QTXXQYXF) binding to c-Srcclose C-terminal tail residues. The dynamic correlation analysis demonstrated a positive correlation for SHP-1 PTP with KD, SH3, and the C-terminal tail of c-Srcclose. In the case of the c-Srcopen-SHP-1 complex, SH3 and SH2 domains of c-Srcopen were correlated to C-SH2 and the C-terminal tail of SHP-1. Our findings reveal that SHP1-dependent c-Src activation through dephosphorylation relies on the conformational shift in the inhibitory C-terminal tail that may ease the recruitment of the N-SH2 domain to phosphotyrosine residue, resulting in the relieving of the PTP domain. Collectively, this study delineates the intermolecular interaction paradigm and underlying conformational readjustments in SHP-1 due to binding with the c-Src active and inactive state. This study will largely help in devising novel therapeutic strategies for targeting cancer development.

Study on the allosteric activation mechanism of SHP2 via elastic network models and neural relational inference molecular dynamics simulation.

As a ubiquitous protein tyrosine phosphatase, SHP2 is involved in PD-1/PD-L1 mediated tumor immune escape and undergoes substantial conformational changes. Therefore, it is considered an ideal target for tumor intervention. However, the allosteric mechanisms of SHP2 binding PD-1 intracellular ITIM/ITSM phosphopeptides remain unclear, which greatly hinders the development of novel structure-based anticancer allosteric inhibitors. In this work, the open and closed structural models of SHP2 are first constructed based on this knowledge; next their motion modes are investigated via elastic network models such as the Gaussian network model (GNM), anisotropic network model (ANM) and adaptive anisotropic network model (aANM); and finally, a possible allosteric signaling pathway is proposed using a neural relational inference molecular dynamics (NRI-MD) simulation embedded with an artificial intelligence (AI) strategy. In GNM and ANM, the N-SH2, C-SH2 and PTP domains all exhibit distinct dynamics partitions, and the N-SH2/C-SH2 regions show a rigid rotation relative to PTP. According to a series of intermediate snapshots given by aANM, N-SH2 is first identified with pY223 specifically, inducing a D'E-loop to change from β-sheets to random coils, and then, C-SH2 serves as a fulcrum to drive N-SH2 to rotate 110° completely away from the original active sites of PTP. Finally, a possible allosteric signaling-transfer path for SHP2, namely R220-R138-T108-R32, is proposed based on NRI-MD sampling. This work provides a possible allosteric mechanism of SHP2, which is helpful for the following design of novel allosteric inhibitors and is expected to be used in clinical synergies with PD-1 monoclonal antibody.

Experiment-guided molecular simulations define a heterogeneous structural ensemble for the PTPN11 tandem SH2 domains.

SHP2 plays an important role in regulating cellular processes, and its pathogenic mutations cause developmental disorders and are linked to cancer. SHP2 is a multidomain protein, comprising two SH2 domains arranged in tandem, a catalytic PTP domain, and a disordered C-terminal tail. SHP2 is activated upon binding two linked phosphopeptides to its SH2 domains, and the peptide orientation and spacing between binding sites are critical for enzymatic activation. For decades, the tandem SH2 has been extensively studied to identify the relative orientation of the two SH2 domains that most effectively binds effectors. So far, neither crystallography nor experiments in solution have provided conclusive results. Using experiment-guided molecular simulations, we determine the heterogeneous structural ensemble of the tandem SH2 in solution in agreement with experimental data from small-angle X-ray scattering and NMR residual dipolar couplings. In the solution ensemble, N-SH2 adopts different orientations and positions relative to C-SH2. We suggest that the intrinsic structural plasticity of the tandem SH2 allows SHP2 to respond to external stimuli and is essential for its functional activity.

PTPN11 mutations in adult acute myeloid leukaemia: Prevalence and clinical implications in the context of NPM1 mutation

More than 90% of NPM1-mutated acute myeloid leukaemia (NPM1mut AML) patients have been determined to harbour other known concurrent mutations. However, there is limited data on the frequency of PTPN11 and its clinical impact in NPM1mut AML. Next-generation sequencing(NGS) was performed retrospectively on 112 genes in 254 patients with NPM1mut AML. Overall, 33 PTPN11 mutations were detected in 29 of the 254 patients (11.42%) screened. PTPN11 alterations were exclusively missense mutations affecting residues located within the N-SH2 (n = 25) and PTP (n = 8) domains and clustered overwhelmingly in exon 3 (n = 25). NPM1mut AML patients with mutant PTPN11 had significant associations with mutations in epigenetic regulators(TET2, IDH1/2, and DNMT3A) (72.41% vs. 46.67%, P = 0.009) and cohesins (RAD21,SMC1A, and SMC3)(10.34% vs. 1.33%, P = 0.02) compared to patients with wild-type PTPN11. Among the patients treated with intensive chemotherapy, the differences in disease-free survival (DFS) and overall survival (OS) between those with mutant and wild-type PTPN11 were not significant (P = 0.4, 0.83, respectively). In conclusion, PTPN11 mutations were frequently identified in NPM1mut AML patients and clustered in exon 3. PTPN11 mutations more frequently co-occurred with mutations involving epigenetic regulators but had no impact on prognosis.

Abstract 13869: The Impact of Genotype on Aortic Dilation in Pediatric Patients With RASopathy

Introduction: Proximal aortic dilation is an important cause of morbidity and mortality secondary to rupture, dissection, and complications of repair. It has been described as a rare finding in RASopathy, but data is limited regarding prevalence, severity, and genetic factors. This study aims to examine outcomes and genotype associations in pediatric patients with aortic dilation and RASopathy. Methods: A retrospective review performed at a single pediatric referral center. Study population included children with a molecularly confirmed RASopathy diagnosis evaluated between January 2000 and August 2019. Baseline data included demographics, genetic data, cardiac diagnostic data, clinical course, and mortality outcome. Results: Ninety patients were identified (mean age 10.4, female 48%). Aortic dilation was found in 28%. Of these, 68% had mild dilation, while moderate and severe dilation were each seen in 16%. Aortic dilation occurred in all major RASopathy types (Noonan, Noonan with Multiple Lentigenes, Costello, and Cardiofaciocutaneous). Hypertrophic cardiomyopathy was seen in 30% and pulmonary stenosis in 53% of RASopathy patients, with similar prevalence among those with aortic dilation (p=1 and 0.45, respectively). Aortic dilation was more prevalent in RAS ( KRAS , HRAS , NRAS ) variants (4/9 versus 12/81, p=0.0494). It was also seen in 16 of 60 patients with PTPN11 variants in association with all three commonly involved regions (exon 3 of N-SH2 region and exons 8 and 13 of PTP domain). The two patients with extreme dilation (z-score &gt; 6) had variants of amino acids 61 and 63 in exon 3 of PTPN11 . No surgical interventions targeting aortic dilation were performed in any patient, and there were no reports of dissection or rupture. There was no clear progression of dilation, and two patients had complete resolution with time. Discussion: Aortic dilation occurred in all four major types of RASopathy and was associated with most of the affected genes, with distribution along the PTPN11 gene being similar to the distribution of RASopathy. The dilation was not progressive and there were no complications or interventions. RAS genes and exon 3 of the PTPN11 gene demonstrated a possible higher association with aortic dilation and warrant further investigation.

Exploring the Distinct Binding and Activation Mechanisms for Different CagA Oncoproteins and SHP2 by Molecular Dynamics Simulations.

CagA is a major virulence factor of Helicobacter pylori. H. pylori CagA is geographically subclassified into East Asian CagA and Western CagA, which are characterized by the presence of a EPIYA-D or EPIYA-C segment. The East Asian CagA is more closely associated with gastric cancer than the Western CagA. In this study, molecular dynamic (MD) simulations were performed to investigate the binding details of SHP2 and EPIYA segments, and to explore the allosteric regulation mechanism of SHP2. Our results show that the EPIYA-D has a stronger binding affinity to the N-SH2 domain of SHP2 than EPIYA-C. In addition, a single EPIYA-D binding to N-SH2 domain of SHP2 can cause a deflection of the key helix B, and the deflected helix B could squeeze the N-SH2 and PTP domains to break the autoinhibition pocket of SHP2. However, a single EPIYA-C binding to the N-SH2 domain of SHP2 cannot break the autoinhibition of SHP2 because the secondary structure of the key helix B is destroyed. However, the tandem EPIYA-C not only increases its binding affinity to SHP2, but also does not significantly break the secondary structure of the key helix B. Our study can help us better understand the mechanism of gastric cancer caused by Helicobacter pylori infection.

Exploring the dynamic mechanism of allosteric drug SHP099 inhibiting SHP2E69K.

The E69K mutation is one of the most frequent protein tyrosine phosphatase-2 (SHP2) mutations in leukemia, and it can cause the increase in the protein activity. Recent studies have shown that the E69K mutation was fairly sensitive to the allosteric inhibitor of SHP2 (SHP099). However, the molecular mechanism of the allosteric drug SHP099 inhibiting SHP2E69K remains unclear. Thus, the molecular dynamic simulations and the post-dynamics analyses (RMSF, PCA, DCCM, RIN and the binding free energies) for SHP2WT, SHP2WT-SHP099, SHP2E69K and SHP2E69K-SHP099 were carried out, respectively. Owing to the strong binding affinity of SHP099 to residues Thr219 and Arg220, the flexibility of linker region (residues Val209-Arg231) was reduced. Moreover, the presence of SHP099 kept the autoinhibition state of the SHP2 protein through enhancing the interactions between the linker region and Q loop in PTP domain, such as Thr219/Val490, Thr219/Asn491, Arg220/Ile488 and Leu254/Asn491. In addition, it was found that the residues (Thr219, Arg220, Leu254 and Asn491) might be the key residues responsible for the conformational changes of protein. Overall, this study may provide an important basis for understanding how the SHP099 effectively inhibited the SHP2E69K activity at the molecular level.

Exploring the mechanism of the potent allosteric inhibitor compound2 on SHP2 WT and SHP2F285S by molecular dynamics study

Abnormal activation of Ras/MAPK signaling pathway could trigger excessive cell division. Src-homology 2 (SH2) domain-containing protein tyrosine phosphatase (SHP2) could promote Ras/MAPK activation by integrating growth factor signals. Thus, SHP2 inhibitors had become a hot topic in the treatment of cancer. SHP2F285S, mutation in SHP2, was detected in leukemia variants. The compound 2 (3-benzyl-8-chloro-2-hydroxy-4H-benzo[4,5]thiazolo[3,2-a]pyrimidin-4-one) had been reported that it was a potent allosteric inhibitor of both SHP2 wild type (SHP2WT) and the F285S mutant (SHP2F285S). However, the mechanism of inhibition remained to be further discovered. Herein, molecular docking and molecular dynamic (MD) simulation were performed to explain the inhibition mechanism of compound 2 on SHP2WT and SHP2F285S. Overall, the molecular docking analysis revealed that compound 2 maintained the “close” structure of SHP2 protein probably by locking the C-SH2 and PTP domain. Next, post-analysis demonstrated that compound 2 might make TYR66-GLU76 of D’E-loop in N-SH2 and GLU258-LYS266 of B’C-loop, HIS458-ARG465 of P-loop, VAL497-THR507 of Q-loop in PTP domain regions tightly connect and much easier maintain “self-inhibited” conformation of SHP2F285S−compound2 than that of SHP2WT-compound2. Importantly, GLU76 of D’E-loop could play a vital role in inhibition of SHP2WT-compound2 and SHP2F285S−compound2. This work provided a reliable clue to develop novel inhibitors for leukemia related to SHP2F285S.