Phase Separation of Disease-Associated SHP2 Mutants Underlies MAPK Hyperactivation.

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

Restraining of glycoprotein VI- and integrin α2ß1-dependent thrombus formation by platelet PECAM1.

The platelet receptors, glycoprotein VI (GPVI) and integrin α2ß1 jointly control collagen-dependent thrombus formation via protein tyrosine kinases. It is unresolved to which extent the ITIM (immunoreceptor tyrosine-based inhibitory motif) receptor PECAM1 and its downstream acting protein tyrosine phosphatase PTPN11 interfere in this process. Here, we hypothesized that integrin α2ß1 has a co-regulatory role in the PECAM1- and PTPN11-dependent restraint of thrombus formation. We investigated platelet activation under flow on collagens with a different GPVI dependency and using integrin α2ß1 blockage. Blood was obtained from healthy subjects and from patients with Noonan syndrome with a gain-of-function mutation of PTPN11 and variable bleeding phenotype. On collagens with decreasing GPVI activity (types I, III, IV), the surface-dependent inhibition of PECAM1 did not alter thrombus parameters using control blood. Blockage of α2ß1 generally reduced thrombus parameters, most effectively on collagen IV. Strikingly, simultaneous inhibition of PECAM1 and α2ß1 led to a restoration of thrombus formation, indicating that the suppressing signaling effect of PECAM1 is masked by the platelet-adhesive receptor α2ß1. Blood from 4 out of 6 Noonan patients showed subnormal thrombus formation on collagen IV. In these patients, effects of α2ß1 blockage were counterbalanced by PECAM1 inhibition to a normal phenotype. In summary, we conclude that the suppression of GPVI-dependent thrombus formation by either PECAM1 or a gain-of-function of PTPN11 can be overruled by α2ß1 engagement.

Dependence of peripheral T-cell lymphoma on constitutively activated JAK3: Implication for JAK3 inhibition as a therapeutic approach.

Peripheral T-cell lymphoma (PTCL) is a clinically heterogeneous group that represents 10%-15% of all lymphomas. Despite improved genetic and molecular understanding, treatment outcomes for PTCL have not shown significant improvement. Although Janus kinase-2 (JAK2) plays an important role in myeloproliferative neoplasms, the critical role of JAK isoforms in mediating prosurvival signaling in PTCL cells is not well defined. Immunohistochemical analysis of PTCL tumors (n = 96) revealed high levels of constitutively active JAK3 (pJAK3) that significantly (p < 0.04) correlated with the activation state of its canonical substrate STAT3. Furthermore, constitutive activation of JAK3 and STAT3 positively correlated, at least in part, with an oncogenic tyrosine phosphatase PTPN11. Pharmacological inhibition of JAK3 but not JAK1/JAK2 significantly (p < 0.001) decreased PTCL proliferation, survival and STAT3 activation. A sharp contrast was observed in the pJAK3 positivity between ALK+ (85.7%) versus ALK-negative (10.0%) in human PTCL tumors and PTCL cell lines. Moreover, JAK3 and ALK reciprocally interacted in PTCL cells, forming a complex to possibly regulate STAT3 signaling. Finally, combined inhibition of JAK3 (by WHI-P154) and ALK (by crizotinib or alectinib) significantly (p < 0.01) decreased the survival of PTCL cells as compared to either agent alone by inhibiting STAT3 downstream signaling. Collectively, our findings establish that JAK3 is a therapeutic target for a subset of PTCL, and provide rationale for the clinical evaluation of JAK3 inhibitors combined with ALK-targeted therapy in PTCL.

Co-occurrence of Erdheim-Chester disease and clonally evolving acute myeloid leukemia with FLT3-ITD and PTPN11 mutations.

Erdheim-Chester disease (ECD) is a rare histiocytosis that tends to co-exist with other myeloid malignancies. Here, we use genetic and transcriptomic sequencing to delineate a case of co-occurring BRAFV600E-mutated ECD and acute myeloid leukemia (AML), followed by AML remission and relapse. The AML relapse involved the extinction of clones with KMT2A-AFDN and FLT3-ITD, and the predominance of PTPN11-mutated subclones with distinct transcriptomic features. This case report has highlighted the screening for other myeloid malignancies at the diagnosis of ECD and the clinical significance of PTPN11-mutated AML subclones that require meticulous monitoring.

Concomitant deletion of Ptpn6 and Ptpn11 in T cells fails to improve anticancer responses.

Anticancer T cells acquire a dysfunctional state characterized by poor effector function and expression of inhibitory receptors, such as PD-1. Blockade of PD-1 leads to T cell reinvigoration and is increasingly applied as an effective anticancer treatment. Recent work challenged the commonly held view that the phosphatase PTPN11 (known as SHP-2) is essential for PD-1 signaling in T cells, suggesting functional redundancy with the homologous phosphatase PTPN6 (SHP-1). Therefore, we investigated the effect of concomitant Ptpn6 and Ptpn11 deletion in T cells on their ability to mount antitumour responses. In vivo data show that neither sustained nor acute Ptpn6/11 deletion improves T cell-mediated tumor control. Sustained loss of Ptpn6/11 also impairs the therapeutic effects of anti-PD1 treatment. In vitro results show that Ptpn6/11-deleted CD8+ T cells exhibit impaired expansion due to a survival defect and proteomics analyses reveal substantial alterations, including in apoptosis-related pathways. These data indicate that concomitant ablation of Ptpn6/11 in polyclonal T cells fails to improve their anticancer properties, implying that caution shall be taken when considering their inhibition for immunotherapeutic approaches.

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.

SHP2 clinical phenotype, cancer, or RASopathies, can be predicted by mutant conformational propensities.

SHP2 phosphatase promotes full activation of the RTK-dependent Ras/MAPK pathway. Its mutations can drive cancer and RASopathies, a group of neurodevelopmental disorders (NDDs). Here we ask how same residue mutations in SHP2 can lead to both cancer and NDD phenotypes, and whether we can predict what the outcome will be. We collected and analyzed mutation data from the literature and cancer databases and performed molecular dynamics simulations of SHP2 mutants. We show that both cancer and Noonan syndrome (NS, a RASopathy) mutations favor catalysis-prone conformations. As to cancer versus RASopathies, we demonstrate that cancer mutations are more likely to accelerate SHP2 activation than the NS mutations at the same genomic loci, in line with NMR data for K-Ras4B more aggressive mutations. The compiled experimental data and dynamic features of SHP2 mutants lead us to propose that different from strong oncogenic mutations, SHP2 activation by NS mutations is less likely to induce a transition of the ensemble from the SHP2 inactive state to the active state. Strong signaling promotes cell proliferation, a hallmark of cancer. Weak, or moderate signals are associated with differentiation. In embryonic neural cells, dysregulated differentiation is connected to NDDs. Our innovative work offers structural guidelines for identifying and correlating mutations with clinical outcomes, and an explanation for why bearers of RASopathy mutations may have a higher probability of cancer. Finally, we propose a drug strategy against SHP2 variants-promoting cancer and RASopathies.

Human Genetics of Semilunar Valve and Aortic Arch Anomalies.

Lesions of the semilunar valve and the aortic arch can occur either in isolation or as part of well-described clinical syndromes. The polygenic cause of calcific aortic valve disease will be discussed including the key role of NOTCH1 mutations. In addition, the complex trait of bicuspid aortic valve disease will be outlined, both in sporadic/familial cases and in the context of associated syndromes, such as Alagille, Williams, and Kabuki syndromes. Aortic arch abnormalities particularly coarctation of the aorta and interrupted aortic arch, including their association with syndromes such as Turner and 22q11 deletion, respectively, are also discussed. Finally, the genetic basis of congenital pulmonary valve stenosis is summarized, with particular note to Ras-/mitogen-activated protein kinase (Ras/MAPK) pathway syndromes and other less common associations, such as Holt-Oram syndrome.

Human Genetics of Atrial Septal Defect.

Although atrial septal defects (ASD) can be subdivided based on their anatomical location, an essential aspect of human genetics and genetic counseling is distinguishing between isolated and familiar cases without extracardiac features and syndromic cases with the co-occurrence of extracardiac abnormalities, such as developmental delay. Isolated or familial cases tend to show genetic alterations in genes related to important cardiac transcription factors and genes encoding for sarcomeric proteins. By contrast, the spectrum of genes with genetic alterations observed in syndromic cases is diverse. Currently, it points to different pathways and gene networks relevant to the dysregulation of cardiomyogenesis and ASD pathogenesis. Therefore, this chapter reflects the current knowledge and highlights stable associations observed in human genetics studies. It gives an overview of the different types of genetic alterations in these subtypes, including common associations based on genome-wide association studies (GWAS), and it highlights the most frequently observed syndromes associated with ASD pathogenesis.

Human Genetics of Ventricular Septal Defect.

Ventricular septal defects (VSDs) are recognized as one of the commonest congenital heart diseases (CHD), accounting for up to 40% of all cardiac malformations, and occur as isolated CHDs as well as together with other cardiac and extracardiac congenital malformations in individual patients and families. The genetic etiology of VSD is complex and extraordinarily heterogeneous. Chromosomal abnormalities such as aneuploidy and structural variations as well as rare point mutations in various genes have been reported to be associated with this cardiac defect. This includes both well-defined syndromes with known genetic cause (e.g., DiGeorge syndrome and Holt-Oram syndrome) and so far undefined syndromic forms characterized by unspecific symptoms. Mutations in genes encoding cardiac transcription factors (e.g., NKX2-5 and GATA4) and signaling molecules (e.g., CFC1) have been most frequently found in VSD cases. Moreover, new high-resolution methods such as comparative genomic hybridization enabled the discovery of a high number of different copy number variations, leading to gain or loss of chromosomal regions often containing multiple genes, in patients with VSD. In this chapter, we will describe the broad genetic heterogeneity observed in VSD patients considering recent advances in this field.

[FLT3-ITD mutation-positive acute myeloid leukemia undergoing clonal transition with PTPN11 mutation at relapse].

A 28-year-old man was diagnosed with acute myelomonocytic leukemia. He achieved complete remission (CR) after two cycles of induction therapy. However, after consolidation therapy, bone marrow aspiration performed to prepare for allogeneic hematopoietic stem cell transplantation revealed disease relapse. Companion diagnostics confirmed the presence of the FLT3-ITD mutation. The patient received gilteritinib monotherapy and achieved CR. Subsequently, he underwent unrelated allogeneic bone marrow transplantation. One year after transplantation, the patient relapsed, and gilteritinib was resumed. However, the leukemia progressed, and panel sequencing using a next-generation sequencer showed that the FLT3-ITD mutation disappeared. A mutation in PTPN11, which regulates the RAS/MAPK signaling pathway, was also detected. Gilteritinib was discontinued, and the patient achieved CR with salvage chemotherapy. He underwent related haploidentical peripheral blood stem cell transplantation but died of relapse. This was a case in which genetic analysis revealed clonal transition and acquisition of resistance to treatment.

Discovery of novel furanylbenzamide inhibitors that target oncogenic tyrosine phosphatase SHP2 in leukemia cells.

Disturbance of the dynamic balance between tyrosine phosphorylation and dephosphorylation of signaling molecules, controlled by protein tyrosine kinases and protein tyrosine phosphatases (PTPs), is known to lead to the development of cancer. While most approved targeted cancer therapies are tyrosine kinase inhibitors, PTPs have long been stigmatized as undruggable and have only recently gained renewed attention in drug discovery. One PTP target is the Src-homology 2 domain-containing phosphatase 2 (SHP2). SHP2 is implicated in tumor initiation, progression, metastasis, and treatment resistance, primarily because of its role as a signaling nexus of the extracellular signal-regulated kinase pathway, acting upstream of the small GTPase Ras. Efforts to develop small molecules that target SHP2 are ongoing, and several SHP2 allosteric inhibitors are currently in clinical trials for the treatment of solid tumors. However, while the reported allosteric inhibitors are highly effective against cells expressing WT SHP2, none have significant activity against the most frequent oncogenic SHP2 variants that drive leukemogenesis in several juvenile and acute leukemias. Here, we report the discovery of novel furanylbenzamide molecules as inhibitors of both WT and oncogenic SHP2. Importantly, these inhibitors readily cross cell membranes, bind and inhibit SHP2 under physiological conditions, and effectively decrease the growth of cancer cells, including triple-negative breast cancer cells, acute myeloid leukemia cells expressing either WT or oncogenic SHP2, and patient-derived acute myeloid leukemia cells. These novel compounds are effective chemical probes of active SHP2 and may serve as starting points for therapeutics targeting WT or mutant SHP2 in cancer.

Signal regulatory protein alpha is a conserved marker for mouse and rat spermatogonial stem cells†.

Characterization of spermatogonial stem cells (SSCs) has been hampered by their low frequency and lack of features that distinguish them from committed spermatogonia. Few conserved SSC markers have been discovered. To identify a new SSC marker, we evaluated SIRPA expression in mouse and rat SSCs. SIRPA was expressed in a small population of undifferentiated spermatogonia. SIRPA, and its ligand CD47 were expressed in cultured SSCs. Expression of both SIRPA and CD47 was upregulated by supplementation of GDNF and FGF2, which promoted SSC self-renewal. Sirpa depletion by short hairpin RNA impaired the proliferation of cultured SSCs, and these cells showed decreased MAP2K1 activation and PTPN11 phosphorylation. Immunoprecipitation experiments showed that SIRPA associates with PTPN11. Ptpn11 depletion impaired SSC activity in a manner similar to Sirpa depletion. SIRPA was expressed in undifferentiated spermatogonia in rat and monkey testes. Xenogenic transplantation experiments demonstrated that SIRPA is expressed in rat SSCs. These results suggest that SIRPA is a conserved SSC marker that promotes SSC self-renewal division by activating the MAP2K1 pathway via PTPN11.

PTPN11 variant may be a prognostic indicator of IDH-wildtype glioblastoma in a comprehensive genomic profiling cohort.

PURPOSE: Glioblastoma (GBM) is the most common type of primary malignant brain tumor and has a poor prognosis. Identifying novel targets and stratification strategies is urgently needed to improve patient survival. The present study aimed to identify clinically relevant genomic alterations in IDH-wildtype GBM using data from comprehensive genomic profiling (CGP) assays performed nationwide in Japan. METHODS: The CGP assay results of 392 IDH-wildtype GBM cases performed between October 2019 and February 2023 obtained from the Center for Cancer Genomics and Advanced Therapeutics were retrospectively analyzed. RESULTS: The median patient age was 52.5 years, and 207 patients (53%) were male. In the 286 patients for whom survival information was available, a protein-tyrosine phosphatase non-receptor type 11 (PTPN11) variant detected in 20 patients (6.8%) was extracted as the gene associated with significantly shorter overall survival (p = 0.002). Multivariate analysis demonstrated that the PTPN11 variant and poor performance status were independent prognostic indicators. In contrast, no prognostic impact was observed in the cohort in The Cancer Genome Atlas data. The discrepancy in the prognostic impact of the PTPN11 variant from these two pools might have resulted from differences in the biases affecting the survival of patients who underwent a CGP assay, including left-truncation and right-censored bias. However, survival simulation done to adjust for these biases showed that the prognostic impact of the PTPN11 variant was also significant. CONCLUSIONS: The PTPN11 variant was a negative prognostic indicator of IDH-wildtype GBM in the patient cohort with the CGP assay.

Cardiac features of Noonan syndrome in Japanese patients.

BACKGROUND: Cardiovascular disease is one of the most important problems in long-term follow-up for Noonan syndrome. We examined cardiovascular issues and clinical manifestations, with a focus on the cardiovascular disease and prognosis of patients with Noonan syndrome. METHODS: This single-centre study evaluated patients who were clinically and genetically diagnosed with Noonan syndrome. RESULTS: Forty-three patients diagnosed with Noonan syndrome were analysed. The most prevalent responsible mutation was found in PTPN11 (25/43). The second and third most prevalent causative genes were SOS1 (6/43) and RIT1 (5/43), respectively, and 67.4% of genetically diagnosed patients with Noonan syndrome had structural cardiovascular abnormalities. Pulmonary valve stenosis was prevalent in patients with mutations in PTPN11 (8/25), SOS1 (4/6), and RIT1 (4/5). Hypertrophic cardiomyopathy was found in two of three patients with mutations in RAF1. There was no difference in the cardiovascular events or cardiovascular disease prevalence in patients with or without PTPN11 mutations. The proportion of RIT1 mutation-positive patients who underwent intervention due to cardiovascular disease was significantly higher than that of patients with PTPN11 mutations. Patients who underwent any intervention for pulmonary valve stenosis exhibited significantly higher pulmonary flow velocity than patients who did not undergo intervention, when they visited our hospital for the first time. All patients who underwent intervention for pulmonary valve stenosis had a pulmonary flow velocity of more than 3.0 m/s at first visit. CONCLUSIONS: These findings suggest that genetic information can provide a clinical prognosis for cardiovascular disease and may be part of genotype-based follow-up in Noonan syndrome.

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.

Lateralized overgrowth with vascular malformation caused by a somatic PTPN11 pathogenic variant: Another piece added to the puzzle of mosaic RASopathies.

Lateralized/segmental overgrowth disorders (LOs) encompass a heterogeneous group of congenital conditions with excessive body tissue growth. Documented molecular alterations in LOs mostly consist of somatic variants in genes of the PI3KCA/AKT/mTOR pathway or of chromosome band 11p15.5 imprinted region anomalies. In some cases, somatic pathogenic variants in genes of the RAS/MAPK pathway have been reported. We present the first case of a somatic pathogenic variant (T507K) in PTPN11 causing a LO phenotype characterized by severe lateralized overgrowth, vascular proliferation, and cerebral astrocytoma. The T507K variant was detected in DNA from overgrown tissue in a leg with capillary malformation. The astrocytoma tissue showed a higher PTPN11 variant allele frequency. A pathogenic variant in FGFR1 was also found in tumor tissue, representing a second hit on the RAS/MAPK pathway. These findings indicate that RAS/MAPK cascade overactivation can cause mosaic overgrowth phenotypes resembling PIK3CA-related overgrowth disorders (PROS) with cancer predisposition and are consistent with the hypothesis that RAS/MAPK hyperactivation can be involved in the pathogenesis of astrocytoma. This observation raises the issue of cancer predisposition in patients with RAS/MAPK pathway gene variants and expands genotype spectrum of LOs and the treatment options for similar cases through inhibition of the RAS/MAPK oversignaling.

Protein Tyrosine Phosphatase SHP2 Controls Interleukin-8 Expression in Breast Cancer Cells.

Treatment of metastasis remains a clinical challenge and the majority of breast cancer-related deaths are the result of drug-resistant metastases. The protein tyrosine phosphatase SHP2 encoded by the proto-oncogene PTPN11 promotes breast cancer progression. Inhibition of SHP2 has been shown to decrease metastases formation in various breast cancer models, but specific downstream effectors of SHP2 remain poorly characterized. Certain cytokines in the metastatic cascade facilitate local invasion and promote metastatic colonization. In this study, we investigated cytokines affected by SHP2 that could be relevant for its pro-tumorigenic properties. We used a cytokine array to investigate differentially released cytokines in the supernatant of SHP2 inhibitor-treated breast cancer cells. Expression of CXCL8 transcripts and protein abundance were assessed in human breast cancer cell lines in which we blocked SHP2 using shRNA constructs or an allosteric inhibitor. The impact of SHP2 inhibition on the phospho-tyrosine-proteome and signaling was determined using mass spectrometry. From previously published RNAseq data (Aceto et al. in Nat. Med. 18:529-37, 2012), we computed transcription factor activities using an integrated system for motif activity response analysis (ISMARA) (Balwierz et al. in Genome Res. 24:869-84, 2014). Finally, using siRNA against ETS1, we investigated whether ETS1 directly influences CXCL8 expression levels. We found that IL-8 is one of the most downregulated cytokines in cell supernatants upon SHP2 blockade, with a twofold decrease in CXCL8 transcripts and a fourfold decrease in IL-8 protein. These effects were also observed in preclinical tumor models. Analysis of the phospho-tyrosine-proteome revealed that several effectors of the mitogen-activated protein kinase (MAPK) pathway are downregulated upon SHP2 inhibition in vitro. MEK1/2 inhibition consistently reduced IL-8 levels in breast cancer cell supernatants. Computational analysis of RNAseq data from SHP2-depleted tumors revealed reduced activity of the transcription factor ETS1, a direct target of ERK and a transcription factor reported to regulate IL-8 expression. Our work reveals that SHP2 mediates breast cancer progression by enhancing the production and secretion of the pro-metastatic cytokine IL-8. We also provide mechanistic insights into the effects of SHP2 inhibition and its downstream repercussions. Overall, these results support a rationale for targeting SHP2 in breast cancer.

SHP2's gain-of-function in Werner syndrome causes childhood disease onset likely resulting from negative genetic interaction.

Prompt diagnosis of complex phenotypes is a challenging task in clinical genetics. Whole exome sequencing has proved to be effective in solving such conditions. Here, we report on an unpredictable presentation of Werner Syndrome (WRNS) in a 12-year-old girl carrying a homozygous truncating variant in RECQL2, the gene mutated in WRNS, and a de novo activating missense change in PTPN11, the major Noonan syndrome gene, encoding SHP2, a protein tyrosine phosphatase positively controlling RAS function and MAPK signaling, which have tightly been associated with senescence in primary cells. All the major WRNS clinical criteria were present with an extreme precocious onset and were associated with mild intellectual disability, severe growth retardation and facial dysmorphism. Compared to primary fibroblasts from adult subjects with WRNS, proband's fibroblasts showed a dramatically reduced proliferation rate and competence, and a more accelerated senescence, in line with the anticipated WRNS features occurring in the child. In vitro functional characterization of the SHP2 mutant documented its hyperactive behavior and a significantly enhanced activation of the MAPK pathway. Based on the functional interaction of WRN and MAPK signaling in processes relevant to replicative senescence, these findings disclose a unique phenotype likely resulting from negative genetic interaction.

miR-6071 inhibits hepatocellular carcinoma progression via targeting PTPN11.

Hepatocellular carcinoma (HCC) is a deadly malignancy. Liver cancer stem cells (LCSCs) participated in HCC progression and caused failure of chemotherapy. However, the underlying mechanism for the LCSCs regulation was unclear. In this study, we found that miR-6071 expression was decreased in LCSCs. Gain-of-function assays showed that miR-6071 overexpression repressed LCSCs self-renewal and tumorigenesis and inhibited HCC cells proliferation and migration. In mechanism, bioinformatics and luciferase reporter assay demonstrated that miR-6071 targeted 3'UTR of PTPN11 mRNA. Pearson analysis revealed a negative correlation between miR-6071 expression and PTPN11 levels in HCC tissue samples. Further study showed that PTPN11 interference and specific inhibitors IACS-13909 abrogated the discrepancy of self-renewal ability, proliferation, migration and tumorigenicity capacity between miR-6071 overexpression HCC cells and control cells. Moreover, upregulation of miR-6071 sensitized HCC cells to lenvatinib treatment. Clinical cohort analysis revealed that HCC patients with high miR-6071 expression got more survival benefit from postoperative lenvatinib treatment than patients with low miR-6071 levels. In conclusion, our study demonstrated a regulation mechanism of LCSCs, a target against LSCSs, and a biomarker for postoperative lenvatinib treatment.