Faculty Bibliography

Catalytically activating mutations inPtpn11, which encodes the protein tyrosine phosphatase SHP2, cause 50% of Noonan syndrome (NS) cases, whereas inactivating mutations inPtpn11are responsible for nearly all cases of the similar, but distinct, developmental disorder Noonan syndrome with multiple lentigines (NSML; formerly called LEOPARD syndrome). However, both types of disease mutations are gain-of-function mutations because they cause SHP2 to constitutively adopt an open conformation. We found that the catalytic activity of SHP2 was required for the pathogenic effects of gain-of-function, disease-associated mutations on the development of hydrocephalus in the mouse. Targeted pan-neuronal knockin of aPtpn11allele encoding the active SHP2 E76K mutant resulted in hydrocephalus due to aberrant development of ependymal cells and their cilia. These pathogenic effects of the E76K mutation were suppressed by the additional mutation C459S, which abolished the catalytic activity of SHP2. Moreover, ependymal cells in NSML mice bearing the inactive SHP2 mutant Y279C were also unaffected. Mechanistically, the SHP2 E76K mutant induced developmental defects in ependymal cells by enhancing dephosphorylation and inhibition of the transcription activator STAT3. Whereas STAT3 activity was reduced inPtpn11

Activation of the RAS/ERK and its downstream signaling components is essential for growth factor-induced cell survival, proliferation, and differentiation. The Src homology-2 domain containing protein tyrosine phosphatase 2 (SHP2), encoded by protein tyrosine phosphatase, non-receptor type 11 (Ptpn11), is a positive mediator required for most, if not all, receptor tyrosine kinase-evoked RAS/ERK activation, but differentially regulates the PI3K/AKT signaling cascade in various cellular contexts. The precise mechanisms underlying the differential effects of SHP2 deficiency on the PI3K pathway remain unclear. We found that mice with myelomonocytic cell-specific [Tg(LysM-Cre);Ptpn11fl/fl mice] Ptpn11 deficiency exhibit mild osteopetrosis. SHP2-deficient bone marrow macrophages (BMMs) showed decreased proliferation in response to M-CSF and decreased osteoclast generation. M-CSF-evoked ERK1/2 activation was decreased, whereas AKT activation was enhanced in SHP2-deficient BMMs. ERK1/2, via its downstream target RSK2, mediates this negative feedback by negatively regulating phosphorylation of M-CSF receptor at Tyr721 and, consequently, its binding to p85 subunit of PI3K and PI3K activation. Pharmacologic inhibition of RSK or ERK phenotypically mimics the signaling defects observed in SHP2-deficient BMMs. Furthermore, this increase in PI3K/AKT activation enables BMM survival in the setting of SHP2 deficiency.-Wang, L., Iorio, C., Yan, K., Yang, H., Takeshita, S., Kang, S., Neel, B.G., Yang, W. An ERK/RSK-mediated negative feedback loop regulates M-CSF-evoked PI3K/AKT activation in macrophages.

Programmed cell death-1 (PD-1) is an essential inhibitory receptor in T cells. Antibodies targeting PD-1 elicit durable clinical responses in patients with multiple tumor indications. Nevertheless, a significant proportion of patients do not respond to anti-PD-1 treatment, and a better understanding of the signaling pathways downstream of PD-1 could provide biomarkers for those whose tumors respond and new therapeutic approaches for those whose tumors do not. We used affinity purification mass spectrometry to uncover multiple proteins associated with PD-1. Among these proteins, signaling lymphocytic activation molecule-associated protein (SAP) was functionally and mechanistically analyzed for its contribution to PD-1 inhibitory responses. Silencing of SAP augmented and overexpression blocked PD-1 function. T cells from patients with X-linked lymphoproliferative disease (XLP), who lack functional SAP, were hyperresponsive to PD-1 signaling, confirming its inhibitory role downstream of PD-1. Strikingly, signaling downstream of PD-1 in purified T cell subsets did not correlate with PD-1 surface expression but was inversely correlated with intracellular SAP levels. Mechanistically, SAP opposed PD-1 function by acting as a molecular shield of key tyrosine residues that are targets for the tyrosine phosphatase SHP2, which mediates PD-1 inhibitory properties. Our results identify SAP as an inhibitor of PD-1 function and SHP2 as a potential therapeutic target in patients with XLP.

Protein tyrosine phosphatase PTP1B is a critical regulator of signaling pathways controlling metabolic homeostasis, cell proliferation and immunity. In this study, we report that global or myeloid-specific deficiency of PTP1B in mice decreases lifespan. We demonstrate that myeloid-specific deficiency of PTP1B is sufficient to promote the development of acute myeloid leukemia (AML). LysM-PTP1B-/- mice lacking PTP1B in the innate myeloid cell lineage displayed a dysregulation of bone marrow cells with a rapid decline in population at midlife and a concomitant increase in peripheral blood blast cells. This phenotype manifested further with extramedullary tumors, hepatic macrophage infiltration and metabolic reprogramming, suggesting increased hepatic lipid metabolism prior to overt tumor development. Mechanistic investigations revealed an increase in anti-inflammatory M2 macrophage responses in liver and spleen, as associated with increased expression of arginase I and the cytokines IL-10 and IL-4. We also documented STAT3 hypersphosphorylation and signaling along with JAK-dependent upregulation of anti-apoptotic proteins Bcl2 and BclXL. Our results establish a tumor suppressor role for PTP1B in the myeloid lineage cells, with evidence that its genetic inactivation in mice is sufficient to drive acute myeloid leukemia.

BCR-ABL1-targeting tyrosine kinase inhibitors (TKIs) have revolutionized treatment of Philadelphia chromosome-positive (Ph⁺) hematologic neoplasms. Nevertheless, acquired TKI resistance remains a major problem in chronic myeloid leukemia (CML), and TKIs are less effective against Ph⁺B-cell acute lymphoblastic leukemia (B-ALL). GAB2, a scaffolding adaptor that binds and activates SHP2, is essential for leukemogenesis by BCR-ABL1, and a GAB2 mutant lacking SHP2 binding cannot mediate leukemogenesis. Using a genetic loss-of-function approach and bone marrow transplantation models for CML and BCR-ABL1⁺B-ALL, we show that SHP2 is required for BCR-ABL1-evoked myeloid and lymphoid neoplasia. Ptpn11 deletion impairs initiation and maintenance of CML-like myeloproliferative neoplasm, and compromises induction of BCR-ABL1⁺B-ALL. SHP2, and specifically, its SH2 domains, PTP activity and C-terminal tyrosines, are essential for BCR-ABL1⁺, but not WT, pre-B-cell proliferation. The mitogen-activated protein kinase kinase (MEK) / extracellular signal-regulated kinase (ERK) pathway is regulated by SHP2 in WT and BCR-ABL1⁺pre-B cells, but is only required for the proliferation of BCR-ABL1⁺cells. SHP2 is required for SRC family kinase (SFK) activation only in BCR-ABL1⁺pre-B cells. RNAseq reveals distinct SHP2-dependent transcriptional programs in BCR-ABL1⁺and WT pre-B cells. Our results suggest that SHP2, via SFKs and ERK, represses MXD3/4 to facilitate a MYC-dependent proliferation program in BCR-ABL1-transformed pre-B cells.

Chondrocytes and osteoblasts differentiate from a common mesenchymal precursor, the osteochondroprogenitor (OCP), and help build the vertebrate skeleton. The signaling pathways that control lineage commitment for OCPs are incompletely understood. We asked whether the ubiquitously expressed protein-tyrosine phosphatase SHP2 (encoded by Ptpn11) affects skeletal lineage commitment by conditionally deleting Ptpn11 in mouse limb and head mesenchyme using "Cre-loxP"-mediated gene excision. SHP2-deficient mice have increased cartilage mass and deficient ossification, suggesting that SHP2-deficient OCPs become chondrocytes and not osteoblasts. Consistent with these observations, the expression of the master chondrogenic transcription factor SOX9 and its target genes Acan, Col2a1, and Col10a1 were increased in SHP2-deficient chondrocytes, as revealed by gene expression arrays, qRT-PCR, in situ hybridization, and immunostaining. Mechanistic studies demonstrate that SHP2 regulates OCP fate determination via the phosphorylation and SUMOylation of SOX9, mediated at least in part via the PKA signaling pathway. Our data indicate that SHP2 is critical for skeletal cell lineage differentiation and could thus be a pharmacologic target for bone and cartilage regeneration.

PTEN inactivation occurs commonly in human cancers and putatively activates the PI3K/AKT/ mTOR pathway. Activation of this pathway has been involved in resistance to chemotherapy or anti-EGFR/HER2 therapies. We evaluated the combination of PI3K-mTOR inhibitors with chemotherapy or the pan-HER inhibitor dacomitinib in PTEN-deficient patient-derived tumor xenografts (PDX). Three PDXs were selected for their lack of PTEN expression by immunohistochemistry: a triple-negative breast cancer (TNBC), a KRAS G12R low-grade serous ovarian cancer (LGSOC), and KRAS G12C and TP53 R181P lung adenocarcinoma (LADC). Two dual PI3K-mTOR inhibitors were evaluated-PF-04691502 and PF-05212384-in combination with cisplatin, paclitaxel, or dacomitinib. The addition of PI3K-mTOR inhibitors to cisplatin or paclitaxel increased the activity of chemotherapy in the TNBC and LGSOC models; whereas no added activity was observed in the LADC model. Pharmacodynamic modulation of pS6 and pAKT was observed in the group treated with PI3K-mTOR inhibitor. Our research suggests that the addition of a PI3K-mTOR inhibitor may enhance tumor growth inhibition when compared to chemotherapy alone in certain PTEN-deficient PDXs. However, this benefit was absent in the KRAS and TP53 mutant LADC model. The role of PTEN deficiency in the antitumor activity of these combinations should be further investigated in the clinic.

Loss-of-function mutations in TET2 occur frequently in patients with clonal hematopoiesis, myelodysplastic syndrome (MDS), and acute myeloid leukemia (AML) and are associated with a DNA hypermethylation phenotype. To determine the role of TET2 deficiency in leukemia stem cell maintenance, we generated a reversible transgenic RNAi mouse to model restoration of endogenous Tet2 expression. Tet2 restoration reverses aberrant hematopoietic stem and progenitor cell (HSPC) self-renewal in vitro and in vivo. Treatment with vitamin C, a co-factor of Fe2+ and alpha-KG-dependent dioxygenases, mimics TET2 restoration by enhancing 5-hydroxymethylcytosine formation in Tet2-deficient mouse HSPCs and suppresses human leukemic colony formation and leukemia progression of primary human leukemia PDXs. Vitamin C also drives DNA hypomethylation and expression of a TET2-dependent gene signature in human leukemia cell lines. Furthermore, TET-mediated DNA oxidation induced by vitamin C treatment in leukemia cells enhances their sensitivity to PARP inhibition and could provide a safe and effective combination strategy to selectively target TET deficiency in cancer.

Reactive oxygen species are produced transiently in response to cell stimuli, and function as second messengers that oxidize target proteins. Protein-tyrosine phosphatases are important reactive oxygen species targets, whose oxidation results in rapid, reversible, catalytic inactivation. Despite increasing evidence for the importance of protein-tyrosine phosphatase oxidation in signal transduction, the cell biological details of reactive oxygen species-catalyzed protein-tyrosine phosphatase inactivation have remained largely unclear, due to our inability to visualize protein-tyrosine phosphatase oxidation in cells. By combining proximity ligation assay with chemical labeling of cysteine residues in the sulfenic acid state, we visualize oxidized Src homology 2 domain-containing protein-tyrosine phosphatase 2 (SHP2). We find that platelet-derived growth factor evokes transient oxidation on or close to RAB5+/ early endosome antigen 1- endosomes. SHP2 oxidation requires NADPH oxidases (NOXs), and oxidized SHP2 co-localizes with platelet-derived growth factor receptor and NOX1/4. Our data demonstrate spatially and temporally limited protein oxidation within cells, and suggest that platelet-derived growth factor-dependent "redoxosomes," contribute to proper signal transduction.Protein-tyrosine phosphatases (PTPs) are thought to be major targets of receptor-activated reactive oxygen species (ROS). Here the authors describe a method that allows the localized visualization of oxidized intermediates of PTPs inside cells during signaling, and provide support for the "redoxosome" model.

Noonan syndrome (NS) is caused by mutations in RAS/ERK pathway genes, and is characterized by craniofacial, growth, cognitive and cardiac defects. NS patients with kinase-activating RAF1 alleles typically develop pathological left ventricular hypertrophy (LVH), which is reproduced in Raf1L613V/+ knock-in mice. Here, using inducible Raf1L613V expression, we show that LVH results from the interplay of cardiac cell types. Cardiomyocyte Raf1L613V enhances Ca2+ sensitivity and cardiac contractility without causing hypertrophy. Raf1L613V expression in cardiomyocytes or activated fibroblasts exacerbates pressure overload-evoked fibrosis. Endothelial/endocardial (EC) Raf1L613V causes cardiac hypertrophy without affecting contractility. Co-culture and neutralizing antibody experiments reveal a cytokine (TNF/IL6) hierarchy in Raf1L613V-expressing ECs that drives cardiomyocyte hypertrophy in vitro. Furthermore, postnatal TNF inhibition normalizes the increased wall thickness and cardiomyocyte hypertrophy in vivo. We conclude that NS-cardiomyopathy involves cardiomyocytes, ECs and fibroblasts, TNF/IL6 signalling components represent potential therapeutic targets, and abnormal EC signalling might contribute to other forms of LVH.